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Advances in Genetics Incorporating Molecular Genetic Medicine Edited by Jeffery C. Hall Jay C. Dunlap Department of B...

Author: Jeffrey C. Hall | Jay C. Dunlap | Theodore Friedmann | Francesco Giannelli

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Advances in Genetics Incorporating Molecular Genetic Medicine Edited by

Jeffery C. Hall

Jay C. Dunlap

Department of Biology Brandeis University Waltham, Massachusetts

Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire

Associate Editors

Theodore Friedmann

Francesco Giannelli

Department of Pediatrics Center for Molecular Genetics School of Medicine University of California, San Diego La Jolla, California

Division of Medical and Molecular Genetics United Medical and Dental Schools of Guy’s and St. Thomas’ Hospital London Bridge, London SEl 9RT United Kingdoin

Academic Press San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper.

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Copyright 0 1997 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1997 chapters are as shown on the title pages, if no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2660197 $25.00

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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

DavidJ. Allan School of Life Science, Queensland University of Technology, Brisbane, Queensland 4000, Australia (35) Shawn 1. Anderson National Science Foundation Center for Biological Timing, Department of Biology, University of Virginia, Charlottesville, Virginia

22903 (1) Carol A. Bascom-Slack Department of Microbiology and Molecular Biology, Tufts University, Boston, Massachusetts 021 11 (253) Aman S. Coonar Cardiological Sciences, St. George’s Hospital Medical School, London SW17 ORE, United Kingdom (285) Dean S. Dawson Department of Microbiology and Molecular Biology, Tufts University, Boston, Massachusetts 021 11 (253) George Dickson School of Biological Sciences, Division of Biochemistry, Royal Holloway College, University of London, Egham, Surrey TW20 OEX, United Kingdom (117) Ariberto Fassati School of Biological Sciences, Division of Biochemistry, Royal Holloway College, University of London, Egham, Surrey TW20 OEX, United Kingdom; and Department of Experimental Pathology, UMDS Guy’s Hospital, London Bridge, London SE1 9RT, United Kingdom (117) Elizabeth M. C. Fisher Neurogenetics Unit and Department of Biochemistry and

Molecular Genetics, Imperial College School of Medicine at St. Mary’s, London W2 IPG, United Kingdom (155) Brian V. Harmon School of Life Science, Queensland University of Technology, Brisbane, Queensland 4000, Australia (35) Steve A. Kay National Science Foundation Center for Biological Timing, Department of Biology, University of Virginia, Charlottesville, Virginia 22903 (1) William J. McKenna Cardiological Sciences, St. George’s Hospital Medical School, London SW 17 ORE, United Kingdom (285)

Contributors

X

Stephen Murphy School of Biological Sciences, Division of Biochemistry, Royal Holloway College, University of London, Egham, Surrey TW20 OEX, United Kingdom ( 117)

Luigi 0. Notarangelo Department of Pediatrics, University of Brescia, 1-25123 Brescia, Italy (57) Lyle 0. ROSSInstitute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030 (253) Allen Shearn Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 (207)

C. 1. Edvard Smith Department of Clinical Immunology and Center for BioTechnology, Department of Bioscience at Novum, Karolinska Institute, S-141 57 Huddinge, Sweden (57) Lisa Timmons Department of Biology, The Johns Hopkins University, Baltimore, Maryland 2 1218 (207)

Phototransduction and Circadian Clock Pathways Regulating Gene Transcription in Higher Plants Shawn 1. Anderson and Steve A. Kay

National Science Foundation Center for Biological Timing Department of Biology, University of Virginia Charlottesville,Virginia 22903

1.MTRODUCTION Temporal regulation of biological functions is of particular importance in plants that must respond to their environment in situ. Thus, many plant functions exhibit rhythms in activity in response to diurnal changes in the environment. Cellular and physiological functions that continue to oscillate rhythmically under constant environmental conditions with a period of approximately 24 hr are termed circadian rhythms and are under the control of an endogenous circadian clock. Regulation by the circadian clock serves to synchronize and optimize cellular and physiological processes in anticipation of periodic changes in the plant’s environment. Plants have long been an amenable biological system for the study of circadian rhythms (Sweeney, 1987). Over 250 years ago, de Mairan (1729) first demonstrated the endogenous nature of circadian rhythms when he observed that the rhythmic leaf movements of Mimosa persisted in the absence of a light-dark cycle. Research on plant systems since that time has described, at the phenomenological level, a wide range of plant activities in diverse species that are regulated by the circadian clock, including enzyme synthesis and activity, photosynthetic capacity, cell division, phototaxis, stomata1 opening, flower opening, and photoperiodic control of flowering. Molecular genetics studies have revealed circadian clock regulation of plant gene expression. Circadian regulation of plant gene expression has been demonstrated at the levels of transcription (Kay, 1993), translation (Morse et al., 1990), and posttranslational modification (Nimmo et al., 1987). Several articles provide compendiums of plant genes whose expression Advances in Genetics, Val. 35

Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved.

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is regulated by the circadian clock, and the interested reader is directed to these excellent reviews (Piechulla, 1993; Beator and Kloppstech, 1994; McClung and Kay, 1994). Although the molecular basis of circadian rhythmicity has not been defined for any single biological system, conceptually the circadian clock consists of three basic components: (1) input signal transduction pathways from the environmental cues (primarily light-dark signals), which entrain the activity of the circadian oscillator and determine the phase of the free-running rhythm; (2) a circadian oscillator that generates the rhythm; and (3) output signal transduction pathways for the temporal regulation of specific biological processes (Takahashi, 1993). Plants, in particular the study of the regulation of CAB gene expression, serve as one of the best model systems for the investigation of the integration of the phototransduction and circadian clock pathways regulating gene transcription. The use of combined biochemical, molecular, and genetic approaches has advanced our understanding of each of the three components of the circadian system involved in the regulation of plant gene transcription. In the present chapter, the identity and function of plant photoreceptors, the input signal transduction pathways from the photoreceptors, the analysis of the circadian oscillator by genetic screens for mutants with altered regulation of CAB expression, and the characterization of the output signal transduction pathways starting from the identification of circadian clock- and light-responsive cis-acting CAB promoter elements will be considered.

II. THE CAB GENE: A MODEL FOR CIRCADIAN CLOCK AND LIGHT REGULATION OF TRANSCRIPTION Expression of the plant CAB gene family, encoding the chlorophyll a and b binding proteins of the light-harvesting complex of photosystem 11, is one of the most extensively studied plant gene families whose expression is circadian clock-regulated at the level of transcription (Fejes et al., 1990; Millar and Kay, 1991; Nagy et al., 1988; Paulsen and Bogorad, 1988; Piechulla, 1993; Wehmeyer et al., 1990). Analysis of CAB gene expression has revealed that a network of regulatory pathways controls CAB transcription. In addition to regulation by the circadian clock, CAB expression is cell-type-specific (Edwards and Coruzzi, 1990), regulated developmentally (Brusslan and Tobin, 1992; Ha and An, 1988), and is regulated by hormones (Chang and Walling, 1991;Flores and Tobin, 1988) and by sugars (Jang and Sheen, 1994), and photoregulated by blue light (Marrs and Kaufman, 1991) and by phytochrome (Karlin-Neumann et al., 1988). The potential for the combinatorial interaction of a multiplicity of inputs to CAB regulation provides a means of generating complex expression patterns. Moreover, the identification of multiple inputs to CAB regulation makes it likely that signal transduction inter-

1. Gene Transcription in Higher Plants

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mediates and cis- and trans-acting elements will be identified, which may mediate multiple controls. Therefore, an improved understanding of CAB regulation will require analysis in terms of the regulatory network. This is of particular importance in the case of light and clock regulation, because light-mediated pathways have already been demonstrated to be involved in the regulation of the phase and period of the circadian clock (Millar et al., 1 9 9 5 ~ ) . In plants, as in most circadian systems, light is a primary signal for resetting the phase of the circadian clock, entraining its activity to the daily light-dark cycle (Giuliano et al., 1988; Nagy et al., 1993), and regulating the period of the . have evolved at least three phocircadian oscillation (Millar et al., 1 9 9 5 ~ )Plants toreceptor systems for perceiving and responding to changes in light fluence and wavelength in their environment, as defined by the wavelengths absorbed by the photoreceptor: ( 1) the family of red and far-red-light-absorbing photoreceptors, the phytochromes (Furuya, 1993); (2) blue-UV-A photoreceptors (Ahmad and Cashmore, 1993); and (3) UV-B photoreceptors (Senger and Schmidt, 1994). Discussion will be restricted here to the phytochromes and blue-light photoreceptors and their roles in mediating the regulation of CAB gene expression. Plant responses to UV-B have been reviewed elsewhere (Stapleton, 1992) and will not be considered further here. In plants grown from seed in complete darkness (etiolated seedlings), little or no CAB mRNA can be detected. Upon transfer of etiolated seedlings to the light, CAB transcription is induced to a high level with a concomitant increase in CAB protein levels. A brief pulse of red light is sufficient for the induction of CAB expression in etiolated seedlings. The red-light induction of CAB expression is attenuated by subsequent far-red illumination, demonstrating that phytochrome mediates this response (Quail, 1991). Similarly, a single pulse of blue light acting through a nonphytochrome photoreceptor system induces CAB expression in etiolated pea seedlings (Marrs and Kaufman, 1991). Gao and Kaufman (1994) observed blue-light induction of CAB1 gene expression, but not of CAB2 or CAB3, in etiolated Arabidopsis seedlings by RNase protection assays. In green plants grown under a light-dark (LD) cycle, CAB expression shows a complex oscillation. The CAB transcript begins to increase in abundance prior to the onset of light to a maximum in late morning and declines to a minimum in late evening. Increased CAB transcription in anticipation of the D to L transition suggests that light is not required for the cyclic component of the expression pattern. The persistence of the oscillation in CAB mRNA abundance with a period of ca. 24 hr in plants transferred from LD to continuous light (LL), a condition under which phytochrome is constantly activated, is evidence for light-independent circadian clock regulation of CAB expression. These data are also consistent with the hypothesis that the clock is a permissive regulator of CAB expression, allowing phytochrome induction of CAB gene transcription at defined times during the day.

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Members of the Kay laboratory (Millar et al., 1992a,b) have developed transgenic tobacco and Arabidopsis lines containing reporter gene fusions of the Arabidopsis CAB2 promoter to the firefly luciferase (Lw)coding region (cab2: :luc). CAB expression is assayed in these lines by spraying the plants with luciferin and measuring light emission with a low-light video camera and photon-counting image processor. The luciferase-catalyzed bioluminescence arising from the reporter gene construct in the transgenic plants accurately reflects the transcription pate terns of the endogenous CAB gene in both tobacco and Arabidopsis. By monitoring expression of the cab2::luc transgene, Millar et al. (199213) demonstrated that CAB2 transcription oscillates with a low amplitude in etiolated tobacco seedlings prior to light treatment. This result indicates that the circadian clock regulates CAB transcription before apparent photoreceptor activation. The period of the oscillation in bioluminescence in etiolated seedlings is ca. 30 hr (Millar et al., 1992a). In LD-grown plants transferred to continuous darkness (DD), the period of the oscillation in CAB transcription lengthens to 30-36 hr (Millar et al., 199513).The longer period for the CAB oscillation in etiolated seedlings or in LDgrown plants transferred to DD, relative to ca. 24 hr in plants transferred to LL, suggests that light modulates the period of the circadian oscillation. In the bioluminescent marine alga Gonyaulax polyedra, constant illumination has been shown to modulate the free-running period of the bioluminescent glow rhythm in a manner dependent upon both wavelength and intensity (Roenneberg and Hastings, 1988).Specifically, blue light shortens and red light lengthens the period of the glow rhythm. The degree of period shortening or lengthening is directly dependent upon the intensity of blue or red light, respectively. In Gonyaulax the effect of constant light on the period of the glow rhythm appears to reflect the entrainment pathways to the clock and implicates the involvement of two photoreceptors in period modulation (Roenneberg and Hastings, 1988). In a similar experiment, Arabidopsis seedlings containing the cab2: :lw transgene were grown in LD and transferred to constant red or blue light to assay the entrainment pathways regulating the period of the circadian oscillation in CAB2 gene expression. Both constant red and blue light shortened the period to almost the same extent as white LL, indicating that both phytochrome and blueresponsive phototransduction pathways modulate the period of the circadian oscillation (Millar et al., 1995b). The threshold intensity of white light sufficient for shortening of the cab2: :lwexpression rhythm to ca. 24 hr is on the order of at least 1-2 pmol m-2 s-l (C. Strayer and S. Kay, unpublished results), and increasing the light intensity from 30 to 600 pmol mP2 s-l did not induce any further period shortening (Millar et al., 1 9 9 5 ~ )The . observation that a broad range of light intensities results in a similar period for the cab2::luc expression rhythm is ecologically relevant, providing evidence that the period of the circadian oscillation does not change with the normal fluctuations in light intensity (i.e.,

5

1. Gene Transcription in Higher Plants Photomorphogenic Development

+ CAB

Blue Light --+ Photoreceptors

++

Photomorphogenic Development

Figure 1.1. Model of the proposed phototransduction and circadian clock pathways that regulate CAB transcription. Light signals mediated by phytochrome and the blue-light photoreceptor system regulate several aspects of the photomorphogenic development of &cot seedlings, including inhibition of hypocotyl elongation, leaf expansion, and chloroplast differentiation. Pathways from these photoreceptors to the clock must also exist for regulating the phase and period of the circadian oscillation in CAB gene expression. In addition, phytochrome- and blue-light photoreceptor-mediated pathways are proposed to regulate positively CAB gene expression at the level of transcription. The circadian clock subsequently regulates the timing of CAB gene expression generating the circadian oscillation in CAB transcription. The circadian clock is proposed to function as a permissive regulator of CAB transcription, allowing phytochrome induction of CAB transcription during a certain period of the day (Kay, 1993).

changes due to cloud and foliage cover and the angle of the incident light) during the regular daylight period. A model describing the interactions between photic and circadian clock signals to regulate CAB transcription based on the preceding observations of the effect of different light regimes on the oscillation in CAB expression is presented in Fig. 1.1. Light- and circadian clock-mediated pathways can function independently to regulate the amplitude and timing of CAB transcription, respectively, and phytochrome- and blue-light-mediated signals can regulate CAB expression indirectly by modulating the phase and period of the circadian oscillator. Several aspects of the plant model not yet described include: (1) Which form or forms of phytochrome modulate the period of the circadian oscillation and regulate the amplitude of CAB gene expression in green plants and in etiolated plants in response to a red-light flash?( 2 ) Which form or forms of blue-light photoreceptors modulate the period of the circadian oscillation and regulate the

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amplitude of CAB gene expression in green plants and in etiolated plants in response to a blue-light flash? (3) What are the molecular components of the signal transduct ion pathways from the clock, phytochrome, and blue-light photoreceptor? (4) What are the molecular components of the circadian oscillator? (5) Do the signal transduction pathways from the clock, the phytochrome, and a bluelight photoreceptor regulate transcription through distinct cis-acting elements (i.e., independent pathways), or do they converge to act upon the same regulatory sequence? In the following sections, we review advances made with respect to these outstanding questions.

111. PLANT PHOTORECEPTORS A. Phytochrome The red- and far-red-light photointerconvertible phytochromes are the predominant and best-characterized plant photoreceptors and have been the topic of several extensive review articles (Furuya, 1993; Vierstra, 1993; Furuya and Song, 1993; Quail, 1994a). Phytochromes exist as a dimer of two ca. 120-kDa apoproteins each with a covalently linked, linear tetrapyrrole chromophore (Furuya and Song, 1993). The phytochrome holoprotein (phy) (see Quail et al., 1994, for a review of phytochrome nomenclature) is synthesized in the red-absorbing form, Pr, which is biologically inactive for most phytochrome-mediated responses. Upon the absorption of red light (A,, ca. 666 nm), Pr is photoconverted to the biologically active far-red-absorbing form, Pfr (Amax ca. 730 nm). The formation of Pfr initiates a signal transduction cascade that ultimately regulates multiple cellular functions, including the transcriptional regulation of nuclear genes for several chloroplast-localized proteins. The effect of red-light irradiation can be reversed for many Pfr-regulated processes by the absorption of far-red light, which largely converts Pfr back to Pr. Phy therefore is often described as a light-regulated molecular switch for the regulation of plant development and gene expression (Gilmartin et ul., 1990; Quail, 1991). Physiological, spectroscopic, and immunological studies have revealed that plants contain two operationally defined types of phytochrome: type I, a light-labile species most abundant in etiolated tissue, and type 11, a light-stable species most abundant in lightsgrown tissue. Molecular studies have shown that the phytochrome apoproteins are encoded by a small multigene family in angiosperms. Arubidopsis thliana contains five phytochrome genes (PHYA-PHYE) (Quail, 1994b). The PHYA gene, encoding the type I phytochrome species, is highly expressed in etiolated plants and is repressed by Pfr (Somers and Quail, 1995). Furthermore, the phyA protein is rapidly degraded in the light via a ubiquitin-dependent pathway (Jabben et al., 1989). PHYB encodes a type I1 phy-

1. Gene Transcription in Higher Plants

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tochrome species. A comparison of the expression of phytochrome B gene promoter-p-glucuronidase (PHYB::GUS) gene fusions in transgenic Arabidopsis demonstrated that PHYB expression in etiolated seedlings is repressed twofold by white light (Somers and Quail, 1995). However, phytochrome B is stable in the Pfr form (Somers et al., 1991), and as a result phyB is the most abundant species in light-grown plants (Quail, 1994a). The identification of multiple PHY genes, their differential expression, and the differential stabilities of the encoded proteins thus provide a clear molecular basis for the operationally distinct phytochrome species. Furthermore, the multiple phytochrome species may have specialized and/or overlapping regulatory roles in plant development. Physiological, molecular, and genetic approaches have been applied to determine which phytochrome species is associated with specific developmental processes. Of these, the analyses of CAB expression in phytochrome-overexpressing lines, in mutants with ieduced levels of all phy species, and in type-specific Arabidopsis phytochrome mutants provide the most powerful tools for the assignment of specific roles for the various phy species in aspects of circadian clock and light regulation of CAB gene regulation. Brief red-light treatment of etiolated seedlings induces a cyclic CAB expression pattern with a transient peak at ca. 4 hr after the onset of light treatment, a second broader peak at ca. 20 hr. (Millar et al., 1992a; Axlerson et al., 1994), and subsequent peaks observed up to 32 hr later (Millar et al., 1992a; Nagy et al., 1993). The transient peak in CAB expression at 4 hr is an acute response to phytochrome activation independent of the circadian clock, with the subsequent peaks corresponding to the high-amplitude cyclic oscillation in CAB expression induced by phytochrome activation (Kay 1993). Evidence of the requirement for photic input for the acute transient increase in CAB expression comes from studies of the effect of cyclic heat-shock treatments on CAB mRNA expression in etiolated barley. Beator et al. (1992) observed that cyclic heat-shock treatments could induce and synchronize the rhythmic oscillation in CAB expression in etiolated barley, with the exception that the acute transient increase in CAB expression was not observed in the heat-shock-treated plants. They interpret this result as evidence for a direct effect of red-light input to induce the acute peak in CAB expression. It has been hypothesized that the acute transient peak is driven by light-labile phyA, with the subsequent peaks driven by a more stable form of phytochrome (Kay, 1993), such as phyB. hyl (long hypocotyl) is a chromophore mutant of Arabidopsis that contains little or no spectrophotometrically detectable levels of all phy species in etiolated seedlings (Chory et al., 1989b). By assaying the acute red-light induction of CAB expression in two hyl alleles, Chory et al. (1989b) compared the accumulation of CAB mRNA 2 hr after the red-light treatment to that in etiolated wild-type seedlings. In etiolated seedlings of the hy l -21.84N allele, containing -4% of the spectrophotometrically detectable phytochrome of wild type, CAB

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mRNA accumulated to only 40% of that in wild type and was photoreversible by far-red light. These results demonstrate that one or more phy species are required for the acute induction of CAB expression in response to red-light treatment. In addition, in the hyl-d412 allele, containing ca. 25% of the spectrophotometrically detectable phy of wild type, CAB mRNA accumulated to levels similar to that in wild type following an inductive red-light flash, demonstrating that photoreversible phytochrome levels 25% of that of wild type are sufficient for wildtype levels of CAB induction in etiolated Arabidopsis (Chory et al., 198913).This result also illustrates that caution must be exercised when interpreting results from experiments using mutants that are not null alleles. Although the hyl alleles showed photomorphogenic phenotypes associated with decreased spectrophotometrically detectable phytochrome (i.e., long hypocotyls and incomplete chloroplast and leaf development), the effect of the mutations on the photoregulation of CAB expression in etiolated seedlings was not dramatic. No difference in CAB mRNA accumulation was observed in high-fluence-rate white light-grown hy 1 seedlings compared to that in wild type, suggesting that very low levels of phy are sufficient for wild-type expression levels in light-grown plants or, alternatively, that phytochrome does not play a significant role in CAB expression in green plants (Chory et al., 1989b). The conclusion that phytochrome does not play a significant role in CAB expression in green plants is unlikely, as evidenced by the damping of the CAB oscillation under DD (Kay, 1993). In green plants transferred from a LD cycle to DD, a condition of declining phytochrome activation, a rapid reduction in the peak levels of the CAB oscillation (or damping) is observed. The rate of damping of CAB expression in DD directly correlates with treatments that increase or decrease Pfr decay (Kay, 1993). Damping is attenuated in transgenic tobacco plants overexpressing the rice PHYA gene and containing 5 - to 10-fold more phytochrome than wild-type plants (Stockhaus et al., 1992). Rice phyA is more stable than tobacco phyA (Stockhaus et al., 1992) and, when present in elevated amounts, contributes to maintaining the level of CAB expression in green plants in DD. These results are consistent with the conclusion that phytochrome activates high-level CAB expression in green tissue. In the Kay laboratory, the cab2: :lucmarker tobacco mosaic virus translational enhancer) has been crossed into the hyl-100 mutant and tested for the involvement of phytochrome in period control of the circadian clock (Millar et al., 1 9 9 5 ~ )The . low-light imaging system was used to follow the activity of the cab2::R::luctransgene in the hyl mutant background relative to wild type when grown under a LD cycle and transferred to DD, continuous red light (R), or continuous white light (LL). Upon transfer to R, expression of the cab2::R::luctransgene is reduced by ca. 40% relative to LL in both wild type and hyl , consistent with the conclusion that wavelengths of light other than red also contribute to the level of CAB expression. In addition, the oscillation does not damp in R as in DD, suggesting that one or more phytochrome species contribute to the ampli-

:a:

(a,

1. Gene Transcription in Higher Plants

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tude of CAB expression observed in LL (Millar et al., 1 9 9 5 ~ )In . wild type, expression of the cab2::Q::Iuc transgene oscillates with a period of 30-36 hr in DD and 24.7 hr in LL. Under R, the period in wild type is shortened to ca. 25 hr, demonstrating that red-responsive photoreceptors alone can mediate shortening of the period relative to DD. In hy J the period of the oscillation is close to that of wild type under LL, but is significantly longer than that of wild type under R. The hyf -100 allele is a leaky mutant; therefore, the low level of phytochrome remaining may be sufficient for period shortening relative to DD. Moreover, these results clearly show that one or more phytochrome species contribute to period control of the circadian oscillation in CAB expression. By using a type-specificphyA null mutant and a phyB null mutant of Arabidopsis, Reed et al. (1994) demonstrated phytochrome induction of CAB expression in etiolated seedlings of the mutants 4 hr after an inductive red-light pulse that was photoreversible by far-red light. CAB mRNA accumulated in these mutants to a level similar to that observed in wild-type seedlings. These results suggest that either phyA and phyB have overlapping roles in the acute phytochrome induction of CAB expression and/or an additional phy species can mediate the induction of CAB expression by red light in etiolated tissue. Similarly, Sun and Tobin (1990) observed equivalent levels of CAB mRNA in etiolated seedlings of both wild type and an Arabidopsis phyB mutant 2 hr after an inductive red-light pulse. However, it is not clear whether a null phyB allele was used in this particular study. In contrast to the phytochrome induction of CAB expression observed in the single phyA and phyB mutants, substantially less CAB mRNA accumulated 4 hr after a red-light pulse in etiolated seedlings of the phyA:phyB double mutant relative to wild type, but a peak in CAB accumulation was observed 12 hr after the light pulse in the phyA:phyB mutant (Reed e t al., 1994). The absence of the acute transient increase in CAB expression in the phyA:phyB double mutant, although this response is observed in the individual phyA and phyB mutants, is evidence that phyA and phyB may have redundant roles in mediating the acute transient increase in CAB expression and that a species other than phyA or phyB (i.e., phyC, -D, or -E) may mediate the subsequent peaks in CAB expression. I t should prove informative to assay circadian clock and phytochrome regulation of expression of the cab2::R: :luc transgene in phyA, phyB, and the phyA:phyB double mutant at high time resolution and in more detail, particularly in view of the fact that much of the preceding work on CAB expression in these mutants did not take into account the possibility of genespecific differences in CAB regulation by phytochrome (Karlin-Neumann et al., 1988; Kay and Millar, 1992).

B. Blue-light photoreceptors Blue light potentiates a number of important developmental responses, including phototropism, inhibition of hypocotyl elongation, regulation of stomata1 aper-

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ture, and induction of gene expression. Because the absorption spectrum for bluelight-mediated responses overlaps with the phytochrome absorption spectrum, it is important to investigate blue-light responses under rigorously controlled conditions that preclude a contribution from phytochrome. For example, the specific effect of blue-light treatments is often assayed in seedlings grown in continuous dim red light to saturate phytochrome responses or in etiolated seedlings pulsed with blue light followed by far-red light to test for the participation of photoreversible phytochrome in the response. hyl , phyA, phyB, the phyA:phyB double mutant, and other phytochrome mutant lines provide potentially unparalleled systems for the assay of phytochrome-independent, blue-light-mediated gene transcription. Like the phytochrome family of photoreceptors, which may participate in both overlapping and unique regulatory pathways, the possibility exists for multiple blue-light photoreceptors in plants (Liscum and Hangarter, 1994; Short and Briggs, 1994; Kaufman, 1993; Briggs, 1993). The availability of several Arabidop sis mutants with altered response to blue light provides a genetic approach for the identification of specific roles in CAB gene regulation for the blue-light-regulated pathways defined by these mutations. The hy4 (Ahmad and Cashmore, 1993) photomorphogenic mutant of Arabidopsis lacks blue-light-dependent inhibition of hypocotyl elongation, hut shows normal phytochrome-mediated inhibition of hypocotyl growth by far-red light. The gene corresponding to the HY4 locus has been cloned and shown to encode the apoprotein of a putative blue-light photoreceptor involved in the inhibition of hypocotyl growth (Ahmad and Cashmore, 1993). The HY4 protein contains significant homology in the amino terminus to microbial DNA photolyases, a class of proteins that catalyze the blue-light-dependent repair of UVB-induced thymidine dimers (Sancar, 1990). The photolyases contain two functional blue-light-absorbing chromophore moieties, a reduced flavin and a pterin or deazaflavin derivative. The highest homology of the HY4 protein with the photolyases is in the chromophore binding regions. The HY4 protein (CRY1) has been shown to hind flavins in vivo (Lin et al., 1995) demonstrating that HY4 indeed encodes a blue light photoreceptor. Gao and Kaufman (1994) demonstrated blue-light induction of total CAB mRNA levels to the same extent in etiolated hy4 and wild-type seedlings, suggesting that the putative HY4 photoreceptor is not involved in the blue-light regulation of CAB gene expression. Millar et al. (1995~) examined the role of blue light in the regulation of both the level of CAB gene expression and the period of the circadian oscillation. The cab2: :luc expression levels in transgenic wildtype and hyl seedlings transferred to continuous blue light (B) were reduced relative to the levels observed in LL to an extent similar to that in R. This suggests that blue-light-mediated pathways also contribute to the level of CAB expression observed in LL. The period of the CAB2 oscillation in B is shortened to nearly

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the same extent in both wild type and hyl , whereas continuous red light was not as effective in hyl as in wild type at shortening the period of the oscillation. The absence of a differential effect for B in hyl relative to wild type demonstrations that blue light does not function through the phytochrome photoreceptor to regulate period and implicates the specific involvement of a blue-responsive photoreceptor(s) in the control of the period of the circadian oscillation. Several Arabidopsis mutant lines (nphl JK218, JK224, and JK229) have been identified that exhibit reduced phototropic curvature in response to unilateral blue-light irradiation (Liscum and Briggs, 1995; Khurana and Poff, 1989). Of these, nphl and JK224, an anallele of nphl, have been proposed to be blue-light photoreceptor mutants. Interestingly, both nphl and JK224 are deficient in a ca. 120-kDa plasma membrane protein (Reymond et al., 199213) previously shown to be rapidly phosphorylated in a blue-1ight.dependent fashion and proposed to be an early step in the phototropism signal transduction pathway (Reymond et al., 1992a). These results suggest that the putative photoreceptor defined by the nphllJK224 locus either may indeed be the blue-light-dependent phosphoprotein and/or may represent the blue-light photoreceptor that mediates this phosphorylation event. The phototropism mutants JK224 and JK218 exhibit normal inhibition of hypocotyl growth (Liscum et al., 1992), while the hy4 mutant shows a normal phototrophic response (Chory, 1992), demonstrating that blue-light-dependent inhibition of hypocotyl growth and phototropism are genetically separable responses. Therefore, the observation of any changes in the expression of the transgene in any or each of these mutants would allow us to begin to identify which if either of these genetically defined blue-light signal transduction pathways may interact with the circadian clock and may be involved in the regulation of CAB gene expression.

IV. IDENTIFICATION OF SIGNAL TRANSDUCTION INTERMEDIATES The mechanism by which the plant photoreceptors transduce light signals remains largely unknown. Studies based on biochemical (described in this section) and genetic (described in Section V) approaches, however, have dramatically advanced our understanding of both phytochrome and blue-light signal transduction. A blue-light-activated G protein associated with the plasma membranes isolated from the apical buds of dark-grown peas has been identified (Warpeha et al., 1991). The activation of this G protein appears to be blue-light-specific, as no GTPase activity was observed upon irradiation with an equal fluence of red light. The fluence threshold for G protein activation is similar to that for blue-light-induced CAB gene expression in pea, suggesting G protein involvement in bluelight-mediated CAB transcription. Fluorescence studies indicate that the pho-

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toreceptor mediating blue-light activation of the G protein is likely to be a plasma-membrane-associatedflavoprotein (Warpeha et al., 1992) and is probably distinct from the photoreceptor involved in blue-light-mediated phototropism and phosphorylation on the basis of differences in their sensitivity to quenchers of activated flavins, tissue localization, and light sensitivity (reviewed in Short and Briggs, 1994). Results from several studies have also demonstrated the involvement of heterotrimeric GTP-binding regulatory proteins (G proteins) in the phytochrome regulation of gene expression. Treatment of etiolated oat seedlings with cholera toxin, an activator of G proteins, induced CAB gene expression and inhibited PHYA expression (Romero et al., 1991). Similarly, cholera and pertussis toxin modulation of G protein activity in a dark-adapted photoautotrophic soybean cell culture was used to show that G proteins participate as an early step in the phytochrome-dependent induction of CAB gene expression (Romero and Lam, 1993). An elegant series of experiments involving the microinjection of various putative signaling molecules, as well as agonists and antagonists of these molecules, into single hypocotyl cells of the aurea mutant of tomato, which lacks functional phytochrome A (Sharma et al., 1994), has been used to further identify signaling molecules functioning in the phytochrome-mediated pathway controlling gene expression. Injection of purified oat phyA into hypocotyl cells of the aurea mutant induces three phytochrome-regulated responses in a cell-autonomous manner: chloroplast development, induction of the expression of a reporter gene construct (CAB::GUS) consisting of a wheat CAB gene promoter fused to the pglucuronidase gene (GUS), and anthocyanin production (Neuhaus et al., 1993). Coinjection of the G protein antagonists GDP-P-S and pertussis toxin together with phyA blocked the phytochrome-mediated responses. The G protein agonists GTP-yS and cholera toxin induced a cellular response indistinguishable from that mediated by the injection of phyA, confirming that one or more G proteins function early in the phytochrome signaling pathway. Results from several studies demonstrate that both a Ca2+/calmodulindependent step (Lam et al., 1989; Romero and Lam, 1993; Neuhaus et al., 1993) and cGMP (Bowler et al., 1994a) function in the phytochrome signal transduction pathway. Injection of either Ca2+or Ca2+-activatedcalmodulin is sufficient to induce the expression of a CAB::GUS reporter gene construct and partial chloroplast development, including the accumulation of components of photosystem I1 (PSII), the light-harvesting complexes I and I1 (LHC I and II), ATP synthase, and rubisco (ribulose bisphosphate carboxylase/oxygenase). However, Ca2+/calmodulininjection did not induce anthocyanin biosynthesis (Neuhaus et al., 1993). Injection of Ca2+/calmodulininhibitors blocked the G-protein-mediated induction of CAB: :GUS expression and partial chloroplast development (Bowler et al., 1994a). In comparison, injection of cGMP induces both anthocyanin production and the expression of a reporter gene construct consisting of

1. Gene Transcription in Higher Plants

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the promoter of the chalcone synthase gene (CHS) fused to GUS (CHS::GUS), but does not induce CAB::GUS expression or chloroplast maturation (Bowler et al., 1994a).Anthocyanin production and induction of CHS::GUS expression mediated by G protein activation are inhibited by coinjection of the cGMP inhibitor Rp-cGMPS. These results demonstrate that both Ca2+/calmodulin and cGMP function downstream of the G proteins in separate pathways to mediate subsets of the phytochrome-induced responses (Bowler et al., 1994a,b). Furthermore, in the absence of phyA, injection of both Ca2+/calmodulinand cGMP is required to fully induce chloroplast maturation, including the production of PSI and cytochrome bdcomponents, and the induction of expression of a reporter gene construct con. taining the promoter of the ferredoxin NADP’ oxidoreductase gene (encoding an extrinsic PSI-associated protein) fused to GUS (Bowler et al., 1994a). O n the basis of the preceding results, a model for phytochrome signal transduction pathways has been proposed in which the Pfr form of PHYA activates one or more G proteins, which in turn activate downstream signaling pathways regulating subsets of phytochrome-mediated responses (Fig. 1.2) (Bowler et al., 1994a). In one pathway, G-protein-mediated increases in cGMP levels result in the activation of genes encoding proteins involved in anthocyanin production. In the second pathway, G protein activation results in increased cellular Ca2+levels, producing Ca2+-activatedcalmodulin, which subsequently induces the transcription of PSII, ATPase structural genes, CAB and RBCS and partial chloroplast development. Signals from both the Ca2+/calmodulinand cGMP pathways must converge at some point and are required for the transcription of genes encoding ,cGMP phytochrome A +G

-j--f;hocyanin

protein(s) Cyt-bfif

‘Caz+-b

CaM

PSI1 LHCVll (CAB) ATP synthase RUBISCO

Chloroplast Development

Figure 1.2. Proposed model of the biochemical pathways for phytochrome A signal transduction based on microinjection experiments with the phyA-deficient aurea mutant of tomato (modified from Bowler eta!. , 1994a). phyA signaling involves the activarion of one o r

more heterotrimeric G proteins and the subsequent participation of three different pathways to regulate subsets of phyA-mediated responses. The cGMP-dependent pathway regulates anthocyanin biosynthesis. The Ca2+/calmodulin(CaM) pathway regulates the synthesis of one subset of components [PSII,LHCI/II (i.e., CAB gene expression), ATP synthase, and rubisco] required for chloroplast development. Signals from both the cGMP and Ca2+/CaMpathways converge at an as yet undefined point and are required to regulate the synrhesis of another suhset of chloroplast components [PSI and cytochrome bJ (Cyt.h,tJ]. The Ca’+/cGMP pathway mediates complete chloroplast development.

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components of the PSI and cytochrome b6f complexes and for complete chloroplast development. The downstream targets of these three positively acting pathways are divided along functional lines, with the cGMP pathway controlling the production of photoprotective anthocyanins and the Ca2+- and Ca2+/cGMP-dependent pathways controlling the expression of genes encoding chloroplast-localized proteins and chloroplast development. Differential regulation of the respective pathways has been proposed as a mechanism for regulating the relative levels of photosynthetic complexes and photoprotective anthocyanins (Bowler et al., 199413). Accordingly, manipulation of signal flow through these respective pathways has been used to demonstrate that high cGMP levels negatively regulate the Ca2+and Ca2+/cGMP-dependent pathways and, conversely, that high Ca2+/calmodulin levels negatively regulate the cGMP pathway. Termed reciprocal control (Bowler et al., 1994b), the negative interactions between these pathways may be both ecologically and physiologically relevant. For example, the negative regulation of the Ca2+-and Ca2+/cGMP-dependent pathways by high cGMP may prevent the development of photosynthetic competency prior to the production of sufficient photoprotectants, and the Caz+/calmodulinpathway may suppress the cGMP pathway once high levels of anthocyanins are no longer required. Moreover, the Ca2+/cGMP-dependentpathway has a ca. 10-fold lower requirement for cGMP than does the cGMP-dependent pathway, which would allow for chloroplast development in the absence of anthocyanin biosynthesis (Bowler et al., 199413). Phosphorylation events downstream of cGMP and Ca2+/calmodulinare likely to play a critical role in transducing the phytochrome signal. In animal signaling pathways, cGMP and Ca2+/calmodulinfunction by the activation of regulatory molecules such as kinases and phosphorylases, and changes in phosphorylation state have been well-documented as a means to alter the activity of transcriptional regulators (Hunter, 1995). The involvement of phosphorylation-dephosphorylation has also been described for regulating the binding activity of trans-acting factors to the promoters of light-regulated plant genes. For example, phosphorylation prevents the binding of the pea nuclear protein AT-1 to the promoters of the CAB and RBCS genes (Datta and Cashmore, 1995), whereas casein kinase 11-mediated phosphorylation of the Arabidopsis G-box binding factor (GBF) stimulates DNA binding activity. Interestingly, light-induced phosphorylation and binding activity of a GBF also correlate with the light-modulated translocation of GBF from the cytoplasm to the nucleus (Harter et al., 1994). Light-dependent chlorophyll accumulation and activation of RBCS (ribulose bisphosphate carboxylase/oxygenase small subunit gene) and C4 pyruvate orthophosphate dikinase gene expression are specifically and effectively inhibited in maize by the protein phosphatase inhibitor okadaic acid, demonstrating that protein phosphatase functions in transmitting signals for light-induced gene expression (Sheen, 1993).

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V. GENETIC ANALYSIS OF PLANT PHOTOTRANSDUCTION PATHWAYS The genetic dissection of the signaling pathways controlling plant development and the expression of light-regulated genes encoding chloroplast-localized proteins have been described in several excellent reviews (Ang and Deng, 1994; Chory and Susek, 1994; Millar et al., 1994). These studies have been based on an alternative approach to identify signal components of light-regulated pathways in plants by the isolation mutants that show a light-grown morphology when germinated and grown in the dark. Mutations in three det (deetiolated) loci (Chory et af., 1989a) and eight cop (constitutively photomorphogenic) (Deng et al., 1991) loci identified by this screen result in pleiotropic phenotypes, including dark-grown seedlings with a short hypocotyl, expanded cotyledons and leaves, altered patterns of cell differentiation, and derepression of light-regulated gene expression. Mutations in these loci are recessive, suggesting that their wild-type gene products function to repress photomorphogenic traits, including CAB transcription in the dark, and that light reverses this repression. Epistasis studies place det (Chory, 1992, 1993) and cop (Deng et al., 1992) downstream of both the phytochromes and blue-light photoreceptor systems. Therefore, some signal pathways from both the phytochromes and the blue-light photoreceptors must converge prior to the common regulatory steps defined by the det and cop mutations. Of the mutants identified, detf , cop1 , cop8, cop9, coplo, and copf f are allelic to the previously identified fusca (fus)seedling lethal mutants (Misera et al., 1994; Castle and Meinke, 1994) and exhibit the most pleiotropic phenotypes. This suggests that the corresponding wild-type alleles participate in early signal transduction steps prior to branching to pathways controlling unique aspects of plant development. In contrast, det2, det3, cop2, cop3, and cop4 define loci controlling subsets of light-mediated responses, and the corresponding wild-type loci therefore probably function downstream of the more pleiotropic loci or in separate pathways, as in the case of DET2 (Chory and Susek, 1994). Several of the DET and COP loci have been cloned and characterized at the molecular level. DETl encodes a ca. 62-kDa hydrophilic protein with two regions of similarity to bipartite nuclear localization signals (Pepper et al., 1994). GUS-DET1 fusions are localized predominantly to the nucleus demonstrating functional nuclear localization signals in DETl ; however, there is no evidence for DNA binding activity by DETl (Pepper et al., 1994). No differences in DETl mRNA levels were observed between light- or dark-grown plants or in several photomorphogenic mutants, suggesting that DETl functions in photomorphogenesis after posttranscriptional modification and/or by protein-protein interaction. Several transcriptional repressors have been identified that do not bind DNA directly, but rather regulate transcription via protein-protein interactions mediated by amphipathic helices [e.g., SIN3 (Wang and Stillman, 1993), SSN6 (Keleher et al., 1992), and Id (Benezra et al., 1990)]. DETl contains 25% am-

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phipathic helices, consistent with the hypothesis of the requirement for protein-protein interactions for DETl function. The structure of the deduced COPl protein provides several possible mechanisms for the mode of action of COPl in plant phototransduction. The 76.2-kDa COPl protein contains an amino-terminal zinc binding motif, a middle domain with a potential coiled-coil structure, and a carboxy-terminal portion with homology to the 6-subunit of heterotrimeric G proteins (Deng et al., 1992). The similarity of COPl to the 6-subunit of trimeric G proteins is consistent with the demonstrated participation of heterotrimeric G proteins in phytochrome and blue-light regulation of gene expression. The domain with homology to the G, proteins consists of multiple WD-40 repeats, which are likely to be involved in protein-protein interactions in a number of regulatory proteins (van der Voorn and Ploegh, 1992). COPl may, therefore, interact with other proteins via the WD-40 repeats and/or the coiled-coil domain. For example, the yeast factor TUPl interacts through its WD-40 domain with the CYC8 protein and, via interaction with sequence-specific DNA binding proteins, functions as a global transcriptional repressor, suggesting a second mode of action for the repression of photomorphogenesis by COPl. Furthermore, it has been proposed that COPl may function in light-regulated RNA splicing (Meyerowitz, 1995), as WD-40 repeats are also found in proteins required for RNA splicing. TAF,,80, a component of the TFIID complex of Drosophila, shows high sequence similarity to COPl (Dynlacht et al., 1993), suggesting that COPl alternatively may mediate transcriptional repression in the dark by directly interacting with the TFIID complex (Deng, 1994). COP1 lIFUS6 has been cloned and also shown to encode a novel hydrophilic protein of 50.5 kDa (Castle and Meinke, 1994). COP9 encodes a ca. 23kDa protein with no significant homology with other protein sequences, but does contain two putative phosphorylation sites (Wei et al., 1994). Interestingly, COP9 is associated in a large complex (>560 kDa) that requires COP8 and COPl 1/FUS6for either formation or stability of the complex, which also shows some light modulation indicated by changes in elution profiles upon size fractionation. That COP8 and COPl 1/FUS6 are required for the formation or stability of the COP9 complex is consistent with the hypothesis that the products of the DETl, COPl, COP8, COP9, COPIO, and COPl 1 loci function not only in the same pathway but also potentially as part of a complex mediating light regulation of development (Wei et al., 1994). Several Arabidopsis mutants were isolated in a screen to identify components that define downstream branches in the light-regulated signal transduction pathway (Li et al., 1994). The screen was based on the identification of mutants that overexpressed a transgene construct containing two CAB3 promoters fused to two different reporter genes. In this way, mutants were isolated that had altered expression of light-regulated genes in the absence of morphological changes, demonstrating that genetically separable pathways control morphological changes and light-regulated gene expression. The recessive mutations identified by this

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screen define three loci designated docl, 2, and 3 (for dark overexpression of CAB). The doc mutants comprise two phenotypic classes. Mutants of the first class (docl) have elevated expression levels for both CAB and RBCS in the dark, and the mutants of the second cIass (doc2, doc3) specifically derepress CAB expression, but not RBCS expression. The doc phenotypic classes demonstrate that the pathway of CAB expression can be further separated from the regulation of RBCS gene expression.

As the genes for D O C , other DET and C O P loci, and other loci not described here [i.e., HY5, and GUN1 , 2 , and 3 (Chory and Susek, 1994)]are cloned and studied, they will undoubtedly contribute to our understanding of the mechanisms of light signal transduction. The cab2::R::luc marker has been crossed into the d e t l , det2, and cop1 mutants and tested for the involvement of these loci in the control of the circadian oscillation in CAB2 transcription (Millar et al., 1 9 9 5 ~ )The . activity of the transgene was monitored in the mutant backgrounds relative to wild type (WT) when grown under a LD cycle and transferred to DD, B, R, and LL. Damping of the oscillation in the expression of the cab2::R::luc transgene upon transfer to DD is abrogated in detl and to a lesser extent in cop1 and det2. The period of the oscillation in detl is very short (ca. 18 hr) in DD relative to (30-36 hr) WT. Similarly, det2 and cop1 have shortened periods in DD relative to wild type, but not to the same extent as in detl . These data are consistent with the observed activity of phototransduction pathways, independent of light in the det and cop mutants. Shortening of the period of the circadian oscillation in R, B, and LL to 19-20 hr, relative to ca. 24 hr in wild type, was also observed for detl . This suggests that DETl contributes to period control in the light and that detl bypasses or negates a period-lengthening factor. Taken together, these results demonstrate the involvement of the phototransduction pathway defined by detl ,det2, and c5pi in the control of both the amplitude and the period of the circadian oscillation in CAB2 gene expression. The observation that the detl mutation shortens the period in both DD and LL to a greater extent than that observed for the transfer of wild type to LL (Millar et al., 1995c) rules out any secondary effects of photosynthesis on the regulation of the period of the oscillation in CAB gene expression. Since some signal transduction pathways from both the phytochromes and blue-light photoreceptors converge at or prior to the regulatory steps defined by det and cop, it is likely that DET1, DET2, and COP1 function upstream of the circadian clock.

VI. GENETIC ANALYSIS OF THE CIRCADIAN CLOCK IN PLANTS AND CYANOBACTERIA The identification of circadian clock mutants and the molecular genetic analysis of clock genes have proven to be the most informative approach for the elucidation of the molecular basis of circadian rhythmicity. Genetic analysis has identi-

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fied several loci involved in the regulation of the periodicity of circadian rhythms. These include loci in Chlamydomonas, mouse, and hamster and the best-characterized clock loci: frq in Neurospora (Dunlap et al., 1993) and per (Rosbash and Hall, 1989) and tim (Sehgal et al., 1994) in Drosophila. fiq (Aronson et al., 1994) and per (Hardin et al., 1990,1993) have been demonstrated to be components of the feedback loop that constitutes the circadian clock in these respective organisms. In addition, tim is likely to be involved in the Drosophila clock feedback loop as the tim mutation abolishes both the rhythmic oscillation of per mRNA (Sehgal et al., 1994) and the localization of PER to the nucleus (Vosshall et al., 1994).

A. Identification of plant circadian clock mutants No sequences with similarity to per or frq have been identified in Arabidopsis (G. Teakle and S. Kay, unpublished results), suggesting that genes homologous to per and frq are not components of the circadian oscillator in plants. However, Millar et al. (1995a) used in vivo assays of CAB2-driven expression of luciferase as a circadian phenotype for the isolation of circadian clock mutants. M2 populations of EMS-mutagenized Arabidopsis lines containing the cab2: :R::luc marker were screened for aberrant patterns of cyclic bioluminescence. Twenty-six timing of CAB (toc) lines were identified, representing at least 21 independent mutations from a screen of ca. 10,000 M2 plants. Mutants with both long (11 lines with periods of 26-28 hr) and short (7 lines with periods of 21-22.5 hr) periods, and a single line with reduced amplitude but wild-type period, were identified. A single mutant line, tocl , with a short period (20.9 hr mean period) was characterized in the most detail. As mutations that affect phototransduction pathways also affect the period of the CAB2 oscillation (Millar et al., 1995c) the photomorphogenic phenotypes of tocl were compared to those of the pleiotropic detl mutant. In all phenotypes examined, including hypocotyl length, pigmentation, flowering, cab2::R: :luc luminescence levels, and amplitude of red-light induction, tocl is indistinguishable from the wild-type transgenic line. The tocl phenotype is not due to a mutation in the reporter gene construct since the tocl mutation functioned in trans to shorten the period of oscillation of the endogenous CAB2 and CAB3 genes as well (A. Millar and S. Kay, unpublished results). Furthermore, the tocl mutation segregates from the cab2: :luc transgene, maps to a different chromosome from both the transgene and the detl and det2 genes, and shortens the period of a separate clock-controlled output in plants, the rhythm in leaf movements. Interestingly, like most of the period mutants in other organisms (Hall, 1990; Dunlap, 1993), tocl is semidominant.

:a:

6. Identification of Cyanobacterial Circadian Clock Mutants Circadian clock regulation in cyanobacteria has been demonstrated for dinitrogen fixation (Grobbelaar et al., 1986; Mitsui et al., 1986; Huang et al., 1990) and

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for photosynthetic gene expression (Kondo er al., 1993).The identification of circadian clock regulation in cyanobacteria is significant for several reasons related to the prokaryote unicellular organization. First, circadian regulation of biological processes is now recognized as an important adaptive mechanism for prokaryotes, as exquisitely exemplified by the temporal separation of photosynthetic 0, evolution in the light and the clock-regulated, 02-labile dinitrogen fixation process in the dark phase in some unicellular cyanobacteria. Second, potentially simpler prokaryotic models for the clock mechanism can now be considered for eukaryotic organisms. Third, the genetic investigation of circadian rhythms is also potentially simplified in a prokaryotic system; cyanobacteria have a much smaller genome than eukaryotes, and in appropriate readily transformable strains, genes can be easily cloned by complementation. Finally, circadian clock regulation of gene expression in cyanobacteria provides a potentially powerful model system for the study of clock regulation of chloroplast gene expression from the viewpoint of a cyanobacterial-like symbiotic progenitor for higher plant chloroplasts. Cyanobacteria have long served as an excellent model system for the elucidation of structural and functional requirements for higher plant photosynthetic electron transport. Several chloroplast-localized genes have been reported to be regulated by the circadian clock (e.g., RBCL and OEC) (Piechulla, 1988, 1993). Therefore, it will be of interest to examine, from both a molecular and an evolutionary perspective, whether the cyanobacterial clock mechanism is related to the mechanism of chloroplast circadian clock regulation, whether localized wholly or partially in either the chloroplast or nuclear genomes. A genetic screen based on the expression of a circadian-clock-regulated photosystem I1 gene promoter (psbAI) fused to a bacterial luciferase gene set (luxAB)was used to identify clock mutants in the cyanobacterium Synechococcus sp. strain PCC 7942 (Kondo er al., 1994). The psbA1::luxAB-containing strain was mutagenized with EMS, and survivors were subcultured for 20-60 generations. Upon screening 150,000 clones, 17 clock mutant phenotypes were identified, 12 resulting in altered periods and 5 with disrupted rhythmicity. Kondo and co-workers (1994) demonstrated for six of the mutant strains that the EMS-induced mutations were not due to damage to the reporter gene construct, as the altered rhythmic phenotype was not changed by replacement of the luciferase reporter construct with a non-EMS-treated construct. Furthermore, they were able to readily clone one of the loci by complementation of a short period mutation strain by conjugation with an Escherichia coli library of wild-type Synechococcw DNA. While no indication of the degree of saturation of mutagenesis was reported, and allowing for the loss of some mutants during the extensive suhculture phase postmutagenesis, the fact that at least two clones for each phenotype were recovered suggests that the screen was saturating as performed. Complementation of the 2690-kb genome of Synechococcus 7942 with a library containing 2-4-kb inserts (Tsinoremas et al., 1994) would require the screening of only ca. 600-1500 clones per each mutant line, a process made remarkably simple by the automated

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assay for lux expression. Hence, it is expected that all of the loci identified by this screen will be cloned in short order, and elucidation of the circadian clock mechanism in this cyanobacterium may be close at hand.

VII. CIS- AND TRANSACTING ELEMENTS REQUIRED FOR LIGHTAND CLOCK-REGULATED CAB EXPRESSION The observation that CAB transcription is regulated by both phytochrome and the circadian clock prompts the investigation of whether regulation is mediated through distinct cis-acting domains or whether the phytochrome and circadian clock signal transduction pathways converge to act upon the same cis regulatory domain. As would be expected for a gene whose transcription is regulated by multiple environmental and cellular regulatory inputs, a number of different protein factors have been shown to interact with CAB promoters from various plant species. In many cases, the function of these protein-CAB promoter interactions in regulated expression has not been examined. However, functional roles for several protein-CAB promoter interactions have been characterized in sufficient detail, allowing us to begin to define the genomic targets for phytochrome- and clock-responsive signal transduct ion pathways. Schindler and Cashmore (1990) identified at least five protein factors that bind the 1.5-kb tobacco CAB-E upstream sequence in uitro; some factors bind at multiple sites within the promoter. Many of the factors binding the CAB-E promoter also bind other photoregulated and nonphotoregulated promoters. Differences in the binding of these factors to the CAB-E sequences were not detected between extracts prepared from light-grown or dark-adapted plants, indicating that photoregulation cannot be explained simply by the presence or absence of binding activity in extracts. The function of these DNA binding proteins in the circadian regulation of CAB-E expression was not examined. The CAB genes of Arabidopsis represent the best-characterized plant promoters regulated by both light and the circadian clock. Arabidopsis contains three well-characterized copies of CAB encoding identical mature proteins (Leutweiler et al., 1986), as well as several other CAB gene family members (Green et al., 1991; McGrath et al., 1992). The 5' flanking sequences of CABl, 2 , and 3 show a high degree of sequence conservation, particularly between - 198 and 1 (+ 1 = transcription start site), suggesting that functionally important elements exist within this region of the CAB promoter (Mitra et al., 1989). Sun et al. ( 1993) reported the identification of an Arabidopsis DNA binding protein, CA-1, that interacts with - 138 to - 111 of the light-responsive CAB1 (CAB140 in that publication) promoter sequence, which contains a highly conserved ACGT motif. The ACGT motif is found in the 5' upstream region of many plant genes regulated by a variety of environmental and physiological

+

1.

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Gene Transcription in Higher Plants

stimuli and is bound by members of a family of bZIP DNA binding proteins (Schindler et al., 1992a,b; Williams et al., 1992). Characterization of the CA-1 binding site demonstrated that although the binding site overlaps the ACGT motif, CA-1 does not require the ACGT sequence for binding. Analysis of the expression of cab1 promoter deletions fused to cat (encoding chloramphenicol acetyltransferase) in transgenic tobacco showed that deletion from - 183 to -88 of cab1 , which spans the CA-1 binding site, resulted in the loss of both detectable cat expression and phytochrome responsiveness, suggesting that sequences downstream of - 183 of the CAB1 promoter are required for phytochrome-regulated expression (Sun et al., 1993). Interaction of CA-1 with either the CAB2 or CAB3 promoter has not been reported; however, a tobacco nuclear protein binding from - 139 to - 115 of the Arabidopsis CAB2 sequence (the region of the CAB2 promoter approximately corresponding to the CA- 1 binding site of the CAB I promoter) has been identified and designated CUF-1 (for CAB upstream factor-1) (Anderson et al., 1994). CUF-1 requires the ACGT motif in this region of the CAB2 promoter for binding (Anderson et al., 1994) and does not bind to any other potentially redundant sites within -322 to 1 of the CAB2 promoter (S. Anderson and S. Kay, unpublished results). Gel mobility shift and competition assays demonstrate that CUF- 1 has binding specificity for two types of ACGT-containing motifs previously identified in plant promoters, the G-box and TGACGT/C motifs, and therefore may be related to other bZIP proteins with affinity for both types of ACGT motif (i.e., CPRF-2). Moreover, the tobacco nuclear factor CUF-1 is distinct from the Arabidopsis factor CA-1 as CUF-1 is competed by the A2 m-1 mutant oligonucleotide (Anderson et d., 1994) that conserves the ACGT motif hut is unable to compete CA-1 binding (Sun et al., 1993). Deletion of the CUF-1 binding site in cab2::R::luc fusions expressed in transgenic tobacco does not alter the phase or period of the endogenous oscillation in luc expression when assayed in green seedlings or the timing of the acute response to red-light irradiation in etiolated seedlings (Anderson et al., 1994). However, deletion of the CUF-1 binding site does significantly reduce the level of cab2: :R::luc expression in both green and etiolated seedlings and lowers the amplitude of the circadian oscillation in green tissue. O n the basis of the expression patterns from only the CUF-1 deletion constructs, CUF-1 is required for high-level CAB2 expression in both green and etiolated seedlings and appears to contribute to the amplitude of the circadian oscillation observed in light-grown plants. Site-directed mutagenesis to specifically interrupt the CUF-1-CAB2 interaction in the context of the native CAB2 promoter was used as a more accurate assay of CUF-1 contribution to CAB2 gene expression (Anderson and Kay, 1995). The abolition of CUF-1 binding in vitro by mutation of the core of the CUF- 1 binding site from ACGT to AATT (CUF-M) in cab2: :luc fusions correlates with reduced expression levels in the absence of any change in the pattern

+

:a:

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(i.e., timing or amplitude) of the circadian oscillation or phytochrome-responsive luc expression in transgenic tobacco in uivo. This result conclusively demonstrates that CUF-1 is a general positive transcription factor that increases CAB2 expression and is not required for clock- or phytochrome-regulated CAB2 transcription (Anderson and Kay, 1995). A cis-acting domain, from - 111 to -38, of the CAB2 promoter has been shown to be sufficient for both circadian-clock- and phytochrome-regulated expression and for function as an enhancer to confer clock- and phytochrome-regulated transcription upon the heterologous -90 to +8 CaMV (cauliflower mosaic virus) 3 5 s promoter (Anderson et al., 1994). This 73-bp domain contains a highly conserved, repeated GATA motif (Grob and Stuber, 1987) bound by the tobacco nuclear factor CGF-1 (for CAB GATA factor-1) (Anderson et al., 1994). The type I PSI1 CAB promoters from several species contain 2-3 GATA repeats with similar spacing (GATAN,GATAN,GATA) and positioning between the CAAT and TATA boxes (Castresana et al., 1987; Gidoni et al., 1989). A t least two distinct classes of GATA binding proteins have been identified in tobacco. GAF- 1 binds the GATA motif of the phytochrome-regulated, but non-clock-regulated -189 to +1 RBCS-3A promoter (Gilmartin et al., 1991). ASF-2 binds a GATA motif in the 35s promoter (Lam and Chua, 1989).GA-1 binds to the triple GATA repeat in the tobacco CAB-E gene and to the GATA motif of the 35s promoter (Schindler and Cashmore, 1990), suggesting that GA-1 and ASF-2 are related. Competition studies with the as-2 binding site have demonstrated that the GAF-1 binding activity is distinct from that of ASF-2 (Gilmartin et al., 1990). Results from gel mobility shift and competition assays demonstrate that although CGF-1 requires the CAB2 GATA sequences for binding, CGF-1 will not bind the GAF-1 and ASF-2 binding sites and therefore may represent a GATA binding factor distinct from GAF-1 and GA-l/ASF-2 (Anderson et al., 1994). A tetramer of the GT-1 binding site, termed box I1 and which contains a core recognition sequence of GGTTAA, has been demonstrated to confer light-regulated transcription upon a heterologous promoter (Lam and Chua, 1990). The box I1 tetramer sequence does effectively compete the - 111 to -38 CAB2 sequence for CGF-1 binding, whereas a box I1 mutant tetramer is not bound by CGF-1 (Anderson et al., 1994). In a reciprocal experiment, Hiratsuka and co-workers (Hiratsuka et al., 1994) demonstrated that the cloned recombinant GT-1 protein will bind to both the - 111 to -38 CAB2 fragment and a trimer of the wild-type CGF1 binding site, but not to a trimer of a mutated CGF-1 binding site in which each GAT of the three GATA repeats is mutated to CCC. Furthermore, CGF-1 and GT- 1 have similar physical and chemical properties and are immunologically related, as antibodies to the cloned recombinant GT-1 cross-react with the CGF1/CAB2 complex (G. Teakle and S.Kay, unpublished results). CGF-1 has also been characterized in relationship to two tomato nuclear factors that bind to another GATA-containing motif, termed the I box (GATAA-

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Pu), found in several light- and circadian-clock-regulated plant promoters (Borello et al., 1993). IBF-2a is a circadian-regulated factor and IBF-2b is a constitutive factor, both of which bind the I box of the clock- and light-regulated tomato nitrate reductase (NIA) promoter. Three protein/DNA complexes between tobacco nuclear extracts and an oligonucleotide probe containing the I box sequence were detected in gel mobility shift assays (G. Teakle and S. Kay, unpublished results). Complex 1 was identical to CGF-l/GT-l, whereas complexes 2 and 3 showed properties similar to those of IBF-2b. These data suggest that CGF-1/GT1, as well as several other GATA factors, can bind to the I box oligonucleotide, and more than one protein factor may be responsible for all of the regulatory aspects of this single cis-acting element. Multiple factor interaction with a single cisacting element has been described for several plant promoters. For example, the R and B proteins of maize regulate anthocyanin biosynthesis in different tissues (Ludwig and Wessler, 1990), and it has been proposed (Schindler et al., 1992a) that the distribution of G-box factor (GBF) homo- and heterodimeric proteins may account for the differential expression of GBF-regulated genes in different tissues and under different conditions. The functional roles for IBF-2a, IBF-2b, or CGF-l/GT-l in the regulation of tomato NIA transcription, however, have not been reported. The function of CGF-1 binding to the Arabidopsis CAB2 promoter in the regulation of transcription, however, has been assayed by the construction of a site-directed mutation of the CGF-1 binding site in the context of the CAB2 promoter sequence fused to the luc reporter gene. Although the CGF-G3M mutation, in which each G of the triple GATA repeat was mutated to C, only moderately, but specifically, attenuated CGF-1 binding in vitro, this mutation has a dramatic effect on luc expression in vivo (Anderson and Kay, 1995). The CGFG3M mutation resulted in a reduction in mean luc expression level in both green and etiolated seedlings and a quantitative reduction in the amplitude of the circadian oscillation, although, quite importantly, the oscillation does persist in these mutant lines. The reduced amplitude in the CGF-G3M lines mimics the damping of the oscillation in CAB expression observed in green seedlings transferred to DD (Nagy etal., 1988; Kay etal., 1989) and, thus, suggests that the CGFG3M mutation disrupts light input to CAB2 gene expression. Moreover, the CGF-G3M mutation dramatically attenuates the acute peak in transcription 4 hr after phytochrome activation in etiolated seedlings. CGF- 1 therefore has been proposed to function as a phytochrome-responsive transcription activator (Anderson and Kay, 1995). Tobin and co-workers (Kehoe et al., 1994) have also demonstrated the functional requirement for two regions of a Lemna Lhcb gene promoter (cabAB 19) necessary for phytochrome regulation, one of which contains a CCAAT motif and the other contains a single GATA motif. However, the cognate protein factors interacting with these cabAB I9 sequence elements have not been described.

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Another property of the contribution of CGF-1 to the regulation of CAB2 expression examined was the potential for redundant CGF-1 binding and function. Two nonoverlapping CAB2 promoter fragments upstream of - 111 are able to compete with the -74 to -42 region of the CAB2 promoter (the original CGF-1 binding site identified) for CGF-1 binding. These fragments therefore define at least three redundant CGF-1 binding sites within the CAB2 promoter: -322 to -199, -199 to -74, and -74 to -42 (G. Teakle, S. Anderson, and S. Kay, unpublished results). The CAB2 fragment from -199 to -74 has been shown to be fully functionally redundant in t h o , with the ability to confer both circadian clock and phytochrome regulation upon the heterologous truncated CaMV 35s promoter fused to luc (S. Anderson and S. Kay, unpublished results). Results from gel mobility shift and competition assays demonstrate that the redundant CGF-1 binding site within the - 199 to -74 domain can be further localized to ca. - 142 to - 1l l and overlaps the CUF-1 binding site (S. Anderson and S. Kay, unpublished results). The localization of a second CGF-1 binding site in this region of the CAB2 promoter is interesting from several viewpoints. CGF-1 and CUF-1,each with their distinct binding site requirements, may be expected to compete for binding to the same region of the CAB2 promoter. Moreover, the observation that a phytochrome-responsive transcription activator binds from ca. - 142 to - 111 explains the difference in the luc expression pattern observed for the 5' deletion from - 142 to - 111 versus for the site-directed mutation that specifically disrupts CUF- 1 binding in the region. The functional delineation of the CAB2 control sequences and corresponding factors specifically responsible for general activation (CUF-1) and phytochrome-mediated transcription activation (CGF-1) implies that the circadian clock may interact with a distinct cis-acting element(s) within - 111 to -38 of the CAB2 promoter to regulate the timing of CAB2 transcription. Furthermore, this suggests that potentially separable light- and clock-mediated pathways exist for the regulation of the amplitude and timing of CAB2 expression, respectively, as is the case for light and clock regulation of gene expression in Neurospora crassa. In Neurospora, the rhythmically expressed clock-controlled gene 2 (ccg-2) (Loros et al., 1989)is also directly regulated by light (Arpaia et al., 1993).In mutants blind to blue light (wc-1 and wc-2), ccg-2 expression is not induced by light, whereas photoinduction of ccg-2 is retained in a clock-null strain (bd;frq9). Therefore, the regulation of ccg-2 expression in response to light and the circadian clock appears to involve independent pathways in Neurospora (Arpaia et al., 1993). The model for the clock- and phytochrome-regulated pathways controlling CAB gene transcription presented in Fig. 1.1 can be modified slightly to reflect the characterization of CGF-1 as a phytochrome-responsive transcription factor. Specifically, phytochrome appears to regulate the phase and period of CAB2 expression by modulating the circadian clock via a distinct phytochromeresponsive pathway interacting directly with the clock itself. The amplitude of light-induced expression is regulated, in turn, by an independent phytochrome-

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regulated pathway interacting directly with the CAB2 promoter via the specific phytochrome-responsive transcription factor CGF-1. In this case, a binding site mutation that interferes with the genomic target of the phytochrome pathway interacting directly with the CAB2 promoter should have no effect on the clock regulation of CAB transcription (i.e., phase or period). The CGF-G3M mutation did not alter the phase or period of the circadian oscillation in the expression in green seedlings relative to wild type, consistent with this prediction. This result then provides evidence for distinct and independent pathways for clock and phytochrome regulation of CAB2 expression. Four additional DNA binding activities, distinct from CGF-1, that interact with the - 111 to -65 region of the CAB2 promoter have been identified in Arubidopsis whole cell extracts, one of which is a potential candidate for a circadian clock transcriptional activator. CUF-2 and CUF-3 bind sequences between - l l 1 and -94, and Tac and Tic bind between -94 and -65 of the CAB2 promoter (Carre and Kay, 1995), a region of the CAB2 promoter containing the two 5’-most GATA repeats. Similar to the binding of several GATA factors to the I box described earlier, Tic binds in close proximity to CGF-1 and Tac binds upstream of Tic, although their binding sites also partially overlap. A CAB2 fragment from -95 to -65 containing a mutation of the two GATA sites from GAT to CCC has a reduced ability to compete for Tic binding relative to the wild-type fragment, demonstrating that the GATA sequences are also important for Tic binding. In vivo assays of transgenic Arubidopsis containing deletions of the cub2 promoter fused to luc demonstrate that sequences from - 74 to -55, including the binding sites for both Tic and CGF-1, are required for circadian clock regulation (Carre and Kay, 1995). This suggests that either Tic, CGF-1, or both are required for rhythmic transcription. But, as described earlier, CGF- 1 appears to function in transgenic tobacco solely as a phytochrome-responsive transcription activator. Therefore, functional assays will be required to determine the role of Tic binding in the regulation of CAB2 gene expression and to establish whether Tic may be a circadian clock transcriptional regulator functioning in close proximity to, and perhaps modulating the activity of, the PHY-responsive transcription activator CGF-1. Furthermore, any trans-acting factor binding the CAB2 promoter, which is posited to function as a circadian clock transcriptional regulator, must satisfy the requirement for redundant interaction and function at least within the -322 to - 111 CAB2 domain, which has been shown to be functionally redundant with respect to clock regulation (S. Anderson and S. Kay, unpublished results).

VIII. CONCLUSIONS AND FUTURE PERSPECTIVES The biochemical, molecular, and genetic studies described here have facilitated our understanding of the phototransduction and circadian clock pathways in plants. These studies have provided answers to some of the questions concerning

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photoreceptor involvement and the identity of signaling components and have provided inroads to the elucidation of the circadian clock mechanism itself. Results from molecular analyses of the in vivo expression of cab: :luc fusions in transgenic plants provide the basis for a model describing the light- and clock-regulated pathways controlling CAB gene expression. This model includes the identification of pathways from both phytochrome and a blue-light photoreceptor to control the period of the circadian oscillation in CAB gene expression, a distinct pathway from phytochrome, which interacts with the CAB promoter via a phytochrome-responsive transcription factor, to regulate the amplitude of the oscillation in CAB expression, and, by implication, a distinct pathway from the clock to the CAB promoter for the regulation of the timing of CAB gene expression. Presently, the definitive identification of a clock-responsive CAB promoter cisacting element and cognate trans-acting factor remains a major research focus. The eventual identification of the respective cis- and trans-acting elements involved in blue-light regulation of CAB gene expression will be required to establish whether the photic pathways regulating the amplitude of CAB gene expression by both red and blue light merge prior to interaction with the CAB promoter or are in fact distinct pathways interacting with unique CAB promoter elements. It is likely that the model for the regulation of CAB gene expression will be refined in the near future to the point of the identification of the specific form or forms of phytochrome responsible for regulating the period of the circadian oscillation and for regulating the acute induction of CAB expression in etiolated seedlings. For example, initial studies of the cab::luc expression patterns in etiolated phyA, phyB, and phyA:phyB double mutant seedlings pulsed with red light confirm that phytochromes A and B have overlapping roles in mediating the induction of CAB expression (S. Anderson and Kay, unpublished results). The biochemical and genetic studies of light signal transduction have provided exciting insights into the identity of some of the messengers that regulate light-mediated gene expression and plant development. However, several aspects of the light-mediated signal transduction pathways are yet to be established. By virtue of the experimental approaches used for their identification, the genetically defined pathways consist of negative regulators of plant development and gene expression that are abrogated in the light, whereas the biochemically defined pathways are composed of positive regulators of light-mediated gene expression and chloroplast development. The relationships between the genetically and biochemically defined pathways are at present unknown. Several different models based on epistasis analyses have been proposed for the hierarchical relationships between the phytochrome and blue-light photoreceptors and the DET-COP-FUS loci, as well as others (Ang and Deng, 1994; Chory and Susek, 1994). Clearly, light signals mediated by phytochrome and a blue-light photoreceptor converge at or prior to DETl and COP1, 8, 9, 10, and 11, which represent relatively early common steps in the signaling pathway, with

s.

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DET3, DOC1,2, and 3, and other loci not described here (such as GUN1,2, and 3 and potentially HY5) functioning in farther downstream branches of the signal transduction pathways (Chory and Susek, 1994). The primary events following photoreceptor stimulation remain relatively uncharacterized, but are likely to lie upstream of DET/COP. It is well-established in animal systems that membrane-localized serpentine receptors interact with membrane-associated G proteins to mediate signal transduction. By analogy to such models for animal signal transduction pathways, an activated G protein step would be expected to function just downstream of the activated photoreceptor. Phytochrome, however, is localized in the cytoplasm (Quail, 1991). Therefore, a mechanism must be identified for the transduction of signals from phytochrome to the G proteins. For instance, upon photoconversion to Pfr, phytochrome may transiently translocate to the plasma membrane, where it activates the G protein. Upon the absorption of red light and photoconversion to Pfr, phytochrome undergoes a rapid relocalization into large aggregates, but there is no evidence for the association of phytochrome with membranes (Quail, 1991). Alternatively, another molecular intermediate(s) may transmit the signal from Pfr to a membrane-associated G protein (Neuhaus et al., 1993). One such example in animal systems for the involvement of a signaling intermediate is the Grb2-mediated translocation of the nucleotide exchange factor Sos to the plasma membrane upon activation of the epidermal growth factor receptor (Egan et al., 1993). In comparison, the blue-light signal transduction system may be mechanistically more similar to the membrane-associated receptor4 protein systems found in animals, since the photoreceptor thought to mediate the regulation of gene expression appears to be localized in the plasma membrane (Warpeha et al., 1992). It will also be important to determine how light signaling specificity is conferred by pathways composed of downstream effectors such as G proteins, Ca2+,calmodulin, and cGMP. Studies suggest that, in animal systems receptors, G proteins and their effectors are more highly organized than previously thought and may be localized in domains associated with the cytoskeleton mediated by proteinorganizing factors (Neubig, 1994). Specifically, P-adrenergic receptors (Raposo et al., 1989), several types of G proteins (Sargiacomo et al., 1993),and Ca2+release channels (Fujimoto, 1993; Fujimoto et al., 1992) are enriched in noncoated pits or caveolae, suggesting that caveolae may be sites for the assembly of signal-transducing complexes. Hence, cell biology may well be the experimental field that next yields the most information concerning light signaling in plants. With respect to the mechanism of the circadian clock in plants, the continued application of luciferase technology will undoubtedly expedite the identification of additional loci involved in the clock mechanism. The ability to rapidly identify a number of loci involved in clock regulation of plant gene expression is likely to place plant systems in the forefront of circadian clock studies. Moreover, as exemplified by the studies described herin, plants represent one of the best

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model systems for the investigation of the interactions between the phototransduction and circadian clock transduction pathways to regulate gene transcription.

Acknowledgments We thank the members of the Kay lab for their helpful discussions during the preparation of this manuscript and Isabelle Carre, Carl Strayer, and Graham Teakle for communicating results prior to publication. Support was provided by grants to S.A.K. from the National Science Foundation (MCB9316756), the Human Frontiers Science Programs, and the National Science Foundation Center for Biological Timing. S.A.K. is supported by an award from the W. M. Keck Foundation. S.L.A. was supported by a National Science Foundation Postdoctoral Research Fellowship in Plant Biology (BIR9104325).

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Millar, A. J., McGrath, R. B., and Chua, N.-H. (1994). Phytochrome phototransduction pathways. Annu. Rev. Genet. 28:325-349. Millar, A. J., Carre, I. A,, Strayer, C. A,, Chua, N.-H., and Kay, S. A. (1995a). Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science 267: 1161-1 163. Millar, A. J., Straume, M., Chory, J., Chua, N., and Kay, S. A. (1995b). Phytochrome and blue-responsive photoreceptors regulate circadian period in Arabidopsis thaliana. Science 267:1161-1163. Millar, A. J., Straume, M., Chory, J., Chua, N.-H., and Kay, S.A. (1995~).The regulation of circadian period by phototransduction pathways in Arabidopsis. Science 267: 1163-1 166. Misera, S., Muller, A. J., Weiland-Heidecker, U., and Jurgens, G. (1994). The FUSCA genes of Arabidopsis: negative regulators of light responses. Mol. Gen. Genet. 244:242-252. Mitra, A,, Choi, H. K., and An, G. (1989). Structural and functional analyses of Arabidopsis thuliana chlorophyll a/b-binding protein (cab) promoters. Plant Mol. Biol. 12:169-179. Mitsui, A., Kumazawa, S., Takahashi, A., Ikemoto, H., Cao, S., and Arai, T. (1986). Stategy by which nitrogen-fixing unicellular cyanobacteria grow photoautotrophically. Nature 323:720-722. Morse, D. S., Fritz, L., and Hastings, 1. W. (1990). What is the clock?Translational regulation of circadian bioluminescence. Trends Biochem. Sci. 15:262-265. Nagy, F., Kay, S. A., and Chua, N.-H. (1988). A circadian clock regulates transcription of the wheat Cab-1 gene. Genes Dev. 2376-382. Nagy, E, Fejes, E., Wehmeyer, B., Dallman, G., and Schafer, E. (1993). The circadian oscillator is regulated by a very low fluence response of phytochrome in wheat. Proc. Natl. Acad. Sci. USA 90~6290-6294. Neubig, R. R. (1994). Membrane organization in G-protein mechanisms. FASEB J. 8:939-946. Neuhaus, G., Bowler, C., Kern, R., and Chua, N.-H. (1993). Calcium/calmodulin-dependentand -independent phytochrome signal transduction pathways. Cell 73:937-952. Nimmo, G. A., Wilkins, M. B., Fewson, C. A., and Nimmo, H. G. (1987). Persistent circadian rhythms in the phosphorylation state of phosphoenolpyruvate carboxylase from Bryophyllum fedtschenkoi leaves and in its sensitivity to inhibition by malate. Planta 170:408-415. Paulsen, H., and Bogorad, L. (1988). Diurnal and circadian rhythms in the accumulation and synthesis of mRNA for the light-harvestingchlorophyll &binding protein in tobacco. Plant Physiol. 88: 1104-1 109. Pepper, A., Delaney, T., Washburn, T.,Poole, D., and Chory, J. (1994). DETI, a negative regulator of light-mediated development and gene expression in Arabidopsis, encodes a novel nuclear-localized protein. Cell 78:109-116. Piechulla, B. (1988). Plastid and nuclear mRNA fluctuations in tomato leaves-diurnal and circadian rhythms during extended dark and light periods. Plant Mol. Biol. 11:345-353. Piechulla, 8.( 1993). 'Circadian Clock'directs the expression of plant genes. Plant Mol. Biol. 22633-542. Quail, P. H. (1991). Phytochrome: a light-activated molecular switch that regulates plant gene expression. Annu. Rev. Genet. 25:389-409. Quail, P. H. (1994a). Photosensory perception and signal transduction in plants. Curr. Opin. Genet. Dew. 4:652-661. Quail, P. H. (1994b). Phytochrome genes and their expression. In Photomorphogenesis in plants (R. E. Kendrick and G. H. M. Kronenberg, Eds.), pp. 71-104. Kluwer Academic Publishers, Dordrecht, The Netherlands. Quail, P. H., Briggs, W. R., Chory, J., Hangarter, R. P., Harberd, N. P., Kendrick, R. E., Koomneef, M., Parks, B., Sharrock, R. A., Schafer, E., Thompson, W. F., and Whitelam, G. C. (1994). Spotlight on phytochrome nomenclature. Plant Cell 6:468-471. Raposo, G., Dunk, I., Delvaier-Klutchko, C., Kaveri, S., Strosberg, A. D., and Benedetti, E. L. (1989). Internalization of P-adrenergic receptor in A43 1 cells involves non-coated vesicles. Eur. J. Cell Biol. 50:340-352. Reed, J. W., Nagatani, A., Elich, T. D., Fagan, F., and Chory, J. (1994). Phytochrome A and phy-

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tochrome B have overlapping but distinct functions in Arabidopsis development, Plant Physiol. 104~1139-1149. Reymond, P., Short, T. W., and Briggs, W. R. (1992a). Blue light activates a specific protein kinase in higher plants. Plant Physiol. 100:655-661. Reymond, P., Short, T. W., Briggs, W. R., and Poff, K. L. (1992b). Light-induced phosphorylation of a membrane protein plays an early role in signal transduction for phototropism in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 89:4718-4721. Roenneherg, T., and Hastings, J . W. (1988).Two photoreceptors control the circadian clock of a unicellar alga. Naturwisschalten 75:206-207. Romero, L. C., and Lam, E. (1993). Guanine nucleotide binding protein involvement in early steps of phytochrome-regulated gene expression. Proc. Natl. Acad. Sci. USA 90: 1465-1469. Romero, L. C., Sommer, D., Gotor, C., and Song, P.3. (1991). G-proteins in etiolated Awenu seedlings: Possible phytochrome regulation. FEBS Lett. 282:341-346. Rosbash, M., and Hall, J. C. (1989). The molecular biology ofcircadian rhythms. Neuron 3:387-398. Sancar, G. B. (1990). DNA photolyases: physical properties, action mechanism, and roles in dark repair. Mutat. Res. 236:147-160. Sargiacorno, M., Sudol, M., Tang, Z.-L., and Lisanti, M. P. (1993). Signal transducing molecules and glycosyl-phosphatidylinositol-linkedproteins from a caveolin-rich insoluble complex in MDCK cells. J. Cell Biof. 122:789-807. Schindler, U., and Cashmore, A. R. (1990). Photoregulated gene expression may involve ubiquitous DNA binding proteins. EMBOJ. 9:3415-3427. Schindler, U., Menkens, A. E., Beckmann, H., Ecker, J. R., and Cashmore, A. R. (1992a). Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF hZIP proteins. EMBOJ. 11:1261-1273. Schindler, U., Terzaghi, W., Beckrnann, H., Kadesch, T, and Cashmore, A. R. (1992b). DNA binding site preferences and transcriptional activation properties of the Arabidopsis transcription factor GBF1. EMBOJ. 11:1275-1289. Sehgal, A., Price, J. L., Man, B., and Young, M. W. (1994). Loss of circadian behavioral rhythms and per RNA oscillations in the Drosophila mutant timekss. Science 263: 1603-1605. Senger, H., and Schmidt, W. (1994). Diversity of photoreceptors. In Photomorphogenesisin Plants, 2nd ed. (R. E. Kendrick and G. H. M. Kronenherg, Eds.), pp. 301-325. Kluwer Academic Publishers, Dordrecht, The Netherlands. Sharma, R., Lopez-Juez, E., Nagatani, A,, and Furuya, M. (1994).Identification ofphoto-inactive phytochrome A in etiolated seedlings and photo-active phytochrome B in green leaves of the aurea mutant of tomato. Plant J. 4:1035-1042. Sheen, J. (1993). Protein phosphatase activity is required for light-inducible expression in maize. EMBO J. 12:3497-3505. Short, T. W., and Briggs, W. R. (1994). The transduction of blue light signals in higher plants. Annu. Rev. Plant Physiol. Plant Mof. Biol. 45:143-171. Somers, D. E., and Quail, P. H. (1995). Phytochrome-mediated light regulation of PHYA- and PHYBGUS transgenes in Arabipopsis thaliana seedlings. Plant Physiol. 107:523-534. Somers, D. E., Sharrock, R. A,, Tepperman, J . M., and Quail, P. H. (1991). The hy3 long hypocotyl muntant of Arabidopsis is deficient in phytochrome B. Plant Cell 3:1263-1274. Stapleton, A. E. (1992). Ultraviolet radiation and plants: burning questions. Plant Cell 4:1353-1358. Stockhaus, J., Nagatani, A., Halfter, U., Kay, S., Furuya, M., and Chua, N.-H. (1992). Serine-to-alanine substitutions at the amino-terminal region of phytochrome A result in an increase in biological activity. Genes Dev. 6:2364-2372. Sun, L., and Tohin, E. M. (1990). Phytochrome-regulated expression of genes encoding Light-harvesting chlorophyll a/b-protein in two long hypocotyl mutants and wild-type of Arabidopsis thalianu. Photochem. Photobiol. 52:5 1-56.

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Sun, L., Doxsee, R. A,, Harel, E., and Tobin, E. M. (1993). CA-1, a novel phosphoprotein, interacts with the promoter of the cab140 gene in Arabidopsis and is undetectable in detl mutant seedlings. Plant Cell 5:109-121. Sweeney, B. M. (1987). Rhythmic phenomena in plants. Academic Press, San Diego. Takahashi, J. S. (1993). Circadian-clock regulation of gene expression. C u r . Opin. Genet. Dew 3:301-309. Tsinoremas, N. F., Kutach, A. K., Strayer, C. A,, and Golden, S.S. (1994). Efficient gene transfer in Synechococcus sp. strains PCC 7942 and PCC 6301 by interspecies conjugation and chromosomal recombination. J. Bacteriol. 176:6764-6768. van der Voom, L., and Ploegh, H. L. (1992). The WD-40 repeat. FEBS Lett. 307:131-134. Vierstra, R. D. (1993). llluminating phytochrome functions. Plant Physiol. 103:679-684. Vosshall, L. B., Price, J. L., Sehgal, A., Saez, L., and Young, M. W. (1994). Block in nuclear localization of period protein by a second clock mutation, timeless. Science 263:1606-1609. Wang, H., and Stillman, D. (1993). Transcriptional repression in Saccharomyces cerevisiae by a SIN3LexA fusion protein. Mol. Cell. Biol. 13:1805-1814. Warpeha, K. M. E, Hamm, H. E., Rasenick, M. M., and Kaufman, L. S. (1991). A blue-light-activated GTP-binding protein in the plasma membranes of etiolated peas. Proc. Natl. A c d . Sci. USA 88:8925-8929. Warpeha, K. M. E, Kaufman, L. S., and Briggs, W. R. (1992). A flavoprotein may mediatiate the blue light-activated binding of guanosine 5'-triphosphate to plasma membranes of Piscum sativum L. Photochem. Photobiol. 55:595-603. Wehmeyer, B., Cashmore, A. R., and Schafer, E. (1990). Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-l,5-bisphosphacecarboxylase in etiolated seedlings of Lycopersicon escukntum (L.) and Nicotiana tabacum (L.) Plant Physiol 93:990-997. Wei, N., Chamovitz, D. A., and Deng, X.-W. (1994). Arabidopsis COP9 is a component of a novel signaling complex mediating light control of development. Cell 78:117-124. Williams, M. E., Foster, R., and Chua, N.-H. (1992). Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. Plant Cell 4 4 8 5 4 9 6 .

Brian V. Harmon and David J. Allan

School of Life Science, Queensland University of Technology Brisbane, Queensland 4000, Australia

I. INTRODUCTION In the past, cell death has been a very unfashionable area of research. Changes observed in dying cells were generally thought of as being a series of passive degenerative events that occurred in a cell after it had been killed. These degenerative events were equated by many to the postmortem autolytic changes that occur in the human body after death, a topic unlikely to inspire much interest. T h e long-standing belief among cell and tissue biologists that cell death was the province of pathologists investigating disease states and was of no relevance to “normal” processes occurring in the body reinforced the general apathy surrounding this topic. In 1972, three pathologists, John Kerr, Andrew Wyllie, and Alistair Currie,I challenged the long-standing cell death doctrine when they put forward their ‘John F. R. Kerr graduated in medicine from the University of Queensland, Australia, in 1958. He completed his Ph.D. at the University of London in 1964 under the supervision of Sir Gordon Roy Cameron and was Professor of Pathology at the University of Queensland from 1974 until his retirement early in 1995. Andrew H. Wyllie graduated in medicine from the University of Aberdeen in 1967 and completed his Ph.D. at the same university in 1975 under the supervision of Professor Alistair Currie. He completed postdoctoral training at Cambridge University in England before rejoining Alistair Currie at the University of Edinburgh and has been Professor of Experimental Pathology at that university since 1992. He continues to work at the forefront of apoptosis research. Alistair R. Currie graduated in medicine from the University of Glascow in 1944. He served as Professor of Pathology at the Imperial Cancer Research Fund Laboratories in Oxford, England, the University of Aberdeen, and the University of Edinburgh and chaired a number of major scientihc committees in the United Kingdom. He was awarded a knighthood for his service to the medical sciences in 1979. His interest in apoptosis continued in retirement until his death early in 1994 Advances in Genetics, Vol. 35

Copyright 0 1997 hy Academic Press All rights of reproduction in any form reserved

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revolutionary concept of the “apoptotic” death of cells (Kerr et al., 1972). They suggested that death by apoptosis of selected individual cells within a tissue might be an active process mediated by the cell’s own biochemical mechanisms. Moreover, they proposed that the deletion of cells by this process might play an important role opposite to that of mitosis over a wide range of physiological and pathological circumstances. It took more than 20 years for their views on cell death to finally gain widespread acceptance. With this acceptance, we have been forced to revise our thinking on a host of medically and biologically important processes. To give just a few examples, oncogenes can no longer be thought of only in terms of cell proliferation; their potential to prevent apoptosis must also be considered. Likewise, many growth factors now need to be viewed as having both a mitogenic and an “antiapoptotic” effect. Finally, a reduced apoptotic rate might be just as important in the development of cancer as enhanced proliferation. In 1990, most of the leading apoptosis researchers of the day gathered at the Cold Spring Harbor Laboratory to discuss molecular approaches to studying the mechanism and regulation of apoptosis. The application of molecular biology techniques to the study of apoptosis was only in its infancy at that stage, but important new findings concerning the apoptotic mechanism already were beginning to emerge. In the 5 years since that symposium, interest in apoptosis has literally exploded. Molecular biologists have been at the forefront of this new wave of enthusiasm, and with each new discovery, the implications of apoptosis for medicine and biology have assumed even greater proportions. It would not be possible in a historical overview such as this to cover all of the exciting discoveries that have been made in the last few years. We therefore will confine our account to an examination of the events leading up to the discovery of apoptosis by Kerr, Wyllie, and Currie (in 1972) and then follow its evolution as a concept until just after the 1990 Cold Spring Harbor Symposium when apoptosis was finally accepted for what it is: one of the most revolutionary and exciting ideas of 20th century science.

II. GENESIS OF THE APOPTOSIS CONCEPT A. Identification of “shrinkage necrosis” The genesis of the apoptosis concept can be traced back to the early 1960s and the identification of “shrinkage necrosis.’’John Ken-, a Pathology Registrar from the Royal Brisbane Hospital in Australia, went to England in 1962 to undertake a Ph.D. at the University College Hospital Medical School under the supervision of one of the most distinguished pathologists of that time, Sir Gordon Roy Cameron. Like many other Australian scientists of his generation, Cameron had left Australia permanently to work overseas. He is best remembered for his monumental monograph, “Pathology of the Cell” (Cameron, 1952), a work likened

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by some as “one to which Virchow himself might have been pleased to have set his name” (Singer and Underwood, 1962).Cameron suggested that ischaemic liver injury produced in rats by ligating branches of the portal vein would be an interesting topic for Kerr to study. However, with his health already in decline, the task of supervising the young Australian scientist fell largely on Cameron’s deputy, Joe Smith.

1. Shrinkage necrosis in ischaemic liver-cell

useful role

death playing a

At the time, lysosomes had only just been described by DeDuve and his colleagues in Belgium, and there was much speculation that the release of lysosomal enzymes following their rupture might be the critical event in the production of cell death, a theory that has not in fact been borne out by subsequent studies. Nevertheless, Smith believed that this idea was worthy of study in the ischaemic liver model that Cameron had suggested, and h e introduced Kerr to the newly developed histochemical methods for demonstrating lysosomal enzymes in frozen sections. It was these methods that were to highlight a n interesting change in the ischaemic livers that in all likelihood would have been missed had only the more conventional haematoxylin and eosin (H&E)-stained sections been employed. In the centraI parts of ischaemic liver lobules, patches of confluent coagulative necrosis developed. As expected, the lysosomes had ruptured in these areas and their enzymes were dispersed evenly throughout the swollen necrotic cells. In areas bordering necrotic regions and in the periportal parenchyma, however, small shrunken masses of cell cytoplasm that still contained intact, discretely staining lysosomes were found to be present. These shrunken masses, which by H&E staining seemed to represent the remains of dying cells, were scattered throughout the viable parenchyma and occurred either singly or in small clusters. Interestingly, once the liver lobes with reduced blood supply had become moderately reduced in size, the small shrunken (cellular) masses were no longer observed. This was interpreted by Kerr as evidence that cell death was performing the useful role of adjusting the liver mass to a new stable state of lower organ mass that could be sustained by the reduced blood supply. Kerr believed that the features of this type of death (i.e., the shrunken appearance, lysosomal integrity, and apparently useful function) were sufficiently distinct from those of coagulative necrosis to warrant a separate name. He proposed the term “shrinkage necrosis’’ for this process (Kerr, 1965), the marked shrinkage that characterized this process contrasting with the swollen appearance of cells undergoing coagulative necrosis.

2. Ultrastructural studies of shrinkage necrosis in liver Upon completion of his Ph.D., Kerr returned to Australia and took up a position as Senior Lecturer in Pathology at the University of Queensland Medical School.

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Among the equipment Kerr had at his disposal in the department was a newly acquired Hitachi HS-7S electron microscope, one of the first to be installed in Australia. Kerr used this microscope to examine the ultrastructural features of shrinkage necrosis and coagulative necrosis in the ischaemic liver model that he had been studying and in liver treated with the hepatotoxins, heliotrine, and albitocin (Kerr, 1969,1970,1971). The sequence of morphological events that he observed ultrastructurally in cells undergoing shrinkage necrosis in all three models was quite striking and consistent. There was marked overall condensation and budding of the hepatocytes into clusters of membrane-bound cell fragments. These fragments were then phagocytosed by neighboring cells and degraded within phagolysosomes. In contrast to classical necrosis, organelle integrity was maintained until the fragments were phagocytosed and digested by surrounding cells. There was no associated inflammation as occurred in areas of coagulative necrosis. Two striking features were recognized by Kerr. First, the occurrence of shrinkage necrosis was not limited to pathological circumstances-he observed small numbers of hepatocytes undergoing this process in untreated control livers. Second, hepatocytes participated along with Kupffer cells in the phagocytic process. Kupffer cells were known to be specialized phagocytic cells lining the hepatic sinusoids, so that their participation was not surprising. The phagocytosis of fragments of dead cells by liver epithelial cells (the hepatocytes), on the other hand, was most surprising as it was at odds with the biological convention of the day that only specialized cells are capable of phagocytosis. Many scientists were later to be skeptical of the entire apoptosis concept as a result of the claim that parenchymal cells could phagocytose apoptotic bodies, even though conclusive evidence had been presented. The ultrastructural features of the dying cells convinced Kerr more than ever that this was indeed a mode of cell death distinct from that of coagulative necrosis, and he published a number of papers on shrinkage necrosis over the next 6 years (Kerr, 1969, 1970, 1971).

3. Shrinkage necrosis in other normal tissues and in cancer Once the light and electron microscopic appearances of shrinkage necrosis had been clearly established, Kerr was able to look for similar appearances in a wide range of histopathological preparations that he was screening routinely in his role as a pathologist. He noticed the occasional cell undergoing this process in a range of normal tissues. In a major breakthrough, Kerr and his colleague Jeffrey Searle (a Doctor of Medicine candidate in the Pathology Department) found that shrinkage necrosis was widespread in basal cell carcinomas (BCC),the most common form of skin cancer in Australia. The important role that spontaneous cell loss plays in the kinetics of tumor growth had only just been recognized (Iversen, 1967; Refsum and Berdal, 1967; Steel, 1967), and Kerr and Searle (1972) suggested that much of the spontaneous loss of tumor cells in BCCs might be due to shrinkage necrosis. Furthermore, they proposed that this might explain the para-

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doxically slow growth rate that these tumors exhibit even though their mitotic index is high (Kerr and Searle, 1972).

6. From shrinkage necrosis to apoptosis As interesting as shrinkage necrosis was turning out to be, it is unlikely that it would have made any significant impact on the wide scientific community had it not been for a fortuitous encounter Kerr was to have with Alistair (later to become Sir Alistair) Currie, Professor of Pathology at the University of Aberdeen in Scotland. In 1970, Currie came to Australia to spend 1 month as the Mayne Guest Professor in Pathology at the University of Queensland. Kerr had the opportunity to discuss with Currie at length his findings on shrinkage necrosis and to show him light and electron micrographs of cells dying by this process. Currie was excited by these findings a5 he had seen similar changes in the adrenal cortices of rats given prednisone-a model being used by one of his Ph.D. students at the time, a medical graduate by the name of Andrew Wyllie. Before returning to Aberdeen, Currie extended an invitation to john Kerr to spend a 12- non nth study leave in the pathology department at the University of Aberdeen a t his earliest convenience, where they could look further into this increasingly interesting phenomenon. Kerr took up Currie’s offer in the followiiLgyear, 1971.

1. Shrinkage necrosis regulated by hormones In Aberdeen, electron microscopic studies carried out by Kerr (1972) confirmed that dying cortical cells in the adrenals of prednisone-treated rats were undergoing the same stereotyped sequence of morphological events that he had observed in liver and tumors [prednisone had a cortisol-like action in inhibiting adrenocorticotropic hormone (ACTH) release by the pituitary gland]. Interestingly, death could be prevented if ACTH were given to rats at the same time as the prednisone treatment. The implications of these findings were profound. Kerr, Wyllie, and Currie realized they were now dealing with a type of cell death that was controllable, one that could be switched on (or off) in an endocrine dependent tissue by a specific trophic hormone. This concept was completely at odds with the widely accepted view, at the time, that cell death (or necrosis as it was generally called) was the inevitable outcome of irreversible injury to a cell and not a process subject to any control mechanisms. They were soon to realize how flawed these long-held views on cell death really were.

2. T h e link between shrinkage necrosis and programmed cell death Kerr, Wyllie, and Currie were helped at this point by a lucky twist offate that landed a zoologist, Alison Crawford, in the University of Aberdeen pathology de-

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partment. Crawford, who was undertaking a Ph.D. at the time, provided Kerr, Wyllie, and Currie with a key piece of the emerging jigsaw puzzle when she recognized the similarity between the cell death they had been studying and programmed cell death in the embryo. She alerted them to the existence of extensive literature on this subject. While the seminal work of Glucksman had established the important and essential role that programmed cell death played in embryogenesis as early as 1951 (Glucksmann, 1951), few outside the field of embryology had been aware of its occurrence. The descriptions given by Glucksmann, however, and the few poor-quality electron micrographs that had been published were enough to convince Kerr, Wyllie, and Currie that programmed cell death and shrinkage necrosis were one and the same process. The work of Glucksman (1951) is worthy of further mention at this point as there is no doubt that it was of great assistance to Kerr, Wyllie, and Currie in the formulation of their revolutionary cell death concept. Glucksmann (195 1) realized that the morphological appearance of degenerating cells had often been misinterpreted and went on to give a description of the type of cell death that he said was most frequently encountered in normal vertebrate embryos. The morphological features detailed included the separation of chromatic from nonchromatic material in the nucleus and the precipitation and coalescence of chromatin into a single mass. Shrinkage of the nucleus and cytoplasm ensued as a result of the loss of fluid, and the dying cells were sometimes phagocytosed and degraded by neighboring cells. While this description is not nearly as comprehensive as that subsequently published by Kerr in his shrinkage necrosis papers (Kerr, 1969, 1970, 1971), it is clear that Glucksmann had recognized many of the key morphological features of the process. Moreover, he found that, as well as occurring in embryos at developmental stages of short duration, the same type of death was produced in tadpoles by X irradiation and by some poisons. He also observed this form of cell death in chicken fibroblasts growing in tissue culture and included some kinetic data on the process. Glucksmann thus had described key morphological features of a mode of cell death that occurred under both physiological and pathological circumstances. He failed, however, to realize that the implications of this mode of cell death extended far beyond the field of embryology. The clearly defined ultrastructural features of the death were the key that allowed Kerr, Wyllie, and Currie to bring together into a unified concept what had initially been three separate fields of investigation: Kerr’s shrinkage necrosis in the liver, Wyllie and Currie’s hormone dependent cell death in the adrenal cortex, and Glucksmann’s programmed cell death in embryonic development.

C. Apoptosis defined Kerr, Wyllie, and Currie set about looking for the occurrence of this “shrinkage” type of cell death in a wide range of situations, and the implications of their emerg-

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ing concept widened with each new discovery that they made. As well as being involved in cell turnover in many healthy adult tissues and being responsible for the elimination of redundant cells during normal embryonic development, it was found to participate in some types of tumor regression and to be involved in both physiological involution and pathological atrophy of tissues. A paper was written for and published by the British Journal of Cancer proposing the name “apoptosis” for this “hitherto little recognized mechanism of controlled cell deletion which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations” (Kerr et al., 1972). T h e term shrinkage necrosis had undesirable connotations as it suggested that the new type of cell death was simply another variant of necrosis. T h e decision to abandon the term shrinkage necrosis and to look for a more appropriate name was taken largely as a result of Alistair Currie’s promptings. The term apoptosis was suggested by the Professor of Greek at the University of Aberdeen at the time, James Cormack. It is used in Greek to describe the dropping off or falling off of petals from flowers or leaves from trees (Kerr et al., 1972). Apoptosis affects scattered single cells within a tissue in much the same way that scattered single leaves fall from a tree. T h e morphological features of apoptosis suggested that it was a n active, inherently programmed process. It was clearly understood by Kerr, Wyllie, and Currie at the time that understanding the factors that regulated this mode of cell death would be of great importance.

D. Morphological features of apoptosis T h e morphological description of apoptosis in the 1972 paper was essentially the same as had been previously reported by Kerr for shrinkage necrosis (Kerr, 1971). T h e changes were described as taking place in two discrete stages. The first comprised nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments. In the second, these fragments, which they termed apoptotic bodies, were phagocytosed by surrounding cells and degraded within phagolysosomes. While more detailed descriptions of the morphology of apoptosis were subsequently published by Kerr, Wyllie, and Currie, especially in relation to nuclear changes (Wyllie et al., 1980; Kerr et al., 1987, 1994a; Walker et al., 1988), the key morphological features of the process were all accurately identified in the original apoptosis publication (Kerr et al., 1972). Despite the many new techniques that have since been developed as markers of apoptosis (DNA ladders, flow cytometry, in situ nick translation analysis, etc.), morphological changes still provide the most reliable criteria for identifying the process (Kerr et al., 199413). Before proceeding to describe the subsequent development of the apoptosis concept and its final acceptance by the wider scientific community, we will give a brief account of the current cardinal morphological features of apoptosis. T h e contrasting ultrastructural features

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of necrosis will not be described; details on this topic can be found in the many published comprehensive reviews (Wyllie et al., 1980; Kerr et al., 1987, 1994a; Walker et al., 1988; Kerr and Harmon, 1991). The sequence of ultrastructural changes that characterize apoptosis occurring in a tissue is shown in stylized form in Fig. 2.1. Electron micrographs de-

Figure 2.1. Stylized diagram illustrating the sequence of ultrastructural changes in cells undergoing apoptosis. A n early apoptotic cell (2) is shown adjacent to a number of normal cells ( I ) . Early apoptosis is characterized by compaction and margination of nuclear chromatin into sharply circumscribed masses that abut the nuclear envelope, convolution of the nuclear outline, overall condensation of the cell, and, in tissues, separation of the dying cell from its neighbors. In the next phase (3), the nucleus fragments, extensive cell surface protrusions develop, and membrane-bound apoptotic bodies of various size and composition are formed (4). Apoptotic bodies formed in wioo are rapidly phagocytosed by specialized mononuclear phagocytes ( 5 ) or neighboring cells (6) and degraded within phagolysosomes.The structural integrity of organelles within apoptotic cells or bodies is usually maintained in wioo until the process of lysosomal degradation begins.

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Figure 2.2. Early apoptotic cell present in a rat liver lobe undergoing atrophy following ligation of its hepatic portal vein. Nuclear chromatin has condensed and marginated into sharply delineated masses abutting the nuclear envelope, and the cytoplasm is markedly condensed. This ischaemic liver model was used hy John Kerr in his Ph.D. thesis and in the original shrinkage necrosis piiblication (Kerr, 197 1) (original magnification X 5500).

picting various stages of the process occurring both in vivo and in vitro are shown in Figs. 2.2-2.8. Apoptosis characteristically affects scattered single cells in tissues rather than large numbers of contiguous cells, and there is n o associated inflammation. T h e first evidence that a cell is beginning to undergo apoptosis is observed in the nucleus. The chromatin condenses and becomes segregated into sharply delineated masses that abut on the inner nuclear envelope (Fig. 2.2). T h e chromatin changes are usually followed by convolution of the nuclear outline. Simultaneously with these nuclear changes, the cytoplasm condenses and protuberances form o n the cell surface. Further convolution of nuclear and cell outlines is followed by budding of the nucleus to produce a number of nuclear fragments that are still enclosed by a double-layered membrane and by budding of the cell as a whole to produce a number of membrane-bound apoptotic bodies (Fig. 2.3). Some of the apoptotic bodies contain one or more nuclear fragments, while others comprise cytoplasmic material only (Fig. 2.3). T h e number, size, and structure of the apoptotic bodies formed are extremely variable and appear to be dependent at least in part on the cell type involved. Thus, cells with a relatively voluminous cytoplasm often undergo extensive budding, with the production of numerous apoptotic bodies (Figs. 2.3 and 2.4). Budding in cells with only a thin rim of cy-

Figure 2.3. Apoptosis occurring in murine EMT6 sarcoma nodule 2 h after heating at 44°C for 30 min. Fragmentation of the nucleus and budding of the cell as a whole into well-preserved membrane-bound apoptotic bodies is shown clearly (original magnification X 11,800).

Figure 2.4. Apoptosis occurring spontaneously in a culture ofNS-1 mouse myeloma cell line. Note the extensive convolution of the cell surface and nucleus and the sharply delineated chromatin abutting the nuclear envelope (original magnification X 8000). 44

Figure 2.5. Apoptosis occurring in rat thymocytes 4 h after hydrocortisone treatment (concentration, 10 pM). Marked condensation of nuclear chromatin is apparent, hut no budding is apparent. In cells like thymocytes with only a thin rim of cytoplasm, hudding is relatively restricted (original magnification x 10,000).

Figure 2.6. Multiple apoptotic bodies present in a rat liver lohe undergoing atrophy following ligation of its hepatic portal vein. Most of the bodies have been phagocytosed by neighhoring hepatocytes and are in the process of being degraded within phagolysosomes (original magnification X 3800). 45

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Figure 2.7. Moderately degraded apoptotic body within an intraepithelial macrophage in a rat ventral prostate 2 days after castration. The apoptotic origin of the body can still be recognized by the presence of typical nuclear fragments (original magnification X 8500).

toplasm, on the other hand, is generally restricted (Fig. 2.5). The cytoplasmic organelles within newly formed apoptotic bodies remain well preserved (Fig. 2.3). Apoptotic bodies formed in tissues are usually rapidly phagocytosed by neighboring cells (including epithelial cells; Fig. 2.6) or by resident macrophages (Fig.2.7) and degraded within phagolysosomes (Figs. 2.6 and 2.7). Apoptotic bodies developing in single-layered lining epithelium may escape phagocytosis and be shed from the surface. Apoptotic bodies formed in vitro are rarely phagocytosed and eventually undergo degenerative changes resembling autolysis (Fig. 2.8); the term secondary necrosis has been used to describe this change (Wyllie et al., 1980). The apoptotic origin of degenerating bodies can still be determined if they contain nuclear remnants with recognizable “apoptotic-like” chromatin changes (Fig. 2.8). If degenerative changes in dying cells are too advanced for the mode of cell death occurring in a particular model to be identified, it may be necessary to repeat the experiment and examine samples taken at earlier time points.

E. Further development of the apoptosis concept The first apoptosis paper (Kerr et al., 1972) went virtually unnoticed by the wider scientific community and may have remained as “just an interesting point of view”

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Figure 2.8. Apoptosis occurring spontaneously in a culture of NS-1 mouse myeloma cell line. The lower apoptotic cell is in an early stage of the process and shows the condensation and margination of nuclear chromatin and the fragmentation of the nucleus into multiple masses of condensed chromatin. The upper apvptotic body shows the supervention of degenerative change similar to those found in necrosis (secondary necrosis), but a nuclear fragment with apoptotic-type chromatin segregation is still recognizable within it (original magnification X 6000).

on cell death had it not been for the firm belief of Kerr, Wyllie, and Currie that this was an incredibly important concept with wide-ranging implications for medicine and biology. It was this belief and their unstinting enthusiasm for the concept that enabled them to keep working in the field when many others would have given up. It was to be another 20 years before their views on cell death were to gain widespread acceptance.

1. Kerr and colleagues extend the known range of occurrence of apoptosis At the end of his period of study leave in Aberdeen, Kerr returned to the Pathology Department in Brisbane and embarked on a series of studies designed to extend the known range of occurrence of apoptosis. He was helped in these studies by a number of local “converts” to the apoptosis concept. Apoptosis was shown to be the mechanism responsible for the deletion of cells during both castration-

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induced involution of the rat prostate (Kerr and Searle, 1973) and regression of the tadpole tail during metamorphosis (Kerr et al., 1974). These studies highlighted the controlled nature of the apoptotic process and the biologically meaningful role that it was capable of playing. Further studies carried out by Kerr showed that apoptosis was induced in normal and neoplastic cells by cancer chemotherapeutic agents (Searle et al., 1975), that it was enhanced in liver allograft rejection (Searle et al., 1977),and that it was the mechanism of T-cell killing (Don et al., 1977). Wyllie and Currie, who both moved to the University of Edinburgh, maintained a keen interest in apoptosis, but did not publish as extensively (Wyllie et al., 1973a,b; Wyllie, 1974) as Kerr in the first 8 years after the first apoptosis paper (Kerr et al., 1972).

2. First apoptosis paper published in Nature In 1977, Chris Potten of the Paterson Institute for Cancer Research, UK, provided a much needed boost to the field of apoptosis when he reported the existence of a small group of cells in adult intestinal crypts that were extremely sensitive to apoptotic induction by irradiation. Jeffrey Searle (a colleague of John Kerr) had alerted Potten to the existence of the apoptosis concept when he visited Potten’s laboratory in Manchester, UK, the previous year. Potten’s work (1977) was the first on apoptosis to be accepted for publication in the influential journal Nature, and as such is one of the important early milestones in apoptosis reseqrch. Furthermore, Potten was the first well-known scientist from outside the Kerr or Wyllie-Currie research groups to publish on apoptosis.

3. Wyllie’s biochemical marker for apoptosis The next apoptosis paper to be published in Nature came some 3 years later. Andrew Wyllie (1980) reported on the association between apoptosis and endogenous endonuclease activation and proposed that activation of this enzyme was responsible for the cleavage of DNA at the linker regions between nucleosomes into oligonucleosomal sized fragments. This resulted in the formation of a characteristic “ladder” pattern in agarose gels stained with ethidium bromide. The DNA ladder was the first biochemical marker of the process and convinced many of the skeptics that apoptosis was more than just an unusual morphological quirk. The paper also marked the introduction by Wyllie of the in vitro model that was to play an important role in studies of the apoptotic mechanism, glucocorticoid-treated rodent thymocytes (Fig. 2.5). Wyllie, Kerr, and Currie reunited that same year to publish an updated review on apoptosis for the International Review of Cytology (Wyllie et al., 1980). Perhaps the most important new finding stressed in the review was the fact that active mRNA and protein synthesis appeared to be required in many circumstances for apoptosis to proceed. With these two publications

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(Wyllie, 1980; Wyllie et al., 1980), apoptosis had ceased to be solely a morphological phenomenon. It was now argued that apoptosis was a gene-directed mode of cell death with a biochemical mechanism distinct from that of necrosis. This, taken together with the wide-ranging implications of its occurrence, marked apoptosis as a topic worthy of study by the biochemists and, later on, the molecular biologists who were to enter the field.

4.

Other cell death studies that helped extend the apoptosis concept

It should be pointed out that while Kerr, Wyllie, and Currie are rightfully credited with developing the apoptosis concept, the published works of a number of other scientists were of considerable assistance to them. Once the apoptotic process had been clearly defined and the circumstances under which it occurred established, considerable insight into the process could be gained by reinterpreting the published results of others. The important role that Glucksmann’s work on programmed cell death played in helping Kerr, Wyllie, and Currie bring together the apoptosis concept in the first place has already been mentioned (Section IIA). However, the contributions of a number of other scientists are worthy of note. Inhibitors of protein synthesis such as cycloheximide had been known to prevent cell death from occurring over a range of different circumstances (Lieberman et al., 1970; Ben-Ishay and Farber, 1975; Pratt and Greene, 1976). Once Kerr, Wyllie, and Currie had established that the cell death occurring in these situations was of the apoptotic type, it was then relatively easy to go on and show that these inhibitors were capable of preventing apoptosis (Wyllie et al., 1980). Similarly, the reported degradation of DNA into regular-sized fragments in irradiated lymphocytes (Skalka et al., 1976) would have assisted Wyllie (1980) to formulate his views on the association between apoptosis and internucleosomal DNA cleavage had he been aware of this work. While Skalka and colleagues (1976) did not show that the DNA degradation was associated with apoptosis, it was known by Kerr, Wyllie, and Currie at that time that lymphocytes were extremely sensitive to apoptotic induction by irradiation (Searle et al., 1975).

W T H E SClENTlfiC REVOLUTION GAINS MOMENTUM A. New investigators enter the field As previously mentioned, the extension of the apoptosis concept from a strictly morphological phenomenon to a gene-directed type of cell death with a distinct biochemical marker (internucleosomal DNA cleavage) made apoptosis a far more interesting research proposition to groups other than those of Kerr and Wyllie. It

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was not long before new researchers began to enter the field, and many did so after encountering the striking DNA ladder in their own fields of investigation. The roles of apoptosis in the immune system and in cancer were to be the main driving forces in apoptosis research over the next decade. John Cohen and Richard Duke from Colorado were among the first of the “new” researchers to enter the field. They were to make significant contributions to our understanding of the role of apoptosis in the immune system in the early- to mid-1980s (Duke et al., 1983; Cohen and Duke, 1984; Cohen et al., 1985; Duke and Cohen, 1986) and have continued in the field to this day. In the field of cancer, an important development in 1984 was the finding that apoptosis was markedly enhanced in preneoplastic foci and nodules developing in liver after the administration of carcinogens (Bursch et al., 1984; Columbano et al., 1984). The enhanced apoptosis in these nodules was thought to indicate an attempt by the body to eliminate damaged cells before they became neoplastic. Kerr and Wyllie continued their active participation in apoptosis research during this period. A notable contribution by Kerr was the suggestion that phagocytosis and degradation of apoptotic bodies without the release of their contents might be an important factor in the containment of viral infections (Clouston and Ken; 1985). Wyllie and his group, on the other hand, showed that the cellular condensation observed morphologically in apoptosis was associated with an increase in density (Wyllie and Morris, 1982).They also suggested that changes observed in the nature of carbohydrates exposed on the surface of apoptotic bodies might be responsible for their rapid phagocytosis (Duvall et al., 1985). While most of Kerr’s efforts were concentrated on studies seeking to extend the known range of the occurrence of apoptosis, Andrew Wyllie began to focus more on the molecular mechanism of apoptosis.

B. Application of molecular biology techniques to the study of apoptosis By the mid-l980s, interest in apoptosis had reached the stage where the hunt to

identify genes that carried the cell death instructions had begun. Horvitz and colleagues were one of the first groups to become involved in this area of research. They used the nematode, Caenorhabditis elegans, as a model in which to study the genetic control of programmed cell death (Ellis and Horvitz, 1986; Yuan and Horvitz, 1990). One of the most significant breakthroughs in this regard, however, came in 1987 when Andrew Wyllie and his colleagues reported that increased apoptosis in tumors could result from processes intrinsic to the tumor cells themselves; different apoptotic levels were found in otherwise similar fibroblast tumors expressing different oncogenes (Wyllie et al., 1987). The important implications of these findings were not widely appreciated at the time. Nevertheless, this study was the forerunner of one of the most exciting areas of apoptosis research in the

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1990s, the role of oncogenes and tumor suppressor genes in the regulation of apoptosis. As such, it represents a major landmark in apoptosis research. Moreover, it can be considered to mark the beginning of a new era in apoptosis research, the application of molecular biology techniques to the study of apoptosis. From this time on, studies using these techniques were to rapidly advance our understanding of the apoptotic mechanism. In 1987, Fesus and his colleagues reported that tissue transglutaminase was induced and activated during the apoptotic process. The following year, the occurrence of castration-induced apoptosis in the rat ventral prostate was reported to be associated with a cascade induction of c-fos, c-myc, and heat-shock 70K transcripts (Buttyan et al., 1988).Interestingly, Kerr and Searle had shown some 14 years earlier that the deletion of cells in this model occurred by apoptosis. It could be argued that given the small number of cells undergoing apoptosis at any one time in involuting prostate, this may not have been the ideal system in which to attempt to correlate gene expression with the occurrence of apoptosis. Nevertheless, subsequent studies carried out by other scientists seem to have confirmed a role for these genes in at least some situations [reviewed by Kerr et al. ( 1994a,b)]. A number of papers were published around this time by McConkey and colleagues that suggested a role for elevated cytosolic Ca2+ in triggering apoptosis (McConkey et al., 1988, 1990). While there subsequently have been other studies showing that elevated Ca2+is not always associated with apoptosis (Bansal et a / . , 1990; Lennon et al., 1992), the work of McConkey and colleagues attracted considerable attention at the time and undoubtedly helped to generate interest in the process. Other notable publications included studies of proteins associated with programmed cell death (Wadewitz and Lockshin, 1988), inhibition of apoptosis by phorbol esters (Kizaki et al., 1989), a possible role for the APO-1 antibody in apoptosis (Trauth et a [ . , 1989), and the role of the endonuclease in apoptosis (Arends et al., 1990). However, the findings that generated most interest in the late 1980s were those relating to the physiological role that apoptosis plays in the immune system [reviewed by Cohen (1991) and Golstein et al. (1991)l. In two separate studies on this topic published in Nature, it was shown that apoptosis was responsible for the deletion of autoreactive T-cells in the thymus during the development of self-tolerance (Smith et al., 1989) and for the selection of B-cells in lymphoid germinal centers during the humoral immune response (Liu et al., 1989). The first international meeting to highlight apoptosis, “Modulating factors in multistage chemical carcinogenesis,” was held in Sardinia in September 1989. The first dedicated apoptosis meeting took place the following year at the prestigious Cold Spring Harbor Laboratory. David Tomei and his colleague Fred Cope (both from Columbus, OH) had been working the field of apoptosis for only a relatively short time when they decided to organize the Cold Spring Harbor meeting to explore molecular approaches to the mechanism and regulation of apoptosis. A select group made up of most of the leading apoptosis researchers of

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the day were invited to attend. Many of the participants were given the opportunity to contribute a chapter to the book “Apoptosis: the molecular basis of cell death,” which was published the following year (Tomei and Cope, 1991). The Cold Spring Harbor Symposium set the scene for the upcoming scientific revolution that apoptosis has become. Hockenberry and his colleagues (1990) ignited this revolution when they showed that the bcl-2 protooncogene operated in a manner completely different from other oncogenes by blocking apoptosis. Vaux et al. (1988) had reported some 2 years earlier that bcl-2 cooperated with another oncogene, c-myc, to immortalize pre-B-cells,but had not shown that it did so by blocking apoptosis. There now appears to be little doubt that the prolonged survival of cells overexpressingbcl-2 is a factor in predisposition to malignancy (Hockenberry, 1991; Korsmeyer, 1992). Moreover, bcl-2 appears to increase the resistance of tumor cells to anticancer drugs (Collins et al., 1992; Lotem and Sachs, 1992; Miyashita and Reed, 1992). The bcl-2 findings and those published soon after suggesting a role for the p53 tumor suppressor gene in the regulation of apoptosis (Yonish-Rouach et al., 1991) generated enormous interest in apoptosis. This is clearly reflected in the number of apoptosis publications coming out: the handful each year in the mid-1970s has now turned into a flood of publications each month.

IV. CONCLUSIONS In the last 3-5 years there has been an incredible surge of interest in apoptosis, and our knowledge of the mechanisms involved and its regulation has grown enormously. The involvement of apoptosis in the deletion of lymphocytes in AIDS and in the pathogenesis of cancer and autoimmune diseases has been clarified, at least to some extent, and studies attempting to link apoptosis with neurodegenerative diseases and even aging are now underway. It seems incredible looking back that it took more than 20 years for the wider scientific community to finally recognize the importance of apoptosis and the critical role it plays in so many aspects of medicine and biology. The introduction of the science of molecular biology and the application of these techniques to the study of apoptosis undoubtedly played a major role in hastening this acceptance. Once concrete examples of the successful application of the apoptosis concept were known and the powerful techniques of molecular biology developed, the explosion of apoptosis research was inevitable. The revolution in late 20th century life science has been characterized by the recognition that cell death by apoptosis is a fundamental factor in the biology and pathology of cells and tissue. Cell and tissue biology and pathology cannot be understood solely on the basis of cell proliferation (mitosis) and differentiation, as has been the prevailing model for most of the 20th century. As cell and

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tissue biology and pathology enter the 21st century, we will be concerned with understanding and controlling three fundamental cellular processes-mitosis, differentiation, and apoptosis. The concept of apoptosis has not simply promoted a reinterpretation of empirical evidence, it has brought about a transformation in the way in which we think about biological systems and also actively influenced the kind of evidence we are now seeking. The excitement of the pioneering apoptosis research in the 1970s and 1980s is now making way for the intensive search for the molecular mechanisms underlying the process and the means by which these mechanisms can be manipulated. Development of the apoptosis concept by Kerr, Wyllie, and Currie critically depended on the breadth of their understanding of biological and pathological processes combined with their passionate pursuit of meaning in the interpretation of observations of hitherto unrecognized phenomena. Are there still further phenomena of fundamental biological significance that will be dependent on an equally broad understanding of principles for their recognition? If so, will the rise of specialization in biological science adequately equip modern scientists for the task of identifying these phenomena?

Acknow Ied gme nts The excellent technical contribution made by Mr. Clay Winterford of the University iif Queensland Pathology Department Electron Microscope Unit, whose help with electron microscopy and photography was invaluable, is gratefully acknowledged. This work was supported hy a Research Encouragement Grant from the Queensland University of Technology.

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Skalka, M., Matyisovi, J., and Cejkovi, M. (1976). DNA in chromatin of irradiated lymphoid tissues degrades in vivo into regular fragments. FEBS Lett. 72:271-274. Smith, C. A., Williams, G. T.,Kingston, R., Jenkinson, E. J., and Owen, J. J. T. (1989). Antibodies to CD3E-cell receptor complex induce cell death by apoptosis in immature T cells in thymic cultures. Nature 337:181-184. Steel, G. G. (1967). Cell loss as a factor in the growth rate of human tumours. Eur. J. Cancer 3:381-387. Tomei, L. D., and Cope, F. 0. (1991). “Apoptosis: the molecular biology of cell death.” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Trauth, B. C., Klas, C., Peters, A. M. I., Matzku, S., Moller, P., Falk, W., Debatin, K.-M., and Krammer, P. H. (1989). Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245:301-305. Vaux, D. L., Cory, S., and Adams, J. M. (1988). Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335:440-442. Wadewitz, A. G., and Lockshin, R. A. (1988). Programmed cell death: Dying cells synthesise a co-ordinated, unique set of proteins in two different episodes of cell death. FEBS Lett. 241:19-23. Walker, N. I., Harmon, B. V., Gobe, G. C., and Kerr, J. F. R. (1988). Patterns of cell death. Meth. Achiev. Exp. Pathol. 13:18-54. Wyllie, A. H. (1974). Death in normal and neoplastic cells. 1. Clin. Pathol. Suppl. (R. Coll. Pathol.) 7:35-42. Wyllie, A. H. ( 1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284:555-556. Wyllie, A. H., and Morris, R. G. (1982). Hormone-induced cell death. Purification and properties fo thymocytes undergoing apoptosis after glucocorticoid treatment. Am. J. Pathol. 109:78-87. Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1973a). Cell death in the normal neonatal rat adrenal cortex. J. Pathol. 111:255-261. Wyllie, A. H., Kerr, 1. F. R., Macaskill, I. A. M., and Currie, A. R. (1973b). Adrenocortical cell deletion: the role of ACTH. J. Pathol. 111:85-94. Wyllie, A. H., Kerr, J. F. R., and Currie, A. R. (1980). Cell death: the significance of apoptosis. Int. Rev. Cytol. 68:251-306. Wyllie, A. H., Rose, K. A,, Morris, R. G., Steel, C. M., Foster, E., and Spandidos, D. A. (1987). Rodent fibroblast tumours expressing human myc and ras genes: growth, metastasis and endogenous oncogene expression. Br. J. Cancer 56:251-259. Yonish-Rouach, E., Resnitzky, D., Lotem, J., Sachs, L., Kimchi, A,, and Oren, M. (1991). Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352:345-347. Yuan, J., and Horvitz, H. R. (1990). The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death. Dew. Biol. 138:33-41.

Molecular Basis for X-Linked Immunodeficiencies C. I. Edvard Smith

Department of Clinical Immunology and Center for BioTechnology Department of Bioscience at Novum Karolinska Institute, S-141 57 Huddinge, Sweden

Luigi D. Notarangelo Department of Pediatrics University of Brescia 1-25123 Brescia, Italy

1. INTRODUCTION Many developments have enabled the identification of several genes defective in immunodeficiency. This chapter discusses four X-linked disorders: X-linked agammaglobulinemia (XLA), X-linked severe combined immunodeficiency (XSCID), Wiskott-Aldrich (WAS), and hyper-IgM (HIGM) syndromes. These disorders all result in an increased susceptibility to infections, with each disease having its typical spectrum. The defective genes were reported within 18 months and were isolated by positional cloning in XLA and WAS and by the candidate gene approach in HIGM and XSCID. At least three of the gene products are involved in signal transduction, representing receptors (HIGM, XSCID) or cytoplasmic transducers (XLA). Large numbers of mutations have been identified worldwide and these have proven instrumental in deciphering the function of affected proteins. International study groups for mutation analysis have been established for these disorders, with the corresponding databases being accessible to any investigator. Mutation analysis has also allowed the inclusion of disease forms with milder symptomatology, previously anticipated to represent separate disorders. Genetic approaches to develop future treatment modalities are under way and several animal models have been described. Of note is that mutations in animal genes Advances in Genelics, Vol. 35 Copyright 0 1997 by Academic Press

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frequently result in phenotypes differing in many respects from the corresponding human immunodeficiencies.

II. BRIEF HISTORY OF IMMUNODEFICIENCIES A. X-l inked agammaglo buIinemia In 1952 a paper was published that heralded a new era in immunology, namely the study of immunodeficiencies. This paper defined a new disease, agammaglobulinemia, by describing its phenotype in terms of its susceptibility to infections, with its primary cause being the lack of immunoglobulins, and introduced substitution with gammaglobulins as a novel treatment (Bruton, 1952). This disease, XLA, was reviewed in detail in Sideras and Smith (1995). In their classical survey of immunodeficiencies, Rosen, Cooper, and Wedgwood refer to XLA as the prototype for immunodeficiency diseases (Rosen et al., 1984), and through the years a very large number of reviews have been written over XLA (Good and Zak, 1956; Firkin and Blackburn, 1957; Gitlin and Janeway, 1957; Good et al., 1962; Rosen and Janeway, 1966; Burgio and Ugazio, 1982; Lederman and Winkelstein, 1985; Conley and Puck, 198813;Hendriks and Schuurman, 1991; Timmers et al., 1991; Conley, 1992; Hermaszewski and Webster, 1993; Kinnon et al., 1993; Rawlings and Witte, 1994; Smith et al., 1994a; Notarangelo, 1996; Rawlings and Witte, 1995; Sideras and Smith, 1995; Mattsson e t al., 1996; Ochs and Smith, 1996; Vihinen and Smith, 1996). The basis for the identification of XLA was the combination of clinical observations and the application of new technology. Arne Tiselius of Uppsala University in Sweden developed electrophoresis technology in the 1930s (Tiselius, 1937; Tselius and Kabat, 1939), and this method was used to identify the defect in XLA. At a meeting organized by Robert A. Good in 1968, Ogden C. Bruton describes how serum electrophoresis was applied in the initial case:

I [O. C. Bruton] heard several times after that, that up at the Walter Reed Institute [in Washington, DC] they had a new machine that could fractionate proteins, the Tiselius moving boundary machine, so I went up and asked them if they would do his [the patient’s] serum and they said they would be delighted. So I sent some blood to them and the next day the technician called me up and said she was sorry but there was something wrong with the machine, that it did not show the boy to have any gammaglobulin, would I mind sending up some more blood. I sent up some more blood, and the same report came back, no gammaglobulins; can’t build antibodies.

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We were very new in our thinking in those days. This sounds so simple now that it seems to me hardly worth repeating, but I thought, Well if he doesn’t have any gammaglobulins, maybe we could try treating him with gammaglobulin.” (Bruton, 1968) Bruton’s idea materialized as a result of his initiating treatment with gammaglobulin (Bruton, 1952), which was available through the procedures developed by Cohn et al. (1940). As previously discussed in more detail (Sideras and Smith, 1995), it has been argued that the patient originally described by Bruton may not have had X-linked agammaglobulinemia (Conley and Puck, 1988b; Burgio et al., 1993). However, although certain features of Bruton’s original patient are unusual for XLA, they do exist, also in patients with mutation-verified XLA, and it cannot be excluded that the patient indeed had XLA. Furthermore, later in 1952 Bruton, together with Apt, Gitlin, and Janeway, described other patients with agammaglobulinemia demonstrating the typical X-linked inheritance (Bruton et al., 1952). Evidence for a sex-linked disease was mainly provided by Janeway and his associates, who identified several informative families (Janeway et al., 1953; Janeway, 1954). The conceptual importance of the original publication (Bruton, 1952) is reflected by the effect it had on research. Within 10 years after Bruton’s description, more than 300 patients with agammaglobulinemia had been reported worldwide, and several new immunodeficiency diseases had been characterized based on the analysis of immunoglobulins (Good et al., 1962).

B. Severe combined immunodeficiency (SCID) Two years prior to Bruton’s description of agammaglobulinemia, a report by the Swiss investigators Glanzmann and Riniker (1950) depicted a congenital form of lymphocyte deficiency. The authors referred to this disease as “essentielle Lymphocytophtise” [Greek phtisis, from phthiein to decay], i.e., an idiopathic lymphocyte-wasting syndrome. Alymphocytosis in an adult had already been published in 1929 (Grote and Fischer-Wasels, 1929). Their patient initially had normal lymphocyte counts but developed complete alymphocytosis within 2 months. At the autopsy, atrophy of the lymph nodes and spleen was found. Glanzmann and Riniker ( 1950) described children in two families, in one of which both a brother and a sister were affected. In Glanzmann’s own words: Based on the observation of two identical cases in early infancy a fatal infection with oidum albicans [Candida albicans] is described under the name “essential lymphocytophthisis” which does not only attack the oro-pharynx, but stretches into the larynx and the lungs, the whole

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length of the oesophagus, stomach, and bowel. At an early time there is lymphopenia in the blood which towards the end goes down to a few percent (alymphocytosis as an analogon to agranulocytosis). The lymphocytophthisis on the one hand and panmyelophthisis on the other hand in the same family point to a constitutional weakness of the blood-forming organs. (Glanzmann and Riniker, 1950) As the disease name indicates, the authors interpreted their findings as if the lymphocytopenia developed over time. Riniker hypothesized that “. . .Besides the constitutional factor leading to lymphocytophthisis disturbance of intestinal resorption on account of an infection with monilia albicans [Candida albicans] producing necrosis has to be made responsible for the lethal course of the disease,” but as an alternative possibility suggested that substances from the fungal infection could be directly lymphotoxic. Despite the fact that these authors described a hereditary severe combined immunodeficiency, they mention that the defect may develop after birth rather than being present from the beginning. Furthermore, as bone marrow transplantation techniques had not yet been developed, it was not possible to correct the defect as was made using gammaglobulin in Bruton’s patient (Bruton, 1952). Thus, Bruton’s report had a much greater impact as it convincingly demonstrated the role of antibodies in the immune defense and formed the basis for the development of immunodeficiency as a discipline of its own. During the 1950s, several additional reports appeared describing patients with SCID [Donohue, 1953; Keidan e t al., 1953; Kozinn et al., 1955; Hitzig e t al., 1958; Tobler and Cottier (1958) described brothers and sisters of the two infants previously reported by Glanzmann and Riniker (1950); reviewed in Hitzig and Willi (1961) and Gitlin and Craig (1963)l. When serum electrophoresis was applied in the analysis of these patients, the absence of gammaglobulins in this syndrome was established. However, in patients described in the 1950s, the isolated absence of gammaglobulins was sometimes not distinguished from agammaglobulinemia as being part of a SCID syndrome. Thus, the patient described by Keidan e t al. (1953) was frequently referred to as having agammaglobulinemia (Martin, 1954; Elphinstone e t al., 1956; Wallenborn, 1960), despite most likely suffering from an autosomal recessive form of SCID as she was the child of first cousins, developed fatal vaccinia, and was found to have both agammaglobulinemia and lymphocytopenia. As several of the publications describing SCID patients emanated from Switzerland (Glanzmann and Riniker, 1950; Hitzig et al., 1958; Tobler and Cottier, 1958; Hitzig and Willi, 1961), this disease was frequently referred to as the Swiss type of agammaglobulinemia. In 1959, de Vaal and Seynhaeve in Amsterdam described a congenital absence of leukocytes and lymphoid organs in a male twin pair. They named this disease “reticular dysgenesia,” and despite both penicillin and gammaglobulin re-

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placement therapy, both twins died within 8 days after birth under septic conditions, demonstrating the severity of this condition (de Vaal and Seynhaeve, 1959). In comparison, the two children with SCID described by Glanzmann and Riniker (1950) died at the age of 5 and 6 months, respectively. Initially, the existence of autosomal recessive as well as X-linked forms of SCID was not appreciated. In fact, as late as 1968, Walter H. Hitzig, summarizing the work in this field, would not accept this subgroup classification as definitive. This is examplified by the following discussion (Hitzig, 1968): “Dr. Good: We now feel that experience with five families establishes, as well as genetic bases can be established, the sex-linked nature of one form of thymic-deficiency syndrome or thymic dysplasia. . . . Dr.Hitzig: I would like to stress again that I was extremely strict in the cases I would accept.” As 70 well-documented, and an additional 74 very probable, SCID patients were known at the time (Hitzig, 1968), this enigma is even more surprising in view of the X-linked form being the most common type of SCID (Fischer et al., 1996; Notarangelo, 1996). However, different opinions existed (Rosen et al., 1962; Gitlin and Craig, 1963), as illustrated by the remark made by Gitlin and Craig (1963): “The marked difference in the sex incidence of the disorder [SCID] between Europe and United States is not clear, but there would appear to be two different modes of inheritance involved, one sex-linked. . . .” Irrespective of Hitzig’s reluctance to accept sex-linked forms as a disease entity, in their classical paper from 1958, Hitzig et al. most likely describe a patient with an X-linked form of SCID, as a nephew displaying the same disease phenotype was born in 1980 (Hitzig, 1993/94; W. H. Hitzig, personal communication). Although mutation analysis has not yet been performed in this family (W. H. Hitzig, personal communication), the inheritance pattern is highly suggestive of an abnormality in the gene for the interleukin receptor common y (yc)chain (synonymously interleukin 2 receptor y-chain), which in 1993 was shown to be defective in this disease (Noguchi e t d . , 1993~). Several other forms of SCID have been identified (Fischer et d., 1995), but will not be the subject of this chapter.

C. Wiskott-Aldrich syndrome The first complete description of clinical and hematological features of WAS appeared in 1937, when similar manifestations (hemorrhages associated with thrombocytopenia, infections, and eczema) were reported in all three male siblings of a single family by a German investigator (Wiskott, 1937). However, the author incorrectly attributed these symptoms to a familial form of Werlhof‘s disease, nor did he perceive the X-linked pattern of inheritance, since he thought that the father’s nephew (who had episodes of diarrhea, with occasionally bloody stools) suffered from a mild form of the disease. Nonetheless, Wiskott realized the fundamental role of platelet abnormalities in WAS (authors’ translation):

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In spite of a certain incompleteness of the examinations, it can be assumed that the basis of the hemorrhagic diathesis [a congenital susceptibility or liability to certain diseases] is mainly a problem of the platelet compartment. One could be tempted to use the thrombopenic component in children B and C for diagnosis of a familial Werlhof‘s disease, especially as the section of the last child . . . demonstrated the main megakaryocytic form to be some pathological giant bone marrow cells, which completely lack any granula. This description is similar to an essential thrombocytopenia described by Franck. However, some results (from Table 1) argue for a functional abnormality of the platelets, especially the almost normal, or slightly prolonged bleeding time, the almost normal number of platelets in case A, who had already started to bleed, and further, the completely missing retraction with a platelet number of 80,000 . . . and finally, the missing clamping of those platelets in the peripheral blood smear. It seems to me that in some blood smears of child C, low or no granules are present in the platelets, similar to what was observed in Glanzmann’s disease (Wiskott, 1937) It was only in 1954 that the X-linked inheritance of WAS was fully appreciated when, starting from an affected male infant (who presented with typic cal signs: “draining ears, eczematoid dermatitis and bloody diarrhea”), Aldrich could trace a family with 16 affected male infants in three generations (Aldrich et al., 1954). As is often the case, it was the family itself that provided extremely valuable information that helped define the inheritance pattern:

From the onset it was clear that the infant’s illness was a familial story to the family. In the initial interview the mother stated that other related male infants had died under somewhat similar circumstances. Fortunately for the present purposes, the family has kept a careful record of births and deaths going back to the couple that originally migrated to this country from Holland six generations before. While definition of the X-linked pattern of inheritance was instrumental in the search for affected males with this disease in other families, the authors failed to recognize the primary nature of the platelet defect, since they suggested “that the basic deviation is a genetically determined increased liability to infection and that the thrombocytopenic purpura observed in the proband was secondary to this” (Aldrich et al., 1954). However, once the X-linked inheritance and the main clinical features of WAS were established, more than 90 affected males were identified within a decade (Cooper et al., 1968), all regularly expressing the clinical triad of thrombocytopenia, intractable eczema, and extraordinary susceptibility to infections. The demonstration that, even when kept in a

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pathogen-free environment, affected males still manifest severe bleeding tendency (Cooper et al., 1968) ruled out the possible secondary nature of the platelet defect, and thus unified the clinical triad as a primary manifestation of the disease. Nonetheless, despite extensive characterization of morphological and biochemical defects of platelets and lymphoid cells, the pathogenesis of such disparate clinical symptoms has long remained elusive and still largely is, even following the cloning of the responsible gene (Derry et al., 1994). The description of a large number of patients has led researchers to establish that WAS is a severe disease: in 1980, Perry found that mean survival had increased from 8 months for patients born before 1935 to 6.5 years for those born after 1964, the main causes of death being infections, hemorrhages, and tumors (especially lymphoid malignancies) (Perry et al., 1980). Yet some male patients with prolonged survival, and no or little eczema or evidence of increased susceptibility to infections, were reported (Vestermark and Vestermark, 1964; Canales and Mauer, 1967; Chiaro and Dharmkrong-at, 1972; Cohn et al., 1975). In this group of patients, a diagnosis of X-linked thrombocytopenia (XLT) was established. The relationship between XLT and WAS has been debated until molecular genetics provided evidence for coincident gene mapping (Donner et al., 1988) and for defects in a single gene (Derry et al., 1995a; Villa et al., 1995; Kolluri et al., 1995). Thus, these observations have further enlarged the clinical spectrum of WAS. Finally, in contrast to the well-recognized X-linked inheritance of WAS/XLT, some female patients have been reported that share typical clinical symptoms and laboratory abnormalities of WAS (Lin and Hsu, 1984; Conley et al., 1992), raising the possibility of an autosomal variant of the disease.

D. X-linked hyper-lgM syndrome X-linked immunodeficiency with hyper-IgM is a rare disorder, characterized by the absence of serum IgG, IgA, and IgE, whereas IgM levels are normal to elevated [reviewed in Notarangelo et al. (1992)l. The original description of the disease dates back to 1960, when unique, previously undescribed immunological features were reported by French investigators in a 15-year-old male who suffered from recurrent pneumonia, meningitis, and lymphoadenopathy. Immunoelectrophoresis revealed “severe diminishment of the y-globulins. The ones that were present showed distinct peculiarities, in that they had a rapid mobility (PZ), whereas in severe, typical agammaglobulinemia only traces of low-mobility y-globulins were found. [In addition, immunoelectrophoresis showed] almost lack of PZA-globulins [and] markedly increased P2-macroglobulin (that is almost always missing in agammaglobulinemia).” Persistence of isoagglutinins was demonstrated, which are usually missing in XLA. In the authors’ words, “the association of severe hypogammaglobulinemia with increase of P2-macroglobulin is surprising. We are entering an area that has yet to be explored” (Israel-Asselain et al., 1960).

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One year later, similar immunological findings were described by Rosen and his associates (1961) in Boston in two children with a history of recurrent bacterial infections. Using different techniques (immunoelectrophoresis, sucrose gradient ultracentrifugation, and analytical ultracentrifugation), Rosen showed that “in the patients reported, the concentrations of 19s gamma-globulin in serum were greater than normal, the concentrations of 7s gamma-globulin in serum were approximately 60 and 100 mg/100 ml, and P2A-globulin was not detectable. This dissociation in gamma-globulin synthesis between the 19s and the 7s gamma-globulinswe have termed dysgammaglobulinemia.” Rosen also provided evidence for a disturbed structure of lymphoid tissues in hyper-IgM, since “the lymph nodes showed poorly organized structure, absence of secondary or germinal centers and absence of plasma cells” (Rosen et al., 1961). Although the original description concerned male patients whose clinical course was undistinguishable from that of XLA, Hong et al. (1962) first noticed that HIGM often has unique features. In particular, these authors appreciated the association with neutropenia, recurrent stomatitis, and increased susceptibility to Pneumocystis carinii infection: The clinical course [of a child affected with hyper-IgM] was notable for the absence of severe bacterial infections . . . [and] was characterized by recurrent bouts of stomatitis in association with persistent neutropenia, and at 27 months of age he contracted alveolar proteinosis upon which was superimposed a Pneumocystis carinii infection. (Hong et al., 1962) Hong et al. (1962) also extended Rosen’s observations on lymph node pathology in HIGM: “the lymph nodes were seen to be devoid of reticuloendothelial or reactive centers and to contain only sparse, unevenly distributed lymph follicles composed of mature lymphocytes.” The distinctive nature of HIGM (originally referred to as dysgammaglobulinemia) versus XLA was thus recognized. Over the following years, several patients with gammaglobulin deficiency and increased 19s macroglobulins were identified. This group of patients proved heterogeneous: although the majority of affected individuals were males and their clinical symptoms were already apparent early in childhood (pointing to a congenital disorder with an X-linked pattern of inheritance), a few girls and adult women with similar immunological abnormalities were reported (Rosen and Bougas, 1963; Hinz and Boyer, 1963; Kyong et al., 1978), indicating the existence of other genetic variants as well as of acquired forms of the disease. In addition to HIGM, other forms of dysgammaglobulinemia (in which levels of one or more, but nor all, immunoglobulin isotypes are markedly different from normal values) were reported in the 1960s, indicating the need for a sub-

7s

3. X-Linked Immunodeficiencies

65

classification of dysgammaglobulinemias. However, because this resulted in rather confusing terminology, the HIGM was variably referred to as “dysgammaglobulinemia type I” (Rosen and Janeway, 1966) or “dysgammaglobulinemia type 11” (Hobbs et al., 1967). It was only in 1974 that the disease was defined as “immunodeficiency with hyper-IgM” (Cooper et al., 1974). The genetic heterogeneity, confusion in terminology, and the rather limited number of patients reported (around 120) (Notarangelo et al., 1992) have resulted in major difficulties in the elucidation of the major biological features and the pathogenetic mechanisms. Figure 3.1 (color plate) shows some of the investigators involved in the original description of X-linked immunodeficiency diseases.

111. PHENOTYPE AND CELLULAR BASIS OF XLA The boy described by Bruton experienced pyogenic infections, including several episodes of septicemia (Bruton, 1952). In XLA, the onset of symptoms is normally within the first year of age with a mean age of diagnosis of 2.5 years in familial cases and 3.5 years in nonfamilial (Lederman and Winkelstein, 1985; Hansel et al., 1987). These bacterial infections (Lederman and Winkelstein, 1985; Stiehm et al., 1986; Spickett et al., 1991;Ochs and Smith, 1996), as well as enteroviral disease (Lederman and Winkelstein, 1985; McKinney et al., 1987; Ochs and Smith, 1996), are hallmarks for XLA as reviewed in Hermaszewski and Webster (1993) and Sideras and Smith (1995). Table 3.1 summarizes the clinical features and some other characteristics of XLA. Analysis of the half-life of gammaglobulin revealed that there was no decrease in XLA (Bruton, 1952; Janeway er al., 1953; Lang et al., 1954). Studies of patients with agammaglobulinemia, including XLA, demonstrated hypoplasia of secondary lymphoid organs lacking germinal centers and having ill-defined follicles and the absence of plasma cells, thus further supporting the notion that these cells are the main source of antibodies (Olhagen, 1953; Craig et al., 1954; Good, 1954; Good and Zak, 1956). These studies were compatible with a defective synthesis rather than increased catabolism as the cause of antibody deficiency. Additional studies utilized the transplantation of cells and tissues to further decipher the origin of XLA (Sideras and Smith, 1995). However, it was not until Naor et al. (1969) analyzed the binding of antigens to cell surface structures that the cellular basis for XLA was further defined. These investigators noticed the absence of cells with this capacity in peripheral blood of XLA patients. In the early 1970s, when techniques used to measure cell surface immunoglobulin were employed, numerous reports appeared showing a highly significant reduction of these cells in peripheral blood and tissue samples (Grey et al., 1971; Froland et al., 1971; Siegal et al., 1971; Cooper and Lawton, 1972; Choi et al., 1972; Frdand and Natvig, 1972; Yata and Tsukimoto, 1972; Preud’Homme et al., 1973; Geha et al., 1973; Aiuti et al., 1973).

Table 3.1. Disease Characteristics in X-Linked Immunodeficiencies' ~

XLA

Features lncidence/lOS

Cells displaying nonrandom X inactivation Age of o w t of of symptoms Age of death prior to thvrapy Susceptihilityto infections

XSCID

WAS

HIGM

T,B,NK~&'~

T,B,NK, granulocpes,

< XLA',' Not. found17

2.7 months19

CD34- ~ e h " - ' ~ <1 (2) V e ~ s ~ ~ ' ~

<0.5 years"

90% t before

7 monthsL9

8m ~ n t h ~ ~ ~ J ~

90% t before

Pyagenic bacrcria,

All agents, including opportunistic organisms'9J7

S. aureus ahscesxs, pyo-

0.5-1'

12.'

B7-9

.syrarj' 2 2 entem"Ims'

.18:21-26

0.44

megakanjocy ces,

genic bacteria, viral and opportunistic infectionP

Other features

-

-

Thromhcytopenia.

Tumor incidence

Miruir increase in

-

cczeina, 13%, meail nnwt

tumor formation at young age',"

9.5 ycdrS'"

'

ailolcsccnce2 I'yogcnic bacteria,

P. carinii , Cvpto-

sw&um21

26

Autoimmuniq; neutropenia" Increase in lymphoid liver, and gasrtointcstinal riimor52LJ8

"Kcfcrcnces: 'Sidcras and Smith (1995); 'Celfmd and Dosch (1983);3Fischer (1993); 4Perry e l al. (1980); 5Fasth (1982); 6Luzi er td. (1983); 7Crmleyct al. 1985); "earon c i al.(1987);5C~nlcyand I ' d (1988a); 'Ol'uckrtd. (1987);'Wonlcy ct d. (19R8); 'Yhndship crnl. (1991); "Gcaly C t al. (1980); 141'rchal e t d . ( 1 9 W '5Fearnnctd. (1988); '%onley (1992): l7Hendriksetd. (1990); '"Ledtnnanand Winkclstcin (1985); "Hitzig (196s); "'SullivancraI. (1994); 21Norarangcloet d.(19Y2);"biensink et al. (1984); 7'+Aldrichet d.(1954); 241 lermaszewski and Wehster (1993);W c h s and Smith (1996); "%pickett et al. (1991); "Fisher et d . (1995); Worarangelo (unpublished).

3. X-Linked Immunodeficiencies

67

The small number of B cells present in peripheral blood in XLA patients (less than 1% versus 5-15% in normal individuals) demonstrated an immature phenotype (Tsuchiya et al., 1980; Conley, 1985; Golay and Webster, 1986). In the late 1970s a progenitor in normal B lymphocyte development, designated the preB cell, expressing cytoplasmic immunoglobulin p heavy chains, but lacking light chains and surface immunoglobulins, was identified (Raff et al., 1976; Hayward et al., 1977; Gathings et al., 1977; Burrows et al., 1979). The presence of pre-B cells in XLA patients indicated that the differentiation block occurred in the transition from pre-B to B lymphocytes (Pearl et al., 1978; Fu et al., 1980). Pearl et al. (1978) also provided evidence for a reduced proliferative capacity of these cells, and Conley (1985) obtained results indicating that the defect could also be exerted at later stages. Studies of lymphocyte subpopulations enabled Campana et al. ( 1990) to further delineate the differentiation defect, demonstrating fewer cell cycling precursors and an increased pro-B to pre-B cell ratio. As will be further described in the following sections on the other Xlinked immunodeficiencies, the analysis of X-chromosome usage enabled investigators to determine the cellular origin of most of these defects. These studies were based on the original observations by Lyon (1966) who showed that one of the X chromosomes in cells from females is always inactivated and that this is a random phenomenon established early during embryogenesis. When cell survival/differentiation is dependent on the expression of a nonmutated gene, this will enable the identification of cells selectively (nonrandomly) expressing one of the X chromosomes, as initially demonstrated in the Lesch-Nyhan syndrome (Lesch and Nyhan, 1964; Nyhan et al., 1970). This technique made it possible for Nahm et al. (1983) to demonstrate nonrandom X-chromosome usage in B cells from the mouse strain CBA/N carrying the xid mutation, a defect later shown to be caused by an abnormality in a mouse gene corresponding to the one defective in humans with XLA (Thomas et al., 1993; Tsukada et al., 1993). In XLA, a similar selective usage in B lymphocytes was demonstrated by employing various technologies (Conley et al., 1986; Fearon et al., 1987; Conley and Puck, 1988a). These studies clearly showed that the defect in XLA is intrinsic to the B-cell lineage, simplifying future attempts to clone the defective gene. A schematic representation of the differentiation block in XLA is depicted in Figure 3.2 (color plate).

IV. CLONING OF THE XLA GENE AND SPECTRUM OF MUTATIONS A. Mapping and cloning of the XLA gene In order to map the location of the XLA gene on the X chromosome, Xg blood group determinants were investigated initially (Sanger and Race, 1963; Rosen et al., 1965; Adam et al., 1971) and later the enzyme G6PD (Prchal et al., 1980).

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These markers are located on the end of the short and long arms, respectively, and were uninformative. As reviewed in greater detail in Sideras and Smith (19954, the first linkage data were obtained using the polymorphic markers DXS3, DXS17, and DXS94, localizing the XLA gene to the midportion of Xq (Kwan et al., 1986; Ott et al., 1986; Mensink et al., 1986; Malcolm et al., 1987), as depicted in Figure 3.3. In two later reports, a crucial finding was the demonstration of the tight

Figure 3.3. Schematic representation of the X chromosome depicting the location of genes defective in primary immunodeficiencies.

3. X-Linked Immunodeficiencies

69

linkage of XLA to the marker DXS178, setting the order of the genes: centromer-DXS3-(XLA, DXS178)-DXS17-telomer (Guioli et al., 1989; Kwan et

al., 1990).

Use of yeast artificial chromosomes (Yacs) hybridizing to DXS178 enabled Vetrie and his collaborators at the Karolinska Institute, Huddinge (Stockholm), Sweden, and the University of UmeA, UmeA, Sweden, to isolate the XLA gene (Vetrie et al., 199313).The technique employed was direct cDNA selection (Lovett et al., 1991; Parimoo et al., 1991), which allowed an enrichment of more than 102-fold using Yacs (Vetrie et al., 199313; Vorechovsky et al., 1994). This technique was utilized in the beginning of 1992 using two cDNA libraries derived from a Burkitt’s lymphoma and an EBV-immortalized cell line from fetal liver (Sideras et al., 1992). In addition to the XLA gene, four ubiquitously expressed novel genes were isolated using this technology (Vorechovsky et al., 1994), and a member of the PKC family was also found to map to this region (Mazzarella et al., 1996). Using a subclone of one of the isolated cDNAs as a probe, abnormalities in Southern blots were first seen in TqI-digested DNA from two XLA patients both carrying point mutations in this gene (Vetrie et al., 1993~). The XLA gene was also identified in another collaborative effort using a different strategy. Tsukada and associates (1993) were searching for novel kinases as these were believed to play crucial roles in B lymphocyte development. By probing a mouse B lineage progenitor cDNA library with a kinase domain clone, they identified a clone that contained a long 5’ region. Mapping studies located the corresponding gene to the X chromosome, and XLA became the candidate disease. The corresponding protein and mRNA were lacking in some XLA patients, compatible with this being the affected gene. These two investigations, both published in January 1993, thus complemented each other and established that the identified gene was defective in XLA. Several investigators provided additional mapping data (Lovering et al., 1993a,b; Parolini et al., 1993; O’Reilly et al., 1993a,b; Vetrie et al., 1993a), and in 1994 a 6.5-Mb Yac contig incorporating 33 DNA markers was reported (Vetrie et al., 1994). The first gene ever identified using a positional strategy was the one mutated in X-linked chronic granulomatous disease (Royer-Pokora et al., 1986). XLA became the second immunodeficiency identified by positional cloning and was the 17th gene isolated by this approach (Collins, 1995). As described in Section VIII, the gene defective in WAS was identified by a similar approach in 1994 (Derry et al., 1994). Certain characteristics of the XLA gene are presented in Table 3.2.

B. Btk, a novel PTK encoded by the XLA gene The XLA gene was found to encode a novel cytoplasmic protein-tyrosine kinase, designated Btk [Bruton’s agammaglobulinemia tyrosine kinase, formerly Atk

Table 3.2. Gciic and Molecular Characteristics in X-Linked Immunodeficiencies"

Rature Locus dcsignation'

Mendelian inheritance[ in man (MIM) numkr Gene product Genbank accession number Size of gene (kb) Number nf e x m s Sire of mRNA (kb) Length of protein (ammo ~ L I A ) MW ( k h )

XLA

XSCID

HIGM

WAS

-

AGMXl 300300

SCIDX1 3083RO 3WOb

WAS 301000 31 3 9 w

HEM1

Bruton's agammaglobulinemia tyrosine kinase, Btk2" X58957

Common y-chatn. yc/IL2RG5.6

Wiskott-Aldrich syndrome protein, WASP7 U12707

CD4@ligand, CD40L""

D11@86

X67878; S50586; X68550; 549008 12.5'O

97.19 127.19 2.0 (4.2)7 5027.19

37.5 3-16 1913-16 2.724 659L4 773.4

308230

520

2.321.21 26I21.22

54 (predi~ccd)~

645.17

-_

..

~.

~.

uReferences:'Pearsonetd. (1994);2Vctricetd1.(IY93c); 'Tsukadactd. (1993);"SiderasandSmith(1995);'Noguchietd. (19'33~);~Noguchierd. (lYY3h); ' D c q e t al. (1994); "Allen ct al. (19Y3a);'Kotthaucr el d.(1993); "'DiSanto t i d.(1991); 'iAniffr>er d.(1993); lZFuleihan et al. (1Y93); ''Sidcras er nl. (1994); '+Hagemam e r d . (1994); '50htaet nl. (1994); 16RohrerEL al. (1944); "Noguchi et d.(1W3a); 'BPuck e t a l . (1993); "Kwan et d.(1994); 2oVilla r t d . (19948); ZIGnfk,rnl.(1992); 22Hollenhaughetal. (1Y92);Z3Ledemanetd.(1992). bDenotcsagammaglohulinemia,Swiss type. '¬es X-linked thrombocytopenia.

3. X-Linked Immunodeficiencies

71

(Vetrie et al., 1993c) or Bpk (Tsukada et al., 1993)], depicted in Figure 3.4 (color plate). This enzyme is composed of five domains designated, from the N terminus, Pleckstrin homology (PH), Tec homology (TH), Src homology 3 (SH3), SH2, and kinase (also described as SH1) (Vetrie et al., 1993c; Tsukada et al., 1993; Smith et al., 199413; Vihinen et al., 1994a; reviewed in Rawlings and Witte, 1994; Smith et al., 199413; Sideras and Smith, 1995; Mattson et al., 1996). Despite resembling the Src family kinases, the Btk family differs in several important aspects, clearly distinguishing Btk and the other members, Tec, Itk/Tsk, and Bmx, belonging to this family (Smith et al., 1994b; Sideras and Smith, 1995; Mattsson et al., 1996). The gene encoding the Bmx kinase is also located on the X chromosome, Xp22.2, but there is no obvious candidate disease mapping to the relevant region (Tamagnone et al., 1994). The exact role of Btk in signal transduction remains elusive, despite the fact that several putative targets, including membrane IgM and its accessory protein Ig-a, protein kinase C (PKC), Src family tyrosine kinases, the proto-oncogene c-cbl, and heterotrimeric G protein subunits, have been identified (Touhara e t al., 1994; Aoki et al., 1994; Kawakami et al., 1994; Saouaf et al., 1994; de Weers et al., 1994a; Cheng et al., 1994; Tsukada et al., 1994; Yao et al., 1994; Cory et al., 1995; Li et al., 1995; Yang et al., 1995; Rawlings et al., 1996; Park et al., 1996; Torres et al., 1996; Mattsson et al., 1996). The kinase domain has catalytic activity, whereas the other domains are thought to locate the protein to relevant subcellular regions in a phosphorylation-regulated fashion. Recently, Btk has been implicated as a mediator of radiation-induced apoptosis (Uckun et al., 1996).

C. Genomic organization of the BTK gene and mutation analysis in XLA Mutation analysis first identified missense mutations affecting the kinase domain in a Swedish and a British patient, respectively (Vetrie et al., 1 9 9 3 ~ )The . initial analysis was based on RT-PCR and Southern blotting (Vetrie et al., 1993c; Vorechovsky et al., 1993a). This was later followed by the use of DNA-based techniques, enabling the direct detection of mutations contingent on the elucidation of the genomic organization of the Btk gene (Sideras et al., 1994; Ohtaet al., 1994; Hagemann et al., 1994; Rohrer et al., 1994), as depicted in Table 3.2 and Figure 3.4. Recently, 69 kb of this chromosomal region, encompassing the Btk gene, have been sequenced (Oeltjen et al., 1995). The promoter has been partially characterized and contains binding sites for Spl and Spi-l/PU.l transcription factors (Himmelmann et al., 1986; Muller et al., 1996). No mutations confined to the promoter region have been reported so far. More than 20 mutation reports have been published and are listed in the legend of Figure 3.4. The single largest pa. tient material analyzed to date comprises 26 unrelated individuals from 11 countries (Vorechovsky et al., 1996). To handle the large number of patients analyzed, an international study

72

Smith and Notarangalo

group was formed in 1994 and a database designated BTKbase is now available through the Internet (http://www.helsinki.fi/science/signal/btkbase.html). The first report of the study group lists 188 mutations identified in 157 patients displaying 122 unique molecular events (Vihinen et al., 1995a), the second report lists 148 different mutations (Vihinen et al., 1996b),and the third update lists 175 unique molecular events (Vihinen et al., 1996a). Each patient is given a unique patient identity number (PIN) and the database contains information about the mutations and patients: immunoglobulin levels, B-cell numbers, age of diagnosis, symptoms, mRNA and protein data, and putative structural consequences of the mutations. Similar international study groups have subsequently been established for the other diseases discussed in this chapter, with the first edition of the corresponding databases being published in a separate issue of the journal lmmunology Today (Notarangelo et al., 1996; Puck et al., 1996; Schwarz et al., 1996). Mutations are scattered over the entire Btk gene, but some coding regions, TH, SH3, and the ATP-binding, upper region of the kinase domain, are more resistant to missense mutations, indicating functional redundancy (Vihinen et al., 1995a). CpG doublets, known to be mutational hot spots (Duncan and Miller, 1980), were affected in all sites that were mutated in three or more unrelated families (Vihinen et al., 1995a, 1996a,b). A broad spectrum of different forms of mutations have been found, distributed as missense (36%), nonsense (20%), small deletions (16%), splice site (15%), insertions (9%), and gross deletions (3%) (Vihinen et al., 1996a). In order to interpret the consequences of mutations in a structural context, molecular modeling has been used for the kinase (Vihinen et al., 1994c), SH2 (Vihinen et al., 1994a), SH3 (Zhu et al., 1994a), and PH domains (Vihinen et al., 1995b). An example of this approach is given in Figure 3.8 (color plate) showing the location of mutations in the kinase domain.

D. Agammaglobulinemia with complex phenotypes Patients with complex phenotypes have been reported. In 1980, a familial form of mild agammaglobulinemia, interpreted as XLA, and concomitant growth hormone deficiency (XLA/GHD) was published (Fleisher et al., 1980). A deletion extending from the XLA gene into an adjacent gene could explain this phenotype, but instead point mutations were seen in some XLA/GHD patients (Duriez et al., 1994; Vihinen et al., 1 9 9 4 ~Vorechovsky ; et al., 1994; Conley et al., 1994). Furthermore, by analyzing the DXSl78 marker, which is located close to the XLA gene (Vorechovsky et al., 1994) and previously found to cosegregate with XLA in all investigated pedigrees (Guioli et al., 1989; Kwan et al., 1990; Lovering et al., 1993a; Sideras and Smith, 1995), a recombinant was observed in the original growth hormone-deficient family (Stewart et al., 1995). Moreover, as normal Btk mRNA as well as protein levels were expressed, and as no mutation was found in

3. X-Linked Immunodeficiencies

73

the Btk-coding sequence in affected individuals in this family, it has been suggested that there are additional genes on the X chromosome that are essential for B-cell development and growth hormone production (Stewart et al., 1995). In one XLA family with a deletion extending centromerically from the Btk gene into the flanking expressed sequence, DXSl274E, cosegregating Xlinked torsion dystonia and sensorineural deafness were observed (Vorechovsky et al., 1994). The sequence of DXS1274 shows limited homology to the mouse proto-oncogene, int-3 (Robbins et al., 1992).

E. Animal models for XLA In 1972, a mouse strain, CBA/N, was identified that displayed an inability to respond to certain polysaccharide antigens (Amsbaugh et al., 1972).The defect was named X-linked immunodeficiency, xid, and is also characterized by low serum IgG3 levels and an absence of the B1 subset of B lymphocytes in the peritoneal cavity (Perlmutter et al., 1979; Slack et al., 1980; Scher, 1982; Sideras and Smith, 1995). In 1993, two groups independently demonstrated that the xid mouse has a mutation in the Btk gene that results in a missense mutation in the PH domain affecting a CpG site (Thomas et al., 1993; Rawlings et al., 1993). Initially, the milder phenotype in the xid mouse was anticipated to be secondary to a less severe effect of the mutation (Vorechovsky et al., 199313).However, de Weers et al. (199413) were the first to show that when the same amino acid is mutated in humans, it causes the more severe XLA. This finding has been confirmed in other patients (Ohta et al., 1994; Vihinen et al., 1995a,b; Vorechovsky et al., 1996).Interestingly, targeting of the mouse Btk gene results in mice with a phenotype resembling xid (Khan et al., 1995; Kemer et al, 1995), further emphasizing the existence of species differences.

V. PHENOTYPE AND CELLULAR BASIS OF SCID Section I1 briefly outlined the phenotype of SCID as described in the original publications. Summarizing the clinical findings of 70 SCID cases, Hitzig (1968) found that therapy-resistant infections were identified within 3 months of age; the main symptoms and signs were diarrhea, pulmonary infections, and failure to thrive (Table 3.1). Prior to bone marrow transplantation the disease was invariably fatal. In the early 1950s, when SCID was first described, the existence of different lymphocyte subsets was not established. The combined efforts in characterizing various immunodeficiencies and experimental work in animal systems, such as bursectomy (Glick et al., 1956) and thymectomy (Good et al., 1962; Miller et al., 1962; Waksman et al., 1962), eventually allowed better understanding of these processes. However, the existence of normal or elevated peripheral B

74

Smith and Notarangelo ~~

~

~

lymphocytes, despite hypogammaglobulinemia, further distinguishing XSCID from XLA, and markedly reduced T-cell numbers were not appreciated until the mid-1970s (Buckley et al., 1976; Griscelli et al., 1978; Yount et al., 1978; Conley et al., 1984). Engraftment of T lymphocytes, but not of B cells, after bone marrow transplantation further demonstrated that these subsets behave differentially in SCID (Griscelli et al., 1978; Buckley et al., 1986). Furthermore, the analysis of lymphocyte subsets in SCID is profoundly complicated by the engraftment of maternal cells (Pollack et al., 1982), and infections may also alter clinical and laboratory findings (Paschal1 et al., 1984). Lymphopenia, although frequently present, is not a constant phenomenon, and the severe diminution of peripheral lymphocytes is mainly seen in end-stage patients (Hitzig, 1968). The examination of thymic tissue revealed the dramatically altered architecture in SCID, including XSCID (Tobler and Cottier, 1958; Gitlin and Craig, 1963; Hitzig, 1968),demonstrating an almost complete absence of lymphoid cells and Hassall’s corpuscles. Other lymphoid organs such as lymph nodes and gut lymphoid tissue also displayed gross abnormalities, clearly distinguishing XLA from XSCID (Hitzig et al., 1958; Gitlin and Craig, 1963). However, it was not until the study of X-chromosome inactivation that the cellular defect was identified in more detail. These investigations allowed Puck and Conley to demonstrate a differentiation block in early T-cell development, whereas only mature B lymphocytes are similarly affected (Puck et al., 1987; Conley et al., 1988; Goodship et al., 1991a,b). Studies have shown a lack of Tcell receptor p gene rearrangement in XSCID thymocytes, compatible with a differentiation block at the pro-T-cell level (Sleaseman et al., 1994). In B lymphocytes, restricted junctional and combinatorial diversity and few somatic mutations have been reported (Minegishi et al., 1996). Peripheral blood NK cells are undetectable in XSCID patients, and a skewed inactivation pattern has been described in XSCID carriers (Gougeon et al., 1990; Goodship et al., 1991b; Wengler et al., 1993). A schematic representation of the differentiation defect is given in Figure 3.2, and the effect on lymphocytes and lymphoid tissue is summarized in Table 3.3.

VI. CLONING OF THE XSCID GENE AND SPECTRUM OF MUTATIONS A. Mapping and cloning of the XSCID gene In 1987 the XSCID gene was mapped to Xqll-13 by linkage analysis within affected pedigrees (de Saint Basile et al., 1987), as depicted in Figure 3.3, and the location was subsequently refined (Puck et al., 1989, 1993; Markiewicz et al., 1993). In 1992, Sugamura and collaborators cloned a constitutively expressed human gene that encodes a third polypeptide chain forming the interleukin 2 re-

75

3. X-Linked Immunodeficiencies

Table 3.3. Phenytypic Characteristics of XSCID and Jak3 Deficiency in Different Species"

Parameter Peripheral T cells Peripheral CD4/CD8 ratio Peripheral B cells B1 B cells Ig levels NK cells y8 intestinal intraepithelial lymphocytes/dendritic epidermal T cellsc Lymph node size Amount of gut-associated lymphoid tissueC Spleen germinal centersC Thymus histology/ sizeC Hassall's corpuscles' Thymic CD4/CD8 ratioC

Human XSCID or Jak3 deficiencyI4

Mouse yc Mouse Jak3 deletion deletion m ~ t a n t l ~ - ~ mutant16-'" ~

Dog XSCID9-I2

o or 1

1,age

-b

-

-

t

T

1'

dependent

Normal or 1'

Normal or '?

-

-

All isotypes 1

IgM normal IgG 1,IgA

1

1

1

-

-

-

Present IgM normal or 1,IgE od 0 1or O

1or o 1

1 1or o

Lor0 0

0

-

Present, large blastlike cells Dysplastic

0

0

1

1

Present Normal

Present Normalor'?

Variable

Dysplastic/ lymphoid atrophy

0

-

1

(normal)d

0

1or o 1

1'

"References: IHitzig (1968); 2Gelfand and Dosch (1983); 3Conley et al. (1990); 4Gougeon et al. (1990); 5Conley (1991); 6Fischer et al. (1995); 7Macchi et al. (1995a,b);8Russell et al. (1995); qezyk et al. (1989); 'OFelsburg et al. (1992); "Somberg et al. (1994); 12Somberget al. (1996); I3DiSanto et al. (1995); I4Cao et al. (1995); I5Ohho et al. (1996); 16Thomiset al. (1995); 17Nosakaet al. (1995); '"ark et al. (1995). hNot reported. 'Not analyzed in Jak3 deficiency patients. dEndogenous IgG not measurable because of transfer of maternal IgC.

ceptor, designated IL-2 receptor y chain (IL-2Ry) (Takeshita et al., 1992). The IL-2R has served as a prototype receptor for interleukins [reviewed in Minami et al. (1993) and Sugamura et al. (19931; the inducibly expressed a-chain was isolated in 1984 (Leonard et al., 1984; Nikaido et al., 1984) and the constitutively expressed P-chain 2 years later (Sharon et al., 1986; Tsudo et al., 1986). The iden-

76

Smith and Notarangelo

tification of the IL-2Ry gene allowed Leonard and associates to assign its location to the X-chromosomal region implicated in XSCID, perform linkage analysis, and subsequently characterize mutations in the human IL-2Ry gene in three unrelated patients with XSCID (Noguchi et al., 1 9 9 3 ~ )Certain . characteristics of the XSCID gene are presented in Table 3.2 and in Figure 3.4.

B. Cytokine receptor signaling As the phenotype of patients with XSCID is more severe than what is seen in both IL-2-deficient humans (Weinberg and Parkman, 1990) and mice (Schorle et al., 1991), it was anticipated that the IL-2Ry chain also participated in the signaling from other receptors. This was found to be true as the IL-2Ry chain is also part of the receptors for IL-4 (one subtype), IL-7, IL-9, and IL-15, but not of the receptor for IL-13 (Kondo et al., 1993; Noguchi et al., 199313; Russell et al., 1993; Giri et al., 1994; He and Malek, 1995; Kimura 2t al., 1995; Mathews et al., 1995; Obiri et al., 1995; Izuhara 2t al., 1996). It was therefore proposed that the name be changed to the common y-chain, y, (Noguchi et al., 1993b). In 1993 the signaling pathways from several cytokine receptors were deciphered (as reviewed in Ihle et al., 1994,1995; Sugamuraet al., 1995; Ihle, 1995, 1996; Ihle and Kerr, 1995). Subsequently it was found that a cytoplasmic (nonreceptor) kinase designated Jak3 (Janus kinase/”Just another kinase”) is involved in the downstream signaling from the yc-chain and that mutations in y, could affect this interaction (Miyazaki et al., 1994; Russell et al., 1994; Witthuhn et al., 1994). Another member of this family, Jakl, associates with the IL-2 receptor pchain or the ligand-specific a-chains of the yc-containing interleukin receptors. Jak3 only binds to the yc-chain, and ligand-induced receptor aggregation brings Jakl and Jak3 together, allowing transphosphorylation, activation of catalytic activity, and subsequent phosphorylation of substrates (Ihle, 1995). Jak3 exists in different splice variants, some of which are mainly expressed in hematopoietic cells (Lai et al., 1995). This signaling pathway also involves Stat (signal transducers and activators of transcription) proteins (Ihle et al., 1995; Lin et al., 1995; Ihle, 1996), but other substrates also exist and the specificity is controlled by the receptor complex (Ihle, 1996). As an example, the activation of Stat5 by interleukin 2 is nonessential for the activation of proliferation and is dependent on the carboxyterminal region of the IL-2Rp chain (Fujii et al., 1995). Stats can dimerize, contain an SH2 domain, allowing regulation by phosphorylation, and carry a DNAbinding region located in the middle (Ihle, 1996). Stats may also interact directly with tyrosine-based motifs in the cytokine receptors (Stahl et al., 1995). In T cells, IL-2, -4, and -9-induced stimulation of Jakl and Jak3 activates Stats 3 , 5, and 6 (Yin et al., 1995; reviewed in Ihle, 1995). Transformation with the v-abl oncogene results in the constitutive activation of Jakl and Jak3 inducing Stat signal-

3. X-Linked Immunodeficiencies

77

ing, thus indicating that these pathway could be involved in tumor formation (Danial et al., 1995). Mutations in Jak3 have been found to cause an autosomal recessive form of SCID, in which immunological and clinical features are indistinguishable from those observed in XSCID (Macchi et al., 199513; Russell et al., 1995), as depicted in Table 3.3. These observations further emphasize the importance of the yc/Jak3 pathway in cytokine signaling in hematopoietic cells. Corollaries to this highly interesting finding will most likely be found in both XLA and HIGM and may explain some of the non-X-linked forms displaying similar phenotypes.

C. Genomic organization of the y,-chain gene and mutation analysis in XSCID The genomic organization of the human XSCID gene was soon characterized, as depicted in Table 3.2 and Figure 3.5 (color plate) (Noguchi et al., 1993a; Puck et al., 1993),enabling DNA-based mutation analysis. The mouse gene has a similar organization (DiSanto et al., 1994).Several mutation reports have been published (see the legend of Figure 3 . 5 ) and the location of individual mutations is given in Figure 3.5. As can be seen, mutations are scattered all over the gene, but hot spots containing CpG sites exist (Pepper et al., 1995). Missense mutations have been found that affect conserved residues, such as extracellular cysteines and the WS motif. Mutation analysis has also provided evidence that XCID, a mild form of XSCID previously anticipated to represent a separate disease entity (Brooks et al., 1990; de Saint Basile et al., 1992a), is caused by mutations in the XSCIDl locus (DiSanto et al., 1994a,b; Schmalsteig et al., 1995). The promoter sequence has also been analyzed, and feedback mechanisms also seem to exist, as the addition of IL-2 results in decreased transcription (Ohbo et al., 1995). The promoter contains an Ets-binding site, and the Ets transcription factor is needed for the survival of T cells in the mouse (Bories et al., 1995; Muthusamy et al., 1995).

D. Animal models for XSCID First recognized in Basset hounds, an X-linked form of canine SCID was reported by Jezyk et al. (1989). Although similar in many respects to human XSCID, certain differences exist (Table 3.3). In 1994, a 4-bp deletion in the canine yJIL2RG gene was found to cause the disease (Henthorn et al., 1994). A second form of canine XSCID was identified in a cardigan Welsh corgi puppy carrying an insertion of a cytosine following nucleotide 582 that also resulted in a premature stop codon (Somberg et al., 1995).The phenotype differed from the described canine XSCID in that the Welsh corgi had more peripheral T cells, albeit nonfunctional. Furthermore, it has been pointed out (Somberg et al., 1996) that T cells developed

78

Smith and Notarangelo

over time in a human SCID patient that was kept in a gnotobiotic environment (Mukhopadhyay et al., 1978) and the phenotype in young (as compared to old) XSCID dogs resembled human SCID more closely. In contrast to humans, there is no engraftment of maternal T cells in canine XSCID (Somberg et al., 1996). Contingent on the cloning of the human XSCID gene, attempts were made to target the corresponding mouse gene to create a deletion mutant. Three groups have reported that they had achieved successful inactivation of the y, gene (DiSanto et al., 1995; Cao et al., 1995; Ohbo et al., 1996). These animal models also differed from their human counterpart. However, of note is the fact that the mouse XSCID mutants show quite striking differences in that the number of T cells may be less affected, whereas B cells are severely diminished (Table 3.3). However, T lymphocytes are functionally impaired. Furthermore, mice with targeted deletions of Jak3 develop similar lesions asiny,knockout mice (Nosakaetal., 1995; Parketal., 1995;Thomisetal., 1995). The results from the knockout experiments (Table 3.3) support the notion of Jak3 being involved in the downstream signaling from the interleukin receptors and corroborates the phenotypic species difference between mice and humans elicited in this transduction pathway.

VII. CELLULAR BASIS OF WAS Although WAS was originally described as a clinical triad of thrombocytopenia, eczema, and immunodeficiency, figures indicate that the complete clinical spectrum of the disease is present in less than a third of affected individuals, with the remaining presenting with only one or two of the main clinical manifestations (Sullivan et al., 1994). The clinical course of the disease is variable, but the prognosis remains generally severe; the average age of surviving males is 11 years (Sullivan et al., 1994). Patients with severe thrombocytopenia ( <10,000/mm3) at diagnosis are at high risk of significant bleeding episodes. In addition, patients with autoimmune manifestations are at high risk of developing malignancies (Sullivan et al., 1994). The main clinical features of WAS are shown in Table 3.1. Identification of a common pathogenetic mechanism that accounts for both platelet and lymphocyte abnormalities in WAS has been problematic. The defective production of isoagglutinins (Krivit and Good, 1959), low serum IgM, and increased IgA and IgE levels (Eitzman and Smith, 1960; West et d., 1962; Blaese et al., 1968; Ochs et al., 1980) are characteristic immunological features, associated with protein hypercatabolism and an increased synthesis rate (Blaese et al., 1971). The hypercatabolism of immunoglobulins is not due to fecal or urinary losses, but most likely reflects the expansion of macrophageerelated reticular cells in lymphoid tissues (Krivit and Good, 1959; Wolff, 1967; Cooper et al., 1968) that may accelerate immunoglobulin catabolism through Fc receptor-mediated endocytosis. Because these reticular cells tend to replace lymphocytes in

3. X-Linked Immunodeficiencies

79

lymphoid organs, progressive lymphoid depletion (ultimately leading to lymphopenia) is observed in lymph nodes, spleen, and thymus (Cooper et al., 1968; Blaese etal., 1968; Ochs etal., 1980). In vivo delayed-type hypersensitivity (DTH) tests are often negative (Root and Speicher, 1963; Cooper et al., 1968). Th'IS contrasts with a normal in vitro proliferative response to PHA; however, the response to immobilized anti-CD3 (Molina et al., 1993) and to allogenic cells (Ochs et al., 1980) is defective. Normal in vivo antibody production is achieved upon challenge with protein antigens (e.g., tetanus toxoid), but lack of an antibody response to polysaccharide antigens is characteristically observed (Blaese et al., 1968; Cooper et al., 1968; Ochs et al., 1980). A defective antibody response to polysaccharides and negativity of DTH tests are in contrast with a normal capability to produce immunoglobulins of various isotypes and a normal lymphocyte count (at least early in the course of the disease). In other words, while the effector mechanisms of immunity (e.g., the immunoglobulin-producing B-cell lineage and effector T lymphocytes) appear preserved, dissociated immune responses are observed in uivo. It was therefore hypothesized that WAS results from a defect in the afferent limb of immunity, possibly as the consequence of ineffective processing/presentation of polysaccharide antigens (Blaese et al., 1968; Cooper et al., 1968). Thrombocytopenia is another characteristic feature of WAS. Although the quantity and morphology of megakaryocytes in the bone marrow are normal (Ochs et al., 1980) quantitative and qualitative defects of platelets have been reported. Reduced platelet volume is the main consistent feature (Kenney, 1990). Controversial data have been reported concerning defects of a-granules, dense bodies, and mitochondria (Grottum et al., 1969; Baldini et al., 1969; Murphy et al., 1972; White, 1990), as well as of platelet aggregation in response to epinephrine (Kuramoto et al., 1970; Murphy et al. , 1972). However, WAS platelets show altered energy consumption that appears beyond the normal range in resting platelets, whereas little increase is observed following in vitro stimulation (Verhoeven et al., 1989). Intrinsic platelet defects are probably the main cause for the shortened survival of autologous platelets in WAS (Murphy et al., 1972) as they are rapidly removed from circulation in the spleen. Following splenectomy, an increase up to normalization of the platelet count is frequently observed (Corash et al., 1985; Mullen et al., 1993). Splenectomy may also lead to an increased mean platelet volume that, however, remains smaller compared to that of normal individuals. In keeping with the hypothesis of accelerated splenic destruction of intrinsically abnormal platelets in WAS is the observation that the survival of transfused homologous platelets is normal (Pearson et al., 1966; Krivit et al., 1966). Similar quantitative and qualitative platelet abnormalities (reduced number and volume, shortened survival) have also been identified in patients with X-linked isolated thrombocytopenia (Murphy et al., 1972). The first possible unifying defect that might account for both hemato-

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logical and immunological abnormalities in WAS was identified in the reduced expression and/or aberrant glycosylation pattern of sialoglycoprotein CD43, as well as of 0-glycosylated platelet-associated molecules, gpIa and gpIb (Parkman et al., 1981; Remold-O’Donnell et al., 1984). Because sialophorin is involved in signal transduction in lymphocytes (Mentzer et al., 1987), it was originally hypothesized that it could represent the primary defect in WAS. This hypothesis was rejected when the sialophorin gene was assigned to chromosome 16 (Pallant et al., 1989). Similarly, defects of glycosyltransferases,which might lead to aberrant sialophorin glycosylation patterns, have been postulated (Piller et al., 1991), and then excluded when it was found that these abnormalities are not expressed by long-term T-cell lines from WAS patients (Molina et al., 1992), suggesting that aberrant or defective glycosylation of sialophorin or sialophorin expression is a secondary phenomenon that occurs in vivo in WAS patients. A role for calpain (a Ca2+-dependent protease) in inducing the accelerated cleavage of gpIb and asialo-CD43 was postulated based on the hypothesis that increased procalpain activation may occur due to the abnormal cytoskeletal architecture of lymphocytes and platelets in WAS (Remold-O’Donnell et al., 1992). Indeed, morphological abnormalities have been found not only in platelets (reduced volume), but also in lymphocytes, as indicated by a decreased size and density of microvilli surface projections (Kenney et al., 1986; Molina et al., 1992). Aberrant cytoskeletal organization might also account for the defective in vitro proliferative response to CD3 (Molina et al., 1993); in fact, it is well known that, in contrast to other mitogens, response to anti-CD3 requires the bundling of cytoskeletal actin (Parsey and Lewis, 1993). Studies support the hypothesis that the pleiomorphic manifestations of WAS may be reconciled under a common cytoskeleton abnormality. The Wiskott-Aldrich syndrome protein (WASP), which is mutated in WAS, has in fact been shown to interact with a small GTP-binding protein, Cdc42 (Aspenstrom et al., 1996; Symons et al., 1996; Kolluri et al., 1996), that is involved in cytoskeleton organization (Kozma et al., 1995; Nobes and Hall, 1995). Aberrant cytoskeleton organization in WAS may lead to defective cell polarization and possibly to ineffective cooperation between immune cells. Similarly, defective actin polymerization might cause the typical platelet abnormalities. In addition to Cdc42, other proteins may interact with WASP. Nck, an adaptor molecule that also binds to tyrosine kinase receptors (Li et al., 1992), interacts with the polyproline stretch of WASP (Rivero-Lezcanoet al., 1995); this association may be important in cell signaling. WASP defects might also therefore prevent the interaction with Nck (and possibly with other protein partners), and thus also affect cellular activation events. While biochemical and molecular studies will ultimately unravel the pathophysiology of WAS, two independent lines of evidence demonstrated that the disease results from a direct involvement of the hematopoietic tissue. First, bone marrow transplantation represents a definitive cure of all clinical, hemato-

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logical, and immunological abnormalities (Bachetal., 1968; Parkman et al., 1981; Fischer et al., 1994). Second, the study of obligate carriers of WAS has shown that they nonrandomly inactivate the X chromosome in all hematopoietic cell lineages (Gealy et al., 1980; Prchal et al., 1980; Fearon et al., 1988; Greer et al., 1989; de Saint Basile et al., 1992b),but not in nonhematopoietic cells (e.g., fibroblasts). Wengler et al. (1995a) have found that the nonrandom pattern of X inactivation is already apparent in CD34+ cells, indicating that the process of selection against precursors expressing the mutated X chromosome as the active X is already operating during early stages of hematopoietic cell differentiation. This finding, together with the proposed functional role of WASP in cytoskeleton organization and cell-to-cell interaction, has led to the hypothesis that WASP may play a role in the interaction of marrow stromal cells with the hematopoietic stem cell (Kirchhausen and Rosen, 1996). Thus, in carrier females of WAS, only those stem cells that express the wild-type WASP may associate with the stromal cells and would then be selected for further differentiation, resulting in a skewed pattern of X-chromosome inactivation in all blood cell lineages. Representation of the effects of WAS mutation on hematopoietic differentiation is shown in Figure 3.2. The demonstration that carrier females of XLT also exhibit a nonrandom pattern of X-chromosome inactivation in blood cells (de Saint Basile et al., 1991; Notarangelo et al., 1991; Kolluri et al., 1995) has provided further evidence for similarity between WAS and XLT that these are indeed two allelic disorders has also been established following the demonstration of WASP gene mutations in XLT families (Villa et al., 1995; Kolluri et al., 1995; Derry et al., 1995a).

VIII. CLONING OF THE WAS GENE AND SPECTRUM OF MUTATIONS A. Mapping and cloning of the WAS gene In 1987, the WAS gene was mapped by linkage analysis to the pericentromeric short arm of the human X chromosome (Peacocke and Siminovitch, 1987), as shown in Figure 3.3. This location was subsequently confirmed and refined (Kwan et al., 1988). Strong association was identified with the highly polymorphic marker DXS255 (Kwan et al., 1989; de Saint Basile et al., 1989; Greer et al., 1990). This information was particularly useful for genetic counseling to WAS families: in fact, because DNA marker DXS255 is methylated differently on the active vs the inactive X chromosomes (Hendriks et al., 1992), the cosegregation of alleles and the methylation pattern at the DXS255 locus were used to attempt carrier detection and prenatal diagnosis in WAS families (Goodship et al., 1991a; de Saint Basile et al., 199213;Notarangelo et al., 1993). Linkage analysis with the use of additional polymorphic markers allowed to finely map the WAS gene at Xp11.22-p11.23, so that the following order was

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established: cen-DXS255-(TFE3, WAS, OATL1)-TIMP-DXS7-Xpter (Greer et al., 1990, 1992; Kwan et al., 1991; Cremin et al., 1993). The WAS gene was identified by positional cloning in 1994 (Derry et al., 1994). A contig was constructed in Xp11.22-p11.23, bounded by DXS255 and TIMP loci. Several cDNAs that are expressed in hematopoietic cells were challenged as candidate genes; one of them was not expressed or was mutated in four unrelated WAS patients (Derry et al., 1994). This gene was designated WASP ( Wiskott-Aldrich syndrome protein). As shown in Figure 3.6 (color plate), the gene is organized in 12 exons and spans approximately 9 kb of genomic DNA. It encodes a protein of 502 amino acids. Expression of WASP mRNA is abundant in thymus and spleen, where two mRNA species (a predominant 2.0-kb band and a weaker 4.2-kb band) are identified. These transcripts are present in a number of T and B lymphocyte cell lines, as well as in megakaryocytic and erythroleukemic cell lines (Derry et al., 1994). The sequence of the WASP cDNA (and, thus, the predicted amino acid sequence of the encoded protein), as originally reported and subsequently corrected (Derry et al., 1994; Kwan et al., 1995a), revealed a proline-rich protein. No distinct similarities to other known proteins were originally recognized. The WASP protein does not contain any obvious hydrophobic membrane-spanning region. In contrast, as shown in Figure 3.6, some regions of potential functional importance have been identified (Derry et al., 1994). Based on these findings, it was originally hypothesized that WASP might be involved in the regulation of gene expression (as suggested by the putative nuclear-localization signal) or that it might bind (through its proline-rich residues) to SH3 domain-containing cytoplasmic proteins, thus perhaps participating in activation processes and/or cytoskeleton organization. The use of monoclonal antibodies to WASP is in keeping with the latter hypothesis, as the protein is detected in the cytoplasm, but not in the nucleus (Stewart et al., 1996). Evidence for an in vivo interaction between the polyproline stretch of WASP and the SH3 domain of Nck has been provided (Rivero-Lezcano et al., 1995). Furthermore, a GTPase-binding domain (GBD) has been identified in WASP (amino acid residues 238-257); this domain is sufficient and essential for Cdc42 binding and is encoded by part of exons 7 and 8 (Symons et al., 1996). Finally, an extensive search of similarity with other proteins has identified two additional sequences in the N and C-terminal regions of WASP that are conserved in other proline-rich proteins involved in cytoskeleton organization. These domains have been named WASP homology 1 (WH1, residues 47-137) and WH2 (residues 423449) (Symons et al., 1996).

B. Mutation analysis in WAS Elucidation of the WASP gene organization (Derry et al., 1994) has facilitated mutation analysis at the DNA level. Screening methods based on SSCP and het-

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eroduplex analysis have been developed that allow the identification of the molecular defect and carrier detection (Kwan et al., 1995a; Kolluri et d., 1995; Derry et al., 1995a; Wengler et al., 1995a). Although distributed across the entire WASP gene, mutations are preferentially located in the first two exons (Figure 3.6). A predominance of premature terminations and frameshift mutations have been detected among patients with either full-blown (i.e., thrombocytopenia, eczema, and infections) or attenuated (i.e., thrombocytopenia associated with mild eczema and/or minor infections) WAS. Missense mutations are also preferentially distributed in the first two exons (Schindelhauer et al., 1996); although this region has not yet been attributed any specific functional property, it has been hypothesized that it may interact with other proteins (Derry ee al., 1995a). In contrast, the proline-rich region is not frequently mutated, suggesting that proline redundancy may compensate for single proline mutations (Derry et al., 1995a).This may be a general characteristic of proline-rich regions as a similar redundancy was seen in the proline-rich TH domain in XLA (Vihinen et al., 1995a, 1996a,b).

C. Mutation analysis in XLT Identification of the WASP gene has prompted mutation analysis in XLT families, aiming to establish whether XLT is an allelic disorder to WAS. Villa et al. ( 1995) have identified three distinct mutations in males with isolated thrombocytopenia, two of which had single amino acid substitutions. These results which demonstrate that XLT is allelic to WAS, have been subsequently confirmed in a larger cohort of XLT patients (Kolluri et al., 1995; Derry et al., 1995a; Kwan et al., 1995a; Zhu et al., 1995). Interestingly, at variance with what was observed in WAS, a predominance of missense mutations or in-frame deletions/insertions have been detected in XLT, implying that the milder phenotype may be related to the maintenance of some functional properties by the WASP protein (Villa et al., 1995; Kolluri et al., 1995; Derry et al., 1995a; Zhu et al., 1995). However, one single missense mutation was identified in three boys with typical WAS and in one male with isolated thrombocytopenia, suggesting that background genetic or even epigenetic factors may also play a role in determining the clinical phenotype (Kolluri et al., 1995).

D. Animal model of WAS The X-chromosome-linked scurfy (sf) mouse mutant has been proposed as a naturally occurring animal model of WAS (Lyon et al., 1990). Scaliness of the skin, anemia, thrombocytopenia (with severe gastrointestinal bleeding), increased occurrence of infections, and early death are characteristic features of the sf mouse that are similar to WAS. However, scurfy differs from WAS in that sf males are consistently hypogonadal. Although the murine Wasp gene lies in the same re-

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gion of the sf locus, no abnormalities of Wasp mRNA size or amount have been detected in sf mice (Derry et al., 199513).

IX. CELLULAR BASIS OF HIGM HIGM is characterized by the defective production of IgG, IgA, and IgE, whereas the synthesis of IgM and IgD is not affected. Circulating B lymphocytes are normal in number, but they uniquely express surface membrane IgM and/or IgD (Levitt et al., 1983; Cooper et al., 1984). In addition, when challenged repeatedly with T-dependent antigens, HIGM patients mount a normal, IgM-restricted primary antibody response, but fail to develop secondary antibody responses (Kyong et al., 1978; Nonoyama et al., 1993). The underlying defect was originally interpreted as a failure of the patients’ B lymphocytes to undergo the process of immunoglobulin isotype switching (Geha et al., 1979; Levitt et al., 1983). The demonstration of a random pattern of X-chromosome inactivation in mature IgG and IgA expressing B lymphocytes from carrier females of HIGM indicated that the immunoglobulin heavy chain class switch machinery is intact, even in those cells that express the mutated X chromosome as the active one, thus suggesting that the disease is not caused by an intrinsic B-cell defect (Hendriks et al., 1990). These findings have been confirmed by Hollenbaugh et al. (1994). Although circulating T cells number and function (as assessed by the conventional in vitro proliferative response to mitogens and in vivo DTH tests) are largely normal (Geha et al., 1979; Benkerrou et al., 1990), the hypothesis of a primary T-cell defect was strongly supported by the observation that a coculture of HIGM B cells with helper T lymphoblasts from a patient with a Sezary-like syndrome, in the presence of pokeweed mitogen, led to isotype switching (Mayer et al., 1986). This finding pointed to a helper T-cell defect in HIGM. In the course of experiments aiming to characterize signals involved in helper T-cell activity and regulation of isotype switching in normal individuals, monoclonal antibodies to CD40 (a surface membrane molecule, mainly expressed by B cells) were shown to induce an immunoglobulin isotype switch if appropriate costimulatory signals (represented by cytokines) are also provided (Zhang et al., 1991; Rousset et al., 1991). In vitro IgE production was also obtained when anti-CD4O monoclonal antibody and IL-4 were added to B lymphocytes from HIGM patients; in contrast, the patients’ T cells were unable to support the isotype switch in vitro when cocultured with autologous or allogenic B cells in the presence of IL-4 (Fuleihan et al., 1993; Durandy et al., 1993). These data confirmed the integrity of the B-cell isotype switch machinery in HIGM and suggested that the molecular defect resides in the appropriate delivery of helper Tcell-derived signals that would normally lead to B-cell differentiation via CD40.

3. X-Linked Immunodeficiencies

85

This hypothesis was confirmed upon the identification of mutations in the CD40 ligand (CD40L) gene in HIGM patients (Aruffo et al., 1993; Korthauer et al., 1993; DiSanto et al., 1993; Allen et al., 1993; Fuleihan et al., 1993). In addition to defective T-B-cell cooperation, other cellular interactions are also probably impaired in HIGM. In particular, it is known that stimulation of monocytes with the anti-CD40 monoclonal antibody results in cellular activation (Alderson et al., 1993) and secretion of IL-12 (Shu et al., 1995). It has been shown that the immune defense to intracellular pathogens (such as Leishmania and Listeria) is dependent on the interaction between CD40+ monocytes and activated CD40L+ T cells, leading to IL- 12 secretion by monocytes, and the subsequent release of IFN-y by T lymphocytes (reviewed in Noelle, 1996). Based on this finding, it is tempting to speculate that an increased susceptibility to some infections (Cryposporidium, Pneumocystis carinii) in HIGM is contributed to by inefficient interaction between monocytes/macrophages and activated T cells. Finally, CD40L is also expressed by medullary thymocytes (Lederman et al., 1992). Defective CD4OL expression might therefore interfere with thymocyte differentiation/function; however, no distinct abnormalities of T-cell function have yet been described in HIGM patients. A schematic representation of the effects of CD40L mutations on the hematopoietic differentiation is shown in Figure 3.2.

X. CLONING OF THE HIGM GENE AND SPECTRUM OF MUTATIONS A. Mapping and cloning of the HIGM gene The HIGM gene was originally assigned by linkage analysis to Xq24-q27 (Mensink et al., 1987). As shown in Figure 3.3, this location was subsequently refined to Xq26-27, closely linked to the HPRT locus (Padayachee et al., 1992a,b). However, identification of the molecular defect was obtained with the candidate gene approach. Using a soluble CD40-Ig fusion protein, it was found that the CD40L protein is expressed by activated murine and human T lymphocytes (Armitage et al., 199213; Lane et al., 1992; Noelle et al., 1992). The cDNA encoding murine CD40L was isolated (Armitage et al., 1992a), and the corresponding gene was mapped to the mouse X chromosome, in proximity to the h p t locus (Allen et al., 1993). Thereafter, the human equivalent CD40L gene was also cloned (Hollenbaugh et al., 1992; Graf et al., 1992) and mapped to Xq26 (Graf et al., 1992; Allen et al., 1993; Aruffo et al., 1993; Pilia et al., 1994). Certain characteristics of the CD40L gene are shown in Table 3.2 and in Figure 3.7 (color plate). As shown in Figure 3.7, the human CD40L gene encodes a transmembrane type I1 protein of 261 amino acids that belongs to the family of tumor necrosis factor a (TNFa). Based on coincident mapping data and on predicted CD40L-mediated

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functional abnormalities in HIGM, the CD40L gene was challenged as a candidate gene. Five different groups independently and simultaneously demonstrated that activated T cells from HIGM patients fail to bind CD40-Ig chimeric molecules (i.e., they lack functional CD40L expression); mutations in the CD40L cDNA were identified in this series of patients (Aruffo et al., 1993; Korthauer et al. , 1993; DiSanto et al., 1993; Allen et al., 1993; Fuleihan et al., 1993).

6. Mutation analysis in HIGM The genomic organization of the CD40L gene has been characterized, as shown in Table 3.2 and in Figure 3.7 (Villa et al., 1994a; Shimadzu et al., 1995; Seyama et al., 1996), enabling mutation analysis at the genomic level. Several reports (listed in the legend to Figure 3.7) have been published that analyze the mutation location in HIGM families. As shown in Figure 3.7, although the mutations are scattered throughout the gene, most of them affect the TNFa homology domain (amino acid residues 123-261 ). Three-dimensional models of murine and human CD40L proteins have been developed (Peitsch and Jongeneel 1993; Hammarstrom et al., 1993; Bajorath et al., 1995a,b; Karpusas et al., 1995), and critical residues involved in CD40 binding have been identified. Interestingly, only some of the missense mutations reported in HIGM patients involve CD40-binding epitopes, whereas others disrupt the CD40L structure or prevent trimer formation (Bajorath et al., 1995a,b, 1996; Karpusas et al., 1995; Notarangelo et al., 1996). Mutations involving CpG sites, frequently affected in genetic disease, seem to be underrepresented in HIGM (Notarangelo et al., 1996).

C. Animal models of HIGM While no naturally occurring animal models of HIGM are known, a number of experimental animal models have been developed. The first series of animal models consisted of mice in which the CD40-CD40L interaction was prevented by the injection of soluble substances. Injection of the anti-CD40L monoclonal antibody into mice reduces both primary and secondary responses to T-dependent, but not T-independent, antigens (Foy et al., 1993). In addition, it blocks germinal center formation and the development of antigen-specific memory B cells (Foy et al., 1994). In contrast to these findings, injection of the soluble CD40-Ig chimeric molecule inhibits memory B-cell development, but not germinal center formation (Gray et al., 1994).These observations may be reconciled based on the fact that the anti-CD40L monoclonal antibody has a stronger affinity than CD40-Ig for CD40L target protein. Thus, human HIGM is better mimicked by injection of the anti-CD40L monoclonal antibody than by injection of CD40-Ig; indeed, germinal centers are characteristically missing or abortive in HIGM (Facchetti et al., 1995). Two groups have developed CD40L-deficient (CD40L/-)mice using ho-

Figure 3.1. Some of the original investigators in the field of X-linked immunodeficiency: ( A ) Dr. Ogden C. Bruton (sitting) in 1993 when one of the authors met with him after the cloning of the XLA gene, (standing) Dr. Jeffrey D. Thomas (left), ClES (center), and Mrs. Kathryn D. Bruton (right); (B) Dr. Albert Wiskott; (C) Dr. Robert A. Aldrich; (D) Dr. Walter H. Hitzig; and (E) Dr. Fred S. Rosen. (Figures 3.1B-3.1E are on next page.)

Figure 3.2. Schematic representation of the cellular defects in X-linked immunodeficiencies. The figure depicts the cellular stages affected by the functional defects (noncrossing arrow) with or without concomitant differentiation block (crossing arrow; solid arrow represents total block whereas dotted arrow represents partial block). In XLA, mRNA and protein expression of the Btk gene occurs in more undifferentiated progenitor cells compared to the defective cell srages (de Weers er al., 1993; Smith et al., 1994a,b).

Figure 3.4. Schematic representation of the XLA/Rtk gene/protein structure and mutations. The physical map across the gene displays adjacent markers (top); boxes denote genes or markers. The genomic organization is shown below, boxes identifying exons; the 5’ and 3’ untranslated regions are shown as white hoxes, coding in color. The colored large box denotes the protein. Boxes represent functional regions or domain structures. Functionally important regions or individual residues are indicated by the corresponding amino acid number. The exon boundaries are indicated by vertical lines below the protein box. The top numbers (gray shaded) represent the nuinher of families having mutations in exons. Exon numbering is depicted helow (nonshaded). The number of families having intron mutations (splice site) is shown in the hottoin (grey shaded), helow the line indicating intron boundaries. The references serving as a basis for the schematic representations are given below. The protein is composed of the following domains: PH (pleckstrin homology), TH (Tec homology), SH3 (Src homology 3), SH2, and Kinase (SHl). The references serving as basis for the schematic are as follows: Genomic map and gene structure: Vetrie et al.(1993h); Ohta et a[. (1994); Hagemann et al. (1994); Rohrer et al. (1994); Sideras et al. (1994); Vorechovsky et al. (1994); Oeltjen et al. (1995); Mazzarella et al. (1996). Mutation analysis: Vetrie et al. ( 1 9 9 3 ~ ) ; Bradley et al. (1994); Conley et al. (1994); de Weers et al. (1994); Lhriez et al. (1994); Genevier et al. (1994); Hagemann et al. (1994); Ohta et al. (1994); Saffran et al. (1994); Vihinen et al. (19944; Zhu et al. (1994a,b);Gaspar et al. (1995); Hageinann et al. (1995a,h); ]in et al. (1995); Ohashi et al. (1995); Vihinen et al. (1995a,h); Vorechovsky et al. (1995a,b,1996);Vihinen et al. (1996a,b).

Figure 3.5. Schematic representation of the XSCID/ILZRG/y,-chain gene/protein structure and inutations. T he general outline of the figure is given in the legend to Figure 4. The protein contains a signal peptide (S),an extracellular domain (EC),transrnemhrane region (TM), and an intracellular region (IC).The location of four comerved cysteines, the WS-motif, and an SH-2 like (“SH2”) domain (hatched) are denoted. The references serving as a basis for the schematic are as follows: Genomic map and gene structure: Noguchi et al. (1993b,c); Pucket ul. (1993). Mutation analysis: Noguchi et al. ( 1 9 9 3 ~ ) ; Puck et al. (1993); DiSanto et al. (1994a,h); Ishii et al. (1994); Markiewicz et al. (1994); Russell et al. (1994); Clark et al. (1995); Kumaki et al. (1995); Matthews et al. (1995); Minegishi et al. (1995); Pepper et al. (1995); Puck et al. (1995); Schmalstieg rt nl. (1995); Tasaara et al. (1995); Hacien-Bey et al. (1996); lzuhara et al. (1996); Puck et al. (1996).

Figure 3.6. Schematic representation of the WASP gene/protein structure and mutations. The general outline of the figure is given in the legend to Figure 4. The protein contains a WASP homology domain 1 (WHI), a GTPase-binding domain (GDB), a prolinerich domain (PRD), and a WASP homology domain 2 (WH2). Functionally important regions or individual residues are indicated by the corresponding amino acid number. The references serving as a basis for the schematic are as follows: Genomic map and gene structure: Derry et al. (1994); Kwan et al. (1995a); Symons et al. (1996). Mutations analysis: Derry et al. (1994, 1995a); Kolluri et al. (1995); Kwan et al. (1995a,b); Villa e t al. (1995); Wengler et al. (1995b); Zhu et al. (1995); Notarangelo et al., unpublished; Schindelhauer et al. (1996); Stewart et al. (1996).

Figure 3.7. Schematic representation of the CS4OL gene/protein structure and mutations. The general outline of the figure is given in the legend to Figure 4. Functionally important regions or individual residues are indicated by the corresponding amino acid number. The protein contains an intracellular (IC), transmembrane (TM), and an extracellular region (EC). The hatched area within the EC region identifies the TNF homology domain. The references serving as basis for the schematic are as follows: Cenomic map and gene structure: Padayachee et al. (1992a,b);Pilia et al. (1994); Villa et al. (1994a); Mutation analysis: Allen et al. (1993); Aruffo et al. (1993); Korthauer et al. (1993); Ramesh et al. (1993); Callard et al. (1994); Chu et al. (1995); DiSanto et al. (1993); Fuleihan et al. (1993); Hollenbaugh et al. (1993); Iseki et nl. (1994); Kroczek et al. (1994); Ramesh et al. (1994); Villa et al. (1994b); Bajorath et al. (1995a,b,1996); Karpusas et al. (1995); Kraakman et al. (1995); Lane et al. (1995); Macchi et al. (1995a); Ramesh et al. (1995); Saiki et al. (1995); Shimadzu et al. (1995); Sayama et al. (1996); Lin et al. (1996); Notarangelo et al. (1996); Notarangelo et al., unpublished.

Figure 3.8. Model of the three-dimensional structure of the Btk kinase domain. The polypeptide is shown as a ribbon running along the backbone. Side chains are shown only for missenst. mutations (yellow). The locations of the nonsense mutations (red), insertions (gold), and deletions (cyan) are shown in the ribbon. ATP (green) and Mg2+ ions (red) are also presented. The missense mutations are mainly located on one face of the domain, indicating structural clustering of functionally important residues. The model is based on Vihinen et al. ( 1 9 9 4 ~and ) is reproduced from Vihinen et al. (1995a).

3. X-Linked Immunodeficiencies

a7

mologous recombination in embryonic stem cells (Xu et al., 1994; Renshaw et al., 1994). These gene-targeted mice exhibit several features of HIGM (hypogammaglobulinemia, lack of germinal centers and memory B-cell development, inability to mount secondary antibody responses to T-dependent antigens, and preservation of antibody responses of all isotypes to T-independent antigens). Characteristically, if kept in a germ-free environment, they fail to exhibit increased IgM serum levels that might represent a secondary phenomenon, even in human HIGM. Similar to HIGM1 patients, CD40L-1-mice have an increased susceptibility to infections sustained by intracellular pathogens, such as Leishmania (Soong et al., 1996) and Pneumocystis carinii (Noelle, 1996). In addition, treatment of mice with the anti-CD40L antibody results in a diminished ability to clear Pneumocystis (Wiley and Harmsen, 1995). Altogether, these observations reinforce the crucial role that the interaction between activated, CD40L+ T cells and macrophages plays in the response against intracellular pathogens. Finally, analysis of the effect of a disrupted CD40L-CD40 interaction has also been analyzed in CD40-deficient mice (Kawabe et al., 1994; Castigli et al., 1994). Immunological features of these mice are similar to those of CD40L-deficient mice. Both IgM serum levels and neutrophil counts are within the normal range; however, if kept in nonpathogen-free conditions, these mice have a reduced neutrophil count compared to wild-type mice, suggesting that neutropenia in HIGM may also be secondary to defective reactive granulopoiesis (Kawabe et al., 1994). The major characteristics of mouse animal models of HIGM, as compared to the human disease, are summarized in Table 3.4.

XI. GENE THERAPY IN PRIMARY IMMUNODEFICIENCY Gene therapy for an inherited disease was first attempted in an immunodeficiency. In 1991,two children with adenosine deaminase (ADA) deficiency were treated at the National Institutes of Health, Bethesda, Maryland, receiving autologous peripheral blood T lymphocytes that were transduced (infected) with a retroviral vector harboring the gene for ADA. Since then, other children with SCID due to ADA deficiency have received gene therapy (although in all cases, gene therapy was associated with enzyme replacement). The long-term clinical and biological results of this study and a similar Italian investigation have been reported (Blaese et al., 1995; Bordignon et al., 1995). In human ADA deficiency the enzyme defect results in the accumulation of metabolites exerting a toxic effect on mainly T lymphocytes (reviewed in Markert, 1994; Fischer et al., 1995) whereas the corresponding targeted inactivation in mouse gives a different phenotype (Migchielsen et al., 1995; Wakamiya et al., 1995). For many years this has been the prototype disease for gene therapy, despite being an extremely rare autosomal recessive disorder. The reason for choosing ADA deficiency was the advantage provided by the selective pressure on nontransduced T lymphocytes, as well as the

Table 3.4. Phenotypic Characteristics of HIGM in Humans and in Animal Models"

Parameter Lymph node or spleen T cells Lymph node or spleen B cells IgG, IgA, IgE levels IgM levels Primary antibody response to T-dependent Ag Secondary antibody response to T-dependent Ag Antibody response to T-independent Ag Lymph node germinal centers FDC in the lymph node Memory B-cell generation Neutropenia

Human'.* Normal

Mice injected with anti-CMOL MAb'

Mice injected with CD40-lg4

-b

CD4O-deficient nice^,^

Normal (ceIIularity J6) Normal (ceIIuiarity L6)

Normal

Normal or ? ' IgM response T

Normal

1

Normal Normal7 or 1"

Normal

1

1

Normal

Normal

Normal

1

CMOL-deficient rni~e~,~

-

1

Largely normal'

1

Normal or t Normal IgM response

-

Normal

Normal

0

0

Present

0

0

1

Normal Inhibited

-

-

-

Inhibited

Inhibited No

Inhibited Nod

1

1

Inhibited Yes

-

-

"References: 'Notarangelo et al. (1992); 2Nonoyamaet d.(1993); 3Foy et al. (1994); 4Gray et al. (1994); 5Xu et al. (1994); 6Renshawet d. (1994); 7Kawabe et al. (1994); Tastigli et al. (1994). bNot reported. 'Reduced number of sIgD'"IgMh' observed by Kawabe et al. (1994). dLack of reactive granulopoiesis in a nonpathogen-free environment observed by Kawabe et al. (1994).

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fact that ADA is a well-tolerated protein, not known to cause any adverse effects when overexpressed. A similar selective advantage would presumably apply in the case of XLA, as affected males virtually lack B lymphocytes. In XSCID the situation may be more complex, as bone marrow transplantation is affected by residual maternal cells that frequently repopulate the patient (Pollak et al., 1982;Fischer et al., 1995). However, in vitro transfer of the y, gene into lymphoblastoid cell lines from XSCID patients restores high-affinity IL-2R receptor expression and function (Candotti et al., 1996b; Hacein-Bey et al., 1996; Taylor et al., 1996). Retroviral vectors have also been used to transfer the y, gene into mouse fibroblasts or human CD34-enriched bone marrow cells (Quazilbash et al., 1995). Similarly, IL-2induced in vitro proliferation is increased in gene-corrected vs mutant Jak3-deficient B-cell lines (Candotti et al., 1996a). These data offer an important basis for a potential in vivo expansion of gene-corrected cells following gene transfer in these diseases. Based on the pattern of X-chromosome inactivation in hematopoietic cells from carrier females, it could be anticipated that a selective growth/differentiation advantage would also apply in WAS, whereas in HIGM this is not expected (Figure 3.2). However, several putative problems exist apart from the generally existing difficulties in achieving long-term expression of transferred genes. Thus, signal-transducing molecules could potentially be harmful when overexpressed, of note for XLA, XSCID, and HIGM. Because the WASP protein seems to be critically involved in the control of cytoskeleton organization and cell activation, potential hazards due to the function of this protein presently cannot be fully evaluated. Another possible caveat in HIGM is caused by the inducible expression of this ligand, in that constitutive expression may prove harmful. However, despite these potential problems, the easy access of bone marrow for gene transfer has made therapy for hematopoietic diseases into one of the most developed areas in gene therapy. To this end, the existence of animal models for XLA, XSCID, and HIGM will be of importance for the study of gene transfer. In the event of future successful achievements in obtaining culture conditions for hematopoietic stem cells, novel therapeutic approaches could be applied immediately. This would not only open the possibility for transfer of an extra therapeutic gene copy, but potentially allow targeting and repair of defective genes, presumably obviating the need to characterize gene regulatory elements to achieve normal expression of the gene.

XII. CONCLUDING REMARKS The human immunodeficiency genes have remained elusive for many years. However, the cloning of several of the classical X-linked immunodeficiency genes has enabled rapid developments in the field of immunology and has far-reaching im-

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plications. Because many of the gene products are involved in receptor-mediated signal transduction, this has resulted in a better understanding of signaling pathways. These findings also add to the notion that signaling molecules frequently are targets for human disease. DNA-based mutation analysis has been carried out in large numbers of immunodeficiency patients, and the generation of databases simplifying the handling of this vast amount of information has been initiated. A set of reports by international study groups have compiled 453 unique mutations in 625 unrelated individuals in the four diseases reviewed in this chapter (Smith and Vihinen, 1996; Vihinen et al., 1996b; Puck et al., 1996; Schwarz et al., 1996; Notarangelo et al., 1996). In addition, the study group for another X-linked disease, X-linked chronic granulomatous disease, reported almost 300 patients with 187 unique molecular events (Roos et al., 1996). CpG sites were frequently altered, with the exception of HIGM. The analysis of the defective genes has allowed the characterization of mutations causing mild or restricted phenotypes. Thus, patients previously classified as having a second form of X-linked SCID have now been found to carry mutations in the XSCIDl (yc/IL2RG) gene, and patients with X-linked thrombocytopenia have been reported to carry mutations in the WAS gene. An autosomal recessive form of SCID, affecting the same signaling pathway, but a different molecule as the one defective in XSCID, has been observed. We expect many such locus heterogeneities to be identified in the not too distant future. In XLA and XSCID, the corresponding mouse models have demonstrated milder and different phenotypes, respectively. Despite these shortcomings, such animals are likely to be instrumental in the development of new treatment modalities. Our knowledge on the function of the proteins affected in X-linked immunodeficiencies is likely to soon become much more comprehensive as the unraveling proceeds. However, the history of these immunodeficiencies, their original descriptions, and the discoveries leading to the cloning of the corresponding genes will not change. This chapter has tried to briefly survey these issues. Unfortunately, it is not possible to do justice to everyone involved in this process extending more than half a century in time. The authors therefore apologize to those investigators whose contributions were not adequately cited.

Acknow Iedgme nts The authors thank Drs. Genevieve de Saint Basile, Hopital Necker-Enfants-Malades, Paris, James N. Ihle, St. Jude Children’s Research Hospital, Memphis, Christine Kinnon, Institute of Child Health, London, David L. Nelson, National Institutes of Health, Bethesda, Maryland, and Jennifer Puck, National Institutes of Health, Bethesda, Maryland, for providing information and unpublished material. The help of Drs. Mohsen Moini, and Mohammad R. Abedi, Department of Clinical Immunology, and Susanne Muller, Department of Bioscience, Karolinska Institute, in the preparation of this chapter is

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gratefully acknowledged. This work was supported by the Swedish Medical Research Council, the Swedish Cancer Society, The Magn. Bergvall Foundation, Telethon (Grant A. 42) and the European BIOMED concerted action “PL1321.”

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I

Gene Therapy of Duchenne Muscular Dystrophy

Ariberto Fassati,*+ Stephen Murphy,* and George Dickson*

*School of Biological Sciences Division of Biochemistry Royal Holloway College, University of London Egham, Surrey TW20 OEX, United Kingdom tDepartment of Experimental Pathology UMDS Guy’s Hospital London Bridge, London SEl 9RT, United Kingdom

1. DUCHENNE MUSCULAR DYSTROPHY A. Clinical and pathological features Duchenne muscular dystrophy (DMD) is an X-linked disorder affecting about 1:3500live male births (Emery, 1993). The disease was first described by Duchenne in 1868 as a progressive, degenerative process involving skeletal muscle, although in recent years it has become apparent that DMD is a more complex disease involving the heart and the central nervous system (CNS), as well as peripheral nerves and smooth muscle (Roberts, 1995; Comi et al., 1995). DMD is generally noticed between the ages of 2 and 5 years (Emery, 1993). Affected children do not walk until 18 months of age or later and often have a waddling gait and difficulty in climbing stairs with an early involvement of the proximal musculature. The classical clinical feature of DMD is the Gower’s sign: affected boys are too weak to completely raise themselves from the floor so they raise on their lower limbs first and than use their arms to help extend the girdle. Other signs include enlarged calf muscles due to deposition of connective and adipose tissue (pseudohypertrophy), the tendency to walk on the toes to compensate for the shortening of the Achilles tendon, winged scapulae, and gross muscle atrophy. Confinement to a wheelchair is inevitable by the age of 12 years due to severe muscle atrophy and weakness. As a consequence of immobilization, there are contractures throughout the skeletal Advances in Genetics, Vol. 35 Copyright 0 1997 by Academic Press

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musculature, causing skeletal deformities. Generally, the facial mimic musculature, the extraocular musculature, and the muscles for phonation are spared, although a loss in facial mimic expressivity has been reported in some cases of old patients. Progressive weakness of the diaphragm and intercostal muscles eventually leads to fatal respiratory insufficiency (Adams and Victor, 1985). The allelic Becker muscular dystrophy (BMD; Becker and Kiener, 1955) is similar to DMD, but with a later manifestation of symptoms related to muscle defects, sometimes referred as myalgia and cramps. The clinical course is milder and patients are often able to walk into adulthood. In association with skeletal muscular symptoms and signs, DMD and BMD patients often present severe cardiomyopathy with right ventricular dilatation and a conduction defect (Miyashita et al., 1993; Melacini et al., 1993; Piccolo et al., 1994), indeed X-linked dilated cardiomyopathy may be the prevailing pathological finding in some families (Mirabellaetal., 1993; Muntoni et al., 1993). Most DMD and BMD patients have dysfunctions of the retina, especially in response to light stimulation of dark-adapted rods and cones (Pillers e t al., 1993; Sigesmund et al., 1994). Furthermore, approximately one-third of DMD patients are mentally retarded (Karagan, 1979), with deficits in verbal reasoning and sentence comprehension (Anderson et al., 1988; Billard et al., 1992). Skeletal muscle is the tissue with the most obvious pathology in DMD, as witnessed by the very high levels of muscle creatine kinase (MCK) in patients sera (up to 50 times normal; Zatz et al., 1991). In younger patients there is a great variation in fiber diameter and many fibers are centrally nucleated, indicating degeneration and regeneration of the muscle. In older patients, fiber necrosis is associated with cell infiltration and phagocytosis and there is deposition of adipose and connective tissue resulting in the disruption of the normal muscle architecture (Emery, 1993). Light and electron microscopic studies showed the presence of breakages and abnormalities of the sarcolemma, although the basal lamina is generally maintained and is sometimes reduplicated (Mokri and Engel, 1975; Carpenter and Karpati, 1979). Anatomical studies of the brains of some affected boys showed extensive Purkinje cell loss, pachygyria, and mononuclear perivascular cuffing with cortical and subcortical gliosis (Rosman, 1970; Jagadha and Becker, 1988). More recent studies using magnetic resonance spectroscopy indicate that DMD patients have significant higher values than controls in the brain ratios of inorganic phosphate to ATE to phosphomonoesters, and to phosphocreatine. Since such a metabolic profile is also present in skeletal muscle, it has been suggested that there might be similar bioenergetic alterations in the two tissues in DMD patients (Tracey et al., 1995).

6. The DMD gene The DMD gene is located on the short arm of the X chromosome (Xp21) and is the largest gene ever characterized, spanning about 3 Mb and encoding at least 79 exons (Roberts et al., 1993).

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There are at least seven different promoters that regulate transcription in a tissue-specific manner. The first promoter contains standard basal transcription elements (such as a TATA box at position -24) in addition to muscle-specific elements and drives the transcription in muscle of a 14-kb mRNA (Klamut et al., 1990). This was the first identified transcript of the DMD locus (Monaco et al., 1986). The 14-kb transcript encodes for a 427-kDa protein called dystrophin and is present in low abundance in skeletal muscle (0.02-1% of total mRNA) (Chelly e t al., 1988). The 14-kb dystrophin mRNA is not present in cultured myoblasts, but is readily detectable after myoblast fuse to form myotubes. This may be due to positive and negative regulatory elements present in the promoter itself conferring a developmentally regulated expression of the 427-kDa dystrophin (Gilgengrantz et al., 1992). The size of the gene itself may also act as a regulatory mechanism, reducing the quantity of dystrophin mRNA available for translation in dividing cells. Indeed it has been calculated that more than 16 hr are needed to complete transcription, a period of time that causes a high rate of abortive transcription events, especially if it overlaps with the process of cell division (Tennyson et al., 1995). Another promoter is located about 100 kb upstream to the muscle promoter and it lacks standard regulatory elements, including the TATA box (Boyce et al., 1991; Makover e t al., 1991). This promoter drives the transcription of a 14kb mRNA in neurons of the cerebral cortex (frontal lobe), in the hypocampus (Lidov et al., 1990; Gorecki et al., 1992), and in the retina (Pillers et al., 1993). This 14-kb mRNA differs from the muscle mRNA in the first exon and in the alternative splicing of the 3’ region encoding the C-terminal of dystrophin (Nude1 et al., 1988; Feener e t al., 1989). A second brain promoter that has to be characterized is able to transcribe a 14-kb mRNA encoding a dystrophin isoform in cerebellar Purkinje cells (Gorecki et al., 1992). Two further promoters are located in the distal half of the dystrophin gene. The first has a structure similar to housekeeping promoters (Lederfein et al., 1993) and ubiquitously transcribes a 4.6-kb mRNA encompassing exons 63-79 and having a novel first exon, with exons 71 and 78 alternatively spliced (apodystrophin 1 or Dp71) (Lederfein et al., 1992). The second promoter transcribes a 5.2-kb mRNA which starts 1 kb upstream of exon 56 and is specifically expressed in Schwann cells of the peripheral nerve (apo-dystrophin 2 or Dpll6) (Byers et al., 1993). A different transcript of about 2.2 kb originating from the distal half of the dystrophin gene is detected in fetal muscle and the lungs and has been called apo-dystrophin 3 (Tinsley et al., 1993). A novel 7.5-kb transcript has also been identified which probably contains an uncharacterized promoter upstream to exon 45. This transcript encodes for a 140-kDa dystrophin isoform present in the piaarachnoid, both at the brain surface and along penetrating blood vessels as well as in the olfactory bulbs and it is likely to be the glial equivalent of apo-dystrophin 2 in peripheral nerve (Lidov et al., 1995). A potential seventh promoter has been identified about 500 kb upstream

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from the brain promoter, driving the transcription of an mRNA of unknown length in which the muscle, brain, and Purkinje promoters as well as exon 2 are spliced out. This mRNA appears to encode a dystrophin isoform (L-dystrophin) expressed in lymphocytes and probably lacks actin-binding activity (Nishio et al., 1994). The different promoters present in the dystrophin gene are not the only transcriptional regulatory elements, and the 3’-untranslated region of the DMD gene, highly conserved between humans and chickens (Lemaire et al., 1988), is likely to have a functional regulatory role by stabilizing the mRNA. Therefore, it appears that spatial and temporal regulation of the DMD gene is a complex phenomenon involving different promoters and enhancers for different protein isoforms, 3‘euntranslated regions, 3‘ alternative splicing, and more structural mechanisms such as the time that the RNA Pol11 complex takes to transcribe such a huge template. The discovery of the DMD gene and the identification of the corresponding mRNA and protein gave great insight into the molecular basis of DMD and BMD. It was soon understood that DMD and BMD are caused by mutations in the dystrophin gene, which has an unusually high rate of intragenic recombination (about 10-12% in normal pedigrees) (Abbs et al., 1990; Oudet et al., 1991). Recombination events appeared to occur mostly in two hotspots located between exon 1-8 and exons 44-51 (Oudet et al., 1992). Molecular studies in DMD and BMD patients showed that approximately two-thirds of mutations are caused by deletions and the remainder are due to duplications or point mutations (den Dunnen, 1989; Roberts et d., 1991, 1992). However, although the kind of mutation affecting the patient was known, there was no obvious correlation between the size of the mutation and the severity of the clinical symptoms. To explain such a lack of correlation between the mutation size and the severity of the phenotype, Monaco etal. (1988) proposed that if the mutation disrupted the reading frame, the patient most likely would have developed a DMD phenotype, whereas if the reading frame was maintained, the patient would have probably developed a BMD phenotype. This “frameshift hypothesis” would predict the absence of the protein in DMD patients and the presence of shorter forms of dystrophin in BMD patients. Hoffman et al. (1988) showed that this was indeed the case since the skeletal muscle of DMD patients was almost completely devoid of dystrophin whereas BMD muscle had dystrophin at >40% of normal levels, although the protein size was reduced. The frameshift hypothesis has since been confirmed by mutation analysis in a large number of patients and was proved to be correct in 90% of cases (Koenig et al., 1989; den Dunnen et al., 1989). In more than two-thirds of DMD patients it is possible to find rare dystrophin-positive myofibers in which an alternative splicing mechanism probably allows skipping of the deleted exons and restoration of the reading frame (Nicholson, 1993 and references therein). Splicing modulation of a mutated dystrophin

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transcript has been obtained in vitro by synthetic oligonucleotides, resulting in the specific inhibition of incorrect exon splicing and restoration of the normal length of the dystrophin mRNA (Takeshima et al., 1995). This approach could be used in viwo to specifically restore the reading frame of the dystrophin gene in selected cases of DMD patients and therefore ameliorate the phenotype.

C. Dystrophin and the apodystrophins The muscle 427-kDa dystrophin contains four structurally distinct domains, namely ( i ) the amino-terminal acting-binding domain (Hemmings et al., 1992; Levine et al., 1990, 1992; Corrado et al., 1994), (ii) a large spectrin-like rod domain, (iii) a cysteine-rich domain, and (iv) a carboxyl-terminal domain (Ahn and Kunkel, 1993). The N-terminal domain is associated with the actin-based membrane cytoskeleton. The rod domain constitutes three-quarters of the protein and comprises 24 spectrin-like repeats which confer a long, flexible, rod-like shape to the central region of the protein (Koenig et al., 1988; Cross et al., 1990). Electron microscopy studies suggest that the molecule can associate as homodimers or tetramers by antiparallel alignment along this rod domain (Sato et al., 1992).The cysteine-rich domain is reminiscent of the calcium-binding motif found in calmodulin, a-actinin, and P-spectrin, although there is no conclusive evidence for calcium-binding activity in dystrophin (Ahn and Kunkel, 1993).The carboxyl (C)-terminal has homology to only utrophin and, together with the cysteine-rich domain, binds to the large membrane-spanning oligomeric complex of dystrophin-associated glycoproteins (DAGs) and proteins (DAPs) that are linked to the extracellular matrix (Ervasti and Campbell, 1991; Suzuki et al., 1992, 1994; Ahn and Kunkel, 1993). The C-terminal can also bind syntrophins if exon 74 is not alternatively spliced (Ahn and Kunkel, 1995; Suzuki et al., 1995). The brain and Purkinje dystrophin isoforms differ from the muscle form in a few amino acids at the beginning of the molecule. Apo-dystrophin 1 is a 71-kDa protein and comprises a cysteine-rich and a carboxyl-terminal domain very similar to dystrophin (Lederfein et al., 1992). Apo-dystrophin 2 is a 116-kDa protein comprising the last 2.5 spectrine-like repeats followed by the C-terminal domain (Byers et al., 1993). In skeletal muscle, dystrophin is localized beneath the sarcolemma (Bonilla et al., 1988; Arahata et al., 1988; Carpenter et al., 1990). In the brain, dystrophin appears to be localized in the postsynaptic densities of particular neuronal populations (Lidov et al., 1990, 1993).

D. The dystrophin-associated glycoprotein complex As previously mentioned, dystrophin links the subsarcolemmal cytoskeleton through the interaction with F-actin at its N-terminal domain whereas the C-terminal domain binds to a complex of proteins and glycoproteins linked to the ex-

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Figure 4.1. Schematic model of the dystrophin-associated glycoprotein complex (DAGC) indicating the dystroglycan (DG) and sarcoglycan (SG) components as well as syntrophins and the neuronal-type nitric oxide synthetase (nNOS).

tracellular matrix (Figure 4.1). The complex includes a-dystroglycan (156 kDa DAG), which is located outside the sarcolemma and binds to merosin, the muscle isoform of laminin (Ibraghimov-Beskrovnayaet al., 1992; Ervasti and Campbell, 199313; Sunada et al., 1994), as well as to a number of other integral and cytoplasmic membrane proteins: P-dystroglycan, a-sarcoglycan, P-sarcoglycan, y-sarcoglycan, syntrophins, and nitric oxide synthetase (Figure 4.1). The DAGs thus provide a physical link and potentially a signaling pathway between the sarcolemmal cytoskeleton and the extracellular matrix [reviewed by Campbell (1995) and Worton (19991. The C-terminal domain of dystrophin appears to be essential for the correct assembly of the complex at the sarcolemmal region, as all the DAGs are dramatically reduced in the sarcolemma of DMD patients with deletions of the C-terminal domain (Matsumura et al., 1993b). In DMD carriers, only fibers positive for dystrophin are also positive for the DAGs, indicating the specificity of this loss (Campbell, 1995). The disruption of this linkage between cytoskeleton and extracellular matrix provided by dystrophin and the DAGs complex is associated with a variety of human diseases: the absence of merosin causes a non-Fukuyama type of congenital muscular dystrophy (CMD; Tome, et al., 1994) characterized by muscle weakness and hypotonia with very early onset,

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delayed motor milestones, severe and early contractures, and often joint deformities (Campbell, 1995). In addition, various autosomal recessive and so-called limb-girdle muscular dystrophies have now been shown to involve mutations in the sarcoglycan components (Worton, 1995).These findings and other studies on transgenic animals (see later) underline the potential importance of restoring the linkage between the myofiber cytoskeleton and the extracellular matrix if gene therapy of DMD is to be effective.

II. THE BASIS OF GENE THERAPY FOR DMD A. Animal models Animal models are becoming increasingly important in testing potential therapeutic approaches for DMD. Although dystrophin deficiency has been identified in cats and chicken, research in the field of gene therapy has concentrated mainly on the dystrophic mouse and dog. Dystrophic mice were first identified during a biochemical screening of a colony of C57BL/10 due to the high levels of serum MCK. The phenotype was inherited as an X-linked trait and these mice were called m d x (Bulfield et al., 1984). Mdx mice show histopathological changes similar to the ones found in very young DMD patients: variation in fiber size, central nucleation, cellular infiltration, and phagocytosis, indicating the presence of muscle degeneration-regeneration with a peak between 2 and 6 weeks of age (Carnwath and Shotton, 1987). Unlike human disease, however, m d x do not show interstitial fibrosis and adipose infiltration, suggesting a more effective regenerative process (or a less severe degenerative process) (Anderson et al., 1988). An exception is represented by the diaphragm and intercostal muscles of old animals (>6 months) which become fibrotic and resemble DMD muscle (Stedman et al., 1991). The murine homolog of the DMD gene has been identified and mapped on the X chromosome (Chamberlain et al., 1987).Dystrophin is absent in mdx skeletal muscle (Hoffman et al., 1987; Chamberlain et al., 1988), and a point mutation at position 3185 was discovered to be the cause of premature translation termination and ribosome release (Sicinski et al., 1989). A mutant dystrophic mouse, called m d ~ ~has ~ "been , obtained by artificially induced mutagenesis using N-ethyl-N-nitrosourea. The mutation in the disrupts both 14-kb and apo-dystrophin 1 reading frames, but the only differences seen in comparison to the m d x are a different proportion of spontaneous dystrophin-positive fibers (so-called "revertant fibers") and a reduced breeding rate (Cox et al., 199313). X-linked muscular dystrophy has been reported in golden retrievers (xmd dogs) (Cooper et al., 1988). These dogs present with stiffness of gait at 8-10 weeks of age and weakness and atrophy of the glutei muscles. Affected animals are even-

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tually unable to support their body weight and die from respiratory failure. Muscle pathology is very similar to DMD, including fibrotic and adipose substitution of necrotic myofibers (Valentine, 1990). Dystrophin is absent in mnd, and carrier females have a mosaic pattern of dystrophin expression in muscle (Cooper et al., 1990). The mutation involving the dystrophin gene is in the 3' consensus splice site within intron 6 which causes skipping of exon 7 and disruption of the reading frame at exon 8 (Sharp et al., 1992). Although mnd are difficult and expensive to breed, they would represent a better preclinical model since the phenotypic features of the disease and the underlying muscle pathology are very close to DMD, thus potentially allowing a more clinically oriented assessment of the efficacy of gene transfer.

B. Transgenic animals The maintenance of the link between the intracellular cytoskeleton and the extracellular matrix through dystrophin and DAGs complex is likely to be an essential requirement for proper muscle growth and function. Transgenic mice are of extreme importance not only to test this hypothesis, but also to evaluate the therapeutic efficacy of a variety of different dystrophin constructs and to determine the minimal essential requirement for dystrophin function in uivo. Full-length dystrophin cDNA under the control of the MCK promoterenhancer has been expressed in transgenic m d x , resulting in a complete correction of the histopathological changes present in the dystrophic mouse. Moreover, serum MCK measurements and an assessment of the contractile performance of diaphragm muscle both indicated a complete correction of normal muscle function (Cox et al., 1993a; Wells et al., 1995). Overexpression of dystrophin in either skeletal or cardiac muscles showed no toxic effects, suggesting that a tight control of dystrophin expression levels may not be a primary target for gene therapy of DMD (Cox et al., 1993a). Although these data strongly suggest that dystrophin gene replacement may be an effective strategy to cure DMD, the size of the dystrophin cDNA precludes its insertion into common recombinant viral vectors, which represent one of the most efficient and promising systems for gene transfer into skeletal muscle (Dickson et al., 1991). Alternative recombinant dystrophin cDNAs have been described and isolated from BMD patients with a very mild phenotype. England et al. (1990) reported a case of a BMD patient with minor dystrophic symptoms that expressed a 229-kDa dystrophin form with a large inframe deletion of the rod domain, extending from exon 17 to exon 48. The presence of a mild phenotype in this patient and his relatives, together with a correct sarcolemmal localization, suggested that the protein was at least partially functional, although histopathological examination of patient muscle showed relatively severe atrophy and fibrotic infiltration. Moreover, the size of the mRNA (6.3 kb) was compatible with

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its use in recombinant viral vectors (England et al., 1990). Several other cases of mild BMD patients have since been described, all having large inframe deletions of the rod domain, up to 50% of the coding sequence (Love et al., 1991; Matsumura et al., 1993a; Passos-Bueno et al., 1994). To evaluate the therapeutic potential of the 229-kDa dystrophin, the corresponding 6.3-kb cDNA under the control of the MCK or human skeletal action (HSA) promoter-enhancer sequences was expressed in transgenic mdx mice. Histopathologic analysis of the diaphragm revealed a high degree of protectivity of the 229-kDa protein even in the long term (6 months), although occasional groups of centrally nucleated fibers were observed (Wells et al., 1995; Phelps et al., 1995). Restoration of the DAGs complex at the sarcolemma was also observed in these transgenic mice, consistent with the situation found in Becker patients having inframe deletions of the dystrophin gene involving the rod domain, in which the DAGs complex is expressed at near-normal levels (Matsumura et al., 1993a, 1994). The 229-kDa dystrophin, however, was found to be less stable than the 427-kDa dystrophin in membrane association, suggesting that higher expression levels will probably be necessary to achieve the same therapeutic effect as the 427-kDa dystrophin (Wells et al., 1995). Truncated forms of dystrophin, which have a deletion of the N-terminal domain but which retain the C-terminal domain, however, can restore the DAGs complex at the sarcolemma but fail in preventing the dystrophic changes in muscle. One more such conclusion was reached using transgenic mdx mice in which a modified apo-dystrophin I or the wild type apo-dystrophin I was expressed in skeletal muscle (Cox et al., 1994; Greenberg et al., 1994). The histopathologic appearance of the transgenic muscle was similar, if not worse, than the mdx, indicating that restoring the C-terminal linkage to merosin is not sufficient for the prevention of muscular dystrophy. The pathology shown by these mice therefore clearly indicates the functional importance of linking the DAGs complex to the cytoskeleton through dystrophin. This concept is supported by BMD patients and transgenic animals expressing deleted dystrophin forms, yet preserving both the actin-binding and the carboxyl-terminal domains in which only mild pathologic signs are present.

C. Assessing the efficacy of dystrophin gene transfer A n important issue is the evaluation of the efficacy of dystrophin gene replacement both in transgenic animals and in mice subjected to somatic dystrophin gene transfer. Although the exact function of dystrophin is not known, a number of tests have been developed which aim to evaluate the phenotypic and functional consequences of dystrophin absence (and replacement) in skeletal muscle. Histopathologic analysis of skeletal muscle appears to be the easiest and most reliable way of assessing the efficacy of dystrophin gene replacement, par-

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ticularly if the analysis is carried out in the diaphragm. Injecting mdx mice with two different adenovirus vectors in skeletal muscle, one expressing the Escherichia coli P-galactosidasegene (LacZ) and the other expressing the 229-kDa dystrophin, proved to be particularly useful. A time course analysis showed that the number of LacZ-positive fibers progressively decreased as the treated animals aged, possibly due to the continuous process of degeneration-regeneration that is ongoing in mdx mice. O n the contrary, the number of dystrophin-positive fibers remained constant throughout the time course analysis. In older animals injected with both vectors, all myofibers expressing LacZ were also expressing dystrophin, suggesting that expression of dystrophin protected myofibers from necrosis and degeneration (Vincent et al., 1993). The evaluation of the ratio of centrally nucleated fibers to peripherally nucleated fibers can be of value: in the mdx mouse many fibers appear to be centrally nucleated (Bulfield et al., 1984) and dystrophin expression has been shown to stabilize the myofiber and promote the positioning of the nuclei at the periphery (Acsadi et al., 1991; Cox et al., 1993a; Vincent et al., 1993; Phelps et al., 1995; Wells et al., 1994). However, injecting m d x skeletal muscle with vectors carrying P+galactosidasedoes not change the ratio central/peripheral nuclei and treatment with steroids increases the ratio, possibly worsening the degenerative process in mdx muscle (Kuhan and Anderson, 1994). The distribution of the area of single fibers has also been used as an index of muscle pathology in the mdx as regenerating fibers are smaller (Krahn and Anderson, 1994). Measurement of serum MCK is also a good, indirect method to monitor the muscle degenerative rate (Cox et al., 1993a; Wells 1992, 1995). Other tests have been devised to evaluate the effects of dystrophin ab+ sence (or replacement) in a more dynamic situation. Diaphragm myofibers of m d x have been shown to be more susceptible to contraction-induced sarcolemma1 rupture; the damage is proportional to the magnitude of mechanical stress (Petrof et al., 1993). The method for such an assessment is relatively straightforward: strips of diaphragm to be analyzed are subjected to eccentric or isometric contraction or to passive lengthening in the presence of a low molecular weight dye and subsequently sectioned to allow visualization and counting of damaged fibers in which the dye penetrated (Petrof et al., 1993). Comparisons of the contractile properties of diaphragm from normal and m d x mice showed that active tension and maximum speed of contraction were both decreased in mdx muscle and that tension was reduced in Soleus and estensor digitorum longus (EDL) muscles of mdx mice compared to normal mice (Dupont-Versteegden and McCarter, 1992). The measurement of diaphragm contractile properties has proved to be a good method in assessing the efficacy of dystrophin gene therapy (Cox et al., 1993a). More recently it has been shown that the membranes of cultured m d x myotubes are less stiff and more susceptible to deformation than control myotubes, suggesting a structural role of dystrophin in reinforcing the myotube membrane skeleton (Pasternak et al., 1995). This method may prove useful to evalu-

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ate some of the phenotypic changes following dystrophin restoration in skeletal muscle.

C. How much dystrophin and where? What can we learn from patients? An important point to be taken into account for any gene transfer approach is the quantity of recombinant protein that is needed for a therapeutic effect. Given the particular histologic organization of skeletal muscle and given that dystrophin is a structural protein, it is important to consider how much protein is needed in a single fiber to prevent degeneration and how many dystrophin-positive fibers are needed in a muscle to prevent symptoms. With regard to the first issue, studies with transgenic animals showed that a wide range of dystrophin gene expression levels can be tolerated without toxicity and that the 427-kDa protein appears to be protective when expressed at 20% of endogenous levels in diaphragm, whereas higher levels of expression are probably needed in quadriceps muscle (Phelps et al., 1995). The 229-kDa dystrophin also appears to be protective in the mdx mouse diaphragm when expressed at >30% of endogenous dystrophin levels (Wells et al., 1995; Phelps et al., 1995). These data are supported by studies examining BMD patients. Although the kinds of mutations affecting the dystrophin gene appear to be crucial in these patients, (Koenig et al., 1989), very mild BMD phenotypes have been reported which expressed 30% of endogenous dystrophin levels (Angelini et al., 1994; Uchino et al., 1994). DMD carriers also provide a useful model to assess the relation between the appearance of clinical symptoms and the distribution of dystrophin-positive fibers in skeletal muscle. In female DMD carriers, only one X chromosome encodes for dystrophin whereas the other contains a mutation in the gene. Because of the randomness of X chromosome inactivation early in life, half of the nuclei in a myofiber should be dystrophin positive and half should be negative. Although this is not always the case, symptomatic carriers do have a mosaic expression of dystrophin in their muscles, and the relative percentage of positive fibers may vary widely [26-48% in one study by Arahata et al. (1989)l. A DMD carrier with a mild phenotype (bilateral weakness in proximal muscle with slight Gowers’ sign at age 42) has been described having less than one-third dystrophin-positive fibers in a muscle biopsy (Arahata et al., 1989). In general, in DMD carriers it is possible to draw a correlation between the relative proportion of dystrophin-positive fibers and clinical severity: if the proportion is less than one-third, the clinical phenotype is usually severe, if the proportion is more than three-quarters, the clinical phenotype is mild or very mild (Hoffman et al., 1992). Moreover, the distribution of dystrophin-negative fibers is important, as carriers with large dystrophin-negative areas in their muscles show an increased local weakness compared to carriers with the same percentage of dystrophin-negative fibers but having a more even distribution (Hoffman et al., 1992).

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A relevant phenomenon for gene therapy is the “normalization” that occurs in skeletal muscle of DMD carriers; in other words, the tendency for the muscle to become more dystrophin positive with time (Hoffman et al., 1992; Pegoraro et al., 1995). Two different mechanisms are responsible for dystrophin normalization in muscle: overexpression of dystrophin in positive nuclei and subsequent protein diffusion can compensate for neighboring negative nuclei within a fiber (biochemical normalization) (Pegoraro et al., 1995). Biochemical normalization has also been observed in m d x female carriers (Watkins et al., 1989; Karpati et al., 1990). Alternatively, dystrophin-negative myonuclei can be substituted by dystrophin-positive ones as a consequence of a continuous process of degeneration-regeneration and selection for dystrophin-positive muscle satellite cells (genetic normalization). Genetic normalization seems to happen mainly in DMD carriers with nonrandom X inactivation and can only partially compensate for dystrophin deficiency (Pegoraro et d., 1995). Both biochemical and genetic normalization are encouraging physiopathological processes from the gene therapy point of view. I t is reasonable to hypothesize that if a patient could be treated early in life with a vector unable to integrate into the cell genome or with a procedure such as direct DNA injection, skeletal muscle may show at least some degree of biochemical normalization whereas integrating vectors may allow some degree of both biochemical and genetic normalization. This is probably the case with a nonintegrating vector such as adenovirus, as will be discussed later. It is likely that the treatment of older DMD patients will have no substantial effects given that muscle degeneration is an irreversible phenomenon followed by a reduction in the amplitude of motor unit potentials, as detected by electromyographic analysis (Adams and Victor, 1985). As such, the sooner the patient is treated, the better. The central problem for gene therapy of DMD is that of preventing muscle degeneration and restoring normal muscle morphology and function. In addition there is also the necessity to ensure that such therapeutic effects persist even if the muscle subsequently undergoes changes and remodelment as a consequence of normal growth. Most vertebrates show a huge increase in the total muscle weight between birth and maturity; because the number of myofibers is fixed at birth in many mammalian species, including humans, hypertrophy of the existing fibers is the major cause of muscle growth (Montgomery, 1962; Moss, 1968a,b). In mice, rats, and chickens, both longitudinal and diameter growth of muscle fibers occur in conjunction with a proportional increase in DNA content brought about by an increase in the number of nuclei per fiber (Allen et al., 1979; Moss, 1968a; Williams and Goldspink, 1971). Although skeletal muscle fibers constituted of postmitotic syncitia of fused embryonic myoblasts, mononucleate cells lie beneath the fiber basal lamina which, under appropriate stimuli, are able to proliferate and fuse with existing fibers (Allbrook, 1981). Such “stem cells” are called muscle satellite cells. It appears that proliferation and fusion of satellite cells are the mechanisms that regu-

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late muscle growth by adding new DNA and nuclei to existing muscle fibers (Allen et al., 1979). The importance of using a vector that can infect muscle satellite cells and integrate into the cell genome appears therefore obvious, as it will probably allow juxtaposition of new dystrophin competent nuclei to a n existing myofiber during the process of normal muscle growth. Biochemical normalization may also play a role, as suggested by long-term studies in mice following adenoviral-mediated dystrophin gene transfer into skeletal muscle (Vincent et al., 1993). Genes transferred to cells by adenoviral vectors do not integrate in the cell genome and tend to be lost with time. Since the process of muscle growth is determined by the fusion of activated satellite cells with existing fibers, it is likely that dystrophin-positive nuclei within a fiber compensated biochemically for the fusion of dystrophin-negative satellite cells, thus allowing the number of dystrophin-positive fibers to remain constant from shortly after birth for up to 6 months.

A. Adenoviral vectors Viruses have evolved highly efficient mechanisms of entering cells, evading the host immune defense, and delivering their genetic material to the nucleus. A n immense amount of research has been directed at harnessing these properties to deliver foreign genes using genetically modified viruses, and our present understanding of the molecular genetics of many viruses makes possible their manipulation as cloning vectors for gene transfer both in cell culture and in uiuo. Because the major objective is long-lasting gene transfer, a deletion of the key regulatory viral genes is essential in manipulating the genetic program of the virus and in ensuring that infection of the target cell does not lead to cell death. Care must also be taken in the construction of viral cloning vectors to ensure replication deficiency such that wild-type propagation is not initiated, causing infection of the surrounding cells and tissues. In terms of gene transfer to muscle, adenoviral and retroviral (see later) vectors have been the most thoroughly examined. Adenoviruses are attractive vehicles for gene therapy as they are highly stable, exhibit wide trophism, and can infect quiescent as well as dividing cells. Respiratory epithelium is the primary target of normal infection, but other major sites include the eye and the gastrointestinal and urinary tracts (Perricaudet and Stratford-Perricaudet, 1995). Most adenoviral infections are, however, subclinical but do result in antibody formation (Perricaudet and Stratford-Perricaudet, 1995).The safety of clinical adenovirus administration has been verified since the mid-1970s with more than 10 million human subjects having received oral vaccines of live virus with n o detectable toxicity (Top et al., 1971). Additional advantageous properties of adenovirus vectors are that the vi-

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ral particles can be purified and concentrated to high titer (- 10” pfu ml-l), making systemic clinical administration feasible, plus the genome is relatively easy to manipulate and does not normally undergo rearrangement at a high rate (Celis and Celis, 1995). The adenovirus genome rarely integrates into the host chromosoine and replicates in an extrachromosomal state. This greatly reduces the risk of insertional mutagenesis, and in the case of skeletal muscle, the postmitotic nature and stability of muscle fibers appear to permit the long-term expression of transferred genes, even in the extrachromosomal form. Adenoviruses are found worldwide as 47 different human serotypes which are grouped according to their ability to cause tumors in newborn hamsters (Perricaudet and Stratford-Perricaudet, 1995). The adenovirus genome consists of a double-stranded linear DNA molecule of -36 kb in length, conventionally divided into 100 map units (mu), which codes for at least 30 mRNA species (Figure 4.2A). The genome is functionally divided into two major noncontiguous overlapping regions, early and late, defined by the onset time of transcription after infection (Perricaudet and Stratford-Perricaudet, 1995). There are four distinct early regions (El to E4) and one major late region (MLR) with five principal coding units (Ll-L5), plus several minor intermediate and/or late regions that are less well characterized. At the extremities of the viral genome are inverted terminal repeats (ITRs) of 100-140 bp that are essential for viral replication. Specific encapsidation signal (+) sequences located adjacent to the 5’-ITR are essential for the packaging of viral DNA into capsids (Kremer and Perricaudet, 1995). The adenovirus capsid consists of nonenveloped “spiked” icosahedron of 60-90 nm enclosing an inner DNA-protein core, which accounts for the relatively high stability of the virus and its insensitivity to complement-mediated inactivation. Three loosely defined sets of proteins are integral to the adenovirus structure (Perricaudet and Stratford-Perricaudet, 1995). First, the outer coat or capsid proteins, which contribute to the isosahedral structure, are composed of a total of 720 hexon and 60 penton protein subunits together with 360 monomers of polypeptide IV, 240 monomers of polypeptide IX, and 60 trimer fiber proteins. Associated with each penton subunit is a fiber protein that protrudes from the capsid and mediates the initial attachment of the virus to a target cell via an undetermined receptor. Following binding, virus internalization involves secondary interactions between penton bases and vitronectin-binding integrins, avP,/p5 (Nemerow et al., 1994; Whickam et al., 1993; White, 1993). The second set of viral structural proteins stabilize the capsid and consist of polypeptide IX involved in the packing of adjacent hexons, polypeptide IIIa, which spans the capsid linking hexons of adjacent faces, and polypeptide VI, which connects capsid proteins to the core. Finally, the third set of viral structural proteins are the core DNAbinding proteins that are involved in genome function and stability and include polypeptides pV, pVII, k, and the 55-kDa terminal protein that is covalently

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Figure 4.2. Adenovirus-based gene transfer vectors. A simplified transcriptional map of the 36-kb adenovirus genome is shown ( A ) which is conventionally divided into 100 map units. Adenovirus exhibits an exceptionally complex and regulated transcriptional cascade involving multiple alternative splicing events. Transcription ofEl products activates all the other early gene regions, and late transcription then occurs following replication of the viral genome. Recombinant adenoviral vectors often incorporate foreign DNA into the deleted E l region (B) to yield replication-defective particles. Alterations in E2, E3, and E4 regions to increase vector capacity or further inhibit replication potential have also been utilized. (C) A minimal vector genome is represented in which only the 3'- and 5'inverted terminal repeats (ITRs), which contain the replication and packaging signals of adenovirus, are retained and all structural genetic material has been replaced by foreign DNA. These latter so-called pseudo-adenoviral vectors offer the potential of high insert capacity and no viral structural gene expression in transduced tissues.

linked at each 5' end of the genome and primes DNA replication as the 89-kDa precursor form, pTP (Kremer and Perricaudet, 1995). The construction of adenovirus vectors is based on the replacement of specific regions of the viral genome with foreign DNA, containing the gene of interest, by the direct ligation of adenovirus DNA or by homologous recombinations in uitro (see Figure 4.2B). First, generation adenovirus vectors have deletions of the E l region which render them replication defective in most cell types (Bett et al., 1993). These replication-incompetent, El-deleted adenoviruses are isolated and replicated in 293 cells which complement in trans for E l gene products (Graham et al., 1977). However, it is essential that flanking subregions of the E l region, essential for replication and packaging, are retained in an El-deleted virus, namely the ITR, W,and the PIX sequences (Bett et al., 1994). One current limitation of adenovirus vectors is a relatively tight upper

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size limit for DNA packaging of approximately 105% of the wild-type genome. Above this, sharp reductions in viral yields and stability are observed due to rapid rearrangements (Bett et al., 1993). In order to extend cloning capacity, the E3 region that is not essential for adenovirus replication in cell culture is also commonly deleted (Bett et al., 1993). Even with El/E3-deleted adenoviral vectors, the capacity for the foreign DNA insert is restricted to about 7.8 kb (Bett et al., 1994) and hence cannot accommodate full-length dystrophin cDNAs. Nevertheless, as discussed earlier, it is possible to use the 6.3-kb human minidystrophin cDNA isolated from one patient with very mild clinical manifestations of BMD (England et al., 1990). A number of groups have now constructed El/E3-deleted recombinant adenovirus vectors containing this 6.3-kb Becker minidystrophin cDNA driven by various promoter elements (Ragot et al., 1993; Acsadi et al., 1995; Dickson et al., unpublished). In vivo studies involving intramuscular injections into neonatal mdx mice have shown that these adenovirus vectors can efficiently direct the synthesis of very significant amounts of the 229-kDa minidystrophin with 5-50% of fibers showing correct sarcolemmal localization. In one study, positive transduced fibers showed only 12% central nucleation compared with over 60% in negative fibers. Under these conditions, dystrophin expression was reported up to 6 months after virus injection with no reported evidence of histopathological alterations or cytotoxic immune responses (Vincent et al., 1993). Quantin et al. ( 1992) constructed a recombinant El/E3-deleted adenovirus containing a pgalactosidase reporter gene under the control of muscle-specific regulatory sequences, This construct was reported to efficiently direct expression in vitro in both myoblasts and postmitotic myotubes from a rodent cell line, but not in 3T3 mouse fibroblasts. In vivo studies in neonatal mice confirmed this specificity, revealing very strong histochemical staining detectable in muscle around the injection site up to 75 days after infection with no expression in nonmuscle tissues. They then went on to construct a recombinant adenovirus vector containing the functional minidystrophin with the same regulatory elements which was shown to exclusively express in myofibers with the correct localization (Alameddine et al., 1994). The route of adenovirus administration is a major factor in terms of transduction efficiencies to different tissues. Following systemic administration of a recombinant adenovirus vector, expression of transgenes in a variety of different organs and tissues is generally found. Kass-Eisler and colleagues (1994) have reported that the administration of recombinant adenovirus into rats via six different routes resulted in the infection of all eight tissues tested, but to varying degrees. The most effective and widespread method of gene transfer was surprisingly found to be via intracardiac injections. In terms of gene transfer to skeletal muscle, intramuscular injection resulted in equivalent efficiencies as via the intracardial route. In terms of adenoviral-mediated gene therapy for muscular dystrophy, complex targeting strate-

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gies are likely to be required, including local administration to selected muscle groups, and intraarterial or intrapleural administration for heart, diaphragm, and respiratory muscles. It has also been shown that the infectivity of muscle and other organs by adenovirus vectors is very much dependent on the maturity of the tissue and/or age of the treated animal. Immature muscle cells are efficiently transduced, but mature muscle fibers are not, even at high titers (Dickson et al., unpublished; Acsadi et al., 1994a,b). Quantitative analyses using luciferase expression demonstrated a 20 times higher gene expression in immature muscle as compared to fully developed muscle (Acsadi et al., 1995).This may correlate with a higher surface density of the available adenovirus internalization receptor in immature muscle cells, which could possibly be due to the aVP3/P5 receptors being blocked in myofibers by the extracellular matrix protein vitronectin (Acsadi et al., 1994a). However, immunological factors most probably play a dominant role. Significantly increased efficiencies have been observed in adult mice if abundant muscle regeneration is induced and immunodeficient animals are used (Dickson et al., unpublished). It is believed that antigen-specific lymphocytic immune responses to virus-infected cells, involving CD4+ and CD8+ T-cell infiltrations, are responsible for diminished transgene expression. Yang and colleagues ( 1995) demonstrated that CD4+ T cells alone are not capable of complete target cell elimination whereas CD8+ cytotoxic T cells are sufficient. However, T-cell responses alone may not be solely responsible for the elimination of transduced cells, and minor nonantigen-specific destruction via natural killer cells or macrophages may also be involved. In addition, humoral neutralizing antibody production is likely to underlie the block to reinfection with a repeated dosage (Yang et al., 1995). Zabner and colleagues (1994) have reported, however, that despite an antibody response to a recombinant adenovirus expressing CFTR and delivered to the respiratory epithelium of cotton rats and rhesus monkeys, there was no evidence of a local or systemic inflammatory response after repeated administration. These authors concluded that a repetitive administration of low doses may be tolerated. A similar report by Bout et al. (1994) reported only mild multifocal perivascular and peribronchial lymphocytic infiltrates found upon histopathological analysis after the administration of high doses of recombinant adenoviruses. Rechallenging resulted again, albeit at lower levels in gene transfer to the rhesus monkey lung and airway at all levels, without evidence of additional histopathological changes. Yei and colleagues (1994) also showed significant reductions of gene expression in the respiratory tract of cotton rats upon repeated adenoviral dosage which correlated inversely with neutralizing antibody titers. Several regions of the adenovirus genome are known to express proteins which generate immune responses in vivo, particularly the late region gene products (Yang et al., 1995). Despite the fact that first-generation adenovirus vectors are defective in early region function (Ela/b), they have been found to lead to the

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induction of host cellular immune responses and the clearance of transduced cells. Second-generation adenovirus vectors are being designed to ablate the expression of immunodominant viral proteins focusing on the E2a gene. Yang et al. (199413) have reported longer-lasting recombinant gene expression and significantly reduced inflammatory responses from a n E2a-defective vector. Recent data also suggest that gp19 encoded by the E3 region is important in avoidance of the host immune surveillance (Gooding, 1992), and vectors reincorporating this function are now available. Furthermore, in terms of clinical safety, the deletion of more than one essential regulatory element in viral vectors would be advantageous in minimizing recombination events leading to replication-competent adenovirus. Presently, a number of laboratories are working toward the production of complementing 293-based cells that supply both E l and E4 products in trans (Kremer and Perricaudet, 1995). Furthermore, attempts are underway to develop vectors in which much larger portions (Mitani et al., 1995) or indeed the entire adenoviral structural gene complement (Dickson et al., unpublished) has been deleted, leaving minimal sequence elements necessary for replication and packaging (Figure 4.2C). Such vectors have been proposed to potentially provide up to a 35kb capacity which would allow the use of complex promoter elements and linked reporter genes, e.g., the full-length dystrophin cDNA. In addition, minimal vector systems would, in theory, completely avoid secondary cell-mediated immune responses to infected cells. A t present, such systems require a so-called helper virus for capsid assembly and packaging, but complementation strategies using packaging-defective viral genomes may be possible.

B. Retroviral vectors Recombinant retroviruses based on mouse C-type retroviruses are widely used vectors for gene transfer in animals and humans (Figure 4.3). Several features render recombinant retroviruses suitable for gene transfer in skeletal muscle. T h e retroviral genome is stably integrated into host cell DNA and as such has the potential for indefinite expression of the recombinant protein, even if muscle undergoes changes as in normal growth. Moreover, skeletal muscle transfected by retroviral vectors may theoretically show both biochemical and genetic normalization. Once integrated, the viral promoter and enhancer can be used to drive transcription of the inserted genes in many cell types, including myoblasts and myotubes. These vectors can be genetically engineered so that all the proteins necessary for viral replication can be provided in trans,generating infectious particles that do not produce viral antigens once infection occurred. T h e main limitations of retroviral vectors are the limited insert size that they can accommodate (9-10 kb) and the fact that only cells that are in, or close to, mitosis can be transduced (Miller et al., 1990). The retrovirus genome consists of two dimerized strands of RNA that are

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+ L m H

+LTR H

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*

Foreian-t-e Nee? LTR

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u

8

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Replication-defective viral particles

-8 Infect target cells or tissues

Figure 4.3. Retrovirus-based gene transfer vectors. The genome of rnurine leukemia virus (MLV) is represented in A showing the viral long terminal repeats (LTRs), which direct replication, integration, and packaging, and the GAG, POL, and ENV structural genes, which encode core and envelope proteins and reverse transcriptase/integrase activities. In MLVbased vectors, GAG, POL, and ENV regions are replaced by foreign DNA (B), and retroviral plasmid constructs are used to transfect packaging cells which supply the viral proteins in trans (C) and release replication-defective retroviral vector particles.

reverse transcribed in a double-stranded DNA provirus. Such a provirus is defined at both 5’ and 3’ ends by identical untranslated direct repeats called long terminal repeats (LTR) that contain the viral transcription regulatory regions (Weiss et al., 1985). T h e provirus also contains a packaging signal and three reading frames for viral structural genes: gag, for viral nucleocapsid, capsid, and matrix proteins; pol, for viral protease, reverse transcriptase, and integrase; and enu, for viral envelope glycoproteins (Varmus et al., 1982).After virus entry into the cell, the RNA genome is reverse transcribed in the cytoplasm and the resulting viral DNA is transposed to the nucleus as part of a nucleoprotein complex and is eventually integrated into the host cell genome. T h e integrated provirus is then transcribed by cellular RNA polymerase, and transcripts are in part translated to give viral proteins and in part packaged into viral particles (Fassati et al., 1995a). Recombinant retroviruses have defective genomes in which the coding regions have been replaced by a recombinant gene. These retroviruses can repli-

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cate only if appropriate structural proteins are provided in trans in the same cell. Cells that encode the proteins necessary for viral replication are called packaging cells. Retroviral vectors can be transfected into the packaging cells, and the transfected provirus is transcribed and packaged into new particles which, lacking all coding sequences, are infectious but not replication competent (Mann et al., 1983; Weiss et al., 1985). Because the retroviral genome is able to integrate into host cell chromatin, both ex vivo and in vivo approaches are feasible for gene transfer in skeletal muscle. The ex vivo approach utilizes infections in vitro by recombinant retroviruses of target cells from the patients and autologous transplantation (Dai et al., 1992; Barr and Leiden, 1991; Dhawan et al., 1991). Retroviral vectors have been used to infect muscle cells in vitro, and expression of the transgene has been detected after myoblasts fusion into myotubes, indicating that profound changes in the cell transcription pattern do not necessarily affect retroviral gene expression (Smith et al., 1990). Following this initial in vitro study performed with a reporter gene, a recombinant retrovirus carrying the 229-kDa dystrophin cDNA has been constructed and used to infect mdx myoblasts, resulting in the expression and correct localization of the protein at the sarcolemma of the skeletal muscle myotubes (Dunckley et al., 1992). Since recombinant retrovirus expression in most cases is not developmentally regulated, the study also confirmed that dystrophin expression in myoblasts does not interfere with the normal process of fusion and myotube formation. Studies in various mouse models showed that myoblasts transduced in vitro with retroviral vectors are able to fuse with preexisting myofibers when transplanted in vivo and are able to produce the recombinant protein (Dai et al., 1992; Barr and Leiden, 1991; Dhawn et al., 1991; Naffakh et al., 1994). Human myoblasts, transduced in vitro with a reporter gene, have been successfully transplanted in a mouse model as a method for potential ex vivo gene therapy for DMD (Salvatori et al., 1993). However, the expression of the recombinant gene after implantation in vivo is somehow transient in primary myoblasts, and sustained expression has been obtained only using heterologous enhancer-promoter sequences (Dai et al., 1992). Direct in vivo administration of recombinant virus has also been attempted (Dunckley et al., 1993). Although myofibers are postmitotic syncitia and refractory to infection with retroviruses, activated satellite cells can be targeted by recombinant retroviruses during the process of muscle regeneration. Experimental degeneration of muscle can be artificially induced in normal mice. Alternatively, mdx mice can be used, in which spontaneous muscle degeneration-regeneration is ongoing (Dunckley et al., 1993). However, the direct injection of retroviral-containing supernatants into regenerating mouse skeletal muscle results in relatively low efficiency of transduction, possibly due to the limited temporal availability of dividing satellite cells at the precise time of infection and to

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the interference by the immune system, which reduced the already short half-life of retroviruses in vivo (Fassati et al., 199513). Some of these problems have been overcome by the direct injection of mitotically inactivated retroviral producer cells into regenerating skeletal muscle, resulting in a much higher efficiency of transduction and in the infection of satellite cells which contributed to new fiber formation in the long term (Fassati et al., 1996). Direct in vivo infection of satellite cells by retroviral vectors results in long-term expression of the recombinant gene compared to the ex vivo approach, although the molecular basis for this difference remains as yet to be defined. A n area of growing interest is the targeting of retroviral vector infection to specific cell types, particularly for in vivo applications. The incorporation of heterologous protein domains that recognize specific receptors into the envelope glycoproteins of retroviral vectors has been reported to modify viral tropism toward the cell type bearing the receptor (Kashahara et al., 1994; Somia et al., 1995),although such manipulations may severely affect viral infectivity (Cosset et al., 1995). Alternatively, the insertion of muscle-specific enhancers within the wildtype enhancer may result in a muscle-specific transcription of the vector (Ferrari et al., 1995). Major issues still remain to be solved before retroviral-mediated gene transfer to skeletal muscle can be attempted in humans. With regard to the ex vivo approach, long-term expression of the recombinant protein after myoblast transplantation is necessary, as well as the capability of growing clinically useful quantities of fusion-competent myoblasts. With regard to the in vivo approach, the efficiency of transduction needs to be further improved, and animal models such as xmd dogs, in which the rate of spontaneous muscle degeneration-regeneration is probably more close to DMD, may be of particular value. Moreover, murine retroviruses are sensitive to human complement-mediated lysis (Welsh et al., 1975). Different strategies have been proposed to overcome the inactivation of retroviral vectors by human complement. Transfection of a murine retroviral vector in human packaging cells expressing RD114 feline envelope glycoproteins instead of the usual amphotropic or ecotropic glycoproteins results in the production of a retroviral pseudotype resistant to inactivation by human complement (Takeuchi et al., 1994). Alternative, but perhaps more invasive, strategies include the use of complement inhibitory drugs or specifically designed monoclonal antibodies able to bind and inactivate specific complement factors (Rother et al., 1995; Matis and Rollins, 1995).

C. Direct DNA injection In terms of gene therapy, the simplest, most inexpensive and possibly the safest procedure would be the introduction of pure circular DNA by direct injection into a desired tissue. Intense research was generated into this technique following the

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findings of Wolff and colleagues (1990),who showed that it was possible to transfer and express recombinant genes in mouse skeletal muscle in vivo by the direct intramuscular injection of supercoiled plasmid DNA. They used promoter-reporter gene constructs, including P-galactosidase, luciferase, and chloramphenicol 0-acetyltransferase (CAT), which all resulted in readily detectable reporter gene expression localized by myofibers. Similar results have since been reported in adult and neonatal rat (Wolff et al., 1991), primate skeletal muscle (Acsadi et al., 1991), and cardiac muscle (Jiao et al., 1991). Wolff and colleagues (199213) later expanded their studies and reported the expression of the foreign gene for at least 19 months after the initial injection. Although the injected DNA remains extrachromosomal, the postmitotic nature and longevity of the muscle fibers allow the long-term expression of transferred genes (Wolff et al., 1990, 199213). The efficiency of reporter gene expression after direct DNA transfer has been reported to be highly variable, especially with small injection volumes, predominantly due to the highly variable distribution of the injected substance. Davis et al. ( 199313) reported that a preinjection of a relatively large volume of hypertonic (25%) sucrose solution reduced this variability due to the fibers being forced apart by the hydrostatic pressure created by both the injection and the subsequent hypertonic-induced edema, therefore increasing intramuscular diffusion. However, the injection of such large volumes results in significant damage to the muscle fibers. Ideally, this damage should be kept to a minimum, as fibers expressing the foreign gene have been shown not to be those damaged during the injection procedure (Davis et al., 199313). Furthermore, in the case of DMD patients where the muscle structure and repair functions are already compromised, any further damage by the treatment would be quite detrimental to the cause. Pretreatment with the mytotoxic local anesthetic bupivicaine, which experimentally induces regenerative activity in muscle tissue, has been shown to significantly enhance direct gene transfer (Danko et al., 1994; Vitodello et al., 1994; Wells, 1993). The fact that regenerating muscle has a higher efficiency of transfection may be advantageous for gene therapy in DMD as regenerating fibers are numerous in the early stage of the disease. Striated muscle tissue seems particularly efficient at plasmid DNA uptake compared to other tissues (Acsadi et al., 1991). However, the mechanism by which the naked DNA enters the striated muscle remains undefined, but it does not appear to be the result of overt injury to the sacrolemmal membrane (Davis and Jasmin, 1993). Wells and Goldspink (1992) demonstrated that the uptake efficiency in mice is dependent on the age and sex of the recipient. Moreover, Davis and colleagues (1993b) showed that pure plasmid DNA was unable to transfect mononucleated muscle cell precursors in toxin-treated muscle and that transfection only becomes efficient at certain levels of fiber maturity. They postulated that this may be associated with the presence of t-tubules, which are unique to striat-

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ed muscle and rise and mature over several days during normal myogenesis (Edge, 1970). Further evidence supplied by electron microscopy studies showed that injected plasmid DNA conjugated to colloidal gold is associated with t-tubules (Wolff et al., 1992a). It has been reported that subepidermysial injection of the diaphragm is safe and results in effective gene transfer while causing minimal myofiber damage (Davis andlasmin, 1993).Also, the level of expression was comparable to that in skeletal muscle pretreated with hypertonic sucrose or with induced regeneration, most probably due to the endomysium being less dense and hence facilitating diffusion. This observation may aid the evaluation of the efficacy of dystrophin gene transfer by direct DNA injection, as the diaphragm of the mdx mouse has been reported to reflect the pathology of DMD (Stedman et al., 1991). Several groups have reported the stable expression of Becker-like and full-length dystrophins in m d x muscle after direct intramuscular injections of expression plasmids. The proteins have been reported to be correctly localized in approximately 1% of fibers (Acsadi et al., 1991) and appeared to protect mdxfibers from degeneration (Danko et al., 1993). However, although the direct injection of plasmid DNA has been reported in one study to be superior to viral vectors for direct gene transfer into adult mouse skeletal muscle (Davis et al., 1993a), the technique is currently constrained by low efficiencies and poor reproducibility. In terms of direct physical gene transfer, a number of mechanisms to enhance DNA uptake have been proposed. These include DNA-coated particle bombardment (Schofield and Caskey, 1995), polycations (Kawai and Nishizawa, 1984), and receptor-mediated gene delivery involving complexing plasmid DNA to specific targeting proteins (Wu et al., 1989) or coupling transferrin-polylysine/DNA complexes to chemically inactivated adenoviruses (Wagner et al., 1992). Intense interest has also focused on the use of cationic liposomes as vehicles for the transfer of recombinant genes into a variety of tissues, both in vitro and in vivo (Alton et al., 1993;Zhu et al., 1993;Trivedi and Dickson, 1995).This technique relies on the electrical charge properties of polycationic lipids which spontaneously bind polyanionic DNA or RNA (Felgner e t al., 1987; Mannino and Gould-Fogerite, 1988). The electrical charge attraction and the fusion properties of the resulting DNA-liposome complexes enable them to efficiently absorb into cell membranes and deliver genes directly to the cytoplasm, bypassing lysosomal degradation pathways. Zhu and colleagues (1993) have reported that a single systemic injection of an expression plasmid/cationic liposome complex into adult mice efficiently transfected many different tissues. Trivedi and Dickson ( 1995) showed an efficient transfer of dystrophin cDNA into primary muscle cultures using liposome vehicles, suggesting the possibility of gene therapy of DMD via direct liposome-mediated intramuscular gene transfer or via autologous transplantation of transfected, dystrophin-positive myoblasts.

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0. Myoblast transplantation Pioneering studies by Partridge et al. (1978) and Lipton and Schultz (1979) showed that myogenic precursor cells, isolated and cultivated in uitro, retain their fusogenic capacity and are able to participate in the regeneration or growth of existing myofibers when implanted in uiuo. The potential therapeutic value of muscle and myoblast transplantation has subsequently been evaluated in dystrophic mice and in mice with an inherited deficiency of the enzyme phosphorylase kinase (PhK; Law and Yap, 1979; Law, 1982; Law and Goodwin, 1986; Watt et al., 1984; Morgan et al., 1988). In the latter case it was shown that implanted myoblasts fused into preexisting myofibers, as detected by analysis of the glucose-6phosphate isomerase isoenzymes, but PhK activity was found only in a small number of mice in which extensive degeneration and implantation of high numbers of myoblasts were performed (Morgan et al., 1988). A similar approach was used to correct dystrophin deficiency in the mdx mouse by injecting normal, dystrophin-competent, primary myoblasts in spontaneously regenerating muscle, which resulted in the expression and correct localization at the sarcolemma of dystrophin in m d x myofibers. A patchy profile of dystrophin immunostaining was present within individual fibers, suggesting the existence of a mosaicism of dystrophin-competent and -incompetent nuclei, as in mdx female carriers (Partridge et al., 1978). The implantation of dystrophin-competent myoblasts in a mdx mouse model that had been previously irradiated to reduce spontaneous muscle regeneration restored fiber morphology and reduced fiber loss, indicating a functional recovery ofpathological muscle (Morgan et al., 1990). Also, implanted myoblasts were observed to survive as muscle precursor cells and could be recovered from the muscles of the recipient mice, even months after implantation (Yao and Kurachi, 1993). The results obtained in the mdx mouse model encouraged several groups to attempt myoblasts allotransplantation in humans. The results, however, have been disappointing. Although the presence of some dystrophin-positive fibers could be demonstrated after grafting and transient strength increases have been measured in a minority of treated DMD patients, the overall efficiency of the procedure was extremely low and no long-term clinical improvement could be observed, even if patients were immune depressed to avoid rejection of implanted cells or were monozygote twins (Huard et al., 1992;Karpati et al., 1993; Law et al., 1990; Tremblay et al., 1993a). Although the reasons for such disappointing results are not completely clear, it seems likely that the immune system played a relevant role. Antibodies against grafted myoblasts and fused myotubes were observed, some of them were able to induce complement-mediated lysis of foreign cells (Tremblay et al., 1993b). More recent studies in mice revealed that an appropriate immune-suppressive regimen with FK 506 or rapamycin greatly increased the efficiency and duration of myoblasts graft, although irradiation and extensive muscle degeneration were required (Kinoshita et al., 1994; Vilquin et

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al., 1995). However, it seems that in addition to the interference of the immune system and the difficulty in growing clinical useful quantities of myoblasts, other factors are capable of limiting the efficiency of this procedure, such as the major loss of myoblasts that appears to occur 48 hr after implantation and the lack of spread of injected cells from the site of injection (Huard et al., 1994).

The dystrophin gene has proved to be far more complex than originally expected, containing multiple alternative promoter elements and splicing pathways and giving rise to multiple protein isoforms that are spatially and temporally regulated. Skeletal muscle is the tissue with the most striking pathology in patients lacking dystrophin, but heart and central nervous system (CNS)may also be affected. No data are yet available from transgenic animals on the effects of dystrophin restoration in heart and CNS or on the minimal structure-function requirements for dystrophin to be functional in these tissues. In this regard, however, it seems unlikely that the mdx mouse will be of great help. Another important point to consider is the possibility that CNS dysfunction in some cases may be manifest with a much more subtle or slower developing phenotype than muscle dysfunction. Moreover, the precise function of dystrophin is still not clearly defined nor is the physiopathological processes that lead to failure in muscle regeneration and subsequent fibrotic and adipose infiltration. These considerations underline some of the additional problems involved in the design of gene therapy approaches for DMD. Whether the treatment of selected muscle groups, for example, to avoid respiratory failure or to maintain a certain degree of physical mobility, will be appropriate remains a clinical uncertainty. It is clear that a rapid transfer of techniques from mdx mice to DMD patients is not a realistic option as clinical trials for myoblasts transplantation have shown. Any gene therapy approach needs to be fully tested in both small and large animal models. All studies on dystrophin gene transfer in mdx mice have so far been restricted to single muscles (e.g., tibialis anterior) or a limited group of muscles (e.g., quadriceps). Such a n experimental scenario is of use to evaluate the efficiency of transfection with particular vector formulations, but it does not address the question of widespread transgenesis of the somatic musculature. Techniques that may allow the delivery of the vectors to large numbers of muscles need to be developed and evaluated in mouse and canine animal models. It is likely that more or less invasive surgical procedures, as well as some sort of temporary or permanent modulation of the immune system, will be required to achieve clinically useful results. However, advances in vector design and delivery are a major focus of academic and industrial research activity. In the next few years, studies in experimental animals will yield proof-of-

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concept analyses and data in relation to skeletal muscle gene transfer. Only at this stage can the logistics and feasibility of gene therapy for DMD be critically analyzed and taken if appropriate to clinical trial.

Acknowledgments This work is supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Leopold Muller Trust, the Medical Research Council, the Genzyme Corporation, and Boehringer Mannheim. We thank Dr. S.C. Brown, Professor G. Scarlato, and Professor F. S.Walsh for very helpful discussions.

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Phelps, S.F.,Hauser, M. A,, Cole, N . M., Rafael, 1. A., Faulkner, 1. A., and Chamberlain, J. S.(1995). Prevention of muscular dystrophy by full-length and internally truncated dystrophins. Hum. Mol. Genet. 4:1251-1258. Piccolo, G., Azan, G., Tonin, P., Arbustini, E., Gavazzi, A., Banfi, P., Mora, M., MOrandi, L., and Tedeschi, S. ( 1994). Dilated cardiomyopathy requiring cardiac transplantation as initial manifestation of Xp2 1 Becker type muscular dystrophy. Neuromusc. Disord. 4:143-146. Pillers, D.-A. M., Bulman, D. E., Weleher, R. C., Sigesmund, D. A., Musarella, M. A., Powell, B. R., Murphey, W. H., Westall,C., Panton, C., Becker, L. E., Worton, R. G., and Ray, l? N. (1993). D y strophin expression in the human retina is required for normal function as defined by electroretinography. Nature Genet. 4:82-86. Prosser, E. J., Murphy, E. G., and Thompson, M. W. (1989). Intelligence and the gene for Duchenne muscular dystrophy. Arch. Dis. Child.44:221-230. Quantin, B., Perricaudec, L. D., Tajakhsh, S., and Mandel, J. L. (1992). Adenovirus as an expression vector in muscle cells in vivo. Proc. Natl. Acad. Sci. USA 89:2581-2584. Ragot, T., Vincent, N., Chafey, P., Vigne, E., Gilgenkrantz, H., Couton, D., Cartaud, J., Bridnd, P., Kaplan, J. C., Perricaudet, M., and Kahn, A. (1993). Efficient adenovirus-mediated transfer of a human mini-dystrophin gene to skeletal muscle of mdx mice. Nature (London) 361:647-650. Roberts, R. G., Bardy, T. F. M., Manners, E., Bobrow, M., and Bentley, D. R. (1991). Direct detection of dystrophin gene rearrangements by analysis of dystrophin mRNA in peripheral blood lymphocytes. Am. J. Hum. Genet. 49:298-310. Roberts, R. G., Bobrow, M., and Bentley, D. R. (1992). Point mutations in the dystrophin gene. Proc. Natl. Acad. Sci. USA 692331-2335. Roberts, R. G., Coffey, A. J., Bobrow, M., and Bentley, D. R. (1993). Exon structure of the human dystrophin gene. Genomics 16:536-538. Roberts, R. G. (1995). Dystrophin, its gene and the dystrophinopathies. Adu. Genet. 33:177-231. Rosman, N. P. (1970). The cerebral defect and myopathy in Duchenne muscular dystrophy: A comparative clinicopathological study. Neuroloa 20:324-335. Rother, R. P., Squinto, S.P., Mason, J. M., and Rollins, S. A. (1995). Protection of retroviral vector particles in the human blood through complement inhibition. Hum. Gen. Ther. 6:429435. Salvatori, C., Ferrari, G., Mezzogiorno, A., Servidei, S., Coletta, M., Tonali, P., Giavazzi, R., Cossu, G., and Mavilio, F. (1993). Retroviral vector-mediated gene transfer into human primary myogenic cells leads to expression in muscle fibers in vivo. Hum. Gene Ther. 4:713-722. Sato, O., Nonomurd, Y.,Kimura, S., and Maruyamd, K. (1992). Molecular shape of dystrophin. 1. Biochem. 112:631-636. Schofield, J. P., and Caskey, C. T.(1995). Non-viral approaches to gene therapy. Brit. Med. Bull. 5 1:56-7 I. Sharp, N. J. H., Kornegay, J . N., VanCamp, S. D., Herbstreith, M. H., Secore, S. L., Kettle, S., Hung, W.-Y., Constantinou, C. D., Dykstra, M. J., Roses, A. D., and Bartlett, R. J. (1992). An error indystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy. Genomics 13:llS-121. Sicinski, P., Geng, Y., Ryder-Cook, A. S., Barnard, E. A., Darlison, M. G., and Barnard, I? J. (1989). The molecular basis of muscular dystrophy in the mdx mouse: A point mutation. Science 244: 1578- 1580. Smith, B. E, Hoffman, R. K., Giger, U., and Wolfe, J. H. (1990). Genes transferred hy retrovirus vectors into normal and mutant myohlasts in primary cultures are expressed in myotubes. Mol. Cell. Hiol . 10: 3268-3 27 1. Somia, N. V., Zoppe, M., and Verma, 1. M. (1995). Generation of targetted retroviral vectors by using a single-chain Varidde fragments-an approach to in vivo delivery. Proc. Natl. Acad. Sci. USA 16:7570-7574. Stedman, H. H., Sweeney, H. L., Shrager, J. B., Maguire, H. C., Panettieri, R. A., Petrof, B., Naru-

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I

The Contribution of the Mouse to Advances in Human Genetics Elizabeth M. C. Fisher

Neurogenetics Unit and Department of Biochemistry and Molecular Genetics Imperial College School of Medicine at St. Mary’s London W2 lPG, United Kingdom

1. INTRODUCTION Research into all eukaryotic genetics benefits from multiorganism study. T h e analysis of different biological systems demands slightly different approaches and each system has different advantages and can provide different insights. As a result, we gain different types of information that can be cross-referenced and integrated to fill in our understanding of eukaryotic biology as a whole. Research into the genetics of any organism is only constrained by the genomic resources that are available. The Human Genome Project has exponentially increased these resources for the study of ourselves, and recent progress in the genomics of other organisms is ensuring that the mouse and other mammals are fast catching up. In consequence the mouse will have the genomic accessibility of human, combined with the advantages of research with the ideal mammal for studies of heritable traits. In addition, once a gene of interest has been isolated, the mouse also provides a system for making specific genetic changes and therefore helping our attempts to understand gene function and the relationships between genotype and phenotype, and mutation and disease. T h e mouse has already contributed much to advances in human genetics and will contribute considerably more as we go into the next millennium and realize the potential of this model mammal for functional analysis. Thus the two species, human and mouse, are continuing a close relationship that has already existed for thousands of years. Advances in Genetics, Vol. 35

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Research into mammalian genetics benefits from a multiorganism approach because different model systems confer different advantages and provide different biological information. Genetics research is only constrained by genomic resources and, for the mouse, these are rapidly becoming as good as human. Once a gene has been isolated, the mouse also provides a system for understanding gene function, how genotype results in phenotype, and how mutation causes disease.

II. HUMANS DISCOVER MOUSE GENETICS The history of mouse genetics can be traced back to an early oriental interest in mice with unusual coats or behaviors; we know that spotted mice were described over 3000 years ago in China (Ginsburg, 1992). Animals with rare coats or strange behaviors became collector’s items and, particularly in the Imperial courts of Japan, “fancy” mice became highly prized. Reports on the breeding of mice and the inheritance of coat color were published in Europe in the 18th century, and by the 19th century many fancy mice had been imported into Europe and the United States (Ginsburg, 1992). Mendel and Darwin were aware of these animals and, in fact, Mendel bred his own white and gray mice in his rooms (Ginsburg, 1992). However, the scientific study of mouse genetics really took shape at the beginning of the 20th century in the United States. Two of the major influences on the development of this new field were Dr. William Castle and Miss Abbie Lathrop (Morse, 1978). William Castle finished his Ph.D. in 1895 and in 1897 he took up a teaching post at Harvard (where he stayed until he retired). Shortly after his appointment, a series of classic papers was published that rediscovered Mendel’s research and profoundly influenced Castle’s thinking (Morse, 1978). In 1902, William Castle began studying inheritance in fancy mice and, during the next three decades, he trained an influential and productive founder population of mouse geneticists. Abbie Lathrop was born the year after William Castle, in 1868, and although she did not work in a university laboratory, she made a very large contribution to mouse genetics. Abbie Lathrop trained as a school teacher, but at the turn of the century she was forced to give up her career because she contracted pernicious anemia (from which she died of in 1918). As an alternative she tried poultry farming, which was unsuccessful and then she hit on the idea of selling mice as pets (Shimkin, 1975). She started her enterprise with just two animals: a pair of waltzing mice (Morse, 1978). Slowly the business took off and large orders for mice started to come in from laboratories and research institutes. As her mouse colony grew, Abbie Lathrop noticed that, like humans, her mice would occasionally develop tumors. She embarked on a series of collaborative studies with laboratories studying cancer and these led to an important set of papers analyz-

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ing the incidence of mammary tumors in different mouse families. These mouse families were the ancestors of many of the inbred strains that are studied all over the world today. Although the American laboratories are responsible for laying much of the foundations of mouse genetics, especially the inbred lines, some significant contributions were also made by European laboratories in the first half of this century. In 1902, CuCnot working in France started to publish a series of papers on the inheritance of coat color mutation in the mouse (and so applied Mendel’s laws to mammals), and in 1915 in the United Kingdom the two coat color mutations albino and pink-eyed dilution were identified by Haldane and colleagues as forming the first linkage group in the mouse (Cuenot, 1902, 1903, 1905; Haldane et al., 1915; Copeland et al., 1993; Dietrich et al., 1995; Silver, 1995). This was achieved only 2 years after genetic mapping had been described in Drosophila (Sturtevant, 1913; Dietrich et al., 1995). Another significant contributor to the field was Hans Gruneberg who fled Nazi Germany in the early 1930s and came to work at University College in London. Gruneberg characterized many of the important mutations we still study, particularly those with developmental defects (Gruneberg, 1943). The first mouse genetics establishment of any size was created in 1929 when Clarence Little, a student of William Castle, became the first director of the Jackson Laboratory in Maine (named after Roscoe B. Jackson, head of the Hudson Motor Car Company in Michigan and a major contributor to the funding of the laboratory). Almost 70 years later the Jackson Laboratory continues to be an international center for mouse genetics and has been joined by two other world class research facilities: Oak Ridge in Tennessee, founded in 1946, and Hanvell near Oxford, founded in 1947. As the end of the 20th century approaches, these three great institutes will be joined by a new European center at Monterotondo in Italy, which will probably be engaged in functional studies of mice with specific germ line mutations. The investment in this new institute indicates that as we go into the 21st century the mouse is becoming even more important to the study of human genetics and human biology. An early oriental interest in fancy mice led to early mouse genetics in the United States, including the setting up of inbred lines. Three major mouse genetics institutions exist currently: the Jackson Laboratory, Oak Ridge, and Harwell. A new European institute with a slightly different remit is being created at Monterotondo.

111. THE MOUSE IS A MODEL MAMMAL All vertebrates share a common ancestor and therefore have a common set of genes and common biological processes and pathways. Therefore, to understand

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human biology and pathology, we can study another mammal as a model to which we are very similar. The mouse is our model because-like other mammals-we have the same developmental and disease processes, and because-unlike most other mammals-we have been studying and breeding mice for thousands of years and mice thrive in the confines of the laboratory environment. Because all stages of development and all tissues are accessible in the mouse, by analyzing development in the mouse instead of in humans we can study an in vivo system in detail at well-defined time points with a variety of different controls. Many such studies would be impossible in humans and therefore large areas of mammalian biology would be closed off if we confined our investigations to ourselves. Almost everything we know of very early mammalian embryonic development comes from research carried out in the mouse. The very limited studies that have been carried out in humans suggest that similar pathways are operating in both species at similar developmental stages. This kind of research into the processes of normal development allows us to better understand human birth defects and developmental abnormalities. Similarly, in the mouse, all stages of disease and all tissues are accessible, with unaffected controls. For example, to understand the progressive neurological damage that results from certain degenerative disorders in humans, we need to study the complete course of disease pathophysiology. This is generally impossible in humans as patients usually present in the later stages of neurodegenerative injury and so the early progression is missed. In addition, human neurological tissue is generally inaccessible to investigation in vivo and tissue culture studies are no help because neurons are postmitotic cells and cannot be grown in culture. However, because the mouse is another mammal that succumbs to progressive neurological disease, mice can help us understand the pathogenesis of neurological and other disorders that may be impossible to study otherwise. In trying to understand how genes exert effects o n mammalian development and disease, the study of mouse genetics excellently complements human genetic research because our common set of genes gives similar instructions in both organisms. T h e DNA sequences are sufficiently related so that most human genes and proteins will function in a mouse, and regulation of gene expression is highly conserved. For studying genetic diseases, mice also have the same patterns of heritability as humans: single gene Mendelian traits, polygenic traits, imprinted traits, chromosomal disorders, and so on all occur and give rise to a n abnormal phenotype in mice as they d o in humans, thus allowing us to model the genotype, phenotype, and modes of inheritance of human genetic disease. In addition, for the diseases that are not necessarily heritable, but in which genes play a role, such as cancer or susceptibility to infectious disease, the mouse suffers the same types of illness as do humans (e.g., Vidal et al., 1993; Sands et al., 1995). Therefore, by making use of the accessibility of the mouse for genetic studies, we are also contributing to advances in human genetics because the information from each species can be cross-referenced.

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A. The mouse is an ideal mammal for genetic studies T h e ideal mammal for genetic studies would have a short gestation and weaning time, would reach sexual maturity soon after birth, and would produce multiple generations in 1 year. Each litter would reliably contain a large number of progeny. For pragmatic reasons this ideal organism would be sociable and not mind living with its fellows, small and not take up large amounts of space, inexpensive to maintain, and have relatively simple needs. Our ideal mammal would be robust and thrive in a laboratory environment with carefully regulated matings. T h e captive population would be maintained either as a genetically homogenous stock or, at the other extreme, as a stock with the highest possible genetic variation. T h e animal would have a similar genetic makeup as humans so that the same gene products would be involved in the same physiological and cell biological pathways. For medical research purposes our ideal mammal would succumb to the genetic diseases that occur in humans. T h e organism would also be accessible to different types of germ line manipulation so that new genetic mutations might be produced and analysis of gene function might be undertaken. Humans clearly do not fit any of these criteria, but mice fulfil all the requirements of our model mammal for genetic studies. Mice can breed from about 2 months old when they become sexually mature. As soon as a male has mated he can be moved o n to the next receptive female, and so one male could sire tens of litters. Gestation time is approximately 3 weeks and litter sizes are generally between 5 and 10 pups, although this varies greatly between different mouse strains. After the litter is born the male can stay in the cage with the female and pups without harming them, and after weaning brothers can be kept together without fighting, which is not the case with many mammals. Laboratory mice have a life span of up to 2 years and are relatively straightforward to keep and breed, providing they are cared for properly. Mice are of immense value in genetic studies because they thrive in situations of controlled mating. As a result, geneticists have been able to instigate a system of brother-sister mating for over 20 generations that has produced different populations of mice that are genetically identical within the population, but genetically divergent from other populations. These mice are members of the various inbred strains. Within a n inbred strain each mouse is homozygous at all loci and has the same set of alleles as all the other members of the inbred strain. Thus each inbred strain has characteristic genetically determined traits such as coat color or visual acuity or response to certain drugs. Intrastrain homogeneity and interstrain heterogeneity are extremely useful to geneticists and other researchers. For example, intrastrain homogeneity means that there will be n o different results due to differing genetic backgrounds of the mice providing any testing is carried out with members of the same inbred strain. Therefore differing test results are due to either a mutation (which can be mapped and cloned) or environmental factors. This can be helpful to our understanding of human health, e.g., in ather-

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osclerosis research to delineate the role of environment (dietary-saturated fat) and genetic factors (Paigen, 1995). Interstrain heterogeneity is useful for highlighting the effects of genetic background: if a number of inbred strains are assayed identically and give divergent results, these differences are due to genetic variance between the strains. The causal genes can then be mapped and isolated. For some genetic studies, such as linkage analysis, the maximum possible genetic variation is needed within a family in order to follow the segregation of alleles through the generations. Here the mouse is again the ideal mammal because not only can laboratory mice (Mus musculus) be mated to outbred M. musculus populations, they can also be mated to entirely different Mus species (Avner et al., 1988). In the wild, different species generally do not interbreed, usually because of geographical or behavioral barriers. However, in the laboratory, two mouse species such as M. musculus and M. spretus, which is a wild mouse from Spain and north Africa, can be crossed to produce hybrid animals with a very high level of genetic heterogeneity between their homologous chromosome pairs (Avner et al., 1988). This provides the greatest possible number of informative markers for linkage analysis to map traits or genes easily, and standard DNA marker assays that can be automated and multiplexed have been developed for such crosses (Copeland et al., 1993; Breen et al., 1994; Dietrich et al., 1995).

B. Mouse mutations Mutations allow us to learn about biological systems by showing us what happens when a genotype is varied, and by studying a mutation in any gene we garner more information about gene function and malfunction. Mutations in mouse genes that correspond to and model human mutations provide us with systems for investigating the pathology of human genetic diseases and their amelioration. Mice were first kept by humans because of their unusual coat colors or behavior and these spontaneously occurring mutations are well documented. However, the number of mouse mutations that arose spontaneously is relatively low (hundreds) compared to those seen in humans (thousands). Unless a mutation is visually striking, it may not be spotted in a mouse colony and it is thought that only approximately 1 gamete in 100,000 is likely to carry a detectable spontaneous mutation at any single locus (Silver, 1995). In studying the mouse as a biological and genetic model mammal, it became clear that more mutations would be useful by providing new insights into development and disease and by indicating the presence of previously unknown genes of importance for different aspects of mammalian biology. Some of the earliest studies in mutation of the mouse genome came out of experiments from the 1920s to understand the effect of radiation on reproduction and development (Silver, 1995). After World War 11, various programs were set up to understand the effects of radiation on humans using mice as a model

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mammal. These programs induced mutation at 20- to 100-fold more than the spontaneous rate and provided a new pool of mice with mutations (Green and Roderick, 1966; Rinchik, 1991; Silver, 1995). The progeny of mice exposed to high doses of X rays tend to have random chromosome deletions, duplications, translocations, or more complex rearrangements that can give rise to new phenotypes. For example, many of the reciprocal translocations found in the mouse have been produced from radiation experiments (Searle, 1989; Silver, 1995). Although radiation exposure is still being studied, more recent experiments to increase the availability of mouse mutants have used chemical treatment, which can produce higher rates of DNA change and can have different mutagenic effects than radiation. Many chemicals are mutagenic, but experiments have shown that N-ethyl-N-nitrosourea (ENU) and chlorambucil (CHL) are extremely effective for mutagenizing mouse male premeiotic and postmeiotic germ cells, respectively (Silver, 1995). ENU tends to induce point mutations whereas CHL tends to induce chromosome rearrangements (Russell et al., 1979; Rinchik et al., 1993). With either chemical, mutation rates with an average per locus frequency of greater than one per thousand have been reported (for further discussion see Silver, 1995). Mouse models of a variety of disorders have been produced from ENU and CHL mutagenesis, including phenylketonuria, X-linked muscular dystrophy, hemoglobinopathy, and polycystic kidney disease (Chapman et al. , 1989; McDonald et al., 1990; Jones and Peters, 1991; Flaherty et al., 1995). Other studies that have increased the pool of mouse mutants have, for example, employed viruses that integrate into the mouse genomic DNA and cause heritable changes (Soriano et al., 1987). In a proportion of all these experiments, animals are bred with DNA changes that give rise to phenotypes, some of which model human genetic diseases and other forms of human pathology. All mutagenesis protocols produce heritable changes, but the changes induced by radiation, chemicals, and viruses as described earlier are essentially, although not totally, random. The alterations cannot be targeted to genes or specific regions of interest using these methods, but are used to create phenotypes of interest so that we can learn about the biological pathways involved and uncover the effects of previously unknown genes. Since the 1980s it has been possible to produce specific and targeted changes in the DNA of a mouse; these transgenic approaches are discussed in detail in Section VII,A.

All mammals have common biological pathways, common genes, and a com-

mon ancestor. The mouse is a model mammal for study because it shares these attributes with other mammals and thrives in a laboratory environment. All stages of development and disease are accessible in the mouse for phenotypic analysis. The mouse is a model mammal for genetic studies because of short breeding time, large litter size, controlled matings, inbred strains, and interspecific crosses.

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The mouse germ line can be mutated to produce new mutants that allow us to study different biological systems and their genes.

IV. THE MOUSE HUMAN COMPARATIVE MAP: A GENETIC ROSETTA STONE We share a common biology with the mouse, we share common genes, and we have a comparative genome map. Therefore, if we map a gene in one species and define its function, we have a good idea of where that gene maps in the other species and what it does. This is important because any advance in the genetics of one species is therefore an advance in the genetics of the other species. A candidate gene in one species is a candidate for a mutated locus in an homologous position in the other species. Thus we can study the genetically tractable mouse to learn more about the human condition, and equally learn from human genetics about the mouse. Humans and mice diverged from a common ancestor approximately 80 million years ago. Over the course of evolution the ancestral genome rearranged to produce humans, who carry 23 pairs of nuclear chromosomes, and mice, who carry 20 pairs of nuclear chromosomes. Human chromosomes are metacentric, submetacentric, and acrocentric whereas mouse chromosomes are almost all thought to be acrocentric. Both species have maintained a very similar DNA content-approximately 3 X lo9 bp per haploid genome-and both species appear to have a very similar gene content, probably around 65,000-80,000 genes with a similar gene density (Fields et al., 1994; Collins, 1995). As far as we can tell, almost all human genes have their mouse equivalent, derived from the same ancestral sequence. The same gene derived from the same ancestor in each species is referred to as a homolog. There are a few examples of genes without homologs, i.e., genes that are present in one species and not in the other, but these appear to be the exceptions, e.g., the human RPS4Y gene has no equivalent on the mouse Y chromosome (Zinn et al., 1994). Also, none of the few unique sequences found on the short arms of the human acrocentric chromosomes, such as chromosome 21, has yet been found to have a homolog in the mouse. Over 1500 genes have been localized in both human and mouse and clearly there is a relationship in the gene maps of the two species. The remains of the ancestral genome are visible in the form of groups of genes that have not been separated by chromosomal rearrangements and are still clustered together in both organisms. By looking at the mapping positions of homologous genes in human and mouse, the comparative gene map has been produced. The terminology of the comparative map is well defined. A “conserved synteny” is “the syntenic association of two or more homologous genes in two separate species irrespective of gene

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order or interspersing of noncontiguous asyntenic segments between the two markers.” A “conserved segment” is “the syntenic association of two or more homologous genes in two separate species that are contiguous (not interrupted by differentchromosome segments) in both species.” A “conserved ordered segment” is “the demonstration that three or more homologous genes are chromosomally linked and in the same order in two separate species.” (Definitions are from the Human Genome Organisation Comparative Genomics Workshop, Fraser Island, Australia, 1995.) The density of the human and mouse gene maps is increasing dramatically, and as more homologs are mapped in both species the density of the comparative map increases concomitantly. Currently over 3500 genes have been mapped in the mouse and approximately 40% of these have been mapped in the human (Dietrich et al., 1995). The present comparative map shows approximately 113 blocks of synteny conservation (including stretches that cross centromeres), and these blocks cover approximately 85% of the mouse autosomal genome; the X chromosome map shows almost total synteny (Mouse Genome Database; Dietrich et al., 1995). Although the comparative map is incomplete, we can see that conserved linkage groups may extend over several cM, e.g., the conserved linkage group on mouse chromosome 11 that has homology to human chromosome 17 extends almost 50 cM in the mouse and crosses the human centromere (Figure 5.1) (Lossie et al., 1994). The comparative map can also provide data for another type of mapping: paralogy mapping (Rabin et al., 1986; Nadeau, 1991; Lundin, 1993; Katsanis et al., 1996). The mammalian genome is thought to have increased in size and complexity by tetraploidization and other processes, including sequence duplication and transposition (Ohno, 1970; Lundin, 1993), so multigene families of paralogous genes have been generated, prior to the divergence of human and mouse. Transcription map data for human and mouse genomes are allowing us to examine the evolutionary relationships between paralogous segments of the mammalian genome. Knowledge of these relationships may have practical application for gene isolation by the process of paralogy mapping, e.g., the finding of a region of triplication in human and mouse genomes led to the prediction of two more members of the PBX and NOTCH gene families in the human genome. By screening the appropriate regions of genomic DNA, this paralogy mapping prediction was verified and new sequences from the human PBX2 gene and the human NOTCH2 gene were isolated (Katsanis et al., 1996). The comparative map does not show in detail the sites of gametic imprinting in human and mouse, although this phenomenon is known to play a role in human diseases such as Prader Willi syndrome or Angelman syndrome (Buiting et al., 1995). However, from current mapping and gene expression data it appears that imprinted regions in one species have imprinted homologous regions

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Figure 5.1. Consensus linkage map of some of the genes on mouse chromosome 11. Distance from the centromere is marked in cM, mouse genes (named according to the Mouse Genome Database) are named on the right, and the position of their homologs in the human genome is shown on the left. Mouse chromosome 11 contains genes that map to at least six human chromosomes.

in the other species (Barlow, 1995; Williamson et al., 1995). There may be exceptions to this statement, but the exact boundaries of genome imprinting are not well defined and therefore a high resolution comparison cannot yet be made between mouse and human (Searle et d.,1994). However, defining the boundaries of imprinting in each organism will likely provide new insights into the molecular basis of imprinting. The comparative map shows us common genes in conserved linkage groups. The comparative map indicates where homologous genes and disease loci are

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likely to map. Therefore, knowledge of location and function may indicate if a gene in one species is a candidate for a disease locus in the other species.

V. MOUSE GENOMICS Any advances in human or mouse molecular genetics are subject to the available genomics resources. To create the various kinds of genome maps and, for example, to carry out positional cloning projects, both human and mouse geneticists are building up a full gamut of genomic and cDNA clones under the auspices of the Human and Mouse Genome Projects. When the Human Genome Project was being planned it was recognized that to study humans alone would he to miss an enormous amount of “value added” information that would come from comparisons with other model organisms (Collins, 1995). Various genome projects have been set up to sequence and provide resources for organisms as diverse as bacteria through to rat (Jacob et d . , 1995). However, because the mouse has been an important model system for so many human disorders and because it is the most genetically well-defined mammal other than ourselves, a priority of the Human Genome Project has been to produce high resolution genetic and physical maps of the mouse (Copeland et al., 1993). T h e bedrock of mouse genomics resources is the mouse genetic map. Because of the ease of genetic mapping in the mouse, this has usually been the method of choice for mouse mapping projects. Because interspecific crosses work so successfully, backcross DNA resources for genetic mapping have been available to the biomedical research community for some time. For example, a collaborative European project has bred a large M. musculus X M. spretus backcross with almost 1000 progeny, and DNAs from the cross are freely available for scientists to carry out genetic mapping studies (Breen et al., 1994). A large set of microsatellite markers has been mapped within this backcross and therefore any new sequence can be positioned relative to these. A number of signal transduction genes have been speedily localized in the mouse genome using DNAs from this resource (Yulug et al., 1993; Hernandez et al., 1994; Hoyle et al., 1994; Yulug et al.,1994), as have many other loci. A t least five other large interspecific backcrosses are available for gene mapping studies, including a backcross at the National Cancer Institute in Frederick, Maryland, and the Jackson Laboratory mapping panels (Copeland and Jenkins, 1991; Copeland et al., 1993). Further information regarding these resources is available at Whitehead Institute/MIT

http://www-genome.wi.-mit.edu/

Frederick (NCI)

http://www. inf0rmatics.j ax.org/

Jackson Laboratory

http://www. inf0rmatics.j ax.org/

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European Collaborative Interspecific Backcross

http://www.hgmp.mrc.ac.uk/

Seldin cross

http://www.informatics.j ax.org/

Kozak cross

emai1:[email protected]/

The integrated maps for the Whitehead/MIT and Frederick mapping data can be accessed through the Internet at http://www-genome.wi.-mit.edu/. Currently the mouse genetic map contains over 15,000 mapped markers (Mouse Genome Database; Dietrich et al. , 1995); this figure includes 700 mutant loci (greater than 900 mutations have been reported) and over 3500 cloned genes (Dietrich et al., 1995). Over 6500 simple sequence-length polymorphisms (SSLPs), such as microsatellites or CA repeats, are also present on the genetic map (Dietrich et al., 1996). Other markers, such as isozyme loci, viral insert sites, restriction landmark genome scanning, and interspersed repetitive sequences, are also positioned on the map (Dietrich et al., 1995). With a genetic haploid genome size of 1600 cM and 15,000 markers, this averages to a marker approximately 0.1 cM, which is an average equivalent of 1 per 200 kb. The genetic map forms the basis for the physical maps of the mouse genome by providing a scaffold of ordered markers. The current physical mapping resources are not as comprehensive as the genetic mapping resources, but this will soon be rectified by the Mouse Genome Mapping Project-notably by creating a yeast artificial chromosome (YAC) contig of the mouse genome and large banks of expressed sequence tags (ESTs). There are several large insert and small insert YAC libraries that are freely available for physical mapping. In total the entire mouse genome is present with an approximately 20.fold coverage from all the libraries combined (Chartier et al., 1992; Kusumi et al., 1993; Larin e t al., 1993). A n ordered approach is now being taken to assemble a complete YAC contig from SSLPs spaced every 450 kb through the genome, with YACs that have an average size of 800 kb (Dietrich et al., 1995). Other cloned resources such as bacterial artificial chromosomes (BACs) have been developed for physical mapping in the mouse, and cosmid and phage libraries are well established (Smoller et al., 1991; Pierce et al., 1992; loannou et al., 1994; Schalkwyk et al., 1995). The EST resources for the mouse are also being developed and this is where mouse has a considerable advantage over human for genetic investigation: because cDNA and ESTs can be produced from all tissues at all stages, all transcripts are accessible for use in gene cloning strategies. A proportion (approximately 12%) of human ESTs will amplify in mouse DNAs and therefore might be physically mapped in the mouse genome by polymerase chain reaction (PCR); most likely a larger number will cross-hybridize on Southern blots, and presum-

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ably vice versa-mouse sequences will amplify and hybridize to human DNA. Therefore, ESTs derived, for example, from early developmental stages in the mouse may be particularly valuable for mapping in human when the cloned human transcript is unavailable. The importance of the mouse and other EST efforts to human genetics has been acknowledged by the creation of a database, XREFdb, that is dedicated to “cross-referencing the genetics of model organisms with mammalian phenov types and accelerating the identification of genes mutated in human diseases.” This database indicates similar protein translations for all available eukaryotic genes and ESTs, establishes potential map positions, and cross-references these to mammalian phenotypes. The XREF database can be reached via the Internet at

http://www.ncbi.nlm.nih.gov/XREFdb/.

Currently the total mouse DNA sequence in Genbank is approximately 15Mb (mostly cDNA) of the 3000 Mb that exists (Dietrich et al., 1995). Com-

paring human and mouse sequence is a valuable tool for highlighting areas of functional interest in both coding regions and noncoding regions. For example, comparison of the human and mouse BRCAl gene that is mutated in forms of human breast cancer has indicated important conserved protein motifs (Abel et al., 1995; Sharan et al., 1995). In addition, comparative sequencing efforts clearly yield considerable information about regulatory sequences that may be obscure from sequencing the human genome only. For example, in a comparative sequencing study of nearly 100 kb of human and mouse T-cell receptor loci, many new regulatory regions became obvious by their conservation between the two species (Koop and Hood, 1994). However, sequencing is still an intensive activity and currently there is no concerted worldwide effort to sequence the mouse genome; it is possible that only particularly gene-dense regions of the mouse genome may be sequenced over any great distance. Another type of genomics resource, which is very well developed in the mouse, is the bioinformatics databases that are available on the Internet. The Mouse Genome Database lists, for example, different types of maps, mouse loci, PCR markers, and mammalian homologs and is extensively referenced. This site is accessed at the Jackson laboratories on http://www.informatics.jax.org/. A n entirely different type of resource, but again one that furthers our understanding of human biology, comes from a computerized database that illustrates gene expression patterns in the mouse. The “Gene Expression Information Resource” combines graphical displays with text and has three main components: a gene expression database that gives spatial and temporal information about transcripts from individual genes, an anatomy database that is both a dictionary and lists cell fates, and a three-dimensional atlas that is constructed from serial sections of embryos at different developmental stages. Further information, including the address of the start-up version, is available through the Internet at the Mouse Genome Database site using the previously given address.

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Mouse genomic resources are rapidly catching up with those for human to give us a complete YAC contig of the mouse genome and extensive EST maps. Mouse EST banks give us access to tissues and stages of development that are inaccessible in human. The informatics resources for the mouse genome cover genetic, physical, transcriptional, and other maps. The databases are comprehensive, are frequently updated, and are freely available over the Internet.

VI. THE CONTRIBUTION OF THE MOUSE TO ADVANCES IN UNDERSTANDING HUMAN GENETIC DISEASE A. Single gene disorders (recessive, dominant, sex linked) The power of mouse genetics for analyzing single gene traits and advancing human genetics has been demonstrated in many biological pathways. Two recent examples that resulted in the isolation of genes in mouse that have great clinical, commercial, and possibly social implications for the genetic control of obesity and body weight in humans are provided by the cloning of the leptin gene from the obese (ob) mouse, which suffers from an inherited form of obesity (Zhang et al., 1994), and the cloning of the tubby gene from mice that suffer from maturity onset obesity combined with other deficits (Coleman and Eicher, 1990; NobenTrauth et al., 1996). The cloning of any disease locus in a single gene disorder can be achieved by a variety of approaches. Currently we have three general routes to the “end game” of mutant gene identification in mammals: functional cloning, candidate gene cloning, and positional cloning/positional candidate cloning. The method of choice depends on the information we have about that locus, the availability of affected individuals, and the genomics resources that can be accessed. Some of these factors will be slightly different in mouse than in human and we can capitalize on these when designing cloning strategies. The different advantages of mouse and human are particularly apparent in the early stages of mutant gene identification and isolation projects: for functional cloning and candidate gene cloning the advantage of the mouse may be marginal depending on the disease, but for positional cloning the mouse is the system of choice. In the latter stages of these projects, the end game, gene identification protocols are essentially the same for human and mouse. Once the disease gene has been isolated, both functional and pathogenetic studies often move into the mouse because of our ability to manipulate the mouse germ line and because of the accessibility of mouse tissues, particularly those that cannot be cultured such as those containing postmitotic cells.

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1. Functional cloning of disease loci Functional cloning entails isolating an unknown disease gene solely on the basis of knowing what the defective protein product is likely to do. The protein is isolated and from this the gene sequence and the mutation are identified. A typical functional cloning project would start with purification of the protein, from which partial amino acid sequence can be derived. Degenerate oligonucleotides can be designed based on the amino acid sequence and likely codon usage, and these can be used to screen cDNA libraries and isolate the gene. Alternatively, for example, antibodies to the protein can be used for screening expression libraries to isolate the cDNA. A recent application of functional cloning that has led to the isolation of a gene of clinical and commercial importance in humans is the cloning of the leptin receptor in mice (Tartaglia et al., 1995). In this example the leptin protein was radiolabeled and a range of mammalian cell lines and tissues were assayed for leptin binding. Regions of the mouse brain, including the choroid plexus, were found to take up both the radiolabeled leptin and subsequently a leptin fusion protein. A cDNA expression library was constructed from choroid plexus and screened with the leptin fusion protein, resulting in the isolation of a leptin receptor cDNA (Tartaglia et al., 1995). It was then shown that the mouse diabetes mutation is due to a defective leptin receptor (Chen etal., 1996;Lee et al., 1996). The functional cloning strategy has led to disease gene isolation in both mouse and human; however, as the genomic resources of these and other organisms increase, this is likely to become a less used approach.

2. Candidate gene cloning of disease loci Candidate gene cloning again requires some knowledge of disease pathology. A known gene encoding a known protein that is thought likely to be defective in the system under study can be sequenced and assessed for mutation. One example with significance for human genetics is the isolation of the gene responsible for the retinaldegeneration (rd) mutation in the mouse (Boweset al., 1990).In mice that are homozygote for the rd mutation, the retinal rod photoreceptor cells begin degenerating approximately 8 days after birth. By 1 month old the animals have no photoreceptors. Before the rd mutation was cloned, cyclic GMP was known to be the key messenger mediating between photon absorption and neuronal signaling in mammals. The cCMP pathway between photon absorption and closure of the cGMP-gated ion channels that activate signaling involves four proteins: rhodopsin, transducin, a 48K rhodopsin-deactivating protein, and an enzyme, cCMP-phosphodiesterase. Studies of the first three proteins indicated no difference between wild-type and rd homozygous mice. However, in affected mice, cGMP-phosphodiesterase was found to have low activity and to be present in reduced concentrations compared to wild-type mice. Therefore, subunits of the

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tetrameric cGMP-phosphodiesterase complex were excellent candidates for the

rd mutation. Analysis of the p subunit of the enzyme showed a defective transcript and subsequently this was shown to be the cause of the rd phenotype. After this

investigation, mutations in the homologous gene in humans were shown to cause forms of hereditary retinitis pigmentosa, which is characterized by retinal degeneration (McLaughlin et al., 1993).

3. Positional cloning and positional candidate cloning of disease loci

Unlike the first two methods, positional cloning requires no information at all about gene function and depends entirely on finding linkage to the inherited trait, then creating a very high resolution genetic map followed by physical map of the region of interest. Finally this region is trawled for genes, e.g., by exon trapping, cDNA selection, or sequencing and “software trapping,” and suitable candidates are assessed for mutation. A gene will be a candidate depending on its expression pattern or potential function. An example of a positional cloning approach in the mouse has resulted in finding a new deafness locus that is mutated in the human population. Genetic deafness is surprisingly common in humans and affects approximately 1 in 2000 births, although large single families suitable for linkage analysis are uncommon (Kain et al., 1995). Many of these human genetic syndromes include primary abnormalities of the sensory neuroepithelia of the inner ear, as do several of the mutations that give rise to hearing impaired mice, suggesting that similar pathways might be affected in both species. The shaker-I mouse mutation has typical neuroepithelial-type cochlear defects and shaker- I homozygous animals show hyperactivity, head tossing, and circling that is thought to be due to vestibular dys. function (Gibson et al., 1995). To identify the shaker-1 (shl) gene, a positional cloning project was initiated by setting up an intraspecific backcross that generated over 1000 progeny segregating the shl mutation (Brown et al., 1992). The size of the cross allowed a high resolution definition of the region of interest and indicated that the shl gene likely mapped within 0.1 cM of the Omp gene on mouse chromosome 7. This is equivalent to approximately 200 kb if the average figure of 1 cM of mouse DNA equivalent to 1.8 Mb is taken into account. A YAC contig was started to span this region, and exon trapping was undertaken to search for genes in the contig. One of the exons that was isolated was used to screen a mouse inner-ear cDNA library, and a clone encoding a myosin-like protein was identified. This turned out to be myosin-type VII, a member of an unconventional family of myosin molecules (Gibson et al., 1995). Subsequent analysis of all seven available alleles at the shl locus proved that a mutation in this gene is responsible for the shl phenotype. This research directly advanced human genetics: the shl gene maps to a region of mouse chromosome 7 that has homology to the region of human chro-

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mosome 11 that includes the locus for Usher syndrome type 1. This is an autosoma1 recessive deafness syndrome, like shaker-1 ; however, it also includes a slow and progressive form of retinitis pigmentosa. Despite this difference in phenotype, mutations in the human myosin VII homolog are responsible for the Usher syndrome type-1 phenotype (Weil et al., 1995). Probably the only route to identifying the genes involved in most human or mouse genetic disease is by positional cloning because it is extremely difficult to work out the primary protein lesion in most disorders. Therefore, positional cloning has become a method of choice for disease gene identification. Currently this process is both time-consuming and labor-intensive; however, positional cloning projects are being greatly facilitated by the cDNA, EST, and informatics resources from the Human and Mouse Genome Projects-to such an extent that a new term, “positional candidate cloning,” has been used to describe the combination of fine mapping a disease locus then assaying cDNAs or ESTs that map to the critical region (Ballabio, 1993). This hybrid approach is likely to be the way in which most mutated loci are cloned in the future. Fine mapping will localize the mutated locus and then the EST databases from different tissues will provide candidate genes for the region. An example of a positional candidate cloning approach using mapped candidate sequences is provided again by the leptin receptor. Mutations in the mouse diabetes (db) gene give rise to a syndrome that resembles human morbid obesity (including the diabetes component) (Chen et al., 1996; Lee et al., 1996). The db locus was fine mapped on mouse chromosome 4 and the mutation was localized to a 300-kb interval that was cloned within two BACs. Exon-trapped and cDNA selected clones were isolated from the clone contig and were used to screen a cDNA library. cDNAs were isolated that turned out to have identity to the leptin receptor, which had just been isolated and mapped to the same region. The leptin receptor was pursued as a good candidate for the db mutation because it both mapped to the same site as db and functionally it seemed an appropriate protein for an obesity mutation phenotype (Tartaglia et d.,1995;Chen et al., 1996; Chua et al., 1996; Lee et al., 1996). Subsequently, abnormal splicing of the leptin receptor was found in db mice (Chen et al., 1996; Lee et al., 1996). Over 100 mutant loci have been cloned in the mouse and most of these have been identified by the positional candidate approach, indicating the power of combining high resolution mouse genetic maps with genomics resources, including cDNA and ESTs.

4. Advantage of the mouse for cloning single gene trait disease loci and assaying for genes

For functional cloning there is probably little to choose between mouse and human to optimize the process, except if, for example, expression libraries need to

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be made from a tissue that is inaccessible from human. Similarly, for candidate gene studies, mouse and human are probably equally appropriate systems if the necessary assays can be performed. Where the mouse has a large advantage over human is in positional cloning projects. These investigations are still labor resource intensive, but are probably the best route to identifying the sequences involved in much human genetic disease. However, identifying human disease genes in mouse depends on the presence of a scorable phenotype at the homologous mouse locus. The chief constraint in efficient positional cloning is the size of the critical region. If the critical region is too large, creating and physically mapping a YAC contig can be very time-consuming and a gene search will likely produce an unrealistically high number of candidate sequences to assay for mutation. By keeping the critical region as small as possible, we reduce the time needed to make the clone contig and we will have to assay a limited number of candidates cDNAs. Confirming that a candidate gene is the causal gene for the mutant phenotype can be labor-intensive and time-consuming so the fewer candidates the better. By analyzing the largest possible number of informative meioses, we can map the disease gene to the smallest possible critical region; the number of informative meioses depends on family size and variation between individuals. Most human single gene disorders are rare and generally only a few small affected families will be available. If families are pooled, we will still not have a large enough sample of meioses to make a direct measurement of genetic distance and we may also introduce problems with genetic heterogeneity. Even in a large extended family the resolution provided by the genetic analysis means that we have to deal with statistical formulae to get a probability of gene and marker order. The mouse is the ideal mammal for positional cloning projects because laboratory mice can produce several litters per year and the typical size of an interspecific cross in a positional cloning project can be over 1000progeny, which potentially limits the critical region to 0.1 cM or approximately 200 kb. Thus we can have a direct measure of recombination fraction and marker order to a high resolution, and the critical region may be contained with one or two large genomic clones. The mouse can also address specific difficulties that may be a problem in investigating particular human diseases. For example, if there is uncertainty about ascertainment, perhaps because of the genetic background effects that occur in many human diseases, then the homologous mouse mutation can be bred onto a n inbred strain and scored with certainty. Scoring one single mutation in a large backcross also overcomes difficulties with genetic heterogeneity. In addition, for the human diseases that are progressive and late onset, there may be no opportunity to collect multiple generation families if, for example, the disease state does not manifest until affected individuals are in their 50s and 60s. In the mouse it is possible to collect three generation progressive disease families within a year or two of starting a breeding project.

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The mouse has advantages in the latter stages of cloning projects when genes are being isolated from the critical region. Transgenesis can be used to check genomic regions for genes that will correct a recessive defect, thereby narrowing the region of interest for gene isolation; equally this technique can also indicate the presence of genes with dominant effects (Strauss et al., 1993; see Section VI1,A). A more widespread advantage of the mouse for gene isolation lies in protocols such as cDNA selection that rely on access to transcripts from a tissue of interest. Not all human tissues are available as fresh material, in large enough quantities, or as cell lines, and cell lines do not necessarily correspond to the in vivo state. However, all tissues at all stages are accessible in the mouse. At this end stage of disease gene identification, even if a mutation cloning project has taken place solely in human, it is still worth accessing the mouse genome because a candidate gene may be mapped to an homologous position in the mouse although it has not been mapped in human. The reverse is true for mouse gene cloning projects where the human transcript map can provide valuable comparative mapping information. A n example of this is provided by the identification of the mouse weaver gene that encodes a G protein coupled inwardly rectifying the potassium channel which gives rise to ataxia and cerebellar defects when mutated (Patil et al., 1995). The gene was first identified in a cDNA selection experiment on the weaver homologous stretch of DNA from human chromosome 21. When the mouse homolog was analyzed in mutant and control mice, it was found to have a missense mutation in weaver mice (Patil et d., 1995). The isolation of the shaker-] mouse mutation illustrates the power of mouse genetics in positional cloning projects because of the fine genetic mapping provided by large backcrosses and because of the accessibility of tissues and cDNAs that would be difficult to produce from human samples (Gibson et al., 1995; Weil et al., 1995).

5. Determining if the candidate gene is the causal gene The common point at the end of all gene cloning projects is verifying that the candidate gene is the causal gene responsible for a mutant phenotype. This is not necessarily a trivial task and, in the case of disease gene identification, a number of different approaches may be needed. DNA sequencing of the gene from expected heterozygous or homozygous affected individuals may reveal obvious differences compared to unaffected individuals. However, in an outbred population, many of these differences could be nonpathological protein polymorphisms. Therefore a statistically significant population must be sampled of affected and unaffected family members, along with members of the general population, to determine that the mutation segregates with the disease trait. Differences in penetrance and genetic heterogeneity can confound the analysis, and here inbred mouse lines have the advantage in that all

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affected individuals will carry the mutation; this will be the only DNA sequence difference between affected and unaffected animals, in which case the two lines are known as “co-isogenic.” Not all genetic disease is due to simple point mutations in the coding region of a gene, there are many other sites of mutation. If no differences in gene sequence are identified, then hybridization of gene probes to a Southern blot containing DNA from affected and unaffected individuals may indicate genomic differences that do not affect the coding region. Similarly, hybridization to a Northern blot will indicate changes in gene expression, e.g., in transcript size or abundance, and protein levels may also need to be checked in affected compared to wild-type individuals. The mouse is particularly helpful for gene expression studies because of the accessibility of both mutant and wild-type tissues. A very good indicator that the candidate gene is the causal gene is the presence of multiple alleles. Once a candidate gene is cloned, then this sequence can be investigated in different allelic mice, and if each is mutated then the candidate gene is almost certainly the correct gene. For example, the microphthalmia-associated transcription factor (MITF) was clearly shown to be responsible for the microphthalmia (mi) mutation when two independent transgene insertions into this gene gave mi phenotypes (Moore, 1995). For some diseases, very different phenotypes arise from mutations at the same locus, e.g., mutations in the RET tyrosine kinase gene in humans cause the neural crest disorder Hirschsprung’s disease and multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (Edery et al., 1994; Romeo et al., 1994; Bolino et al., 1995). In human i t is diffiicult to show that different diseases arise from the same gene, without cloning the gene. In mouse, because of the density of the genetic map and our ability to mate different mutant animals and look for complementation in the progeny, we can be fairly sure which phenotypes are allelic. Comparative analysis between human and mouse of homologous mutations is also strong evidence that the causal gene has been found as, for example, was the case with the myosin VII gene responsible for forms of human and mouse deafness, as discussed earlier (Gibson et al., 1995; Weil et al., 1995), or the Gli3 zinc finger containing gene that is responsible for the limb and craniofacial development disorder known as Grieg syndrome in humans and extra toes in mouse (Vortkamp et al., 1991; Schimmang et al., 1992; Hui and Joyner, 1993). Mutations in species other than the mouse and human may be useful in this regard, and the XREF database provides the type of comparative gene mapping-phenotype information that will be very helpful for future disease gene isolation. Finally, a rigorous test of a gene’s candidacy is to recreate the mutation on a wild-type background and see if this recreates the phenotype. If the phenotype can be assayed in tissue culture cells, then this can be done in vitro. If not,

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then the phenotype can be recreated in the whole animal through germ line manipulation. For dominant diseases we may see a phenotype in a heterozygous mouse. For recessive diseases we can breed a mouse that carries homozygous mutations and so manifests the phenotype. If a wild-type copy of the candidate gene is then manipulated into the genome, the recessive phenotype may be “rescued” back to the normal state, in which case this is a powerful indicator that we have the causal gene (e.g., see Tobler et al., 1996; see Section VII,A for further details on germ line manipulation).

B. Identifying genes Involved in chromosomal syndromes and other chromosomal aberrations Chromosomal aneuploidies are the most common cause of genetic abnormality in humans. The frequency of these “chromosomal syndromes” is 4 per 1000 live births, and 50% of all spontaneous abortions have chromosomal abnormalities. Despite chromosomal disorders making a large contribution to genetic disease, we have extreme difficulty in isolating the genes underlying these syndromes because we cannot use techniques such as linkage analysis or positional cloning. The problem in determining which sequences are responsible for chromosomal syndromes lies in identifying which normal genes give rise to a phenotype when their dosage is subtly changed from 2 to 3 (in the case of trisomies) or 2 to 1 (in the case of monosomy), The regulation of gene expression through transcriptional, posttranscriptional, or posttranslational control probably ensures that very few genes exert a pathological effect when present in aberrant copy number, although this is a difficult assertion to test. In general, we also have very little molecular data on the importance of gene dosage to the mammalian cell and the whole organism. A n example of where the study of the mouse may be helpful in assaying for genes involved in chromosomal syndromes, i.e., genes that exert a dosage effect, is provided by mice that are trisomic for regions that correspond to human chromosome 2 1. Trisomy 2 1, which results in Down syndrome (DS), is the most common aneuploidy in humans and is the most common genetic cause of mental retardation, with a frequency of 1 in 600 live births. Patients have specific abnormalities, such as hypotonia and mental retardation, and have variable features, such as cardiovascular defects, an increased risk for leukemia, and premature aging. Overall, the development of many tissues is affected. Although DS is usually caused by trisomy for the whole of chromosome 2 1, occasionally, in rare patients, DS results from trisomy of only part of the chromosome. This has allowed the definition of regions of chromosome 2 1 that probably contain dosage-sensitive genes that are responsible for proportions of the variance of different DS features (Delabar e t al., 1993; McCormick et al., 1993; Korenberg e t al., 1994). However, because the number of patients involved in defining these regions is relatively small

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(approximately 50)) limited human data are available. A mouse model would be useful in studying the effects of dosage of specific genes on different genetic backgrounds, particularly during development in organs such as the brain. A region of human chromosome 2 1 that is thought to contain dosagesensitive genes involved in many key features of DS has homology to a portion of mouse chromosome 16 and so a mouse that is trisomic for this region may exhibit features of DS. Therefore a first approach to generating a mouse model for DS was provided by studies of the trisomy 16 mouse, which can be produced by breeding strategies employing mice with chromosome 16 Robertsonian translocations (Epstein, 1986). However, this animal does not survive beyond birth and consequently the postnatal development of structures such as the brain cannot be studied (Epstein, 1986). In addition, mouse chromosome 16 is larger than human chromosome 21 and likely contains genes that give rise to a phenotype, but which do not map to human chromosome 21. Mouse chromosome 16 has stretches of synteny on at least human chromosomes 3, 16, and 22 (Davisson et al., 1990; Reeves et al., 1995). Instead of creating a whole chromosome trisomy model of Down syndrome, another approach has been to analyze partial chromosomal trisomy. The Ts65Dn mouse is trisomic for a small portion of mouse chromosome 16 that corresponds to a region of human chromosome 2 1 which likely contains dosage-sensitive genes involved in DS mental retardation and other features (Reeves et al., 1995). The mouse does not exhibit many of the components of DS (at least on the genetic backgrounds investigated), but it does exhibit certain behavioral deficits, including spontaneous locomotor hyperactivity and impaired learning and memory abilities (Reeves et al., 1995). Although currently we cannot extrapolate at all between mouse and human behavior, these studies show that three doses of specific genes in the trisomic region do result in a phenotype in mouse. Therefore we know that genes from this region are dosage sensitive in mouse, and through transfer experiments with individual genes we have a handle for dissecting the region and determining exactly which genes are of interest. These genes may play a role in human DS, but even if human and mouse biology is different in this respect, they will tell us more about gene dosage effects themselves. The gene transfer, or “transgenic,” approach to understanding chromosomal aneuploidy is to produce mice that carry an extra copy of specific regions of the chromosome or specific genes. The main difficulty of this approach lies in reproducing the subtle dosage differences found in the human chromosomal syndromes. However, transgenesis involving YACs, for example, allows genes or groups of genes to be transferred in one copy with all their regulatory sequences into the mouse genome. For example, to investigate DS, a panel of transgenic mice was constructed that in total contain a 2-Mb set of YAC and P1 clones from human chromosome 21 (Smith et al., 1995).The mouse panel has been described as an “invivo library” of human sequences and assaying for phenotypes in the pan-

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el will help determine a fine mapping position for dosage-sensitive genes. By making transgenic mice with smaller and smaller regions from individual YACs, human sequences which confer specific learning difficulties on the mice have been mapped to a region of less that 0.5 Mb that includes the mouse homolog of the Drosophila minibrain gene which is involved in the development of the nervous system (D. Smith, personal communication). Transgenesis involving single genes may provide information about gene dosage effects for individual genes from human chromosome 21. For example, the ETSZ protooncogene maps to human chromosome 2 1 and is overexpressed in human DS individuals. Ets2 is a transcription factor that is expressed in many cell types, including newly forming embryonic cartilage. A n Ets2 transgene has been transferred into a mouse and is expressed at levels of less than twofold overexpression (Sumarsono et al., 1996). This fairly subtle difference from wild type is sufficient to give rise to mice with shortened snouts, abnormally shaped heads, and other characteristics reminiscent of certain features of DS (Sumarsono et al., 1996). Therefore it is possible that some morphometric abnormalities seen in DS are due to overexpression of human ETSZ. New technology such as chromosome engineering (discussed in Section VII,A) will allow us to create specific chromosome aberrations, including deletions, duplications, and inversions, that will model human chromosomal syndromes in the mouse (Ramirez-Solis et al., 1995). Such technology is useful for understanding another type of chromosomal aberration that also results in disease: the somatic cell changes that can lead to tumorigenesis. A feature of many types of neoplasia is loss of heterozygosity; using chromosome engineering, large targeted deletions can now be produced, allowing us to model loss of heterozygosity for specific regions. For example, a 3- to 4-cM region of mouse chromosome 11 has been deleted that is syntenic with a portion of human chromosome 179,which contains the familial breast cancer gene, BRCAl , and possibly other tumor suppressor genes, and a mouse containing this deletion has been bred (Ramirez-Solis et al., 1995).

C. Polygenic disease: the most common human genetic cause of death The most clinically expensive, resource demanding, and biggest killers of humans are diseases such as heart disease or schizophrenia that have a genetic component which is provided by more than one gene. These polygenic disorders or complex traits are the bulk of human genetic disease and affect every system of the body and mind. Polygenic diseases are encoded by a number of loci and where these have measurable effects, for example, hypertension, they are known as quantitative trait loci (QTLs) (Weeks and Lathrop, 1995). These genetic traits are difficult to dissect in the human population because it is outbred which greatly limits the types of statistical analysis that can be performed on samples (Weeks and Lath-

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rop, 1995).Nonparametric approaches such as affected sib pair analysis may work well for some diseases, but these require very large numbers of affected individuals (Hugot et al., 1996; Ott, 1996).The numbers required grow exponentially but in inverse proportion to the phenotypic effect of the locus; for example, mapping a disease gene to within a region of approximately 1 cM requires a median of 700 sib pairs, if the disease allele causes a twofold increase in risk of being affected (Weeks and Lathrop, 1995). Realistically, genes with less effect can probably only be mapped to a region of 2-5 cM in most studies (Weeks and Lathrop, 1995). Environmental variation can also be extreme and can affect the analysis. In addition, there are often difficulties in ascertaining who is affected as multiple genes are involved and differences between individuals may be small. Another problem with ascertainment, particularly for psychiatric disorders, can be lack of agreed defined measurable characteristics that determine if an individual is classified as affected or not. The study of complex traits in mice started in 1903 with tumor transplantation studies (Klein, 1975; Frankel, 1995). There is often a great variability of phenotype between the different inbred strains, or mouse species, and this genetic divergence combined with the phenotypic extremes make the mouse a powerful system for analyzing polygenic traits. Controlled matings can be set up to isolate animals at the phenotypic extremes, and then their genotypes can be analyzed for the differences that give rise to the phenotype to establish a tight linkage and to define a critical region. It is only because of the phenotypic and genotypic variation in the mouse and the density of DNA markers in the mouse genome that polygenic disease studies have forged ahead in this animal. A n example of polygenic disease mapping using an interspecific cross between laboratory mice (M. musculus) and M. spretus is given in a study that found and mapped three different loci involved in determining if skin tumors are benign (M. spret u s ) or malignant ( M . musculus) (Nagase et al., 1995). A n intraspecific cross was analyzed for loci involved in epilepsy, which is the second most common human neurological lesion after stroke (Rise et al., 1991). Another important human disease, type I diabetes, has also been dissected through QTL analysis of an in+ traspecific mouse cross (Prins et al., 1991). The advantage of analyzing polygenic disease in the mouse is especially apparent for approaches in determining which are the causal genes. Once a critical region has been established, in human the next step is to assay for candidate genes over what may be physically a large region of interest. There may not be an obvious candidate gene or, far worse, there could be many obvious candidate genes, each requiring careful analysis of coding and regulatory sequences in affected compared to unaffected individuals. A t this stage, projects investigating mouse QTLs either could look for candidate genes or could breed congenic strains (Frankel, 1995). Congenics (another innovation provided by the Jackson Laboratory) are mouse strains in which all the genome is derived from one strain ex-

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One cross of strain

s m2

strain 1

Then several generations of breeding to strain 1 and selecting for the phenotype of strain 2 on the genotype of strain 1

)

B

phenoG of strain 2 is confimed to a small region on the chromosomes of strain 1

Figure 5.2. Breeding congenic strains. Mice from two different strains are crossed once. The progeny are bred back to strain 1 over several generations so that a congenic strain is produced that has all chromosome regions derived from strain 1, except the region encoding the trait from strain 2. A chromosome of the appropriate genotype is shown under each mouse.

cept the locus of interest, which is derived from another strain (Frankel, 1995). If the two strains are at the phenotypic extremes of the quantitative trait, for example, one likes alcohol the other hates alcohol, then the mice are bred according to a scheme which maximizes the amount of genotype of one strain, but always chooses animals that have the phenotype of the second strain. After several generations a mouse line is bred that has all chromosome regions derived from strain 1, except the region encoding the trait from strain 2 (Figure 5.2). Physical analysis of polymorphic markers within the congenic strain will reveal the outer limits of the region of interest and this can then be assayed as a critical region for candidate genes. In addition, knowledge of the origin of the strains under study and therefore their likely genotypes can be used to find and evaluate candidate genes (Frankel, 1995). Where the mouse has also been particularly helpful is in indicating the existence of modifier loci. These loci exert variable effects and are difficult to detect in outbred populations such as humans. In the mouse they can be obvious: if

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a trait is bred onto a different genetic background and as a result the phenotype alters, then there is probably a modifier locus (loci) that modulates the phenotype. One of the earliest examples of the identification of a modifier gene came from the mouse, through research into human familial colon cancer syndromes (Dietrich et al., 1993; MacPhee et al., 1995). Colon cancer is one of the most prevalent cancers in the Western world, and mutations of the adenomatous polyposis coli (APC) gene are known to cause both sporadic and familial forms of the disease, which is inherited as a dominant autosomal disorder. The APC gene is also mutated in the ArcMinmouse that has similar clinical features to human families with APC mutations. However, the phenotype of the APCMinmouse varies greatly depending on the genetic background carrying the mutation. QTL analysis of phenotypically extreme mice on different genetic backgrounds indicated a region of mouse chromosome 4 that accounted for approximately 50% of the genetic variation (Dietrich et al., 1993). A candidate gene encoding secretory phospholipase A2 (Pla2s) was mapped within this region and subsequently it was shown that tumor-susceptible strains carry a different allele of this gene than do resistant mouse strains (MacPhee et al., 1995). The human homolog of Ph2s maps to a region of the human genome that is associated with loss of heterozygosity in tumors. Polygenic disease studies in the mouse are directly relevant to the human condition. Although gene homologs may be responsible for different percentages of the phenotypic variation in each species, a candidate gene from the mouse is a candidate gene for human. Recently a modifier locus has been identified in the mouse that affects the severity of phenotype in animals that have been targeted at the cystic fibrosis transmembrane conductance regulator gene, and which therefore model aspects of cystic fibrosis (Rozmahel et al., 1996). Modifying loci for cystic fibrosis were suspected in humans, but proved very difficult to map because of genetic heterogeneity and small sample sizes. In the mouse a modifier locus has been mapped, and identification of the gene is likely to provide insights into the heterogeneity of symptoms seen in cystic fibrosis patients, into the pathology and possible treatments for the disease (Rozmahel et al., 1996).

D. Mice and gametic imprinting Gametic imprinted genes are differentially expressed depending on their parental origin (Barlow, 1995). Clues about the existence of imprinting originally came from the mouse, e.g., in breeding studies that resulted in aberrant development in mice with diploid chromosomal contributions from one parent only and none from the other, or in transgenic studies in which transgenes were expressed depending on which parent they were inherited from (DeLoia and Solter, 1990; Ferguson-Smith et al., 1991; Latham et al., 1995).Studies of human genetics then also highlighted the importance of imprinting in normal development and human

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disease. The expression of imprinted genes can vary spatially and temporarily, and full investigation of the transcription pattern of an imprinted human gene in all tissues at all stages would be extremely difficult. However, it seems likely that mostly the same genes are imprinted and play similar roles in embryogenesis and pathology in both human and mouse (Searle et al., 1994; Williamson etal., 1995). Therefore the mouse provides us with a system for studying the relationship of imprinted genes to development and disease in a model mammal. During normal embryogenesis in mouse the insulin-like growth factor 2 (IGF2) gene, for example, is transiently expressed from the paternal allele whereas the maternal allele is inactive. The converse is true for the IGF2 receptor, which is expressed from the maternal allele only during embryogenesis. Without access to early embryonic tissue from the mouse, this relationship between the IGF2 and the IGFZR genes could not be investigated. The importance of access to early embryonic tissues is also shown in experiments to determine the pattern of expression of another imprinted sequence at the Xist locus, which is expressed only from inactive X chromosomes (Kay et al., 1993, 1994). As X inactivation occurs very early in the embryo, studies of this gene in humans are limited and are therefore carried out in the mouse. A n example of genetic disease and imprinting is provided by the cyclindependent kinase inhibitor p57KIP2 that is located on human chromosome llp15.5 and the homologous position on distal mouse chromosome 7 and is imprinted in both species. This gene is a candidate tumor suppressor gene and may be responsible for the sporadic and familial cancers (associated with Beckwith-Wiedemann syndrome) that arise in humans from mutation in this genomic region (Hatada and Mukai, 1995). The effects of imprinting and disease can be seen at the chromosome level as well as the gene level, in both human and mouse. Uniparental disomy for whole chromosomes or chromosome regions can give rise to retardation in human growth and development (e.g., see Kotzot et al., 1995) and is modeled in mouse by breeding strategies using animals with Robertsonian or reciprocal translocations (Cattanach and Beechey, 1990; Ferguson-Smith et al., 1991).

E. Position effects, triplet repeat mutation, and other human genetic diseases In organisms such as Drosophila and mouse that are studied as genetic paradigms, many different phenomena associated with genes and their regulation have been described that have then been found in humans. One example is the discovery of X inactivation (lyonization) in the mouse which was then shown to occur in humans (Lyon, 1962). Another genetic phenomenon that is found in mouse and human but was first described in Drosophila is the position effect, in which the causal muta-

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tion can be as far as several hundred kilobases from the coding region of the gene involved. These mutations are extremely difficult to pin down and often only the existence of other alleles that give rise to a mutant phenotype will indicate which gene is involved. Therefore in these cases as many mutant alleles as possible are helpful. Position effects are increasingly documented in humans, e.g., in campomelic dysplasia, resulting from a position effect mutation affecting the SOX9 gene, and in mouse, e.g., from a mutation affecting the quaking locus (Bedell et al., 1996; Ebersole et al., 1996). Currently not all mutations found in humans have yet been seen in the mouse; at the time of writing there were no mouse mutations for mitochondria1 disease. However, this is likely to be due to a chance effect of not spotting such mutations in animal houses rather than their lack of occurrence or lack of phenotype in mice. Types of human genetic disease that have also not been found in mice but that have devastating effects in humans are the triplet repeat expansion diseases. These are a group of neurodegenerative disorders characterized by expansions in the number of repeating trinucleotide units (Ross, 1995). The diseases generally show anticipation between successive generations, and in those described so far, the length of the repeat correlates inversely with the age of onset and disease severity. One set of genes involved in the central nervous system degeneration diseases have expansions of a CAG trinucleotide, but because they share no other regions of homology than this repeat, it is thought that the diseases may arise from a common mechanism of pathogenesis (Ross, 1995). Because they are late onset neurological diseases, it is impossible to fully model their pathology in tissue culture systems and therefore the mouse is a very valuable system for investigation as the molecular mechanisms by which CAG repeat expansion leads to losses of different specific subsets of neurons in each disease are currently unknown. Attempts are being made to study triplet repeat mutations in the mouse, and the relationship of mouse and human biology to the etiology of these diseases needs to be elucidated. To model these dominant disorders of the central nervous system, researchers are creating transgenic mice that carry numbers of CAG repeats that would cause disease in humans. Spinocerebellar ataxia type 1 (SCA1) is an adult onset autosomal dominant disorder in which the expansion of an unstable CAG repeat in the coding region of the ataxin-I gene gives rise to the degeneration of cerebellar Purkinje cells, spinocerebellar tracts, and certain brain stem neurons. SCAl is generally an adult onset disorder characterized by ataxia, dysarthria, wasting, and neuropathy. The size of the repeat in unaffected alleles ranges from 6 to 40 units. Individuals who are affected by S C A l have been described with CAG repeats of 40 to 83 units (Banfi et al., 1994). The human and mouse SCAl genes are highly homologous, and the mouse gene contains 2 CAG units at the corresponding site to the human CAG repeats. The human SCAI gene was expressed in transgenic mice as either the normal gene or containing an

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expanded CAG tract of 82 triplets. Mice with a nonexpanded transgene showed no pathology. Those with the expanded gene developed ataxia and Purkinje cell degeneration, indicating that although CAG repeat expansion was not seen in any of the transgenic animals, the pathology of progressive specific neuron degeneration is reproducible in mice with a human adult onset disease transgene. The mouse model of S C A l is likely to provide insights into the other CAG triplet repeat expansion diseases because they probably share a common mechanism of pathogenesis ( Burright et al., 1995).

F. Advancing research into human disease involving genes With the advent of DNA-based diagnostic techniques such as Southern blotting and the polymerase chain reaction, we can detect quite subtle differences between the genetic makeup of individuals. The more these techniques are used, the more prevalent unusual cases turn out to be, and these human disease states can also be modeled in the mouse. Taking the example of mosaicism, somatic mosaicism may have clinical relevance in the live born individual; germ line and placental mosaicism can be a serious problem for prenatal diagnosis. The mouse can model and provide insight into many aspects of human mosaicism. For example, chromosome mosaicism is sometimes confined to only part of the conceptus, and the reason for this is unknown. Tetraploid-diploid chimeric mouse embryos can be produced and the cell line contribution to the different lineages of the embryo and extraembryonic tissues can then be studied to determine cell lineage preference and ultimately to provide more accurate prenatal diagnosis of chromosomal abnormalities (James and West, 1994). Another different example of human defects that involve genes is provided by triploidy and tetraploidy which combined may account for up to 20% of all human spontaneous abortions with a numerical chromosomal defect. Despite this significant contribution to pregnancy loss, little is known about the early embryogenesis and phenotypic features of this phenomenon. Mouse models of early postimplantation stages of development of triploid and tetraploid embryos allow us to examine the relationship between genotype and developmental potential (Kaufman, 1991). For disease gene identification the mouse can provide high resolution genetic maps to increase the speed of positional cloning; inbred strains to make phenotypic scoring reliable; access to all tissues for cDNA for candidate gene identification; and comparative mapping that may indicate good candidates in an homologous mapping position in another species. Advantages of mouse for confirming a candidate gene is causal are comparing candidate gene sequences for multiple alleles and in affected and unaffected

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individuals on inbred line backgrounds; and germ line manipulation, e.g., phenotypic rescue or creating homologous mutations (see Section VILA). In studying chromosomal syndromes the mouse allows us to create subtle gene dosage effects for individual genes and thereby assay for genes involved in chromosomal disease, including loss of heterozygosity in neoplasia. Advantages of mouse for understanding quantitative trait loci and modifying loci: the contribution and map position of these genes are very difficult to investigate on noninbred background such as humans, but are relatively straightforward to analyze in the mouse. Congenic strains then allow us to determine small critical regions in which to assay for candidate genes. The mouse can model other forms of human disease involving genes for advancing our understanding of how these disorders arise and how to treat them.

VII. MODELING HUMAN DISORDERS IN THE MOUSE Right from the first studies in mammalian genetics earlier this century, the mouse has provided a parallel system for human genetic disease research; for example, Abbie Lathrop’s studies on the susceptibility of mice to different tumors indicated the involvement of heritable factors in cancer. Since then, the mouse has provided important models of human diseases, including diabetes, neurological disorders, birth defects, and many other anomalies (Copeland et al., 1993). These models are often vital because cell culture systems, which can be useful for much research, cannot mimic the whole animal, e.g., when developmental processes need to be investigated, when the systemic effects of new drugs need to be elucidated, or when new behavior or activities need to be observed. Therefore, once a disease gene is cloned and a genetics project metamorphoses into a cell biology and physiology project, then many investigations of genetic disease move into the mouse, whether or not they started in humans. Recently, mouse models have also become particularly important for testing strategies for gene therapy. The mouse models of cystic fibrosis have been a valuable asset in testing approaches to vector design and delivery (Alton et al., 1993; Grubb et al., 1994; Zhou et al., 1994) and a mouse model of hereditary tyrosinemia type I has demonstrated a potentially successful pathway for human gene therapy by retroviral gene transfer into hepatocytes (Overturf e t al., 1996). Mouse models for human disease may exist in the current population of mutants, which includes those occurring naturally and those produced from mutagenesis experiments. All these mutants are catalogued and described in the Mouse Genome Database. However, many more human genetic diseases exist than mouse mutants, but as soon as the human disease gene has been isolated, we

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can manipulate the mouse germ line to create specific stably inherited mutations in the homologs of human disease genes.

A. Mouse germ line manipulation A revolution in the study of mammalian genetics came in the early 1980s with experiments designed to produce specific heritable change in the DNA of the

mouse germ line. Therefore the results of specific mutations in specific genes could be studied in vivo to model human disease, to test if certain mutations are disease causing, or to try and dissect the normal role of these genes in the living organism. The first rounds of manipulation led to the production of what are referred to as “transgenic” mice. A very few years later new types of transgenics, the “gene targeted” (or “knockout”) animals, were produced. The mid-1990s has shown another wave of new techniques. These include methods for modeling chromosomal disorders in the mouse and methods designed to produce specific changes not only in the mouse germ line, but also in subsets of somatic cells. All these techniques are being used to further our understanding of human genetics by allowing us to create mouse models of human disease and to functionally dissect different genetic domains, thus enabling us to study the often obscure connections between genotype and phenotype and between mutation and disease. In addition, these methods are the basis of DNA transfer and manipulation in the somatic cells of humans with genetic diseases-in other words, human gene therapy. There are now so many transgenic and targeted animals that a database, Tbase, has been set up to provide information about genotype and phenotype. This can be accessed at http://www.gdb.org/Dan/tbase/.

1. Transgenic mice Transgenic mice are animals whose genome contains foreign DNA, usually extra copies of a mouse or human gene. The most common route for producing a transgenic mouse is to inject the foreign DNA into the pronucleus of a fertilized mouse egg. Injected eggs are placed into a “foster” mother where they develop to term and pups are born carrying the extra foreign DNA. This DNA has stably integrated at an essentially random site in a mouse chromosome. When the transgenic mouse mates and produces progeny, the transgene is inherited in the same way as any other chromosomal DNA and a line of transgenic mice can be bred that carry the extra DNA (Figure 5.3) (Hogan et d., 1994). Insertion of a transgene has two possible outcomes. In the majority of cases the insertion results in a new phenotype because of gene expression from the transgene rather than any disturbance at the site of insertion. The transgene exerts a dominant effect and therefore transgenic animal may model dominant hu-

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Figure 5.3. Creating transgenic mice. The DNA construct to be injected is linearized and purified. The DNA is then injected through a microneedle into the pronucleus of a fertilized mouse egg. The transgene integrates stably and randomly into one chromosome, as indicated by the black bar on one of the two homologous chromosomes shown. Putative transgenic embryos are placed back into the uterus of a foster mother, where they develop to term. Transgenic mice are born that are heterozygotes for the transgene.

man genetic traits. In a small proportion of cases the transgene insertion will occur at a site that disrupts endogenous gene function and therefore gives rise to a new mutation. If a phenotype results from this mutation it may be inherited as a dominant or recessive trait depending on the biology of the disrupted gene. For example, an insertional mutation caused by a transgene interrupting sequences on mouse chromosome 18 resulted in a mouse with vestibular and craniofacial abnormalities in the heterozygote and prenatal death in the homozygote (Ttng, 1994). Transgenes are often present in multiple copies because of the mechanisms by which foreign DNA integrates into a host chromosome. As transgenic mice have their own genome intact plus extra copies of a foreign gene, the mice may have multiple “doses” of the gene product of interest. Sometimes the phenotype or clinical presentation of the mouse varies with the number of doses of the transgene and in accordance with how much protein is produced from the transgene. For example, mice that carry the human apolipoprotein B gene have

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plasma concentrations of human apoB that are related to gene copy number. Those with the highest transgene copy numbers have the greatest LDL cholesterol amounts (Callow et al., 1994). Similarly, transgenic mice that express mutated forms of human Cu, Zn superoxide dismutase (SOD1) that cause amyotrophic lateral sclerosis in humans have a more severe phenotype of motor neuron degeneration depending on the copy number of the transgene (Gurney et

al., 1994).

The DNA that is injected into the fertilized mouse egg may be manipulated so that the transgene need not necessarily contain its own regulatory and noncoding sequences. It may be driven by the promoter from another gene or it may be a cDNA sequence that consists of just coding sequences without the introns. Minigene constructs consisting of the coding region and the first one or two introns (which may contain important regulatory sequences) can be injected if the gene spans a large genomic region that is difficult to handle as one single cloned DNA fragment. However, because new technologies are allowing very large pieces of DNA to be cloned, the complete genomic sequence of big genes or adjacent groups of genes may be put into the mouse germ line. The vectors that carry large pieces of genomic DNA include cosmids, yeast artificial chromosomes (Lamb and Gearhart, 1995), and P1 phagemid vectors (Callow et al., 1994). Bacterial artificial chromosomes and mammalian artificial chromosomes (MACs) have also been developed (Brown, 1992; Schalkwyk et al., 1995). YACs have been introduced into the mouse germ line by a variety of methods, including pronuclear injection, lipofection, and sphereoplast fusion (Lamb and Gearhart, 1995). These technologies allow us to produce mice carrying extra copies of unusually large genes, such as the amyloid plaque protein precursor gene (APP) (Lamb et al., 1993; Pearson and Choi, 1993), and therefore to model defects in large genes or to biologically assay the roles of large genes. In addition to modeling specific diseases, transgenic mice also allow the dissection of gene expression by allowing us to “cut and paste” potential regulatory sequences and examining their effect on transcription. Whole animal systems indicate subtleties of expression that cannot be studied in tissue culture; for example, the regulatory sequences required for expression of the Glut4 glucose transporter gene have been partially mapped by experiments in both an adipocyte cell line and a transgenic mice (Ezaki et al., 1993). In addition, such “cut and paste” experiments may indicate which portions of a protein are nonessential for certain functions. This information may have a direct bearing on human disease. For example, dystrophin is a huge gene that spans 2 Mb and is mutated in Duchenne and Becker muscular dystrophy in humans. This gene has a very complicated pattern of splicing and expression, nevertheless a dystrophin minigene that encodes a truncated protein was able to protect muscle fibers against degeneration in mdx mutant mice that lack dystrophin protein (Vincent et al. , 1993). These sorts of results are important for gene therapy strategies, for example, indicating that it

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may not always be necessary to correct the human defect by replacing the whole gene.

2. Transgenesis: Gene targeted or “knock out” mice Although the transgenesis described earlier allows the addition of extra genes to the mouse genome and the modeling of human dominant disorders, most human diseases are recessive and are caused by relatively subtle changes in genes, such as the alteration of 1 bp in a region coding for a particular protein (Wilkie, 1994). A single base pair mutation can have a variety of effects ranging from changing the properties of the protein to stopping its production altogether. Therefore to truly model human disease and to understand the precise mechanisms of protein function in uiuo, researchers needed to target and create specific mutations in single genes of interest out of the approximately 70,000 genes in the mouse genome. This feat became possible in the late 1980sby the development of embryonic stem (ES) cells. Some of the first examples of the creation of animal models by mutation in specific genes come from gene targeting experiments that produced mouse models of cystic fibrosis, which is the most common autosomal recessive disease of Caucasians and for which no model had previously existed (Dorin et al., 1992; Snouwaert e t al., 1992; O”ea1 et al., 1993). ES cells are derived from very early mouse embryos, they are pluripotential and therefore have the capacity to form any kind of tissue including eggs and sperm, and they can be grown indefinitely in tissue culture. To make a mutation in a gene of interest-”gene targeting”-scientists use a combination of molecular genetic and tissue culture techniques to alter one of the two endogenous loci in mouse ES cells. The mutant cell line is then manipulated to form part of a mouse embryo, which develops to term in a foster mother. A live mouse is born that can then be bred to produce a strain of animals who carry the mutation (Hogan et al., 1994). There is more than one route to gene targeting; a widely used method typically starts with the construction of a DNA clone that contains genomic sequences from the gene of interest along with some drug resistance sequences. The DNA construct is transfected into ES cells growing in culture medium that includes a selective drug (usually an antibiotic) that kills normal ES cells. Because the construct contains a gene for selection (usually an antibiotic resistance gene), then ES cells that have stably incorporated the DNA construct can be positively selected and the rest of the cells will die. The surviving ES cells carry the DNA clone as a sequence that has integrated into the chromosomal DNA. However, in the vast majority of cases, this whole sequence has integrated randomly and is no use to the researcher because the specific gene of interest has not been targeted. In a very small (this can be as few as one in lo7 cells or less) number of cells the DNA construct will have integrated at its cognate sequence in the gene of inter-

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est. This type of integration, the “swapping” of two almost identical pieces of DNA, is known as “homologousrecombination” and is a rare event in mammalian cells. When it happens a second selective gene that lies at one end of the construct is swapped out of the integrating DNA sequence because the ends of the DNA construct exchange almost perfectly with their cognate endogenous sequences. As a result the rare ES cell lines containing construct DNA that has integrated by homologous recombination do not contain this second selective gene. The majority of the ES cells, which contain the randomly integrated DNA construct, do contain the second selective gene. The next step in the gene targeting process is to select for the minority of homologous recombinants; these are the targeted clones. Selection is achieved by applying a second drug that kills cells containing the second selective gene and allows only the population of homologous recombinant ES cells to survive. This selection process is known as “positive-negative’’ selection (Figure 5.4) (Mansour et al., 1988). The process of selection of ES cells in tissue culture is a very important stage of the protocol. Because homologous recombination is a rare event in mammalian cells, the whole gene targeting protocol can often be very inefficient. Therefore, after introducing the DNA construct into the cells, millions of individual ES cells are selected for the very few that contain the correct gene targeting event. DNA is made from individual clonal populations of the cells and is assayed by techniques including amplification in polymerase chain reactions and Southern blotting to check that one of the homologous pair of genes of interest does contain the desired mutation. Therefore the cell line is heterozygous for the mutation. Once an ES cell line has been produced in which the gene of interest has been correctly targeted, this ES cell line is injected into mouse blastocysts that are then placed back in a female mouse. Usually the ES cell line and the blastocyst will have come from different colored mice, e.g., the targeted cell line will have been derived from a white mouse line and the blastocyst line will have come from a black mouse. The ES,cells and the host blastocyst cells develop normally, and pups that are born are chimeras, made up of both cell lines (Figure 5.5). The extent of chimerism is easily visible because the mice have patches of black and white fur. The pups are crossed and in some chimeric mice the gonads will have been formed by the targeted cells, in which case these cells will form the germ cells. Therefore the egg and sperm that contribute to future generations will be heterozygous for the targeted gene. As with any other heterozygous mutation, the offspring have a 50% chance of inheriting the mutant allele. Thus a specific heritable change has been introduced in a gene of interest and gene targeted mice can be bred to produce homozygous animals in which both alleles of the gene are mutated and which may model human recessive disease. Many gene targeting experiments are designed to stop production of protein by the gene of interest so the gene function is “knocked out” and a strain of knockout mice is produced (Smithies, 1993; Hogan et al., 1994).

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DNA construct: contains (i) sequences from the region being targeted ‘genomicDNA;(ii) a drug resistance gene; (iii) a drug sensitivity gene.

Gene targeting construct is transfected into mouse embryonic stem cells (ES cells) Drug 1 is applied. Only those ES cells that have stably incorporated the construct survive. These cells a~ a mix of non-homologous and homologous recombinants.

genomic DNA

Non-homologous recombinants are ES cells that contain the DNA construct which has incorporated randomly, as shown here. When Drug 2 is applied, the cells are therefore sensitive to the drug, and they die. genomic DNA

flanking eenomic DNA

A

(

1

genomic DNA

endogenousgenomic target DNA

1

A

flanking eenomic DNA

Homologous recombination

Homologous recombinants are ES cells that contain the DNA construct which has recombined exactly at its cognate sequences in the genome. Therefore the non-homologous ends of the construct containing the ‘drug sensitivity gene’ are lost. The new genomic DNA includes the ‘drugresistance gene‘; this can interrupt the function of the target gene. Figure 5.4. Creating homologous recombinant mouse embryonic stem cells using “positive-negative” selection.

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Homologous recombinant ES cells containing one wild type and one targeted copy of the locus under study.

Es cells are injected into

a host blastocyst from a different mouse strain

The chimaeric blastocyst is placed in a foster mother, and the mice deveop to term Mice are born that are chimaeras of the gene targeted ES cell line and the host blastocyst cell line chimaeric mice are crossed; the gonads contain cells derived from the gene targeted ES line and the blastocyst line. Animals produced from ES line derived germ cells will contain the targeted gene. Animals produced from blastocyst derived germ cells will be wild type.

Figure 5.5. Creating gene targeted mice from homologous recombinant mouse embryonic stem cells.

3. Recent innovations in targeting DNA sequences Gene targeting is poised to become considerably more flexible with three recent innovations: “knockin” mutations, “conditional” mutations, and “chromosome engineering.” Knockin mice are being produced in which the gene of interest is targeted and either additional DNA, such as that encoding a new protein domain, is added into the gene or an endogenous domain may be exchanged for a different exogenous domain. Knockin mice will help in understanding the biological roles of, for example, closely related proteins in which subtle changes of related DNA domains may have large changes in protein function. Conditional mutations are being produced in which the gene of interest

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can be mutated in specific tissues only and remains wild type in all other tissues, for example, the gene of interest might be ablated in a subset of motor neurons only, and be producing protein as usual in all other tissues in the mouse. Conditional mutants are produced by a number of methods, often based on inducing promoters that are responsive to heavy metals or steroids, for example (for a review see Spencer, 1996).A recent innovation in producing conditional mutations in mouse uses standard gene targeting techniques to place two 34-bp pieces of DNA with a particular sequence known as the IoxP sequence on either side of the gene that is being mutated. Therefore a mouse is produced that contains loxP sites flanking the gene of interest. O n their own the presence of the loxP sites will not alter the DNA in any way or give rise to any phenotype. To create the conditional mutation the IoxP bearing mouse is bred to a mouse that carries a transgene which produces the bacteriophage enzyme Cre recombinase. Progeny mice that carry the IoxP sites without the Cre recombinase transgene, or the transgene without the loxP sites, are phenotypically normal (except in cases where the extra DNA resulted in an insertion mutation). A proportion of the progeny mice will carry both the IoxP sites and the Cre recombinase transgene and these are the conditional mutant mice. The Cre recombinase enzyme that is produced in these mice specifically recognizes the loxP sites and catalyzes recombination between the sites. As a consequence the DNA that lies between the loxP sites is deleted and so the gene of interest is disabled or ablated (Figure 5.6). The key to the tissue specificity of this Cre-IoxP system lies in which promoter drives the recombinase because DNA excision will only happen between the loxP sites in the presence of the Cre recombinase enzyme. Therefore, for example, a promoter that only allowed Cre recombinase expression in hepatocytes would result in the DNA between the loxP sites being excised in hepatocytes only and remaining unaffected in other cells. By using the Cre-loxP system we can subtly alter gene expression in subsets of cells in the whole animal to learn about the biology of the gene in which we are interested. An example of the use of the Cre-bxP system is provided by the T-cellspecific deletion of a portion of the DNA polymerase p gene (Gu et al., 1994). This shows the tissue-specific targeting of a ubiquitously expressed gene. This system will probably be very helpful in modeling diseases in which widely expressed genes cause tissue-specific pathology, such as mutations in the ubiquitously expressed SOD1 gene that affect motor neurons only (Deng et al., 1993; Rosen et al., 1993), and in modeling diseases such as cancer or possibly late onset sporadic (rather than familial) neurodegenerative diseases that arise from somatic mutation and somatic mosaicism (Lakso et al., 1992). Targeting the disruption of ubiquitously expressed genes in specific tissues only will help our understanding of why pathology is apparently confined to a few tissues even though the gene product appears to be necessary in all tissues. The Cre-loxP system is versatile, and the way the DNA between the loxP

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Transgenic mouse carrying for example, an ear specific promotor driving a Cre recornbinase gene

I

Gene targeted mouse in which a gene of interest is flanked by two loxP sites in direct repeats

I

These two mice are crossed ear specific urnmotor Cre recombinase gene loxp targetgene loxp

1 -

-t

A proportion of the progeny will carry the Cre recombinase transgene (inherited from parent 1) and the target gene flanked by loxP sites (inherited from parent 2).

Y

In these mice, the Cre recombinase will be activated in the ear cells only, and will delete the target gene in these cells only. Figure 5.6. Conditional mutation using the Cre recombinase-IoxP system. Transgenic mice bearing a Cre recombinase gene that is driven by a tissue-specific promoter are bred to gene targeted animals that have a normal wildtype copy of the gene of interest, flanked by IoxP sites. A proportion of the progeny of these mice will have both the Cre transgene and the loxP flanked target gene. At the appropriate time and in the appropriate tissue only, the promoter will activate the Cre recombinase which then deletes the sequences between the two loxP sites. Thus tissue-specific gene ablation has occurred.

sites behaves in the presence of the Cre recombinase depends on the orientation of the sites. “Chromosome engineering” makes use of the versatility of the Cre-loxP system to make targeted deletions, inversions, duplications, and translocations, and these rearrangements can span several megabases of DNA (RamirezSolis e t al., 1995; Van Deursen et al., 1995). In the first use of the Cre-loxP system for chromosome engineering in mice, loxP sites were targeted in ES cells to the ends of a large interval of interest (Ramirez-Soliset al., 1995).A Cre recombinase transgene with a promoter that is active in ES cells was transfected into the loxP containing cells. The Cre recombinase was expressed, the enzyme recognized the loxP sites, and the resulting cell lines contained a chromosome with specific deletions, inversions, or duplications that extended up to 3-4 cM (probably up to approximately 7 Mb). These ES cell lines were injected into blastocysts, and

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chimeric mice were born that have been bred to produce lines of animals with defined chromosomal aberrations. These mice and this technology are particularly important for furthering our understanding of a large section of human genetic disease: the chromosomal anomalies, which are responsible for much fetal loss and for other diseases ranging from the chromosomal aneuploidy syndromes such as Down syndrome through to cancers arising from translocations and internal chromosomal deletions.

B. Mice are not human With all mouse models of human disease we need to remember that mice are not

human. Their biology is different in many aspects and their response to stimuli, including drugs, may be different from that of primates. Exact gene function may differ between us, and even an exactly homologous mutation will not give rise to an exactly identical disease. However, while the rate-limiting step or biological significance of a particular pathway may be different in mouse compared to hue man, in almost all cases the same gene products will be involved in the same pathways, therefore the mouse can serve as a helpful model for human disease by ale lowing us to experiment with, and thereby understand, fundamental biological pathways and disease processes. These pathways may be sensitive to mutation at different points in each organism, depending on subtle differences in enzyme activities or protein-binding affinities,for example, but the basic biology is the same. In other words, mouse models allow us to dissect mechanism and but not outcome. An example of the differences in mouse phenotypes from those intended by gene targeting experiments is provided by the mouse models of human TaySachs and Sandhoff diseases (Cohen-Tannoudji et al., 1995; Sango et al., 1995; Phaneuf et al., 1996). This example also illustrates that genotypic but nonphenotypic models can still be extremely helpful in broadening our understanding of the disease process and how to treat it. These diseases are both autosomal recessive lysosomal storage disorders, resulting from an inability to break down G,, ganglioside. Tay-Sachs disease is due to mutations in the H E X A gene that encodes the ci subunit of P-hexosaminidase, the enzyme which catabolyzes G,, ganglioside. Sandhoff disease arises from mutations in the HEXB gene that encodes the P subunit of the enzyme. Tay-Sachs disease and Sandhoff disease present a similar clinical picture of severe early onset neurodegeneration and early death; Sandhoff disease also includes a visceral pathology. The Hexa and Hexb genes were both targeted in ES cells, and homozygous mutant mice were produced to model both diseases (Cohen-Tannoudji et d., 1995; Sango et d., 1995; Phaneuf et al., 1996). Both types of mouse accumulate G,, ganglioside in the brain, as expected. However, the Hexa targeted homozygotes that model TaySachs disease have no obvious neurological or behavioral deficits. The Hexb targeted Sandhoff disease mice have a rapid progressive and fatal neurodegenerative disease. Therefore the Hexa targeted mice do not appear to mimic human

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Tay-Sachs disease whereas the Hexb targeted mice appear to be a good model for Sandhoff disease. Although the Hexa targeted mice at first appear to be of limited help in studies of Tay-Sachs disease, they in fact illustrate the importance of targeted gene mutation in vivo for defining the biology of the system under investigation; a study of the biochemistry of these mice and similar gene targeted animals indicates that the mouse sialidase enzyme is much more important in this catabolic pathway than was previously thought (Cohen-Tannoudji et al., 1995; Sango et al., 1995; Phaneuf et al., 1996). Therefore, modulating human sialidase is a new potential therapeutic approach to humans with Tay-Sachs disease. The different emphasis in usage of the enzymes involved in ganglioside break down in human, and the mouse has provided us with a new insight into a way of controlling the pathways involved. Compensatory pathways may provide us with new avenues for therapy, and the relevance and possible future use of sialidase would not have been apparent without this targeting experiment. Mouse phenocopies of human disease-in which the disease genes in the mouse are different from those in humans-can also help our understanding of pathological processes. For example, psoriasis is a hyperproliferative skin disorder that is thought to affect about 2% of the population across the world. Transgenic mice that ectopically express specific integrins develop a phenotype that resem. bles psoriasis. These mice may greatly assist our comprehension of the biology of this disease and the affected pathways that lead to cell proliferation, although human sufferers do not appear to have the same genetic lesions (Carroll et al., 1995). Mouse models allow us to investigate the basic biology of linking genotype with phenotype, and mutation with disease. Mouse models allow us to design and test both conventional and gene therapies. We can create mouse models for human genotypes that are involved in disease. Because mouse biology is not identical to human biology, we may see different phenotypes associated with mutation than are seen in humans: these provide new information about the affected biological pathway. Specific methods of germ line manipulation include the various different forms of transgenesis; recent innovations in germ line manipulation include “knockin” gene targeting, creation of conditional mutants, and chromosome engineering.

VIII. THE FUTURE CONTRIBUTION OF THE MOUSE TO ADVANCES IN HUMAN GENETICS Many more phenotypes that arise from genetic differences have been described in humans than in any other mammal because we study ourselves in detail and catalogue what we find. The tally of human genetic diseases is currently running at

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over 4000 different single gene traits, most of which are rare and are genotypically undefined. In the mouse we have the ideal mammal for genetic analysis and a mammal that has almost an identical gene content to humans. However, because only about 300 mutations are known in the mouse at loci that are associated with genetic disease in humans (Searle and Selley, 1996), a “phenotype gap” exists, defined as “the lack of phenotypes in the mouse that result from mutation in loci that are responsible for human genetic disease” (S. Brown, personal communication). If we had these mutations they would provide us with resources from which to clone new human disease genes and with mouse models for designing therapeutic strategies. One example of the power of multiple new mutations in different genes has come from the field of epilepsy research where a range of germ line modifications has given rise to mice that have seizures due to cerebral dysfunction. These mice have provided many new insights into epilepsy, the causes, disease process, and potential approaches to treatment. Without such mutations these insights would not have been possible (Noebels, 1996). In addition, multiple mutations at one locus provide us with basic biological information about gene function and disruption. Our lack of knowledge of gene function is becoming apparent as the genome projects send exponentially increasing numbers of cDNAs and genes into the databases, but only in a very small proportion of cases do we know what the gene products do. For all these reasons, we need more mouse mutants. Therefore, one likely future area of interest will be projects to increase the numbers of mouse mutants available, focusing on phenotypes of interest to individual laboratories, e.g., mice with skeletal abnormalities or mice with diabetes. These projects will use random mutagenesis techniques because we have no a priori information about which genes are involved and generally little information about where interesting phenotypes map in the human or mouse genomes for most of the rarer disorders. Once the phenotypes are in place, then identifying the responsible genes will eventually become routine as the full resources of the human and mouse genome Projects come on line. With a large pool of mouse mutants we may be able to understand the progression of some biological pathways by breeding studies involving different mutations, using strategies that are familiar to Drosophila geneticists. Double gene targeted mutant animals are already proving helpful in studies of cellular signal transduction pathways (Williams e t al., 1994). A large mutant pool will also provide multiple alleles of single loci. One example of the usefulness of multiple genotypes in furthering our understanding of gene function is again provided by the microphthalmia locus. In addition to the two transgene mutations described earlier, at least 13 other mi mutations are known, 9 of which have been cloned (Moore, 1995). Determining the molecular genetic lesion of each mi mutation and correlating these with the wide-ranging phenotypes that result allow the functional dissection of the various motifs in the MITF protein in uiuo. This has direct relevance for human disease because mutations in MITF result in Waardenburg’s syndrome

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type 11, which is a dominantly inherited syndrome with many effects, particularly hearing loss and pigmentary disturbances (Tassabehji et al., 1994; Moore, 1995). Another area of interest in the mouse that will have an impact on human genetics is the increased study of polygenic disease and the increasing sophistication of the statistical techniques used to analyze such diseases, such as multiple regression analysis and composite interval mapping (Frankel, 1995). A t the time of writing, many projects in mouse and human have identified areas of interest in both genomes for different polygenic diseases, and the next step is to identify the causal genes. Information from such projects will be relevant to disease inheritance, affected pathways, and pathological processes in both species, but causal genes are likely to be easier to identify in the mouse and can only be targeted for functional studies in the mouse. Currently, over 20 complex traits have been mapped in the mouse that directly correlate with human disease ( Frankel, 1995). These include epilepsy, various tumors, obesity, neural tube defects, autoimmune diseases, gallstones, and diabetes (Frankel, 1995). Future studies that combine polygenic disease investigations with a very important phenotype for human disease are the behavioral studies. The rat has traditionally been the model mammal for behavioral studies, but now attention is focusing on behavioral testing in the mouse because of the genetic resources provided by this animal (Moran et al., 1996). Such studies are relevant not only for aberrant behavior such as substance abuse-certain inbred strains are well-known morphine addicts, whereas others choose not to consume opiates-but are also relevant to normal behavior and emotion (Berrettini et al., 1994; Flint et al., 1995). Humans and mice have been living together for a very long time. We have learned a lot from this animal but most of the biological knowledge is still waiting to be unearthed. At last we should be able to start to combine our molecular biology resources with new phenotypic resources so that we can go into the next millennium prepared for the functional studies have have resulted from thousands of years of looking at heritable traits in mice.

Acknowledgments I am extremely grateful to Nessan Bermingham, Steve Brown, Diana Hernandez, and Elizaheth M. Simpson for providing critical comments and to Paula Reynolds for generous assistance in preparing this manuscript. The reader is referred to Silver (1995) as an excellent guide to mouse genetics. Readers are also referred to the extremely helpful and comprehensive review by Meisler. Meisler, M. H. (1996). The role of the laboratory mouse in the human genome project. Am. 1. Hum. Genet. 59, 764-771.

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Ting, C. N. (1994). Insertional mutation on mouse chromosome 18 with vestibular and craniofacial abnormalities. Genetics 136:247-254. Tobler, I., Gaus, S. E., Deboer, T., Achermann, P., Fischer, M., Rulicke, T., Maser, M., Oesch, B., McBride, P. A., and Mason, J. C. (1996). Altered circadian activity rhythms and sleep in mice devoid of prion protein. Nature 380:639-642. Van Deursen, J., Fomerod, M., Van Rees, B., and Grosveld, G. (1995). Cre-mediated site specific translocation between non homologous mouse chromosomes. Proc. Natl. Acad. Sci. USA 92:7376-7380. Vidal, S. M., Malo, D., Vogan, K., Skamene, E., and Gros, P. (1993). Natural resistance to infection with intracellular parasites: Isolation of a candidate for Bcg. Cell 73:469485. Vincent, N., Ragot, T., Gilgenkrantz, Z., Couton, D., Chafey, P., Gregoire, A., Briand, P.,Kaplan, J. C., Kahn, A., and Perricaudet, M. (1993). Long term correction of mouse dystrophic degeneration by adenovirus mediated transfer of a minidystrophin gene. Nat. Genet. 5:130-134. Vortkamp, A., Gessler, M., and Grzeschik, K. H. (1991 1. GL13 zinc-finger gene interrupted by translocations in Greig syndrome families. Nature 352:539-540. Weeks, D. E., and Lathrop, D. M. (1995). Polygenic disease: Methods for mapping complex disease traits. Trends Genet. 11:513-519. Weil, D., Blanchard, S., Kaplan, J., Guilford, P., Gibson, E, Walsh, J., Mburu, P., Varela, A., Levilliers, J., Weston, M. D., Kelley, P. M., Kimberling, W. J., Wagenaar, M., Levi-Acobas, E, Larget-Piet, D., Munnich, A., Steel, K. P., Brown, S. D. M., and Petit, C. (1995). Defective myosin VlIA gene responsible for Usher syndrome type 1B. Nature 374:60-61. Wilkie, A. 0. (1994). The molecular basis of genetic dominance. J. Med. Genet. 31:89-98. Williams, B. O., Remington, L., Albert, D. M., Mukai, S., Bronson, R. T., and Jacks, T. (1994). Cooperative tumorigenic effects of germline mutations in Rb and p53. Nat. Genet. 7:480484. Williamson, C. M., Dutton, E. R., Abbott, C. M., Beechey, C. V., Ball, S. T., and Peters, J . (1995). Thirteen genes (Cebpb, E2f1, Tcf4, Cyp24, Pckl, Acra4, Edn3, Kcnbl, M d r , Ntsr, Cd40, Plcgl and Rcad) that probably lie in the distal imprinting region of mouse chromosome 2 are not monoallelically expressed. Genet. Res. 65:83-93. Yulug, 1. G., Egan, S. E., Pollock, P. M., and Fisher, E. M. C. (1993). A homologue of the Drosophila Son of Sevenless gene maps to mouse chromosome 17. Genomics 18:733-734. Yulug, I. G., Egan, S. E., See, C. G., and Fisher, E. M. C. (1994). Mapping GRBZ, a signal transduction gene in the human and the mouse. Genomics 22:313-318. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J . M. (1994). Positional cloning of the mouse obese gene and its human homologue. Nature 372:425431. Zhou, L., Dey, C. R., Wert, S. E., DuVall, N. D., Frizzell, R. A,, and Whitsett, J, A. (1994). Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR. Science 266:1705-1708. Zinn, A. R., Alagappan, R. K., Brown, L. G., Wool, I., and Page, D. C. (1994). Structure and function of ribosomal protein S4 genes on the human and mouse sex chromosomes. Mol. Cell Biol. 14:2485-2492.

I

prune/Killer of prune: A Conditional Dominant lethal Interaction in Drosophila Lisa Timmons and Allen Shearn

Department of Biology The Johns Hopkins University Baltimore, Maryland 21218

T h e prunelKiller ofprune interaction of Drosophila melanoguster is a n example of a rare genetic phenomenon: conditional dominant lethality. This curious genetic interaction was first observed by A. H. Sturtevant in 1955 when a mating of prunelprune females to males from a laboratory stock produced only daughters and no sons. Because the prune gene is o n the X chromosome, this cross should have produced daughters heterozygous for prune with wild-type colored eyes and sons hemizygous for prune with prune colored eyes. T h e laboratory stock was determined to be homozygous for a third chromosome mutation which Sturtevant named Kilkr of prune. T h e interaction is conditional because it is the combination of the two mutations (prune and Kilkr of prune) that is lethal; individually, neither mutation is lethal when homozygous. T h e interaction is dominant because only one copy of the Killer ofprune mutation is required for lethality of prune mutant individuals. T h e explanation for why this combination of otherwise viable mutations is lethal has remained elusive. This chapter summarizes what has been learned about prune, Kilkr of prune, and the pune/Killer of prune lethal interaction since Sturtevant’s discovery.

1. DISCOVERY OF THE pruneKMer of prune INTERACTION A. H. Sturtevant, a protege of Thomas Hunt Morgan, participated in the early studies using Drosophila as a model organism to learn the principles of genetics (Lewis, 1995). During that period, many stocks were recovered with mutations giving rise to eye phenotypes. One of these, Star, was a dominant mutation on the Advances in Genetics, Vol. 35

Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved.

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Lisa Timmons and Alien Shearn

Figure 6.1. prune/Killer of prune genetic interaction. Females homozygous for mutations in prune when mated to males homozygous for Killer of prune produce viable female progeny and male progeny which die during the third larval instar.

second chromosome that affected the shape of the eye. A mutation on the tip of the left arm of the third chromosome named Enhancer of Star was later recovered due to its ability to dominantly enhance the phenotype of Star. It was in this Star/+;Enhancer of Star/+ stock that the Killer ofprune mutation was first observed (parental male in Figure 6.1) (Sturtevant, 1956). Sturtevant must have been looking for an interaction between either of these two dominant eye mutations and mutations in other genes affecting the phenotype of the eye, and the result he obtained with the prune mutation must have surprised him. T h e prunelKiller ofprune interaction was observed to be independent of the Star and Enhancer of Star mutations. These mutations were removed by standard genetic crosses, and a homozygous Killer of prune stock devoid of other mutations was obtained. Killer of prune was mapped to the tip of the right arm of chromosome 3 by recombination mapping. Sturtevant demonstrated that the Killer of prune mutation did not have a lethal interaction with a variety of other mutations, including vermilion, cinnabar, scarlet, brown, white, zeste, claret, chocolate, sepia, and purpleoid, and it did not modify the eye color caused by these mutations. In retrospect, the discovery of the Killer ofprune mutation was quite a remarkable accident and was made possible by the coincidence of several fortunate genetic circumstances. T h e lethal effect of the Killer of prune mutation is 100% penetrant, and this contributed to its ready discovery. In addition, the Star/+;Enhancer of Star/+ males which Sturtevant crossed to prune- females (Figure 6.1) were homozygous for the Killer of prune mutation on the third chromosome; thus all the male progeny from this cross died. If the Star/+;Enhancer of Star/+ stock

6. prunefliller of prune

209

had been heterozygous for the Killer of prune mutation, half the male progeny would have survived. Furthermore, the fact that prune is a sex-linked recessive mutation greatly facilitated the discovery of the prune/Kilkr of prune interaction. If any of these circumstances had not been met-if the lethal effects of Killer of prune had not been completely penetrant, if the Killer ofprune mutation had been heterozygous, or if the prune gene had been an autosomal-the lethal interaction between the prune mutation and this heretofore unknown Killer ofprune mutation may have gone unnoticed. Thus, as Sturtevant himself noted, many conditional dominant lethal interactions may exist in a variety of different organisms, but the specific requirements for each of their discoveries have not been met.

II. THE awdGENE A. Early genetic studies of the prune/Ki//er of prune interaction: recovery of revertants Lifschytz and Falk ( 1969b) undertook a genetic analysis of the prune/Kilkr ofprune interaction in an attempt to understand the mechanism of action of a dominant mutation. Their approach was to screen for revertants of the prune/Killer of prune interaction by exposing Killer of prune males to gamma rays or to ethylmethane sulfonate (EMS), mating (similar to the first cross outlined in Figure 6.2) them to prune females, and looking for surviving prune/Killer ofprune sons (Figure 6.2). In their screens, the sons could have survived as a result of (1) incomplete penetrance of the prune/Killer of prune interaction, (2) induced dominant mutations which could suppress the prune/Kilkr of prune interaction, ( 3 ) spontaneous mutations in the mutant prune gene which by the nature of their screen would have had to affect the prune/Kilkr of prune interactions but not the prune eye phenotype, or (4) a reversion at the Killer ofprune locus. Ofthe 27 fertile prune/Killer of prune revertant sons recovered in their screen, all fell into category 4, all revertants were lethal when homozygous, and all failed to complement each other. Lifschytz and Falk (1969b) correctly concluded that the gene in which Killer ofprune is a mutation had a vital function. That vital function was not affected by the Killer of prune mutation but was affected by the revertant mutations which they interpreted to be loss of function at the Killer ofprune locus. This interpretation agreed with Sturtevantk suggestion that the Killer of prune mutation is neomorphic.

B. Killer ofprune is an allele of awd The phenotypes of the homozygous lethal revertants of Killer of prune were never reported and the stocks were discarded (E. Lifschytz, personal communication). Nearly 20 years later Dearolf et al. (1988a) identified a gene which was named ab-

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9

E s/:

prune’. prune’’

prune’; prune

y RAYS

kar e kar e

mwh red e awdKRs6 TM3,Sb 6 Ser

Figure 6.2. prune/Killer of prune revertant screen. Surviving male progeny from the standard prune/Killer of prune cross can be produced by mutagenesis of atudKpnmales. All of the revertants which have been recovered are allelic to awd.

normal wing discs (awd) in a screen for mutations which affect the development of imaginal discs in Drosophila. The original allele of awd, awdb3,contained a P-element insertion, and the gene was cloned by transposon tagging (Dearolf et al., 198813).The awd gene was mapped to the tip of the right arm of chromosome 3 close to the position of Killer ofprune. Because the Killer ofprune revertants (some of which were presumed to be deletions) produced by Lifschytz and Falk no longer existed, a screen for gamma ray-induced revertants of the prune/Kilkr of prune lethal interaction was undertaken with the hope of obtaining a deletion of the awd gene (Biggs et al., 1988). Unexpectedly, all of the 12 revertants recovered in this screen failed to complement the lethality of the original awdb3allele. Thus it was reasonable to suspect that the Killer of prune mutation was either in the awd gene itself or in a closely linked gene. This was tested by germline transformation using an awd transgene cloned from Killer of prune homozygotes. This transgene caused lethality of prune individuals (Biggs et al., 1988) and rescued the lethality of awd null homozygotes, which indicated that indeed Killer ofprune is a mutation in awd. Kilter ofprune is the consequence of a point mutation resulting in a single amino acid substitution: proline at residue 97 to serine (Lascu et al., 1992; Timmons et al., 1995). The Killer of prune mutation will now be referred to as an allele of awd , awdKp”.

6. prune/l(lller of prune

21 1

C. The phenotype of awdmutants Imaginal discs are groups of epithelial cells that are set aside during embryogenesis and which eventually give rise to the adult structures of the fly (Figure 6.5). During metamorphosis, most of the larval cells are histolyzed or are removed by other processes and are completely replaced by imaginal tissues. The dramatic result is that a n adult animal emerges from the pupal case which is completely different in appearance from the larva it once was. Metamorphosis occurs during the pupal stage, and it is during this stage that the differentiation of imaginal discs into recognizable adult structures with the patterns of pigmentation, bristle, etc. characteristic for that adult structure proceeds. Homozygous awd null mutant animals do not form a pupal case, and the animals die during third instar. All of the imaginal discs from third instar homozygous awd null alleles are very reduced in size; they are not larger than imaginal discs from second instar wild-type larvae. Imaginal discs can differentiate into adult structures when transplanted into metamorphosing host larvae; however, imaginal discs from larvae that are homozygous for a null mutation in the awd gene, awdKRS6(Timmons e t al., 1995), never differentiate when transplanted (Hersperger and Shearn, unpublished observation). Thus the failure of awd mutants to differentiate is disc autonomous. T h e original awdb3 P-element insertion allele is also lethal during the third instar stage as a homozygote. However, the phenotype of awdb3 imaginal discs is not as severe as that of null awdKRS6imaginal discs: imaginal discs from homozygous awdb3individuals are variable in size, and some are even wild type in appearance. These results indicate that awdb3has some residual, yet variable, expression of awd product; therefore, awdb3is a severe hypomorphic mutation, not a null. Further support for this conclusion comes from ovary transplantation experiments. Ovaries from wild-type larvae can develop into normal, functional ovaries when transplanted into female sterile owoD1host larvae. (The ovaries from female sterile owoD1 hosts normally do not generate functional ovaries.) When ovaries from homozygous awdb3mutants are transplanted into female sterile hosts, the hosts develop egg chambers that are defective (Dearolf et al., 198813). When ovaries from homozygous awdKRr6mutants are transplanted into female sterile hosts, the hosts do not develop egg chambers at all (Xu et al., 1996). These results indicate that awd' function is required during oogenesis and that the amount of awd product produced from the awdb3allele is enough to improve the phenotype of the ovary in comparison to the awdKRs6null allele. Still, this amount is not enough to provide for complete rescue of the ovary phenotype. awdb' homozygotes have been demonstrated to produce a larger mRNA by Northern blot analysis, and this mRNA also hybridizes to P-element sequences (Dearolf et al., 198813).Perhaps, some functional protein can be produced from this altered-size transcript even though the P-element is inserted in codon 12. Other hypomorphic awd alleles have been isolated (Table 6.1))but none

Table 6.1. awd Mutant Alleles Name of allele

Size of Pstl fragmentn

Protein accumulation

NDP kinase activityb

awd+

2.2 kb

+

3.2

Wild type

awdQ"

2.2 kb

+

0.92

Pro97Ser

auldb3

2.7 kb

+/-

awdKR7 awdKKI4

Absent 2.2 kb

NA'

NA 0.05

awdKKI7 awdKR2' awdKRA awdmn awdKR"' awdmm2

1.4 kb Absent Absent Absent Absent 2.2 kb

NA NA NA NA

NA NA NA NA 0.04

+I-

-

awd gene alteration

Insettion of 0.5-kb defective Pelement >S-kb deletion Nucleotide change 105 bases upstream of ATG 0.8-kb deletion Deletion of 10K-D Deletion of 1 0 K - D Deletion of 10K-D >5-kb deletion Pro97Ser and MetlLys

Ref. Dearolf et al. (1988b); Timmons et al. (1995) Lascu et al. (1992); Timmons et al. (1995) Dearolf et al. (198813) Biggs et al. (1988) Biggs et al. (1988); Timmons et al. (1995) Biggs et al. (1988) Biggs et al. (1988) Unpublished Unpublished Timmons et al. (1995) Timmons et al. (1995)

auldKRn3 auldKRm4 awdKRm5 awdKRs6

auldKRm7 auldKRd auldKR"' .,dKRmI

0

.,dKRmJ

I

awdKRmlZ awdKRm I 3 awdKRml 4 awdKRml 5 .,dKRmI

6

awdKRm I 7 awdKRm 18

2.2 kb 2.2 kh 2.2 kh 1.4 kh 2.2 kh 2.2 kb 2.2 kh 2.2 kh Absent 2.2 kb 2.2 kh Absent 2.2 kh 2.2 kh 2.2 kh

+ +

NA c

+

NA -

+

0.03 0.03 0.04 0.06 0.03 0.04 0.19 0.05 NA 0.17

Pro97Ser and Arg89Cys Pro97Ser and Glu 130Lys Pro97Ser and Aspl5Asn 788-bp deletion Pro97Ser and Argl06Cys Pro97Ser and MetlVal Pro97Ser and Alal27Thr Pro97Ser and Aspl5Asn >5-kb deletion Pro97Ser and Aspl22Asn

0.03 NA 0.02 0.03 0.39

Pro97Ser and Arg89His

"In wild-type Drosophila the entire auld gene is contained in a 2.2-kh Pstl restriction. "NDP kinase activity was measured in auldKRs6 transheterozygotes. 'Not applicable.

N r

W

Timmons et al. (1995) Timmons et al. (1995) Timmons et al. (1995) Timmons et al. (1995) Timmons et al. (1995) Xmmons et al. (1995) Timmons et al. (1995) Timmons et al. (1995) Timmons et al. (1995) Tirnmons et al. (1995) Unpublished Unpuhlished Unpuhlished Unpuhlished Unpublished Unpuhlished

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Lisa Timmons and Allen Shearn

are as severe as awdb3. As hemizygotes with awdKRs6,the imaginal discs of all these hypomorphic alleles have wild-type appearance; yet some are lethal during third instar, some are lethal as pupae, one is lethal as a pharate adult (with some viable adult escapers), and one is homozygous viable. The fact that some hypomorphs are viable suggests that the fly produces more AWD activity than is required for viability. All of these hypomorphic alleles were generated in awdKW revertant screens and will be discussed in subsequent sections.

D. The awdgene is similar to nm23 When the awd gene was cloned and the protein coding sequence determined, no proteins with significant similarity were found in the existing databases. However, 1 year later, Rosengard et al. (1989) reported that the predicted AWD protein was 78% identical to the predicted human NM23 protein. Human NM23 is 94% identical to mouse NM23, which was isolated in a differential screen comparing patterns of gene expression in melanoma cells with little metastatic potential to melanoma cells with high metastatic potential. The mouse nm23 transcript was consistently expressed at higher levels in the poorly metastatic cells, which suggested that NM23 might act as a metastasis supressor (Steeg et al., 1988a). Reduced levels of nm23 mRNA accumulation in several kinds of cancers, such as mouse melanoma (Steeg et al., 1988a), transformed rat embryo fibroblasts (Steeg et al., 1988b), and human breast carcinomas (Bevilacqua et al., 1989),were highly correlated with increased metastatic potential. The same inverse correlation of nm23 accumulation with metastasis in human breast cancers (Barneset aE., 1991; Hennesy et al., 1991; Hirayama et al., 1991) and in human melanomas (Florenes et al., 1992) was found in several independent studies. One study reported not finding an inverse correlation of nm23 accumulation with metastasis of human breast cancers (Sastre-Garau et al., 1992). To examine whether the relationship between metastases and nm23 expression goes beyond the observed correlation and is actually causal, Steeg and co-workers transfected mouse melanoma cells of high metastatic potential with nm23 linked to an SV40 promoter and demonstrated that an increased level of nm23 expression could suppress the metastatic phenotype (Leone et al., 1991). Similar transfection studies with cultured human breast cancer cells revealed that an increased level of nm23 expression could suppress the metastatic phenotype (Leone et al., 1993). Whereas an increased metastatic potential in human breast cancers and melanomas is correlated with a low level of nm23 accumulation, in human neuroblastomas two independent studies have concluded that advanced stage disease is correlated with high levels of NM23/NDP kinase accumulation (Keim et al., 1992; Leone et al., 1992). Humans have a second gene that is quite similar in sequence to nm23. The nm23 gene (now renamed nm23-HI) encodes a subunit that is 88% identical to the subunit encoded by the second gene which is called nm23-H2 (Stahl et

6 . prunefliiler of prune

215

al., 1991).The only study which used probes that could distinguish between these

two gene products supported the idea that accumulation of nm23-HI transcript was inversely proportional to metastatic potential and that accumulation of nm23-H2 transcript did not vary with metastatic potential (Stahl e t al., 1991). Evidence suggests that NM23-H2 can bind to a specific sequence in the myc regulatory region (Postel et al., 1993) and more generally can bind single-stranded DNA that is pyrimidine rich (Hildebrandt et al., 1995). The relationship between awd gene expression and tumor invasiveness in Drosophila has also been investigated (Timmons et al., 1993). awd gene expression was monitored in wild-type and in lethal (2) giant larvae, l(2)gl, brains. l(2)gl encodes a novel protein which localizes to epithelial cell junctions (Strand et al., 1994), and brains from l(2)gl mutant larvae appear overgrown and highly disorganized. When brains are removed from homozygous mutant l(2)gl larvae and transplanted into normal hosts, mutant donor cells invade host tissues and ultimately kill the hosts (Gateff and Schneiderman, 1974). This invasive behavior of mutant Drosophila cells is analogous to the behavior of mammalian metastatic cancer cells. Like mutant l(2)gl-induced Drosophila brain tumors, mammalian brain tumors rarely metastasize in situ but give rise to highly metastatic tumors when transplanted (Katz and Liotta, 1986).These overgrown mutant l(2)gl brains and tumors derived from such brains contained a greater amount of AWD/NDP kinase as a consequence of an increase in the proportion of awd' expressing cells. The relationship between the awd gene and the tumor suppressor genes of Drosophila has been extended by the analysis of double mutants using both the awdb3severe hypomorphic allele and the awdKRS6 null allele. Doubly homozygous 1(2)gl-;awdb3 individuals live until the third instar [as do singly homozygous l(2)gl- and awdb' mutants], and transplanted brain tissue from these double homozygotes does not invade host tissue. In contrast, doubly homozygous l(2)glF; awdKRS6 individuals die during the embryonic stage, even though singly homozygous awdKRsdindividuals also die during third instar (Woodhouse, unpublished). The maternal contribution of the awd+ and l(2)gl' product in the singly homozygous awdKRs6or awdb3animal and in the homozygous l(2)gl- animal allows these mutants to survive until third instar. Even though these doubly homozygous embryos have the same maternal contributions of awd' and l(2)gl' product as the singly homozygous animals, the double homozygotes die much earlier. Furthermore, the small amount of awd expression from the severe awdb3hypomorph is enough to allow the doubly homozygous 1(2)gl-;awdb3 individual to live until third instar. These genetic analyses were continued using other Drosophila tumor supressor genes and similar results were found. discs large (dlg) is a membrane-bound protein of the MAGUK family (Woods and Bryant, 1991). dlg mutants die during third instar and have overgrown brains and discs. brain tumor (brat) encodes a novel 99-kDa protein (Hankins, 1993);mutations in brat die during third instar

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Lisa Timmons and Allen Shearn

and have overgrown discs and brains. However, as in lg(2)l-;awdKRs6doubly homozygous animals, animals doubly homozygous for dlg-;awdKRs6 and hat-; awdKRs6die during the late embryonic stage (Woodhouse, unpublished). These intriguing genetic interactions between awd and tumor suppressor genes solidify the notion that NDP kinase activities and tumor supressor activities influence the same cellular process.

E. The awdgene encodes a nucleoside diphosphate kinase In 1990, two independent studies led to the conclusion that AWD has nucleoside diphosphate kinase (NDP kinase) activity. Based on the mitotic phenotype observed in brains from awdb3homozygotes, Biggs et al. (1990) reasoned that AWD might be associated with microtubules. The same antibodies directed against NDP kinases that copurified with bovine brain microtubules (Nickerson and Wells, 1984) also cross-reacted with AWD, which suggested that AWD encodes an NDP kinase. This was confirmed by showing that awd mutant larvae have less than 2% of the NDP kinase activity of nonmutant larvae of the same stage. At about the same time, Gipl7 was isolated in a screen for proteins from Dictyostelium discoideum which bind GTP (Lacombe et al., 1990). The Gipl7 protein was suspected to be a nucleoside diphosphate kinase (NDP kinase; EC 2.7.4.6) based on its size, amino acid composition, and ability to bind GTP. Functional assays of bacterially expressed Dictyostelium Gipl7 protein proved that it indeed had NDP ki. nase activity. The predicted Gipl7 amino acid sequence is 60% identical to the predicted AWD sequence.

F. What are NDP kinases? NDP kinases were originally isolated over 40 years ago from a wide variety of tis-

sue sources, including human erythrocytes, spinach leaves, yeast, bovine liver, and brain (Parks and Agarwal, 1973). The enzymatic properties of these purified enzymes have been well characterized. NDP kinases catalyze the reversible conversion of nucleoside diphosphates to nucleoside triphosphates (Figure 6.3). NDP kinases show little preference toward sugar or base residue of the nucleoside substrate: NDP kinase can utilize both purine and pyrimidine ribo- and deoxyriNlTP N2DP

+ +

E E-P

N1TP t N2DP

-

NlDP N2TP

+

+ ~

N1DP t

E-P E ~

N2TP

Figure 6.3. Reaction catalyzed by nucleoside diphosphate kinas-

(E). The enzyme is transiently phosphorylated on an active site histidine.

es

6. pruneKiiler of prune

217

bonucleoside diphosphates as well as some nucleoside analogs (Parks and Agarwal, 1973).The reaction proceeds via a ping-pong mechanism and the enzyme itself is phosphorylated on a histidine residue during phosphate transfer (Edlund et

d.,1969).

X-ray crystallography of NDP kinases from different species provides an explanation for the broad range of substrate specificity. NDP kinases have no glycine-rich phosphate-binding loop or “Rossmann fold” (Rossmann et al., 1975) and the enzyme has few direct contacts with the base. The nucleoside substrate enters the active site phosphate first. The active site histidine is in a cleft, and conserved residues lining the cleft primarily interact with the phosphate and sugar groups of nucleoside substrates. This active site configuration is conserved among all the NDP kinases which have been crystallized so far: Myxococcus (Williams et al., 1993); Dictyostelzum (Dumas et al., 1992); Drosophila (Chiadmi et al., 1993; Morera et al., 1994a,b); and human (Webb et al., 1995). cDNAs encoding NDP kinases from a variety of species have been obtained. In several species, two distinct cytosolic NDP kinases have been cloned: human nm23-HI and nm23-H2 (Steeg et al., 1988a; Stahl et al., 1991), rat (Y and p NDPK (Kimura et al., 1990; Shimada et al., 1993), mouse nm23-MI and nm23M2 (Steeg et al., 198813; Urano et al., 1992), and spinach NDPK-1 and NDPK-11 (Nomura et al., 1992; Zhang et al., 1993). The two human cytosolic NDP kinases can exist as heterohexamers in addition to homohexamers (Gilles et al., 1991). It is not known whether the different hexameric forms have different subcellular localizations or distinct functions. This question is being vigorously pursued by several investigators. The cytosolic forms have a striking degree of sequence identity: the two human cytosolic NDP kinases are 88% identical; the awd cytosolic NDP kinase is 78% identical to each of the two human NDP kinases; the Dictyostelium NDP kinase is 60% identical to the AWD protein; and the Dictyostelium cytosolic NDP kinase is 58% identical to the Pisum sativum cytosolic NDP kinase. A n additional human NDP kinase has been cloned (Venturelli et al., 1995) which has approximately 70% homology toNM23-H1 and NM23-H2. The subcellular localization of this form has not been determined, nor has the ability of this form to multimerize with the NM23-H1 and NM23-H2 forms been tested. In addition, five different NDP kinase cDNAs have been cloned from Xenopus laevis which may correspond to as many as four different genes in the pseudotetraploid frog. The amino acid sequences are 82 to 87% identical to the human NM23-H1 and NM23-H2 proteins (Ouatas and Mazabraud, 1995). In addition to the cytosolic NDP kinase forms, a nuclear-encoded mitochondrial NDP kinase has been cloned from Dictyostelium (Troll et al., 1993), and chloroplast NDP kinase has been cloned from the garden pea, Pisum sativum (Lubeck and Soll, 1995). Only one cytosolic NDP kinase gene has been isolated from these species, which is also the case for Drosophila. The Dictyostelium mitochondrial NDP kinase gene contains four introns, whereas the cytosolic form con-

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Lisa Timmons and Allen Shearn

tains two introns. The positions of the second mitochondrial intron and the first cytoplasmic intron are identical, as are the positions of the fourth mitochondrial intron and the second cytoplasmic intron. This suggests that the two genes diverged from a common ancestor (Troll et al., 1993). The Dictyostelium mitochondrial NDP kinase is 58% identical to the Dictyostelium cytosolic NDP kinase, 45% identical to Escherichia coli NDP kinase, and 40% identical to the Pisum sativum chloroplast NDP kinase.

G. Molecular analysis of the awdKPnmutation The Pro97 residue is conserved among all NDP kinases isolated, and crystal structure data reveal that this residue is located in a conserved structure called the “Kpn loop.” NDP kinases isolated from a variety of species are hexamers with the exceptions of Myxococcus (Williams et al., 1993), E. coli (Almaula et al., 1995), and other prokaryotic forms which are tetramers. (The crystal structure of the Myxococcus tetrameric NDP kinase also has the conserved “Kpn loop.”) The NDP kinase hexamer can be divided into a ‘‘top”trimer and a “bottom” trimer. The Kpn loops from the “top” trimer are positioned on the top of the hexamer whereas the three Kpn loops of the “bottom”trimer reside on the bottom of the hexamer. Some residues of the Kpn loop are located at trimer subunit interfaces and may therefore play a role in stabilization of the hexamer. Indeed, the purified KPN mutant protein of Drosophila is less stable, as measured by its ability to refold properly into a functional protein after heat or urea denaturation (Lascu et al., 1992).The KPN protein denatured at much lower temperatures than wild-type AWD, and while AWD recovered up to 40% of its enzymatic activity after urea denaturation, KPN was essentially unable to recover from this treatment. After denaturation, the mutant KPN protein did not recover its quaternary structure as efficiently as wildtype AWD protein and accumulated as folded monomers. These results suggest that the relatively minor substitution of Pro97Ser in the KPN protein causes a dramatic change in the tertiary or quaternary structure of the KPN protein. However, crystallographic analysis of Dictyostelium NDP kinase containing the equivalent mutation (Pro100Ser) revealed only minor changes in the hexameric structure from that of the wild-type structure (Karlsson et al., 1996). A disruption of the interaction between Asp115 (Gln112 of Drosophila) and the C terminus of another subunit was noted. Although this residue is not strictly conserved among all the NDP kinases cloned, in the wildtype AWD crystal structure, the G l n l l 2 amide group hydrogen bonds to the carboxy-terminal Glu153 (a conserved residue) of the adjacent subunit. Thus, it appears that the amino acid residue at this position is responsible for intersubunit contacts. Like Drosophila KPN, the Dictyostelium KPN thermal stability and stability in 2 M urea are much reduced with respect to wild-type protein (Karlsson et al., 1996). Thus the relatively minor structural changes caused by ProlOOSer substitution lead to a major change in thermostability of the enzyme. The fact that

219

6. prune/Kiler of prune

awdKPll homozygous stocks do not fare as well at 27°C as well as wild-type stocks (which is evident by reduced fecundity of awdKm homozygotes at this temperature) might be a reflection of these observations. It would be interesting to know

whether the Pro97Ser substitution of Drosophila has a more drastic effect on the structure of the active site than the Dictyostelium ProlOOSer mutation. The KPN mutation has also been introduced by site-directed mutagenesis into the human NM23-HI protein and transfected into human breast carcinoma cells (MacDonald, 1996). Because the endogenous nm23-HI and nm23-H2 genes were also present in this cell line, any phenotypes induced by the Pro96Ser substitution must be dominant. The phenotype which was monitored was cell motility, measured in Boyden chambers in the presence of chemoattractants. The motility of transformed cell lines containing a wild-type nm23-HJ transgene was reduced, whereas cell lines transfected with the nm23-HI KPN mutant exhibited similar or slightly elevated motility levels in comparison to untransfected cell lines. Thus no dominant phenotype was induced by NM23-H1/KPN expression with respect to control cells in this system; yet the motility behavior of NM23Hl/KPN-expressing cells was different from that of cell lines expressing NM23Hl/wild-type protein-the NM23-H1/KPN protein, unlike the NM23-Hl/wildtype protein, did not have the ability to suppress the motility of these cells. In order to investigate the effects of other amino acid substitutions at this site in the Drosophila NDP kinase, the Pro97 residue of AWD was mutated to Gly by site-directed mutagenesis (J. Xu and A. Shearn, unpublished observations) and inserted into awd null mutants via P-element-mediated transformation (Spradling and Rubin, 1982).Wild-type copies of the awd cDNA regulated by 600 bp of awd upstream region rescued the lethality caused by awd null mutations (Timmons et al., 1993).The Pro97Ser KPN protein expressed from this same promoter also rescued awd null lethality and caused lethality of prune- individuals (L. Timmons and A. Shearn, unpublished observations). However, the Pro97Gly mutation did not rescue awd null lethality and did not cause lethality of prune- individuals. The Pro97Gly mutant protein acted as a severe hypomorph: awd null animals expressing Pro97Gly died at a slightly later stage than awd null mutants and the phenotype of the imaginal discs from lethal Pro97Gly individuals was wild type. Furthermore, the NDP kinase activity of Pro97Gly transformants was much reduced with respect to that of Pro97Ser transformants. This is an indication that the neomorphic character of the Pro97Ser KPN mutation requires the activity of the mutant protein and that this residue is important for the activity of the wild-type AWD protein. Further illustrations of this point are described in the next section.

H. Models of the neomorphic character of the

mutation

The molecular mechanism by which the neomorphic uwdKpn mutation causes lethality in prune- individuals is not known. In light of the earlier discussion, one could propose a model in which AWD and Prune proteins participate in a com-

220

Lisa Timmons and Allen Shearn

plex which provides an essential activity, and that disruption of this complex would then necessarily lead to lethality of the organism. In one such model, Prune would regulate or stabilize the complex, which could be composed of either NDP kinase subunits alone (the AWD hexamer) or the AWD hexamer plus other protein subunits. If one supposes that the less stable KPN protein is disruptive to such a complex, then the absence of Prune might further disrupt the integrity of the complex or might lead to further misregulation of the complex, and this would eventually lead to lethality of the organism. Thus the neomorphic character of KPN would essentially be derived from a loss-of-function event which occurs only in the absence of Prune. Although this kind of model is attractive, not much support for it exists. According to this model, one should be able to “titrate” the “poisonous” effects of the KPN protein on the complex by increasing the amount of AWD relative to KPN in prunelKilkr of prune lethal organisms, and this should lead to improved viability. In lethal prune/KilEer of prune animals, one copy of a wild-type awd gene and one copy of the awdKPn gene are present, and therefore the relative amounts of AWD and KPN proteins are equal. Attempts to “dilute” the KPN concentration in prune/Kilkr of prune individuals by engineering these individuals to contain additional transgenic copies of wild-type awd under the regulation of its own promoter or under the regulation of the HSP70 promoter have failed to rescue the lethality of these engineered prunelKilkr ofprune animals (Biggs et al., 1988; L. Timmons and A. Shearn, unpublished observations). Likewise, reducing the amount of KPN protein in prune- animals by expression of KPN from a weaker promoter relative to that of the promoter driving the endogenous wild-type AWD still results in a lethality (L. Timmons and A. Shearn, unpublished observation). Although prune and awdKPninteract genetically, there is no evidence that the proteins interact; therefore, this kind of model is not likely to be a correct interpretation of the prunelKilkr of prune interaction. Because the Kpn loop has some residues in the active site of the mole. cule (Arglo6 of Drosophila hydrogen bonds with the p phosphate of ADP), another model for prune/Kilkr of prune lethality can be proposed which is based on an altered activity of the mutant enzyme. The Pro97Ser substitution is not drastic enough to render the active site nonfunctional, but might confer more flexibility to the active site with respect to the substrates it will accept. By this reasoning, the neomorphic character of the KPN mutation is a function of the altered enzyme activity of the KPNNDP kinase hexamer which can accept (an) additional substrate(s) in comparison to the AWDNDP kinase hexamer. Further support for the model that the KPN mutation affects substrate specificity comes from analysis of awdKPn revertants. So far, 19 null alleles have been obtained in prune/Kilkr of prune revertant screens: 7 are deletions, 9 have the awdKpn point mutation and one additional point mutation, and 3 are uncharacterized. Two hypomorphic alleles have been obtained in revertant screens: both have the awdKpnmutation and an additional amino acid substitution. One,

6. pruneMiller of prune

221

~

awdKRn9,is a viable allele when hemizygous with awdKRS6, whereas awdKRmJ2 is lethal at the late pupal stage. (The lethal stage for awd null alleles is third instar.)

O n e additional hypomorphic allele has been obtained which has a point mutation in the regulatory region in addition to the awdKp" point mutation. This allele, awdKR14,drastically reduces the level of KPN enzymatic activity. The awdKRJ4mutation is a point mutation approximately 105 bp upstream from the start of transcription. The site of this point mutation does not correspond precisely to any known transcription factor binding site; however, southwestern blot analysis using radiolabeled oligonucleotides corresponding to the mutated region revealed that a protein does bind at this site (L. Timmons and A. Shearn, unpublished observations). Most of the homozygous awdKR14animals die during the pupal stage and a few survive to adulthood, especially when reared at lower temperatures. awdKRi4homozygotes have drastically reduced NDP kinase-specific activities at all developmental stages, and therefore are hypomorphs. In situ mRNA and immunohistochemical hybridizations performed on awdKRl4 homozygous third instar larvae revealed some residual expression of awd mRNA and protein, with the most expression in the imaginal discs. This residual expression is sufficient to allow the animal to survive, but not enough to be lethal in the absence of Prune+ (because awdKRJ4was isolated as a awdKpn revertant, the awdKm mutation is still present). T h e important observation from these hybridization experiments is that not only is the amount of Kl" expression drastically reduced in a n awdKRJ4mutant, but the tissue distribution of KPN is also affected. The NDP kinase activity was measured in all awdKW revertants as hemizygotes at the third instar stage and was found to range between 0 and 20% of the activity of hemizygous awdKp third instar larvae (Timmons et al., 1995).Thus a compensatory mutation which reverts the KPN active site to a wild-type configuration with wild-type levels of enzymatic activity has not been found. Instead, the recovered revertants contain mutations which reduce KPN activity by deletion of the awdKpnallele, by severely reducing transcription of awdKpn,or by further reducing the enzymatic activity of KPN/NDP kinase by affecting residues which by X-ray crystallography are known to be important for substrate binding. T h e 27 additional revertants isolated by Lifschytz and Falk (1969b) are n o longer available for analysis. These investigators reported that all these revertants were lethals as homozygotes, which suggests that these alleles were also loss of function. The highest activity measured in a hemizygous revertant is 6% of wild-type activity at the third instar stage. Similar levels of activity were measured in a n awd null mutant containing a single copy of a n awdKPntransgene, yet this transgene was able to rescue the awd null mutation and conferred lethality to prune- individuals (L. Timmons and A. Shearn, unpublished observation). This result, along with the revertant analysis, is further confirmation that KPNWDP kinase has a neomorphic activity and that a certain threshold level of KPN/NDP kinase activity is required for prune/Killer of prune lethality.

222

Lisa Timmons and Alien Shearn

111. THE prune GENE A. Phenotypes and genetics of prune mutations The original prune mutant allele discovered by 0.W. Beadle (Beadle and Ephrussi, 1936) was a spontaneous mutation which caused the eye color to appear dark brownish-red as opposed to the brick red color of wild-type eyes. Since then, many other alleles of prune have been recovered, most were viable alleles whose only phenotype was the altered eye color. Table 6.2 lists all the prune alleles that have been reported; unfortunately, many of these stocks are no longer available for analysis. The prune alleles can be grouped into three categories: those exhibiting the “classic” dark brownish-red eye color which are also lethal in the presence of awdKpn,those exhibiting a lighter eye color which are insensitive to the lethal affects of awdKPn, and lethal alleles of prune. The alleles which are homozygous lethal probably represent deletions of neighboring essential genes, as discussed later. Heteroallelic combinations of these lethal alleles with viable alleles of prune (i.e., prunelethal/prune”iable) exhibit the same prune eye color phenotype and awdKpnlethality as those of the more “classic”viable prune alleles. The “classic” prune eye color has been observed in heteroallelic combinations of all prune alleles (e.g., prune1/prune2)with very few exceptions. The combination of p r ~ n e ~ ~ - ~ / p rinu heteroallelic ne~~~ females was reported to produce wild-type eye color and to be insensitive to the lethal effect of awdKP” (Wagenberg and Burdick, 1969), unlike homozygous p r ~ n e ~ ~ - ~ / p r uand n e ho~~-~ mozygous prune68b/prune68b individuals which were sensitive to the lethal affect of awdKpnand which exhibited dark brownish-red (prune) eyes. In addition, the class of alleles generated by Orevi and Falk (1975) had light brown eye colors when grown at a restrictive temperature (29”C),but wild-type eye color when grown at a permissive temperature (20°C). Furthermore, the pmnets-e light brown eye phenotype was dominant over a more “classic” prune- mutant (prunetS-e/prune-)at restrictive temperature. In addition, these ts mutants were insensitive to awdKpn at any temperature. However, another temperaturesensitive prune allele, resembled a “classic” prune allele at the restrictive temperature with respect to both the eye phenotype and the sensitivity to awdKPn.All this indicates that the “classic” brownish-red prune- eye phenotype cannot be separated from the awdKpnsensitivity.

B. Function of the prune gene The prune gene has been cloned and encodes a protein of 45 kDa with little similarity to any previously cloned gene (Frolov et al., 1994;Teng et al., 1991;Timmons and Sheam, 1996).The prune gene is located in a transcriptionally dense region; six mRNAs have been detected in a span of 30 kb between the wings-apart

Table 6.2. prune Mutants prune

mutant

18a 12c A PWZ 38 77c33 1-hl 24-hl 1 2 3 5 12 20 25 26 36

40 45 55

62 69 la

26-20

Ref."

Mutagen

1 1 1 2 3 1 1 1 4 5 6 4,7 8 9 89 83 89 89 89 10 11 12

Ethyl methanesulfonate Ethyl methanesulfonate Ethyl methanesulfonate P-element P-element P-element Ethyl methanesulfonate Ethyl methanesulfonate Spontaneous X-ray

9 13

X-ray X-ray X-ray X-ray X-ray X-ray X-ray X-ray Spontaneous X-ray N-Methyl-"-nitro-Nnitrosoguanidine X-ray X-ray

Mutation

Phenotype Prune eyes Prune eyes Prune eyes Prune eyes Prune eyes Prune eyes Homozygous lethal Homozygous lethal Prune eyes Prune eyes Prune eyes Prune eyes Homozygous lethal Prune eyes Homozygous lethal Homozygous lethal Homozygous lethal Homozygous lethal Homozygous lethal Prune eyes Prune eyes Prune eyes Homozygous lethal Prune eyes

Premature stop, ccdon 186 8-bp deletion/frameshift, codon 223 Amino acid substitutions, C154Y and S165F P-element insertion. codon 290 P-element insertion, codon 89 P-element (active) insertion, codon 62

Protein accumulation?

No No No

Yes

No No ? ?

Insertion of 422 transposable element 4-hp insertion, codon 245 Insertion

No No No ? I

? ? ? ? ? ? ? ? ?

? ?

(continued)

Table 6.2. (Continued) mutant

Ref."

Mutagen

27-9 27-22 2a 3a 51b 51h 59j 63d 68b AA 1 FG FS 1 MS2 tr (40 alleles) ts-e (9 alleles)

13 13 89 89 14 15 16 17 18 19,20 20 21 20 21 21

Sulfur mustard Sulfur mustard x-ray X-ray P-32 X-ray Spontaneous X-ray X-ray Diethyl sulfate Spontaneous Ethyl methanesulfonate X-ray Ethyl methanesulfonate Ethyl methanesulfonate

ts-ek ~

Ethyl methanesulfonate

21 ~~~~

~

Mutation

Phenotype Prune eyes Prune eyes Homozygous lethal Homozygous lethal Prune eyes Prune eyes Prune eyes Prune eyes Prune eyes Prune eyes Prune eyes Prune eyes Prune eyes Prune eyes Light brown eyes-temperature sensitive-Kpn insensitive Prune eyes-temperature sensitive ~~~

Protein accumulation? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

~

"Key to references: (1) Timmons and Sheam (1996). (2,3) no history available, (4) Beadle and Ephrussl(1936), (5) Beadle (1937), (6) Wagenberg and Burdick (1969), (7) Glass (1934), (8) Ilyina (1980), (9) Slobodyanyuk and Serov (1983), (10) Clancey (1959), (11) Petty (unpublished), (12) Kaufman (1970), (13) Sobels (1958), (14) King (1951), (15) Baker (1956). (16) Narayanan and Weir (1964), (17) Mittler (1967), (18) Wagenberg and Burdick (1969), (19) Lifschytz and Falk (1968), (20) Lifschytz and Falk (1969b), (21) Orevi and Falk (1975).

6. pnmeNiller of prune

225

(distal) and prune (proximal) loci. The direction of prune transcription is from telomere to centromere. An additional mRNA located proximal to the prune transcript is transcribed in the opposite direction with respect to that of prune, and the 3’ ends of the two transcripts overlap. Two mRNAs transcribed from the region between prune and wings apart encode members of the cytochrome P450 family-4 group of heme-binding monooxygenases (CYP4D1 and CYP4D2) (Frolov and Alatortsev, 1994; Frolov et al., 1994; Ghandi et al., 1992) which are enzymes involved in the oxidative metabolism of a wide variety of drugs, chemical carcinogens, mutagens, and natural substrates including steroids, fatty acids, prostaglandins, leukotrienes, biogenic amines, pheromones, and plant metabolites (Nebert and Gonzalez, 1987). Other members of the cytochrome P450 family might also be located near this locus as CYP4D2 cDNA probes recognize additional genomic DNA fragments located proximal to prune (Frolov and Alatortsev, 1994). Cytochrome P450 genes are members of an ancient superfamily of genes which have divergently evolved, and subfamilies of P450 genes often appear as a cluster of tightly linked genes (Nelsonet al., 1993). The Prune protein does not contain the highly conserved cysteinyl-containing peptide sequence involved in heme binding of P450 enzymes and is not a member of this family of genes. One approach to solving the mystery of the function of a novel protein is to look for sequence changes affecting the coding region in mutant versions of that protein. A functionally important domain might be identified if the mutant protein has amino acid substitutions or small deletions within that domain. With that goal in mind, the Prune coding sequences in eight independently arising “classic”prune mutations were determined (Timmons and Shearn, 1996). These mutations are summarized in Table 6.2. Some of the alleles analyzed were found to contain point mutations causing amino acid substitutions. Unfortunately, these mutant proteins, and all the other prune mutant proteins analyzed except PrunePw2,failed to accumulate as analyzed by immunoblotting. The effect of most of these mutations was to cause Prune to be unstable so it is not possible to speculate about the effects of the mutations on the function of the protein. Since the mutant immunoblot was performed on extracts of whole pupae, it may be that a small amount of mutant protein is present, but is not concentrated enough in the pupal extracts to be detected on immunoblots. This might especially be the case if Prune protein is expressed in only a few tissue types in the animal. If a detectable amount of mutant Prune is present in some tissues then structure/function conclusions may be drawn from the sequence analysis of these mutants. However, the mutant protein would have to be expressed in a tissue exhibiting a phenotype, such as the eye or the lethal focus of the prunelKiller of prune interaction. Immunohistochemistry of both wildtype and mutant individuals is currently being used to investigate this possibility. The fact that a variety of defects in the Prune coding region (insertions, frameshifts, amino acid substitutions, and one premature stop codon) produce

226

Lisa Timmons and Allen Shearn

identical eye color and awdKPnsensitivity phenotypes suggests that "classic" prune mutations are nulls. Most of the mutants analyzed fail to accumulate protein on immunoblots, which also supports this conclusion. Thus, null mutation in prune lead to the prune eye color and to lethality in the presence of awdKPn. With this conclusion in mind, the prunetspetemperature-sensitive class of mutants, which produced a less extreme eye phenotype and were insensitive to the lethal effects of awdKPn,were probably hypomorphs, and the amount of functional Prune remaining in these mutants must have been enough to rescue them from awdKPn lethality. A similar explanation would hold for the ~ r u n e ~ ~ - ~ / p r uheteroallelic n e ~ ~ " combination of alleles; however, in this case, it is the combination of the two alleles which produces a less severe phenotype than each allele in isolation. One model which could explain this curious result is that Prune is a multimeric protein. Even though homozygous and homozygous Prune68bmultimers are nonfunctional, a P r ~ n e ~ ~ - ~ / P r uheteromultine~~" mer could have enough wild-type Prune activity to improve the null phenotypes, provided that the two kinds of mutations can compensate for each other within the Prune heteromultimer. Of course, it would be very interesting to know the molecular nature of the defects in prune6*",and mutants; unfortunately, they no longer exist.

C. Rescue of the prune/Ki/er ofprune interaction by a prune transgene Rescue of both the prune eye phenotype and the prune/Killer of prune lethal condition was obtained by expression of a wild-type prune cDNA from a HSP70 promoter (Timmons and Shearn, 1996). So little Prune+ was required for rescue of both these phenotypes that heat induction was not necessary, and the amount of Prune+ produced in this manner was undetectable on an immunoblot. These data lend credence to the hypothesis that the prunetspe class of alleles and the p r ~ n e ~ ~ - ~ / p r combination u n e ~ ~ " were hypomorphic for Prune function. The fact that such a small amount of Prune+ protein can rescue both the prune eye phenotype and the PrunelKiller of prune interaction suggests that Prune (like AWD/KPN) is an enzyme or perhaps an enzyme regulator. The small amount of Prune+ required to rescue the null phenotype is reminiscent of other Drosophila enzymes, e.g., awd, maroonlike, and cinnamon. An uninduced HSP70 promoter can supply enough awd product to rescue the lethality of homozygous null awd mutants (Timmons et al., 1993). Mutations in maroonlike and cinnamon also cause the eye to appear dark brownish-red. However, in homozygous or hemizygous mutants the eye color is wild type if the mothers of these mutants were heterozygous for the mutation (Glassman and McLean, 1962). Thus the wild-type mRNA or protein product of maroonlike and cinnamon deposited into the egg is sufficient to rescue the phenotype of a homozygous mutant adult eye!

6. pruneNiiler of prune

227

D. Developmental expression of Prune Immunoblot anaylsis of Prune protein expression using a polyclonal anti-Prune antibody demonstrated the presence of Prune at all stages of development. Maximum accumulation of Prune protein occurred during pupal and adult stages. T h e smallest quantity of Prune protein relative to total protein was present during the third larval instar stage (Xmmons and Shearn, 1996), the stage during which prune/Killer ofprune animals die. Genetically, prune-/Y;awdKPn/awd+progeny from the traditional prune/Kilkr of prune cross (Figure 6.1) have n o functional maternal stores of prune+ mRNA or protein as the mothers in this cross are prune-. In daughters of this same cross, a functional prune+ product is derived via transcription of the paternally derived prune+ gene, and therefore rescue of prune/Killer of prune lethality and the prune eye phenotype occurs. Thus it seems imperative to the survival of the animal that prune+ is expressed during third instar to counteract the lethal affects of awdKPnwhich is also expressed at this stage. This conclusion also holds true if the awdKpnproduct is supplied at a n earlier stage. If a different cross is performed in which both prune+ and awdKpnproducts are maternally deposited into the egg, the genetically prune-/Y;awdKpn/awd+ progeny derived from this egg also die during late third instar; they do not die earlier (during embryonic stages) due to the maternal deposition of prune+. So prune/Killer of prune animals with Prune+ maternally deposited, or with both Prune+ and KPN maternally deposited, die during third instar. The only combination of maternally deposited products remaining-maternal deposition of awdKPnbut no maternal deposition of prune+ product-is not possible, as this is a lethal prune/Killer of p u n e condition and these animals die during third instar. However, this combination can be generated in adult female ovaries using somatic recombination techniques; this is discussed in the last section. In summary, a small amount of prune+ product is required to rescue both the prune eye phenotype and the prune/Killer of prune interaction, and the protective effects of prune+ can occur at embryonic as well as later larval stages of development. Maternally deposited prune+ prevents embryonic lethality, whereas zygotic prune+ prevents late larval lethality in awdKpprogeny.

E. prune mutants have less drosopterin pigments The pigments that compose the wild-type eye include ommochromes and pteridine derivatives. Ommochromes are brown pigments that are biosynthesized from tryptophan and contain the structural group 1,2-pyridino-3H-phenoxazine. Pteridines are red pigments with the structural group 2-amino-3H-4-oxopteridine. Mutations resulting in a reduction of the amount of pigmented ommochromes cause the eye to appear more red than wild type, whereas mutations resulting in a reduction of the amount of pteridine pigments cause the eye to ap-

228

Lisa Timmons and Allen Shearn

pear more brown. Both pigment classes are present in membrane-bound pigment granules in primary and secondary pigment cells on the perimeter of each ommatidia in the fly eye. The function of Drosophila pigments in the eye is not well understood; both ommochromes and pteridines probably act as absorption filters, surrounding and protecting each ommatidium from the laterally deflected light of its neighbor (Ziegler and Harmsen, 1969; Phillips and Forrest, 1980). The red pteridine pigments found in fly eyes are ubiquitous, heterocyclic compounds first isolated from the wings of butterflies because of their beautiful and interesting spectral properties (Hopkins, 1889), but not all pteridines are visible or fluorescent. Included in this large family of compounds are folic acid, a water-soluble vitamin which is also a 6-substituted pterin derivative, and tetrahydrobiopterin, a cofactor for phenylalanine hydroxylase, tryptophan hydroxylase, tyrosine hydroxylase (Alcathiz et al., 1995; Morales et al., 1990; Neckameyer and White, 1992), and nitric oxide synthase (Mayer et al., 1991; Regulski and Tully, 1995)-all of which are enzymes involved in neurotransmitter biosynthesis. Tetrahydrobiopterin and other nonpigmented pteridines are biosynthesized from GTP in pathways utilizing enzymes common to the biosynthesis of pigmented pteridines. Figure 6.4 is a composite of some of the pathways leading to pteridine production that have been elucidated in Drosophila. Few of the enzymes which catalyze these reactions in Drosophila have been unambiguously correlated to known genes that have been identified by mutations. Those gene products identified by mutations which have been associated with catalytic activities are shown in parentheses; those activities identified biochemically are also listed for each reaction. The bright red component of the Drosophila eye color is due to the presence of “drosopterin” pigments. Four different “drosopterin” pigments have been identified on the basis of their red color, similar Rf migrations on thin-layer chromatography, and similar patterns of accumulation in various eye color mutants: aurodrosopterin, neodrosopterin, drosopterin, and isodrosopterin. (Because “drosopterin” refers to both a specific pigment and a class of pigments, the quotation marks will be used to denote the class of pigments.) The amounts of all these pigments are reduced in prune- mutants (Evans and Howells, 1978; Ferre et al., 1986; Hadorn and Mitchell, 1951; Lifschytz and Falk, 1969a; Narayanan and Weir, 1964). Aurodrosopterin is especially reduced, and neodrosopterin is reduced the least (Schwinck, 1975). In the mutant class, whose eye colors were light brown and were insensitive to the lethal effects of awdKPn,the “drosopterin” levels were higher than in “classic” prune mutants, but still less abundant than wild type. However, the mutant, whose eye phenotype and awdKpnsensitivity was identical to the more “classic”prune- mutants, was measured to have “drosopterin” levels identical to prune- mutants when reared at restrictive temperature and similar to wild-type levels at permissive temperature (Orevi and Falk, 1975). This further supports the notion that the class of mutants were

6. prunefliller of prune

229

hypomorphs. In addition, these data show a direct correlation between reduction of “drosopterin” pigments and both the prune- eye phenotype and PrunelKiller of prune lethality.

F. The role of Prune in the pteridine biosynthesis pathway Data presented so far suggest that Prune is an enzyme or regulator of an enzyme which, when absent, causes a reduction in the amount of “drosopterin)’pigments. The fact that “drosopterin” accumulation is reduced in prune- mutants is not necessarily an indication that Prune is directly involved in “drosopterin” biosynthesis. There are several examples of mutations in genes encoding enzymes that are not directly responsible for “drosopterin” biosynthesis which nonetheless affect the accumulation of “drosopterins.” Mutations in rosy, for example, reduce the amount of “drosopterins,” yet rosy is the structural gene for xanthine dehydrogenase/xanthine oxidase (Forrest et al., 1956; Keith et al., 1987). Similarly, mutations in maroonlike, cinnamon, and low xanthine dehydrogenuse, which are involved in the biosynthesis of molybdopterin, the molybdenum-binding cofactor for xanthine dehydrogenaselxanthine oxidase (Kamdar et al., 1994; Schott et ul., 1986; Wahl et al., 1982))result in a brownish-red eye color. Also, mutations in raspberry, burgundy, and other genes which encode enzymes in the de novo purine biosynthesis pathway affect “drosopterin” accumulation. In contrast, flies homozygous for null mutations of sepia, which encodes an enzyme that is directly involved in “drosopterin” biosynthesis, the amounts of “drosopterins” and of PDA-a precursor of “drosopterins”-are undetectable. Because null mutations in sepia are not lethal, it can be concluded that “drosopterin” pigments are not required for viability, and therefore any further reductions of these pigments in prune- mutants would not be a direct cause for lethality in prune/Kiilkr ofprune animals. Clues to the role of Prune in the pteridine/”drosopterin” biosynthesis pathway have been obtained from analysis of animals doubly homozygous for prune- and other genes which cause eye phenotypes. One gene tested in combination with prune was rosy, the structural gene for xanthine dehydrogenase/xanthine oxidase. Homozygous null rosy- mutants are viable and fertile; they have no detectable amounts of isoxanthopterin, decreased levels of “drosopterins,” and increased levels of pterin, dihydrobiopterin, and tetrahydrobiopterin. When prune- was introduced into a homozygous rosy- background, the levels of “drosopterins” were reduced into comparison to the amount in rosy- alone, whereas the levels of other pteridines did not change significantly (Lifschytz and Falk, 1969a). The authors suggested that this result is an indication that the loss of prune+ disrupted “drosopterin” biosynthesis at a step which precedes that affected by loss of rosy+. However, it is also plausible that the reverse is true: “drosopterin” accumulation could be reduced by mutations in rosy at an earlier step but to a lesser degree than mutations in prune, and the greater effect on

l.t

W

0

H2 NEOPTERIN P

3

6 H2 PTERIN

CPYRUVOYL H4 PTERIN

"DROSOPTERINS DROSOPTERIN TETRAHYDROBIOPTERIN

(Xanlhmo

SEPlAPTERlN H2 XANTHOPTERIN

XANTHOPTERIN

ISOXANTHOPERIN

Figure 6.4. Biochemical pathways of pteridine biosynthesis in Drosophila. IMP, inosine monophosphate; XMP, xanthine monophosphate; GMP, guanosine monophosphate; GDP, guanosine diphosphate; GTP, guanosine triphosphate; H2 neopterin P3, 6-(o-eqthro-I ',2',3'-trihydroxypropyl)-7.8-dihydropterin triphosphate; H2 pterin, 7,8-dihydropterin; pterin, 2-amino3H-4(oxo)-pteridine; H2 xanthopterin, 6(OH)-7,8-dihydropterin; xanthopterin, 6(0H)-pterin; isoxanthopterin, 7(0H)-pterin; PDA, 2-amino-4-0x0- 6-acetyl-7,8-dihydro-3H,9H-pyrimido~[4,5~h]~[l,4]diazepine; 6-pyruvoyl H4 pterin, 6-(~-e~ythro-l'oxo-2'oxo-propyl)-5,6,7,8-tetrahydropterin; 6-lactoyl H4 pterin, 6-(~-e~ythro-l'oxo-2'-hydroxypropyl)-5,6,7,8-tetrahydropterin;X, 6-(~-eythro-l'hydroxy-2'oxopropyl)-5,6,7,8-tetrahydropterin; sepiapterin, 6-(~-erythro-l'oxo-2'hydroxy-propyl)-7,8-dihydropterin;oxidized sepiapterin, 6-(~-erythro-l'oxo-2'hydroxy-propyI)-pterin; biopterin, 6-(~-elythro-l',2'dihydroxypropyl)-pterin;7,8 H2 biopterin, 64~-erythro-1'2'dihydrox~ropyl)-7,8-dihydropterin; tetrahydrobiopterin, 6-(~-erythro-l'2'dihydroxypropyl)-5,6,7,8-tetrahydropterin. Each reaction catalyzed by a Drosophila enzyme is referenced according to the reaction number. Names of mutant Drosophila genes encoding enzymes catalyzing the reaction are provided in parentheses where known. ( 1 ) Nash et al. (1994).Slee and Bownes (1995);(2) Chomey and Nash (1996).Johnstone et al. (1985);(3) soluble guanylate kinases have been isolated in E. coli: Gentry et al. (1993);yeast: Berger et al. (19891,Konrad (1992),Moriguchi et d.(1981).and Stehle and Schulz (1992); and human: Aganval et af.(1978);yet not from Drosophila; (4) Biggs et al. (1990);(5) Mackay and O'Donnell(1983). O'Donnell et al. (1989),Weisberg and O'Donnell(l986);(6)Yim et al. (1978,1981);(7)Fan and Brown (1979),Silva et al. (1991),Unnash and Brown (1982);(8)Rembold and Gutensohn (1968),Yen and Glassman (1966);(9) Remhold and Gutensohn (1968),Silva et al. (1991). Dihydropterin oxidase activity is presumed to catalyze this reaction based on the similar activities catalyzed in reactions 7, 17,and 20;(10) Forrest et al. (1956),Keith et al. (1987).Yen and Glassman (1966);(1 1 ) Dorsett and Jacobson (19821,Kim and Yim (1995),Krivi and Brown (1979),Nar et al. (1994),Park et al. (1990), Switchenko and Brown (19851,Wiederrecht et al. (1984),Wiederrecht and Brown (1984),Wilson and Jacobson (1977); (12) Krivi and Brown (19791,Park et al. (19901,Switchenko and Brown (1985);(13) Primus and Brown (1994), Switchenko et al. (1984).(14) Evidence for this pathway in Drosophila was obtained hy Primus and Brown (1994).The structure of compound " X has not been determined in Drosophila; the structure indicated was determined in bovine adrenal and rat liver extracts hy Curtius et al. (1985)and Smirh (1987);(15) Curtius et al. (1986).Primus and Brown (1994), Switchenko et al. (1984);(16) Switchenko and Brown (1985);(17) Fan and Brown (1979),Silva rtal. (1991).Unnasch and Brown (1982);(18)Fan and Brown (19791,Primus and Brown (1994);(19)Fan and Brown (1979),Primus and Brown (1994);(20) Fan and Brown (1979).Silva et al. (19911,Unnash and Brown (1982);(21) DHFR has been isolated in Drosophila: Fan and Brown (19791,Hao et al. (1994),Rancourt and Walker (1990);and the methocrexate-sensitive conversion of H2 biopterin to H4 biopterin has been demonstrated in other systems: Curtius et al. (1985),Webber and Whiceley (1985).Williams and Morrison (1992);(22) Dorsett and Jacobson (1982),Wiederrecht et al. (1984).Wiederrecht and Brown (1984); (23) Dorsett et al. (1979).Wiederrecht et af. (1981).N o enzymes have been isolated which catalyze these reactions.

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“drosopterin” biosynthesis caused by mutations in prune would mask the effects caused by mutations in rosy. A prune-;sepia- double mutant was also constructed. The Sepia protein is suspected to be part of the structural enzyme for PDA synthase (Wiederrecht and Brown, 1981; Wiederrecht et al., 1984). Homozygous sepia- mutants are viable and fertile and have no detectable “drosopterins,”increased pterin, dihydro-, and tetrahydrobiopterin levels, and enormous amounts of sepiapterin which oxidize to a yellow pigment (see Figure 6.4). In pune-;sepia- double homozygotes, the amount of sepiapterin and biopterin was 10 times lower with respect to the levels in sepia- alone (Lifschytzand Falk, 1969a).The conclusion drawn from this analysis was that loss of prune+ action affects pteridines other than “drosopterins” and probably interferes at a step preceding the divergence in the pathways to “drosopterin” and sepiapterin biosynthesis. Lifschytz and Falk (1969a) also analyzed uric acid accumulations in whole animals as a function of increasing age in sepia- homozygotes and pune-;sepia- double homozygotes. The purines xanthine and hypoxanthine normally accumulate during metamorphosis due to the breakdown of larval tissues and DNA and RNA. These compounds are reduced to uric acid via xanthine oxidase (rosy) and then excreted. In addition to encoding xanthine oxidase, the rosy locus also encodes xanthine dehydrogenase, a NADH-linked dehydrogenase, which catalyzes the conversion of dihydropterin to dihydroxanthopterin and pterin to isoxanthopterin (Figure 6.4). Isoxanthopterin is found in large quantities in insects and may be a storage form of nitrogen. Isoxanthopterins and “drosopterins” have no vital functions in Drosophila; their complete absence in rosy and sepia mutants, respectively, is not a lethal condition. The xanthine oxidase activity may be derived from proteolytic cleavage of xanthine dehydrogenase (Amaya et al., 1990; Shinoda and Glassman, 1968). The rationale behind measuring uric acid levels in sepia- and in pune-;sepia- was that if an early step in the conversion of guanosine into pteridines was defective, then a progressive increase in the products of guanosine catabolism might be seen. Indeed, a progressive increase in uric acid relative to the amount found in wild type was observed in prune- mutants and in sepia- mutants. In prune-;sepia- double mutants, the amount of uric acid accumulated was even more than than measured in prune- or sepia- homozygotes (Lifschytz and Falk, 1969a). In addition to the increased levels of uric acid seen in prune mutants, the levels of isoxanthopterins were increased with respect to wild type (Evans and Howells, 1978). These increases suggest that the activity of xanthine dehydrogenase/xanthine oxidase in pune mutants is increased with respect to that of wild type, which may be a reflection of an increase in GTP concentrations. However, direct measurements of GTP concentrations have yet to be performed in prune-

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or in prune/Kilkr of prune larvae; furthermore, whole organism concentrations of

GTP may prove meaningless if the affect of mutations in prune is tissue specific [which is the case for the prune/Killer of prune interaction (section IV)].

In rosy- mutants, the lack of xanthine dehydrogenase/xanthine oxidase should cause hypoxanthine to accumulate instead of uric acid. In pune-;rosydouble mutants, 55% more hypoxanthine was seen to accumulate over the amount in rosy- homozygotes, indicating that the assays for uric acid were indeed measuring an increase in the activity of the catabolic path of guanosine. However, it does not follow that increased GTP catabolism would lead to increased levels of hypoxanthine since Drosophila does not have a GMP reductase activity (D. Nash, personal communication). One would have to assume that the effect of accumulating GTP might lead to increased catabolism of unconverted IMP, causing the increase in hypoxanthine/uric acid.

G. “Drosopterin” biosynthesis is dependent on de novo biosynthesis of GTP It has been suggested that one function of the isoxanthopterin and “drosopterins” compounds is to provide an efficient method for the removal of purines which accumulate during metamorphosis via histolysis of larval tissues. However, for the case of “drosopterin” biosynthesis, this may not be true. Mutations in the de novo GTP biosynthetic pathway result in decreased levels of “drosopterins.” For example, hypomorphic mutations in both raspberry and burgundy cause a reduction in the level of “drosopterins” (Ferre et al., 1986), and both mutants have dark ruby eye colors (null mutations in either of these genes are lethal). raspberry encodes an inosine monophospate dehydrogenase, which is the first enzyme in the de novo purine biosynthetic pathway specific for guanylate biosynthesis (Nash et al., 1994; Sifri et al., 1994; Slee and Bownes, 1995). burgundy encodes a protein with similarity to GMP synthase (Chomey and Nash, 1996), another enzyme involved in the de novo biosynthesis of guanylate. Furthermore, semilethal mutations in Prat, Drosophila ade2, and Drosophila ade3, which together encode enzymes catalyzing the first 5 reactions of de novo purine biosynthesis, are purine auxotrophs which also have reduced red eye pigments (Clark, 1994; Tiong et al., 1989; Tiong and Nash, 1990). These data suggest that de now-synthesized GTP, not GTP derived from the salvage pathway, is utilized in the biosynthesis of “drosopterins.” Further support for the notion that “drosopterin” biosynthesis is dependent on de novo biosynthesis of GTP comes from the fact that salvage of guanine residues is poor in Drosophila. There is essentially no guanine-phosphoribosyltransferase (or hypoxanthine-phosphoribosyltransferase) activity in fly extracts (Becker, 1978; Becker, 1980; Johnson et al., 1980; Moiseenko and Kakpakov, 1974). In addition guanosine kinase activity is minimal (D. Nash, personal com-

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munication). Therefore, by assumption, most of the GTP found in flies is made de novo from IMP, and therefore there is little option but to utilize the de now0 guany-

late pathway for pteridine biosynthesis. If “drosopterin” pigments are exclusively derived from GTP synthesized via the de now biosynthesis pathway rather than from the salvage pathway, then reduced biosynthesis of “drosopterins” should increase the general pool of GTP, unless the block in “drosopterin” biosynthesis precedes the biosynthesis of GTP. Assuming that this de nova synthesized GTP is not quantitatively converted into “drosopterins” in prune- mutants, the increase in uric acid and isoxanthopterin concentrations measured in prune- mutants and the increase in hypoxanthine concentrations measured in prune-;rosy- mutants may be an indication that prune+ activity is downstream of GTP biosynthesis. However, as mentioned previously, GTP concentrations in prune mutants have yet to be determined directly.

H. Reductions in “drosopterin” pigment accumulation have many causes Mutations in more than 50 different genes affect the accumulation of “drosopterin” pigments. Those mutations which affect enzymes directly involved in the biosynthesis of “drosopterins,”e.g., sepia and clot, produce an eye color phenotype. Mutations affecting processes farther upstream to the direct biosynthesis of “drosopterins,” e.g., purple and Punch, also produce an eye color phenotype. Null mutations in these two genes are lethal, and in the case of some Punch mutants, the eye color phenotype is a tissue-specific effect of the mutation. Perhaps “drosopterin” biosynthesis is sacrificed at the expense of the vital tetrahydrobiopterin biosynthetic pathway in situations where neopterin triphosphate is lime icing. (This may also be the case for mutations in raspberry, burgundy, and other mutations in the de now biosynthetic pathway discussed in the previous section.) More likely, the lethal phenotype and eye color phenotypes caused by different mutations in Punch and perhaps other enzymes in this pathway may be a reflection of the dual function of this metabolic system. One (essential) function of the pathway may be to produce pteridines which are utilized in housekeeping roles (e.g. cofactors for essential enzymes), while another (developmental) functions may lead to the production of pteridine eye pigments. Why rosy mutants have an eye color phenotype has long been a mystery since neither the substrates (dihydropterin, pterin, hypoxanthine, or xanthine) nor the products (dihydroxanthopterin, isoxanthopterin, xanthine, or uric acid) are pigmented, and rosy is not likely to catalyze a reaction which is directly involved in “drosopterin” biosynthesis. [Actually Reaume et al., (1989) has proposed that “XDH might serve as a carrier molecule bringing an eye pigment precursor in the form of an enzyme substrate to the eye at the time of pigment formation.” However, this issue has not been resolved.]

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Mutations in cinnamon, maroonlike, and low xmthine dehydrogenase also cause reductions in “drosopterin” accumulation. Mutations in these genes affect the biosynthesis of the essential, nonpigmented pteridine molybdenum cofactor required for rosy activity (Kamdar et al., 1994; Schott et al., 1986; Stilvaletta et al., 1988; Wahl et al., 1982).The eye color phenotypes of these mutants may simply be due to the loss of rosy activity, and the reduction in “drosopterin” accumulation in these mutants might than be mechanistically similar to the reduction of “drosopterins” in rosy mutants. It is interesting to note that mutations affecting the biosynthesis of one class of pteridine compound (molybdenum cofactor) can affect the biosynthesis of another class of pteridine compound (“drosopterins”), which is synthesized via a different branch of the metabolic pathway. The pteridine biosynthetic pathways which have been elucidated in Drosophih are depicted in Figure 6.4. The biosynthesis of all these pteridine compounds proceeds from GTP via a dihydroneopterin triphosphate intermediate. Less is known about the biosynthesis of molybdenum cofactors; it is not known if their biosynthesis also proceeds from GTP via a dihydroneopterin triphosphate intermediate (Rajagopalan and Johnson, 1992). Alternatively, the reduction of “drosopterin” pigments in rosy mutants may be due to feedback inhibition by pterin or dihydropterin. These xanthine dehydrogenase substrates also accumulate in cinnamon, maroonlike, and low xunthine dehydrogenase mutants. GTP cyclohydrolase from mammalian sources and E. coli has been demonstrated to be inhibited by pterins. Thus inhibition by pterin or dihydropterin might reduce the available pool of dihydroneopterin triphosphate, which might then be utilized in the tetrahydropterin synthesis pathway at the expense of “drosopterin” biosynthesis. However, little isoxanthopterin mutants (Figure 6.4) have reduced pterin accumulation with respect to that of wild type (Ferre et al., 1986), yet “drosopterin” levels are also reduced in these mutants. A variety of mutated genes can indirectly affect the accumulation of “drosopterin” pigments. Haw mutations in prune affect the accumulation of “drosopterins” is not known. It is interesting to note that prune mutants, like raspberry mutants, have a reduced rate of activity of GTP cyclohydrolase (Punch) as analyzed in extracts of fly heads (Mackay and O’Donnell, 1983). The eye color phenotype of raspberry mutations was suggested by Nash et al., (1994) to be a reflection of guanine nucleotide deficit due to reduced IMP hydrogenase (raspberry) activity, which causes the activity of GTP cyclohydrolase to be reduced, which in turn reduces the level of pteridine biosynthesis specifically “drosopterin” biosynthesis. The reduction of GTP cyclohydrolase activity may operate at the level of enzyme production or, as proposed by Nash et al., (1994), by a “gating” mechanism which, for example, might favor the biosynthesis of the essential pteridine compounds over the biosynthesis of “drosopterins.” Such a “gating” effect might especially be noticed in the eye, since the pigment production in this tissue is enormous. The reductions in GTP concentrations in raspberry mutants may also

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be eye- or tissue-specific. Indeed Nash et al., (1994) have evidence that IMP dehydrogenase, an essential enzyme, is regulated in a tissue-specific manner. In contrast, the analysis of some prune mutations suggests that prune is not an essential gene and that prune mutants are nulls. Therefore, the effects of mutations in prune may likely affect all the tissues in which prune is normally expressed. Answers to how or if prune influences GTP cyclohydrolase activity awaits the identification of the function ofprune, and how or if this observation is related to theprunelKiller of prune interaction has yet to be determined. Accumulated biochemical data from a variety of different systems reveal a considerable evolutionary similarity among the enzymes which utilize pteridine cofactors and among the enzymes which synthesize them. The pteridine biosynthesis pathways are tightly regulated, with feedback inhibition and/or induction by products from other branches of the pteridine pathway or from other catabolic/metabolic pathways. In addition to examples mentioned previously, Drosophila sepiapterin reductase is inhibited by N-acetyl serotonin (Primus and Brown, 1994) which is a biogenic amine whose synthesis is dependent on the tetrahydrobiopterin-binding enzyme tyrosine hydroxylase. The mammalian GTP cyclohydrolase is inhibited by tetrahydrobiopterin [as is the E. coli GTP cyclohydrolase (Schoedon et al., 1992)] and other pterins and is stimulated by phenylalanine (Harada et al., 1993; Shen et al., 1988). Thus the reduction of “drosopterin” pigments in prune mutants may be due to the loss of an enzyme responsible for their biosynthesis, to the loss of a positive regulator of an enzyme responsible for “drosopterin” biosynthesis, or to the loss of an enzyme not directly involved in “drosopterin” biosynthesis but whose accumulated substrate causes feedback inhibition of “drosopterin” biosynthesis.

IV. THE pruneRMer of prune LETHAL GENETIC INTERACTION prune/Killer ofprune animals die during the third larval instar. The animals live for an extended period of time in third instar, as long as 3 weeks at 20°C as opposed to 5 days for a wild-type larvae, and do not undergo pupariation. [Some references report an earlier stage of lethality for prunelKilkr of prune animals (Hackstein, 1975; Lifschytz and Falk, 1969a; Sturtevant, 1956). This discrepancy may simply be a reflection of the media upon which these animals are reared.] During this extended third instar period, fat body reabsorption persists, causing the larvae to appear transparent, and melanotic pseudotumors accumulate randomly within the larvae. Melanotic pseudotumors are normally formed as a result of a host cellular immune response. Invading microorganisms or damaged host tissues are encapsulated and subsequently melanized by hemocytes. Melanotic pseudotumors do not necessarily result from an uncontrolled overproliferation of hemocytes.

6. prunefliller of prune

23 7

The melanin deposits of these pseudotumors are due to the activity of phenol oxidase which is released by hemocytes and which possess both tyrosinase (monophenol monooxygenase; monophenol L-dopa:oxygen oxidoreductase; EC 1.14.18.1) activity and o-diphenol oxidase (1,2-benzenediol:oxygen oxidoreductase; EC 1.10.3.1) activity. Melanin biosynthesis is used by a wide variety of plants and animals in an evolutionarily conserved pathway to fight off invaders (Prota, 1992) and for pigmentation. (In insects, phenol oxidase is also involved in sclerotization of the cuticle, which is necessary for protection and support of invertebrate tissues.) Melanotic pseudotumors can form as a result of normal immune response to damaged host tissue or as a result of an aberrant immune response due to defects in hemopoietic organs or hemocytes. The mechanism by which the melanotic psuedotumors of pune/Killer of prune lethal larvae arise is not known, and it is not known whether the aberrant immune response is a direct cause of lethality of prune/Kilkr of prune larvae. Extracts of wild-type larvae form an insoluble, black pigment when exposed to air. Extracts of uwdKpnhomozygous mutant larvae also produce this pigment, which accumulates to greater concentrations than that of wild-type extracts. Homozygous prune extracts accumulate this melanin pigment, but to a lesser degree than wild type. However, prune/Killer of prune lethal larvae do not accumulate melanin (Hackstein, 1992; L. Timmons and A. Shearn, unpublished). This defect in melanin production may be due to the inhibition of phenol oxidase (Hackstein, 1992), yet the melanization process in the hemocytes which form melanotic pseudotumors is not affected in prune/Killer of prune larvae. The black, insoluble pigment was observed in extracts of whole wild-type, prune, and awdKPn larvae, whereas melanotic tumors are observed in intact animals. Hence, these observations may reflect tissue-specific inhibition of phenol oxidase in prune/Killer of prune larvae.

A. The prune/l(//er of prune interaction is not lethal for all cell types Several groups have performed genetic analyses of the prune/Kilkr of prune lethal interaction in order to determine what tissue types in the fly are sensitive to killing by this combination of genotypes. The first such study was by Lifschytz and Falk (1969a). The technique they chose was to induce small patches of prune/Killer of prune tissue by radiation-induced somatic recombination. Larvae of the genotype y m sc prune sn / ; / cauwdKPnwere irradiated at different developmental stages. Somatic recombination of the X chromosome in these heterozygous cells should resolve into clones homozygous for prune- and sister clones which are wild type. Because the irradiated cells were also heterozygous for awdKPn,the prune/Killer of prune lethal genotype was thus produced in recombined clones, which were marked with yellow and singed to facilitate their identification in the wild-type background of the adult cuticle. The size and locations of homozygous yellow

++

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singed patches (prune/Killer ofprune cells) on the adult cuticle were noted. Two results were significant in this analysis. First, prune/Killer of prune clones were noted on adult cuticular structures. This indicates that either the prunelKiller of prune genotype is not lethal for imaginal cells or the prune/Killer of prune lethal condition is nonautonomous-that the prune/Killer of prune cells were supported by the surrounding wild-type epithelia. Second, the size of some of the prune/Kilkr of prune cuticular clones was noted to be large. This is an indication that the X chromosome recombination event took place early in the life of the imaginal disc. Lifschytz and Falk (1969a) performed the same experiment as just &scribed utilizing an unstable X chromosome, Xc2, In(]) wWCf,which is frequently lost during mitosis. Loss of an X chromosome by a Drosophila female cell results in male-specific development, which is not a lethal condition. Thus in a female of the genotype Xc2, In( 1) wvcf/prune2 ; / ca awdKpn,gynandromorphs are produced when the Xc2 chromosome is lost in somatic cells, and the male cells in these gynandromorphs are genotypically prune/Killer of prune. The advantage of this method over radiation-induced recombination is that the autosomal chromosomes remain unaffected, so there is no chance of producing prune- homozygous clones that are not awdKPn;also, even larger clones can be produced. Indeed the investigators found half-body gynandromorphs, indicating that very early in the life of imaginal discs (two cell stage), these clones are insensitive to the lethal effects of the prunelKiller of prune genotype. prune- eye imaginal discs transplanted into metamorphosing awdKPn hosts were autonomous with respect to eye color and pteridine accumulation. The same was also true for awdKpneye imaginal discs transplanted into metamorphosing prune- hosts (Grell, 1958). prunelKiller of prune discs transplanted into wildtype metamorphosing hosts also differentiate into recognizable adult structures (Hersperger and Shearn, unpublished obversation). This is a meaningful result as prune/Kilkr ofprune animals normally do not live past the third instar stage and, therefore, discs of these animals never have the opportunity to undergo metae morphosis. The results from these experiments also imply that the prune/Killer of prune genotype is not lethal for all cell types or perhaps the lethality can be rescued by the wild-type cells of the host. Several methods are now being employed in order to determine which tissues in the larvae are sensitive to the prunelKiller of prune lethal condition and which of these is responsible for the death of the entire organism (the “lethal focus”). One method is simply to determine the tissues in which both Prune and AWD/KPN proteins are expressed. Because AWD is expressed in most tissues of the third instar larvae and because very small amounts of Prune protein are required to rescue the prunelKiller of prune interaction, any conclusion based on this method would require verification by another method. So another method, the yeast GAL4 targeted expression system (Brand and Perrimon, 1993), is being employed to reproduce the pune/Killer of prune lethal genotype in a subset of tissues

+

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6. prune/KilIer of prune

ENHANCER-GAL4 LINES 74 (Shearn) 76 (Shearn) 2-371 (Shearn) 718 (Perrlmon)

GENOTYPE:

y prune-w

.’

7

f

+

[

ENHANCER-GAL4

UAS-awdKpn~

Figure 6.5. Mosaic analysis of the prunelKilkr of prune interaction. Individuals that are genetically prune- which also express awdKononly in imaginal discs survive. awdKPnexpression in imaginal discs is obtained using the imaginal disc-specific yeast GAL,-4 enhancer traps listed. These GAL-4 enhancer traps are used to drive awdKPnexpression from a awdKl’n cDNA-containing transgene under the regulatton of GAL4 promoter elements.

in an otherwise viable animal. In this system, expression of the yeast GAL4 transcription factor is under the control of chromosomal enhancers which are near the insertion site of a GAL4-containing transgene. The enhancer-driven GAL4 transcription factor can then be used to drive expression of awdKpnwhich is under the control of a GAL4 promoter in a prune- fly. Our preliminary results also demonstrate that imaginal-derived tissues are not susceptible to the prune/Killer of prune lethal genotype: enhancer traps which result in GAL4 expression, and thus awdKmexpression, only in imaginal tissues of prune- larvae are completely viable (Figure 6.5). If, however, the enhancer causes expression of GAL4 (and thus awdKpn)in the “lethal focus” of this prune- fly, the animal will die. Other GAL4 enhancer traps have been obtained which confer lethality to prune- larvae by GAL4-directed expression of awdK@ in a subset of cells of the larvae. This is an indication that the grune/KilIer ofprune interaction is cell autonomous, and by analyzing a large number of such GAL4 enhancer trap expression patterns, the “lethal focus” can be identified. It has been suggested that this interaction might also be a useful tool for inducing lethality in cells of Drosophila in order to monitor the developmental

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consequences of the loss of these cells. However, this kind of induced lethality is not a tool that is applicable to all cell types as the prune/Killer of prune genotype is not lethal for all cell types. Creating the PrunelKiller of prune genotype by genetic engineering will only be useful in those tissues that are sensitive to the lethal interaction (cells of the "lethal focus"). Because prune/Killer of prune lethality is 100%penetrant, this system is a convenient tool for the analysis of X chromosome nondisjunction events. All the progeny from the standard prune/Killer of prune cross are females, but occasionally a few spontaneous awdKpn revertant males are observed. These males have prune eye color, are fertile, and contain mutations in the awdKPngene that do not complement awd null alleles. In addition, some male progeny with wild-type eye color are observed which are not fertile and which represent X chromosome nondisjunction events that occurred in the prune mother. The numbers of nondisjoined males from this cross are comparable to the numbers of nondisjoined events originally noted by Morgan. A third-site mutation which increases the rate of Xchromosome nondisjunction could easily be scored in the prune/Killer of prune cross.

B. When is prune/Killer of prune lethal? prune/Killer of prune males generated from the cross in Figure 6.1 die during third instar. These lethal males contain a maternally supplied prune+ product. If prune/Killerofpune males are derived from mothers who are heterozygous for both prune and awdKPnmutations, lethality also occurs during third instar, even though

these males as embryos contained a maternally supplied awdKpn product. These latter males do not die earlier than third instar as they also have a maternal supply of prune+. In order to test earlier stages for sensitivity to the prune/Killer of prune interaction, it is necessary to avoid maternal deposition of prune+. One method that has been employed to test for earlier sensitivity of the prune/KiEEer of prune interaction was to generate prune- homozygous clones in the ovaries of prune-/prune+ ovoD;awdKpn/awd+heterozygous females by somatic recombination. m o D is a dominant female sterile mutation, and ovaries containing the ovoD mutation do not develop egg chambers. Somatic recombination of the X chromosome in these heterozygous females will not only provide for removal of the ovoD mutation, but will also produce homozygous prune- sister clones which should also contain the awdKPnmutation. Thus, any eggs produced by somatic recombination in these females will have a maternal supply of awdKP" but no maternal supply of prune product, and after fertilization of these eggs, the lethal stage can be assessed. Embryos derived from these PrunelKiller of prune eggs do not survive and development of these embryos never proceeds beyond a few nuclear divisions (L. Ttmmons and A. Shearn, unpublished observations). This is true even for prunelKiller of prune eggs that have been fertilized with a wild-type sperm,

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which therefore have the potential to express prune+ product zygotically. In addition, the somatically recombined females produce prune/Kilkr of prune ovarioles which appear normal by DAPI staining and the eggs which are laid are fertilized. The nature of the defects in these prune/Kilkr of prune embryos is under investigation.

C . Why is prune/Ki//er of prune lethal? Several observations related to the prunelKilkr of prune lethal interaction are key to the mechanistic understanding of the lethal interaction. Reversion of the prune/Kilkr of prune interaction has been accomplished only by eliminating or severely reducing the activity of the KPN protein. This, in addition to the fact that awdKpnhomozygotes are completely viable, indicates that the mutant KPN enzyme has a neomorphic activity, which causes lethality only in a prune mutant background, not in a wild-type background. The neomorphic activity induces a toxic condition in some, but not all, of the tissues of a prune mutant which eventually leads to death of the animal. Furthermore, this toxic condition can be generated in early embryos. An important missing piece in the prune/Kilkr of prune puzzle is the function of Prune. The results of all the experiments on prune and prune/Kilkr of prune suggest the following model. It is possible that in prune/Kilkr ofprune lethal animals some biochemical pathway becomes abnormally activated or inhibited by a prune/Kilkr of prune-generated “toxin” to elicit cellular responses that eventually cause lethality. A “toxin” which accumulates in prunelKilkr of prune animals has not been identified, and this will be key to identifying the biochemical pathways which produce this “toxin” and which are sensitive to this “toxin.” W h y prune+ animals are not sensitive to the neomorphic activity of KPN is another part of the puzzle. The neomorphic activity of KPN might be thwarted subsequent to KPN action by “detoxification” of an abnormal KPN product or prior to KPN action by preventing the accumulation of a potential substrate which can be utilized by KPN (but not AWD) and converted to a toxin. Two models of the prune/Kilkrofprune lethal interaction can be proposed (Figure 6.6). The first model favors the notion that prune encodes an enzyme whose substrate accumulates and inhibits “drosopterin” production. According to this model, an accumulated Prune substrate (X) is converted into a “toxin” by neomorphic KPN activity. In prune+ animals, the substrate (X) is efficiently converted to product (Y) and no substrate (X) accumulates. One attractive feature of this model is that the prune eye color phenotype and the tissue specificity of the prune/Killer of prune interaction can be explained. Only those tissues which express KPN, Prune, and enzyme (2)-a hypothetical enzyme responsible for the production of Prune substrate (X)-will produce the “toxin,” and only those tissues expressing factors sensitive to the “toxin” will be lethal in prune/Kilkrofprune

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animals. The KPN/Prune/enzyme 2-containing cells and “toxin”-sensitive cells may be identical. In the second model, the Prune enzyme performs its normal activity and also can “detoxify”an aberrant product of the neomorphic activity of KPN (Figure 6.6). According to the model, this aberrant KPN “toxin” is similar enough to the normal Prune substrate (X) that it is recognized by Prune. The tissue specificity of the prune/Kilkr of prune interaction can also be rationalized by this model. Only those tissues which both produce the substrate for the aberrant KPN activity (i.e., express enzyme Z) and are also sensitive to the KPN “toxin” would be affected. In both models, enzyme Z is present in cells also expressing Prune; otherwise the awdKPnmutant would be lethal in a prune+ background. The fact that so little Prune is required to rescue prune/Kilkr of prune lethality indicates that, according to Model 1, the Prune enzyme is either highly processive or the Prune substrate is not abundant or, according to Model 2, the Prune enzyme is either highly processive in “detoxifying”the KPN “toxin” or that the “toxin” is not abundant and is therefore very potent in the absence of Prune. The imaginal cells and other cells in the larvae that are genotypically prune/Killer ofprune do not die, do not cause lethality of the entire animal, do not represent the “lethal focus,” and, accordingly to this model, would not contain the essential components of the biochemical pathway which produce the prune/Killer of prune “toxin.” The most likely biochemical pathway responsible for producing such a “toxin” is the pteridine biosynthetic pathway as both awd and prune are likely to be enzymes which catalyze reactions in this pathway: awd, the biosynthesis of GTP, and prune, an enzyme with unknown functions whose absence causes a reduction of “drosopterins.” Examples of noncompetitive inhibition by an aberrant pteridine pigment exist in mammalian systems: in human and rat cells, “7-tetrahydrobiopterin,” a nonenzymatically rearranged version of accumulated “6-tetrahydrobiopterin,” acts as a potent inhibitor of amino acid hydroxylases, a condition which can cause vitelligo (Davis eta!., 1991, 1992; Davis and Kaufman, 1991; Schallreuter et al., 1994). Inhibition of amino acid hydroxylases could be a lethal condition in Drosophila; however, this is not likely to be the cause of lethality for prune/Kilkr ofprune larvae. Hackstein ( 1992) measured the concentrations of free amino acids of third instar prune/Kilkr ofprune lethal males and their viable sisters. The concentrations of all of the amino acids measured were either similar for the two types of larvae or higher in lethal larvae than in nonlethal larvae, except for tyrosine, phenylalanine, and arginine concentrations which were 80,27, and 35% reduced, respectively. Since these three amino acids are substrates for tyrosine hydroxylase, phenylanine hydroxylase, and nitric oxide synthase (tryptophan concentrations were not measured), the reduction in their concentrations indicates that these enzymes are functional in lethal larvae and may even have increased activity with respect to nonlethal larvae. Therefore, tetrahydrobiopterin

Figure 6.6. Models of the p u n e / K i k r of pune genetic interaction

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biosynthesis is not likely to be affected in lethal larvae. The levels of isoxanthopterins in prune/Kilkr of Dune lethal larvae, however, are drastically decreased with respect to those of nonlethal larvae (Hackstein, 1975, 1992) whereas levels of isoxanthopterins are increased in prune mutants, as mentioned previously. Complete loss of isoxanthopterins is not a lethal condition; however, this aberrant metabolism of isoxanthopterin in lethal larvae is an indication that pteridines other than “drosopterins” are affected in lethal larvae. In 1956, when Sturtevant originally discovered pune/Killer of pruneeinduced lethality, he proposed a model which could explain the lethality of these individuals. Sturtevant’s original proposal was that Killer of prune had a neomorphic activity which caused lethality ofprune mutants. Although not rich in mechanistic detail, his model has not been disproven and was based on careful analysis of the progeny of only a few genetic crosses. Analysis of the prune/Killer ofprune interaction subsequent to Sturtevant’s discovery has been and will be to define the nature of the “toxin” and the factors which are affected by the “toxin.” The end results of these kinds of analyses may provide further information about the pteridine biosynthetic pathway, its regulation, and its effect on other pathways.

Acknowledgments The authors thank Dr. Gene Brown and Dr. David Nash for critical reading of the manuscript prior to publication and Dr. Nash in particular for enlightening discussions of purines in Drosophila.

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Chiasmata, Crossovers, and Meiotic Chromosome Segregation Carol A. Bascom-Slack,' Lyle 0. Ross1.* and Dean S. Dawson3

Department of Microbiology and Molecular Biology Tufts University Boston, Massachusetts 02 111

I. INTRODUCTION In most diploid organisms, genetic exchange is essential for successful sexual reproduction. Reciprocal recombination leading to exchange between homologous chromosomes plays a critical role in their partitioning in meiosis I. T h e goal of this chapter is to consider the ways in which meiotic reciprocal recombination might contribute to the fidelity of chromosome segregation. Meiosis is a specialized division that results in the production of haploid cells during sexual reproduction. Premeiotic cells replicate their genetic material so that they are 4C. In meiosis I, homologous chromosomes pair and segregate away from each other to opposite poles of the cell (Fig. 7.la). In most eukaryotes, homologous chromosome pairs (bivalents) experience meiotic exchange following DNA replication, but prior to meiosis I segregation [see Hawley (1988) for a list of exceptions]. In meiosis 11, replicated sister chromatids segregate away from each other so that four haploid genomes are obtained. Studies performed in a number of experimental organisms have shown that mutations that alter the frequency and distribution of meiotic exchanges result in chromosome segregation errors at meiosis I [reviewed by Baker et al. (1976); Hawley, 19881. These meiotic missegregations result in aneuploid gametes and, as a consequence, greatly reduced fertility. Two types of meiotic segregation errors that occur at elevated frequencies in the absence of 'The contribution to this chapter by these authors was equal. 2Current address: Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030. 3T0 whom correspondence should be addressed.

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Figure 7.1. Chromosome segregation in meiosis, (a) Correct disjunction of a pair of homologous chromosomes. (b) Meiosis I nondisjunction. The absence of crossovers between homologous chromosomes allows them to segregate randomly at meiosis I. When the homologous chromosomes are linked (by a chiasma), they are referred to as a bivalent. When they are unlinked they are called univalents. The result (for the model chromosome shown) is two cells with In 1 chromosomes and two cells with In - 1 chromosomes. (c) Precocious separation of sister chromatids.

+

meiotic exchange are nondisjunctions (Fig. 7. lb) and precocious separation of sister chromatids (Fig. 7 . 1 ~ see ; Baker et al., 1976). In meiosis I nondisjunction, the homologous chromosomes segregate randomly with respect to each other at meiosis 1. In precocious sister separation, sister chromatids disjoin in meiosis I, instead of meiosis 11. Our current understanding of the role of meiotic recombination in segregation would not be possible were it not for our ability to assay the consequences of recombination both genetically and cytologically. For the purposes of this chapter, we will use the term crossover to refer to the breaking and rejoining of DNA strands that results in a reciprocal exchange between a pair of homologous chromosomes. The term chiasma will refer to the cytological evidence of a meiotic crossover or exchange. An exchange is a meiotic crossover between chromatids of homologues and involves two of the four chromatids present after DNA replication. The idea that chiasmata are the cytological outcome of exchanges was not always widely accepted. Chiasmata were originally described by Janssens ( 1909) as the cytologically observable “crosses” or nodes between the arms of chromosome pairs during late prophase I. From this, Janssens developed the idea that chiasmata are formed at the sites of genetic exchange. This idea, called the chias-

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matype theory, sparked a debate that continued for over half a century. A n accumulation of indirect evidence in favor of the chiasmatype theory generally settled the controversy [fora comprehensive review, see Whitehouse ( 1969)l. These data primarily came from two types of experiments. In one, the products of exchange between homologous chromosomes with heterozygous inversions were monitored cytologically. In these experiments, exchange within the inversions generated diagnostic chromosomal products (i.e., acentric and dicentric products). The frequency with which these were observed in anaphase I corresponded to the frequency with which chiasmata were observed within the inversion. Experiments of the second type were meticulous comparisons of chiasma number and distribution with exchange number and distribution. Chiasma data were gathered by observing cells fixed in meiosis I, whereas information regarding exchanges could be obtained by examining cells fixed in meiosis 11. These experiments were performed by using organisms with cytologically distinguishable homologous chromosomes. For example, if one chromosome has a terminal deletion that makes it recognizably shorter than its homologue, then evidence that an exchange has occurred will be provided by the appearance in meiosis I1 of two univalents, each with one long and one short chromatid. By comparing the chiasma and exchange data sets, it was clear that they occurred not only at the same frequency but also in similar positions on homologous chromosomes [one elegant example of this type of study is found in Brown and Zohary (1955)l. The most convincing evidence that chiasmata are sites of exchange was provided by Tease and Jones (1978). By using techniques to differentially label sister chromatids, they observed that chiasmata were sites where chromatids were broken and rejoined to nonsister chromatids. The majority of now visible crossovers coincided exactly with the positions of chiasmata in the bivalents obe served. Thus, the correlation between chiasmata and meiotic exchange was clearly demonstrated.

II. EXCHANGES ENHANCE THE FIDELITY OF MEIOTIC CHROMOSOME SEGREGATION Throughout the first half of this century, much evidence was accumulated that chiasmata are necessary for proper chromosome segregation to ensue; since that time, several excellent reviews of the supporting data have been published. In this section we will summarize that work. The following sections will focus on the manner in which exchange contributes to proper chromosome segregation. As chiasmata were shown to be the cytological manifestations of crossovers, it became apparent that crossovers (exchanges) must play a role in ensuring disjunction. The data that demonstrate this point have been accumulated from many sources. Most notably, many mutants with decreased levels of exchange and sub-

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sequent increases in meiosis I nondisjunction have been identified (Hawley, 1988). Additionally, the recombination frequencies of spontaneous, nondisjoined chromosomes in wild-type organisms have been compared to the recombination frequencies of properly disjoined chromosomes (Merriam and Frost, 1964). In these experiments, it was observed that nondisjoined chromosomes either had not undergone exchange or had greater numbers of exchanges than disjoined chromosomes. These results led to the idea that exchanges are necessary to ensure chromosome disjunction. Cytological studies have also allowed the observation of the fate of achiasmate chromosomes in mutant organisms. In general, achiasmate chromosomes fail to remain paired and segregate randomly with respect to each other at anaphase I. The ability to make model chromosomes and more easily manipulate natural ones has provided a vehicle for investigators to ask questions that previously could not be addressed in natural systems.

A. Use of recombination mutants The accumulation of a wealth of mutations in a multitude of organisms has allowed investigators to observe the consequences of removing exchange from meiosis. The list of such mutations is too long to review here; however, clear discussions of several recombination-defective mutants have been presented by Baker et al. (1976) and Hawley (1988). In most organisms, mutations that result in a reduction in meiotic exchange are correlated with reductions in the viability of the gametes produced. This is the expected outcome if, in fact, exchanges are necessary for correct chromosome disjunction, and if aneuploidies, due to missegregations in the absence of exchange, are generally lethal. Decreases in gamete viability make the study of many meiotic mutations extremely difficult. This problem has been circumvented in various ways. In maize, for example, nondisjoined chromosomes can be assayed for reciprocal recombination cytologically. Strains in which one (or more) homologue is heterozygous for a distal “knob” of cytologically visible heterochromatin can be monitored cytologically for exchanges. For example, Miller (1963) demonstrated that a class of mutants in maize, called asynaptic (as), led to univalents at diakinesis that were never heterozygous for the knob, whereas normally segregating bivalents were heterozygous. This indicates that, in the absence of exchange, nondisjunctions are likely. Drosophila has been a productive experimental organism for studying exchange and segregation mutations, in part because it has only four chromosomes. This small number of chromosomes, even when segregating randomly, will result in a reasonable number of gametes with a correct complement of chromosomes. Furthermore, Drosophila can tolerate aneuploidies for two of its four chromosomes, which further increases the number of “viable” gametes that result in the absence of exchange. In yeast, the viability problem can sometimes be circumvented by tak-

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ing advantage of spoJ3 mutations (Malone and Esposito, 1981; Klapholz et al., 1985).In meiosis, spo13 strains undergo normal levels of recombination, but then undergo an equational (mitotic-like) rather than a reductional division, resulting in two diploid spores. Bypassing the first meiotic division obviates the need for some gene products that are essential for meiosis 1. For example, yeast with mutations in certain genes involved in the initiation of meiotic recombination (for example rad50 or spof I ; Malone and Esposito, 1981; Klapholz et al., 1985) or synaptonemal complex formation (for example hopJ or red1 ; Rockmill and Roeder, 1988; Hollingsworth and Byers, 1989) that exhibit low spore viability will produce high levels of viable diploid spores if they carry a spol3 mutation as well. Analysis of the resulting diploid spores reveals that they have experienced decreased levels of meiotic recombination. This approach has made it possible to distinguish between certain categories of meiotic recombination mutations. Mutations that allow initiation, but prevent repair, of meiotic recombination events often result in a meiotic arrest that is not rescued by a spoJ3 mutation (for example, r&OS or dmcJ ; Alani et al., 1990, Bishop et al., 1992).

B. Observations of spontaneous nondisjunctions in wild-type organisms Another approach that has been used to examine the importance of exchanges in the disjunction process is to observe the behavior of the occasional spontaneous achiasmate chromosome in a wild-type organism. This is difficult for two reasons. First, in most organisms exchanges are distributed nonrandomly across the chromosomes in meiosis. That is, a limited number of exchanges are distributed such that each bivalent experiences one to a few exchanges per meiosis [reviewed by Carpenter ( 1988)].Because of this regulation, the frequency of spontaneous achiasmate chromosomes is low enough to make their behavior difficult to assay. Second, most organisms are intolerant of aneuploidy, so that finding an experimental system amenable to this sort of analysis can be difficult. In Drosophila, Merriam and Frost (1964) examined the disjunction of X chromosomes in 45,112 female progeny. They found that bivalents with one exchange showed the highest fidelity of segregation [bivalents with one exchange nondisjoined 0.07% (23/29,600) of the time; bivalents with no exchanges nondisjoined 1.1% (24/2095) of the time; and bivalents with two or more exchanges nondisjoined 0.35% (47/13,417) of the time]. X homologues were prone to missegregation under two conditions. First, bivalents with multiple exchanges were 5-fold more likely to missegregate than homologues with single exchanges, possibly due to chromosome entangling. Second, homologous chromosomes without exchanges were 16-fold more likely to nondisjoin than single-exchange bivalents. The level of X homologue nondisjunction would have been higher were it not for the existence of a back-up achiasmate segregation pathway in Drosophila females

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that correctly segregates the nonexchange chromosome four pair and other occasional nonexchange chromosomes [Grell, 1976; reviewed by Hawley and Theurkauf ( 1993)]. The advent of methods for detecting DNA polymorphisms along the length of chromosomes has made it possible to test the correlation between exchange and the nondisjunction of several different human chromosomes. These studies have also begun to shed light on the basis of the correlation between increased maternal age and increased incidence of trisomic births in humans. These studies have shown that while autosomal trisomies can arise through failure in any of a few steps in gametogenesis and embryogenesis (meiosis I and I1 errors in maternal or paternal gametogenesis and postzygotic mitotic errors), the predominant manner by which trisomies are generated is through segregation errors during maternal meiosis I [reviewed in Hassold and Jacobs, 1984; Hassold et al., 1993; Abruzzo and Hassold, 19951. Studies of those chromosome 21 trisomies attributable to maternal meiosis I missegregation have shown that while many errant chromosomes have experienced exchange, a significant fraction probably have not (Sherman etal., 1994).Similarly, although chromosome 18 trisomies are most often due to meiosis I1 failure, in those cases caused by meiosis I missegregation chromosome 18 shows reduced levels of crossing over (Fisher et al., 1993,1995). Additionally, cases of Klinefelter’s syndrome (47,XXY), often attributable to a paternal contribution of both an X and a Y chromosome, have been correlated with reduced exchange between the X and Y during spermatogenesis (Hassold et al., 1991). These studies suggest that, in humans, as in more traditional experimental organisms, exchange is used to enhance the fidelity of meiotic chromosome segregation. It should be noted here that, for some autosomal trisomies, there is no obvious correlation between meiosis I missegregation and reduced crossing over (Hassold et al., 1991; Robinson et al., 1993). In these cases, it may be the failure of the chiasmata to maintain the linkage of homologues until anaphase I that is the culprit (see the following sections). The basis of the elevated incidence of trisomic birth with increasing maternal age remains somewhat mysterious. A comparison of the genetic map lengths of maternal chromosome 21’s from the normal progeny of “younger” and “older” mothers showed that the chromosomes contributed by older mothers had experienced somewhat lower levels of crossing-over at telomeres and pericentromeric regions, indicating an age-related difference in crossover frequency and distribution (Tanzi et al., 1992). An analysis of maternal meiosis I missegregated chromosomes from trisomy 21 progeny, showed that these chromosomes had experienced considerably less recombination in maternal meiosis than had properly segregated control chromosomes. The missegregated chromosomes were less likely to have experienced multiple exchanges and more likely to have zero or one exchange (Sherman et al., 1994). Crossover frequencies along the length of the missegregated chromosomes were reduced when compared to controls, except for

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the distal region of 219. This finding is consistent with the model that chromosomes with internal exchanges are biased to segregate properly, whereas those with a terminal exchange are not. When the data were analyzed as a function of maternal age, a correlation between decreased recombination with increased age was noted. These studies are consistent with the model that the probability of a chromosome being achiasmate and consequently missegregating is elevated if the chromosome is in a mature ovum produced by an older woman. These results are perplexing given the fact that, in humans, meiotic crossing-over in the developing oocytes occurs in the fetal ovary, years before ovulation begins. One model used to explain how the chromosomes in eggs ovulated later in life might be different from those produced earlier is the production line hypothesis (Henderson and Edwards, 1968).The model, which suggests that the order of entry of germ cells into meiosis dictates the order in which they are released as mature ova, has been difficult to test (Polani and Crolla, 1991). Another model proposed to explain the maternal age-dependent nondisjunction of autosomes in humans suggests that two or more factors contribute to a nondisjunction event (Hawley et al., 1994). By this model the ability to produce a fully functional spindle decreases with increasing maternal age. The combination of a compromised spindle and a chromosome pair with zero or only a terminal exchange would lead to an elevated probability of missegregation. Other factors that might decay with time and thus contribute to elevated levels of missegregation include loss of sister chromatid cohesion (which might preferentially affect bivalents with terminal exchanges) or checkpoint mechanisms that monitor bipolar spindle attachment of the chromosomes.

C. Cytological evidence that crossovers are necessary for proper chromosome segregation The stages of meiosis became well defined in the 1930s via cytological examinations in a number of plant and animal species (Fig. 7.2). Although there is some variation from one organism to the next, chromosomes generally condense in early meiosis, with structures referred to as axial elements assembling along individual chromosomes. The axial elements of homologous chromosomes are then synapsed (often concurrently with axial element formation) to form a structure called the synaptonemal complex. Although recombination is initiated early in meiotic prophase (Fig. 7.2), it is not until the end of prophase, at diplotenediakinesis when the synaptonemal complex disappears,that chiasmata joining the bivalent pair are revealed. Soon after these initial observations, investigators began cytological examinations of plant strains with decreased fertility. A class of mutants, referred to as “asyndetic,”defined by the presence of univalents at metaphase I was described.

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Figure 7.2. Generalized time line of meiosis. Events described in this time line have been observed in numerous organisms with the exceptions mentioned. Meiosis begins with a replication of the diploid genome to make a 4C cell. In leptotene, the replicated homologous chromosomes (each chromosome has two identical

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There are two types of asyndetic mutants, asynaptic and desynaptic. Asynaptic mutants have improper synapsis, showing a range of synaptonemal complex formation from no synapsis to almost complete synapsis, whereas desynaptic mutants apparently have normal synapsis, but the bivalents prematurely dissociate at diplotene-diakinesis. It should be noted that these definitions have not always been strictly adhered to (Soost, 1951). Asyndetic mutants often result in achiasmate bivalents, and furthermore, decreases in chiasmata correspond to increases in univalents [reviewed by Baker et al. (1976)l. Observations of cytologically distinguishable homologous chromosomes, for example, one with a knob on one end and the other without, were used to show that in asynaptic maize the observed univalent chromosomes had not experienced an exchange (Miller, 1963). A large number of experiments utilizing different asyndetic mutants all give results consistent with the notion that achiasmate bivalents separate at diplotene-diakinesis, allowing missegregation and chromosome loss [reviewed by Baker et al. (1976)l.

D. Meiotic behavior of model chromosomes in Saccharomyces cerevisiae Most studies of the segregation of natural chromosomes that have failed to undergo exchange are subject to one or more limitations. First, because natural chromosomes carry essential genes, their missegregation leads to dead or less vital gametes and offspring, making it difficult to score missegregations. Second, in most organisms, chromosomes that usually experience exchange do so reliably; in other words, the chances of such chromosomes failing to exchange are very low. The sister chromatids; Fig. 7.1 ) begin to condense, coiling along a cytologically visible proteinaceous structure called the axial element. In Saccharomyces cerevisiae, coincident with axial element formation, double-strand breaks in the DNA, precursors to meiotic recombination, appear ( Padmore et al., 1991). In zygotene, a central core structure begins to form, linking the axial elements of the homologues into a tripartite structure called the synaptonemal complex. Recombination nodules (not shown), which are large proteinaceous structures believed to be responsible for meiotic recombination, begin appearing in zygotene (early nodules) coincident with the appearance of tripartite synaptonemal complex. In pachytene, the synaptonemal complex is complete and late nodules are present. Mature recombinants between homologous chromosomes have been observed in Saccharomyces cerevisiae at the end of or just after pachytene when the first meiotic division occurs (Padmore et al., 1991). In diplotenediakinesis, the synaptonemal complex has been removed, revealing the individual homologues linked by chiasmata, and sister chromatids become apparent. In developing mammalian oocytes, meiosis arrests in a prolonged diplotene, called dictyotene, for a period of many years until the oocyte is released from the ovary sometime following puberty. Prometaphase begins as the bivalents begin to move to the metaphase plate (congression) in a microtubule-dependent fashion. Metaphase begins when all of the bivalents have achieved a bipolar attachment to the spindle. Anaphase is marked by the separation of the bivalents, followed by univalent disjunction to opposite poles of the cell.

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low frequency of nonexchange homologues can be elevated by performing experiments in strains with recombination-deficient mutations. These mutant studies have yielded invaluable contributions to the field, but are limited because they lead to missegregation of chromosomes and, hence, increased inviability. The use of model chromosomes has made it possible to circumvent these difficulties. The advantage of these model chromosomes is that they carry no essential genes required for cell viability, so that when they missegregate it is not necessarily a lethal event. Furthermore, because many of the model chromosomesexperience low levels of exchange, it is possible to examine the segregation of large numbers of noncrossover or single-crossover chromosomes. Experiments using model chromosomes have provided clear evidence supporting the notion that exchanges act to ensure correct chromosome disjunction.

1. Exchange enhances the segregation of model chromosomes Simple tests of the meiotic roles of exchanges in the yeast Sacchromyces cereuisiue have been made possible with the advent of model chromosomes in yeast. Three kinds of linear model chromosomes have been useful in these experiments: teleocentric yeast artificial chromosomes (YACs) composed primarily of human DNA (Sears et al., 1992), bacteriophage A-based artificial chromosomes (YLps) (Dawson et al., 1986; Ross et al., 1992), and shortened versions of yeast chromosome III (mini III) (Fig. 7.3) (Dawson et al., 1986; Ross et al., 1992). In all three cases, pairs of model chromosomes can be established that undergo low levels of exchange, such that the genetic consequences of nonexchange and singleexchange bivalents can be examined by tetrad analysis. In all three cases, nonexchange model chromosomes disjoin in 85-95% of meioses. In cases where the model chromosomes nondisjoin, two spores receive two copies of the model chromosome and two spores receive none (because the YLps, YACs, and mini III’s are nonessential, all spores are viable). By utilizing genetic approaches it is possible to determine the linkage of markers on the nondisjoined model chromosomes and, hence, whether they have undergone meiotic exchange. Model chromosomes that experience exchange show much lower levels of nondisjunction than nonexchange chromosomes, but exchange chromosomes do occasionally nondisjoin. The conclusion from these experiments is that exchange greatly enhances but does not ensure the segregation of model chromosomes.

2. Disjunction of chromosomes in the presence of a third pairing partner Diploid yeast cells that contain three nonhomologous, achiasmate chromosomes partition them as equal partners at meiosis I (Dawson et al., 1986; Guacci and Kaback, 1991; Ross et al., 1996a). This chromosome behavior is reminiscent of

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YACs

Mini Ilk

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YLps

0pBR322 I I yeast DNA in non-native environment

5 kb

large block of native contiguous yeast DNA Mhuman DNA bacteriophage lambda DNA

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Figure 7.3. Scale drawings of model chromosomes used in yeast meiotic segregation studies. (a) YACs (Burke et al., 1987). YACs are constructed by inserting genomic DNA between yeast telomeric sequences that have selectable markers, an autonomous replication sequence (ARS), and a centromere. The YACs used in the studies described here contained human or mouse DNA, were from 50 to 360 kb in length, and were marked with URA3SUPJ J and HIS3, LYS2 and TRPl , or TRPl and URA3. (b) Mini IIJ's are constructed by inserting telomeres at varying distances from the chromosome III centromere by homologous recombination. The mini Ill's used in the studies described here were 72 kb in length (from LEU2 to PGKJ ) and had yeast markers with pBR322 sequences added to their ends (Murray and Szostak, 1983; Dawson et al., 1986). (c) The YLps were constructed by inserting centromeres, telomeres, an ARS, and genetic markers into a bacteriophage A backbone (Murray and Szostak, 1983; Dawson et al., 1986). The YLps are 70 kb in length.

that exhibited by certain sets of three achiasmate chromosomes in the meiosis of female Drosophila (Grell, 1976; Hawley and Theurkauf, 1993). That is, each of the three chromosomes segregates away from the other two with equal frequency (Fig. 7.4).

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1

2 3

33%

1 3

33%

2

3

1 2

33%

Figure 7.4. Segregation patterns expected if three chromosomes act as equal partners in meiosis I.

The ability of exchanges and gene conversion to bias segregation has been monitored in yeast by using strains with three chromosomes that in the absence of exchange act as equal pairing partners. By constructing strains with two homologous model chromosomes, marked such that exchanges between them can be scored, and one nonhomologous model chromosome, it has been possible to explore the role played by homology and recombination in meiotic chromosome segregation. In one such experiment with two YLps and one nonhomologous mini III, when the YLps did not undergo exchange, they nondisjoined at meiosis I 33% of the time. This experiment shows that homologous bacteriophage X sequences do not bias the segregation of nonexchange YLps (Dawson et al., 1986). In a similar experiment with two 70-kb mini III chromosomes and a third metacentric artificial chromosome composed of X phage DNA (Ross et al., 1996a), the mini 111's nondisjoined in 25% of meioses when they did not experience exchange and in 2% when they did. Although 25% nondisjunction is a high level compared to the 2% for exchange chromosomes, it is significantly lower than 33%. This indicates that the homologous yeast sequences bias segregation of nonexchange mini III chromosomes, contrary to the bacteriophage lambda sequences in the YLps. In another experiment with two 70-kb YLps composed of bacteriophage A DNA with a recombinogenic 12.5-kb insert of yeast chromosome VIII and a mini 111, exchanges between the YLps improved their disjunction fidelity from 33% nondisjunction in the absence of exchange to 3% in the presence of exchange. In this experiment, the exchanges between the YLps were largely attributable to the 12.5-kb insert. This insert had genetic markers known to undergo meiotic gene conversion (nonreciprocal recombination). In meioses where the markers showed gene conversion, but not exchange, the disjunction of the YLps was not enhanced, which is consistent with the idea that exchanges and not gene conversions lead to chiasmata (Carpenter, 1984). These experiments show that, for model chromosomes in yeast, homologous yeast sequences influence chromosome disjunction at a low level, while a

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single exchange can greatly elevate the disjunction of model chromosomes at meiosis I.

111. THE ROLES OF RECOMBINATION IN ENHANCING DISJUNCTION What are the roles of meiotic recombination events that allow them to contribute to the fidelity of meiotic chromosome segregation?In meiosis I, homologous chromosomes undergo a complex series of interactions that culminate in their positioning on the meiotic spindle such that at anaphase I they move to opposite poles. Briefly, early in meiosis, chromosomes distinguish their homologues from the other chromosomes in the cell, and they pair tightly (synapse) through the assembly of the synaptonemal complex. In most eukaryotes, the synaptonemal complex is dissolved in diplotene, at which time the chiasmata become visible. Subsequently, in prometaphase, the chromosomes are moved to the metaphase plate where they remain attached to the spindle (Fig. 7.5a) until homologue separation occurs at anaphase I. T h e roles of meiotic recombination events that might contribute to the fidelity of these events are considered in the following sections.

A. The role of recombination in partner recognition and pairing How do homologous chromosomes align in meiosis? Premeiotic chromosomes do not appear to have a random configuration within the nucleus. Centromeres and

Figure 7.5. (a) Bipolar spindle attachment of a homologous pair of chromosomes (a bivalent) joined at a chiasma. The open circles are kinetochores and the thin black lines represent spindle fibers. (b) Unipolar spindle attachment of a bivalent.

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telomeres are maintained in specific locations, resulting in a general alignment of similar regions of the chromosome arms [reviewed by Fussell (1987)l. Furthermore, in at least two cases, Drosophila and Sacchromyces cerevisiue, homologous chromosomes appear to be closely juxtaposed prior to meiosis (Carpenter, 1979; Weiner and Kleckner, 1994). Such arrangements of chromosomes may allow easier comparisons of sequences in homology searches. For the purpose of this chapter, we refer to this early juxtaposition of chromosomes as pairing. The intimate association of homologous chromosomes by synaptonemal complex formation will be referred to as synapsis. The relationship between meiotic chromosome synapsis and recombination has been an area of active discussion. The assembly of a synaptonemal complex occurs in two stages: the formation of axial elements along the sister chromatids and the synapsis of the axial elements with the addition of a central element. The question of whether chromosome synapsis, via synaptonemal complex formation is required for recombination or vice versa has been a longstanding one. Yeast has proven especially useful for studying this question because of its excellent genetics and because of the fact that it can be grown in synchronous meiotic cultures amenable to the study of recombination using physical analyses and chromosome synapsis using constantly improving cytological techniques. The results of experiments in yeast are consistent with the notion that synaptonemal complex formation is not necessary for meiotic recombination. Most notably, in dpl mutants, which do not form a central element, meiotic exchange occurs at near wild-type levels in the absence of synaptonemal complex formation (Sym and Roeder, 1994). Also, a redl mutant shows no tripartite synaptonemal complex or axial elements (Rockmill, and Roeder, 1990), yet redl spol.? diploids cross over at about 25% of wild-type levels. A temporal study comparing meiotically induced DNA double-strand breaks (DSBs), thought to be one of the initiating events in meiotic recombination, and the deposition of a synaptonemal complex showed that the average time of DSB formation is before that of the tripartite structure of the synaptonemal complex (Padmore et al., 1991). Additionally, it was revealed that meiotic DSBs occur in experiments in which the timing and frequency of DSB formation was measured in the absence of a homologue (de Massy et d., 1994) or in the absence of potential pairing sites on a homologue (Gilbertson and Stahl, 1994). de Massy et al. concluded that the occurrence of DSBs in meiosis is not dependent on the presence of a homologue, yet such DSBs form at a slower rate and lower frequency than in the presence of a homologue. Loidl et al. (1991) have demonstrated that extensive synaptonemal complex is formed in yeast haploid meiosis; therefore, it is not possible to say that, in these experiments, DSBs occurred in the absence of the synaptonemal complex. The reverse question, of whether recombination or pairing interactions that precede strand cleavage (see the following) are necessary for synapsis (i.e.,

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synaptonemal complex formation), has not been fully resolved. The idea that events necessary for recombination contribute to synaptonemal complex formation is supported by cytological analyses of the colocalization of the synaptonema1 complex and structures referred to as recombination nodules (RN) (see von Wettstein et al., 1984; Zickler et al., 1992). RNs are dense, cytologically detectable structures distributed along chromosomes in meiotic prophase. In many organisms, two types of RNs have been observed: early RNs appear during zygotene coincident with synapsis, and late RNs occur during pachytene when synaptonemal complex formation is complete. The number and distribution of late RNs in pachytene suggest that they lead to the formation of chiasmata [reviewed by Carpenter ( 1988)l. Early RNs, which are more numerous and have morphologies different from those of late nodules, have been speculated to be involved in events that lead to simple gene conversion (i.e., gene conversion in the absence of exchange) (see Carpenter, 1988). Experiments examining the sites at which axial elements have begun synapsing to make a tripartite synaptonemal complex showed that this synapsis usually occurs adjacent to a recombination nodule. These observations revealed a strong correlation between synaptonemal complex formation and recombination nodules, but did not establish a causal relationship (Zickler et al., 1992). Studies on the cellular localization of a meiotic recombination protein of yeast, Dmclp, are also consistent with the notion that recombination initiation precedes synaptonemal complex formation. DMCl and RAD.51 are genes encoding proteins structurally related to recA of Escherichia coli and are essential for meiotic recombination (Bishop et al., 1992; Shinohara et al. , 1992). Indirect immunofluorescence experiments have shown that, in wildtype cells, Dmclp and Rad5 l p colocalize to “complexes” distributed along the lengths of chromosomes prior to synaptonemal complex assembly (Bishop, 1994). Strains deleted for DMC1 make, but do not process, DSBs and have incomplete synapsis. Axial elements are formed, but the central element, while initiated, does not appear to be completed (Bishop et al., 1992). This observation is consistent with a model that requires a specific recombination intermediate or the processing of recombinant precursors in order to complete synapsis. A role for recombination in the early events of meiosis has been proposed by Smithies and Powers (1986) and Carpenter (1987). The model suggests that events that resolve as simple gene conversions might be part of a mechanism by which the alignment of homologous sequences is checked prior to synapsis. The model provides an elegant meiotic function for simple gene conversion that is alternative to the assumption that gene conversion is an incidental consequence of exchange. In yeast, double strand break formation is probably not the first step in homologue pairing. Instead, the earliest steps of homology searching may involve intimate pairing prior to strand cleavage through interactions such as paranemic joint formation (Bianchi et al., 1983; Kleckner et ul., 1991; Goyon and Lichten,

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1993; Kleckner and Weiner, 1993; Hawley and Arbel, 1993; Xu and Kleckner, 1995;Rocco and Nicolas, 1996). Evidence consistent with pairing preceding DSB formation is the demonstration that a DSB site on one homologue is cleaved less frequently if the site on the other homologue contains heterologies or is absent (Xu and Kleckner, 1995; Rocco and Nicolas, 1996). One early role of recombination might be to lock paired homologous chromosomes in alignment (Hawley and Arbel, 1993).

B. The role of recombination in maintaining bivalents in meiosis I In many organisms, from the time synapsis is initiated until anaphase I, homologous chromosomes remain joined. When synapsis is completed, the chromosomes continue to condense and thicken. In yeast, although chiasrnata are not visible at this stage, physical analysis of yeast DNA has shown that recombination has been initiated in the form of DSBs (Padmore et al., 1991). Further processing of the DNA ends occurs during this stage of meiosis, with resection of the 5' ends of the termini to expose 3' single-strand tails up to several hundred bases long (Sun et al., 1991. In experiments utilizing two-dimensional gel electrophoresis of DNA, an interaction between the homologous strands has been observed in pachytene during the time when double-strand ends are being processed (Collins and Newlon, 1994; Schwacha and Kleckner, 1994). Analysis of these joint molecules has demonstrated they are recombination intermediates composed of duplexes from homologous chromosomes that are joined by even numbers (likely two) of Holliday junctions (Schwacha and Kleckner, 1995). The kinetics with which joint molecules appear and disappear suggest that they are formed soon after DSB formation, coincident with synaptonemal complex formation, and persist until just before the dissolution of the synaptonemal complex. It is at approximately this time that mature crossover DNA molecules are first observed. Consistent with the notion that the recombining DNA molecules are evolving during pachytene is the observation of DNA synthesis at recombination nodules during that period of meiosis in Drosophila (Carpenter, 1981). In many organisms, following pachytene, the synaptonemal complex breaks down, revealing chiasmata [reviewed by von Wettstein et al. (1984)] as the contact points between the homologues. When the synaptonemal complex has disappeared (diplotene), the homologous chromosomes move away from each other, bowing out from the connections that hold them together at chiasmata, as if repulsed by as yet poorly understood forces. It is at this time in meiosis that noncrossover univalents are first observed in cytological experiments (Miller, 1963), suggesting that crossovers between homologues become critical for maintaining the bivalent when the synaptonemal complex is removed. The fact that univalents typically are not seen until diplotene is consistent with the model that crossovers are not needed to keep homologues paired until that time.

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A great deal of chromosome movement occurs in prometaphase as the chromosomes are positioned on the spindle. Observations of these movements in mitotic cells have shown that chromosomes are moved toward the poles by virtue of attachments of their kinetochores to spindle fibers (Merdes and De Mey, 1990; Rieder and Alexander, 1990; Rieder et al., 1993).Another force, the “polar wind,” pushes against chromosome arms, forcing them away from the poles toward the metaphase plate [reviewed by Carpenter (1991); Rieder et al., 1986; Salmon, 19891. The ability of bivalents to remain intact in the face of these forces may be dependent upon the establishment of suitable chiasmata.

C. The role of exchanges in establishing proper attachment to the meiotic spindle Chromosomes attain a stable attachment to the meiotic spindle when fibers from one pole attach to the kinetochore of one homologue and fibers from the other pole attach to the kinetochore of the other homologue (Nicklas, 1988). Observations of this process in living cells suggests that, in most instances, if one kinetochore attaches to fibers from one pole, then the other kinetochore will attach to fibers from the other pole, achieving what is referred to as bipolar attachment (Ostergren, 1951; Bauer et al., 1961; Nicklas, 1967). Once all of the bivalents have achieved a bipolar attachment, anaphase I can proceed. Improper unipolar attachments are rectified by detachment of one or both kinetochores from the spindle, followed by reattachment (Hughes-Schrader, 1943; Nicklas, 1967). This cycle can be repeated until bipolar attachment has been achieved (Dietz, 1958; Nicklas and Ward, 1994). In many organisms a bipolar meiotic spindle assembles in the absence of centrosomes. Instead, disorganized microtubules assemble on condensed chromatin then are organized into bipolar arrays [reviewed in Rieder et al., (1993); McKim and Hawley, 19951. The demonstration that prokaryotic DNA injected into Xenopus extracts and plasmid coated beads in Xenopus egg extracts can serve as sites of assembly of bipolar spindles demonstrates that in this system the chromosomes play a simple structural role in this process (Karsenti eta!. , 1984; Heald et al., 1996). Experiments in Drosophila oocytes, have shown that recombination is important for the assembly of the meiotic spindle around the condensed chromosomes. The experiments showed that mutants lacking a chromokinesin, nod (Afshar et al., 1995), a chromatin-associated motor protein that pushes the chromosomes towards the center of the spindle, could assemble bipolar spindles as could recombination mutants. However, double mutants never developed spindles (McKim and Hawley, 1993). nod and crossovers both serve to keep chromosomes in close proximity; crossovers do so by physically preventing them from moving apart, whereas nod pushes them together. nod and crossovers probably play an unglamorous role in meiotic spindle assembly, that of holding the chro-

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matin mass together, so that other elements of the meiotic machinery can shape the microtubules that assemble around the chromatin into a spindle. Chiasmata have been suggested to be important for bipolar attachment in two regards. First, kinetochores probably are assembled in an outward-facing arrangement such that if one faces one pole, the other will face the opposite pole (Ostergren, 1951; Nicklas, 1974). The association of homologues by chiasmata may, in some cases, lend a rigidity to the bivalent structure, which aids in keeping the kinetochores of the homologous chromosomes from pointing to the same pole (Nicklas, 1971). The second role of chiasmata in this process is as part of the mechanism to signal that bipolar attachment has been achieved. The nature of this signal was revealed by a series of remarkable experiments in living grasshopper spermatocytes [reviewed by Nicklas (1985)l. Chromosomes manipulated to have unipolar attachment (Fig. 7.5b) were observed to detach and reattach to spindle fibers until they achieved a bipolar orientation (Nicklas and Koch, 1969). However, chromosomes attached to a single pole were stable if a micromanipulator was used to apply tension by pulling toward the pole that had no fiber attachment. These experiments provided evidence that correct spindle fiber attachment during metaphase I resulted in tension across the bivalent, which is the signal to the segregation machinery that proper microtubule attachment has occurred. In order for tension to be transmitted across the bivalent, the homologous chromosomes must be linked. Chiasmata are the sole linkages between homologues at this point in meiosis [reviewed by Jones (1987)] and must serve as the bridge across which tension is transmitted. Indeed, in mantids, the chromosomes show stretching between the chiasma and the kinetochore when the kinetochores have attached to spindle fibers (Swanson, 1942; Hughes-Schrader, 1943). Chiasmata are not only involved in signaling mechanisms that affect individual chromosomes, but also have been shown to be involved in transmitting signals that affect passage of the cell through meiosis [reviewed by McKim and Hawley ( 1995)l. In certain organisms, failure to establish or maintain chiasmata leads to an arrest or delay of meiosis. One demonstration of this phenomenon comes from analysis of praying mantid spermatocytes, which normally contain a sex chromosome trivalent of two X’s and one Y. The trivalent achieves a bipolar spindle attachment with the X’s oriented away from the Y. In about 10% of the spermatocytes, the trivalent disassembles to yield an XY bivalent and an X univalent which can only achieve a unipolar spindle attachment. These cells delay entry into anaphase for several hours. However, when a micromanipulation needle is used to apply tension to a univalent that is attached to one pole, the cells resume meiosis (Li and Nicklas, 1995). Experiments using antibodies specific for phosphorylated kinetochore proteins have shown that in grasshopper spermatocytes, when a chromosome is placed under tension by achieving bipolar spindle attachment or through use of a micromanipulation needle, kinetochore dephos-

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phorylation occurs (Nicklas et al., 1995). Thus, the mechanical forces that depend upon chiasma formation are converted to chemical signals at the kinetochore. Some meiotic cells, notably oocytes of many species, normally arrest prior to anaphase I. Experiments in Drosophila have shown that tension transmitted across a single exchange is sufficient to induce the meiosis I arrest (Jang et al., 1995). Mutants defective for recombination fail to arrest (McKim et al., 1993). A recombination proficient strain carrying only compound chromosomes also fails to arrest. In this strain exchanges occur between the homologous arms of the compound chromosomes, but no tension is placed on the chromosome because the exchanges fail to link two kinetochores that can attach to opposite poles. Replacement of one compound chromosome with a pair of normal homologues resulted in a strain that showed normal meiotic arrest (Jang et al., 1995). These examples suggest that tension on kinetochores, transmitted across chiasmata, is likely a common mechanism for monitoring meiotic cell cycle progression.

IV. THE ROLE OF CHIASMA BINDER IN ENSURING DISJUNCTION Crossovers are necessary to link homologous chromosomes, but they are insufficient to accomplish this task alone. The diagram in Fig. 7.6a illustrates that a resolved crossover between homologues is not a knot. In the simplified situation shown in this figure, if outward forces were applied to the exchange bivalent, the pair would come apart. An activity termed “chiasma binder” has been proposed to act in conjunction with exchanges to enable them to stably join homologous chromosomes (Darlingtnn, 1932; Maguire, 1974, 1995). The chiasma binder

a.

b.

C.

d.

Figure 7.6. Models for chiasma binder activity. ( a ) A pair of homologues joined by a resolved crossover. Without a chiasma binder, recombined homologous chromosomes can become unlinked. (b) Chiasma binder is represented as cohesion between sister chromatids. (c) Chiasma binder acts at the site of the crossover to fix the chiidsma in place. (d) Chiasma binder is provided by base pairing between DNA strands when the crossover remains unresolved.

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could stabilize a chiasma by maintaining an association of sister chromatids distal to the chiasma (Fig. 7,6b), by acting directly at a chiasma (Fig. 7 . 6 ~or ) ~by a combination of these activities. A requirement for sister-chromatid cohesion in meiosis I was first proposed by Darlington (1932), and the idea of sister-chromatid cohesion as chiasma binder was formalized by Maguire (1974). Sister-chromatid cohesion during meiosis is defined cytologically. Sister chromatids remain tightly juxtaposed from the time they are generated by premeiotic DNA replication until they lose their association at anaphase I. A role for sister-chromatid cohesion in providing chiasma binder activity is suggested by a number of observations. Two models have been proposed to explain the basis of sister-chromatid cohesion: linking via DNA structure perhaps through intertwining or cohesion by chromatinlinking proteins [reviewed by Miyazaki and Orr-Weaver ( 1994)) Evidence that sister-chromatid cohesion is supplied by proteinaceous structures comes from the observation that synaptonemal complex or synaptonemal complex-like material remains between sister chromatids after desynapsis in orthopterans (Esponda and Kramer, 1979; Moens and Church, 1979).Consistent with the chiasma binder being a protein matrix, in mice antibodies against meiotic proteins immunolocalize between sister chromatids at metaphase I, after synaptonemal complex removal (Dobson et al., 1994). Genetic analysis has identified several genes whose products are important for both chromatid cohesion and meiosis I chromosome disjunction. A subset of mutant alleles that show increased levels of precocious separation of sister chromatids (PSSC),consistent with but not proving a defect in sister-chromatid cohesion, includes DISI-I (Rockmill and Fogel, 1988) and medl -1 (Rockmill and Roeder, 1994) in Saccharornyces cerevisiae. Cytological analysis has shown that defects in dyl and dsyl in maize (Maguire, 1978) (it is not known whether these are allelic) andmei-S332 (Davis, 1971;Ken-ebrocket al., 1992) and ord (Mason, 1976; Goldstein, 1981; Miyazaki and Orr-Weaver, 1992) in Drosophila lead to defects in sister-chromatid cohesion (Miyazaki and Orr-Weaver, 1992; Carpenter, 1994; Maguire, 1995). The potential roles of these and other genes in providing chiasma binder activity have been reviewed in more detail elsewhere (Carpenter, 1994; Maguire, 1995). Intertwining of sister chromatids has also been suggested as a possible mechanism for maintaining their association [discussed by Murray and Szostak (1985)l. Topoisomerase I1 is necessary for passage through meiosis in yeast, which is consistent with the model that, in meiosis, sister chromatids are intertwined. In vegetatively growing yeast cells, following replication sister chromatids are intertwined in a fashion that requires resolution by topoisomerase I1 before they can be segregated at anaphase (DiNardo et al., 1984; Holm et al., 1985). Although this intertwining may contribute to sister association in mitosis, it is not essential for proper segregation of the sister chromatids. In yeast cells blocked in G2, centromere plasmids are not linked by catenation and segregate properly when the

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block is removed (Koshland et al., 1985; Guacci and Koshland, 1994). Nonetheless, the presence of topoisomerase 11 along mitotic metaphase chromosomes prior to disjunction and the need for decatenation prior to disjunction in meiosis suggest that the topological intertwining of sisters may play a critical role in meiosis 1 [reviewed by Miyazaki and Orr-Weaver (1994)]. In lieu of sister-chromatid cohesion, it is not difficult to imagine that components of recombination nodules or the synaptonemal complex in the vicinity of an exchange could lock it in place. Remnants of the synaptonemal complex and recombination nodules have indeed been observed at chiasmata in some organisms [Holm and Rasmussen, 1980; Holm et al., 1981; reviewed by von Wettstein et al. (1984)]. Relevant to these models, experiments in maize testing the movement of acentric fragments, resulting from the breakage of chromosomes with heterozygous inversions, are inconsistent with chiasma binder occurring exclusively at crossovers [McClintock, 1938; Maguire, 1982, 1985; reviewed by Maguire (19991.

V. RECOMBINATION INTERMEDIATES AND CHIASMA FORMATION The structure of the DNA component of chiasmata may contribute to chiasma function. Experiments designed to probe the structure of recombining DNA in yeast have provided insights into this aspect of chiasma formation. These experiments have been made possible by the fact that cultures of yeast can be induced to undergo rapid and synchronous meiosis, by the discovery that yeast chromosomes have sites at which meiotic recombination events are initiated at high frequency, by the development of techniques that have made it possible to use cytological approaches to examine meiotic chromosome behavior, and by the identification of a collection of genes essential for meiotic chromosome segregation. Over the past 30 years, a number of models have been proposed to describe the events that culminate in the formation of a crossover. In 1964, Holliday proposed that recombinants form a four-strand intermediate that could be resolved to give either crossover or non-crossover products. Most current working models for describing recombination in yeast suggest that the process is initiated by the cleavage of both strands of a DNA duplex (Fig. 7.7a; Resnick and Martin, 1976; Szostak et al., 1983). A wealth of genetic and physical evidence suggests that double-strand breaks in the DNA ( DSBs) are the initiating event in crossover formation in yeast. Some of the experiments and key observations that led to this conclusion include the following: (1) Genetic analyses of strains with deletions in a region exhibiting high frequencies of gene conversion were performed to map a site required for high levels of recombination initiation (Nicolas et al., 1989). ( 2 ) Physical analysis of DNA from synchronous meiotic yeast cultures was used

a

b

C

d

t 3’

. \ * / , 5

‘y t

.....,

f

t

Figure 7.7. Model of meiotic recombination. The model shown invokes the creation of double-strand breaks in the DNA as an early event in the recombination process (Szostak et al., 1983). (a) A doublestrand break forms in one sister chromatid of four. (b) Shown are the two nonsister chromatids participating in the recombination reaction. Resection at the 5’ end leaves 3’ single strands of DNA. (c) A free 3’ end invades a nonsister chromatid, displacing one of the strands, which forms a D loop. (d) DNA synthesis using the 3’ ends as primers. (e) The ends are healed, forming two Holliday junctions. Each Holliday junction can be cleaved in one of two orientations. The arrows shown give the orientations in which the junctions were cut to give the structure seen in (f). (f) The recomhinant strands are resolved to give a crossover.

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to demonstrate that DSBs appear concurrent with the time that cells commit to performing meiotic recombination. These DSBs occur at chromosomal locations deemed to be recombination initiation sites by genetic tests for meiotic initiation sites (Sun et al., 1989; Cao et al., 1990). (3) Physical analysis of large DNA molecules by pulsed-field gel electrophoresis demonstrated that the number and distribution of DSBs along the length of Saccharomyes cerevisiue chromosomes are consistent with DSBs playing a role in the initiation of meiotic recombination (Game, 1992; Zenvirth et al., 1992). (4) Mutations that prevent the formation of double-strand breaks were shown to confer defects in meiotic recombination (Cao et al., 1990; Ivanov et al., 1992; Johzuka and Ogawa et al., 1995; Rockmill et al., 1995). (5) Artificially induced breaks were shown to efficiently promote recombination in both mitotic (Orr-Weaver et al., 1981; Nickoloff et al., 1986; Connoly et al., 1988; Rudin and Haber, 1988; Rudin et al., 1989; Plessis et al., 1992 and premeiotic (Klar and Miglio, 1986; Kolodkin et al., 1986) cells. In a landmark study by Padmore et al. (1991), the temporal placement of recombination intermediates in the context of synaptonemal complex formation was recorded. Their results showed that DSB formation occurs concomitant with the appearance of synaptonemal complex precursors. These DSBs are quickly converted into recombination intermediates that link homologues (Schwacha and Kleckner, 1995). These and other studies (Goyon and Lichten, 1993; Nag and Petes, 1993)have shown that the interval from initiation until the resolution of meiotic recombinants extends for a considerable length of time. Heteroduplex DNA formation is predicted from most models for meiotic recombination (Fig. 7.7) and was first demonstrated by genetic analysis to occur in yeast meiosis (Esposito, 1971). One technique for detecting heteroduplexes in DNA harvested from meiotic yeast cells depends upon the altered mobility in acrylamide gels of DNA fragments bearing mismatched base pairs (Lichten et al., 1990; Goyon and Lichten, 1993). Another approach involves inserting heterozygous palindromic sequences adjacent to a recombination initiation site. Heteroduplex formation between the two homologues creates a cruciform that is resistant to restriction digestion with enzymes able to cut the homoduplex (Nag and Petes, 1993). By using either approach, heteroduplex molecules are not detected in synchronous meiotic cells until just prior to the time that mature recombinant molecules are detected. These results together imply that either heteroduplex DNA does not form until relatively late in the recombination process or if it does occur early, it does so in such a way that it cannot be detected using these techniques. For example, the early occurrences of heteroduplex DNA might be very short, only extending to detectable sizes by branch migration just prior to resolution of the recombinant (Goyon and Lichten, 1993; Nag and Petes, 1993). These studies, and the characterization of joint molecules described above (Collins and Newlon, 1994; Schwacha and Kleckner, 1994; Schwacha and Kleckner, 1995), demonstrate that the generation of mature recombinants is a

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slow process that is initiated prior to the formation of tripartite synaptonemal complex and is completed approximately at the time of its disappearance. The structure of recombination intermediates suggests that they might also contribute to maintaining the association of homologous chromosomes. Figure 7.6d shows a homologue pair linked by a recombination intermediate with a pair of Holliday junctions. The pairing of DNA strands from one homologue to complementary strands from the other homologue will contribute to their linkage until those strands are cut.

Most studies concerning the ability of exchanges to enhance the fidelity of chro. mosome segregation have been of a global nature. The extent to which individual exchanges contribute to chromosome segregation has been studied less extensively. Studies of chromosome segregation in two Drosophila mutants have provided evidence that not all exchanges are equally able to enhance chromosome segregation fidelity. Mutations in the nod gene almost exclusively result in defects in the segregation of achiasmate chromosomes in Drosophila females (Carpenter, 1973; Rasooly et d.,1991). The disjunction of exchange bivalents is generally unaffected in nod mutants. However, chromosomes that have experienced exchange in centromere distal intervals (that is, near the telomeres) show higher frequencies of nondisjunction than chromosomes with exchanges occurring centromere proximal. Similar observations have been reported in studies of Drosophila harboring the dominant Dub mutation, which leads to strong defects in the segregation of achiasmate chromosomes as well as to lesser defects in the segregation of exchange chromosomes (Moore et al., 1994). These results led to the model that distal chiasmata may be less able to maintain the association of homologues than more internal ones, such that some terminal chiasmata fail, leaving these homologues to be partitioned by the backup system (Carpenter, 1973; Rasooly et al., 1991; Moore et al., 1994). Additional evidence consistent with the idea that distal exchanges have reduced ability to ensure disjunction comes from studies of Drosophila ord' mutants, showing that X chromosomes with distal exchanges are more likely to nondisjoin than those with exchanges elsewhere (Mason, 1976). A systematic study of the ability of individual exchanges to enhance disjunction has been performed in yeast. These experiments monitored the ability of individual exchanges, between pairs of model chromosomes, to enhance their segregation. The ability of exchanges to enhance disjunction increased with distance from the telomeres of the model chromosomes (Ross et al., 1996b). Why are some exchanges less effective at ensuring disjunction? One possibility is that the exchanges themselves may be inadequate in enhancing disjunction. Exchanges ini-

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tiated or resolved at inappropriate times or mediated by enzymes different from those used to establish meiotic recombination nodules and chiasmata may be unable to influence segregation. Alternatively, exchanges that do not ensure disjunction may be adequate, but not supported by chiasma binder activity in such a way that allows the chiasma to maintain the linkage of homologues until anaphase I. The failure of exchanges near telomeres to ensure disjunction is consistent with the model that the chiasma binder is less effectively established near telomeres. It is also consistent with the model that the ability of sister-chromatid cohesion to hold a chiasma in place is proportional to the distance between the exchange and the telomere: greater distance results in greater cohesion. Note that if the extent of sister chromatid cohesion, distal to a crossover, is important for ensuring disjunction, then closely spaced two strand double crossovers should not enhance disjunction; the length of sister cohesion holding the homologues together is that between the exchanges (Nilsson and Sall, 1995). The hypothesis that ord' plays a role in sister-chromatid cohesion in Dosobhila (Miyazaki and Orr-Weaver, 1992) suggests that the inability of distal exchanges to ensure chromosome disjunction in ord mutants (Mason, 1976) is due to a decrease in chromatid cohesion between exchanges and telomeres. In the yeast model chromosome experiments, single exchanges that occur centromere proximal, while greatly enhancing correct chromosome segregation, do not always ensure disjunction. Yeast natural chromosomes typically experience three or more exchanges per meiosis (Mortimer et al., 1989). Perhaps the low frequency of nondisjunction of exchange yeast chromosomes reflects the combined abilities of the individual exchanges to maintain the linkage of homologues until anaphase I. A n analysis of the distributions of crossovers on versions of human chromosome 2 1 that had experienced meiosis I missegregations, leading to trisomy 2 1 showed that among those that had recombined, a disproportionate number had experienced a single crossover in the interval adjacent to the telomere (Sherman et al., 1994). These observations are strikingly reminiscent of the yeast and Drosophila experiments described earlier and raise the possibility that in humans too terminal chiasmata are less able to enhance segregation fidelity and may show a failure rate proportional to maternal age.

VII. SUMMARY Meiotic recombination events are probably critical for the completion of several meiotic processes. In addition, recombination is likely to be involved in the events that lead up to synapsis of homologues in meiotic prophase. Recombination events that ultimately become resolved as exchanges are needed for the formation of chiasmata. Chiasmata maintain the association of paired homologues follow-

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ing loss of the synaptonemal complex and participate in the mechanism that signals that the bivalent has attached to the spindle in a bipolar orientation that will result in meiosis I disjunction.

Ac know I edgme nts We are deeply grateful to Adelaide Carpenter, Terry L. Orr-Weaver, and Marjorie Maguire for their

thoughtful and illuminating comments on this chapter. Their substantial efforts enhanced every one of its pages. We also thank Allyson Holmes and Rebecca Maxfield for their comments on the manuscript. Finally, we thank all those who provided us with preprints, reprints, and other forms of direction in the preparation of this chapter.

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Polani, P. E., and Crolla, J. A. (1991). A test of the production line hypothesis of mammalian oogenesis. Hum. Genet. 88:64-70. Rasooly, R. S., New,C. M.,Zhang, P., Hawley,R. S., andBaker, B. S. (1991).The kthal(l)TW-6”rnutation of Drosophila melanogaster is a dominant antimorphic allele of nod and is associated with a single base change in the putative ATP-binding domain. Genetics 129:409422. Resnick, M. A., and Martin, P. (1976). The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerewisiae and its genetic control. Mol. Gen. Genet. 143:119-129. Rieder, C. L., and Alexander, S.P. (1990). Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to the spindle in newt lung cells. J. Cell Biol. 110:81-96. Rieder, C. L., Davison, E. A., Jensen, L. C. W., Cassimeris, L., and Salmon, E. D. (1986). Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half-spind1e.J. CeU Biol. 103581-591. Rieder, C. L., Ault, J. G., Eichenlaub-Ritter, U., and Sluder, G. (1993). Morphogenesis of the mitotic and meiotic spindle: conclusions obtained from one system are not necessarily applicable to the other. In “Chromosome segregation and aneuploidy” (B. K. Vig, Ed.), Vol. 72, pp. 183-197. Springer-Verlag, Berlin, Heidelberg. Robinson, W. P., Bernasconi, F., Mutirangura, A., Ledbetter, D. H., Langlois, S., Malcolm, S., Morris, M. A,, and Schinzel, A. A. (1993). Nondisjunction of chromosome 15: origin and recombination. Am. J. Hum. Genet. 53:740-751. Rocco, V., and Nicolas, A. (1996). Sensing of DNA non-homology lowers the initiation of meiotic recombination in yeast. Genes Cell 1, in press. Rockmill, B., and Fogel, S. (1988). DISI: A yeast gene required for proper meiotic chromosome disjunction. Genetics 119:261-272. Rockmill, B., and Roeder, G. S.(1988). REDI: A yeast gene required for the segregation of chrornosoma during the reductional division of meiosis. Proc. Natl. Acad. Sci. USA 85:60574061. Rockmill, B., and Roeder, G. S. (1990). Meiosis in asynaptic yeast. Genetics 126563-574. Rockmill, B., and Roeder, G. S. (1994). The yeast medl mutant undergoes both meiotic homolog nondisjunction and precocious separation of sister chromatids. Genetics 136:65-74. Rockmill, B., Engebrecht, J., Scherthan, H., Loidl, J., and Roeder, G. S. (1995). The yeast MER2 gene is required for chromosome synapsis and the initiation of meiotic recombination. Genetics 141:49-59. Ross, L. O., Treco, D., Nicolas, A., Szostak,J. W., and Dawson, D. (1992). Meiotic recombination on artificial chromosomes in yeast. Genetics 131:541-550. Ross, L. O., Rankin, S., Flatters, M., and Dawson, D. (1996a). The effects of homology, size and exchange on the meiotic segregation of model chromosomes in Sacchuromyces cerewisiae. Genetics 142: 79-89. Ross, L. O., Maxfield, R., and Dawson, D. (1996h). Exchanges are not equally able to enhance meiotic chromosome segregation in yeast. Proc. Natl. Acad. Sci. USA 93:4979-4983. Rudin, N., and Haber,J. E. (1988). Efficient repair of HO-induced chromosomal breaks in Sacchromyces cereuisiae by recombination between flanking homologous sequences. Mol. CeU. Biol. 8:3918-3928. Rudin, N., Sugarman, E., and Haber, J. E. (1989). Genetic and physical analysis ofdouble-strand break repair and recombination in Saccharomycescereuisiae. Genetics 122:519-534. Salmon, E. D. (1989). Microtubule dynamics and chromosome movement. In “Mitosis” (1. S. Hymans and B. R. Brinkley, Eds.), pp. 119-181. Academic Press, New York. Schwacha, A,, and Kleckner, N. (1994). Identification of joint molecules that form frequently between homologs hut rarely between sister chromatids during yeast meiosis. Cell 7 6 5 1-63. Schwacha, A,, and Kleckner, N. (1995). Identification of double Holliday junctions as intermediates in meiotic recombination. Cell 83:783-791. Sears, D. S., Hegemann, J. H., and Hieter, P. (1992). Meiotic recombination and segregation of hu-

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I

Molecular Genetics of Familial Cardiomyopathies

Aman S. Coonar and William J. McKenna

Cardiological Sciences St. George’s Hospital Medical School London SW 17 ORE, United Kingdom

1. INTRODUCTION Since any classification is necessarily incomplete and acts as a bridge between complete ignorance and total understanding in any biological system, further modification and changes are likely to occur as knowledge advances. . . . The frontiers of cardiomyopathy. (Goodwin, 1982) “Cardiomyopathy” was originally defined by Brigden in 1957 as a heart muscle disease of unknown cause, distinguished from myocardial disease arising from known cause, e.g., hypertension, coronary artery disease, and valve disease. Since that time, this stance has been reiterated (Goodwin, 1961; WHO/ISFC, 1980), although not without controversy (Abelmann, 1984; Keren and Popp, 1992;Johnson, 1982). Classifications have been developed and modified in the light of new information. Therefore, definitions that were originally largely clinical, and made by the exclusion of other causes, were adapted to include the positive presence of various criteria, such as echocardiographic findings. With an expanding number of disease syndromes recognized as having significant myocardial involvement (McKusick, 1996), along with a rapid elucidation of their molecular and biochemical basis, such a definition based on unknown cause is increasingly recognized as being unsatisfactory, effectively only including the common end phenotypes of different disease processes. In the near future, it is likely that the definition and classification of faAdvances in Genetics, Vol. 35

Copyright 0 1997 by Academic Press All rights of reproduction in any form reserved

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milial cardiomyopathies will become clarified in an organization based on etiology, reflecting their origins in the specific gene mutations. Accurate clinical classification will continue to be needed, as the vital prerequisite to the determination of any genetic abnormality remains the correct recognition of the disease phenotype. New clinical classifications will be modified, illuminated by advances in clinical diagnosis, themselves aided by new technologies such as ultrafast magnetic resonance imaging and computerized tomography. Of notable interest has been the elucidation of specific mutations producing hypertrophic cardiomyopathy, as well as the identification of disease loci in familial dilated cardiomyopathy and arrythmogenic right ventricular cardiomyopathy. The identification of specific mutations allows us to determine the functional role of particular proteins and thus to determine their significance in cardiac and general homeostasis. It also paves the way for the implementation of screening based on molecular genetic diagnosis. Such screening is now in the first phase of implementation. The major cardiomyopathies are hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrythmogenic right ventricular cardiomyopathy/dysplasia (ARVC), and restrictive cardiomyopathy. This chapter focuses on these four disease entities. Because of the dramatic advances that have been recently achieved in hypertrophic cardiomyopathy, particular attention will be focused on this disease.

II. HYPERTROPHIC CARDIOMYOPATHY A. Definition and clinical syndrome HCM is currently defined as a “heart muscle disease of unknown cause” that is “characterized by disproportionate hypertrophy of the left ventricle and occasionally also of the right ventricle which typically involves the septum more than the free wall but occasionally is concentric. “Typically the left ventricular volume is normal or reduced (Figures 8.1 and 8.2). Systolic gradients are common” (WHO/ISFC, 1980). However, for the reasons discussed earlier, such a definition is increasingly considered incomplete. Specifically, it was based on a pattern of disease recognized in selected tertiary centers and reflects the status of the technology available. Recent, rapid advances in determining the molecular basis of the disease, accompanied by improvements in clinical diagnostic methods, have delivered the concept of a much broader, and often subtle, phenotype, which may reflect incomplete gene penetrance as well as variable disease expression. The concept of the molecular basis to this disease is to be included in a new classification of the cardiomyopathies (WHO/ISFC, 1996).

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Figure 8.1. Section through the heart of a patient with hypertrophic cardiomyopathy. Scale shows 2cm line for comparisons.

Figure 8.2. Microscopic view of a section of heart from a patient with hypertrophic cardiomyopathy. Note myocyte and myofihrillar disarray.

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B. Epidemiology Hypertrophic cardiomyopathy has been observed in all the major racial groups. Prevalence data are mainly limited to European and North American populations. Depending on methodology, surveys identified rates of between 3.2 and 33/100,000 (Bennett, 1987; Bjarnason, 1982). In Minnesota, a survey of a defined population found a prevalence of 20/100,000 (Codd, 1989). If these numbers were representative, the prevalence would be approximately 1/5000. This estimate is probably conservative because a proportion of mildly affected individuals and families would have been missed with the diagnostic criteria applied. Indeed, Spirito et al. (1989) reported that HCM patients in an outpatient population were much less severely affected or even asymptomatic as compared to patients investigated in referral institutions. Using better methodology and strict diagnostic criteria, a prevalence of cardiac hypertrophy of about 1 in 500 was reported (Maron et al., 1995). This study was based on echocardiographic analyses of men and women between 23 and 35 years of age selected from the general population. Seven of 41 11 individuals fulfilled diagnostic criteria for HCM. A n additional 5 had left ventricular hypertrophy which was interpreted to be the consequence of systemic hypertension. These figures suggest that HCM is much more common in young adults than previously recognized. Furthermore, this rate is likely to be an underestimate, for it excludes the younger age groups which are known to suffer a high mortality and does not account for those gene carriers who have little or no mutation expression. This rate may therefore rise further with increasing recognition of hypertrophic cardiomyopathy as a cause of stillbirth and death in early childhood (Maron, 1982), recognition of unsuspected “latent” disease in adults (Lewis and Maron, 1994, 1989), and as molecular-genetic screening becomes available as a diagnostic tool. Evidence also suggests that disease expression may vary considerably between ethnic groups (Sakamoto et al., 1976; Anastakis, 1995; Ando et al.,

1990).

C. Clinical features Clinically, patients may be entirely asymptomatic or may be affected by varying degrees of dyspnea, chest pain, palpitation, or syncope. Rhythm disturbances are common and include atrial fibrillation, supraventricular tachycardia, and ventricular tachycardia. Clinical risk stratification is now well advanced, and in adults a family history of sudden death and a personal history of syncope or documented nonsustained ventricular tachycardia are predictors of adverse prognosis. Unfortunately, a proportion of patients who ultimately have a poor prognosis will not be positive for one of these risk markers. In addition, there are relatively few reliable markers of poor prognosis in children. Risk stratification is likely to benefit

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from the elucidation of the genetic defect and the results of genotype-phenotype correlation.

D. Clinical genetics The commonest inheritance pattern is autosomal dominant with variable penetrance and expression. Sporadic cases are also recognized and de novo germ-line mutations have been identified. A few cases of recessive transmission have also been reported (Emanuel et al., 1971; Yamaguchi et al., 1979; Branzi, 1985). However, these latter cases were diagnosed on criteria less stringent than now and convincing evidence of recessive transmission is not supported. Preliminary evidence suggests that the phenotype may differ for different genes and mutations. Expression of HCM is age related and clinical manifestations typically develop during periods of growth (McKenna et al., 1981;Maron et al., 1983, 1986; Spirit0 and Maron, 1989). HCM is infrequent prior to adolescence. Even adult populations may show partial or incomplete expression as obligate gene carriers without clinical manifestations have been recognized (Watkins et al., 1995). The fact that disease morphology and severity may vary greatly within families and even in monozygotic twins (Reid et al., 1989) suggests that expression of the phenotype is also significantly influenced by environmental and other genetic factors.

E. Molecular genetics Early reports suggested a linkage of hypertrophic cardiomyopathy to the HLA region on chromosome 6 (Matsumori et al., 1979, 1981; Kishimoto et al., 1983). This proposal, however, was never confirmed (Jarchoet al., 1989; Solomon et al., 1990a,b; Tanigawa et al.,1990). Rather, investigation now illustrates that up to 50-60% of HCM may be accounted for by mutations in four contractile protein genes. These are p-cardiac myosin (cardiomyopathy, hypertrophic 1; CMH 1) at chromosome 14q11-12 (Jarcho et al., 1989; Solomon et al., 1990a; Geisterfer et al., 1990; Watkins et al., 1992a), troponin T (CMH2) at chromosome lq3 (Watkins et al., 1993a), a-tropomyosin (CMH3) at chromosome 1592 (Thierfelder et al., 1993), and, most recently, myosin-binding protein C (CMH4) (Watkins, 1995b; Carrier, 1995) on chromosome 1lp13-q13 (Carrier et al., 1993). The P-myosin heavy chain participates directly in myosin-actin crossbridge formation, whereas the other three proteins control or modulate cardiac contractility. It has therefore been suggested that HCM should be reclassified as a disease of the sarcomere (Thierfelder et al., 1994). In addition to the four etiological genes identified as causing HCM, linkage analysis has also identified a region of chromosome 7q3 which is strongly linked to HCM in a family in whom members had familial HCM and/or the

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Wolff-Parkinson-White syndrome (CMH6) (Macrae, 1995). Furthermore, as other families are known not to be linked to the five reported loci, there is the implication of further genetic heterogeneity and the presence of at least one more gene locus, provisionally designated CMH5 (Hengstenberg, 1995).

F. p-cardiac myosin mutations (Table 8.1) The identification of a French-Canadian family with 44 affected members (21 living and 23 dead) and 58 unaffected by hypertrophic cardiomyopathy permitted linkage analysis. In 1989 a DNA locus D14S26 mapping to chromosome 14s (Jarcho et al., 1989) was found to cosegregate with the disease. A lod score of +9.37 (8 = 0) strongly suggested that a gene responsible for familial hypertrophic cardiomyopathy mapped to this region. This part of chromosome 14 included the genes for both the a-and P-myosin heavy chains (Matsuoka et al., 1988, 1989). These two myosin heavy chain subtypes belong to a multiprotein family of at least Table 8.1. Cardiac p Myosin Heavy Chain Gene Mutations (Chromosome 14s CMH1) Identified in Hypertrophic Cardiornyopathy ~~

~~

Amino acid

Nucleotide

Exon

Charge change

Arg249Gln Arg403Gln

G832A G1294A

9 13

-1 -1

Arg453Cys Phe513Cys Gly584Arg Va1606Met Asn615Lys Gly716Arg Arg7 19Trp Arg723Cys Leu908Val Glu924Lys Glu949Lys Gly741Arg Gly256Glu Arg403Trp Arg403Leu Asp778Gly Asn232Ser Gly1208Ala Arg403*** Hybrid gene 3’deletion

C1443T T1624G G1836C G1902A G1931C G2232A C2241T C2253T C2808G G2856A G2931A G741C G256A C403T G403T A778G A232G G 1208A

14 15 16 16 16 19 19 20 23 23 23 20 9 13 13 21 8 13 13

a/P hybrid

-1

0

+1 0 +1 +I -1 -1 0 +2 +2 +1 -1 -1 -1

0 0 0

Reference Rosenzweig et al. (1991) Watkins et al. (1992); Epstein et al. (1992); Perryman et al. (1992) Watkins et al. (1992) Anan et al. (1994) Watkins e t al. (1992) Watkins e t al. (1992) Nishi et al. (1994) Anan et al. (1994) Anan et al. (1994) Watkins et al. (1992) Epstein et al. (1992) Watkins et al. (1992) Watkins e t al. (1992) Fananapazir et al. (1993) Fananapazir et al. (1993) Dausse et al. (1993) Dausse et al. (1993) Harada et al. (1993) Dufour e t al. (1994) Moolman et al. (1993) Moolman et al. (1993) Tanigawa et al. (1990) Marian et al. (1992)

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eight members that are functionally expressed in striated muscle. The genes for cardiac muscle are located on chromosome 14 and for skeletal muscle on chromosome 17 (Saez et al., 1987). The a-isoform is expressed at high levels in atrial myocytes, whereas the p-chain is the major adult ventricular isoform. The P-isoform is also present in the myofibers of slow skeletal muscles (Sinha e t al., 1982; Lompre et al., 1984; Lichter, 1986). Myosin heavy chains constitute the major component of myofibril thick filaments in striated muscle, are intrinsic to muscle contraction, and are present in various isoforms which are differentially expressed in various tissues and through development. A restriction fragment length polymorphism (RFLP) within intron 28 of the P-myosin heavy chain gene (Siewertsen et al., 1990) was used to reassess linkage of the disease to the myosin heavy chain locus. A two-point lod score of 4.62 (0 = 0) supported the hypothesis that cardiac myosin heavy chain genes were disease genes for hypertrophic cardiomyopathy. Further evidence was the identification of an a/p-chain rearrangement of the myosin cluster on chromosome 14 in another HCM family (Tanigawa et al., 1990). This rearrangement may have arisen from an unequal meiotic crossover. These two highly homologous genes lie closely spaced on chromosome 14 (about 5 kb apart). The hybrid a/P-myosin heavy chain allele was informative because in this family it segregated only with disease, but not in any of 200 unrelated persons. However, subsequent investigation revealed that the rearranged allele was probably only a tightly linked genomic marker rather than the disease gene. This conclusion was based on the observation that affected members also carried a missense mutation in the nonrearranged @-myosin heavy chain gene on the same chromosome. Furthermore) subsequent identification of unrelated HCM patients who had the same missense mutation) but not the hybrid a/@-allele,made it unlikely that the rearrangement contributed to the phenotype (Watkins et al., 1992a). Sequencing of cloned genomic DNA from HCM patients of the French-Canadian family identified a CGG403CAG missense mutation in exon 13 of the P-myosin heavy chain gene (Geisterfer et al., 1990). The consequence at the level of the polypeptide is Arg403Gln. Following identification of this mutation, several groups, using a range of different techniques, have determined several different etiological mutations (Figure 8.3). RNA-based techniques have found a particular role as the cardiac P-myosin heavy chain gene is large, consisting of 40 exons composed of more than 23,000 bp. Fortunately) the encoded mRNA is only 6000 bp. Low-level ectopic mRNA transcription occurs in other tissues, including peripheral blood lymphocytes (Rosenzweig et al., 1991), which therefore have been utilized to indirectly determine mutations expressed at the level of the adult heart. Techniques utilized included RNase A protection (MacRae et al., 1994), as well as single-strand conformation polymorphism (Nishi et al., 1994), chemical cleavage (Epstein et al., 1992a,b), heteroduplex analysis (Dausse et al., 1993; Dausse and Schwartz, 1993), and DNA cycle sequencing (Thierfelder et al., 1994).

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The most frequent base exchanges identified have been G to A transitions in the majority of cases involving a CpG dinucleotide. It has been suggested that this preference indicates that oxidative deamination of methylated cytosine residues may in part be responsible for the mutational events (Coulondre et al., 1978; Youssoufian et al., 1988; Green et al., 1990; Rideout et al., 1990). Several mutations have been found in more than one family, e.g., Arg403Gln or Va1606Met, whereas others have been found to be unique to particular pedigrees. Haplotype analysis in families with identical mutations supports the hypothesis of independent mutational events rather than founder effects (Watkins et al., 1993b). This implies that many mutations were of relatively recent origin, and in conjunction with the identification of de novo mutations (Watkins et al., 1992b; Greve et al., 1994), a relatively high mutation rate of this gene can be assumed. The most frequent mutation is of codon 403. Investigation of eight unrelated families found that the same CGG403CAG mutation (Arg403Gln) was observed. In addition, a G to T transversion and a C to T transition have been detected in the same codon (Dausse et al., 1993; Moolman et al., 1993), suggesting a mutational hotspot in triplet 403. In codon 741, two different base changes have resulted in identical replacements (Gly-Arg) (Fananapazir et al., 1993). Three mutations in @-myosinheavy chain genes have been reported that do not appear to be disease related. One is the a/@-hybridallele discussed earlier. A second is a 2.4-kb deletion removing the terminal exon 40, the 3 primeeuntranslated region including the polyadenylation signal (required for correct transcriptional termination), and part of the intergenic region between the a-and the @-myosingene (Marian et al., 1992). This deletion, however, does not apparently cosegregate with the disease or else has age+relatedexpression in late adulthood. Finally, a nonsense mutation in codon 54 has been described in a Japanese family that creates a potential null allele of the @-gene(Nishi et al., 1994). Because this allele is found in clinically healthy adult probands, it is not linked to disease, at least not in the heterozygous state. In the homozygous state, as is discussed later in the section on molecular pathogenesis, such a mutation would probably have severe consequences. In summary, disease-related mutations of the P-myosin heavy chain gene have all been missense mutations.

G. Discovery and characteristics of other etiological gene mutations The discovery that @-myosinheavy chain mutations cause hypertrophic cardiomyopathy suggested that the disease may also be due to abnormalities of other sarcomeric elements. Subsequently, linkage analysis led to the identification of disease loci on chromosome lq3 (Watkins et al., 1993a), 15q2 (Thierfelder et al., 1993), and 1 1 ~ 1 3 4 1 3(Carrier et al., 1993). These have been identified as tro-

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ponin T (CMHZ), a-tropomyosinTPM1 (CMH3) (Thierfelder et al., 1994), and cardiac myosin-binding protein C (CMH4), respectively.

H. a-Tropomyosin (TPM1) mutations (Table 8.2) Linkage analysis was performed in two unrelated families of German origin. Using short sequence repeats (SSR) (Weber and May, 1989, Litt, 1989; Weber, 1990a,b), approximately 50% of the genome was excluded before a disease locus was assigned to D15S108 on chromosome 15q2. Multipoint linkage calculations yielded a peak lod score of 4.16. (penetrance at 0.95; with a penetrance of 0.5 the lod score was 5.19). In the second smaller family with a more severe form of the disease, a lod score of 2.28 (0 = 0) for the chromosome 15q2 locus was found. A comparison of the lod score values by the HOMOG program, which is used to determine the probability of genetic heterogeneity, supported the hypothesis that a common gene was involved in both families (Thierfelder et al., 1993). Identification of the 1592 locus was not associated with a strong candidate gene. The cardiac actin gene mapping close to this site was considered (Gunning et al., 1984) but was rapidly excluded by linkage analysis. Instead, identification of the a-tropomyosin gene was mediated by genomic map information obtained from the mouse. In this mammal the a-tropomyosin gene was assigned Table 8.2. Troponin T Gene (Chromosome lq3 CMHZ) Mutations Identified in Hypertrophic Cardiomyopathy

Amino acid

Nucleotide

Exon

Charge change

Arg92Gln

G287A

9

-1

Ile79Asn

T248A

8

0

Intronl5 G l - t A

G1A (intron)

lntron 15 splice site

NA

Phel lOIle

T340A

9

0

Glu163Lys

G499A

11

+2

Glu244Asp

(37441

14

0

Aglu160”

AGAG

11

+1

Ref. Thierfelder et al. (1994) Thierfelder et al. (1994) Thierfelder et AI. (1994) Watkins et al. (1995) Watkins et al. (1995) Watkins et al. (1995) Watkins et al. (1995)

“Deletion of three nucleotides corresponding to an entire glutamic acid codon and therefore does not cause a frameshift mutation. Frameshift mutation=mutation causing a change in the reading frame in which triplets are translated into protein.

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Aman S. Coonar and William J. McKenna

to chromosome 9, a region syntenic with part of human chromosome 15 (Schleef al., 1993). Because the sequence of the human a-tropomyosin gene was not known at that time, DNA clones and polymorphic SSR therein were isolated in order to establish linkage of the disease to this gene in the two families. With linkage confirmed, the a-tropomyosin gene was screened by individual exon polymerase chain reaction amplification and direct sequencing. Two different missense mutations in exon 5 were identified in the two families. The two mutations were closely spaced, but in different positions (codons 175 and 180) of the same domain of the protein (Thierfelder e t al., 1994). The vertebrate a-tropomyosin TPMl gene consists of 15 exons. There is a high level of conservation through evolution: human and rat muscle atropomyosin share 99.6% amino acid homology, and there is high identity between human a- and @-tropomyosingenes. Both mutations are in highly conserved regions and result in single amino acid exchanges accompanied by an appropriate charge change. The region affected lies close to the site of the calcium-dependent troponin T binding domain, a critical protein in cardiac contraction. For these three reasons it was hypothesized that these mutations were likely to produce significant cardiac dysfunction. a-Tropomyosin is expressed in many cell types, and indeed mutations in another tropomyosin, the a-tropomyosin TPM3 gene at 1q22-23, are the cause of nemaline myopathy, a generalized skeletal myopathy. It is unclear why the two identified mutations produce hypertrophic cardiomyopathy alone. It may be that exon 5 is of critical importance in cardiac striated muscle only or there may be other cardiac-specific differences in the isoforms of a-tropomyosin-associated molecules (i.e., @-myosin,troponin C, I) or other local cardiac factors. et

1. Troponin T mutations (Table 8.3) The two a-tropomyosin mutations constitute part of a domain of the molecule known to interact with troponin T in a calcium-dependent manner (Zot and Potter, 1987; White et al., 1987). This therefore raised the possibility that troponin T was a disease gene. The previously unknown genomic location of this gene had to be determined and therefore it was mapped to chromosome lq31 (Watkins et

Table 8.3. a-Tropomyosin Gene (Chromosome 15q2 CMH3) Mutations Identified in Hypertrophic Cardiomyopathy Amino acid

Nucleotide

Exon

Charge change

Ref.

Asp 175Asn Glul80Gly

G579A A595G

5 5

+I +1

Thierfelder et al. (1994) Thierfelder et al. (1994)

295

8. Molecular Genetics of Familial Cardiomyopathies

al., 1993a). Linkage of this gene to hypertrophic cardiomyopathy in a family previously unlinked to the chromosome 14 and 15 loci was confirmed in a family of 42 members with a lod score of 6.66 (8 = 0). Three families were mapped to this region on chromosome 1 before the cardiac troponin T gene itself was localized (Watkins et al., 1993a). To date, eight nonconservative mutations have been identified in the troponin T gene in 11 families (Thierfelder et al., 1994; Watkins, 1994). Six are

missense mutations, one is a 3 nucleotide deletion (codon 160), and one is a splice site mutation. The vertebrate troponin T gene sequence is highly conserved through evolution, and mutations cause structural and charge changes. The 5’ splice site mutation, in particular, would result in a markedly aberrant cardiac troponin T mRNA transcript. The troponin T region involved by the missense mutations (Ile79Asn, Arg92Gln, PhellOIIe, AGlu160, Glu163Lys) encodes a section the functional role of which is thought to involve calcium-insensitive atropomyosin binding (Brissonet al., 1986; Pearlstone et al., 1986; Pan et al., 1991, 1986). The other mutations (intronl5 Gl+A, Glu244Asp, Arg278Cys) could alter the carboxyl terminus of troponin T, a region contributing to tropomyosin interaction as well as to interaction with troponin I and troponin C. Indeed these latter mutations may interfere with calcium-dependent binding to a-tropomyosin (Ishii and Lehrer, 1991). The cardiac troponin T isoform is not expressed in adult skeletal muscle (Anderson et al., 1991, 1995; Ma!ouf et al., 1992) and this may explain the tissue-specific effects of this mutation.

J. Cardiac myosin-binding protein C mutations (Table 8.4) A fourth locus harboring a HCM disease gene was demonstrated on chromosome 11 in a French family unlinked to chromosome 14 (Carrier et al., 1993). Twopoint lod scores (8 = 0) for two marker loci on chromosome 11 (D11S905 and DllS986, with a distance of 6 cM between them) were 3.81 and 4.98. A strong candidate gene has been mapped to this locus. It codes for the cardiac-specific myosin-binding protein C (MyBP-C) (Gautel et al., 1995). Table 8.4. Cardiac Myosin-Binding Protein C (Chromosome 1lpl1.2 CMH4) Mutations Identified in Hypertrophic Cardiomyopathy Nucleotide

G5 intron M/N

18-bp tandem duplication 11-bp deletion in splice acceptor site 140-bp deletion in splice acceptor site

Exon

P

Ref. Watkins et nl. (1995) Watkins et al. (1995) Bonne (1995) Bonne (1995)

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Aman S. Coonar and William J. McKenna

MyBP-C is a component of the sarcomere. It is arrayed transversely in sarcomere A bands and binds myosin heavy chain in thick filaments and titin in elastic filaments. Phosphorylation of MyBP-C may modulate cardiac contraction. Titin is

a giant protein that is thought to be involved in the organization of muscle filaments (Labeit et al., 1992; Trinick, 1994). Shortly after this discovery two families were described with two different mutations in MyBP-C: (1) an internal partial duplication of 18 bp predicted to lead to the introduction of 6 amino acids in the mutant protein (Watkins, 1995) and (2) a splice site mutation in the C-terminal region predicted to lead to the loss of the terminal 213 amino acid residues, with a frameshift mutation encoding 37 novel amino acid residues followed by premature termination (Watkins, 199%). The following letter in the same journal describes an identical splice site mutation in exon 5 of the gene in two unrelated (or on the basis of haplotype analysis possibly distantly related) French families. Both mutations lead to a frameshift followed by a premature stop codon. This mutation conceivably leads to a truncated version of the affected protein devoid of its myosin-binding domain or alternatively a null allele (Bonne, 1995).

K. Other loci Despite the impressive advances in the molecular characterization of this disease, it appears that at most only 50% of HCM is accounted for by identified mutations. Linkage analysis continues, and Macrae (1995) has identified a locus at chromosome 7q3 in a large Irish family in whom members had familial hypertrophic cardiomyopathy and/or the Wolff-Parkinson-White syndrome. This finding suggests the presence of a single gene, the mutation of which may cause either disease or both, suggesting a common pathogenesis. Other chromosomal loci tentatively suggested to cosegregate with HCM are on chromosome 16 (Italian pedigree) (Ferraro et al., 1990) and on chromosome 18 (Japanese pedigree) (Hejtmancik et al., 1991; Machida, 1994). Screening of Japanese pedigrees suggests that other chromosomal loci may be more important in the etiology of HCM than those identified in pedigrees of European descent (Machida, 1994). These findings, however, await further confirmation.

L. Are the identified mutated genes etiological? This conclusion rests on the following arguments: 1. Mutated genes code for essential cardiac functions. 2. Mutations have been found only within HCM families and not as polymor-

8. Molecular Genetics of Familial Cardiomyopathies

3.

4.

5. 6.

7. 8.

297

phisms in the control populations evaluated. A few polymorphisms have been reported that do not cosegregate with the disease in affected families (Greve et al., 1994; Watkins et al., 1995; Watkins, 1995a). Within families, all those phenotypically affected have the appropriate mutation. Obligate carriers with the appropriate mutation have been identified. Mutations are located in evolutionary conserved regions. Most of the mutational amino acid changes confer an altered charge to the protein, and are therefore likely to have a major impact on protein stoichiometry and function. Analysis has shown that de novo mutations in P-myosin heavy chain genes (Watkins et al., 199213;Greve et al., 1994) and the a-tropomyosin gene (Watkins et al., 1995) are transmitted together with the disease. In vitro analysis of P-myosin heavy chains and contractile fibers isolated from muscle biopsies of patients (slow skeletal muscle biopsies) has demonstrated that mutated P-myosins are functionally impaired with respect to contractile properties and filament assembly (see later).

M. Molecular pathogenesis Detrimental effects of the identified mutations on cardiac function can be expected at different biological levels. A basic level is the organization and function of thick and thin filaments within the myocyte. The next level is the development of these cells and their interaction with other cardiac components (fibroblasts and the constituent cells of small vessel walls). A tertiary level is the altered metabolic and functional behavior of hypertrophied and relatively disorganized tissue (Figure 8.2) with regard to cardiac contraction, relaxation, and coordination of electrical activity. Given the relatively recent identification of etiological mutations, the determination of these consequences remains at a very early stage. The @-myosin heavy chain has been most investigated. The @-chainis 1935 amino acids long. The myosin promoter has a molecular mass of about 500,000 D. It consists of two identical heavy chains (in cardiac myocytes either type a or P, each about 220,000 D) and four light chains of two different isotypes (18,000 to 24,000 D). The heavy chains are highly asymmetric with a globular head at the N terminus and a long filamentous tail. The head containing the ATP-binding site and the domain involved in cross-bridge formation with actin extends from amino acid 1 to 866. The junction between head and rod is the site for light chain attachment. The light chains are modulators of the heavy chain activity. The filamentous rod, which comprises about half of the molecule, extends to the C terminus. In the na-

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Aman S. Coonar and William J. McKenna

tive monomeric myosin molecule, the two rods are coiled around each other. Rods are involved in the formation of multimeric thick myosin filaments within the contractile cells. Mutational amino acid replacements may be either conservative or nonconservative depending on the assignment of respective residues to one of the four main classes of amino acids (hydrophobic, neutral polar, positively charged, and negatively charged). Conservative mutations are generally assumed to be less disruptive for the protein structure and function than nonconservative changes. A change in charge is therefore considered to have a major impact on the stability of a protein. Most of the known @-myosinheavy chain mutations are accompanied by a change in charge. Although the consequences of such changes are unknown, suggestions have been made that a change in charge of the P-myosin molecule has in general a more detrimental effect on cardiac functions than an electrochemically neutral exchange. Truly conservative exchanges have been observed only in a minority of replacements (in position 124,232,606,870, and 908). Early clinical observations indicate that mutations associated with charge changes predispose to a more severe disease (Anan et al., 1994). Mutations have also been analyzed in terms of their location. The altered sites have been restricted to the globular head or the head-rod junction. Within this domain they may occur over a wide range. Five of the known mutations are close to or within the sites controlling ATP binding and actin interaction. The corresponding amino acid residues are in positions 232, 249, 256, and 403. Mutated residues Asn232Ser, Arg249Gln, and Gly256Glu could be part of the ATPbinding site; the residue Arg403-Gln/Trp/Leu is probably part of the region directly involved in cross-bridge formation with actin filaments (Rayment et al., 1993a,b, 1995; Schroder et al., 1993; Rayment and Holden, 1994; Fisher et al., 1995). For these exchanges, an effect on a distinct function may be expected. In contrast, other amino acid replacements cannot satisfactorily be related to defined sites or functions of the myosin head. However, it is conceivable that many of the mutations could disturb proper folding from a distance, with some of them changing a particular function related to ATP, actin, or myosin light chain binding or having more general effects. The mutations detected so far have been heterozygous. This results in mutations and their abnormal protein products coexisting with copies of the normal gene and protein in affected persons. The precise mechanism by which the disease is produced, however, remains unknown. It is postulated that the mutant protein may act as a “poison peptide” by incorporation into the sarcomere structure in such a way as to subsequently disrupt structure or function. Alternatively, a mutation may act to functionally deactivate a gene, potentially reducing peptide concentrations by 50%.

8. Molecular Genetics of Familial Cardiomyopathies

299

The interaction between the thick and the thin filaments of muscle is a dynamic process reliant on energy-dependent changes in the stoichiometry of the relevant molecules. Small changes at one site are capable of large overall changes in molecular conformation and activity. Abnormal conformation or binding properties may therefore seriously affect force generation, relaxation, and molecular stability. Further compensatory mechanisms may act to produce as secondary phenomena myocardial disarray, electrical instability, and hypertrophy. Hypertrophy may arise from the induction of oncogenes or other isoforms of the sarcomeric contractile proteins, as has been seen in models of secondary hypertrophy (Parker and Schneider, 1991; Parker, 1993). The splice donor mutation in cardiac troponin T may act as a null allele. The human mutation is analogous to a 5’ splice donor site mutation in intron 7 of the Drosophila rnelanoguster flight muscle troponin T, resulting in the mutant fly upheld2 (Fyrberg et al., 1990). This results in a truncated polypeptide product. Homozygous upheld2 flies have no troponin T in their flight muscles and have virtually no thin filaments. Heterozygote flies, analogous to the human situation, have a disrupted myofibrillar architecture with a disordered architecture of thick and thin filaments in the outer half of each myofibril (Mogami and Hotta, 1981; Mogami et al., 1982). It is predicted that the effect of this splice site mutation is to produce an aberrantly shortened mRNA transcript with a resultant peptide product. Under in vivo conditions, however, the situation is probably more complex, e.g., the apparent null mutation in the human p myosin heavy chain gene mentioned earlier does not appear to be associated with a disease phenotype in the heterozygous state (Nishi et al., 1994). In cases where both normal and mutant proteins are produced, the effects may result from a dominant negative or poison peptide mechanism. The incorporation of wild-type and mutated proteins into multisubunit structures such as thick filaments might induce a distorted function even if the normal product is present in excess. The difficulties may begin with filament assembly and could extend to enzymatic and other functions. For example, in the nematode Caenorhabditis elegans, mutations in the unc-54 gene cause autosomal dominant paralysis (Bejsovec and Anderson, 1988, 1990).These are missense mutations in the globular head and head-rod junction regions of a myosin heavy chain, a proportion of which result in a charge change in the amino acid residue (Dibb e t al., 1985, 1989; Mitchell e t al., 1989). Mutant heavy chains are incorporated into thick filaments and subsequently disrupt filament and sarcomere assembly. Indeed, quantitative assay in the nematode has shown that as little as 2% of expressed mutant protein is sufficient to disrupt thick filament and sarcomere assembly. I t has been proposed that a similar process may occur in humans. The finding of mutated pmyosin mRNA and protein in cardiac tissue of patients lends support to this concept (Perryman et al., 1992; Yu e t al., 1993).

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In the nematode, a gene-dose effect has also been observed; when two mutant genes are present, the pattern of inheritance becomes recessive and the phenotype is lethal. In humans, it has also been observed that the presence of mutations affecting both alleles of the P-MHC gene is associated with a more severe phenotype (Nishi et d.,1994). In a consanguineous Japanese family, Nishi et al. (1994) found a missense mutation due to a G to A mutation in codon 935, leading to a replacement of Glu935Lys. In the family studied, those members who were heterozygous manifest either cardiac hypertrophy or were clinically normal. However, the proband and his elder brother were homozygous for the mutation and displayed a more severe phenotype than heterozygotes from the same family. These observations tentatively suggest a gene-dose effect of the mutant cardiac P-myosin heavy chain gene on the clinical manifestation of familial hypertrophic cardiomyopathy. Marian et al.(1995) sought to determine whether the expression of mutant P-myosin heavy chain in adult feline cardiac myocytes induces sarcomere disarray. A full-length P-myosin heavy chain cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the P-myosin heavy chain cDNA by site-directed mutagenesis. Infection of COS-1 cells with the P-myosin heavy chain construct resulted in the expression of a fulllength myosin protein. The efficiency of infection of isolated adult cardiac myocytes was >95%. Expression of the P-myosin heavy chain constructs into mRNA at 48 hr after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The subsequent study of transformed myocytes revealed a disruption of sarcomere assembly, analogous to that seen in the hearts of persons with hypertrophic cardiomyopathy. The conclusion being that mutated P-myosin heavy chain, as one of the consequences, does genuinely lead to sarcomeric disarray. It also has been proposed that abnormal sarcomeric proteins lead to an abnormal force velocity relationship following sarcomere excitation (Cuda et al., 1993). Mutant P-myosin, also shown to be present in skeletal muscle by Western blot analysis, has been investigated. In one study, P-myosins containing the mutations Arg403Gln and Leu908Val were tested. These myosins were isolated from slow fibers taken from soleus muscle biopsies. They were analyzed for their ability to promote in vitro motility of thin actin filaments (Cuda e t al., 1993). A decrease in motility was seen with both mutations. The Arg403Gln mutation produced motility of 20% of normal, whereas the Leu908Val mutation produced motility of 40% of normal. One should note, however, that in vitro motility studies describe an experimental situation that is not directly comparable to physiological events in vertebrate skeletal or cardiac muscle. In vitro motility is not coupled to mechanical work, as it would be in muscle, and the myosin molecules are not assembled in a manner comparable to native thick filaments. In a second study of altered function, in vitro motility was tested with a recombinant rat a-myosin heavy chain mutated in position 403 (Sweeney et al., 1994). Again, the corollary of the mutation was slowed in vitro motility of actin filaments. In addition, the

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301

ATPase activity of the altered P-myosin molecules was greatly reduced as compared to wild-type molecules. A third investigation using single fibers of slow muscle biopsies demonstrated altered contractile properties, such as decreased isometric force generation and a depressed shortening velocity with the mutated P-myosins Arg403Gln and Gly741Arg. In contrast, the properties of mutation Gly256Glu were indistinguishable from normal (Lankford et al., 1995).Thus, single amino acid changes in P-myosin may result in abnormal force generation, which supports the primary role of missense mutations in the P-myosin heavy chain gene in the etiology of some cases of hypertrophic cardiomyopathy. Such a model would have an impairment of contractile protein interaction as the primary abnormality, with disorganized growth, fibrosis, small vessel abnormalities, electrical instability, and hypertrophy being secondary phenomenon. There is a wide range of disease expression even within related individuals with identical gene mutations. Several theories have attempted to explain this. Interest in the role of the renin-angiotensin system as a disease modifier has arisen. Particular attention has focused on the role of the angiotensin-converting enzyme (ACE). ACE is present in tissue bound, as well as forms secreted into for example, the circulation and cerbrospinal fluid. ACE is upregulated in pressure overload-induced cardiac hypertrophy as well as heart failure (Schunkert et al., 1993; Danser et al., 1995a,b). In addition to catalyzing the conversion from angiotensin I to angiotensin 11, ACE has greater catalytic power for the conversion of the vasodilator bradykinin to kinins. Angiotensin I1 is a potent vasoconstrictor and has significant trophic and mitogenic properties. It is a growth factor for cardiac myocytes, inducing cardiac hypertrophy independent of hernodynamic or neurohumoral effects (Sadoshima and Izumo, 1993). Inhibition of the angiotensin-converting enzyme induces the regression of cardiac hypertrophy independent of load (Linz et al., 1992) and prevents dilation and adverse remodeling of the ventricle following myocardial infarction (Pfeffer et al., 1992; Lindpaintner et al., 1995; Pfeffer, 1995). A n insertion/deletion polymorphism (I/D) in the ACE gene due to the presence or absence of a 287-bp A h repeat in intron 16 of the ACE gene has been described (Rigat et al., 1992). The I/D polymorphism results in three genotypes: DD,ID, and 11. The DD genotype is associated with plasma levels of ACE twice that of genotype I1 (Rigat et al., 1990). Lechin et al. (1995) investigated whether the ACE genotype influenced the phenotypic expression of hypertrophy in 183 patients with HCM. In genetically independent patients, measures of left ventricular hypertrophy were significantly greater in persons with the DD genotype when compared with those with the ID and I1 genotypes (P < 0.05-0.005). Regression analysis showed that ACE genotypes accounted for up to 6.5% of the variability in left ventricular hypertrophy. In 26 patients from a single family, left ventricular hypertrophy was also greater in patients with DD than in those with ID and I1 genotypes. The authors concluded that ACE genotypes significantly influence the phenotypic expression of hypertrophy in HCM (Lechin et al., 1995).

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Because P-myosin is a constituent of slow skeletal muscle, the question has arisen whether skeletal muscle is also affected by mutations in this protein. It has been shown that the mutant protein is expressed in these fibers (Yu et al., 1993; Cuda et al., 1993; Lankford et al., 1995). Muscle symptoms are, however, rare in HCM patients. Conceivably, the minimal effects of mutated myosin in skeletal muscle are explained by the special role of slow twitch muscles in the body. They are primarily involved in the control of body posture rather than in the development of force for moving. In addition, it may also be that cardiac dysfunction simply masks a weakness in muscles. A definite conclusion about the extent to which skeletal muscle function is impaired in HCM patients can presently not be drawn.

N. Genotype and phenotype correlation The correlation available at this time can only be considered as preliminary because relatively few pedigrees and mutations to date have been identified. Considerably more assessment is required to determine the prognostic significance of a given mutation and how it may be influenced by modifying genetic and environmental factors.

0. p-Cardiac myosin heavy chain Different mutations within the P-cardiac MHC gene appear to correlate with significantly different rates of survival (figure 8.3) (Epstein et al., 1992a; Watkins et al., 1992a; Anan et al., 1994). However, they have not been shown to correlate convincingly with differences in morphology (Solomon et al., 1993). The mutations Arg403Gln, Arg453Gln, and Arg7 19Trp have been particularly associated with a significantly worse or “malignant” prognosis (Watkins et al., 1992a). Mutations in this gene appear to be fully penetrant.

P. a-Tr o po my 0sin In contrast, due to a-tropomyosin mutations in HCM, significantly less cardiac hypertrophy was associated with Glul80Gly than with Asp1 75Asn, although this was not of prognostic significance and the life expectancy of patients with either mutation was similar (Thierfelder et al., 1993, 1994). To date, only two pedigrees with mutations in this gene have been discovered.

Q. Troponin T The troponin T mutations Ile79Asn, Arg92Gln, AGlu160, and intron 15GlA were associated with a significantly shortened life expectancy, similar to that seen with “malignant” P-MHC mutations (Watkins etal., 1995) (figure 8.4). There was also a significantly higher rate of sudden death before the age of 30 as compared to

8. Molecular Genetics of Familial Cardiomyopathies

303

I00 Va1606Met

90

Benign

80

70 Cumulative 60 Survival

50

(”/I

40

1

30 20 10

:0

20

40

60

80

Age (Years) Figure 8.3. Survival by P-MHC gene mutations. Comparison of survival by specific P-MHC gene mutations. Note the difference in survival conferred by a so-called “benign” mutation Va1606Met in contrast to the “malignant” pattern seen with Arg403Gln, Arg453Cys, and Arg249Gln. Data from Watkins et al., (1992a).

P-MHC mutations (Watkins et al., 1995). T h e degree of cardiac hypertrophy associated with cardiac troponin T mutations was significantly less than that associated with p-MHC mutations (mean maximal wall thickness 16.7 ? 5.5 mm with cardiac troponin T mutations versus 23.7 -+ 7.7 mm with P-MHC mutations). Each cardiac troponin T mutation produced a similar increase in the maximal thickness of the left ventricular wall (mean range 13.4-19.8 mm). Clinical evaluation of family members of probands also identified several genetically affected but otherwise apparently normal relatives (asymptomatic, no detectable signs, and normal ECG and echocardiogram). This gave an estimate of gene penetrance for troponin T mutations as 75% in contrast to the 95% penetrance associated with P-MHC mutations associated with a comparable malignant phenotype. T h e disparity between the severity of the degree of cardiac hypertrophy and prognosis in mutations of the cardiac troponin T gene again illustrate the relative shortcomings of diagnosis and risk evaluation based on just clinical criteria. It is expected that a proportion of apparently unaffected individuals as assessed by current clinical criteria will ultimately suffer sudden cardiac death. Their prognosis may be significantly improved by the potential interventions arising from a molecular diagnosis. These results also indicate that a correlation may exist between the mutation and the prognosis of familial HCM caused by altered P-myosin heavy chain

Aman S. Coonar and William J. McKenna

304

100 90

80 70 Cumulative 60 Survival

50

(W

40

30 20 10 0

0

20

40

60

80

Age (Years) Figure 8.4. Survival by troponin T gene mutation. Comparison of survival by troponin T mutation with superimposed differences in survival conferred by so-called “benign” and “malignant” P-MHC gene mutations. Note the poor prognosis conferred by the mutations intront5GtA, AtgQZGln,AGlul60, and lle79Asn, similar to that seen in “malignant” pMHC gene mutations. Data from Watkins et al., (1995).

and cardiac troponin T genes. However, the number of families studied remains small. In addition, discordant observations have been communicated for the mutation Va1606Met occurring in different families (Fananapazir and Epstein, 1994). Furthermore, in one large family (46 persons affected with good prognosis), the cause was the missense mutation Gly256Glu accompanied by a change in charge (Fananapazir and Epstein, 1994). Thus, studies of more families are required to determine whether genotyping will significantly aid in identifying high-risk families and individuals.

A. Definition and clinical syndrome Dilated cardiomyopathy is clinically recognized (WHO/ISFC, 1980) by uni- or biventricular dilatation, is accompanied by impairment of contractile function, and is associated with rhythm disturbances, thromboembolism, and sudden death. The early phase of the syndrome has been little investigated, and the primary functional abnormality is unknown. Following the onset of contractile dysfunction at the molecular and cellular level, compensatory mechanisms are adequate and there

-

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8. Molecular Genetics of Familial Cardiomyopathies

Main classes Genetic

- X-linked dystrophin

-unknown, linkage to l p l - l q l , lq32,3p22-25,9q13-22 lschaemic Valvular Hypertensive Toxic

- ie: alcohol, adriamycin

Peripartum

Minor cardiac dilatation and/or contractile dysfunction

- ie: thyroid disease, diabetes, storage disorders - ie: sarcoid, haemachromatosis,SLE Muscular dystrophy - ie: Duchenne/Becker, myotonic dystrophy Metabolic

lnfiltrativelgeneral system disease

Neuromuscular ie: Noonan' syndrome

*

Significantcardiac dilatation and/or contractile dysfunction meeting diagnosticcriteria for DCM

Inflammatory/Autoimmune ie: myocarditis Others

Figure 8.5. Hypothesized heterogeneity of dilated cardiomyopathy,

is no obvious abnormality, but later cardiac dilatation with overt contractile failure develops. It is very likely that the syndrome recognized as DCM is highly heterogeneous in etiology and that the poorly dilated contractile heart is merely the end point of several different disease processes (figure 8.5). Diagnosis is by exclusion of other causes of cardiac failure, such as hypertension, coronary artery disease, valve disease, and specific heart muscle disease (WHO/ISFC, 1980).Principal causes of dilated cardiomyopathy are summarized in Figure 8.5 and are recognized in a new classification of the cardiomyopathies (WHO/ISFC, 1996). The major pathological finding is of increased heart weight with dilatation of both ventricles (Roberts et al., 1987; Manolio et al., 1992; Davies and McKenna, 1995; Roberts, 1989).Secondary dilatation of valvular annuli is frequently observed. Microscopic features include hypertrophy and degeneration of myocytes, varying degrees of interstitial fibrosis, and a variable inflammatory infiltrate.

B. Epidemiology Dilated cardiomyopathy is seen worldwide, but epidemiological information has been assessed only in a few countries. In the United States, a prevalence of 36 per 100,000 has been estimated, with an annual incidence of 5-8 per 100,000(Codd, 1989; Manolio et al., 1992). However, it is likely that these are underestimates, for in most cases DCM has a long preclinical phase during which patients are asymptomatic (Gillum, 1994). This is discussed further in the following section.

C. Clinical genetics A number of families have been published with an X-linked pattern of inheritance, and it is in this group that the most advances have been made in deter-

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mining the nature of the molecular abnormality. Sporadic, autosomal dominant and autosomal recessive patterns of inheritance have also been identified. Because these studies were based on retrospective assessments and/or on selective screening of families suspected to have familial disease, they were unable to determine the prevalence of familial DCM. Recent prospective studies suggest that familial disease may be present in at least 20% of probands. In the Mayo Clinic study (Michels et al., 1992), famd ilies of 59 consecutive unrelated DCM patients were screened for affected relad rives. DCM was diagnosed in 18 of 325 relatives (5.5%) from 12 families with a familial prevalence of 20% (12/59). This study demonstrated the importance of family screening; 83% of affected relatives were asymptomatic and would have been missed by screening based on the presence of symptoms. In all cases, a mode of transmission most consistent with autosomal dominant inheritance was deter, mined. In another study, 40 families of consecutive DCM probands were evaluated in the United Kingdom (Keeling, 1995): 25/236 (10.6%) relatives from 10families were identified as being affected with a familial prevalence of 25% (10/40). Segregation analysis again was most consistent with autosomal dominant trans. mission in familial DCM, with penetrance estimated to be approximately 65%. In many of these families there was a high rate of structural and/or functional cardiac abnormalities that were significantly greater than in a control population screened using identical methodology, but did not meet the formal criteria of DCM. Segregation analysis was repeated including these family members. The pattern of inheritance was now strongly in favor of a pattern of autosomal dominant inheritance with high gene penetrance but variable expression (Coonar, 1995). It is likely that the majority of familial DCM will be eventually explained thus, but probably with significant genetic heterogeneity.

D. Molecular genetics

1. X linked Berko (1987) described a five-generation pedigree of 63 persons. Affected males with DCM had early onset, rapid progression, and severe disease in contrast to females with DCM who had later onset, slow progression, and milder disease. There was no male-to-male transmission and all affected females had sons who died of DCM. No patient had evidence of skeletal myopathy or neuromuscular disease, although all the affected males had elevated serum creatine kinase levels, suggesting a process involving muscle damage. The consistently different pattern of disease by gender suggested a pattern of X-linked inheritance which suggested the role of mutations affecting either the X chromosome or the mitochondria1 genome. Towbin et al. (1993), investigating two unrelated families, identified linkage of X-linked DCM to Xp21, the site of the dystrophin gene, with a maximum lod score of +4.33 (0 = 0) with DXS206 using two-point linkage and +4.81 at the same

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locus with multipoint linkage analysis. Abnormalities of cardiac dystrophin were shown by Western blotting with a N-terminal dystrophin antibody, whereas skeletal muscle dystrophin was normal, suggesting primary involvement of the dystrophin gene with a preferential involvement of cardiac muscle. The authors concluded that X-linked DCM was due to an abnormality within the centromeric half of the dystrophin gene region in the heart. This abnormality could be accounted for by a mutation in the 5’ region of the dystrophin-codingsequence preferentially affectingcardiac function, splicing abnormalities which resulted in an abnormal cardiac protein, or a cardiac-specificpromoter mutation. Subsequently, Muntoni et al. (1993, 1995) showed in a large X-linked pedigree that a deletion removing the dystrophin muscle promoter, the first muscle exon, and part of intron 1 caused a severe dilated cardiomyopathy with no associated skeletal muscle weakness. Dystrophin transcription and expression in the heart of one member of this family were then studied. In contrast to skeletal muscle, dystrophin transcription and expression were absent in the heart, with the exception of the distal Dp71 dystrophin isoform. The 43- and 50-kDa dystrophin-associated proteins were severely reduced in the heart, despite the presence of Dp7 1, but not in skeletal muscle. The authors argue that the absence of dystrophin and the downregulation of the dystrophin-associated proteins in the heart accounted for the severe cardiomyopathy in this family. The presence of cardiac-specific disease with other mutations has also been described (Yoshida et al., 1993). The determinants of whether a dystrophin mutation causes cardiomyopathy, skeletal myopathy, or both are not yet fully known. It is been shown that dystrophin gene expression is driven by at least five distinct promoters with independent cell-type specificity (Ahn and Kunkel, 1993).Conceivably, specific mutations exist which preferentially affect the heart and not muscle. This remains to be further assessed, and has been further discussed in an earlier chapter in this series. Because both the Becker- and the Duchenne-type muscular dystrophies arising from mutations in the dystrophin gene located at Xp21 have significant cardiac involvement, in a pattern similar to dilated cardiomyopathy, the identification of the causal gene in these diseases led to speculation that these mutations were also responsible for cases of idiopathic non-X-linked dilated cardiomyopathy. Dystrophin gene deletion analysis was performed in two independent studies; in 27 unselected DCM patients (Michels et al. 1993) and in 33 male DCM patients with sporadic DCM (Coonar, 1996). In neither case were dystrophin gene defects found, indicating that dystrophin mutations are probably a rare cause of idiopathic dilated cardiomyopathy.

2. Autosomal dominant Linkage analysis using short sequence repeat polymorphisms has identified at least four loci for the autosomal dominant form of the disease: lpl-lql (Kass et al., 1994), 9q13-22 (Krajinovic et al., 1995), lq32 (Durand, 1995), and 3~22-25 (Olson, 1996).

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In a large pedigree characterized by Graber e t al. (1986) in whom dilated cardiomyopathy was associated with a progressive cardiac conduction disease, Kass et al. (1994) demonstrated linkage of the disease locus to the locus Ipl-lql with a maximum multipoint lod score of 13.2 for the interval between DlS305 and DlS176. Based on the disease phenotype and the map location the gap junction protein connexin 40 ((2x40) was proposed as a candidate gene. Durand (1995) studied a 46 member family with four generations and found linkage to lq32. They identified a peak multipoint lod score of 6.37 at DlS414 and suggested that MEF-2D (myocyte enhancer factor 2D), FMOD (flavin-containing monooxygenase), PCMCA4, renin, and helix-loop-helix DNA-binding protein MYF-4 were candidate genes. Krajinovic e t al. (1995) studied a large six-generation kindred and two other families with an autosomal dominant pattern of transmission. All three families were unrelated and had identical clinical features. Linkage was found for chromosome 9q13-q22, with a maximum multipoint lod score of 4.2. Genetic heterogeneity was not supported. The locus was placed in the interval between loci D9S153 and loci D9S152. The authors hypothesize as candidates the FRDA/FARR Friedreich ataxia gene, CAMP-dependent protein kinase, and tropomodulin, a tropomyosin-modulating protein. Linkage has also been identified in a family with dilated cardiomyopathy to 31322-25 (Olson, 1996). A maximum two-point lod score of 6.09 was identified at D3S2303. A number of candidate genes map to this locus and, using single-strand conformational polymorphism, excluded linkage to five genes in this interval. These were a G-protein (GNAlZ), a calcium channel (CACNL 1A2), a sodium channel (SCN5A), an inositol triphosphate (ITPRl), and the gap junction connexin 45. Identification of linkage also allowed the demonstration of appropriately inherited haplotypes, as gene mutation markers, in obligate carriers. By correlating genetic and phenotypic data in this family, it was then possible to suggest that sinus bradycardia may be an early clinical marker of disease manifestation. This pedigree has a similar phenotype (dilated cardiomyopathy with conduction disease) to the family characterized by Graber e t al. (1986) and discussed earlier as mapping to chromosome lpl-lql (Kass et al., 1994). This locus was specifically excluded in the pedigree studied by Krajinovic and hence this investigation further demonstrates genetic heterogeneity within DCM.

3. Recessive There are a only a few reports of true familial DCM occurring in a pattern consistent with autosomal recessive inheritance, and as yet no specific locus has been assigned. In a study of 165 consecutive patients (Mestroni et al., 1990), inheritance was autosomal dominant in seven families and recessive in four. Koike et al. (1987) in an investigation of the role of the HLA system, identified a single family in

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which inheritance was most probably autosomal recessive. Goldblatt ( 1987) identified dilated cardiomyopathy occurring in three members of a consanguineous Madeira Portuguese family which followed a recessive pattern of inheritance. These reports should be reviewed in light of our current understanding of DCM. Since we now recognize a prolonged presymptomatic phase during which cardiac abnormalities may be present but not yet overt, it may well be that cases initially characterized as being recessive in inheritance are actually not, What, however, is likely is that DCM is a highly heterogeneous disorder, representing the end stage of a number of different disease processes.

E. Pathogenesis The proposed mechanisms by which the dystrophin gene or related mutations result in disease have been extensively reviewed in a previous chapter in this series, As for nondystrophin-related dilated cardiomyopathy, the primary or initiating events are unknown. A popular mechanism of pathogenesis has been an enteroviral trigger (coxsackie virus type B) (Kandolf et d.,1993;Keelinget al., 1992) with chronic immune-mediated damage (Caforio et al., 1990a,b,c, 1992; Caforio and McKenna 1990) occurring in the genetically predisposed individual (Zachara et al., 1993; Keeling et al., 1995; Coonar, 1995). This hypothesis has arisen from the experience of animal models of viral- and immune-mediated myocarditis and heart failure (Sole and Liu, 1993; Kodama et al., 1994). Although raising the role of immune system genes as disease candidates, confirmation of such a hypothesis to explain human-dilated cardiomyopathy as yet has not been forthcoming.

IV. ARRHYTHMOGENIC RIGHT VENTRICULAR CARDIOMYOPATHY A. Definition and clinical syndrome Until recently, arrhythmogenic right ventricular cardiomyopathy (ARVC), previously known as arrhythmogenic right ventricular dysplasia (ARVD), was not included as a cardiomyopathy in the last WHOlICSF task classification, and it was then unclear as to whether it was merely a subset of dilated cardiomyopathy. However, the disease has become well recognized as a distinct entity since the mid1980s and is to be formally classified with the cardiomyopathies in the forthcoming WHO classification (WHO/ISFC, 1996). ARVC is characterized by fibro-fatty replacement of the myocardium with a marked predilection for the right ventricle. Initially the disease tends to be patchy, but more diffuse right ventricular involvement, with or without left ventricular abnormalities, may occur later (Marcus and Fontaine, 1995; Davies, 1994; McKenna et al., 1994). Clinical features include palpitation, syncope, sudden

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death, and systolic heart failure, although the latter is an unusual late complication of this disease. Definitive diagnosis of ARVC is based on the histological demonstration of transmural fibro-fatty replacement of the right ventricular myocardium at either necropsy or surgery. In the absence of this finding, diagnosis is based on clinical presentation; structural, functional, or electrocardiographic abnormalities; and family history. Recently, criteria for diagnosis based on a classification of clinical features into major and minor criteria have been proposed (McKenna et al., 1994) and are under prospective evaluation.

8. Clinical genetics Systematicpedigree studies have not been performed for most probands. In most cases, however, preliminary observations suggest a familial pattern with autosomal dominant inheritance (Laurent et al., 1987; Nava, 1987; Canciani et al., 1992). There are also a number of well-characterized pedigrees with an autosomal recessive pattern of inheritance (Protonotarios et al., 1986 and manuscript in preparation).

C. Epidemiology There is no good population-based evidence upon which to base reliable estimates of incidence or prevalence. However, retrospective pathological studies suggest that it may be second only to hypertrophic cardiomyopathy as a cause of sudden death in apparently healthy young athletes (Fontaine et al., 1989; Fontaine et al., 1992; Kenny and Shapiro, 1992). In addition, a single survey from the Veneto area revealed that it is the leading cause of sudden death in the young (Corrado et al., 1990).

D. Molecular genetics To date, three loci have been reported for the autosomal dominant form of ARVC. Rampazzo et al. (1994) found a maximum lod score of 6.04 for linkage with the polymorphic marker D14S42 (14q23-24) (ARVD1) in two families of Italian origin, one of which had 82 subjects (19 affected) in four generations. Subsequent work refined the map position to 14q24.3 in close proximity to the a-actinin 1 (ACTN1) gene which was hypothesized as a candidate gene for this disease, although confirmation of this is still needed. The same group illustrated genetic heterogeneity in the disorder by demonstrating that other families with ARVC were not linked to this locus. Then, using the hypothesis that a-actinin was the etiological gene, they went on to search for linkage to the gene coding for a-actinin 2 (ACTN2), an isoform of a-actinin 1. Using this strategy, they successfully identified linkage in a pedigree demonstrating linkage at lq32 with a marker in close proximity to the map position of the a-actinin 2 gene.

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Another group investigated three unrelated European families of different ethnic origin (Italian, Slovenian, and Belgian). Of 37 members, 13 were diagnosed as having ARVC. They excluded linkage to D14S42 (ARVDl), but recently identified linkage to the same chromosome at 14q12-22 with the marker D14S252 with a cumulative two-point lod score of3.26 (9 = 0) (Severini, 1996). A number of families originating from an isolate (Protonotarios et al., 1986) have been reported with an autosomal recessive form of the disease (A. S. Coonar, unpublished data). A total of 177 individuals were originally characterized, but only 37 individuals with 13 affected from a total of eight families were subsequently available for molecular genetic analysis. In this family the cardiac defect cosegregated perfectly with a characteristic keratoderma and woolly hair. This raised the possibility of either a contiguous gene syndrome or a single gene that specifically produces its effects in myocardium, hair, and skin. Linkage analysis in this family is being pursued using a strategy of homozygosity mapping, searching for shared haplotypes identical by descent. Such a strategy relies on the hypothesis that a single copy of the disease gene was introduced into the population as a founder effect. In an isolate, therefore, there is a statistical probability that the disease gene becomes enriched in frequency and may become manifest as a recessively inherited phenotype. To date, linkage to the three loci mentioned earlier has been excluded (A. S. Coonar, unpublished data). This implies the presence of at least four loci for ARVC, including a novel one for the recessive form of the disease.

V. RESTRICTIVE CARDIOMYOPATHY Definition and clinical syndrome Restrictive cardiomyopathy was included in the WHO/ISFC classification as a cardiomyopathy. However, with this disorder there is probably the greatest degree of controversy regarding diagnosis and debate as to whether it truly represents a distinct myocardial disorder. It is probable that the majority of cases are the consequence of a nonmyocardial pathological process and thus it is not a primary myocardial disease. Diagnosis of the condition (be it primary or secondary) is clinical and relies on the demonstration of abnormal diastolic function, characterized by a restrictive filling pattern, accompanied by a reduced diastolic volume of either or both ventricles. Systolic function and myocardial wall thickness are normal or near normal. A few reports indicate that restrictive cardiomyopathy can be inherited as a primary cardiac disease (Aroney et al., 1988; Fitzpatrick et al., 1990). A father and daughter with idiopathic restrictive cardiomyopathy have been described. The hemodynamic profile was characteristic and there was echocardiographic evidence of diastolic dysfunction and atrial enlargement without ventricular dilatation. The distinction between restrictive cardiomyopathy and endomyocar-

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dial fibroelastosis is also unclear. Whereas the former is primarily a clinical defin. ition, the latter is pathological. Endomyocardial fibrosis is characterized by thickening of the endocardium with a deposition of fibrous and elastic tissue. Endomyocardial fibroelastosis has been observed in primary genetic forms as well as in sporadic cases. Congenital cases which frequently do not show other cardiac anomalies may account for up to 10% of childhood death from heart disease. The clinical symptoms are of biventricular cardiac failure. There is no effective therapy, with the possible exception of cardiac transplantation. Genetic studies of families with endomyocardial fibroelastosis have revealed that X-linked inheritance is the most frequent pattern of transmission (Hodgson et al., 1987). In addition, a few reports have suggested that autosomal recessive inheritance may also occur (Hallidie and Olsen, 1968). However, whether fibroelastosis itself should be classified as a restrictive or dilated cardiomyopathy is questioned since the pathological changes of fibroelastosis can be found in the hearts of persons with autosomal dominant dilated cardiomyopathy (Ross et al., 1978). In these cases it is not known whether the endocardia1changes are primary, representing a separate disease, or whether they are a feature of the underlying cardiomyopathy. The structural and functional effects of infiltrative disorders are variable, but often produce a secondary form of restrictive cardiomyopathy.

VI. OTHER PRIMARY HEART MUSCLE DISEASES A number of other familial disorders have been described which meet the crite-

ria of being primary myocardial disorders and not part of a greater disease syndrome. For reasons that reflect the earlier lack of knowledge, as well as the segregation of diseases to different clinical subspecialties, these were not considered as cardiomyopathies per se. Pending a reclassification based on molecular etiology, it is now clear that to some extent they must fall into the same category.

Long QT syndrome Prominent among these disorders is the idiopathic or “primary”long QT syndrome. This is diagnosed by the demonstration of a pathologically prolonged corrected QT interval on the ECG (a measure of the rate of ventricular depolarization and repolarization) in the absence of other secondary causes, including, for example, certain anti-arrhythmic drugs and electrolyte disturbances. Studies of cellular electrophysiology have suggested an abnormality at the level of myocyte repolarization. The disorder predisposes the heart to serious rhythm disturbances such as ventricular tachycardia, torsade de pointes, and ventricular fibrillation. Despite pharmacological therapy, mortality can be high, and sudden cardiac death is a frequent outcome. Because the electrical conducting system of the heart consists of modified myocytes and because selective insulation is derived from fibro-blasts and the extracellular

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matrix substance which they produce, it has long been hypothesized that the idiopathic long QT syndrome may represent a primary myocardial disorder. A pattern of Mendelian inheritance has also been recognized in these disorders, with the syndrome occurring in isolation or as part of a greater disease syndrome. The eponymous name Romano-Ward syndrome and the Jervell-Lange-Nielsen syndrome are used for an autosomal dominant form and for a form associated with deafness and autosomal recessive inheritance, respectively. Considerable progress has been made in the clinical and molecular genetic understanding of the long QT syndrome. Three genes have been identified as being responsible for different forms of the Roman-Ward syndrome. The first to be identified, HERG, is situated at 7q35-36 (LQT2) (Curran et al., 1995) and encodes a potassium channel related to the imaginatively named “ether-a-go-go” gene in Drosophila. The function of HERG was further defined by the expression of the protein in Xenopus oocytes (Sanguinetti et d., 1995). The properties of expressed HERG were shown to be nearly identical to the rapidly activating delayed rectifier K + current (IKr) in cardiac myocytes. The HERG current is K + selective, declines with depolarization above 0 mV, is activated by extracellular K+, and is blocked by lanthanum. Interestingly, the HERG current is not blocked by drugs that specifically block IKr in cardiac myocytes. This data indicates that HERG proteins form IKr channels, but that an additional subunit may be required for drug sensitivity. Since blocking IKr is a known mechanism for drug-induced cardiac arrhythmias, the finding that HERG encodes IKr channels provides a fascinating link between certain forms of inherited and acquired LQT. LQT3 is accounted for by a different gene (SCNSA) which codes for a sodium channel and maps to 31121-24 (Wang et al., 1995). The deleted sequences reside in a region important for channel inactivation. The effect of three mutations in the sodium channel SCN5A were studied in Xenopus oocytes. It was demonstrated that these mutations result in a prolonged inward sodium current following depolarization and that this may in part be due to late “bursts” of multiple channel reopening (Bennett et al., 1995). Wang et al. (1996) has identified mutations in a novel potassium channel gene (KVLQT1) as causing LQTl which was previously mapped to llp15.5 and thought earlier to be due to a mutation in the harvey ras proto-oncogene (Keating et al., 1991a,b), which has now been excluded. This is the commonest form of the long QT syndrome and probably accounts for at least 50% of the phenotype. A fourth locus (LQT4) (Schott et al., 1995) has also been mapped to chromosome 4q25-27, but no mutated gene has been identified as yet. Because these ion channel proteins are functionally expressed at the cell surface, it is worth considering that we may anticipate targeted therapeutic strategies to be developed more rapidly than in disorders in which the functional protein is intracellular, such as the mutated contractile proteins in hypertrophic cardiomyopathy.

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VII. FAMILIAL SYNDROMES WHICH INCLUDE MYOCARDIAL INVOLVEMENT The introduction of this chapter mentioned that a great number of syndromes are considered as having significant myocardial involvement. These include diseases due to abnormalities in the mitochondria1 genome, storage disorders, deficiency disorders, and infiltrative disorders such as amyloid, generalized myopathies, and neurological disease. In addition, many familial diseases are explained in terms of single gene mutations. A detailed discussion of these diseases is beyond the scope of this chapter, but readers are recommended to further reading (McKusick, 1996).

VIII. CONCLUSION Considerable advances have been made with regard to hypertrophic cardiomyopathy. Significant new findings have been made with respect to dilated cardiomyopathy, arrythmogenic right ventricular cardiomyopathy, and the long QT syndrome. The commonly used definition of cardiomyopathy has been shown to be nearing the limits of its useful life and will soon be modified (WHO/ISFC, 1996). A new classification will have to encompass the large number of diseases identified as being primary myocardial disorders. This classification will no longer be negative, based on the exclusion of all other causes, but instead will be positive and based on specific molecular etiology. Such a system will have to integrate with a workable clinical classification useful to the diagnosis, screening, and management of patients and their families, and in such a way will be tested. This profound debate is already well underway.

Ac know I ed gments Many thanks to E. W. A. Needham and V. S. Mohan-Ram for critical review of the manuscript.

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Aman S. Coonar and William J. McKenna

Watkins, H. E. A. (1995). A mutated cardiac myosin binding protein-C gene on chromosome 11causes familial hypertrophic cardiomyopathy. Nat. Genet. 11:435437. Weber, J. L. (1990a). Human DNA polymorphisms and methods of analysis. Curr. Opin. Biotechnol. 1:166-1 7 1. Weber, J. L. (1990b). Informativeness of human (dC-dA)n.(dG-dT)n polymorphisms. Genomics 7524-530. Weber, J. L., and May, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44:388-396. White, S. P., Cohen, C., and Phillips, G. 1. (1987). Structure of co-crystals of tropomyosin and troponin. Nature 325:826-828. WHO/ISFC (1980). Report of the WHO/ISFC task force on the definition and classification of cardiomyopathies. Br. Heart J. 44:672-673. WHO/ISFC (1996). Report of 1995 WHO/ISFC Task Force on the definition and classification of cardiomyopathies. Circulation 93(5):84 1-2. Yamaguchi, H., Ishimura, T., Nishiyama, S., Nagasaki, F., Nakanishi, S.,Takatsu, E, Nishijo, T., Umeda, T., and Machii, K. (1979). Hypertrophic nonobstructive cardiomyopathy with giant negative T waves (apical hypertrophy): Ventriculographic and echocardiographic features in 30 patients. Am.J. Cardiol. 44:401-412. Yoshida, K., Ikeda, S., Nakamura, A., Kagoshima, M.,Takeda, S.,Shoji, S., and Yanagisawa, N. (1993). Molecular analysis of the Duchenne muscular dystrophy gene in patients with Becker muscular dystrophy presenting with dilated cardiomyopathy. Muscle Nerve 16:1161-1166. Youssouhan, H., Antonarakis, S. E., Bell, W., Griffin, A. M., and Kazazian, H. J. (1988). Nonsense and missense mutations in hemophilia A: Estimate of the relative mutation rate at CG dinucleotides. Am. J. Hum. Genet. 42, 718-725. Yu, Q. T., lfegwu, J., Marian, A. I., Mares, A. J., Hill, R., Perryman, M. B., Bachinski, L. L., Roberts, R., and Marian, A. J. (1993). Hypertrophic cardiomyopathy mutation is expressed in messenger RNA of skeletal as well as cardiac muscle Circulation 87:406-412. [Published errata appear in Circulation 87(5):1775 and 87(6):2070] Zachara, E., Caforio, A. L., Carboni, G. P., I’ellegrini, A., Pompili, A., Del, P. G., Sciarra, A., Bosman, C., Boldrini, R., Prati, P. L., et al. (1993). Familial aggregation of idiopathic dilated cardiomyopathy: Clinical features and pedigree analysis in 14 families. Br. Heart J. 69:129-135. Zot, A. S., and Potter, J. D. (1987). Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction. Annu. Rev. Biophys. Biophys. Chem. 16635-559.

Adenosine deaminase deficiency disorder, gene therapy, 87-89 Adenoviral vectors, Duchenne muscular dystrophy gene therapy, 129-134 Agammaglobulinemia cloning studies, 67-73 animal models, 73 Bruton’s agammaglobulinemia tyrosine kinase role, 69-7 1 gene map, 67-69 mutation analysis, 71-72 future research directions, 89-90 historical perspective, 58-59 overview, 57-58 phenotypes, 65-67,72-73 Animal models, see specific model Apodystrophins, Duchenne muscular dystrophy role, 121 Apoptosis, historical perspective, 35-53 acceptance, 49-53 applications, 50-52 concept development, 46-49 definition, 40-41 morphology, 41-46 overview, 35-36 shrinkage necrosis, 36-40 Arrhythmogenic right ventricular cardiomyopathy, 310-312 awd Gene, punelKilles of prune interaction, 209-221 awdKF mutation analysis, 21t%221 early studies, 209 Killer ofpmne allele, 209-210 neomorphic character, 219-22 1 nm23 gene comparison, 2 14-2 16 nucleoside diphosphate kinase activity, 216-218 phenotypes, 211-214

Becker muscular dystrophy, 118, 120, 124 Biochemical markers

apoptosis detection, 4 8 4 9 expressed sequence tags, mouse libraries, 166-1 68, 191- 194 Blue-light photoreceptors, plant transcription regulation, 9-1 1 Bruton’s agammaglobulinemia tyrosine kinase, x-linked cloning studies, 69-72

CAB gene, plant transcription regulation, circadian clock pathways, 1-28

cis- and trans-acting element requirements,

20-25

cyanobacteria model, 17-20 future research directions, 25-28 gene characteristics, 2-6 overview, 1-2 photoreceptors, 6-1 1 blue-light receptors, 9-1 1 phytochrome, 6 9 phototransduction pathway analysis, 15-17 signal transduction intermediates, identification, 11-14 Calmodulin, plant transcription regulation, 12-14,27 Cancer apoptosis, 50-52 shrinkage necrosis, 38-39 p-Cardiac myosin hypertrophic cardiomyopathy role, 290-292, 302 Cardiac myosin-binding protein C hypertrophic cardiomyopathy role, 295-296 Cardiomyopathy, see Familial cardiomyopathies cGMP, plant transcription regulation, 12-14, 27 Chiasmata, see Chromosomes, segregation fidelity Chromosomal syndromes, mouse models, 175-177 Chromosomes libraries

325

326

index

Chromosomes (cont.) human genes, see Human Genome Project mouse genomics, 165-168 yeast artificial chromosome libraries, 166, 168 segregation fidelity, meiotic reciprocal recombination, 253-278 chiasma formation, 273-276 chromosome segregation studies, 276-277 disjunction enhancement, 265-27 1 bivalents, 268-269 chiasma binder, 271-273 pairing, 265-268 partner recognition, 265-268 spindle attachment, 269-27 1 exchange contributions, 255-265 crossover cytology, 259-261 recombination mutant use, 256-257 spontaneous nondisjunctions, 257-259 yeast chromosome model, 261-265 overview, 253-255 recombination intermediates, 273-276 Circadian rhythms, plant transcription regulation, 1-28 CAB gene characteristics, 2-6 cis- and trans-acting element requirements, 20-25 cyanobacteria model, 17-20 future research directions, 25-28 overview, 1-2 photoreceptors, 6-1 1 blue-light receptors, 9-1 1 phytochrome, 6-9 phototransduction pathway analysis, 15-1 7 signal transduction intermediate identification, 11-14 Conditional dominant lethality, see prune/Killer of prune Interaction Crossovers, see Chromosomes, segregation fidelity Currie, Alistair, apoptosis concept development, 35-53 acceptance, 49-53 applications, 50-52 definition, 40-41 morphology, 41-46 overview, 35-36 shrinkage necrosis, 36-39 studies, 46-49 Cyanobacteria, plant transcription regulation, circadian regulation pathways, 17-20

Cytokine receptor, signaling properties, severe combined immunodeficiency cloning studies, 76-77

Dilated cardiomyopathy, 305-3 10 clinical syndrome, 305-306 definition, 305-306 epidemiology, 306 genetics, 306-309 autosomal dominant form, 308-309 recessive inheritance, 309 X-linked inheritance, 307-308 pathogenesis, 309-310 Diphosphate kinase, see Nucleoside diphosphate kinase Disjunctions, see Chromosomes, segregation fidelity Drosophiki mehnogusrer, prunelKilkr of prune interaction, 207-209 awd gene, 209-221 awdKPnmutation analysis, 218-221 early studies, 209 Kilkr ofprune allele, 209-210 neomorphic character, 219-221 nm23 gene comparison, 214-216 nucleoside diphosphate kinase activiry, 2 16-2 18 phenotypes, 21 1-214 discovery, 207-209 lethal interactions, 237-244 mechanisms, 241-244 sensitivity, 238-240 timing, 240-241 prune gene, 222-236 developmental expression, 227 drosopterin pigment, 227-236 function, 222-226 phenotypes, 222 prune/Kilkr of prune interaction rescue, 226 pteridine biosynthesis pathway, 229-234 Drosopterin pigments accumulation reduction, 234-236 GTP role, 233-236 prune gene mutations, 227-229 pteridine biosynthesis pathway, 229-233 Duchenne muscular dystrophy, gene therapy, 117-142

327

Index animal models, 123-125 disease characteristics, 11 7-123 apodystrophins, 121 clinical features, 117-1 18 DMDgene, 118-121 dystrophin, 121-1 23, 127-1 29 dystrophin-associated glycoprotein complex, 121-123 future research directions, 141-142 gene transfer efficacy, 125-127 methodology, 129-141 adenoviral vectors, 129-134 direct DNA injection, 137-139 myoblast transplantation, 140-14 1 retroviral vectors, 134-137 Eystrophin, 12 1-1 23, 127-1 29

Expressed sequence tags, mouse libraries, 166- 168,191- 194

Familial cardiomyopathies, 285-3 15 arrhythmogenic right ventricular cardiomyopathy, 310-312 dilated cardiomyopathy, 305-3 10 clinical syndrome, 305-306 definition, 305-306 epidemiology, 306 genetics, 306-309 autosomal dominant form, 308-309 recessive inheritance, 309 X-linked inheritance, 307-308 pathogenesis, 309-3 10 future research directions, 3 15 hypertrophic cardiomyopathy, 28&305 clinical syndrome, 286-287 definition, 286-287 epidemiology, 288 features, 288-289 genetics, 289-290 genotype-phenotype correlation, 302 mutation characteristics (3-cardiac myosin, 290-292, 302 cardiac myosin-binding protein C, 295-296 etiology. 296-297

a-tropomyosin, 293-294,302-303 troponin T, 294-295,303-305 pathogenesis, 297-302 long QT syndrome, 313-312 overview, 285-286 restrictive cardiomyopathy, 3 12-3 13

Gametic imprinting, mouse models, 180-181 Gene libraries human genes, see Human Genome Project mouse genomics, 165-168 yeast artificial chromosome libraries, 166, 168 Gene therapy Duchenne muscular dystrophy, 117-142 animal models, 123-1 25 disease characteristics, 117-1 23 apodystrophins, 121 clinical features, 117-1 18 DMDgene, 118-121 dystrophin, 121-123,127-129 dystrophin-associated glycoprotein complex, 121-123 future research directions, 141-142 gene transfer efficacy, 125-127 methodology, 129-141 adenoviral vectors, 129-1 34 direct DNA injection, 137-139 myoblast transplantation, 140-141 retroviral vectors, 134-137 primary immunodeficiency disorders, 87-89 Genetic disorders, see specific disorders Genomic resources human genes, see Human Genome Project mouse genomics, 165-168 yeast artificial chromosome libraries, 166, 168 yc-Chain gene, severe combined immunodeficiency cloning studies, 77 G protein, plant transcription regulation, 11-13, 27 GTP, drosopterin biosynthesis, 233-236

Heart disease, see Familial cardiomyopathies Human genetic disorders, see Mouse models; specifif disorders

328 Human Genome Project genomic resources, 165-168 mouse comparisons, 155, 162-165 Hyper-immunoglobulin M syndrome characteristics, 84-85 cloning studies, 85-87 animal studies, 86-87 gene map, 85-86 mutation analysis, 86 future research directions, 89-90 historical perspective, 63-65 overview, 57-58 Hypertrophic cardiomyopathy, 286-305 clinical syndrome, 286-287 definition, 286-287 epidemiology, 288 features, 288-289 genetics, 289-290 genotype-phenotype correlation, 302 mutation characteristics P-cardiac myosin, 290-292,302 cardiac myosin-binding protein C, 295-296 etiology, 296-297 a-tropomyosin, 293-294,302-303 troponin T, 294-295,303-305 pathogenesis, 297-302

Immunodeficiency disorders, see X-linked immunodeficiency disorders Immunoglobulin M, see Hyper-immunoglobulin M syndrome Internet resources, mouse genomics, 165-168

Kerr, John, apoptosis concept development, 35-53 acceptance, 49-53 applications, 50-52 definition, 40-41 morphology, 4 1 4 6 overview, 35-36 shrinkage necrosis, 36-40 studies, 46-49 Killer ofprune gene, see pune/Kilkr ofprune Interaction

Index Libraries human genes, see Human Genome Project mouse genomics, 165-168 yeast artificial chromosome libraries, 166, 168 Liver cells, shrinkage necrosis, 37-38 Long QT syndrome, 3 13-3 12

Markers apoptosis detection, 48-49 expressed sequence tags, mouse libraries, 166-168, 191-194 Meiotic reciprocal recombination, chromosome segregation fidelity, 253-278 chiasma formation, 273-276 chromosome segregation studies, 276-277 disjunction enhancement, 265-27 1 bivalents, 268-269 chiasma binder, 271-273 pairing, 265-268 partner recognition, 265-268 spindle attachment, 269-271 exchange contributions, 255-265 crossover cytology, 259-261 recombination mutant use, 256-257 spontaneous nondisjunctions, 257-259 yeast chromosome model, 261-265 overview, 253-255 recombination intermediates, 273-276 Models, see specific model Mouse models, 155-197 future research directions, 183-184, 195-197 genomic resources, 165-168 historical perspective, 156-157 human disorder modeling, 184-195 model differences, 194-195 mouse germ line manipulation, 185-194 DNA sequence targeting, 191-194 gene targeting, 188-191 transgenic animals, 185-188 human genetic disease advances, 168-184 chromosomal syndromes, 175-177 future research directions, 183-184 gametic imprinting, 180-181 polygenic disease, 177-180 position effects, 181-183 sin& gene disorders, 168-175 assays, 171-173

Index candidate gene cloning, 169-1 70 causal gene determination, 173-1 75 functional disease loci cloning, 169 positional cloning, 170-171 triplet repeat mutation, 181-183 model attributes, 157-162 genetic studies, 159-160 mutations, 160-162 mouse-human comparative map, 162-1 65 overview, 155-156 Muscular dystrophy, see Becker muscular dystrophy; Duchenne muscular dystrophy Mutation analysis agammaglobulinernia cloning studies, 7 1-72 hyper-immunoglobulin M syndrome, 86 mouse models, 160-162 prunelKiller of prune interaction, awd gene, 2 18-22 1 severe combined immunodeficiency disorder,

77

Wiskott-Aldrich syndrome, 82-83 Myoblasts, Duchenne muscular dystrophy gene therapy, 140-141 Myosin hypertrophic cardiomyopathy role, 290-292, 302

nm23 Gene, prune/Kilkr of prune interaction, awd gene comparison, 214-216 Nucleoside diphosphate kinase awd gene, 216 awdKW mutation analysis, 218-221 characteristics, 216-218

Phototransduction pathways future research directions, 25-28 genetic analysis, 15-1 7 Phytochrome, plant transcription regulation, 6-9 Plants, transcription regulation, circadian clock pathways, 1-28 CAB gene characteristics, 2-6 cis- and nans-acting element requirements, 20-25 cyanobacteria model, 17-20

329

future research directions, 25-28 overview, 1-2 photoreceptors, 6-1 1 blue-light receptors, 9-1 1 phytochrome, 6-9 phototransduction pathway analysis, 15-1 7 signal transduction intermediate identification, 11-14 Polygenic disease, mouse models, 177-180 Programmed cell death apoptosis, see Apoptosis shrinkage necrosis, 3 9 4 0 pune/Kilkr of prune Interaction, 207-209 awd gene, 209-221 awdKb" mutation analysis, 218-221 early studies, 209 Kilkr ofprune allele, 209-210 neomorphic character, 219-221 nm23 gene comparison, 2 14-2 16 nucleoside diphosphate kinase activity, 216-218 phenotypes, 2 11-2 14 discovery, 207-209 lethal interactions, 237-244 mechanisms, 241-244 sensitivity, 238-240 timing, 240-241 prune gene, 222-236 developmental expression, 227 drosopterin pigment accumulation reduction, 234-236 content, 227-229 GTP role, 233-236 pteridine biosynthesis pathway, 229-234 function, 222-226 phenotypes, 222 prune/Kilkr of prune interaction rescue, 226 Pteridine, biosynthesis pathway, 229-234

Recessive inheritance dilated cardiomyopathy, 309 Recombination, see Meiotic reciprocal recombination Restrictive cardiomyopathy, 312-3 13 Retroviral vectors, Duchenne muscular dystrophy gene therapy, 134-137

330 Smchromyces cereuisiae, meiosis model, chro-

mosome segregation fidelity, 261-265 Severe combined immunodeficiency disorder cloning studies, 74-78 animal models, 77-78 cytokine receptor signaling, 76-77 gene map, 74-76 yc-chain gene organization, 77 mutation analysis, 77 future research directions, 89-90 historical perspective, 59-61 overview, 57-58 phenotype, 73-74 Shrinkage necrosis, apoptosis concept development, 3 6 4 0 hormone regulation, 39 identification, 36-39 cancer cells, 38-39 ischaemic liver cells, 37-38 normal tissues, 38-39 programmed cell death, 39-40 Single gene disorders, mouse models, 168-175 assays, 171-173 candidate gene cloning, 169-170 causal gene determination, 173-1 75 functional disease loci cloning, 169 positional cloning, 170-171 Skeletal muscles, disorders, see Duchenne muscular dystrophy

Transcription, regulation, plant circadian clock pathways, 1-28 CAB gene characteristics, 2-6 cis- and trans-acting element requirements, 20-25 cyanobacteria model, 17-20 future research directions, 25-28 overview, 1-2 photoreceptors, 6-1 1 blue-light receptors, 9-1 1 phytochrome, 6-9 phototransduction pathway analysis, 15-17 signal trwsduction intermediate identification, 11-14 Transgenics chromosomal aneuploidy study, 176-177 human disease models, mouse germ line manipulation, 185-1 97

Index DNA sequence targeting, 191-194 future research directions, 195-197 gene targeting, 188-191 transgenic animals, 185-188 Triplet repeat mutations, mouse models, 181-1 83 a-Tropomyosin hypertrophic cardiomyopathy role, 293-294, 302-303 Troponin T hypertrophic cardiomyopathy role, 294-295, 303-305

Wiskott-Aldrich syndrome characteristics, 78-81 cloning studies, 81-84 animal models, 83-84 gene map, 81-82 mutation analysis, 82-83 future research directions, 89-90 historical perspective, 61-63 overview, 57-58 World Wide Web sites, mouse genomics, 165-1 68 Wyllie, Andrew, apoptosis concept development, 35-53 acceptance, 49-53 applications, 50-52 definition, 40-41 morphology, 4 1 4 6 overview, 35-36 shrinkage necrosis, 36-40 studies, 46-49

X-linked disorders, see Dilated cardiomyopathy ; Duchenne muscular dystrophy; X-linked immunodeficiency disorders X-linked immunodeficiency disorders future research directions, 89-90 gene therapy, 87-89 hyper-immunoglobulin M syndrome characteristics, 84-85 cloning studies, 85-87 animal studies, 86-87 gene map, 85-86 mutation analysis, 86 historical perspective, 63-65

Index overview, 57-58 severe combined immunodekiency cloning studies, 74-78 animal models, 77-78 cytokine receptor signaling, 76-77 gene map, 74-76 yc-chain gene organization, 77 mutation analysis, 77 historical perspective, 59-61 phenotype, 73-74 Wiskott-Aldrich syndrome characteristics, 78-8 1 cloning studies, 81-84 animal models, 83-84 gene map, 81-82 mutation analysis, 82-83 historical perspective, 61-63

33 1 x-linked agammaglohulinemia cloning studies, 67-73 animal models, 73 Bruton’s agammaglobulinemia tyrosine kinase role, 69-71 gene map, 67-69 mutation analysis, 71-72 historical perspective, 58-59 phenotypes, 65-67, 72-73

Yeast, meiosis model, chromosome segregation fidelity, 261-265 Yeast artiticial chromosomes libraries, 166, 168 transgenesis, 176-177

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