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ILAR Journal V39(2/3) 1998
Comparative Gene Mapping

Gene Maps of Marsupials
Paul B. Samollow and Jennifer A. Marshall Graves
Paul B. Samollow, Ph.D., is Associate Scientist in the Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas. Jennifer A. Marshall Graves, Ph.D., is Professor and Head of the School of Genetics and Human Variation, La Trobe University, Bundoora, Melbourne, Australia.

INTRODUCTION

Increased interest in the structural characteristics of mammalian genomes, together with rapid advances in mapping technology, have led to the explosive expansion of gene mapping activity in recent years. Species for which gene mapping data were virtually nonexistent just a decade ago now possess substantial and serviceable gene maps that are enabling the localization of economically and biomedically relevant loci and are contributing to a fuller understanding of genome evolution and the relationships between genome structure and gene function. Recent progress in the compilation and effective application of gene mapping data has been particularly evident in the fascinating group of species that comprise the mammalian infraclass Metatheria, better known as marsupial mammals.

As late as 1988, only 22 marsupial genes had been reported as mapped by any method in any species (a gene is considered mapped if it adheres to any of the following criteria: (1) assigned to a specific chromosome; (2) a member of a linkage group; (3) autosomal in marsupials but known to be X or Y linked in eutherians). These few gene assignments were achieved by a variety of methods, and were scattered piecemeal among a dozen different species (this number excludes 14 additional species in which 1 or more ribosomal RNA (RNR) genes were the only loci mapped [Hayman and Rofe 1977; Hayman and Sharp 1981; Young and others 1982]). As a consequence, few species had more than 2 or 3 genes mapped, and only I species could boast as many as 9 gene assignments. Currently, at least 142 loci have been assigned to physical locations or linkage groups in marsupials, and more than 15 species have at least 3 gene assignments. Most important, 2 distantly related species, Macropus eugenii (tammar wallaby) with 70 loci mapped and Monodelphis domestica (gray, short-tailed opossum) with 69 loci mapped, have emerged as focal species for intensive gene mapping studies.

The successes of the Human Genome Project and ancillary mapping efforts in mouse and various livestock species have contributed to the growing recognition of the unique value of marsupial genome data for comparative genome analyses. This heightened awareness has undoubtedly spurred interest in marsupial gene mapping research, but the primary stimuli of recent progress in this area have been fundamental improvements in mapping technology and the development of new animal resources. The advent and application of sophisticated physical mapping tools based on DNA hybridization methods, and the emergence and availability of tractable animal resources (highly productive, pedigreed marsupial colonies) for family studies and linkage analyses, have finally enabled meaningful examinations of the organization and evolution of chromosome structures and gene arrangements in species that had previously been inaccessible to traditional genetic analysis.

Marsupials in the Mammalian Scheme

The class Mammalia has 3 distinct branches: the subclass Prototheria (monotremes) and the infraclasses Metatheria (marsupials) and Eutheria (so-called placental mammals), which together comprise the subclass Theria. Molecular data documenting the earliest radiation of the 3 branches are inconclusive (reviewed by Hope and others 1990), but traditional considerations of anatomical, reproductive, and developmental features strongly imply that the prototherian lineage diverged from the common metatherian/eutherian line some 160 to 200 million yr ago (MYA1). Results from molecular analyses of globin gene sequences suggest that the ancestors of modem marsupial and eutherian mammals arose later, by means of a split in the metatherian/eutherian lineage approximately 140 to 165 MYA (Hope and others 1990). Extensive analysis of DNA hybridization data led Kirsch and others (1997) to place the metatherian/eutherian divergence considerably later, at 101 to 107 MYA. A recent cladistic analysis based on mtDNA sequence data (Janke and others 1997) challenges both of these traditional schemes by suggesting that the eutherian lineage arose first, at approximately 130 MYA, leaving a common metatherian/prototherian line that split again some 15 million yr later (approximately 115 MYA). Additional evidence would be needed to corroborate such an unconventional branching topology, but in any case there is no doubt that the 3 mammalian lineages have had long, independent evolutionary histories during which they have evolved many distinctive morphological, physiological, and genetic features. Of these, none so thoroughly distinguish the 3 groups as do their unique reproductive characteristics.

Egg laying sharply differentiates modern prototherians (the platypus and 2 species of echidnas) from all other living mammals. Although marsupials and eutherians share the live-bearing habit, there are important differences in early developmental events that clearly define these latter groups as well. Most obvious is the difference in the relative proportions of in utero versus ex utero development. By contrast to eutherians, marsupial fetuses are born at an extremely early stage of development after a brief gestation. For species in which placental development occurs, it is minimal and of short duration. Marsupials complete the majority of their "fetal" development subsequent to birth, attached to a teat, and often, but not always, within a protective pouch. Thus, whereas eutherian development is gestationally intensive and occurs internally, primarily via the placental attachment, marsupial development is lactationally intensive and occurs external to the mother.

Extant marsupials represent less than 6% of the more than 4,600 mammalian species currently recognized (Wilson and Reeder 1993). Despite this impoverished species count, the ecological and morphological diversity of marsupials has led to the recognition of 15 (or more) families in 3 orders (Nowak and Paradiso 1983; Wilson and Reeder 1993). Marsupials are found on both American continents (approximately 83 species) and in Australasia (Australia, Tasmania, New Guinea, and associated small islands; approximately 170 species). They inhabit cool temperate, hot xeric, and tropical habitats and range from tiny, mouse-like predators of the family Dasyuridae to large grazing and browsing herbivores such as kangaroos and other members of the Macropodidae. Their lifestyles parallel nearly all trophic levels and modes found among eutherian groups.

Marsupial Genomes

More than half of all marsupial species, including members of all families, have been examined cytologically (Hayman 1990; Hayman and Martin 1974; Hayman and Role 1977; Sharman 1974). Marsupial genomes are similar in size to those of eutherians but are considerably less diverse in chromosome number and structural arrangements. Chromosome numbers range from 2n--10 to 2n=32, but more than 90% of the species examined exhibit 2n=14 to 22. The most common number, 2n=14, is seen in both American and Australasian species from 10 families. In almost every case this 2n=14 "basic complement" (Role and Hayman 1985) comprises 3 large metacentric chromosome pairs, 2 pairs of intermediate-sized metacentrics, 1 small submetacentric pair, and the sex chromosomes, X and Y. Judged from studies of representative Australasian and American species, the G-banding morphologies of the chromosomes in most 2n= 14 species are extremely similar, manifesting only minor deviations that are easily explained by simple internal rearrangements (Rofe and Hayman 1985). More recent analyses of groups with different chromosome numbers indicate that most other arrangements are also easily derivable by simple centromeric fissions and/or fusions accompanied by minor internal rearrangements (Eldridge and Close 1993; Hayman 1990).

The strong karyotypic conservation evident in marsupial genomes implies a parallel conservation in gene arrangements and syntenic relationships among species, both within and among marsupial families. If this idea is correct, it might be possible to establish a representative marsupial gene map that could be used to predict the locations of large syntenic gene blocks in many or all marsupials through examination of cytologically detectable differences in chromosome structures and internal rearrangements. Assessing the prospects for such a basic gene map will require the detailed mapping of many homologous genes in 2 or more distantly related marsupial species.

In this article, we summarize the progress that has been made in mapping marsupial genomes, discuss the methodologies that are enabling the quickening pace of marsupial gene mapping, and look ahead to potential uses of gene maps of the tammar wallaby and the gray, short-tailed opossum, species that have been developed as focal organisms for basic genetic and biomedical research.

REASONS FOR MAPPING THESE SPECIES

Of little agricultural or commercial importance (except as crop pests and wildlife attractions), marsupials have insufficient economic significance to justify gene mapping research for genetic improvement purposes. Similarly, the perennial absence of a marsupial equivalent of the laboratory mouse has long precluded the intensive use of marsupials in many kinds of basic research, thus obviating the need to map any such species for enhancement of its research potential. Consequently, the primary impetus for genetic mapping studies in marsupials has traditionally related to comparative analyses of gene locations in marsupial genomes relative to those on the detailed maps of eutherian species (particularly human and mouse).

Resemblances between the genome structures of marsupial and eutherian mammals bear witness to their common ancestry, while differences reflect changes that have accumulated during their lengthy evolutionary separation and thereby comprise important quanta of phylogenetic information. Differences in the regulation of gene expression and mechanisms of gene interaction that underlie phylogenetic distinctions in biochemical, physiological, reproductive, and developmental characteristics of these taxa also must have evolved as modifications of ancient genetic mechanisms that were present in the common therian (mammals, exclusive of prototherians) ancestor. The genic and chromosomal differences between these taxa thus represent parallel, and potentially interrelated, evolutionary variants of basic molecular mechanisms and structures that can be exploited to gain insight concerning fundamental genetic processes common to all mammalian species.

The possibility of relating evolutionary modifications in gene synteny or gene order relationships to differences in gene function has driven the comparative mapping effort far beyond the tracing of phylogenetic relatedness. Analyses of taxon-specific differences in genetic processes and distinctions in genome structure have led to new insights and novel hypotheses regarding (1) the evolution of sex chromosomes and the genetic bases of sex-determining mechanisms (reviewed by Graves 1995, 1996a; Graves and Foster 1994); (2) the evolution of gene regulation and expression patterns (reviewed by Graves 1995, 1996b; see also Delbridge and others 1997; D'Esposito and others 1997; Mitchell and others 1998; Toder and others 1996); (3) the evolution of gene structure (O'Neill and others 1998); and (4) the multilevel nature of X chromosome inactivation (reviewed by Cooper and others 1990, 1993; Graves and Foster 1994; Riggs 1990).

The immature state of the newborn marsupial, together with the extended lactational developmental period, facilitates many kinds of fetal manipulations that are impractical or impossible with eutherian embryos. For example, marsupials are used extensively as models for studying normal and regenerative development of the central nervous system and peripheral nervous structures (for example, Breckenridge and others 1997; Harman 1997; Leblond and Cabana 1997; Luque and others 1998; Shapiro and others 1997), normal and chemically perturbed skin development (for example, Adelson and others 1997; Armstrong and Ferguson 1995), development of endocrine secretion and receptor systems (for example, Buaboocha and Gemmell 1997; Sernia and others 1997; Sonea and others 1997; Xie and others 1998), chemical and ultraviolet radiation-induced skin and eye carcinogenesis (for example, Robinson and Dooley 1995; Robinson and others 1994; VandeBerg and Robinson 1997), normal and experimentally disturbed patterns of secondary sexual differentiation (for example, Lucas and others 1997; Ryhorchuk and others 1997; Whitworth and others 1996, 1997), and much more. The attractiveness of marsupial models for these and other kinds of research has stimulated repeated attempts, involving more than a dozen species, to establish self-perpetuating laboratory colonies of various marsupial species (discussed by Hope 1993; VandeBerg 1990; see also Godfrey 1969; Godfrey and Crowcroft 1971; Hinds and others 1990; Jurgelski 1974; Jurgelski and Porter 1974; Jurgelski and others 1974; Woolley 1971, 1973). Only recently, however, have pedigreed marsupial colonies been sufficiently successful to enable the distribution of genetically characterized animals for research purposes (Hinds and others 1990; Hope 1993; VandeBerg 1990; VandeBerg and Robinson 1997). With this success has come expanded utilization of marsupials in divers areas of research and increasing interest in the establishment of gene maps to enhance their usefulness as research models.

Successful colonies of 2 species now comprise the fastest growing group of marsupials used for basic biomedical and genetic research purposes. In Australia, the tammar wallaby (M. eugenii, a member of the kangaroo family: Macropodidae) has emerged as the preeminent model marsupial for studies of reproduction, fetal development, lactation, and a variety of genetically related topics including X-chromosome inactivation and the molecular basis and evolution of sex determination (Hinds and others 1990; McKenzie and others 1993, 1995). Despite its success as a research model, physical and reproductive limitations related to its size (4.5 to 8.5 kg) and relatively low fecundity (litter size of 1; but can be manipulated to produce multiple offspring annually) will preclude the broad distribution and breeding of this species and thus impede its full development as a laboratory animal. The colony is maintained at Maquarie University in Sydney.

The gray, short-tailed opossum (M. domestica) is a South American marsupial (family: Didelphidae) that has been fully adapted to laboratory conditions (VandeBerg 1990; VandeBerg and Robinson 1997). It is small (100 to 150 g), highly prolific (mean litter size of 8; up to 3 litters annually), and housed and bred in small cages under conditions that compare favorably with those used for laboratory rodents. Initiated from a small founding stock in 1979, the research colony at the Southwest Foundation for Biomedical Research (SFBR1; San Antonio, Texas) has been repeatedly infused with new genetic material from natural populations and exhibits very high genetic diversity (VandeBerg 1990; VandeBerg and Robinson 1997; van Oorschot and others 1992b). Tens of thousands of animals, including members of outbred and partially inbred strains, have been produced from the SFBR colony, and the distribution of experimental animals and breeding stock to other research laboratories has resulted in the establishment of several colonies worldwide, making M. domestica the predominant laboratory-bred research marsupial in the world today. It serves a broad spectrum of basic and biomedical research topics including normal and abnormal physiology, cellular and organismal development, gene regulation, DNA repair, photobiology, evolutionary genetics, and more.

The range and extent of research focused on marsupial animals cannot be surveyed adequately in this review, but recent inspection of a biomedically oriented literature database (PubMed) revealed 519 publications pertaining to marsupials in the 24-mo period ending September 1, 1998. Of these, 111 articles involved the use of a few widely distributed marsupial kidney cell lines (OK and PtK lines). Of the remaining 408 studies, those involving M. domestica and M. eugenii accounted/'or the largest numbers of papers (n=66 and n=51, respectively) in which the target species were unambiguously identified. A breakdown of research topics involving these 2 species included fetal development; neurobiology; reproductive biology and gametogenesis; general physiology; biochemistry and metabolism; endocrinology; comparative anatomy and biomechanics; and a host of genetic topics (gene and genomic evolution; gene structure, regulation, and expression; sex determination; DNA repair and cancer genetics; gene mapping; and more).

The expanding use of M. eugenii and M. domestica in basic research applications has generated a need to explore the fundamental genetic characteristics of these species to enhance their research utility. The establishment of basic gene maps is among the most important objectives. The availability of such maps would facilitate the localization of genes that contribute to physiologically relevant variation in complex characteristics such as developmental anomalies and disease susceptibilities, and would provide additional insight regarding the conservation of gene synteny and linkage organization between diverse marsupial species, and between marsupial and eutherian genomes. This latter point is particularly important from a biomedical viewpoint because knowledge of the degree of conservation between marsupial and human genomes will expedite the chromosomal localization, identification, and molecular characterization of genes in the human genome that are possible homologues of genes that become identified as disease-related factors in marsupial models.

CURRENT MAP STATUS

Until the early 1980s, gene mapping data from marsupial species were usually acquired as by-products of research on patterns of gene expression of putatively X-linked loci and the inheritance of particular protein variants detected in species under examination for other purposes. Intensive family-based linkage studies were rarely conducted because of the dearth of colonies of pedigreed animals that could be bred in large numbers. Early attempts to map genes by somatic cell hybrid approaches met with limited success, but progress was inhibited by the inherent instability of marsupial chromosomes in rodent x marsupial cell hybrids (reviewed by Graves and others 1990). By 1988, 6 genes had been mapped to the X chromosome, and 5 of them were mapped in at least 4 or more species. Sixteen loci were found to be autosomal, either by exclusion from the X chromosome (7 loci), or by localization to specific autosomes (9 loci); however, only 4 were mapped in more than a single species.

The situation improved dramatically as modern physical mapping methodologies were brought to bear on the mapping issue (see Approaches Used to Develop the Maps) and as mapping efforts converged on a few key species. Presently there are multiple gene assignments for 17 marsupial species representing 3 Australasian families (Macropodidae, Dasyuridae, and Phalangeridae) and 1 American (Didelphidae) family. Fifteen species have more than 4 gene assignments each, and 8 have more than 10 genes mapped. This progress in marsupial gene mapping is summarized in Tables 1 and 2. In Table 1, locations are displayed of genes in the 8 best-mapped species from each of the 4 families relative to their homologues on the human map. In Table 2, additional mapping information is listed for species in which a smaller amount of work has been done.

Inspection of Table I reveals that the gene maps of M. eugenii and M. domestica exceed those of any other marsupial species in terms of the number of loci mapped. It is equally clear from the kinds of mapping data compiled for M. eugenii and M. domestica that the mapping strategies applied to these 2 species have differed considerably.

Mapping progress in M. eugenii has proceeded primarily by physical approaches based on in situ hybridization using radiolabeled heterologous and homologous probes (Graves and others 1990; Maccarone and others 1994), and more recently through more sensitive, fluorescence in situ hybridization (FISH1) methods (for example, Delbridge and others 1997; D'Esposito and others 1997; Pask and others 1997; Spurdle and others 1997; Toder and Graves 1998; Toder and others 1996, 1997a). The data are primarily in the form of chromosome or chromosome arm assignments that indicate synteny affiliations, but there is little information on linkage relationships. Because much of this information was accumulated by less sensitive in situ hybridization methods using radiolabeled heterologous probes, gene ordering is still problematic for many loci. Newer assignments based on FISH have yielded increasingly precise localizations, enabling more confident gene ordering. Due to the emphasis on expressed genes, few anonymous sequences have been mapped in this or any other Australian species.

In contrast, the majority of mapping information available for M. domestica is in the form of linkage data, and many of these data pertain to anonymous loci detected by random amplified polymorphic DNA (RAPD1) and microsatellite approaches. Of the 58 loci presently assigned to autosomal linkage groups, 24 are expressed genes and 34 are anonymous loci. An additional 3 coding genes are known to be X linked and 1 is Y linked. Nesterova and others (1997) recently published the results of the first mapping studies utilizing M. domestica x rodent cell hybrids. Nine genes were assigned to 4 autosomes and the X chromosome by co-occurrence of M. domestica chromosomes and M. domestica-specific isozyme expression in the M. domestica x rodent somatic cell hybrid panel. Unfortunately, there is little overlap between the current linkage data and the physical mapping data, but the provisional assignment of TK to chromosome 5 by Nesterova and others (1997) and the tentative placement of this locus in linkage group (LG1) 3 by linkage analysis (Sokolova and others 1997) define the first probable chromosomal localization of a large syntenic block to the M. domestica genome.

APPROACHES USED TO DEVELOP THE MAPS

Pioneering Studies

The earliest information bearing on the chromosomal locations of marsupial genes arose from an interest in a form of gene regulation that is peculiar to marsupial mammals: paternal X-chromosome inactivation. X-chromosome inactivation is the process whereby most of the genes on 1 of the X chromosomes in each cell of a female embryo are coordinately and permanently silenced early in embryogenesis. This phenomenon, which results in equivalent dosage of X-linked gene products in the XX female and XY male, occurs in all therian species; however, whereas inactivation occurs randomly with regard to the parental origin of the X chromosomes in eutherian embryos, in marsupials it is invariably the paternally derived X chromosome that is repressed (reviewed by Cooper and others 1990, 1993; VandeBerg and others 1987). The initial detection of this marsupial-specific form of X-inactivation at the G6PD and PGK1 loci of several kangaroo species (Cooper and others 1971; Richardson and others 1971 ) was followed by a flurry of investigations of other loci known to be X linked in eutherian mammals. These investigations led to the establishment, via pedigree studies, of the X-linked inheritance of 4 genes (G6PD, PGK1, HPRT, and GLA) in several marsupial species (Cooper and others 1990, 1993; VandeBerg and others 1987). These findings were consistent with Susumo Ohno's well-known prediction that the gene content of the X chromosome would be conserved among all mammalian species (Ohno 1967). Ohno's "law" appeared inviolate in eutherian mammals (although rare exceptions are now known), but several cases were detected in marsupials wherein the expectation of X linkage was refuted (for example, Cooper and others 1984; Sinclair and others 1988; Spencer and others 1991a), resulting in the first exclusions of gene locations from the marsupial X chromosome.

Physical Mapping in Australian Marsupials

Although the earliest gene localizations in marsupials resulted from family-based studies, limitations imposed by the perennial lack of laboratory animal resources quickly led to a search for physical mapping methods that did not require the breeding of pedigreed animals. The application of somatic cell hybridization genetics and subsequent use of conventional in situ hybridization methods using radiolabeled probes (ISHR1) signaled the beginning of serious gene mapping studies in marsupials (reviewed by Graves and others 1990). Initial attempts to establish marsupial × rodent hybrid cell lines were enormously problematic. The fusions themselves often failed and, when successful, produced unstable clones containing highly fragmented marsupial chromosomes. In many cases no marsupial chromosomes were detectable even though the hybrid cells clearly retained marsupial genes. Continued experimentation with novel species combinations led to improved stability and enabled the first successful mapping by somatic cell hybrid (SCH1) methods--the establishment of synteny among G6PD, PGK1, and HPRT in the wallaroo, Macropus robustus (Graves and others 1979).

These groundbreaking SCH analyses were based on the co-occurrence of marsupial chromosome fragments with species-specific enzyme products as revealed by electrophoretic analyses of SCH clone homogenates. The advent of Southern hybridization techniques and availability of human and mouse probes enabled the detection of marsupial genes (rather than gene products) in the hybrid cells and vastly expanded the range of genes that could be studied by these approaches. Additional refinement of cytological detection methods eventually led to the identification of SCH clones with intact marsupial chromosomes or segments of chromosomes and to success in the mapping of a small number of genes to the X chromosomes and autosomes of several species.

By the time in situ hybridization methods emerged as powerful tools for eutherian gene mapping, Southern blotting studies had already shown that many mouse and human gene probes would hybridize to marsupial DNA under reduced stringency. Thus, ISHR quickly supplanted the SCH approach and led to the detection of the majority of genes now mapped in M. eugenii and in several other Australian marsupials (Graves and others 1990, 1993; Maccarone and others 1994).

However, the ISHR approach (which is dependent on the exposure of silver grains in photographic emulsion overlays of chromosome spreads that have been labeled with radioactive DNA probes) was not without difficulties. First, there is an uncertainty factor due to the sheer size of silver grains relative to the size of chromosomes. Under the best of conditions, a gene can be assigned to the distal, medial, or proximal region of a chromosome arm, and often only to one arm or the other. This limitation has enabled only the crudest ordering of loci within a chromosome arm.

More important are problems related to the reduced stringencies required by the use of heterologous probes. To discriminate hybridization signal from background exposure (noise), a statistical approach is employed to detect regions of the genome that show exposure above background level (Ewens and others 1992). The reduced stringencies exacerbate the signal-to-noise problem, and often no clear signal can be detected because of overall high background. Even more serious is cross-hybridization of heterologous probes to nontarget sequences, such as other members of gene families. This problem, together with the possibility that more than one homologue of the eutherian gene may actually exist in a given marsupial genome, has resulted in ambiguous localizations (that is, multiple chromosomal assignments for an individual probe) of some marsupial genes. Examples include AMEL (Watson and others 1992), CYBB (Spencer and others 1991a), UBE1 (Mitchell and others 1992), and ZFY (Sinclair and others 1988). This ambiguity ultimately requires the investigator to make mapping assignments based on the assumption that the strongest signal represents the true homologue of the eutherian gene probe.

Marsupial DNA fragments are being cloned with increasing frequency. Currently there are 704 marsupial DNA sequences listed in the GenBank database, including 90 for M. eugenii and 76 for M. domestica. Growth in marsupial DNA resources has permitted the utilization of homologous, or at least closely related, DNA probes for gene mapping and thereby encouraged a shift to FISH methods (Lichter and (others 1988). The FISH approach has improved the specificity and precision of physical mapping in marsupials to the point where gene ordering is now practical and reliable (for example, Toder and others 1996), and has replaced ISHR for gene mapping studies of M. eugenii and other Australian species. It has not yet been applied to M. domestica mapping.

Most recently, the availability of advanced in situ hybridization techniques has enabled the application of chromosome painting methods for the analysis of higher level structural rearrangements of marsupial chromosomes (reviewed by Toder and others 1998). This approach has led to novel insights regarding the evolution of marsupial sex chromosomes that were not obvious at the level of individual gene localizations (for example, Toder and others 1997a,b).

Linkage Studies in M. domestica

Owing to its favorable reproductive characteristics, the availability of genetically characterized, pedigreed animals, and use in biomedically related research (wherein the ability to localize phenotypic effects by linkage analysis may be important), gene mapping in M. domestica has been conducted almost exclusively by breeding studies aimed at defining linkage relationships. The first linkage studies of this species capitalized on the discovery (during a systematic survey of the SFBR colony for protein polymorphisms) of individual animals that were heterozygous at multiple protein-coding loci. These multiply heterozygous individuals were crossed to appropriate homozygous mates, and offspring were examined to detect patterns of cosegregation of alleles among the relevant loci. These early studies detected the first evidence of linkage and defined the first 2 linkage groups in this species (van Oorschot and others 1992a, 1993).

Current mapping studies in M. domestica are aimed at the establishment of a framework linkage map composed of a large number of anonymous DNA markers interspersed with the locations of functional genes including homologues of traditional mammalian anchor loci (O'Brien and others 1993), other human genes, and sequences of unknown homology derived by the cloning of M. domestica and other marsupial genes. Anonymous markers have been developed using 2 approaches: arbitrary priming methods (RAPD polymorphisms: Williams and others 1990) and microsatellite detection methods (short tandem repeat polymorphisms: Edwards and others 1991; Hughes 1993; Weber and May 1989). Functional gene markers are detected by means of protein electrophoresis, Southern blotting, and polymerase chain reaction amplification approaches. Linkage is sought by examining allelic cosegregation patterns among loci in a panel of backcross families produced by crossing descendants of animals derived from geographically distinct populations. (The rationale is that animals derived from distant populations should exhibit different allelic frequencies and fixations at many loci and therefore produce an F1 generation that is heterozygous at more loci than would be expected from an intrapopulation cross. Backcrossing such F1 offspring to either parental population produces a highly informative backcross generation.) Locus by locus linkage and most probable gene orders are determined by pairwise and multipoint analyses using maximum likelihood methods that incorporate data from all generations of the crossing scheme.

A Question of Strategies

The emphasis on physical mapping in Australian marsupials versus the linkage approach in M. domestica is partly a result of the different availability of animal resources; but differences in the underlying objectives of mapping research in these 2 species are equally important. For Australian species, the proximate objective is to achieve a level of map saturation, based on recognized mammalian type I anchor loci (Graves and others 1995; O'Brien and others 1993) that will enable in-depth comparisons of genome structure between marsupial and eutherian gene maps. The ascendant objective is to better understand genome evolution and to exploit eutherian/metatherian differences as probes to explore the phylogenetic nuances of sex determination, gene expression, and other questions pertaining to the evolution of gene structure and function. These objectives are entirely consistent with those envisioned for a M. domestica gene map, but an added impetus for M. domestica mapping is the continuing development of this species as a laboratory animal for basic biological and biomedical research (VandeBerg 1990; VandeBerg and Robinson 1997). Accordingly, the gene map is of interest not only as a comparative tool, but equally for its intrinsic value in localizing genes and examining their functions within the species itself. For example, if a gene influencing susceptibility to a complex disease condition (such as ultraviolet radiation-induced skin or eye cancer, or diet-induced hypercholesterolemia) becomes identified in the M. domestica model, it would be of primary interest to localize the position of the gene within the M. domestica genome. The fastest route to this goal is through linkage analysis. This has not been a major motivation in mapping the M. eugenii genome.

Another difference in emphasis lies in the relative importance of anonymous markers for gene map construction. Of the 70 loci listed in Table 1 for M. eugenii, all but 4 are homologues of human genes, and only 2 are anonymous DNA fragments. In contrast, almost half of the 69 loci listed for M. domestica are anonymous RAPD or microsatellite loci. This difference reflects the discordant objectives of physical and linkage mapping approaches as presently applied to these species. The essence of comparative mapping is the assumption that loci being compared among species were derived from the last common ancestral gene; that is, they are orthologous genes. Anonymous DNA sequences are virtually useless in this regard, because the means by which they are identified are completely arbitrary with respect to homologies between species. Nevertheless, anonymous sequences are highly valuable for the construction of linkage maps because they create a framework on which the locations of functional (RNA-coding) genes can be superimposed. Anonymous markers such as microsatellites are usually highly heterozygous relative to coding genes and possess greater potential for linkage detection by statistical analyses based on maximum likelihood methods. Thus, anonymous marker loci serve to fill gaps between more distantly spaced coding genes that might otherwise fail to show linkage because of great interlocus distance and/or low heterozygosity. Moreover, the accuracy of recombination estimates over longer distances rises with increasing marker density due to the increasing probability of detecting multiple recombination events between loci. Hence, the use of anonymous markers can generate more accurate linkage data among coding loci even if linkage between them is detectable without such markers.

The objectives of physical and linkage mapping are entirely compatible and ultimately identical: to determine the locations of, and distances between, genes on chromosomes. The development of a detailed genetic map thus furnishes the tools both for tracing genome evolution and for localizing genes that influence normal and abnormal physiologic and developmental processes. Physical mapping alone cannot provide the information on genetic distances between loci that is necessary to position genes whose influences are detected by experimental studies of phenotypic variation. To the extent that knowledge of recombination frequencies between loci is important in understanding the inheritance of disease susceptibility or the genesis of complex variation in a species, linkage mapping is uniquely able to provide the necessary information. However, the utility of linkage information could be vastly enhanced by the ability to extrapolate the physical locations of genes between species. For example, if the position of a gene that influences a phenotype of interest becomes determined on the M. domestica linkage map, it might be desirable to infer the potential location of its homologue in the human genome. Knowledge of the correspondence between the physical features (such as banding landmarks) and gene locations in each species could be used to identify potentially homologous regions between the marsupial and human genomes and suggest the likely location of the human homologue of the M. domestica gene. Thus, linkage data alone cannot localize linkage groups to individual chromosomes and are insufficient for understanding the complete picture of genome structure differences among species. The problem is amply illustrated by the mapping data available for M. domestica (Table 1) wherein only a single linkage group has been tentatively assigned to an autosome. Fortunately, promising results from recent M. domestica x rodent cell hybridization studies suggest that the physical anchoring of linkage groups may soon be practical using somatic cell genetic approaches. As mentioned previously, Nestrova and others (1997) have produced somatic cell hybrid panels using M. domestica x Chinese hamster (Cricetulus griseus) and M. domestica × vole (Microtus subarvalis) cell fusions. The clones in this panel contain intact M. domestica chromosomes and appear sufficiently stable to permit mapping studies through standard correlational and exclusional criteria applied to M. domestica genes and gene products. The success of these hybridizations apparently results from the matching of appropriate eutherian species as fusion partners, rather than any unique quality of M. domestica itself, because fusions with cells derived from other eutherian species resulted in chromosome fragmentation and loss reminiscent of that previously observed in the construction of Australian marsupial x rodent SCH panels.

SCIENTIFIC CONTRIBUTIONS OF THE MAPS

Information obtained from marsupial gene mapping research has contributed materially to the understanding of patterns and mechanisms of mammalian genome evolution and has fostered a deeper appreciation of the complex history of evolutionary changes that have resulted in the contemporary array of mammalian genome structures. However, the most significant benefit derived from the establishment of marsupial gene maps has been the acquisition of new tools that are enabling expanded opportunities to explore the structure, regulation, function, and evolution of genes themselves.

Conservation of Syntenic Blocks

The arrangements of genes in the genomes of eutherian mammals are remarkably conservative. Despite bewildering variation in chromosome numbers, sizes, and shapes, a mounting body of evidence from physical and linkage mapping--and more recently from chromosome painting approaches--indicates that the diversity of eutherian chromosomes is an outcome of rearrangements among a relatively few large blocks of syntenic gene groups (reviewed by O'Brien and others 1988; CGOW 1996; Edwards 1994; Lundin 1993). For example, the genomes of species as distinct as humans and felids may differ by as few as 30 block rearrangements (Rettenberger and others 1995a), whereas human and porcine or bovine genomes appear somewhat more scrambled, requiring up to 50 rearrangements (Rettenberger and others 1995b; Solinas-Toldo and others 1995). The mouse genome is particularly rearranged and appears to differ from the human genome by approximately 90 block changes (Edwards 1994). As might be expected, the genomes of species within orders are generally highly conserved, differing by only a few obvious translocations or inversions, although exceptions occur in almost all groups. Perhaps the most remarkable finding is not that large chromosome blocks can be identified physically, but that the gene content (although not necessarily gene order) of these blocks is also conserved as judged from linkage and physical mapping studies (for example, DeBry and Selden 1996). Mapping data from marsupials suggest that this strong conservation of gene synteny relationships may extend to the Metatheria as well.

Karyotypically, the genomes of eutherian and marsupial mammals appear massively rearranged, but at the genic level, there is growing evidence of strong conservation in syntenic relationships that has persisted through at least 100 to 150 million yr of evolution. Several examples, evident in Table 1, indicate that despite many rearrangements, substantial conservation of synteny remains between the human and marsupial genomes. Seven genes located on HSA (Homo sapiens) 3p are located together on MEU (Macropus eugenii) 2q (Miller and others 1994). Five HSA11p genes are arranged in 2 clusters on MEU 2p and MEU 3q (Sinclair and Graves 1991; Toder and others 1996), and 3 of these are also syntenic in dasyurids (Bennett and others 1986; Sinclair and Graves 1991; Wainwright and Hope 1985). Five HSA 21p genes are found in 2 clusters on MEU 3/4q (chromosomes 3 and 4 were indistinguishable) and MEU 7, as well as in the dasyurid Sminthopsis macroura (Brookes and others 1992; Maccarone and others 1992). Six HSA 17q loci appear to be located within a single linkage group in M. domestica (Samollow and others 1998; Sokolova and others 1997). Two marsupial linkage relationships, GPI - PI in M. eugenii (van Oorschot and Cooper 1989) and C6 - C7 in M. domestica (van Oorschot and others 1993) also occur in humans and other eutherians.

The converse perspective can also be informative. For example, MEU I q shares gene content with at least 4 human chromosomes. Two of these chromosomes exhibit syntenic gene clusters that have remained intact since the divergence of the metatherian and eutherian lineages: HSA 10 (ILRA2, HK1, PLAU; Spurdle and others 1997) and HSA 15 (SNRPN, ZNF127; Toder and others 1996). Additional insight into genome evolution and conservation was derived from the discovery that a block of genes on HSA 21, which are syntenic in all eutherians examined, are found in 2 separate autosomal blocks in marsupials. When combined with mapping data from various eutherian species, these observations led to a novel hypothesis concerning the evolution of HSA 3 and HSA 10 homologues during early eutherian evolution (Maccarone and others 1992). Another highly conserved block--HBB, HRAS, LDHA, CAT--was also found intact in marsupials but was disparate in mouse, suggesting that the mouse condition is derived, whereas human and other eutherian species share the ancestral state (Sinclair and Graves 1991 ).

Sex Chromosome Evolution

Homologues of eutherian X-linked genes have received considerably more attention in marsupial gene mapping than autosomal loci, and have been the object of numerous reviews (for example, Graves 1996a,b; Graves and Foster 1994). This special interest is attributable to the discovery that marsupials do not conform to Ohno's law regarding the conservation of X-chromosome gene content.

Mapping studies of human X-linked gene homologues indicate that with rare exceptions in the pseudoautosomal (X-Y pairing) region, the gene content of the eutherian X is completely conserved. Genes located on the long arm of the human X chromosome (HSA Xq) and the HSA Xp pericentric region are also uniformly X linked in marsupials, but genes located on HSA Xp11.23-Xpter are not. Rather, they are autosomal in all marsupials examined. For example, they cluster on 2 separate autosomes (1 and 5) in M. eugenii and appear to lie on 3 different autosomes in the brushtail possum (Trichosurus vulpecula) (Table 1).

The autosomal locations of most HSA Xp genes in marsupials (and prototherians: see review by Graves 1998) suggest that the human Xp is a relatively young addition to an earlier, smaller version of the X (proto-X), which was similar in size and gene content to the modern marsupial X chromosome (Graves 1995, 1996a; Graves and Foster 1994; Watson and others 1993). Judged from the locations and syntenic relationships among marsupial homologues of these HSA Xp genes, the current eutherian X evolved in 2 or more distinct stages by addition of autosomal segments that became part of a pseudoautosomal portion of both the proto-X and proto-Y chromosomes in the early eutherian lineage. The mapping of human Y-linked homologues in marsupials (reviewed by Graves 1995; see also Delbridge and others 1997; Pask and others 1997; Toder and Graves 1998) has helped to refine this hypothesis by revealing that human and mouse Y chromosomes bear homologues of HSA Xp genes that are posited to have been recruited to the proto-X during early eutherian evolution. Thus, it appears that both the X and Y have grown larger during eutherian evolution by the repeated addition of autosomal genes, followed by eventual recruitment of these genes into the X-inactivation system and subsequent (concomitant?) silencing and loss of these genes on the Y.

Results from recent chromosome painting studies suggest that a parallel addition/attrition process may have occurred in marsupials as well. Painting of M. eugenii chromosomes with homologously derived, microdissected sex chromosome probes has revealed homologous sequences on the X and Y chromosomes that may represent recent additions of autosomal material to the macropodid sex chromosomes (Toder and others 1997b). These sequences do not appear homologous to any eutherian X/Y shared regions, suggesting that the sex chromosomes of macropodids have grown by the addition of autosomal material subsequent to the eutherian/metatherian divergence.

Chromosome painting methods have also been used to examine sex chromosome evolution among closely related marsupial species. The swamp wallaby (Wallabia bicolor) possesses an unusual karyotype that includes an X/Y1/Y2 male sex chromosome system. Results from cross-species chromosome painting experiments using chromosome paints derived from M. eugenii (which has a typical macropodid karyotype) imply that the current X chromosome of W. bicolor probably evolved by a fusion of 2 autosomes to the original X (and Y), followed by a fission event involving the newly expanded Y that created 2 novel Y chromosomes (Toder and others 1997a).

Hypothesis Testing

The comparative use of marsupial gene mapping information has provided a powerful tool for examining the evolution of sex determination and the potential involvement of particular genes in sex determination and spermatogenesis. ZFY, a gene that encodes a zinc-finger protein, was believed to be the long-sought testis-determining factor (TDF1) due to its location within a region of the Y chromosome that was implicated in sex reversals when deleted (Page and others 1987). However, comparative mapping studies showed that ZFY was autosomal in marsupials, suggesting that it was not testis determining in this group of mammals and perhaps was not the eutherian TDF either (Sinclair and others 1988). When the actual sex-determining gene, SRY, was discovered a short time later, it was found to be restricted to the Y chromosome in both eutherians and marsupials, as would be expected for the TDF gene (reviewed by Goodfellow and Lovell-Badge 1993).

Mapping of the marsupial SRY gene led to the detection of a homologous sequence with very strong similarity on the marsupial X chromosome and the subsequent identification of eutherian homologues on the human (SOX3) and mouse (Sox3) X chromosomes (Graves 1995). Sequence comparisons suggested that SOX3 and SRY were once homologues on the autosomal proto-X and proto-Y chromosomes, and that SRY evolved its sex-determining function secondarily, after the Y had already taken on a sex-determining function and become isolated from the X (Graves 1995). This hypothesis receives support from the observation that no other vertebrates, including prototherian mammals, have sex-specific SRY homologues. However, the hypothesis raises questions concerning the identity of genes that may have been involved in sex determination before this function was usurped by SRY. Information from the mapping of marsupial genomes has helped to narrow the possibilities. DAX1 (also known as AHC) is a gene on human Xp that has been proposed as an ancient sex-determining gene because it triggers sex reversals when duplicated in XY individuals. The marsupial homologue of DAX1 lies among a cluster of autosomal genes that appear to have been added to the eutherian X since the divergence of the marsupial and eutherian branches. This indicates that its function as a sex-determining gene could not have predated that of SRY (Pask and others 1997).

Marsupial mapping information similarly eliminated DAZ as a possible ancient Y-borne spermatogenesis gene. Deletions in a specific region of the Y chromosome are known to lead to azoospermia in humans. Based on their appropriate locations on the Y chromosome, as well as the similarities of their expression patterns and gene products, RMB1 and DAZ are both potential candidates for an azoospermia factor gene. However, the marsupial DAZ homologue is autosomal, whereas the position of RMB1 is conserved on the marsupial Y. This suggests that RMB1 is the better candidate for the azoospermia factor (Delbridge and others 1997).

Another example of the utility of comparative applications of marsupial gene maps centers on 4 imprinted genes--IGF2, H19, SNRPN, and ZNF127. These genes are located in 2 clusters that lie on a single autosome in mouse but are divided between 2 autosomes in human. Scenarios proposed concerning the origin of imprinting include the simultaneous evolution of imprinting capability of a syntenic block of genes, possibly in connection with, or even as part of, the X-chromosome inactivation system on the proto-X chromosome (discussed in Toder and others 1996). Which of the 2 present states of gene synteny is ancestral--the mouse with 2 syntenic clusters or the human with 2 nonsyntenic clusters--is crucial to this question. Evidence from marsupial gene mapping studies is quite clear on the issue. These 4 genes lie in 2 clusters on separate autosomes in M. eugenii, just as in the human genome (Toder and others 1996). The mouse configuration is the derived one; hence, there is little chance that the 4 genes were once syntenic on the ancestral X chromosome, or any other chromosome, in a recent common ancestor of mice and humans.

X-Chromosome Inactivation

Early gene mapping studies in marsupials provided support for Ohno's predicted conservation of mammalian X-chromosome gene content but also revealed a pattern of X-chromosome inactivation (XCI1) in marsupials that differed substantially from the familiar eutherian pattern. Not only was inactivation of the X chromosome nonrandom, it also was incomplete and locus specific; some loci on the inactivated paternal X were partially expressed, while others were completely silent (VandeBerg and others 1987). Moreover, the locus specificity of partial expression was also species and tissue specific, leading to 2 distinct hypotheses about the marsupial XCI process. One suggested that inactivation was accomplished piecemeal, on a locus by locus basis. The alternative hypothesis posited that inactivation spread from an inactivation center on the X chromosome, with the distance of genes from the inactivation center determining the extent and stability of the inactive state.

The latter hypothesis predicts that locus-specific patterns of partial expression should differ in species with different X-linked gene orders (Graves and Dawson 1988). The order of G6PD, PGK1, HPRT, and GLA are different in dasyurid and macropodid marsupials (Graves and Dawson 1988), but because X-linked gene expression patterns have not been closely examined in any dasyurid species, the hypothesis remains untested. However, the G6PD locus of Didelphis virginiana, an American didelphid, lies on the opposite arm of the X chromosome from PGK1 (Driscoll and Migeon 1988), making this the only marsupial known in which all 4 of the loci mentioned are not on the same chromosomal arm. Interestingly, this is also the only marsupial in which G6PD is expressed in many tissues while PGK1 is uniformly silent (Samollow and others 1987, 1995). In all Australian marsupials examined, G6PD is silent in all tissues, being expressed only in cultured fibroblasts, whereas PGK1 is expressed at various levels in many tissues and cultured cells of several species (Cooper and others 1993; VandeBerg and others 1987).

Recombination Rates

An important outcome of linkage analyses in marsupials has been the detection of striking differences in sex-specific rates of recombination in some species. Although sex-specific recombination rates are common in mammals (Burl and others 1991; Dunn and Bennett 1967), the magnitudes of the marsupial differences were extraordinarily large and ran counter to prevailing observations that female recombination maps tend to be larger than male maps among eutherian species (Davisson and others 1989; Ellegren and others 1994; Morton 1991).

Inheritance studies of several protein polymorphisms in the fat-tailed dunnart (Sminthopsis crassicaudata), a dasyurid marsupial, detected evidence of linkage among 2 groups of 3 genes, defining 2 autosomal linkage groups (Bennett and others 1986). Surprisingly, females exhibited substantially reduced recombination among all of these genes relative to male values. A concurrent cytological examination (Bennett and others 1986) revealed strongly discordant patterns of chiasma distribution as well, with female chiasmata clustered at chromosome termini, whereas male chiasmata were more uniformly distributed. These findings encouraged speculation of a causal relationship between chiasma distribution and genetic recombination (Bennett and others 1986). Subsequent studies also revealed severely reduced female recombination among genes in 2 linkage groups in M. domestica (van Oorschot and others 1992a, 1993). As in S. crassicaudata, the linkage data were consistent with cytological evidence of reduced chiasma frequency in cells from female, as compared with male, M. domestica and a restriction of chiasmata primarily to telomeric regions in female cells (Hayman and others 1988). Such similar findings in 2 distantly related marsupials prompted the cautious speculation that female marsupials have generally lower recombination rates than their male counterparts (Hope 1993; van Oorschot and others 1992a, 1993).

More extensive linkage data from M. domestica (Perelygin and others 1996; Samollow and others Forthcoming) indicate that females of this species do, indeed, exhibit much less recombination than males; but these data also reveal that recombination rates are both sex and linkage group specific. In some linkage groups, females exhibit greatly reduced recombination rates, and in others, male and female rates are virtually identical. However, cytological data from marsupials in other families (Bettongia penicillata, family Potoroidae; and Trichosurus vulpecula, family Phalangeridae) have revealed little difference in sex-specific chiasma number or distribution (Hayman and Rodger 1990; Hayman and others 1990). In addition, a report by McKenzie and others (1995) indicates that female M. eugenii (family Macropodidae) exhibit recombination rates that are equal to, or higher than, those in males. Apparently then, reduced female recombination does occur in some species, to an extent unparalleled among eutherian mammals. Overall however, there seems no compelling evidence that it is a fundamental marsupial characteristic.

ANTICIPATED FUTURE CONTRIBUTIONS OF THE MAPS

Comparative Mapping

Compared with the current pace of mapping human, mouse, and other mammalian genomes, gene mapping in marsupials is still a cottage industry. In view of the embryonic states of even the most advanced marsupial maps, it is likely that important advances in the near future will derive mainly from the continued mapping of highly conserved anchor loci by means of physical and linkage techniques. Increasing the density of such loci will improve the utility of the maps for comparative applications in molecular phylogenetic analyses of eutherian groups, and for advancing our understanding of the evolution of genes and gene families. Insightful explorations of differences in eutherian and metatherian gene structure and function will continue to illuminate fundamental mechanisms of gene regulation and expression, such as the molecular basis of XCI, autosomal imprinting, and the evolution of sex-determining genes and molecular mechanisms of sex determination pathways.

Recent adoption of FISH approaches has enhanced the accuracy and speed of physical mapping in Australian species and will surely be adapted for future use in M. domestica. We anticipate that the application of cross-species chromosome painting techniques could similarly increase the resolution of interspecific chromosome conservation and rearrangements. The coupling of high-resolution gene maps with high-resolution chromosome maps would engender a new level of refinement in hypotheses regarding the co-conservation of chromosome structure and gene content in marsupial genomes, and would enable more informative contrasts between eutherian and metatherian genome structures.

Biomedical Applications

In addition to physical mapping, continued linkage mapping will soon enable the localization of individual genes that are found to contribute to variation in phenotypes related to physiology, development, and disease susceptibility in the M. domestica model. This concept will be especially valuable when linkage data are finally combined with physical mapping approaches (for example, Nesterova and others 1997) to anchor linkage maps on particular chromosomal regions. Attainment of this goal would enable the direct comparison of regions of the M. domestica genome to homologous regions in the genomes of eutherian species--especially humans wherein they may be of use in the study of normal and anomalous developmental processes and susceptibilities to common diseases. Specifically, such an ability would be important in determining the locations of genes that may become discovered through ongoing studies of ultraviolet radiation-induced melanoma and corneal carcinomas and genes that contribute to differential response to dietary fat and cholesterol (VandeBerg and Robinson 1997).

USES OF THE MAPS AND ACCESSIBILITY

As described in foregoing sections, the immediate utility of marsupial gene maps lies in their comparative function. As the sister group of eutherians, marsupials represent the natural outgroup for many kinds of comparative analyses, especially those relating to the genetic aspects of phylogeny, the molecular evolution of genes and gene families, and the evolution of gene structure and function. As more data become available from mapping the M. domestica genome, there will be the added utility of exploiting this map to locate genes that influence phenotypic variation (that is, quantitative trait loci) through linkage analysis, and the possibility of extrapolating the locations of such genes to maps of other marsupials and to eutherian species, especially humans.

The need for a central electronic repository of marsupial gene mapping information became strikingly apparent during the preparation of this review. The growing number of genes being mapped among marsupial species is becoming increasingly difficult to track, and a regularly updated, publicly available site must soon become reality. In the interim, our advice for researchers requiring up-to-date information on the state of the gene maps of particular species is to contact the main laboratories involved. For information on the tammar wallaby (M. eugenii), other macropod species, and dasyurid species:

And

For information on the status of the M. domestica map:

CONCLUSION

Marsupials have long suffered under a form of eutherian chauvinism that relegated them to the status of secondary mammals. Viewed as primitive and strange--as much for their low species number as for their distinctive reproductive characteristics--their use as research animals was restricted to specialized areas of developmental biology that took advantage of the immature state at birth and the ability to experimentally manipulate the external "fetuses." This view has changed substantially due to the broadening recognition that marsupials are perfectly good mammals that have their own distinctive modifications of fundamental mammalian designs (Graves 1996a). Marsupials and eutherians have been evolving along unique pathways for more than 100 million yr (possibly much more) and represent alternative, rather than inferior or superior, evolutionary solutions to the basic mammalian way of life. The essential similarities among metatherian and eutherian mammals--the legacy of their common ancestry--far outnumber and outweigh their differences. Indeed, their differences represent superficial variants on common mammalian themes that are best viewed as potential probes for the study of underlying molecular processes shared by all mammalian taxa.

With this emerging recognition of marsupials as alternative mammals has come an increased interest in the utilization of unique marsupial variations as instruments for investigating elemental mammalian mechanisms of gene evolution, regulation, and expression. Exemplary products of this expanding interest include a clearer understanding of the history of mammalian genome evolution, an evolving picture of the origins and evolution of sex chromosomes and sex-determining genes, a growing body of evidence concerning mechanisms of sex determination, and alternative models for examining the essential determinants of X-linked gene expression. The development of M. domestica as a model for biomedical research may furnish the means to utilize more effectively the features of marsupial ontogeny that have long been coveted by developmental biologists and expand the use of marsupials as models for a broader range of basic research applications.

Past genetic studies of marsupials have been a hodgepodge of opportunistic and ad hoc investigations that took advantage of newly detected genetic variants whenever and in whatever species they were detected. Today, with the sharpening focus of genetic studies on 2 key species, prospects for the development of detailed marsupial gene maps are excellent. As these maps grow in density and extent, the largely untapped potential of marsupial models for basic and applied biological research can finally be realized.

1Abbreviations used in this paper: FISH, fluorescence in situ hybridization; ISHR, in situ hybridization methods using radiolabeled probes; LG, linkage group; MYA, million years ago; RAPD, random amplified polymorphic DNA: SCH, somatic cell hybrid; SFBR, Southwest Foundation for Biomedical Research; TDF, testis-determining factor; XCI, X-chromosome inactivation.

ACKNOWLEDGMENTS

Support for the acquisition and compilation of data, and for the preparation of this manuscript, was provided in part by National Institutes of Health grant P01 RR09919 and a grant from the Samuel Roberts Noble Foundation, Inc.

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TABLE 1 Gene assignments in marsupials in relation to human map locationsa



TABLE 2 Gene assignments in other marsupialsa





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