In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/Dmrt1bY genes of the teleost fish medaka have been characterized as sex-determining genes that trigger formation of the testis. However, the molecular mechanism of the ZZ/ZW-type system in vertebrates, including the clawed frog Xenopus laevis, is unknown. Here, we isolated an X. laevis female genome-specific DM-domain gene, DM-W, and obtained molecular evidence of a W-chromosome in this species. The DNA-binding domain of DM-W showed a strikingly high identity (89%) with that of DMRT1, but it had no significant sequence similarity with the transactivation domain of DMRT1. In nonmammalian vertebrates, DMRT1 expression is connected to testis formation. We found DMRT1 or DM-W to be expressed exclusively in the primordial gonads of both ZZ and ZW or ZW tadpoles, respectively. Although DMRT1 showed continued expression after sex determination, DM-W was expressed transiently during sex determination. Interestingly, DM-W mRNA was more abundant than DMRT1 mRNA in the primordial gonads of ZW tadpoles early in sex determination. To assess the role of DM-W, we produced transgenic tadpoles carrying a DM-W expression vector driven by Ϸ3 kb of the 5-flanking sequence of DM-W or by the cytomegalovirus promoter. Importantly, some developing gonads of ZZ transgenic tadpoles showed ovarian cavities and primary oocytes with both drivers, suggesting that DM-W is crucial for primary ovary formation. Taken together, these results suggest that DM-W is a likely sex (ovary)-determining gene in X. laevis.T he sexual fate of metazoans is determined genetically or by environmental factors, such as temperature. In the former case, heterogametic sex chromosomes determine the male (XY() or female (ZW&) fate in many species of vertebrates. In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/Dmrt1bY gene of the teleost fish medaka have been characterized as sex-determining genes that initiate testis formation, leading to male sexual development (1-5). In contrast, the molecular mechanism for the ZZ/ZW sex-determining system remains unclear, because no sexdetermining genes have been isolated.The Drosophila melanogaster doublesex (dsx) and Caenorhabditis elegans male abnormal (mab)-3 genes are known to control sexual development in these animals (6, 7). The two genes encode proteins containing a zinc finger-like DNA-binding motif called the DM domain. In vertebrates, the DM-domain gene DMRT1 is implicated in sexual development. In the mouse, DMRT1 is essential for postnatal testis differentiation (8, 9). In some other vertebrates, such as the chicken and turtle, DMRT1 expression is connected to testis formation in undifferentiated gonads (10-12). As mentioned above, the medaka fish gene DMY/Dmrt1bY, which is a coorthologue of DMRT1, causes testis formation as a sex-determining gene (3-5). In the chicken, which has the ZZ/ZW system, DMRT1 is located on the Z chromosome, suggesting that gene dosage may induce male development (...

All snake species exhibit genetic sex determination with the ZZ͞ZW type of sex chromosomes. To investigate the origin and evolution of snake sex chromosomes, we constructed, by FISH, a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 109 cDNA clones. Eleven of the 109 clones were localized to the Z chromosome. All human and chicken homologues of the snake Z-linked genes were located on autosomes, suggesting that the sex chromosomes of snakes, mammals, and birds were all derived from different autosomal pairs of the common ancestor. We mapped the 11 Z-linked genes of E. quadrivirgata to chromosomes of two other species, the Burmese python (Python molurus bivittatus) and the habu (Trimeresurus flavoviridis), to investigate the process of W chromosome differentiation. All and 3 of the 11 clones were localized to both the Z and W chromosomes in P. molurus and E. quadrivirgata, respectively, whereas no cDNA clones were mapped to the W chromosome in T. flavoviridis. Comparative mapping revealed that the sex chromosomes are only slightly differentiated in P. molurus, whereas they are fully differentiated in T. flavoviridis, and E. quadrivirgata is at a transitional stage of sex-chromosome differentiation. The differentiation of sex chromosomes was probably initiated from the distal region on the short arm of the protosex chromosome of the common ancestor, and then deletion and heterochromatization progressed on the sex-specific chromosome from the phylogenetically primitive boids to the more advanced viperids.comparative map ͉ chromosome homology ͉ FISH ͉ sex-determining gene ͉ reptile A ll snake species are subject to genetic sex determination with sex chromosomes, as are mammals and birds, and they have female heterogamety (ZZ males and ZW females). Comparative gene mapping between human and chicken revealed that human XX͞XY and chicken ZZ͞ZW sex chromosomes have no homologies (1, 2), suggesting that the sex chromosomes of mammals and birds were derived from different pairs of autosomes of the common ancestor. Beçak et al. (3) found that there is close karyological similarity between snakes and birds, such as distinct differentiation of macro-and microchromosomes and constant occurrence of ZW-type sex chromosomes. This finding leads us to predict the presence of homology between ophidian and avian sex chromosomes. However, no attempts have yet been made to investigate the conservation of the linkage homologies of snake chromosomes to human and chicken chromosomes by comparative gene mapping, although this approach would provide fundamental information on the genome evolution and the origin of sex-chromosome differentiation in amniotes. In another study (4), we constructed a preliminary cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 52 EST clones, which were isolated from the cDNA library of the brain tissue and were identified as snake homologues of human and chicken orthologous genes by a search of the DNA database. Of 52 EST clones, two genes, T...

The karyotypes of birds, turtles and snakes are characterized by two distinct chromosomal components, macrochromosomes and microchromosomes. This close karyological relationship between birds and reptiles has long been a topic of speculation among cytogenetists and evolutionary biologists; however, there is scarcely any evidence for orthology at the molecular level. To define the conserved chromosome synteny among humans, chickens and reptiles and the process of genome evolution in the amniotes, we constructed comparative cytogenetic maps of the Chinese soft-shelled turtle (Pelodiscus sinensis) and the Japanese four-striped rat snake (Elaphe quadrivirgata) using cDNA clones of reptile functional genes. Homology between the turtle and chicken chromosomes is highly conserved, with the six largest chromosomes being almost equivalent to each other. On the other hand, homology to chicken chromosomes is lower in the snake than in the turtle. Turtle chromosome 6q and snake chromosome 2p represent conserved synteny with the chicken Z chromosome. These results suggest that the avian and turtle genomes have been well conserved during the evolution of the Arcosauria. The avian and snake sex Z chromosomes were derived from different autosomes in a common ancestor, indicating that the causative genes of sex determination may be different between birds and snakes.3

Recent progress of chicken genome projects has revealed that bird ZW and mammalian XY sex chromosomes were derived from different autosomal pairs of the common ancestor; however, the evolutionary relationship between bird and reptilian sex chromosomes is still unclear. The Chinese soft-shelled turtle (Pelodiscus sinensis) exhibits genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes have been identified. In order to investigate this further, we performed molecular cytogenetic analyses of this species, and thereby identified ZZ/ZW-type micro-sex chromosomes. In addition, we cloned reptile homologues of chicken Z-linked genes from three reptilian species, the Chinese soft-shelled turtle and the Japanese four-striped rat snake (Elaphe quadrivirgata), which have heteromorphic sex chromosomes, and the Siam crocodile (Crocodylus siamensis), which exhibits temperature-dependent sex determination and lacks sex chromosomes. We then mapped them to chromosomes of each species using FISH. The linkage of the genes has been highly conserved in all species: the chicken Z chromosome corresponded to the turtle chromosome 6q, snake chromosome 2p and crocodile chromosome 3. The order of the genes was identical among the three species. The absence of homology between the bird Z chromosome and the snake and turtle Z sex chromosomes suggests that the origin of the sex chromosomes and the causative genes of sex determination are different between birds and reptiles.

To define the process of karyotypic evolution in the Galliformes on a molecular basis, we conducted genome-wide comparative chromosome painting for eight species, i.e. silver pheasant (Lophura nycthemera), Lady Amherst’s pheasant (Chrysolophus amherstiae), ring-necked pheasant (Phasianus colchicus), turkey (Meleagris gallopavo), Western capercaillie (Tetrao urogallus), Chinese bamboo-partridge (Bambusicola thoracica) and common peafowl (Pavo cristatus) of the Phasianidae, and plain chachalaca (Ortalis vetula) of the Cracidae, with chicken DNA probes of chromosomes 1–9 and Z. Including our previous data from five other species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and blue-breasted quail (Coturnix chinensis) of the Phasianidae, guinea fowl (Numida meleagris) of the Numididae and California quail (Callipepla californica) of the Odontophoridae, we represented the evolutionary changes of karyotypes in the 13 species of the Galliformes. In addition, we compared the cytogenetic data with the molecular phylogeny of the 13 species constructed with the nucleotide sequences of the mitochondrial cytochrome b gene, and discussed the process of karyotypic evolution in the Galliformes. Comparative chromosome painting confirmed the previous data on chromosome rearrangements obtained by G-banding analysis, and identified several novel chromosome rearrangements. The process of the evolutionary changes of macrochromosomes in the 13 species was in good accordance with the molecular phylogeny, and the ancestral karyotype of the Galliformes is represented.

Palaeognathous birds (Struthioniformes and Tinamiformes) have morphologically conserved karyotypes and less differentiated ZW sex chromosomes. To delineate interspecific chromosome orthologies in palaeognathous birds we conducted comparative chromosome painting with chicken (Gallus gallus, GGA) chromosome 1-9 and Z chromosome paints (GGA1-9 and GGAZ) for emu, double-wattled cassowary, ostrich, greater rhea, lesser rhea and elegant crested tinamou. All six species showed the same painting patterns: each probe was hybridized to a single pair of chromosomes with the exception that the GGA4 was hybridized to the fourth largest chromosome and a single pair of microchromosomes. The GGAZ was also hybridized to the entire region of the W chromosome, indicating that extensive homology remains between the Z and W chromosomes on the molecular level. Comparative FISH mapping of four Z-and/or W-linked markers, the ACO1/IREBP, ZOV3 and CHD1 genes and the EE0.6 sequence, revealed the presence of a small deletion in the proximal region of the long arm of the W chromosome in greater rhea and lesser rhea. These results suggest that the karyotypes and sex chromosomes of palaeognathous birds are highly conserved not only morphologically, but also at the molecular level; moreover, palaeognathous birds appear to retain the ancestral lineage of avian karyotypes.2

Salmonid fishes are among the few animal taxa with a probable recent tetraploid ancestor. The present study is the first to compare large (>100 kb) duplicated genomic sequence fragments in such species. Two contiguous stretches with major histocompatibility complex (MHC) class I genes were detected in a rainbow trout BAC library, mapped and sequenced. The MHC class I duplicated regions, mapped by fluorescence in situ hybridization (FISH), were shown to be located on different metaphase chromosomes, Chr 14 and 18. Gene organization in both duplications is similar to that in other fishes, in that the class I loci are tightly linked with the PSMB8, PSMB9, PSMB10 and ABCB3 genes. Whereas one region, Onmy-IA, has a classical MHC class I locus (UBA), Onmy-IB encodes only non-classical class Ib proteins. The nucleotide diversity between the Onmy-IA and Onmy-IB noncoding regions is about 14%. This suggests that the MHC class I duplication event has occurred about 60 mya close to the time of an hypothesized ancestral tetraploid event. The present article is the first convincing report on the co-existence of two closely related MHC class I core regions on two different chromosomes. The interchromosomal duplication and the homology levels are supportive of the tetraploid model.

In order to construct a chicken (Gallus gallus) cytogenetic map, we isolated 134 genomic DNA clones as new cytogenetic markers from a chicken cosmid DNA library, and mapped these clones to chicken chromosomes by fluorescence in situ hybridization. Forty-five and 89 out of 134 clones were localized to macrochromosomes and microchromosomes, respectively. The 45 clones, which localized to chicken macrochromosomes (Chromosomes 1–8 and the Z chromosome) were used for comparative mapping of Japanese quail (Coturnix japonica). The chromosome locations of the DNA clones and their gene orders in Japanese quail were quite similar to those of chicken, while Japanese quail differed from chicken in chromosomes 1, 2, 4 and 8. We specified the breakpoints of pericentric inversions in chromosomes 1 and 2 by adding mapping data of 13 functional genes using chicken cDNA clones. The presence of a pericentric inversion was also confirmed in chromosome 8. We speculate that more than two rearrangements are contained in the centromeric region of chromosome 4. All 30 clones that mapped to chicken microchromosomes also localized to Japanese quail microchromosomes, suggesting that chromosome homology is highly conserved between chicken and Japanese quail and that few chromosome rearrangements occurred in the evolution of the two species.