Evolutionary history of novel genes on the tammar wallaby Y chromosome: Implications for sex chromosome evolution.

We report here the isolation and sequencing of 10 Y-specific tammar wallaby (Macropus eugenii) BAC clones, revealing five hitherto undescribed tammar wallaby Y genes (in addition to the five genes already described) and several pseudogenes. Some genes on the wallaby Y display testis-specific expression, but most have low widespread expression. All have partners on the tammar X, along with homologs on the human X. Nonsynonymous and synonymous substitution ratios for nine of the tammar XY gene pairs indicate that they are each under purifying selection. All 10 were also identified as being on the Y in Tasmanian devil (Sarcophilus harrisii; a distantly related Australian marsupial); however, seven have been lost from the human Y. Maximum likelihood phylogenetic analyses of the wallaby YX genes, with respective homologs from other vertebrate representatives, revealed that three marsupial Y genes (HCFC1X/Y, MECP2X/Y, and HUWE1X/Y) were members of the ancestral therian pseudoautosomal region (PAR) at the time of the marsupial/eutherian split; three XY pairs (SOX3/SRY, RBMX/Y, and ATRX/Y) were isolated from each other before the marsupial/eutherian split, and the remaining three (RPL10X/Y, PHF6X/Y, and UBA1/UBE1Y) have a more complex evolutionary history. Thus, the small marsupial Y chromosome is surprisingly rich in ancient genes that are retained in at least Australian marsupials and evolved from testis-brain expressed genes on the X.

How the gene content of human sex chromosomes evolved.

The X and Y chromosomes of humans and other mammals both have very atypical gene contents. The degenerate Y bears only a handful of genes that are specialized for male sex and reproduction. Now it seems that the X over-represents genes controlling reproductive traits and intelligence. This is hard to explain in terms of function but makes excellent sense in terms of evolution. Comparisons between the gene content of the X and Y in humans, distantly related mammals, and other vertebrates, define the evolutionary past of our sex chromosomes and suggest how special selective forces act on the X and Y.

Identification of cryptic sex chromosomes and isolation of X- and Y-borne genes.

Comparative molecular cytogenetics provides a powerful tool for deciphering the evolutionary history of vertebrate sex chromosomes. We have adapted cell culture and molecular cytogenetic techniques to study the sex chromosomes of many exotic mammals, birds, and reptiles. Here we describe differential chromosome banding and staining techniques that distinguish sex chromosomes in species with no morphologically distinct XY or ZW chromosome pairs. We describe a method to isolate, identify, and map genomic BAC clones from the Y chromosome, and we also identify strategies for isolating candidate sex chromosome genes.

Characterizing the chromosomes of the Australian model marsupial Macropus eugenii (tammar wallaby).

Marsupials occupy a phylogenetic middle ground that is very valuable in genome comparisons of mammal and other vertebrate species. For this reason, whole genome sequencing is being undertaken for two distantly related marsupial species, including the model kangaroo species Macropus eugenii (the tammar wallaby). As a first step towards the molecular characterization of the tammar genome, we present a detailed description of the tammar karyotype, report the development of a set of molecular anchor markers and summarize the comparative mapping data for this species.

Marsupial anti-Mullerian hormone gene structure, regulatory elements, and expression.

During male sexual development in reptiles, birds, and mammals, anti-Müllerian hormone (AMH) induces the regression of the Müllerian ducts that normally form the primordia of the female reproductive tract. Whereas Müllerian duct regression occurs during fetal development in eutherian mammals, in marsupial mammals this process occurs after birth. To investigate AMH in a marsupial, we isolated an orthologue from the tammar wallaby (Macropus eugenii) and characterized its expression in the testes and ovaries during development. The wallaby AMH gene is highly conserved with the eutherian orthologues that have been studied, particularly within the encoded C-terminal mature domain. The N-terminus of marsupial AMH is divergent and larger than that of eutherian species. It is located on chromosome 3/4, consistent with its autosomal localization in other species. The wallaby 5' regulatory region, like eutherian AMH genes, contains binding sites for SF1, SOX9, and GATA factors but also contains a putative SRY-binding site. AMH expression in the developing testis begins at the time of seminiferous cord formation at 2 days post partum, and Müllerian duct regression begins shortly afterward. In the developing testis, AMH is localized in the cytoplasm of the Sertoli cells but is lost by adulthood. In the developing ovary, there is no detectable AMH expression, but in adults it is produced by the granulosa cells of primary and secondary follicles. It is not detectable in atretic follicles. Collectively, these studies suggest that AMH expression has been conserved during mammalian evolution and is intimately linked to upstream sex determination mechanisms.