A human candidate spermatogenesis gene, RBM1, is conserved and amplified on the marsupial Y chromosome.

Three genes, RBM1, DAZ and TSPY, map to a small region of the long arm of the human Y chromosome which is deleted in azoospermic men. RBM1, but not DAZ or TSPY, has a Y-linked homologue in marsupials which is transcribed in the testis. This suggests that RBM1 has been retained on the Y chromosome because of a critical male-specific function. Marsupial RBM1 is closely related to human RBM1, but, like the related autosomal gene hnRNPG, lacks the amplification of an exon. This suggests that RBM1 evolved from hnRNPG at least 130 million years ago and has undergone internal amplification in primates, as well as independent amplification in several therian [corrected] lineages.

Conservation of a chromosome arm in two distantly related marsupial species.

Marsupials, which diverged from eutherian mammals 150 million years ago (MYA), occupy a phylogenetic position that is very valuable in genome comparisons of mammal and other vertebrate species. Within the marsupials, the Australian and American clades (represented by the tammar wallaby Macropus eugenii, and the opossum Monodelphis domestica) diverged about 70 MYA. G-banding and chromosome painting suggest that tammar wallaby chromosome 6q has homology to opossum chromosome 7q. We tested this conservation by physically mapping the tammar wallaby orthologs of opossum chromosome 7q genes. We isolated 28 tammar wallaby BAC clones that contained orthologs of 16 opossum chromosome 7q genes. We used fluorescence in situ hybridization (FISH) to show that they all mapped specifically to the tammar wallaby chromosome 6q in nearly the same order as their orthologs on opossum chromosome 7q. Thus this chromosome arm is genetically, as well as cytologically, conserved over the 55-80 million years that separate kangaroos and the opossum.

Mapping platypus SOX genes; autosomal location of SOX9 excludes it from sex determining role.

In the absence of an SRY orthologue the platypus sex determining gene is unknown, so genes in the human testis determining pathway are of particular interest as candidates. SOX9 is an attractive choice because SOX9 deletions cause male-to-female sex reversal in humans and mice, and SOX9 duplications cause female-to-male sex reversal. We have localized platypus SOX9, as well as the related SOX10, to platypus chromosomes 15 and 10, respectively, the first assignments to these platypus chromosomes, and the first comparative mapping markers from human chromosomes 17 and 22. The autosomal localization of platypus SOX9 in this study contradicts the hypothesis that SOX9 acts as the sex determining switch in platypus.

Sequence analysis, and chromosomal localization of a gene encoding a cystatin-like protein from Drosophila melanogaster.

Using polyclonal antibodies raised against a Drosophila Ca2(+)-binding protein (DCABP-23), clones were isolated from a Drosophila head cDNA library constructed in the expression vector lambda gt11. Two non-homologous clones have been isolated and are being subjected to sequence analysis. One of these clones, though not encoding DCABP-23, does encode a Drosophila cystatin-like protein. This presumed Drosophila cystatin shows homology to mammalian cystatins, chicken egg white cystatin and the rice oryzacystatin. The Drosophila cystatin has been mapped, by in situ hybridization, to region 88C on the right arm of the third chromosome.

Sex determination in platypus and echidna: autosomal location of SOX3 confirms the absence of SRY from monotremes.

In eutherian ('placental') mammals, sex is determined by the presence or absence of the Y chromosome-borne gene SRY, which triggers testis determination. Marsupials also have a Y-borne SRY gene, implying that this mechanism is ancestral to therians, the SRY gene having diverged from its X-borne homologue SOX3 at least 180 million years ago. The rare exceptions have clearly lost and replaced the SRY mechanism recently. Other vertebrate classes have a variety of sex-determining mechanisms, but none shares the therian SRY-driven XX female:XY male system. In monotreme mammals (platypus and echidna), which branched from the therian lineage 210 million years ago, no orthologue of SRY has been found. In this study we show that its partner SOX3 is autosomal in platypus and echidna, mapping among human X chromosome orthologues to platypus chromosome 6, and to the homologous chromosome 16 in echidna. The autosomal localization of SOX3 in monotreme mammals, as well as non-mammal vertebrates, implies that SRY is absent in Prototheria and evolved later in the therian lineage 210-180 million years ago. Sex determination in platypus and echidna must therefore depend on another male-determining gene(s) on the Y chromosomes, or on the different dosage of a gene(s) on the X chromosomes.

Construction of a highly enriched marsupial Y chromosome-specific BAC sub-library using isolated Y chromosomes.

The Y chromosome is perhaps the most interesting element of the mammalian genome but comparative analysis of the Y chromosome has been impeded by the difficulty of assembling a shotgun sequence of the Y. BAC-based sequencing has been successful for the human and chimpanzee Y but is difficult to do efficiently for an atypical mammalian model species (Skaletsky et al. 2003, Kuroki et al. 2006). We show how Y-specific sub-libraries can be efficiently constructed using DNA amplified from microdissected or flow-sorted Y chromosomes. A Bacterial Artificial Chromosome (BAC) library was constructed from the model marsupial, the tammar wallaby (Macropus eugenii). We screened this library for Y chromosome-derived BAC clones using DNA from both a microdissected Y chromosome and a flow-sorted Y chromosome in order to create a Y chromosome-specific sub-library. We expected that the tammar wallaby Y chromosome should detect approximately 100 clones from the 2.2 times redundant library. The microdissected Y DNA detected 85 clones, 82% of which mapped to the Y chromosome and the flow-sorted Y DNA detected 71 clones, 48% of which mapped to the Y chromosome. Overall, this represented a approximately 330-fold enrichment for Y chromosome clones. This presents an ideal method for the creation of highly enriched chromosome-specific sub-libraries suitable for BAC-based sequencing of the Y chromosome of any mammalian species.

Regulatory mutants of the aroF-tyrA operon of Escherichia coli K-12.

The regulatory region of the aroF-tyrA operon was fused to the chloramphenicol acetyltransferase (cat) gene on a plasmid vector. Expression of the cat gene was subject to repression by tyrR+. This fusion was used to isolate regulatory mutants with increased expression of the cat gene in which repression by tyrR+ was affected. Nucleotide sequencing of these mutants has led to the identification of three sites involved in the repression of aroF by tyrR+. The existence of a functional promoter divergently transcribing from the aroF regulatory region was also demonstrated by using the cat fusion vector. The expression of this promoter is also regulated by tyrR+.

Mammalian Y chromosome evolution and the male-specific functions of Y chromosome-borne genes.

All mammals have an XY chromosomal sex determining system, in which a small Y chromosome triggers male development, and contains genes required for spermatogenesis. The X and Y chromosomes were originally homologous, but diverged during evolution as the Y chromosome was degraded progressively. Comparisons among the sex chromosomes of different mammal groups indicate that the X and Y chromosomes received additions of material from other chromosomes. Genes on the Y chromosome originated from the ancient X-Y pair, or from these additions, or were copies of genes on one of the autosomes. Only genes with important male-specific functions, such as sex determination and spermatogenesis, are selected for and retained on the differential region of the Y chromosome. The mammalian sex determining gene, SRY, controls the testis determination pathway, which includes at least one related gene. Several candidate spermatogenesis genes have been identified, but so far the only one that is conserved on the Y chromosome of all therian mammals is RBM (RNA-binding motif gene, Y chromosome).

The X--a sexy chromosome.

There is new and convincing evidence that the mammalian X chromosome, as well as the Y chromosome, contains an atypically high proportion of genes involved in sex and reproduction (SRR genes). Here we consider alternative explanations for this concentration. One possibility is that a particularly well-endowed autosome was "chosen" for a career as a sex chromosome. Alternatively, the high concentration of SRR genes may have resulted from the accumulation of these genes on the X after the degradation of the Y, either by transposition of autosomal SRR genes to a "selfish X", or by acquisition of SRR functions by widely expressed genes on the X. We suggest experiments to distinguish these possibilities, and speculate on the implications of gathering evidence that genes with other functions, too, are not distributed uniformly over the genome.

The human Y chromosome derives largely from a single autosomal region added to the sex chromosomes 80-130 million years ago.

Mapping of human X-borne genes in distantly related mammals has defined a conserved region shared by the X chromosome in all three extant mammalian groups, plus a region that was recently added to the eutherian X but is still autosomal in marsupials and monotremes. Using comparative mapping of human Y-borne genes, we now directly show that the eutherian Y is also composed of a conserved and an added region which contains most of the ubiquitously expressed Y-borne genes. Little of the ancient conserved region remains, and the human Y chromosome is largely derived from the added region.

De novo insertion of an intron into the mammalian sex determining gene, SRY.

Two theories have been proposed to explain the evolution of introns within eukaryotic genes. The introns early theory, or "exon theory of genes," proposes that introns are ancient and that recombination within introns provided new exon structure, and thus new genes. The introns late theory, or "insertional theory of introns," proposes that ancient genes existed as uninterrupted exons and that introns have been introduced during the course of evolution. There is still controversy as to how intron-exon structure evolved and whether the majority of introns are ancient or novel. Although there is extensive evidence in support of the introns early theory, phylogenetic comparisons of several genes indicate recent gain and loss of introns within these genes. However, no example has been shown of a protein coding gene, intronless in its ancestral form, which has acquired an intron in a derived form. The mammalian sex determining gene, SRY, is intronless in all mammals studied to date, as is the gene from which it recently evolved. However, we report here comparisons of genomic and cDNA sequences that now provide evidence of a de novo insertion of an intron into the SRY gene of dasyurid marsupials. This recently (approximately 45 million years ago) inserted sequence is not homologous with known transposable elements. Our data demonstrate that introns may be inserted as spliced units within a developmentally crucial gene without disrupting its function.

Chromosome painting in marsupials: genome conservation in the kangaroo family.

In order to deduce the ancestral genome arrangement in the karyotypically diverse marsupial family Macropodidae, and to assess chromosome change in this family, chromosome-specific paints from the tammar wallaby (2n = 16) were hybridized to metaphase spreads from the two species proposed to represent the 2n = 22 ancestral karyotype, as well as species with derived 2n = 20 and 2n = 14 karyotypes. Identical patterns were observed in the two 2n = 22 species, from which the rearrangements to form the three derived karyotypes may be easily deduced to be 1, 3 and 4 different fusions, respectively. The identical Thylogale and Dorcopsis genomes may both be used to represent the pleisiomorphic macropodid chromosome complement. Variation in the X chromosome was also investigated by hybridizing an X-Y shared tammar wallaby 12-kb repeat element to chromosomes from the other four macropodid species, finding that it hybridized only to the most closely related species, and therefore is of recent origin.

Expression and conservation of processed copies of the RBMX gene.

RBMX and RBMY are members of an ancient pair of genes located on the sex chromosomes that encode RNA-binding proteins involved in splicing. These genes have differentiated and evolved separately on the X and Y Chromosomes. RBMY has acquired a testis-specific function, whereas, as shown here, RBMX is ubiquitously expressed and is subject to X inactivation. We have also found that multiple processed copies of RBMX are present in the human genome. RBMX-like sequences (RBMXLs) located on human Chrs 1, 4, 6, 9 (9p13 and 9p24), 11, 20, and X lack introns and thus probably result from retroposition events. We found RBMXLs to be conserved in primates and great apes at corresponding chromosomal locations, indicating that they arose prior to the divergence of human. Some of the RBMXLs show insertions, deletions, and stop codons, which would probably result in nonfunctional proteins. The RBMXL on Chr 20 is deleted in some individuals. Two of the largely intact RBMXLs, located on Chrs 1 and 9p13, are expressed in different tissues and may encode novel proteins involved in splicing in a tissue-specific manner. The RBMXL located at 9p13 is specifically expressed in testis, and to a lesser extent in brain, and may therefore play a role in testis function. This autosomal, testis-specific copy of RBMX could potentially compensate for RBMX that is presumably inactivated in male germ cells, in a manner analogous to autosomal retroposed copies of other X-linked genes.