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.

Interspecies comparative genome hybridization and interspecies representational difference analysis reveal gross DNA differences between humans and great apes.

Comparative chromosome G-/R-banding, comparative gene mapping and chromosome painting techniques have demonstrated that only few chromosomal rearrangements occurred during great ape and human evolution. Interspecies comparative genome hybridization (CGH), used here in this study, between human, gorilla and pygmy chimpanzee revealed species-specific regions in all three species. In contrast to the human, a far more complex distribution of species-specific blocks was detected with CGH in gorilla and pygmy chimpanzee. Most of these blocks coincide with already described heterochromatic regions on gorilla and chimpanzee chromosomes. Representational difference analysis (RDA) was used to subtract the complex genome of gorilla against human in order to enrich gorilla-specific DNA sequences. Gorilla-specific clones isolated with this technique revealed a 32-bp repeat unit. These clones were mapped by fluorescence in situ hybridization (FISH) to the telomeric regions of gorilla chromosomes that had been shown by interspecies CGH to contain species-specific sequences.

De novo inverted duplication of chromosome 7(q21.3-->q35): cytogenetic diagnosis confirmed by FISH analysis.

We report on a newborn female patient with a de novo pure partial duplication of 7q. The clinical features are compared with those of 19 cases from the literature with pure partial duplication of different segments of 7q. Conventional cytogenetic investigation led to the diagnosis of duplication of bands q21.3 to q35. This was confirmed by chromosome painting and by fluorescence in situ hybridization with different YAC probes from the duplicated region.

CSF2RA, ANT3, and STS are autosomal in marsupials: implications for the origin of the pseudoautosomal region of mammalian sex chromosomes.

The X and Y Chromosomes (Chrs) of eutherian ("placental") mammals share a pseudo-autosomal region (PAR) that pairs and recombines at meiosis. In humans and other eutherians, the PAR contains several active genes and has also been thought to be critical for pairing and fertility. In order to explore the origin of the PAR, we cloned and mapped three human or mouse pseudoautosomal genes in marsupials, a group of mammals that diverged from eutherians about 130 (MYrBP). All three genes were autosomal in marsupials, and two co-localized with other human Xp genes on an autosome. This implies that the human PAR, like most of human Xp, represents a relic of an autosomal region added to both X and Y Chrs between 80 and 150 MYrBP.

The minimal mammalian Y chromosome - the marsupial Y as a model system.

The mammalian X and Y chromosomes are very different in size and gene content. The Y chromosome is much smaller than the X and consists largely of highly repeated non-coding DNA, containing few active genes. The 65-Mb human Y is homologous to the X over two small pseudoautosomal regions which together contain 13 active genes. The heterochromatic distal half of the human Yq is entirely composed of highly repeated non-coding DNA, and even the euchromatic portion of the differential region is largely composed of non-coding repeated sequences, amongst which about 30 active genes are located. The basic marsupial Y chromosome (about 10 Mb) is much smaller than that of humans or other eutherian mammals. It appears to include no PAR, since it does not undergo homologous pairing, synaptonemal complex formation or recombination with the X. We show here that the tiny dunnart Y chromosome does not share cytogenetically detectable sequences with any other chromosome, suggesting that it contains many fewer repetitive DNA sequences than the human or mouse Y chromosomes. However, it shares several genes with the human and/or mouse Y chromosome, including the sex determining gene SRY and the candidate spermatogenesis gene RBMY, implying that the marsupial and eutherian Y are monophyletic. This minimal mammalian Y chromosome might provide a good model Y in which to hunt for new mammalian Y specific genes.

Comparative cytogenetic studies in tree shrews (Tupaia).

Through use of BrdU replication, RBA-banded karyotypes of Tupaia belangeri, T. chinensis, and T. glis were obtained. A chromosome number of 2n = 62 for T. belangeri is described here for the first time and is confirmed for T. chinensis. All chromosomes between these two phenotypically different species appear to have identical RBA banding patterns; in addition, there is no difference between T. belangeri and T. chinensis in the number and position of nucleolus organizer regions (NORs). The reduced chromosome number of 2n = 60 in T. glis can be explained by a Robertsonian translocation between two acrocentric chromosome pairs, Nos. 10 and 13, of T. belangeri and/or T. chinensis, resulting in the metacentric chromosome pair 1 of T. glis. Furthermore, two chromosome pairs each of T. glis and T. belangeri and/or T. chinensis are not homoeologous, as judged by their RBA patterns. Differences were also found in the number and position of NORs; whereas T. glis displays eight positively stained NORs after AgNO3 staining, there are only four silver-stained NORs in both T. belangeri and T. chinensis. The possibility of geographical isolation as an explanation for the lack of chromosomal differentiation between T. belangeri and T. chinensis is discussed.

Gene dosage in the evolution and function of mammalian sex chromosomes.

Ohno's early suggestions about the origin of sex chromosomes and the consequences of alterations of dosage of X and Y genes have provided an important framework for understanding sex chromosome organization, function and evolution. Here we review evidence that heteromorphic sex chromosomes evolved from an autosomal pair, and that one of the consequences of X-Y differentiation is the evolution of dosage compensation by X inactivation and upregulation of the active X, which in turn, has selected for a highly conserved X chromosome.

The origin and evolution of the pseudoautosomal regions of human sex chromosomes.

The human X and Y chromosomes share two homologous pseudoautosomal regions (PARs) which pair and recombine at meiosis. PAR1 lies at the tips of the short arms, and the smaller PAR2 at the tips of the long arms. PAR1 contains several active genes, and has been thought to be critical for pairing and fertility. The inconsistent gene content of the PARs between different species of eutherian ('placental') mammals suggests that gene content is immaterial to function, and the failure to detect a PAR at all in some rodents and all marsupials implies that homologous pairing is not universally essential for fertility. The autosomal localization of marsupial homologues of human PAR1 genes and their co-localization with human Xp22 genes implies that the human PAR1 represents a relic of part of an autosomal region added to both X and Y chromosomes between 80 and 130 MYrBP. The same argument may be made for part of PAR2. Independent additions to the sex chromosomes of macropodid marsupials and monotremes can also be inferred from comparative mapping. We conclude that the PARs are relics of differential additions, loss, rearrangement and degradation of the Y chromosome in different mammalian lineages.

Species-specific evolution of repeated DNA sequences in great apes.

DNA sequencing reveals that the genomes of the human, gorilla and chimpanzee share more than 98% homology. Comparative chromosome painting and gene mapping have demonstrated that only a few rearrangements of a putative ancestral mammalian genome occurred during great ape and human evolution. However, interspecies representational difference analysis (RDA) of the gorilla between human and gorilla revealed gorilla-specific DNA sequences. Cloning and sequencing of gorilla-specific DNA sequences indicate that there are repetitive elements. Gorilla-specific DNA sequences were mapped by fluorescence in-situ hybridization (FISH) to the subcentromeric/centromeric regions of three pairs of gorilla submetacentric chromosomes. These sequences could represent either ancient sequences that got lost in other species, such as human and orang-utan, or, more likely, recent sequences which evolved or originated specifically in the gorilla genome.

Comparative mapping of SRY in the great apes.

Cytogenetic studies of the primate Y chromosomes have suggested that extensive rearrangements have occurred during evolution of the great apes. We have used in situ hybridization to define these rearrangements at the molecular level. pHU-14, a probe including sequences from the sex determining gene SRY, hybridizes close to the early replicating pseudoautosomal segment in a telomeric or subtelomeric position of the Y chromosomes of all great apes. The low copy repeat detected by the probe Fr35-II is obviously included in Y chromosomal rearrangements during hominid evolution. These results, combined with previous studies, suggest that the Y chromosome in great apes has a conserved region including the pseudoautosomal region and the testis-determining region. The rest of the Y chromosome has undergone several rearrangements in the different great apes.

ANT3 and STS are autosomal in prosimian lemurs: implications for the evolution of the pseudoautosomal region.

Comparative in situ hybridization in various primate species has revealed a pseudoautosomal location for the human ANT3 gene and an X-specific location for the steroid sulfatase (STS) gene throughout the higher primate species up to the New World monkeys. However, ANT3 and STS map together on an autosome of two prosimian species of the genus Lemur and Eulemur. These results suggest an autosome-to-X/Y translocation after the simians radiated from the prosimians, resulting in a pseudoautosomal location of genes such as ANT3 and STS. In simian primates, STS then became X-specific by a pericentric inversion in the Y chromosome followed by mutational inactivation of the Y allele.

An X-Y homologous pairing segment in tree shrews (Tupaia).

High-resolution early replication banding of tupaia metaphase chromosomes revealed a synchronous early replicating segment in the short-arm telomeric regions of the active and inactive X chromosomes and in the long-arm telomeric region of the Y chromosome. Hybridization with the human-derived pseudoautosomal probe 113F (STIR) showed that this repeat is conserved and specifically localized within these synchronously early replicating segments of the X short arm and the Y long arm of all three tupaia species (Tupaia belangeri, T. chinensis, and T. glis) investigated. Moreover, meiotic studies demonstrated that a synaptonemal complex is formed at one telomeric end of the XY bivalent during the pachytene stage of meiosis in a male T. glis specimen. Thus, apart from the mouse, the tupaias are the first nonprimate mammals for which cytogenetic and molecular evidence is provided that their highly heteromorphic X and Y chromosomes share a conserved homologous segment in the telomeric position, a location that is compatible with pairing and crossing-over in male meiosis. Taken together, these observations strongly, albeit indirectly, suggest that this chromosome segment at the tip of a sex-chromosome arm might behave pseudoautosomally.

The candidate sex-reversing DAX1 gene is autosomal in marsupials: implications for the evolution of sex determination in mammals.

The human X-linked DAX1 gene was cloned from the region of the short arm of the human X found in duplicate in sex-reversed Xdup Y females (E. Zanaria et al., 1994, Nature 372: 635-641). DAX1 is suggested to be required for ovarian differentiation and to play an important role in mammalian sex determination or differentiation pathways. Its proposed dose-dependent effect on sexual development suggests that DAX1 could represent an evolutionary link with an ancestral sex-determining mechanism that depended on the dosage of an X-linked gene. Furthermore, DAX1 could also represent the putative X-linked switch gene, which independently controls sexual dimorphisms in marsupial mammals in an X-dose-dependent manner (D.W. Cooper et al., 1993, Semin. Dev. 4: 117-128). If DAX1 has a present role in marsupial sexual differentiation or had an ancestral role in mammalian sex determination, it would be expected to lie on the marsupial X chromosome, despite the autosomal localization of other human Xp genes. We therefore cloned and mapped the DAX1 gene in the tammar wallaby (Macropus eugenii). DAX1 was located on wallaby chromosome 5p near other human Xp genes, indicating that it was originally autosomal and that it is not involved in X-linked dose-dependent sex determination in an ancestral mammal nor in marsupial sexual differentiation.

Shared synteny between human chromosome 10 and chromosome 1 of the marsupial tammar wallaby, Macropus eugenii.

Marsupial homologs of the human chromosome 10 loci IL2RA, HK1, and PLAU have been cloned and mapped by fluorescence in situ hybridization to chromosome 1q of the tammar wallaby, Macropus eugenii. Relative distance measurements of the hybridization signals on M. eugenii chromosome 1 show that marsupial homologs of human (HSA) 10p IL2RA and 10q HK1/PLAU flank the marsupial homologs of the human 5q gene IL5 and the human 15q imprinted genes SNRPN and ZNF127. The shared synteny, therefore, does not necessarily mean that HSA 10 represents an ancestral grouping; rather, it suggests that HSA 10p and HSA 10q represent two different ancestral mammalian units which fused directly in primates and were incorporated independently into two different regions of the same chromosome in marsupials.

Mapping the distribution of the telomeric sequence (T2AG3)n in the Macropodoidea (Marsupialia), by fluorescence in situ hybridization. I. The swamp wallaby, Wallabia bicolor.

Thylogale spp. (pademelons) retain the plesiomorphic (ancestral) 2n = 22 karyotype for the marsupial family Macropodidae (kangaroos and wallabies). The swamp wallaby, Wallabia bicolor, has the most derived macropodid karyotype with the lowest chromosome number (2n = 10 female, 11 male), and a multiple sex chromosome system (XX female, XY1Y2 male). All but one of the W. bicolor chromosomes are fusion chromosomes. Two of these chromosomes, the X chromosome and chromosome 1, are composed of three plesiomorphic Thylogale-like chromosomes. The distribution of the vertebrate telomeric sequence (T2AG3)n was examined by fluorescence in situ hybridization (FISH) in both species and a 'map' of non-telomeric (T2AG3)n sites on W. bicolor chromosomes relative to Thylogale chromosomes was constructed. (T2AG3)n signals were observed at six fusion sites in the four fusions chromosomes examined, indicating that the (T2AG3)n sequence is consistently retained during fusions. The distribution of the interstitial signals on the long arm of chromosome 1 of W. bicolor and the X chromosome suggests how a combination of inversions, fusions and centromeric transpositions have resulted in interstitial telomeric sequence.

Isolation and characterization of marsupial IL5 genes.

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.

The human/mouse imprinted genes IGF2, H19, SNRPN and ZNF127 map to two conserved autosomal clusters in a marsupial.

The four genes IGF2, H19, SNRPN and ZNF127 are imprinted in mouse and human. IGF2 and H19 form one conserved cluster on the distal part of mouse chromosome 7 and human chromosome 11p15.5, whereas SNRPN and ZNF127 form another on the middle of mouse chromosome 7 and on human chromosome 15q11-13. We have explored the evolution of these imprinted regions by cloning and mapping IGF2, H19, SNRPN and ZNF127 homeologues in marsupials. Specifically, we wished to determine whether the arrangements were shared in eutherian and marsupial mammals, and to determine whether they lay on autosomes, or on the X, as might be predicted by the hypothesis that imprinting evolved from X inactivation. Using fluorescence in situ hybridization, we localized the marsupial homeologues of IGF2 and H19 to the distal part of tammar wallaby chromosome 2p and the marsupial homeologues of SNRPN and ZNF127 to the middle of chromosome 1q. Thus, these genes were originally organized in two separate autosomal clusters in the therian ancestor 180 million years ago, the conservation of which may suggest a functional relationship. The autosomal location of these clusters does not suggest a recent evolutionary relationship between imprinting and X chromosome inactivation.

A retrospective CISS hybridization analysis of a case with de novo translocation t(18;22) resulting in an 18p- syndrome.

An unbalanced de novo translocation t(18;22) leading to a severely malformed liveborn girl with 18p- syndrome is described. Using the chromosomal in situ suppression (CISS) hybridization technique on 4-year-old G-banded chromosome preparations, it could be demonstrated that the translocation chromosome is composed of the long arm including the centromere of a chromosome 22 and the long arm of a chromosome 18. Consequently, the patient described here has lost the short arm including the centromere of chromosome 18. The possibility of restudying cytogenetically unsolved cases in clinical cytogenetics using older G-banded chromosome preparations with the fluorescence in situ hybridization techniques is pointed out.

Inverted and satellited Y chromosome in the orangutan (Pongo pygmaeus).

An inverted and satellited Y chromosome of almost acrocentric appearance was detected in seven of 14 male orangutans. In the remaining seven animals a submetacentric Y chromosome without NORs occurred. The high frequency with which the satellited Y chromosomes were associated with acrocentric autosomes and the positive AgNO3-staining of their satellite stalks clearly indicate the active state of the NOR on the Y chromosomes. DNA fingerprinting in two orangutan families showed that the inverted and satellited Y chromosomes in carrier orangutan males do not interfere with normal fertility. Within our sample of male orangutans studied, the inverted and satellited Y chromosome is restricted to Sumatran animals; all Bornean specimens possessed the submetacentric Y chromosome. The question arises whether these two kinds of Y chromosome differ constitutively between the Pongo pygmaeus subpopulations.