Genomic organization of a cDNA (QM) demonstrating an altered mRNA level in nontumorigenic Wilms' microcell hybrid cells and its localization to Xq28.

Using a cosmid clone derived from human Xq28 as a probe which shows cross-species homology, we isolated cDNA clones and the nucleotide sequence analysis of the cDNA revealed that the cDNA is identical to QM cDNA. The QM cDNA has recently been reported as a cDNA with down-regulation in tumorigenic Wilms' tumor microcell hybrid. Comparison of the nucleotide sequences of the cDNA with those of the genomic DNA allowed us to determine the genomic organization of the QM gene. The QM gene consists of at least 7 exons and is located at Xq28. Southern blot analysis of a somatic cell hybrid panel indicates that the QM genes are scattered at least to chromosome 2, 3, 6, 14, 16, and possibly to other chromosomes. Northern blot analysis demonstrated the QM gene is expressed in all the examined adult human tissues as well as cell lines including HeLa cells, fibroblasts, and somatic cell hybrids with increased expression in liver, spleen, testis, and adrenal gland. The results suggest that the QM gene belongs to a new multi-gene family with yet undetermined function.     

COMMENTS

Human spermatogenesis is regulated by a network of genes located on autosomes and on sex chromosomes, but especially on the Y chromosome. Most results concerning the germ cell function of the Y genes were obtained by genomic breakpoint mapping studies of the Y chromosome of infertile patients. Although this approach has the benefit of focussing on those Y regions that contain most likely the Y genes of functional importance, its major drawback is the fact that fertile control samples were often missing. In fertile men, molecular and cytogenetic analyses of the Y chromosome has revealed highly polymorphic chromatin domains especially in the distal euchromatic part (Yq11.23) and in the heterochromatic part (Yq12) of the long arm. In sterile patients cytogenetic analyses mapped microscopically visible Y deletions and rearrangements in the same polymorphic Y regions. The presence of a Y chromosomal spermatogenesis locus was postulated to be located in Yq11.23 and designated as AZoospermia Factor (ZF). More recently, molecular deletion mapping in Yq11 has revealed a series of microdeletions that could be mapped to one of three different AZF loci: AZFa in proximal Yq11 (Yq11.21), AZFb and AZFc in two non‐overlapping Y‐regions in distal Yq11 (Yq11.23). This view was supported by the observation that AZFa and AZFb microdeletions were associated with a specific pathology in the patients' testis tissue. Only AZFc deletions were associated with a variable testicular pathology and in rare cases AZFc deletions were even found inherited from father to son. However, AZFc deletions were found with a frequency of 10–20% only in infertile men and most of them were proved to be “de novo”, i.e. the AZFc deletion was restricted to the patient's Y chromosome. Based mainly on positional cloning experiments of testis cDNA clones and on the Y chromosomal sequence now published in GenBank, a first blueprint for the putative gene content of the AZFc locus can now be given and the gene location compared to the polymorphic DNA domains. This artwork of repetitive sequence blocks called AZFc amplicons raised the question whether the AZFc chromatin is still part of the heterochromatic domain of the Y long arm well known for its polymorphic extensions or is decondensed and part of the Yq11.23 euchromatin? We discuss also the polymorphic DAZ gene family and disclose putative origins of its molecular heterogeneity in fertile and infertile men recently identified by the analyses of Single Nucleotide Variants (SNVs) in this AZFc gene locus.     

Polymorphic DAZ gene family in polymorphic structure of AZFc locus: Artwork or functional for human spermatogenesis?

Human spermatogenesis is regulated by a network of genes located on autosomes and on sex chromosomes, but especially on the Y chromosome. Most results concerning the germ cell function of the Y genes were obtained by genomic breakpoint mapping studies of the Y chromosome of infertile patients. Although this approach has the benefit of focussing on those Y regions that contain most likely the Y genes of functional importance, its major drawback is the fact that fertile control samples were often missing. In fertile men, molecular and cytogenetic analyses of the Y chromosome has revealed highly polymorphic chromatin domains especially in the distal euchromatic part (Yq11.23) and in the heterochromatic part (Yq12) of the long arm. In sterile patients cytogenetic analyses mapped microscopically visible Y deletions and rearrangements in the same polymorphic Y regions. The presence of a Y chromosomal spermatogenesis locus was postulated to be located in Yq11.23 and designated as AZoospermia Factor (ZF). More recently, molecular deletion mapping in Yq11 has revealed a series of microdeletions that could be mapped to one of three different AZF loci: AZFa in proximal Yq11 (Yq11.21), AZFb and AZFc in two non-overlapping Y-regions in distal Yq11 (Yq11.23). This view was supported by the observation that AZFa and AZFb microdeletions were associated with a specific pathology in the patients' testis tissue. Only AZFc deletions were associated with a variable testicular pathology and in rare cases AZFc deletions were even found inherited from father to son. However, AZFc deletions were found with a frequency of 10-20% only in infertile men and most of them were proved to be "de novo", i.e. the AZFc deletion was restricted to the patient's Y chromosome. Based mainly on positional cloning experiments of testis cDNA clones and on the Y chromosomal sequence now published in GenBank, a first blueprint for the putative gene content of the AZFc locus can now be given and the gene location compared to the polymorphic DNA domains. This artwork of repetitive sequence blocks called AZFc amplicons raised the question whether the AZFc chromatin is still part of the heterochromatic domain of the Y long arm well known for its polymorphic extensions or is decondensed and part of the Yq11.23 euchromatin? We discuss also the polymorphic DAZ gene family and disclose putative origins of its molecular heterogeneity in fertile and infertile men recently identified by the analyses of Single Nucleotide Variants (SNVs) in this AZFc gene locus.     

Characterization of the genomic organization, localization and expression of four PRY genes (PRY1, PRY2, PRY3 and PRY4).

PRY (PTP-BL related on the Y chromosome) has been proposed as a candidate spermatogenesis gene. We report the characterization of the genomic structure, the number of copies on the Y chromosome and the expression of the gene. By comparison of the cDNA sequence with the genomic sequence, five exons were identified. Analysis of GenBank-derived clones on the Y chromosome revealed the presence of two full-length copies in azoospermia factor region b (AZFb) (PRY1 and PRY2) and two shorter versions of the PRY gene containing exons 3, 4 and 5 in AZFc (PRY3 and PRY4). A clone containing sequences homologous to exons 3, 4 and 5 is located in area 5L (between AZFa and AZFb), a clone containing a sequence homologous to exon 5 is located in area 5M (in AZFb) and a clone containing a fragment homologous to exon 3 is located in 6F. A repeat structure of exons 1 and 2 is present on the short arm of the Y chromosome as well as on the long arm. PRY1 and PRY2, two gene copies that are located in AZFb, a region often deleted in patients with severe male infertility, were shown to be expressed in the testis. PRY may therefore play an important role in spermatogenesis.     

The human Y chromosome genes BPY2, CDY1 and DAZ are not essential for sustained fertility

Deletions of the AZFc interval of the human Y chromosome are found in >5% of male patients with idiopathic infertility and are associated with a severely reduced sperm count. The most common deletion type is large (>1 Mb) and removes members of the Y-borne testis-specific gene families of BPY2, CDY1, DAZ, PRY, RBMY2 and TTY2, which are candidate AZF genes. Four exceptional individuals who have transmitted a large AZFc deletion naturally to their infertile sons have, however, been described. In three cases, transmission was to an only son, but in the fourth case a Y chromosome, shown to be deleted for all copies of DAZ, was transmitted from a father to his four infertile sons. Here we present a second family of this latter type and demonstrate that an AZFc-deleted Y chromosome lacking not only DAZ, but also BPY2 and CDY1, has been transmitted from a father to his three infertile sons. Polymerase chain reaction (PCR) and Southern blot analyses revealed no difference in the size of the AZFc deletion in the father and his sons. We propose that the father carries rare alleles of autosomal or X-linked loci which suppress the infertility that is frequently associated with the absence of AZFc.     

The human autosomal gene DAZLA: testis specificity and a candidate for male infertility

The DAZ (Deleted in AZoospermia) and DAZLA (DAZ-like autosomal) genes may be determinants of male infertility. The DAZ gene on the long arm of the human Y chromosome is a strong candidate for the 'azoospermia factor' (AZF). Its role in spermatogenesis is supported by its exclusive expression in testis, its deletion in a high percentage of males with azoospermia or severe oligospermia, and its homology with a Drosophila male infertility gene boule. No DAZ homologous sequences have been found on the mouse Y chromosome. Instead, a Dazla gene was isolated from mouse chromosome 17 and has been considered to be a murine homologue of DAZ. However, the homology between human DAZ and mouse Dazla is not strong, and Dazla contains only one of the seven DAZ repeats found in DAZ. We report the isolation of the human DAZLA gene by screening a human testis cDNA library with a DAZ cDNA clone. DAZLA encodes only one DAZ repeat and shares high homology with the mouse Dazla, indicating that these two genes are homologues. Using a panel of rodent-human somatic cell lines and fluorescence in situ hybridization, the DAZLA gene was mapped to 3p24, a region not known to share homology with mouse chromosome 17. The DAZLA gene may be involved in some familial cases of autosomal recessive male infertility.      

A SPGY copy homologous to the mouse gene Dazla and the Drosophila gene boule is autosomal and expressed only in the human male gonad

We have isolated a series of human testis poly(A) cDNA clones by cross- hybridization to SPGY1, a Y gene homologous to DAZ. Their sequence analysis revealed an identical nucleotide composition in different 'full-length' clones, suggesting that all were encoded by the same gene. We mapped this gene to the short arm of chromosome 3 and designated it SPGYLA (SPGY like autosomal). Comparison of the SPGYLA cDNA sequence with the cDNA sequences of DAZ and SPGY1 revealed two prominent differences. The tandem repetitive structure of 72 bp sequence units (DAZ repeats) is absent. SPGYLA contains only one 72 bp sequence unit. Downstream of it, a specific 130 bp sequence domain is present which is absent in DAZ and SPGY1 but present in the mouse gene Dazla and in the Drosophila gene boule. SPGYLA encodes an RNA binding protein expressed only in the human male gonad. The data presented give strong evidence that not DAZ but SPGYLA is the functional human homologue of Dazla and boule.      

The Y chromosome of the Okinawa spiny rat, Tokudaia muenninki, was rescued through fusion with an autosome

The genus Tokudaia comprises three species, two of which have lost their Y chromosome and have an XO/XO sex chromosome constitution. Although Tokudaia muenninki (Okinawa spiny rat) retains the Y chromosome, both sex chromosomes are unusually large. We conducted a molecular cytogenetic analysis to characterize the sex chromosomes of T. muenninki. Using cross-species fluorescence in situ hybridization (Zoo-FISH), we found that both short arms of the T. muenninki sex chromosomes were painted by probes from mouse chromosomes 11 and 16. Comparative genomic hybridization analysis was unable to detect sex-specific regions in the sex chromosomes because both sex probes highlighted the large heterochromatic blocks on the Y chromosome as well as five autosomal pairs. We then performed comparative FISH mapping using 29 mouse complementary DNA (cDNA) clones of the 22 X-linked genes and the seven genes linked to mouse chromosome 11 (whose homologue had fused to the sex chromosomes), and FISH mapping using two T. muenninki cDNA clones of the Y-linked genes. This analysis revealed that the ancestral gene order on the long arm of the X chromosome and the centromeric region of the short arm of the Y chromosome were conserved. Whereas six of the mouse chromosome 11 genes were also mapped to Xp and Yp, in addition, one gene, CBX2, was also mapped to Xp, Yp, and chromosome 14 in T. muenninki. CBX2 is the candidate gene for the novel sex determination system in the two other species of Tokudaia, which lack a Y chromosome and SRY gene. Overall, these results indicated that the Y chromosome of T. muenninki avoided a loss event, which occurred in an ancestral lineage of T. osimensis and T. tokunoshimensis, through fusion with an autosome. Despite retaining the Y chromosome, sex determination in T. muenninki might not follow the usual mammalian pattern and deserves further investigation.     

<Emphasis Type="Italic">AZFc</Emphasis> region of the Y chromosome shows singular structural organization

Owing to clonal inheritance, haploid status and lack of recombination, structural polymorphism in the human Y chromosome is more prevalent than that in the remaining parts of the genome. We studied structural organization of the AZFc region, assessed microdeletions therein and studied copy number variation (CNV) of several candidate genes in 750 Indian males. FISH mapping of 13 Y-specific BAC/cosmid clones uncovered a hitherto unreported AZFc configuration showing inter-DAZ gene sequence onto the Yp instead of Yq region. Such inter-DAZ gene arrangements were also detected in five German males (European Y). In 40–50% males, partial u3 and one of the green amplicons, g1, g2 or g3 was present on the Yp in addition to Yq, suggesting an alteration in the IR3 region. Among other AZFc candidates, complete TTY3 and partial CDY1 BAC sequences were detected on the proximal 5p and distal 15q regions, respectively, in both the sexes. However, primers deduced from these clones showed male specific amplification of TTY3 and CDY1 exons suggesting (re)organization of their flanking sequences between Y and autosomes. Importantly, ∼5% males showed CNV of various Y-linked genes, and ∼3%, random microdeletions across the AZF region. Present study demonstrates hitherto unreported singular structural organization with respect to DAZ, TTY3 and CDY1 genes highlighting organizational complexities of the human Y chromosome in the global context.     

Identification of testis development and spermatogenesis-related genes in human and mouse testes using cDNA arrays

We have constructed cDNA microarrays from the human testis large insert cDNA library, containing 9216 genes, together with several housekeeping genes. The cDNA microarrays were used to identify gene expression differences between human fetal and adult testes. Of >8700 hybridized clones, 731 exhibited significant differential expression characteristics. About 7500 genes were identified when the same cDNA microarrays were used for hybridization with cDNA probes from mouse testis, with 256 genes having significant differential expression between the age of 1-4 weeks. Among these genes, 101 were identified as critically related to testis development and possibly to spermatogenesis since they were found in both human and mouse testes, and expressed differentially at different stages of testis development. Of the 101 development-related genes, 59 full-length cDNAs have been sequenced previously, while the full-length cDNAs of the other 42 genes have not been published. We have obtained 11 full-length sequences of the 42 genes and deposited them in the GenBank. The conserved testis development-related genes found in both human and mouse testes may include genes that are likely to be involved in testicular functions, especially spermatogenesis, thus providing a basis for further functional characterization of the genes in mouse models.     

A deletion of a novel heat shock gene on the Y chromosome associated with azoospermia

Deletions of the Y chromosome are a significant cause of spermatogenic failure. Three major deletion intervals have been defined and termed AZFa, AZFb and AZFc. Here, we report an unusual case of a proximal AZFb deletion that includes the Y chromosome palindromic sequence P4 and a novel heat shock factor (HSFY). This deletion neither include the genes EIF1AY, RPS4Y2 nor copies of the RBMY1 genes. The individual presented with idiopathic azoospermia. We propose that deletions of the testis-specific HSFY gene family may be a cause of unexplained cases of idiopathic male infertility. This deletion would not have been detected using current protocols for Y chromosome microdeletion screens, therefore we recommend that current screening protocols be extended to include this region and other palindrome sequences that contain genes expressed specifically in the testis.     

The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5' expressed sequence tags (5' expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ~40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5' ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.     

Sequence homology of surface membrane proteins of Babesia rodhaini

Four surface membrane proteins of Babesia rodhaini have previously been shown to induce a degree of protective immunity, and to carry both unique and cross-reactive determinants. cDNA clones for two of the genes coding for these proteins have been isolated and used as probes to isolate a single large genomic DNA fragment which contained all four genes. DNA sequence of two of the genes and their predicted amino acid sequences confirmed that the proteins had hydrophobic sequences at their N- and C-termini, an observation consistent with their proposed cell surface location. Homologies in both amino acid and nucleotide sequences were found at the 3'and at the 5' ends, but considerable sequence variations existed elsewhere in the genes and their products. The genes coding for these four proteins were tandemly arranged along a single relatively short length of chromosome, and such structures, because of their sequence homologies, probably could have arisen by gene duplication. The extensive variation suggested that there may be a functional need for these proteins to be different or capable of varying, although computer analysis implied that the extent of this variation may be constrained by structural requirements. This variation could be indicative of a role for these proteins in the host-parasite relationship or immune evasion.