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The human DAZ (deleted in azoospermia) gene family on the Y chromosome and an autosomal DAZ-like gene, DAZL1, encode RNA-binding proteins that are expressed exclusively in germ cells. Their role in spermatogenesis is supported by their homology with a Drosophila male infertility gene boule and sterility of Daz11 knock-out mice. While all mammals contain a DAZL1 homologue on their autosomes, DAZ homologues are present only on the Y chromosomes of great apes and Old World monkeys. The DAZ and DAZL1 proteins differ in the copy numbers of a DAZ repeat and the C-terminal sequences. We studied the interaction of DAZ and DAZL1 with other proteins as an approach to investigate functional similarity between these two proteins. Using DAZ as bait in a yeast two-hybrid system, we isolated two DAZAP (DAZ-associated protein) genes. DAZAP1 encodes a novel RNA-binding protein that is expressed most abundantly in the testis, and DAZAP2 encodes a ubiquitously expressed protein with no recognizable functional motif. DAZAP1 and DAZAP2 bind similarly to both DAZ and DAZL1 through the DAZ repeats. The DAZAP genes were mapped to chromosomal regions 19p13.3 and 2q33-q34, respectively, where no genetic diseases affecting spermatogenesis are known to map.

A gene, GS2 (DXS1283E), was isolated from a CpG island located approximately midway between the steroid sulfatase (STS) and the Kallmann syndrome (KAL1) loci on the distal short arm of the human X chromosome. DNA sequencing of a GS2 cDNA clone revealed an open reading frame for a basic protein of 253 amino acid residues. A polymorphic CT dinucleotide repeat was found in the 3' untranslated region. The GS2 gene is expressed in all human tissues examined, including heart, brain, placenta, lung, liver, muscle, kidney, pancreas, and spleen. Several GS2 transcripts, ranging in size from 1.1 to 5.8 kb, were found among different tissues, suggesting tissue-specific processing of the GS2 transcript. Characterization of GS2 genomic clones revealed that the gene consists of 7 exons spanning over 26 kb, with a CpG island located in the first intron. The GS2 gene is transcribed toward Xpter, in the same direction as KAL1 but opposite to that of STS.

The human Y chromosome is unique in that it does not engage in pairing and crossing over during meiosis for most of its length. Y chromosome microdeletions, a frequent finding in infertile men, thus occur through intrachromosomal recombination, either within a single chromatid or between sister chromatids. A recently identified polymorphism associated with increased risk for spermatogenic failure, the gr/gr deletion, removes two of the four Deleted in Azoospermia (DAZ) genes in the AZFc region on the Y-chromosome long arm. We found the likely reciprocal duplication product of gr/gr deletion in 5 (6%) of 82 males using a novel DNA-blot hybridization strategy and confirmed the presence of six DAZ genes in three cases by FISH analysis. Additional polymorphisms identified within the DAZ repeat regions of the DAZ genes indicate that sister chromatid exchange plays a significant role in the genesis of deletions, duplications, and polymorphisms of the Y chromosome.

Deletion interval 6 (DI6) of the human Y chromosome, located at the distal end of the long arm euchromatic region, is required for normal spermatogenesis. About 10% of males with idiopathic azoospermia or oligospermia have microdeletions in this region. Six gene families, including RBMY (RNA binding motif, Y chromosome), DAZ (deleted in azoospermia), and four recently isolated genes, have been mapped to this interval. Genes from all of these families show testis-specific expression and are thus candidates for azoospermic factor (AZF). DI6 is also rich in Y-specific repetitive sequences, which may be responsible for its frequent deletion. To understand the sequence organization of this region, a 5-Mb restriction map was constructed based on YAC clones and was partially verified on genomic DNA. The locations of five gene family members, as well as numerous STSs, were determined. The map shows several inverted and direct repeats several hundred kilobases in size. The restriction map of DI6 will facilitate future mapping of deletion breakpoints in infertile males and elucidation of mechanisms behind frequent deletions.

The RBMY (RNA-binding motif, Y chromosome) gene family encodes a germ-cell-specific nuclear protein implicated in spermatogenesis. It consists of approximately 30 genes and pseudogenes, found on both arms of the Y chromosome. RBMY shares high homology with an autosomal hnRNPG gene that contains an RNA-binding motif and one of the four SRGY repeats found in RBMY. One proposal is that RBMY represents an ancestral hnRNPG gene, transposed to the Y chromosome and then amplified. We characterized seven RBMY genes in interval 6 of the Y chromosome long arm. Four have the normal structure with 12 exons spanning 15 kb, whereas one lacks the first 3 exons, therefore representing a pseudogene. The remaining two genes belong to a different subfamily, resembling the autosomal hnRNPG gene with only one SRGY repeat. We also found that most RBMY genes in interval 6 are arranged in tandem. The structure and organization of the Y-linked RBMY genes support the transposition-amplification hypothesis.

The RBM (RNA-binding motif) gene family on the human Y chromosome encodes proteins with an RNA-binding domain. Its exclusive expression in germ cells and its partial deletion in some azoospermic or severely oligospermic males provide evidence of a role for RBM genes in spermatogenesis. There are approximately 30 RBM genes, found on both arms of the Y chromosome. Two RBM cDNA clones with slightly different sequences have been reported. To investigate the number of functional genes, we studied RBM expression by use of RT-PCR of RBM transcripts and by characterizing numerous RBM cDNA clones. A total of 27 RT-PCR and 19 cDNA clones were sequenced. Whereas the RT-PCR clones pointed to the existence of at least six RBM subfamilies (RBMI to RBMVI), the cDNA clones indicated that only RBMI is actively transcribed and encodes functional proteins. A total of six RBMI genes were identified, which produce four polypeptides due to some silent base substitutions. The transcripts of each gene are alternatively spliced to generate protein isoforms with three or four SRGY boxes, thus greatly increasing the complexity of the products of the RBM gene family. We also provide evidence suggesting that a 5-bp deletion in a previously reported RBM cDNA clone represents a processing irregularity.

The majority of patients with steroid sulfatase deficiency have a deletion of the entire STS gene located on the distal short arm of the human X chromosome; however, two patients with partial gene deletions have been identified. We now report the sequences at the breakpoints of a deletion of the 3′ end of the STS gene. The deletion starts within intron 7 of the gene and extends over 150 kb downstream toward the centromere. Analysis of sequences flanking the deletion breakpoints revealed 3 bp of homology. The 3′ flanking sequence provides a new probe for isolation of YAC clones and for studying patients with deletions in this region of the X chromosome.