TSPY may represent such a function in monkey testis. The putative nucleic-acid-binding motif within SRY and its testis specific expression is consistent with that of SRY which has a role in the developmental regulation of the testis ( Sinclair et al., 1990). Such a feature was already appeared in the high mobility group (HMG) box, a main functional domain, of the sex determining region Y (SRY) ( Jantzen et al., 1990). This finding allows us to speculate that these residues may be implicated in the DNA binding property as a structural evidence of the Arg and Lys-rich region. The region was well conserved between monkey, human, and bovine TSPY, and positively charged residues were concentrated in the lined box ( Fig. Interestingly, the 26-residue segment (residues 79-105) had an abundance of basic residues such as Arg and Lys. One of the characteristic features of this protein was its hydrophilic nature, and three hydrophilic regions (residues 42-51, 79-105, and 202-207) were clearly identified ( Fig. The sum of Glu and Asp was higher than that of basic residues such as Arg and Lys being consistent with the monkey TSPY which was an acidic protein. Amino acids contained at high levels in monkey TSPY were Glu (10.6%), Ala (9.4%), and Arg (7.3%). The analysis of the deduced amino acid sequence indicates that the gene product is a slightly acidic protein with an isoelectric point of pH 5.35 and has a molecular mass of 28 kDa. A polyadenylation site, AATAAA, was not included in the sequence since we used an oligonucleotide containing this site as a PCR primer. The ATG triplet indicated as nucleotides 1-3 was an initiation codon of the open reading frame and stop codon appeared in nucleotides 739-741 as a TGA triplet. The cDNA consisted of 976 nucleotides, which included an open reading frame of 741 nucleotides encoding 246 amino acids. The nucleotide sequences were the same in three independent clones of RT-PCR products, being almost certain that the sequence was free from the mutated ones during amplification. The nucleotide and deduced amino acid sequences of monkey TSPY cDNA are shown in Fig. Molecular cloning of TSPY cDNA and structure analysis In the present study, we cloned and sequenced a full-length of the TSPY cDNA from Japanese monkey testis RNA, described its molecular characterizations, and examined the expression of the TSPY gene in various tissues. To date, however, the complete structure of a TSPY gene is not known except for the human gene. Since in primates it is possible that TSPY genes evolve some structural differentiations and cause specificities in reproductive systems, it is necessary to clarify the structures of TSPY genes in various primates. In our succeeding report, we compared restriction patterns and chromosomal localizations of TSPY genes in man, gibbons, and Old World monkeys, and found variations of gene structures among them ( Kim et al., 1996). We have sequenced exon 1, exon 2, and the first intron of the TSPY gene of great apes and the baboon and determined phylogenetic relationship among them ( Kim and Takenaka, 1996). The number of TSPY genes and related genes are highly amplified especially in primates ( Kim and Takenaka, 1996 Kim et al., 1996). (1995) showed that TSPY gene-related sequences are conserved and Y chromosome specific in hominoids. Using the chromosomal in situ hybridization technique, Schempp et al. DYZ5 sequences have been shown to be conserved on the Y chromosome of the great apes by Southern blot and in situ hybridization ( Guttenbach et al., 1992). Human TSPY gene-related sequences are organized as constitutive parts of DYZ5 repeat units ( Manz et al., 1993) which are located on the short arm of the Y chromosome ( Tyler-Smith et al., 1988). The gene contained six exons with five introns and was estimated to be approximately 2.7 kb. A genomic clone for human TSPY was also isolated successively ( Zhang et al., 1992). A cDNA clone for human TSPY was isolated from an adult human testis cDNA library ( Arnemann et al., 1991). Although the exact function of TSPY is still unknown, this protein might play a role in DNA replication ( Schnieders et al., 1996). The expression of the TSPY gene has been shown to be restricted to testicular tissue ( Arnemann et al., 1991) and appeared to be confined to germ cells of the spermatogonial and early spermatocyte stages in adult human males ( Chandley and Cooke, 1994 Schnieders et al., 1996). Testis-specific protein Y (TSPY) is known to be a product of the Y-chromosome specific gene.
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