Analysis of mechanisms of male differentiation with sex determining genes in mammals

克隆了在牛胚胎发育过程中诱导雄性性腺分化的Y基因性别决定区(BSRY)，并对其编码蛋白进行了鉴定。SRY最初是通过在人类Y染色体区域内的定位克隆来鉴定的，并在尚未确定功能的睾丸中高水平表达。在所有测试的哺乳动物中，都发现了一个在SRY上保守的序列。因此，我们尝试以人SRY序列为引物，用AGPC法从牛睾丸组织中提取总RNA，利用RT-PCR技术分离出bSRY的cDNA克隆。序列分析表明，该克隆全长317个核苷酸，含有SRY基因的保守区，并尝试用5‘RACE方法测定其上游区域，从而进一步确定bSRYcDNA1上游101bp的序列，经5’RACE方法克隆得到的基因命名为bSRYcDNA2。令人惊讶的是，当比较bSRYcDNA1和bSRYcDNA2的核苷酸序列时，发现从保守区上游108bp开始的5‘方向的序列不同，这一点发现了潜在的剪接受体的特征。因此，序列的差异可能归因于bSRY mRNA的环状结构，正如在MUSE SRY中所描述的那样。接下来，为了鉴定编码多肽的bSRY-1与β-半乳糖苷酶的融合蛋白，将pETSRYlacZ质粒导入BL21细胞。经IPTG诱导后，融合蛋白在细胞中得到高效表达。我们正在尝试用β-半乳糖苷酶亲和层析柱分离融合蛋白，并且我们发现从bSRYcDNA1衍生的引物用于牛胚胎囊胚期的性别鉴定是有用的。

Cloning of bovine sex determining region of Y gene (bSRY), which is supposed to induce male differentiation of gonad during bovine embryogenesis, and characterization of bSRY-coding proteins were designed. The SRY was first identified by positional cloning within the region of the human Y chromosome and expressed at high levels in the testes where no function has been determined. A sequence has been found that is conserved on SRY of all mammals tested. We therefore attempted to isolate a cDNA clone of bSRY using RT-PCR with human SRY sequence as primers and total RNA extracted by AGPC method from bovine testes. Sequence analysis of this clone, designated as bSRYcDNA-l revealed that it consisted of a total 317 nucleotides, which contained conserved region of SRY.We next attempted to determine the upstream region of bSRYcDNA using 5'RACE.As a result, we could further determine the 101 bp upstream sequence of bSRYcDNA-1, the cDNA cloned by 5'RACE was designated as bSRYcDNA-2. Surprisingly, when the nucleotide sequences of bSRYcDNA-1 and bSRYcDNA-2 were compared, it was found that the sequences in 5'direction from the 108 bp upstream of the conserve region were different, at which point the characteristic of a potential splice acceptor was found. Therefore, the difference of the sequence may be attributed to the circular structure of bSRY mRNA,as has been described in muse SRY.Next to characterize a fusion proteins of bSRY-1 encoding polypeptides and beta-galactosidase, pETSRYlacZ plasmid was transfected into BL21 cells. The fusion proteins were highly expressed in the cells with IPTG induction. We are now trying to isolate the fusion proteins using beta-galactosidase affinity column.Moreover, we could find that the primers derived from bSRYcDNA-1 were useful for sexing of bovine embryos at the blastocyst stage using PCR method.