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

在牛胚胎发育过程中诱导性腺雄性分化的Y基因性别决定区（bSRY）的克隆，以及bSRY编码蛋白的鉴定。SRY最初是通过在人类Y染色体区域内的位置克隆发现的，并在睾丸中高水平表达，但尚未确定其功能。在所有测试的哺乳动物的SRY中发现了一个保守的序列。因此，我们尝试以人SRY序列为引物，利用AGPC法从牛睾丸中提取总RNA，采用RT-PCR方法分离bSRY cDNA克隆。该克隆被命名为bsrycdna - 1，序列分析显示该克隆共包含317个核苷酸，其中包含SRY的保守区。接下来，我们尝试使用5'RACE来确定bSRYcDNA的上游区域。结果，我们进一步确定了bSRYcDNA-1上游101 bp的序列，通过5'RACE克隆的cDNA被命名为bSRYcDNA-2。令人惊讶的是，当比较bSRYcDNA-1和bSRYcDNA-2的核苷酸序列时，发现在保守区上游108 bp的5'方向上的序列不同，在这一点上发现了潜在剪接受体的特征。因此，序列的差异可能归因于bSRY mRNA的圆形结构，正如缪斯SRY中所描述的那样。接下来，将pETSRYlacZ质粒转染到BL21细胞中，以表征编码多肽和β -半乳糖苷酶的bSRY-1融合蛋白。融合蛋白在IPTG诱导下高表达。我们现在正尝试用-半乳糖苷酶亲和柱分离融合蛋白。此外，我们发现bSRYcDNA-1引物可用于用PCR方法对囊胚期牛胚胎进行性别鉴定。

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.