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

哺乳动物性别决定基因雄性分化机制分析

基本信息

批准号：
04454107
负责人：
SAKAI Hiroshi
金额：
$ 2.94万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454107/
关键词：
Male specific DNA Bovine SRY gene Male sex differentiation bSRY fusion proteins 性決定遺伝子 SRY 胚の性判別ウシ PCR法デオキシ鎖反応性分化 BOV97M

项目摘要

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.

本研究克隆了牛Y基因性别决定区（bSRY）基因，并设计了bSRY基因编码蛋白。SRY首先通过在人类Y染色体区域内的定位克隆来鉴定，并且在睾丸中以高水平表达，其中没有确定功能。已经发现在所有测试的哺乳动物的SRY上保守的序列。因此，我们尝试以人SRY基因序列为引物，用AGPC法从牛睾丸中提取总RNA，通过RT-PCR方法克隆bSRY基因。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，并对bSRYcDNA-1的上游序列进行了5 'RACE分析，确定了bSRYcDNA-1上游101 bp的序列，将5' RACE克隆的cDNA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，发现从保守区上游108 bp开始的5 '方向的序列不同，在该点上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，接下来，为了表征编码bSRY-1的多肽和β-半乳糖苷酶的融合蛋白，将pETSRYlacZ质粒转染到BL 21细胞中。经IPTG诱导，融合蛋白在细胞中得到高效表达。本研究利用β-半乳糖苷酶亲和层析柱对融合蛋白进行分离纯化，并利用PCR方法对bSRYcDNA-1进行性别鉴定。