Studies on the Regulatory Genes of Gonadal Differentiation from the Viewpoint of Utilization in Animal Resource Science.

在赠款任期内取得的代表性成果如下。1)克隆了柴巴山羊(Capra Hircus Var Shiba)全长4.9kb的全基因组SRY(Y染色体性别决定区)DNA。2)将人和小鼠SRY DNA分别导入小鼠Sertoli细胞的TM-4细胞，建立了转化细胞系。奇怪的是，转基因小鼠SRY DNA诱导了P450芳香族基因的表达。目前正在研究该细胞系，以确定构成SRY级联的基因。3)从胎儿睾丸cDNA文库中分离得到的牛SRY基因具有环状构象。4)克隆了山羊c-kit基因，并进行了序列测定。结果表明，山羊c-kit的胞内结构域含有一个额外的丙氨酸插入片段，这在迄今所研究的绵羊以外的任何物种中都没有描述过。这一插入似乎仅限于山羊和绵羊。…正在进行尝试以更多地确定插入物的生物学意义。5)在患有性别分化障碍的患者中发现了有趣的突变基因。为了阐明位于Y染色体上的基因的功能，并推测该基因可能促进身体的生长。6)对W^f和W^&lt；sh&gt；小鼠突变体的表型进行了详细的分析，揭示了一些有趣的特征，这些特征可能有助于深入了解c-kit/S1系统在反刍动物中的功能。7)发现了可能与大鼠卵巢黄体细胞凋亡相关的新基因。8)在卵泡细胞凋亡变化中的作用进行了研究。9)研究了细胞周期蛋白在卵母细胞成熟分裂中的作用。10)分析了WAP启动子的组织特异性，发现WAP启动子在除乳腺外的许多组织中都有功能。11)为提高转基因动物的生产效率，采用限制性内切酶和聚合酶链式反应相结合的方法，筛选出能成功整合转基因基因的胚胎。较少

Representative results obtained during the tenure of the grant are as follows. 1) Coplete genomic SRY (sex determining region on Y chromosome) DNA of Shiba goat (Capra hircus var Shiba) spanning 4.9 kb was cloned and sequenced. 2) Transformed cell lines were established by transfecting TM-4 cells derived from mouse Sertoli cells, with human and mouse SRY DNA.Curiously, as it turned out, the expression of P450aromatese genes was induced by the transfected mouse SRY DNA.This cell line is currently investigated in order to identify the genes which constitute the putative SRY cascade. 3) Bovine SRY cDNA isolated from a fetal testicular cDNA library is confirmed to possess circular conformation. 4) Caprine c-kit cDNA was cloned and sequenced. It turned out that the intracellular domain of caprine c-kit possess one extra alanine insert which have not been described in any other species except sheep so far examined. The insert appears to be limited to goats and sheep. Attempts are being made … More to determine the biological meaning of the insert. 5) Interesting mutant genes were identified in human patients suffering from the disorders of sexual differetiation. Investigations were made to clarify the function of the gene located on Y chromosome and putatively promote the growth of the body. 6) Detailed analysis of phenotypes of W^f and W^<sh> mouse mutants revealed several interesting features which may be useful to approach the understanding of functioning of c-kit/Sl system in ruminants. 7) Novel genes which might be responsible for the apoptosis of lutcal cells in the rat ovary, were identified. 8) Functions of oncognenes in apoptotic changes in the follicles have been investigated. 9) Role of cyclins in maturation division of oocytes has been investigated. 10) Tissue specificity of the WAP promoter was analyzed, and found to be a 'lcaky' promoter functioning in many tissues other than the mammary gland. 11) Attempts were made to improve efficiency of the production of transgenic animals by selecting embryos which successfully incorporated the transgenes into their genome by combined treatment with restrictions cnzymes and PCR analysis. Less