FUNCTIONS OF THE TESTIS DETERMINING GENES

人和睾丸中Y基因座、睾丸决定因子(TDF)的研究 在其他哺乳动物中，Y(TDY)负责开启 睾丸分化。TDF/TDY需要其他合作伙伴的相互作用 完成这一分化过程的因素。隔绝了 TDF/TDY的候选基因性别决定区Y(SRY/Sry)具有 提供了一个极好的机会，通过以下方式进一步定义该机制 SRY/Sry和其他睾丸决定基因介导性 决心。最近，我们发现了 几种家鼠Sry等位基因的谷氨酰胺富集区 与XY动物性反转程度相关的菌株 将它们的Y染色体导入近交系C57BL/6J (B6)，基因组背景。我们假设这个富含谷氨酰胺的区域 参与了Sry和另一个共同的蛋白质之间的蛋白质相互作用 Sry相互作用蛋白(Sip)，形成转录复合体 对Sry的生物学功能至关重要。这种多态会影响 家蝇Sry和B6 sip之间的这种相互作用，从而 导致B6.Y/DOM性反转。使用包含以下内容的标记融合蛋白 Sry-Glu(Sry-Glu)结构域在远蛋白质印迹杂交中的应用 我们确实检测到Sry-Glu结构域和Sry-Glu结构域之间的特定结合 睾丸提取液中含有约110kD的蛋白质。三个可实现的具体目标 在本申请中提出的目的是为了检验这一假设 性别决定。首先，我们将把基因类型和表型联系起来。 在B6.Y/DOM动物中使用胚胎干细胞和 嵌合小鼠构建策略。在这些研究中，Sry编码 B6或129Sv(相同Sry等位基因)ES细胞的序列将为 取而代之的是来自几个国内菌株的菌株，这些菌株表现出不同的 B6.Y/随机性反转程度。它们在性别决定中的作用 将对产生的嵌合小鼠进行评估并将其与它们的 不同的基因类型。第二，我们将在体外和体内使用几种 分离sip基因的蛋白质-蛋白质相互作用筛选方法 从原核或酵母克隆系统中的表达cDNA文库中获得。 分离的sip cdna将根据它们的dna和 蛋白质序列、染色体位置、系统发育保守性和 与Sry的互动。第三，对sip的功能进行了进一步的评价。 通过胎鼠和成年鼠的表达研究和报告基因分析 转录调控中与Sry的相互作用。基因敲除 将进行实验，以确定其与Sry和Sry的确切关系 性别决定。拟议的研究将提供重要的 哺乳动物性别决定和性别决定的分子机制 辨证论治病因学和临床上的宝贵信息 男性性腺发育不全和不育症的处理。

The Y-located locus, testis determining factor (TDF) in humans and testis determining Y (Tdy) in other mammals, is responsible for switching on the testis differentiation. TDF/Tdy requires the interactions of other co- factors to complete this differentiation process. The isolation of the candidate gene, sex determining region Y (SRY/Sry) for TDF/Tdy has provided an excellent opportunity to further define the mechanism by which the SRY/Sry and other testis determining genes mediate sex determination. Recently, we have identified a polymorphism within the glutamine-rich domain of the Sry alleles of several domesticus mouse strains that correlates with the degrees of sex reversal in XY animals when their Y chromosome is introduced into an inbred strain, C57BL/6J (B6), genomic background. We hypothesize that this glutamine-rich domain is involved in a protein-protein interaction between Sry and another co- factor, Sry interactive protein (Sip), to form a transcriptional complex essential for the biological function of Sry. The polymorphism affects such an interaction between the domesticus Sry and the B6 Sip, thereby causing B6.Y/Dom sex reversal. Using a labeled fusion protein containing the Sry-glutamine-rich (Sry-glu) domain as a probe in farwestern blotting we have indeed detected a specific binding between the Sry-glu domain and a protein of about 110 kD in testis extracts. Three achievable specific aims are proposed in the present application to test this hypothesis of sex determination. First, we will correlate the genotypes and phenotypes in B6.Y/Dom animals using gene targeting in embryonic stem (ES) cells and chimeric mouse construction strategies. In these studies, the Sry coding sequence of B6 or 129sv (of the same Sry allele) ES cells will be replaced with those from several domesticus strains that manifest various degrees of B6.Y/dom sex reversal. Their effects in the sex determination of the resulting chimeric mice will be evaluated and correlated to their respective genotypes. Second, we will use several in vitro and in vivo protein-protein interaction screening approaches to isolate the Sip cDNAs from expression cDNA libraries in prokaryotic or yeast cloning systems. The isolated Sip cDNAs will be characterized in terms of their DNA and protein sequences, chromosomal locations, phylogenetic conservation and interaction with Sry. Third, the function of Sip is further evaluated by expression studies in fetal and adult mice and reporter gene assays on its interaction with Sry in transcription regulation. Gene knockout experiments will be performed to define its exact relationship to Sry and sex determination. The proposed studies will provide significant insights on the molecular mechanisms of mammalian sex determination and differentiation and invaluable information on the etiology and clinical management of gonadal dysgenesis and infertility in man.