FUNCTIONS OF THE TESTIS DETERMINING GENES

人和睾丸中的睾丸决定因子（TDF）和Y定位基因座 在其他哺乳动物中决定Y（Tdy），负责打开 精巢分化 TDF/Tdy需要其他共- 完成这一分化过程。 的孤立 TDF/Tdy的候选基因，性别决定区Y（SRY/Sry）， 提供了一个极好的机会，进一步界定机制， SRY/Sry和其他睾丸决定基因介导性别 保持战略定力 最近，我们发现了一种多态性， 几种仓鼠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 当它们的Y染色体被引入近交系C57 BL/6 J时， (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和另一种辅蛋白之间的蛋白质-蛋白质相互作用， Sry相互作用蛋白（Sip），以形成转录复合物 对Sry的生物学功能至关重要。 多态性影响 这种相互作用之间的arcticus Sry和B6 Sip，从而 导致B6.Y/Dom性别逆转 使用标记的融合蛋白， 富含Sry-谷氨酰胺（Sry-glu）结构域作为远蛋白印迹探针 我们确实检测到了Sry-Glu结构域和 睾丸提取物中约110 kD的蛋白质。 三个可实现的具体 在本申请中提出的目的是检验这种假设 性别决定首先，我们将基因型和表型 在B6.Y/Dom动物中使用胚胎干（ES）细胞中的基因靶向， 嵌合小鼠构建策略。 在这些研究中，Sry编码 B6或129 sv（具有相同的Sry等位基因）ES细胞的序列将是 用来自几种表现出各种 B6.Y/dom性别逆转的程度。 它们在性别决定中的作用 的嵌合小鼠将被评估，并与它们的 各自的基因型。 其次，我们将使用几种体外和体内 分离Sip cDNA的蛋白质-蛋白质相互作用筛选方法 来自原核或酵母克隆系统中的表达cDNA文库。 分离的Sip cDNA将根据它们的DNA进行表征， 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，进一步评价了Sip的功能 通过在胎鼠和成年小鼠中的表达研究以及报告基因测定 在转录调控中与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 哺乳动物性别决定的分子机制的见解， 分化和宝贵的信息，病因和临床 男性性腺发育不全和不育症的管理。

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.