FUNCTIONS OF SRY AND SRY INTERACTIVE PROTEIN GENES

小鼠Y染色体上的性别决定基因(Sry)编码一个含有两个主要结构域的蛋白质：氨基末端HMG盒和富含羧基谷氨酰胺(Q)结构域。HMG盒已被证明以序列特异性的方式与DNA结合。富Q区域的函数是未知的。它主要通过CAG重复序列的帧内插入来编码。最近，利用远蛋白质印迹技术，我们证明了这个Sry Q区是一个蛋白结合域，并与存在于成人睾丸和胚胎组织中的三种主要蛋白(SiP)特异性地相互作用。进一步的研究表明，在性别决定时，SIPs优先在成年睾丸的体细胞、早期胚胎组织和胎儿性腺中表达。我们假设SIPs在睾丸决定中作为Sry的辅助因素起着关键作用(S)。在这项申请中提出了三个具体的目标来检验这一假设。首先，我们将使用转基因小鼠的方法来解决有关HMG盒和富含Q的结构域在Sry功能中的重要性的问题。将构建嵌合的Sry基因，以容纳来自不同品系的HMG盒和富Q结构域的各种组合，这些品系表现出Sry基因之间的不亲和性。将根据它们在转基因XX动物中调解睾丸测定的能力进行评估。我们计划确定是HMG盒还是富Q结构域导致了这种不兼容性。其次，将使用交互式克隆技术、亲和层析和蛋白质显微测序来分离SiP的cDNA。它们编码的蛋白质与Sry Q富含结构域的相互作用特性将独立得到确认。第三，我们将从基因序列、表达模式、蛋白质结构、相互作用结构域(S)、染色体位置和进化保守性等方面对sip基因进行特征描述。我们将研究它们在不同肌肉和家养品系之间可能的多态。我们希望将这种多态与它们在Sry相互作用中的兼容性联系起来。基因敲除策略进一步描述了它们的功能。了解小鼠性别决定的机制可能会对人类性别决定的机制有所启发，并最终可能导致性腺发育不全和不育的临床诊断和治疗的发展。

The sex determining gene (Sry) on the mouse Y chromosome encodes a protein with two major domains: an amino terminal HMG box and a carboxyl glutamine-rich (Q-rich) domain. The HMG box has been demonstrated to bind to DNA in a sequence-specific manner. The function for the Q-rich domain is unknown. It is primarily encoded by an in-frame insertion of a CAG repetitive sequence. Recently, using the farwestern blotting technique, we have demonstrated that this Sry Q-rich domain is a protein-binding domain and interacts specifically with 3 major proteins (Sips) present in adult testis and embryonic tissues. Further studies demonstrated that the Sips are preferentially expressed in somatic cells of the adult testis and early embryonic tissues and fetal gonads at the time of sex determination. We hypothesize that the Sips play a critical role(s) as co-factors of Sry in testis determination. Three specific aims are proposed in this application to test this hypothesis. First, we will address the question regarding the importance of the HMG box and Q-rich domains in Sry function using transgenic mouse approaches. Chimeric Sry genes will be constructed to harbor various combinations of HMG box and Q-rich domain from different mouse strains that exhibit incompatibility among their Sry genes. They will be evaluated in terms of their ability to mediate testis determination in transgenic XX animals. We plan to determine whether the HMG box or the Q-rich domain is responsible for such incompatibility. Second, interactive cloning techniques, affinity chromatography and protein microsequencing will be used to isolate the cDNAs for the Sips. The interactive properties of their encoded proteins to the Sry Q-rich domain will be confirmed independently. Third, we will characterize the Sip genes in terms of their cDNA sequences, expression patterns, protein structures, interactive domain(s), chromosomal locations, and evolutionary conservation. We will investigate their probable polymorphisms among different musculus and domesticus strains. We hope to relate such polymorphisms to their compatibility in Sry interactions. Their functions are further delineated by gene knockout strategies. Understanding the mechanisms of sex determination in mouse may shed insights on those in humans, and eventually may lead to developments of clinical diagnosis and treatment of gonadal dysgenesis and infertility.