FUNCTIONS OF SRY AND SRY INTERACTIVE PROTEIN GENES

小鼠Y染色体上的性别决定基因（Sry）编码一种具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-rich）结构域。HMG盒子已被证明以序列特异性的方式与DNA结合。富q域的函数是未知的。它主要通过帧内插入CAG重复序列进行编码。最近，我们利用最先进的印迹技术证明了这个富含Sry q的结构域是一个蛋白质结合结构域，并与存在于成年睾丸和胚胎组织中的3种主要蛋白（Sips）特异性相互作用。进一步的研究表明，Sips在成体睾丸、早期胚胎组织和胎儿性腺的体细胞中优先表达。我们假设Sips作为Sry的辅助因子在睾丸测定中起关键作用。在本应用程序中提出了三个具体目标来验证这一假设。首先，我们将使用转基因小鼠方法解决有关HMG盒和富q域在Sry功能中的重要性的问题。嵌合Sry基因将被构建为包含来自不同小鼠品系的HMG盒和富q域的各种组合，这些组合在它们的Sry基因之间表现出不相容。将根据其介导转基因XX动物睾丸测定的能力对其进行评估。我们计划确定是HMG盒还是富q域导致了这种不兼容。其次，利用交互式克隆技术、亲和层析技术和蛋白质微测序技术分离Sips的cdna。他们编码的蛋白与Sry q -富结构域的相互作用特性将被独立确认。第三，我们将从cDNA序列、表达模式、蛋白质结构、相互作用结构域、染色体位置和进化保守性等方面对Sip基因进行表征。我们将研究它们在不同的肌肉和家养菌株之间可能的多态性。我们希望将这种多态性与它们在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。了解小鼠的性别决定机制可能有助于了解人类的性别决定机制，并最终导致性腺发育不良和不孕症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.