PAX6 FUNCTION AND ANIRIDIA

Pax-6调节从水母到海蜇等动物眼睛的发育。 人类敲除研究表明，除了眼睛外，Pax 6还 这是大脑和胰腺正常分化所必需的。杂合 Pax 6的突变导致人类无虹膜和啮齿动物的小眼睛。无虹膜 是一种先天性双眼疾病，其特征是完全或部分 没有虹膜。白内障会逐渐丧失视力， 青光眼发作和角膜混浊。Pax 6的功能是转录 该因子具有两个DNA结合结构域（N末端的配对结构域和N末端的配对结构域）， 中间的同源结构域），一个富含甘氨酸的区域， 两个DNA结合结构域，和在C末端的反式激活结构域。那里 是Pax 6的两种主要的选择性剪接形式， 或不存在配对结构域中间的14个氨基酸， 都是由一个叫做5a的外显子编码的+5a亚型特异于并完全 在脊椎动物中是保守的。可变剪接区突变 会导致明显的眼部异常Pax 6中包含外显子5a 蛋白质改变了DNA结合位点的特异性，这表明， 这两种形式可能调节不同的靶基因。我们的研究表明 这两种异构体的行为不同，即使同源结构域DNA结合 网站.本建议的重点是（a）确定误解的影响 Pax-6上的突变在体外和体内发挥作用， （B）确定具有功能异常类型的突变， Pax-6的+5a同种型的功能。长期目标是了解 Pax-6在眼睛发育级联中的作用。5a外显子缺失的小鼠模型 Pax-6基因中的一个基因。外显子5a缺失的影响将是 通过组织的组织学分析确定。的表达模式 在发育过程中，将在不同组织中测定+5a亚型， +5a同种型特异性抗体。体外反式激活研究， 将结合定点诱变和DNA结合试验， 为了理解Pax-6和+5a同种型的DNA结合，进行了研究。 RT-PCR分析将用于鉴定其他可变剪接形式的 圣神六号这项工作将提供深入了解+5a亚型的作用， Pax-66在发育和分化中的作用，并确定特定的 在无虹膜患者中发现的突变。

Pax-6 regulates the development of the eye in animals ranging from jellyfish to humans. Knockout studies have shown that in addition to eyes Pax6 is also required for the normal differentiation of brain and pancreas. Heterozygous mutations in Pax6 cause aniridia in humans and small eye in rodents. Aniridia is a congenital bilateral disorder of the eye marked by complete or partial absence of the iris. Vision is progressively lost through cataracts, early onset of glaucoma, and corneal opacification. Pax6 functions as a transcription factor and has two DNA binding domains (a paired domain at the N terminus and a paired like homeodomain in the middle), a glycine-rich region that links the two DNA-binding domains, and a transactivation domain at the C terminus. There are two major alternatively spliced forms of Pax6 that differ by the presence or absence of 14 amino acids that are in the middle of the paired domain and are coded by an exon known as 5a. The +5a isoform is specific to and fully conserved among vertebrates. Mutation in the alternatively spliced region appear to cause a distinct eye abnormality. The inclusion of exon 5a in Pax 6 protein changes the specificity of the DNA-binding site, which indicates that the two forms may regulate different target genes. Our studies have indicated that the two isoforms behave differently even with the homeodomain DNA-binding sites. The focus of this proposal is (a) to determine the effects of missense mutations on Pax-6 function in vitro and in vivo to correlate the position of mutation with the type of functional abnormality and (b) to determine the function of the +5a isoform of Pax-6. The long term goal is to understand the role of Pax-6 in the cascade of eye development. A mouse model lacking exon 5a in the Pax-6 gene will be developed. The effect of loss of exon 5a will be determined by histological analysis of the tissues. The expression pattern of the +5a isoform will be determined in different tissues during development with +5a isoform specific antibodies. In vitro transactivation studies with combination of site-directed mutagenesis and DNA-binding assays will be performed to understand the DNA-binding of the Pax-6 and the +5a isoform. RT-PCR analysis will be used to identify other alternatively spliced forms of Pax-6. This work will provide insight into the role of the +5a isoform of Pax-66 in development and differentiation and define the effects of specific mutations found in aniridia patients.