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PAX6 FUNCTION AND ANIRIDIA

PAX6 功能和无虹膜

基本信息

批准号：
6797371
负责人：
GRADY F SAUNDERS
金额：
$ 30万
依托单位：
UNIVERSITY OF TX MD ANDERSON CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-08-01 至 2005-08-31
项目状态：
已结题

项目摘要

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.

Pax-6 调节从水母到动物的眼睛发育人类。敲除研究表明，除了眼睛之外，Pax6 还大脑和胰腺正常分化所需的。杂合子 Pax6 突变会导致人类无虹膜和啮齿动物小眼。无虹膜属是一种先天性双侧眼睛疾病，其特征为完全或部分虹膜缺失。早期因白内障逐渐丧失视力青光眼发作和角膜混浊。 Pax6 发挥转录作用因子并具有两个 DNA 结合域（N 末端的配对域和像中间的同源域一样配对），一个富含甘氨酸的区域，连接两个 DNA 结合域，以及 C 末端的反式激活域。那里是 Pax6 的两种主要的选择性剪接形式，其不同之处在于存在或配对结构域中间缺少 14 个氨基酸，并且由称为 5a 的外显子编码。 +5a 同种型是特定的并且完全在脊椎动物中保存。选择性剪接区域的突变似乎会导致明显的眼睛异常。 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。我们的研究表明即使有同源结构域 DNA 结合，两种异构体的行为也不同网站。该提案的重点是（a）确定错义的影响 Pax-6 的突变在体外和体内功能与位置相关突变与功能异常的类型和（b）确定 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。缺乏外显子5a的小鼠模型 Pax-6基因中的将被开发。外显子 5a 丢失的影响将是通过组织的组织学分析确定。的表达模式为 +5a 同工型将在发育过程中在不同组织中确定 +5a 亚型特异性抗体。体外反式激活研究定点诱变和 DNA 结合测定的结合将旨在了解 Pax-6 和 +5a 同工型的 DNA 结合。 RT-PCR 分析将用于鉴定其他可变剪接形式帕克斯-6。这项工作将深入了解 +5a 同工型的作用 Pax-66 在发育和分化中的作用并定义了特定的作用在无虹膜患者中发现的突变。