PAX6 FUNCTION AND ANIRIDIA

Pax-6调节从水母到水母的各种动物的眼睛发育 人类。基因敲除研究表明，除了眼睛，Pax6还 大脑和胰腺的正常分化所必需的。杂合子 Pax6基因的突变会导致人类无虹膜和啮齿动物的小眼睛。无虹膜 是以完全性或部分性为特征的先天性双眼疾患？ 虹膜缺失。早期白内障会导致视力逐渐丧失 青光眼的发病，以及角膜混浊。Pax6作为转录因子发挥作用 该因子具有两个DNA结合域(N端的配对结构域和 像中间的同源结构域一样配对)，一个富含甘氨酸的区域，连接着 在C末端有两个DNA结合域和一个反式激活结构域。那里 是Pax6的两种主要的可选剪接形式，它们的存在方式不同 或者在配对结构域的中间没有14个氨基酸， 由一个被称为5a的外显子编码。+5a亚型是特异的和完全的 在脊椎动物中保存。选择性剪接区的突变 似乎会导致明显的眼睛异常。Pax 6基因外显子5a的包含 蛋白质改变了DNA结合部位的特异性，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 即使在同源结构域DNA结合的情况下，这两种异构体的行为也不同 网站。这项建议的重点是(A)确定误解的影响 体内和体外Pax-6功能突变与肿瘤发生部位的关系 突变与功能异常的类型和(B)确定 Pax-6+5a亚型的功能。我们的长期目标是理解 Pax-6在眼发育级联反应中的作用一种缺失外显子5a的小鼠模型 在Pax-6基因中将被开发出来。外显子5a缺失的影响将是 通过对组织的组织学分析确定。的表达方式 +5a亚型将在发育过程中的不同组织中被确定 +5a亚型特异性抗体。体外反式激活研究 定点突变和DNA结合分析的结合将是 以了解Pax-6和+5a亚型的DNA结合。 RT-PCR分析将用于鉴定其他选择性剪接形式的 帕克斯-6。这项工作将提供对+5a亚型的作用的洞察 Pax-66在发育和分化中的具体作用及定义 在无虹膜患者中发现突变。

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.