MOLECULAR GENETIC ANALYSIS OF THE PAX6 GENE IN ANIRIDIA

这项建议的长期目标是研究 一种名为PAX6的基因，它是正常人类眼睛发育所必需的， 并确定受PAX6调控的基因。Pax6是一种 属于配对盒子家族的基因，因此很可能起到 转录因子。小鼠Pax-6基因在小眼球中发生突变 哪些杂合子(Sey/+)有小眼球，无前房 房室，视网膜异常折叠，小晶状体或无晶状体。 纯合Sey/Sey小鼠完全没有眼睛和鼻子。一直以来 提出人类的一种称为无虹膜的全眼病是同源的。 对小鼠小眼睛的突变，因为相似的、半显性的模式 遗传、相关表型和同源染色体位置。 为了检测人类PAX6基因在无虹膜和眼部疾病中的作用 总的来说，我们已经克隆了PAX6并鉴定了几个 无虹膜家系中PAX6的独立点突变，从而提供 PAX6导致无虹膜的确凿证据。中国的表型 无虹膜和小眼睛可能是由晶状体损伤引起的 诱导，由于前房结构的发展， 包括虹膜，在很大程度上依赖于镜头的影响。 此外，该模型与PAX6的强烈表达一致 在视泡中，一种已知的晶状体分化的诱导剂。这个 发现PAX6基因剂量减少50%会导致表型 表明该基因产物控制着一个关键的限速步骤 在眼睛发育方面。 进一步了解PAX6不同部分的功能 蛋白质，我们将分析无虹膜的其他PAX6突变。我们会 解决SSCP分析的可靠性问题，方法是对多个 SSCP阴性样本的每个外显子的等位基因。在……里面 此外，我们将测试PAX6在其他潜在的 眼睛发育的等位基因障碍，如人的彼得畸形和 小鼠的缺陷性突变(Cm)。的功能后果 PAX6的可识别突变将通过凝胶位移和足迹进行测试 测定它们对DNA结合的影响，以及它们对 抄写。为此，我们将开发一种细胞培养系统来 剖析配对结构域和同源结构域的作用及其转录 调节C末端的活性。我们建议识别基因 在眼球发生过程中由PAX6调节的基因，通过测试小鼠和 果蝇基因的人类同源基因是已知的 果蝇PAX6同源物，配对。我们还将采用替代方案 策略包括免疫沉淀基因组DNA或染色质， 和cDNA差减。最后，我们将检测PAX6基因的充分性 逆转录病毒载体下注射诱导晶状体的表达 雏鸡胚胎发育中的表面外胚层。可提供的 克隆的人类和小鼠眼部缺陷相关基因提供了 一个强大的系统，在其中不仅研究基础胚胎学和 眼睛发育的遗传学，但也是一种视力下降的障碍 在人类身上起作用。

The long term objectives of this proposal are to study the function of a gene called PAX6, which is required for normal human eye development, and to identify genes which in turn are regulated by PAX6. PAX6 is a gene belonging to the paired box family and thus likely acts as a transcription factor. the mouse Pax-6 gene is mutated in small-eye, in which heterozygotes (Sey/+) have microphthalmia, absent anterior chambers, abnormally folded retinas, and small or absent lens. Homozygous Sey/Sey mice lack eyes and noses entirely. It has been proposed that a panocular disorder in man, called aniridia, is homologous to the mouse Small-eye mutation, because of similar, semi-dominant modes of inheritance, related phenotypes, and homologous chromosomal locations. to test the involvement of the human PAX6 gene in aniridia, and in eye development in general, we have cloned PAX6 and identified several independent point mutations in PAX6 in aniridia families, thus providing firm evidence that PAX6 is responsible for aniridia. The phenotype in aniridia and small-eye is potentially explained by an impairment in lens induction, since the development of anterior chamber structures, including the iris, is critically dependent on the influence of the lens. Moreover, this model is consistent with the intense expression of PAX6 in the optic vesicle, a known inducer of lens differentiation. The finding that a 50% reduction in PAX6 gene dosage results in a phenotype indicates that this gene product controls a critical, rate-limiting step in eye development. To gain further insight into the function of different parts of the PAX6 protein, we will analyze additional PAX6 mutations in aniridia. We will address the reliability of the SSCP analysis by sequencing multiple alleles of each exon from samples which are negative by SSCP. In addition, we will test the involvement of PAX6 in other potentially allelic disorders of eye development such as Peter's anomaly in man and the Coloboma mutation (Cm) in the mouse. the functional consequences of identifiable mutations in PAX6 will be tested by gel shift and footprint assay for their effect on DNA binding, and also for their effects on transcription. to this end, we will develop a cell culture system to dissect the role of the paired and homeo domains and the transcription regulating activity of the C-terminus. We propose to identify genes which are regulated by PAX6 during oculogenesis, by testing mouse and human homologs of Drosophila genes which are known targets for the Drosophila PAX6 homolog, paired. We will also employ alternative strategies including immuno-precipitation of genomic DNA or chromatin, and cDNA subtraction. Finally, we will test the sufficiency of PAX6 gene expression for lens induction by injecting a retroviral vector underneath surface ectoderm in the developing chick embryo. The availability of a cloned gene associated with ocular defects in both man and mouse provides a powerful system in which to study not only the basic embryology and genetics of ocular development, but also a disorder of decreased visual function in man.