MOLECULAR GENETIC ANALYSIS OF THE PAX6 GENE IN ANIRIDIA

无虹鳟 PAX6 基因的分子遗传学分析

• 批准号: 2163827
• 负责人: RICHARD L MAAS
• 金额: $ 11.07万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2163827
• 关键词: DNA footprinting; cell differentiation; chick embryo; developmental genetics; developmental neurobiology; eye; eye disorder; gel mobility shift assay; gene expression; gene mutation; genetic disorder; genetic regulation; genetic regulatory element; human subject; laboratory mouse; lens; molecular cloning; molecular genetics; nonmammalian vertebrate embryology; polymerase chain reaction; protein structure function; regulatory gene; reporter genes; subtraction hybridization; tissue /cell culture; transcription factor; transfection /expression vector

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.

这项建议的长远目标是研究 一种叫做PAX 6的基因，它是人类眼睛正常发育所必需的， 并鉴定反过来受PAX 6调控的基因。 PAX 6是一个 属于配对盒家族的基因，因此可能充当 转录因子 小鼠Pax-6基因在小眼中发生突变， 哪些杂合子（Sey/+）有小眼症， 室，异常折叠的视网膜和小或缺失的透镜。 纯合子Sey/Sey小鼠完全没有眼睛和鼻子。 已经 他提出，人类的一种叫做无虹膜的全眼球疾病， 小眼突变的小鼠，因为相似的，半显性模式， 遗传、相关表型和同源染色体位置。 为了测试人类PAX 6基因在无虹膜和眼睛中的作用， 在一般的发展中，我们克隆了PAX 6，并确定了几个 无虹膜家族中PAX 6的独立点突变，从而提供 PAX 6是导致无虹膜的有力证据。 中的表型 无虹膜和小眼可能是由于透镜受损所致 由于前房结构的发展， 包括虹膜的光学特性严重依赖于透镜的影响。 此外，该模型与PAX 6的强烈表达一致。 在视泡中，一种已知的透镜分化诱导物。 的 发现PAX 6基因剂量减少50%会导致表型 表明这种基因产物控制着一个关键的限速步骤 在眼睛发育中。 进一步了解PAX 6不同部件的功能 蛋白质，我们将分析其他PAX 6突变无虹膜。 我们将 通过对多个序列进行测序来解决SSCP分析的可靠性 SSCP阴性的样本中每个外显子的等位基因。 在 此外，我们还将测试PAX 6在其他潜在的 眼睛发育的等位基因疾病，如人类的彼得异常， 小鼠中的Coloboma突变（Cm）。 的功能性后果 PAX 6中可识别的突变将通过凝胶位移和足迹来测试。 测定它们对DNA结合的影响，以及它们对 转录。 为此，我们将开发一种细胞培养系统， 剖析配对结构域和同源结构域以及转录的作用 调节C末端的活性。 我们建议识别基因 在眼发生过程中由PAX 6调节，通过测试小鼠和 果蝇基因的人类同源物，这些基因是已知的 果蝇PAX 6同源物，配对。 我们还将采用替代 包括基因组DNA或染色质的免疫沉淀的策略， 和cDNA消减。 最后，我们将测试PAX 6基因的充分性， 通过将逆转录病毒载体注射到下面来诱导透镜的表达 发育中的鸡胚的表面外胚层。 的可用性 克隆的与人类和小鼠眼睛缺陷相关的基因提供了 一个强大的系统，不仅可以研究基本的胚胎学， 眼睛发育的遗传学，但也是一种视力下降的疾病， 人的功能。