GENETIC AND MOLECULAR ANALYSES OF MUTATIONS

描述：(改编自调查人员摘要)这是一场竞争性的 在第三个供资周期续签RO1，要求五年 支持识别和分析由基因重排发现的基因， 影响p基因。申请人之前的工作定义了两个 补充组-矮小/干裂/不育(RJS)和腭裂-哪一个 分别位于p的近端和远端。在上一个供资周期中， 申请人确定了RJS基因的一个非常强大的候选人，证明 GABRB3基因的缺失是导致腭裂的原因，在 此外，还描述了一种逆转等位基因p100H，它可以破坏Sox6基因和 引起一种不同寻常的肌肉疾病，让人想起埃默里-德莱弗斯肌肉 营养不良。目前的申请提议延长所有三个领域的工作。 RJS虚证的分子发病机制有待进一步研究。 基因和蛋白质表达的特征，通过鉴定 相互作用的蛋白质，以及与其他人合作，生化和/或 可能在蛋白质泛素化中发挥作用的RJS结构域的细胞生物学分析 和鸟嘌呤核苷酸交换。此外，loxP侧翼的RJS等位基因将是 创建是为了调查RJS引起的多效性表型 缺陷反映了几个特定组织缺陷的总和。 初步研究表明，GABRB3缺乏引起的腭裂 反映了中枢神经系统之外的要求，指向 GABA信号在腭部形态发生中的潜在新作用。这些 对GABRB3转基因基因的进一步研究将证实这一数据 神经元特异性烯醇化酶启动子。此外，GABRB3基因和GABRB3基因的 蛋白质表达和GABA结合位点将在 非神经性组织，特别注意发育中的腭部。 由p100H突变引起的肌肉疾病的发病机制将是 通过对一种新发现的SOX6亚型的进一步表征进行研究 在肌肉中高表达，通过成肌细胞/肌细胞培养的发展 来自突变动物的系统，并通过体内和/或Sox6基因拯救实验 在试管中。此外，差异显示、cDNA差减和/或基因 将使用表达谱来比较突变和非突变组织在 试图识别Sox6目标。

DESCRIPTION: (Adapted from investigator's abstract) This is a competitive renewal for an RO1 in its third funding cycle that requests five years of support to identify and analyze genes uncovered by genetic rearrangements that affect the p gene. Previous work by the applicant has defined two complementation groups -- runty/jerky/sterile (rjs) and cleft palate -- which lie proximal and distal to p, respectively. In the previous funding cycle, the applicant identified a very strong candidate for the rjs gene, demonstrated that deletion of the Gabrb3 gene was responsible for cleft palate, and, in addition, described an inversion allele, p100H, that disrupts the Sox6 gene and causes an unusual muscle disease reminiscent of Emery-Dreyfuss muscular dystrophy. The current application proposes to extend work in all three areas. The molecular pathogenesis of rjs deficiency will be investigated by further characterization of gene and protein expression, by identification of interacting proteins, and, in collaboration with others, biochemical and/or cell biologic assays of rjs domains that may function in protein ubiquitination and guanine nucleotide exchange. In addition, a loxP-flanked rjs allele will be created to investigate whether the pleiotropic phenotype caused by rjs deficiency reflects the sum of several tissue-specific defects. Preliminary studies suggest that cleft palate caused by deficiency for Gabrb3 reflects a requirement outside the central nervous system, pointing to a potentially novel role for GABA signaling during palate morphogenesis. These data will be confirmed by further studies of a Gabrb3 transgene controlled by the neuron-specific enolase promoter. In addition, the sites of Gabrb3 gene and protein expression, and GABA binding sites, will be characterized in non-neuronal tissues with special attention to the developing palate. The pathogenesis of muscle disease caused by the p100H mutation will be investigated by further characterization of a newly recognized Sox6 isoform highly expressed in muscle, by development of myoblast/myocyte cell culture systems from mutant animals, and by Sox6 gene rescue experiments in vivo and/or in vitro. In addition, differential display, cDNA subtraction, and/or gene expression profiling will be used to compare mutant and non-mutant tissues in an attempt to identify Sox6 targets.