GENETIC AND MOLECULAR ANALYSES OF MUTATIONS

描述：（改编自研究者摘要）这是一项竞争性研究 在第三个供资周期延长一个RO 1， 支持识别和分析基因重排发现的基因， 影响p基因。申请人以前的工作已经确定了两个 互补组-矮小/干/不育（rjs）和腭裂-其中 分别位于p的近端和远端。在上一个供资周期， 申请人鉴定了RJS基因的非常强的候选者， Gabrb 3基因的缺失是导致腭裂的原因， 此外，还描述了一个倒位等位基因p100 H，它破坏了Sox 6基因， 会导致一种不寻常的肌肉疾病， 营养不良目前的申请建议在所有三个领域扩大工作。 rjs缺乏症的分子发病机制将进一步研究 基因和蛋白质表达的表征，通过鉴定 相互作用的蛋白质，并与其他生物化学和/或 可能在蛋白质泛素化中起作用的RJS结构域的细胞生物学测定 和鸟嘌呤核苷酸交换。此外，loxP侧翼的rjs等位基因将是 研究是否由RJS引起的多效性表型 缺陷反映了几种组织特异性缺陷的总和。 初步研究表明，Gabrb 3缺乏引起的腭裂 反映了中枢神经系统以外的要求，指向一个 GABA信号在腭形态发生中的潜在新作用。这些 这些数据将通过进一步研究由以下基因控制的Gabrb 3转基因来证实： 神经元特异性烯醇化酶启动子。此外，Gabrb 3基因和 蛋白质表达和GABA结合位点，将在 非神经元组织，特别注意发育中的腭。 由p100 H突变引起的肌肉疾病的发病机制将是 通过进一步表征新识别的Sox 6同种型进行研究 在肌肉中高度表达，通过成肌细胞/肌细胞培养物的发育 系统，并通过Sox 6基因拯救实验在体内和/或 体外此外，差异显示、cDNA消减和/或基因修饰也是一种有效的方法。 表达谱将用于比较突变和非突变组织， 尝试识别Sox 6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.