MOLECULAR BASIS OF HOMEOTIC GENE REGULATION

同源基因调控的分子基础

• 批准号: 2179715
• 负责人: Peter J. Harte
• 金额: $ 21.14万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179715
• 关键词: DNA binding protein; Drosophilidae; RNase protection assay; alleles; cytogenetics; developmental genetics; enzyme linked immunosorbent assay; gel electrophoresis; gene dosage; gene expression; gene interaction; gene mutation; genetic mapping; genetic promoter element; homeobox genes; immunoprecipitation; molecular cloning; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; protein structure function; regulatory gene; thorax

DESCRIPTION: This is an amended proposal to continue a study of the mechanisms that are responsible for the stable, long-term maintenance of homeotic gene expression during Drosophila development. It focuses on the trithorax gene, which has been shown to be required for the maintenance of the expression patterns of the genes of the BX-C and ANTP-C and to counterbalance the effects of the Polycomb group genes, which act as negative regulators of homeotic gene expression. Through a detailed genetic and molecular genetic analysis of the trx gene, and through a detailed functional dissection of the trx protein, Dr. Harte proposes an ambitious experimental attack on questions whose resolution likely involves the relationship between chromatin conformation and state of transcription activation. This is an aspect of transcriptional regulation that has fascinated biologists for many years, and remains poorly understood, but is likely to attract increasing interest during the next several years. During the previous grant period, a significant effort was devoted to a detailed description of the varied responses to trx mutation by numerous genes and to the cloning and molecular characterization of the trx gene. The trx transcription unit was found to be large and to produce a complex array of RNA products and proteins that are estimated to range in size between 360 and 420 kd. The heroic efforts to characterize this gene (including transformation with rescue constructs containing a 34 kb genomic fragment) have been rewarded by the discovery of several recognizable sequence domains in the protein coding sequence, including a presumed steroid receptor family DNA binding domain and a region homologous to the human ALL-1 proteins which have been implicated in childhood Acute Lymphocytic Leukemias. The trx protein has been shown to bind to DNA in vitro, to bind to 63 specific sites on polytene chromosomes, and to functionally associate with a related trx-G protein, ash-1, and functional trx response elements have been identified. The experimental goals of the proposed research constitute an extensive and detailed analysis of trx. High resolution mapping of trx chromosome binding sites will be pursued using germ line transformation of reporter constructs to map both chromosomal binding sites and the ability of these sequences to affect expression of linked genes. Characterization of in vitro DNA binding will be pursued, buoyed by the recent success in showing that a fragment of trx protein is active in a mobility shift assay with a 660 bp core PRE. These studies will be extended to better define binding affinities and specificities, and in collaboration with Renato Paro, to map sites to which trx protein binds in vivo. To identify other proteins with which trx associates, several approaches are described, including co-immunoprecipitation, further characterization of the apparent association between trx and ash-1, and use of the polytene binding assay in various mutant backgrounds. Trx will be overexpressed with heat shock transgenic constructs and by increasing its gene dosage to determine the effects on homeotic gene expression and to test models of competition between Pc-G and trx-G proteins. Finally, structure/function studies of the trx protein will be pursued by defining the mutations present among the large collection of extant trx alleles and by generating a variety of mutations in genes that will be transformed into the genome.

描述：这是一项修订的建议，以继续研究 负责稳定，长期维护的机制 果蝇发育过程中的同源基因表达。它专注于 Trithorax基因已被证明是 维持BX-C和BX-C和 ANTP-C并平衡Polycomb基因的影响， 它充当同源基因表达的负调节剂。通过 TRX基因的详细遗传和分子遗传分析，以及 通过TRX蛋白的详细功能解剖，Harte博士 提出对解决方案的雄心勃勃的实验攻击 可能涉及染色质构象与状态之间的关系 转录激活。这是转录的一个方面 使生物学家着迷多年的监管，并且仍然存在 理解不佳，但可能会引起越来越多的兴趣 接下来的几年。 在上一个赠款期间，大力致力于 详细描述了对TRX突变的各种响应 基因以及TRX基因的克隆和分子表征。 发现TRX转录单元很大并产生 复杂的RNA产物和蛋白质的复杂阵列估计为范围 大小在360至420 kd之间。为了表征这一点的英勇努力 基因（包括使用包含34的救援结构的转换 KB基因组片段已被发现得到了奖励 蛋白质编码序列中的可识别序列域，包括 假定的类固醇受体家族DNA结合结构域和一个区域 与人类全1蛋白是同源的 儿童急性淋巴细胞性白血病。 TRX蛋白已显示 在体外结合与DNA结合，与聚二烯上的63个特定位点结合 染色体，并在功能上与相关的TRX-G蛋白相关联， ASH-1和功能性TRX响应元件已被鉴定。 拟议研究的实验目标构成了广泛的 和TRX的详细分析。 TRX染色体的高分辨率映射 绑定位点将使用记者的种系变换来追求 构造以绘制染色体结合位点的构造和 这些序列影响连接基因的表达。表征 在最近的成功中，将追求体外DNA结合的体外DNA结合 表明TRX蛋白的片段在迁移率移动中活跃 用660 bp核心的测定。这些研究将扩展到更好 定义具有约束力的亲和力和特殊性，并与 renato paro，将TRX蛋白在体内结合的位点绘制位点。到 识别与哪种trx联合的其他蛋白质，几种方法是 描述的，包括共免疫沉淀，进一步表征 TRX和ASH-1之间的明显关联，以及使用聚烯 在各种突变背景中的结合测定。 TRX将过表达 使用热休克转基因构建体并增加其基因剂量 确定对同源基因表达和测试模型的影响 PC-G和TRX-G蛋白之间的竞争。最后， TRX蛋白的结构/功能研究将由 定义大量现存的TRX中存在的突变 等位基因，并通过在基因中产生各种突变 转变为基因组。