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：这是一项修订提案，旨在继续研究 负责稳定、长期维护的机制 果蝇发育过程中的同源异型基因表达。它专注于 trithorax 基因，已被证明是 维持 BX-C 基因的表达模式和 ANTP-C 并抵消 Polycomb 组基因的影响， 作为同源异型基因表达的负调节因子。通过 trx 基因的详细遗传和分子遗传分析，以及 Harte 博士对 trx 蛋白进行了详细的功能剖析 提出了一项雄心勃勃的实验性攻击，解决其解决方案 可能涉及染色质构象和状态之间的关系 转录激活。这是转录的一个方面 多年来一直让生物学家着迷的监管，并且仍然存在 知之甚少，但可能会在期间引起越来越多的兴趣 未来几年。 在上一个资助期间，我们投入了大量精力 详细描述了许多人对 trx 突变的不同反应 基因以及 trx 基因的克隆和分子表征。 发现 trx 转录单位很大并且产生 一系列复杂的 RNA 产物和蛋白质，估计范围 尺寸在 360 至 420 kd 之间。刻画这一特征的英勇努力 基因（包括用含有 34 kb 基因组片段）已因几个发现而受到奖励 蛋白质编码序列中可识别的序列域，包括 推测的类固醇受体家族 DNA 结合结构域和区域 与人类 ALL-1 蛋白同源，该蛋白与 儿童急性淋巴细胞白血病。 trx蛋白已被证明 体外与 DNA 结合，与多线上的 63 个特定位点结合 染色体，并与相关的 trx-G 蛋白功能相关， ash-1 和功能性 trx 反应元件已被鉴定。 拟议研究的实验目标构成了广泛的 以及trx的详细分析。 trx 染色体的高分辨率作图 将使用报告基因的种系转化来追踪结合位点 构建图谱染色体结合位点和能力 这些序列影响连锁基因的表达。表征 受最近成功的推动，体外 DNA 结合将被追求 表明 trx 蛋白片段在迁移率变化中具有活性 使用 660 bp 核心 PRE 进行测定。这些研究将进一步扩大 定义结合亲和力和特异性，并与 Renato Paro，绘制了体内 trx 蛋白结合位点的图谱。到 识别与 trx 相关的其他蛋白质，有几种方法 描述，包括免疫共沉淀，进一步表征 trx 和 ash-1 之间的明显关联以及聚乙烯的使用 各种突变背景中的结合测定。 Trx会过度表达 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的影响并测试模型 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 trx 中存在的突变 等位基因并通过在基因中产生各种突变 转化到基因组中。