MOLECULAR BASIS OF DROSOPHILA HOMEOTIC GENE

果蝇同源基因的分子基础

• 批准号: 3296067
• 负责人: Peter J. Harte
• 金额: $ 12.11万
• 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 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3296067
• 关键词: DNA; Drosophilidae; RNA; alleles; body regions; complementary DNA; cytogenetics; developmental genetics; enzyme linked immunosorbent assay; fluorescent dye /probe; gel electrophoresis; gene complementation; gene expression; gene interaction; gene mutation; genetic mapping; genetic promoter element; genetic regulation; genetic transcription; head; homeobox genes; in situ hybridization; molecular cloning; molecular genetics; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; radionuclides; regulatory gene; thorax; transposon /insertion element

The long term goal of this research is to understand the molecular mechanisms underlying the regulation of the homeotic genes during development. Specifically, we will investigate the regulation of the homeotic genes by the trithorax locus (trx) in Drosophila melanogaster. Genetic and molecular evidence suggests that trx encodes a positive trans-acting regulator of many of the homeotic genes in the Bithorax Complex (BX-C) and Antennapedia Complex (ANT-C). The trx gene product is required to establish and maintain the normal level of expression of these genes in their appropriate segments. We will investigate how trx exerts its effects on the homeotic genes by molecular cloning of the trx DNA sequences and by characterizing the structure of its RNA and protein products. We will also determine the effects of trx mutations on the expression of selected homeotic gene products. The major strategy for cloning trx will be chromosomal walking. Determination of the location and physical extent of the locus will involve molecular mapping of trx mutations and rescue of trx mutations by P element-mediated transformation. The primary structure of the trx gene and its RNA and protein products will be determined by Northern analysis, S1 nuclease protection, primer extension and DNA sequencing of trx cDNA clones and corresponding genomic DNA. Temporal and spatial expression patterns of trx will be determined by in situ hybridization of trx gene probes to tissue sections and by immunofluorescent staining of embryos with anti-trx antibodies. The effects of trx mutations on homeotic gene expression will also use these methods, with the available probes for selected homeotic gene products. The results of these studies will provide the foundation for further experiments to determine whether trx protein(s) interacts directly with the homeotic genes and address how trx functions in the positive regulation of these genes to effects their proper spatial expression during development.

这项研究的长期目标是了解分子 同源基因调控的机制 在开发过程中。 具体来说，我们将调查 三胸位点（trx）对同源异型基因的调节 黑腹果蝇。 遗传和分子证据 表明 trx 编码一个正反式作用调节因子 Bithorax Complex (BX-C) 中的许多同源异型基因和 触角复合体（ANT-C）。 需要 trx 基因产物 建立并维持这些基因的正常表达水平 基因在其适当的片段中。 我们将研究如何 trx 通过分子克隆对同源基因发挥作用 trx DNA 序列并通过表征其结构 RNA 和蛋白质产品。 我们还将确定以下影响 trx突变对选定同源基因表达的影响 产品。 克隆 trx 的主要策略是染色体步移。 确定轨迹的位置和物理范围 将涉及 trx 突变的分子图谱和 trx 的拯救 P元件介导的转化引起的突变。 初级 trx基因及其RNA和蛋白质产物的结构将是 通过 Northern 分析、S1 核酸酶保护、引物确定 trx cDNA 克隆的延伸和 DNA 测序以及 相应的基因组DNA。 时间和空间表达 trx的模式将通过trx的原位杂交来确定 对组织切片进行基因探针并通过免疫荧光染色 具有抗trx抗体的胚胎。 trx突变的影响 同源异型基因表达也将使用这些方法，其中 针对选定的同源异型基因产物的可用探针。 这些研究结果将为进一步的研究奠定基础 确定 trx 蛋白是否直接相互作用的实验 与同源基因并解决 trx 在 这些基因的正调控以影响其适当的空间 发育过程中的表达。