MECHANISM OF SELECTIVE EXPRESSION OF TRNA GENES

TRNA基因选择性表达机制

• 批准号: 2176759
• 负责人: KAREN U SPRAGUE
• 金额: $ 31.54万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176759
• 关键词: Bombycidae; DNA footprinting; gel mobility shift assay; genetic promoter element; genetic regulation; genetic transcription; laboratory rabbit; molecular cloning; recombinant proteins; tissue /cell culture; transcription factor; transfer RNA

The goal of the proposed experiments is to determine the mechanism of tissue-specific regulation of tRNA transcription. We will focus on two tRNA/A1a genes of silkworms, one of which (C) is transcribed constitutively, and the other (SG) only in silkglands. We will use both in vitro and in vivo assays of promoter function, and will analyze in detail protein-DNA complexes that include the upstream segment of C and SG promoters. This segment is interesting because it controls differential transcription from the two promoters in vitro, in homologous transcription systems. The upstream promoters for both genes are contained within -35 bp DNA segments immediately upstream of their transcription initiation sites, and promoter function for the C gene is conferred by two AT-rich boxes within this segment. The specific sequence or structural distinction that is responsible for differential transcription from C and SG promoters is not known. We plan the following experiments to understand how this critical promoter segment functions: We will use both in vitro and in vivo assays (in cultured cells and in intact silkglands) to identify the sequence and/or structural features that functionally distinguish the upstream promoters of C and SG genes, and that confer silkgland specificity on the SG promoter. We will compare the interaction of TFIIIB with C and SG upstream promoters, using TFIIIB isolated either from silkglands or from a tissue (ovaries) in which the SG promoter is inactive. Based on this analysis, we will identify and purify candidate polypeptides that are likely to be key discriminators between C and SG promoters. Polypeptides that are found to regulate SG transcription in vitro will be functionally tested in vivo by determining their ability to regulate SG transcription when ectopically expressed in cultured Drosophila and silkworm cells and in intact, differentiated silkworm tissues.

所提出的实验的目标是确定 tRNA转录的组织特异性调节。我们将重点关注两个 家蚕的tRNA/A1 a基因，其中之一（C）是转录的 另一种（SG）仅存在于丝腺中。我们将使用两者 启动子功能的体外和体内测定，并将在 详细的蛋白质-DNA复合物，包括C的上游片段， SG启动子。这一部分很有趣，因为它控制着 在体外，在同源 转录系统。两个基因的上游启动子是 包含在紧邻其上游的~ 35 bp DNA片段内， 转录起始位点，C基因的启动子功能是 由这一部分中的两个AT丰富的盒子赋予。特定序列 或结构上的区别， C和SG启动子的转录是未知的。 我们计划进行以下实验，以了解这种关键性的 启动子片段功能： 我们将使用体外和体内试验（在培养的细胞和在培养的细胞中）。 完整的丝腺）以鉴定序列和/或结构特征 在功能上区分C和SG基因的上游启动子， 并赋予SG启动子丝腺特异性。 我们将比较TFIIIB与C和SG上游的相互作用 启动子，使用从丝腺或组织分离的TFIIIB （卵巢），其中SG启动子失活。根据这一分析， 我们将鉴定和纯化候选多肽， C和SG启动子之间的关键区别。 发现在体外调节SG转录的多肽将 通过测定其调节细胞内蛋白质的能力 SG在培养的果蝇中异位表达时的转录， 家蚕细胞和完整的分化的家蚕组织中。