MOLECULAR BASIS FOR SELECTIVE EXPRESSION OF TRNA GENES

TRNA 基因选择性表达的分子基础

• 批准号: 3282013
• 负责人: KAREN U SPRAGUE
• 金额: $ 11.34万
• 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 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282013
• 关键词: Bombycidae; DNA directed RNA polymerase; DNA footprinting; alanine; binding proteins; cell differentiation; cell free system; developmental genetics; eukaryote; gene expression; genetic manipulation; genetic transcription; genome; immunochemistry; microinjections; molecular cloning; natural gene amplification; nucleic acid probes; nucleic acid sequence; radiotracer; transfer RNA

To analyze the molecular mechanisms involved in selective gene expression in differentiated eukaryotic cells, we are studying the alanine tRNA genes from the silkworm, Bombyx mori. These genes are interesting because their products, alanine tRNAs, are accumulated in a tissue-specific fashion. One species of alanine tRNA (tRNACAla) is present in all tissues and is therefore designated constitutive, while the other species (tRNASGAla) is found only in the silkgland. Our goal is to discover the molecular basis for the tissue-specific appearance of alanine tRNA in the silkworm. An investigation into the mechanism responsible for this phenomenon addresses the general problem of gene control during eukaryotic cellular differentiation. We think it likely that tissue-specific regulation of polymerase III transcription is the basis of the differential accumulation of the two alanine tRNAs. We have discovered both cis-acting and trans-acting elements that differentially affect transcription of these templates. Because these effects are large, and because they can be analyzed in vitro, we are in a strong position to determine the precise molecular basis of differential tRNAAla gene expression. We propose a detailed in vitro analysis of the components that interact in trans with the tRNAASG1a gene. In brief, we will define the transcription component(s) that differentially affects transcription of tRNACAla and tRNASGAla genes. We will ask whether the gene-specific component exerts its effect by binding to the gene itself, or by binding to part of the general transcription apparatus, and we have proposed experiments to determine what step in the overall transcription reaction is differentially affected on the two templates. To determine the relevance of our in vitro analysis to the situation in vivo, we will use in vivo footprinting to examine transcription complexes formed in intact silkworm cells. In addition, with antibodies raised against gene-specific transcription components, we will determine the tissue distribution of these factors to see if modulations in their level could account for the activity of tRNASGAla genes in the silkgland.

分析选择性基因的分子机制 在分化的真核细胞中表达，我们正在研究 家蚕的丙氨酸tRNA基因。 这些基因 有趣的是，它们的产物，丙氨酸转移RNA， 以组织特异性的方式。 一种丙氨酸tRNA （tRNACAla）存在于所有组织中，因此被命名为 组成型，而另一种（tRNASGAla）仅在 丝腺 我们的目标是发现 家蚕中丙氨酸tRNA的组织特异性表现。 一个 调查造成这一现象的机制 解决了真核生物过程中基因控制的一般问题 细胞分化 我们认为组织特异性的聚合酶调节 III转录是差异积累的基础， 两个丙氨酸转移RNA 我们已经发现了顺式作用和 差异影响转录的反式作用元件 这些模板。 因为这些影响是巨大的，因为它们 可以在体外进行分析，我们有能力确定 差异tRNAAla基因精确分子基础 表情 我们提出了一个详细的成分在体外分析， 与tRNAASG1a基因相互作用。 简而言之，我们将 定义差异影响的转录组分 tRNACAla和tRNASGAla基因的转录。 我们会问， 基因特异性成分通过结合到 基因本身，或通过结合到一般转录的一部分 仪器，我们已经提出了实验，以确定什么步骤 在整个转录反应中受到不同程度的影响 在两个模板上。 为了确定我们的体外 针对体内情况分析，采用体内足迹法 检测完整家蚕细胞中形成的转录复合物。 此外，随着针对基因特异性抗体的产生， 转录成分，我们将确定组织分布 看看它们的水平是否可以解释 tRNASGAla基因在丝腺中的活性。