SOMATIC GENETICS OF A CLONED HUMAN RIBOSOMAL PROTEIN

克隆人核糖体蛋白的体细胞遗传学

• 批准号: 3295741
• 负责人: DONALD J ROUFA
• 金额: $ 12.42万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295741
• 关键词: cell transformation; chemical carcinogen; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic transcription; hamsters; messenger RNA; molecular cloning; molecular genetics; mutant; nucleic acid probes; nucleic acid sequence; point mutation; protein biosynthesis; ribosomal RNA; ribosomal proteins; ribosomes; structural genes; transfection

The project described in this application employs mammalian somatic cell and molecular genetics to recognize and characterize a novel set of human structural gene and regulatory mutations. As for all mutant surveys, the ultimate value of this project depends directly on the nature of mutants isolated. Because many factors that usually constrain the spectrum of viable mutants observed are obviated by genetic design of this experimental system, and because a comprehensive set of available nucleic acid reagents facilitate the required molecular analyses, mutants isolated are likely to comprise a novel and informative genetic resource. Application of current molecular genetic technology to several important medical (as well as agricultural and industrial) problems awaits significantly better understanding of mechanisms regulating mammalian gene expression. Information expected from this project is likely to contribute significantly to the required body of knowledge. A set of "wildtype" cell lines that contain a coresident pair of functional, ribosomal protein (RPS14) alleles has been constructed using DNA transfection. The genes used are unique in that their expression results in selectible drug resistance phenotypes and their complete nucleic acid sequences are known. RNA and protein products of the two alleles can be resolved by convenient molecular assays. Thus, mutations which alter either gene's product (structural gene mutations) can be distinguished from mutations that disrupt their normal patterns of expression (regulatory mutations). Because the "wildtype" cells maintain two functionally regulated copies of the gene, they can express mutations which in normal cells would be lethal. One copy of the gene can be rescued from mutant clones; and these can be mapped to specific nucleic acid sequences. Substantial biochemical and genetic data from lower eu- and prokaryotic systems indicate that ribosomal protein genes are stringently regulated by the combined action of transcriptional and post-transcriptional mechanisms. Experiments to distinguish which of these operate on a human ribosomal protein gene and to discern cis-active regulatory sites within the human gene are described.

本应用程序中描述的项目采用哺乳动物 识别和表征的体细胞和分子遗传学 一组新型的人类结构基因和调节突变。 至于所有突变调查，该项目的最终价值 直接取决于孤立的突变体的性质。 因为很多 通常会限制可行突变体频谱的因素 通过该实验的遗传设计观察到观察到的 系统，以及一组全面的可用核酸 试剂促进所需的分子分析，突变体 孤立的可能包括一个新颖而有益的遗传 资源。 当前分子遗传技术的应用 几种重要的医学（以及农业和工业） 问题等待着更好地理解机制 调节哺乳动物基因表达。 预期的信息 这个项目可能会为 所需的知识。 一组“野生型”细胞系，其中包含一对核心 功能性，核糖体蛋白（RPS14）等位基因已构造 使用DNA转染。 所使用的基因是独一无二的 表达导致可选的耐药性表型和 它们的完整核酸序列是已知的。 RNA和 两种等位基因的蛋白质产品可以通过方便解决 分子测定。 因此，改变了基因的突变 产品（结构基因突变）可以区分 破坏其正常表达方式的突变 （调节突变）。 因为“野生型”细胞维持两个 在功能调节基因的副本中，它们可以表达 正常细胞中的突变将是致命的。 一份 基因可以从突变克隆中救出；这些可以映射 特定的核酸序列。 实质性的生化和 来自欧盟较低和原核生物系统的遗传数据表明 核糖体蛋白基因受到组合的严格调节 转录和转录后机制的作用。 实验以区分这些对人的运作 核糖体蛋白基因和辨别顺式活性调节位点 描述了人类基因内。