COMPLEXES THAT REGULATE 5S RNA GENE EXPRESSION

调节 5S RNA 基因表达的复合物

• 批准号: 3271130
• 负责人: DONALD D BROWN
• 金额: $ 29.47万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 1975
• 资助国家: 美国
• 起止时间: 1975-09-01 至 1990-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271130
• 关键词: DNA; DNA directed RNA polymerase; Escherichia coli; RNA; Xenopus; antibody formation; binding proteins; cell transformation; developmental genetics; early embryonic stage; endonuclease; gene complementation; gene expression; genetic manipulation; genetic mapping; genetic promoter element; genetic regulation; genetic transcription; germ cells; laboratory rabbit; molecular cloning; molecular genetics; nucleic acid hybridization; nucleic acid sequence; operon; radiotracer; scintillation counter

Two kinds of 5S RNA multigene families exist in the genome of Xenopus. The oocyte-type 5S RNA genes function only in the oocyte and are repressed in somatic cells. The somatic 5S RNA genes are transcribed actively in both cocytes and somatic cells. The expression of these genes can be assayed by the synthesis of 5S RNA in vitro, in vivo, or with isolated chromatin. The goal of these experiments is to reproduce in vitro with purified components the same differential control of gene activity that is seen in vivo. In somatic cells of Xenopus, active 5S RNA genes are associated with transcription factors to form stable transcription complexes. Repressed genes are not associated with these factors but rather complexed with nucleosomes that keep transcription factors from forming complexes with the gene. Both the active and repressed state of 5S RNA genes are stable. One purpose of this project is to identify the molecules associated with active and repressed complexes and characterize the details of how they interact with each other and with DNA signals in and around the 5S RNA gene. The gene for the 5S-specific positive transcription factor (TFIIIA) will be cloned, expressed in E. coli, and mutants prepared in order to map further the functional amino acids of the protein that bind to critical regions of the internal control region of the 5S RNA gene and to other components of the transcription complex. A transient developmental expression assay is sought that will reproduce the control of the two kinds of 5S RNA genes that occurs during early embryogenesis. From this assay, the exact DNA sequences in the 5S RNA gene involved in the developmental control, as well as the molecules in embryos that are responsible for reading the DNA signals, will be elucidated. In addition, this assay will be designed to determine how discrimination between two genes is established and then perpetuated in early embryogenesis. It is suggested that these stable transcription complexes might play a role in the phenomenon of determination or "commitment" of genes in development. Some experimental schemes will test this proposal.

爪蟾基因组中存在两种5S RNA多基因家族。 这 卵母细胞型 5S RNA 基因仅在卵母细胞中发挥作用，并且在 体细胞。 体细胞 5S RNA 基因在两种细胞中均活跃转录 核细胞和体细胞。 这些基因的表达可以通过以下方式测定 5S RNA 的体外、体内或与分离的染色质的合成。 这 这些实验的目标是用纯化的成分进行体外复制 与体内观察到的相同的基因活性差异控制。 在 爪蟾体细胞中，活跃的 5S RNA 基因与 转录因子形成稳定的转录复合物。 压抑 基因与这些因素无关，而是与 阻止转录因子与核小体形成复合物 基因。 5S RNA 基因的活性和抑制状态都是稳定的。 一 该项目的目的是识别与活性相关的分子 和被压抑的情结并描述它们如何相互作用的细节 彼此之间以及与 5S RNA 基因内部和周围的 DNA 信号。 这 5S特异性正转录因子（TFIIIA）的基因将是 克隆，在大肠杆菌中表达，并制备突变体以进一步作图 蛋白质的功能性氨基酸与关键区域结合 5S RNA 基因的内部控制区域和其他成分 转录复合物。 瞬时发育表达测定是 寻求重现两种5S RNA基因的控制 发生在早期胚胎发生期间。 从这个测定中，准确的 DNA 5S RNA 基因中的序列也参与发育控制 作为胚胎中负责读取 DNA 的分子 信号，将予以阐明。 此外，该测定将被设计为 确定如何建立两个基因之间的区分，然后 在早期胚胎发生过程中持续存在。 建议这些稳定的 转录复合物可能在以下现象中发挥作用 发育中基因的决定或“承诺”。 一些实验性的 计划将测试该提案。