MINICHROMOSOMES INTRODUCED IN CELL NUCLEI

细胞核中引入微型染色体

• 批准号: 3294098
• 负责人: MASARU RYOJI
• 金额: $ 13.34万
• 依托单位: SCRIPPS CLINIC AND RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 1990-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294098
• 关键词: Xenopus; cell nucleus; chromatin; chromosomes; circular DNA; conformation; egg /ovum; electron microscopy; electrophoresis; fresh water environment; gene expression; genetic manipulation; genetic mapping; genetic recombination; genetic transcription; microinjections; nucleic acid sequence; nucleosomes; protein structure; radiotracer

Our goal is to understand how the structure of chromatin is involved in the regulation of gene expression and replication. Several specific questions will be asked by employing minichromosomes that are assembled on DNA injected in frog oocytes and eggs. Firstly, the structure of the peculiar "dynamic" minichromosomes will be studied. These minichromosomes carry unconstrained DNA supercoils, and are thought to represent transcriptionally competent chromatin. Dynamic minichromosomes will be separated from the conventional "static" minichromosomes, and their structure will be characterized by nuclease digestions, by electrophoretic identification of protein composition, and by electron microscopic observations. Taking advantage of the small size of the 5S RNA gene, further efforts will directed to isolate and analyze a fraction of dynamic minichromosomes that are actually engaged in transcription. Secondly, replication of dynamic minichromosomes will be studied with respect to whether they are preferentially replicated relative to the conventional, static minichromosomes, and whether the dynamic structure can be directly propagated to the progeny minichromosomes. The efficient assembly of dynamic minichromosomes on the preformed complex of 5S DNA and transcription factor TFIIIA will be exploited to examine such a possibility. Thirdly, nucleosome positions in inactive, static minichromosomes carrying a gene for either 5S RNA or histone H4 will be examined in order to test the hypothesis that the gene inactivation could be a result of blockage of promoter sequences by a nucleosome core. Minichromosomes carrying 32P label at a unique restriction site will be used to map nucleosome positions.

我们的目标是了解染色质的结构如何参与 基因表达和复制的调节。 几个具体问题 将通过使用组装在 DNA 上的微型染色体来询问 注射到青蛙卵母细胞和卵子中。 首先，奇特的“动态”微型染色体的结构将是 研究过。 这些微型染色体携带不受约束的 DNA 超螺旋，并且 被认为代表具有转录能力的染色质。 动态的 微型染色体将与传统的“静态”分离 微型染色体，其结构将由核酸酶表征 通过电泳鉴定蛋白质成分进行消化，以及 通过电子显微镜观察。 利用小尺寸的优势 5S RNA基因，进一步的努力将针对分离和分析 实际参与的动态微型染色体的分数 转录。 其次，将研究动态微型染色体的复制 关于它们是否相对于 常规的静态微型染色体，以及动态结构是否可以 直接繁殖到后代微型染色体。 高效的 动态微型染色体在 5S DNA 预制复合体上的组装 将利用转录因子 TFIIIA 来检验这种可能性。 第三，核小体在不活跃的静态微型染色体中的位置，携带 将检查 5S RNA 或组蛋白 H4 的基因以进行测试 假设基因失活可能是由于 由核小体核心组成的启动子序列。 携带 32P 的微型染色体 独特限制位点处的标签将用于绘制核小体位置。