STRUCTURE OF INTERPHASE AND METAPHASE CHROMOSOMES

间期和中期染色体的结构

• 批准号: 3278030
• 负责人: Martin A Gorovsky
• 金额: $ 21.23万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-12-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278030
• 关键词: DNA; DNA binding protein; DNA gyrase; Xenopus; aldehyde dehydrogenases; cell cycle; chromatin; chromosomes; density gradient ultracentrifugation; electron microscopy; fresh water environment; gel electrophoresis; gene expression; genetic manipulation; genetic mapping; genetic transcription; histones; molecular cloning; nonhistone nucleoprotein; nucleic acid sequence; nucleoproteins; nucleosomes; radiotracer; tissue /cell culture

We shall continue our analysis of the chromatin assembly reaction catalyzed by the high speed supernatant of Xenopus laevis oocytes (S-150). The fate of the histones and non-histone proteins during the reaction will be monitored by immunoblots with specific histone antibodies and by T25I labelling of the DNA-bound proteins in the nascent chromatin. We shall conduct parallel studies on the composition of nascent chromatin in a kidney cell line of Xenopus laevis, to ascertain whether the in vitro process of assembly which we have characterized reflects the in vivo reaction that takes place during chromatin replication. We will characterize the dynamic (torsionally strained), transcriptionally active chromatin assembled with TFIIIA, the positive transcription factor of the 5S RNA gene. The composition of the dynamic minichromosomes will be examined by immunoblots with the histone antibodies and by 125I labeling. The dynamic, "open" nucleosomes will be characterized by electron microscopy and by MNase (micrococcal nuclease) and DNase I digestion. We will continue the purification of the factors required for the assembly of transcriptionally active chromatin. In particular, we shall purify and characterize the eucaryotic DNA gyrase, an enzyme which plays a key role in the production and maintenance of the torsionally strained supercoils in dynamic chromatin. These studies should illuminate the mechanisms which govern gene expression in eucaryotes.

我们将继续我们的分析染色质组装反应催化 非洲爪蟾卵母细胞（S-150）的高速离心上清液。 命运 组蛋白和非组蛋白蛋白在反应过程中将 通过特异性组蛋白抗体的免疫印迹和T25 I 标记新生染色质中的DNA结合蛋白。 我们将 对新生染色质的组成进行平行研究， 非洲爪蟾肾细胞系，以确定是否在体外 我们所表征的组装过程反映了体内 染色质复制过程中发生的反应。 我们将描述动态（扭转应变），转录 活性染色质与正转录因子TFIIIA组装 5S RNA基因。 动态微型染色体的组成将是 用组蛋白抗体免疫印迹和125 I标记检测。 动态的、“开放的”核小体的特征是电子 通过显微镜和MNase（微球菌核酸酶）和DNase I消化来测定。 我们将继续净化大会所需的因素 转录活跃的染色质。 特别是，我们将净化和 描述真核DNA旋转酶的特征，这种酶在 扭转应变超螺旋的生产和维护 动态染色质 这些研究应该阐明 控制着真核生物的基因表达。