DNA REPLICATION AND CHROMOSOME STRUCTURE IN YEAST

酵母中的 DNA 复制和染色体结构

• 批准号: 3274402
• 负责人: VIRGINIA A. ZAKIAN
• 金额: $ 14.37万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3274402
• 关键词: Saccharomyces; affinity chromatography; chromosomes; fungal genetics; gel electrophoresis; genetic manipulation; genetic transcription; meiosis; plasmids; radiotracer; ribosomal DNA; ribosomal RNA; synchronous cell division

Experiments in this grant focus on sites used for initiation of chromosomal DNA replication using the lower eukaryote S. cerevisiae as a model system. ARSs (autonomously replicating sequences) are putative initiation sites for DNA replication recognized by their ability to support the self-replication of plasmid DNAs. The behavior of 14 different ARSs will be examined during mitosis and meiosis. For the mitotic studies, the presence and copy number of ARS plasmids will be monitored by a visual assay sensitive to plasmid copy number. These studies will provide a detailed description of the efficiency with which an individual ARS promotes the inheritance of plasmid DNAs during mitosis and meiosis. It will also be determined if trans-acting factors encoded by 2 Mum DNA can effect the mitotic stability of either synthetic or authentic yeast chromosomes. DNA contained within small replication bubbles and therefore, by definition, containing an origin of DNA replication, will be isolated from yeast chromosomal DNA after selective labelling with 4-thiouridine. Single-stranded DNA containing thio-substituted residues will be purified by affinity chromatography and used to screen a library of yeast DNA. If this origin-enriched DNA is enriched in ARSs, the experiment will establish a correlation between ARSs and replication origins in yeast chromosomes. Regardless of the outcome in terms of ARSs, the procedure will be valuable for isolating early replicating sequences which can be used in nuclear matrix experiments. The influence of nuclear sub-structure on the organization of yeast DNA, especially in terms of DNA replication, will be examined. Low salt extraction procedures will be developed to test if association of replication forks with the nuclear matrix is observed with different isolation methods. The interaction of replication origins with the nuclear matrix will be examined in populations of cells arrested at different points in the cell cycle: these experiments will determine if origins are permanently or transiently associated with the matrix. As a longer range goal, the association of other structural DNAs with the matrix will be examined. The experiments in this proposal will provide insights into mechanisms involved in control of entry into S-phase and the establishment of a temporal program for chromosome replication. Detailed knowledge of these events is important for understanding how they can be perturbed in abnormal cells.

本次资助的实验重点关注用于染色体起始的位点 使用低等真核生物酿酒酵母作为模型系统进行 DNA 复制。 ARS（自主复制序列）是假定的起始位点 DNA 复制因其支持自我复制的能力而受到认可 质粒 DNA。 14 种不同 ARS 的行为将在 有丝分裂和减数分裂。 对于有丝分裂研究，存在和拷贝数 ARS 质粒将通过对质粒敏感的视觉测定进行监测 拷贝数。 这些研究将详细描述 单个ARS促进质粒遗传的效率 有丝分裂和减数分裂期间的DNA。 还将确定是否 2 Mum DNA编码的反式作用因子可影响有丝分裂稳定性 合成或真实的酵母染色体。 内含DNA 小复制气泡，因此，根据定义，包含 DNA复制起点，将从酵母染色体DNA中分离出来 用4-硫尿苷选择性标记后。 单链DNA 含有硫代残基的残基将通过亲和力纯化 色谱法并用于筛选酵母 DNA 文库。 如果这个 起源富集的DNA在ARS中富集，该实验将建立一个 ARS 与酵母染色体复制起点之间的相关性。 无论 ARS 的结果如何，该程序都将很有价值 用于分离早期复制序列，可用于核 矩阵实验。 核亚结构对核子结构的影响 酵母 DNA 的组织，特别是 DNA 复制方面，将 检查了。 将开发低盐提取程序来测试是否 观察到复制叉与核基质的关联 不同的隔离方法。 复制起点与复制起点的相互作用 将在捕获的细胞群中检查核基质 细胞周期的不同点：这些实验将确定是否 原点与矩阵永久或暂时相关。 作为一个 更远距离的目标，其他结构 DNA 与基质的关联 将接受检查。 本提案中的实验将提供见解 进入 S 期控制所涉及的机制 建立染色体复制的时间程序。 详细的 了解这些事件对于理解它们如何发生非常重要 异常细胞受到干扰。