DNA REPLICATION AND CHROMOSOME STRUCTURE IN YEAST

这项资助的实验集中在用于启动染色体的位点上。 利用低等真核生物S.酿酒酵母作为模型系统。 ARS（自主复制序列）是一个假定的起始位点， DNA复制通过其支持自我复制的能力来识别 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， 将通过对质粒敏感的目视试验监测ARS质粒 副本编号。 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 μ m DNA编码的反式作用因子影响有丝分裂的稳定性 合成或真实的酵母染色体。 其中含有的DNA 小的复制气泡，因此，根据定义， DNA复制起点，将从酵母染色体DNA中分离 在用4-硫代尿苷选择性标记后。 单链DNA 含有硫代取代的残基的化合物将通过亲和层析纯化， 将其通过柱层析纯化并用于筛选酵母DNA文库。 如果这 来源富集的DNA在ARS中富集，该实验将建立一个 酵母染色体中ARS和复制起点之间的相关性。 无论ARS的结果如何，该程序都是有价值的 用于分离早期复制序列， 矩阵实验 原子核亚结构对核分裂的影响 酵母DNA的组织，特别是在DNA复制方面， 考察 将开发低盐浸提程序，以测试 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 核基质将在细胞群中进行检查， 细胞周期的不同点：这些实验将确定 原点与矩阵永久或暂时相关。 作为 更长远的目标，其他结构DNA与基质的关联 将被审查。 该提案中的实验将提供见解 进入S期的控制机制， 建立一个时间程序染色体复制。 详细 对这些事件的了解对于理解它们是如何 在异常细胞中受到干扰。

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.