EUKARYOTIC CHROMOSOME REPLICATION

该提案的重点是在酵母中进行染色体复制 酿酒。特别令人感兴趣的是复制的机制 相关事件在细胞周期中的整合，以及如何处理 发生在S阶段的是有时间规律的。重大实验 工具是培养密度转移，平衡密度带到 分离新旧DNA，一维和二维凝胶电泳法 从完全复制的数据解析复制中间结构 用克隆探针进行DNA-DNA和RNA-DNA杂交 染色体序列，以及新型质粒的构建。该项目 将研究：沿延伸的复制的时间模式 以获取ARS(自主复制)的证据 序列)元素是复制的起源；混合的影响 早期和晚期复制染色体序列，作为一种确定 决定复制时间的顺式作用元素；复制时间和性质 着丝粒和端粒在细胞周期中的复制；是否“渗漏” 在cdc7突变细胞中复制特定序列是 特定的ARS元件；不同的ARS元件对质粒复制的影响 复制起点和转录单位的组合，作为一种方法 理解如何解决拓扑冲突；可能的 原生2分离和扩增的机制 MU质粒；以及识别染色体基因的可能性 可以抑制有缺陷的ARS元素，作为理解 ARS函数的性质。这些实验的结果应该是，在 最基本的是，对哺乳动物的相关机制产生了可测试的想法 并最终有助于理解某些人类 病理学。

The proposal focuses on chromosome replication in the yeast Saccharomyces cerevisiae. Of particular interest is the mechanisms by which replication related events are integrated within the cell cycle, and how processes taking place in the S phase are temporally regulated. Major experimental tools are culture density transfers, equilibrium density banding to separate old and new DNAs, one and two dimensional gel electrophoresis to resolve replication intermediate structures from completely replicated ones, DNA-DNA and RNA-DNA hybridizations using cloned probes for chromosomal sequences, and the construction of novel plasmids. The project will examine: the temporal pattern of replication along extended stretches of chromosomes to obtain evidence whether ARS (autonomous replication sequence) elements are origins of replication; the effect of intermixing early and late replicating chromosome sequences, as an approach to defining cis-acting elements that determine replication time; the time and nature of centromere and telomere replication in the cell cycle; whether "leakage" replication of specific sequences in cdc7 mutant cells is a property of specific ARS elements; the effect on plasmid replication of different combinations of replication origins and transcription units, as an approach to understanding how the topological conflicts are resolved; possible mechanisms involved in the segregation and amplification of the native 2 Mum plasmid; and, the possibility of identifying chromosomal genes which can suppress defective ARS elements, as an approach to understanding the nature of ARS function. The results of these experiments should, at the very least, yield testable ideas about related mechanisms in mammalian cells and ultimately contribute to an understanding of some human pathologies.