EUKARYOTIC CHROMOSOME REPLICATION

该提案的重点是染色体复制在酵母 啤酒。 特别感兴趣的是复制的机制 相关事件整合在细胞周期中，以及过程如何 在S期发生的变化是暂时调节的。 主要实验 工具是文化密度转移，平衡密度带， 分离新旧DNA，一维和二维凝胶电泳， 从完全复制中解析复制中间结构 使用克隆探针进行DNA-DNA和RNA-DNA杂交， 染色体序列和新型质粒的构建。 项目 我将研究：复制的时间模式沿着延伸延伸 以获得是否ARS（自主复制）的证据 序列）元素是复制的起点;混合的效果 早期和晚期复制染色体序列，作为一种方法来定义 决定复制时间的顺式作用元件; 细胞周期中的着丝粒和端粒复制;是否“渗漏” CDC 7突变细胞中特异性序列的复制是 特异性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， 理解拓扑冲突是如何解决的;可能的 参与分离和扩增的天然2 Mum质粒;以及鉴定染色体基因的可能性， 可以抑制有缺陷的ARS元件，作为理解 ARS功能的性质。 这些实验的结果应该，在 至少，在哺乳动物的相关机制方面， 细胞，并最终有助于了解一些人类 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.