DNA REPLICATION IN SACCHAROMYCES CEREVISIAE

酿酒酵母中的 DNA 复制

• 批准号: 3288699
• 负责人: CAROL S. NEWLON
• 金额: $ 21.23万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1990-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288699
• 关键词: Saccharomyces cerevisiae; chromosome aberrations; chromosome deletion; cytogenetics; electron microscopy; endonuclease; fungal genetics; genetic manipulation; genetic mapping; molecular cloning; nucleic acid sequence; plasmids

The overall objective of this project is to understand in detail how a eukaryotic chromosome replicates and segregates. To this end a 185 kilobase (kb) circular derivative of yeast chromosome III has been cloned and a restriction map constructed. Since the average spacing of replication origins, measured by electron microscopy of replicating chromsomal DNA, is 36 kb, this chromosome is expected to have 5-7 origins of replication. This chromosome and derivatives of it will be used to study the location, structure and efficiency of replication origins, to study the temporal sequence of replication, and to identify genes whose products are necessary for the replication of this chromosome. This particular proposal is addressed to the following issues. Sequences on chromosome III which are good candidates for replication origins have been cloned and mapped. These autonomously replicating sequences (ARS's) allow the extrachromosomal replication of plasmids in yeast. These sequences will be studied to determine whether replication initiates at these sites in vivo, whether these sequences are necessary for normal chromosome function and what DNA sequences are essential for ARS function. Genes whose products interact with ARS's will be identified using a novel selection scheme. Cloned fragments of chromosome III will be used in several experiments designed to determine whether there is a fixed temporal order of replication on the chromosome. If so, then sequences in which the information that specifies the temporal order will be identified. These studies should greatly increase our understanding of a fundamental cellular process, DNA replication. These questions cannot be approached in higher eukaryotes at the present time because their chromosomal DNA's are too large to isolate without breakage and because no autonomously replicating chromosomal sequences have been identified which function in the cells from which they were isolated. Knowledge we gain from the yeast system should be applicable to higher cells, and may help lead to an understanding of the basis of unscheduled DNA synthesis, which is characteristic of malignant cells.

该项目的总体目的是详细了解 真核染色体复制并分离。 为此结束185 酵母染色体III的千目标（KB）圆形衍生物已克隆 以及构建的限制图。 由于平均间距 复制起源，通过复制的电子显微镜测量 染色体DNA，为36 kb，该染色体有望具有5-7个起源 复制。 这种染色体和衍生物将用于 研究复制起源的位置，结构和效率， 研究复制的时间序列，并鉴定其基因 产品对于复制该染色体是必需的。 该特定的提议涉及以下问题。 序列 关于复制起源的好候选染色体III 被克隆和映射。 这些自主复制序列（ARS） 允许酵母中质粒的质体外复制。 这些 将研究序列以确定复制是否启动 这些位点在体内，这些序列是否需要正常 染色体功能和什么DNA序列对于ARS功能至关重要。 产品与ARS相互作用的基因将使用小说识别 选择方案。 染色体III的克隆碎片将用于 旨在确定是否有固定时间的几个实验 染色体上的复制顺序。 如果是这样，那么序列 指定时间顺序的信息将被标识。 这些研究应大大增加我们对基本的理解 细胞过程，DNA复制。 这些问题无法解决 目前较高的真核生物，因为它们的染色体DNA是 太大而无法分离而没有破裂，因为没有自主 已经确定了复制染色体序列 它们分离的细胞。 我们从酵母中获得的知识 系统应适用于较高的细胞，并可能有助于导致 理解不定期的DNA合成的基础，这是 恶性细胞的特征。