DNA REPLICATION IN SACCHAROMYCES CEREVISIAE

酿酒酵母中的 DNA 复制

• 批准号: 3288703
• 负责人: CAROL S. NEWLON
• 金额: $ 33.21万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288703
• 关键词: DNA replication; DNA replication origin; Saccharomyces cerevisiae; chromosome aberrations; chromosome deletion; computer program /software; cytogenetics; electron microscopy; endonuclease; fungal genetics; gel electrophoresis; genetic manipulation; genetic mapping; molecular cloning; nucleic acid sequence; plasmids; protein transport

The long-term objective of this project is to understand how a eukaryotic chromosome replicates and segregates. To this end a 200 kilobase (kb) circular derivative of Saccharomyces cerevisiae chromosome III that includes 60% of the chromosome has been cloned and restriction mapped. This chromosome is being used to study location, structure, and pattern of use of replication origins and to identify genes whose products are necessary for the replication of this chromosome. This proposal addresses three issues related to the replication of this chromosome. First, replication origins used in vivo are being mapped using 2D gel techniques, and positions of origins are being compared with known positions of ARS elements, which promote autonomous replication of plasmids. Replication origins will be deleted and moved to determine how many are required for normal chromosome stability and whether the temporal patterns of replication or the pattern of origin usage is perturbed by changes in origin spacing or changes in chromosome context. Second, the DNA sequences required for origin function will be further defined by isolating additional mutant origins using in vitro mutagenesis techniques and characterizing their function both in plasmids and in their normal chromosomal context. Third, proteins that interact with ARS elements will be identified using both novel genetic screens and biochemical approaches. 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.

该项目的长期目标是了解真核如何 染色体复制并分离。 为此结束200千座（KB） 酿酒酵母染色体III的圆形衍生物 包括60％的染色体被克隆并制定限制。 该染色体用于研究位置，结构和模式 使用复制起源并确定其产品的基因 复制该染色体所必需的。 该提案解决了与此复制有关的三个问题 染色体。 首先，正在使用体内使用的复制起源 将2D凝胶技术和原始位置与已知的 ARS元素的位置，促进自主复制的位置 质粒。 复制起源将被删除并移动以确定如何 正常染色体稳定性需要许多 复制的模式或原始用法的模式受到干扰 原点间距的变化或染色体环境中的变化。 第二， 原点功能所需的DNA序列将进一步定义 使用体外诱变技术隔离其他突变体的起源 并在质粒和正常状态中表征它们的功能 染色体上下文。 第三，与ARS元素相互作用的蛋白质将 可以使用新型的遗传筛选和生化方法来识别。 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。