REGULATION OF RDNA REPLICATION IN TETRAHYMENA

四膜虫 RDNA 复制的调控

• 批准号: 3467616
• 负责人: DRENA L DOBBS
• 金额: $ 9.94万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467616
• 关键词: DNA binding protein; DNA replication; DNA replication origin; Tetrahymena; cell free system; cell growth regulation; eukaryote; genetic mapping; genetic transcription; microinjections; natural gene amplification; nucleic acid sequence; ribosomal RNA; transcription factor

The precise control of DNA replication is a prerequisite for faithful transmission of genetic information in all dividing cells. The molecular mechanisms that regulate DNA replication in eukaryotic cells are not understood. The goal of this project is to investigate the molecular details of DNA replication control in eukaryotes, by focusing on the replication of the amplified rRNA genes (rDNA) in the single-celled protozoan, Tetrahymena thermophila. The proposed experiments investigate the contrdl of rDNA replication in two ways. One is the delineation of cis-acting DNA sequence signals required for accurate and efficient replication of the rDNA. The other is the identification of proteins or other trans-acting factors that act at these sites. Specifically, cis- acting control regions will be identified by: 1) determining the nucleotide sequence changes responsible for rDNA replication defects in two mutant Tetrahymena strains; 2) mapping precisely the in vivo origins of replication in these and additional previously characterized replication mutants; and 3) assaying the replication properties of altered rDNA constructs both in vitro, using cell- free replication extracts, and in vivo, by microinjection of Tetrahymena cells. The trans-acting factors that mediate regulation of rDNA replication will be investigated by: 1) identifying specific DNA binding proteins that interact with the origin of replication and other critical control sites identified above; and 2) testing the possibility that specific RNA transcripts are involved in regulating rDNA replication and copy number. Analysis of the molecular mechanisms that ensure faithful DNA replication is essential from understanding the fundamental processes that regulate normal cell growth and division, and should provide use full insights into the causes of aberrant growth control.

DNA复制的精确控制是DNA复制的前提 所有分裂细胞中遗传信息的忠实传递。 调控DNA复制的分子机制 真核细胞尚不清楚。 该项目的目标是 研究 DNA 复制控制的分子细节 真核生物，通过关注扩增的 rRNA 的复制 单细胞原生动物四膜虫中的基因（rDNA） 嗜热菌。 拟议的实验研究了 rDNA 的控制 复制有两种方式。 一是顺式作用DNA的描绘 准确高效复制所需的序列信号 rDNA 的。 另一个是蛋白质或其他的鉴定 在这些位点起作用的反式作用因子。 具体来说，顺式 行动控制区域将通过以下方式确定： 1) 确定 负责 rDNA 复制的核苷酸序列变化 两个突变四膜虫菌株的缺陷； 2）精确映射 这些和其他先前的体内复制起点 特征复制突变体； 3) 分析复制 改变的 rDNA 构建体的特性均在体外，使用细胞- 自由复制提取物，并在体内，通过显微注射 四膜虫细胞。 介导的反式作用因子 rDNA 复制的调控将通过以下方式进行研究：1) 识别与DNA相互作用的特定DNA结合蛋白 确定复制起点和其他关键控制位点 多于; 2) 测试特定RNA转录的可能性 参与调节 rDNA 复制和拷贝数。 分析确保 DNA 忠实的分子机制 复制对于理解基本原理至关重要 调节正常细胞生长和分裂的过程，并且应该 提供对异常生长原因的全面见解 控制。