REGULATION OF RDNA REPLICATION IN TETRAHYMENA

精确控制DNA复制是 在所有分裂的细胞中忠实地传递遗传信息。 细胞内DNA复制调控的分子机制 真核细胞还不为人所知。这个项目的目标是 为了研究DNA复制控制的分子细节 真核生物，通过专注于复制扩增的rRNA 单细胞原生动物四膜虫的基因(RDNA) 嗜热菌。 建议的实验研究rdna的控制。 以两种方式进行复制。其一是顺式作用DNA的描绘。 准确高效复制所需的序列信号 RDNA的基因。另一个是蛋白质或其他物质的鉴定 在这些部位起作用的反式作用因子。具体而言，独联体- 行动控制区域将通过以下方式确定：1)确定 与rDNA复制有关的核苷酸序列变化 两个四膜虫突变株的缺陷；2)精确定位 在这些和其他以前的体内复制起点 表征复制突变体；以及3)检测复制 改变的rDNA结构的性质在体外，使用细胞- 无复制提取物，并在体内通过微量注射 四膜虫细胞。调停的交易作用因素 RDNA复制的调节将通过以下方式进行调查：1) 鉴定与细胞因子相互作用的特定DNA结合蛋白 复制来源和已确定的其他关键控制点 以及2)测试特定的RNA转录物 参与调节rDNA复制和拷贝数。 确保DNA忠实的分子机制分析 复制是理解基础知识的关键 调节正常细胞生长和分裂的过程，并且应该 提供对异常增长原因的全面洞察 控制力。

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.