REGULATION OF RDNA REPLICATION IN TETRAHYMENA

DNA复制的精确控制是 在所有分裂的细胞中忠实地传递遗传信息。 调节DNA复制的分子机制 真核细胞尚不清楚。 这个项目的目标是 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的rDNA基因 嗜热菌 所提出的实验研究rDNA的控制 复制有两种方式。 一个是顺式作用DNA的描述 准确和有效复制所需的序列信号 的rDNA。 另一个是蛋白质或其他 在这些位点起作用的反式作用因子。 具体来说，顺- 将通过以下方式识别作用控制区域：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）精确定位 在这些和其他先前的体内复制起点 表征的复制突变体;和3）测定复制突变体的复制能力， 改变的rDNA构建体的性质，无论是在体外，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。 这些反式作用因子 rDNA复制的调节将通过以下方式进行研究：1） 鉴定与DNA相互作用的特异性DNA结合蛋白， 确定的复制起点和其他关键控制位点 2）测试特定RNA转录物 参与调节rDNA复制和拷贝数。 分析确保DNA忠实的分子机制 复制对于理解基本的 调节正常细胞生长和分裂的过程， 提供对异常增长原因的充分了解 控制

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.