DNA REPLICATION INITIATION SITES IN MAMMALIAN CELLS

哺乳动物细胞中的 DNA 复制起始位点

• 批准号: 3305179
• 负责人: CARL L SCHILDKRAUT
• 金额: $ 29.34万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-08 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3305179
• 关键词: DNA binding protein; DNA replication origin; Epstein Barr virus; animal tissue; cell cycle proteins; enzyme inhibitors; gel electrophoresis; gene deletion mutation; gene expression; helicase; human tissue; immunoglobulin genes; ion exchange chromatography; nucleic acid probes; nucleic acid repetitive sequence; phosphorylation; polymerase chain reaction; radiotracer; tissue /cell culture; virus genetics; virus replication

We will use two model systems to study DNA replication in human cell lines. 1. The role of the Epstein-Barr virus (EBV) nuclear antigen I (EBNA-1) in the formation of a replication barrier at the EBV family of repeated sequences in human cell lines will be studied using an in vitro replication system. We will determine the minimum number of repeats that are required to produce a barrier to replication in vitro and determine whether EBNA-1 binding to the family of repeats inhibits helicase activity of the T antigen. We will then determine whether cellular proteins can also produce a replication barrier by binding to the EBV family of repeats. 2. We will identify replication origins in the rDNA gene cluster that replicate early or late during the S phase. We will determine the location of the sites at which the replication of the immunoglobulin heavy chain multigene family initiates. This will be determined in cell lines in which one or more of these genes are expressed and in cell lines in which they are transcriptionally silent. We will use deletion analysis to determine the sequences that are critical for these immunoglobulin replication origins to function. Targeted integration mediated by homologous recombination will be used to modify the chromosomal origin region.

我们将使用两个模型系统来研究人类细胞系中的DNA复制。 1. EB病毒（EBV）核抗原I（EBNA-1）在 在EBV家族中形成复制屏障， 将使用体外复制方法研究人类细胞系中的序列。 系统 我们将确定所需的最小重复次数 以在体外产生复制屏障，并确定EBNA-1 与重复序列家族的结合抑制T细胞的解旋酶活性， 抗原的 然后我们将确定细胞蛋白质是否也能产生 通过与EBV重复序列家族结合而形成复制屏障。 2.我们将 确定rDNA基因簇中复制较早的复制起点 或晚于S期。 我们将确定复制 免疫球蛋白重链多基因家族起始。 这将是 在表达一种或多种这些基因的细胞系中确定 以及它们在转录上沉默的细胞系中。 我们将使用 缺失分析，以确定对这些关键的序列 免疫球蛋白复制起点到功能。 靶向整合 通过同源重组介导的基因修饰将用于修饰染色体 原产地。