HUMAN C MYC GENE REPLICATION

人类 C MYC 基因复制

• 批准号: 2668524
• 负责人: Michael LEFFAK
• 金额: $ 15.07万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2668524
• 关键词: DNA footprinting; DNA replication; DNA replication origin; HeLa cells; adeno associated virus group; chromatin; gel mobility shift assay; gene deletion mutation; genetic enhancer element; genetic promoter element; mutant; nucleic acid structure; nucleosomes; plasmids; polymerase chain reaction; protooncogene; regulatory gene; transcription factor; transfection

The broad goals of our work are to identify the structures which define a mammalian DNA replication origin, and to understand the mechanism of origin activation. The focus of our experiments is the replication origin of the human c-myc gene. The c-myc origin is one of a limited number of chromosomal origins identified in complex eucaryotes, and the only metazoan origin to initiate autonomous replication in vitro and in transfected cells at the chromosomal initiation sites. c-myc is an immediate early response gene, activated by many mitogenic pathways in normal cells. Multiple promoter and enhancer elements are located in the region of the c-myc replication origin, 5' to the c-myc gene. Depending on growth conditions the c-myc protein can stimulate DNA synthesis and cell division or promote apoptosis, whereas aberrant c-myc gene expression can contribute to oncogenesis. These observations suggest that the c-myc replication origin region may be an intersection point for cellular networks of transcription and growth control. As such, the c-myc origin may be a site of action of oncogenic viral or chemical pathogens. Understanding the function of DNA elements in the c-myc replication origin is likely therefore to give new insight into mechanisms which control the cell division in normal and pathological states. Three Specific Aims will test the hypothesis that the c-myc origin comprises start sites for DNA synthesis, and cis-acting origin elements which regulate replication initiation. c-myc origin mutants will be constructed which have site-specific deletions or DNA substitutions. Aim 1 will examine the replication of the c-myc origin constructs targeted to specific chromosomal integration sites by adeno-associated virus vectors or the yeast FLP recombines. To contrast the activity of the c-myc origin in plasmids and in the chromosome, Aim 2 will use these constructs to identify sequences which affect the ability of plasmids to replicate autonomously in 293S cell extracts, and in transfected HeLa cells. These Aims will test the relative efficiencies of the origin constructs, and map the locations of initiation events. Also in Aims 1 and 2, nuclease digestion will be used to probe the chromatin structure of the active and inactive origin constructs. Aim 3 will identify protein binding sites in elements which influence the activity of the c-myc replication origin. c-myc origin fragments identified by mutation as important for plasmid ARS activity or chromosomal origin activity, and supercoiled c-myc origin constructs, will be incubated with 293S cell extracts in vitro under replication initiation conditions. Specific protein:DNA complexes will be analyzed by electrophoretic mobility shift and DNase I footprinting.

我们工作的广泛目标是确定定义A的结构 哺乳动物DNA复制起源，并了解起源的机理 激活。 我们实验的重点是复制起源 人C-Myc基因。 C-MYC起源是有限数量的 在复杂桉树中鉴定出的染色体起源，而唯一的后生动物 起源以在体外和转染细胞中启动自主复制 在染色体起始位点。 C-MYC是一个直接的早期反应基因，由许多有丝分裂激活 正常细胞中的途径。 多个启动子和增强子元素是 位于C-Myc复制起源区域，5'至C-Myc 基因。 根据生长条件，C-MYC蛋白可以刺激DNA 合成和细胞分裂或促进凋亡，而异常C-MYC 基因表达可以有助于造成肿瘤。 这些观察表明 C-MYC复制起源区域可能是 转录和生长控制的细胞网络。 因此，C-Myc 起源可能是致癌病毒或化学病原体的作用部位。 了解C-MYC复制起源中DNA元素的功能 因此，很可能会对控制机制有新的见解 正常和病理状态的细胞分裂。 三个具体目标将检验C-Myc起源的假设 包括用于DNA合成的开始位点和顺式作用的起源元素 调节复制启动。 c-myc起源突变体将是 构建具有特定位点缺失或DNA取代的构造。 目的 1将检查针对的C-Myc起源结构的复制 通过腺相关病毒载体或 酵母FLP重组。 与C-Myc起源的活性对比 质粒和染色体中，AIM 2将使用这些结构来识别 影响质粒自主复制能力的序列 293S细胞提取物，在转染的HeLa细胞中。 这些目标将测试 原点构造的相对效率，并绘制位置 启动事件。 同样在目标1和2中，核酸酶消化将是 用于探测活性和非活性起源的染色质结构 构造。 AIM 3将在元素中识别蛋白质结合位点 影响C-MYC复制起源的活性。 C-Myc起源 通过突变鉴定的片段对于质粒ARS活性很重要或 染色体起源活动和超螺旋的C-MYC起源结构将 在复制启动下与293S细胞提取物一起孵育 状况。 特异性蛋白质：DNA复合物将通过 电泳移动性转移和DNase I足迹。