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一个 哺乳动物DNA复制起点，并了解起源的机制 activation. 我们的实验重点是复制起点的 人c-myc基因。 c-myc基因的起源是一个有限的数目， 在复杂的真核生物中发现了染色体起源， 在体外和转染细胞中启动自主复制的起点 在染色体起始位点。 c-myc是一种立即早期反应基因，由许多促有丝分裂因子激活。 在正常细胞中。 多个启动子和增强子元件是 位于c-myc复制起点区域，c-myc的5'端 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 基因表达可促进肿瘤发生。 这些观察结果表明 c-myc复制起始区可能是 转录和生长控制的细胞网络。 因此，c-myc 起源可以是致癌病毒或化学病原体的作用位点。 了解c-myc复制起点中DNA元件的功能 因此，有可能对控制这些疾病的机制提供新的见解。 正常和病理状态下的细胞分裂。 三个具体目标将检验c-myc起源于 包括DNA合成的起始位点和顺式作用起始元件 调控复制起始的基因。 c-myc起源突变体将是 构建了具有位点特异性缺失或DNA取代的基因。 目的 1将检查c-myc起始构建体的复制， 通过腺相关病毒载体的特异性染色体整合位点，或 酵母FLP重组。 为了对比c-myc起源的活性， 在染色体中，Aim 2将使用这些构建体来识别 影响质粒自主复制能力的序列， 293 S细胞提取物和转染的HeLa细胞中。 这些目标将受到考验 原点构造的相对效率，并绘制位置 初始化事件。 此外，在目的1和2中，将核酸酶消化作为目标。 用于探测活性和非活性起源的染色质结构 结构。 目标3将确定蛋白质结合位点的元素， 影响c-myc复制起点的活性。 c-myc起源 通过突变鉴定为对质粒ARS活性重要的片段，或 染色体起源活性和超螺旋c-myc起源构建体， 在复制起始条件下与293 S细胞提取物体外孵育 条件 特异性蛋白质：DNA复合物将通过 电泳迁移率偏移和DNA酶I足迹法。