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.

我们工作的主要目标是确定定义一个 哺乳动物DNA复制的起源，并了解起源的机制 激活。我们实验的重点是 人类c-myc基因。C-myc的起源是有限数量的 染色体起源于复杂的真核生物，也是唯一的后生动物 在体外和转基因细胞中启动自主复制的起源 在染色体起始点。 C-myc是一种即刻早期反应基因，由许多有丝分裂原激活。 正常细胞中的通路。多个启动子和增强子元件 位于c-myc复制起始区，5‘端至c-myc 吉恩。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细胞凋亡，而c-myc基因异常 基因表达可以促进肿瘤的发生。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 起源可能是致癌病毒或化学病原体的作用部位。 了解DNA元件在c-myc复制起始点中的作用 因此，很可能给出对控制 正常和病理状态下的细胞分裂。 三个特定的目标将检验c-myc起源的假设 包括DNA合成的起始点和顺式作用的起源元素 它们调节复制的启动。C-myc来源的突变体将是 构建了具有特定位点缺失或DNA替换的。目标 1将检查针对以下目标的c-myc起始构建体的复制 通过腺相关病毒载体或 酵母FLP重组。为了对比c-myc起源于 在染色体上，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细胞提取液，并转入HeLa细胞。这些目标将考验 原点构造的相对效率，并绘制位置图 入会活动。同样在目标1和目标2中，核酸酶消化将是 用于探测活性来源和非活性来源的染色质结构 构造。目标3将确定元素中的蛋白质结合部位 影响c-myc复制起始点的活性。C-myc起源 通过突变确定的对质粒ARS活性重要的片段或 染色体起源活性和超螺旋c-myc起源结构，将 在复制启动下与293S细胞提取物体外孵育 条件。特定蛋白质：DNA复合体将通过 电泳迁移率改变和DNase I足迹。