HUMAN C MYC GENE REPLICATION
人类 C MYC 基因复制
基本信息
- 批准号:2668524
- 负责人:
- 金额:$ 15.07万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 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
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Michael LEFFAK的其他文献
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{{ truncateString('Michael LEFFAK', 18)}}的其他基金
Mechanisms of Replication-Dependent Microsatellite Instability in Human Disease
人类疾病中复制依赖性微卫星不稳定性的机制
- 批准号:
10004155 - 财政年份:2017
- 资助金额:
$ 15.07万 - 项目类别:
Second-site genetic modifiers of CTG/CAG microsatellite stability
CTG/CAG 微卫星稳定性的第二位点遗传修饰剂
- 批准号:
8652473 - 财政年份:2012
- 资助金额:
$ 15.07万 - 项目类别:
Second-site genetic modifiers of CTG/CAG microsatellite stability
CTG/CAG 微卫星稳定性的第二位点遗传修饰剂
- 批准号:
8870378 - 财政年份:2012
- 资助金额:
$ 15.07万 - 项目类别:
Second-site genetic modifiers of CTG/CAG microsatellite stability
CTG/CAG 微卫星稳定性的第二位点遗传修饰剂
- 批准号:
8218826 - 财政年份:2012
- 资助金额:
$ 15.07万 - 项目类别:
Second-site genetic modifiers of CTG/CAG microsatellite stability
CTG/CAG 微卫星稳定性的第二位点遗传修饰剂
- 批准号:
8464166 - 财政年份:2012
- 资助金额:
$ 15.07万 - 项目类别:
The Role of the DNA Unwinding Element Binding Protein, DUE-B, in DNA Replication
DNA 解旋元件结合蛋白 DUE-B 在 DNA 复制中的作用
- 批准号:
7846744 - 财政年份:2009
- 资助金额:
$ 15.07万 - 项目类别:
Analysis of the Human c-myc Gene Replication Origin
人类c-myc基因复制起点分析
- 批准号:
7032445 - 财政年份:1996
- 资助金额:
$ 15.07万 - 项目类别:
Analysis of the Human c-myc Gene Replication Origin
人类c-myc基因复制起点分析
- 批准号:
7226647 - 财政年份:1996
- 资助金额:
$ 15.07万 - 项目类别:
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