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。
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其他文献
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:
6869317 - 财政年份:1996
- 资助金额:
$ 15.07万 - 项目类别:
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