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REGULATION OF AAV DNA REPLICATION

AAV DNA 复制的调控

基本信息

批准号：
6180225
负责人：
KENNETH I. BERNS
金额：
$ 23.24万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-05-01 至 2002-04-30
项目状态：
已结题

项目摘要

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome has been constructed by insertion of the SV40 regulatory region (nt 5171-5243-270) into a deletion in the AAV genome from nt 144-264. The deletion removes the leftward most AAV promoter and about 100 bases upstream, but leaves intact in the hybrid genome the cap site of the transcript from the deleted AAV promoter. The inserted sequence contains the SV40 origin of replication (ori). However, when transfected into cells that constitutively express the SV40 T-antigen the plasmid replicates very poorly. We have discovered the inhibition of replication requires a trans-acting product from the AAV rep gene (an open reading frame in the left half of the genome whose products are necessary for replication) and two cis-acting target sequences which are within the inverted terminal repeats of the AAV genome. This proposal describes experiments in cell culture and in vitro to characterize the mechanism of this negative regulation. In cell culture experiments we will determine: 1) the exact parameters of the target sequence, 2) whether the distance between SV40 ori and the target sequence is important, 3) whether there is a critical ratio of oris to target sequences, 4) whether inhibition can be overcome by excess T-antigen, 5) which part of the AAV rep gene encodes the inhibitory product and 6) whether inhibition can be overcome by viral oncogene expression, or treatment of cells with physical or chemical carcinogens. We plan to use the established in vitro assay for SV40 DNA replication as an assay during fractionation of the inhibitory activity from infected cells. Experiments are described to isolate that AAV rep gene products from either infected cells or by means of expression vectors. There will also be an attempt to synthesize active rep gene products in vitro. By these means we hope to gain insight into the mechanisms underlying the negative regulation of AAV DNA replication.

腺相关病毒(AAV)/猴病毒40(SV40)混合型病毒通过插入SV40调节基因构建了基因组将AAV基因组(5171-5243-270)插入缺失。 NT 144-264。删除会移除最左侧的AAV 启动子和上游约100个碱基，但在杂交基因组从缺失的转录本的帽子位置 AAV启动子。插入的序列包含SV40原点复制(ORI)。然而，当被转染到细胞中时，原始性表达SV40T抗原的质粒复制很差。我们已经发现了复制的抑制作用需要来自AAV rep基因的反式作用产物(开放的基因组左半部分的读框，其产物是复制所必需的)和两个顺式作用靶序列它们位于AAV的反向终端中继器内基因组。这项建议描述了细胞培养和体外研究这种负性反应的机制监管。在细胞培养实验中，我们将确定：1) 目标序列的准确参数，2)距离是否 SV40 ORI和靶序列之间的关系很重要，3) 是否存在ORIS与靶序列的临界比例，4) 是否可以被过量的T抗原克服抑制，5) 部分AAV rep基因编码抑制产物和6) 病毒癌基因表达能否克服抑制，或用物理或化学致癌物治疗细胞。我们计划使用已建立的SV40DNA体外检测方法复制作为抑制物分离过程中的一种分析方法受感染细胞的活性。描述了分离的实验 AAV rep基因产物来自受感染的细胞或由表达载体的方法。还将有一种尝试体外合成具有活性的rep基因产物。通过这些手段，我们希望能深入了解负面情绪背后的机制 AAV DNA复制的调控。

项目成果

期刊论文数量（17）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Modulation of the cellular phenotype by integrated adeno-associated virus.

整合腺相关病毒对细胞表型的调节。

DOI：
10.1016/0042-6822(92)91218-j
发表时间：
1992
期刊：
Virology
影响因子：
3.7
作者：
Winocour,E;Puzis,L;Etkin,S;Koch,T;Danovitch,B;Mendelson,E;Shaulian,E;Karby,S;Lavi,S
通讯作者：
Lavi,S

Parvovirus replication.

细小病毒复制。