Temporal Regulation of Baculovirus Gene Expression

杆状病毒基因表达的时间调控

• 批准号: 8804732
• 负责人: Linda Guarino
• 金额: $ 23.84万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1992-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8804732&HistoricalAwards=false
• 关键词: Temporal; Regulation; Baculovirus; Gene; Expression

The polyhedrin gene of Autographa californica nuclear polyhedrosis virus is maximally expressed very late in infection, after the release of extracellular virus. It is believed that temporal regulation of polyhedrin requires the sequential expression of an immediate early gene(s) to activate delayed early gene expression; a delayed early gene(s) to turn on DNA synthesis and large gene expression; and a late gene(s) to stimulate polyhedrin, a very large gene. I have devised a complementation assay to map the viral factors which control the temporal regulation of polyhedrin expression. A plasmid, polcat, was constructed that contains the bacterial gene chloramphenicol acetyltransferase (CAT) under the control of the polyhedrin promoter. CAT is expressed upon cotransfection of permissive insect cells with polcat and viral DNA, but not with viral DNA which has previously been digested with restriction enzymes. The inability of digested DNA to support polcat expression is apparently due to inactivation of one or more essential genes by restriction digest. The location of these genes can be mapped by complemenation with cloned PstI-restriction fragments. Preliminary results indicated that twelve of the PstI clones complement different digests of viral DNA, indicating that at least twelve genes are involved in the temporal cascade. I intend to map and determine the function of several of these factors in temporal regulation of polyhedrin expression. The ultimate goal of this project is to define a limited set of viral genes which permits high level expression of polyhedrin in insect cells. Although it may not be possible to identify all of the factors during the grant period, this research should increase our understanding of the mechanisms of temporal regulation of baculovirus gene expression.

苜蓿银纹夜蛾核型多角体蛋白基因 多角体病毒最大表达的时间很晚， 感染后，细胞外病毒的释放。 是 认为多面体蛋白的时间调节需要 立即早期基因的顺序表达， 激活延迟的早期基因表达;延迟的早期 启动DNA合成和大基因表达的基因; 和一个晚期基因来刺激多角体蛋白，一个非常大的 基因 我设计了一种互补分析来绘制 病毒因子控制的时间调节 多角体蛋白表达 构建了质粒polcat 含有氯霉素的细菌基因 多角体蛋白控制下的乙酰转移酶（CAT） 启动子 CAT在共转染 允许昆虫细胞与polcat和病毒DNA，但没有 病毒DNA已经被消化 限制酶 消化后的DNA不能 支持polcat表达显然是由于失活 一个或多个必需基因的限制性酶切。 的 这些基因的位置可以通过与 克隆的Pst I-限制性片段。 初步结果 表明12个Pst I克隆互补不同的 病毒DNA的序列，表明至少有12个基因 参与了时间级联。我打算绘制地图， 确定其中几个因素的功能， 多角体蛋白表达的时间调节。 这个项目的最终目标是定义一个有限的集合 允许高水平表达的病毒基因 昆虫细胞中的多角体蛋白 尽管不可能 为了确定赠款期间的所有因素， 研究应该增加我们对这些机制的理解， 杆状病毒基因表达的时间调节。