Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression
Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression
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晚期基因表达所需的加州纹夜蛾核多角体病毒基因的功能图谱
DOI：
10.1128/jvi.62.2.463-471.1988
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发表时间：
1988
影响因子：
5.4
通讯作者：
M. Summers
中科院分区：
文献类型：
--
作者：
L. Guarino;M. Summers
构建了含有细菌氯霉素乙酰转移酶（CAT）基因的质粒，该基因受加州银纹夜蛾核型多角体病毒（AcNPV）晚期基因启动子控制。该质粒（pL 2cat）还含有AcNPV hr 5增强子元件。瞬时表达实验表明，晚期启动子在共转染pL 2cat和AcNPV DNA的草地贪夜蛾细胞中有活性，但单独转染pL 2cat时没有活性。在pL 2cat和pIE-1DNA共转染的细胞中观察到低水平的CAT活性。然而，CAT活性在缺乏顺式连接的增强子元件的类似质粒中不被诱导，表明该增强子是晚期基因表达所需的。AcNPV DNA的pPstI克隆的共转染作图表明，病毒DNA的pPstI-G克隆含有进一步刺激晚期基因表达3至10倍的因子。pPstI-G亚克隆的瞬时表达分析将反式因子定位于3.0-丝氨酸蛋白酶XbaI片段。测定了该片段的核苷酸序列，发现含有三个潜在的开放阅读框。对蛋白质数据库的计算机辅助搜索没有发现密切相关的蛋白质。其中一个预测的氨基酸序列含有潜在的金属结合结构域类似于那些发现在核酸结合蛋白。亚克隆和随后的CAT分析表明，两个开放阅读框是激活pL 2 cat所必需的。感染和转染RNA的核酸酶S1作图表明，两个开放阅读框被转录为延迟早期基因。定量核酸酶S1分析和回收质粒的差异DNA消化表明，pL 2cat的激活不是由于病毒DNA的mRNA复制的稳态水平的增加。
A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.