Hybridization Selection and In Vitro Translation of Autographa californica Nuclear Polyhedrosis Virus mRNA-猫眼课题宝

苜蓿银纹夜蛾核型多角体病毒mRNA的杂交选择和体外翻译

DOI：

发表时间：

1981

期刊：

Journal of Virology

影响因子：

5.4

通讯作者：

中科院分区：

医学2区

文献类型：

作者：

J. Vlak;Gale E. Smith;M. Summers

我们从感染苜蓿银纹夜蛾核型多角体病毒的细胞的细胞质中分离出多腺苷酸化RNA，该细胞在感染后后期（感染后21小时）。当时，细胞内蛋白质合成几乎完全针对受感染的细胞特异性蛋白质。感染后21小时细胞质中含有聚腺苷酸的RNA序列在体外进行放射性标记，并与加利福尼亚州核型多角体病毒DNA限制性片段杂交。含有聚腺苷酸的RNA源自代表整个病毒基因组的区域。通过与病毒DNA和含聚腺苷酸的RNA杂交选择的细胞质RNA在网状细胞无细胞蛋白质合成系统中的翻译产生了几乎相同的多肽模式，这表明感染后后期，感染细胞中存在的几乎所有细胞质含聚腺苷酸的RNA都是病毒来源的。通过免疫沉淀和比较分子量来鉴定翻译产物中的多角体蛋白（分子量，33,000）和许多病毒体结构蛋白。此外，还检测到一些暂定的非结构性感染细胞特异性蛋白。使用杂交选择技术，我们确定与多角体蛋白的信息编码互补的序列位于加利福尼亚州核多角体病毒DNA的EcoRI片段I上，而编码假定的非结构蛋白（分子量，39,000）的序列位于EcoRI片段J上。

We isolated polyadenylated RNA from the cytoplasm of cells infected with Autographa californica nuclear polyhedrosis virus late after infection (21 h postinfection). At that time intracellular protein synthesis was directed almost exclusively toward infected cell-specific proteins. The polyadenylic acid-containing RNA sequences in the cytoplasm at 21 h postinfection were radiolabeled in vitro and hybridized to A. californica nuclear polyhedrosis virus DNA restriction fragments. The polyadenylic acid-containing RNA was derived from regions representing the entire viral genome. Translation in a reticulocyte cell-free protein-synthesizing system of cytoplasmic RNA selected by hybridization to viral DNA and polyadenylic acid-containing RNA produced almost identical polypeptide patterns, suggesting that late after infection almost all of the cytoplasmic polyadenylic acid-containing RNA present in infected cells was of viral origin. Polyhedrin protein (molecular weight, 33,000) and a number of virion structural proteins were among the translation products which were identified by immunoprecipitation and by comparing molecular weights. In addition, some tentative nonstructural infected cell-specific proteins were also detected. Using the hybridization selection technique, we determined that sequences complementary to the message coding for polyhedrin were located on EcoRI fragment I of A. californica nuclear polyhedrosis virus DNA, whereas sequences coding for a putative nonstructural protein (molecular weight, 39,000) were on EcoRI fragment J.

Immunoautoradiographic detection of proteins after electrophoretic transfer from gels to diazo-paper: analysis of adenovirus encoded proteins.