Yamanaka K, Ogasawara N, Yoshikawa H, Ishihama A, Nagata K
Department of Molecular Genetics, National Institute of Genetics, Shizuoka, Japan.
Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5369-73. doi: 10.1073/pnas.88.12.5369.
A system for the expression of a foreign gene derived from negative polarity RNA was developed using influenza virus, a negative-stranded RNA virus. From cDNA for the influenza virus RNA genome segment 8, the region coding for the nonstructural protein was deleted and replaced by the chloramphenicol acetyltransferase (CAT) gene. The resulting DNA sequence was placed under the control of the promoter of T7 RNA polymerase such that the antisense RNA to CAT mRNA was produced when transcribed by T7 RNA polymerase. Transfection of HeLa cells with this antisense CAT RNA in the presence of the helper ribonucleoprotein cores led to no significant production of the CAT. In contrast, when the RNA was covered with purified nucleoprotein prior to transfection, the CAT gene was efficiently expressed. This indicated that the viral RNA polymerase transcribed the RNA transfected as the RNA-nucleoprotein complexes. In addition, this system was used for analysis of the cis-acting region in transcription and the promoter structure of the viral RNA genome.
利用负链RNA病毒——流感病毒，开发了一种用于表达源自负极性RNA的外源基因的系统。从流感病毒RNA基因组片段8的cDNA中，删除了编码非结构蛋白的区域，并用氯霉素乙酰转移酶（CAT）基因取代。将所得的DNA序列置于T7 RNA聚合酶启动子的控制下，使得当由T7 RNA聚合酶转录时，产生与CAT mRNA互补的反义RNA。在辅助核糖核蛋白核心存在的情况下，用这种反义CAT RNA转染HeLa细胞，未导致CAT的显著产生。相反，当RNA在转染前用纯化的核蛋白覆盖时，CAT基因得到有效表达。这表明病毒RNA聚合酶转录转染的RNA时，是以RNA-核蛋白复合物的形式进行的。此外，该系统还用于分析转录中的顺式作用区域和病毒RNA基因组的启动子结构。
