Krappa R, Knebel-Mörsdorf D
Institute of Genetics, University of Cologne, Federal Republic of Germany.
J Virol. 1991 Feb;65(2):805-12. doi: 10.1128/JVI.65.2.805-812.1991.
We have started to identify early viral RNAs that are transcribed at 1 h after inoculation to investigate the mechanism involved in the regulation of early gene expression of Autographa californica nuclear polyhedrosis virus (AcNPV). Cloned viral DNA fragments were hybridized to Northern (RNA) blots of polyadenylated RNA isolated from Spodoptera frugiperda cells at 1, 2, and 6 h postinfection to localize very early transcripts. Subsequently we prepared a cDNA library of polyadenylated RNA transcribed at 1 h after inoculation to analyze the cDNA clones corresponding to the major early RNAs. We identified a gene located upstream of the immediate-early gene IE-N extending in the opposite direction. Because of the very early expression during AcNPV infection and the transient expression in uninfected cells, we conclude that we found an immediate-early gene, designated PE-38. The determination of the nucleotide sequence of PE-38 revealed one open reading frame potentially encoding a gene product of 38 kDa. Results of in vitro translation experiments suggest that a PE-38-specific polypeptide of approximately 38 kDa can be expressed. We have evidence from computer analyses that the predicted amino acid sequence includes two putative DNA-binding motifs, a zinc finger, and a leucine zipper.
我们已开始鉴定接种后1小时转录的早期病毒RNA,以研究苜蓿银纹夜蛾核型多角体病毒(AcNPV)早期基因表达调控所涉及的机制。将克隆的病毒DNA片段与感染后1、2和6小时从草地贪夜蛾细胞中分离的聚腺苷酸化RNA的Northern(RNA)印迹杂交,以定位极早期转录本。随后,我们制备了接种后1小时转录的聚腺苷酸化RNA的cDNA文库,以分析与主要早期RNA对应的cDNA克隆。我们鉴定出一个位于立即早期基因IE-N上游且方向相反的基因。由于该基因在AcNPV感染期间表达非常早,且在未感染细胞中瞬时表达,我们得出结论,我们发现了一个立即早期基因,命名为PE-38。PE-38核苷酸序列的测定揭示了一个潜在编码38 kDa基因产物的开放阅读框。体外翻译实验结果表明,可以表达一种约38 kDa的PE-38特异性多肽。我们从计算机分析中获得的证据表明,预测的氨基酸序列包括两个假定的DNA结合基序、一个锌指和一个亮氨酸拉链。