Ozsahin Emine, Sezen Kazım, Demirbag Zihni
Department of Biology, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey.
Arch Virol. 2014 Oct;159(10):2541-7. doi: 10.1007/s00705-014-2096-1. Epub 2014 May 6.
The open reading frame (ORF) amv133 of Amsacta moorei entomopoxvirus, encodes a putative lipase gene. Its temporal expression pattern was characterized by RT-PCR and found to start at 6 h postinfection (h p.i.) and reach a maximum level at 48 h p.i. While the ORF has a late promoter motif, the inhibition of viral DNA synthesis by Ara-C failed to inhibit transcription, but a general inhibitor of protein synthesis prevented its transcription, indicating that amv133 is an intermediate gene. 5'-RACE analysis showed that transcription was initiated at position -77 relative to the translational start site. To determine the size of the promoter, several truncations were generated and cloned upstream of the firefly luciferase reporter gene. The resulting constructs were tested in a dual assay. A fragment that contained 115 bp relative to the transcription start site exhibited optimum promoter length.
摩尔伊蚊昆虫痘病毒的开放阅读框（ORF）amv133编码一个假定的脂肪酶基因。通过逆转录聚合酶链反应（RT-PCR）对其时间表达模式进行了表征，发现其在感染后6小时（h p.i.）开始表达，并在感染后48小时达到最高水平。虽然该ORF具有晚期启动子基序，但阿糖胞苷（Ara-C）对病毒DNA合成的抑制未能抑制转录，但蛋白质合成的一般抑制剂可阻止其转录，这表明amv133是一个中间基因。5'-cDNA末端快速扩增（5'-RACE）分析表明，转录起始于相对于翻译起始位点的-77位置。为了确定启动子的大小，构建了几个截短片段并克隆到萤火虫荧光素酶报告基因的上游。在双重检测中对所得构建体进行了测试。相对于转录起始位点包含115 bp的片段表现出最佳的启动子长度。
