Jiabo Ding, Zhizhong Cui, Shijin Jiang, Sanjay Reddy
Animal Science and Technology College, Shandong Agricultural University, Taian 271018, China.
Sci China C Life Sci. 2006 Feb;49(1):53-62. doi: 10.1007/s11427-004-0119-y.
There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene and 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP and pP(1.8-kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF) and pp38-deleted derivative rMd5deltapp38 infected CEF (rMd5deltapp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF, and no EGFP could be detected in uninfected CEF or rMd5deltapp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5deltapp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5deltapp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24 and EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38 and pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.
马立克氏病病毒（MDV）基因组中，38 kd磷酸化蛋白（pp38）基因与1.8-kb mRNA转录本基因家族之间存在一个双向启动子。在本研究中，在该双向启动子的两个方向上构建了增强绿色荧光蛋白（EGFP）报告质粒pP(pp38)-EGFP和pP(1.8-kb)-EGFP。将这两个质粒分别转染至未感染的鸡胚成纤维细胞（CEF）、感染MDV克隆rMd5的CEF（rMd5-CEF）以及感染pp38缺失衍生物rMd5deltapp38的CEF（rMd5deltapp38-CEF）。转染分析表明，EGFP仅在rMd5-CEF中表达，在未感染的CEF或rMd5deltapp38-CEF中未检测到EGFP，这意味着pp38是影响该启动子活性的一个因素。构建了表达pp38的重组质粒pcDNA-pp38，与pP(pp38)-EGFP或pP(1.8-kb)-EGFP共转染CEF或rMd5deltapp38-CEF。在这种情况下，仅在rMd5deltapp38-CEF中检测到EGFP，而在未感染的CEF中仍未检测到，这意味着pp38需要其他蛋白质共同作用以实现完整的反式作用活性。将另一个MDV基因，24 kd磷酸化蛋白pp24基因克隆到pcDNA3.1中，构建成表达pp24的重组质粒pcDNA-pp24。当未感染的CEF与pcDNA-pp38、pcDNA-pp24以及表达EGFP的质粒pP(pp38)-EGFP或pP(1.8-kb)-EGFP共转染时，可检测到EGFP。这些结果表明，pp38和pp24共同作用时可增强启动子的活性。DNA迁移率变动分析表明，尽管pp38和pp24单独都不影响启动子DNA的迁移率，但在pp24共存时，pp38会与双向启动子结合。上述所有结果表明，MDV pp38与pp24结合时可反式激活双向启动子。结果还表明，启动子在1.8-kb mRNA方向的活性明显强于pp38方向的活性。
