Jones E V, Puckett C, Moss B
J Virol. 1987 Jun;61(6):1765-71. doi: 10.1128/JVI.61.6.1765-1771.1987.
Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A.
制备了针对痘苗病毒颗粒中多亚基DNA依赖性RNA聚合酶的抗血清，以进行遗传学研究。该抗血清选择性地抑制了病毒聚合酶的活性，但对小牛胸腺RNA聚合酶II没有影响。通过从解离的病毒颗粒中免疫沉淀RNA聚合酶亚基，进一步证明了抗血清的特异性。通过体外翻译确定了痘苗病毒感染细胞中编码聚合酶亚基的mRNA的存在。当将早期mRNA添加到网织红细胞提取物中时，产生了分子量约为135,000、128,000、36,000、34,000、31,000、23,000、21,000、20,000和17,000的可免疫沉淀多肽。这些亚基由痘苗病毒基因组编码，这通过与痘苗病毒DNA杂交的早期mRNA的翻译得到证明。亚基基因的位置最初通过RNA与一系列重叠的40千碱基对DNA片段杂交来确定，这些片段克隆在黏粒载体中。通过克隆的HindIII限制性片段进行了进一步的定位。这些研究结果表明，RNA聚合酶亚基基因在感染早期被转录，并分布在痘苗病毒基因组高度保守的中央部分的HindIII片段E、J、H、D和A中。
