Miner J N, Weinrich S L, Hruby D E
Department of Microbiology, Oregon State University, Corvallis 97331-3804.
J Virol. 1988 Jan;62(1):297-304. doi: 10.1128/JVI.62.1.297-304.1988.
Promoter elements responsible for directing the transcription of six tightly clustered vaccinia virus (VV) late genes (open reading frames [ORFs] D11, D12, D13, A1, A2, and A3) from the HindIII D/A region of the viral genome were identified within the upstream sequences proximal to each individual locus. These regions were identified as promoters by excising them from the VV genome, abutting them to the bacterial chloramphenicol acetyl transferase gene, and demonstrating their ability to drive expression of the reporter gene in transient-expression assays in an orientation-specific manner. To delineate the 5' boundary of the upstream elements, two of the VV late gene (A1 and D13) promoter: CAT constructs were subjected to deletion mutagenesis procedures. A series of 5' deletions of the ORF A1 promoter from -114 to -24 showed no reduction in promoter activity, whereas additional deletion of the sequences from -24 to +2 resulted in the complete loss of activity. Deletion of the ORF A1 fragment from -114 to -104 resulted in a 24% increase in activity, suggesting the presence of a negative regulatory region. In marked contrast to previous 5' deletion analyses which have identified VV late promoters as 20- to 30-base-pair cap-proximal sequences, 5' deletions to define the upstream boundary of the ORF D13 promoter identified two positive regulatory regions, the first between -235 and -170 and the second between -123 and -106. Background levels of chloramphenicol acetyltransferase expression were obtained with deletions past -88. Significantly, this places the ORF D13 regulatory regions within the upstream coding sequences of the ORF A1. A high-stringency computer search for homologies between VV late promoters that have been thus far characterized was carried out. Several potential consensus sequences were found just upstream from RNA start sites of temporally related promoter elements. Three major conclusions are drawn from these experiments. (i) The presence of promoters preceding each late ORF supports the hypothesis that each is expressed as an individual transcriptional unit. (ii) Promoter elements can be located within the coding portion of the upstream gene. (iii) Sequence homologies between temporally related promoter elements support the notion of kinetic subclasses of late genes.
负责指导病毒基因组HindIII D/A区域六个紧密聚集的痘苗病毒（VV）晚期基因（开放阅读框[ORF] D11、D12、D13、A1、A2和A3）转录的启动子元件，在每个单独基因座近端的上游序列中被鉴定出来。通过从VV基因组中切除这些区域，将它们与细菌氯霉素乙酰转移酶基因邻接，并在瞬时表达测定中证明它们以方向特异性方式驱动报告基因表达的能力，从而将这些区域鉴定为启动子。为了确定上游元件的5'边界，对两个VV晚期基因（A1和D13）启动子：氯霉素乙酰转移酶（CAT）构建体进行了缺失诱变程序。对ORF A1启动子从-114到-24进行的一系列5'缺失显示启动子活性没有降低，而从-24到+2的序列进一步缺失导致活性完全丧失。从-114到-104删除ORF A1片段导致活性增加24%，表明存在一个负调控区域。与之前将VV晚期启动子鉴定为20至30个碱基对的帽近端序列的5'缺失分析形成鲜明对比的是，用于确定ORF D13启动子上游边界的5'缺失鉴定出两个正调控区域，第一个在-235和-170之间，第二个在-123和-106之间。删除超过-88的序列可获得氯霉素乙酰转移酶表达的背景水平。重要的是，这使得ORF D13调控区域位于ORF A1的上游编码序列内。对迄今为止已表征的VV晚期启动子之间的同源性进行了高严格度计算机搜索。在时间相关启动子元件的RNA起始位点上游发现了几个潜在的共有序列。从这些实验中得出了三个主要结论。（i）每个晚期ORF之前存在启动子支持了每个ORF作为一个单独转录单元表达的假设。（ii）启动子元件可以位于上游基因的编码部分内。（iii）时间相关启动子元件之间的序列同源性支持晚期基因动力学亚类的概念。
