TRANSCRIPTION ANALYSIS AND SEQUENCE OF THE PUTATIVE MURINE CYTOMEGALOVIRUS DNA-POLYMERASE GENE
TRANSCRIPTION ANALYSIS AND SEQUENCE OF THE PUTATIVE MURINE CYTOMEGALOVIRUS DNA-POLYMERASE GENE
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DOI：
10.1016/0042-6822(91)90765-4
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发表时间：
1991-11-01
期刊：
影响因子：
3.7
通讯作者：
SPECTOR, DH
中科院分区：
文献类型：
--
作者：
ELLIOTT, R;CLARK, C;SPECTOR, DH
疱疹病毒DNA聚合酶的保守性使得交叉杂交研究可以用于病毒基因组的鉴定和定位。利用含有人巨细胞病毒（HCMV） DNA聚合酶基因的DNA片段作为杂交探针，我们能够将鼠巨细胞病毒（MCMV） DNA聚合酶基因定位到MCMVEcoRI片段B中横跨hindiii片段D和h的hindiii位点的区域，该位点与HCMV菌株AD169 DNA聚合酶基因共线。为了证实该区域编码MCMV DNA聚合酶基因，我们测序了从hindiii片段D的Pstl位点到hindiii片段H的aBg/ isite的5131个核苷酸片段。启动inindiii片段D并延伸到hindiii片段H的是一个长1097个氨基酸的开放阅读框（ORF），与HCMV、单纯疱疹病毒和eb病毒的DNA聚合酶具有广泛的同源性。ORF聚合酶的上游是一个阅读框，与人疱疹病毒糖蛋白B基因的羧基末端有相当大的同源性。在感染的早期，我们可以用一个代表ORF聚合酶部分的探针检测到两个3 '共末端转录物，长度分别为3.9 kb和1.7 kb。S1核酸酶和外切酶VII分析表明，这两个转录本都是非剪接的，并在hind ii片段d的独立位点上启动。通过引物延伸，我们能够精确地将3.9 kb RNA的5 '端定位到DNA聚合酶ORF开始上游186个核苷酸的位置。
The conservation of the herpesvirus DNA polymerases has allowed cross-hybridization studies to be used for their identification and mapping on the viral genome. With the use of a DNA fragment containing the DNA polymerase gene of human cytomegalovirus (HCMV) as a hybridization probe, we were able to localize the DNA polymerase gene of murine cytomegalovirus (MCMV) to a region within MCMVEcoRI fragment B which spans theHindIII site separatingHindIII fragments D and H. This site is colinear with the HCMV strain AD169 DNA polymerase gene. To confirm that this region encoded the MCMV DNA polymerase gene, we sequenced a 5131 nucleotide fragment from the Pstl site inHindIII fragment D to aBg/IIsite inHindIII fragment H. Initiating inHindIII fragment D and extending intoHindIII fragment H was a long open reading frame (ORF) 1097 amino acids in length with extensive homology to the DNA polymerases of HCMV, herpes simplex virus, and Epstein-Barr virus. Upstream of the polymerase ORF was a reading frame with considerable homology to the carboxy terminal half of the glycoprotein B gene of human herpesviruses. At early times in the infection, we could detect with a probe representing part of the polymerase ORF two 3′ coterminal transcripts, 3.9 kb and 1.7 kb in length. S1 nuclease and exonuclease VII analyses indicated that both transcripts were unspliced and initiated at independent sites inHindIII fragment D. By primer extension, we were able to map precisely the 5′ end of the 3.9-kb RNA to a site 186 nucleotides upstream of the beginning of the DNA polymerase ORF.