INVITRO TRANSCRIPTION OF THE HEPATITIS-B VIRUS GENE BY NUCLEAR EXTRACTS OF HUMAN HEPATOMA-CELLS
INVITRO TRANSCRIPTION OF THE HEPATITIS-B VIRUS GENE BY NUCLEAR EXTRACTS OF HUMAN HEPATOMA-CELLS
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DOI：
10.1016/0042-6822(91)90595-3
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发表时间：
1991-06-01
期刊：
影响因子：
3.7
通讯作者：
SHIKATA, T
中科院分区：
文献类型：
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作者：
NISHIZONO, A;MAENO, M;SHIKATA, T
利用人肝癌细胞株核提取物研究了乙型肝炎病毒DNA （HBV DNA）的体外转录。在HepG2和PLC/PRF/5细胞的核提取物中观察到pre-St启动子控制下的RNA聚合酶ii依赖性pre-S mRNA的流出转录，并且这些提取物的效率显著高于非肝细胞（如HeLa， Molt-4和Ehrlich）的核提取物。以HBV DNA缺失突变体为模板，合成寡核苷酸为竞争对手，对st前启动子的径流转录本进行分析，结果表明，肝细胞核因子1对于s前mRNA的体外转录起始是必要的。还检测了前基因组RNA的径流转录本并确定了其起始位点。肝癌细胞和非肝细胞的核提取物均具有体外前基因组rna转录活性。然而，在该系统中未观察到S mRNA和X mRNA的径流转录本。这些结果提示HBV DNA转录在体外和体内的机制存在一定差异。这种体外转录系统可以很容易地分析核因子的生化和功能特征，有助于阐明HBV DNA的转录调控机制。
In vitrotranscription of hepatitis B virus DNA (HBV DNA) was studied using nuclear extracts of human hepatoma cell lines. RNA polymerase II-dependent run-off transcription of pre-S mRNA under the control of pre-St promoter was observed in nuclear extracts obtained from HepG2 and PLC/PRF/5 cells, and the efficiencies in these extracts were significantly higher than those in nuclear extracts of non-liver cells such as HeLa, Molt-4, and Ehrlich. Analysis of run-off transcripts by the pre-St promoter, using deletion mutants of HBV DNA as templates and synthetic oligonucleotides as competitors, showed that hepatocyte nuclear factor 1 was necessary for initiation ofin vitrotranscription of pre-S mRNA. The run-off transcript of pregenome RNA was also detected and its initiation site was determined. Nuclear extracts of not only hepatoma cells but non-liver cells were active in transcription of pregenome RNAin vitro. However, run-off transcripts of S mRNA and X mRNA were not observed in this system. These results suggest that there were some differences between the mechanisms of HBV DNA transcriptionin vitroandin vivo. Thisin vitrotranscription system will be useful for clarifying the mechanism regulating transcription of HBV DNA since the biochemical and functional characteristics of the nuclear factors can readily be analyzed.