ANALYSIS OF UPSTREAM REGION OF HEPATITIS-B VIRUS CORE GENE USING IN-VITRO TRANSCRIPTION SYSTEM

Transcription of the core (C) gene of hepatitis B virus DNA (HBV-DNA) was studied by an in vitro transcription system using nuclear extracts of human liver cell (HepG2) and non-liver cell (HeLa) origins. RNA po lymerase II-dependent run-off transcription of 3.5-kb (C) mRNA was obs erved in both nuclear extracts; but the efficiency was much higher in the HepG2 nuclear extract. Analysis of run-off transcripts using upstr eam deletion mutants of HBV-DNA demonstrated that there are two transc ription start sites located at approximately nucleotide numbers (nt) 1 ,797 +/- 5 and 1,815 +/- 5. This analysis also suggested that the mini mum core promoter sequence and a cis-acting and liver-specific element for C mRNA transcription are located in the downstream region from -8 0 and -110 (HincII site) of transcription start sites, respectively. D NA-binding protein assays using synthetic double-stranded oligonucleot ide probes corresponding to three regions in the upstream region (nt f rom 1,401 to 1,760) of transcription start sites revealed that there a re some liver cell-specific and non-specific DNA-binding proteins in b oth nuclear extracts. The amount of those proteins was generally highe r in the HepG2 nuclear extract. However, no obvious correlation was ob served in the present study between the presence of DNA-binding protei ns and transcription activity of nuclear extracts in our system. The p ossible causes of this discrepancy are discussed. (C) 1994 Wiley-Liss, Inc.