Characterization of the role of individual protein binding motifs within the hepatitis B virus enhancer I on X promoter activity using linker scanning mutagenesis.
A combination of linker scanning mutagenesis and deletional analyses has been used to determine the role of individual DNA-protein binding sites on expression from the hepatitis B virus (HBV) enhancer I-X promoter (map position (mp) 1042-1354, HBV adw2). Linker scanning mutation of the EF-C site caused a 67.5% drop in X promoter activity in HuH7 cells, but had no effect in HepG2 or HepSK cells. Mutation of the E element resulted in an approximately 50% reduction in X promoter activity in HuH7, HepG2, and HepSK cells. Deletional analysis showed that sequences upstream of the EF-C site (mp 1163) were required for full X promoter activity and implicated the NF-1a site as being sufficient for basal X promoter activity. However, PCR-directed linker scanning mutation of the NF-1a site did not cause a reduction in X promoter activity, indicating that this site was not an essential component of the X promoter. Taken together, these results indicated that multiple, partially redundant protein:DNA interactions in the enhancer I are essential for full X promoter activity. The lack of an essential basal promoter element supports the suggestion that the two separate HBV enhancer elements (enhI and enhII) were created by integration of the X gene into a primordial enhancer element.
Gustin
K
K
Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461.
Shapiro
M
M
Lee
W
W
Burk
R D
RD
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005814:Genes, Viral; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008099:Liver; D008113:Liver Neoplasms; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014407:Tumor Cells, Cultured
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase