Identification of a novel transcription factor, ACF, in cultured avian fibroblast cells that interacts with a Marek's disease virus late gene promoter.
Interactions between factors in duck and chick embryo fibroblast (DEF and CEF, respectively) nuclear extracts and the Marek's disease virus (MDV) gp57-65 gene promoter were investigated. Results of in vitro transcription and gel mobility-shift assays indicated that multiple cellular factors interact with 5'-flanking sequences of the MDV gp57-65 gene. One sequence-specific DNA binding activity (termed ACF for avian cell factor(s)) was identified by interaction of DEF and CEF nuclear extract proteins with a particular site (nucleotides -193 to -177) in the MDV gp57-65 gene promoter. Binding of ACF to its apparent recognition sequence, contained within the 17-bp oligonucleotide 5'-CTAGTTTACTTGTTTGT-3' (ACF-12), was highly sequence-specific. Radiolabeled ACF-12 oligonucleotide bound significant ACF protein in the presence of a 400-fold molar excess of unlabeled nonspecific competitor DNA. A similar amount of specific competitor completely abolished ACF binding to probe DNA. Deletion of the ACF binding site from MDV gp57-65 gene promoters linked to a chloramphenicol acetyltransferase (CAT) reporter gene reduced expression of CAT activity by twofold relative to that seen with a gp57-65 promoter-CAT construct containing an intact ACF binding site. Transfection inhibition assays using double-stranded ACF binding site competitors reduced steady-state levels of gp57-65 mRNA in MDV infected cells by over twofold relative to those in control infected cells. Introduction of a similar amount of nonspecific double-stranded oligonucleotide had no adverse effect on gp57-65 mRNA levels. These data suggest that ACF is important for efficient expression of gp57-65.
Coussens
P M
PM
Department of Animal Science, Michigan State University, East Lansing 48824.
Tieber
V L
VL
Mehigh
C S
CS
Marcus
M
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002478:Cells, Cultured; D002642:Chick Embryo; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002872:Chromosome Deletion; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins; D004372:Ducks; D004625:Embryo, Nonmammalian; D005347:Fibroblasts / Q000502:physiology; D005814:Genes, Viral; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D008381:Herpesvirus 2, Gallid / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D015500:Chloramphenicol O-Acetyltransferase