Deletion analysis of the polyomavirus late promoter: evidence for both positive and negative elements in the absence of early proteins.
We have been interested in understanding more about the sequences that constitute the polyomavirus late promoter. Our approach has been to target specific deletions to the viral intergenic region by oligonucleotide-directed mutagenesis. Wild-type and mutant promoter cassettes with defined deletions were then inserted into a promoterless expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene (cat). Plasmids were introduced into mouse NIH 3T3 cells by transfection, and promoter activities were assessed by quantitation of both CAT enzyme and cat mRNA levels. In this report, we present the results of experiments designed to map promoter elements which affect late transcription in the absence of early viral proteins and viral DNA replication. Using this approach, we mapped two major cis-acting elements (a positive and a negative one) which affect transcription in our transient expression system. The first, positive, element coincided with the enhancer A element, which is known to be important for early transcription and viral DNA replication. Removal of this element reduced late transcription by 50- to 100-fold. The second element was a negative one; removal of 89 base pairs that included two high-affinity large-T-antigen-binding sites just to the early side of the inverted repeat structure within the replication origin resulted in a 5- to 10-fold increase in late promoter activity. The implications of these findings for late promoter function and regulation are discussed.
Cahill
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KB
Department of Microbiology, University of Connecticut Health Center, Farmington 06032.
Carmichael
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GG
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics; D002872:Chromosome Deletion; D004742:Enhancer Elements, Genetic; D051379:Mice; D008969:Molecular Sequence Data; D011120:Polyomavirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic
D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase