Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids.

Abstract

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neor colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 ElA core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.The 5.3-kilobase (kb) genome of polyomavirus contains a noncoding region of approximately 460 base pairs (bp) that includes the viral origin of replication as well as sequences involved in the regulation of viral transcription. These sequences include an enhancer region that is required in cis for both early region gene expression and viral DNA replication (13,41,47,55). The necessity of this enhancer region for replication is independent of the effects of this region on early region transcription since provision of the viral large T antigen in trans fails to induce DNA replication in mutants that lack this region (14).The enhancer region is located within a 244-bp segment of polyomavirus DNA between the BclI and the PvuII site at nucleotide 5262 (13). This region contains two nonoverlapping elements that have homology to two other viral enhanc...

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

“…The replication in permissive cells of many of the mutants used in these studies has been previously described (13,31,32,52). In this study, several of the mutants were either tested for the first time or reexamined for their ability to replicate in WOP mouse cells (13).…”

“…Also, the relative affinities of Py and SV40 T Ags for binding sites within their respective ori regions are apparently dissimilar (12,14,28,47). Moreover, while both viruses encode enhancer elements, the Py enhancer is almost directly adjacent to the late boundary of the ori region and consists of a series of discrete elements that bear homologies to other enhancer elements including those of SV40, adenovirus Ela, and mouse immunoglobulin genes (2,22,23,26,31,38,46,51,53). By contrast, the SV40 enhancer, a 72-bp repeated sequence (see reference 22 and references therein), is separated from the core ori by a series of G+C-rich sequences (the 21-bp repeats) that form a distinct transcriptional control element to which the cellular transcription factor, Spl, binds (17) and that is lacking in the Py genome.…”

“…Immunopurified Py T Ag was stored at -20°C after dialysis against buffer containing 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.5), 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 50% glycerol (T Ag dialysis buffer) and retained activity for at least 2 months. FM3A cell extracts were prepared from suspension cultures of mouse FM3A cells grown to a density of 5 x 105 cells per ml in Dulbecco modified Eagle medium plus 10% calf serum, according to procedures developed for the preparation of HeLa cell extracts for in vitro replication of SV40 DNA (56 Superhelical circular duplex (RF1) plasmid DNAs were prepared by published procedures (13,31,52) and were generally subjected to two rounds of CsCI equilibrium density centrifugation. DNAs were quantified both by measuring A260 and by comparison with standards after agarose gel electrophoresis and ethidium bromide staining.…”

A unique subpopulation of murine DNA polymerase alpha/primase specifically interacts with polyomavirus T antigen and stimulates DNA replication.

“…Here, by using affinity columns containing either DNA polymerase ac/primase or Py T antigen, we present evidence that supports this suggestion. Furthermore (24), and pSVS contains the entire SV40 genome inserted at the BamHI site of pBR322. The incubations were carried out at 33 and 37°C for Py and SV40 replication, respectively.…”

Enhancer and promoter elements from simian virus 40 and polyomavirus can substitute for an upstream activation sequence in Saccharomyces cerevisiae.

“…(ii) An enhancer region, originally defined as the 244 bp between the BcII (bp 5021) and PvuII (bp 5264) restriction enzyme sites (17,81), has been subdivided into two unrelated but functionally redundant elements, enhancer A and enhancer B, both of which are capable of stimulating transcription of heterologous genes in an orientation-and position-independent manner (42). Studies with gel retention and nuclease mapping assays have identified several cellular protein factors that bind specifically to the B enhancer (7,26,70 (9,38) and in vivo (2,32,40) and that at least some of the elements of the polyomavirus early promoter, including portions of the enhancer, may regulate the levels of transcription from the late promoter of that virus (8,13,(52)(53)(54).…”