Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

Keller, Janis; Alwine, James C.

doi:10.1128/mcb.5.8.1859

Cited by 129 publications

(142 citation statements)

References 67 publications

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“…Alternatively, in pRC4CATori the cytochrome c promoter is positioned 2.5 kilobases downstream from the late side of the SV40 origin, and T antigen may trans-activate this promoter, as it does the SV40 late genes, through specific origin sites (5,6,18,19). To distinguish between these T-antigendependent activities, a replication-defective origin, identical to that in pRC4CATori except for a 4-bp deletion in Tantigen-binding site 11 (12), was used for the construction of pRC4CAToriA, a replication-defective derivative.…”

Section: Resultsmentioning

confidence: 99%

“…It is unlikely that T antigen itself augments these cellular promoters by direct binding, because the pure protein did not bind to the cytochrome c promoter under conditions in which the interaction of T antigen with known binding sites in the SV40 origin was clearly detected. Likewise, activation of the SV40 late genes by T antigen can occur in the absence of T-antigen-binding sites (19). Recently, the transcription factor that mediates the zinc-dependent induction of the human metallothionein II promoter has been shown to be required for maximal activity of the SV40 enhancer (37).…”

Section: Discussionmentioning

confidence: 99%

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Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.

Evans

Scarpulla

1988

Mol. Cell. Biol.

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To investigate the transcriptional control of nuclear-encoded respiratory genes in mammals, we have performed a deletional analysis of cis-acting regulatory sequences in the rat somatic cytochrome c gene. Three major regions are required for maximal expression of the transfected gene in kidney cell lines CV-1 and COS-1. One of these, region III (+71 to + 115 from the transcription initiation site), is an unusual intragenic controlling element found in the 5' end of the first intron, while the other two, region I (-191 to -165) and region II (-139 to -84), define the upstream promoter. Region II contains two consensus CCAAT boxes and mediates a constitutive level of expression in both cell lines. In contrast, regions I and III are both required for the increased promoter activity observed in COS-1 cells compared with promoter activity observed in CV-1 cells, and the regions function individually as competitors with the full promoter for trans-acting factors or complexes. Region III contains a perfect octanucleotide homology with region I in addition to a consensus Spl-transcription-factor-binding site. Promoter stimulation in COS-1 cells can be duplicated in CV-1 cells by cotransfecting with a T-antigen-producing vector, but purified T antigen does not bind anywhere in the cytochrome c promoter. A control promoter from the mouse metallothionein I gene is similarly activated in T-antigen-producing cells only in the presence of zinc, which activates its upstream regulatory sites. We conclude that T antigen stimulates these cellular promoters through the activation or induction of cellular factors or complexes that mediate their effects through promoter-specific regulatory elements. Cytochrome c promoter regions activated in this system may play a physiological role in controlling gene expression.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.

Evans

Scarpulla

1988

Mol. Cell. Biol.

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“…The plasmid that served as a template for the RNA probe for RNase protection assays, named pGEM2-PAS, was constructed by isolating the SV40 polyadenylation signal- The plasmid used as a source of T antigen in cotransfection experiments was p6-ldL, described elsewhere (25).…”

Section: Methodsmentioning

confidence: 99%

Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

Carswell¹,

Alwine²

1989

Mol. Cell. Biol.

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141

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The late polyadenylation signal of simian virus 40 functions with greater efficiency than the early polyadenylation signal, in turn affecting steady-state mRNA levels. Two chloramphenicol acetyltransferase (CAT) transient expression vectors, pL-EPA and pL-LPA, that differ only in their polyadenylation signals were constructed by using the early and late polyadenylation signals, respectively. In transfections of Cos, CV-1P, or HeLa cells and subsequent Northern blot analysis of CAT-specific RNA, approximately five times more steady-state CAT mRNA was produced in transfections with pL-LPA than with pL-EPA. The basis for this difference was not related to the specific promoter used or to RNA stability. Overall, the difference in steady-state mRNA levels derived from the two plasmids appeared to be attributable to intrinsic properties of the two polyadenylation signals, resulting in distinctly different cleavage and polyadenylation efficiencies. Additionally, we found that the utilization of the late polyadenylation site was dramatically reduced by deletion of sequences between 48 and 29 nucleotides 5' of the AAUAAA hexanucleotide. This reduction of mRNA levels was shown not to be caused by altered stability of mutant precursor RNAs or mRNAs, suggesting that these upstream sequences constitute an element of the late polyadenylation signal and may cause, at least to some extent, the greater efficiency of utilization of the late polyadenylation site.Most eucaryotic mRNAs are cleaved postranscriptionally at a specific site in the 3' untranslated region between 10 and 30 nucleotides downstream of a consensus AAUAAA sequence (42). Subsequent to cleavage, a polyadenosine chain with an average length of about 200 to 300 nucleotides is covalently attached to the RNA at the cleavage site (reviewed in reference 8). The purpose of 3'-end cleavage and polyadenylation is not clearly understood. However, studies with mutants have implicated polyadenylation as an essential process for the production of stable mRNA (reviewed in reference 5). In the present study, we compared the relative efficiencies of polyadenylation at the simian virus 40 (SV40) early and late polyadenylation sites. We found that the late polyadenylation signal is substantially more efficient than the early one in vivo. The findings in this work support the existence of a gene expression mechanism by which the relative efficiency of polyadenylation site utilization affects the steady-state levels of mRNA. We also found that the SV40 late polyadenylation signal contains an unusual element upstream of its AAUAAA hexanucleotide that is required for efficient usage of the late polyadenylation site.SV40 is a temporally regulated DNA virus, with early and late phases of infection. During a lytic infection of permissive monkey cells, early RNA is transcribed and the major early protein, T antigen, is made. T antigen is a multifunctional protein that enables replication of viral DNA as well as down-regulation of its own transcription (reviewed in reference 1) and activa...

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“…Recent studies have suggested that in the absence of DNA replication, T antigen also plays a significant role, both direct and indirect, in activating late transcription (10)(11)(12)(13).…”

mentioning

confidence: 99%

Carboxyl-terminal mutants of the large tumor antigen of simian virus 40: a role for the early protein late in the lytic cycle.

Khalili¹,

Brady²,

Pipas³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Simian virus 40 (SV40) mutants d11066 and d11140 contain deletions within the region encoding the carboxyl terminus of the large tumor (T) antigen. Although these mutations have little effect on the efficiency of viral DNA replication, they decrease the yield of infectious virus particles by 3-4 orders of magnitude [Pipas, J. (1985) J. Virol. 54,[569][570][571][572][573][574][575]. Here we show that the level of late RNA is lower by a factor of 5-15 in CV-1P monkey cells infected with these mutants compared to cells infected with wild-type SV40. Consistent with this decrease in RNA, synthesis of late viral structural proteins VP1 and VP3 decreases by a factor of 5-15. In contrast, the synthesis of SV40 agnoprotein decreases by a factor >100. Intercistronic complementation of these mutants with pm1493 and d1121, two SV40 mutants that are defective in agnoprotein but encode wild-type T antigen, results in an increased synthesis of agnoprotein in the infected cells. These results suggest that the carboxyl-terminal portion of T antigen participates in the posttranscriptional regulation of agnoprotein.Simian virus 40 (SV40) is a small DNA tumor virus that has served as a model system for studies of eukaryotic gene regulation (1). The virus produces a lytic infection of African green monkey kidney cells in which its genes are expressed temporally in two phases. Prior to the onset of DNA replication, an early set of genes encoding large (T) and small (t) tumor antigens is expressed. After the onset of DNA replication the major transcriptional program shifts to the expression of the late genes, which encode the agnoprotein and the viral structural proteins VP1, VP2, and VP3 (1-3). While the function of the 61 amino acid agnoprotein is not entirely clear, it appears to play a role in encapsidation, in assembly of the mature viral particles, or in release of virus particles from infected cells (4-6). In addition, the late agnoprotein may play a role in regulation of late gene expression (7-9).The role of T antigen in activating the late phase of the lytic cycle relates in part to its autoregulation of early gene transcription and the initiation of rounds of DNA replication that increases the SV40 template number. Recent studies have suggested that in the absence of DNA replication, T antigen also plays a significant role, both direct and indirect, in activating late transcription (10-13).Mutations in the carboxyl terminus of T antigen (14,15) have an interesting phenotype in that, although a minimal decrease in the efficiency of viral DNA replication is observed, under appropriate conditions they decrease the yield of infectious virus particles by 3 orders of magnitude (14).This reduction of plaque formation occurs on BSC-40 cells at 32°C or on CV-1P cells at 32°C or 37°C. The present study was conducted to determine the effect of these T-antigen mutants on the levels of late viral RNA or capsid protein. MATERIALS AND METHODSCells and Viruses. The cells used in these experiments were established CV-1P and BSC-40 A...

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Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

Cited by 129 publications

References 67 publications

Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.

Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.

Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

Carboxyl-terminal mutants of the large tumor antigen of simian virus 40: a role for the early protein late in the lytic cycle.

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