Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules.

Download Full-text

Sequential transcription-translation of simian virus 40 by using mammalian cell extracts

Molecular and Cellular Biology ◽

10.1128/mcb.1.10.919-931.1981 ◽

1981 ◽

Vol 1 (10) ◽

pp. 919-931

Author(s):

C L Cepko ◽

U Hansen ◽

H Handa ◽

P A Sharp

Keyword(s):

Transcription Initiation ◽

Simian Virus 40 ◽

Restriction Enzymes ◽

Simian Virus ◽

T Antigen ◽

Coding Regions ◽

Cell Extracts ◽

Initiation Of Translation

Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of small t mRNA synthesized in vitro than of small t mRNA synthesized in vivo. The transcripts that served as mRNA's for the various polypeptides were identified by using the following two criteria. (i) The sensitivity of synthesis of a given protein to digestion of the template DNA with restriction enzymes allowed the localization of the promoter and coding regions. (ii) Translation of size-fractionated RNA allowed confirmation of the transcript-mRNA assignments. With these techniques we found that VP2, VP3 and, in some cases, VP1 synthesis resulted from the initiation of translation at internal AUG codons. In fact, families of polypeptides were produced by initiation of translation at AUG codons within sequences coding for VP1 and T, presumably as a result of transcription initiation events that generated 5' ends immediately upstream from these AUGs. Application of this technology for the identification of coding regions within cloned DNA fragments is discussed.

Download Full-text

Sequential transcription-translation of simian virus 40 by using mammalian cell extracts.

Molecular and Cellular Biology ◽

10.1128/mcb.1.10.919 ◽

1981 ◽

Vol 1 (10) ◽

pp. 919-931 ◽

Cited By ~ 4

Author(s):

C L Cepko ◽

U Hansen ◽

H Handa ◽

P A Sharp

Keyword(s):

Transcription Initiation ◽

Simian Virus 40 ◽

Restriction Enzymes ◽

Simian Virus ◽

T Antigen ◽

Coding Regions ◽

Cell Extracts ◽

Initiation Of Translation

Ribonucleic acids (RNAs) transcribed in vitro by using the whole-cell extract system of Manley et al. (Proc. Natl. Acad. Sci. U.S.A. 77:3855-3859, 1980) were tested for their efficiency and fidelity in directing protein synthesis in reticulocyte lysates. Simian virus 40 deoxyribonucleic acid (DNA), cleaved by various restriction endonucleases, was used as the template. Successful translation of the small tumor antigen t, as well as the capsid proteins VP1, VP2, and VP3, was detected by immunoprecipitation analysis. Although no synthesis of large T antigen was detected, use of this technology allows detection of large T synthesis resulting from the correct splicing of as little as 0.2% of the in vitro RNA transcripts, making it ideal for use as an in vitro splicing assay. Transcripts synthesized in vitro were used as messages at least as efficiently as were viral messenger RNA's (mRNA's) synthesized in vivo; and in the case of small t, there was more efficient translation of small t mRNA synthesized in vitro than of small t mRNA synthesized in vivo. The transcripts that served as mRNA's for the various polypeptides were identified by using the following two criteria. (i) The sensitivity of synthesis of a given protein to digestion of the template DNA with restriction enzymes allowed the localization of the promoter and coding regions. (ii) Translation of size-fractionated RNA allowed confirmation of the transcript-mRNA assignments. With these techniques we found that VP2, VP3 and, in some cases, VP1 synthesis resulted from the initiation of translation at internal AUG codons. In fact, families of polypeptides were produced by initiation of translation at AUG codons within sequences coding for VP1 and T, presumably as a result of transcription initiation events that generated 5' ends immediately upstream from these AUGs. Application of this technology for the identification of coding regions within cloned DNA fragments is discussed.

Download Full-text

The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2317-2323.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2317-2323

Author(s):

D Zarkower ◽

P Stephenson ◽

M Sheets ◽

M Wickens

Keyword(s):

Hela Cell ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

Polyadenylation Site ◽

Single Base ◽

Cleavage And Polyadenylation ◽

Cell Nuclear Extract

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.

Download Full-text

Replication of cloned DNA containing the Alu family sequence during cell extract-promoting simian virus 40 DNA synthesis

Molecular and Cellular Biology ◽

10.1128/mcb.4.8.1476-1482.1984 ◽

1984 ◽

Vol 4 (8) ◽

pp. 1476-1482

Author(s):

H Ariga

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Rna Polymerase Iii ◽

Simian Virus 40 ◽

Cell Extract ◽

Simian Virus ◽

T Antigen ◽

Sv40 T Antigen ◽

Sv40 Dna

The replicating activity of several cloned DNAs containing putative origin sequences was examined in a cell-free extract that absolutely depends on simian virus 40 (SV40) T antigen promoting initiation of SV40 DNA replication in vitro. Of the three DNAs containing the human Alu family sequence (BLUR8), the origin of (Saccharomyces cerevisiae plasmid 2 micron DNA (pJD29), and the yeast autonomous replicating sequence (YRp7), only BLUR8 was active as a template. Replication in a reaction mixture with BLUR8 as a template was semiconservative and not primed by a putative RNA polymerase III transcript synthesized on the Alu family sequence in vitro. Pulse-chase experiments showed that the small-sized DNA produced in a short-term incubation was converted to full-length closed circular and open circular DNAs in alkaline sucrose gradients. DNA synthesis in extracts began in a region of the Alu family sequence and was inhibited 80% by the addition of anti-T serum. Furthermore, partially purified T antigen bound the Alu family sequence in BLUR8 by the DNA-binding immunoassay. These results suggest that SV40 T antigen recognizes the Alu family sequence, similar to the origin sequence of SV40 DNA, and initiates semiconservative DNA replication in vitro.

Download Full-text

Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.75-83.1990 ◽

1990 ◽

Vol 10 (1) ◽

pp. 75-83

Author(s):

Y Berko-Flint ◽

S Karby ◽

D Hassin ◽

S Lavi

Keyword(s):

De Novo ◽

Chinese Hamster ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Dna Molecules ◽

Viral Origin ◽

Cell Extracts ◽

Origin Region

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3694-3704.1987 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3694-3704

Author(s):

C Prives ◽

Y Murakami ◽

F G Kern ◽

W Folk ◽

C Basilico ◽

...

Keyword(s):

Dna Replication ◽

Simian Virus 40 ◽

T Antigen ◽

Early Gene ◽

Wild Type ◽

The Core ◽

Cell Extracts ◽

Sequence Requirements

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

Download Full-text

BMP9 Can Induce Schwann Cells Expressing Simian Virus 40 T Antigen to Differentiate into Fat and Bone In Vivo and In Vitro

Cellular Reprogramming ◽

10.1089/cell.2020.0069 ◽

2021 ◽

Vol 23 (2) ◽

pp. 108-116

Author(s):

Rui-Fang Li ◽

Guo-Xin Nan ◽

Dan Wang ◽

Chang Gao ◽

Juan Yang ◽

...

Keyword(s):

Schwann Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen

Download Full-text

Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3815 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3815-3825 ◽

Cited By ~ 41

Author(s):

R S Decker ◽

M Yamaguchi ◽

R Possenti ◽

M L DePamphilis

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Early Gene ◽

Initial Direction ◽

Dna Polymerase Alpha

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

Download Full-text

Short donor site sequences inserted within the intron of beta-globin pre-mRNA serve for splicing in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4484 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4484-4491 ◽

Cited By ~ 17

Author(s):

A Mayeda ◽

Y Ohshima

Keyword(s):

Consensus Sequence ◽

Donor Site ◽

Simian Virus 40 ◽

Nuclear Extract ◽

Simian Virus ◽

T Antigen ◽

Beta Globin ◽

Vitro Assay ◽

Active Donor

We constructed SP6-human beta-globin derivative plasmids that included possible donor site (5' splice site) sequences at a specified position within the first intron. The runoff transcripts from these templates truncated in the second exon were examined for splicing in a nuclear extract from HeLa cells. In addition to the products from the authentic donor site, a corresponding set of novel products from the inserted, alternative donor site was generated. Thus, a short sequence inserted within an intron can be an active donor site signal in the presence of an authentic donor site. The active donor site sequences included a 9-nucleotide consensus sequence, 14- or 16-nucleotide sequences at the human beta-globin first or second donor, and those at simian virus 40 large T antigen or small t antigen donor. These included 3 to 8 nucleotides of an exon and 6 to 8 nucleotides of an intron. The activity of the inserted donor site relative to that of the authentic donor site depended on the donor sequence inserted. The relative activity also strongly depended on the concentrations of both KCl (40 to 100 mM) and MgCl2 (1.6 to 6.4 mM). At the higher KCl concentrations tested, all the inserted, or proximate, donor sites were more efficiently used. Under several conditions, some inserted donor sites were more active than was the authentic donor site. Our system provides an in vitro assay for donor site activity of a sequence to be tested.

Download Full-text