Transcription of Simian virus 40 VI. SV40 DNA-RNA polymerase complex isolated from productively infected cells transcribed in vitro

Virology ◽  
1976 ◽  
Vol 75 (2) ◽  
pp. 346-354 ◽  
Author(s):  
Orgad Laub ◽  
Yosef Alonii
Keyword(s):  
Rna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polymerase Complex ◽  
Infected Cells ◽  
Sv40 Dna

scholarly journals Template Structural Requirements for Transcription In Vivo by RNA Polymerase II

1982 ◽  
Vol 2 (12) ◽  
pp. 1595-1607 ◽  
Author(s):  
Timothy J. Miller ◽  
Janet E. Mertz
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Structural Requirements ◽  
Infected Monkey ◽  
Full Complement ◽  
Sv40 Dna

Purified simian virus 40 (SV40) DNA is reconstituted into chromatin and transcribed by endogenous RNA polymerase II when microinjected into nuclei ofXenopus laevisoocytes. We have correlated the kinetics of chromatin reconstitution with that of accumulation of virus-specific RNA in this system. A delay of approximately 3 h was found in the appearance of appreciable numbers of both fully supercoiled molecules and transcriptionally active templates. SV40 minichromosomes, isolated from virus-infected monkey cells with 0.2 M NaCl, also exhibited this lag in onset of transcriptional activity when microinjected into oocytes. These findings indicate that neither purified SV40 DNA nor SV40 DNA containing a full complement of nucleosomes can function as a template for transcription in vivo before association with appropriate cellular nonhistone chromosomal factors has taken place. In addition, the gradual degradation of linear SV40 DNA in oocytes was not sufficient to account for the fact that it was much less transcriptionally active than circular SV40 DNA. Taken together, these results indicate that the conformational state of the DNA can affect its ability to function as a template for transcription in vivo by RNA polymerase II. In contrast, transcription by RNA polymerase III of purified, circularized cloned DNAs encoding genes for 5S rRNA was detectable long before the injected DNAs had time to reconstitute into chromatin. Therefore, the template structural requirements for transcription in vivo by RNA polymerases II and III are different.

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

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.

scholarly journals Transcription of hepatitis B virus by RNA polymerase II.

1983 ◽  
Vol 3 (10) ◽  
pp. 1766-1773 ◽  
Author(s):  
L B Rall ◽  
D N Standring ◽  
O Laub ◽  
W J Rutter
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Simian Virus 40 ◽  
Virus Genome ◽  
Simian Virus ◽  
Free System ◽  
B Virus

We employed an in vitro cell-free transcription system to locate RNA polymerase II promoters on the hepatitis B virus genome. The strongest promoter precedes the surface antigen (HBsAg) gene, which is comprised of a long (500 base pairs) presurface region as well as the mature HBsAg coding sequence. The origin of this transcript was localized by using truncated templates and S1 endonuclease mapping. The activity of the promoter was confirmed in transfection experiments in which the complete HBsAg gene was introduced into monkey kidney cells via a simian virus 40 expression vector. A second RNA polymerase II promoter preceding the HBcAg gene was also active in the cell-free system. The presence of multiple promoters in the hepatitis B virus genome suggests that the relative levels of viral-specific proteins detected in liver and serum may reflect differential or regulated promoter efficiency.

Transfer of nucleosomes from parental to replicated chromatin

1991 ◽  
Vol 11 (12) ◽  
pp. 6257-6267
Author(s):  
T Krude ◽  
R Knippers
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Replication Process ◽  
Two Dimensional Gel Electrophoresis ◽  
Sv40 Origin ◽  
Dna Strands ◽  
Topological State ◽  
High Concentrations ◽  
Sv40 Dna

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.

Evaluation of Topoisomerase Inhibitors as Potential Antiviral Agents

1993 ◽  
Vol 4 (2) ◽  
pp. 85-91 ◽  
Author(s):  
B. Maschera ◽  
E. Ferrazzi ◽  
M. Rassu ◽  
M. Toni ◽  
G. Palù
Keyword(s):  
Nalidixic Acid ◽  
Topoisomerase Ii ◽  
Antiviral Agents ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Etoposide Treatment ◽  
Cross Links ◽  
Infected Cells ◽  
Cellular Dna ◽  
Sv40 Dna

Anti-eukaryotic topoisomerase drugs, Camptothecin and Etoposide, were tested for their ability of selectively interfering with the replication of simian virus 40 (SV40) DNA. Nalidixic acid was also assayed for a comparison, since the compound has been previously reported to affect papoyavirus growth. Our results indicate that anti-eukaryotic topoisomerase drugs significantly inhibit viral DNA replication but at concentrations that are also toxic for uninfected cells. Etoposide treatment produced a relatively higher number of DNA-protein cross-links in virus-infected cells as compared to uninfected control cells. Nalidixic acid displayed some degree of selectivity for inhibiting SV40 DNA synthesis more effectively than synthesis of cellular DNA without appreciable reduction of cell growth. This activity does not appear to depend on DNA damage or interference with topoisomerase II and deserves further evaluation.

scholarly journals Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase.

1996 ◽  
Vol 16 (1) ◽  
pp. 94-104 ◽  
Author(s):  
F Stadlbauer ◽  
C Voitenleitner ◽  
A Brückner ◽  
E Fanning ◽  
H P Nasheuer
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Polymerase Alpha ◽  
Cell Extracts ◽  
Human Dna ◽  
Sv40 Dna ◽  
Hybrid Complexes

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.

scholarly journals Primer-DNA formation during simian virus 40 DNA replication in vitro.

1993 ◽  
Vol 13 (5) ◽  
pp. 2882-2890 ◽  
Author(s):  
D Denis ◽  
P A Bullock
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Antigen ◽  
Origin Region ◽  
Sv40 Dna ◽  
Proliferating Cell

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.

Simian virus 40 minichromosomes contain torsionally strained DNA molecules

1985 ◽  
Vol 5 (11) ◽  
pp. 3048-3057
Author(s):  
J Barsoum ◽  
P Berg
Keyword(s):  
Topoisomerase I ◽  
Simian Virus 40 ◽  
Staphylococcal Nuclease ◽  
Simian Virus ◽  
Infected Cells ◽  
Complete Digestion ◽  
Sv40 Genome ◽  
Negative Supercoiling ◽  
Sv40 Dna ◽  
Nuclease Sensitivity

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.

Transcription of hepatitis B virus by RNA polymerase II

1983 ◽  
Vol 3 (10) ◽  
pp. 1766-1773
Author(s):  
L B Rall ◽  
D N Standring ◽  
O Laub ◽  
W J Rutter
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Simian Virus 40 ◽  
Virus Genome ◽  
Simian Virus ◽  
Free System ◽  
B Virus

We employed an in vitro cell-free transcription system to locate RNA polymerase II promoters on the hepatitis B virus genome. The strongest promoter precedes the surface antigen (HBsAg) gene, which is comprised of a long (500 base pairs) presurface region as well as the mature HBsAg coding sequence. The origin of this transcript was localized by using truncated templates and S1 endonuclease mapping. The activity of the promoter was confirmed in transfection experiments in which the complete HBsAg gene was introduced into monkey kidney cells via a simian virus 40 expression vector. A second RNA polymerase II promoter preceding the HBcAg gene was also active in the cell-free system. The presence of multiple promoters in the hepatitis B virus genome suggests that the relative levels of viral-specific proteins detected in liver and serum may reflect differential or regulated promoter efficiency.

Induction of specific transcription by RNA polymerase III in transformed cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3068-3076
Author(s):  
M F Carey ◽  
K Singh ◽  
M Botchan ◽  
N R Cozzarelli
Keyword(s):  
Rna Polymerase ◽  
Gene Family ◽  
Rna Polymerase Iii ◽  
Simian Virus 40 ◽  
In Vitro Transcription ◽  
Simian Virus ◽  
Intrinsic Signals ◽  
Polymerase Iii ◽  
Pol Iii

RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation-inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA.

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