• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
J Virol. Aug 1979; 31(2): 360–369.
PMCID: PMC353458

Regulation of Simian Virus 40 Transcription: Sensitive Analysis of the RNA Species Present Early in Infections by Virus or Viral DNA

Abstract

We have examined the discrete species of simian virus 40 (SV40) RNA present very early in infection of monkey cells with wild-type virus, with mutant tsA58 virus, and with the corresponding DNAs to distinguish between two classes of models for control of late transcription: (i) positive control mediated by large-T antigen and (ii) negative control mediated by a repressor protein associated with viral DNA in the virion. Total cytoplasmic or nuclear polyadenylated RNAs from infected cells were denatured with glyoxal, separated by electrophoresis on agarose gels, and transferred to diazobenzyloxymethyl paper. The positions of specific early and late RNA species were determined with region-specific SV40 DNA probes. The technique can detect individual RNAs present at the level of less than one copy per cell. After 9.5 h at 37°C, appreciable amounts of two early RNAs (2.6 kilobases [kb] and 2.9 kb) were present in the cytoplasm of cells infected with wild-type virus or DNA, along with much smaller amounts of two late RNAs, 1.6 kb (16S) and 2.5 kb (19S). The amounts of the late RNAs were reduced, but they were still synthesized in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. In comparable infections with tsA58 virus or DNA at nonpermissive temperature (41°C), substantial amounts of the two early RNAs were again present, but the two late RNAs could not be detected. However, small amounts of the late RNAs were found when infections with tsA58 virus or DNA were prolonged to 30 h at 41°C. These results are not consistent with negative control of late transcription through the action of a repressor and, taken together with other data, suggest that T antigen has an active role in late RNA synthesis. Specific early and late viral RNAs were also detected in the nuclear poly(A)+ fractions and were similar in size to the RNA species found in the cytoplasmic polyadenylated fractions. The late nuclear RNAs (1.8 and 2.9 kb) were significantly larger than the late cytoplasmic species, possibly because they are precursors. The 2.6- and 2.9-kb early RNAs found in the cytoplasm are probably the messengers for large-T and small-t antigens, respectively.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.1M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Alwine JC, Kemp DJ, Stark GR. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. [PMC free article] [PubMed]
  • Alwine JC, Reed SI, Stark GR. Characterization of the autoregulation of simian virus 40 gene A. J Virol. 1977 Oct;24(1):22–27. [PMC free article] [PubMed]
  • Avila J, Saral R, Martin RG, Khoury G. The temperature-sensitive defect in SV40 group D mutants. Virology. 1976 Aug;73(1):89–95. [PubMed]
  • Aviv H, Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. [PMC free article] [PubMed]
  • Berk AJ, Sharp PA. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. [PubMed]
  • Berk AJ, Sharp PA. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. [PMC free article] [PubMed]
  • Birkenmeier EH, Radonovich MF, Shani M, Salzman NP. The SV40 DNA template for transcription of late mRNA in viral nucleoprotein complexes. Cell. 1977 Jul;11(3):495–504. [PubMed]
  • Chou JY, Martin RG. DNA infectivity and the induction of host DNA synthesis with temperature-sensitive mutants of simian virus 40. J Virol. 1975 Jan;15(1):145–150. [PMC free article] [PubMed]
  • Cowan K, Tegtmeyer P, Anthony DD. Relationship of replication and transcription of Simian Virus 40 DNA. Proc Natl Acad Sci U S A. 1973 Jul;70(7):1927–1930. [PMC free article] [PubMed]
  • Crawford LV, Cole CN, Smith AE, Paucha E, Tegtmeyer P, Rundell K, Berg P. Organization and expression of early genes of simian virus 40. Proc Natl Acad Sci U S A. 1978 Jan;75(1):117–121. [PMC free article] [PubMed]
  • Estes MK, Huang ES, Pagano JS. Structural polypeptides of simian virus 40. J Virol. 1971 May;7(5):635–641. [PMC free article] [PubMed]
  • Ferdinand FJ, Brown M, Khoury G. Characterization of early simian virus 40 transcriptional complexes: late transcription in the absence of detectable DNA replication. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5443–5447. [PMC free article] [PubMed]
  • Fiers W, Contreras R, Haegemann G, Rogiers R, Van de Voorde A, Van Heuverswyn H, Van Herreweghe J, Volckaert G, Ysebaert M. Complete nucleotide sequence of SV40 DNA. Nature. 1978 May 11;273(5658):113–120. [PubMed]
  • Frost E, Williams J. Mapping temperature-sensitive and host-range mutations of adenovirus type 5 by marker rescue. Virology. 1978 Nov;91(1):39–50. [PubMed]
  • Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. [PubMed]
  • Green MH, Brooks TL. Recently replicated simian virus 40 DNA is a preferential template for transcription and replication. J Virol. 1978 May;26(2):325–334. [PMC free article] [PubMed]
  • Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. [PubMed]
  • Khoury G, Byrne JC, Martin MA. Patterns of Simian Virus 40 DNA transcription after acute infection of permissive and nonpermissive cells. Proc Natl Acad Sci U S A. 1972 Jul;69(7):1925–1928. [PMC free article] [PubMed]
  • Khoury G, Howley P, Nathans D, Martin M. Posttranscriptional selection of simian virus 40-specific RNA. J Virol. 1975 Feb;15(2):433–437. [PMC free article] [PubMed]
  • Khoury G, May E. Regulation of early and late simian virus 40 transcription: overproduction of early viral RNA in the absence of a functional T-antigen. J Virol. 1977 Jul;23(1):167–176. [PMC free article] [PubMed]
  • Lai CJ, Dhar R, Khoury G. Mapping the spliced and unspliced late lytic SV40 RNAs. Cell. 1978 Aug;14(4):971–982. [PubMed]
  • Lai CJ, Nathans D. A map of temperature-sensitive mutants of simian virus 40. Virology. 1975 Jul;66(1):70–81. [PubMed]
  • Laub O, Aloni Y. Transcription of simian virus 40. V. Regulattion of simian virus 40 gene expression. J Virol. 1975 Nov;16(5):1171–1183. [PMC free article] [PubMed]
  • Laub O, Bratosin S, Horowitz M, Aloni Y. The initiation of transcription of SV40 DNA at late time after infection. Virology. 1979 Jan 30;92(2):310–323. [PubMed]
  • Lindstrom DM, Dulbecco R. Strand orientation of simian virus 40 transcription in productively infected cells. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1517–1520. [PMC free article] [PubMed]
  • Manteuil S, Girard M. Inhibitors of DNA synthesis: their influence on replication and transcription of simian virus 40 DNA. Virology. 1974 Aug;60(2):438–454. [PubMed]
  • Manteuil S, Kopecka H, Caraux J, Prunell S, Girard M. Letter to the editor: In vivo incorporation of cytosine arabinoside into simian virus 40 DNA. J Mol Biol. 1974 Dec 25;90(4):751–756. [PubMed]
  • McDonell MW, Simon MN, Studier FW. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. [PubMed]
  • McMaster GK, Carmichael GG. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. [PMC free article] [PubMed]
  • Mertz JE, Berg P. Defective simian virus 40 genomes: isolation and growth of individual clones. Virology. 1974 Nov;62(1):112–124. [PubMed]
  • Paucha E, Harvey R, Smith AE. Cell-free synthesis of simian virus 40 T-antigens. J Virol. 1978 Oct;28(1):154–170. [PMC free article] [PubMed]
  • Reed SI, Ferguson J, Davis RW, Stark GR. T antigen binds to simian virus 40 DNA at the origin of replication. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1605–1609. [PMC free article] [PubMed]
  • Reed SI, Stark GR, Alwine JC. Autoregulation of simian virus 40 gene A by T antigen. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3083–3087. [PMC free article] [PubMed]
  • Rigby PW, Dieckmann M, Rhodes C, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. [PubMed]
  • Robb JA, Martin RG. Genetic analysis of simian virus 40. 3. Characterization of a temperature-sensitive mutant blocked at an early stage of productive infection in monkey cells. J Virol. 1972 Jun;9(6):956–968. [PMC free article] [PubMed]
  • Sambrook J, Sharp PA, Keller W. Transcription of Simian virus 40. I. Separation of the strands of SV40 DNA and hybridization of the separated strands to RNA extracted from lytically infected and transformed cells. J Mol Biol. 1972 Sep 14;70(1):57–71. [PubMed]
  • Tabak HF, Flavell RA. A method for the recovery of DNA from agarose gels. Nucleic Acids Res. 1978 Jul;5(7):2321–2332. [PMC free article] [PubMed]
  • Takemoto KK, Kirschstein RL, Habel K. Mutants of simian virus 40 differing in plaque size, oncogenicity, and heat sensitivity. J Bacteriol. 1966 Oct;92(4):990–994. [PMC free article] [PubMed]
  • Tegtmeyer P. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J Virol. 1972 Oct;10(4):591–598. [PMC free article] [PubMed]
  • Tegtmeyer P, Schwartz M, Collins JK, Rundell K. Regulation of tumor antigen synthesis by simain virus 40 gene A. J Virol. 1975 Jul;16(1):168–178. [PMC free article] [PubMed]
  • Tjian R. The binding site on SV40 DNA for a T antigen-related protein. Cell. 1978 Jan;13(1):165–179. [PubMed]
  • Villarreal LP, White RT, Berg P. Mutational alterations within the simian virus 40 leader segment generate altered 16S and 19S mRNA's. J Virol. 1979 Jan;29(1):209–219. [PMC free article] [PubMed]
  • Zimmerman SB, Sandeen D. The ribonuclease activity of crystallized pancreatic deoxyribonuclease. Anal Biochem. 1966 Feb;14(2):269–277. [PubMed]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...