Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1967 Aug;1(4):771–778. doi: 10.1128/jvi.1.4.771-778.1967

Examination of the Qβ-Replicase Reaction by Sucrose Gradient and Gel Electrophoresis

N R Pace a,1, D H L Bishop a,2, S Spiegelman a
PMCID: PMC375352  PMID: 4912234

Abstract

The products of an in vitro synthesis with the Qβ replicase purified from Escherichia coli infected by the ribonucleic acid (RNA) bacteriophage Qβ were examined by sucrose gradient centrifugation and polyacrylamide gel electrophoresis. It was found that, in contrast to sucrose gradients, gel electrophoresis clearly resolved four classes in the newly synthesized RNA. The product was found to contain mature Qβ-RNA and small-molecular-weight RNA. In addition, two species were identified which corresponded to the structures found in vivo by Francke and Hofschneider and by Franklin. All of the participants implicated in RNA replication by in vivo studies have now been synthesized in vitro and isolated from the reaction mixture.

Full text

PDF
771

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bishop D. H., Claybrook J. R., Pace N. R., Spiegelman S. An analysis by gel electrophoresis of Q-beta-RNA complexes formed during the latent period of an in vitro synthesis. Proc Natl Acad Sci U S A. 1967 May;57(5):1474–1481. doi: 10.1073/pnas.57.5.1474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bishop D. H. Ribonucleic acid synthesis by Escherichia coli C 3000/L after infection by the ribonucleic acid coliphage ZIK/1, and properties of the coliphage-induced double-stranged ribonucleic acid. Biochem J. 1966 Sep;100(3):601–613. doi: 10.1042/bj1000601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Francke B., Hofschneider P. H. Infectious nucleic acids of e. Coli bacteriophages, ix. Sedimentation constants and strand integrity of infectious m12 phage replicative-form RNA. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1883–1890. doi: 10.1073/pnas.56.6.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Francke B., Hofschneider P. H. Uber infektiöse Substrukturen aus Escherichia coli Bakteriophagen. VII. Formation of a biologically intact replicative form in ribonucleic acid bacteriophage (M12)-infected cells. J Mol Biol. 1966 Apr;16(2):544–552. doi: 10.1016/s0022-2836(66)80190-0. [DOI] [PubMed] [Google Scholar]
  5. Franklin R. M. Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1504–1511. doi: 10.1073/pnas.55.6.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. HARUNA I., NOZU K., OHTAKA Y., SPIEGELMAN S. AN RNA "REPLICASE" INDUCED BY AND SELECTIVE FOR A VIRAL RNA: ISOLATION AND PROPERTIES. Proc Natl Acad Sci U S A. 1963 Nov;50:905–911. doi: 10.1073/pnas.50.5.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Haruna I., Spiegelman S. Specific template requirments of RNA replicases. Proc Natl Acad Sci U S A. 1965 Aug;54(2):579–587. doi: 10.1073/pnas.54.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Loening U. E. The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem J. 1967 Jan;102(1):251–257. doi: 10.1042/bj1020251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mills D. R., Pace N. R., Spiegelman S. The in vitro synthesis of a noninfectious complex containing biologically active viral RNA. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1778–1785. doi: 10.1073/pnas.56.6.1778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Pace N. R., Spiegelman S. In vitro synthesis of an infectious mutant RNA with a normal RNA replicase. Science. 1966 Jul 1;153(3731):64–67. doi: 10.1126/science.153.3731.64. [DOI] [PubMed] [Google Scholar]
  11. Pace N. R., Spiegelman S. The synthesis of infectious RNA with a replicase purified according to its size and density. Proc Natl Acad Sci U S A. 1966 Jun;55(6):1608–1615. doi: 10.1073/pnas.55.6.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Spiegelman S., Haruna I., Holland I. B., Beaudreau G., Mills D. The synthesis of a self-propagating and infectious nucleic acid with a purified enzyme. Proc Natl Acad Sci U S A. 1965 Sep;54(3):919–927. doi: 10.1073/pnas.54.3.919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. WEISSMANN C., BORST P., BURDON R. H., BILLETER M. A., OCHOA S. REPLICATION OF VIRAL RNA, III. DOUBLE-STRANDED REPLICATIVE FORM OF MSW PHAGE RNA. Proc Natl Acad Sci U S A. 1964 Apr;51:682–690. doi: 10.1073/pnas.51.4.682. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES