Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1972 Feb;9(2):207–215. doi: 10.1128/jvi.9.2.207-215.1972

Isolation of ΦX174 Specific Messenger Ribonucleic Acids In Vivo and Identification of Their 5′ Terminal Nucleotides

Marie N Hayashi 1, Masaki Hayashi 1
PMCID: PMC356284  PMID: 4552415

Abstract

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) hybridization in formamide at low temperature was applied to hybridization of ΦX174 replicative form DNA and in vivo ΦX174 specific messenger RNA (mRNA) with some modification. We found that ΦX174 mRNA up to molecular weight 1.2 × 106 could be hybridized to and eluted from DNA without apparent breakage of phosphodiester bonds and the 5′ terminal guanosine triphosphate and adenosine triphosphate of the RNA. By alkali hydrolysis of the purified in vivo ΦX174 mRNA and subsequent thin-layer chromatography of the digest, we isolated the 5′ terminal nucleotides and identified them as 2′- or 3′-monophosphate guanosine 5′-triphosphate (pppGp) and 2′- or 3′-monophosphate adenosine 5′-triphosphate (pppAp). By comparing the in vitro and in vivo synthesized ΦX174 mRNA, a difference in the pppAp-pppGp ratio was observed. In the in vitro RNA, this ratio was 1.5, whereas in the in vivo RNA it was 5.5.

Full text

PDF
207

Images in this article

212Image
on p.212
213Image
on p.213

Selected References

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

  1. Bonner J., Kung G., Bekhor I. A method for the hybridization of nucleic acid molecules at low temperature. Biochemistry. 1967 Dec;6(12):3650–3653. doi: 10.1021/bi00864a005. [DOI] [PubMed] [Google Scholar]
  2. Bremer H., Konrad M. W., Gaines K., Stent G. S. Direction of chain growth in enzymic RNA synthesis. J Mol Biol. 1965 Sep;13(2):540–553. doi: 10.1016/s0022-2836(65)80116-4. [DOI] [PubMed] [Google Scholar]
  3. Cashel M., Lazzarini R. A., Kalbacher B. An improved method for thin-layer chromatography of nucleotide mixtures containing 32P-labelled orthophosphate. J Chromatogr. 1969 Mar 11;40(1):103–109. doi: 10.1016/s0021-9673(01)96624-5. [DOI] [PubMed] [Google Scholar]
  4. Gelfand D. H., Hayashi M. Electrophoretic characterization of phiX174-specific proteins. J Mol Biol. 1969 Sep 28;44(3):501–516. doi: 10.1016/0022-2836(69)90376-3. [DOI] [PubMed] [Google Scholar]
  5. Gillespie D., Spiegelman S. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol Biol. 1965 Jul;12(3):829–842. doi: 10.1016/s0022-2836(65)80331-x. [DOI] [PubMed] [Google Scholar]
  6. Glynn I. M., Chappell J. B. A simple method for the preparation of 32-P-labelled adenosine triphosphate of high specific activity. Biochem J. 1964 Jan;90(1):147–149. doi: 10.1042/bj0900147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Goldstein A., Kirschbaum J. B., Roman A. Direction of synthesis of messenger RNA in cells of Escherichia coli. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1669–1675. doi: 10.1073/pnas.54.6.1669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hayashi M. N., Hayashi M. The stability of native DNA-RNA complexes during in vivo phiX-174 transcription. Proc Natl Acad Sci U S A. 1968 Nov;61(3):1107–1114. doi: 10.1073/pnas.61.3.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jorgensen S. E., Buch L. B., Nierlich D. P. Nucleoside triphosphate termini from RNA synthesized in vivo by Escherichia coli. Science. 1969 May 30;164(3883):1067–1070. doi: 10.1126/science.164.3883.1067. [DOI] [PubMed] [Google Scholar]
  10. Kuwano M., Schlessinger D., Apirion D. Ribonuclease V of Escherichia coli. IV. Exonucleolytic cleavage in the 5' to 3' direction with production of 5'-nucleotide monophosphates. J Mol Biol. 1970 Jul 14;51(1):75–82. doi: 10.1016/0022-2836(70)90271-8. [DOI] [PubMed] [Google Scholar]
  11. Maitra U., Hurwitz H. The role of DNA in RNA synthesis, IX. Nucleoside triphosphate termini in RNA polymerase products. Proc Natl Acad Sci U S A. 1965 Sep;54(3):815–822. doi: 10.1073/pnas.54.3.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. McConaughy B. L., Laird C. D., McCarthy B. J. Nucleic acid reassociation in formamide. Biochemistry. 1969 Aug;8(8):3289–3295. doi: 10.1021/bi00836a024. [DOI] [PubMed] [Google Scholar]
  13. Morikawa N., Imamoto F. Degradation of tryptophan messenger. On the degradation of messenger RNA for the tryptophan operon in Escherichia coli. Nature. 1969 Jul 5;223(5201):37–40. doi: 10.1038/223037a0. [DOI] [PubMed] [Google Scholar]
  14. Roberts J. W. Termination factor for RNA synthesis. Nature. 1969 Dec 20;224(5225):1168–1174. doi: 10.1038/2241168a0. [DOI] [PubMed] [Google Scholar]
  15. Scherberg N. H., Weiss S. B. Detection of bacteriophage T4- and T5-coded transfer RNAs. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1164–1171. doi: 10.1073/pnas.67.3.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Weiss S. B., Hsu W. T., Foft J. W., Scherberg N. H. Transfer RNA coded by the T4 bacteriophage genome. Proc Natl Acad Sci U S A. 1968 Sep;61(1):114–121. doi: 10.1073/pnas.61.1.114. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES