Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1978 Feb;5(2):591–598. doi: 10.1093/nar/5.2.591

Enzymatic synthesis of a segment of bacteriophage Qbeta coat protein gene.

Y Kikuchi, K Sakaguchi
PMCID: PMC342004  PMID: 416426

Abstract

The oligoribonucleotide, A-A-A-C-U-U-U-Gp, constituting a segment of RNA bacteriophage Qbeta coat protein gene was efficiently synthesized at a milligram scale by a combination of enzymatic methods using bacteriophage T4 RNA ligase and the thermophilic polynucleotide phosphorylase. A-A-A-Cp was synthesized from A-A-A and pCp by the newly developed mononucleotide addition method using T4 RNA ligase in a yield of 83%, followed by dephosphorylation with bacterial alkaline phosphatase to obtain A-A-A-C. pU-U-U-Gp was synthesized from pU-U-U and GDP by the simultaneous action of polynucleotide phosphorylase and RNase T1 in a yield of 32%. finally, the two oligonucleotides (A-A-A-C and pU-U-U-Gp) were ligated with T4 RNA ligase and the octanucleotide, A-A-A-C-U-U-U-Gp, was obtained in a yield of 85%.

Full text

PDF
591

Selected References

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

  1. Cranston J. W., Silber R., Malathi V. G., Hurwitz J. Studies on ribonucleic acid ligase. Characterization of an adenosine triphosphate-inorganic pyrophosphate exchange reaction and demonstration of an enzyme-adenylate complex with T4 bacteriophage-induced enzyme. J Biol Chem. 1974 Dec 10;249(23):7447–7456. [PubMed] [Google Scholar]
  2. GAREN A., LEVINTHAL C. A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim Biophys Acta. 1960 Mar 11;38:470–483. doi: 10.1016/0006-3002(60)91282-8. [DOI] [PubMed] [Google Scholar]
  3. Hishinuma F., Hirai K., Sakaguchi K. Thermophilic polynucleotide phosphorylase from Thermus thermophilus. Purification and properties of an altered form of enzyme which lacks phosphorolytic activity to polynycleotide. Eur J Biochem. 1977 Aug 1;77(3):575–583. doi: 10.1111/j.1432-1033.1977.tb11701.x. [DOI] [PubMed] [Google Scholar]
  4. Kaufmann G., Kallenbach N. R. Determination of recognition sites of T4 RNA ligase on the 3'-OH and 5' -P termini of polyribonucleotide chains. Nature. 1975 Apr 3;254(5499):452–454. doi: 10.1038/254452a0. [DOI] [PubMed] [Google Scholar]
  5. Kikuchi Y., Hirai K., Sakaguchi K. Novel monofunctional substrates of polynucleotide phosphorylase. The "single-addition" of 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide. J Biochem. 1975 Feb;77(2):469–472. doi: 10.1093/oxfordjournals.jbchem.a130747. [DOI] [PubMed] [Google Scholar]
  6. Kikuchi Y., Hirai K., Someno K., Sakaguchi K. Enzymatic synthesis of oligonucleotides of defined sequence. The "single addition" of 2(3)-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide catalyzed by a thermophilic polynucleotide phosphorylase. J Biochem. 1976 Jul;80(1):33–37. doi: 10.1093/oxfordjournals.jbchem.a131254. [DOI] [PubMed] [Google Scholar]
  7. Last J. A., Anderson W. F. Purification and properties of bacteriophage T4-induced RNA ligase. Arch Biochem Biophys. 1976 May;174(1):167–176. doi: 10.1016/0003-9861(76)90335-0. [DOI] [PubMed] [Google Scholar]
  8. Ohtsuka E., Nishikawa S., Sugiura M., Ikehara M. Joining of ribooligonucleotides with T4 RNA ligase and identification of the oligonucleotide-adenylate intermediate. Nucleic Acids Res. 1976 Jun;3(6):1613–1623. doi: 10.1093/nar/3.6.1613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Silber R., Malathi V. G., Hurwitz J. Purification and properties of bacteriophage T4-induced RNA ligase. Proc Natl Acad Sci U S A. 1972 Oct;69(10):3009–3013. doi: 10.1073/pnas.69.10.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Sninsky J. J., Last J. A., Gilham P. T. The use of terminal blocking groups for the specific joining of oligonucleotides in RNA ligase reactions containing equimolar concentrations of acceptor and donor molecules. Nucleic Acids Res. 1976 Nov;3(11):3157–3166. doi: 10.1093/nar/3.11.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Snopek T. J., Sugino A., Agarwal K. L., Cozzarelli N. R. Catalysis of DNA joining by bacteriophage T4 RNA ligase. Biochem Biophys Res Commun. 1976 Jan 26;68(2):417–424. doi: 10.1016/0006-291x(76)91161-x. [DOI] [PubMed] [Google Scholar]
  12. Steitz J. A. Oligonucleotide sequence of replicase initiation site in Q RNA. Nat New Biol. 1972 Mar 22;236(64):71–75. doi: 10.1038/newbio236071a0. [DOI] [PubMed] [Google Scholar]
  13. Sugino A., Snoper T. J., Cozzarelli N. R. Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates. J Biol Chem. 1977 Mar 10;252(5):1732–1738. [PubMed] [Google Scholar]
  14. Uhlenbeck O. C., Cameron V. Equimolar addition of oligoribonucleotides with T4 RNA ligase. Nucleic Acids Res. 1977 Jan;4(1):85–98. doi: 10.1093/nar/4.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Walker G. C., Uhlenbeck O. C., Bedows E., Gumport R. I. T4-induced RNA ligase joins single-stranded oligoribonucleotides. Proc Natl Acad Sci U S A. 1975 Jan;72(1):122–126. doi: 10.1073/pnas.72.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES