Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1977 Jul;4(7):2467–2475. doi: 10.1093/nar/4.7.2467

Restoration by T4 ligase of DNA sequences sensitive to "flush" cleaving restriction enzyme.

M Mottes, C Morandi, S Cremaschi, V Sgaramella
PMCID: PMC342579  PMID: 198743

Abstract

Fouteen "flush"-ended segments originate from the action of the restriction endonuclease Hae III of Haemophilus aegiptius on the DNA of the colicinogenic factor ColE 1 (A. Oka and M. Takanami, Nature, 264, 191, 1976). They are joined by the T4 polynucleotide ligase. The reaction can be monitored by gel electrophoresis, electron microscopy and resistance to phosphatase of the 5'-32P labelled ends. The joined products are a random recombination of the original segments, and can be cleaved by the same Hae III endonuclease to restore the exact electrophoretic pattern of the Hae III-cut ColE 1 DNA. In a properly diluted mixture of 5'-32P segments treated with T4 ligase, the level of phosphatase resistance is very close to the frequency of circle-formation as determined by electron microscopy: thus, the joining of the "flush"-ends involves the formation of circular structures covalently closed in both strands.

Full text

PDF
2467

Images in this article

2470Image
on p.2470
2472Image
on p.2472

Selected References

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

  1. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cohen S. N., Miller C. A. Multiple molecular species of circular R-factor DNA isolated from Escherichia coli. Nature. 1969 Dec 27;224(5226):1273–1277. doi: 10.1038/2241273a0. [DOI] [PubMed] [Google Scholar]
  3. Dugaiczyk A., Boyer H. W., Goodman H. M. Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. J Mol Biol. 1975 Jul 25;96(1):171–184. doi: 10.1016/0022-2836(75)90189-8. [DOI] [PubMed] [Google Scholar]
  4. Inman R. B., Schnös M. Partial denaturation of thymine- and 5-bromouracil-containing lambda DNA in alkali. J Mol Biol. 1970 Apr 14;49(1):93–98. doi: 10.1016/0022-2836(70)90378-5. [DOI] [PubMed] [Google Scholar]
  5. Jeppesen P. G. A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels. Anal Biochem. 1974 Mar;58(1):195–207. doi: 10.1016/0003-2697(74)90458-8. [DOI] [PubMed] [Google Scholar]
  6. Knopf K. W. Simple isolation method and assay for T4 DNA ligase and characterization of the purified enzyme. Eur J Biochem. 1977 Feb 15;73(1):33–38. doi: 10.1111/j.1432-1033.1977.tb11289.x. [DOI] [PubMed] [Google Scholar]
  7. Middleton J. H., Edgell M. H., Hutchison C. A., 3rd Specific fragments of phi X174 deoxyribonucleic acid produced by a restriction enzyme from Haemophilus aegyptius, endonuclease Z. J Virol. 1972 Jul;10(1):42–50. doi: 10.1128/jvi.10.1.42-50.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Oka A., Takanami M. Cleavage map of colicin E1 plasmid. Nature. 1976 Nov 11;264(5582):193–196. doi: 10.1038/264193a0. [DOI] [PubMed] [Google Scholar]
  9. Richardson C. C. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. Proc Natl Acad Sci U S A. 1965 Jul;54(1):158–165. doi: 10.1073/pnas.54.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Scheller R. H., Dickerson R. E., Boyer H. W., Riggs A. D., Itakura K. Chemical synthesis of restriction enzyme recognition sites useful for cloning. Science. 1977 Apr 8;196(4286):177–180. doi: 10.1126/science.847463. [DOI] [PubMed] [Google Scholar]
  11. Sgaramella V. Enrichment of Bacillus subtilis DNA in the sequences for ribosomal RNA. Biochim Biophys Acta. 1969 Dec 16;195(2):466–472. doi: 10.1016/0005-2787(69)90653-4. [DOI] [PubMed] [Google Scholar]
  12. Sgaramella V. Enzymatic oligomerization of bacteriophage P22 DNA and of linear Simian virus 40 DNA. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3389–3393. doi: 10.1073/pnas.69.11.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sgaramella V., Khorana H. G. CXII. Total synthesis of the structural gene for an alanine transfer RNA from yeast. Enzymic joining of the chemically synthesized polydeoxynucleotides to form the DNA duplex representing nucleotide sequence 1 to 20. J Mol Biol. 1972 Dec 28;72(2):427–444. doi: 10.1016/0022-2836(72)90155-6. [DOI] [PubMed] [Google Scholar]
  14. Sgaramella V., Van de Sande J. H., Khorana H. G. Studies on polynucleotides, C. A novel joining reaction catalyzed by the T4-polynucleotide ligase. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1468–1475. doi: 10.1073/pnas.67.3.1468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Weiss B., Jacquemin-Sablon A., Live T. R., Fareed G. C., Richardson C. C. Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J Biol Chem. 1968 Sep 10;243(17):4543–4555. [PubMed] [Google Scholar]

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

RESOURCES