Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate
- PMID: 1068464
- PMCID: PMC431149
- DOI: 10.1073/pnas.73.10.3524
Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate
Abstract
A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found.
Similar articles
-
Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis.Proc Natl Acad Sci U S A. 1977 Mar;74(3):851-5. doi: 10.1073/pnas.74.3.851. Proc Natl Acad Sci U S A. 1977. PMID: 191836 Free PMC article.
-
In vitro genetic recombination of bacteriophage lambda.Proc Natl Acad Sci U S A. 1974 Jun;71(6):2496-9. doi: 10.1073/pnas.71.6.2496. Proc Natl Acad Sci U S A. 1974. PMID: 4526306 Free PMC article.
-
In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.Proc Natl Acad Sci U S A. 1975 Sep;72(9):3416-20. doi: 10.1073/pnas.72.9.3416. Proc Natl Acad Sci U S A. 1975. PMID: 1103146 Free PMC article.
-
Molecular mechanisms in genetic recombination.Annu Rev Genet. 1973;7:87-111. doi: 10.1146/annurev.ge.07.120173.000511. Annu Rev Genet. 1973. PMID: 4593311 Review. No abstract available.
-
Restriction endonucleases in the analysis and restructuring of dna molecules.Annu Rev Biochem. 1975;44:273-93. doi: 10.1146/annurev.bi.44.070175.001421. Annu Rev Biochem. 1975. PMID: 166604 Review. No abstract available.
Cited by
-
Torsional stress and local denaturation in supercoiled DNA.Proc Natl Acad Sci U S A. 1979 Aug;76(8):3870-4. doi: 10.1073/pnas.76.8.3870. Proc Natl Acad Sci U S A. 1979. PMID: 226985 Free PMC article.
-
Enzymatic breakage of the cohesive end site of phage lambda DNA: terminase (ter) reaction.Proc Natl Acad Sci U S A. 1978 Sep;75(9):4199-203. doi: 10.1073/pnas.75.9.4199. Proc Natl Acad Sci U S A. 1978. PMID: 279909 Free PMC article.
-
An allele of gyrA prevents Salmonella enterica serovar Typhimurium from using succinate as a carbon source.J Bacteriol. 2006 Apr;188(8):3126-9. doi: 10.1128/JB.188.8.3126-3129.2006. J Bacteriol. 2006. PMID: 16585773 Free PMC article.
-
Biology of bacterial deoxyribonucleic acid topoisomerases.Microbiol Rev. 1984 Dec;48(4):273-89. doi: 10.1128/mr.48.4.273-289.1984. Microbiol Rev. 1984. PMID: 6097803 Free PMC article. Review. No abstract available.
-
Novel template requirements of N4 virion RNA polymerase.Proc Natl Acad Sci U S A. 1978 Jul;75(7):3220-4. doi: 10.1073/pnas.75.7.3220. Proc Natl Acad Sci U S A. 1978. PMID: 356050 Free PMC article.
References
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
