Biomaterial Database

Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate. 1976

K Mizuuchi, and H A Nash

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.

UI	MeSH Term	Description	Entries
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D009537	Nicotinamide Mononucleotide	3-Carbamoyl-1-beta-D-ribofuranosyl pyridinium hydroxide-5'phosphate, inner salt. A nucleotide in which the nitrogenous base, nicotinamide, is in beta-N-glycosidic linkage with the C-1 position of D-ribose. Synonyms: Nicotinamide Ribonucleotide; NMN.	Mononucleotide, Nicotinamide
D011118	Polynucleotide Ligases	Catalyze the joining of preformed ribonucleotides or deoxyribonucleotides in phosphodiester linkage during genetic processes. EC 6.5.1.	Polynucleotide Synthetases,Ligases, Polynucleotide,Synthetases, Polynucleotide
D011995	Recombination, Genetic	Production of new arrangements of DNA by various mechanisms such as assortment and segregation, CROSSING OVER; GENE CONVERSION; GENETIC TRANSFORMATION; GENETIC CONJUGATION; GENETIC TRANSDUCTION; or mixed infection of viruses.	Genetic Recombination,Recombination,Genetic Recombinations,Recombinations,Recombinations, Genetic
D002874	Chromosome Mapping	Any method used for determining the location of and relative distances between genes on a chromosome.	Gene Mapping,Linkage Mapping,Genome Mapping,Chromosome Mappings,Gene Mappings,Genome Mappings,Linkage Mappings,Mapping, Chromosome,Mapping, Gene,Mapping, Genome,Mapping, Linkage,Mappings, Chromosome,Mappings, Gene,Mappings, Genome,Mappings, Linkage
D003090	Coliphages	Viruses whose host is Escherichia coli.	Escherichia coli Phages,Coliphage,Escherichia coli Phage,Phage, Escherichia coli,Phages, Escherichia coli
D004262	DNA Restriction Enzymes	Enzymes that are part of the restriction-modification systems. They catalyze the endonucleolytic cleavage of DNA sequences which lack the species-specific methylation pattern in the host cell's DNA. Cleavage yields random or specific double-stranded fragments with terminal 5'-phosphates. The function of restriction enzymes is to destroy any foreign DNA that invades the host cell. Most have been studied in bacterial systems, but a few have been found in eukaryotic organisms. They are also used as tools for the systematic dissection and mapping of chromosomes, in the determination of base sequences of DNAs, and have made it possible to splice and recombine genes from one organism into the genome of another. EC 3.21.1.	Restriction Endonucleases,DNA Restriction Enzyme,Restriction Endonuclease,Endonuclease, Restriction,Endonucleases, Restriction,Enzymes, DNA Restriction,Restriction Enzyme, DNA,Restriction Enzymes, DNA
D004270	DNA, Circular	Any of the covalently closed DNA molecules found in bacteria, many viruses, mitochondria, plastids, and plasmids. Small, polydisperse circular DNA's have also been observed in a number of eukaryotic organisms and are suggested to have homology with chromosomal DNA and the capacity to be inserted into, and excised from, chromosomal DNA. It is a fragment of DNA formed by a process of looping out and deletion, containing a constant region of the mu heavy chain and the 3'-part of the mu switch region. Circular DNA is a normal product of rearrangement among gene segments encoding the variable regions of immunoglobulin light and heavy chains, as well as the T-cell receptor. (Riger et al., Glossary of Genetics, 5th ed & Segen, Dictionary of Modern Medicine, 1992)	Circular DNA,Circular DNAs,DNAs, Circular
D004274	DNA, Recombinant	Biologically active DNA which has been formed by the in vitro joining of segments of DNA from different sources. It includes the recombination joint or edge of a heteroduplex region where two recombining DNA molecules are connected.	Genes, Spliced,Recombinant DNA,Spliced Gene,Recombinant DNA Research,Recombination Joint,DNA Research, Recombinant,Gene, Spliced,Joint, Recombination,Research, Recombinant DNA,Spliced Genes
D004279	DNA, Viral	Deoxyribonucleic acid that makes up the genetic material of viruses.	Viral DNA