Purification of bacteriophage T7 DNA-membrane complex and its application to the in vitro recombination reaction.
In order to construct an in vitro recombination system of T7 DNA, the reaction products of which resemble those in vivo in structure, T7 DNA-membrane complex which is free from concomitant DNase activity was purified from T7 phage-infected cells. T7-infected cells were lysed with T4 lysozyme/Brij58, and T7 DNA-membrane complex was purified through three successive density gradient centrifugations. The properties of the complex on exposure to defined nucleases and observation of the complex by electron microscopy revealed that in T7 DNA-membrane complex, both ends of a linear T7 DNA are bound with membrane components. A mixture of 32P-labeled T7 DNA-membrane complex and BU-labeled T7 DNA-membrane complex was incubated with T7 exonuclease and T7 DNA-binding protein, and the reaction products with intermediate density were purified. Most of the products were found to have structures similar to that of the recombination intermediate found in T7-infected cells upon electron microscopic examination.
D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000032:analysis* / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism; D008854:Microscopy, Electron; D011995:Recombination, Genetic; D013604:T-Phages / Q000032:analysis* / Q000235:genetics / Q000648:ultrastructure; D014764:Viral Proteins / Q000378:metabolism
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D003851:Deoxyribonucleases; D005090:Exodeoxyribonucleases; C046264:gene b exonuclease