[Cloning of bacteriophage T5 DNA fragments in the plasmid pBR322. Analysis of recombinant plasmids by the method of bonding with RNA-polymerase from Escherichia coli on nitrocellulose filters].
DNA of bacteriophage T5 was hydrolyzed with restriction endonucleases HindIII and BamHI, and subjected to the combined hydrolysis with BamHI+EcoRI and BamHI+ +HindIII. Fragments obtained were cloned in the plasmid pBR322. About 17% of T5 genome were recovered in recombinant plasmids. Cloned fragments were localized on the physical map of the phage by restriction analysis and Southern hybridization. With the aim of direct cloning of T5 promoters, PstI/HindIII fragments were inserted into pBR322 followed by selection of recombinants on ApsTCr phenotype. Binding of BsuRI and AluI fragments of hybrid plasmids with E. coli RNA polymerase was studied by nitrocellulose filter assay. The fragments, which were capable to form heparin resistant complexes were identified.
D001483:Base Sequence; D003001:Cloning, Molecular; D003101:Collodion; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005814:Genes, Viral; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D013604:T-Phages / Q000235:genetics*; D014462:Ultrafiltration
D004274:DNA, Recombinant; D004279:DNA, Viral; D003101:Collodion; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes
Klonirovanie fragmentov DNK bakteriofaga T5 v plazmide pBR322. Analiz rekombinantnykh plazmid metodom sviazyvaniia s RNK-polimerazoĭ Escherichia coli na nitrotselliuloznykh fil'trakh.