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 fragment...

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Bibliographic Details
Published inMolekuliarnaia biologiia Vol. 16; no. 6; p. 1253
Main Authors Krutilina, A I, Ksënzenko, V N, Kamynina, T P, Kriukov, V M, Baev, A A
Format Journal Article
LanguageRussian
Published Russia (Federation) 01.11.1982
Subjects
Base Sequence
Cloning, Molecular
Collodion
DNA Restriction Enzymes
DNA, Recombinant - metabolism
DNA, Viral - genetics
DNA-Directed RNA Polymerases - metabolism
Escherichia coli - enzymology
Escherichia coli - genetics
Genes, Viral
Nucleic Acid Hybridization
Plasmids
T-Phages - genetics
Ultrafiltration
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Summary: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.
ISSN:0026-8984