Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis |
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Authors: | Fujio Kawamura Hiuga Saito and Yonosuke Ikeda |
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Institution: | (1) Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, 113 Tokyo, Japan |
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Abstract: | Summary We attempted to use Bacillus subtilis phage 1 as a gene-cloning vector since the 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, 1E1 and 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant 1E2 1 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage 11 DNA, that 1E2 1 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten 1 clones isolated from independent transfectants and found that six of them carried 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the 11 DNA portion, whereas the parental 1E2 1 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases. |
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