Kawamura F, Saito H, Ikeda Y
Mol Gen Genet. 1980;180(2):259-66. doi: 10.1007/BF00425837.
We attempted to use Bacillus subtilis phage phi 1 as a gene-cloning vector since the phi 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A phi 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, phi 1E1 and phi 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant phi 1E2 delta 1 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage rho 11 DNA, that phi 1E2 delta 1 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten phi 1 clones isolated from independent transfectants and found that six of them carried rho 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 and the rho 11 DNA portion, whereas the parental phi 1E2 delta 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.
由于发现枯草芽孢杆菌噬菌体phi 1基因组在用几种限制性内切酶消化时切割位点很少，我们试图将其用作基因克隆载体。由J. Ito（美国亚利桑那大学图森分校）提供的phi 1菌液包含两种噬菌体，phi 1E1和phi 1E2，它们的基因组中分别有一个和两个EcoRI切割位点。从后一种分离物中诱导出缺失突变体phi 1E2 delta 1，以扩大可克隆DNA片段的大小范围。通过与噬菌体rho 11 DNA进行体外重组实验证明，phi 1E2 delta 1可用于克隆各种大小的EcoRI片段。我们分析了从独立转染子中分离出的十个phi 1克隆的DNA，发现其中六个携带插入到两个EcoRI切割位点之一的rho 11 DNA片段。发现一些杂交噬菌体DNA被BamHI和HaeIII内切酶切割，而rho 11 DNA部分，而亲本phi 1E2 delta 1 DNA对这些酶均不敏感。因此，这些杂交噬菌体将是用于克隆由BamHI或HaeIII内切酶切割产生的外源DNA片段的有用载体。
