Biomedical subjects
Publications and source records attributed to F Kawamura.
HaeIII, BalI, StuI, BamHI, SlaI, and EcoRII did not cut the genome of Bacillus subtilis phage phi 1 at all, whereas ThaI, BglII, EcoRI, SalI, and Bsu1247I cut the genome once or twice. The physical map of the phi 1 genome was constructed with the latter restriction endonucleases.
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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.
A cleavage map of Bacillus subtilis temperate phage rho 11 was constructed with restriction endonucleases SalI, BamHI and BglII, which cut the genome into 6, 7 and 21 fragments, respectively. The molecular weight of the rho 11 genome was calculated to be 78 x 10(6). Among other endonucleases tested, PvuII, EcoRI and XbaI cleaved the genome into more than 25 fragments, while HaeIII, StuI, BalI and BamNx did not cut the genome at all. The rho 11-coded thymidylate synthetase gene, thyP11, was found to be located in the SalI-D fragment, which was in the central region of the genome.
Chromosomal DNA of Bacillus subtilis 168 (trpC2) prepared from defective phage P BSX was digested by restriction endonuclease Eco RI and ligated in vitro with DNA fragments of page phi 105C digested by the same endonuclease. The ligated DNA was used to transform a competent culture of B. subtilis (trpC2 lys3 metB10) which was lysogenic for phi 105, and transformants of the auxotroph markers were selected. The bacterial DNA ligated to the phage DNA fragments could be integrated into the prophage genome by transformation. The transformants in toto were treated with mitomycin C and the lysate was used to transduce B. subtilis (trpC2 lys3 metB10). Among metB+ transductants, one clone appeared to be a double lysogen carrying both plaque forming and metB+ transducing phage genomes. The latter defective phage was designated phi 105dmetB. Physical mapping of these phages was carried out by agarose gel electrophoresis of the restriction endonuclease digests and also by electron microscopic analysis of heteroduplex DNA. These results indicate that two adjacent fragments Eco RI-G and E of phi 105 DNA had been substituted with a foreign fragment Eco RI-M in phi 105dmetB DNA. Transformation experiments showed that the metB+ gene resided on the fragment Eco RI-M. This fragment was found to have a BamHI-sensitive site. The transforming activity for the metB marker, however, was not affected by the treatmment with BamHI.
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DNA from a temperate phage rho 11 and chromosomal DNA of Bacillus subtilis 168 were digested with endonuclease EcoRI and then ligated with T4 polynucleotide ligase. The ligated DNA fragments were used to transform a lysogenic strain, B. subtilis spoA12 lys21 hisA1 leuA8 p11, and Lys+, His+ or Leu+ transformants were selected. The cells of each type were then mixed, grown and treated with mitomycin C; the induced phages were tested for abilities abilities to form plaques and to tranduce the auxotrophic marker. Various types of plaque-forming or defective phages which transduce hisA or lys marker at considerably high frequencies were thus obtained.
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The phi29 early mRNA's synthesized in infected Bacillus subtilis were studied by using sedimentation velocity analysis, polyacrylamide gel electrophoresis, and hybridization of phi29 DNA fragments generated by the restriction endonuclease Eco RI. Viral RNAs synthesized in vivo in the resence of chloramphenicol were found to hybridize to Eco RI-A, -C, and -D fragments, but not to Eco RI-B and -E fragments, of the viral genome. Major early mRNA sedimenting as 16S material in neutral sucrose gradients was examined in detail. Radioactive phi29 RNA, purified by sucrose gradient centrifugation, was hybridized to either the Eco RI-A or Eco RI-C DNA fragment. The RNA was eluted from the hybrids and then tested for complementary hybrid formation with Eco RI-A and -C fragments. RNA eluted from the Eco RI-A fragment annealed only to the Eco RI-A fragment and not to the Eco RI-C fragment. Similarly, RNA eluted from the Eco RI-C fragment hybridized to the Eco RI-C and -D fragments. Viral RNAs synthesized in vitro using B. subtilis RNA polymerase hybridized to both Eco RI-A and -C DNA fragments. Furthermore, RNA initiated with [gamma-(32)P]GTP also hybridized to both Eco RI-A and -C fragments. These results indicate that there are at least two efficient promotors for early transcription on the phi29 chromosome. In addition, a low-molecular-weight RNA initiated with [gamma-(32)P]ATP was found to hybridize exclusively with the Eco RI-A fragment. Kinetic studies of phi29 mRNA synthesis during the lytic cycle have shown that viral RNAs hybridizable to the Eco RI-A and -C fragments are synthesized immediately after phage infection. On the other hand, mRNA specific for the Eco RI-B fragment was not synthesized for several minutes after phage infection. Based on the results of the in vivo and in vitro transcription studies, a transcription map of the phi29 chromosome is proposed.
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