Essential structure in the cloned transforming DNA that induces gene amplification of the Bacillus subtilis amyE-tmrB region.
Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.
D001412:Bacillus subtilis / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis*; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; D004352:Drug Resistance, Microbial; D005784:Gene Amplification; D008970:Molecular Weight; D009154:Mutation; D014415:Tunicamycin / Q000494:pharmacology; D000516:alpha-Amylases / Q000378:metabolism
D004269:DNA, Bacterial; D014415:Tunicamycin; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII; D000516:alpha-Amylases