PURPOSE: To obtain the subject insertion factor, present adjacent to a gene capable of coding a chromosomal alcohol dehydrogenase of a strain belonging to the genus Bacillus, containing a structural gene of transposase and utilizable for expressing various structural genes.
CONSTITUTION: The objective insertion factor is obtained by culturing a Bacillus stearothermophilus NCA1503 strain in a culture medium at 55°C for 16hr by shaking culture, then centrifuging the resultant culture, recovering microbial cells, preparing a chromosomal DNA according to a conventional method, partially hydrolyzing the chromosomal DNA with a restriction enzyme Sau3AI, providing a DNA fragment of 5-7kb, linking the resultant DNA fragment to a plasmid vector pTB524 treated with a restriction enzyme BamHI with a T4DNA ligase, constructing an expression vector, transducing the expression vector into a Bacillus subtilis MI113 strain, carrying out transformation, culturing the obtained transformant, selecting tetracycline resistance, extracting a plasmid DNA according to a conventional method and treating the extract with a restriction enzyme. The above-mentioned insertion factor has a specific base sequence.
SAKODA HISAO
INUI MASAHIRO
TAKEUCHI TOSHIO
