Cloning of the phospho-beta-galactosidase gene in Escherichia coli from lactose-negative mutants of Streptococcus mutans isolated following random mutagenesis with plasmid pVA891 clone banks.
In order to mutagenize Streptococcus mutans a marker rescue plasmid, pVA891, was employed. The plasmid was ligated with Sau3AI digested chromosomal DNA fragments from S. mutans GS-5IS3 and the resultant plasmids were amplified in Escherichia coli. These plasmids were then randomly integrated into the chromosome of strain GS-5IS3 following transformation. Lactose-negative transformants were isolated as white colonies on lactose-BTR-Xgal agar plates containing erythromycin. Six lactose-negative mutants representing three different chromosomal sites of integration were isolated from about eight thousand transformants. Mutant chromosomal DNA fragments flanking the plasmids were recovered by a marker-rescue method in E. coli and exhibited phospho-beta-galactosidase activity.
Sato
Y
Y
Department of Biochemistry, Tokyo Dental College, Chiba, Japan.
Yamamoto
Y
Y
Kizaki
H
H
Kuramitsu
H K
HK
D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D005819:Genetic Markers; D006026:Glycoside Hydrolases; D007785:Lactose / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D013295:Streptococcus mutans / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics*
D005819:Genetic Markers; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C022361:6-phospho-beta-galactosidase; D007785:Lactose