Sato Y, Yamamoto Y, Kizaki H, Kuramitsu H K
Department of Biochemistry, Tokyo Dental College, Chiba, Japan.
FEMS Microbiol Lett. 1992 Mar 15;70(3):219-24. doi: 10.1016/0378-1097(92)90701-o.
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.
为了诱变变形链球菌，使用了一种标记拯救质粒pVA891。该质粒与经Sau3AI酶切的变形链球菌GS-5IS3染色体DNA片段连接，所得质粒在大肠杆菌中扩增。然后，这些质粒在转化后随机整合到GS-5IS3菌株的染色体中。在含有红霉素的乳糖-BTR-Xgal琼脂平板上，乳糖阴性转化体被分离为白色菌落。从大约八千个转化体中分离出六个代表三个不同染色体整合位点的乳糖阴性突变体。通过标记拯救方法在大肠杆菌中回收了质粒两侧的突变体染色体DNA片段，并表现出磷酸β-半乳糖苷酶活性。
