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.

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.
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使用质粒 pVA891 克隆库进行随机诱变后,从变形链球菌乳糖阴性突变体中克隆大肠杆菌中的磷酸-β-半乳糖苷酶基因。

DOI:
10.1016/0378-1097(92)90701-o
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发表时间:
1992
影响因子:
2.1
通讯作者:
Kuramitsu,HK
Kuramitsu,HK
中科院分区:
生物学4区
文献类型:
--
作者:
Sato,Y;Yamamoto,Y;Kizaki,H;Kuramitsu,HK

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为了诱变变形链球菌,使用了标记拯救质粒pVA 891。将该质粒与Sau 3AI酶切的S.突变体GS-5IS 3的质粒在大肠杆菌中进行扩增。然后在转化后将这些质粒随机整合到菌株GS-5IS 3的染色体中。在含有红霉素的乳糖-BTR-Xgal琼脂平板上分离乳糖阴性转化体作为白色菌落。从约8000个转化体中分离出代表三个不同染色体整合位点的六个乳糖阴性突变体。质粒两侧的突变体染色体DNA片段通过标记拯救法在E. coliand具有磷酸-β-半乳糖苷酶活性。
In order to mutagenizeStreptococcus mutansa marker rescue plasmid, pVA891, was employed. The plasmid was ligated withSau3AI digested chromosomal DNA fragments fromS. mutansGS-5IS3 and the resultant plasmids were amplified inEscherichia 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 inE. coliand exhibited phospho-β-galactosidase activity.
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