Cloning of the phospho-β-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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Authors: | Yutaka Sato Yasuhito Yamamoto Harutoshi Kizaki Howard K Kuramitsu |
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Institution: | Department of Biochemistry, Tokyo Dental College, Chiba, Japan;Department of Pediatric Dentistry, University of Texas Health Science Center, San Antonio, TX, U.S.A. |
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Abstract: | Abstract In order to mutagenize Streptococcus mutans a marker rescue plasmid, pVA891, was employed. The plasmid was ligated with Sau 3AI 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-β-galactosidase activity. |
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Keywords: | Random mutagenesis Phospho-β-galactosidase Streptococcus mutans |
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