Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp. lactis and Escherichia coli.
Promoters were cloned from the chromosomal DNA of Lactococcus lactis subsp. lactis NP4510 by using promoter-probe vector pGKV210. N-Methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis of L. lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced expression. Subcloning and sequencing of the mutated plasmid designated pBV415 revealed that the mutation is located within the PstI-HindIII fragment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine). A set of otherwise identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by site-directed mutagenesis. These plasmids containing cat-86 derivatives displayed a significant variation in cat expression in L. lactis and E. coli. The data suggest that cat expression is dependent on the secondary structure of the cat mRNA. New cat-86 derivatives described here can be used in lactococci, in which they provide additional flexibility for promoter cloning.
Institute of Molecular Genetics and Genetic Engineering, Belgrade, Yugoslavia.
B
B
Bojovic
G
G
Djordjevic
A
A
Banina
L
L
Topisirovic
D000595:Amino Acid Sequence; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002702:Chloramphenicol Resistance / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D018387:Codon, Initiator; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D013294:Lactococcus lactis / Q000235:genetics*; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins
D018387:Codon, Initiator; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008769:Methylnitronitrosoguanidine; D015500:Chloramphenicol O-Acetyltransferase