Efficient cloning of a mutant adenylate-kinase-encoding gene from Escherichia coli.
An optimized system has been developed for the transfer of a mutant gene from the Escherichia coli chromosome to a plasmid carrying the wild type (wt) allele. The wt allele was first cloned into a low-copy-number, self-transmissible plasmid with a single EcoRI, HindIII, and BamHI site. The plasmid was then transferred to a mutant strain that had been previously transformed with a high-copy-number plasmid carrying the recA+ gene to allow efficient homologous recombination. A 15% frequency of homogenotization was obtained during cloning of an adk gene that encodes a temperature-sensitive adenylate kinase (AK). The mutant AK had decreased mobility on sodium dodecyl sulfate-polyacrylamide gels compared with the wt enzyme. This was due to a point mutation that changed leucine-107 in the wt enzyme to glutamine-107 in the mutant enzyme as determined by nucleotide sequencing.
Liang
P
P
Department of Biochemistry, University of Illinois, Urbana 61801.
Glaser
M
M
D000263:Adenylate Kinase / Q000096:biosynthesis / Q000235:genetics*; D015153:Blotting, Western; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000302:isolation & purification; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D010770:Phosphotransferases / Q000235:genetics*; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics; D013696:Temperature
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D010770:Phosphotransferases; D000263:Adenylate Kinase; D011938:Rec A Recombinases