The cloned polC gene of Bacillus subtilis: characterization of the azp12 mutation and controlled in vitro synthesis of active DNA polymerase III.


Wild type (wt) Bacillus subtilis polC and polCazp12, a mutant ...
Wild type (wt) Bacillus subtilis polC and polCazp12, a mutant derivative specifying a form of DNA polymerase III resistant to hydroxyphenylazopyrimidines, were cloned as genomic fragments approximating the length required to encode the entire polymerase. The cloned DNA fragments were subjected to restriction and partial sequence analysis to locate the 5' end of the polC-specific coding sequence and the azp12 mutation, which was identified as a T----G transversion specifying replacement of serine with alanine. The cloned wt and azp12-coding sequences were recloned in an Escherichia coli expression vector with their respective 5' ends under the control of the bacteriophage lambda PL promoter and cIts857-encoded repressor. In response to induction, the wt- and azp12-specific recombinant plasmids expressed active DNA polymerases indistinguishable from the native enzymes derived from the respective B. subtilis hosts.




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