Cloning of the exonuclease III gene of Escherichia coli.
Overproducers of exonuclease III (exo III) were found within a colony bank containing ColE1-Escherichia coli hybrid plasmids. Through the enzymatic ligation of restriction enzyme fragments, the exo III gene, xth, was transferred to a thermoinducible, integration-proficient lambda phage and to a chimeric ColE1-lambda plasmid that was thermoinducible for lambda-directed DNA replication. Transfer of the xth gene was facilitated by a technique involving prior selection for Tn5 insertions into plasmid, thereby linking the gene to additional restriction sites and to a selectable (drug resistance) marker. After heat induction, cells bearing the thermoinducible ColE1-lambda-xth plasmid produced 120-fold more exo III than did plasmid-free cells. Enzyme production was not further enhanced by any of the following chromosomal mutations: dnaA, recBC, tob, or nusA snu. Several observations suggested that enzyme over-synthesis was the result primarily of lambda-detected replication rather than lambda-directed transcription.
D003086:Bacteriocin Plasmids; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D003851:Deoxyribonucleases / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005090:Exodeoxyribonucleases; D005092:Exonucleases / Q000096:biosynthesis / Q000235:genetics*
D004251:DNA Transposable Elements; D003851:Deoxyribonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C020133:exodeoxyribonuclease III