Targeted mutagenesis in vitro: lac repressor mutations generated using AMV reverse transcriptase and dBrUTP.
We have cloned the gene for the lac operon repressor (lacI) of Escherichia coli into the M13 related phage f1. Mutagenesis of the lacI gene was performed in vitro by filling dsDNA molecules gapped over the lacI gene with Avian Myeloblastosis Virus (AMV) reverse transcriptase. LacI mutants are found at a frequency of 1 in 10(4) using a genetic screen in vivo. For two-thirds of the 60 mutants, lesions were identified within the first 400 bases of lacI, by dideoxy sequencing. An unexpectedly wide range of different lesions were observed, including transitions, transversions, and deletions (of which the most common were the removal of single base pairs). The replacement of dTTP by dBrUTP in the filling reaction resulted in a doubling of deletions in the sample population as well as the anticipated T to C and C to T transitions. Although the lacI gene has been extensively studied in vivo, the power of this technique for mutagenesis in vitro is demonstrated by the generation of three previously undescribed lacI mutations.
D000595:Amino Acid Sequence; D001354:Avian Leukosis Virus / Q000201:enzymology*; D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014500:Uracil Nucleotides / Q000633:toxicity*; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000633:toxicity*
D004269:DNA, Bacterial; D012097:Repressor Proteins; D014157:Transcription Factors; D014500:Uracil Nucleotides; D012194:RNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; D014544:Uridine Triphosphate