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 fl. Mutagenesis of the lacI gene was performed in vitro by filling dsDNA molecules gapped over the lacI gene with Avian Myeloblastosis Virus (AMV) reverse transcriptese. LacI mutants are found...

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Bibliographic Details
Published in:Nucleic acids research 1984-05, Vol.12 (10), p.4139-4152
Main Authors: Mott, John E., Van Arsdell, Janelle, Platt, Terry
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Avian Leukosis Virus - enzymology
Avian myeloblastosis virus
Avian Myeloblastosis Virus - enzymology
Base Sequence
Cloning, Molecular
DNA Restriction Enzymes
DNA, Bacterial - genetics
EScherichia coli
Escherichia coli - genetics
Genes, Bacterial
Mutation
Phage f1
Repressor Proteins - genetics
repressors
RNA-Directed DNA Polymerase - metabolism
site-directed mutagenesis
Transcription Factors - genetics
Uracil Nucleotides - toxicity
Uridine Triphosphate - analogs & derivatives
Uridine Triphosphate - toxicity
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Summary:We have cloned the gene for the lac operon repressor (lacI) of Escherichia coli into the M13 related phage fl. Mutagenesis of the lacI gene was performed in vitro by filling dsDNA molecules gapped over the lacI gene with Avian Myeloblastosis Virus (AMV) reverse transcriptese. LacI mutants are found at a frequency of 1 in 104 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 aa 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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/12.10.4139