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. 1984 May 25;12(10):4139–4152. doi: 10.1093/nar/12.10.4139

Targeted mutagenesis in vitro: lac repressor mutations generated using AMV reverse transcriptase and dBrUTP.

J E Mott, J Van Arsdell, T Platt
PMCID: PMC318822  PMID: 6203097

Abstract

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.

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Selected References

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  1. Battula N., Loeb L. A. The infidelity of avian myeloblastosis virus deoxyribonucleic acid polymerase in polynucleotide replication. J Biol Chem. 1974 Jul 10;249(13):4086–4093. [PubMed] [Google Scholar]
  2. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boeke J. D. One and two codon insertion mutants of bacteriophage f1. Mol Gen Genet. 1981;181(3):288–291. doi: 10.1007/BF00425599. [DOI] [PubMed] [Google Scholar]
  4. Busby S., Dreyfus M. Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation. Gene. 1983 Jan-Feb;21(1-2):121–131. doi: 10.1016/0378-1119(83)90154-3. [DOI] [PubMed] [Google Scholar]
  5. Calos M. P., Lebkowski J. S., Botchan M. R. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. doi: 10.1073/pnas.80.10.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Everett R. D., Chambon P. A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA. EMBO J. 1982;1(4):433–437. doi: 10.1002/j.1460-2075.1982.tb01187.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Farnham P. J., Platt T. A model for transcription termination suggested by studies on the trp attenuator in vitro using base analogs. Cell. 1980 Jul;20(3):739–748. doi: 10.1016/0092-8674(80)90320-7. [DOI] [PubMed] [Google Scholar]
  8. Farnham P. J., Platt T. Rho-independent termination: dyad symmetry in DNA causes RNA polymerase to pause during transcription in vitro. Nucleic Acids Res. 1981 Feb 11;9(3):563–577. doi: 10.1093/nar/9.3.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fersht A. R. Fidelity of replication of phage phi X174 DNA by DNA polymerase III holoenzyme: spontaneous mutation by misincorporation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4946–4950. doi: 10.1073/pnas.76.10.4946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gopinathan K. P., Weymouth L. A., Kunkel T. A., Loeb L. A. Mutagenesis in vitro by DNA polymerase from an RNA tumour virus. Nature. 1979 Apr 26;278(5707):857–859. doi: 10.1038/278857a0. [DOI] [PubMed] [Google Scholar]
  11. Heidecker G., Messing J., Gronenborn B. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene. 1980 Jun;10(1):69–73. doi: 10.1016/0378-1119(80)90145-6. [DOI] [PubMed] [Google Scholar]
  12. Hibner U., Alberts B. M. Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex. Nature. 1980 May 29;285(5763):300–305. doi: 10.1038/285300a0. [DOI] [PubMed] [Google Scholar]
  13. Müller W., Weber H., Meyer F., Weissmann C. Site-directed mutagenesis in DNA: generation of point mutations in cloned beta globin complementary dna at the positions corresponding to amino acids 121 to 123. J Mol Biol. 1978 Sep 15;124(2):343–358. doi: 10.1016/0022-2836(78)90303-0. [DOI] [PubMed] [Google Scholar]
  14. Peden K. W., Nathans D. Local mutagenesis within deletion loops of DNA heteroduplexes. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7214–7217. doi: 10.1073/pnas.79.23.7214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Platt T., Files J. G., Weber K. Lac repressor. Specific proteolytic destruction of the NH 2 -terminal region and loss of the deoxyribonucleic acid-binding activity. J Biol Chem. 1973 Jan 10;248(1):110–121. [PubMed] [Google Scholar]
  16. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shortle D., Koshland D., Weinstock G. M., Botstein D. Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5375–5379. doi: 10.1073/pnas.77.9.5375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Traboni C., Ciliberto G., Cortese R. A novel method for site-directed mutagenesis: its application to an eukaryotic tRNAPro gene promoter. EMBO J. 1982;1(4):415–420. doi: 10.1002/j.1460-2075.1982.tb01184.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Traboni C., Cortese R., Ciliberto G., Cesareni G. A general method to select for M13 clones carrying base pair substitution mutants constructed in vitro. Nucleic Acids Res. 1983 Jun 25;11(12):4229–4239. doi: 10.1093/nar/11.12.4229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Zakour R. A., Loeb L. A. Site-specific mutagenesis by error-directed DNA synthesis. Nature. 1982 Feb 25;295(5851):708–710. doi: 10.1038/295708a0. [DOI] [PubMed] [Google Scholar]
  21. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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