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. 1982 Feb 11;10(3):821–832. doi: 10.1093/nar/10.3.821

Oligonucleotide-directed mutagenesis of gene IX of bacteriophage M13.

G F Simons, G H Veeneman, R N Konings, J H van Boom, J G Schoemakers
PMCID: PMC326205  PMID: 6278437

Abstract

The synthetic oligodeoxyribonucleotide pCGAAAGACTACAC has been applied as a site-specific mutagen to introduce a T leads to G transversion mutation at nucleotide position 1223 of the M13 DNA sequence. The in vitro-induced conversion of a TAT codon into a TAG at this position resulted in gene IX mutants with an amber mutant character thereby confirming that this reading frame defines a gene of an essential phage protein. The gene IX amber mutants obtained grew well on SuI (Ser) and SuIII (Tyr) suppressing strains but could not be propagated on SuII (Gln) and SuVI (Leu) strains. Complementation studies show that amber mutants in genes V and VII exert a polar effect on gene IX expression suggesting that these three contiguous genes form an operon. In addition, we demonstrate the in vitro synthesis of gene IX-protein in a coupled transcription-translation system.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baas P. D., van Teeffelen H. A., Teertstra W. R., Jansz H. S., Veeneman G. H., van der Marel G. A., van Boom J. H. Restoration of the biological activity of in vitro synthesized phi X DNA by transfection of ung- spheroplasts or dUTPase treatment. FEBS Lett. 1980 Jan 28;110(1):15–20. doi: 10.1016/0014-5793(80)80012-3. [DOI] [PubMed] [Google Scholar]
  2. Beck E., Sommer R., Auerswald E. A., Kurz C., Zink B., Osterburg G., Schaller H., Sugimoto K., Sugisaki H., Okamoto T. Nucleotide sequence of bacteriophage fd DNA. Nucleic Acids Res. 1978 Dec;5(12):4495–4503. doi: 10.1093/nar/5.12.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gillam S., Astell C. R., Smith M. Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent phi X174 mutant, with a specific nucleotide deletion, at very high efficiency. Gene. 1980 Dec;12(1-2):129–137. doi: 10.1016/0378-1119(80)90023-2. [DOI] [PubMed] [Google Scholar]
  5. Gillam S., Jahnke P., Astell C., Phillips S., Hutchison C. A., 3rd, Smith M. Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens. Nucleic Acids Res. 1979 Jul 11;6(9):2973–2985. doi: 10.1093/nar/6.9.2973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gillam S., Smith M. Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum ologodeoxyribonucleotide length. Gene. 1979 Dec;8(1):81–97. doi: 10.1016/0378-1119(79)90009-x. [DOI] [PubMed] [Google Scholar]
  7. Grant R. A., Lin T. C., Konigsberg W., Webster R. E. Structure of the filamentous bacteriophage fl. Location of the A, C, and D minor coat proteins. J Biol Chem. 1981 Jan 10;256(1):539–546. [PubMed] [Google Scholar]
  8. Hulsebos T., Schoenmakers J. G. Nucleotide sequence of gene VII and of a hypothetical gene (IX) in bacteriophage M13. Nucleic Acids Res. 1978 Dec;5(12):4677–4698. doi: 10.1093/nar/5.12.4677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hutchison C. A., 3rd, Phillips S., Edgell M. H., Gillam S., Jahnke P., Smith M. Mutagenesis at a specific position in a DNA sequence. J Biol Chem. 1978 Sep 25;253(18):6551–6560. [PubMed] [Google Scholar]
  10. Jay E., Bambara R., Padmanabhan R., Wu R. DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping. Nucleic Acids Res. 1974 Mar;1(3):331–353. doi: 10.1093/nar/1.3.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Konings R. N., Hulsebos T., Van den Hondel C. A. Identification and characterization of the in vitro synthesized gene products of bacteriophage M13. J Virol. 1975 Mar;15(3):570–584. doi: 10.1128/jvi.15.3.570-584.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Konings R. N. Template function of restriction enzyme fragments of phage M13 replicative form DNA. Methods Enzymol. 1980;65(1):795–811. doi: 10.1016/s0076-6879(80)65074-5. [DOI] [PubMed] [Google Scholar]
  13. Lin T. C., Webster R. E., Konigsberg W. Isolation and characterization of the C and D proteins coded by gene IX and gene VI in the filamentous bacteriophage fl and fd. J Biol Chem. 1980 Nov 10;255(21):10331–10337. [PubMed] [Google Scholar]
  14. Lyons L. B., Zinder N. D. The genetic map of the filamentous bacteriophage f1. Virology. 1972 Jul;49(1):45–60. doi: 10.1016/s0042-6822(72)80006-0. [DOI] [PubMed] [Google Scholar]
  15. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Marvin D. A., Schaller H. The topology of DNA from the small filamentous bacteriophage fd. J Mol Biol. 1966 Jan;15(1):1–7. doi: 10.1016/s0022-2836(66)80204-8. [DOI] [PubMed] [Google Scholar]
  17. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  18. Meyer T. F., Geider K. Bacteriophage fd gene II-protein. II. Specific cleavage and relaxation of supercoiled RF from filamentous phages. J Biol Chem. 1979 Dec 25;254(24):12642–12646. [PubMed] [Google Scholar]
  19. Miller J. H., Ganem D., Lu P., Schmitz A. Genetic studies of the lac repressor. I. Correlation of mutational sites with specific amino acid residues: construction of a colinear gene-protein map. J Mol Biol. 1977 Jan 15;109(2):275–298. doi: 10.1016/s0022-2836(77)80034-x. [DOI] [PubMed] [Google Scholar]
  20. Model P., Zinder N. D. In vitro synthesis of bacteriophage f1 proteins. J Mol Biol. 1974 Feb 25;83(2):231–251. doi: 10.1016/0022-2836(74)90389-1. [DOI] [PubMed] [Google Scholar]
  21. Razin A., Hirose T., Itakura K., Riggs A. D. Efficient correction of a mutation by use of chemically synthesized DNA. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4268–4270. doi: 10.1073/pnas.75.9.4268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Simons G. F., Beintema J., Duisterwinkel F. J., Konings R. N., Schoenmakers J. G. Genes VI, VII and IX of bacteriophage M13: identification of their products as minor capsid proteins. Prog Clin Biol Res. 1981;64:401–411. [PubMed] [Google Scholar]
  23. Simons G. F., Konings R. N., Schoenmakers J. G. Genes VI, VII, and IX of phage M13 code for minor capsid proteins of the virion. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4194–4198. doi: 10.1073/pnas.78.7.4194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Simons G. F., Konings R. N., Schoenmakers J. G. Identification of two new capsid proteins in bacteriophage M13. FEBS Lett. 1979 Oct 1;106(1):8–12. doi: 10.1016/0014-5793(79)80683-3. [DOI] [PubMed] [Google Scholar]
  25. Sugimoto K., Sugisaki H., Okamoto T., Takanami M. Studies on bacteriophage fd DNA. IV. The sequence of messenger RNA for the major coat protein gene. J Mol Biol. 1977 Apr 25;111(4):487–507. doi: 10.1016/s0022-2836(77)80065-x. [DOI] [PubMed] [Google Scholar]
  26. Taketo A. Sensitivity of Escherichia coli to viral nucleic acid. V. Competence of calcium-treated cells. J Biochem. 1972 Oct;72(4):973–979. doi: 10.1093/oxfordjournals.jbchem.a129988. [DOI] [PubMed] [Google Scholar]
  27. Van Den Hondel C. A., Weijers A., Konings R. N., Schoenmakers J. G. Studies on bacteriophage M13 DNA. 2. The gene order of the M13 genome. Eur J Biochem. 1975 May 6;53(2):559–567. doi: 10.1111/j.1432-1033.1975.tb04099.x. [DOI] [PubMed] [Google Scholar]
  28. Wilson G. G., Murray N. E. Molecular cloning of the DNA ligase gene from bacteriophage T4. I. Characterisation of the recombinants. J Mol Biol. 1979 Aug 15;132(3):471–491. doi: 10.1016/0022-2836(79)90270-5. [DOI] [PubMed] [Google Scholar]
  29. van Wezenbeek P. M., Hulsebos T. J., Schoenmakers J. G. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene. 1980 Oct;11(1-2):129–148. doi: 10.1016/0378-1119(80)90093-1. [DOI] [PubMed] [Google Scholar]

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