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. 1984 Jan 11;12(1 Pt 2):777–787. doi: 10.1093/nar/12.1part2.777

Introduction of restriction enzyme sites in protein-coding DNA sequences by site-specific mutagenesis not affecting the amino acid sequence: a computer program.

R Arentzen, W C Ripka
PMCID: PMC321092  PMID: 6320109

Abstract

Structure/function relationship studies of proteins are greatly facilitated by recombinant DNA technology which allows specific amino acid mutations to be made at the DNA sequence level by site-specific mutagenesis employing synthetic oligonucleotides. This technique has been successfully used to alter one or two amino acids in a protein. Replacement of existing DNA sequence coding for several amino acids with new synthetic DNA fragments would be facilitated by the presence of unique restriction enzyme sites in the region of interest. This computer program provides a means of searching the DNA sequence of interest for restriction enzyme sites that could be introduced by site-specific mutagenesis not affecting the amino acid sequence of the protein. Alternately, the program will also allow single amino acid changes to be made.

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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., Teertstra W. R., van Mansfeld A. D., Jansz H. S., van der Marel G. A., Veeneman G. H., van Boom J. H. Construction of viable and lethal mutations in the origin of bacteriophage 'phi' X174 using synthetic oligodeoxyribonucleotides. J Mol Biol. 1981 Nov 15;152(4):615–639. doi: 10.1016/0022-2836(81)90120-0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Carmichael G. G., Schaffhausen B. S., Dorsky D. I., Oliver D. B., Benjamin T. L. Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3579–3583. doi: 10.1073/pnas.79.11.3579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Charles A. D., Gautier A. E., Edge M. D., Knowles J. R. Targeted point mutation that creates a unique Eco RI site within the signal codons of the beta-lactamase gene without altering enzyme secretion or processing. J Biol Chem. 1982 Jul 25;257(14):7930–7932. [PubMed] [Google Scholar]
  6. Dalbadie-McFarland G., Cohen L. W., Riggs A. D., Morin C., Itakura K., Richards J. H. Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6409–6413. doi: 10.1073/pnas.79.21.6409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Gillam S., Smith M. Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: II. In vitro selection of mutant DNA. Gene. 1979 Dec;8(1):99–106. doi: 10.1016/0378-1119(79)90010-6. [DOI] [PubMed] [Google Scholar]
  9. Gingeras T. R., Roberts R. J. Steps toward computer analysis of nucleotide sequences. Science. 1980 Sep 19;209(4463):1322–1328. doi: 10.1126/science.6251542. [DOI] [PubMed] [Google Scholar]
  10. Hsiung H., Inouye S., West J., Sturm B., Inouye M. Further improvements on the phosphotriester synthesis of deoxyribooligonucleotides and the oligonucleotide directed site-specific mutagenesis of E. coli lipoprotein gene. Nucleic Acids Res. 1983 May 25;11(10):3227–3239. doi: 10.1093/nar/11.10.3227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Inouye S., Hsu C. P., Itakura K., Inouye M. Requirement for signal peptide cleavage of Escherichia coli prolipoprotein. Science. 1983 Jul 1;221(4605):59–61. doi: 10.1126/science.6344218. [DOI] [PubMed] [Google Scholar]
  13. Inouye S., Soberon X., Franceschini T., Nakamura K., Itakura K., Inouye M. Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3438–3441. doi: 10.1073/pnas.79.11.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Khorana H. G. Total synthesis of a gene. Science. 1979 Feb 16;203(4381):614–625. doi: 10.1126/science.366749. [DOI] [PubMed] [Google Scholar]
  15. Kramer W., Schughart K., Fritz H. J. Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments. Nucleic Acids Res. 1982 Oct 25;10(20):6475–6485. doi: 10.1093/nar/10.20.6475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kudo I., Leineweber M., RajBhandary U. L. Site-specific mutagenesis on cloned DNAs: generation of a mutant of Escherichia coli tyrosine suppressor tRNA in which the sequence G-T-T-C corresponding to the universal G-T-pseudouracil-C sequence of tRNAs is changed to G-A-T-C. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4753–4757. doi: 10.1073/pnas.78.8.4753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Larson G. P., Itakura K., Ito H., Rossi J. J. Saccharomyces cerevisiae actin--Escherichia coli lacZ gene fusions: synthetic-oligonucleotide-mediated deletion of the 309 base pair intervening sequence in the actin gene. Gene. 1983 Apr;22(1):31–39. doi: 10.1016/0378-1119(83)90061-6. [DOI] [PubMed] [Google Scholar]
  18. Laski F. A., Belagaje R., RajBhandary U. L., Sharp P. A. An amber suppressor tRNA gene derived by site-specific mutagenesis: cloning and function in mammalian cells. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5813–5817. doi: 10.1073/pnas.79.19.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Matteucci M. D., Heyneker H. L. Targeted random mutagenesis: the use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5' of an ATG initiation codon. Nucleic Acids Res. 1983 May 25;11(10):3113–3121. doi: 10.1093/nar/11.10.3113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Miyada C. G., Soberón X., Itakura K., Wilcox G. The use of synthetic oligodeoxyribonucleotides to produce specific deletions in the araBAD promoter of Escherichia coli B/r. Gene. 1982 Feb;17(2):167–177. doi: 10.1016/0378-1119(82)90070-1. [DOI] [PubMed] [Google Scholar]
  21. Montell C., Fisher E. F., Caruthers M. H., Berk A. J. Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site. Nature. 1982 Feb 4;295(5848):380–384. doi: 10.1038/295380a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Roberts R. J. Restriction and modification enzymes and their recognition sequences. Nucleic Acids Res. 1983 Jan 11;11(1):r135–r167. [PMC free article] [PubMed] [Google Scholar]
  24. Rossi J. J., Kierzek R., Huang T., Walker P. A., Itakura K. An alternate method for synthesis of double-stranded DNA segments. J Biol Chem. 1982 Aug 25;257(16):9226–9229. [PubMed] [Google Scholar]
  25. Rossi J. J., Soberon X., Marumoto Y., McMahon J., Itakura K. Biological expression of an Escherichia coli consensus sequence promoter and some mutant derivatives. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3203–3207. doi: 10.1073/pnas.80.11.3203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sigal I. S., Harwood B. G., Arentzen R. Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7157–7160. doi: 10.1073/pnas.79.23.7157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Simons G. F., Veeneman G. H., Konings R. N., van Boom J. H., Schoemakers J. G. Oligonucleotide-directed mutagenesis of gene IX of bacteriophage M13. Nucleic Acids Res. 1982 Feb 11;10(3):821–832. doi: 10.1093/nar/10.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sutcliffe J. G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. doi: 10.1073/pnas.75.8.3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Temple G. F., Dozy A. M., Roy K. L., Kan Y. W. Construction of a functional human suppressor tRNA gene: an approach to gene therapy for beta-thalassaemia. Nature. 1982 Apr 8;296(5857):537–540. doi: 10.1038/296537a0. [DOI] [PubMed] [Google Scholar]
  30. Vlasuk G. P., Inouye S., Ito H., Itakura K., Inouye M. Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli. J Biol Chem. 1983 Jun 10;258(11):7141–7148. [PubMed] [Google Scholar]
  31. Wallace R. B., Johnson P. F., Tanaka S., Schöld M., Itakura K., Abelson J. Directed deletion of a yeast transfer RNA intervening sequence. Science. 1980 Sep 19;209(4463):1396–1400. doi: 10.1126/science.6997991. [DOI] [PubMed] [Google Scholar]
  32. Wallace R. B., Schold M., Johnson M. J., Dembek P., Itakura K. Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res. 1981 Aug 11;9(15):3647–3656. doi: 10.1093/nar/9.15.3647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wasylyk B., Chambon P. A T to A base substitution and small deletions in the conalbumin TATA box drastically decrease specific in vitro transcription. Nucleic Acids Res. 1981 Apr 24;9(8):1813–1824. doi: 10.1093/nar/9.8.1813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wasylyk B., Derbyshire R., Guy A., Molko D., Roget A., Téoule R., Chambon P. Specific in vitro transcription of conalbumin gene is drastically decreased by single-point mutation in T-A-T-A box homology sequence. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7024–7028. doi: 10.1073/pnas.77.12.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wilkinson A. J., Fersht A. R., Blow D. M., Winter G. Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. Biochemistry. 1983 Jul 19;22(15):3581–3586. doi: 10.1021/bi00284a007. [DOI] [PubMed] [Google Scholar]
  36. Winter G., Fersht A. R., Wilkinson A. J., Zoller M., Smith M. Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding. Nature. 1982 Oct 21;299(5885):756–758. doi: 10.1038/299756a0. [DOI] [PubMed] [Google Scholar]
  37. Zarucki-Schulz T., Tsai S. Y., Itakura K., Soberon X., Wallace R. B., Tsai M. J., Woo S. L., O'Malley B. W. Point mutagenesis of the ovalbumin gene promoter sequence and its effect on in vitro transcription. J Biol Chem. 1982 Sep 25;257(18):11070–11077. [PubMed] [Google Scholar]
  38. 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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