Summary
Two complementary 24 base single stranded oligonucleotides containing randomly located inosine residues were synthesized in vitro. Once annealed, the two oligonucleotides were cloned into derivatives of ColE1 and transformed into Escherichia coli. Sequence analysis of 157 clones yielded 305 mutations. The pattern of the mutations revealed the following: (1) The frequency of inosine induced mutations was significantly less than predicted from its content in the oligonucleotides; (2) Inosine incorporation resulted almost exclusively in base changes to guanine; (3) The mutation distribution is biased towards A/T to G/C substitutions; (4) There were reproducible position biases; and (5) There was a reproducible strand bias which was independent of the cassette orientation with respect to the plasmid origin of replication.
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References
Beaucage SL, Coruthers MH (1981) Deoxynucleoside phophoramidite — a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahed Lett 22:1859–1962
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Burns PA, Gordon AJ, Glickman BW (1987) Influence of neighboring base sequence of N-methyl-N′-nitro-N-nitrosoguanidine in the lacI gene of Escherichia coli. J Mol Biol 194:385–390
Chen EY, Seeburg PY (1985) Supercoiled sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4:165–170
Fazakerley GV, Quignard E, Woisard A, Guschlbauer W, Van der Marel GA, Van Boom JH, Jones M, Radman M (1986) Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system. EMBO J 5:3697–3703
Fix DF, Glickman BW (1987) Asymmetric cytosine deamination revealed by spontaneous mutational specificity in an Ung - strain of Escherichia coli. Mol Gen Genet 209:78–82
Karran P, Lindhal T (1978) Enzymatic excision of free hypoxanthine from polydeoxynucleotides and DNA containing deoxyinosine monophosphate residues. J Biol Chem 253:5877–5879
Lathe R, Skory S, Kieny MP (1984) Inserting new restriction sites by linker tailing. Focus 6:1–6
MacClain WH, Foss K, Mittelstadt KL, Schneider J (1986) Variants in clones of gene-machine-synthesized oligodeoxynucleotides. Nucleic Acids Res 14:6770
Makris JC, Nordmann PL, Reznikoff WS (1988) Mutational analysis of IS50 and Tn5 ends. Proc Natl Acad Sci USA 85:2224–2228
Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Maniatis T, Tritsch EF, Sambrook J (1982) In: Molecular cloning. A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 93–94
Martin FH, Castro MM, Aboul-Ela F, Tinoco IJ (1985) Base pairing involving deoxyinosine: implication for probe design. Nucleic Acids Res 13:8927–8938
Matteucci MD, Coruthers MH (1981) Synthesis of deoxyoligonucleotides on a polymer support. J Am Chem Soc 103:3185–3191
Miller JH (1977) In: Experiments in Molecular Genetics, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, p 433–434
Myrnes B, Guddal PH, Krokan H (1982) Metabolism of dITP in Hela cell extracts, incorporation into DNA by isolated nuclei and release of hypoxanthine from DNA by a hypoxanthine-DNA glycosylase activity. Nucleic Acids Res 10:3693–3701
Ohtsuka E, Matsuki S, Ikehara M, Takahaski Y, Masubara K (1985) An alternative approach to deoxyoligonucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J Biol Chem 260:2605–2608
Reed J, Hutchinson F (1987) Effect of the direction of DNA replication on mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine in adapted cells of Escherichia coli. Mol Gen Genet 208:446–449
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Singer B, Kusmierck JR (1982) Chemical mutagenesis. Annu Rev Biochem 51:655–693
Smith M (1985) In vitro mutagenesis. Annu Rev Genet 19:423–462
Staudenbauer WL, Scherzinger E, Lanka E (1979) Replication of the colicin E1 plasmid in extracts of Escherichia coli: uncoupling of leading strand from the lagging strand synthesis. Mol Gen Genet 177:113–120
Swartz ME, Oberholtzer JE (1987) Compositional confirmation of synthetic prepared oligodeoxyribonucleotides by LC. Biochromatography 2:98–100
Wells JA, Vasser M, Powers DB (1985) Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites. Gene 34:315–323
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Communicated by B.J. Kilbey
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Nordmann, P.L., Makris, J.C. & Reznikoff, W.S. Inosine induced mutations. Mol Gen Genet 214, 62–67 (1988). https://doi.org/10.1007/BF00340180
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DOI: https://doi.org/10.1007/BF00340180