Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria.
Mutagenic specificity of 2-acetylaminofluorene (AAF) has been established in mammalian cells and several strains of bacteria by using a shuttle plasmid vector containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene (C8-dG-AAF) adduct. The nucleotide sequence of the gene conferring tetracycline resistance was modified by conservative codon replacement so as to accommodate the sequence d(CCTTCGCTAC) flanked by two restriction sites, Bsm I and Xho I. The corresponding synthetic oligodeoxynucleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)-fluorene (AAAF), forming a single dG-AAF adduct. This modified oligodeoxynucleotide was hybridized to its complementary strand and ligated between the Bsm I and Xho I sites of the vector. Plasmids containing the C8-dG-AAF adduct were used to transfect simian virus 40-transformed simian kidney (COS-1) cells and to transform several AB strains of Escherichia coli. Colonies containing mutant plasmids were detected by hybridization to 32P-labeled oligodeoxynucleotides. Presence of the single DNA adduct increased the mutation frequency by 8-fold in both COS cells and E. coli. Over 80% of mutations detected in both systems were targeted and represented G.C----C.G or G.C----T.A transversions or single nucleotide deletions. We conclude that modification of a deoxyguanosine residue with AAF preferentially induces mutations targeted at this site when a plasmid containing a single C8-dG-AAF adduct is introduced into mammalian cells or bacteria.
Moriya
M
M
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794.
Takeshita
M
M
Johnson
F
F
Peden
K
K
Will
S
S
Grollman
A P
AP
D015073:2-Acetylaminofluorene / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured / Q000502:physiology; D002522:Chlorocebus aethiops; D004249:DNA Damage; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation / Q000187:drug effects*; D010957:Plasmids; D014170:Transformation, Genetic / Q000187:drug effects
D015073:2-Acetylaminofluorene