Proc. Nati. Acad. Sci. USA Vol. 85, pp. 1586-1589, March 1988 Genetics Targeted mutations induced by a single acetylaminofluorene DNA adduct in mammalian cells and bacteria (shuttle vector/mutagenesis) MASAAKI MORIYA*, MASARU TAKESHITA*, FRANCIS JOHNSON*, KEITH PEDENt, STEPHEN WILL*, AND ARTHUR P. GROLLMAN *Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794; and tHoward Hughes Medical Institute Laboratory, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205 Communicated by Richard B. Setlow, November 2, 1987 ABSTRACT Mutagenic specificity of 2-acetylamino- lems were obviated by initiating mutagenesis with a defined fluorene (AAF) has been established in mammalian cells and DNA adduct introduced at a specific site in the genome and several strains of bacteria by using a shuttle plasmid vector by detecting the full spectrum of mutations by oligodeoxy- containing a single N-(deoxyguanosin-8-yl)acetylaminofluo- nucleotide hybridization. rene (C8-dG-AAF) adduct. The nucleotide sequence of the Viral and plasmid vectors containing specifically located gene conferring tetracycline resistance was modified by con- DNA adducts have been described (5-8); some ofthese have servative codon replacement so as to accommodate the se- been tested for mutagenic properties in bacteria (8-10). We quence d(CCTTCGCTAC) flanked by two restriction sites, have constructed a shuttle plasmid containing a single Bsm I and Xho I. The corresponding synthetic oligodeoxynu- "bulky" adduct, N-(deoxyguanosin-8-yl)acetylaminofluo- cleotide underwent reaction with 2-(N-acetoxy-N-acetylamino)- rene (C8-dG-AAF). This modified plasmid was allowed to fluorene (AAAF), forming a single dG-AAF adduct. This replicate in mammalian cells and bacteria. In both experi- modified oligodeoxynucleotide was hybridized to its comple- mental systems, the adduct generated a mutagenic response mentary strand and ligated between the Bsm I and Xho I sites consisting primarily of transversions and single-base dele- of the vector. Plasmids containing the C8-dG-AAF adduct tions targeted to the site of chemical modification. were used to transfect simian virus 40-transformed simian kidney (COS-l) cells and to transform several AB strains of MATERIALS AND METHODS Escherichia coli. Colonies containing mutant plasmids were detected by hybridization to 32P-labeled oligodeoxynucleo- Construction of Plasmid Vector and 2-Acetylaminofluorene tides. Presence of the single DNA adduct increased the muta- (AAF)-Modified Oligodeoxynucleotide. The shuttle plasmid tion frequency by 8-fold in both COS cells and E. coli. Over pAG75 was constructed from the bacterial plasmid pKP772 80% of mutations detected in both systems were targeted and by inserting the simian virus 40 origin of replication (11), represented G-C -- CG or G-C- T-A transversions or single nucleotides 5155-119, at the EcoRI site. Plasmid pKP772 nucleotide deletions. We conclude that modification of a was derived from pBR322 by deletion of nucleotides deoxyguanosine residue with AAF preferentially induces mu- 1332-2520 (K.P., unpublished data). Oligonucleotide- tations targeted at this site when a plasmid containing a single directed mutagenesis (12) created unique Mlu I (nucleotide C8-dG-AAF adduct is introduced into mammalian cells or 945) and Xho I (nucleotide 978) sites in the tetracycline- bacteria. resistance (TcR) gene (Fig. 1). The 33-base-pair sequence between these restriction sites was replaced by a chemically synthesized oligonucleotide containing sites for SnaBI Chemical mutagenesis often involves formation of a covalent (nucleotide 956), Nhe I (nucleotide 964), and Bsm I (nucle- adduct between the mutagen and DNA (1, 2). Such lesions otide 968), creating pGM86 (Fig. 1). After digestion with activate cellular mechanisms involved in DNA repair and, Bsm I and Xho I, pGM86 was used as vector for the muta- unless repaired prior to replication, may lead to nucleotide genesis studies described in this paper. Plasmid pGM87 was substitutions, deletions, and chromosome rearrangements constructed by replacement of the short Bsm I/Xho I frag- (3). Chemical mutagens are also reported to activate cellular ment with another synthetic oligodeoxynucleotide, protooncogenes (4). d(CCTTCGCTAC) (G-10). Base substitutions introduced by Primary chemical structures have been established for these procedures conserve the original amino acid sequence; different DNA adducts (1, 2). However, such information thus pGM86 and pGM87 retain a functional gene conferring alone does not distinguish between potential mutagenic TcR. This property is lost in plasmids containing a single- species; in fact, so called "minor" adducts may be more base deletion or G -+ C mutation at position 974, the site of important in this respect than those detected in much larger the dG-AAF adduct. quantities (1). Oligodeoxynucleotide G-10 and its complementary strand, Experiments in which cells are exposed to a given chem- d(TCGAGTAGCGAAGGCT) (C-16), were synthesized by ical mutagen involve a number of factors, including trans- solid-state methods, using conventional phosphoramidite port, metabolism, and the site of adduct formation. These chemistry (13). After removal from the solid support, oligo- processes complicate interpretation of subsequent muta- deoxynucleotides were purified as their 5'-dimethyltrityl genic events. Furthermore, many methods used for detec- derivatives, using reversed-phase HPLC on a Bio-Sil ODS- tion of mutations, although sensitive and convenient, intro- 5S column (Bio-Rad) (300 x 4.5 mm), eluting with a linear duce selective bias into the mutagenic response and/or fail gradient of 15-30% acetonitrile in 0.05 M triethylammonium to detect silent mutations. In our experiments, these prob- acetate buffer (pH 7.0). The main UV-absorbing fraction was The publication costs of this article were defrayed in part by page charge Abbreviations: AAAF, N-acetoxy-2-(acetylamino)fluorene; AAF, payment. This article must therefore be hereby marked "advertisement" 2-acetylaminofluorene; C8-dG-AAF, N-(deoxyguanosin-8-yl)ace- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tylaminofluorene; TcR, tetracycline resistance. 1586 Downloaded by guest on September 29, 2021 Genetics: Moriya et al. Proc. Natl. Acad. Sci. USA 85 (1988) 1587 from plates containing ampicillin (80 ,ug/ml), were analyzed for TcR. Mutant plasmids were detected by oligodeoxynu- cleotide hybridization. Mutagenesis in Mammalian Cells. Simian virus 40- transformed simian kidney cells (COS-1) (17) were grown at 370C under 5% C02/95% air in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were seeded at 3 x 105 cells per 60-mm plate. After 24 hr, cells were transfected with ligation mixture (50 ng of DNA) using Pst (2423)- DEAE-dextran (18). Forty-eight hours after transfection, plasmid DNA was extracted from the cells, digested with proteinase K for 4 hr, and then treated sequentially with phenol, phenol/chloroform, and chloroform (19). After eth- anol precipitation, the DNA was treated with Dpn I to degrade input DNA (19) and used to transform competent E. coli DH5 cells. Transformants were screened for mutations by oligodeoxynucleotide hybridization. Asp A/a Leu G1Y Rr Vol Lou Leu A/a Phe A/a 7hr/iR G/Y Oligodeoxynucleotide Hybridization to Bacterial Colonies. Ampicillin-resistant colonies were replica-plated onto What- pAG75 944GAC GCGCTG GGC TAC GTC TTG CTG GCG TTC GCGACG CGAGGC985 man 3MM paper and incubated overnight at 370C. After pGM86 GAC GCG TTG GGC TAC GTA TTG CTA GCA TTC GCG ACT CGA GGC fixation of plasmids, filters were baked at 80'C under vac- p~~~~~~~~~p GM87 GM7IGAC GCG TTG GGC TAC GTA TTG CTA GCC rrc GCT ACT CGA GGC uum for 2 hr, hybridized at 420C or 450C for 16 hr with 32P-labeled oligodeoxynucleotide probes (12), washed three FIG. 1. Structure of pGM86 and partial sequence of the modified times at 40'C with 6 x SSC buffer (20), dried, and subjected TcR genes of pAG75 and pGM87. The nucleotide sequence and to autoradiography. Oligodeoxynucleotides used to detect corresponding amino acid sequence from residues 287-300 are mutations in these experiments were identical to C-16 except shown for pAG75 together with the nucleotide substitutions of the nucleotide at position 974 was changed to deoxyguano- pGM86 and pGM87. The bracketed sequence represents the in- serted G-10 oligonucleotide. The site of AAF modification (nucleo- sine (G-16), deoxyadenosine (A-16), or deoxythymidine (T- tide 974) and nucleotides changed are indicated. SV40, simian virus 16), or it was deleted (D-16). Colonies that failed to hybridize 40; ori, origin; Ap, ampicillin; Tet, tetracycline; bp, base pairs. to these probes were tested with L-16 and R-15, oligodeoxy- nucleotides that contain sequences complementary to the collected and then rechromatographed as the deprotected junctions between insert and vector. This procedure detects species, eluting with 5-15% acetonitrile. all mutant plasmids containing correctly inserted sequences. G-10 (0.1 mg) and N-acetoxy-2-(acetylamino)fluorene Plasmid DNA was prepared (21) from the mutants, including (AAAF) (0.5 mg) were dissolved in 1.0 ml of sodium citrate colonies that hybridize to L-16 and R-15 but fail to hybridize buffer containing 10%6 ethanol and allowed to stand for 90 to C-16, G-16, A-16, T-16, or D-16, and the DNA sequence min at 370C under nitrogen in the dark. HPLC analysis of the was determined by the dideoxynucleotide chain-termination reaction products, eluted over 10 min with a linear 0-20% method (22). gradient of acetonitrile in triethylammonium acetate buffer followed by an isocratic 20o elution, revealed a new UV- RESULTS absorbing peak, eluting at 23.5 min, which was clearly resolved from unmodified G-10, eluting at 21.6 min. The UV Transformation Efficiency and Mutation Frequencies of spectrum of this modified exhibited an AAF-Modified Plasmids. The presence of a single AAF oligodeoxynucleotide trans- maximum at 270 nm with a shoulder at 300 nm.