Eighteen mutants deficient in dihydrofolate reductase (DHFR) activity were induced with 0.5 microM N-hydroxy-aminofluorene in four separate experiments. This carcinogen dose killed approximately 80% of the treated cells and resulted in a mutational frequency approximately 3 x 10(-6). The nature of the induced changes in each of the mutants was determined by direct sequencing following polymerase chain reaction amplification, or in one instance, by Southern blot analysis. Nearly all (15/17) of the mutations were single base changes. Consistent with the binding specificity of this chemical, all mutations were targeted to guanine bases. The predominant change was G:C-->T:A transversion which was evident in 11/15 mutants. A single dG-AF mutational hotspot was noted at a site in the DHFR coding sequence of exon 4; one-third of the induced point mutations arose at this position. These results are compared with our previous analyses of mutants induced with the related aromatic amine, N-2-acetoxy-2-acetyl-aminofluorene.

Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3a,4fi-dihydroxy-la,2a-epoxy-1,2,3,4-tetrahydrobenzo[cJphenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 ,AM yielded a 46% survival of the treated population and an induced frequency of mutation of 1.7 X 10-, 10_-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A'T -+ T-A and G'C -+ T-A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG (the underlined base is the target). Surprisingly, 42% of the mutations created mRNA splicing defects (16/38) Fig. 1. We determined the induced sequence changes in 38 DHFR-mutants. A remarkably large proportion (16/38) of the induced mutations occurred at pre-mRNA consensus splice sites, resulting in splicing defects. MATERIALS AND METHODSMaterials. Reagents, media, and materials used in this work were the same as indicated (8) sulfoxide) for 90 min; they were then treated with trypsin, counted, and plated on 15 150-mm dishes. Selection for DHFR-clones (14) followed a 6-day expression period. Individual colonies were isolated and tested for glycine, thymidine, and hypoxanthine auxotrophy. Subclones of auxotrophs were expanded and assayed for ability to bind [3H]methotrexate (15). Three colonies (designated A, B, and C) per dish were analyzed. Mutants arising on the same dish were deemed independent if DNA sequencing showed different base changes for each mutant. Presumed sisters were discarded (six altogether).DNA Sequence Analysis. DHFR fragments from genomic DNA or DHFR cDNA from each mutant were amplified by the polymerase chain reaction (PCR) (16) as described (9,17 5464The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

We evaluated the formation and removal of (+)-3a,48-dihydroxy-la,2a-epoxy-1,2,3,4-tetrahydroben- 2). A similar strand bias for BPDE-induced mutations in the hypoxanthine phosphoribosyl transferase gene (HPRT) of human fibroblasts was described where 77% of the premutagenic guanine bases were also on this strand (3). Assuming that carcinogen modification of DNA in vivo occurs with similar frequency on both strands, several possible explanations for the apparent strand selectivity of induced mutations can be considered and tested. One explanation for the strand bias of mutations is that the selection for dihydrofolate reductase-deficient mutants is very stringent, causing mutations to occur on one strand preferentially (4). We recently devised a reversion assay to address this possibility (5). BcPHDE-induced forward mutations were transversions favored at triple purine sequences where the 5' base was altered (2). The assay used DHFRnonsense mutants that carry this target sequence on both strands at each of two stop codons, and all purine thymine mutations yield amino acid substitutions compatible with dihydrofolate reductase activity. Randomly induced mutations at these sites would produce a 2:1 ratio of substitutions on the nontranscribed versus the transcribed strand. Sequence analysis of 66 independent BcPHDE-induced revertants showed that the combined ratio of mutations affecting purines on the nontranscribed strand was 15:1. Hence, the strand bias here was independent of phenotypic selection.Analysis of induced mutations in rodent HPRT led to the suggestion that strand-biased mutations relate to the polarity of the marker gene with respect to DNA replication (6). Since strand-biased UV-induced mutations were found in the absence of DNA repair on the transcribed strand of HPRT (7), the misinsertion rate differences for replicative polymerases a and 6 may also contribute to the strand-bias phenomenon.

DNA strand-specific mutations induced by (+)-3a,4.6-dihydroxy1a,2a-epoxy-1,2,3,4-tetrahydrobenzo[c] ABSTRACT We previously showed that the preferred mutation induced by (±)-3a,4.8-dihydroxy-la,2a-epoxy-1,2,3,4-tetrahydrobenzo[cJphenanthrene (BcPHDE) in the dihydrofolate reductase gene in Chinese hamster ovary cells was a purine to thymine transversion on the nontranscribed strand at the sequence 5'-RRR-3' (R is a purine and the mutated base is underlined). To determine whether the observed mutational strand specificity was due to bias in the phenotypic selection, we designed a nonsense-codon reversion assay in which a triple

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