In order to study mutagenesis in mammalian cells, stable mouse L-cell lines were established with multiple copies of a X phage vector that contains the supF gene of Escherichia coli as a target for mutagenesis. Rescue of viable phage from high molecular weight mouse cell DNA using X in vitro packaging extracts was efficient (5 phage per ,ug of cell DNA per copy) and yielded a negligible background of mutant phage (0 out of 54,605). From mouse cells exposed to 254-nm ultraviolet light (12 J/m2), 78,510 phage were rescued, ofwhich 8 were found to have mutant supF genes. DNA sequence analysis of the mutants suggests that the primary site of UV mutagenesis in mammalian cells is at pyrimidine-cytosine (Y-C) sequences, and that the most frequent mutation at this site is a C--T transition.

When a shuttle vector containing a tyrosine suppressor tRNA (supF) gene as a target for mutagenesis replicated in a monkey kidney cell line, the frequency of SupF+ mutations was 2.3 +/- 0.5 x 10(-3). When the host cells were treated with ethyl methanesulfonate 40 h before transfection, a 10-fold increase in SupF+ mutation frequency was observed. These results supported the hypothesis that a damage-inducible mutagenic pathway exists in mammalian cells and also demonstrated the utility of this shuttle vector for the study of mutagenesis in mammalian cells.

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. colh strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmnid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesiumcontaining buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideox nucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.The repair of base pair mismatches in the DNA of an organism plays an important role in reducing the frequency of mutations and in preserving the genetic integrity of the organism. Mismatches can occur in several ways. Recombination events can generate heteroduplex regions in DNA, and the process of gene conversion is thought to involve heteroduplex structures as intermediates. In DNA replication errors can occur which produce mismatched bases that must be corrected to avoid a high rate of spontaneous mutagenesis.In Escherichia coli DNA mismatch repair has been studied extensively. Transfection experiments with heteroduplex bacteriophage DNA (for a review, see reference 11) have demonstrated that E. coli has an efficient system for mismatch repair. The power of procaryotic genetic analysis has allowed identification of mutants that are deficient in various aspects of the repair process, and this has led to an understanding of some of the mechanisms that are involved. The products of the mutH, mutL, mutS, and mutU loci all seem to play a role in mismatch repair. Mutations at these loci produce strains that undergo a high rate of spontaneous mutagenesis because they cannot repair DNA mismatches effectively (11). Experiments involving the transfection of E. coli with hemimethylated A DNA coupled with the identification of mutants with altered function in the methylas...

Purified human B lymphocytes were examined for transcriptional expression of c-myc in response to mitogenic stimulation by the method of in situ hybridization using S-labeled DNA probes. The level of c-myc expression increased 10-to 20-fold within 2 hr after the addition of ant-la, formalinized Staphylococcus aureus Cowan strain I, or B-cell growth factor, as compared to resting B cells. After 72-96 hr of mitogenic stimulation, c-myc expression remained elevated 5-fold, but expression among individual cells had become more heterogeneous than at early time points. To determine whether c-myc expression in human B lymphocytes is phase specific within the cell cycle, mitogen-stimulated cells were sorted by DNA content into populations of cells in Go/Gig S and G2/M phases of the cell cycle. Examination of c-myc expression in phase-specific cells revealed that c-myc expression was elevated in all phases of the cell cycle, but it appeared to be maximally expressed in S phase. These studies suggest that c-myc expression in normal human B lymphocytes is cell-cycle dependent and remains elevated in all phases of the cycling cell.Although altered expression of the c-myc gene has been implicated in the genesis of human B-cell malignancies (1-5), the regulation of c-myc expression in normal human B cells remains unknown. Recent evidence suggests that c-myc expression is cell-cycle dependent in normal cells. Transcripts of the c-myc gene have been shown to increase sharply after the addition of mitogens to resting cultures of murine spleen cells or fibroblasts and then decline before the onset of DNA synthesis (6). Based on this observation, it has been inferred that c-myc expression is specific to the G1 phase of the cell cycle. However, other investigators have shown that the level of c-myc transcripts in proliferating avian fibroblasts and several transformed cell lines is constant throughout the cell cycle (7,8). Thus c-myc expression appears to be linked to the cell cycle, but its precise regulation during cell cycling remains uncertain.To assess the role ofaltered regulation of c-myc expression in the transformation of human B cells, it is important to understand the regulation ofthe c-myc gene in normal human B cells. Recent advances in our understanding of the sequence of events that trigger human B-cell activation, proliferation, and differentiation have provided the framework for the study of c-myc expression in normal human B lymphocytes (9-13). Human B-cell activation as measured by increased cellular volume (9, 10), enhanced synthesis ofRNA and DNA (9-13), or altered cell-surface antigens (9, 12, 13) can be induced in vitro by substances that cross-link surface immunoglobulin such as anti-Ig antibodies or the protein A component of Staphylococcus aureus Cowan strain I. Subsequent proliferation and differentiation of activated B cells requires soluble T-cell-derived factors (9-13). Recent studies have suggested that one or more T-cell-derjved factors, called B-cell growth factor(s) (BCGF), specifically fun...

When a shuttle vector containing a tyrosine suppressor tRNA (supF) gene as a target for mutagenesis replicated in a monkey kidney cell line, the frequency of SupF+ mutations was 2.3 +/- 0.5 x 10(-3). When the host cells were treated with ethyl methanesulfonate 40 h before transfection, a 10-fold increase in SupF+ mutation frequency was observed. These results supported the hypothesis that a damage-inducible mutagenic pathway exists in mammalian cells and also demonstrated the utility of this shuttle vector for the study of mutagenesis in mammalian cells.