Non-phenotypic selection of N-methyl-N′-nitro-N-nitrosoguanidine-directed mutation at a predicted hotspot site

The striking mutational specificity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) exhibited in the lacI gene in Escherichia coli allows comment on the phenotypic consequences of mutation at specific sequences that are not recovered after MNNG mutagenesis. We predict that the I⁺ phenotype is maintained when such silent positions are substituted by amino acids whose codons are generated by the MNNG-directed G:C → A:T transition. We chose the mutationally silent Gly200 codon (an MNNG hotspot motif sequence) to test this prediction. Through MNNG mutagenesis we have generated, identified and isolated a G:C → A:T transition at position 627 (5′-GG-C3′) under non-selective conditions which creates the Gly200→Asp substitution. The I⁺ phenotype is retained for this altered repressor.     

N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli

274 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations in the lacI gene of an Escherichia coli RecA- strain were cloned and sequenced. Base substitutions accounted for 264 mutations and consisted of 261 G:C----A:T transitions (including one double mutant with two G:C----A:T transitions separated by 25 base pairs), two A:T----G:C transitions and one A:T----T:A transversion. Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O6-methylguanine, and O4-methylthymine residues during DNA synthesis. The source of the transversion is not known. The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis. The observed distribution of mutations recovered is identical to that found in a RecA+ background indicating little involvement of RecA function in MNNG-induced mutation. Analysis of neighbouring base sequence revealed that the G:C----A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine. A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein. Within this domain the great majority of mutations generate nonsense codons or alter Gly codons.     

A Tester System for Detecting Each of the Six Base-Pair Substitutions in Saccharomyces Cerevisiae by Selecting for an Essential Cysteine in Iso-1-Cytochrome C

A collection of isogenic yeast strains that is specifically diagnostic for the six possible base-pair substitutions is described. Each strain contains a single, unique base-pair substitution at the Cys-22 codon of the CYC1 gene, which codes for iso-1-cytochrome c. These mutations encode replacements of the functionally critical Cys-22 and render each strain unable to grow on media containing nonfermentable carbon sources (Cyc-). Specific base-pair substitutions, which restore the Cys-22 codon, can be monitored simply by scoring for reversion to the Cyc+ phenotype. These strains revert spontaneously at very low frequencies and exhibit specific patterns of reversion in response to different mutagens. Only true (CYC1+) revertants were recovered after 7 days on selection medium. The following mutagen specificities were observed: ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine, G.C----A.T; 4-nitroquinoline-1-oxide, G.C----T.A and G.C----A.T; diepoxybutane, A.T----T.A, A.T----G.C and G.C----T.A; 5-azacytidine, G.C----C.G. Methyl methanesulfonate induced all six mutations, albeit at relatively low frequencies, with preference for A.T----T.A and A.T----G.C. Ultraviolet light was the most inefficient mutagen used in this study, consistent with its preference for transition mutations at dipyrimidine sequences reported in other systems. This tester system is valuable as a simple and reliable assay for specific mutations without DNA sequence analysis.     

N-Methyl-N′-nitro-N-nitrosoguanidine induced DNA sequence alteration; non-random components in alkylation mutagenesis

Our approach to the study of how the molecular nature of DNA modulates the behavior of mutational sites involves the characterisation of distributions of mutations. The Escherichia coli lacI genetic/M13 cloning system allows the comparison of base substitution frequencies at a large number of sites. The observed distribution of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced G:C → A:T transition (the predominant event), and A:T → G:C transition (a relatively rare event), is strikingly non-random. Some sites of G:C → A:T mutation are almost 100 times more often mutated by MNNG than the least susceptible sites. Sites of mutation, however, do not display a continuum of mutability, but rather can be strictly demarcated by their 5′ flanking base. Sites with a high frequency of occurrence share a common sequence motif, namely 5′-R-G-N-3′, which is the sole apparent feature that distinguishes them from sites less commonly mutated (i.e. 5′-Y-G-N-3′). A corollary of this defined site specificity is the absence of a strand bias in MNNG-induced lacI^−d mutation. The availability of specific or non-specific alkylation-repair systems does not appear to alter the distribution of mutation, which suggests that the observed mutational distribution is a direct reflection of the initial damage distribution. MNNG does not belong to that class of compounds typified by ultraviolet light or 4-nitroquinoline-N-oxide which exhibit both random and non-random components of mutagenesis.     

Description of a new amplifiable shuttle vector for mutagenesis studies in human cells: application to N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation spectrum.

In order to analyze the mechanisms of mutagenesis in human cells, we have established a human 293 cell-derived line containing a permanent mutagenesis target, the bacterial lacZ' gene, on an episomal EBV/SV40-based shuttle vector. This plasmid was maintained at a low copy number per cell which rendered it closer to an endogenous gene as compared to the usual transient shuttle vectors. Transient amplification of vectors, inside the host cell due to expression of the SV40 T-antigen, allowed the recovery of a large number of bacterial colonies transformed by plasmids extracted from human cells. Mutations produced in human host cells on the lacZ' locus were easily and rapidly scored and identified in bacteria using the blue/white color assay. Over a 6-month period in culture, we have shown that the lacZ' gene exhibited a low background frequency of point mutations (< 4.8 x 10(-6)). The efficiency of our system for detecting genotoxic-induced mutations was investigated by treating cells with a potent mutagen, the direct alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). A significant increase (< 230-fold) in the frequency of single-base substitutions was observed after MNNG treatment. In total, 63 MNNG-induced independent mutations were characterized. All substitutions but one involved G:C base pairs with 89% being G:C to A:T transitions which is consistent with the MNNG mutagenic specificity already reported in bacteria and mammalian cells. Mutations were distributed along the two strands of the lacZ' gene and there was no obvious influence of either the 5' or the 3' flanking base near the G:C to A:T transition sites. The low spontaneous point mutation frequency on the mutagenesis locus and the ability to detect induced point mutations indicate that this system could be readily used in human mutagenesis studies at the molecular level.     

Role of neighbouring bases and assessment of strand specificity in ethylmethanesulphonate and N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis in the SUP4-o gene of Saccharomyces cerevisiae

A total of 318 forward mutations induced by ethylmethanesulphonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the SUP4-o gene of the yeast Saccharomyces cerevisiae was characterized by DNA sequence analysis. Only base-pair substitutions were detected among the mutations examined and, for both agents, the majority (greater than 96%) were G.C to A.T. transitions. The remaining changes included A.T to G.C transitions and transversions at G.C sites. For EMS, two of the transversions were accompanied by nearby G.C to A.T transitions. There was considerable overlap of the sites within the SUP4-o gene that were mutated by EMS and MNNG and of the sites that each agent failed to mutate. However, EMS and MNNG mutagenesis differed with respect to the frequencies at which mutations were recovered at G.C pairs where the guanine is flanked (5') by a purine or pyrimidine. EMS exhibited no preference for either type of site, whereas a G.C site was 12-fold or fivefold more likely to be mutated by MNNG if preceded by a 5' adenine or guanine, respectively, than if flanked by a 5' pyrimidine. Finally, neither EMS nor MNNG mutagenesis showed a preference for G.C sites having the guanine on the non-transcribed strand.     

Differential enhancement of spontaneous transition mutations in the lacI gene of an Ung- strain of Escherichia coli

In this communication, the contribution of cytosine deamination to spontaneous mutagenesis in the lacI gene of E. coli was examined. In a wild-type strain, 75% of the amber mutations recovered were G:C----A:T transitions and 60% of these were at the 5-methylcytosine spontaneous hotspots Am6, Am15 and Am34. In a strain deficient for uracil-DNA glycosylase (Ung-), 96% of the amber mutations were G:C----A:T transitions while only 15% of these occurred at the hotspot sites. This shift in the mutational distribution demonstrates that cytosine deamination is a potent mutagenic process, which is enhanced in the absence of glycosylase. Moreover, some amber sites were greatly enhanced in the Ung- strain while others were only slightly enhanced. This result suggests that the rate of cytosine deamination at individual sites may be influenced by surrounding base composition. Therefore, we examined the neighboring sequences and found a strong correlation between the fold-increase in mutation and the A/T richness of the surrounding sequence. It is suggested that A/T-rich regions denature more often, forming transient single strands in which cytosine residues would be expected to deaminate more readily.     

DNA Sequence Analysis of Mutagenicity and Site Specificity of Ethyl Methanesulfonate in Uvr+ and UvrB - Strains of ESCHERICHIA COLI

EMS-induced mutations within a 180 base pair region of the lacI gene of E. coli were cloned and sequenced. In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced. The specificity of EMS-induced mutagenesis was very similar in the two strains; G:C----A:T transitions accounted for all but three of the mutants. The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain. This demonstrates, at the DNA sequence level, that the presumed premutagenic lesion, O6-ethylguanine, is subject to repair by the uvrABC excision repair system of E. coli. An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that O6-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machinery than those not adjacent to A:T base pairs.     

Codon usage changes and sequence dissimilarity between human and rat

Summary This paper reports on the relationship between the number of silent differences and the codon usage changes in the lineages leading to human and rat. Examination of 102 pairs of homologous genes gives rise to four main conclusions: (1) We have previously demonstrated the existence of a codon usage change (called the minor shift) between human and rat; this was confirmed here with a larger sample. For genes with extreme C+G frequencies, the C+G level in the third codon position is less extreme in rat than in human. (2) Protein similarity and percentage of positive differences are the two main factors that discriminate homologous genes when characterized by differences between rat and human. By definition, positive differences result from silent changes between A or T and C or G with a direction implying a C+G content variation in the same direction as the overall gene variation. (3) For genes showing both codon usage change and low protein similarity, a majority of amino acid replacements contributes to C+G level variation in positions I and II in the same direction as the variation in position III. This is thus a new example of protein evolution due to constraints acting at the DNA level. (4) In heavy isochores (high C+G content) no direct correlation exists between codon usage change (measured by the dissymmetry of differences) and silent dissimilarity. In light isochores the opposite situation is observed: modification of codon usage is associated with a high synonymous dissimilarity. This result shows that, in some cases, modification of constraints acting at the DNA level could accelerate divergence between genomes.     

Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escherichia coli lacI gene for a wild-type and an excision-repair-deficient strain

We have analyzed the DNA sequence changes in a total of 409 ultraviolet light-induced mutations in the lacI gene of Escherichia coli: 227 in a Uvr+ and 182 in a UvrB- strain. Both differences and similarities were observed. In both strains the mutations were predominantly (60 to 75%) base substitutions, followed by smaller contributions of single-base frameshifts, deletions and frameshift hotspot mutations. The base substitutions proved largely similar in the two strains but differences were observed among the single-base frameshifts, the deletions and the hotspot mutations. Among the base substitutions, both transitions (72.5%) and transversions (27.5%) were observed. The largest single group was G.C----A.T (60% of all base substitutions). The sites where G.C----A.T changes occurred were strongly correlated (97.5%) with sequences of adjacent pyrimidines, indicating mutation targeted ultraviolet photoproducts. Comparable amounts of mutation occurred at cytosine/cytosine and (mixed) cytosine/thymine sites. From an analysis of the prevalence of mutation at either the 5' or 3' side of a dipyrimidine, we conclude that both cyclobutane dimers and (6-4) lesions may contribute to mutation. Despite the general similarity of the base-substitution spectra between the wild-type and excision-defective strains, a number of sites were uniquely mutable in the UvrB- strain. Analysis of their surrounding DNA sequences suggested that, in addition to damage directly at the site of mutation, the potential for nearby opposite-strand damage may be important in determining the mutability of a site. The ultraviolet light-induced frameshift mutations were largely single-base losses. Inspection of the DNA sequences at which the frameshifts occurred suggested that they resulted from targeted mutagenesis, probably at cyclobutane pyrimidine dimers. The prevalence of frameshift mutations at homodimers (TT or CC) suggests that their formation involves local misalignment (slippage) and that base-pairing properties are partially retained in cyclobutane dimers. While the frameshift mutations in the Uvr+ strain were distributed over many different sites, more than half in the UvrB- strain were concentrated at a single site. Ultraviolet light-induced deletions as well as frameshift hotspot mutations (+/- TGGC at positions 620 to 632) are considered to be examples of untargeted or semitargeted mutagenesis. Hotspot mutations in the Uvr+ strain showed an increased contribution by (-)TGGC relative to (+)TGGC, indicating that ultraviolet light may specifically promote the loss of the four bases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells.

A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia coli) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G . C----A . T transitions; the other transition (A . T----G . C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.     

Induction of transversion mutations in Escherichia coli by N-methyl-N''-nitro-N-nitrosoguanidine is SOS dependent.

Escherichia coli alkA mutants, which are deficient for an inducible DNA glycosylase, 3-methyladenine-DNA glycosylase II, are sensitive to mutagenesis by low doses of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). As many as 90% of the alkA-dependent mutations induced by MNNG are also umuC+ dependent and thus are due to DNA lesions that are substrates for the mutagenic functions of the SOS response. A great number of these mutations are base substitutions at A . T sites, particularly A . T transversions. We discuss which DNA lesions may be responsible for these mutations. Our results show that the induction of 3-methyladenine-DNA glycosylase II, which occurs as part of the adaptive response to alkylating agents such as MNNG, significantly reduces the mutagenicity as well as the lethality of alkylation damage.     

Mechanism of mutation by thymine starvation in Escherichia coli: clues from mutagenic specificity.

To probe the mechanisms of mutagenesis induced by thymine starvation, we examined the mutational specificity of this treatment in strains of Escherichia coli that are wild type (Ung+) or deficient in uracil-DNA-glycosylase (Ung-). An analysis of Ung+ his-4 (ochre) revertants revealed that the majority of induced DNA base substitution events were A:T----G:C transitions. However, characterization of lacI nonsense mutations induced by thymine starvation demonstrated that G:C----A:T transitions and all four possible transversions also occurred. In addition, thymineless episodes led to reversion of the trpE9777 frameshift allele. Although the defect in uracil-DNA-glycosylase did not appear to affect the frequency of total mutations induced in lacI by thymine deprivation, the frequency of nonsense mutations was reduced by 30%, and the spectrum of nonsense mutations was altered. Furthermore, the reversion of trpE9777 was decreased by 90% in the Ung- strain. These findings demonstrate that in E. coli, thymine starvation can induce frameshift mutations and all types of base substitutions. The analysis of mutational specificity indicates that more than a single mechanism is involved in the induction of mutation by thymine depletion. We suggest that deoxyribonucleoside triphosphate pool imbalances, the removal of uracil incorporated into DNA during thymine starvation, and the induction of recA-dependent DNA repair functions all may play a role in thymineless mutagenesis.     

DNA damage caused by ascorbate in the presence of Cu2+ induces mutations

The DNA damage induced by ascorbate in the presence of Cu2+ was analyzed by sequencing, and the mutagenic consequences of damages to plasmid pUC18 lacZ' were assayed in a forward repairing system in E. coli JM109 in vivo. Ascorbate induced two classes of DNA damage in the presence of Cu2+, one being non-base-specific direct strand cleavage, and the other being sequence-specific base modification labile to alkali treatment. Radicals generated from ascorbate hydroperoxide were involved in DNA damaging reactions. Ascorbate and Cu2+ caused mutations in pUC18 lacZ' gene. The mutation frequency by this method was about 10(-4) at 18% survivors when measured as a loss of alpha-complementation. All the mutations found were single-base substitutions that occurred in the structural part of the lacZ' gene. They were predominantly G:C----A:T transitions.     

Ultraviolet mutagenesis in human lymphocytes: The effect of cellular transformation

We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells. To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189. Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058). pZ189 was treated with uv and introduced into the four cell lines by electroporation. Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli. Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines. Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%). In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells. Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared. Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia.     

Identification of a frameshift mutation responsible for the silent phenotype of human serum cholinesterase, Gly 117 (GGT----GGAG).

A frameshift mutation that causes a silent phenotype for human serum cholinesterase was identified in the DNA of seven individuals of two unrelated families. The mutation, identified using the polymerase chain reaction, causes a shift in the reading frame from Gly 117, where GGT (Gly)----GGAG (Gly+ 1 base) to a new stop codon created at position 129. This alteration is upstream of the active site (Ser 198), and, if any protein were made, it would represent only 22% of the mature enzyme found in normal serum. Results of analysis of the enzymatic activities in serum agreed with the genotypes inferred from the nucleotide sequence. Rocket immunoelectrophoresis using alpha-naphthyl acetate to detect enzymatic activity showed an absence of cross-reactive material, as expected. One additional individual with a silent phenotype did not show the same frameshift mutation. This was not unexpected, since there must be considerable molecular heterogeneity involved in causes for the silent cholinesterase phenotype. This is the first report of a molecular mechanism underlying the silent phenotype for serum cholinesterase. The analytical approach used was similar to the one we recently employed to identify the mutation that causes the atypical cholinesterase variant.     

Activation and induction of glycine N-methyltransferase by retinoids are tissue- and gender-specific

Betaine-homocysteine S-methyltransferase (BHMT; EC2.1.1.5) is a zinc metalloenzyme that catalyzes the transfer of a methyl group from betaine to homocysteine to produce dimethylglycine and Met, respectively. This enzyme is a member of a family of zinc-dependent methyltransferases that use thiols or selenols as methyl acceptors and which contain the following motif: G[ILV]NCX(20, 100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I. We recently reported that the three cysteine residues within this motif function as ligands to zinc in BHMT because changing any of them to alanine abolished zinc-binding and enzyme activity (A. P. Breksa, III, and T. A. Garrow, 1999, Biochemistry 38, 13991-13998). To determine if other amino acid residues in this motif were critical for enzyme function, the two regions defined by the motif in human BHMT, GVNCH(218) and VRYIGGCCGFEPYHI(307), were subjected to semirandom and random site-directed mutagenesis. Mutant enzymes were classified as either active or inactive based on their ability to complement the Met auxotrophy of Escherichia coli strain J5-3. The Gly residue at position 214 was found to be absolutely essential for complementation. The positions occupied by Gly297, Gly298, and Gly301 favored substitutions of small amino acids like Ala and Ser. We hypothesize that these Gly residues provide the necessary flexibility to the Zn-binding region to permit coordination of the metal.     

Specificity of spontaneous mutation in the lacI gene cloned into bacteriophage M13

We have studied the specificity of spontaneous mutation in the lacI gene of Escherichia coli cloned into bacteriophage M13. The comparison of the spectrum of 85 spontaneous mutations with that of the lacI gene carried on an E. coli F' episone revealed the following characteristics: (i) base substitution was predominant, accounting for 80% of spontaneous events compared with only 11% on the F' episome; (ii) among the base substitutions, the majority were G:C----A:T transitions (86%); (iii) not one mutation recovered on M13 corresponded to a mutation at the spontaneous hotspots seen in the F' spectrum (i.e., neither the addition or deletion of the tetramer 5'-CTGG-3' at position 620-631 nor the A:T----G:C transition at position +6 of lacO were recovered). The enhanced rate of cytosine deamination in single-stranded DNA, the unique replication mechanism and the refractory nature of single-stranded DNA to excision-repair processes present likely explanations for the observed mutational spectrum.     

Ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts

In order to examine possible cell-type specificity in mutagenic events, a shuttle-vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in Epstein-Barr virus transformed lymphoblastoid cell lines from a patient, XP12BE, with xeroderma pigmentosum (XP), group A, and a normal control. XP is a skin-cancer-prone disorder with UV hypersensitivity and defective DNA repair. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of E. coli. An earlier report on this data [Seetharam et al., (1990) J. Mol. Biol., 212, 433] indicated lower survival and higher mutation frequency with the UV-treated plasmid passed through the XP12Be(EBV) line. In the present report, sequence analysis of 198 mutant plasmids revealed a predominance of G:C----A:T transitions with both lymphoblastoid cell lines. This finding is consistent with the bias of polymerases toward insertion of an adenine opposite non-coding photoproducts (dinucleotides or other lesions). Transversion mutagenesis, non-adjacent double mutations, and triple-base mutations may involve other mechanisms. These results were compared to similar data from a fibroblast line from the same patient [Bredberg et al., (1986) Proc. Natl. Acad. Sci. (U.S.A.), 83, 8273]. The frequency of G:C----A:T transitions was higher, and there were fewer plasmids with multiple-base substitutions and with transversion mutations with both XP lymphoblasts and fibroblasts than with the normal lymphoblasts and fibroblasts. There were no significant differences in classes or types of mutations in the UV-treated plasmid replicated in the XP lymphoblasts and the XP fibroblasts. This suggests that the major features of UV mutagenesis in different cell types from the same individual are similar.     

Heat mutagenesis in bacteriophage T4: another walk down the transversion pathway.

Extracellular nonreplicating bacteriophage T4 particles accumulate mutations as functions of temperature, time, pH, and ionic environment via two mechanisms: 5-hydroxymethylcytidine deamination produces G.C----A.T transitions while a guanosine modification produces transversions. Neither frameshift mutations nor mutations at A.T base pairs are appreciably induced. We now show that heat induces G.C----T.A transversions which we suggest may arise via a G*.A mispair, in which G* is a modified guanosine that has experienced a glycosylic bond migration. The rate of this reaction at 37 degrees C is sufficient to present a genetic hazard, particularly to large genomes; thus, the lesion is probably efficiently repaired in cellular genomes.