Abnormal processing of transfected plasmid DNA in cells from patients with ataxia telangiectasia.

In order to assess spontaneous mutability and accuracy of DNA joining in ataxia telangiectasia, a disorder with spontaneous chromosome breakage, the replicating shuttle vector plasmid, pZ189, was transfected into SV40 virus-transformed fibroblasts from ataxia telangiectasia patients. The ataxia telangiectasia fibroblasts showed elevated frequency of micronuclei, a measure of chromosome breakage. The spontaneous mutation frequency was normal with circular plasmids passed through the ataxia telangiectasia line. These results were compared to those with transformed fibroblasts from a patient with xeroderma pigmentosum, and from a normal donor. Mutation analysis revealed spontaneous point mutations and deletions in the plasmids with all 3 cell lines, however, insertions or complex mutations were only detectable with the ataxia telangiectasia line. To assess DNA-joining ability, linear plasmids which require joining of the DNA ends by host cell enzymes for survival, were transfected into the cells. We found a 2.4-fold less efficient DNA joining in ataxia telangiectasia fibroblasts (p = 0.04) and a 2.0-fold higher mutation frequency (p less than 0.01) in the recircularized plasmids than with the normal line. Plasmid DNA joining and mutation frequency were normal with the xeroderma pigmentosum fibroblasts. These findings with the ataxia telangiectasia fibroblasts of abnormal types of spontaneous mutations in the transfected plasmid and inefficient, error-prone DNA joining may be related to the increased chromosome breakage in these cells. In contrast, an EB virus-transformed ataxia telangiectasia lymphoblast line with normal frequency of micronuclei showed normal types of spontaneous mutations in the transfected plasmid and normal frequency of DNA joining which was error-prone. These data indicate that mechanisms that produce chromosome breakage in ataxia telangiectasia cells can be reflected in processing of plasmid vectors.     

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.     

Mutation spectrum in gamma-irradiated shuttle vector replicated in ataxia-telangiectasia lymphoblasts.

Cells from ataxia-telangiectasia (AT) patients are hypersensitive to the lethal effects of ionizing radiation. To assess radiation mutagenesis in these cells, the SV40-based shuttle vector, pZ189, was used to analyze gamma-ray-induced mutations following the plasmid's replication in AT lymphoblasts. Progenies from the AT line GM2783 exposed to 50 Gy showed a mutation frequency of 7.6 x 10(-3), 63-fold over background; surviving plasmids were 3.4% of control. Both values were essentially the same as those of irradiated plasmids replicated in a normal lymphoblast line, GM606. In addition, pZ189 exposed to 25 Gy of gamma radiation and replicated in another normal lymphoblast line and in cells of two additional AT lymphoblast lines showed similar mutation frequencies and percentages of surviving plasmids. Qualitative comparison of plasmid mutations from AT and normal cells showed no significant differences, indicating that the damaged DNA was repaired with similar fidelity in AT and normal cells. These studies suggest that there is no correlation between the enhanced sensitivity of AT cells to killing by ionizing radiation and gamma-radiation-induced mutagenesis of plasmid DNA processed in these cells.     

UV-induced base substitution mutations in a shuttle vector plasmid propagated in group C xeroderma pigmentosum cells.

To assess the contribution to mutagenesis of human DNA repair defects, the UV-irradiated shuttle vector plasmid pZ189 was propagated in fibroblasts derived from a xeroderma pigmentosum (XP) patient in DNA repair complementation group C. In comparison to results with DNA repair-proficient human cells (WI-38 VA13), UV-irradiated pZ189 propagated in the XP-C (XP4PA(SV)) cells showed fewer surviving plasmids and a higher frequency of mutated plasmids. Base sequence analysis of 67 mutated plasmids recovered from the XP-C cells revealed similar classes of point mutations and mutation spectrum, and a higher frequency of G:C to A:T transitions along with a lower frequency of transversions among plasmids with single or tandem mutations compared to plasmids recovered from the normal line. Most single-base substitution mutations (83%) occurred at G:C base pairs in which the 5'-adjacent base of the cytosine was thymine or cytosine. These results indicate that the DNA repair defects in XP-C, in comparison to data previously reported for XP-A, XP-D and XP-F, result in different UV survival and mutation frequency but in similar types of base substitution mutations.     

UV mutation spectra in cell lines from patients with Cockayne's syndrome and ataxia telangiectasia, using the shuttle vector pZ189.

We have used the SV40-based shuttle vector pZ189 to determine ultraviolet mutation spectra in SV40-transformed cell lines from two patients with Cockayne's syndrome (CS) and ataxia telangiectasia (AT). The shuttle vector was UV-irradiated, transfected into the cells and recovered two days later, after many rounds of replication had occurred. Plasmid DNA was used to transform indicator bacteria in which plasmids containing a mutation in the supF gene resulted in white colonies. Mutant plasmids were analysed both by agarose gels and by DNA sequencing. In contrast to published spectra for xeroderma pigmentosum cells, the types of mutation induced by UV mutation in the CS and AT cell lines were similar to each other and to published spectra for normal cell lines. There were however, some differences in the sequence distribution of the mutations.     

An SV40-transformed xeroderma pigmentosum group D cell line: establishment, ultraviolet sensitivity, transfection efficiency and plasmid mutation induction

Fibroblasts from a patient with xeroderma pigmentosum complementation group D were treated with Simian virus 40 to establish a transformed cell line suitable for studies of DNA-mediated gene transfer. After progressing through 2 crises, a stable line, XP6Be(SV40), was established and cultured for more than 1 year. This line retains the characteristic xeroderma pigmentosum ultraviolet hypersensitivity and is able to complement a SV40-transformed group A line when fused and assayed for ultraviolet radiation inhibition of colony-forming ability. XP6Be(SV40) expressed high levels of transfected chloramphenicol acetyltransferase activity (0.1 nmole X mg-1 X min-1) in a transient expression assay, showed stable expression of transfected gpt or neo genes (frequency 1-20 X 10(-5)), and permitted replication of the mutagenesis shuttle vector plasmid, pZ189. Ultraviolet treatment (500 J X m-2) of pZ189 prior to replication in XP6Be(SV40) resulted in a large reduction in plasmid yield (5% survival) and a 60-fold increase in the mutation frequency, reflecting the reduced ability of these cells to repair ultraviolet-damaged transfecting DNA. This cell line provides the opportunity to utilize transfection studies in cells with the xeroderma pigmentosum group D defect in excision repair.     

Repair and replication of plasmids with site-specific 8-oxodG and 8-AAFdG residues in normal and repair-deficient human cells.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.     

Mutation of potassium permanganate- and hydrogen peroxide-treated plasmid pZ189 replicating in CV-1 monkey kidney cells

We examined the effect of the oxidation of plasmid pZ189 by KMnO4, which does not produce free radicals, and H2O2/Fe(2+)-diethylenetriaminepentaacetic acid (DTPA), which does, on the mutation frequency of pZ189 transfected into monkey kidney CV-1 cells. Treatment with 1.5 mM KMnO4 increased the content of certain modified bases, principally Thy and Cyt modified at C-5 and C-6, by up to 300-fold, as measured by GC/MS; however, the mutation frequency increased only 5-fold above background. 1.0 mM H2O2/0.1 mM Fe(2+)-DTPA treatment, which increased the mutation frequency 10-fold above background, increased the content of certain modified bases by up to 4-fold. Sequence analysis revealed both deletions and point mutations, with a predominance of C-G substitutions, among H2O2/Fe(2+)-DTPA-associated mutations. These data suggest that KMnO4-modified DNA is only weakly mutagenic in DNA replicating in mammalian nuclei, despite substantial production of Thy glycol and other base modifications, whereas H2O2/Fe(2+)-DTPA-modified DNA is more mutagenic. H2O2/Fe(2+)-DTPA generated mutations occur predominantly at C-G base pairs.     

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.     

Requirement for p53 in ionizing-radiation-inhibition of double-strand-break rejoining by human lymphoblasts.

Ionizing radiation (IR) triggers apoptosis, cell-cycle arrest, and DNA-repair induction in mammalian cells. These responses are mediated by proteins, including p53, which are activated or induced by IR. To determine the role of p53 in double-strand break (DSB) repair following irradiation of mammalian cells, we compared the abilities of unirradiated and irradiated TK6 human lymphoblast line and its derivatives TK6-E6-20C and TK6-E6-5E to repair restriction-enzyme-linearized shuttle pZ189 and the luciferase-reporter plasmid pGL3-control. TK6-E6-20C expresses wild-type p53 like the parental TK6 line, while TK6-E6-5E is p53 null. DSB-rejoining capacity was determined from the ratio of viable progenies arising from DSB-containing plasmids (linDNA) to the number of viable progenies from undamaged, supercoiled plasmids (scDNA). The ratio from the p53wt hosts was two- to three-fold higher than that from the p53null host, using either pZ189 or pGL3-control plasmid. After exposure of both hosts to 0.5 Gy gamma-radiation, DSB-rejoining capacity of p53null increased two-fold compared to unirradiated null controls, if transfection occurred immediately after irradiation. In contrast, the DSB-rejoining capacity of p53wt was unaffected by irradiation. If transfection was delayed for 2 h following irradiation, however, DSB-rejoining declined in both p53wt and p53null hosts. Irradiation also altered DSB-rejoining fidelity, measured from the mutation frequencies, among progenies of pZ189 linDNA. But, unlike rejoining capacity, changes in DSB-rejoining fidelity were similar in p53wt and p53null hosts. Changes in cell-cycle distribution in p53wt and p53null hosts were also similar following irradiation. These findings show that IR increases DSB-rejoining capacity in mammalian cells without functional p53, suggesting that p53 participates in suppressing DSB-rejoining following exposure of mammalian cells to IR.     

The spectrum of mutations generated by passage of a hydrogen peroxide damaged shuttle vector plasmid through a mammalian host.

Treatment of a plasmid shuttle vector (pZ189) with a combination of hydrogen peroxide and a ferric iron/EDTA complex prior to transfection and passage in simian (CV-1) cells increases the frequency of mutations at the supF locus by up to 60-fold over the spontaneous background. This increase in mutation frequency is abolished when the inhibitors desferrioxamine, superoxide dismutase, catalase or dimethyl sulfoxide are included in the initial reaction or when the iron/EDTA complex is omitted, a strong indication that the premutagenic damage arises as a result of direct attack by hydroxyl radical generated in a superoxide driven Fenton reaction. DNA sequence analysis of the mutated plasmids shows that 1) Deletions occuring in combination with base-substitutions arise in 22.5 percent of the induced mutants compared with only 3 percent of spontaneous mutants 2) Sixty percent of all induced deletion mutations involve the loss of a single base and 77 percent of these (20 out of 26) occur at two adenine-containing sites 3) The base-change spectrum of mutants arising in the treated plasmid population is marked by the predominance of mutants containing a single base-change and by an increase in changes at AT base pairs. These results provide direct information concerning the nature of mutations arising in mammalian cells as a result of hydroxyl radical mediated DNA damage.     

A vector for recombinant DNA in Staphylococcus aureus

Staphylococcal plasmids pS194 and pSC194 which confer streptomycin and streptomycin-chloramphenicol resistance respectively have been used as vectors for construction of recombinant DNA, since they each carry one single recipient site for endonuclease EcoRI. Hybrid DNA does not express streptomycin resistance, a marker which is present in both vectors, presumably because the marker gene is cleaved by EcoRI. A chloramphenicol marker present in pSC194 was used for positive hybrid selection. Hybrid plasmids generated by joining pSC194 with one or more of the four EcoRI fragments of the large (18.1-10(6) daltons) staphylococcal plasmid pI258 were constructed and permitted us to develop a physical map for pI258.     

Test of models for the sequence specificity of UV-induced mutations in mammalian cells

We have used mathematical modeling and statistical analysis to examine the correlation between UV-induced DNA damage and resulting base-substitution mutations in mammalian cells. The frequency and site specificity of UV-induced photoproducts in the supF gene of the pZ189 shuttle vector plasmid were compared with the frequency and site specificity of base-substitution mutations induced upon passage of the UV-irradiated vector in monkey cells. The hypothesis that the observed mutational spectrum is due to a preferential insertion of adenosine opposite UV photoproducts in the DNA template was found to best explain the mutational data. Models in which it was postulated that only (6-4) photoproducts, and not cyclobutane dimers, are mutagenic, or that the relative frequency of photoproduct formation does not influence mutation frequencies, fit the data much less well. This analysis demonstrates that molecular mechanisms of mutagenesis in mammalian cells can be deduced from mutational data obtained with a shuttle vector system.     

Overproduction of Escherichia coli replication proteins by the use of runaway-replication plasmids.

A derivative of the runaway-replication plasmid was constructed. This plasmid, pSY343, has the gene for kanamycin resistance and single sites for EcoRI, BamHI, HindIII, KpnI, and XhoI that can be used as cloning sites without inactivating the kanamycin resistance gene or the replication genes. Three replication genes of Escherichia coli were cloned on the plasmid. The activity of dnaA, dnaZ, and ssb gene products were 200-, 90-, and 60-fold greater, respectively, in the cells containing these plasmids than in normal cells.     

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.     

Mutation spectra induced by alpha-acetoxytamoxifen-DNA adducts in human DNA repair proficient and deficient (xeroderma pigmentosum complementation group A) cells

Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.     

Comparing the frequency and spectra of mutations induced when an SV-40 based shuttle vector containing covalently bound residues of structurally-related carcinogens replicates in human cells

An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene (supF) was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into the human embryonic kidney cell line 293 and allowed to replicate. The progeny plasmids were rescued and assayed for the frequency of supF mutants by being used to transform indicator bacteria carrying an amber mutation in the beta-galactosidase gene. The agents tested were the 7,8-diol-9,10-epoxide of benzo[a]pyrene (BPDE); 1-nitrosopyrene (1-NOP); N-acetoxy-2-acetylaminofluorene (N-AcO-AAF); and its trifluoro-derivative (N-AcO-F3-AAF) which yields deacetylated adducts. With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies as high as 20 x 10(-4) to 40 x 10(-4), with a background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid, BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately 4 times more mutagenic than 1-NOP, N-AcO-AAF and N-AcO-F3-AAF, which bind principally or exclusively to the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule. It could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least 3 times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of 35 mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base-substitutions. DNA-sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmids and 60 from 1-NOP-treated plasmids indicated that 60% and 80%, respectively, contained a single base-substitution, 5-10% had two base-substitutions, and 4-10% had small insertions or deletions (one or two base pairs).(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrogen peroxide induces G:C to TA and G:C to C:G transversions in the supF gene of Escherichia coli

A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe³⁺/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:C→C:G (28 cases) and G:C→T:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5′-PuGA-3′ was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5′PyGN3′. G:C→T:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:C→C:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.     

A novel method for rapid isolation of plasmid DNA

A new disposable chromatographic column, pZ523, has been developed for separating plasmid DNA from bacterial chromosomal DNA. Use of pZ523 spun columns eliminates the need for ethidium bromide-cesium chloride density gradients which require long centrifugation times. pZ523 purified plasmids have been shown to be of purity suitable for restriction analysis, ligation, transfection of mammalian cells and transformation of bacteria. Unlike the traditional ultracentrifugation method, pZ523 offers an extremely rapid alternative method for purifying large amounts of plasmid DNA (2.5 mg to 4.5 mg) from cleared bacterial lysates in only 25 minutes.