Influence of ammonium sulfate and sodium chloride on ethyl methanesulfonate-induced chromosomal aberrations and HPRT mutations in V79 hamster cells

V79 hamster cells were mutagenized with ethyl methanesulfonate (EMS) and immediately afterwards posttreated with hypertonic solutions of sodium chloride or ammonium sulfate. The posttreatment had a clear effect on chromosomal aberrations, but TGr mutations were only enhanced by ammonium sulfate, but not by sodium chloride posttreatment. It is suggested that hypertonic salt posttreatment leads to conformational changes in the DNA, resulting in an increase in TGr mutations and chromosomal aberrations.     

Chromosomal aberrations induced by the restriction endonuclease Alu I in Chinese hamster ovary cells: influence of duration of treatment and potentiation by cytosine arabinoside

Induction of chromosomal aberrations by the restriction endonuclease Alu I in Chinese hamster ovary cells (CHO) has been studied. Treatment of cell pellets with Alu I for a time as short as 1 min was found to induce significant increase in the frequency of chromosomal aberrations. Alu I was found to be effective both in trypsinized cells as well as in cells which were collected with a rubber policeman, indicating that trypsinization of cells is not a prerequisite for the entry of the enzyme into the cells. Treatment of cells with Alu I in the presence of 1-beta-D-arabinosylcytosine (ara C) led to an increase in the induced frequency of aberrations, most probably due to the inhibition of ligation of DNA-strand breaks by ara C.     

Linkage disequilibrium among RFLPs at the insulin-receptor locus despite intervening Alu repeat sequences.

Multiple mutations of the insulin receptor (INSR) gene have been identified in individuals with extreme insulin resistance. These mutations have included recombination events between Alu repeat units in the tyrosine kinase-encoding beta-chain region of the gene. To evaluate the influence of Alu and dinucleotide repetitive sequences on recombination events within the insulin receptor gene, I examined the degree of linkage disequilibrium between RFLP pairs spanning the gene. I established 228 independent haplotypes for seven RFLPs (two each for PstI, RsaI, and SstI and one for MspI and 172 independent haplotypes which included an additional RFLP with BglII) from 19 pedigrees. These RFLPs span > 130 kb of this gene, and my colleagues and I previously demonstrated that multiple Alu sequences separate RFLP pairs. Observed haplotype frequencies deviated significantly from those predicted. Pairwise analysis of RFLP showed high levels of linkage disequilibrium among RFLP in the beta-chain region of the insulin receptor, but not between alpha-chain RFLPs and those of the beta-chain. Disequilibrium was present among beta-chain RFLPs, despite separation by one or more Alu repeat sequences. The very strong linkage disequilibrium which was present in sizable regions of the INSR gene despite the presence of both Alu and microsatellite repeats suggested that these regions do not have a major impact on recombinations at this locus.     

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells

AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.     

The chromosome-breaking activity of the restriction endonuclease Alu I in CHO cells is independent of the S-phase of the cell cycle

The restriction endonuclease Alu I induces chromosomal aberrations in living Chinese hamster ovary (CHO) cells. Multiple fixation times reveal that the chromosome-breaking activity of Alu I is similar to that of ionizing radiation in that it is independent of the S-phase of the cell cycle. These results indicate that DNA double-strand breaks are the ultimate lesions for the production of chromosomal aberrations in all stages of the cell cycle.     

Effect of heat treatment on chromosomal aberrations induced by the alkylating agent trenimon or the restriction endonuclease Alu I in Chinese hamster ovary (CHO) cells

Heat treatment of CHO cells in the G1-phase of the cell cycle leads to chromatid-type aberrations in first posttreatment metaphases. Posttreatment of heat-treated cells with the alkylating agent trenimon leads to a synergistic effect on the production of chromatid-type exchanges. These results indicate that heat induces lesions which like the lesions produced by trenimon give rise to chromatid-type aberrations during the first posttreatment S-phase, and that these lesions can interact with each other to produce chromatid-type exchanges. Treatment of CHO cells in the G1-phase of the cell cycle with the restriction endonuclease Alu I induces chromosomal aberrations. Pretreatment of cells with heat leads to a reduction of Alu I induced chromosome-type aberrations. When cells are allowed to recover after heat treatment for 22 h, the aberration frequencies produced by Alu I are the same as in cells not treated with heat. These findings can be explained by assuming that heat-induced accumulation of accessory proteins in the chromatin protects the DNA from being cut by Alu I, and that the cells recovered from the heat-induced protein accumulation after 22 h.     

The restriction endonuclease Alu I induces chromosomal aberrations in human peripheral lymphocytes in vitro

The restriction endonuclease Alu I (recognition site AG/CT) produces chromosomal aberrations in isolated human peripheral lymphocytes in vitro. The aberrations are of the chromosome-type when the cells are treated in G1 and of the chromatid-type when the cells are treated in late S, early G2. Additional treatment with ammonium sulphate leads to higher aberration frequencies than treatment with Alu I alone.     

Elevation of Alu I-induced frequencies of chromosomal aberrations in Chinese hamster ovary cells by Neurospora crassa endonuclease and by ammonium sulfate

The frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa (EC 3.1.30.1), or with ammonium sulfate in which the Neurospora endonuclease is suspended. These data indicate that Alu I does not produce DNA single-strand breaks in the chromatin of living cells, which can be recognized by the Neurospora endonuclease. The salt may induce conformational changes in the chromatin which make more recognition sites available for Alu I. Experiments with recovery times between the treatments with Alu I and the salt indicate that Alu I can act in the nucleus for at least 40 min.     

Enhanced mutability at the tk locus in the radiosensitive double-strand break repair mutant xrs5

The thymidine kinase locus (tk) has been utilised as the target locus to measure the induced mutation frequency following X-irradiation in the X-ray-sensitive xrs5 mutant and its parent CHO K1 line of Chinese hamster cells. Mutations to tk- cells were measured by plating cells in selective medium containing trifluorothymidine after a post-irradiation expression time of 4 days. Our results show that the mutation frequency was 3-4 times higher in the xrs5 mutant than in the CHO K1 cell line. This enhanced mutation frequency in xrs5 is though to result from the deficiency in DNA double-strand break repair in this cell line which also results in the enhanced cell killing and higher frequencies of chromosomal aberrations in response to X-irradiation. The findings of the present study suggest that DNA double-strand break is a critical lesion leading to mutations in irradiated cells.     

A chromosome study of 6-thioguanine-resistant mutants in T lymphocytes of Hiroshima atomic bomb survivors

Cytogenetic characterizations were made of lymphocyte colonies established from somatic mutation assays for 6-thioguanine (TG) resistance in Hiroshima atomic bomb survivors. G-banded chromosomes were analyzed in both TG-resistant (TGr) and wild-type colonies. Included were 45 TGr and 19 wild-type colonies derived from proximally exposed A-bomb survivors, as well as colonies from distally exposed control individuals who did not receive a significant amount of A-bomb radiation (18 TGr and 9-wild type colonies). Various structural and numerical chromosome abnormalities were observed in both TGr and wild-type colonies. Aberrations of the X chromosome, on which the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus is present, were found in 6 colonies: 2 resistant colonies from controls (45,X/46,XX; 46,X,ins(X)), 3 resistant colonies (45,X/46,XX/46,X, + mar; 46,X,t(Xq +;14q-); 46,Y,t(Xq-;5q +)), and 1 wild-type colony (45,X/47,XXX) from proximally exposed persons. In cases with exchange aberrations, each of the break points on the X chromosome was situated proximally to band q26 where the HPRT locus is known to be assigned. DNA-replicating patterns were also studied, and it was found that abnormal X chromosomes showed early replicating patterns, while normal X chromosomes showed late replicating patterns.     

Evidence for a receptor-mediated endocytosis of Alu I in Chinese hamster ovary cells

Pretreatment of Chinese hamster ovary cells with proteases or with NaN3 leads to less chromosomal aberrations when the cells are posttreated with Alu I compared to the treatment of cells with Alu I alone. The same result is obtained when the cells are treated with Alu I at 0 degree C instead of 37 degrees C. The cells recover from the protease treatment when they are kept in medium before treatment with Alu I. These results are interpreted to mean that Alu I is bound by surface receptors and that the Alu I-receptor complexes are internalized by an energy-dependent endocytotic process.     

Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts

The mutagenic specificities of ethylnitrosourea (ENU), X-rays (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene (BPDE), ICR-191, and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) were analyzed and compared in diploid human fibroblasts and Salmonella typhimurium. In the human fibroblasts, we compared the frequency of diphtheria toxin (DT)-resistant mutants, presumably induced in the gene coding for elongation factor-2, with the frequency of 6-thioguanine (TG) resistance induced by mutations in the gene coding for hypoxanthine(guanine)phosphoribosyltransferase (HPRT). Recovery of DT-resistant (DTr) cells requires that the mutant EF-2 retain the ability to carry on protein synthesis since the normal EF-2 will be inactivated by DT selection. Therefore, the DTr mutation cannot involve major changes in the gene. In contrast, cells can acquire TG resistance by any mechanism which eliminates HPRT activity, e.g., base substitution, frameshift, deletion, loss of chromosomes. Each agent was assessed by calculating the ratio of the slopes of the dose-response plots (induced variant frequency as a function of dose of the agent used) for the two markers (DTr/TGr variants.). In S. typhimurium we examined the reversion frequency in four histidine-requiring strains bearing forward mutations of the frameshift (TA1538, TA98) or missense (TA1535, TA100) type. ENU, which was predominantly a base substitution mutagen in the bacteria, gave a ratio of DTr to TGr variants of 1.5. As expected of an agent inducing gross chromosomal changes, X-rays induced no revertants in bacteria and in human cells gave a ratio of 0.1. ICR-191 which was predominantly a frameshift mutagen in bacteria gave a ratio of 0.15. In the set of bacterial strains containing the plasmid pKM101, BPDE reverted both frameshift and base substitution mutations. It did not cause reversions in the other set of strains. In human cells BPDE gave a response similar to ENU, i.e., a ratio of DTr/TGr variants of 1.5. As reported by others, N-AcO-AAF was predominantly a frameshift mutagen in bacteria. However, in the human cells it gave a ratio of DTr/TGr variants of 1.5, similar to ENU and BPDE. These results suggest that in human cells, BPDE and N-AcO-AAF, like ENU, yield predominantly base substitutions, while ICR-191 and X-rays largely produce mutations by mechanisms which result in more extensive alterations in the gene.     

Induction of mutation in V79 Chinese hamster cells by tetrachlorohydroquinone, a metabolite of pentachlorophenol

Tetrachlorohydroquinone (TCHQ) and tetrachlorocatechol (TCC), two metabolites of the environmental mutagen and carcinogen pentachlorophenol, were tested without exogenous activation in V79 Chinese hamster cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus to 6-thioguanine resistance (TGr) and at the Na/K-ATPase locus to ouabain resistance (OuaR). Treatment was for 24 h at 37 degrees C. TCHQ produced statistically significant increases in the frequency of TGr mutants. The lowest observed effective dose (LOED) was 20 microM, where the relative cloning efficiency was 63%. The relationship between the dose of TCHQ and the frequency of TGr mutants was approximately linear over the range of 0-60 microM with an estimated slope (+/- 95% confidence limits) of 1.1 +/- 0.3 mutants per 10(6) clonable cells per microM. At the highest tested dose of TCHQ, 60 microM, the relative cloning efficiency was reduced to 7%. In contrast to TCHQ, TCC was unable to induce TGr mutants at doses up to 120 microM. The relative cloning efficiency at this dose was 5%. Both TCHQ and TCC were unable to induce OuaR mutants. The results suggest that TCHQ is at least partly responsible for the genotoxic activity of pentachlorophenol. TCHQ can produce reactive oxygen species, which may cause large genetic damage such as deletions, resulting in mutation to TGr but not to OuaR.     

In situ nick translation of human chromosomes using Alu I: unmasking of recognition sites by proteinase K pretreatment

Human chromosomes prepared according to routine methods were treated with the restriction endonuclease Alu I followed by staining with Giemsa solution or fluorescent dyes. This procedure results in a C-band-like appearance of the chromosomes due to removal of DNA from euchromatic chromosomal regions. The resistance of heterochromatic regions against cleavage by the enzyme has mainly been interpreted by the absence or rareness of recognition sites for this particular enzyme in these regions. Proteinase K pretreatment followed by a nick translation procedure with Alu I was combined to check this hypothesis. The results show that heterochromatic chromosomal regions can also be labelled. Thus, they are not characterized by a lack of recognition sites. Gradual deproteinisation of chromosomes changes the labelling pattern from a reverse C-banding pattern to a C-band-like appearance. The resistance of heterochromatic chromosomal parts revealed by the technique is mainly due to local chromatin configuration rather than to the underlying DNA sequence itself.     

DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus

The level of DNA supercoiling is crucial for many cellular processes, including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toc and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.     

Studies of mutagenicity and clastogenicity of 5-azacytidine in human lymphoblasts and Salmonella typhimurium

5-Azacytidine (5-AzaC) induced mutation in the TK^+/− human lymphoblastoid line, TK6, at both the thymidine kinase (tk) locus as measured by resistance to trifluorothymidine (F₃TdR), and the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus, as measured by resistance to 6-thioguanine (6TG). F₃TdR^R and 6TG^R mutant fractions induced by 5-AzaC were observed after a normal phenotypic expression time and remained stable. Interestingly, 5-AzaC was 5–10 times more mutagenic at the tk locus than the hgprt locus. However, F₃TdR^R colonies from 5-AzaC-treated cultures behaved like TK-deficient mutants induced by other chemical mutagens.

The TK or HGPRT phenotype had no effect on the toxicity of 5-AzaC, thus eliminating differential toxicity as a potential cause for the observed higher mutability at the tk locus. 5-AzaC did not induce F₃TdR^R cells in the parental TK^+/+ lymphoblastoid line, indicating that 5-AzaC-induced F₃TdR^R variants were not due to a dominant alteration in gene expression. 5-AzaC did not induce chromosomal aberrations in TK6 cells, eliminating clastogenic events as a potential cause for the higher mutability at the tk locus.

5-AzaC was also found to be mutagenic in a forward mutation assay to 8-azaguanine resistance in Salmonella typhimurium.     

Genetics of actinomycin C production in Streptomyces chrysomallus

Three distinct classes of mutations affecting the biosynthesis of actinomycin have been established in Streptomyces chyrsomallus by crossing various actinomycin-nonproducing mutants with each other by protoplast fusion. In crosses between members of different classes of mutations, actinomycin-producing recombinant progeny arose, whereas in crosses between members of the same class, no actinomycin-producing recombinants were seen. Biochemical examination of a number of mutants revealed that the expression of all actinomycin synthetases was reduced by about 1 order of magnitude in mutants belonging to class II. In mutants of class I, the specific activities of the actinomycin synthetases were comparable with those measured in their actinomycin-producing parents. Feeding experiments with 4-methyl-3-hydroxyanthranilic acid (4-MHA), the biosynthetic precursor of the chromophore moiety of actinomycin, with representative mutants of the three genetic classes revealed formation of actinomycin in minute amounts by mutants of class I. It is suggested that mutants belonging to class I are mutated at a genetic locus involved in the biosynthesis of 4-MHA. Mutants belonging to class II appear to carry mutations at a locus involved in the regulation of the expression of the actinomycin synthetases. The role of the locus in class III mutations could not be assigned. Mapping studies in S. chrysomallus based on conjugal matings revealed the chromosomal linkage of all three loci. Mutations belonging to classes I and III were closely linked. Their genetic loci could be localized in a map interval of the chromosomal linkage group which is significantly distant from the gene locus represented by mutations belonging to class II.     

The induction of chromosome aberrations by restriction endonucleases that produce blunt-end or cohesive-end double-strand breaks

Restriction endonucleases have been used to study the involvement of specific types of DNA damages in the production of chromosome aberrations. In this study restriction endonucleases were introduced into viable CHO cells using osmolytic shock of pinocytic vesicles. We compared two cohesive-end cutters, Msp I (CCGG-2-base overlap) and Sau3A I (GATC-4-base overlap) with two blunt-end cutters, Alu I (AGCT) and Rsa I (GTAC). All 4 enzymes were effective at inducing aberrations. The 4-base overlap cohesive-end cutter Sau3A I was approximately as effective as the blunt-end cutter Alu I. We present evidence that cutting frequency rather than cut end-structure is important in determining efficiency of aberration induction. There is over-dispersion of the distribution of dicentrics and rings among cells, and the data could be fitted to a Neyman Type A distribution, a modified Poisson, that indicates that there is a probability distribution both for the entry of the enzyme into a cell nucleus and for the induction of aberrations once the enzyme has entered a cell nucleus. In addition, we used Alu I to determine the sensitivity of cells to aberration induction in the different stages of the cell cycle. Alu I induced aberrations in all stages of the cycle, chromatid-type in S/G2 and chromosome-type in G1. In agreement with data of others, there were variations in sensitivity with cycle stage, and changes in the proportions of the different aberration classes for chromatid-type aberrations.     

Identification of a new locus,mus115, in Drosophila melanogaster

Previous screens for autosomal genes that are necessary to resistance to DNA cross-linking agents but not to monofunctional agents have produced 6 mutations; all of which fall within the third chromosomal gene mus308. In an effort to identify analogous sex-linked genes, a screen of mutagenized X-chromosomes has been conducted for mutations that confer hypersensitivity to nitrogen mustard. This search has identified a new locus, mus115, through the recovery of a mutant that is strongly hypersensitive to nitrogen mustard but marginally sensitive to methyl methanesulfonate.