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.     

Detection of deletion mutations in pSV2gpt-transformed cells.

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.     

Quantitative and molecular analyses of radiation-induced mutation in AS52 cells

pSV2gpt-Transformed and wild-type Chinese hamster ovary (CHO) cell lines have been used to study radiation-induced mutation at the molecular level. The transformant, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line and contains a single, functional copy of the Escherichia coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. AS52 and wild-type CHO-K1-BH4 cells exhibit similar cytotoxic responses to uv light and X rays; however, significant differences occur in mutation induction at the gpt and hprt loci. A number of HPRT and XPRT mutants which arose following irradiation were analyzed by Southern-blot hybridization. Most XPRT (21/26) and all HPRT (23/23) mutants induced by uv light exhibited hybridization patterns indistinguishable from their parental cell lines. In contrast, all XPRT (26/26) and most HPRT mutants (15/21) induced by X irradiation contained deletion mutations affecting some or all of the gpt and hprt loci, respectively. These results indicate that X rays induce predominantly deletion mutations, while uv light is likely to induce point mutations at both loci.     

Quantitative and molecular analyses of ethyl methanesulfonate- and ICR 191-induced mutation in AS52 cells

A pSV2gpt-transformed Chinese hamster ovary (CHO) cell line has been used to study mutation at the molecular level. This cell line, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line, and has been previously shown to contain a single, functional copy of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. In this study, conditions for its use in the study of mammalian cell mutagenesis have been stringently defined. The spontaneous mutation rate (2 X 10(-6)/cell division) and phenotypic expression time (7 days) of the gpt locus compare favorably with those of the hprt locus in wild-type CHO-K1-BH4 cells. While both cell lines exhibit similar cytotoxic responses to ethyl methanesulfonate (EMSO and ICR 191, significant differences in mutation induction were observed. Ratios of XPRT to HPRT mutants induced per unit dose of EMS and ICR 191 are 0.70 and 1.6, respectively. Southern blot hybridization analyses revealed that most XPRT mutant cell lines which arose following treatment with EMS (20/22) or ICR 191 (20/24) exhibited no alterations of the gpt locus detectable by this technique. Similar observations were made for the hprt locus in EMS-(21/21) and ICR 191-induced (22/22) HPRT mutants. In contrast, most spontaneous gpt mutants (14/23) contained deletions, while most spontaneous hprt mutants (18/23) exhibited no detectable alterations. Results of this study indicate that the AS52 cell line promises to be useful for future study of mutation in mammalian cells at the DNA sequence level.     

Analyses of mutation in pSV2gpt-transformed CHO cells

We have developed a system to study mutations which affect expression of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) in hypoxanthine-guanine phosphoribosyl transferase-deficient (HPRT-) Chinese hamster ovary (CHO) cells that have been transformed by the plasmid pSV2gpt. Several gpt-transformed cell lines have been isolated and characterized with respect to integrated pSV2gpt sequences, expression of the gpt gene, and cytotoxic and mutagenic responses to UV light. While the gpt-transformed CHO and wild-type CHO-K1-BH4 cell lines have similar cytotoxic responses to UV light, the gpt-transformed cell lines respond differently from the parental CHO-K1-BH4 cell line in terms of mutation induction. As with CHO-K1-BH4 HPRT mutants, spontaneous or induced XPRT mutants derived from the gpt+ cell lines can be selected for 6-thioguanine resistance (TGr). Analysis of cell-free extracts from a number of these TGr clones indicates that the mutant phenotype is due to the absence of XPRT activity. One transformant, designated AS52, has previously been described in limited detail. Here we describe additional characteristics of this cell line, as well as several related transformants.     

Gene recombination in X-ray-sensitive hamster cells.

Recombination was measured in Chinese hamster ovary (CHO-K1) cells and in the X-ray-sensitive mutants xrs1 and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the different cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-K1 and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-K1, but variation in the data was considerable. A much larger difference between CHO-K1 and the xrs mutants was found when the DNA concentration dependence of transformation was explored. While the transformation frequency of CHO-K1 increased linearly with DNA concentration, no such increase occurred with the xrs mutants irrespective of whether complete plasmids or pairs of deletion plasmids were transferred. The fraction of cells taking up DNA, assayed autoradiographically, was similar in all cell types. Therefore we suggest that while homologous recombination of plasmid molecules may not be substantially reduced in the xrs mutants,processes involved in the stable integration of plasmid DNA into genomic DNA are significantly impaired.     

Suppressive effects of selected antioxidants on the activated leukocytes-induced mutagenesis in the co-culture assay systems

We recently established a novel co-culture assay system using activated inflammatory cells and AS52 Chinese hamster ovary cells, and demonstrated that reactive oxygen and nitrogen species (RONS) generated from activated inflammatory leukocytes induce mutations in the gpt recorder gene in AS52 cells. In this study, we examined the inhibitory effects of 19 agents with antioxidative properties on RONS generation in cultured inflammatory cells and on mutagenesis in AS52 cells co-cultured with activated inflammatory cells. The results demonstrate that there is a linear correlation between the ability of these agents to suppress RONS production in activated inflammatory cells and to inhibit mutation in AS52 cells.     

Lead and mercury mutagenesis: Type of mutation dependent upon metal concentration

Lead and mercury are toxic metals that are widely distributed in the atmosphere, soil, and groundwater. It is estimated that 2–4 × 10⁴ tons of these metals are released annually into the environment by natural and industrial processes. Therefore, human exposure to low relatively nontoxic concentrations of these metals is unavoidable. However, the possible health effects of such exposure remain controversial. We have previously reported that low, subthreshold concentrations (0.1–1 μM) of these metals are mutagenic in the transgenic Chinese hamster ovary cell line AS52. The purpose of the present study is to determine the types of mutations induced in the gpt gene in AS52 cells. Using multiplex polymerase chain reaction and southern blot analyses, we characterized the 138 lead‐induced, 192 mercury‐induced, 29 reactive oxygen radical‐induced, and 20 spontaneously arising mutants for point and deletion mutations in the gpt gene. Similar levels of point mutations were observed in the lead‐ and mercury‐induced populations (47.8 and 53.6, respectively), which was significantly less than that occurring in the spontaneously arising and reactive oxygen intermediate‐induced mutants. However, further examination of the data revealed that at concentrations of the metals of equal to or less than 0.4 μM, the majority of the mutations in the gpt gene were point mutations, while at higher concentrations, deletions (partial and complete) were the predominant type of mutation. These results are consistent with the hypothesis that lead and mercury induce mutations in eukaryotic cells by at least two distinct mechanisms. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 107–112, 1999     

Construction of an EBV-derived shuttle vector for studying the influence of transcription on mutagenesis

We have constructed an EBV-derived shuttle vector, pF1-EBV, which replicates in human cells as an extrachromosomal element. The structural sequences of the gene encoding the bacterial xanthine-guanine-phosphoribosyltransferase (gpt) were fused to the promoter and presumptive control region of the mouse metallothionein I (MT-I) gene. Human 293 cells transformed with the recombinant plasmid synthesized gpt mRNA and the expression of the gene was inducible by zinc. The gpt gene offers a convenient system of selection for mutant plasmids by transformation into the appropriate gpt- E. coli strain. A clonal cell line created by establishment of the pF1-EBV shuttle vector showed a spontaneous gpt- frequency of 2.10(-5). An increase in mutation frequency above background was induced by mutagenizing this cell line with the alkylating agent N-methyl-N-nitrosourea (MNU). The recombinant molecule that we have constructed should provide a tool for studying the role of gene expression in DNA repair and mutagenesis.     

High spontaneous mutation frequency in shuttle vector sequences recovered from mammalian cellular DNA.

The recombinant shuttle vector pSV2gpt was introduced into V79 Chinese hamster cells, and stable transformants expressing the Escherichia coli gpt gene were selected. Two transformants carrying tandem duplications of the plasmid at a single site were identified and fused to simian COS-1 cells. Plasmid DNA recovered from the heterokaryons was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. The high frequency of Gpt- plasmids (ca. 1%) was similar to that observed when plasmid was recovered from COS-1 cells which had been transfected with pSV2gpt. Most of the mutant plasmids had rearrangements in the region containing the gpt gene.     

Ethylnitrosourea-induced mutation and molecular analysis of transgenic mice containing the gpt shuttle vector

Novel transgenic mice were developed in order to study the in vivo mutagenesis. The transgenic mice carried pCGK shuttle vector, which contained the Escherichia coli gpt gene as a mutational target, the kanamycin-resistant gene (Kanr) and cos region derived from bacteriophage lambda. The shuttle vector can be recovered from the transgenic mouse genome into the gpt-deficient E. coli by an in vitro packaging method and is selectable as a Kanr phenotype. Mutations induced at the gpt gene can be easily detected with a selective agent, 6-thioguanine (6-TG). In the previous study, the pCGK shuttle vector was incorporated into Chinese hamster CHL/IU cells and the resultant transgenic cell line was shown to be a useful system to study in vitro mutagenesis at the gpt gene. Therefore, an advantage of the shuttle vector is that in vivo mutational data obtained from the transgenic mouse can be compared with those of transgenic cell line in vitro. A transgenic CD-1 mouse line, designated as #128, that carried approximately 50 copies of pCGK shuttle vectors, was selected among 4 transgenic mouse lines. To investigate the sensitivity of the #128 line, the transgenic mice were treated with a single intraperitoneal injection of 250 mg/kg of N-ethyl-N-nitrosourea (ENU) or with 50 mg kg-1 day-1 of ENU for 5 consecutive days, and bone marrow, spleen and liver were dissected to investigate their mutational responses. The background mutant frequency was between 18x10(-6) and 75x10(-6) among all tissues tested. ENU induced significant increases in the mutant frequency above the background level in all three tissues at 14 days after single or 5-day treatment with the chemical. The increases in the mutant frequencies in bone marrow, spleen and liver were 6.4- to 6.8-fold, 3.0- to 5.6-fold and 3.0- to 3.3-fold, respectively. The shuttle vector DNA was recovered from the bone marrow of both spontaneous and ENU-treated mice and the gpt gene was amplified by polymerase chain reaction. The amplified DNA was subject to DNA sequence analysis. Out of 79 spontaneous and 52 ENU-induced mutants, the gpt gene could be amplified from 28 spontaneous and 46 ENU-induced mutants. DNA sequence analysis showed that predominant mutations were identified as A:T to T:A transversions (22 out of 46 sequenced mutants) and G:C to A:T transitions (9/46) in ENU-induced mutants, whereas G:C to T:A transversions (7 out of 28 sequenced mutants) were predominant in spontaneous mutants. These results demonstrate that this transgenic mouse, in combination with the transgenic CHL/IU cell line, is a useful system to study in vivo and in vitro mutational events at the same target gene.     

Studying DNA mutations in human cells with the use of an integrated HSV thymidine kinase target gene

A shuttle vector carrying the origin of SV40 replication, the thymidine kinase (tk) gene of herpes simplex virus and the E. coli xanthine guanine phosphoribosyl transferase (gpt) gene has been introduced into human TK- cells. A transformed cell line containing only one stably integrated copy of the shuttle vector was used to study mutations in the introduced tk gene at the molecular level. Without selection for gpt expression, spontaneous TK- mutants arose at a frequency of approximately 10(-4)/generation, and were caused by deletion of plasmid sequences. However, when selection for expression of the gpt gene was applied, the background level of mutations at the tk gene was below 4.10(-6). From this cell line, TK- mutants were obtained after treatment with N-ethyl-N-nitrosourea (ENU). COS fusion appeared to be an efficient method for rescue and amplification of the integrated shuttle vector from the human chromosome. After further amplification and analysis in E. coli, rescued tk genes were easily identified and were shown to be physically unaltered by the rescue procedure. In contrast to rescued tk genes from TK+ cells, those obtained from the ENU-induced TK- mutants were unable to complement thymidine kinase-negative E. coli cells. Two such tk mutations were mapped in E. coli by marker rescue analysis. A GC----AT transition was the cause of both mutations. We show here that plasmid rescue by COS fusion is a reliable system for studying gene mutations in human cells, since no sequence changes occurred in rescued DNA except for the 2 ENU-induced sequence changes.     

Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimutagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1(-)) HCT116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1-3 weeks) treatment of HCT116 cells with VAN at minimally toxic concentrations (0.5-2.5mM) reduced the spontaneous HPRT mutant fraction (MF, mutants/10(6) survivors) in a concentration-related manner by 19-73%. A similar treatment with CIN at 2.5-7.5microM yielded a 13-56% reduction of the spontaneous MF. Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimutagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116+chr3) and deficient (HCT116) cells at concentrations that were antimutagenic in HCT116 cells. There were 64 genes whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for four selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN are antimutagenic against spontaneous mutations in mammalian (human) cells. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair, which reduces spontaneous mutations.     

Molecular characterization of methyl methanesulphonate (MMS)-induced HPRT mutations in V79 cells

Spontaneous and methyl methanesulphonate-induced HPRT-deficient mutants were analysed for changes in the hprt gene structure using multiplex polymerase chain reaction. The PCR amplification pattern of 21 MMS-induced mutations revealed one total deletion of the hprt coding exons and one small deletion within exon 5, while 19 mutants showed the V79 wild-type pattern. Molecular analysis of 30 spontaneous mutations revealed no mutants with amplification patterns which differed from those of wild-type cells. We further analysed MMS-induced mutants in a different V79 cell line with a high (40%) spontaneous deletion frequency. MMS caused a dose-dependent increase in the mutant frequency but the incidence of deletions was reduced to 6% at 2 × 10⁻⁴ M and to 13% at 5 × 10⁻⁴ M indicating that mainly point mutations were induced. The repair inhibitor cytosine arabinoside (araC) enhanced mutation induction by MMS but did not change the proportion of deletions in the mutation spectrum. The results indicate that different V79 cell lines spontaneously produce different amounts of deletion mutations. The frequency of MMS-induced deletions does not depend on the frequency of spontaneous deletions in a given cell line. The MMS-induced mutation spectrum seems to be unchanged even at high concentrations with a strong cytotoxic effect. Deletions are not increased as a consequence of araC-inhibited repair of MMS-inducd lesions.     

Chromium(III) tris(picolinate) is mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase locus in Chinese hamster ovary cells.

Chromium trispicolinate (CrPic) is a popular dietary supplement that is not regulated by the Food and Drug Administration. We are using this compound as a bio-available model to explore the role of Cr(III) in Cr(VI)-induced cancers. The ability of CrPic to cause mutations at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus of CHO AA8 cells has been measured after a 48 h exposure. The highest dose tested was 80 microg/cm(2) CrPic, which, if fully soluble, would be equivalent to 1mM or 0.44 mg/ml CrPic, and would correspond to 1mM Cr(III) or 52 microg/ml Cr(III). This exposure resulted in 68+/-16% cell survival based on 48 h cell counts, and 24+/-11% survival by 7-day colony formation. Exposure of CHO cells to CrPic produced a statistically significant increase in 6-thioguanine (6-TG)-resistant cells over the dose range tested. The 80 microg/cm(2) CrPic exposure resulted in an average induced mutation frequency (MF) of 58 per 10(6) surviving cells, or an average 40-fold increase in hprt mutants relative to untreated cells. An equivalent dose of 3mM Pic was highly cytotoxic and did not yield hprt mutants. The dose range of 0.375-1.5mM Pic produced a slight increase in hprt mutants, but the increase was not statistically significant. An equivalent dose of 1mM chromic chloride yielded an induced MF of 9 per 10(6) surviving cells, or a 10-fold increase in mutants with cell survivals of >100%. The coordination of Cr(III) with picolinic acid may make the metal more genotoxic than other forms of Cr(III). In light of the current results and the known ability of Cr(III) and CrPic to accumulate in tissues, as well as the growing evidence of Cr(III) involvement in Cr(VI)-induced cancers, we caution against ingestion of large doses of CrPic for extended periods.     

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.     

Effects of an inhibitor and a mimic of superoxide dismutase on bleomycin mutagenesis in Chinese hamster ovary cells

We have investigated the roles of reactive oxygen species (ROS) in bleomycin (BLM)-induced gene mutations in Chinese hamster ovary (CHO) cells using a superoxide dismutase (SOD) inhibitor, triethylenetetramine (TRIEN), and a SOD mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), to lower and increase intracellular “SOD activity”, respectively. Pretreatment of CHO cells with TRIEN (1 mM) for 1 h enhanced the mutagenic response of BLM (5–50 μg/ml, 1 h treatment) in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in CHO cell clone K1-BH4 (CHO/HPRT assay) and the xanthine-guanine phosphoribosyltransferase (gpt) gene in a CHO-K1 cell derivative AS52 (AS52/GPT assay). Pretreatment with TEMPOL (1 mM) for 1 h decreased the BLM (20–100 μg/ml, 1 h treatment) mutagenicity in the AS52/GPT assay. The mutagenic response of BLM appears to be modulated by the intracellular level of ‘SOD activity’ and hence the intracellular level of ROS. These data provide further evidence for the involvement of ROS in bleomycin mutagenesis in mammalian cells.     

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.     

The histone deacetylase inhibitor trichostatin A reduces nickel-induced gene silencing in yeast and mammalian cells

We have previously reported that nickel (Ni)-silenced expression of the URA3 gene in yeast (Saccharomyces cerevisiae) and gpt transgene in G12 Chinese hamster cells. In both cases, close proximity to a heterochromatic region was required for gene silencing. Yeast exposed to Ni exhibited reduced acetylation of the lysine residues in the N-terminal tail of histone H4. Ni-induced silencing of the gpt gene in mammalian cells involved hypermethylation of promoter region DNA. Yeast do not employ DNA methylation to silence gene expression. To determine if histone deacetylation participates in Ni-induced silencing of the URA3 and gpt genes, we exposed yeast and G12 hamster cells to the histone deacetylase inhibitor trichostatin A (TSA) prior to and concurrently with Ni. Treatment of yeast cells with 0.2-0.6mM NiCl(2) resulted in reduced expression of the URA3 gene as assessed by increased resistance to 1g/l 5-fluorotic acid (5-FOA). This effect was lessened when yeast were pre-treated with 50 microg TSA/ml. Similarly, treatment of G12 cells with 5 ng/ml TSA during and after exposure to 0.3 microg Ni(3)S(2)/cm(2) reduced silencing of the gpt gene as gauged by resistance to 10 microg/ml 6-thioguanine (6-TG). The ability of TSA alone and in combination with the DNA-demethylating agent (5-AzaC) to reactivate the gpt gene in Ni-silenced variants was also assessed. Although treatment with 100 ng/ml TSA for 48 h was partially effective in reactivating the gpt gene, treatment with 5 microM 5-AzaC was more efficacious. The greatest gpt gene reversion frequencies were observed following a sequential 5-AzaC/TSA treatment. Taken all together, our data from mammalian cells suggests that both DNA methylation and histone deacetylation participate in Ni-induced silencing of the gpt gene with DNA hypermethylation playing the more dominant role in maintaining the silenced state.     

Escherichia coli mutants deficient in guanine-xanthine phosphoribosyltransferase.

We studied the purine phosphoribosyltransferases (PRTases) of Escherichia coli and were able to isolate a mutant that is defective in its ability to convert guanine and xanthine to their respective ribonucleotides. The affected gene (gpt) lies between metD and proA and is 78.6% co-transducible with proA. Both this point mutant and a strain with a pro-lac deletion contain less than 2% of wild-type xanthine PRTase activity, yet still contain about 30% of wild-type guanine PRTase activity. Thus, the gpt gene is only one of at least two genes responsible for guanine PRTase activity in E. coli.