Temperature-Sensitive Mutants of a Chinese Hamster Cell Line. I. Selection of Clones with Defective Macromolecular Biosynthesis

Temperature-sensitive clones have been selected from a mutagenized culture of Chinese hamster lung cells by a procedure involving bromodeoxyuridine (BrdU) incorporation and irradiation with black light. The selection procedure used in these studies was adapted from methods developed by others to yield mutants that cease DNA replication within a short time after they are transferred to nonpermissive temperature. After mutagenesis with ethyl methanosulfonate ten clones survived the selection procedure. Three of the clones (mutants) were temperature-sensitive as measured by growth properties. Two mutants ceased DNA synthesis within six hours of being shifted to 39degrees and the third mutant continued to synthesize DNA at nonpermissive temperature at a reduced rate for at least 24 hours. Thus, all three mutants survived the selection procedure for understandable reasons, since each was unable to incorporate sufficient BrdU at 39degrees to lethally protosensitize its DNA during the standard exposure period. The two mutants that cease DNA synthesis at high temperature (clones 115-47 and 115-53) also stop incorporating radioactive amino acids and uridine within six hours at 39degrees. Their complex phenotype, i.e. defective DNA, RNA and protein biosynthesis, is reversible. When these mutants were returned to 33 degrees after 8 hours at 39 degrees, both resumed DNA synthesis immediately (less than 1 hour). Reversal of defective DNA synthesis in both mutants were sensitive to drugs that inhibit protein biosynthesis specifically. Those same drugs, as well as toxic amino acids analogs, also effected a striking mutant phenocopy in wild-type cells. The phenocopy produced by amino acid analogs that are incorporated into mammalian proteins suggested that one or more proteins must be synthesized continuously to support mammalian cells engaged in programmed DNA replication.     

Replication and repair of UV-induced mutational lesions in chemostat cultures of Escherichia coli WP2 Hcr

Mutation to resistance to bacteriophage T5 was studied in chemostat cultures of Escherichia coli strain WP2 Hcr exposed to ultraviolet radiation (UV). The results are in generally good agreement with those obtained earlier by Bridges and Munson for UV-induced reversion to tryptophan independence in exponentially growing cultures of the same strain: expressed mutant yields followed a dose-squared response, mutations were not expressed before approximately one generation after exposure to UV, there was a slow disappearance of dimers especially noticeable in slowly growing and stationary cultures, and the first replication gave rise to duplex mutants in both strands. Several new results were also obtained. In addition to expressed mutant yields, induction of mutational capacity was also observed to follow a dose-squared response, indcating that the response is not an artifact of selection or repair. Induction also increased with growth rate, apparently as the square of the number of genes for T5-sensitivity per cell. It is suggested that mutagenesis is proportional to the number of genes per cell, that recombination is also proportional to the number of genes per cell, and that the number of mutational lesions is proportional to the product of the two. These results also provide evidence that DNA replication occurs near the end of the cell cycle in slowly growing cultures. Under all growth conditions, latent mutant concentrations mutational capacity) decreased by a factor of two with each successive division. Latent mutants were, however, photoreversible for only the first two generations. If mutagenesis occurs as a recombinant event between two mutational lesions, then the results also indicate that these lesions are separated, on the average, by no more than a single cistron.     

Evidence that accumulation of mutants in a biofilm reflects natural selection rather than stress-induced adaptive mutation

The accumulation of mutant genotypes within a biofilm evokes the controversy over whether the biofilm environment induces adaptive mutation or whether the accumulation can be explained by natural selection. A comparison of the virulence of two strains of the dental pathogen Streptococcus mutans showed that rats infected with one of the strains accumulated a high proportion (average, 22%) of organisms that had undergone a deletion between two contiguous and highly homologous genes. To determine if the accumulation of deletion mutants was due to selection or to an increased mutation rate, accumulations of deletion mutants within in vitro planktonic and biofilm cultures and within rats inoculated with various proportions of deletion organisms were quantified. We report here that natural selection was the primary force behind the accumulation of the deletion mutants.     

Evidence that most radiation-induced HPRT mutants are generated directly by the initial radiation exposure.

Radiation-induced HPRT mutants are generally assumed to arise directly from DNA damage that is misrepaired within a few hours after X-irradiation. However, there is the possibility that mutations result indirectly from radiation-induced genomic instability that may occur several days after the initial radiation exposure. The protocols that commonly employ a 5-7 day expression period to allow for expression of the mutant phenotype prior to replating for selection of mutants would not be able to discriminate between mutants that occurred initially and those that arose during or after the expression period. To address this question, we performed a fluctuation analysis in which synchronous or asynchronous populations of human bladder carcinoma cells were treated with single doses of X-irradiation. For comparison, radiation was delivered during the expression period, either from an initial dose of 1.0 Gy followed by two 1.0 Gy doses separated by 24 h or from disintegrations resulting from I125dU incorporated into DNA. The mutation frequency observed at the time of replating was used to calculate the average number of mutants in the initial irradiated culture by assuming that the mutants were induced directly at the time of irradiation. Then, this average number was used to calculate the fraction of the irradiated cultures that would be predicted by a Poisson distribution to have zero mutants. There was reasonably good agreement between the predicted poisson distribution and the observed distribution for the cultures that received single doses. Moreover, as expected, when cultures were irradiated during the expression period, the fraction of the cultures having zero mutants was significantly less than that predicted by a Poisson distribution. These results indicate that most radiation-induced HPRT mutations are induced directly by the initial DNA damage, and are not the result of radiation-induced instability during the 5-7 day expression period.     

Evidence that Accumulation of Mutants in a Biofilm Reflects Natural Selection Rather than Stress-Induced Adaptive Mutation

The accumulation of mutant genotypes within a biofilm evokes the controversy over whether the biofilm environment induces adaptive mutation or whether the accumulation can be explained by natural selection. A comparison of the virulence of two strains of the dental pathogen Streptococcus mutans showed that rats infected with one of the strains accumulated a high proportion (average, 22%) of organisms that had undergone a deletion between two contiguous and highly homologous genes. To determine if the accumulation of deletion mutants was due to selection or to an increased mutation rate, accumulations of deletion mutants within in vitro planktonic and biofilm cultures and within rats inoculated with various proportions of deletion organisms were quantified. We report here that natural selection was the primary force behind the accumulation of the deletion mutants.     

Probing the function of individual amino acid residues in the DNA binding site of the EcoRI restriction endonuclease by analysing the toxicity of genetically engineered mutants

We have developed an assay that allows analysis of the activity of EcoRI restriction endonuclease (ENase) and its mutants in vivo. This assay is based on the fact that wild type (wt) EcoRI ENase is toxic for Escherichia coli cells not expressing the EcoRI methyltransferase (MTase). The viability factor defined by the ratio of the viable counts of E. coli cultures having or not having expressed the ecoRIR gene for a defined time is 10(-6) for wt EcoRI ENase and close to one for a totally inactive EcoRI ENase mutant. While the EcoRI MTase (M.EcoRI) provides substantial protection against the toxic effects of the wt EcoRI ENase and several of the mutants, some mutants become more toxic in the presence of M.EcoRI. Twenty-four different DNA-binding-site mutants of EcoRI ENase were characterized in their activity in vivo with this assay. The results obtained allow us to conclude that the structural integrity of the region at and around aa 200 seems to be very critical for the enzymatic function of EcoRI ENase: nonconservative replacements there lead to viability factors of 1-10(-2). While our results indicate that the region around aa 144 and 145 is also involved in the EcoRI ENase-catalyzed reaction, it is also evident that the effects of mutation there are not as large: viability factors of approx. 10(-3) are obtained even for drastic replacements. These results are discussed in the light of the x-ray structure analysis of an EcoRI ENase-DNA recognition complex.     

A mutational assay system for L5178Y mouse lymphoma cells, using hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) -deficiency as marker. The occurrence of a long expression time for mutations induced by X-rays and EMS.

The development of a system for the detection of somatic cell mutation to hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT) (EC 2.4.2.8) deficiency in L5178Y mouse lymphoma cells is described. The selection of mutant cells was not influenced by the concentration of the selective agent 6-thioguanine (6-TG). In addition, all the mutants selected, spontaneous as well as induced ones, showed a complete loss of HGPRT activity. In reconstruction experiments, in which mutant cells were mixed with wild-type cells, the recovery of mutant cells was only markedly influenced when wild-type cells were seeded in a cell density ten times higher than the one, 5-10(4) cells/ml, used in subsequent induction experiments. X-irradiation and treatment with ethyl methanesulfonate (EMS) increased in the mutation rate above the spontaneous background. A clear-cut dose-dependent mutagenic effect after exposure to X-rays was measured. The rate of induced mutations at the HGPRT locus in lymphoma cells was 1-3-10(-7) per R, as determined after exposures of 200, 300, 400, 500 and 600 R. The time the cells needed to express their mutations was much longer than 48 h. Further study of this phenomenon showed that the optimal expression time for TGr-resistant mutants in L5178Y cells was 6 to 7 days. No indication for a dose-dependent effect on the optimal expression of the mutants was found.     

Simplification of the CHO/HGPRT mutation assay through the growth of Chinese hamster ovary cells as unattached cultures

A novel technique for the growth of Chinese hamster ovary (CHO) cells as unattached cells on nontissue culture plates was applied to the CHO/HGPRT mutation assay, using EMS and MNNG as mutagens. The subculturing procedures for the unattached cultures involved less time and effort than those for the conventional attached cultures since trypsinization was not required for cell detachment. No significant difference in the maximum mutation frequency was observed for cells grown as unattached or attached cultures during the expression period. The optimum expression time was, however, shorter for the unattached cells (6 days) than for the attached cells (9 days). No selection for or against the mutant population was observed when mutant and wild-type cells were co-cultivated as unattached cultures, indicating that the procedure does not affect the quantitativeness of the mutation assay. The growth of CHO cells as unattached cells during the expression period thus could decrease the cost and effort involved in the use of the CHO/HGPRT mutation assay in the screening of potential mutagens/carcinogens.     

Mammalian mitochondrial mutants selected for resistance to the cytochrome b inhibitors HQNO or myxothiazol

Mouse LA9 cell lines were selected for increased resistance to either HQNO or myxothiazol, inhibitors of electron transport which bind to the mitochondrial cytochrome b protein. Two phenotypically distinguishable HQNO-resistant mutants were recovered while the myxothiazol-resistant isolates had a common phenotype. All three mutant phenotypes were transmitted cytoplasmically in cybrid crosses. Biochemical studies further established that for all three mutant types, resistance at the cellular level was paralleled by an increase in inhibitor resistance of mitochondrial succinate-cytochrome c oxidoreductase, the respiratory complex containing cytochrome b. As with the previously described mitochondrial antimycin-resistant mutant, the initial biochemical and genetic studies indicated that these mutations occur within the mitochondrial cytochrome b gene. This conclusion was strongly supported by the results of mtDNA restriction fragment analyses in which it was found that one HQNO-resistant mutant had undergone a small insertion or duplication in the apocytochrome b gene. Finally, all four mitochondrial cytochrome b mutants have been analyzed in both cell plating studies and succinate-cytochrome c oxidoreductase assays to determine the pattern of cross-resistance to inhibitors of cytochrome b other than the one used for selection.     

Mutation to ouabain-resistance in Chinese hamster cells: induction by ethyl methanesulphonate and lack of induction by ionising radiation

The spontaneous frequency of mutants resistant to growth inhibition by ouabain (OUAR mutants) was found to be about 5:10(-5) per viable cell in uncloned cultures of Chinese hamster V79-4 cells. In freshly-isolated clones or cultures started from a few cells this frequency was initially reduced to about 1.10(-6) in 1 mM ouabain. No increase in the frequency of OUAR mutants was found in cultures treated with gamma-rays despite exploration of such variables as radiation dose, ouabain concentration, post-treatment interval before selection, cell density in selective medium, and clonal state of the cells at the time of adding ouabain (in situ vs. respreading method). A similar negative result was found for accelerated helium ions, for which the mutagenic effectiveness per unit dose has been shown to be about 10 times higher than gamma-rays for the induction of thioguanine-resistant mutants in these cells. Some evidence was found for an interaction between cellular radiation damage and ouabain-resistance, which may lead to a reduction in the survival of OUAR mutants in irradiated populations, but this damage seemed insufficient to account for inability to detect radiation-induced OUAR mutants. Reproducibly large increases in the frequency of OUAR mutants were found in cultures treated with various concentrations of ethyl methanesulphonate (EMS) by respreading cells in 1 mM ouabain for up to 8 days after EMS treatment. The concentration-OUAR mutant induction curve was approximately linear with low EMS concentrations. Recent evidence is reviewed in support of the suggestion, made in earlier studies, that ionising radiation is unable to induce OUAR mutants because of the severity of the genetic damage it causes.     

The cytotoxic and mutagenic effects of 5-bromodeoxyuridine-plus-black-light treatment in cultured mammalian cells

The cytotoxic and mutagenic effects of the incorporation of 5-bromodeoxyuridine (BrdU)_followed by exposure to black light were investigated with Chinese hamster ovary (CHO) cells in cell culture. Mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hgprt) locus was determined by selection for 6-thioguanine resistant (TG^r) mutants (CHO/HGPRT system). BrdU alone has been shown to be mutagenic only at concentrations of 50 μM or greater. This study was performed in an effort to determine whether BrdU is actually incorporated into the hgprt gene when lower, nonmutagenic concentrations are employed. Neither BrdU (1–20 μM) nor exposure to black light alone was mutagenic, but the combined treatment did result in the induction of TG^r mutants. The mutant frequency increased with increasing light exposure at constant BrdU and with inreasing BrdU at constant light exposure. These results show that BrdU is incorporated into the hgprt gene, but that this does not result in mutation induction in the absence of light exposure. Such a BrdU-plus-light procedure might be applied to studies of DNA repair at this locus, since mutation induction requires both BrdU incorporation and subsequent exposure to black light.     

Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities.

Pseudomonas aeruginosa is considered a strict aerobe that possesses several enzymes important in the disposal of toxic oxygen reduction products including iron- and manganese-cofactored superoxide dismutase and catalase. At present, the nature of the regulation of these enzymes in P. aeruginosa Is not understood. To address these issues, we used two mutants called A4 and C6 which express altered Fur (named for ferric uptake regulation) proteins and constitutively produce the siderophores pyochelin and pyoverdin. Both mutants required a significant lag phase prior to log-phase aerobic growth, but this lag was not as apparent when the organisms were grown under microaerobic conditions. The addition of iron salts to mutant A4 and, to a greater extent, C6 cultures allowed for an increased growth rate under both conditions relative to that of bacteria without added iron. Increased manganese superoxide dismutase (Mn-SOD) and decreased catalase activities were also apparent in the mutants, although the second catalase, KatB, was detected in cell extracts of each fur mutant. Iron deprivation by the addition of the iron chelator 2,2'-dipyridyl to wild-type bacteria produced an increase in Mn-SOD activity and a decrease in total catalase activity, similar to the fur mutant phenotype. Purified wild-type Fur bound more avidly than mutant Fur to a PCR product containing two palindromic 19-bp "iron box" regions controlling expression of an operon containing the sodA gene that encodes Mn-SOD. All mutants were defective in both ferripyochelin- and ferripyoverdin-mediated iron uptake. Two mutants of strain PAO1, defective in pyoverdin but not pyochelin biosynthesis, produced increased Mn-SOD activity. Sensitivity to both the redox-cycling agent paraquat and hydrogen peroxide was greater in each mutant than in the wild-type strain. In summary, the results indicate that mutations in the P. aeruginosa fur locus affect aerobic growth and SOD and catalase activities in P. aeruginosa. We postulate that reduced siderophore-mediated iron uptake, especially that by pyoverdin, may be one possible mechanism contributing to such effect.     

Does chemically induced hepatocyte proliferation predict liver carcinogenesis?

Cell proliferation has long been recognized as having an important role in chemically induced carcinogenesis. Based on findings that certain nongenotoxic chemical carcinogens induced cell proliferation in the same organ that had an increased incidence of tumors, it has been hypothesized that a chemically induced response of enhanced DNA synthesis and cellular division causes cancer by increasing the rate of spontaneous mutations. It was further suggested that there would be no increased human risk of cancer by non-DNA-reactive compounds at doses that do not cause a proliferative response. An evaluation of the literature on the relationship between chemically induced cell proliferation and liver carcinogenesis reveals that very few systematic cell proliferation studies have been conducted over periods of extended exposure, and in many cases the exposure concentrations were not similar to those used in the cancer studies. The proliferative response resulting from exposure to many nongenotoxic carcinogens is not well sustained, whereas the carcinogenic response by these chemicals often requires prolonged exposure. The available literature leads to the conclusion that quantitative correspondences between cellular proliferation and carcinogenic responses have not been demonstrated and do not support the hypothesis that chemically induced cell proliferation is the primary mechanism by which nongenotoxic chemicals cause liver cancer. Studies of liver carcinogenesis in two-stage models point out the need to better understand chemical effects on cell loss as well as on cell replication, and demonstrate that measurements of cell proliferation alone are not sufficient to elucidate mechanisms of tumor development.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in amino acid transport System L

The Chinese hamster ovary cell line CHO-tsH1 is a temperature-sensitive leucyl-tRNA synthetase mutant that shows temperature-dependent regulation of the amino acid transport responsible for accumulating leucine, System L. At nonpermissive temperatures, CHO-tsH1 cells are unable to grow because they are unable to incorporate leucine into protein. As a result, System L activity is increased. We have isolated mutants from CHO-tsH1 that have constitutively de-repressed System L activity. These mutants are temperature-resistant as a result of increased intracellular steady-state accumulations of System L-related amino acids, which compensates for the defective synthetase activity. In this study, we have subjected one of these regulatory mutant cell lines (C11B6) to a tritium-suicide selection, in which L-[3H]leucine was used as a toxic substrate. Three mutant cell lines, C4B4, C5D9, and C9D9 that showed reduced System L transport activity were isolated. The decreases in the initial rates of System L transport activity lead to reduced steady-state accumulations of System L-related amino acids. In contrast to the parental cell line, C11B6, the transport-defective mutants are temperature-sensitive because the reduced intracellular pool of leucine can no longer compensate for the defective synthetase activity.     

Selection of Penicillium funiculosum strains with high glucose oxidase activity

Metabolic inhibitors, riboflavin, and end products of glucose oxidation were shown to hold much promise for the selection of Penicillium funiculosum mutant strains with a high glucose oxidase activity. The incidence of positive mutations was highest in clones resistant to sodium azide, riboflavin, and beta-D-glucono-delta-lactone. Enzyme activity in Penicillium funiculosum mutants was studied under conditions of submerged cultivation. The intensity of glucose oxidase synthesis in seven cultures was 24-56% higher than that in the parent strain of Penicillium funiculosum NMM95.132.     

Tracing the tracks of genotoxicity by trivalent and hexavalent chromium in Drosophila melanogaster

Mutagen sensitive strains (mus) in Drosophila are known for their hypersensitivity to mutagens and environmental carcinogens. Accordingly, these mutants were grouped in pre- and post-replication repair pathways. However, studying mutants belonging to one particular repair pathway may not be adequate for examining chemical-induced genotoxicity when other repair pathways may neutralize its effect. To test whether both pre-and post-replication pathways are involved and effect of Cr(III)- and Cr(VI)-induced genotoxicity in absence or presence of others, we used double mutant approach in D. melanogaster. We observed DNA damage as evident by changes in Comet assay DNA migration in cells of larvae of Oregon R(+) and single mutants of pre- (mei-9, mus201 and mus210) and post- (mei-41, mus209 and mus309) replication repair pathways and also in double mutants of different combinations (pre-pre, pre-post and post-post replication repair) exposed to increasing concentrations of Cr(VI) (0.0, 5.0, 10.0 and 20.0 μg/ml) for 48 h. The damage was greater in pre-replication repair mutants after exposure to 5.0 μg/ml Cr(VI), while effects on Oregon R(+) and post replication repair mutants were insignificant. Post-replication repair mutants revealed significant DNA damage after exposure to 20.0 μg/ml Cr(VI). Further, double mutants generated in the above repair categories were examined for DNA damage following Cr(VI) exposure and a comparison of damage was studied between single and double mutants. Combinations of double mutants generated in the pre-pre replication repair pathways showed an indifferent interaction between the two mutants after Cr(VI) exposure while a synergistic interaction was evident in exposed post-post replication repair double mutants. Cr(III) (20.0 μg/ml) exposure to these strains did not induce any significant DNA damage in their cells. The study suggests that both pre- and post-replication pathways are affected in Drosophila by Cr(VI) leading to genotoxicity, which may have consequences for metal-induced carcinogenesis.     

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

Summary A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10^–9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.     

A method to distinguish between the de novo induction of thymidine kinase mutants and the selection of pre-existing thymidine kinase mutants in the mouse lymphoma assay

The mouse lymphoma assay (MLA) is the most widely used in vitro mammalian gene mutation assay. It detects various mutation events involving the thymidine kinase (Tk) gene in L5178Y/Tk+/- -3.7.2C mouse lymphoma cells. Mutants are detected using a thymidine analogue that arrests the growth of cells containing a functional Tk gene. However, there are a number of potential test chemicals that are thymidine analogues, and there is a problem when using the MLA to evaluate the mutagenicity of these chemicals. Thymidine analogues are activated by Tk before eliciting their toxicity. Therefore, any pre-existing Tk-/- mutants may avoid the toxicity of the test chemical and obtain a growth advantage over the Tk+/- cells, increasing the Tk mutant frequency (MF) in the culture via a selection mechanism. This potential mutant selection effect needs to be distinguished from de novo mutant induction in order to properly evaluate the mutagenicity of these chemicals. Here we describe a simple MLA study design that can differentiate between the selection of pre-existing mutants and de novo mutant induction. Trifluorothymidine (TFT), a thymidine analogue and the selection agent normally used in the MLA, and 4-nitroquinoline-1-oxide (4-NQO), a potent mutagen, were used to treat cells from two different Tk+/- mouse lymphoma cell cultures with different background MFs (approximately 112 and 305x10(-6)). Both agents significantly increased the Tk MFs in both the normal and high background cultures (p<0.01). In 4-NQO-treated cultures, the induced MFs (MF of treated culture-MF of control) for the cultures with different background MFs were about the same (p>0.1), while in TFT-treated cultures, they were significantly different (p<0.01). In TFT-treated cultures, the fold-increases of MF (MF of treated culture/MF of control) for the cultures with different background MFs were about the same (p>0.1), while in 4-NQO-treated cultures, they were significantly different (p<0.01). This study confirms that, when de novo mutations are induced, the induced MF is the same for cultures with normal and artificially high background MFs. In situations where the increase in MF is due solely to selection of pre-existing mutants, the "induced" MF will be a multiple of the background MF and the magnitude of the increase of the induced MF will depend upon the magnitude of the background MF. Our results demonstrate that it is possible, using this experimental design, to distinguish between chemicals acting primarily via the selection of pre-existing Tk mutants and those inducing de novo mutants in the MLA.     

Culture conditions of protoplast-derived cells of nicotiana sylvestris for mutant selection

Large numbers of protoplasts showing reproducible high plating efficiency can be isolated from in vitro propagated, haploid and diploid, plants of Nicotiana sylvestris. Their successful use in the selection of biochemical mutants depends on the establishment of suitable selection parameters: culture medium, cell density, age of cells at selection etc. Plating of protoplasts at low densities as well as simulation and reconstruction experiments of mutant selection were employed to optimize such selection parameters. The results show that some of the principles determined for tobacco protoplast cultures manipulated at low densities or in view of mutant selection are of more general value. However, requirements specific to N. sylvestris protoplast cultures have also been established; they play a decisive part in the successful isolation of resistant mutants in this species.Abbreviations AEC S-aminoethylcysteine - BA benzyl-adenine - NAA napthaleneacetic acid - p-cells or p-colonies protoplast-derived cells or colonies     

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.