Characterization of a cell cycle mutant derived from hamster fibroblast: reversion analysis

K12 is a temperature-sensitive (ts) mutant cell line derived from Chinese hamster fibroblasts. When incubated at the nonpermissive temperature, K12 cells exhibit the following properties: (a) the cells cannot initiate DNA synthesis;o (b) the synthesis of cytosol thymidine kinase is suppressed; and (c) the synthesis of three cellular proteins of molecular weights 94, 78, and 58 kdaltons is greatly enhanced. Here we characterize a spontaneous revertant clone, R12, derived from the K12 cells. We selected the revertant clone for its ability to grow at the nonpermissive temperature. Our results indicate that all the traits which constitute the K12 mutant phenotype are simultaneously reverted to the wild type in the revertant cell line, suggesting that the ts mutation of the K12 cells is of regulatory nature and exerts multiple effects on the expressed phenotypes.     

Hypermutability of a UV-sensitive aphidicolin-resistant mutant of Chinese hamster fibroblasts

An ultraviolet light (UV)-sensitive thymidine auxotroph of Chinese hamster V79 cells that exhibits pleiotropic effects such as a high level of deoxycytidine triphosphate, slow growth, sensitivity to cytidine, and high frequencies of site-specific bromodeoxyuridine-dependent chromosomal aberrations was selected by its resistance to aphidicolin. The UV-induced mutability of this mutant and one of its revertants, which retains some of the phenotypes listed above, was studied in 3 mutation assay systems. The results showed that the mutant was hypermutable for ouabain and diphtheria-toxin-resistant mutations compared to wild-type V79 cells at the same UV dose or the same survival level. The mutant exhibits a delayed expression of maximal frequency of induced 6-thioguanine-resistant mutants. When maximal frequencies are compared at the same UV dose, the mutant also has higher mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase locus. The revertant was similar to the wild-type in UV sensitivity and mutability. Our results showed that UV sensitivity and hypermutability are correlated and controlled by a single gene. Thymidine auxotrophy or nucleotide pool imbalance apparently did not contribute to the UV sensitivity and mutability of the mutant.     

Comparison of the Frequencies of Spontaneous and Chemically-Induced 5-Bromodeoxyuridine-Resistance Mutations in Wild-Type and Revertant Bhk-21/13 Cells

5-bromodeoxyuridine resistance mutations induced by mutagenesis were studied. The average expression time for induced mutations varied with the concentration of the mutagen ethyl methanesulfonate (EMS). However, a constant number of two generation times was necessary for half maximal expression of induced mutations. Also, induced mutation rates were compared under optimal expression conditions for bromodeoxyuridine, fluorodeoxyuridine and azaguanine resistance markers. Ten independent bromodeoxy-uridine-resistant clones were tested for reversion. Two clones reverted-one spontaneously and the other after mutagenesis. The spontaneous rate of mutation to bromodeoxyuridine resistance, estimated by the fluctuation test, was high in revertant clones (4 x 10(-6) / cell / generation) and low in the wild-type cells (< 3.5 x 10(-8) / cell / generation). A comparison of induced mutation frequencies at variable EMS concentrations showed a single-hit curve for revertant clones and a multihit curve for the wild-type cells. Thymidine kinase activities of resistant clones were usually less than 2% of that of the wild-type clone. Inducibility, thermal stability and intracellular localization of the thymidine kinases of the wild-type cells and of a revertant clone were identical. A low, but significant (P < 0.10), Km discrepancy was observed between enzyme extracts of these lines. The genetic implications of these results are discussed.     

Folate-dependent enzymes in cultured Chinese hamster ovary cells: evidence for mutant forms of folylpolyglutamate synthetase

A Chinese hamster ovary auxotroph requiring glycine + adenosine + thymidine (CHO AUXB1) was shown by us previously to lack several folylpolyglutamate synthetase (FPGS) type activities. Two revertants of AUXB1 (one spontaneous and one Pt(S0₄)₂ induced) have been isolated and found to contain altered forms of this enzyme. The revertant enzymes are more sensitive to heat inactivation (37 °C, pH 7.4 or 9.0) than the parent CHO enzyme. Increased sensitivity of revertant FPGS is observed irrespective of whether one assays the specific catalysis of radioactive tetrahydropteroyldi- or tetraglutamate synthesis. ATP and MgCl₂ protect both revertant and parent CHO FPGS against rapid heat denaturation at pH 9.0, but not at pH 7.4. A genetically related auxotroph (CHO AUXB3) contains one-fifth the parent amount of FPGS. AUXB3 FPGS shows a normal sensitivity to 37 °C heat inactivation, but it has an altered substrate saturation and specificity pattern when assayed for tetrahydropteroyldi[U-¹⁴C]glutamate synthesis. Also, unlike the FPGS from parent CHO and a genetically unrelated mutant requiring only glycine (CHO AUXB2), the AUXB3 enzyme specifically lacks tetrahydropteroyltetra[U-¹⁴C]glutamate synthetase activity. These findings and polyethylene glycol fusion data with AUXB2 indicate that AUXB1 and AUXB3 each carry a mutation in the structural gene for a CHO FPGS that catalyzes tetrahydropteroyldi- as well as tetraglutamate formation. The altered form of FPGS in AUXB3 is responsible for its glycine + adenosine auxotrophy under standard culture conditions.     

Repair of DNA damage in shuttle vectors,virus, and chromosomal DNAs may depend on their biological imprinting — a ‘Pygmalion’ effect

We have created a cell line that can repair damage in chromosomal DNA and in herpes virus, while not repairing the same damage in shuttle vectors (pZ189 and pRSVcat). This cell line, a xeroderma pigmentosum (XP) revertant, repairs the minor (6-4)-photoproducts, but not cyclobutane dimers, in chromosomal DNA. The phenotype of this revertant after irradiation with ultraviolet (UV) light is the same as that of normal cells for survival, repair replication, recovery of rates of DNA and RNA synthesis, and sister-chromatid exchange formation, which indicates that a failure to mend cyclobutane dimers may be irrelevant to the fate of irradiated human cells. The two shuttle vectors were grown in Escherichia coli and assayed during transient passage in human cells, whereas the herpes virus was grown and assayed exclusively in mammalian cells. The ability of the XP revertant to distinguish between the shuttle vector and herpes virus DNA molecules according to their ‘cultural background’, i.e., bacterial or mammalian, may indicate that one component of the repair of UV damage involves gene products that recognize DNA markers that are uniquely mammalian, such as DNA methylation patterns. This component of excision repair may be involved in the original defect and the reversion of XP group A cells.     

An Unstable Allele of the am Locus of NEUROSPORA CRASSA

The mutant strain am126 was isolated, using the direct selection procedure, after nitrous acid mutagenesis. It produced neither measurable NADP-dependent glutamate dehydrogenase (GDH) nor immunologically cross-reacting material. That the am126 strain produced some form of GDH product was shown by the fact that it complemented several other am mutant strains. The GDH formed by complementation between am126 and each of two other am mutants was relatively thermolabile, but could not be distinguished from wild-type GDH formed by electrophoresis in polyacrylamide gels. This, together with the relatively high yield of the complementation enzymes, suggest that the am126 product is a polypeptide chain not grossly abnormal in structure. The spontaneous revertant frequency was between 0.3 and 3 prototrophic revertants per 10(5) live cells. This frequency was at least 40 times greater than that for am19, which had the second highest spontaneous revertant frequency among the mutants tested. Neither meiosis nor mutagenesis increased the revertant frequency, nor did incubation at elevated temperatures lower it. Sixty-eight revertant strains were examined for thermostability of their GHD. All appeared to be identical to wild type. Seven of the revertant strains were also tested for instability with regard to forward mutation to am auxtrophy. None was found to be unstable. Models for the genetic instability of the am126 mutation are discussed.     

Mutants of Chinese hamster cells deficient in thymidylate synthetase

Stable mutants of Chinese hamster V79 cells deficient in thymidylate synthetase (TS; E.C. 2.1.1.45) have been selected from cultures grown in medium supplemented with folinic acid, aminopterin, and thymidine (FAT). After chemical mutagenesis, the frequency of colonies resistant to the "FAT" medium increased more than 100-fold over the spontaneous frequency. The optimal expression time of the mutant phenotype was 5-7 days after mutagen treatment. The recovery of FAT-resistant colonies in the selective medium was not affected by the presence of wild-type cells at a density below 9,000 cells per cm2. All 21 mutants tested exhibited thymidine auxotrophy; neither folinic acid nor deoxyuridine could support mutant cell growth. There was no detectable TS activity in all 11 mutants so far examined and only about 50% of wild-type activity in three prototrophic revertants, as measured by whole-cell and cell-free enzyme assays. The apparent Michaelis-Menten constant (Km) for deoxyuridine-5'-monophosphate and inhibition constant (Ki) for 5-fluoro-deoxyuridine-5'-monophosphate, measured by whole-cell enzyme assay, appear to be similar for the wild-type and revertant cell lines. Using 5-fluoro-[6-3H]-2'-deoxyuridine 5'-monophosphate as active site titrant, the relative amounts of TS in crude cell extract from the parental, revertant, and mutant cells were shown to exist in a 1:0.5:0 ratio. Furthermore, the enzymes from two revertants were more heat labile than that of V79 cells. These properties, taken together, suggest that the FAT-resistant, thymidine auxotrophic phenotype may be the result of a structural gene mutation at the TS locus. The availability of such a mutant facilitates studies on thymidylate stress in relation to DNA metabolism, cell growth, and mutagenesis.     

Mutant of herpes simplex virus type 1 conditionally able to transform thymidine kinaseless L cells to a tk+ phenotype.

After nitrous acid mutagenesis of herpes simplex virus type 1 (HSV-1), a mutant, 1093, was isolated which, during productive infection, induced very low levels of thymidine kinase (tk). The mutant virus was found, after UV irradiation, to be unable to transform L cells lacking tk (Ltk-) to a tk+ phenotype as chararcterized by growth of the cells in a modified HAT-selective medium containing 1.6 X 10(-5) M thymidine. Cells transformed by wild-type virus grew vigorously under the same conditions. The mutant was able to transform Ltk- cells if the medium contained 10(-3) M thymidine. These transformed cells maintained their conditional character and would not grow in low concentrations of thymidine in selective medium. Therefore, this mutant is conditional on the thymidine concentration in the selection medium in its ability to transform Ltk- cells to a tk+ phenotype. The conditionally transformed cells could be supertransformed with wild-type UV-irradiated HSV-1 to a phenotype which would grow in low-thymidine selective medium. The frequency of supertransformation closely approximated the frequency of transformation of Ltk- cells by wild-type virus. Supertransformation at high frequency could not be effected by mutant 1093 or the tk- mutant B2006. These results indicate that the presence of HSV-1 genetic information in HSV-1-transformed cells does not preclude the acquisition by these cells of at least one additional HSV-1 gene, that for tk.     

High genome-wide mutation rates in vegetatively propagated bermudagrass

A cascade DNA amplification strategy that generates arbitrary signatures from amplification profiles (ASAP) was used to measure genome-wide mutation rates in bermudagrass (Cynodon). ASAP quantified nucleotide changes that were induced by irradiation, genetic instabilities and normal vegetative growth of cultivars and accessions of sterile interspecific hybrids. DNA sequence divergence between cultivar ‘Tifway’ and its γ radiation-induced mutant ‘Tifway II’ (0.70 ± 0.66%) was comparable to estimates in radiation-induced mutants and spontaneous sports of chrysanthemum (Chrysanthemum morifolium Ramat.). A similar divergence in sequence (0.95 ± 0.20%) was observed in the pairwise comparison of 17 nondisjunctive ‘Tifgreen’ and ‘Tifdwarf’ accessions. Mutation during normal Tifdwarf vegetative growth was evaluated by planting sprigs and sampling their offspring. Somatic sequence divergence levels (0.004 ± 0.007%) resulted in a mutation rate of 1.05 × 10^?8 per nucleotide per generation, assuming that a bermudagrass sprig constitutes a generation of growth. These rates were comparable to those found in germinal cells and individuals of either human or Drosophila melanogaster, supporting the notion that eukaryotic evolution is generation rather than time dependent. The high accumulation of somatic mutations (10 per triploid genome) is consistent with a model whereby mutation load in a population exhibiting obligate vegetative reproduction is substantially higher than in a population under sexual or asexual reproduction. These constraints could be the cause of reported genetic instabilities in the Tifgreen–Tifdwarf complex. Finally, a long-term rate measured across accessions and indicative of the accumulation of mutations in 17 Tifgreen–Tifdwarf populations (µ = 1.02 × 10^?8 per nucleotide per generation) was strikingly congruent with the bermudagrass vegetative mutation rate, suggesting absence of evolutionary constraints in the sampled genomic regions. Mutation rates calculated from across-accesions divergence estimates (5.18 ± 0.53%) indicated that plant material was evolving 100 times faster (3.8 × 10^?7 changes per nucleotide per year) than a molecular clock rate estimate for grasses, probably resulting from the compound effect of clonal growth and life span of the hybrid plant material.     

A single nucleotide mutation in GID1c disrupts its interaction with DELLA1 and causes a GA‐insensitive dwarf phenotype in peach

Plant stature is one important factor that affects the productivity of peach orchards. However, little is known about the molecular mechanism(s) underlying the dwarf phenotype of peach tree. Here, we report a dwarfing mechanism in the peach cv. FenHuaShouXingTao (FHSXT). The dwarf phenotype of ‘FHSXT’ was caused by shorter cell length compared to the standard cv. QiuMiHong (QMH). ‘FHSXT’ contained higher endogenous GA levels than did ‘QMH’ and did not response to exogenous GA treatment (internode elongation). These results indicated that ‘FHSXT’ is a GA‐insensitive dwarf mutant. A dwarf phenotype‐related single nucleotide mutation in the gibberellic acid receptor GID1 was identified in ‘FHSXT’ (GID1c^S191F), which was also cosegregated with dwarf phenotype in 30 tested cultivars. GID1c^S191F was unable to interact with the growth‐repressor DELLA1 even in the presence of GA. ‘FHSXT’ accumulated a higher level of DELLA1, the degradation of which is normally induced by its interaction with GID1. The DELLA1 protein level was almost undetectable in ‘QMH’, but not reduced in ‘FHSXT’ after GA₃ treatment. Our results suggested that a nonsynonymous single nucleotide mutation in GID1c disrupts its interaction with DELLA1 resulting in a GA‐insensitive dwarf phenotype in peach.     

Renin inhibitors as an example of presumptive irrelevant positive findings in the Salmonella/mammalian microsome assay (Ames test)

An increase in the number of mutant colonies in the Ames test is generally taken as a strong indication for a genotoxic (e.g., DNA damaging) property of the test compound or its metabolites. However, a few examples are known in which mechanisms usually related to some sort of growth enhancement will lead to increases in mutant frequencies of spontaneous origin.The renin inhibitor Ro 42-5892 increased the number of mutant colonies of strain TA1538 and to a lesser degree of TA98 in the standard plate incorporation assay (Ames test). Since there is no chemical basis for a ‘DNA reactivity’ of this compound, experiments were performed to obtain information about possible indirect mechanisms of enhancing the number of spontaneous mutant colonies.Circumstantial evidence is presented to attribute the weak activity not to an inherent genotoxic property but rather to an as yet undefined indirect effect on the expression of spontaneous mutants.Since Ro 42-5892 contains a histidine residue it was a reasonable assumption to suspect a growth enhancing property of the test compound. However, none of the strains showed an elevation of the number of revertant colonies or an increase in the density of the background growth. In addition, structurally related non-histidine containing renin inhibitors showed absolutely no increase in the number of revertant colonies. Furthermore, no growth induction (either in liquid or under selective conditions) and no histidine cleave off by a TA1538/TA98 specific metabolism could be shown.A second line of evidence showing parallelism to growth enhancing compounds concerns the comutagenicity of histidine containing renin inhibitors. When Ro 42-5892 was tested in combination with established mutagens, a multiplicative synergism was found. This effect was observed not only in strains TA1538 and TA98 but also in the standard Salmonella tester strains where the spontaneous mutant frequency was not increased by Ro 42-5892. Analogous effects were previously shown for free histidine, isohistidine, phenobarbital and tetracycline and in part explained by molecular mechanisms.     

A mutation in the GTPase domain of the large subunit rRNA is involved in the suppression of a -1T frameshift mutation affecting a mitochondrial gene in Chlamydomonas reinhardtii

The dum19 mutation isolated in Chlamydomonas reinhardtii is due to the deletion of one T at codon 152 of the mitochondrial cox1 gene sequence. Phenotypically, the dum19 mutant is characterized by a lack of cytochrome c oxidase activity and is unable to grow under heterotrophic conditions. A spontaneous pseudo-revertant that grows slowly in the dark was isolated from the dum19 mutant strain. A genetic and molecular analysis allowed us to demonstrate that the revertant phenotype is the consequence of two additional mutations that together act as a frameshift suppressor: an m mutation affecting a mitochondrial gene other than cox1 and an n mutation affecting a nuclear gene. On its own the n mutation does not act as a suppressor, whereas the m mutation very slightly compensates for the effect of the -1T mutation. Sequencing analysis showed that the m mutation affects the GTPase-associated domain of the large subunit (LSU) ofmitochondrial rRNA. Surprisingly, two substitutions, A1090 to G and A1098 to C, were found in the LSU rRNA of the revertant, the latter one being already present in the dum19 mutant strain itself. The A1090 to G substitution is thus involved in the suppression of the frameshift mutation, but it is not clear whether the change at position 1098 is also required for the expression of the suppressed phenotype. To our knowledge, this is the first example of a mutation in the GTPase-associated domain acting as a suppressor of a frameshift mutation.     

Increase in UV Mutagenesis by Heat Stress on UV-Irradiated E. coli Cells

When leu- auxotrophs of Escherichia coli, after UV irradiation, were grown at temperatures between 30 and 47°C, the frequency of UV-induced mutation from leu- to leu+ revertant increased as the UV dose and the temperature increased. For cells exposed to a UV dose of 45 J/m2, the mutation frequency at 47°C was 1.9 times that at 30°C; for a dose of 90 J/m2, it was 3.25 times; and for 135 J/m2, it was 4.8 times. Similar enhancement of reversion frequency was observed when the irradiated cells were grown at 30°C in the presence of a heat shock inducer, ethanol (8% v/v). Heat shock-mediated enhancement of UV mutagenesis did not occur in an E. coli mutant sigma 32 (heat shock regulator protein), but sigma 32 overexpression in the mutant strain (transformed with a sigma 32-bearing plasmid) increased the UV-induced mutation frequency. These results suggest that heat stress alone has no mutagenic property, but when applied to UV-damaged cells, it enhances the UV-induced frequency of cell mutation.     

Mutational tolerance to carbendazim in Botrytis cinerea

No spontaneous mutation for tolerance to the fungicide carbendazim was detected in C. 10⁸ conidia from each of eight carbendazim-sensitive field isolates of Botrytis cinerea. Conidia of B. cinerea were highly insensitive to u.v.-irradiation, although after severe irradiation treatments mutant strains showing the same levels of tolerance as two groups of carbendazim-tolerant field isolates were selected at frequencies of between 10^-9 and 10^-6 of survivors. Mutants with low levels of tolerance (ED₅₀ > 10 μg ml^-1 carbendazim; ‘partially-tolerant’) were selected from irradiated conidia obtained from sensitive field isolates and a further series of mutants capable of growth on 10 000 μg ml^-1 carbendazim (‘fully-tolerant’) were selected from irradiated conidia from either partially-tolerant mutants or from partially-tolerant field isolates. Both mutation steps were confirmed in similar experiments in which tolerance to an unrelated fungicide, 2, 6-dichloro-4-nitroaniline (DCNA), was incorporated as a genetic marker in the parent strains.     

A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA

We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient ρ⁻ or ρ⁰ mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methanesulfonate or ultraviolet light. The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter. When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia. Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron. We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells. Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair. These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron. Received: 26 March 1999 / Accepted: 29 June 1999     

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. II. The effect of posttreatment with non-toxic concentrations of thymidine

The ability of posttreatment exposure to non-toxic concentrations of thymidine (TdR) to enhance the lethal effects of a number of alkylating agents, X-rays and UV and the lethal and mutagenic effects of N′-ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) has been examined in V79 cell lines. TdR posttreatment enhanced the cytotoxic effects of ethyl methanesulphonate (EMS), MNU and ENU but not of UV or X-rays and increased both the spontaneous and MNU- and ENU-induced frequencies of azaguanine resistant (AZ^R) mutants. No significant effect of TdR on the spontaneous frequency of thioguanine resistant (TG^R) mutants was demonstrated but the frequency of MNU-induced mutants to TG^R was enhanced. The effects on expression of both potentially lethal and premutagenic damage were reversed by addition of an equimolar concentration of deoxycytidine (dCdR). The enhancement in spontaneous and induced mutant frequency (IMF) at the HGPRT locus appears to be due to an alteration in the selective efficiency of purine analogous due to alteration in growth kinetics of cells exposed to TdR or treated with alkylated agents or posttreated with thymidine after alkylation damage and not to an alteration in the miscoding potential of alkylated bases.     

Heritable alterations at the adenine phosphoribosyltransferase (APRT) locus in human lymphoblastoid cell lines.

Human lymphoblastoid cell lines derived from WI-L2 exhibit unexpected frequencies of diaminopurine (DAP) resistant mutants. The background mutant fractions of 10(-7) to 10(-8) in untreated cultures are much lower than the frequencies expected for loss of a heterozygous autosomal locus (10(-5) to 10(-6), yet much higher than expected for a homozygous locus (10(-10) to 10(-12). We used aminopterin, adenine and thymidine (AAT) to select DAP-sensitive (DAPS) revertants from one resistant line. The background frequency of DAPR in these revertant cell lines ranged from 3.5 to 6.5 x 10(-4), approximately the square root of 10(-7). Thus these data suggest that both alleles of aprt are inactivated at similarly high frequencies. They also indicate that the DAPS revertants were heterozygotes (aprt +/-) or hemizygotes (aprt +/0) and that WI-L2 was homozygous (aprt+/+). Mutational dose-response studies with X-rays, ethyl methanesulfonate (EMS), and ICR-191 were conducted in 4 of these revertant cell lines. EMS and ICR-191, which induce mainly point mutations, did not induce an increase in mutant fraction. A dose of 200 cGy X-rays, however, induced a frequency of 10(-3). Treatment of DAPR cells with 5-azacytidine induced a significant increase in reversion to DAPS. Southern blot analysis of the aprt gene after digestion with MspI or HpaII also suggests that differential methylation changes may play a major role in the generation of DAP sensitivity and resistance.     

Multiple mutant of Escherichia coli synthesizing virtually thymineless DNA during limited growth.

The dut gene of Escherichia coli encodes deoxyuridine triphosphatase, an enzyme that prevents the incorporation of dUTP into DNA and that is needed in the de novo biosynthesis of thymidylate. We produced a conditionally lethal dut(Ts) mutation and isolated a phenotypic revertant that had a mutation in an unknown gene tentatively designated dus (for dut suppressor). The dus mutation restored the ability of the dut mutant to grow at 42 degrees C without restoring its enzymatic activity or thymidylate independence. A strain was constructed bearing, in addition to these mutations, ones affecting the following genes and their corresponding products: ung, which produces uracil-DNA N-glycosylase, a repair enzyme that removes uracil from DNA; deoA, which produces thymidine (deoxyuridine) phosphorylase, which would degrade exogenous deoxyuridine; and thyA, which produces thymidylate synthase. When grown at 42 degrees C in minimal medium containing deoxyuridine, the multiple mutant displayed a 93 to 96% substitution of uracil for thymine in new DNA. Growth stopped after the cellular DNA had increased 1.6- to 1.9-fold and the cell mass had increased 1.7- to 2.7-fold, suggesting a general failure of macromolecular biosynthesis. DNA hybridization confirmed that the uracil-containing DNA was chromosomal and that new rounds of initiation must have occurred during its synthesis.     

Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant.

We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent.