Effect of the adenine level of a culture medium on the frequency of revertants in haploid yeasts

The frequency of reversion from adenine auxotrophy in the yeast Saccharomyces cerevisiae increases with the decrease of adenine concentration in the medium: for the 8PG-59 strain (a ade2-192 rad2) the reversion frequency is 1.7 X 10(-8), 3.2 X 10(-6) and 1.8 X 10(-5) per cell division, the initial adenine concentrations being 10, 1 and 0.1 mg/l, respectively. An increase of the reversion frequency with the culture age in the stationary phase of growth is demonstrated using an improved method of registration of revertants, with the initial concentration of 0.1 mg/l of adenine in the medium. The reversion frequency was 9.1 X 10(-7) on the 7th day, 1.8 X 10(-6) on the 10th day and 1.8 X 10(-5) on the 14th day.     

The proof of real increase in the frequency of reverse mutations in haploid yeasts in media with decreased adenine content

It has been shown by the direct experiment in the haploid yeast Saccharomyces cerevisiae, strain p192 (a ade2-192), that, if cultivated in the solid media containing 1 and 10 mg/l of adenine, the reversion frequencies (5.6 X 10(-8) and 0.82 X 10(-8), respectively) do not depend on the number of cells (from 10(2) to 10(7)) in colonies growing on either medium. This means that the difference in the reversion frequencies mentioned is not connected with the "selection effect" and is only caused by the varying initial content of adenine in the medium. A method combining the technique of ordered plating and the use of nuclear filters is recommended for the study of mutagenesis in microorganisms.     

Spontaneous mutability in yeast. I. Stability of lysine reversion rates to variation of adenine concentration

Novick and Szilard demonstrated that increasing the concentrations of adenine enhance the mutation rate of E. coli. We have found that the spontaneous mutation rate of the yeast Saccharomyces cerevisiae remains constant over a 200-fold range of adenine concentration.The system that we commonly use for measuring spontaneous mutation rate is reversion of the super-suppressible mutant lys1-1. In this system, growth of the yeast is limited by limiting the amount of lysine in the medium. A reversion to lysine independence will continue to grow. One of the other super-suppressible mutants in the test system is ade2-1, a mutant that causes accumulation of red pigment. By adjusting the concentration of adenine slightly above that of lysine, reversions of super-suppressors produce white colonies and reversions of the lys1-1 locus itself produce red colonies.     

Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.     

Ultraviolet-Induced Reversion of cyc1 Alleles in Radiation Sensitive Strains of Yeast. II. rev2 Mutant Strains

The range of specificity of the rev2-1 mutation, an allele that reduces the frequency of ochre revertants induced by UV in Saccharomyces cerevisiae (LEMONTT 1971a), has been investigated by examining its influence on the reversion of eleven well-defined and contrasting cyc1 mutations. We have shown, in support of a suggestion of LEMONTT (1971a), that the REV2 gene product is concerned only with the reversion of ochre alleles; it plays virtually no role in the reversion of amber, missense or frameshift mutations. We have also shown that its effect is specific and confined to only some highly revertible ochre alleles. The REV2 gene product appears to enhance reversion at these sites by facilitating the conversion of two otherwise nonmutagenic photo-products into a single premutational lesion. UV-induced killing of rev2-1 strains was found to be significantly greater on fermentable rather than on nonfermentable media.     

Fluorescence energy transfer between heterologous active sites of affinity-labeled aspartokinase of Escherichia coli.

The distance between aspartokinase and homoserine dehydrogenase active sites was determined using fluorescence energy transfer between modified substrates. The fluorescent 1,N(6)-ethenoadenosine 5'-triphosphate was bound at the kinase active site by Co(III) affinity labeling. Reduced thionicotinamide adenine dinucleotide phosphate quenched the fluorescence of bound nucleotide. Fluorescence depolarization measurements led to a delimitation of the value of the dipolar orientation factor to the range 0.3 to 2.8. The distance between the fluorescent probe and the quencher was 29 +/- 4 A. In the presence of threonine, this distance increased to 36 +/- 5 A. Threonine binding either increased the intersite distance by ca. 7 A or caused a reorientation of the probe at the dehydrogenase site.     

Regulation of the Biosynthesis of Amino Acids of the Aspartate Family in Coliform Bacteria and Pseudomonads

The control of aspartokinase and homoserine dehydrogenase activities was compared in aerobic and fermentative pseudomonads (genera Pseudomonas and Aeromonas), and in coliform bacteria representative of the principal genera of the Enterobacteriaceae. Isofunctional aspartokinases subject to independent end-product control occur in the Enterobacteriaceae and in Aeromonas. In Pseudomonas, there appears to be a single aspartokinase, subject to concerted feedback inhibition by lysine and threonine. Within this genus, the sensitivity of aspartokinase to the single allosteric inhibitors varies considerably: the aspartokinase of the acidovorans group is little affected by the single inhibitors, whereas that of the fluorescent group is severely inhibited by either amino acid at high concentration. In all bacteria examined, homoserine dehydrogenase activity is inhibited by threonine; inhibition is more severe in aerobic pseudomonads than in the other groups. In most of the bacteria examined, either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate can serve as a cofactor for this enzyme, though the relative activity with the two pyridine nucleotides varies widely. Aerobic pseudomonads of the acidovorans group contain a homoserine dehydrogenase that is absolutely specific for NAD. The taxonomic implications of these findings are discussed.     

Quorum sensing in Vibrio fischeri: evidence that S-adenosylmethionine is the amino acid substrate for autoinducer synthesis.

Synthesis of the autoinducer signal involved in the cell density-dependent activation of Vibrio fischeri luminescence is directed by luxI. The autoinducer is N-(3-oxohexanoyl)homoserine lactone, and little is known about its synthesis. We have measured autoinducer synthesis by amino acid auxotrophs of Escherichia coli that contained luxI on a high-copy-number plasmid. Experiments with cell suspensions starved for methionine or homoserine show that either methionine or S-adenosylmethionine but not homoserine or homoserine lactone is required for autoinducer synthesis. The S-adenosylmethionine synthesis inhibitor cycloleucine blocks methionine-dependent autoinducer synthesis. Thus, it appears that S-adenosylmethionine rather than methionine is the molecule required for autoinducer synthesis. The amount of 15N-labeled methionine incorporated into the autoinducer by growing cultures of a homoserine and a methionine auxotroph was measured by mass spectrometry. The labeling studies show that even in the presence of homoserine, almost all of the autoinducer produced contains the 15N label from methionine. Thus, it appears that S-adenosylmethionine serves as the amino acid substrate in the luxI-dependent synthesis of the V. fischeri autoinducer.     

Non-equivalence of YEPD and synthetic complete media in yeast reversion studies

In yeast reversion studies, assay of the total number of cells is made by plating irradiated cells on agar plates containing yeast extract, peptone and dextrose (YEPD) medium. The number of revertants are scored by plating cells on synthetic complete (SC) medium deficient in the particular nutrient for which the reversion is tested. In this procedure equivalence for cell survival between the YEPD and the SC media is always assumed. However it is shown in this paper that this assumption is valid only up to dose levels where cell killing is not significant. At high doses, survivals on the two media differ significantly from each other for both high and low LET radiations. This difference influences the slope of the reversion frequency curve at high doses. Since the reversion frequency is expressed with reference to the number of survivors after a given radiation dose, it is essential to see that the same chance of survival is offered to the reverted and unreverted cells.Even though reversion is reported to vary linearly with dose, it is found that this linearity is restricted only to dose levels where cell killing is not significant. At higher doses, the reversion frequency varies in a very complex manner with dose for both high and low LET radiations. The complexity depends further on the reference medium chosen.     

Relationship between cell-mediated and humoral immune attack on tumor cells: II. The role of cellular lipid metabolism and cell surface charge in the outcome of immune attack

Treatment of P815 tumor cells with adriamycin increased their sensitivity to killing by anti-P815 antibody plus C, but not by allogeneic P815-sensitized spleen cells. Conversely, mitomycin C treatment enhanced the cells' sensitivity to cell-mediated, but not antibody-C, killing. Hydrocortisone, but not epinephrine, was effective in increasing the resistance of the cells to killing by both antibody-C and cell-mediated attack systems. The ability of the tumor cells to resist antibody-C killing correlated with their ability to incorporate fatty acid into complex cellular lipid; no such correlation was found between the cellular lipid synthesis and tumor cell susceptibility to cell-mediated killing. Drug or hormone-treated tumor cells exhibited unique changes in cellular lipid synthesis and composition and in cell surface physical properties that correlated with their susceptibility to antibody-C or cell-mediated attack. Cells increased in their sensitivity to antibody-C killing exhibited a decreased cholesterol:phospholipid mole ratio. In contrast, cells rendered more sensitive to cell-mediated killing exhibited an increase in polar phospholipid content and a measurable decrease in net negative cell surface charge density. These data implicate unique chemical and/or physical properties of tumor cells to be of fundamental importance for their ability to resist either humoral or cell-mediated immunologic attack; modulation of one or another of these cellular properties results in a change in the cells' susceptibility to immune killing by antibody plus C or by cytotoxic T lymphocytes.     

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12. Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation.

Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment. Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process. Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine. A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds.     

Kinetics of Mutation Induction by Ultraviolet Light in Excision-Deficient Yeast

We have measured the frequency of UV-induced reversions (locus plus suppressor) for the ochre alleles ade2-1 and lys2-1 and forward mutations (ade2 adex double auxotrophs) in an excision-deficient strain of Saccharomyces cerevisiae (rad2-20). For very low UV doses, both mutational systems exhibit linear induction kinetics. However, as the dose increases, a strikingly different response is observed: in the selective reversion system a transition to higher order induction kinetics occurs near 9 ergs/mm2 (25% survival), whereas in the nonselective forward system the mutation frequency passes through a maximum near 14 ergs/mm2 (4.4% survival) and then declines. This contrast in kinetics cannot be explained in any straightforward way by current models of induced mutagenesis, which have been developed primarily on the basis of bacterial data. The bacterial models are designed to accommodate the quadratic induction kinetics that are frequently observed in these systems. We have derived a mathematical expression for mutation frequency that enables us to fit both the forward and reversion data on the assumptions that mutagenesis is basically a "single event" Poisson process, and that mutation and killing are not necessarily independent of one another. In particular, the dose-response relations are consistent with the idea that the sensitivity of the revertants is about 25% less than that of the original cell population, whereas the sensitivity of the forward mutants is about 29% greater than the population average. We argue that this relatively small differential sensitivity of mutant and nonmutant cells is associated with events that take place during mutation expression and clonal growth.     

DNA synthesis and cell survival after X-irradiation of mammalian cells treated with caffeine or adenine.

The expression of the transient depression in the rate of DNA synthesis normally observed after exposure of randomly-dividing Chinese hamster V-79 or Chinese hamster CHO cells to ionizing radiation can be postponed or diminished by a post-irradiation treatment with 1.0 to 1.0 mM adenine or 1.5 mM caffeine. Caffeine may exert its effect by creating additional sites for replication in irradiated cells. Cells treated with caffeine or adenine for 2 or 4 hours after exposure to 3000 rad of 300 kVp X-rays exhibit depressed synthesis only after the removal of caffeine or adenine. These alterations in the timing of the X-ray-induced depression of the rate of DNA synthesis have no effect on X-ray-induced cell killing. Although a 4 hour post-irradiation treatment of randomly-dividing Chinese hamster V-79 cells with 1.0 or 2.0 mM caffeine potentiates X-ray-induced cell killing, this reduction in survival is due primarily to effects on cells in S-phase.     

Characterization of a cell culture model for the study of adenosine deaminase- and purine nucleoside phosphorylase-deficient immunologic disease.

The absence of erythrocytic adenosine deaminase (ADA) or purine nucleoside phosphorylase (PNP) has been associated with severe immunodeficiency disease in children. We have developed a cell culture model to study the possible relationships between purine salvage enzymes and immunologic function using an established T cell lymphosarcoma (S49) and a potent inhibitor of ADA, erythro-9(2-hydroxy-3-nonyl) adenine (EHNA). Wild-type S49 cells are killed by dexamethasone or dbc AMP, and adenosine (5 muM) in the presence of an ADA inhibitor (6 muM EHNA) also prevents the growth of and kills these S49 cells. It has been proposed that adenosine is toxic to lymphoid cells by virtue of its ability to increase the intracellular concentrations of cyclic AMP. We examined the sensitivity of three mutants of S49 cells, with distinctive defects in some component of cyclic AMP metabolism or action, to killing by adenosine and EHNA. All three mutants are resistant to killing by isoproterenol or cholera toxin and two are resistant to dbc AMP itself, but all are sensitive to killing by adenosine and EHNA. Similarly, two dexamethasone-resistant S49 mutants are as sensitive to adenosine and EHNA as are the wildtype cells. We have also simulated the purine nucleoside phosphorylase deficiency in S49 cells by adding inosine and adenosine to the growth medium. In the presence of EHNA or inosine, the toxic effects of adenosine can be partially reversed by addition of (10-20 muM) uridine, an observation suggesting that adenosine is toxic as the result of its inducing pyrimidine starvation.     

Correlation between the ability of tumor cells to resist humoral immune attack and their ability to synthesize lipid.

Agents that increase (certain metabolic inhibitors, chemotherapeutic agents, and x-irradiation), decrease (hormones), or have no effect (hyperthermia) on the susceptibility of line-1 and line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of these tumor cells to synthesize macromolecules. A correlation was found between the drug-induced increase in sensitivity of these cells to antibody-C mediated killing and the loss of their ability to incorporate fatty acids into complex cellular lipids. Similarly, the hormone-induced increase in resistance of the cells to killing was accompanied by an enhancement in complex lipid synthesis by these cells was also observed after the cells were exposed to physical means of insult (x-irradiation or hyperthermia). No correlation was found between the sensitivity of the cells to antibody-C mediated killing and their ability to synthesize DNA, RNA, protein, or complex carbohydrate, or their capacity for de novo lipid synthesis as measured by incorporation of acetate and glycerol into cellular macromolecules. The assembly of free fatty acids into complex lipid moieties is therefore proposed to be of fundamental importance for the ability of the tumor cells to resist humoral immune killing.     

Nucleoside analog cytotoxicity and bystander cell killing of cancer cells expressing Drosophila melanogaster deoxyribonucleoside kinase in the nucleus or cytosol.

We have recently shown that the overexpression of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) in cancer cell lines increases the cells' sensitivity to several cytotoxic nucleoside analogs and the enzyme may accordingly be used as a suicide gene in combined gene/chemotherapy treatment of cancer. To further characterize the enzyme for possible use as a suicide gene, we constructed a replication-deficient retroviral vector that expressed either the wild-type enzyme that localizes to the cell nucleus or a mutant (arg247ser) that localizes to the cytosol. A thymidine kinase-deficient osteosarcoma cell line was transduced with the recombinant virus and we compared the sensitivity and bystander cell killing when the cell lines were incubated with the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-D-arabinofuranosylthymine. In summary, we showed that the cells' sensitivity and the efficiency of bystander cell killing were not dependent on whether Dm-dNK was located in the nucleus or cytosol.     

Relation between the frequency of various types of reverse mutations in yeasts auxotrophic for adenine and the adenine content of the medium

As shown in the haploid yeast Saccharomyces cerevisiae, the strain 769-p192-15B-n4 (a ade2-192 lys5-3), the rates of reversion to adenine prototrophy are 0.36 X 10(-8), 1.7 X 10(-8) and 2.7 X 10(-8), when the medium contains 100, 10 and 1 mg/l adenine, respectively. Two types of revertants were taken into account: those prototrophic both for adenine and lysine, i. e. suppressors, and those prototrophic for adenine only, most of them being locus revertants. The proportion of locus revertants at 100, 10 and 1 mg/l adenine does not exceed 2, 25 and 41%, respectively. It is assumed that excess adenine (100 mg/l) suppresses the activity of the genes controlling its synthesis, including the mutant ade2 gene. A hypothesis is forwarded, according to which the genes being in the "active" state mutate significantly more frequently than "not working" genes.     

Seminal antioxidants in humans: preoperative and postoperative evaluation of coenzyme Q10 in varicocele patients.

Coenzyme Q10 in seminal fluid shows a direct correlation with seminal parameters except in patients with varicocele. To evaluate whether surgical treatment of varicocele could revert CoQ10 abnormalities, we have studied CoQ10 distribution in thirty-three VAR patients, before and 6-8 months after varicocelectomy, twenty patients with idiopathic oligozoospermia, eleven with isolated asthenozoospermia and sixteen normal fertile men. CoQ10 was assayed in total seminal fluid, plasma or cell pellet by HPLC. A significantly higher CoQ10 proportion in seminal plasma in VAR vs. controls (mean +/- SEM: 61.68 +/- 2.41 vs. 41.60 +/- 1.99%, respectively) was present; total CoQ10 correlated with sperm motility in controls, but not in VAR; an inverse correlation between cellular CoQ10 and motility was present in VAR, but not in controls. Postoperatively, a partial reversion was observed, since the plasma-to-total CoQ10 ratio decreased, but the correlation between total CoQ10 and motility was not restored. On the contrary, the peculiar correlation between cellular CoQ10 and motility was no more detectable in postoperative VAR patients. A partial postoperative reversal of abnormalities in CoQ10 distribution and correlation with seminal parameters was therefore present. As seminal plasma CoQ10 reflects an interchange between intracellular and extracellular compartments, its different distribution could cause a greater sensitivity to peroxidative damage and a reduced utilization for energetic purpose.     

Nucleotide pools and mutagenic effects of alkylating agents in wild-type and APRT-deficient Friend erythroleukaemia cells

Wild-type Friend mouse erythroleukaemia cells (clone 707) were compared with adenine phosphoribosyltransferase (APRT)-deficient mutant subclones (707DAP8 and 707DAP10) for sensitivity to cell killing and mutagenesis by ethyl methanesulphonate (EMS) and methyl methanesulphonate (MMS). Cells were exposed to 0-300 micrograms/ml EMS and to 0-20 micrograms/ml MMS for a period of 16 h. A slight difference was found between wild-type cells and the two APRT-deficient subclones in terms of sensitivity to cell killing by both mutagens. The APRT-deficient subclones were, however, significantly more sensitive than wild-type cells to mutagenesis to 5-bromo-2-deoxyuridine resistance and 6-thioguanine resistance by EMS and MMS. The APRT-deficient subclones were found to have significantly decreased levels of dATP and dTTP nucleotides and decreased levels of all four ribonucleoside triphosphates (ATP, GTP, CTP and UTP) relative to wild-type cells. Wild-type Friend cells were found to have insignificant levels O6-methylguanine-DNA methyl transferase and it is suggested that the increased mutagen sensitivity of APRT-deficient cells may be due to imbalance of deoxyribonucleoside triphosphate pools during DNA excision-repair processes, or more probably due to deficiency of ATP for ATP-dependent DNA excision-repair enzymes.     

SOS mutator effect in E. coli mutants deficient in mismatch correction.

We have used bacteriophage lambda to characterize the mutator effect of the SOS response induced by u.v. irradiation of Escherichia coli. Mutagenesis of unirradiated phages grown in irradiated or unirradiated bacteria was detected by measuring forward mutagenesis in the immunity genes or reversion mutagenesis of an amber codon in the R gene. Relative to the wild-type, the SOS mutator effect was higher in E. coli mismatch correction-deficient mutants (mutH, mutL and mutS) and lower in an adenine methylation-deficient mutant ( dam3 ). We conclude that a large proportion of SOS-induced 'untargeted' mutations are removed by the methyl-directed mismatch correction system, which acts on newly synthesized DNA strands. The lower SOS mutator effect observed in E. coli dam mutants may be due to a selective killing of mismatch-bearing chromosomes resulting from undirected mismatch repair. The SOS mutator effect on undamaged lambda DNA, induced by u.v. irradiation of the host, appears to result from decreased fidelity of DNA synthesis.