Elisabeth Bautz Freese

Mutagenic and Inactivating DNA Alterations

typic consequences has greatly benefited from the advancement of biochemical and genetic methods. 1. The alteration of nucleic acid bases can be measured by UV,1 infrared and nuclear magnetic spectra, use of radioactive bases in nucleic acids, chromatography, and straightforward chemistry. Cross-linking can be shown by reversible DNA denaturation, and backbone breakage by decreases in the sedimentation constant, viscosity, or light scattering. To uncover the primary site of attack and the high specificity of some chemicals toward nucleic acids, synthetic oligo- and polynucleotides will have to be increasingly used. 2. The methods for determining the phenotypic consequences of nucleic acid alterations range far afield. Chromosomal breaks and large chromosomal alterations can be determined cytologically or by classical genetic methods. The extent and specificity of small nucleic acid alterations can be analyzed both by genetic fine structure measurements and by the specificity of forward and reverse mutation induction. The correlation between nucleic acid alterations and genetic investigations has been possible mainly by the use of viruses or transforming DNA for which the effect of chemicals can be safely attributed to the direct effect on nucleic acids. Finally, the analysis of mutagenic nucleic acid alterations can be used, together with in vitro experiments on protein synthesis, to determine the base sequence of some genetic regions. The consistency of these results checks in turn the validity of the genetic and biochemical conclusions.

Inactivating DNA alterations induced by peroxides and peroxide-producing agents

Abstract Agents containing a free-NOH group such as hydroxylamine, N -methylhydroxylamine, hydroxyurea, hydroxyurethan, and hydrazines produce H 2 O 2 on exposure to oxygen. All these agents, H 2 O 2 itself, and disuccinyl peroxide, predominantly induce inactivating DNA alterations, whereas their mutagenic effect (per lethal hit) on transformung DNA is small. Chemicals containing NH 2 in place of NOH, such as urea and urethan (= ethylcarbamate) do not inactivate DNA. Formaldehyde and its oxidation products, formic acid, performic acid, and di-hydroxymethyl peroxide do not inactivate or mutate transforming DNA at a significant rate.

DNA damage caused by antidepressant hydrazines and related drugs.

Abstract Antidepressant hydrazines ( e.g. isocarboxazid, nialamide, and phenelzine) and the anti-tubercle agent isoniazid react with oxygen, producing hydrogen peroxide. The radicals formed in this reaction inactivate DNA. These compounds, as well as carbamates that can be enzymatically converted to reactive N -hydroxycarbamates, may therefore cause chromosomal breaks and mutations.

Comparison of mutation and inactivation rates induces by bacteriophage and transforming DNA by various mutagenes

Abstract Inactivation and mutation rates were measured for T 4 phages and Bacillus subtilis transforming DNA treated by low pH, nitrous acid, or hydroxylamine at different temperatures. The frequency of mutants increased linearly with time for all three agents, whereas the logarithm of survival gave a linear plot only for nitrous acid and low pH. An Arrhenius plot showed the same slopes for both inactivation and mutation rates after treatment with low pH or nitrous acid; for the latter agent the slope remained unaltered even when the DNA was treated in the denaturated state. In contrast, mutation rates obtained after the exposure to hydroxylamine differed greatly for native or denatured DNA, phate T 4 being intermediate. Treatment by low pH or nitrous acid interrupted the genetic linkage between tryptophan and histidine, the interrrupting hits being about 1 3 as frequent as lethal hits, independent of the temperature.

Optimal extraction conditions for high-performance liquid chromatographic determination of nucleotides in yeast

Different extraction methods of nucleotides from the yeast Saccharomyces cerevisiae were compared. A new extraction solution--formic acid saturated with 1-butanol--was found to be more effective than the commonly used solutions of trichloroacetic acid, perchloric acid, or formic acid alone. Using this solution the optimal extraction conditions were established. Nucleotide recovery was evaluated by adding standard nucleotides to the extraction medium and carrying them together with the cells through the whole extraction procedure. Nucleotides were separated and quantitated by high-performance liquid chromatography on an anion-exchange column.

The oxygen-dependent reaction of hydroxylamine with nucleotides and DNA

Abstract Hydroxylamine reacts, at low concentrations (10 −2 M), with DNA, as exhibited by the initial increase (melting) and later decline of the extinction at 260 mμ which can be observed at temperatures far below the melting temperature of DNA; the reaction rate increases with the pH. Under the same conditions several nucleotides show a reaction which has not been observed at high hydroxylamine concentrations. All these reactions are oxygen dependent, and they are quenched by catalase, peroxidase and Tris, but most effectively by pyrophosphate. This indirect effect of hydroxylamine is contrasted to the direct effect of high hydroxylamine concentrations on cytosine and uracil and is explained by the production of peroxides and free radicals.

Partial deprivation of GTP initiates meiosis and sporulation in Saccharomyces cerevisiae

SummaryWe have investigated the physiological conditions under which meiosis and the ensuing sporulation of Saccharomyces cerevisiae are initiated. Initiation of sporulation occurs in response to carbon, nitrogen, phosphorus, or sulfur deprivation, and also, when met auxotrophs are partially starved for methionine, but not after starvation of other amino acid auxotrophs. It also occurs after partial starvation of pur or gua auxotrophs for guanine but not after starvation of ura auxotrophs for uracil. Under all these sporulation conditions the concentrations of both guanine nucleotides (GTP) and S-adenosylmethionine (SAM) decrease whereas those of other nucleotides show no trend. We show that the decrease of guanine nucleotides is essential for the initiation of meiosis and sporulation: when a gua auxotroph, also lacking one of the two SAM synthetases, is starved for guanine but supplemented with 0.1 mM methionine, GTP decreases while SAM slightly increases and yet the cells sporulate.

Induction of sporulation in developmental mutants of Bacillus subtilis

SummarySporulation of the standard strain of Bacillus subtilis can be induced by decoyinine (a specific inhibitor of guanosine monophosphate synthesis) in the presence of excess ammonium ions, rapidly utilizable carbohydrates and phosphate. Certain developmental mutants unable to sporulate in usual sporulation media can also be induced to sporulate under these conditions. Among the inducible strains are mutants blocked in the citrate cycle, in phosphoenolpyruvate carboxykinase or one of the glycerolphosphate dehydrogenases (NAD dependent or independent). A mutant lacking phosphoglycerate kinase activity and able to grow on glucose and L-malate but not on either carbon source alone, can also be induced to sporulate in a medium containing both glucose and malate. Among other sporulation mutants whose biochemical deficiency is not known, some are weakly inducible whereas many, blocked at sporulation stages O, II, III, or IV, are not inducible. The results show that the functions of the citrate cycle (used for ATP synthesis) and of gluconeogenesis (used for glycerol phosphate and D-glucosamine-6-phosphate synthesis), normally needed for sporulation under conditions of carbohydrate deprivation, are no longer required when sporulation is induced by decoyinine in the presence of carbohydrates. Mutants in which sporulation is highly inducible can be used as a sensitive tool for the detection of other inducing compounds because their spontaneous (uninduced) background of spores is very low.

Initiation of meiosis and sporulation of Saccharomyces cerevisiae by sulfur or guanine deprivation.

Homothallic Saccharomyces cerevisiae, growing exponentially in a synthetic acetate medium, could be initiated to undergo meiosis and subsequent sporulation by removal of sulfur from the medium or by partial purine deprivation of purine auxotrophs or, most efficiently, by guanine deprivation of a guanine auxotroph. In contrast, partial uracil deprivation of uracil auxotrophs did not cause sporulation. Under any of the above and other sporulation conditions, the intracellular concentrations of GTP and, usually at some time later, S-adenosylmethionine (SAM) decreased; the concentrations of the other nucleoside triphosphates decreased under some but increased under other sporulation conditions. The addition of 1 mM methionine or, more effectively, of SAM or the combination of adenine plus methionine greatly increased the intracellular concentration of SAM and reduced or prevented sporulation, even when GTP decreased. However, differentiation can be inhibited by an excess of many metabolites which do not specifically control the initiation process; in particular, SAM is known to inhibit yeast metabolism (e.g., transamination). Therefore, we cannot yet decide whether the deficiency of GTP or SAM (or related compounds) serves as a signal for the initiation of meiosis/sporulation.

Adaptation of Glucose-grown Saccharomyces cerevisiae to Gluconeogenic Growth and Sporulation

When cells of Saccharomyces cerevisiae growing exponentially on d-glucose as sole carbon source were washed and transferred to buffered yeast nitrogen base containing 100 mM-acetate, they were unable to resume growth for several days whereas they adapted within a few hours to grow (slowly) on ethanol, and within 12 h to grow on pyruvate. After the cell transfer, oxygen consumption and ATP concentration decreased rapidly but recovered within a few hours on ethanol, more slowly on pyruvate, and only after 70 h on acetate. When the acetate culture had lost all detectable ATP, the viable cell titre slowly decreased until after 70 h enough cells had adapted to resume growth. At lower acetate concentrations (optimally 5 to 15 mM), ATP decreased less, and growth resumed within 1 d. After transfer from glucose medium to buffer plus a carbon source, cells sporulated equally well at ethanol concentrations from 20 to 150 mM and at pH 5.5 or 7.0; with dihydroxyacetone, another uncharged carbon source, sporulation was optimal at concentrations between 30 and 50 mM and about equal at pH 5.5 and 7.0. In contrast, after transfer from glucose medium to buffer plus acetate, cells sporulated at pH 5.5 optimally with 15 mM-acetate but not with 50 mM-acetate or more; at pH 7.0 sporulation showed a broader optimum of acetate concentration around 50 mM. The results indicated that in cells not adapted to gluconeogenesis, high concentrations of neutral acetic acid molecules caused complete consumption of intracellular ATP; consequently the cells could not adapt to gluconeogenesis for a long time.