Isolation of relaxed-control mutants of Escherichia coli K-12 which are sensitive to glucose starvation

Mutants of $\text{Escherichia coli}$ K-12 which are sensitive to glucose starvation were isolated by an enrichment procedure using thymine starvation to select for nongrowing cells. Eleven independent isolates were obtained by this method. The mutants are also sensitive to glycerol starvation and to a lesser extent to nitrogen or amino acid starvation. The mutants are more sensitive than the parental strain to inhibitors of protein synthesis but not inhibitors of RNA or DNA synthesis. [³H]-leucine incorporation experiments indicate that protein synthesis is blocked in the mutants during recovery from glucose starvation or chloramphenicol inhibition. Incorporation of [³H]uridine in amino acid-starved cells demonstrates that the mutants are partially relaxed for control of RNA synthesis. Physiological and genetic experiments indicate that these mutants are different from previously isolated relaxed-control mutants.     

In vivo radiation-induced thymine residue release from E. coli DNA

Cells of $\text{E. coli}$ C thy^?321 are examined for thymine residue release from DNA following gamma-irradiation from 5 to 15 krad. Experimental conditions are designed to inhibit enzyme activity that might promote base residue release. Enzyme action is restricted in order to assess the physicochemical action of radiation on cellular DNA, and to this end irradiation is done under O₂, N₂, and N₂O saturating conditions. Both thymine and thymidine release from bacterial DNA are detected and quantitated, and three oxygen effects are noted in comparing yields of these products. No difference in effect is observed between N₂ and N₂O gassing conditions, suggesting that the hydroxyl radical has little effect on thymine or thymidine release from irradiated DNA $\text{in vivo}$.     

A comparison of the utilization of thymine and thymidine for the synthesis of DNA-thymine in novikoff hepatoma cells

A comparison was made between the utilization of thymine and thymidine for the synthesis of DNA in Novikoff hepatoma cells growing in suspension culture. When the cell cultures were switched from exponential growth to a relatively non-growing condition, by resuspending them in culture media minus serum for 18 h, there was an 85% decrease in the rate of thymidine incorporation but only a 15% decrease in the rate of thymine incorporation. Exposure to an alkylating agent (methyl methane sulfonate) resulted in a 79% decrease in thymidine incorporation, while thymine incorporation was decreased only 35%. Thymidine at a concentration equal to its Km for incorporation into DNA (4 × 10⁻⁷ M) had virtually no effect on thymine incorporation. It was not until a thymidine concentration of ten times the K_m was employed that appreciable (40%) decreases in the rate of thymine incorporation were observed. Examination of total cellular DNA or nuclear DNA gave similar results. These studies are interpreted as indicating the presence of multiple precursor pools for the synthesis of DNA-thymine in Novikoff hepatoma cells.     

Excision of thymine dimers by proteolytic and amber fragments of E. coli DNA polymerase I

Excision of thymine dimers from specifically incised ultraviolet irradiated DNA by $\text{E. coli}$ DNA polymerase I is stimulated by concurrent DNA synthesis. The 36,000 molecular-weight “small fragment” obtained by limited proteolysis of DNA polymerase I, which retains only the 5′ → 3′ exonuclease activity, also excises thymine dimers, but at one-tenth the rate of the intact enzyme. However, the rate of excision is increased by addition of the “large” 76,000-molecular weight fragment. With the further addition of the 4 deoxynucleoside triphosphates, permitting DNA synthesis to occur, excision approaches rates observed with the intact enzyme. The same result was obtained with a fragment of DNA polymerase I with 5′ → 3′ exonuclease activity that is present uniquely in polymerase I $\text{amber}$ mutants.     

In vitro release of thymine from DNA by neocarzinostatin.

$\text{In vitro}$ degradation of DNA to acid soluble products was induced by the combined action of neocarzinostatin and sulfhydryl agent as 2-mercaptoethanol, dithiothreitol, or reduced glutathione, but not other reducting agent as ascorbic acid or NaBH₄. From the analysis by Sephadex G-10 gel filtration, acid soluble products were found to be thymine and oligonucleotide, but not thymidylic acid and thymidine. Release of adenine or guanine from DNA was not detected.From these results, it is suggested that DNA chain breakage by the combined action of neocarzinostatin and 2-mercaptoethanol may be due to an indirect phosphodiester bond breakage with release of thymine.     

Inactivation of SV40 replication by derivatives of benzo[a]pyrene.

When African green monkey kidney cell lines, infected with simian virus 40, were exposed to benzo[a]pyrene-7,8-dihydrodiol or $\text{anti}$-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, inhibition of progeny virus formation was observed. Alkylation of SV40 DNA with $\text{anti}$-BPDE inhibits the infectivity of this viral DNA; however, the inactivation does not follow a single-hit mechanism. Studies on [³H]thymidine incorporation indicate that SV40 DNA synthesis is markedly impaired for the first 12 hours following BPDE treatment; 24 to 36 hours later, however, SV40 DNA synthesis is almost normal. These data suggest that the inhibition of SV40 DNA synthesis by BP derivatives is reversible and that the observed reduction in viral titer requires some other explanation.     

Underestimation of DNA Synthesis by [3H]Thymidine Incorporation in Marine Bacteria

A direct comparison of [³H]thymidine incorporation with DNA synthesis was made by using an exponentially growing estuarine bacterial isolate and the naturally occurring bacterial populations in a eutrophic subtropical estuary and in oligotrophic offshore waters. Simultaneous measurements of [³H]thymidine incorporation into DNA, fluorometrically determined DNA content, and direct counts were made over time. DNA synthesis estimated from thymidine incorporation values was compared with fluorometrically determined changes in DNA content. Even after isotope dilution, nonspecific macromolecular labeling, and efficiency of DNA recovery were accounted for, [³H]thymidine incorporation consistently underestimated DNA synthesized by six- to eightfold. These results indicate that although the relationship of [³H]thymidine incorporation to DNA synthesis appears consistent, there are significant sources of thymine bases incorporated into DNA which cannot be accounted for by standard [³H]thymidine incorporation and isotope dilution assays.     

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

Uptake of 125I-iododeoxyuridine in mouse organs during deuteration of body water: Evidence for a thymus-specific effect

The effects of body water deuteration on mammalian DNA synthesis $\text{in vivo}$ during the deuterium equilibration period in the body were studied. Young adult mice were given 15% or 30% D₂O in the drinking water for 4, 10 or 21 days. Control mice were given distilled water. Eighteen hours prior to sacrifice, ¹²⁵IUdR, a conveniently monitored synthetic analogue of the DNA precursor thymidine, was injected intravenously. Although neither radioiodine activity of the total body nor body weight varied significantly among the three groups, thymic radioactivity per g tissue was significantly lower in mice given 30% D₂O and, to a lesser extent, in mice given 15% D₂O than in the control group. In contrast, intestine and hemopoietic bone marrow displayed minor changes in ¹²⁵IUdR incorporation. This reduction of ¹²⁵IUdR incorporation is discussed in relation to the particular importance of thymidine reutilization in the thymus.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Radiation chemistry of nucleic acids: identification of the major hydroperoxy thymine

The major product from γ-radiation of thymine in aerated aqueous solution is shown to be $\text{cis}$-5-hydroxy-6-hydroperoxy-5,6-dihydrothymine by considering its uv, ir, nmr spectral data, the nmr deshielding effect on the OOH proton, and its reduction to $\text{cis}$-thymine glycol. These suggest that the earlier structural assignment [Cadet and Teoule, Biochem. Biophys. Acta., $\text{238}$, 8 (1971)] was in error. Furthermore, HOOH oxidation of either $\text{cis}$- or $\text{trans}$-thymine glycol in acidic condition gives this hydro-peroxide in yields >90%. This result again directly contradicts that reported in the previous paper. Our present findings are readily explained by considering the chemistry of thymine glycols and the reaction of OH radicals with thymine. Also, contrary to the earlier notion that this peroxide should be unstable, we find that it is sufficiently stable for studying the thymine peroxide interaction with chromosomes, bacterial cells and nucleic acid components.     

Increase in dATP pool in aphidicolin-resistant mutants of mouse FM3A cells

Mutants that were resistant to aphidicolin were isolated from mutagenized mouse FM3A cells at a frequency of about 10^?6. Resistance to aphidicolin in these mutants was not due to an effect on [³H]thymidine incorporation into DNA, DNA synthesis in permeabilized cells, or DNA polymerase α.All the mutants showed a greatly increased dATP pool and decreased ability to incorporate [³H]deoxycytidine into DNA. They also showed cross-resistance to both 1-β-D-arabinofuranosyladenine and 1-β-D-arabinofuranosylcytosine.These results indicate that an enzyme involved in production of dATP or its regulation is altered in these mutants. It is suggested that dATP competes with aphidocolin at its killing site or that dATP reverses the effect of aphidicolin by some unknown mechanism $\text{in}\text{vivo}$.     

Correlation between synthesis and methylation of DNA in HeLa cells

For the whole cell cycle the methylation of DNA was studied in synchronized $\text{HeLa}$ cells and in nuclei isolated from them. In the intact cells the methylation of DNA cytosine runs parallel to DNA synthesis. The pattern of DNA cytosine methylation by the isolated nuclei is almost identical to that obtained with the whole cells. Since the isolated nuclei do not synthesize DNA, it is shown that DNA methylation continues for at least 30 min after DNA synthesis is over. No DNA minor thymine is found in the isolated nuclei.     

Adaptation of thymidine utilization to changing rates of DNA synthesis in the cell cycle

Summary In synchronous cultures of P-815 murine mastocytoma and of Chinese hamster ovary (CHO) cells, the relative contribution of exogenous thymidine to DNA synthesis was studied by comparing rates of (³H)thymidine incorporation with the rate of DNA synthesis as derived from incorporation of (³H)thymidine (10^–5 m) in the presence of amethopterin. In synchronous P-815 cultures, time-dependent variations of DNA synthesis rates were in close agreement with those of (³H)thymidine incorporation rates at concentrations of the precursor ranging from 5 × 10^–8 to 10^–5 m. Similarly, in synchronous CHO cell cultures prepared by two different methods, time-dependent changes in DNA synthesis rate were almost identical with those of the rate of incorporation of (³H)thymidine supplied at 5 × 10^–8 m. Thus, at a given thymidine concentration in the medium, the proportion of thymine residues in DNA that were derived from exogenous thymidine remained nearly constant, even though rates of cellular DNA synthesis underwent pronounced changes. This indicates that in the synchronous culture systems used, utilization of exogenous thymidine is efficiently adapted to changing rates of DNA synthesis.In partial fulfillment of the requirements for the degree of Ph.D. by G.G.M.     

Letter: On the source of "minor thymine" in DNA from a Novik off rat hepatoma cell line

To determine if a fraction of DNA-thymine might derive from deamination of DNA-5-methylcytosine in polymeric DNA, the occurrence of tritiated DNA-thymine residues was measured in DNA isolated from Novikoff rat hepatoma cells incubated with varying levels of unlabeled thymidine and with G-³H-labeled deoxycytidine, unlabeled deoxyguanosine, deoxyadenosine, adenosine, serine, glycine and amethopterin. At 0 and 10^?5 M-thymidine, significant incorporation of label was recovered as DNA-thymine. At higher levels of thymidine (5× 10^?5m to 10^?3m) label in DNA-thymine progressively decreased to levels that could be accounted for by deamination of DNA-thymine by the two different two-dimensional Chromatographic systems used to separate free DNA bases. “Minor thymine” in DNA of Novikoff hepatoma cells is thus generated by incomplete amethopterin blockade, insufficient dilution by exogenous thymidine and methodological deamination.     

Further characterization of a ribonucleotide-polymerizing enzyme from Histoplasma capsulatum. II. Possible role in cellular metabolism

An enzyme, ribonucleotide polymerase, isolated from the yeast phase of a fungus, $\text{Histoplasma capsulatum}$ has been found to stimulate the incorporation of dTMP in the reaction catalysed by DNA polymerase from $\text{H. capsulatum}$ and $\text{E. coli}$. The stimulation is dependent on the amount of ribonucleotide polymerase added. The data indicate that protein-protein interaction is responsible for the increase in DNA synthesis. It is suggested that ribonucleotide polymerase may be involved in supplying short RNA primers for DNA polymerase.     

Quantitation of Some DNA Precursor Data

THE work of Kornberg on DNA repair and synthesis^1,2 implicates deoxyribonucleoside 5′-triphosphate as a direct precursor of DNA synthesis. This relationship was questioned by the possibility of alternative replication schemes^3,4. Werner⁵ studied the flux of thymine and thymidine into Escherichia coli DNA to determine the in vivo precursors of replicating DNA. Werner studied the incorporation of ³H labelled thymine into DNA and intracellular nucleotide pools under steady state conditions, in which thymine is converted into thymidine, thymidine monophosphate (TMP), thymidine diphosphate (TDP) and thymidine triphosphate (TTP). Werner measured separately the activities of labelled TMP, TDP, TTP and DNA at various times after E. coli cells had been exposed to a ³H-thymine synthetic medium. From a qualitative consideration of his results, Werner concluded that both TDP and TTP—but not TMP—were possible direct precursors of DNA replication.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

Synthesis of small polynucleotide chains in thymine-depleted bacteria

Bacteria that are depleted of intracellular thymidine nucleotide pools incorporate [³H]thymine at full specific activity, allowing the detection of early intermediates in DNA replication. A short pulse of [³H]thymine is incorporated almost exclusively into very small DNA chains which, during further incorporation of thymine, are converted into larger chains and high molecular weight DNA. The synthesis of these small DNA chains depends on the products of dna genes B, E and G. Analysis of the DNA by gel filtration on Sepharose 2B revealed an abundance of extremely short DNA chains while the frequency of larger chains decreased exponentially with increasing size. This size distribution of small DNA chains suggests a mechanism of DNA replication in which larger chains (Okazaki pieces, Okazaki et al., 1968a) arise through joining of extremely short polynucleotide chains in a process resembling crystallization rather than unidirectional chain elongation.