Concentrations Of Thymine Research Articles

Determination of deoxyribonucleic acid replication time in exponentially growing Escherichia coli B/r

The time necessary to replicate the chromosome (C period) was measured in Escherichia coli B/r (ATCC 12407) and a low-thymine-requiring derivative of that strain. In the Thy- strain, C was measured as a function of growth rate and exogenous thymine concentration either from step-up or chloramphenicol experiments. In the Thy+ parental strain, C was measured only as a function of the growth rate and only by the chloramphenicol method. The C period was found to decrease with growth rate and, in the Thy- strain, the C period also decreased with increasing thymine concentration. It approached a value of approximately 37 min at high growth rates.

Discrimination between bromouracil and thymine for uptake into DNA in drm − and dra − mutants of Escherichia coli K12

The relative efficiency of bromouracil and thymine for uptake into DNA was measured in various thymine-requiring strains of Escherichia coli K12. It was found that: 1. 1. Mutants with genotype thyA − dra − discriminate against bromouracil to much greater extent than do mutants with genotype thyA − drm −. 2. 2. The discrimination in dra − mutants is dependent on thymine concentration, whereas discrimination in drm − mutants is almost independent of thymine concentration. It is suggested that the intracellular level of deoxyribose 5-phosphate affects the efficiency of uptake into DNA of bromouracil relative to thymine.

Initiation and termination of deoxyribonucleic acid replication in bacteria after a stepwise increase in the velocity of replication

The theoretical relations between replication, initiation, termination, and deoxyribonucleic acid (DNA) accumulation were derived for experiments in which the length of the time required for the replication of the bacterial chromosome (C period) can be varied. This theory enables one to determine absolute values of the C period from kinetics of DNA accumulation after a "stepup" with thymine-requiring bacteria that are subjected to a sudden increase in the exogenous thymine concentration. Application of this method of data evaluation to an observed step-up experiment with a thy-derivative of Escherichia coli B/r (ATCC 12407) indicated that the theory describes the observed post-step accumulation of DNA accurately within experimental errors. It is also concluded that changes in the replication velocity (C) do not measurably affect the timing of initiation events in a culture.

Effect of thymine concentration on the mode of chromosomal replication in Escherichia coli K-12

Determination of mutation frequencies after pulse treatment with nitrosoguanidine was used to study the direction of chromosome replication in a thymine-requiring strain of Escherichia coli K-12. With a high thymine concentration, the mode of replication was found to be bidirectional, whereas at low thymine concentration chromosome synthesis was unidirectional. As a consequence, we suggest that termination of replication occurs either when two forks meet or when one fork has completed one round of replication.

The effect of 5-Iodouracil on the growth and biosynthetic processes of bacteriophage T4 td8 in the absence of light

5-Iodouracil (IUra)-substituted progeny bacteriophage T4 td8 were grown under conditions such that, upon CsCl equilibrium isopycnic gradient centrifugation, progeny with density distributions about the median similar to that of unsubstituted phage are obtained. In the absence of light a monotonie relationship exists between decreasing progeny viability and increasing percent IUra substitution. IUra is equivalent to thymine as a growth factor on a molar basis, and at concentrations of IUra plus thymine above that required for maximum particle production, the percent IUra substitution in phage DNA is determined by the mole fraction of IUra in the medium. The lethal effects of 5-iodo-2'-deoxyuridine (IdUrd) and IUra are equivalent, and are not produced by a direct effect on the phage particles. At equivalent percent substitution in phage DNA the order of lethality is IUra > 5-bromouracil (BrUra) > 5-chlorouracil (ClUra). There is no interference with the transfer of thymine from host cell to progeny phage by the presence of IUra in the medium, and IUra affects neither the time of lysis nor the content of phage DNA in the infected cells.

The virulence of trimethoprim-resistant thymine-requiring strains of Salmonella.

A thymine-requiring (thy-), trimethoprim-resistant (tmpr) mutant isolated from the faeces of chickens experimentally infected with salmonella typhimurium and treated with mixture of trimethoprim and sulphadiazine was less virulent for chickens than the parent strain and a thy+tmps revertant prepared in vitro from the mutant. The difference in chicken-virulence was more noticeable when the strains were administered orally than when they were administered subcutaneously. All tmpr mutants prepared in vitro from four other salmonella strains were also thy-; those tested were less virulent for chickens and mice than their parent strains. After oral infection, thy- salmonella organisms were found much less commonly in the alimentary tract of chickens then were thy+ organisms. This was especially so in the caeca, the principal site of colonization of both the thy+ and thy- organisms. Relatiely high concentrations of thymine or related compounds were found in the contents of all regions of the alimentary tract of chickens except the caeca; the caeca usually contained low or undetectable concentrations. The thy- salmonella strains would not grow on one brand of briliant green agar because of its deficiency in thymine; their colonial and appearance on other kinds of media used for isolating salmonellae from clinical material was often 'un-salmonella-like'.

Studies of intracellular thymidine nucleotides. Relationship between the synthesis of deoxyribonucleic acid and the thymidine triphosphate pool in Escherichia coli K12.

The two types of mutant strains which show resistance to T-even phage infection have been isolated and been shown to have either a higher or lower ratio of dTDP-sugar to dTTP than that of the parent strains. The one with a higher ratio of dTDP-sugar to dTTP than the parents has a large dTDP-sugar pool and small dTTP pool, and a high level of dTDPG pyrophosphorylase activity. The other one, with a lower ratio of dTDP-sugar to dTTP than the parents, has a small dTDP-sugar pool and large dTTP pool, and a low or deficient level of this enzyme activity. They form an entirely mucoid colony in the synthetic agar plate. Mutant cells (Ter-6 and Ter-21) which have deficient dTDPG pyrophosphorylase activity show 2 -- 3 times higher activity of UDPG pyrophosphoyrlase than that of parent cells. The dTDPG pyrophosphorylase-deficient mutants (Ter-15 and Ter-21) have a 3 -- 4 times higher concentration of dTTP and a faster rate of DNA synthesis and cell division than those of parent strains in growth with external thymine. The dTDPG pyrophosphorylase constitutive mutant (Ter-4) has a 0.5 -- 0.33 smaller dTTP pool and a slower rate of DNA synthesis and cell division than those of parent cells grown in the same medium. In the Ter-15 and Ter-21 mutants, the intracellular dTTP-dependent DNA synthesis rapidly disappeared in thymine suboptimal concentration, but the Ter-4 mutant maintained its dTTP-dependent DNA synthesis over a 20 muM concentration of external thymine. In high concentration (100 muM) of external thymidine, the thymidine effects on the intracellular dTTP concentration do not significantly appear in these enzyme-deficient mutants (Ter-15 and Ter-21). Also, the concentration of intracellular dTTP in the cell growth with external thymidine is 2.5 times greater than that with external thymine in these enzyme-deficient mutants (Ter-15 and Ter-21).

Relationship between chromosome replication and cell division in a thymineless mutant of Escherichia coli B/r.

The relationship between chromosome replication and cell division was investigated in a thymineless mutant of Escherichia coli B/r. Examination of the changes in average cell mass and DNA content of exponential cultures resulting from changes in the thymine concentration in the growth medium suggested that as the replication time (C) is increased there is a decrease in the period between termination of a round of replication and the subsequent cell division (D). Observations on the pattern of DNA synthesis during the division cycle were consistent with this relationship. Nevertheless, the kinetics of transition of exponential cultures moving between steady states of growth with differing replication velocities provided evidence to support the view that the time of cell division is determined by termination of rounds of replication under steady-state conditions.

The biochemical basis of the antimicrobial action of sulfonamides and trimethoprim in vivo—II. Experiments on the limited thymine and thymidine availability in blood and urine

Escherichia coli 15T −, which undergoes “thymineless death” upon thymine deprivation, is not viable in human blood and urine. The addition of as little as 0·05 μg/ml thymidine results in a distinct growth promoting effect in both fluids whereas the thymine concentrations needed for growth are higher. Growth kinetics with thymine are different from those observed with thymidine. By comparison of the growth of E. coli 15T − in synthetic media that in blood and urine it is assumed that the concentrations of thymidine and thymine in the biological fluids are below 0·05 μg/ml and 0·2 μg/ml, respectively. These concentrations are not sufficient to antagonize the bactericidal action of trimethoprim, sulfonamides or their combinations. From the growth characteristics of E. coli 15 (TAU) − in blood it is concluded that other pyrimidines such as uracil (or uridine) are present and that there is a specific lack of thymine and thymidine. The in vivo conditions for the action of antifolates are discussed.

Purification of thymidine phosphorylase from Escherichia coli and its photoinactivation in the presence of thymine, thymidine, and some halogenated analogs.

Isoelectric focusing was used as the final step in the isolation of thymidine phosphorylase which was found to have an isoelectric point of 4.1. Analytical acrylamide gel electrophoresis showed the purified enzyme preparation contained one major protein band which stained for thymidine phosphorylase activity and usually a minor, faster migrating band devoid of activity. Inactivation of thymidine phosphorylase alone or in the presence of sensitizers by ultraviolet light, primarily at 253.7 nm, followed first order inactivation kinetics. The rate of inactivation of the enzyme was the same at pH 5 and 7.4 and the addition of various pyrimidine bases and nucleosides enhanced the inactivation rate at both pH values, but to a greater extent at pH 5. Linear plots of inactivation rates versus concentrations of thymidine or thymine were the same. At 7.8 mM thymidine or thymine, 11- and 4.4-fold increases in photoinactivation of thymidine phosphorylase were observed at pH 5 AND 7.4 RESPECTIVELY. Parabolic curves were obtained with increasing concentrations of either 5-iodo-2'-deoxyuridine or 5-iodouracil. 5-Iodouracil at 5.2 mM caused 212- (pH 5) and 100- (pH 7.4) FOLD INCREASES IN THE RATES OF PHOTOINACTIVATION OF THYMIDINE PHOSPHORYLASE. However, 5-iodo-2'-deoxyuridine at 5.0mM only enhanced the photoinactivation of enzyme by factors of 83 (pH 5) and 21 (pH 7.4). Neither 5-bromo-2'-deoxyuridine or 5-bromo-uracil was as potent in sensitizing the enzyme as the iodo analogs. Combinations of 5-iodouracil or 5-iodo-2'-deoxyuridine with thymine resulted in higher inactivation rates than the additive inactivation rates of individual compounds, whereas combinations of either iodo analog with thymidine resulted in lower inactivation rates. Increasing concentrations of phosphate or NaCl lessened the photoinactivation rate of thymidine phosphorylase alone and protected the enzyme from the sensitization caused by the different bases and nucleosides. No quantitative changes in the number of primary amino groups in thymidine phosphorylase was evident as a result of irradiation in the presence or absence of 5-iodouracil or 5-iodo-2'-deoxyuridine. Examination of the irradiated enzyme on Sephadex G-150 indicated that a larger protein species is formed and that 5-iodouracil promotes this process.

The biochemical basis of the antimicrobial action of sulfonamides and trimethoprim In vivo—I. : Action of sulfonamides and trimethoprim in blood and urine

Trimethoprim (2 μg/ml), sulfamethoxazole (5 μg/ml) and a combination of these compounds reduced the viable cell count of Escherichia coli in human blood and urine in a bactericidal manner. This bactericidal action can be prevented by thymidine or thymine, the effectiveness of thymidine being considerably greater than that of thymine. Growth kinetics in the presence of trimethoprim, thymidine or thymine differed strikingly. Concentrations of thymidine below 0.05 μg/ml were detectable by their growth promoting effect, which started without a lag. Considerably higher concentrations of thymine, however, did not initially prevent the onset of cell inactivation, but resulted in a higher level of survivors. It is concluded that the concentrations of thymidine and thymine in blood and urine are too low to prevent a bactericidal action of trimethoprim, sulfonamides or a combination of these whereas other compounds, such as amino acids and purines, which support the bactericidal action, are present in sufficient quantities.

Thymineless mutagenesis in Escherichia coli.

To clarify the relationship between thymineless death and thymineless mutagenesis, the induction of arginine revertants of Escherichia coli TAU-bar by thymine starvation was examined in physiological terms. Induced revertants were detectable both on minimal medium lacking arginine and minimal medium supplemented with 1 mug of arginine per ml. Substantial thymineless mutagenesis occurred during the period before the onset of thymineless death. Mutagenesis and loss of viability were observed upon incubation in medium lacking thymine and arginine, and both were inhibited upon incubation in medium lacking thymine and uracil. Mutagenesis also occurred during thymine starvation at 25 C, where there was relatively little loss of viability. At 37 C thymineless mutagenesis did not require complete thymine starvation, and the induction of revertants appeared to be initiated at the same suboptimal thymine concentration at which lethality was first detectable. Mutagenesis was found not to occur preferentially at the growing point of deoxyribonucleic acid replication. These results suggest that thymineless mutagenesis does not involve simply errors in base pairing due to the absence of thymine. The data also suggest that the induction of mutations and thymineless death are due to the same primary event but that mutagenesis is the more sensitive response.

CHARACTERISTICS OF THE PHENOTYPICAL THYMIDINE RESISTANT MUTANTS OF <i>ESCHERICHIA COLI</i> K12

Studies were made on mutants from a deoxyriboside-sensitive Thy- strain selected for their ability to grow on thymidine at high concentration, which still require a low concentration of thymine for growth. They were found to be divided into two groups with regard to the inducibilities of their thymidine phosphorylase and purine nucleoside phosphorylase. In one group these enzymes were inducible (inducible type) and in the other group they were not (non-inducible type). All the mutants showed lower thymidine phosphorylase activity than the parent strain. However, there were striking quantitative differences between non-inducible type strains, in the level of activity of purine nucleoside phosphorylase. The only exception was strain T429 which showed such high purine nucleoside phosphorylase activity that the enzyme seemed to be constitutive. All these thymidine-resistant mutants lacked deoxyriboaldolase. Some non- inducible type mutants also contained no phosphodeoxyribomutase, while others, as well as inducible type mutants contained this enzyme.The gene(s) involved in the mutation causing these changes was mapped preliminary by transduction with phage Plc on a site which was closely linked to thr at the opposite side to leu where the deo-operon is present. This gene was concluded to be in the drm gene in some of the non-inducible type mutants, and not to be in the drm gene in the other non-inducible type mutants and in all inducible type thymidine-resistant utants.

Mutations as possible replication errors in bacteria growing under conditions of thymine deficiency

When Escherichia coli or Bacillus subtilis cells having inhibited thymidylate synthetase activity were incubated for a long time on solid medium supplemented with a limiting concentration of thymine or thymidine (0.1–0.3 μg/ml) most of them became mutants for one or more genetic markers. This “overall mutagenesis” was detected both in Thy − bacteria and in prototrophs for thymine (Thy +) with thymidylate synthetase inhibited by the addition of 5-fluorodeoxyuridine (FUdR) to the growth medium. When thymine (or thymidine) was present in very low amounts (10 −3 μg/ml) or was totally absent, the efficiency of mutagenesis decreased some 100-fold. The solid growth medium is essential because it supports the filamentous cells grown under conditions of limiting thymine. For some of the mutants with identified deficiency their ability to revert under the action of different mutagens was studied. Most efficient was 5-bromouracil (BU). This reversion is the characteristic response of mutations due to AT → GC transitions. In addition to single mutants, many multiple mutants were induced. The repair-defective strain of E. coli pol A1 − and strains Rec A − and Exr A −, which are also defective in UV-induced mutagenesis, showed a high level of mutation induction under the conditions described. All these results are in accord with the hypothesis that overall low-thymine mutagenesis reflects the accumulation of replication errors in DNA under the conditions of a precursor deficiency.

THYMINE HYDRATE FORMED BY ULTRAVIOLET AND GAMMA IRRADIATION OF AQUEOUS SOLUTIONS

Abstract— Evidence is presented for the formation of a thymine hydrate upon ultraviolet (UV) or gamma irradiation of aqueous solutions. The UV quantum efficiency exhibits a dependence on pH similar to that shown for uracil hydration, but the yield is three orders of magnitude smaller than for uracil. Hydration is not affected by wavelength, oxygen, or concentration of thymine. The reversal rate of the photohydrate to thymine is similar to the reversal rates of both isomers of the thymine hydrate formed by γ radiolysis, and depends on pH in the same way as the rate for the uracil photohydrate. The photohydrate of thymine is chromatographically identical to the cis isomer of 6‐hydroxy‐5, 6‐dihydrothymine.

Changes in Cell Size and Shape Associated with Changes in the Replication Time of the Chromosome ofEscherichia coli

Average cell mass is shown to be inversely related to the concentration of thymine in the growth medium of a thy(-) strain of Escherichia coli. The kinetics of the transition from one steady-state average cell mass to another was followed in an attempt to determine the relationship between the chromosome replication time and the time between completion of a round of chromosome replication and the subsequent cell division. Differences in average cell mass are shown to be associated with similar differences in average cell volume. Changes in volume associated with changes in thymine concentration are shown to be due primarily to differences in the width of cells. It is proposed that extension in length of the cell envelope occurs at a linear rate which is proportional to the growth rate and which doubles at the time of termination of rounds of replication. Changes in volume not associated with a change in growth rate are therefore accommodated by a change in cell width. Conditions are described under which average cell mass can continue to increase in successive generations and no steady-state average cell mass is achieved.

Thymineless death and ultraviolet sensitivity in Micrococcus radiodurans.

Thymine-requiring mutants of Micrococcus radiodurans have been isolated by selection on solid medium containing trimethoprim. Strains requiring either high concentrations of thymine (50 mug/ml) or low concentrations (2 mug/ml) for normal growth were obtained. The Thy(-) mutant requiring low thymine concentrations has been characterized. It was shown to retain the high ultraviolet light (UV) resistance typical of wild-type M. radiodurans, but it was not resistant to thymineless death. Preliminary exposure of the cells to thymineless conditions resulted in enhanced UV sensitivity, and this interaction occurred under conditions where "unbalanced growth" was inhibited by the addition of chloramphenicol. Upon addition of thymine to deprived cells, UV resistance was gradually restored, and this recovery took place in the absence of protein synthesis. A model is proposed to account for the similarity of thymineless death in bacteria whose deoxyribonucleic acid repair efficiencies differ widely.

Growth cycles of filamentous Escherichia coli: II. Induced filaments

The effect of inhibition of cell division by gentian violet and thymine deficiency on the course of division of two E. coli strains, growing in batch cultures, was studied. These factors inhibited division and induced filament formation only in the pre-log phase and the exponential phase of growth. In the late logarithmic phase, the division was spontaneously derepressed even in the presence of inhibitor or thymine deficiency, and the filaments broke up into rods. Division of the filaments in the early log phase can be induced in the presence of the dye or thymine deficiency by transfering the filaments to media without any added carbon- or nitrogen source. In the thy − mutant, filament formation was suppressed during growth in a synthetic minimal medium with a low thymine concentration. Derepression of cell division under growth-limiting conditions is not caused by adaptation, selection of mutants or impermeability for the dye. The hypothesis is expressed that inhibition of the cell division, which can be reversed by growth-limiting conditions, acts indirectly on the division mechanism by way of unbalanced macromolecular synthesis. Die Einwirkung von Gentianaviolett und Thyminmangel auf die Hemmung der Zellteilung und den Verlauf derselben von Escherichia coli in Batch-Kulturen wurde studiert. Diese Faktoren hemmen die Zellteilung und induzieren Fadenbildung nur in der pre-logarithmischen und logarithmischen Phase des Wachstums; in der späten logarithmischen Phase wurde die Zellteilung spontan reaktiviert auch unter den Bedingungen eines Thyminmangels oder bei Anwesenheit eines Inhibitors. Die Filamente fragmentieren unter Bildung von Kurzstäbchen. Durch Übertragung der fadenförmigen Zellen aus der frühen logarithmischen Phase in ein Stickstoff- oder Kohlenstoff-freies Minimalmedium konnte Filamentteilung bei Anwesenheit eines Inhibitors oder bei Thyminmangel induziert werden. Die Neuerwerbung der Teilungsfähigkeit in der späten logarithmischen Phase wurde nicht durch Adaptation, Selektion von Mutanten oder Impermeabilität für Gentianaviolett verursacht.

Origin and sequence of chromosome replication in Escherichia coli

Two methods have been used to determine the origin and direction of chromosome replication in Escherichia coli: gradient of marker frequency and sequence of replication in synchronized cultures. In both cases, DNA-DNA hybridization was used to assay for gene dosage. A series of isogenic strains were made lysogenic for phage λ and for phage Mu-1, with phage Mu-1 in a different chromosomal location in each strain. In a first group of experiments, DNA from exponential cultures of the various strains was extracted, denatured, immobilized on filters and hybridized against a mixture of differentially labeled phage λ and phage Mu-1 DNA. This was done for several culture conditions. The ratio of hybridization Mu-1/λ gives a measurement of the dosage of the chromosome region where phage Mu-1 is integrated. A plot of this ratio versus map position reflects the marker frequency distribution. In another group of experiments, several of the strains were synchronized by amino-acid starvation, then restitution of essential amino acids. The cultures thus synchronized were density labeled with bromouracil and their DNA extracted at various times, separated by density on a CsCl gradient, and the amount of phage Mu-1 specific DNA present in each band was measured by DNA-DNA hybridization. The following conclusions have been reached. 1. (1) The origin of replication in near ilv, at 74 minutes on the standard genetic map of Escherichia coli. 2. (2) Replication proceeds simultaneously in the two opposite directions, at approximately the same velocity in both directions. 3. (3) This velocity is influenced, in a thymineless mutant, by the concentration of exogenous thymine. 4. (4) The two growing forks meet at a point diametrically opposed to the origin, near trp (25 min).

Effect of thymine limitation of chromosomal deoxyribonucleic acid synthesis in Proteus mirabilis.

The effects of thymine limitation on the rates of growth, deoxyribonucleic acid (DNA) synthesis, and increase in viable cell number for a thymine auxotroph of Proteus mirabilis were investigated. At thymine concentrations of 1.0 mug/ml and below, these rates were markedly decreased. After a reduction in thymine concentration from 10 mug/ml to 0.2 mug/ml, mass synthesis continued at the preshift rate for several hours. In contrast, the rate of DNA synthesis immediately decreased, resulting in a decrease in the DNA to mass ratio to about one-half of its normal level. Viable counts remained constant for several hours after the reduction in thymine concentration, and enlarged cells and multicellular "snakes" were formed. The rate of DNA synthesis was reduced at thymine concentrations below approximately 1.7 mug/ml. The addition of thymine to cultures which had been completely starved for thymine increased the rate of DNA synthesis to at least twice its normal value; this suggests that extra rounds of chromosome replication can be induced in P. mirabilis as previously observed in Escherichia coli.