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At least 19 recordsLinked to original sources
PubMed1992

Splitting of cis-syn cyclobutane thymine-thymine dimers by radiolysis and its relevance to enzymatic photoreactivation.

The 137Cs-gamma-irradiation of cis-syn thymine-thymine cyclobutane type dimers has been studied in aqueous solution. The mechanism of thymine dimer cleavage by eaq-, CO2.-, OH.,SO4.-,Br2.- and isopropanol radicals was studied using high pressure liquid chromatography (HPLC). Evidence that the one-electron reductants studied induce dimer cleavage partially by a chain reaction is presented. Approximate values for the one-electron reduction potential of thymine-thymine are obtained and thermodynamic calculations are presented in order to predict the direction of electron transfer in the case of enzymatic photoreactivation.

Cesium Radioisotopes↗
PubMed1978

Changes in cell size and shape in thymine-requiring Escherichia coli associated with growth in low concentrations of thymine.

Thymine auxotrophs of three unrelated strains of Escherichia coli (K-12, B/r, and 15) were grown in media containing various concentrations of thymine. During steady-state growth conditions, cell volume increased as thymine concentration decreased but, in contrast to previous reports, this change was due to an increase in cell length without change in cell diameter.

Cell Count↗
PubMed1992

Localization of hepatitis C virus RNA in human liver biopsies by in situ hybridization using thymine-thymine dimerized oligo DNA probes: improved method.

To establish the most proper method of in situ hybridization in detection of HCV-RNA in the liver, various detailed procedures were examined using frozen as well as paraffin-embedded sections of tissue derived from patients. In frozen sections of the liver from hepatitis C patients obtained at autopsy or surgery, HCV-RNA was detectable by in situ hybridization using thymine-thymine dimerized oligonucleotide DNA probes when the sections were treated with ethanol-acetic acid at first, then 0.2 N hydrochloric acid, proteinase K (0.02 u/ml) and DNase. When the paraffin-embedded liver sections were used, more intense proteinase K treatment (0.2-2 u/ml) was required to expose viral RNA and even after that, the positive HCV-RNA signals were less than those in frozen sections, because the cytoplasmic RNA in the routine paraffin-embedded sections was preserved unevenly and less than in frozen sections. These findings indicate that in situ hybridization of HCV-RNA is useful for diagnosing HCV infection and should be a potent tool for monitoring the state of virus activities during therapy. However, the liver biopsy method should be modified so that RNA is retained properly to utilize biopsies more effectively for the routine diagnosis of HCV infection.

Base Sequence↗
PubMed1975

Studies of intracellular thymidine nucleotides. Thymineless death and the recovery after re-addition of thymine in Escherichia coli K 12.

In a thymine-deprived culture, the mutant cells (deficient in dTDP-glucose pyrophosphorylase activity and named Ter-15) lose viability at a faster rate, form longer filaments for the first 60 min and lose thymidine nucleotides and dTDP-sugar pools at a faster rate for the first 15 min than those of the parent cells, but the dTDP-sugar pool in the parent cells is maintained at high concentration for the first 90 min during thymine starvation. In the recovery of cell growth after re-addition of thymine into the thymine-deprived culture, parent cells recommence growth immediately, but the mutant cells (Ter-15) show a lag-phase for 45 min after which time their growth recommences. The rate of dTTP synthesis for the first 10 to 15 min after re-addition of thymine to thymine-deprived cultures of parent and mutant (Ter-15) cells is three-fold higher than that of thymine nondeprived culture (control), but the rates of dTMP and dTDP-sugar syntheses are the same as those of the control. The total DNA synthesis after re-addition of thymine is equal to that of the control, and the period of thymine starvation other than the number of viable cells during thymine starvation plays an important role. After separation of the filament cells from normal-sized cells by sucrose gradient centrifugation, the initial rate of DNA synthesis of filament cells is three-fold faster than that of normal-sized cells. These results show that the dependency of DNA synthesis upon dTTP concentration is maintained after re-addition of thymine into thymine-deprived culture.

Cell Survival↗
PubMed1975

A kinetic study of thymine 7-hydroxylase from neurospora crassa.

The steady-state kinetics of thymine 7-hydroxylase (thymine, 2-oxoglutarate dioxygenase, EC 1.14.11.6) has been investigated. Initial velocity plots were all found to be linear and intersecting. Variation in concentration of two of the substrates, when the third substrate was at a constant high or low concentration, gave initial velocity plots that conform to an ordered sequential mechanism, where thymine is the second substrate to add. With 5-carboxyuracil, which is the end product in the sequential oxygenation of thymine, a competitive inhibition pattern was observed when 2-ketoglutarate was the variable substrate. When either thymine or oxygen was the variable substrate a noncompetitive inhibition pattern was obtained. When either 2-ketoglutarate or thymine was the variable substrate the inhibition patterns observed with bicarbonate were noncompetitive. With succinate noncompetitive inhibition patterns with hyperbolic intercept replots were obtained. These results are consistent with an ordered sequential kinetic mechanism, where 2-ketoglutarate is added first, followed by thymine and oxygen, and the products are released in the order: bicarbonate, succinate, and 5-hydroxymethyluracil. The order of the two last mentioned products, however, is changed in the presence of succinate.

Ketoglutaric Acids↗
PubMed1978

Incorporation of labelled degradation products of radioactive thymine into non DNA material.

When thymine auxotrophs are grown in the presence of methyl labelled [3H] or [14C] thymine which has been stored at 4 degrees C, two classes of material are labelled which are not DNA. One class sediments on neutral sucrose gradients with spontaneously single stranded Okazaki pieces, is unstable in alkali, migrates on alkaline gels as very small material and is digested by ribonucleases and micrococcal nuclease, but not by DNAase I. This class is presumably RNA. The second class sediments more slowly on both neutral and alkaline sucrose gradients than Okazaki pieces, but co-migrates on alkaline gels with DNA whose size is between 700 and 4000 nucleotides. It is not digested by alkali, ribonucleases or deoxyribonucleases. Its identity is unknown. The proportion of the total incorporated counts in these two classes depends on the time of storage of the thymine and is already sufficient to interfere with certain types of experiments when the thymine is only a few weeks old. Thymine is easily purified by paper chromatography and this purified thymine does not label the two non DNA classes of material. It is recommended that radioactive thymine be purified in this way before use.

Carbon Radioisotopes↗
PubMed1977

Thymine 7-hydroxylase from Neurospora crassa. Substrate specificity studies.

A partially purified preparation of thymine 7-hydroxylase (thymine, 2-oxoglutarate : oxygen oxidoreductase (7-hydroxylating), EC 1.14.11.6) from Neurospora crassa was incubated with a number of pyrimidines chemically related to tyymine. 1. Pyrimidines with oxygen or sulfur substituents on atoms Nos. 2 and 4 as well as an alkyl group on atom Nos. 1 or 5 were substrates. 2. Km values were determined for 1-methyluracil, 1-ethyluracil, thymine, 6-azathymine, 1-methylthymine, 1-ethylthymine, 5-formyluracil and 5-hydroxymethyluracil. 3. Uracil was identified as one of the metabolites after incubation with 1-methyluracil. The one-carbon metabolite has not been characterized. 4. Several pyrimidines with polar groups on atoms Nos. 2 and 4 were inhibitory. 5. Addition of 1-methyluracil, 1-methylthymine, 1-ethylthymine or 5-hydroxymethyluracil to incubations with thymine and 2-oxo[1-14C1]glutarate did not result in additional formation of 14CO2, indicating that the same enzyme acts on the different compounds. It has previously been found (Bankel, L., Holme, E., Lindstedt, G. and Lindstedt, S. (1972) FEBS Lett. 21, 135-138) that a mutant strain of N. crassa which is devoid of thymine 7-hydroxylase activity also lacks ability to perform the coupled oxygenation of 2-oxoglutarate and 1-methyluracil, 5-hydroxymethyluracil and 5-formyluracil, respectively. It is concluded that one and the same oxygenase is responsible for the activities studied.

Ketoglutaric Acids↗
PubMed1977

A possible prebiotic synthesis of thymine: uracil-formaldehyde-formic acid reaction.

When uracil is reacted with formaldehyde and formic acid in dilute aqueous solutions at 100-140 degrees C, 5-hydroxymethyluracil (5-HMU), methylenebiuracil (MBU) and thymine are formed. It has been shown that 5-HMU is an intermediate in the formation of MBU and thymine. In the presence of formic acid, 5-HMU gives MBU, thymine and in some cases uracil. The formation of thymine is generally favoured under acidic conditions, although small amounts of this base could also be obtained when the reactions were carried out under mildly basic conditions. A hydride ion transfer mechanism is suggested for some of these reactions. These results have relevance to the formation of thymine under prebiotic conditions.

Chemical Phenomena↗
PubMed1975

Aberrant DNA methylation under conditions of thymine deprivation in Bacillus subtilis.

A study has been made of the levels of 6-methylaminopurine and 5-methyl-cytosine in the DNA of Bacillus subtilis during thymine deprivation. While DNA synthesis was inhibited by thymine deprivation, DNA methylation continued. Base analysis indicated that this aberrant methylation involved an increase solely in the amount of 5-methylcytosine. These aberrant 5-methylcytosine residues were removed from the DNA during continued growth of bacteria in medium lacking thymine. In contrast, 5-methylcytosine residues synthesized during normal growth were relatively unaffected by thymine deprivation. The results are interpreted to indicate that the extensive DNA damage which occurs during thymine deprivation is due in part to exonuclease digestion of regions of DNA containing aberrant 5-methylcytosine residues.

Adenine↗
PubMed1976

Effect of pigment on photomediated production of thymine dimers in cultured melanoma cells.

It was the aim of these studies to determine whether the presence of intracellular melanin quantitatively alters the rate of production of thymine dimers in DNA of irradiated cells in culture. Pigmented and nonpigmented Cloudman mouse melanoma cells were selected assuming that the two cell lines differ primarily in their content of melanin pigment. Cells were cultivated in tritiated thymine in order to label their DNA and were then exposed to ultraviolet (UV) irradiation (260 nm, 500-2000 ergs/mm2). Neither cell line survived these doses of irradiation. DNA was extracted immediately following irradiation and was subjected to acid hydrolysis. The presence of thymine dimers was determined by two-dimensional paper chromatography. The percent of labeled thymine recovered as thymine dimer was calculated and was found to be a linear function of UV dose for both cell lines. The rate of formation of dimers in the nonpigmented cells was nearly twice that in the pigmented cells. These data demonstrate the photoprotective property of intracellular melanin in shielding isolated cells from one type of photomediated injury to DNA.

Animals↗
PubMed1977

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. Mutants with genotype thyA- dra- discriminate against bromouracil to much greater extent than do mutants with genotype thyA- drm-. 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.

Bromouracil↗
PubMed1978

The kinetics of thymine dimer excision in ultraviolet-irradiated human cells.

We have investigated the kinetics of the loss of thymine dimers from the acid-insoluble fraction of several ultraviolet (UV)-irradiated cultured human cell lines. Our results show that UV fluences between 10 and 40 J/m2 produce an average of 21-85 x 10(5) thymine dimers per cell and an eventual maximal loss per cell of 12-20 x 10(5) thymine dimers. The time for half-maximal loss of dimers ranged from 12-22 h after UV irradiation. In contrast, the time for half-maximal repair synthesis of DNA measured by autoradiography was 4.5 h. This figure agrees well with reported half-maximal repair synthesis times, which range from 0.5 to 3.6 h based on our analysis. The discrepancy in the kinetics of the loss of thymine dimers from DNA and repair synthesis is discussed in terms of possible molecular mechanisms of thymine dimer excision in vivo and in terms of possible experimental artifacts.

Cell Line↗
PubMed1979

Sex difference in the accumulation of D-beta-aminoisobutyrate in organs of mouse after thymine loading.

The concentration of D-beta-aminoisobutyric acid (D-BAIB) in the liver and kidney was twice as high and dropped more slowly in the female mouse than in the male after an intraperitoneal injection of thymine. The concentration of beta-alanine, formed from uracil by the same enzyme system catalyzing formation of D-BAIB from thymine, was not different in the liver and kidney of both sexes after an intraperitoneal injection of uracil. After the intraperitoneal injection of D-BAIB, the concentration of BAIB in male liver decreased faster than that in female liver. Inhibition of D-BAIB: pyruvate aminotransferase caused by injection of D-cycloserine resulted in a significant increase in the concentration of BAIB in liver of both sexes after injection of thymine, but the concentration dropped more rapidly in the male. The activity of D-BAIB: pyruvate aminotransferase was not different in the livers of male and female mice. Under the action of probenecid, an inhibitor of active transport systems, the sex difference in accumulation and disappearance of the amino acid in the liver was not observed. This suggested that the excretion of BAIB is more active in the renal tubules of the male mouse than in those of the female. However, the amount of BAIB excreted in the urine after injection of thymine was larger in the female mice than in the male mice. There may be another probenecid-sensitive enzyme for the disposal of BAIB in male mice.

Aminoisobutyric Acids↗
PubMed1979

Deoxythymidine sugars are not direct precursors of DNA-thymine.

A theoretical model for the kinetics of uptake of a putative precursor molecule into nucleotide pools and into replicating DNA has been developed. The relationship between the accumulation of radioactively labeled precursors in the pool and the appearance of radioactivity in DNA is then derived. Experiments have been carried out in bacteria to compare the uptake of radioactive thymine into deoxythymidine triphosphate, deoxythymidine diphosphate sugars, and DNA to test the suitability of either compound as the direct precursor of thymine in DNA. New one-dimensional, thin-layer chromatographic procedures were used to determine the specific activity of deoxythymidine triphosphate and deoxythymidine triphosphate and deoxythymidine diphosphate sugars in growing cultures of 32PO4-labeled Escherichia coli during pulse labeling with [3H]-thymine. A comparison of the experimental data with our theoretical model supports the hypothesis that deoxythymidine triphosphate, but not deoxythymidine sugar, is the direct precursor of thymine in normally replicating DNA in vivo.

DNA Replication↗
PubMed1975

Gamma-ray-induced thymine damage in the DNA in coliphage phi chi 174 and in E. coli.

The radiation chemical reactivity of thymine residues has been compared for single-stranded phi chi 174 DNA free in solution and in the intact bacteriophage, as well as for native E. coli DNA in solution and inside the cell. The gamma-ray-induced release of [3H]H2O from thymine-methyl[3H] was measured to assess the reactivity of the thymine methyl group. Formation of ring-saturated products of the 5,6-dihydroxy-dihydrothymine type (see article) was determined by an alkali-acid degradation assay. It was observed that the formation of that the formation of [3H]H2O was suppressed by a factor of 9 in intact phi chi 174 bacteriophage compared to phi chi 174 DNA, whereas the formation of (see article) was only slightly suppressed in the phage. For E. coli cells the formation of [3H]H29 was reduced 790-fold compared with free E. coli DNA, and (see article) formation was reduced 140-fold. The extents of survival after gamma-irradiation of phi chi 174 phage plaque-forming ability and E. coli colony-forming ability were also determined. Under the conditions used in these experiments, a linear relationship was observed between the loss of biological activity and the formation of thymine damage for both phi chi 174 phage and E. coli cells.

Cesium Isotopes↗