Comparison of radiolysis products of thymine and thymidine with e.s.r. results.

Radicals determined by e.s.r. spectrometry of irradiated thymine or thymidine and radiolytic products generated under tha ction of gamma rays in aerated aqueous solutions have been compared. This comparison lies mainly in the fact that a radical R gives rapidly the corresponding peroxide ROOH. The authors have isolated and characterized twenty peroxides, i.e., the four isomers cis (-), cis (+), trans (-), trans(+) of 6-hydroperoxy-5-hydroxy-5,6-dihydrothymidine; the four isomers cis (-), cis (+), trans (-), trans (+) of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine; 5-hydroperoxy-2-deoxyuridin;cis and trans 6-hydroperoxy-5-hydroxy-5,6-dihydrothymine; cis and trans 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine; 5-hydroperoxymethyl-uracil; 5-hydroperoxy-5,6-dihydrothymine;cis and trans 6-hydroperoxy-5,6-dihydrothymine; 5-hydroperoxy-5-methyl barbituric acid; 5-hydroperoxy-5-methyl hydantoin; trans 5,6-dihydroperoxy-5,6-dihydrothymine. Most of thethymine and thymidine radicals hypothesized or described in the literature were correlated to these peroxides. However, the presence of certain peroxides could not be explained by recognized radicals. Taking advantage of this fact, the existence of new thymine or thymidine radicals so far unknown can be predicted.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

ESR/ENDOR study of guanosine 5'-monophosphate (free acid) single crystals X-irradiated at 10 K

Single crystals of the free base of guanosine 5'-monophosphate were X-irradiated at 10 and at 65 K and investigated between these temperatures and room temperature using K-band ESR and ENDOR spectroscopy. Three free radicals were detected in this temperature range. Two of these were identified as the O6-protonated anion radical and the C8 H-addition radical. Both of these species were present immediately after irradiation at 10 K. The anion radical was formed in two slightly different conformations, of which one decayed at about 150 K and the second at about 250 K. No successor radicals could be detected following the decay of the anion radical. The C8 H-adduct was stable at all temperatures used. The use of partially deuterated crystals confirmed the assignments made and showed that the main pathway for the formation of the C8 H-adduct consisted of addition of a proton from an easily exchangeable site. It is suggested that the C8 H-adduct is formed subsequent to a primary oxidation event localized either at the guanine base or at a nearby water of crystallization. Possible mechanisms for the formation of this product are discussed.     

Photoreactions of thymine and thymidine with N-acetyltyrosine.

We report here the photoinduced formation of a thymine-N-acetyltyrosine adduct. Irradiation of dilute solutions of thymine in the presence of N-acetyltyrosine (NAT) leads to the formation of N-acetyl-4-hydroxy-3-(6-hydrothymin-5-yl)phenylalanine (I), isolated as a mixture of the 5R and 5S diastereoisomers; the photoreaction occurs when irradiation is done either at lambda = 254 nm or at wavelengths of lambda > 290 nm. Irradiation of thymidine in the presence of NAT and of thymine in the presence of tyrosine leads to analogous photoadducts. The photoreaction of thymine with NAT is completely quenched by oxygen and cannot be sensitized by acetone. The likely mechanism involves initial photoionization of the amino acid and deprotonation to form the phenoxyl radical. Thymine then probably captures the released aqueous electron, leading to protonation at C6 of the resulting radical anion. Combination of the phenoxyl and 5,6-dihydrothymin-5-yl radicals would then lead to formation of the final products. The quantum yield for production of the thymine-NAT adduct at pH 7.8 was estimated to be about 5.5 x 10(-4), while a value of 2.3 x 10(-3) was estimated for production of corresponding thymidine adduct at pH 8.1. The dependence of the quantum yield for adduct formation on pH has been determined for both the thymine and thymidine reactions with NAT; the maxima in the quantum yield profiles occur at pH 8-8.5, while appreciable values were measured at pH 7.5. We have also demonstrated that a similar reaction occurs when tyrosine is located within a peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial thymidine kinase and the enzymatic network regulating thymidine triphosphate pools in cultured human cells

In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.     

Sequence-targeted photosensitized reactions in nucleic acids by oligo-alpha-deoxynucleotides and oligo-beta-deoxynucleotides covalently linked to proflavin

Proflavin was covalently linked to the 3'-end or to the 5'-end of an octadeoxythymidylate. This oligonucleotide was synthesized with either the natural beta-anomer of thymidine or its synthetic alpha-anomer. A polymethylene chain was used to link one of the amino groups of proflavin to a terminal thiophosphate group of the oligonucleotide. A 27-mer oligodeoxynucleotide containing an octadeoxyadenylate sequence was used as a target for the proflavin-substituted octadeoxythymidylates. Upon irradiation with visible light, photo-cross-linking reactions induced the formation of branched species that migrated more slowly than the 27-mer on denaturing polyacrylamide gels. Piperidine treatment of the photo-cross-linked species induced strand breaks in the 27-mer. In addition, proflavin induced photosensitized reactions at guanine residues in the 27-mer sequence which were converted to strand breaks following piperidine treatment. Triple-helix formation by the oligothymidylates with their complementary oligodeoxyadenylate sequence at high salt concentration led to photo-cross-linking and cleavage reactions on both sides of the target sequence. These results show that it is possible to target photosensitized reactions to specific sequences on nucleic acids. This opens new possibilities for site-directed mutagenesis and the development of photoactive anti-messenger oligodeoxynucleotides.     

Ability of melanins to protect against the radiolysis of thymine and thymidine

Individuals with black skin rarely get skin cancer, and melanomas, tumors arising from pigmented cells, are generally resistant to radiation therapy. The role of melanin in these two phenomena has not been defined, but oxygen-radical species have been implicated in both effects. These studies were undertaken to determine the ability of various melanins to compete for ionizing radiation-produced radicals which destroy nucleic acid bases. The ability of Sigma eumelanin (S-eumelanin) to protect against the radiolysis of thymidine in buffered solutions was compared to the protective ability of seven amino acids, including melanin precursors; bovine serum albumin, as a model protein; ficoll, as a model polysaccharide; and DNA. Both proteins and polysaccharides are known to scavenge hydroxyl radicals in cells. The concentration of thymidine after exposure to gamma radiation was determined by High Performance Liquid Chromatography (HPLC) analysis after removal of insoluble melanin by acid precipitation. S-eumelanin was more effective at competing with thymidine for free radicals than bovine serum albumin, Ficoll, or DNA, but less effective than certain of the small molecules. Several of the above compounds were also examined for ability to protect against thymine radiolysis. In addition, melanins from other sources were compared to S-eumelanin. Of these, enzymatically synthesized phaeomelanin was the most effective. The results indicate that melanins can compete for base- and nucleoside-damaging free radicals more effectively than other cellular macromolecules. Of the small molecules, the phenolic compounds had the greatest scavenging ability. In vivo, melanins are found in melanosomes bound to protein. Therefore, the relevance of these findings to the photo- and radiobiology of melanins in vivo has yet to be determined.     

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.     

Studies on the utilization of dietary thymidine and thymine by Drosophila melanogaster larvae

Deoxycytidine improves tolerance of Drosophila melanogaster to thymidine block, suggesting the presence of deoxycytidine kinase. At appropriate concentrations, a mixture of thymidine and deoxycytidine allows larvae to tolerate a higher concentration of 5-fluoro-2′-deoxyuridine than is tolerated with either thymidine or deoxycytidine alone. Thus, at this high concentration, 5-fluoro-2′-deoxyuridine appears to act primarily upon thymidylate synthetase, as it does at lower concentrations, rather than upon RNA metabolism, as has been suggested previously. Larvae can also be rescued from 5-fluoro-2′-deoxyuridine-induced death by a high concentration of thymine. The effect is enhanced by the presence of deoxyadenosine. Since this compound is known to increase the intracellular concentration of deoxyribose-1-phosphate, the main effect of thymine is probably due to its salvage utilization as a thymidine source, via the anabolic functioning of thymidine phosophorylase.     

Defective transport of thymidine by cultured cells resistant to 5-bromodeoxyuridine

A line of HeLa cells resistant to 5-bromo-2′-deoxyuridine (BUdR) was established by continuous culture in growth medium containing BUdR; during the selection period, BUdR concentrations, initially 15 μM, were gradually increased to 100 μM. Cells of a clone (HeLa/B5) established from this line were also resistant to 5-fluoro-2′-deoxyuridine (FUdR), but not to the free base, 5-fluorouracil. Although extracts of HeLa/B5 cells exhibited levels of thymidine kinase activity comparable to those of parental cells, rates of uptake of BUdR, FUdR, and thymidine into intact cells were much reduced. The kinetics of uptake of uridine and adenosine, nucleosides which appear to be transported independently of thymidine in HeLa cells, were similar for HeLa/B5 and the parental line (HeLa/0). Relative to thymidine uptake by HeLa/0 cells, that by HeLa/B5 cells was distinctly less sensitive to nitrobenzlthionosine (NBMPR), a specific inhibitor of nucleoside transport in various types of animal cells. Despite this difference in NBMPR sensitivity, both cell lines possessed the same number of high affinity NBMPR binding sites per mg cell protein. The altered kinetics of thymidine uptake and the NBMPR insensitivity of that function in HeLa/B5 cells suggest that resistance to BUdR is due to an altered thymidine transport mechanism.     

The reaction of solvated electrons with cytosine, 5-methyl cytosine and 2'-deoxycytidine in squeous solution. The reaction of the electron adduct intermediates with water, p-nitroacetophenone and oxygen. A pulse spectroscopic and pulse conductometric study.

Using conductivity detection, pulse radiolysis experiments showed that solvent protonation of the electron adducts of cytosine, 5-methyl cytosine and 2'-deoxycytidine occurs with rate constants k greater than or equal to 2 x 10(4) M-1S-1. The protonated electron adducts transfer an electron to p-nitroactetophenone (PNAP) with rate constants ranging from 3.5 x 10(9) to 5.3 x 10(9) M-1S-1. The transfer is quantitative (G = 2.7), as shown by conductometric and spectroscopic measurements. In the presence of O2 no electron transfer to O2 takes place, implying that O2 adds to the protonated electron adduct radicals. No electron transfer from the H- and OH-adducts of the cytosine derivatives, either to PNAP or to O2, takes place near neutral pH. It is suggested that the differences in the reaction behaviour of the H-adduct radicals and the protonated electron adduct radicals towards PNAP can be accounted for if different radicals are formed by H-addition and protonation of the electron adduct. The H atoms most probably add to the C-5-C-6 double bonds, whereas the electron adducts are protonated at N-3 and/or 0-2.     

Altered thymidine metabolism due to defects of thymidine phosphorylase.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease due to mutations in the thymidine phosphorylase (TP) gene. TP enzyme catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxy-D-ribose 1-phosphate. We present evidence that thymidine metabolism is altered in MNGIE. TP activities in buffy coats were reduced drastically in all 27 MNGIE patients compared with 19 controls. All MNGIE patients had much higher plasma levels of thymidine than normal individuals and asymptomatic TP mutation carriers. In two patients, the renal clearance of thymidine was approximately 20% that of creatinine, and because hemodialysis demonstrated that thymidine is ultrafiltratable, most of the filtered thymidine is likely to be reabsorbed by the kidney. In vitro, fibroblasts from controls catabolized thymidine in medium; by contrast, MNGIE fibroblasts released thymidine. In MNGIE, severe impairment of TP enzyme activity leads to increased plasma thymidine. In patients who are suspected of having MNGIE, determination of TP activity in buffy coats and thymidine levels in plasma are diagnostic. We hypothesize that excess thymidine alters mitochondrial nucleoside and nucleotide pools leading to impaired mitochondrial DNA replication, repair, or both. Therapies to reduce thymidine levels may be beneficial to MNGIE patients.     

Preferential uptake of thymidine by thymineless enterobacteria: its significance in DNA labelling.

Minimum satisfactory concentrations of thymine and thymidine were determined for the growth of a high thymine-requirng (thy) mutant to Escherichia coli strain J5-3. Cultures were then grown in the presence of these concentrations of non-radioactive ('cold') pyrimidine together with 5 microCi/ml [methyl-3H)thymine, or [methyl-3H)thymidine (specific activities 5 Ci/m mole), and the uptake of radioactivity into ice cold trichloroacetic acid insoluble material determined. By far the most efficient labelling system was obtained if the label was supplied as radioactive thymidine and growth requirements satisfied by thymine alone. The addition of deoxyadenosine to the labelled thymidine/unlabelled thymine system dramatically reduced uptake of label. The addition of radioactive thymine with either thymine or thymidine to ensure satisfactory growth gave poor labelling. Using the [methyl-3H] thymidine/thymine system it was possible to increase the concentration of thymine from 8 to 64 microgram/ml with only a 25% reduction in label uptake after a 2 h period. The same system was also shown to be most efficient for labelling a thy derivative of another K12 strain, a thymine low-requiring (tir) K12 strain, a thy mutant of Klebsiella aerogenes 418 and a tir derivative of Salmonella typhimurium LT2.     

Mutagenicity and clastogenicity of proflavin in L5178Y/TK +/- -3.7.2.C cells

We evaluated the ability of proflavin to induce specific-locus mutations at the heterozygous thymidine kinase (tk) locus of L5178Y/TK +/- -3.7.2C mouse lymphoma cells, which appears to permit the recovery of mutants due to single-gene and chromosomal mutations. Proflavin was highly mutagenic at the tk locus, producing 724-965 TK mutants/10(6) survivors (background = 56-85/10(6); survival = 29-32%). Most of the mutants were small colonies, which suggested that proflavin may induce chromosomal mutations. The potent clastogenicity of proflavin was confirmed by cytogenetic analysis for chromosomal aberrations. At the highest dose analyzed (1.5 micrograms/ml), proflavin produced 82 aberrations/100 metaphaes (background = 2/100). The large-colony TK mutant frequency produced by proflavin (48-109/10(6) survivors; background = 23/10(6); survival = 57-61%) was similar to published HPRT mutant frequencies produces by proflavin in L5178Y and CHO cells (50-100/10(6) survivors; background = 2-50/10(6); survival = 50-62%). These results lead to the conclusion that proflavin is a potent clastogen and induces a high frequency of small-colony TK mutants; however, it induces a low frequency of HPRT mutants and a low frequency of large-colony TK mutants.     

Induction of thymidine kinase and DNase in varicella-zoster virus-infected cells and kinetic properties of the virus-induced thymidine kinase.

Thymidine kinase (TK), DNA polymerase, and DNase activities were induced in human foreskin fibroblasts after varicella-zoster virus infection. The induced TK and DNase activities have electrophoretic mobilities different from the corresponding host enzymes. Varicella-zoster virus-induced TK was purified and separated from the host enzyme by affinity column chromatography. This enzyme has been shown to have a broader substrate specificity with respect to either the phosphate donor or acceptor as compared with human cytoplasmic and mitochondrial TKs. The best phosphate donor is ATP, with a Km of 16 microM. The Km values of thymidine, deoxycytidine, and 5-propyl deoxyuridine were estimated to be 0.4, 180, and 0.8 microM, respectively. The Ki values for several analogs of thymidine such as 5-iododeoxyuridine, arabinofuranosylthymine, 5-ethyl deoxyuridine, and 5-cyanodeoxyuridine were also examined. TTP acted as a noncompetitive inhibitor with respect to thymidine with a Ki of 5 microM. The kinetic behavior of varicella-zoster virus-induced TK is different from human cytoplasmic, human mitochondrial, and herpes simplex virus type 1- and 2-induced TKs.     

Mutants affecting thymidine metabolism in Neurospora crassa

When (14)C-thymidine labeled only in the ring is administered to Neurospora crassa, the majority of the recovered label is found in the ribonucleic acid (RNA). Three mutants were isolated in which different steps are blocked in the pathway that converts the pyrimidine ring of thymidine to an RNA precursor. Evidence from genetic, nutritional, and accumulation studies with the three mutants shows the pathway to proceed as follows: thymidine --> thymine --> 5-hydroxymethyluracil --> 5-formyluracil --> uracil --> uridylic acid. A mutant strain in which the thymidine to thymine conversion is blocked is unable to metabolize thymidine appreciably by any route, including entry into nucleic acids. This suggests that Neurospora lacks a thymidine phosphorylating enzyme. A second mutation blocks the pathway at the 5-hydroxymethyluracil to 5-formyluracil step, whereas a third prevents utilization of uracil and all compounds preceding it in the pathway. The mutant isolation procedures yielded three other classes of mutations which are proposed to be affecting, respectively, regulation of the thymidine degradative pathway, transport of pyrimidine free bases, and transport of pyrimidine nucleosides.     

Thymidylate nucleotide supply for mitochondrial DNA synthesis in mouse L-cells. Effect of 5-fluorodeoxyuridine and methotrexate in thymidine kinase plus and thymidine kinase minus cells.

The effects of 5-fluorodeoxyuridine and methotrexate on [3H]thymidine and 32P labeling of mtDNA were studied in two lines of mouse L-cells. LMTK- cells, which lack the major cellular thymidine kinase (EC 2.7.1.21) but contain a genetically distinct mitochondrial enzyme, were compared to LA9 cells, which contain both thymidine kinase activities. LMTK- cells were resistant to 5-flurodeoxyuridine by a factor of 200 in comparison to LA9 cells. In both cells lines appropriate drug treatment increased utilization of exogenous thymidine for mtDNA synthesis. The maximum enhancement was 10- to 12-fold for LA9 cells and approximately 20-fold for LMTK- cells when treated with 10 muM methotrexate. The rates of mtDNA and nuclear DNA synthesis during drug treatment were analyzed with 32P labeling and 5-bromo-2'-deoxyuridine density labeling experiments. Synthesis of both mtDNA and nuclear DNA were strongly inhibited by drug treatment of either LA9 or LMTK- cells in the absence of exogenous thymidine. The rate of mtDNA synthesis substantially exceeded that of nuclear DNA in LA9 cells treated with 4 muM 5-fluorodeoxyuridine and less than 5 muM thymidine. Both synthetic rates approached those of untreated LA9 control cultures if 20 muM thymidine was present during 5-fluorodeoxyuridine treatment. In contrast, in LMTK- cells treated with 10 muM methotrexate and 20 muM thymidine, mtDNA synthesis continued at 50 to 60% of the control rate for at least 10 hours while nuclear DNA synthesis was 96% inhibited. Synthesis of mtDNA mass-labeled in both strands with 5-bromouracil occurred when LMTK- cells were incubated for 30 hours with 10 muM methotrexate and 20 muM 5-bromodeoxyuridine. These results indicate that mtDNA synthesis is resistant to a limitation of the thymidine triphosphate supply and is not strictly dependent upon concomitant nuclear DNA synthesis in these cells.     

Inhibition of thymidine uptake in asynchronous HeLa cells by nitrobenzylthioinosine

Nitrobenzylthioinosine (NBMPR), an inhibitor of nucleoside transport by human erythrocytes, was found to be a potent inhibitor of thymidine uptake by asynchronous monolayer cultures of HeLa cells. Rates of thymidine uptake by the cultures at 20 °C were constant between 10 and 40 sec after thymidine addition and were assayed during this interval; TTP was the principal metabolite of thymidine and the thymidine phosphates accumulated at constant rates which extrapolated through time zero. The lack of an effect of NBMPR on thymidine kinase activity, or on the relative proportions of thymidine metabolites in cell extracts, indicated that NBMPR inhibited thymidine transport. When mediated entry (transport) was eliminated by 2 μM NBMPR, a significant diffusional component of thymidine entry was apparent. The mediated component of thymidine uptake exhibited Michaelis-Menten kinetics and apparent K_m and V_max values of 0.5 μM and 10–21 pmoles/min/10⁶ cells were obtained. When NBMPR-treated cells were transferred to NBMPR-free medium, inhibition of thymidine uptake persisted, suggesting that NBMPR was firmly bound to the transport inhibitory sites.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.     

Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates.

One assumption made in bacterial production estimates from [3H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibrio sp. strain D19, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into the cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems.