Effect of Adenosine and Deoxyadenosine on the Incorporation and Breakdown of Thymidine in Escherichia coli K-12

Deoxyadenosine (AdR) and adenosine (AR) enhance the incorporation of thymidine (TdR) into bacterial deoxyribonucleic acid (DNA) by the inhibition of TdR phosphorolysis in vivo. Neither of the purine nucleosides has an effect on the reaction catalyzed by TdR phosphorylase in vitro. AdR induces TdR phosphorylase and both purine nucleosides induce purine nucleoside phosphorylase. AR can stimulate uptake of more TdR into bacterial DNA than AdR.     

Specificity and Efficiency of Thymidine Incorporation in Escherichia coli Lacking Thymidine Phosphorylase

A mutant of Escherichia coli lacking the catabolic enzyme thymidine phosphorylase readily incorporates exogenous thymidine into deoxyribonucleic acid (DNA) even when provided at concentrations as low as 0.2 mug/ml. Incorporation by this prototrophic strain occurs specifically into DNA, since, with radioactively labeled thymidine, (i) more than 98% is incorporated into alkali-stable material, (ii) at least 90% is recovered as thymine after brief formic acid hydrolysis, and (iii) at least 90% is incorporated into material with the buoyant density of DNA. During growth in medium containing thymidine, the bacteria obtain approximately half of their DNA thymines from the exogenous thymidine and half from endogenous synthesis. The thymines and cytosines of DNA can be simultaneously and specifically labeled by thymidine-2-(14)C and uridine-5-(3)H, respectively. The mutant, which does not degrade thymidine, retains the ability to degrade the thymidine analogue 5-bromodeoxyuridine.     

Participation of exogenous thymine and thymidine in deoxyribonucleic acid synthesis in Lactobacillus acidophilus.

The degree of participation (DP) of exogenous thymidine and thymine in overall DNA synthesis was studied in Lactobacillus acidophilus R-26. The DP of thymidine remains constant under a variety of conditions (except at low thymidine concentrations, when the DP is influenced by deoxyribonucleosides and folic acid). A 5-bromodeoxyuridine-resistant mutant was isolated, which displayed cross-resistance to 5-bromouracil and a significantly lower DP of thymidine than the parental strain. Thymine was poorly incorporated in the parental strain even in the presence of deoxyribosides. The results of this investigation would be compatible with the possibility of an alternative pathway for thymidylate synthesis other than the known thymidylate synthase pathway.     

Effect of 2,6-diaminopurine resistance mutations on adenine and adenosine assimilation by cells of an adenine-dependent strain of Escherichia coli K-12]

In purine-requiring strain of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pur, pup) mutants (designated apt) have been obtained that are resistant to 2,6-diaminopurine on guanine-containing medium and incapable to utilize adenine for their growth at 42degreesC, but they are still sensitive to the analogue and can utilize adenine at 28degreesC. It has been shown that the introduction of the corresponding apt mutations in the genome of adenine-requiring strains impaired the ability of these strains to grow on both adenine and adenosine at 42degreesC.     

Incorporation of thymine-containing DNA precursors in wild-type and mutant T4-infected plasmolysed cells

Summary T4-infected cells, plasmolysed 15 min after infection, incorporate low concentrations (>20 M) of deoxythymidine (TdR) into DNA at a significantly greater rate than dTMP, dTTP or thymine. At higher concentrations (>40 M), dTMP incorporation rate is high, approaching that of TdR at 200 M. TdR is selectively incorporated at all concentrations tested, and is not inhibited by the other thymine containing DNA precursors. Incorporation of low concentrations of TdR requires the T4-induced thymidine kinase (tk) and is not significantly affected by the presence or absence of T4-induced thymidylate synthetase (td). We show that, in T4-infected plasmolysed cells, exogenously added TdR is preferentially incorporated into short DNA fragments during short pulse times. To explain these and other data a model is proposed in which thymidine plays a modulatory role between leading and lagging strand precursor feeds.Preliminary accounts of these data were presented at the West Coast Phage Meetings, Evergreen State College 1980, 1981     

The effect of nalidixic acid on the expression of some genes in Escherichia coli K-12.

The synthesis of maltodextrin phosphorylase and the phage λ receptor of Escherichia coli K-12 is substantially inhibited by the presence of 50 μg nalidixic acid/ml in the culture medium. β-galactosidase synthesis is inhibited to a lesser extent and no inhibition of L-tryptophanase synthesis is observed. The inhibition of enzyme synthesis is apparently not due to the effect of nalidixic acid on deoxyribonucleic acid synthesis.     

Role of ribonucleic acid synthesis in conjugational transfer of chromosomal and plasmid deoxyribonucleic acids

A strain of Escherichia coli K-12 containing mutations that allow for the experimental control of RNA and DNA syntheses was constructed to investigate the role that RNA synthesis plays in conjugational DNA transfer when DNA replication is inhibited. The mutations possessed by this strain and its donor derivatives include: (i) thyA, which blocks synthesis of dTMP, causing a requirement for thymine; (ii) deoC, which blocks breakdown of deoxyribose 5-phosphate, permitting growth with low levels of thymine; (iii) pyrF, which blocks synthesis of UMP from OMP, imposing a requirement for uridine; (iv) cdd and pyrG, which block the deamination of cytidine to uridine and the synthesis of CTP from UTP, respectively, causing a requirement for cytidine; (v) codA and codB, which block the deamination of cytosine to uracil and cytosine transport, respectively, preventing the substitution of cytosine for cytidine; and (vi) dnaB, which blocks vegetative but not conjugational DNA replication at 42 degrees C. DNA synthesis can be blocked in the donor strains by the addition of excess uridine when exogenous thymine is not present. We found that RNA synthesis can also be blocked by addition of excess uridine when exogenous cytidine is not present. Blocking RNA synthesis prior to mating, under conditions in which DNA synthesis either is or is not inhibited, depresses DNA transfer. However, under conditions in which DNA synthesis is inhibited, the blocking of RNA synthesis immediately after mating has commenced had no effect on continued conjugational transfer of DNA. Thus, RNA synthesis is needed to initiate but not to continue conjugational DNA transfer.     

Soluble di- and aminopeptidases in Escherichia K-12. Dispensible enzymes.

As part of a study of the peptidase content of Escherichia coli K-12, two peptidase-deficient amino acid auxotrophs isolated and characterized by Miller as pepD- (strain CM17) and pepD- pepN- pepA- pepB- pepQ- (strain CM89) were examined for the presence of several peptidases previously obtained from strain K-12 in this laboratory. The soluble fraction of each mutant was found to lack the broad-specificity strain K-12 dipeptidase DP and the strain CM89 fraction also lacked activity characteristic of the strain K-12 aminopeptidases AP, L, and OP; like strain CM17, strain CM89 contained the tripeptide-specific aminopeptidase TP. Strain CM89 (but not CM17) appeared to contain little if any activity attributable to the ribosome-bound aminopeptidase I of strain K-12. Whereas loss of DP, AP, OP, and aminopeptidase I activity may be attributed to the pepD-, pepB-, pepN-, and pepA- mutations, respectively, the reason for the loss of L activity remains uncertain. Grown responses of strain CM89 in liquid media containing di- or tripeptides were in accord with absence of enzymes catalyzing rapid hydrolysis of dipeptides. In synthetic liquid media supplemented with the required amino acids per se or with peptone, cultures of both CM strains grew more slowly than strain K-12 and produced smaller cell-yields than those produced by strain K-12.     

Double labeling with [3H]thymidine and [125I]iododeoxyuridine as a method for determining the fate of injected DNA and cells in vivo

Mice were injected intravenously and intraperitoneally with preparations of intestinal nucleoprotein, spleen nuclei, mouse thymus cells, or human kidney T cells whose DNA had been labeled with both [3H]thymidine (TdR) and [125I]-iododeoxyuridine (IUdR). Since free TdR is reutilized more efficiently than free IUdR produced by enzymic hydrolysis of the exogenous DNA, the ratio of [3H]TdR/[125I]IUdR in the DNA fraction of the tissues of the recipient mice provides a measure of the amount of intact exogenous DNA in the tissue. In most instances, the doubly labeled exogenous DNA was almost completely hydrolyzed within 1 day injection, but survival of the DNA from whole cells could be demonstrated in some cases.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

The present experiments with [14C]-thymidine (TdR) and [3H]-bromodeoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 microgram g body weight-1 and that of BrdU is about 5.0 micrograms g body weight-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [3H]-TdR doses commonly used in cell kinetic studies this proportion is only 0.1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [3H]-TdR.     

Regulation of Thymidine Metabolism in Escherichia coli K-12: Evidence That at Least Two Operons Control the Degradation of Thymidine

In Escherichia coli K-12, the rise in activity of thymidine phosphorylase, phosphodeoxyribomutase, and deoxyribose-5-phosphate aldolase caused by exogenous thymidine is dependent on the synthesis of new enzyme protein. Phosphodeoxyribomutase is induced by the purine ribonucleosides adenosine and guanosine, whereas the other two enzymes are not. The mutase activity induced by thymidine and by the purine ribonucleosides has been shown to be the same enzyme by four different criteria. This independent induction of phosphodeoxyribomutase suggests that the gene for this enzyme is in an operon different from the one that may contain the genes for thymidine phosphorylase and deoxyribose-5-phosphate aldolase.     

K-252b Potentiation of Neurotrophin-3 Is trkA Specific in Cells Lacking p75NTR

Abstract: K-252b potentiates the neurotrophic effects of neurotrophin-3 (NT-3) in primary cultures of rat central cholinergic and peripheral sensory neurons and in a rat pheochromocytoma PC12 cell line. The ligand and receptor specificity, and role of the low-affinity neurotrophin receptor (p75^NTR) in the potentiation response induced by K-252b, are unknown. To address the issues of ligand and receptor specificity of K-252b potentiation, we have examined neurotrophin-induced DNA synthesis ([³H]thymidine incorporation) in NIH3T3 cells expressing trkA, trkB, or trkC . Neither NT-3 nor K-252b alone could stimulate mitogenic activity in the trkA -overexpressing clone. However, coaddition of K-252b (EC₅₀ of ∼2 n M ) with 10–100 ng/ml NT-3 led to incorporation of [³H]thymidine in trkA expressing cells to a level induced by optimal concentrations of nerve growth factor (NGF). The K-252b- and NT-3-induced [³H]thymidine incorporation correlated with an increase in the tyrosine autophosphorylation of the trkA receptor as well as tyrosine phosphorylation of trk -associated phospholipase C-γ1 and SH2-containing proteins. K-252b did not potentiate submaximal doses of NGF, or maximal doses of brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5) in trkA -expressing cells. Furthermore, K-252b did not potentiate DNA synthesis by submaximal doses of BDNF, NT-4/5, or NT-3 in trkB - or trkC -expressing NIH3T3 cells, suggesting that the potentiation profile for K-252b was specific for NT-3 in trkA -expressing cells. We found no expression of p75^NTR in the trk -expressing NIH3T3 cells. This is the first demonstration that K-252b potentiates a trkA -mediated biological nonneuronal response by NT-3 that occurs independent of p75^NTR and appears to be both ligand and receptor specific.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

Abstract. The present experiments with [¹⁴C]-thymidine (TdR) and [³H]-bromo-deoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 µg g body weight^-1 and that of BrdU is about 5·0 µg g body weight^-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [³H]-TdR doses commonly used in cell kinetic studies this proportion is only 0-1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [³H]-TdR.     

Vitamin B12 enhances the stimulation of DNA synthesis by serum in resting cultures of 3T6 cells

When low levels of serum are used to stimulate resting cultures of 3T6 cells the limiting factor in increasing DNA synthesis is a low molecular weight component, rather than a polypeptide growth factor. The former component is absent from Dulbecco's modified Eagle's medium (DEM) but present in Waymouth medium. We identify the essential component as vitamin B₁₂. Addition of vitamin B₁₂ to DEM increases the effectiveness of low levels of serum, as assessed both by [³H]TdR incorporation and autoradiography. Thus cultures of 3T6 cells in vitamin B₁₂-supplemented DEM offer a useful in vitro system for testing the mitogenic potency of polypeptides and serum fractions.     

Effect of Galactose on β-Galactosidase Synthesis in Escherichia coli K-12

In wild-type strains of Escherichia coli K-12, the rate of thiomethylgalactoside (TMG)-induced beta-galactosidase synthesis is decreased in the presence of galactose or glucose. A spontaneous mutant of a K-12 strain, 58-161, which synthesizes beta-galactosidase at a low rate was isolated. In this mutant, galactose, after a lag of about one generation time, evoked the same final differential rate of enzyme synthesis as did the gratuitous inducer TMG. However, constitutive, TMG-induced and galactose-induced synthesis in the mutant were subject to inhibition by exogenous glucose. It is concluded that repression of beta-galactosidase synthesis derived from glucose is distinct from the inhibition derived from galactose.     

Escherichia coli mut T1. II. Consequences of modification on the association of DNA with the cell membrane.

1. Two isogenic strains of Escherichia coli, K-12 which differ by mutator gene character (mut T1) have been studied. This characteristic caused introduction of a high frequency of undirectional transversions, A-T leads to -CG, into the DNA of the strain harboring it. 2. It had been previously shown that the presence of this gene is accompanied by an alteration of a cell membrane component. Now, the nuclease susceptibility of DNA associated with membrane/DNA/DNA polymerase complexes is reported. DNA of mut T1 membranes is more sensitive towards exogenous nuclease than DNA of membrane complexes from the wild type mut+ strain. 3. Auto-digestion of this DNA by endogenous nuclease associated with the membrane complex is, also, more severe in preparations derived from mut T1 than from the wild-type strain, mut+, but to a lesser extent than observed with exogenous nucleases. 4. Nuclease susceptibility of mut+ membrane bound DNA is markedly influenced by the growth state of the cell. The nuclease susceptibility of membrane bound DNA from mut T1 cells, however, shows no differences between stationary and log states. 5. These differential sensitivities may be due to conformational changes in the membrane introduced as a pleiotrophic consequence of an altered membrane protein. A pertinent role of this protein in a modified replication/repair complex is an attractive suggestion, especially in the context of the mutator character of this strain.     

Accumulation of trehalose by Escherichia coli K-12 at high osmotic pressure depends on the presence of amber suppressors

When grown at high osmotic pressure, some strains of Escherichia coli K-12 synthesized substantial levels of free sugar and accumulated proline if it was present in the growth medium. The sugar was identified as trehalose by chemical reactivity, gas-liquid chromatography, and nuclear magnetic resonance spectroscopy. Strains of E. coli K-12 could be divided into two major classes with respect to osmoregulation. Those of class A showed a large increase in trehalose levels with increasing medium osmolarity and also accumulated proline from the medium, whereas those in class B showed no accumulation of trehalose or proline. Most class A strains carried suppressor mutations which arose during their derivation from the wild type, whereas the osmodefective strains of class B were suppressor free. When amber suppressor mutations at the supD, supE, or supF loci were introduced into such sup0 osmodefective strains, they became osmotolerant and gained the ability to accumulate trehalose in response to elevated medium osmolarity. It appears that the original K-12 strain of E. coli carries an amber mutation in a gene affecting osmoregulation. Mutants lacking ADP-glucose synthetase (glgC) accumulated trehalose normally, whereas mutants lacking UDP-glucose synthetase (galU) did not make trehalose and grew poorly in medium of high osmolarity. Trehalose synthesis was repressed by exogenous glycine betaine but not by proline.     

Inhibition of sporulation in Bacillus subtilis by bromodeoxyuridine and the effect on DNA replication

A 5-bromo-2'-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR:BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.     

Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.