An atypical deoxynucleoside triphosphate inBacillus subtilis infected with bacteriophage SP10c

In mature DNA ofBacillus subtilis phage SP10c, deoxythymidine monophosphate is partially replaced by a hypermodified nucleotide. During the interval of phage replication, infected cells contained greatly reduced levels of deoxythymidine triphosphate. However, an atypical mononucleotide, tentatively identified as 5-hydroxymethyldeoxyuridine triphosphate, was present during the interval of SP10c DNA synthesis. It is proposed that the atypical mononucleotide, and not deoxythymidine triphosphate, is a substrate for SP10c DNA replication.     

Synthesis of deoxythymidylate and the unusual deoxynucleotide in mature DNA of Bacillus subtilis bacteriophage SP10 occurs by postreplicational modification of 5-hydroxymethyldeoxyuridylate

Mature DNA of Bacillus subtilis W23 phage SP10 contains a hypermodified nucleotide (YdTMP) that replaces ca. 20% of the DTMP. SP10 DNA was pulse-labeled for 1 min at 20 degrees C with 32Pi. Among the oxopyrimidine nucleotides, virtually all of the radioactivity was recovered as 5-hydroxymethyldeoxyuridylate (HMdUMP). During the subsequent chase, radioactivity was lost from HMdUMP and recovered as YdTMP. At 37 degrees C, exogenous [6-3H]5-hydroxymethyldeoxyuridine (HMdUrd) was incorporated into SP10 DNA. Label administered as HMdUrd was phosphorylated to HMdUTP in the infected cells, but all radioactivity was recovered from SP10 DNA as YdTMP and dTMP. Two heat-sensitive mutants defective in hypermodification of SP10 DNA are described. In one mutant, HMdUMP replaces YdTMP in DNA. The other mutant generates a DNA containing a novel deoxynucleotide in place of YdTMP. The novel deoxynucleotide seems to consist of PPi esterified to the 5-hydroxymethyl function of HMdUMP (PP-HMdUMP). Both mutants make normal amounts of dTMP. The data are discussed in terms of the following conclusions. (i) Both oxopyrimidine nucleotides in mature SP10 DNA are derived by postreplicative modification of HMdUMP in nascent DNA. (ii) PP-HMdUMP is an intermediate that facilitate formation of a putative exocyclic methylene intermediate which receives the hypermodification. It is also argued that PP-HMdUMP and the same exocyclic methylene intermediate could serve as intermediates in reductive modification to dTMP. (iii) YdTMP is not an intermediate in the formation of dTMP, and reductive modification proceeds independently of hypermodification.     

DNA Synthesis and Gene Expression in Bacillus subtilis Infected with Wild-Type and Hypermodification-Defective Bacteriophage SP10

A hypermodified base (Y-Thy) replaces 20% of the thymine (Thy) in mature DNA of Bacillus subtilis phage SP10. Two noncomplementing hypermodification-defective (hmd) mutants are described. At 30°C, hmd phage carried out a normal program, but at temperatures of ≥37°C, the infection process was nonproductive. When cells were infected at 37°C with hmd phage, DNA synthesis started at its usual time (12 min), proceeded at about half the normal rate for 6 to 8 min, and then stopped or declined manyfold. All, or nearly all, of the DNA made under hmd conditions consisted of fully hypermodified parental DNA strands H-bonded to unhypermodified nascent strands. The reduced levels of DNA synthesis observed under hmd conditions were accompanied by weak expression of late genes. A sucrose gradient analysis of SP10 hmd⁺ replicating DNA intermediates was made. Two intermediates, called VG and F, were identified. VF consisted of condensed DNA complexed to protein; VF also contained negatively supercoiled domains covalently joined to relaxed regions. F was composed of linear concatenates from which mature DNA was cleaved. None of those intermediates was evident in cells infected at 37°C with hmd phage. Shiftup experiments were performed wherein cells infected with hmd phage at 30°C were shifted to 37°C at a time when replication was well under way. DNA synthesis stopped or declined manyfold 10 min after shiftup. The hmd DNA made after shiftup was conserved as a form sedimentationally equivalent to the F intermediate, but little mature DNA was evident. It is proposed that Y-Thy is required for replication and DNA maturation because certain key proteins involved with these processes interact preferentially with hypermodified DNA.     

Polymer-level synthesis of oxopyrimidine deoxynucleotides by Bacillus subtilis phage SP10: characterization of modification-defective mutants.

Bacillus subtilis phage SP10 DNA has two oxopyrimidines, thymidine 5'-monophosphate (dTMP) and its hypermodified analog (YdTMP). Published data suggest that both are synthesized by postreplicational modification of 5-hydroxymethyldeoxyuridylate (HOMedUMP) in nascent DNA by the following pathway: HOMedUMP----PPOMedUMP----dTMP (85%) or YdTMP (15%); PPOMedUMP is 5-(hydroxymethyl-O-pyrophosphoryl)deoxyuridylate, the pyrophosphoric acid ester of the C5CH2OH function of HOMedUMP. This paper describes aberrant DNAs synthesized at nonpermissive temperatures by a complementary series of heat-sensitive, modification-defective (mod) mutants. Collectively, these mutants encompass the major steps in the complete modification of nascent SP10 DNA. DNA produced by modA phage retains HOMedUMP as its sole oxopyrimidine, implying that (i) this mutant is defective in the pyrophosphorylation step and (ii) formation of PPOMedUMP is required for any further modification. Furthermore, studies with double mutants indicated that modA is epistatic for all other mod mutants, which supports the hypothesis that modA controls the earliest step in the modification pathway. Since their DNAs contain no YdTMP, modC and modD are defective in hypermodification (i.e., PPOMedUMP----YdTMP). However, dTMP occupies the entire oxopyrimidine fraction of modC DNA, whereas modD DNA has a normal dTMP content, but the now-missing YdTMP is replaced by either PPOMedUMP or a byproduct of abortive hypermodification. It is proposed that the modD mutants are defective in the catalytic aspects of hypermodification and that modC are defective in some regulatory function that promotes hypermodification at the expense of reductive modification (i.e., PPOMedUMP----dTMP). Reductive modification is defective in modB phage, as evidenced by the absence of dTMP. In contrast to the others, modB DNA has a complex oxopyrimidine content: HOMedUMP, ca. 30%; PPOMedUMP, ca. 40%; and YdTMP, ca. 30%. The expanded level of YdTMP suggests that at certain sites, reductive modification and hypermodification are competing reactions. Interestingly, the PPOMedUMP content of modB DNA seemingly reflects the maximum degree to which phage DNA can be pyrophosphorylated, since the loss of YdTMP from modBmodC and modBmodD DNAs results in a unilateral increase in HOMedUMP content.     

Deoxythymidine nucleotide metabolism in Bacillus subtilis W23 infected with bacteriophage SP1Oc: preliminary evidence that dTMP in SP10c DNA is synthesized by a novel, bacteriophage-specific mechanism.

Despite the fact that mature SP10c DNA contains dTMP, the acid-soluble fraction of infected cells contained no dTTP during the interval of phage replication. However, infected cells contained normal cellular levels of dATP, dGTP, and dCTP. Upon infection of deoxythymidine-starved Bacillus subtilis M160 (a deoxythymidine-requiring mutant of B. subtilis W23), mature phage DNA with a normal dTMP content was made. SP10c codes for an enzyme that seems to catalyze the tetrahydrofolate-dependent transfer of 1-carbon fragments to the 5 position of dUMP. The transfer of 1-carbon fragments is not accompanied by oxidation of tetrahydrofolage to dihydrofolate, implying that the enzyme in question is not a dTMP synthetase. It is proposed that dTMP in mature SP10c DNA is derived by the postreplicational modification of some other nucleotide and not by the direct incorporation of dTTP into DNA.     

Two different electron transfer pathways may involve in azoreduction in Shewanella decolorationis S12

Electron transfer pathways for azoreduction by S. decolorationis S12 were studied using a mutant S12-22 which had a transposon insertion in ccmA. The results imply that there are two different pathways for electron transport to azo bonds. The colony of S12-22 was whitish and incapable of producing mature c-type cytochromes whose α-peak was at 553 nm in the wild type S12. The mutant S12-22 could not use formate as the sole electron donor for azoreduction either in vivo or in vitro, but intact cells of S12-22 were able to reduce azo dyes of low polarity, such as methyl red, when NADH was served as the sole electron donor. Although the highly polar-sulfonated amaranth could not be reduced by intact cells of S12-22, it could be efficiently reduced by cell extracts of the mutant when NADH was provided as the sole electron donor. These results suggest that the mature c-type cytochromes are essential electron mediators for the extracellular azoreduction of intact cells, while the other pathway without the involvement of mature c-type cytochromes, NADH-dependent oxidoreductase-mediated electron transfer pathway can reduce lowly polar sulfonated azo dyes inside the whole cells or highly polar sulfonated azo dyes in the cell extracts without bacterial membrane barriers.     

G2 arrest and differentiation in the petal of Tradescantia clone 4430

The floral organs of Tradescantia clone 4430 were used to investigate, in terms of cell cycle parameters, cellular behaviour during the maturation of a terminally differentiating system. Petals were sampled at different stages of development for (a) cell number; (b) nuclear DNA content by cytophotometry; (c) [³H]thymidine incorporation into nuclei by autoradiography; and (d) pigment production by spectrophotometry. DNA synthesis was confirmed by measurement of [³H]thymidine incorporation into TCA-insoluble material and changes in DNA content by colorimetric estimation of DNA extracts by diphenylamine. The development of the petal involved four sequential steps. First, there was an increase in cell number, an event characterized by mitoses, DNA synthesis, a few cells in G2 and a predominance in G1. Second, there was a cessation of cell division and DNA synthesis when all the cells accumulated in G1. Third, there was a shift of a large proportion of the total cell population from G1 to the G2 stage of the cell cycle and finally, there was pigment production. In addition, cytophotometric analysis of individual tissues in the mature petal revealed tissue specific differences in the proportion of cells in G2.     

Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells

7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [¹⁴C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 10⁶ nucleotides, 5–6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis.     

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by -D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.     

The effect of folic acid and/or methionine deficiency on deoxyribonucleotide pools and cell cycle distribution in mitogen-stimulated rat lymphocytes

Abstract. Folate deficiency will induce abnormal deoxynucleoside triphosphate (dNTP) metabolism because folate-derived one-carbon groups are essential for de novo synthesis of purines and the pyrimidine, thymidylate. Under conditions of methionine deprivation, a functional folate deficiency for deoxynucleoside triphosphate synthesis is induced as a result of the irreversible diversion of available folates toward endogenous methionine resynthesis from homocysteine. The purpose of the present study was to examine the effect of nutritional folate and/or methionine deprivation in vitro on intracellular dNTP pools as related to DNA synthesis activity and cell cycle progression. Primary cultures of mitogen-stimulated rat splenic T-cells were incubated in complete RPMI 1640 medium or in custom-prepared RPMI 1640 medium lacking in folic acid and/or methionine. Parallel cultures, initiated from the same cell suspension, were analysed for deoxyribonucleotide pool levels and for cell proliferation. The distribution of cells within the cell cycle was quantified by dual parameter flow cytometric bromodeoxyuridine/propidium iodide DNA analysis which allows more accurate definition of DNA synthesizing S-phase cells than the traditional DNA-specific staining with propidium iodide alone. Relative to cells cultured in complete RPMI 1640 media, the cells cultured in media deficient in folate, methionine or in both nutrients manifested increases in the deoxythymidylate pool and an apparent depletion of the deoxyguanosine triphosphate pool. Both adenosine triphosphate and nicotinamide adenine diphosphate levels were significantly reduced with single or combined deficiencies of folate and methionine. These nucleotide pool alterations were associated with a decrease in the proportion of cells actively synthesizing DNA and an increase in cells in G₂+ M phase of the cell cycle. Folate deprivation in the presence of adequate methionine produced a moderate decrease in DNA synthesizing cells over the 68 h incubation. However, methionine deprivation, in the presence or absence of folate, severely compromised DNA synthesis activity. These results are consistent with the established ‘methyl trap’ diversion of available folates towards the resynthesis of methionine from homocysteine and away from nucleotide synthesis. The data confirm the metabolic interdependence of folic acid and methionine and emphasize the pivotal role of methionine on the availability of folate one-carbon groups for deoxynucleotide synthesis. The decrease in DNA synthesis activity under nutrient conditions that negatively affect nucleotide biosynthesis suggest a possible role for abnormal dNTP metabolism in the regulation of cell cycle progression and DNA synthesis.     

Protein charge determination and implications for interactions in cell extracts

Decades of dilute‐solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was ～2‐fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were charge‐neutral and “inert” in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge–charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed.     

Selective protection of 5' ... GGCC ... 3' and 5' ... GCNGC ... 3' sequences by the hypermodified oxopyrimidine in Bacillus subtilis bacteriophage SP10 DNA.

The DNA of Bacillus subtilis bacteriophage SP10 is partially resistant to cleavage and methylation in vitro by restriction enzyme R . BsuRI and its cognate methylase even though greater than 20 copies of the target sequence, 5' ... GGCC ... 3', are present on the phage genome. YThy, a hypermodified oxopyrimidine that replaces a fraction of the thymine residues in SP10 DNA, was responsible for this protection, since YThy-free DNA was no longer resistant. Sites that were normally resistant could nevertheless be cleaved or methylated in vitro if the salt concentration was reduced or dimethyl sulfoxide was added to the reaction buffer. Analysis of the termini produced by cleavage suggested that resistant sites occurred in the sequence 5' ... GGCC-YThy ... 3', whereas sensitive sites, of which there were only two per genome, occurred in the sequence 5' ... GGCCG ... 3'. These in vitro results provide an explanation for the in vivo resistance of SP10 to restriction-modification by B. subtilis R. They also suggest ways in which the presence of the atypical base YThy in regions that flank the target might upset critical DNA-enzyme interactions necessary to locate and recognize the specific site of cleavage or methylation. YThy also strongly protected 5' ... GCNGC ... 3' (R . Fnu4HI) sequences on SP10 DNA, but the biological relevance of this protection is unclear.     

Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a γ-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-β- -arabinofuranosyladenine-5′-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2′,3′-dideoxythymidine-5′-triphosphate (IC₅₀>400 μM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase γ. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC₅₀>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).     

Isolation and characterization of a bacteriophage forBacillus licheniformis A5

The isolation of a virulent bacteriophage forBacillus licheniformis A5 is reported. This bacteriophage, designated NLP-1, has an icosahedral head 100 nm in diameter and a contractible tail with a maximum length of 130 nm. Its DNA has a density of 1.741 g/cm³ and aT _m of 78.4°C. Base composition analysis showed that thymine is absent and is replaced by hydroxymethyluracil. NLP-1 appears to belong to theBacillus group of bacteriophages that includes SP01 and SP82. It will infectB. cereus T andB. brevis 8185, but will not infectB. subtilis W23 or 168.     

Sugar utilization by citrus juice cells as determined by [14C]-sucrose and [14C]-fructose feeding analyses

Sugar utilization by mature citrus juice cells was investigated in light of previous reports suggesting the inability of these cells to phosphorylate hexoses. Grapefruit juice sac cells were incubated in solutions of [¹⁴C]-sucrose or [¹⁴C]-fructose for 16 h during which ¹⁴CO₂ evolution was measured by trapping into soluene. After the incubation period, tissue was extracted in 5 % trichloroacetic acid or 80 % ethanol and extracts separated and identified by thin layer chromatography. Fructose was taken up and metabolized more rapidly than sucrose. In both cases, significant amounts of ¹⁴CO₂, [¹⁴C]-pyruvic and [¹⁴C]-citric acid were recovered after incubation. In separate experiments, hexokinase activity in tissue extracts was found to co-sediment with mitochondrial fractions but was not detected in the soluble fractions as previously reported. The data indicated that, contrary to earlier observations, mature citrus fruit juice cells contain the enzymatic machinery to metabolize soluble sugars. This is consistent with the glycolytic utilization of sugars in cells undergoing anaerobic respiration.