Nature of Deoxyribonucleic Acid Synthesis and Its Relationship to Protein Synthesis During Outgrowth of Bacillus cereus T

Deoxyribonucleic acid (DNA) synthesis during early outgrowth of spores of Bacillus cereus T (thy(-)) has been examined. (14)C-thymidine incorporated begins 2 to 5 min after germination and continues at a slow rate up to 30 min, after which the rate of (14)C-thymidine incorporation increases considerably. Early DNA synthesis up to 30 min after germination is dependent upon simultaneous protein synthesis. The examination of the stability of proteins synthesized soon after germination shows that they are susceptible to intracellular degradation. The evidence provided here indicates that protein degradation is the cause of observed dependence of DNA synthesis on simultaneous protein synthesis. The DNA synthesis occurring soon after germination is primarily a repair type synthesis which is followed by the onset of normal replication approximately 30 min after germination.     

Depression by NAD of x-ray-induced repair-type DNA synthesis in toluene-treated Bacillus subtilis.

NAD prevents a DNA repair-type synthesis that is dependent on polymerase I in toluene-treated, X-irradiated Bacillus subtilis. In unirradiated preparations, NAD had little effect on an ATP-dependent, semiconservative synthesis but partially inhibited a repair-type synthesis. In a mutant lacking polymerase I (polA1-), the presence of NAD did not affect dTTP utilization in DNA synthesis. Nicotinamide mononucleotide (NMN) partially reverses the NAD inhibition of repair-type DNA synthesis. NADP and FAD were ineffective as substitutes for NAD. Since NAD is the cofactor for polynucleotide ligase in Bacillus subtilis and NMN is known to discharge AMP from the active AMP ligase complex, it is proposed that activation of DNA ligase reduces dTMP incorporation by reducing sites for, or limiting DNA polymerase I action.     

Early growth of wheat embryonic axes and the synthesis of RNA and DNA

The requirement for the synthesis of RNA and DNA in early germination of wheat (Triticum aestivum var Newana) embryonic axes has been studied by incubating embryos in the presence of appropriate inhibitors and monitoring both embryo growth and the rates of specific metabolic processes. Experiments with 5-fluorouridine showed that both rRNA and DNA synthesis could be curtailed by 60 to 70% without affecting embryo growth to 24 hours. Similarly, the presence of mitomycin C and methotrexate inhibited DNA synthesis 70%, with only a small effect on growth. Experiments with a range of concentrations of cordycepin and α-amanitin indicated that mRNA synthesis could be curtailed by 30 to 40% within the first 8 hours of germination with only a small effect on embryo growth. Thus, at least the initial phases of seed embryo germination are not closely linked to the synthesis of mRNA, rRNA, or DNA. Maximal sensitivity of embryo growth was obtained with cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamide, supporting the idea that protein synthesis is the macromolecular process most closely linked to early germination.     

Superoxide anion and hydrogen peroxide metabolism in soybean embryonic axes during germination.

The total rate of mitochondrial O2- production in the presence of NADH as substrate increased from 200 to 1340 pmol/min per axis between 2 and 30 h of imbibition. The activities of the enzymes involved in hydroperoxide metabolism, e.g., superoxide dismutase, catalase, peroxidase and glutathione and ascorbate peroxidases, markedly changed during the germination of soybean embryonic axes. Superoxide dismutase was the enzymatic activity affected the most during the initial stages of germination. Intracellular O2- steady-state concentration, calculated from the rate of O2- production and superoxide dismutase activity, showed a 2-fold increase from 2 x 10(-8) M to 4 x 10(-8) M in germination phase I, declined in phase II to 2 x 10(-8) M and remained constant over the rest of the incubation period. The reaction of H2O2 and luminol catalyzed by Co2+ was utilized to measure H2O2 diffused out of the soybean axes after 5 to 10 min of incubation. The catalase-sensitive luminol emission of diffusates prepared from axes previously imbibed from 2 to 30 h corresponded to a H2O2 intracellular steady-state concentration in the range of 0.3 to 0.9 microM. The activity of metal-containing antioxidant enzymes was determined in the extracellular fluid. Cell wall peroxidase activity increased from 10 to 300 mumol/min per mg protein and appears as a potentially important pathway for H2O2 utilization. Hydrogen peroxide metabolism in soybean embryonic axes during early inhibition appears to have the following main features: (a) mitochondrial membranes are the most important source of cytosolic O2- and H2O2; (b) H2O2 is regulated at a steady-state concentration of 0.3-0.9 microM; (c) catalase is the main enzyme in terms of H2O2 utilization; (d) H2O2 exo-diffusion is quantitatively important destiny of intracellular H2O2; and (e) extracellular peroxidase located at the cell wall affords an enzymatic system able to use diffused H2O2.     

Deoxyribonucleic acid metabolism and nuclear division during spore germination in Fusarium oxysporum.

Ungerminated microconidia of Fusarium oxysporum have a mean cell DNA content of 0-134 times 10--12 g/cell with a guanine-plus-cytosine composition (%GC) of 50-75%. During germination, the first dry weight increase of the spore population was defected after 3 h incubation and the first germ tube appeared after 4 h. The total DNA of the culture sharply increased after 5 h, followed by a pause at 6 h. At this time the DNA content per nucleus was maximal and the first nuclear divisions were detected. auses in the rise of total DNA of the culture and in the [14C]adenine incorporation pattern suggest that there is partial synchrony in DNA synthesis at the beginning of incubation. This is also supported by the fact that until 8 h, only hyphae with 1, 2 and 4 nucleic were observed. [14C]adenine incorporation into DNA averaged 2-68% of the total taken up in 10 h incubation.     

Production of ochratoxin A by Aspergillus ochraceus NRRL-3174 before and after exposures to 60Co irradiation.

Spores from the toxigenic organism Aspergillus ochraceus NRRL-3174 were exposed to specific levels of gamma irradiation and then allowed to germinate on selected media. Increases in ochratoxin A production by irradiated, compared to non-irradiated, spores were observed after inoculation of spores onto a cracked red wheat or into a synthetic liquid medium. Variations in daily ochratoxin production were also observed for control and irradiated spore-derived cultures developing on both media, with maximum toxin production varying from 7 to 11 days of incubation. The most notable increases in ochratoxin A production occurred from cultures developing from spores having been irradiated with 10, 25, or 50 krad. Exposures to 400 or 600 krad resulted in complete inhibition of spore germination and, consequently, no ochratoxin production. Of the two substrates used, wheat and synthetic, the quantities of ochratoxin A produced were significantly lower in the synthetic media than on the natural substrate. Higher and more rapid toxin production occurred from spores having been irradiated with 10, 25, 50, and 100 krad than occurred from the non-irradiated control spores when grown on synthetic media. Cultures derived from spores having been exposed to 10, 25, 50, and 100 krad produced significantly higher levels of ochratoxin A after 8 days of incubation on natural substrate than did the controls. Analysis of variance revealed that substrate, length of incubation, as well as irradiation levels all affected the time required to produce maximum levels of ochratoxin A.     

Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium.

Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.     

Function of S-adenosylmethionine in germinating yeast ascospores.

Germination and outgrowth of ascospores of Saccharomyces cerevisiae 4579 require both methionine and adenine, whereas leucine is only required for outgrowth. The methionine requirement may be satisfied by S-adenosylmethionine, but this sulfonium compound will not substitute for adenine. Between 30 and 70 min of protein synthesis is initially required for the completion of germination in strain 4579. The inhibition of S-adenosylmethionine synthetase by trifluoromethionine prevents both germination and protein synthesis. During the initial stages of germination, the S-adenosylmethionine synthetase, S-adenosylmethionine decarboxylase, and transfer ribonucleic acid methyltransferases increased significantly, indicating that polyamines and/or the methylation of transfer ribonucleic acid are required for the initiation of germination.     

Temperature-sensitive deoxyribonucleic acid replication in a dnaC mutant of Bacillus subtilis.

A temperature-sensitive mutant of Bacillus subtilis is defective in deoxyribonucleic acid (DNA) synthesis, contains a lesion in the dnaC locus, and is not primarily an initiation mutant. The amount of DNA synthesized by this mutant at temperatures above 40 C decreases with increasing temperature. DNA synthesis resumes within 20 min after the temperature is lowered to 30 C. In the presence of chloramphenical, DNA synthesis begins at a reduced rate after the temperature is lowered to 30 C. Spores germinated at 46 C cannot initiate DNA replication. The capacity for residual DNA synthesis is stable at the restrictive temperature during inhibition of DNA synthesis. When the temperature is lowered to 30 C after a period of incubation at 43 C, DNA synthesis starts at the origin of the chromosome as well as at preexisting growing points. Similar DNA synthesis patterns are found in mutant cells in vivo and after toluene treatment.     

An autoradiographic analysis of the mode of proliferation in the buccal mucosa of rats incubated up to 5 hours

This study was concerned with the course of DNA synthetic activity (3H-TdR-LI-method) in the buccal mucosa of adult male. Sprague Dawley rats over an incubation period of 5 h. Interest was focussed on the influences of different media and, in particular, on temoporary changes in the proliferative activity. 3H-LI were compared in specimens (a) kept in active (= i.e., 3H-thymidine containing) medium throughout their respective incubation period and (b) pre-incubated in inactive medium for varying, but clearly defined periods before being transferred into active medium for 30 min (actual 3H-LI). Independent of the medium used the rate of DNA synthesis was markedly lowered at 60 min, the reduction being more or less significant in the different media. This was due to a temporary inhibition of DNA synthesis, which was restored after 120-180 min. In contrast, the blockade of G2-phase and/or mitosis persisted up to the end of incubation, as indicated by the unchanged number of labelled mitotic figures after 120 min. Addition of glutamine to Mc Coy's 5A markedly enhanced the activity of 3H-thymidine incorporation, but could not prevent the temporary inhibition of DNA synthesis. The biochemical mechanisms relevant for cell proliferation have been reviewed and correlated to the present results.     

Early transcriptional responses of HepG2-A16 liver cells to infection by Plasmodium falciparum sporozoites

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.     

Linkages between deoxyribonucleic acid synthesis and cell division in Myxococcus xanthus.

Addition of chloramphenicol or 0.5 M glycerol to growing Myxococcus xanthus resulted in an immediate cessation of cell division and 40% net increase in deoxyribonucleic acid (DNA). Although the chloramphenicol-treated cells divided in the presence of nalidixic acid after chloramphenicol was removed, glycerol-induced myxospores required DNA synthesis for subsequent cell division. Myxospores prepared from chloramphenicol-treated cells lost this potential to divide in the presence of nalidixic acid. The "critical period" of DNA synthesis necessary for cell division after germination overlapped in time (3 to 5 h) with initiation of net DNA synthesis. The length of the critical period of DNA synthesis was estimated at 12 min, or 5% of the M. xanthus chromosome. The requirement for cell division during germination also involved ribonucleic acid and protein synthesis after DNA synthesis. The data suggest that replication at or near the origin of the chromosome triggers the formation of a protein product that is necessary but not sufficient for subsequent cell division; DNA termination is also required. During myxospore formation, the postulated protein is destroyed, thereby reestablishing and making apparent this linkage between early DNA synthesis and cell division.     

In vitro synthesis of DNA in nuclei isolated from human lung cells infected with herpes simplex type II virus.

An isolated nuclei system prepared from herpes type II- and mock-infected human embryonic lung cells is able to synthesize cellular and viral DNA in the same proportion as in vivo at various times after infection. Incorporation of (3H)TTP in the in vitro reaction mixture requires Mg2 plus and ATP. Overall in vitro DNA synthesis in nuclei isolated from herpes-infected cells is semiconservative as demonstrated by bromodeoxyuridine-substituted DNA density-transfer experiments, but exhibits a significant fraction of repair-type replication. Relative rates of total DNA synthesis in vitro and in vivo are the same any time after infection. Isolated nuclei synthesize cell and viral DNA for a length of time and at a rate dependent upon the incubation temperature, but there are differences in the length of time of linear in vitro DNA synthesis between herpes- and mock-infected cells. The temperature optima for in vitro DNA synthesis differ significantly for herpes- and mock-infected cells, and are the same for cells abortively infected with herpes type II as for mock-infected cells.     

Radioadaptive enhancement of the repair of UV-induced postreplication gaps in Escherichia coli DNA

Postreplication DNA repair (PRR) in UV-irradiated Escherichia coli WP2 uvrA (tryptophan-dependent strain) and K12 AB1886 uvrA6 pre-irradiated by gamma-rays in low doses (radioadaptation, the first stress effect) has been investigated. PRR was found to be more effective after incubation in the growth medium (for 45-60 min) than in non-radioadapted cells: the repair of postreplication gaps increased by 6-15%. If cells of WP2 uvrA strain were incubated after UV-irradiation in media lacking tryptophan or casamin acids (the second stress effect), PRR was seen to increase as early as within 15 min of incubation and it is more effective than at the first stress. After a 30-60 min incubation the double stress effect leads to an increase in postreplication gap repair by 23-45%. In this case almost all the gaps prove to be repaired. The second stress alone exerts no influence on PPR efficiency. It is supposed that a preliminary radioadaptation may stimulate synthesis of a protein (proteins) of the SOS-response (presumably DNA polymerase V). The second stress effect apparently induces synthesis of an unknown factor (or depreesses synthesis of a MmrA-like protein), and this in cooperation with a protein newly synthesized during radioadaptation significantly increases the efficiency of PPR.     

Possible involvement of DNA-repair events in the transdifferentiation of mesophyll cells ofZinnia elegans into tracheary elements

In cultures of isolated mesophyll cells ofZinnia elegans, transdifferentiation into tracheary elements is induced by a combination of auxin and cytokinin and is blocked by inhibitors of DNA synthesis and poly (ADP-ribose) synthesis. During transdifferentiation, a very low level of synthesis of nuclear DNA was found in some cultured cells by microautoradiography after pulse-labeling with [³H]thymidine. Density profiles of nuclear DNA that had been double-labeledin vivo with bromodeoxyuridine (BrdU) and [³H]thymidine indicated that this DNA synthesis was repair-type synthesis. The sedimentation velocity of nucleoids increased during the culture of isolated mesophyll cells and the increase was dependent on phytohormones. This phenomenon may reflect the rejoining of DNA strand breaks after repair-type DNA synthesis during transdifferentiation. Treatment of cells with inhibitors of DNA synthesis or of poly(ADP-ribose) synthesis prevented the increase in the sedimentation velocity of nucleoids. The data suggest the involvement of DNA-repair events in the transdifferentiation of mesophyll cells into tracheary elements.     

Pollen DNA repair after treatment with the mutagens 4-nitroquinoline-1-oxide, ultraviolet and near-ultraviolet irradiation, and boron dependence of rapair

Summary Irradiation of dry, mature pollen from Petunia hybrida with near-ultraviolet light from an erythemal-sunlamp gave rise to a repair-like, unscheduled DNA synthesis during the early stages of in vitro germination. Like that brought about by farultraviolet light from a germicidal lamp, this DNA synthesis is enhanced by hydroxyurea added to the germination medium, and reduced by photoreactivating light given after ultraviolet irradiation and before germination begins. It is concluded that pollen, often receiving considerable exposure to sunlight, has, in addition to the protection afforded by the ultraviolet filtering effect of yellow pigments, also the capacity to repair ultraviolet produced changes in DNA, by both photoreactivation and dark repair processes.Because mature Petunia pollen is arrested at the G₂ stage of the cell cycle, germinating pollen provides us with a highly synchronous plant tissue with a very low background of DNA replicative synthesis suitable for sensitive measurement of DNA repair synthesis. Thus we have shown that 4-nitroquinoline-1-oxide, at concentrations greater than 0.001 mM, gives rise to an unscheduled DNA synthesis which is enhanced by hydroxyurea. Like that induced by ultraviolet radiation, the chemical mutagen brings about DNA repair only during the early stages of pollen germination, and further it has been possible to show that repair ceases at about the time that generative cell division and pollen tube elongation begins.Boron addition enhances both ultraviolet and 4-nitroquinoline-1-oxide induced repair synthesis. By delaying the chemical mutagen initiation of repair until after germination has begun, we have been able to show that boron is most beneficial during the first hour of germination. It is postulated that this is achieved through an as yet unknown effect of boron on the supply of precursors before pollen cell metabolism is fully committed to pollen tube synthesis later in the germination period.     

Repair of Radiation Damage to Deoxyribonucleic Acid in Germinating Spores of Bacillus subtilis

The repair of deoxyribonucleic acid (DNA) in germinating spores was studied in comparison with that in vegetative cells. Radiation-induced single-strand breaks in the DNA of spores and of vegetative cells of Bacillus subtilis were rejoined during postirradiation incubation. The molecular weight of single-stranded DNA was restored to the level of nonirradiated cells. The rate of the rejoining of DNA strand breaks in irradiated spores was essentially equal to that in irradiated vegetative cells. The rejoining in spores germinating in nutrient medium occurred in the absence of detectable DNA synthesis. In this state, normal DNA synthesis was not initiated. Very little DNA degradation occurred during the rejoining process. On the other hand, in vegetative cells the rejoining process was accompanied by a relatively large amount of DNA synthesis and DNA degradation in nutrient medium. The rejoining occurred in phosphate buffer in vegetative cells but not in spores in which germination was not induced. Chloramphenicol did not interfere with the rejoining process in either germinating spores or vegetative cells, indicating that the rejoining takes place in the absence of de novo synthesis of repair enzyme. In the radiation-sensitive strain uvs-80, the capacity for rejoining radiation-induced strand breaks was reduced both in spores and in vegetative cells, suggesting that the rejoining mechanism of germinating spores is not specific to the germination process.