Viability and DNA Maintenance in Nonculturable Spiral Campylobacter jejuni Cells after Long-Term Exposure to Low Temperatures

Survival of Campylobacter jejuni at 4 and 20°C was investigated by using cellular integrity, respiratory activity, two-dimensional (2D) protein profile, and intact DNA content as indicators of potential viability of nonculturable cells. Intact DNA content after 116 days, along with cellular integrity and respiring cells, was detected for up to 7 months at 4°C by pulsed-field gel electrophoresis. Most changes in 2D protein profiles involved up- or down-regulation.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

The role of single-stranded DNA in the integration of SV40 genome into cellular DNA

The integration of temperature-sensitive SV40 mutant DNA (tsA239) into the Chinese hamster cellular genome at an early stage of infection was studied. The content of single-stranded DNA structures in the infected and control cells at a non-permissive temperature (40 degrees C) differed drastically from that in control cells at permissive temperatures (33 degrees C, 37 degrees C). The role of single-stranded structures in the integration of the SV40 genome into cellular DNA was shown by blot hybridization. The integration mechanism is discussed.     

Physiological Effects of Growth of an Escherichia coli Temperature-Sensitive dnaZ Mutant at Nonpermissive Temperatures

The physiological effects of incubation at nonpermissive temperatures of Escherichia coli mutants that carry a temperature-sensitive dnaZ allele [dnaZ(Ts)2016] were examined. The temperature at which the dnaZ(Ts) protein becomes inactivated in vivo was investigated by measurements of deoxyribonucleic acid (DNA) synthesis at temperatures intermediate between permissive and nonpermissive. DNA synthesis inhibition was reversible by reducing the temperature of cultures from 42 to 30 degrees C; DNA synthesis resumed immediately after temperature reduction and occurred even in the presence of chloramphenicol. Inasmuch as DNA synthesis could be resumed in the absence of protein synthesis, we concluded that the protein product of the dnaZ allele (Ts)2016 is renaturable. Cell division, also inhibited by 42 degrees C incubation, resumed after temperature reduction, but the length of time required for resumption depended on the duration of the period at 42 degrees C. Replicative synthesis of cellular DNA, examined in vitro in toluene-permeabilized cells, was temperature sensitive. Excision repair of ultraviolet light-induced DNA lesions was partially inhibited in dnaZ(Ts) cells at 42 degrees C. The dnaZ(+) product participated in the synthesis of both Okazaki piece (8-12S) and high-molecular-weight DNA. During incubation of dnaZ(Ts)(lambda) lysogens at 42 degrees C, prophage induction occurred, and progeny phage were produced during subsequent incubation at 30 degrees C. The temperature sensitivity of both DNA synthesis and cell division in the dnaZ(Ts)2016 mutant was suppressed by high concentrations of sucrose, lactose, or NaCl. Incubation at 42 degrees C was neither mutagenic nor antimutagenic for the dnaZ(Ts) mutant.     

A temperature-sensitive mutation in the WD repeat-containing protein Smu1 is related to maintenance of chromosome integrity

Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell cycle arrest at S and G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 degrees C. To identify the causative mutation, we fused tsTM18 cells with normal human cells to generate hybrids carrying fragments of human chromosomes. Analysis of chromosome content of temperature-resistant transformants and introduction of a bacterial artificial chromosome containing part of human chromosome 9 led to isolation of the human SMU1 gene. Comparison of sequences of the Smu1 gene from wild-type and mutant cells revealed that the mutant phenotype is caused by a G-to-A transition that yields a gly-to-arg substitution at position 489 in hamster Smu1. The substituted glycine is located in the WD-repeat domain of Smu1. Single-stranded DNA accumulated in the nuclei of mutant cells at 39 degrees C. Furthermore, cdc2 kinase was not activated during G2 phase, and there was no chromosome segregation due to incomplete assembly of the spindle during M phase. Thus, Smu1 appears to be involved directly or indirectly in DNA replication, activation of cdc2 kinase, spindle assembly, and maintenance of chromosome integrity, reflecting the important roles of Smu1 in cellular function.     

Simian virus 40 gene A regulation of cellular DNA synthesis. I. In permissive cells.

The kinetics of host cellular DNA stimulation by simian virus 40 (SV40) tsA58 infection was studied by flow microfluorometry and autoradiography in two types of productively infected monkey kidney cells (AGMK, secondary passage, and the TC-7 cell line). Prior to infection, the cell populations were maintained predominantly in G0-G1 hase of the cell cycle by low (0.25%) serum concentration. Infection of TC-7 or AGMK cells by wild-type SV40, viable deletion mutant dl890, or by SV40 tsA58 at 33 degrees C induced cells through S phase after which they were blocked with a 4N DNA content in the G2 phase. The infection of TC-7 cells by tsA58 at 41 degrees C, which was a nonpermissive temperature for viral DNA replication, induced a round of cell DNA synthesis in approximately 30% of the cell population. These cells proceeded through S phase but then re-entered the G1 resting state. In contrast, infection of AGMK cells by tsA58 at 41 degrees C induced DNA synthesis in approximately 50% of the cells, but this population remained blocked in the G2 phase. These results indicate that the mitogenic effect of the A gene product upon cellular DNA is more heat resistant than its regulating activity on viral DNA synthesis and that the extent of induction of cell DNA synthesis by the A gene product may be influenced by the host cell.     

Protein kinase C controls activation of the DNA integrity checkpoint

The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.     

Interaction between exercise training and cold acclimation in rats

Five groups of 10 rats were used. Group A included sedentary rats kept at 24 degrees C, group B exercised-trained rats and group C rats exposed at -15 degrees C for 2 h every day and kept at 24 degrees C for the remaining time. These 3 groups were kept on this regimen for 10 weeks. In addition group D was acclimated to cold (2 h.d-1 at -15 degrees C) for 6 weeks and subsequently deacclimated at 24 degrees C for 4 weeks. Group E was also acclimated to cold for 6 weeks and during the deacclimation, at 24 degrees C period which lasted 4 weeks, the animals were exercised 2 h per day. Following the 10 week experimental period all animals were sacrificed and DNA and protein content of the IBAT as well as its total mass were measured. The results show significant increases in the cold adapted group. Exercise training which had no effect on brown adipose tissue IBAT at room temperature, caused an accelerated reduction in weight, DNA and protein content of the BAT in rats previously acclimated to cold. In spite of this, the thermogenic response to noradrenaline was significantly enhanced in the group which exercised during the deacclimation period. It is suggested that tissues other than IBAT may explain this enhanced heat production capacity.     

Simian virus 40 large T-antigen function is required for induction of tetraploid DNA content during lytic infection.

Infection of quiescent CV-1 cells with simian virus 40 mutant tsA30 at 37 degrees C resulted in the induction of two rounds of cellular DNA synthesis in T-antigen-positive cells, as previously described for wild-type simian virus 40. Following infection with tsA30 at 40.5 degrees C, T-antigen-positive cells were induced into S phase and reached a diploid G2 DNA content; however, a second S phase was not initiated. The failure of tsA30-infected CV-1 cells to enter tetraploid S phase at 40.5 degrees C identifies a T-antigen function, distinct from T-antigen functions responsible for stimulation of cell DNA synthesis, which is required for initiation of a second round of DNA synthesis without mitosis.     

Identification of adenovirus 2 early genes required for induction of cellular DNA synthesis in resting hamster cells.

tsAF8 cells are temperature-sensitive (ts) mutants of BHK-21 cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. When made quiescent by serum restriction, they can be stimulated to enter the S phase by 10% serum at 34 degrees C, but not at 40.6 degrees C. Infection by adenovirus type 2 or type 5 stimulates cellular DNA synthesis in tsAF8 cells at both 34 and 40.6 degrees C. Infection of these cells with deletion Ad5dl312, Ad5dl313, Ad2 delta p305, and Ad2+D1) and temperature-sensitive (H5ts125, H5ts36) mutants of adenovirus indicates that the expression of both early regions 1A and 2 is needed to induce quiescent tsAF8 cells to enter the S phase at the permissive temperature. This finding has been confirmed by microinjection of selected adenovirus DNA fragments into the nucleus of tsAF8 cells. In addition, we have shown that additional viral functions encoded by early regions 1B and 5 are required for the induction of cellular DNA synthesis at the nonpermissive temperature.     

Heat shock proteins and thermotolerance in a cultured cell line from the Mediterranean fruit fly, Ceratitis capitata.

Heat shock proteins (hsps) were identified in a cell line from the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) exposed to elevated temperatures. Cells produced three hsps (Mr 87,000, 69,000, and 34,000) in response to a temperature shift from 26 degrees C to 37 degrees C (30-60 min) with a concomitant decrease in synthesis of most other cellular proteins. Synthesis of low Mr hsps was not evident. The heat shock response is triggered within 30 min at temperatures from 33 degrees C to 41 degrees C. At temperatures greater than 41 degrees C protein synthesis was shut down. Within 2-3 h after return to 26 degrees C, synthesis of proteins repressed at the higher temperatures resumed production while the major hsps disappear. Heat shock proteins were not produced in the presence of actinomycin D. Evaluations on the role of hsps in conferring thermotolerance to the cells showed an increase in cell viability in heat-shocked cells over non-heat-shocked cells (after 3 and 10 days) when subsequently placed at 45 degrees C for 1 h, a normally lethal temperature. Heat shock alone had little effect on subsequent cell viability or growth at 26 degrees C. These results suggest that hsps produced by these cells may aid in the maintenance of cell integrity and thus play a transitory role in thermotolerance.     

Adenovirus-specific DNA-binding protein inhibits the hydrolysis of DNA by DNase in vitro.

The adenovirus-specific DNA-binding protein was isolated from adenovirus type 5-infected KB cells and shown to possess DNase inhibitor activity. The protein decreased the rate of hydrolysis of single-strand DNA proportionately to its concentration in the reaction. Two peaks of activity were obtained upon sedimentation in a glycerol gradient, probably corresponding to the two major adenovirus-specific polypeptides in the preparation (molecular weights, 72,000 and 44,000). The DNase inhibitor activity of the adenovirus DNA-binding protein was distinguishable from that of the cellular DNA-binding protein, which we have described previously (K, Nass and G. D. Frenkel, J. Biol. Chem. 254:3407-3410, 1979), by its pattern of sedimentation and by the effect of temperature on the two activities. For the adenovirus DNA-binding protein, the ratio of DNase inhibitor activity at 43 degrees C to that at 30 degrees C was approximately 14, whereas for the cellular protein this ratio was less than 3. The DNase inhibitor activity with the temperature coefficient of 14 was absent from cells infected with adenovirus type 5 ts125 at 40 degrees C. DNase inhibition is a simple, sensitive, quantitative method for assay of the adenovirus DNA-binding protein.     

Induction of endoreduplication in temperature-sensitive mutants of rat 3Y1 fibroblasts

Four temperature-sensitive mutants of rat 3Y1 fibroblasts belonging to separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly with a 2C DNA content, when cells proliferating at 33.8 degrees C are shifted up to 39.8 degrees C (Ohno et al., 1984). Zaitsu and Kimura (submitted for publication) showed that 3Y1tsF121 cells synchronized in the early S phase were arrested with a 4C DNA content at 39.8 degrees C. We studied the traverse through the S and G2 phases at 39.8 degrees C in the four ts mutants synchronized at the early S phase and found that 3Y1tsG125 and 3Y1tsH203 cells were arrested with a 4C DNA content as 3Y1tsF121, while 3Y1tsD123 cells went through S and G2 phases and underwent mitosis. When 3Y1tsF121 and 3Y1tsG125 mutants arrested at 39.8 degrees C were shifted down to 33.8 degrees C, a substantial fraction of the cells with a 4C DNA content started, with a certain lag period, DNA synthesis without intervening mitosis and underwent the first mitosis with a lag period similar to that in the cells arrested with a 2C DNA content. The tetraploid cells thus generated had a proliferating ability lower than that of diploid cells.     

Translocation of MRE11 from the nucleus to the cytoplasm as a mechanism of radiosensitization by heat.

Zhu, W-G., Seno, J. D., Beck, B. D. and Dynlacht, J. R. Translocation of MRE11 from the Nucleus to the Cytoplasm as a Mechanism of Radiosensitization by Heat. Radiat. Res. 156, 95-102 (2001).Hyperthermia sensitizes mammalian cells to ionizing radiation, presumably by inhibiting the repair of radiation-induced double-strand breaks (DSBs). However, the mechanism by which heat inhibits DSB repair is unclear. The nuclear protein MRE11 is a component of a multi-protein complex involved in nonhomologous end joining (NHEJ) of radiation-induced DSBs. Using one-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blotting, we found that MRE11 is translocated from the nucleus to the cytoplasm when human U-1 melanoma or HeLa cells are heated for 15 min at 45.5 degrees C or when cells are heated after irradiation with 12 Gy of X rays. No such translocation is observed in unheated irradiated cells. The kinetics of migration of MRE11 to the cytoplasm was dependent upon whether the heated cells were irradiated, while the magnitude of redistribution of MRE11 was dependent upon post-treatment incubation time at 37 degrees C. Cytoplasmic MRE11 content reached a maximum 2-4 h after heating; the increase was not due to new protein synthesis. Partial recovery of nuclear MRE11 content was observed when heated cells or heated irradiated cells were incubated for up to 7 h at 37 degrees C after treatment. Western blotting results showing translocation of MRE11 from the nucleus to the cytoplasm after heating and irradiation were confirmed using confocal microscopy and immunofluorescence staining of fixed cells. Our data suggest that radiosensitization by heat may be caused, at least in part, by translocation of the DNA repair protein MRE11 from the nucleus to the cytoplasm.     

Adenovirus early function required for protection of viral and cellular DNA.

Studies were done to characterize a DNA-negative temperature-sensitive (ts) mutant of human adenovirus type 2, H2 ts111. The temperature-sensitive defect, which was reversible on shift-down in the absence of protein synthesis, was expressed as early as 2 h postinfection, and the results of density-labeling experiments are in agreement with at least a DNA replication initiation block. On shift-up, after allowing viral DNA synthesis at permissive temperatures, the newly synthesized viral DNA and the mature viral DNA were cleaved into fragments which sedimented as a broad peak with a mean coefficient of 10-12S. This cleavage was more marked in the presence of hydroxyurea as the DNA synthesis inhibitor. Parental DNA in infected cells was degraded to a much lesser extent regardless of the incubation temperature. In contrast, the parental DNA was strongly degraded when early gene expression was permitted at 33 degrees C before shift-up to 39.5 degrees C. Furthermore, cellular DNA was also degraded at 39.5 degrees C in ts111-infected cells, the rate of cleavage being related to the multiplicity of infection. This cleavage effect, which did not seem to be related to penton base-associated endonuclease activity, was also enhanced when early gene expression was allowed at 33 degrees C before shift-up. The ts111 defect, which was related to an initiation block and endonucleolytic cleavage of viral and cellular DNA, seemed to correspond to a single mutation. The implication of the ts111 gene product in protection of viral and cellular DNA by way of a DNase-inhibitory function is discussed.     

Eumelanin causes DNA strand breaks and kills cells

Synthetic eumelanin prepared by autooxidation of D,L-DOPA causes DNA strand breaks, as determined by alkaline elution after cell lysis with detergent and proteolysis, in B16CL4 mouse melanoma cells. The melanin is toxic to the cells in the range of doses that causes strand breaks. When the melanin was incubated with the cells at 37 degrees C in tissue culture medium, it was maximally effective after 15 to 20 min at causing strand breaks in the DNA. The extent of damage is concentration dependent, but the effect plateaus at 1 mg/ml. The nature of the interaction of the cellular DNA with melanin is consistent with strand breaks, not DNA-DNA crosslinks. The strand break damage is repaired, even in the continued presence of melanin, but repair is more rapid if the cells are washed and the melanin is removed. The form of the melanin is important for obtaining the effect. Sonication for 3 min abrogates the effect to a considerable extent, and repeated cycles of sonication can completely destroy the activity. Lost activity returns slowly with storage at 4 degrees C. Melanin is more effective at damaging DNA in a protein-free medium. It is also DNA-damaging at 4 degrees C, but less so than at 37 degrees C. Preliminary studies indicate that the strand breaks caused by melanin are additive with those caused by ionizing radiation. The extent of DNA strand breaks and alkali-labile sites caused by several other melanins was also determined. Some melanins did not cause frank strand breaks, but were active in causing alkali-labile sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of DNA synthesis in division-arrested mouse C127 cells permissive for bovine papillomavirus DNA amplification.

Spontaneous amplification of bovine papillomavirus type 1 DNA occurs following a prolonged period of serum starvation of wild-type virus-transformed C127 cell lines and is associated with abundant viral E2 protein synthesis and a concomitant induction of viral oncogene (E5 and E6) expression. We show here that a subpopulation of the permissive cells incorporate bromo-deoxyuridine under conditions of cell growth arrest (serum starvation), whereas DNA synthesis is suppressed in the resting population of nonpermissive cells. Flow cytometric measurements of the cellular DNA content of the permissive cell population indicated that it contained predominantly a 4n DNA content, suggesting that these cells were blocked in the G2 phase of the cell cycle. In keeping with the hypothesis that viral DNA amplification is associated with the induction of a cellular S phase, we observed a specific induction of expression of two cell proliferation-related cellular antigens (PCNA and Ki67) in a subpopulation of permissive cells. C127 cell lines transformed by an E5-minus bovine papillomavirus type 1 mutant, which was competent for autonomous plasmid replication in mitotic cells, were completely defective for the induction of DNA synthesis and mutant viral DNA amplification under conditions of serum starvation. Moreover, the E5 protein is shown by immunofluorescence analysis to be expressed at a high level specifically in the permissive cell population. These results imply a dual role for the viral E5 protein in the C127 model system, both as a transforming protein and as a factor required for the induction of viral DNA amplification in postmitotic cells. We suggest that E5 acts at an early step in the induction of this process in C127 cells and may be required to turn on host cell DNA synthesis as a prerequisite for viral DNA amplification.     

Cell death in Escherichia coli dnaE(Ts) mutants incubated at a nonpermissive temperature is prevented by mutation in the cydA gene

Cells of the Escherichia coli dnaE(Ts) dnaE74 and dnaE486 mutants die after 4 h of incubation at 40 degrees C in Luria-Bertani medium. Cell death is preceded by elongation, is inhibited by chloramphenicol, tetracycline, or rifampin, and is dependent on cell density. Cells survive at 40 degrees C when they are incubated at a high population density or at a low density in conditioned medium, but they die when the medium is supplemented with glucose and amino acids. Deletion of recA or sulA has no effect. We isolated suppressors which survived for long periods at 40 degrees C but did not form colonies. The suppressors protected against hydroxyurea-induced killing. Sequence and complementation analysis indicated that suppression was due to mutation in the cydA gene. The DNA content of dnaE mutants increased about eightfold in 4 h at 40 degrees C, as did the DNA content of the suppressed strains. The amount of plasmid pBR322 in a dnaE74 strain increased about fourfold, as measured on gels, and the electrophoretic pattern appeared to be normal even though the viability of the parent cells decreased 2 logs. Transformation activity also increased. 4',6'-diamidino-2-phenylindole staining demonstrated that there were nucleoids distributed throughout the dnaE filaments formed at 40 degrees C, indicating that there was segregation of the newly formed DNA. We concluded that the DNA synthesized was physiologically competent, particularly since the number of viable cells of the suppressed strain increased during the first few hours of incubation. These observations support the view that E. coli senses the rate of DNA synthesis and inhibits septation when the rate of DNA synthesis falls below a critical level relative to the level of RNA and protein synthesis.     

Sublethal heat stress of Vibrio parahaemolyticus.

When Vibrio parahaemolyticsu ATCC 17802 was heated at 41 degrees C for 30 min in 100 mM phosphate-3% NaCl buffer (pH 7.0), the plate counts obtained when using Trypticase soy agar containing 0.25% added NaCl (0.25 TSAS) were nearly 99.9% higher than plate counts using Trypticase soy agar containing 5.5% added NaCl (5.5 TSAS). A similar result was obtained when cells of V. parahaemolyticus were grown in a glucose salts medium (GSM) and heated at 45 degrees C. The injured cells recovered salt tolerance within 3 h when placed in either 2.5 TSBS or GSM at 30 degrees C. The addition of chloramphenicol, actinomycin D, or nalidixic acid to 2.5 TSBS during recovery of cells grown in 2.5 TSBS indicated that recovery was dependent upon protein, ribonucleic acid (RNA, and deoxyribonucleic acid (DNA) synthesis. Penicillin did not inhibit the recovery process. Heat-injured, GSM-grown cells required RNA synthesis but not DNA synthesis during recovery in GSM. Chemical analyses showed that total cellular RNA decreased and total cellular DNA remained constant during heat injury. The addition of [6-3H]uracil, L-[U-14C]leucine, and [methyl-3H]thymidine to the recovery media confirmed the results of the antibiotic experiments.