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1.
Sea urchin egg fertilization studied with a fluorescent probe (ANS)   总被引:2,自引:0,他引:2  
The rates of intracellular DNA synthesis at various temperatures between 39 ° and 31 °C were determined in hamster fibroblasts and HeLa cells by measuring average amounts of 3H-thymidine incorporated per cell in S phase per unit of time. The energy of activation and Q10 for intracellular DNA synthesis were calculated from the slopes of the relative rates of DNA synthesis in HeLa cells and hamster fibroblasts vs. time, plotted on Arrhenius coordinates. In both cell types the incorporation of thymidine into DNA is characterized by an energy of activation of 21 000 calories/mole and a Q10 of 2.94. The absolute rates of DNA synthesis were determined in hamster cells at various temperatures, with values ranging from 1.44 to 0.60 × 10?14 g DNA/ min/cell at 39 ° to 31 °C, respectively. The length of the S phase of the hamster cell was calculated over a 39 ° to 31 °C range, and found to be 5.0 to 11.9 h, respectively. It is concluded that the S phase length is partly determined by the rate of temperature-dependent DNA synthesis.  相似文献   

2.
The rates of intracellular RNA synthesis at various temperatures between 33 and 41 °C were determined in Chinese hamster lung fibroblasts by measuring average amounts of [3H]uridine incorporated per cell per unit of time. The energy of activation and Q20 for intracellular RNA synthesis were calculated from the slopes of the relative rates of RNA synthesis in hamster fibroblasts vs time, plotted on Arrhenius coordinates. The incorporation of uridine into RNA is characterized by an energy of activation of 19 200 calories/mole and a Q10 of 2.71. The absolute rates of RNA synthesis were determined at various temperatures, with values ranging from 1.55 to 0.60 × 10−15 g RNA/min/cell at 41 to 33 °C, respectively.  相似文献   

3.
A.W. Rutherford  A.R. Crofts  Y. Inoue 《BBA》1982,682(3):457-465
A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S2QA state while that at 25°C is attributed to recombination of S2QAQB (and S3QAQB upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S2QAQB and S3QAQB are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to QA and thence to QB or QB) results in predictable changes in the extent of flash-induced thermoluminescence.  相似文献   

4.
The present study reports on effects of different light:dark periods, light intensities, N:P ratios and temperature on the specific growth rate of flagellated cells of Phaeocystis pouchetii in culture. The specific growth rate was estimated by diel changes in cellular DNA content. The cellular DNA content and cell cycle of flagellated cells of P. pouchetii are shown, and the importance of light:dark period in cell division is demonstrated. Diel patterns of the cellular DNA content showed that cell division was confined to the dark period. The cells dealt with more than one division per day by rapid divisions shortly after each other.The specific growth rates (μDNA) based on the DNA cell cycle model were in close agreement with specific growth rates (μCell) determined from cell counts. The temperature affected the specific growth rates (multiple regression, p < 0.01) and were higher at 5 °C (μ ≤ 2.2 d−1) than at 10 °C (μ ≤1.6 d−1). Increasing the light:dark period from 12:12 h to 20:4 h affected the specific growth rate of P. pouchetii at the lower temperature tested (5 °C) (multiple regression, p < 0.01), resulting in higher specific growth rates than at 10 °C. At 10 °C, the effect of light:dark period was severely reduced. Neither light nor nutrients could compensate the reduction in specific growth rates caused by elevated temperature. The specific growth rates was not affected by the N:P ratios tested (multiple regression, p = 0.21). The experiments strongly suggest that the flagellated cells have a great growth potential and could play a dominating role in northern areas at increased day length.  相似文献   

5.
DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G1 and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G1 phase was enriched in origin sequences in comparison with matrix-attached DNA from early G1 phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G1 phase and dissociate after initiation of DNA replication in S phase.  相似文献   

6.
Heat-sensitive (arrested at 39.5°C, multiplying at 33°C) and cold-sensitive (arrested at 33°C, multiplying at 39.5°C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in ‘gently lysed cells’ (obtained by treatment with 0.01% Brij-58) or ‘highly lysed cells’ (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [3H]dTTP efficiently, while highly lysed cells exhibited a low level of [3H]dTTP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extracts reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25 000, but differed in heat sensitivity and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.  相似文献   

7.
Synchronously growing HeLa cells demonstrated a different profile of DNA synthesis to that observed for Chinese hamster V79-379A cells after treatment with cis-Platinum(II) diamminedichloride (cis-Pt(II)) in the G1 phase of the cell cycle. The progression of G1 phase treated cells into the DNA synthetic phase was not affected. The peak rate of DNA synthesis in the first cycle was decreased in a dose dependent manner. However, no displacement in the time of appearance of this peak rate of DNA synthesis was observed in the first cycle as had been observed in Chinese hamster V79-379A cells. The timing of mitosis after the first cycle was delayed in a dose dependent manner and resulted in a concomitant delay in the appearance of the peak rate of DNA synthesis in the second cycle. The peak rate of DNA synthesis in the second cycle was reduced in a dose dependent manner. The ability of cells to divide after the first cycle was not related to their eventual ability to survive. Incubation of HeLa cells with caffeine after treatment with cis-Pt(II) did not increase the toxicity of cis-Pt(II). This was consistent with the lack of effect of caffeine posttreatment on the rate of DNA synthesis in cis-Pt(II) treated synchronously growing HeLa cells. HeLa cells did not show the characteristics of caffeine sensitive replication repair, nor did they show evidence for the presence of an inducible repair system. The rate of DNA synthesis, cell number and survival data were discussed in relation to a mechanism of cell death proposed for Chinese hamster cells.  相似文献   

8.
HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.  相似文献   

9.
The temperature-sensitive S. cerevisiae mutant alg1-1, defective in the N-glycosylation of proteins, shows a first cycle arrest at the non-permissive temperature of 36 °C. The cell number increases by 50% and the absorbance approximately doubles. The budding index of 0.4 at 26 °C drops to 0.15 and DNA synthesis quickly comes to a halt at 36 °C. When the temperature is lowered again, budding and DNA synthesis start after a lag of 2–3 h; α-factor prevents both these processes in cells of mating type a. In addition, cells arrested at 26 °C in G1 with α-factor also do not start budding at the non-permissive temperature after removal of α-factor. The results support recent findings obtained with tunicamycin and suggest that at least one glycoprotein is required for G1-S phase transition in yeast.  相似文献   

10.
Protein tyrosine kinase (PTK) inhibitors have been reported to inhibit proliferation of vascular smooth muscle cells (SMC). To elucidate the made of this inhibition, the effects on the cell cycle of cultured vascular SMC of three PTK inhibitors with different modes of action (methyl 2,5-dihydroxyeinnamate, genistein, and herbimycin A) were studied. Rat aortic SMC were synchronized to the G0 phase of the cell cycle and then released to proceed through the cell cycle by the addition of platelet-derived growth factor (PDGF), and [3H]thymidine incorporation into DNA was measured. The three PTK inhibitors all inhibited PDGF-induced DNA synthesis in a dose-dependent fashion, with IC50 values of 4.7 ± 1.4 μM for methyl 2,5.dihydroxycinnamate, 6.7 ± 2.5 μM for genistein, and 0.17 ± 0.07 μM for herbimycin A. Time course studies suggested that the agents inhibited early G1 phase but not the G0-G1 transition. The lack of effect on the G0-G1 transition was also supported by the finding that the agents did not inhibit the ligand-induced autophosphorylation of PDGF receptor nor the induction of c-fos mRNA at concentrations which were sufficient to inhibit DNA synthesis. PTK inhibitors inhibited progression of the S phase when they were added to SMC that had been arrested at the G1-S border with hydroxyurea. Methyl 2,5-dihydroxyeinnamate also blocked the M phase when it was added to SMC cultured in the presence of 10% fetal calf serum, while genistein and herbimycin A did not inhibit the M phase under the same experimental conditions. In accordance with our previous observation, methyl 2,5-dihydroxycinnamate impaired microtubule networks and formation of the mitotic spindle during the M phase. Our findings indicated that PTK inhibitors inhibit multiple steps of the vascular SMC cell cycle.  相似文献   

11.
1. 1. The 31P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined.
2. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hexagonal (HII) polymorphic phase transition as the temperature is increased through 10°C, and thus prefers the HII) arrangement at 37°C. Rat liver mitochondrial phosphatidylcholine, on the other hand, adopts the bilayer phase at 37°C.
3. 3. Total inner mitochondrial membrane lipids, dispersed in an excess of aqueous buffer, exhibit 31P-NMR spectra consistent with a bilayer arrangement for the majority of the endogeneous phospholipids; the remainder exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca2+ results in hexagonal (HII) phase formation for a portion of the phospholipids, as well as formation of ‘lipidic particles’ as detected by freeze-fracture techniques.
4. 4. Preparations of inner mitochondrial membrane at 4 and 37°C exhibit 31P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca2+ results in an increase in the size of this component, which can become the dominant spectral feature.
5. 5. Intact mitochondria, at 4°C, exhibit 31P-NMR spectra arising from both phospholipid and small water-soluble molecules (ADP, Pi, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37°C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times.
6. 6. It is suggested that the uncoupling action of high Ca2+ concentrations on intact mitochondria may be related to a Ca2+-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.
Keywords: 31P-NMR; Inner mitochondrial membrane; Phosphatidylethanolamine; Ca2+; Hexagonal (HII) phase; Lipidic particle  相似文献   

12.
Nuclei have been isolated from unsynchronized cultures of Chinese hamster fibroblasts after varying intervals of growth following the incorporation of thymidine -3H for 20 min. These nuclei were fractionated by unit gravity sedimentation in a stabilizing density gradient of sucrose, and fractions were analyzed for the concentration of nuclei, DNA, and radioactivity. A more rapidly sedimenting population of nuclei in the G2 phase of the cell cycle was separated from a group of nuclei in the G1 phase, and nuclei in progressive stages of DNA synthesis (S phase) were distributed between these two regions. The fractionation of intact cells by sedimentation according to their position in the cell cycle was found to be less satisfactory than the corresponding separation of nuclei. This probably results from the continuous accumulation of mass within individual cells throughout the entire cell cycle, whereas most of the mass of a nucleus is replicated during a relatively narrow interval of the total cell cycle.  相似文献   

13.
The serine proteinase α-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). 125I-labeled α-thrombin binds rapidly and specifically to CCL39 cells with high affinity (Kd ≈ 4 nM). Binding at 37°C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of α-thrombin receptors on CCL39 cells was identified by covalently coupling 125I-α-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major α-thrombin-binding site of Mr ≈ 150 000 revealed as a 125I-α-thrombin cross-linked complex of Mr ≈ 180 000. Independent of chemical cross-linking, 125I-α-thrombin also formed a covalent complex with a minor, 35 000 Mr, membrane component identified as protease nexin. Two derivatives of α-thrombin modified at the active site are 1000-fold less than α-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native α-thrombin, and affinity-labeled the receptor subunit of Mr 150 000. When present in large excess, during incubation of cells with α-thrombin, these binding antagonists were ineffective in blocking α-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 Mr binding sites that display high affinity for α-thrombin do not mediate induction of the cellular mitogenic response.  相似文献   

14.
The ability of the adenovirus type 5 E1B 55-kDa mutants dl1520 and dl338 to replicate efficiently and independently of the cell cycle, to synthesis viral DNA, and to lyse infected cells did not correlate with the status of p53 in seven cell lines examined. Rather, cell cycle-independent replication and virus-induced cell killing correlated with permissivity to viral replication. This correlation extended to S-phase HeLa cells, which were more susceptible to virus-induced cell killing by the E1B 55-kDa mutant virus than HeLa cells infected during G1. Wild-type p53 had only a modest effect on E1B mutant virus yields in H1299 cells expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39°C, the E1B 55-kDa mutant viruses produced wild-type yields of virus and replicated independently of the cell cycle. In addition, the E1B 55-kDa mutant viruses directed the synthesis of late viral proteins to levels equivalent to the wild-type virus level at 39°C. We have previously shown that the defect in mutant virus replication can also be overcome by infecting HeLa cells during S phase. Taken together, these results indicate that the capacity of the E1B 55-kDa mutant virus to replicate independently of the cell cycle does not correlate with the status of p53 but is determined by yet unidentified mechanisms. The cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in both late gene expression and cell cycle-independent replication leads us to speculate that these functions of the E1B 55-kDa protein may be linked.  相似文献   

15.
The influence of cisplatin, an anticancer agent, on DNA synthesis and cell cycle progression of a cisplatin-resistant cell line was investigated. Cell cycle analysis using flow cytometry showed that cytotoxic concentrations of cisplatin caused a transient inhibition of parental HeLa cells at S phase, followed by accumulation at G2 phase. In contrast, the resistant cells progressed through the cell cycle without being affected by the same treatment. However, cell cycle distributions were the same in the resistant and the parental cells at IC50, the drug concentration inhibiting cell growth by 50%. Studies using a [3H]thymidine incorporation technique also demonstrated a transient inhibition of DNA synthesis in HeLa cells by cisplatin; such inhibition was greatly reduced in the resistant cells. These data argue for the hypothesis that the inhibition of DNA synthesis is important in determining cisplatin-induced cytotoxicity. In addition, the accumulation of cells at G0/G1 by serum starvation was not effective in the resistant cells compared to the parental cells, suggesting that the control of cell cycle exiting is also altered in the resistant cells. Taken together, these results support the notion that alterations in cell cycle control, in particular G2 arrest, are important in determining the sensitivity or resistance of mammalian cells to cisplatin and may have a role in clinical protocols.  相似文献   

16.
The methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G1 phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl-14C] methionine and [methyl-3H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis. A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G1 phase. In both conditions it suppressed 3H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments. The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post-synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.  相似文献   

17.
Isolated, intact rat liver nuclei have high-affiity (Kd=10−9 M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd=10−9 M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (Kd=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (Kd=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.  相似文献   

18.
Monoclonal antibody (mAb) 7E2.2, which recognizes the β subunit of the hamster fibronectin receptor (FnR) (Brown, P. J. and Juliano, R. L. (1988) Exp. Cell Res. 177, 303), was used to examine the distribution of and to quantify the internalization of the FnR and possibly related integrins on adherent fibroblasts. Purified 7E2.2 IgG was iodinated and used in binding and internalization studies. Binding to Chinese hamster ovary cells was saturable with a Km of 0.3 nM and an estimated total number of cell surface β subunits at 2 × 105 per cell. The FnR colocalized with fibronectin at cell adhesion contact sites and also was distributed evenly over the dorsal cell surface as discrete clusters. By using a direct immunocolloidal gold approach, the FnR was not associated with coated pits at 4 °C until internalization followed warming of the labeled cells to 37 °C. A proportion of the FnRs were endocytosed with a half-time of 6.5 min and, consistent with clathrin-mediated uptake, this was sensitive to hypertonic conditions. Receptor-immunocomplexes rapidly became localized within coated pits, small diameter tubules, and peripheral endosomes but the majority remained at the cell surface. At subsaturating concentrations of bound 7E2.2, approximately one-fourth of the total cell receptor population resided intracellularly at any one moment following steady-state; however, appreciable degradation of the iodinated mAb was not detected following accumulation for 4 h at 37 °C. These data showed that at least a portion of the FnR are endocytosed via a receptor-mediated pathway and suggested that these receptors do not immediately enter a degradative compartment.  相似文献   

19.
The effects of sodium butyrate on [3H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S3 cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [5H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [3H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [3H]thymidine incorporation in randomly growing HeLa S3 cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.  相似文献   

20.
The combined effect of mercury (HgCl2) and high temperature on the growth and synthesis of nucleic acid and protein, and on the cell cycle of HeLa S3 cells was investigated. The subsequent growth of the cells was dose-dependently inhibited by mercury at 37.2° and 41.2°C. The inhibitory effect of mercury on subsequent growth was enhanced at the higher temperature. IC50 values for DNA and RNA synthesis but not protein synthesis, at 41.2°C, were significantly lower than those at 37.2°C (P<0.05,P<0.01, respectively). Flow cytometric analysis using synchronous cells indicated the possibility of blocking of cell cycle progression in the early part of S phase by the combined treatment. These results suggest that the cytotoxicity of mercury to cell growth was enhanced at the higher temperature and that this enhancement is related to the increased inhibitory effect of mercury on DNA and RNA synthesis and on the cell cycle at high temperatures.  相似文献   

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