Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and ‘wild-type’ murine P-815 cells

Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.     

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.     

Relationship between sensitivity to 4'-(9-acridinylamino)methanesulfon-m-anisidide and DNA topoisomerase II in a cold-sensitive cell-cycle mutant of a murine mastocytoma cell line

The cold-sensitive (proliferating at 39.5 degrees C, reversibly arrested in GI-phase at 33 degrees C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.     

Formation of mast cell granules in cell cycle mutants of an undifferentiated mastocytoma line: evidence for two different states of reversible proliferative quiescence

A heat-sensitive (hs, arrested at 39.5 degrees C, multiplying at 33 degrees C) and a cold-sensitive (cs, arrested at 33 degrees C, multiplying at 39.5 degrees C) cell cycle variant were isolated from an undifferentiated P-815 murine mastocytoma line. At the respective nonpermissive temperature, both the hs and the cs variant cells were reversibly arrested with a DNA content, typical of G1 phase. The cells of two cs variant subclones, when exposed to the nonpermissive temperature of 33 degrees C, formed metachromatically staining granules with an ultrastructure resembling that of mature mast cells. In addition, the cellular 5-hydroxytryptamine content underwent a marked increase, and the cells responded to compound 48/80 by degranulation as described for normal mast cells. On the other hand, in cells of two hs variant subclones, essentially no mast cell granules were detectable at either 33 or 39.5 degrees C. As previously reported, the cs cell cycle variant phenotype is expressed dominantly in heterokaryons obtained by fusing cs with wild-type cells, whereas hs cell cycle variant cells, similar to other hs mutants, were found to behave recessively under these conditions. Thus the state of proliferative quiescence induced in the cs cells at 33 degrees C is qualitatively different from the state of cell cycle arrest observed in hs cells at 39.5 degrees C and may represent a model for proliferative quiescence of differentiated cells in the intact organism.     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Heat-sensitive (arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (arrested at 33 degrees C, multiplying at 39.5 degrees 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 extract 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.     

Dominant versus recessive behavior of a cold- and a heat-sensitive mammalian cell cycle variant in heterokaryons

A heat-sensitive (hs, arrested at 39.5 degrees C, termed 21-Ta) and a cold-sensitive (cs, arrested at 33 degrees C, termed 21-Fb) clonal cell cycle variant were isolated from the same clone of the P-815 murine mastocytoma line. At the respective nonpermissive temperatures, both the hs and the cs variant were reversibly arrested in G1 phase, and numbers of cells forming colonies upon reincubation at the permissive temperature remained nearly constant for at least 6 days. Cells arrested in G1 by incubation at the respective nonpermissive temperatures were fused to cells of another P-815 clone (31-S) that had been arrested by serum deprivation. Upon reincubation in medium containing 10% serum for 48 h at 39.5 degrees C, 21-Ta x 31-S heterokaryons, similar to 31-S x 31-S homokaryons, entered the S phase, whereas at 33 degrees C, 21-Fb x 31-S heterokaryons, similar to 21-Fb x 21-Fb homokaryons, remained arrested in G1, indicating a recessive expression of the hs and a dominant expression of the cs phenotype.     

Control of DNA polymerase alpha, beta and gamma activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase alpha, beta and gamma activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase alpha activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase alpha activities in arrested cells were as high as 15-40% of control values. After return of arrested cells to the permissive temperature, polymerase alpha activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), the decrease of polymerase alpha during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase alpha is tightly coupled with reentry of cells into S phase. For DNA polymerase beta and gamma activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Relationship between sensitivity to 4′-(9-acridinylamino)methanesulfon-m-anisidide and DNA topoisomerase II in a cold-sensitive cell-cycle mutant of a murine mastocytoma cell line

The cold-sensitive (proliferating at 39.5°C, reversibly arrested in GI-phase at 33°C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.     

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

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.     

Control of DNA polymerase α, β and γ activities in heat- and cold-sensitive mammalian cell-cycle mutants

Two heat-sensitive (arrested in G₁ at 39.5°C) and two cold-sensitive (arrested in G₁ at 33°C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase α, β and γ activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase α activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase α activities in arrested cells were as high as 15–40% of control values. After return of arrested cells to the permissive temperature, polymerase α activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77–85), the decrease of polymerase α during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase α is tightly coupled with reentry of cells into S phase. For DNA polymerase β and γ activities, no obvious correlation with changes in the proliferative state of cells was detected.     

Proliferative quiescence of normal mast cells resembles that of cold-sensitive mutant mastocytoma cells. Dominant expression of the quiescent state in heterokaryons

Normal murine peritoneal mast cells were fused to serum-deprived, non-proliferating cells of a cultured subline (41-SB-4) of the P-815 murine mastocytoma. Upon reincubation in medium containing 10% horse serum for 48 h, mono- and binuclear 41-SB-4 cells reentered S phase of the cell cycle, while mast cell X 41-SB-4 heterokaryons as well as mono- and binuclear mast cells remained in proliferative quiescence, indicating dominant expression of the quiescent state of mast cells. The quiescent state of normal mast cells thus resembles that of cold-sensitive (cs) mutant cells (21-F) of the undifferentiated P-815 mastocytoma: at the non-permissive temperature of 33 degrees C, the 21-F cells were found to enter a state of quiescence which is characterized by its dominant expression in heterokaryons and by morphological differentiation with the formation of metachromatically staining granules similar to those of mast cells. This suggests that the cellular control mechanisms involved in entry into proliferative quiescence and in morphological differentiation of cs 21-F cells may be analogous to those of normal mast cells and/or their precursors.     

Defect in prereplicative phase of G0-specific ts mutant, tsJT60

A temperature-sensitive mutant, tsJT60, grew exponentially at both 34 degrees and 39.5 degrees C, but when stimulated from the resting state it entered S phase at 34 degrees but not at 39.5 degrees C. The mutated function appeared to be a prerequisite throughout from 0 to 9 h following the stimulation, in order that G0-arrested cells would enter S phase. When the arrested cells were stimulated with serum, the amount of and synthesis of protein increased at 34 degrees but not at 39.5 degrees C. The amount of polysome fraction was much smaller in stimulated and unstimulated cells at 39.5 degrees C than in those stimulated at 34 degrees C. Of the events reported to increase shortly after the stimulation, uridine transport increased at both temperatures. Mutation in tsJT60 cells may be concerned with the function prerequisite to induce protein synthesis following serum stimulation, resulting in the blocking of cell cycle progression toward S phase at 39.5 degrees C.     

A temperature-sensitive mutant of the mammalian RNA helicase,DEAD-BOX X isoform,DBX, defective in the transition from G1 to S phase

ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library. X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells. At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days. In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h. The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.     

Activation of the simian virus 40 (SV40) genome abrogates sensitivity to AVP in a rabbit collecting tubule cell line by repressing membrane expression of AVP receptors

To analyze the role of SV40 genome in the phenotypic alterations previously observed in SV40-transformed cell lines, we infected rabbit renal cortical cells with a temperature-sensitive SV40 mutant strain (tsA58) and compared the cell phenotypes at temperatures permissive (33 degrees C) and restrictive (39.5 degrees C) for SV40 genome expression. At both temperatures, the resulting cell line (RC.SVtsA58) expresses cytokeratin and uvomorulin, but epithelial differentiation is more elaborate at 39.5 degrees C as shown by the formation of a well-organized cuboidal monolayer with numerous tight junctions and desmosomes. Functional characteristics are also markedly influenced by the culture temperature: cells grown at 33 degrees C respond only to isoproterenol (ISO, 10(-6) M) by a sevenfold increase in cAMP cell content above basal values; in contrast, when transferred to 39.5 degrees C, they exhibit increased sensitivity to ISO (ISO/basal: 19.1) and a dramatic response to 10(-7) M dDarginine vasopressin (dDAVP/basal: 18.2, apparent Ka: 5 X 10(-9) M) which peaks 48 h after the temperature shift. The latter is associated with membrane expression of V2-type AVP receptors (approximately 50 fmol/10(6) cells) which are undetectable when SV40 genome is activated (33 degrees C). Clonal analysis, additivity studies, and desensitization experiments argue for the presence of a single cell type responsive to both AVP and ISO. The characteristics of the RC. SVtsA58 cell line at 39.5 degrees C (effector-stimulated cAMP profile, lack of expression of brush-border hydrolases and Tamm-Horsfall protein) suggest that it originates from the cortical collecting tubule, and probably from principal cells.     

Isolation of ts mutant cells which arrest in G1/G0 phase at the non-permissive temperature in the presence of appropriate growth factors from a Fischer rat cell line, 3Y1

Two types of cell-cycle-ts mutants were isolated from Fischer rat cell line, 3Y1, and characterized. Clones in one complementation group, tsJT51 and tsJT341, grew at 34 degrees C in the presence of 10% fetal bovine serum (FBS). When the cells growing at 34 degrees C were transferred to 39.5 degrees C, they were arrested alive in G1/G0 phase in the presence of both FBS and epidermal growth factor (EGF), but died in the presence of one of these growth factors. The cells in the other complementation group, tsJT59, tsJT308, tsJT314 and tsJT349, grew at 34 degrees C in the presence of 10% FBS. When the cells growing at 34 degrees C were transferred to 39.5 degrees C, they were arrested alive in G1/G0 phase in the simultaneous presence of FBS, EGF and insulin, but died quickly if one of these growth factors was lacking. Growth-arrested cells at 39.5 degrees C were viable at least one or two weeks and had a potency to resume growth following the shift-down of temperature. Those are assumed to be ts mutant cells which enter and stay in G1/G0 phase from the cell cycle at the non-permissive temperature only in the presence of appropriate growth factors.     

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.     

Cell cycle analysis of p53-induced cell death in murine erythroleukemia cells.

A temperature-sensitive mutant of murine p53 (p53Val-135) was transfected by electroporation into murine erythroleukemia cells (DP16-1) lacking endogenous expression of p53. While the transfected cells grew normally in the presence of mutant p53 (37.5 degrees C), wild-type p53 (32.5 degrees C) was associated with a rapid loss of cell viability. Genomic DNA extracted at 32.5 degrees C was seen to be fragmented into a characteristic ladder consistent with cell death due to apoptosis. Following synchronization by density arrest, transfected cells released into G1 at 32.5 degrees C were found to lose viability more rapidly than did randomly growing cultures. Following release into G1, cells became irreversibly committed to cell death after 4 h at 32.5 degrees C. Commitment to cell death correlated with the first appearance of fragmented DNA. Synchronized cells allowed to pass out of G1 prior to being placed at 32.5 degrees C continued to cycle until subsequently arrested in G1; loss of viability occurred following G1 arrest. In contrast to cells in G1, cells cultured at 32.5 degrees C for prolonged periods during S phase and G2/M, and then returned to 37.5 degrees C, did not become committed to cell death. G1 arrest at 37.5 degrees C, utilizing either mimosine or isoleucine deprivation, does not lead to rapid cell death. Upon transfer to 32.5 degrees C, these G1 synchronized cell populations quickly lost viability. Cells that were kept density arrested at 32.5 degrees C (G0) lost viability at a much slower rate than did cells released into G1. Taken together, these results indicate that wild-type p53 induces cell death in murine erythroleukemia cells and that this effect occurs predominantly in the G1 phase of actively cycling cells.     

Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases

The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.     

Effect of cell cycle position on thermotolerance in Saccharomyces cerevisiae.

We showed that the heat killing curve for exponentially growing Saccharomyces cerevisiae was biphasic. This suggests two populations of cells with different thermal killing characteristics. When exponentially growing cells separated into cell cycle-specific fractions via centrifugal elutriation were heat shocked, the fractions enriched in small unbudded cells showed greater resistance to heat killing than did other cell cycle fractions. Cells arrested as unbudded cells fell into two groups on the basis of thermotolerance. Sulfur-starved cells and the temperature-sensitive mutants cdc25, cdc33, and cdc35 arrested as unbudded cells were in a thermotolerant state. Alpha-factor-treated cells arrested in a thermosensitive state, as did the temperature-sensitive mutant cdc36 when grown at the restrictive temperature. cdc7, which arrested at the G1-S boundary, arrested in a thermosensitive state. Our results suggest that there is a subpopulation of unbudded cells in exponentially growing cultures that is in G0 and not in G1 and that some but not all methods which cause arrest as unbudded cells lead to arrest in G0 as opposed to G1. It has been shown previously that yeast cells acquire thermotolerance to a subsequent challenge at an otherwise lethal temperature during a preincubation at 36 degrees C. We showed that this acquisition of thermotolerance was corrected temporally with a transient increase in the percentage of unbudded cells during the preincubation at 36 degrees C. The results suggest a relationship between the heat shock phenomenon and the cell cycle in S. cerevisiae and relate thermotolerance to transient as well as to more prolonged residence in the G0 state.