Temperature Shift-down Research Articles

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002.

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C18 fatty acids increased in cells grown at lower temperature for all lipid classes, and omega 3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the omega 3 position of C18 fatty acids increased gradually during a 12-h period after a temperature shift-down to 22 degrees C. However, the mRNA levels of the desA (delta 12 desaturase), desB (omega 3 desaturase) and desC (delta 9 desaturase) genes increased within 15 min after a temperature shift-down to 22 degrees C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38 degrees C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22 degrees C and 38 degrees C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.

Cold shock in Bacillus subtilis: different effects of benzyl alcohol and ethanol on the membrane organisation and cell adaptation

A temperature shift-down of Bacillus subtilis from 40 to 20°C induces an 80 min growth lag. Benzyl alcohol reduced this period to 51 min, whereas ethanol prolonged it up to 102 min. The effect of the two alcohols on the membrane state was investigated by measuring the steady-state fluorescence anisotropy and analysing the lifetime distribution of diphenylhexatriene (DPH) and its polar derivative, TMA-DPH. As followed from the fluorescence anisotropy, the two alcohols exerted similar (fluidising) effects on the cytoplasmic membranes of B. subtilis. However, benzyl alcohol significantly shortened the main DPH lifetime component and widened its distribution, while ethanol had no effect. The benzyl alcohol activity was interpreted in terms of an increased membrane hydration due to disordering of the membrane structure. Such an effect imitates the cold shock induced synthesis of unsaturated fatty acids in B. subtilis. The fatty acid analysis revealed that ethanol hindered this adaptive synthesis of fatty acids. At the same time, its effect on the membrane state (membrane order) was very low and could not substitute the physiological response as was the case with benzyl alcohol. It can thus be concluded that the adaptation of the membrane physical state contributes significantly to the cold shock response of B. subtilis.

Stress-Responsive Gene Expression in Tetrahymena

Cells properly respond to extracellular stimuli and circumstantial environment. The unicellular eukaryotic protozoan Tetrahymena is a potentially useful animal cell model system for studying the molecular mechanism of adaptation to environment. Tetrahymena is exposed to fluctuations in temperature, pH, amounts of nutrients and concentration of dissolved gases in natural habitat. For example, the cells adapt to cold environment by increase in unsaturated fatty acids in membrane phospholipids to maintain proper membrane fluidity. To accomplish this modification, the activity of fatty acid desaturase is increased upon a down-shift in temperature. We have cloned delta9 fatty acid desaturase which is involved in this process and shown evidence that its mRNA level increased in response to cold environment. Moreover, in order to examine other genes responsive to clod stress, we have adopted mRNA differential display technique to temperature shift-down of T. thermophila. We have cloned two kinase genes, NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk) and MAP kinase-related kinase (MRK). Interestingly, these genes were also shown to be expressed by the osmotic stress.

A two-stage process with temperature-shift for enhanced anthocyanin production in strawberry cell suspension cultures

A two-stage process with temperature-shift has been developed to enhance the anthocyanin yield in suspension cultures of strawberry cells. The effect of the temperature-shift interval and the shift-time point was investigated for the optimization of this strategy. In this process, strawberry cells were grown at 30 degrees C (the optimum temperature for cell growth) for a certain period as the first stage, with the temperature shifted to a lower temperature for the second stage. In response to the temperature shift-down, anthocyanin synthesis was stimulated and a higher content could be achieved than that at both boundary temperatures but cell growth was suppressed. When the lower boundary temperature was decreased, cell growth was lowered and a delayed, sustained maximum anthocyanin content was achieved. Anthocyanin synthesis was strongly influenced by the shift-time point but cell growth was not. Consequently, the maximum anthocyanin content of 2.7 mg.g-fresh cell(-1) was obtained on day 9 by a temperature-shift from 30 degrees C, after 3-d culture, to 15 degrees C. The highest anthocyanin yield of 318 mg.L-1 on day 12 was achieved when the temperature was shifted from 30 degrees C, after 5-d culture, to 20 degrees C. For a global optimization of both the yield and productivity, the optimum anthocyanin yield and productivity of 272 mg.L-1 and 30.2 mg.L-1.d(-1) on day 9 were achieved by a two-stage culture with a temperature-shift from 30 degrees C after 3 d to 20 degrees C.

Molecular cloning and cell-cycle-dependent expression of a novel NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk) in Tetrahymena cells.

With the intention of investigating the signal-transduction pathway that mediates the cold-stress response in Tetrahymena, we isolated a gene that encodes a novel protein kinase of 561 amino acids, termed Tetrahymena pyriformis NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk), by differential display from Tetrahymena cells exposed to temperature shift-down. TpNrk possesses an N-terminal protein kinase domain that is highly homologous with other NIMA-related protein kinases (Neks) involved in the control of the cell cycle. The TpNrk protein is 42% identical in its catalytic domain with human Nek2, 41% identical with mouse Nek1 and 37% with A. nidulans NIMA. In addition, TpNrk and these NIMA-related kinases have long, basic C-terminal extensions and are therefore similar in overall structure. In order to further explore the function of the TpNrk gene and the association of the cold stress with the cell cycle of Tetrahymena, changes of TpNrk mRNA were determined during the course of the synchronous cell division induced by the intermittent heat treatment. The level of TpNrk transcription increased immediately after the end of the heat treatment, with a peak at 30 min, and declined thereafter reaching the minimum level when nearly 80% of the cells synchronously entered cell division (75 min after the end of heat treatment). The accumulation of TpNrk mRNA starting from 0 min to 30 min after the end of the heat treatment was assumed to be a prerequisite for the start of synchronous cell division. These results suggest that TpNrk may have a role in the cell cycle of Tetrahymena, and that mRNA expression, at least, is under tight cell-cycle control.

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C 18 fatty acids increased in cells grown at lower temperature for all lipid classes, and ω3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the ω3 position of C 18 fatty acids increased gradually during a 12-h period after a temperature shift-down to 22°C. However, the mRNA levels of the desA (Δ12 desaturase), desB (ω3 desaturase) and desC (Δ9 desaturase) genes increased within 15 min after a temperature shift-down to 22°C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38°C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22°C and 38°C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.

Tyrosine phosphorylation of ACK in response to temperature shift-down, hyperosmotic shock, and epidermal growth factor stimulation

The mammalian Cdc42 protein regulates various kinds of cellular responses, including formation of filopodia, polarization of T cells, and cell cycle progression. A non-receptor tyrosine kinase ACK, which specifically binds to the GTP-bound form of Cdc42, was isolated as a putative target of Cdc42. Here we show the induction of tyrosine phosphorylation of ACK in response to temperature shift-down to 25°C, and hypertonic shock, as well as stimulation with epidermal growth factor (EGF) in human embryonic kidney (HEK) 293 cells. The increased tyrosine phosphorylation level upon temperature shift-down was sustained for at least 60 min, whereas reversion of the temperature to 37°C caused rapid tyrosine dephosphorylation to the initial level. The responses to EGF and the high osmolarity were transient. Furthermore, we observed association of ACK with an adaptor protein Grb2, which may suggest the involvement of Grb2 in EGF receptor-mediated tyrosine phosphorylation of ACK.

Effect of benzyl alcohol and ethanol on cold-shock response ofBacillus subtilis

Benzyl alcohol and ethanol were found to exhibit an opposite effect on cold adaptation ofBacillus subtilis. The growth lag induced in bacteria by temperature shift-down was reduced in the presence of benzyl alcohol from 80 to 58 min, whereas ethanol prolonged this interval up to 102 min. In parallel, the physical state of the cytoplasmic membrane in intact bacteria was examined with the help of steady-state fluorescence anisotropy of diphenylhexatriene. Benzyl alcohol decreased the anisotropy as a consequence of membrane fluidization whereas ethanol increased its value, suggesting membrane rigidization. The correlation of growth and fluorescence data indicates that the control of membrane structure represents an important part of bacterial adaptation to cold.

Transcriptional activation of the origin of coliphage λ DNA replication is regulated by the host DnaA initiator function

The initiator of phage λ DNA replication, the λ O protein, is considered to be an analogue of the initiator of DNA replication (DnaA) of its host, Escherichia coli. Both specifically recognize their origins of replication, oriλ and oriC, respectively, and organize the assembly of specific replication complexes. However, DnaA has an additional activation function, acting on oriC-proximal DnaA-boxes, and regulating transcription initiated at promoters in and around oriC. Here, we demonstrate that λ plasmid replication can be synchronized by a temperature shift-down that caused renaturation of the previously denatured DnaAts protein. Moreover, we show that elimination of the activating DnaA function affects transcriptional activation at oriλ. DnaA may act by binding to DnaA-boxes, situated around the λ p R promoter; there are no such sequences in oriλ. Our results begin to explain in molecular terms why λ plasmid replication is DnaA-dependent [Kur et al., J. Mol. Biol. 198 (1987) 203–210] and why the initiation of phage λ DNA replication is blocked (in E. coli devoid of prophage Rac) after inactivation of DnaA [Wggrzyn et al., Genetics (1995) in press]

Heat-Induced DNA Relaxiation In Vitro by Mouse DNA Topoisomerase I in the Presence of Ethidium Bromide

In general, covalently-closed circular DNAs that are relaxed by DNA topoisomerase I at higher temperatures are more negatively (or less positively) supercoiled when analyzed at the same temperature. We found that in the presence of ethidium bromide the reaction products were less negatively supercoiled at higher temperatures. Consequently, the linking number of substrate DNA changed reversibly with temperature-shift: DNA relaxation occurred on temperature shift-up, and re-supercoiling on temperature shift-down. Analyses of the migration of covalently closed circular DNA on agarose gel electrophoresis and of the fluorescence intensity of ethidium bromide bound to DNA indicated decrease in binding of the drug to DNA at higher temperatures. Thus, the thermal effects on the topology of the reaction products of mouse DNA topoisomerase I in the presence of ethidium bromide can be explained by the temperature-sensitive interaction of the drug with DNA.

Establishment of herpes simplex virus latency in vitro with cycloheximide.

Human embryonic lung cells were infected with herpes simplex virus (HSV), treated with 10 micrograms/ml or more of cycloheximide for 24 h, incubated at 37 degrees C, and then shifted to 40.5 degrees C for various periods of time (0 to 40 days) without cycloheximide treatment. No infectious virus was detected after freezing and thawing of the cultures; however, infectious virus was recovered after temperature shift-down to 37 degrees C or superinfection with human cytomegalovirus (HCMV). The time course for formation of infectious centres after temperature shift-down was examined with and without HCMV superinfection during incubation at 40.5 degrees C. Two patterns of latently infected cells were identified: one pattern showed spontaneous reactivation of virus after temperature shift-down, and the second showed reactivation of HSV after superinfection with HCMV. The first pattern showed a rapid decrease in the number of infectious centres with time, whereas the second maintained a steady reactivation rate up to 40 days at 40.5 degrees C. The same tendency was observed for infectious centre formation at 37 degrees C with and without HCMV superinfection in the HSV latency system established with (E)-5-(2-bromovinyl)-2'-deoxyuridine and interferon treatment.

Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes.

Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non-structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T-antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift-down, defined circular DNA molecules were generated and amplified up to 2000-5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20-65-fold in the expression of NS1 and NS2 proteins was observed after temperature shift-down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes.

An amplified genome that may have resulted from recombination within bidirectionally replicating DNA

Temperature shift-down of permissive mouse cells transformed by a temperature-sensitive (ts) polyomavirus (Py) genome has been shown to induce the accumulation of free copies of the viral DNA. We report here on an unusual product from such induction. The structure of this product is that expected from the occurrence of recombination between growing points in a bidirectionally replicating Py-mouse DNA molecule. This observation may be relevant to the mechanism of gene amplification in mammalian cells.

Associations of elements of the Golgi apparatus with microtubules.

The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by immunolabeling of the G protein of the virus that was arrested in the GA during its intracellular passage to the plasma membrane 13 min after the temperature shift-down. Complete disassembly of the cytoplasmic microtubules by nocodazole at the nonpermissive temperature before the temperature shift led to the dispersal of the GA elements, from their normal compact perinuclear configuration close to the microtubule-organizing center (MTOC) into the cell periphery. Washout of the nocodazole that led to the reassembly of the microtubules from the MTOC also led to the recompaction of the GA elements to their normal configuration. During this recompaction process, GA elements were seen in close lateral apposition to microtubules. In cells treated with nocodazole followed by taxol, an MTOC developed, but most of the microtubules were free of the MTOC and were assembled into bundles in the cell periphery. Under these circumstances, the GA elements that had been dispersed into the cell periphery by the nocodazole treatment remained dispersed despite the presence of an MTOC. In cells treated directly with taxol, free microtubules were seen in the cytoplasm in widely different, bundled configurations from one cell to another, but, in each case, elements of the GA appeared to be associated with one of the two end regions of the microtubule bundles, and to be uncorrelated with the locations of the vimentin intermediate filaments in these cells. These results are interpreted to suggest two types of associations of elements of the GA with microtubules: one lateral, and the other (more stable) end-on. The end-on association is suggested to involve the minus-end regions of microtubules, and it is proposed that this accounts for the GA-MTOC association in normal cells.

A post-alpha gene function turns off the capacity of a host protein to bind DNA in cells infected with herpes simplex virus 1.

HEp-2 cell proteins electrophoretically separated in denaturing polyacrylamide gels and electrically transferred to nitrocellulose sheets contain a polypeptide which efficiently binds linear native DNA end labeled with 32P but not denatured DNA. The polypeptide has an apparent molecular weight of ca. 130,000. The activity of the protein was stable, and no appreciable turnover was observed after exposure of uninfected cells to inhibitory concentrations of cycloheximide for intervals of up to 24 h. However, the activity was absent from lysates of cells harvested 6 h or later postinfection with wild-type viruses. To identify the viral function involved in the loss of DNA-binding activity, we tested the lysates of cells infected with several mutants. Thus, the DNA-binding activity was unaffected in cells infected with a temperature-sensitive mutant (herpes simplex virus 1 tsLB2) in the alpha 4 gene and was maintained at a nonpermissive temperature (39 degrees C). Experiments involving (i) temperature shift-down of cells infected with tsLB2 in the presence of cycloheximide, (ii) withdrawal of cycloheximide in the presence and absence of actinomycin D from cells infected with wild-type virus, (iii) infection of cells at 33 and 39 degrees C with herpes simplex virus 1 tsHA1 carrying a temperature-sensitive lesion in the beta 8 gene, and (iv) infection of cells in the presence of inhibitory concentrations of phosphonoacetate led to the conclusion that the viral functions responsible for the loss of DNA-binding capacity were specified by either beta or gamma genes not dependent on viral DNA synthesis for their expression.

Inducible permissive cells transformed by a temperature-sensitive polyoma virus: superinfection does not allow excision of the resident viral genome.

After exposure of mouse embryo cells to the early temperature-sensitive mutant tsP155 of polyoma virus (Py), a transformed cell line (Cyp line) that can be readily induced to synthesize Py by transfer to 33 degrees C was isolated at 39 degrees C (7). Virus production and synthesis of free viral DNA occurring after temperature shiftdown or superinfection with wild-type Py or both were studied in several clonal isolates of the Cyp cell line. Measurements of virus yields indicated that, although some could be induced more effectively than others, all cell clones behaved as highly permissive when subjected to superinfection. We analyzed the origin of free viral DNA accumulating in the superinfected cultures, taking advantage of (i) the unique physical properties of the low-molecular-weight DNA which, in the case of one of the Cyp clones, accumulates during temperature shiftdown, and (ii) the differences between resident and superinfecting viral genomes in their susceptibilities towards restriction endonucleases. At 33 degrees C, both viral genomes were found to accumulate in all clones studied whereas in the case of the clones with lower inducibility, the replication of the resident genome appeared to be enhanced by superinfection. At 39 degrees C, however, accumulation of the superinfecting genome was not accompanied by that of the resident genome, unless it had already been initiated before superinfection. These findings demonstrate that, when routinely cultivated at 39 degrees C, Cyp cells contain few viral DNA molecules readily available for autonomous replication and that, upon transfer to 33 degrees C, therefore, excision must first take place before the resident genome can accumulate as free viral DNA. Our findings also suggest that, unlike the P155 gene product provided by the resident viral genome upon induction, the allelic gene product supplied by the superinfecting genome may be less effective in triggering excision than in promoting replication.

Latency in vitro using irradiated herpes simplex virus.

Human embryonic fibroblasts infected with u.v.-irradiated herpes simplex virus type 2 (HSV-2, strain 186) and maintained at 40.5 degrees C did not yield detectable virus. Virus synthesis was induced by temperature shift-down to 36.5 degrees C. The induced virus grew very poorly and was inactivated very rapidly at 40.5 degrees C. Non-irradiated virus failed to establish latency at 40.5 degrees C in infected cells. Enhanced reactivation of HSV-2 was observed when latently infected cultures were superinfected with human cytomegalovirus (HCMV) or irradiated with a small dose of u.v. light at the time of temperature shift-down. HCMV did not enhance synthesis of HSV-2 during a normal growth cycle but did enhance synthesis of u.v.-irradiated HSV-2. These observations suggest that in this in vitro latency system, some HSV genomes damaged by u.v. irradiation were maintained in a non-replicating state without being destroyed or significantly repaired.

Enhanced production of virus-inhibiting factor (interferon) in human diploid cells by ultraviolet irradiation and temperature shift-down after stimulation with Newcastle disease virus.

The production of the virus-inhibiting factor or interferon (IF) was highest in cells incubated at 37 C after inoculation with Newcastle disease (ND) virus and decreased as the incubation temperature was lowered. Shift-down of incubation temperature to 32 C or 34 C after incubation at 37 C for 4--7 hr enhanced IF production in cell cultures stimulated with ND virus, as compared with cultures incubated continuously at 37 C. Shift-down to 32 C after incubation at 37 C for 6 hr was optimal for this enhancement of IF yield. Enhanced IF production was also observed in cell cultures irradiated by ultraviolet light 4--7 hr after stimulation with ND virus.

Heat shock proteins in Tetrahymena studied under growth conditions

Heat shocks or cold shocks were found to induce characteristic changes in the pattern of protein synthesis in Tetrahymena pyriformis as analysed on sodium dodecyl sulphate (SDS)-polyacrylamide gels. Within 10 min the temperature shift-up induces increased incorporation of [ 35S]methionine into 4–5 protein bands, whereas incorporation into other bands is reduced, The heat-induced proteins appear to be metabolically stable, and no change in protein turnover could be detected at the elevated temperature. The protein induction is not observed when RNA synthesis is inhibited by actinomycin D. The temperature shift-down induces a different pattern of protein synthesis and there is no evidence of a common molecular mechanism underlying synchrony induction with heat shocks and cold shocks.

Phosphoprotein associated with activation of the src gene product in myogenic cells

Summary Chicken mononucleated myoblasts were infected with a temperature sensitive mutant of Rous sarcoma virus (tsNY68) and allowed to develop into myotubes by incubating at 41°C (nonpermissive temperature for the src gene product). Activation of the src gene product by temperature shiftdown to 36°C (permissive temperature) in these myotubes was found to phosphorylate a 36,000 dalton cellular protein which is similar to the one reported by Radke and Martin (1) recently. Phosphorylation of this 36,000 dalton protein was observed within 3 hr after temperature shift. No appreciable phosphorylation of this protein was observed in control myotubes kept at 41°C nor in the uninfected myotubes. These results suggested that this 36,000 dalton protein may be a possible target of the src gene product (pp60 src ) of Rous sarcoma virus for its cellular transformation in myotubes as well as in fibroblasts.