Stage dependent synthesis of heat shock induced proteins in early embryos of Drosophila melanogaster

Summary The synthesis of heat shock proteins (hsp) has been examined during the early embryogenesis of Drosophila melanogaster. Normal protein synthesis stops after heat shock at all developmental stages, while hsp synthesis is induced only after treatment at blastoderm and later stages. The small hsps continue to be synthesised after heat shock for a longer period than the larger ones. Heat shocks at 35°C, 37°C and 40°C were compared for their effect on hsp synthesis and the effect of heat shock on the normal course of development was analysed.     

Immunofluorescence localization of a small heat shock protein (hsp 23) in salivary gland cells of Drosophila melanogaster

Summary An aggregate present in cell-free extracts of Drosophila melanogaster tissue culture cells, sedimenting at 20 to 30S, contains hsps 23, 26 and 27. Hsp 23 was purified from this aggregate and a monospecific antibody was raised against it. Immunofluorescence microscopy showed the presence of hsp 23 preferentially in nuclei after heat shock, while on return to 25° C, hsp 23 was reduced in nuclei and increased in the cytoplasm. Thus the immunofluorescence observations reported here unambignously confirm for hsp 23 earlier reports that heat shock proteins are mainly found in nuclei after heat shock and that upon return to 25° C, they move to the cytoplasm.Abbreviations NP-40 Nonidet P40 - PMSF Phenylmethylsulfonyl-fluoride - SDS Sodium dodecyl sulfate - EDTA Ethylene diamine tetraacetic acid - TCA Trichloroacetic acid. hsp 22, hsp 23 etc.: heat shock proteins of 22,000, 23,000 daltons etc. molecular weight     

Nucleosomal organization of a BPV minichromosome containing a human H4 histone gene

Summary When the body temperature of rats is elevated to 42°C, four heat shock proteins, with the molecular weights of 70000, 71000, 85000, and 100000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), are induced in various tissues of rats (Fujio et al., J Biochem 101, 181–187, 1987). Heat shock proteins are induced by various stresses other than heat in varieties of cultured cells, so we studied whether heat shock proteins are induced in intact rats by different treatments. Analysis of the translation products of poly(A) + RNA isolated from the livers of rats recovering from ischemia of the liver showed that mRNAs for hsp 70, hsp 71, and hsp 85 were induced. These hsp-mRNAs were also induced in the livers of rats 6 h after a partial hepatectomy, and had returned to control levels 24 h after the surgery. These results suggested that heat shock proteins have not only the function of protection against various stresses but also physiological functions in the normal growth and development of animals.     

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.     

The heat shock response inLocusta migratoria

Summary Locusta migratoria adults reared at 27–30°C die after 2 h at 50°C, but they survive this temperature stress if first exposed to 45°C for 0.5 to 4.5 h. Fat bodies from adult females produce a set of at least six specific polypeptides with molecular weights of 81, 73, 68, 42, 28, and 24×10³ in reponse to heat shock (39–47°C for 1.5 h). These molecular weights closely match those of the heat shock proteins (hsps) observed inDrosophila, with the possible exception of the 42 kd protein of locusts. The optimal temperature for induction of hsps in locusts is 45°C, which is one of the highest heat shock temperatures reported in metazoans. The correspondence between the optimal temperature for hsp induction and the temperature at which enhanced heat tolerance is acquired (both 45 °C) suggests that the hsps may be associated with thermal protection in these insects.There appears to be no substantial translational control in the locust heat shock response, since other proteins are produced, albeit with some reduction, under heat shock conditions. Vitellogenin synthesis in fat bodies at 45°C is 55% of that observed at 30°C. The high optimal heat shock induction temperature and the continued synthesis of non-heat shock proteins may be adaptive to the locust's natural environment.     

Synthesis of heat shock proteins inThermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes EM1)

The response to heat stress was examined inThermoanaerobacterium thermosulfurigenes EM1. Upon a temperature shift-up from 50° to 62°C, four heat shock proteins (hsps) were synthesized at an elevated level. Two proteins were found to be immunologically related to theEscherichia coli GroEL protein and theMycobacterium tuberculosis hsp71 (DnaK similar protein), and the correspondinggroE anddnaK homologous sequences were detected in the chromosome ofT. thermosulfurigenes EM1. The heat shock response in this thermophile was transient, with a maximum synthesis of hsps between 10 and 15 min after the shock. The enhanced synthesis of DnaK and GroEL was consistent with increased mRNA levels of the genes, which reached a maximum 15 min after heat treatment.     

Heat Shock Proteins and Their mRNAs in Dry and Early Imbibing Embryos of Wheat

Two-dimensional gels of in vitro translation products of mRNAs isolated from quiescent wheat (Triticum aestivum) embryos demonstrate the presence of mRNAs encoding heat shock proteins (hsps). There were no detectable differences in the mRNAs found in mature embryos from field grown, from 25°C growth chamber cultivated, or from plants given 38°C heat stresses at different stages of seed development. The mRNAs encoding several developmentally dependent (dd) hsps were among those found in the dry embryos. Stained two-dimensional gels of proteins extracted from 25°C growth chamber cultivated wheat embryos demonstrated the presence of hsps, including dd hsps. A study of the relationship of preexisting hsp mRNAs and the heat shock response during early imbibition was undertaken. Heat shocks (42°C, 90 minutes) were administered following 1.5, 16, and 24 hours of 25°C imbibition. While the mRNAs encoding the low molecular weight hsps decayed rapidly upon imbibition, the mRNAs for dd hsps persisted longer and were still detectable following 16 hours of imbibition. After 1.5 hours of imbibition, the mRNAs for the dd hsps did not accumulate in response to heat shock, even though the synthesis of the proteins was enhanced. Thus, an applied heat shock appeared to lead to the preferential translation of preexisting dd hsp mRNAs. The mRNAs for the other hsps, except hsp 70, were newly transcribed at all of the imbibition times examined. The behavior of the hsp 70 group of proteins during early imbibition was examined by RNA gel blot analysis. The mRNAs for the hsp 70 group were detectable at moderate levels in the quiescent embryo. The relative level of hsp 70 mRNA increased after the onset of imbibition at 25°C and remained high through 25.5 hours of prior imbibition. The maximal levels of these mRNAs at 25°C was reached at 17.5 hours of imbibition. Heat shock caused modest additional accumulation of hsp70 mRNA at later imbibition times.     

Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter

We have isolated two sunflower genes, Ha hsp 18.6 G2 and Ha hsp 17.7 G4, that encode small heat shock proteins (sHSPs). RNAse A protection experiments, carried out with RNA probes transcribed from each gene and hybridized to sunflower total RNA, allowed us to distinguish their mRNA accumulation patterns. In sunflower, Ha hsp 17.7 G4 mRNAs accumulated during zygotic embryogenesis at 25°C. In vegetative tissues, these mRNAs accumulated in response to either heat shock (42°C), abscisic acid (ABA), or mild water stress treatments. In all cases, the mRNAs were transcribed from the same initiation site. In contrast, Ha hsp 18.6 G2 mRNAs accumulated only in response to heat-shock. This result demonstrates differential regulation of these two sHSP genes. The complex regulation depicted by the Ha hsp 17.7 G4 promoter has been further analyzed in transgenic tobacco, using G4::GUS translational fusions. Developmental induction of Ha hsp 17.7 G4 during zygotic embryogenesis was faithfully reproduced in the transgenic plants. 5-distal sequences (between -1132 and -395) were required to confer a preferential spatial expression of GUS activity in the cotyledons. More proximal sequences (from -83 to +163) conferred to the chimeric genes most of the developmental regulation, and the responses to ABA and heat shock characteristic of the Ha hsp 17.7 G4 promoter. The water stress response of this gene was not reproduced in transgenic tobacco and, thus, could be uncoupled from its regulation during embryogenesis.     

Heat shock response during anther culture of broccoli (Brassica oleracea var italica)

Embryo formation from microspores of Brassica oleracea var Italica (Broccoli) and other Brassica species is greatly enhanced by an initial incubation at elevated temperatures (eg 35°C) followed by continued incubation of 25°C. In the present study we observed that a three hour high temperature treatment induced the formation of heat shock proteins in cultured anthers. These were identified in two dimensional gels by silver staining, and labelled heat shock proteins were synthesised in vitro from isolated anther RNA. The appearance of heat shock proteins in anthers followed a similar pattern and displayed similar characteristics to that from leaves. Comparison of the heat shock proteins induced in isolated cultured anthers of known highly embryogenic and less embryogenic plans did not reveal obvious qualitative differences.     

Heat and cold shock responses in different strains of Thiobacillus ferrooxidans

Different strains of Thiobacillus ferrooxidans were examined for their ability to produce a heat shock and a cold shock response. Strain A1, heat shocked from 20° to 35°C, acquired thermotolerance, as it showed a 1000-fold reduction in cell mortality when exposed to the supermaximum temperature of 42°C, as compared to a non-heat-shocked control. A heat shock from 25° to 35°C yielded similar results, although a higher degree of thermotolerance was achieved for the shorter exposure times. Cultures heat shocked for 5 h showed a five-log reduction in viable counts after 41 h at 42°C, whereas non-heat-shocked cultures showed a similar reduction in viability in 28 h. Conferred thermotolerance was immediate and sustained for the duration of the exposure to 42°C. Heat-shocked cultures were not significantly protected against loss of viability due to freezing (-15°C for 24 h). Strain S2, cold shocked from 25° to 10°C, and strain D6, cold shocked from 25° to 5°C, were not protected against freezing at-15°C. An analysis of proteins extracted from heat-shocked cells of strain A1 showed the presence of at least one newly induced protein and eight hyper-induced proteins. The molecular weights of the heat shock proteins were in the range of 15–80.3 kDa.     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.     

Genomic approaches to detecting thermal stress in Calanus finmarchicus (Copepoda: Calanoida)

Zooplankton may be subjected to physiological stress as they encounter rapid and large changes in temperature through vertical migration or transfer into different water masses. Induction of one or more heat shock proteins (hsp) is a common protective response to thermal stress in organisms. We looked for evidence for such a response in Calanus finmarchicus. We compared hsp70 expression in copepods exposed to temperature stress with that for non-stressed controls. Partial sequences of the amplified cDNA product were obtained and aligned with known hsp70 sequences to establish the identity of the heat shock protein. In one experiment, animals were transferred from their collection temperature (8 °C) to 20 °C for 30 min, and then returned to 8 °C for 4 h before sampling for gene expression levels. In another, the animals were exposed to 18 °C over 48 h before sampling for molecular analysis. A four-fold induction of hsp70 was measured in both groups of heat shocked animals using quantitative real time polymerase chain reaction (PCR). The experimental temperatures, although high for C. finmarchicus, are within the range of temperatures experienced by this species in their habitat. In addition to confirming an hsp70-mediated response in C. finmarchicus, the findings suggest that a recent history of thermal stress may be assessed in natural populations through a routine molecular assay.     

Basic features of the Streptococcus thermophilus heat shock response

The heat shock response was investigated in the thermophilic acid bacterium Streptococcus thermophilus. The heat resistance (58°C, 30 min) of log-phase cells grown at 42°C was enhanced by pretreatment at 52°C for 15 or 30 min. Concurrently to this acquired thermotolerance, two-dimensional gel electrophoresis indicates that the cells induced the synthesis of at least 22 heat shock proteins after temperature upshift. Furthermore, following SDS-PAGE, Western blotting, and immunological analysis, six proteins were found to be antigenically related to the Escherichia coli heat shock proteins DnaK, DnaJ, GroEL, GrpE, and La and to the Bacillus subtilis ⁴³ factor Among these six proteins, two related to DnaK and GroEL, are clearly overexpressed during this stress. It is concluded that S. thermophilus possesses a heat shock response similar to that known to occur in mesophilic microorganisms.     

Heat shock response in psychrophilic and psychrotrophic yeast from Antarctica

The response to heat stress in six yeast species isolated from Antarctica was examined. The yeast were classified into two groups: one psychrophilic, with a maximum growth temperature of 20°C, and the other psychrotrophic, capable of growth at temperatures above 20°C. In addition to species-specific heat shock protein (hsp) profiles, a heat shock (15°C–25°C for 3 h) induced the synthesis of a 110-kDa protein common to the psychrophiles, Mrakia stokesii, M. frigida, and M. gelida, but not evident in Leucosporidium antarcticum. Immunoblot analyses revealed heat shock inducible proteins (hsps) corresponding to hsps 70 and 90. Interestingly, no proteins corresponding to hsps 60 and 104 were observed in any of the psychrophilic species examined. In the psychrotrophic yeast, Leucosporidium fellii and L. scottii, in addition to the presence of hsps 70 and 90, a protein corresponding to hsp 104 was observed. In psychrotrophic yeast, as observed in psychrophilic yeast, the absence of a protein corresponding to hsp 60 was noted. Relatively high endogenous levels of trehalose which were elevated upon a heat shock were exhibited by all species. A 10 Celsius degree increase in temperature above the growth temperature (15°C) of psychrophiles and psychrotrophs was optimal for heat shock induced thermotolerance. On the other hand, in psychrotrophic yeast grown at 25°C, only a 5 Celsius degree increase in temperature was necessary for heat shock induced thermotolerance. Induced thermotolerance in all yeast species was coincident with hsp synthesis and trehalose accumulation. It was concluded that psychrophilic and psychrotrophic yeast, although exhibiting a stress response similar to mesophilic Saccharomyces cerevisiae, nevertheless had distinctive stress protein profiles. Received: August 7, 1997 / Accepted: October 22, 1997     

Heat shock protein response to thermal stress in the Asiatic clam,Corbicula fluminea

A recent approach to evaluate environmenta induced damages has been damages has been suggested, based on the stress response. The approach involves the detection of stress protein induction in organisms to infer about environmental conditions in their surroundings. However, to be an indicator of adverse biological effects in the environment, the elevation of stress proteins should be compared to a response pattern for the experimental species. JuvenileCorbicula fluminea, collection from a control site, were submited to heat-shock stress in the laboratory to obtain the stress response pattern under normal and extreme conditions. Acclimated to 26°C, the specimens were submited to 29, 32, 35 and 38°C, for 96 h. After 1, 2, 4, 8, 24, 48, 72 and 96 h of exposure, clams were removed from each vial and prepared for stress protein analysis. Animals from the control site were frozen in liquid nitrogen at the time of collection, and prepared for stress protein analysis. Hsp60 and 70 were detected by immunoreactivity after separation on 12.5% polyacrylamide gels and transference to nitrocellulose by western blotting, to determine the stress protein concentrations. The result showed that hsp70 increased at 4h from the beginning of the experiment and progressed over the 96 h experimental period in animals exposed to 35°C. However hsp70 levels decreased between 4 h and 24 h for the clams stressed at their lethal temperature of 38°C. Immunoblotting with hsp60 showed similar reactivity. At 38°C there was an increase in the amount of hsp60 at 4h, reaching a maximum eight-fold level at 8h. By 96h, the amount decreased to levels lower than those observed at 4h. At 38°C the level of hsp60 began to decrease at 8 h and continue to decline to 24 h when the clams died. The data support the hypothesis of increasing concentrations of stress protein until the heat shock approaches the thermal limits for the species. The results of this research suggest the usefulness of using the stress response as a diagnostic in environmental toxicology. They confirm that the sps response may serve as a valid biomonitoring tool under chronic, sublethal exposures when it is still possible to prevent effects at organismal or higher organizational levels.     

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.