Showing papers on "Heat shock protein published in 1982"

Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin

Abstract: The primary base sequence of the protein coding regions of the four small heat shock genes of Drosophila melanogaster present at cytological locus 67B has been determined. A single open reading frame large enough to encode a small heat shock protein is found for each gene. The molecular weights of the predicted proteins are in good agreement with experimentally determined values obtained from gel electrophoresis. The predicted amino acid sequences of the four small heat shock genes show striking homologies over approximately 50% of their lengths. This region of extensive homology extends from about amino acid 85 to amino acid 195 out of a total of approximately 200 amino acids. Comparison of the predicted sequence with the known sequences of other proteins revealed a remarkable similarity between this region of homology and the corresponding region of mammalian alpha-crystallin. The possible functional significance of this structural similarity is discussed.

Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts.

Abstract: Synthesis of a family of proteins called "heat shock" proteins is induced or enhanced in cells in response to various environmental stresses, suggesting that these proteins may perform functions essential to cell survival. Because a brief, nonlethal heat treatment can dramatically induce a transient resistance to a subsequent lethal heat treatment (thermotolerance), we examined the effect of heat treatment (41-46 degrees C) on protein synthesis and cell survival in plateau-phase Chinese hamster fibroblast (HA-1) cells. After heat treatments that either drastically inhibited total protein synthesis (46 degrees C) or did not suppress it (41 degrees C), the synthesis of heat shock proteins was greatly enhanced over that in unheated cells, and cell survival was increased 10(2)- to 10(6)-fold when cells were challenged by a subsequent lethal heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance, and the stability of these proteins correlated well with the persistence of thermotolerance up to 36 hr. Sodium arsenite, hypoxia, and ethanol also induced both the synthesis of heat shock proteins and transient thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis and persistence of polypeptides of Mr 70,000 or 87,000 most closely conformed to the kinetics of thermotolerance.

Synthesis and Degradation of Heat Shock Proteins during Development and Decay of Thermotolerance

Abstract: Morris hepatoma 7777 cells, heat conditioned at 43 degrees for 0.5 hr, become gradually thermoresistant during an incubation at 37 degrees as judged by their ability to form colonies following a second heat challenge. Pulse incorporation of [35S]methionine into proteins at various times after the conditioning treatment and subsequent fractionation of the proteins by polyacrylamide gel electrophoresis indicate that the gradual putative modifications occurring at the cellular level and leading to the thermotolerance state are accompanied by an elevated synthesis above the normal level of a small set of polypeptides with apparent molecular weights of 27,000, 65,000, 68,000, 70,000, 89,000, and 107,000. Both thermotolerance development and protein induction are completed after a 6- to 8-hr period. At the end of this period, thermotolerance is at its maximum level and heat shock protein synthesis is returned to normal. This acquired thermal resistance eventually disappears between 60 and 80 hr following conditioning treatment. In a parallel manner, the heat shock-induced proteins synthesized during the first 4 hr following the conditioning treatment are maintained in the cells at a high level for several hr but become undetectable by 82 hr. The results provide strong circumstantial evidence that heat shock proteins are involved in the acquisition, maintenance, and decay of thermotolerance.

Genetic control of heat-shock protein synthesis and its bearing on growth and thermal resistance in Escherichia coli K-12.

Abstract: When Escherichia coli cells grown at 30 degrees C are transferred to 42 degrees C, synthesis of several polypeptides is markedly and transiently induced. A temperature-sensitive nonsense mutant (tsn-K165) of E. coli K-12 is found to be defective in the induction of these proteins. mRNA for one major heat-shock polypeptide (groE protein) tested is induced in the wild type but not in the mutant upon temperature shift. Hence, the mutation defines a (regulatory) gene, designated hin (heat shock induction), whose product is required for active transcription of a set of heat-inducible operons in E. coli. The results reported herein suggest that the heat-shock polypeptides controlled by the hin gene play an important role in cell growth at high temperature. The possible involvement of the hin gene product in protection against thermal killing is also discussed.

Antibodies to two major chicken heat shock proteins cross-react with similar proteins in widely divergent species

Abstract: Three of the proteins induced by heat shock of chicken embryo fibroblasts have been purified, and rabbit antibodies have been raised against them. These antibodies have been used in radioimmune precipitation reactions and in a solid-phase immune assay to detect antigenic material in non-heat-shocked chicken tissues and in extracts of widely different species ranging from yeast to mammalian tissue culture cells and human erythrocyte ghosts. Antibodies to two of the major chicken heat shock proteins, chsp89 and chsp70, cross-reacted with proteins of similar molecular weights in normal embryonic and adult chicken tissues and in extracts from widely different organisms. These data provide further evidence for the university of the heat shock response and conservation of proteins induced by this type of stress.

A synthetic heat-shock promoter element confers heat-inducibility on the herpes simplex virus thymidine kinase gene.

Abstract: Previous deletion analysis of the Drosophila hsp70 heat-shock promoter has identified a sequence upstream of the TATA box that is required for heat induction. This region contains homology to other heat-shock promoters, and it was proposed that the common sequence is an important element in the regulation of the heat-shock genes. We have constructed sequences similar to the consensus CT-GAA-TTC-AG from synthetic oligonucleotides and placed them upstream of the TATA box of the herpes virus thymidine kinase gene, in place of the normal upstream promoter element. The resultant genes are heat-inducible both in monkey COS cells and in Xenopus oocytes. We conclude that the transcriptional heat-shock response is mediated by some factor that interacts with this sequence.

Heat shock proteins and thermotolerance; a comparison of induction kinetics.

Abstract: The hypothesis that the expression of heat shock proteins following a preliminary hyperthermic treatment is responsible for subsequent thermotolerance to a second heat treatment is examined. CHO cells were given a 12 min, 45 degrees C pretreatment and then incubated for varying intervals at 37 degrees C. The synthesis of certain intracellular proteins was monitored as a function of time post-incubation by using 35S-methionine incorporation as determined in SDS polyacrylamide gel electrophoresis. Cell survival was concurrently measured by challenging the cells with a second heat treatment (45 degrees C/27 min). Major heat shock proteins were observed at 68 000, 89 000 and 110 000 daltons. The synthesis of these proteins was significantly reduced in the presence of cyclohexamide. The total 35S-methionine incorporation into these proteins correlated well with the induction of survival resistance (thermotolerance). An approximate exponential relationship between survival and the amount of each of these proteins may occur. These and other heat shock proteins were also present, in a significantly reduced degree, in control (non-heat shocked) cells maintained under normal culture conditions at 37 degrees C. It is possible that heat shock proteins are responsible for the phenomenon of thermotolerance.

Heat shock and recovery are mediated by different translational mechanisms

Abstract: When Drosophila cells are shifted from 25 degrees C to 37 degrees C, protein synthesis is rapidly redirected from the complex pattern characteristic of normal growth to the simple pattern of heat shock proteins (HSPs). On return to 25 degrees C, synthesis of normal proteins is gradually reactivated and that of HSPs is repressed. In quantifying many different recovery experiments, we found that preexisting mRNAs always behaved as a cohort, with messages for different proteins returning to translation at the same rate. Heat shock mRNAs (HS mRNAs), on the other hand, never behaved as a cohort. Their repression was asynchronous, with translation of hsp70 always the first and translation of hsp82 always the last to be repressed. Although recovery times varied enormously (depending on the severity of the heat treatment), repression of hsp70 was always correlated with restoration of normal synthesis, suggesting a link between the two events, hsp70 repression was not simply due to competition with reactivated 25 degrees C mRNAs. A general decline in the translation efficiency of hsp70 mRNA was not observed. Instead, an increasing number of messages were translationally inactivated, while those remaining in the translational pool retained full ribosome loading. Unlike inactive 25 degrees C mRNAs, which are stable during heat shock, inactive HSP mRNAs are degraded during recovery.

Newcastle disease virus stimulates the cellular accumulation of stress (heat shock) mRNAs and proteins.

Abstract: A biological agent, Newcastle disease virus, stimulated the synthesis of stress proteins in cultured chicken embryo cells. Previously, only physical and chemical agents were known to induce these proteins. The levels of translatable stress mRNAs were elevated in cells infected with avirulent or virulent strains; however, stress protein synthesis was stimulated strongly only in cells infected by avirulent strains. As did several other paramyxoviruses, avirulent strains of Newcastle disease virus stimulated the synthesis of glucose-regulated proteins as well as stress proteins. Possible stimuli of the synthesis of these two sets of proteins in paramyxovirus-infected cells are considered.

Thermotolerance and Heat Shock Proteins in Mammalian Cells

Abstract: One of the more intriguing aspects of thermal biology is the response of heated cells to subsequent exposures at elevated temperatures. It has been clearly demonstrated by Gerner and Schneider (1) and Henle and Leeper (2) that an initial heat dose can induce a temporary state of heat resistance. This state has been termed thermotolerance. If the initial heat dose is at temperatures greater than about 43?C, the transient resistance manifests itself only if an intervening period at 37?C is permitted. Maximum thermotolerance is reached around 6 hr after transfer to 37?C; tolerance maintains itself for some time, decays slowly, and disappears by perhaps 100 hr. Alternatively, if the initial heating is at temperatures lower than 43?C, thermotolerance can develop during the heating period. The crucial role of protein synthesis in the development of thermotolerance has been recognized for some time (3). Protein synthesis is inhibited at temperatures of 43?C or higher, but goes on at lower temperatures. Thermotolerance also cannot develop if protein synthesis is inhibited by means other than heat itself, as, for example, during incubation in medium in which water has been replaced by deuterium oxide (4) or if cells are at low temperature (1, 2). It has been known for a considerable time that heat induces synthesis of a family of proteins, usually referred to as heat shock proteins (hsp) (5). These proteins, perhaps as many as 20 or more, range in molecular weight from less than 20,000 to more than 100,000. Because of the important role that protein synthesis appears to play in the development of thermotolerance, as discussed in the previous paragraph, it is not surprising that experiments were performed to determine whether or not hsp may play a role in conferring heat resistance on cells. Indeed, several studies have now shown that in mammalian cells there is a good temporal correlation between the induction of heat shock proteins and the development of thermotolerance (6, 7). Furthermore, it has been demonstrated that thermotolerance can be

Saccharomyces cerevisiae contains a complex multigene family related to the major heat shock-inducible gene of Drosophila.

Abstract: Saccharomyces cerevisiae contains a family of genes related to the major heat shock-induced gene of Drosophila (hsp 70). Two members of the multigene family (YG100 and YG101) were isolated. The primary DNA sequences of more than one-half of the protein-encoding regions of YG100 and YG101 were determined and compared with the Drosophila hsp 70 gene sequence; the predicted amino acid sequences were 72 and 64% homologous to the sequence of the Drosophila hsp 70 protein, respectively. The predicted amino acid sequences of the yeast genes were 65% homologous. Our results demonstrate a striking sequence conservation of hsp 70-related sequences in evolution. Hybridization of the S. cerevisiae genes to total S. cerevisiae DNA indicated that the multigene family consists of approximately 10 members. Hybridization of labeled RNAs from heat-shocked and control cells suggested that, like transcription of the Drosophila hsp 70 gene, transcription of YG100 or a closely related gene is enhanced after heat shock. However, the amount of RNA sequences homologous to YG101 was reduced after heat shock. A multigene family related to the hsp 70 gene exists in Drosophila; transcription of some members is induced by heat shock, whereas transcription of others is not. Our results suggest that S. cerevisiae, like Drosophila, contains a multigene family of hsp 70-related sequences under complex transcriptional regulation and that the differential control, as well as the nucleotide sequence, has been highly conserved in evolution.

Regulation of heat-shock genes: a DNA sequence upstream of Drosophila hsp70 genes is essential for their induction in monkey cells.

Abstract: Heat-shock genes coding for heat-shock protein 70 (HSP70) in Drosophila melanogaster were subcloned into an SV40/plasmid recombinant capable of replication in permissive monkey COS cells. Following transfection of COS cells, no significant amount of Drosophila hsp70 RNA was detected at 37 degrees C. In contrast, a heat-shock at 43 degrees C or arsenite poisoning at 37 degrees C induced the massive production of Drosophila hsp70 RNA of correct size and faithful 5' ends. After heat-shock, the efficiency of hsp70 transcription in COS cells containing 2-4 X 10(4) gene copies was found to be 15-30% of that measured in Drosophila, on a per gene basis. By testing a series of 5' deletion mutants in this inducible transcription assay it was found that a sequence less than 70 bp long, directly upstream of the hsp70 gene, was essential for the heat or arsenite induction of transcription.

Nonspecific stabilization of stress-susceptible proteins by stress-resistant proteins: a model for the biological role of heat shock proteins.

Abstract: It is demonstrated experimentally that addition of proteins that are themselves resistant to denaturation by heat or ethanol can nonspecifically stabilize other proteins that are ordinarily highly susceptible to inactivation. It is proposed that the diffusion-limited rate with which unfolded protein molecules encounter each other and become irreversibly crosslinked is reduced in the presence of substantial concentrations of an unreactive globular protein. We suggest that one of the functions of heat shock proteins, which are synthesized in large amounts after exposure of cells to increased temperature and other forms of stress, may be to stabilize other proteins kinetically in a similarly nonspecific fashion.

Synthesis of low molecular weight heat shock peptides stimulated by ecdysterone in a cultured Drosophila cell line

Abstract: Treatment of Schneider's line 3 Drosophila cells with the steroid hormone ecdysterone rapidly stimulated the synthesis and accumulation of the polypeptide previously designated p7 [Berger, E. M., Ireland, R. C. & Wyss, C. (1980) Somatic Cell Genet. 6, 119-129]. In this report, p7 is identified as the 23,000-dalton heat shock polypeptide (hsp23). In addition to hsp23, the synthesis of the low molecular weight heat shock polypeptides hsp22, hsp26, and hsp27 was also stimulated by ecdysterone, although to different extents. Hybridization of a nick-translated genomic clone containing the hsp23 gene to a total RNA blot showed that ecdysterone stimulation of hsp23 synthesis was the result of an increase in the hsp23 RNA content of S3 cells. We detected no effect of the hormone on the synthesis of heat shock polypeptides hsp68, hsp70, and hsp83.

Diverse forms of stress lead to new patterns of gene expression through a common and essential metabolic pathway

Abstract: Many eukaryotic organisms respond to heat shock by synthesizing new proteins. We examined the possibility that heat shock proteins represent a particular expression of a general response to stress and that, regardless of the nature of the effective stimulus, the same proteins are synthesized. Accordingly, cardiac stress was applied in the intact rat by four methods: banding the ascending aorta, increasing body temperature to 42 degrees C, reducing body temperature to 18 degrees C, and forcing the rat to swim until exhausted. The hearts were then extirpated and analyzed for new mRNA synthesis. The extracted RNA was translated in a cell-free medium containing [35S]methionine. Translation products were resolved by two-dimensional electrophoresis and visualized by autoradiography. Lactic acid concentration in heart tissue was determined enzymatically. The results showed that two new and distinct proteins of Mr 71,000 and isoelectric points of 5.8 and 6.1 were synthesized in hearts stressed by banding and by heating but not in hearts of exhausted swimmers or in animals at reduced body temperatures. There was no significant difference in cardiac lactic acid concentration between control hearts and hearts from swimmers or cold-treated animals. However, there was a 2-fold increase in lactic acid concentration in hearts of rats with banded aortas compared to controls and a 10-fold increase in heat shocked hearts. We conclude that, under conditions in which the energy requirements of the heart are not completely met by aerobic processes, the resultant lactic acidosis creates an intracellular environment that leads to the selective activation of genes, the production of new mRNA, and the synthesis of a typical group of stress proteins.

Heat shock proteins and effects of heat shock in plants.

Abstract: Soybean seedlings when exposed to a heat shock respond in a manner very similar to that exhibited by cultured cells, and reported earlier [2]. Maximum synthesis of heat shock proteins (HSPs) occurs at 40C. The heat shock response is maintained for a relatively short time under continuous high temperature. After 2.5 hr at 40 C the synthesis of HSPs decreases reaching a very low level by 6 hr. The HSPs synthesized by cultured cells and seedlings are identical and there is a large degree of similarity in HSPs synthesized between the taxonomically widely separated species, soybean and corn. Storage protein synthesis in the developing soybean embryo is not inhibited but is actually stimulated during a heat shock, unlike most other non-HSPs, whose synthesis is greatly reduced. Seedlings respond differently to a gradual increase in temperature than they do a sudden heat shock. There is an upward shift of several degrees in the temperature at which maximum protein synthesis occurs and before it begins to be inhibited. In addition, there appears to be a protection of normal protein synthesis from heat shock inhibition when the temperature increase is gradual. An additional function of the heat shock phenomenon might be the protection of seedlings from death caused by extreme heat stress. The heat shock response appears to have relevance to plants in the field.

Induced thermal tolerance and heat shock protein synthesis in Chinese hamster ovary cells.

Abstract: We have performed experiments to determine the kinetics of induction of thermal tolerance in Chinese hamster HA-1 cells, and the effects of heat treatments on the recovery of protein synthesis, with particular attention to whether heat induces specific proteins, perhaps the heat shock proteins (HSP). The kinetics of the development of thermal tolerance were measured by increases in cellular survival. In parallel experiments, the effects of heat treatment on the recovery of protein synthesis in HA-1 cells were examined. After heating (45°, 20 minutes), some of these cells were immediately labeled with 35 S-methionine (10 μCi/ml) for 1 hour at 37°, while the others were incubated at 37° for 1–8 hours and then labeled. The cell samples were prepared for electrophoresis on a gradient SDS gel. The incorporation of label into HA-1 cell proteins was drastically inhibited by the 45° heat treatment, but recovered gradually during the 8-hour incubation period at 37°C. A comparison of the proteins synthesized following heat shock with those synthesized by non-heated cells showed that the levels of synthesis of certain proteins were greatly enhanced following the 45° treatment. By 8 hours, it was qualitatively apparent that three proteins, with molecular weights of 59K, 70K and 87K, were synthesized in greater amounts than in untreated cells. The kinetics of HSP synthesis were compared to the kinetics of thermal tolerance; these showed good correlation. Overall protein synthesis also increased during this time, although at a rate slower than the synthesis of the HSP. The question of whether the HSP play a causative role in the development of thermal tolerance and if so, what that role might be, has not been answered.

Quantitative analysis of the heat shock response of Saccharomyces cerevisiae.

Abstract: Transient protein synthesis in Saccharomyces cerevisiae, after shift from 21-23 degrees C to 37 degrees C, was quantitatively analyzed. Pulse-labeled proteins were separated by two-dimensional gel electrophoresis, and autoradiograms of the gels were analyzed by a recently described method involving a computer-coupled film scanning system. In this way, the rate of incorporation of L-[35S]methionine into approximately 500 proteins was followed. The synthesis of more than 80 of these proteins was transiently induced at 37 degrees C, with about 20 being classified as major heat shock proteins (defined as those whose rate of labeling was increased at least eightfold at some time during the response). The synthesis of more than 300 of the proteins was transiently repressed at 37 degrees C, and several general temporal patterns of repression could be distinguished. The influence of temperature-sensitive mutations affecting RNA synthesis and transport on the heat shock response was also examined. A protein whose induction in response to heat shock has a post-transcriptional component could be identified. As previously pointed out, the heat shock repression of certain proteins is so rapid that it also must involve post-transcriptional effects.

Thermotolerance and heat shock proteins induced by hyperthermia in rat liver cells

Abstract: Hepatic epithelial cells become thermotolerant when conditioned with a 30 minute heat-treatment at 43°C. The effect reaches a full amplitude after a 4–8 hour period at 37°C and lasts for more than one day at a level corresponding to a 50-fold increase in cellular thermoresistance. During the development period, electrophoretic patterns of proteins from cells incubated in presence of 35 S-methionine reveal an increased synthesis of a small set of proteins with molecular weights of 107, 89, 70, 68 and 27KD. The maximal synthesis of the induced proteins occurs concomitantly with the maximal increase of cell thermotolerance and has returned to normal when thermotolerance levels off. The induction of specific protein synthesis is also observed in other liver epithelial cells of normal and cancerous origins and in freshly isolated hepatocytes. It is suggested that the accumulation of these proteins in the cells plays a role in the process leading to a thermotolerant state.