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1.
Upon heat shock, Drosophila Kc cells still contain normal cellular messenger RNAs in the cytoplasm. The distribution of these 25°C mRNAs between polysomes and the postpolysomal fraction of heat-shocked cells appears unaltered as compared with control cells. The translatability of these normal cellular messages isolated from heat-shocked and non-heat-shocked Kc cells is unaltered when analyzed by in vitro translation in the rabbit reticulocyte lysate. In contrast, homologous cell-free translation systems obtained from Kc cells effectively discriminate between the in vitro translation of normal cellular messages and heat-shock-specific mRNAs. In particular, a cell-free system from heat-shocked Drosophila Kc cells almost completely shuts down the translation of 25°C messenger RNA species, whereas the translatability of heat-shock-specific messenger RNA appears to be unaffected. 相似文献
2.
When Drosophila tissue culture cells are shifted from 25 to 36°C (heat shocked) the pre-existing mRNAs (25°C mRNAs) remain in the cytoplasm but their translation products are underrepresented relative to the induced heat shock proteins. Many of these undertranslated 25°C mRNAs are found in association with polysomes of similar size in heat-shocked and control cells. Furthermore, the messages encoding α-tubulin, β-tubulin, and actin are found associated with one-third to one-half as many total ribosomes in heat-shocked cells as in cells incubated at 25°C. Increased temperature should lead to increased output of protein per ribosome. However, the 25°C proteins are actually synthesized at less than 10% of 25°C levels in heat-shocked cells. Thus, the rates of both elongation and initiation of translation are significantly (15- to 30-fold) slower on 25°C mRNAs than they are on heat shock mRNAs in heat-shocked cells. 相似文献
3.
M E Carbajal J L Duband F Lettre J P Valet R M Tanguay 《Biochimie et biologie cellulaire》1986,64(8):816-825
To gain insight on the possible functions of heat shock proteins (hsp's) in Drosophila, we have purified the 83-kilodalton hsp (hsp 83) from cultured cells and studied its intracellular localization by immunofluorescence in normal, heat-shocked, and recovering cells. The specificity of the antibody was assessed by one- and two-dimensional gel immunoblotting and by partial proteolytic digestion. The anti-hsp 83 antibody does not show any significant cross-reactivity with hsp's of different avian or mammalian cell lines, but cross-reacts with hsp's of similar molecular masses in other dipteran insects. The partial proteolytic peptide maps of Drosophila hsp 83 differ from those of mouse hsp 89 and chicken hsp 84. Immunoblotting of Drosophila Kc cells heat shocked at different temperatures indicates a maximal expression of hsp 83 at 33 degrees C. By immunofluorescence, hsp 83 is shown to have a strictly cytoplasmic localization. In unstressed cells, it is distributed in the entire cytoplasm with a slight enrichment in the perinuclear region. After heat shock, it seems to concentrate at the cell periphery close to the plasma membrane and it gradually redistributes to the whole cytoplasm during cellular recovery at normal temperatures. 相似文献
4.
Regulation of protein synthesis in heat-shocked Drosophila cells. Soluble factors control translation in vitro 总被引:1,自引:0,他引:1
We have developed an in vitro translation system from heat-shocked and normal Drosophila cultured cells. The lysates retain regulation of translation typical of the whole cells from which they were prepared, both when programmed by endogenous mRNA and when RNA-dependent. These systems have been used to investigate the mechanism of shutdown of normal protein synthesis and selection of heat shock mRNAs for translation in heat shock in Drosophila. Supplementation of intact RNA-dependent lysates with separated ribosome or supernatant fractions from normal or heat-shocked translation systems showed the normal supernatant fraction could "rescue" normal protein synthesis in a heat shock lysate. Normal ribosomes had no rescuing activity and neither heat shock fraction affected translation in normal lysates. Reconstitution of the system from separated ribosomes and supernatant in normal and mixed combinations showed heat shock and normal ribosomes were both competent to support normal protein synthesis with normal supernatant. Heat shock supernatant did not support normal protein synthesis with ribosomes from either source. We conclude that the factors regulating translation in heat-shocked Drosophila cells are soluble factors in the lysate and that the soluble factors present in the normal lysate are dominant. 相似文献
5.
Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells 总被引:12,自引:5,他引:7
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《The Journal of cell biology》1981,91(1):175-183
Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures. 相似文献
6.
Heat shock causes the collapse of the intermediate filament cytoskeleton in Drosophila embryos 总被引:2,自引:0,他引:2
Heat shock has a dramatic effect on the organization of the cytoplasm, causing the intermediate filament cytoskeleton to aggregate at the nucleus. This has previously been shown in cultured Drosophila and mammalian cells. In this paper we analyze the heat lability of the intermediate filament cytoskeleton in early Drosophila embryos by indirect immunofluorescence. At all stages of embryogenesis tested, the intermediate filament cytoskeleton, which is maternally provided, is severely disturbed by 30 min heat shock at 37 degrees C. After the nuclei have migrated to the subcortical cytoplasm, it collapses around them. Nuclei in all heat-shocked embryos are considerably enlarged and become displaced. Embryos before cellular blastoderm stage, in which heat shock protein synthesis is not inducible, are irreversibly arrested in development by heat shock. Embryos at or after cellular blastoderm, which do synthesize heat shock proteins in response to stress, are also immediately arrested in development but continue development when returned to 25 degrees C. We discuss the possibility that cytoplasmic events such as the intermediate filament cytoskeleton rearrangement may be involved in heat shock-mediated phenocopy induction. 相似文献
7.
Steroid hormone regulation of the Achlya ambisexualis 85-kilodalton heat shock protein, a component of the Achlya steroid receptor complex. 总被引:2,自引:0,他引:2
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The steroid hormone antheridiol regulates sexual development in the fungus Achlya ambisexualis. Analyses of in vivo-labeled proteins from hormone-treated cells revealed that one of the characteristic antheridiol-induced proteins appeared to be very similar to the Achyla 85-kilodalton (kDa) heat shock protein. Analysis of in vitro translation products of RNA isolated from control, heat-shocked, or hormone-treated cells demonstrated an increased accumulation of mRNA encoding a similar 85-kDa protein in both the heat-shocked and hormone-treated cells. Northern (RNA) blot analyses with a Drosophila melanogaster hsp83 probe indicated that a mRNA species of approximately 2.8 kilobases was substantially enriched in both heat-shocked and hormone-treated cells. The monoclonal antibody AC88, which recognizes the non-hormone-binding component of the Achyla steroid receptor, cross-reacted with Achlya hsp85 in cytosols from heat-shocked cells. This monoclonal antibody also recognized both the hormone-induced and heat shock-induced 85-kDa in vitro translation products. Taken together, these data suggest that similar or identical 85-kDa proteins are independently regulated by the steroid hormone antheridiol and by heat shock and that this protein is part of the Achyla steroid receptor complex. Our results demonstrate that the association of hsp90 family proteins with steroid receptors observed in mammals and birds extends also to the eucaryotic microbes and suggest that this association may have evolved early in steroid-responsive systems. 相似文献
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Robertson SL Ni W Dhadialla TS Nisbet AJ McCusker C Ley SV Mordue W Mordue 'Luntz' AJ 《Archives of insect biochemistry and physiology》2007,64(4):200-208
Cell-proliferation in Drosophila Kc167 cells was inhibited by 50% when cell cultures contained 1.7 x 10(-7) M azadirachtin for 48 h (a tertranortriterpenoid from the neem tree Azadirachta indica). Drosophila Kc167 cells exhibited direct nuclear damage within 6-h exposure to azadirachtin (5 x 10(-7) M and above) or within 24 h when lower concentrations were used (1 x 10(-9) M). Fractionation of an extract of Drosophila Kc167 cells combined with ligand overlay technique resulted in the identification of a putative azadirachtin binding complex. Identification of the members of this complex by Peptide Mass Fingerprinting (PMF) and N-terminal sequencing identified heat shock protein 60 (hsp60) as one of its components. 相似文献
11.
Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold 总被引:156,自引:0,他引:156
Data are presented for sequence-specific chromatin-loop organization in histone-depleted nuclei from Drosophila melanogaster Kc cells. We find one loop for each of the tandemly repeated histone gene clusters. The attachment site is localized in the A + T rich H1-H3 spacer on a 657 bp fragment. In the cluster of the hsp70 heat-shock genes, in both control and heat-shocked cells, we find two attachment sites in close proximity upstream of regulatory elements. The transcribed sequences are not associated with the nuclear scaffold in control or in heat-shocked cells. A family of attachment sites related by hybridization to those of the hsp70 genes was discovered. 相似文献
12.
The heat shock response of Myxococcus xanthus was investigated and characterized. When shifted from 28 to 40 degrees C, log-phase cells rapidly ceased growth, exhibited a 50% reduction in CFU, and initiated the synthesis of heat shock proteins (HTPs). Heat-shocked log-phase M. xanthus cells labeled with [35S]methionine were found to produce 18 major HTPs. The HTPs, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, were characterized with regard to molecular mass, subcellular location (periplasm, membrane, or cytoplasm), and temperature required for expression. Most HTPs were expressed at 36 degrees C, the optimum growth temperature of M. xanthus. Cells preincubated at 36 degrees C for 1 h before being shifted to 40 degrees C demonstrated increased thermotolerance compared with cells shifted directly from 28 to 40 degrees C. The HTPs produced by heat-shocked starvation-induced fruiting cells and glycerol-induced sporulating cells were also analyzed and characterized. Thirteen HTPs were detected in fruiting cells shifted from 28 to 40 degrees C. Six of these HTPs were not seen in vegetative M. xanthus cells. Log-phase cells induced to sporulate by the addition of glycerol produced 17 HTPs after being shifted to 40 degrees C. These HTPs were found to be a mixture of HTPs detected in heat-shocked log-phase cells and heat-shocked fruiting cells. 相似文献
13.
M Bielińska 《Acta biochimica Polonica》1983,30(3-4):355-361
The pattern of polypeptides synthesized in a cell-free protein synthesizing system containing polysomes isolated from heat-shocked (37 C) Drosophila embryos showed significant differences when compared with the pattern obtained when polysomes from normal embryos were used. The synthesis of normal embryonal proteins was reduced and the heat shock proteins were the major products of elongation. After short, 10 min, heat treatment mainly quantitative changes were observed suggesting that normal mRNAs were still present on polysomes, and their products could be completed in vitro in the heterologous cell-free system. The mRNAs coding for normal embryonal proteins were present in almost unchanged amounts in heat-shocked embryos as could be judged from the pattern of proteins synthesized in heterologous cell-free system supplemented with cytoplasmic RNA from normal and heat-shocked embryos. Thus the change in protein synthesis in heat-shocked embryos is not associated with degradation of normal embryonal mRNAs but with their inaccessibility for translation. 相似文献
14.
Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells. 总被引:5,自引:0,他引:5
We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila. 相似文献
15.
J Cervera 《Cell biology international reports》1985,9(2):131-141
The effect of heat shock or cadmium treatment on protein synthesis and cell survival in L-132 cells has been examined. After cadmium treatment, the synthesis of a polypeptide of Mr 68000 (P68) was greatly enhanced over that of untreated cells. Besides P68 the synthesis of another polypeptide of Mr 89000 (P89) was also enhanced in heat-shocked cells. Both heat shock and cadmium treatment induced self-tolerance. The kinetics of the synthesis of induced polypeptides correlated well with the development of self-tolerance. The patterns of peptide maps obtained after partial proteolytic digestion from P68 induced in cadmium-treated and heat-shocked cells were virtually identical. However, neither heat-shocked cells did not confer cadmium tolerance nor did cadmium-treated cells induce thermotolerance. 相似文献
16.
Drosophila Kc cells contain a series of RNA fragments ranging in size from 980 to 4600 bp. The fragments copurify with a virus-like nucleoprotein particle which has a density of 1.384 g/cm3 and is a 62 nm diameter icosahedron. There are 11-13 double stranded RNAs in the particles; they are not homologous with either cultured cell or embryo genomic DNA. The particle also contains a minimum of seven polypeptides, three of which are major, and all of which continue to be synthesized in Kc cells in heat shock when normal cellular protein synthesis is shut down. This virus-like particle occurs in large enough amounts in Kc cells to confuse molecular and physiological studies, however the cells continue to multiply in its presence. 相似文献
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Many recent studies have suggested that heat and other inducers of the heat shock (stress) response in eukaryotic cells might result in the generation of abnormal proteins which would result in the overloading of protein degradation systems and the stabilization of proteins involved in positively regulating heat shock (hs) gene expression. In this study we have examined the effects different heat treatments and other hs inducers have on protein degradation in Chinese hamster ovary (CHO) and Drosophila Kc and Schneider cells. We have found that intermediate temperatures which induced the hs response (42 degrees C in CHO and 34 degrees C in Kc cells) did increase protein degradation rates whereas, higher temperatures which also induced the hs response (45 degrees C in CHO and 37 degrees C in Kc cells) initially increased but then decreased protein degradation rates. While these results are consistent with a model in which the protein degradation system is being overloaded and/or components of it are being depleted, we have found several conditions which induce hs proteins which rule out this mechanism. Exposure of either cell type to amino acid analogs (5 mM canavanine or 5 mM S-aminoethyl cysteine) resulted in the rapid degradation of those proteins which had incorporated the analogs in both CHO and Drosophila cells. However, the addition of analogs had little or no effect on the degradation of preexisting proteins, indicating that the introduction of abnormal proteins probably didn't overload the protein degradation system(s). The addition of 100 microM cadmium sulfate or 100 microM sodium arsenite had little or no effect on protein degradation rates in CHO cells even though both were good inducers of the hs proteins. Thus, exposure to inducers of the hs response does not universally increase protein degradation rates nor does it stabilize preexisting proteins. Therefore, the degradation of abnormal proteins is probably not involved in inducing the hs genes. 相似文献
19.
C M de Sa E Rollet M F de Sa R M Tanguay M Best-Belpomme K Scherrer 《Molecular and cellular biology》1989,9(6):2672-2681
Prosomes and heat shock protein (HSP) complexes isolated from the cytoplasm of Drosophila cells in culture were biochemically and immunologically characterized. The two complexes were found to separate on sucrose gradients, allowing the analysis of their protein constituents by two-dimensional polyacrylamide gel electrophoresis and by reaction with anti-HSP sera and prosome-specific monoclonal antibodies. All of the prosomal proteins were found to be clearly distinct from the HSP; none of the prosomal proteins was synthesized de novo in heat shock. However, an antiprosome (anti-p27K) monoclonal antibody (mouse anti-duck) recognizing the Drosophila p29K prosomal protein allowed immunoprecipitation from a heat-shocked postmitochondrial supernatant of the crude HSP complex, including the low- and the high-molecular-weight components, in particular the 70 x 10(3)-molecular weight HSP. The highly purified small 16S HSP complex still contained this preexistent p29K prosomal protein, which thus also seems to be a metabolically stable constituent of the HSP complex. The significance of this structural and possibly functional relationship between prosomes and HSP, involving the highly ubiquitous and evolutionarily conserved prosomal protein p27/29K, remains to be elucidated. 相似文献
20.
The expression of the sequences encoding the four nucleosomal histone proteins was examined following heat shock of a variety of Drosophila cells and was found to be highly differential. In Drosophila melanogaster KC-O cells grown in suspension culture, there is a continuation of the synthesis of all four of the nucleosomal histone proteins following heat shock. Analysis of RNA from these cells confirms that histone messengers are transcribed and located on polysomes. This exact same pattern of histone protein synthesis occurs in KC-O cells grown to low density on plates. In contrast, KC-O cells grown to high density on plates exhibit a dramatic elevation of H2b protein synthesis relative to the synthesis of the other core histones. Organs from D melanogaster third instar larvae were examined to ascertain whether histone protein synthesis continues following heat shock in the organism. Different tissue types exhibited differential histone synthesis. Imaginal disks excised from heat-shocked larvae continue to synthesize nucleosomal histones in a variable fashion. In contrast, neither fat bodies, brains, nor salivary glands continues to synthesize core histone proteins at a significant level. D hydei plated cell cultures and larval tissues fail to synthesize histones at any detectable level following a heat shock. Based on these observations, we propose that there is a differential synthesis of nucleosomal proteins in Drosophila that is highly dependent on the state of the cells prior to the heat shock. 相似文献