Low Molecular Weight Heat Shock Proteins Research Articles

Effect of ischemia on localization of heat shock protein 25 in kidney

The effects of renal ischemia on the intracellular distribution of the low-molecular weight heat shock protein (HSP)25 were examined using immunofluorescence microscopy. In all kidney zones, ischemia decreased HSP25 in the supernatant of the tissue homogenates and increased it in the pellet fraction (containing mainly nuclei and cytoskeletal components). This was associated with disappearance of HSP25 staining from the brush border of proximal convoluted tubule (PCT) cells. Because no nuclear staining of cortical tubule cells was apparent either in control or ischemic kidneys, ischemia seems to cause a closer association of HSP25 with cytoskeletal components. HSP25 probably participates in the postischemic restructuring of the cytoskeleton of PCT cells.

Identification and characterisation of cultivar‐specific 22‐kDa heat shock proteins from mitochondria of Pisum sativum

Pea plants (Pisum sativum L. cv. Feltham First) exposed to a heat stress of 37°C for 6 h accumulated two low molecular weight (LMW) heat shock proteins (HSPs) of molecular mass 22 kDa. The two LMW HSPs were associated with purified mitochondria. N‐terminal amino acid sequencing analysis indicates that the more basic of these proteins is a novel protein. The response of other cultivars of P. sativum to heat shock revealed that up to three 22‐kDa HSPs were expressed in a cultivar‐specific manner. Evidence presented suggests that the different 22‐kDa HSPs arise as a result of there being multiple 22‐kDa HSP genes. The expression of the most basic novel HSP was studied in the Feltham First cultivar using two dimensional SDS‐PAGE. Treatment of intact plants with chloramphenicol and cycloheximide prior to heat stress treatment indicated that the LMW HSPs were nuclear encoded and de novo synthesised. The response to heat shock was rapid with protein expression detected within 45 min and the protein remained in excess of 6 days following removal of the stress. The protein accumulated to very high levels with maximal expression being 2% of the total mitochondrial protein. The results are discussed in relation to the likely role of LMW HSPs in thermotolerance.

Cortical application of potassium chloride induces the low-molecular weight heat shock protein (Hsp27) in astrocytes.

Spreading depression induces tolerance to ischemic injury, and ischemic tolerance has been associated with expression of heat shock proteins (Hsp). Here we examine Hsp27 expression after KCl-induced spreading depression. Twenty-minute cortical KCl application induced Hsp27 immunoreactivity in glial fibrillary acidic protein-positive astrocytes of the ipsilateral neocortex. Systemic administration of MK-801 (3 mg/kg) suppressed KCl-induced Hsp27 expression in the parietal cortex. Astrocytes in the posterior cingulate and retrosplenial cortex did not express Hsp27 after KCl application but did express Hsp27 after systemic administration of high dose MK-801 (9 mg/kg). Whereas Hsp27 was usually observed in all layers of the parietal cortex after 5-minute application of KCl, in 2 of 6 rats, Hsp27 was seen in clusters of astrocytes or in astrocytes in the superficial layers I to III of the parietal cortex. We conclude that (1) cortical application of KCl triggered Hsp27 astrocytic expression; (2) astrocytes in the cingulate and retrosplenial cortex responded differently compared with astrocytes of the parietal cortex; (3) Hsp27 expression progressed from small clusters of astrocytes throughout superficial layers of the cortex that joined and recruited astrocytes in deeper layers; (4) several mechanisms induced Hsp27 astrocytic expression. We propose that Hsp27 is involved in spreading depression-induced ischemic tolerance through protection of astrocyte function.

Tissue-Type-Specific Heat-Shock Response and Immunolocalization of Class I Low-Molecular-Weight Heat-Shock Proteins in Soybean.

A monospecific polyclonal antibody was used to study the tissue-type specificity and intracellular localization of class I low-molecular-weight (LMW) heat-shock proteins (HSPs) in soybean (Glycine max) under different heat-shock regimes. In etiolated soybean seedlings, the root meristematic regions contained the highest levels of LMW HSP. No tissue-type-specific expression of class I LMW HSP was detected using the tissue-printing method. In immunolocalization studies of seedlings treated with HS (40[deg]C for 2 h) the class I LMW HSPs were found in the aggregated granular structures, which were distributed randomly in the cytoplasm and in the nucleus. When the heat shock was released, the granular structures disappeared and the class I LMW HSPs became distributed homogeneously in the cytoplasm. When the seedlings were then given a more severe heat shock following the initial 40[deg]C -> 28[deg]C treatment, a large proportion of the class I LMW HSPs that originally localized in the cytoplasm were translocated into the nucleus and nucleolus. Class I LMW HSPs may assist in the resolubilization of proteins denatured or aggregated by heat and may also participate in the restoration of organellar function after heat shock.

Differential display-mediated isolation of a genomic sequence for a putative mitochondrial LMW HSP specifically expressed in condition of induced thermotolerance in Arabidopsis thaliana (L.) heynh.

Plants of Arabidopsis thaliana pre-treated at 37 degrees C for 2 h can survive an otherwise lethal heat shock at 45 degrees C. Differential display reverse transcriptase-PCR (DDRT-PCR) was utilized to clone DNA fragments corresponding to mRNAs specifically expressed in conditions of induced thermotolerance or of expression of thermotolerance. One of these DDRT-PCR fragments enabled the isolation of a genomic clone pAt1.3EX, containing the sequence Athsp23.5, the gene for a low-molecular-weight (LMW) heat shock protein (HSP), AtHSP23.5. Athsp23.5 is low- or single-copy in the Arabidopsis genome and its open reading frame is interrupted by a 137 bp intron. Analysis of the sequence suggests AtHSP23.5 is targeted to the mitochondrion. The steady-state level of the AtHSP23.5 mRNA varied significantly according to the heat treatment, increasing on heat shock (transfer from 22 degrees C to 37 degrees C), with a further increase during expression of thermotolerance (transfer from 22 degrees C to 37 degrees C and then to 45 degrees C). Expression was low after an abrupt stress (from 22 degrees C to 45 degrees C). This behaviour was different from that observed for other LMW HSP mRNAs that were present at high level at 37 degrees C, but did not increase significantly in condition of expression of thermotolerance, and reached a considerable steady-state level also during the abrupt stress at 45 degrees C. The retrotranscription of AtHSP23.5 mRNA followed by amplification with two primers encompassing the intron allowed for the isolation of an almost full-length cDNA sequence. The sequence analysis of the two cDNAs obtained from condition 22 degrees C-->37 degrees C and condition 22 degrees C-->45 degrees C suggested that in both cases the intron had been correctly spliced. The importance of correct intron splicing in survival at high temperatures and the role of mitochondrial HSP in induction and expression of thermotolerance are discussed.

Characterization of three heat-shock-protein genes and their developmental regulation during somatic embryogenesis in white spruce [Picea glauca (Moench) Voss

Three cDNAs (PgEMB22, 27 and 29) predicted to encode low-molecular-weight (LMW) heat-shock proteins (HSPs) were cloned and characterized from white spruce [Picea glauca (Moench) Voss] somatic embryo tissues by differentially screening a cotyledonary embryo cDNA library. Clone PgEMB22 is predicted to encode a putative mitochondria-localized LMW HSP, and PgEMB27 and 29 are predicted to encode different cytoplasmic class II LMW HSPs, although they share 84.7% identity within DNA coding regions and 83.0% identity for predicted proteins. They are developmentally regulated during somatic embryo development and subsequent embryo germination, in addition they show strong response to heat-shock stress. Transcripts of the two kinds of hsp genes could be detected in embryogenic tissues before induction of embryo maturation, but subsequently increased, being most abundant at late embryo stages. Gene expression levels were very low or not detectable in germinated plantlets or needle tissues from older plants. Abscisic acid and polyethylene glycol, stimulators for spruce embryo maturation, could also induce the hsp genes.

Induction and Regulation of Heat-Shock Gene Expression by an Amino Acid Analog in Soybean Seedlings.

The effect of the proline analog azetidine-2-carboxylic acid (Aze) on the induction and the regulation of heat-shock (HS) mRNA accumulation and heat-shock protein (HSP) synthesis in soybean (Glycine max) seedlings was studied. Treatment with Aze elicited an HS-like response at the normal growth temperature, 28[deg]C, with seven of nine HS cDNA clones tested. Two cDNA clones, Gm-Hsp22.5 and pFS2033, share 78% identity; however, transcripts hybridizing to GmHsp22.5 but not pFS2033 accumulated with Aze treatment at 28[deg]C. Substantial incorporation of radioactive amino acid into high molecular weight HSPs but not low molecular weight HSPs was observed in vivo during Aze treatment at 28[deg]C. Low molecular weight HSPs were detected using antibodies raised against an abundant member of low molecular weight class I HSPs, indicating that low molecular weight HSPs were synthesized at normal growth temperatures during Aze treatment despite a lack of substantial in vivo radioactive amino acid incorporation. In summary, Aze treatment induced accumulation of most but not all HS mRNAs and HSPs in soybean seedlings; the observations presented here suggest differential regulation among various HS genes at the transcriptional and posttranscriptional levels.

Photodegradation of Tryptophan Residues and Attenuation of Molecular Chaperone Activity in α-Crystallin Are Correlated

The major eye-lens protein α-crystallin is known to possess a remarkable sequence homology to the low molecular weight heat-shock proteins and has been shown to protect several proteins against thermally induced aggregation. In this work we demonstrate that the rapid aggregation of rabbit muscle phosphoglycerate kinase during incubation at 52°C is completely inhibited in presence of 1/3 moles α-crystallin monomer per mole enzyme. Upon irradiation by UV light, tryptophan fluorescence intensity of α-crystallin declines, reflecting the destruction of these residues. A remarkable correlation is revealed between the reduction in α-crystallin fluorescence during UV-irradiation and the loss of its ability to protect phosphoglycerate kinase against aggregation. Since a loss of tryptophan fluorescence in intact eye lenses in vivo has been demonstrated to occur upon exposure to UV light, as well as during aging, it is proposed that the enhanced rate of lens opacification and cataract formation, as well as the increased levels of damaged lens proteins, which accumulate under these conditions, are the result of the gradual loss of the chaperone-protein efficacy of α-crystallin.

Genetic analysis of heat shock proteins in maize

A genetic analysis of heat shock protein (HSP) synthesis was performed in seedling leaf tissue of two maize inbred lines, their F1 hybrid and F2 progeny. Protein synthesis following a high temperature treatment was visualized by [(35)S]-methionine in vivo labelling and two-dimensional gel electrophoresis. The parental lines' HSP synthesis patterns revealed both qualitative and quantitative polymorphisms implicative of differences in HSP structural genes and regulatory factors. The F1 hybrid HSP profile indicated that synthesis of all parental HSPs conformed to dominant inheritance patterns, including complete dominance, over-dominance and co-dominance. Alleles for six low-molecularweight HSPs in F2 progeny assorted according to typical 3∶1 Mendelian ratios for dominant gene expression. There is evidence for unlinked gene loci of four different HSP gene pairs, but data for three other HSP gene pairs were inconclusive, perhaps reflecting linkage for one pair and complex regulatory factor interactions for the other two pairs of genes. These results clearly indicate the existence of genetic variability in HSP synthesis and emphasize the potential of partitioning their roles in thermal tolerance using genetic and molecular analyses.

Cloning and characterization of a cDNA encoding an 18.0-kDa class-I low-molecular-weight heat-shock protein from rice

A novel cDNA clone, Oshsp18.0 cDNA, encoding a rice ( Oryza sativa L. cv. Tainong 67) 18.0-kDa heat-shock protein (HSP), was isolated from a cDNA library of heat-shocked rice seedlings by use of the rice HSP cDNA, Oshsp17.3 cDNA, as a probe. The sequence showed that Oshsp18.0 cDNA contains a 749-bp insert encoding an ORF of 160 amino acids, with a predicted molecular mass of 18.0 kDa and a pI of 7.3. Sequence comparison reveals that Oshsp18.0 cDNA is highly homologous to other low-molecular-weight (LMW) HSP cDNAs. Also, the results of hybrid-selected in vitro translation clearly establish that Oshsp18.0 cDNA is the rice 18.0-kDa LMW HSP-encoding cDNA clone. The recombinant Oshsp18.0 fusion protein produced in Escherichia coli was of the size predicted, and was recognized by the class-I rice 16.9-kDa HSP antiserum. The results suggest that Oshsp18.0 cDNA is an 18.0-kDa class-I LMW HSP- encoding cDNA clone from rice.

The Heat-Shock Response and Expression of Heat-Shock Proteins in Wheat Under Diurnal Heat Stress and Field Conditions

The occurrence of heat-shock proteins (HSPs) in response to high temperature stress is a universal phenomenon in higher plants and has been well documented. However, in agriculturally important species, less is known about the expression of HSPs under natural environments. A review of the heat-shock response in wheat (Triticum aestivum L.) is presented and recent results on the expression of wheat HSPs under diurnal stress and field conditions are reported. In the field experiment, flag leaf blade temperatures were obtained and leaf blades collected for northern blot analysis using HSP 16.9 cDNA as a probe. Temperatures of leaf blades ranged from 32 to 35�C under the tested field conditions at New Deal near Lubbock, Texas. Messenger RNAs encoding a major class of low molecular weight HSPs, HSP 16.9, were detected in all wheat genotypes examined. The results suggested that HSPs are synthesised in response to heat stress under agricultural production, and furthermore, that HSPs are produced in wheats differing in geographic background. In the controlled growth chamber experiment, HSP expression in two wheat cultivars, Mustang (heat tolerant) and Sturdy (heat susceptible) were analysed to determine if wheat genotypes differing in heat tolerance differ in in vitro HSP synthesis (translatable HSP mRNAs) under a chronic, diurnal heat-stress regime. Leaf tissues were collected from seedlings over a time-course and poly (A)+RNAs were isolated for in vitro translation and 2-D gel electrophoresis. The protein profiles shown in the 2-D gel analysis revealed that there were not only quantitative differences of individual HSPs between these two wheat lines, but also some unique HSPs which were only found in the heat tolerant line. This data provides evidence of a correlation between HSP synthesis and heat tolerance in wheat under a simulated field environment and suggests that further genetic analysis of HSPs in a segregating population is worthy of investigation. In conclusion, the results of this study provide an impetus for the investigation of the roles of HSP genes in heat tolerance in wheat.

Expression of Low Molecular Weight Heat-Shock Proteins under Field Conditions.

Heat-shock proteins (HSPs) are known to be expressed in plants experiencing high-temperature stress. We have examined the expression of class I cytoplasmic low molecular weight (LMW) HSPs and find that these HSPs also frequently accumulate in seeds, seed pods, and flowers during a normal growing season. We first examined the expression of class I cytoplasmic LMW HSPs by western blot analysis in a range of seed samples from both commercially grown and wild legumes. LMW HSPs were present in all seed samples, indicating that these HSPs are regularly expressed in these tissues. To examine more specifically conditions under which LMW HSPs were produced during an average growing season, additional studies of Medicago sativa were carried out during the fall season in Tucson, AZ. Plants were irrigated to avoid conditions of water stress, and canopy temperature was monitored throughout the study period. LMW HSP expression in leaves, flowers, and developing seed pods was analyzed by western blotting. Results show that in the field HSPs are frequently produced in flowers and seed pods, even in plants that show no HSP expression in leaves. Parallel greenhouse studies indicate that HSP expression in seeds is in part developmentally regulated. In total our data suggest a more widespread occurrence of HSPs in optimal growth environments and emphasize their potential role during reproduction.

Floral organ-specific and constitutive expression of an Arabidopsis thaliana heat-shock HSP18.2::GUS fusion gene is retained even after homeotic conversion of flowers by mutation.

Organ-specific and constitutive expression of the Arabidopsis HSP18.2 gene under normal growth conditions (22 degrees C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for beta-glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.

Molecular cloning and sequence analysis of cDNAs encoding cytoplasmic low molecular weight heat shock proteins in hexaploid wheat

Heat shock proteins (HSPs) are known to accumulate in plants under high temperature stress conditions. In an effort to study the function of low molecular weight (LMW) HSPs in cultivated wheat, we have cloned and characterized three cDNAs from Triticum aestivum, Tahsp16.9B, and Tahsp17.4, respectively, t at encode peptides having homology to the cytoplasmic LMW HSP family. Nucleotide sequence comparisons indicate that: (1) Tahsp16.9A and Tahsp16.9B belong to the LMW HSP class I; there is 82% identity of whole cDNA sequence between them, but only 58% identity at the 3′ non-coding region. (2) Tahsp17.4 belongs to LMW HSP class II. Amino acid sequence comparisons indicate that there is a 97% similarity between Tahsp16.9A and Tahsp16.9B and 57% between class I and class II (Tahsp17.4) members in wheat. There is a 93% similarity between Tahsp17.4 and Zmhsp17.9 from maize which belong to class II of LMW HSPs. Northern blot hybridization analysis was performed using poly(A) + RNA from green leaf tissues treated at different temperatures (25°, 28°, 31°, 34°, 37° and 40°C) for 60 min or at 37°C for different time periods (0, 7.5, 15, 30, 60, 120 and 240 min). These experiments revealed that the expression of Tahsp16.9B and Tahsp17.4 was induced beginning at 31°C and reached maximum levels at 37°C. The onset of mRNA induction occured within about 7 min, reached a peak at 60 min and declined after 240 min when treated under the optimal heat stress condition (37°C). In vitro hybrid selection and Southern blot analysis data indicate that Tahsp16.9A and Tahsp16.9B belong to a 12-member class I multigene family. Tahsp 17.4 on the other hand belongs to a 14-member class II multigene family. These cDNAs, which encode cytoplasmic LMW HSPs in hexaploid wheat, will provide us with tools to investigate the rolesof HSPs in an agriculturally important cereal crop species.

Localization of small heat shock proteins to the higher plant endomembrane system.

Three related gene families of low-molecular-weight (LMW) heat shock proteins (HSPs) have been characterized in plants. We describe a fourth LMW HSP family, represented by PsHSP22.7 from Pisum sativum and GmHSP22.0 from Glycine max, and demonstrate that this family of proteins is endomembrane localized. PsHSP22.7 and GmHSP22.0 are 76.7% identical at the amino acid level. Both proteins have amino-terminal signal peptides and carboxyl-terminal sequences characteristic of endoplasmic reticulum (ER) retention signals. The two proteins closely resemble class I cytoplasmic LMW HSPs, suggesting that they evolved from the cytoplasmic proteins through the addition of the signal peptide and ER retention motif. The endomembrane localization of these proteins was confirmed by cell fractionation. The polypeptide product of PsHSP22.7 mRNA was processed to a smaller-M(r) form by canine pancreatic microsomes; in vivo, GmHSP22.0 polysomal mRNA was found to be predominantly membrane bound. In vitro-processed PsHSP22.7 corresponded in mass and pI to one of two proteins detected in ER fractions from heat-stressed plants by using anti-PsHSP22.7 antibodies. Like other LMW HSPs, PsHSP22.7 was observed in higher-molecular-weight structures with apparent masses of between 80 and 240 kDa. The results reported here indicate that members of this new class of LMW HSPs are most likely resident ER proteins and may be similar in function to related LMW HSPs in the cytoplasm. Along with the HSP90 and HSP70 classes of HSPs, this is the third category of HSPs localized to the ER.

Heat-shock proteins protect cells from monocyte cytotoxicity: possible mechanism of self-protection.

We have previously shown that major heat-shock protein (hsp 70) protects WEHI-S tumor cells from cytotoxicity mediated by tumor necrosis factor alpha (TNF-alpha) and TNF-beta. In the present study, the effect of altered expression of hsp70 and low molecular weight heat-shock protein, hsp27, on tumor cell sensitivity to monocytes and lymphokine-activated killer (LAK) cells was studied. Constitutive and stable expression of transfected human hsp70 rendered cells almost completely resistant to monocytes. Conversely, inhibition of endogenous hsp70 by expression of antisense hsp70 RNA enhanced the sensitivity of cells to monocyte-mediated killing. Surprisingly, overexpression of human hsp27, which does not protect WEHI-S cells from TNF killing, conferred partial resistance to monocytes. Only approximately 60% of monocyte-mediated killing of WEHI-S cells could be blocked by neutralizing TNF-alpha antibody or immunoglobulin G-TNF receptor chimeric protein, suggesting the presence of both TNF-dependent and TNF-independent lytic mechanisms. As free radicals have been suggested to be mediators of monocyte cytotoxicity, we tested the sensitivity of transfected cells to oxidative stress. Overexpression of either hsp70 or hsp27 rendered cells partially resistant to hydrogen peroxide. No significant changes in the susceptibility of cell lines overexpressing hsp70 or hsp27 to cytotoxicity mediated by LAK cells were observed. Interestingly, monocytes but not LAK cells contained detectable levels of hsp27 and hsp70 in nonstressed conditions. Taken together, these data indicate that hsp70 protects tumor cells from TNF-mediated monocyte cytotoxicity and that both hsp27 and hsp70 confer resistance to TNF-independent, probably free radical-mediated lysis by monocytes. Moreover, hsp27 and hsp70 may provide monocytes with a protective mechanism against their own toxicity.

Molecular and Physiological Analysis of a Heat-Shock Response in Wheat

We have isolated two cDNA clones from wheat (Triticum aestivum L. var Stephens), designated WHSP16.8 and WHSP16.9, that are highly similar in sequence to the low molecular weight heat-shock protein genes previously isolated from soybean. RNA blot analysis confirms that these sequences are present in heat-shocked wheat seedlings, but not in control tissues. The WHSP16.8 and WHSP16.9 cDNAs were isolated by screening a lambda gt11 expression library with antibodies to HMGc (a chromosomal protein of wheat). Immunoblot analysis has demonstrated that the antibodies raised against HMGc also recognize a group of proteins that are induced by heat shock and have molecular weights (estimated by sodium dodecyl sulfate electrophoresis) consistent with the molecular weights of the proteins deduced from the sequences of the cDNAs.

Expression of a Conserved Family of Cytoplasmic Low Molecular Weight Heat Shock Proteins during Heat Stress and Recovery

Plants synthesize several families of low molecular weight (LMW) heat shock proteins (HSPs) in response to elevated temperatures. We have characterized two cDNAs, HSP18.1 and HSP17.9, that encode members of the class I family of LMW HSPs from pea (Pisum sativum). In addition, we investigated the expression of these HSPs at the mRNA and protein levels during heat stress and recovery. HSP18.1 and HSP17.9 are 82.1% identical at the amino acid level and are 80.8 to 92.9% identical to class I LMW HSPs of other angiosperms. Heat stress experiments were performed using intact seedlings subjected to a gradual temperature increase and held at a maximum temperature of 30 to 42 degrees Celsius for 4 hours. HSP18.1 and HSP17.9 mRNA levels peaked at the beginning of the maximum temperature period and declined rapidly after the stress period. Antiserum against a HSP18.1 fusion protein recognized both HSP18.1 and HSP17.9 but not members of other families of LMW HSPs. The accumulation of HSP18.1-immunodetected protein was proportional to the severity of the heat stress, and the protein had a half-life of 37.7 +/- 8 hours. The long half-life of these proteins supports the hypothesis that they are involved in establishing thermotolerance.

Accumulation, stability, and localization of a major chloroplast heat-shock protein.

Diverse higher plant species synthesize low molecular weight (LMW) heat shock proteins (HSPs) which localize to chloroplasts. These proteins are homologous to LMW HSPs found in the cytoplasm of all eukaryotes, a class of HSPs whose molecular mode of action is not understood. To obtain basic information concerning the role of chloroplast HSPs, we examined the accumulation, stability, tissue specificity, and intra-chloroplast localization of HSP21, the major LMW chloroplast HSP in pea. Intact pea plants were subjected to heat stress conditions which would be encountered in the natural environment and HSP21 mRNA and protein levels were measured in leaves and roots. HSP21 was not detected in leaves or roots before stress, but the mature, 21-kD protein accumulated in direct proportion to temperature and HSP21 mRNA levels in both tissues. All of the HSP21 in leaves was localized to chloroplasts; there was no evidence for its transport into other organelles. In chloroplast fractionation experiments, greater than 80% of HSP21 was recovered in the soluble chloroplast protein fraction. The half-life of HSP21 at control temperatures was 52 +/- 12 h, suggesting the protein's function is critical during recovery as well as during stress. We hypothesize that HSP21 functions in a catalytic fashion in both photosynthetic and nonphotosynthetic plastids.

Attenuated induction of heat shock gene expression in aging diploid fibroblasts

We examined the effects of cellular aging on the regulation of heat shock gene expression in IMR-90 human diploid fibroblasts. Heat shock (42-43 degrees C) and canavanine (200-400 micrograms/ml) were used to evoke the heat shock response in these cells. We showed that heat shock induced the synthesis of proteins with apparent molecular weights of 98,000, 89,000, 78,000, 72,000, 64,000, 50,000 and 25,000, with heat shock protein (HSP) 89 and 72 being most prominent. Canavanine induced the synthesis of the four high molecular weight HSPs, particularly HSP 89 and HSP 78, without noticeably enhancing synthesis of the low molecular weight HSPs. We found that, while a similar series of HSPs were induced in the young and old cells, there was a marked decrease in the magnitude of this induction in the old cells. Using cells with defined population doubling levels, we observed a direct correlation of the inducibility of HSP synthesis and the replicative potential of the cells used. Analysis of the amount of translatable and hybridizable mRNA, by the methods of in vitro translation and Northern blot hybridization, demonstrated that the induction of HSPs synthesis can be accounted for by increases in their mRNA. Nuclear runoff transcription provided evidence that the decrease in inducible expression of the HSPs in aging IMR-90 cells was attributable to a transcriptional mechanism. This conclusion was substantiated by analysis of the hsp 70 promoter activity in transient expression assay of the hsp 70 promoter-chloramphenicol acetyltransferase construct. We propose that there is an age-associated dysfunction in the signaling mechanism of the heat shock response.