Expression patterns and organization of the hsp70 genes correlate with thermotolerance in two congener endemic amphipod species (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal

We studied various aspects of heat‐shock response with special emphasis on the expression of heat‐shock protein 70 (hsp70) genes at various levels in two congener species of littoral endemic amphipods (Eulimnogammarus cyaneus and E. verrucosus) from Lake Baikal which show striking differences in their vertical distribution and thermal tolerance. Although both the species studied demonstrate high constitutive levels of Hsp70, the thermotolerant E. cyaneus exhibited a 5‐fold higher basal level of Hsp70 proteins under normal physiological conditions (7 °C) and significantly lower induction of Hsp70 after temperature elevation compared with the more thermosensitive E. verrucosus. We isolated the hsp70 genes from both species and analysed their sequences. Two isoforms of the cytosolic Hsp70/Hsc70 proteins were detected in both species under normal physiological conditions and encoded by two distinct hsp/hsc70 family members. While both Hsp70 isoforms were synthesized without heat shock, only one of them was induced by temperature elevation. The observed differences in the Hsp70 expression patterns, including the dynamics of Hsp70 synthesis and threshold of induction, suggest that the increased thermotolerance in E. cyaneus (compared with E. verrucosus) is associated with a complex structural and functional rearrangement of the hsp70 gene family and favoured the involvement of Hsp70 in adaptation to fluctuating thermal conditions. This study provides insights into the molecular mechanisms underlying the thermal adaptation of Baikal amphipods and represents the first report describing the structure and function of the hsp70 genes of endemic Baikal species dwelling in thermally contrasting habitats.     

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.     

Zebrafish HSC70 promoter to express carp muscle-specific creatine kinase for acclimation under cold condition

Zebrafish (Danio rerio) is used as a model system for in vivo studies. To expand the research scope of physical, biochemical and physiological studies, a cold-tolerant model of zebrafish was developed. The common carp (Cyprinus carpio) muscle form of creatine kinase (CK, EC 2.7.3.2) can maintain enzymatic activity at a temperature of around 15°C. However, a cold-inducible promoter of zebrafish, hsc 70 (heat shock protein 70 cognate), is able to increase the expression of gene product by 9.8 fold at a temperature of 16°C. Therefore, the carp CK gene was promoted by hsc 70 and transfected into zebrafish embryos. Resulting transgenic zebrafish survived and could maintain its swimming behavior at 13°C, which was not possible with the wild-type zebrafish. The swimming distance of the transgenic fish was 42% greater than that of the wild type at 13°C. This new transgenic fish model is ideal for studies of ectothermal vertebrates in low-temperature environments.     

An intron-containing,heat-inducible stress-70 gene in the millipede Tachypodoiulus niger (Julidae,Diplopoda)

The highly conserved part of the nucleotide-binding domain of the hsp70 gene family was amplified from the soil diplopod Tachypodoiulus niger (Julidae, Diplopoda). Genomic DNA yielded 701, 549 and 540 bp sequences, whereas cDNA from heat shocked animals produced only one distinct fragment of 543 bp. The sequences could be classified as a 70 kDa heat shock protein (hsp70), the corresponding 70 kDa heat shock cognate (hsc70) and a glucose-related hsp70 homologue (grp78). Comparisons of genomic and cDNA sequences of hsc70 identified two introns within the consensus sequence. Generally, stress-70 expression levels were low, which hampered successful RT-PCR and subsequent subcloning. Following experimental heat shock, however, the spliced hsc70 was amplified predominantly, instead of its inducible homologue hsp70. This finding suggests that microevolution in this soil-dwelling arthropod is directed towards low constitutive stress-70 levels and that the capacity for stress-70 induction presumably is limited. hsc70, albeit having introns, apparently is inducible and contributes to the stress-70 response.     

Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy

A cell line derived from the tailfin of the marine teleost yellowtail fish Seriola quinqueradiata was established to examine cellular temperature regulation in an ectothermic animal. Three cytosolic members of the HSP70 family, heat-shock cognate proteins HSC70-1, HSC70-2 and heat-shock protein HSP70, were isolated from cultured yellowtail cells as stress-responsive biomarkers. Expression of hsp70 was heat-inducible, in contrast to the hsc70-1 gene product, which was expressed constitutively. In addition, expression of hsc70-2 was only induced under severe heat-shock conditions. Subcellular fractionation and immunocytochemistry showed localization of HSC70/HSP70 in the lysosomes, indicating that chaperone-mediated autophagy is induced by heat shock. Thus, chaperone-mediated autophagy is assisted by HSC70/HSP70, and heat-inducible expression of the genes encoding these proteins may be responsible for survival and adaptation under heat-shock conditions in fish cells.     

Isolation of a cDNA encoding a 70 kDa heat-shock cognate protein expressed in vegetative tissues ofArabidopsis thaliana

A cDNA encoding a 70 kDa heat shock cognate protein (hsc70) was isolated fromArabidopsis thaliana by using a rat hsc70 cDNA as probe. Sequence analysis demonstrated the conservation of functional domains and important amino acid residues among hsc70s in plants and animals. The expression of this gene was stress-inducible, and was found at a substantial level during normal growth in root, stem, leaf and flower tissues, but not in siliques. Multiple copies of this gene exist in theArabidopsis genome.     

热应激和吡虫啉对大豆蚜hsp70和hsc70基因mRNA表达的影响

【目的】昆虫在高温或农药的胁迫下,通过高效表达热休克蛋白(HSP)等建立应激自我保护机制。本研究为从转录组水平上认识大豆蚜Aphis glycines在热应激和吡虫啉胁迫下hsp70和hsc70 mRNA表达分子机制,进而寻找自我保护应激反应中的薄弱环节,为大豆蚜的生物防治提供理论基础。【方法】采用同源克隆、RACE技术和实时荧光定量PCR等方法研究不同热激时间和热激后不同恢复时间及不同吡虫啉浓度对大豆蚜4龄若虫hsp70和hsc70的表达影响。【结果】37℃热激后,大豆蚜4龄若虫中hsp70表达量先上调,1 h时升至对照组的10.36倍（P<0.05）,然后逐渐下降。同样热激后恢复时间的长短对大豆蚜若蚜中hsp70的表达具有显著影响。热激处理后,大豆蚜若蚜中hsp70立即大量表达,表达量为对照组的8.78倍（P<0.05）,随后表达量下降至对照组水平,而hsc70的表达量并没有显著变化（P>0.05）。大豆蚜若蚜受吡虫啉的胁迫时,其hsp70和hsc70的表达量受吡虫啉的浓度及胁迫的时间的影响,呈现先升高后下降的趋势,具有明显的短期效应。【结论】吡虫啉诱导大豆蚜hsp70和hsc70表达量的上调;而热胁迫对hsp70和hsc70 mRNA具有不同的表达模式,高温可以诱导hsp70的表达,但对hsc70没有明显的诱导作用。     

Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix

Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to rodent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations sugest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is coregulated.     

Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon)

We cloned the complementary deoxyribonucleic acid (cDNA) of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon). It was 2207 bp long and included a 1959-bp coding region, a 40-bp flanking region at the 5' end, and a 208-bp flanking region at the 3' end. The deduced, 652-amino acid sequence had a molecular mass of 71 481 Da and an estimated isoelectric point (pI) of 5.2. Based on phylogenetic analysis, the gene is clustered with the hsc70 proteins of invertebrates and vertebrates. In native gel electrophoresis, recombinant P. monodon hsc70 expressed in an Escherichia coli system is tightly associated with carboxymethylated alpha-lactalbumin (CMLA), which indicates that hsc70 probably functions as a chaperone. In an in vitro adenosine triphosphatase assay, recombinant hsc70 hydrolyzed adenosine triphosphate to adenosine-5'-diphosphate and increased hydrolysis activity by binding to unfolded peptide, CMLA. In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp. The expression of hsc70 messenger ribonucleic acid (mRNA) in hemocytes increased 2- to 3-fold at the first hour after shrimp experienced heat shock and 0.5-hour recovery. Hsc70 mRNA decreased gradually to the background level. Cloning and characterizing the P. monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins with the stress or immune responses of shrimp.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.