Heat Shock Cognate Gene Research Articles

Balancing selection at the ATP binding site of heat shock cognate 70 (HSC70) contributes to increased thermotolerance in Artemia franciscana

A number of single-nucleotide polymorphisms (SNPs) in heat shock protein 70 (HSP70) family genes have been reported to be associated with stress tolerance. In this study, the presence of SNPs in either the ArHSP70 and ArHSC70 genes (heat shock cognate gene) was verified in 2 Artemia franciscana populations, either a control population (CF12) or a population selectively bred for increased induced thermotolerance (TF12) over 12 generations. In TF12 animals, a novel non-synonymous SNP was identified at position 171 of the cDNA (C171A; N57K; in the ATP/ADP binding site) in ArHSC70 but not in ArHSP70. Cloning and expression of the 2 allelic forms in yeast (Saccharomyces cerevisiae) confirmed that yeast cells containing an ArHSC70-N57K plasmid could tolerate higher temperatures than yeast cells containing an ArHSC70-wild type plasmid. This strongly suggests that the SNP at C171A (N57K) increases thermotolerance of the TF12 A. franciscana population. Upon analysis of individuals of the CF12 and TF12 populations it appears that the C171A allele frequency increased substantially in the TF12 population. Both populations appear not to be in Hardy-Weinberg equilibrium, with CF12 displaying an apparent lack of heterozygotes, while the TF12 displays a large excess of heterozygotes. It is argued that these observations are a novel case of balancing selection. As such, HSP genes join a group of immune related genes where balancing selection contributes to population fitness.

Cloning and characterization of carboxyl terminus of heat shock cognate 70-interacting protein gene from the silkworm, Bombyx mori

Carboxyl terminus of heat shock cognate 70-interacting protein (CHIP) is an evolutionarily conserved E3 ubiquitin ligase across different eukaryotic species and is known to play a key role in protein quality control. CHIP has two distinct functional domains, an N-terminal tetratricopeptide repeat (TPR) and a C-terminal U-box domain, which are required for the ubiquitination of numerous labile client proteins that are chaperoned by heat shock proteins (HSPs) and heat shock cognate proteins (HSCs). During our screen for CHIP-like proteins in the Bombyx mori databases, we found a novel silkworm gene, Bombyx mori CHIP. Phylogenetic analysis showed that BmCHIP belongs to Lepidopteran lineages. Quantitative reverse transcription-PCR analysis indicated that BmCHIP was relatively highly expressed in the gonad and fat body. A pull-down experiment and auto-ubiquitination assay showed that BmCHIP interacted with BmHSC70 and had E3 ligase activity. Additionally, immunohistochemical analysis revealed that BmCHIP was partially co-localized with ubiquitin in BmN4 cells. These data support that BmCHIP plays an important role in the ubiquitin proteasome system as an E3 ubiquitin ligase in B. mori.

Ex vivo promoter analysis of antiviral heat shock cognate 70B gene in Anopheles gambiae

BackgroundThe Anopheles gambiae heat shock cognate gene (hsc70B) encodes a constitutively expressed protein in the hsp70 family and it functions as a molecular chaperone for protein folding. However, the expression of hsc70B can be further induced by certain stimuli such as heat shock and infection. We previously demonstrated that the An. gambiae hsc70B is induced during o'nyong-nyong virus (ONNV) infection and subsequently suppresses ONNV replication in the mosquito. To further characterize the inducibility of hsc70B by ONNV infection in An. gambiae, we cloned a 2.6-kb region immediately 5' upstream of the starting codon of hsc70B into a luciferase reporter vector (pGL3-Basic), and studied its promoter activity in transfected Vero cells during infection with o'nyong-nyong, West Nile and La Crosse viruses.ResultsSerial deletion analysis of the hsc70B upstream sequence revealed that the putative promoter is likely located in a region 1615–2150 bp upstream of the hsc70B starting codon. Sequence analysis of this region revealed transcriptional regulatory elements for heat shock element-binding protein (HSE-bind), nuclear factor κB (NF-κB), dorsal (Dl) and fushi-tarazu (Ftz). Arbovirus infection, regardless of virus type, significantly increased the hsc70B promoter activity in transfected Vero cells.ConclusionOur results further validate the transcriptional activation of hsc70B during arbovirus infection and support the role of specific putative regulatory elements. Induction by three taxonomically distinct arboviruses suggests that the HSC70B protein may be expressed to cope with cellular stress imposed during infection.

Molecular identification and expression of heat shock cognate 70 (HSC70) in the pacific white shrimp Litopenaeus vannamei

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80-99.6% homology with 12 diverse species' HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multi-tissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of beta-actin. Furthermore, quantitative real-time experiments showed that HSC70 was up-regulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps' cytoprotection.

Adaptive divergence in a high gene flow environment: Hsc70 variation in the European flounder (Platichthys flesus L.)

Little is known about local adaptations in marine fishes since population genetic surveys in these species have typically not applied genetic markers subject to selection. In this study, we used a candidate gene approach to investigate adaptive population divergence in the European flounder (Platichthys flesus L.) throughout the northeastern Atlantic. We contrasted patterns of genetic variation in a presumably neutral microsatellite baseline to patterns from a heat-shock cognate protein gene, Hsc70. Using two different neutrality tests we found that the microsatellite data set most likely represented a neutral baseline. In contrast, Hsc70 strongly deviated from neutral expectations. Importantly, when estimating standardized levels of population divergence (F(ST)'), we also found a large discrepancy in the patterns of structuring in the two data sets. Thus, samples grouped according to geographical or historical proximity with regards to microsatellites, but according to environmental similarities with regards to Hsc70. The differences between the data sets were particularly pronounced in pairwise comparisons involving populations in the western and central Baltic Sea. For instance, the genetic differentiation between geographically close Baltic Sea and North Sea populations was found to be 0.02 and 0.45 for microsatellites and Hsc70 respectively. Our results strongly suggest adaptive population divergence and indicate local adaptations at the DNA level in a background of high levels of gene flow, typically found in many marine fish species. Furthermore, this study highlights the usefulness of the candidate gene approach for demonstrating local selection in non-model organisms such as most marine fishes.

Cloning and characterization of a 70 kDa heat shock cognate gene (HSC70) from two species of Chironomus.

In the present study we carried out the isolation and characterization of an HSC70 gene from two midges, Chironomus tentans and C. yoshimatsui. The HSC70 cDNAs are approximately 2424 (C. tentans) and 2464 bp (C. yoshimatsui) long, and contain 1950 and 1956 bp open reading frames, respectively. Analysis of genomic DNA revealed the presence of two introns in these genes. The 5' untranslated regions of the HSC70 genes are adenosine-rich, a feature found in inducible HSP70 genes. The nucleotide and amino acid sequences exhibit high identity with cytosolic HSC70s from other Dipterans. Northern hybridization indicated that HSC70 is expressed at all developmental stages, from embryo to adult, and Southern hybridization confirmed the presence of multiple HSP70 genes in Chironomus.

Mutations in Drosophila heat shock cognate 4 are enhancers of Polycomb.

The homeotic genes controlling segment identity in Drosophila are repressed by the Polycomb group of genes (PcG) and are activated by genes of the trithorax group (trxG). An F(1) screen for dominant enhancers of Polycomb yielded a point mutation in the heat shock cognate gene, hsc4, along with mutations corresponding to several known PcG loci. The new mutation is a more potent enhancer of Polycomb phenotypes than an apparent null allele of hsc4 is, although even the null allele occasionally displays homeotic phenotypes associated with the PcG. Previous biochemical results had suggested that HSC4 might interact with BRAHMA, a trxG member. Further analyses now show that there is no physical or genetic interaction between HSC4 and the Brahma complex. HSC4 might be needed for the proper folding of a component of the Polycomb repression complex, or it may be a functional member of that complex.

Characterization of a cDNA encoding a 72 kDa heat shock cognate protein (Hsc72) from the Pacific oyster, Crassostrea gigas.

A full-length cDNA encoding a 72 kDa heat shock cognate protein (Hsc72) was isolated from a Crassostrea gigas hemocyte library. This cDNA is 75% identical to a human hsc70 cDNA. C. gigas cDNA contains a 659 amino acid open reading frame encoding a 72 kDa protein which is 87% identical to a human Hsc70 protein. Northern blotting indicated that even if the hsc72 gene was constitutively expressed in oyster hemocytes, it could be stimulated by heat shock in vitro as well as in vivo. Homologies observed both at the cDNA and protein levels, as well as the expression pattern of the hsc72 mRNA, support our conclusion that the isolated cDNA clone corresponds to an oyster heat shock cognate gene.

A member of the hsp70 gene family from the Mediterranean fruit fly, Ceratitis capitata.

A gene from the Mediterranean fruit fly (Medfly) Ceratitis capitata, belonging to the hsp70 family of genes, has been cloned and characterized. This gene appears to be an example of a heat shock cognate (hsc) gene based on a variety of structural and functional criteria. The heat shock cognate genes serve critical roles in chaperoning of cellular proteins, and they are expressed in a more constitutive manner compared to the inducible heat shock genes. We determined the sequence of the entire coding region of this gene (designated Cerhsc1) together with 5' and 3' flanking regions. The coding region exhibits specific structural features that are characteristic of the subclass of heat shock genes producing proteins localized to the cytosol. The 5' regulatory region of Cerhsc1 contains onlythe minimal array of canonical heatshock element sequences, a phenomenon commonly seen in heat shock cognates. Abundant transcripts are detected from this gene in adult tissue compared to the larval stage. In larval tissue, heat shock appears to produce only a slight increase in the level of transcripts detected. Based on overall structure and function, this Medfly gene appears to be an ortholog of heat shock cognate gene 4 (Drohsc4) of Drosophila melanogaster.

Cloning and characterization of hscl +, a heat shock cognate gene of the fission yeast Schizosaccharomyces pombe

A heat shock cognate gene from the fission yeast Schizosaccharomyces pombe (Sp), designated hscl + , was cloned. The putative translation product of hscl + contains 613 aa, with an estimated molecular mass of 67 205 Da, and is more similar to the Saccharomyces cerevisiae (Sc) heat shock cognate protein SSB1 (69% identity) than the Sp heat-inducible sspl + gene product (41% identity). The hscl + mRNA was abundant during steady-state growth at 23°C and decreased upon heat shock. Immunoblot analysis showed that the hscl protein is also abundant and constitutively expressed, however, we could not observe significant change in the protein level upon heat shock. DNA blot analyses indicated that hscl + is localized in Sp chromosome II, and suggested that the Sp genome contains a relatively smaller number of HSP70 genes compared with the Sc genome.

Pollen and the heat shock response

The literature related to the expression of heat shock protein (hsp) genes in developing pollen and in the mature male gametophyte has been surveyed. In general, mature pollen lacks a normal heat shock response. In mature pollen of several species either no hsps are synthesized in response to heat stress or, if synthesized, only a subset are made and the response is weak at both the transcriptional and translational levels, compared to the response in vegetative tissues. In developing pollen, however, a subset of hsps is induced in response to heat shock. In addition, certain hsp genes or heat shock cognate (hsc) genes are activated during normal pollen development in the absence of heat stress, indicating that these genes are likely to have important developmental functions. How the distinctive activation pattern of hsp and hsc genes relates to the known sensitivity of pollen to heat stress is discussed.

Cloning and characterization of a 70 kd heat shock cognate (hsc70) gene from the zebrafish (Danio rerio).

The heat shock 70 family of proteins is one of the most highly conserved among all species. The genes encoding these proteins have been cloned and sequenced from bacterial species to humans with a high degree of homology preserved throughout evolution. Here we describe the cloning and characterization of a cDNA encoding a 70 kd heat shock cognate (hsc70) gene from the zebrafish (Danio rerio). A high degree of conservation is observed among hsc70 genes of other species as shown by phylogenetic analysis. The characterization of a hsc70 gene in the zebrafish provides a marker for studying the role of a constitutively expressed member of the hsp70 family in an important developmental and evolutionary model system.

Cloning, Sequencing, and Mapping of the Human Chromosome 14 Heat Shock Protein Gene (HSPA2)

A genomic clone for the human heat shock protein (HSP) 70 gene located on chromosome 14 was isolated and sequenced. The gene, designated HSPA2, has a single open reading frame of 1917 bp that encodes a 639-amino acid protein with a predicted molecular weight of 70,030 Da. Analysis of the sequence indicates that HSPA2 is the human homologue of the murine Hsp70-2 gene with 91.7% identity in the nucleotide coding sequence and 98.2% in the corresponding amino acid sequence. HSPA2 has less amino acid homology to other members of the human HSP70 gene family, 83.3% to the heat-inducible HSP70-1 gene and 86.1% with the human heat shock cognate gene HSC70. HSPA2 is constitutively expressed in most tissues, with very high levels in testis and skeletal muscle. Significant but lower levels are also expressed in ovary, small intestine, colon, brain, placenta, and kidney. A yeast artificial chromosome (YAC) clone containing HSPA2 (YAC741H4) that also contained the polymorphic marker D14S63 was identified. This 670-kb YAC was mapped to 14q24.1 by fluorescence in situ hybridization (FISH). Subsequent two-color FISH and genetic mapping placed HSPA2/D14S63 proximal to the markers D14S57 and D14S77.

A gene encoding a DnaK/hsp70 homolog in Escherichia coli.

Eukaryotic organisms have been shown to have multiple forms of hsp70-class stress-related proteins, but only a single family member, DnaK, has been found in prokaryotes. We report here the identification of a heat shock cognate gene, designated hsc, in Escherichia coli. The amino acid sequence deduced from hsc predicts a 65,647-Da polypeptide having 41% sequence identity with DnaK from E. coli, and overexpression produces a protein (Hsc66) with properties similar to DnaK. In contrast to dnaK, however, the hsc gene lacks a consensus heat shock promoter sequence, and expression is not induced by elevated temperature. The hsc gene is located near 54 min on the physical map, immediately upstream of the fdx gene, which encodes a [2Fe-2S] ferredoxin; evidence is presented that the hsc and fdx genes make up a bicistronic operon in which expression of the ferredoxin is coupled to that of Hsc66. The function of Hsc66 is not known, but the coregulation of its expression with that of ferredoxin suggests the possibility of a specific role in association with the ferredoxin protein.

Transcriptional regulation in Drosophila during heat shock: A nuclear run-on analysis

We used a nuclear run-on assay as a novel approach to study the changes in transcriptional activity that take place in Drosophila melanogaster during heat shock. In response to a rapid temperature upshift, total transcriptional activity in cultured KC161 cells decreased proportionally to the severity of the shock. After extended stress at 37 degrees C (15 min or more), transcription was severely reduced, and at 39 degrees C most transcription was instantaneously arrested. However, strikingly different responses were observed for individual genes. Transcription of histone H1 genes was severely inhibited even under mild heat shock conditions. Transcription of the actin 5C gene decreased progressively with increasing temperature, while transcription of the core histone genes or of the heat shock cognate genes was repressed only under severe heat shock conditions. Transcriptional activation of the D. melanogaster heat shock genes was also investigated. In unshocked cells, hsp84 was moderately transcribed, while transcriptional activity at the other protein-coding heat shock genes was undetectable (less than 0.2 polymerases per gene). Engaged but paused RNA polymerase molecules were found at the hsp70 and hsp26 genes, but not at the other heat shock genes. The rates of transcription increased with increasing temperature with a peak of expression at around 35 degrees C. At 37 degrees C, induction was less efficient, and no induction was achieved after a rapid shift to 39 degrees C. Increased transcription of the heat shock genes was observed within 1-2 min of heat shock, and maximal rates were reached within 2-5 min. Despite very similar profiles of response, different heat shock genes were transcribed at strikingly different rates, which varied over a 20-fold range. The noncoding heat shock locus 93D was transcribed at a very high rate under non-heat shock conditions, and showed a transcriptional response to elevated temperatures different from that of protein-coding heat shock genes. An estimation of the absolute rates of transcription at different temperatures was obtained.

Expression of a heat-inducible gene of the HSP70 family in human myelomonocytic cells: regulation by bacterial products and cytokines

In this study we have examined the expression of a heat-shock protein (HSP) 70 gene in normal human peripheral blood leukocytes. Northern blot analysis showed that appreciable levels of hsp70 mRNA are present in monocytes and granulocytes, whereas transcript levels were barely detectable or absent in lymphocytes. Monocytes functionally activated by bacterial lipopolysaccharide (LPS) showed an early (15 minutes) increase of hsp70 transcripts that was shown, by actinomycin D blocking and nuclear run-off experiments, to be dependent on transcriptional activation of the gene. LPS did not appreciably affect the hsp70 mRNA half-life. Monocytes exposed to inactivated streptococci, phorbol-12- myristate-13-acetate, and tumor necrosis factor showed augmented levels of hsp70 transcripts, whereas interferon-gamma and monocyte, granulocyte, and granulocyte-monocyte colony-stimulating factors had no effect. Adherence to plastic augmented hsp70 expression in monocytes. S1 protection analysis indicated that the gene expressed in monocytes is indeed a heat-inducible member of the hsp70 gene family rather than a constitutively expressed heat-shock cognate gene. Western blot analysis showed that a heat-inducible HSP72 was present in monocytes and, at augmented levels, in LPS-treated monocytes. LPS-activated monocytes were more resistant to heat shock than unstimulated cells. These data indicate that a heat-inducible hsp70 gene can be efficiently expressed in myelomonocytic cells at physiologic temperatures. Expression of hsp70 genes in monocytes suggests a possible role of heat- inducible genes in the differentiation and/or functional activation of terminally differentiated nonproliferating elements of the myelomonocytic lineage.

Expression of a Heat-Inducible Gene of the HSP70 Family in Human Myelomonocytic Cells: Regulation by Bacterial Products and Cytokines

In this study we have examined the expression of a heat-shock protein (HSP) 70 gene in normal human peripheral blood leukocytes. Northern blot analysis showed that appreciable levels of hsp70 mRNA are present in monocytes and granulocytes, whereas transcript levels were barely detectable or absent in lymphocytes. Monocytes functionally activated by bacterial lipopolysaccharide (LPS) showed an early (15 minutes) increase of hsp70 transcripts that was shown, by actinomycin D blocking and nuclear run-off experiments, to be dependent on transcriptional activation of the gene. LPS did not appreciably affect the hsp70 mRNA half-life. Monocytes exposed to inactivated streptococci, phorbol-12-myristate-13-acetate, and tumor necrosis factor showed augmented levels of hsp70 transcripts, whereas interferon-gamma and monocyte, granulocyte, and granulocyte-monocyte colony-stimulating factors had no effect. Adherence to plastic augmented hsp70 expression in monocytes. S1 protection analysis indicated that the gene expressed in monocytes is indeed a heat-inducible member of the hsp70 gene family rather than a constitutively expressed heat-shock cognate gene. Western blot analysis showed that a heat-inducible HSP72 was present in monocytes and, at augmented levels, in LPS-treated monocytes. LPS-activated monocytes were more resistant to heat shock than unstimulated cells. These data indicate that a heat-inducible hsp70 gene can be efficiently expressed in myelomonocytic cells at physiologic temperatures. Expression of hsp70 genes in monocytes suggests a possible role of heat-inducible genes in the differentiation and/or functional activation of terminally differentiated nonproliferating elements of the myelomonocytic lineage.

Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster.

The Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.

Molecular and Developmental Characterization of the Heat Shock Cognate 4 Gene of Drosophila melanogaster

The Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.

The brain 68-kilodalton microtubule-associated protein is a cognate form of the 70-kilodalton mammalian heat-shock protein and is present as a specific isoform in synaptosomal membranes.

The relationship between the 68-kilodalton microtubule-associated protein (68KMAP) and the major heat-induced protein (HSP70) in rat and human cells was investigated by comparison of their heat induction properties and by tryptic and Cleveland peptide mapping procedures. HSP70 synthesis was induced by heat shock of rat and human cells, whereas 68KMAP was a major synthesised protein in the absence of heat shock, with its synthesis being only slightly increased on heat shock. Tryptic peptide mapping, however, indicated strong peptide homology between the two proteins. These data, therefore, confirm that 68KMAP represents a constitutively expressed, heat-shock cognate gene. Two-dimensional gel electrophoretic analysis of subcellular fractions of rat brain, combined with peptide mapping procedures, indicated that 68KMAP exists as at least two isoforms separable by isofocussing, the more acidic of which (alpha 68KMAP) is present in fractions enriched in microtubules, cytosol, microsomes, synaptosomal plasma membranes, and synaptic vesicles, and the more basic of which (beta 68KMAP) is present predominantly in fractions enriched in synaptic vesicles and synaptosomal plasma membranes. These two forms are distinguishable in terms of changes in Cleveland peptide maps, and we conclude that alpha- and beta 68KMAP, therefore, represent distinct forms. The significance of these findings to the molecular pathogenesis of Down's syndrome in the human brain is discussed.