Hsp70 Gene Family Research Articles

Global changes in protein synthesis during adaptation of the yeast Saccharomyces cerevisiae to 0.7 M NaCl.

Exponentially growing Saccharomyces cerevisiae was challenged to increased salinity by transfer to 0.7 M NaCl medium, and changes in protein synthesis were examined during the 1st h of adaptation by use of two-dimensional gel electrophoresis coupled to computerized quantification. An impressive number of proteins displayed changes in the relative rate of synthesis, with most differences from nonstressed cells being found at between 20 and 40 min. During this period, 18 proteins exhibited more than eightfold increases in their rates of synthesis and were classified as highly NaCl responsive. Only two proteins were repressed to the same level. Most of these highly NaCl-responsive proteins seemed to constitute gene products not earlier reported to respond to dehydration. Applying a selection criterion to subsequent samples of a twofold change in the relative rate of synthesis, 14 different regulatory patterns were discerned. Most identified glycolytic enzymes exhibited a delayed response, and their rates of synthesis did not change until the middle phase of adaptation, with only a minor decrease in the rate of production. A slight salt-stimulated response was observed for some members of the HSP70 gene family. Overall, the data presented indicate complex intracellular signalling as well as involvement of diverse regulatory mechanisms during the period of adaptation to NaCl.

Candidate genes of hypertension with defective environmental expression

Previous studies in our laboratory have demonstrated that the thermosensitivity locus cosegregates with blood pressure and that the elevated expression and restriction fragment length polymorphism of HSP70 gene are associated with hypertension. Cell protection against environmental stressors such as heat and chemicals is often accompanied by up-regulated expression of a wide spectrum of heat shock genes(HSP). To further investigate the interrelation between HSP expression and blood pressure regulation, we employed an effective method of cloning 2 potential hypertension-related HSPs. Synthetic oligonucleotides corresponding either to a highly-conserved region of the known HSP family or a repetitive sequence in the protein encoding gene were used as target primers for polymerase chain reaction (PCR). cDNA prepared from heat-stressed and non-stressed vascular smooth muscle cells (VSMC) of Brown Norway rats (BN.lx) and spontaneously hypertensive rats (SHRp) respectively served as template in the reaction. The PCR products were subsequently analyzed in a single-stranded conformational polymorphism (SSCP) electrophoresing system. Differential gene expression in BN. lx and SHRp was seen on autoradiographs of SSCP gel by comparing the migration patterns of PCR-amplified DNA fragments. Using this technique, we also found that HSP27 and a new member of the large HSP gene family were differentially expressed in BN.1x and SHRp VSMC.

The hsc70 gene which is slightly induced by heat is the main virus inducible member of the hsp70 gene family

We have found that SV40 infection of CV1 cells induces the synthesis of a 72 kDa protein that upon molecular cloning was shown to be the product of the hsc70 gene. The above gene product was found to be mainly virus inducible, in contrast to the hsp70 gene product which was mainly heat inducible. The two genes were found to be cell cycle regulated in a distinctively different manner.

Hsp70 in parasites: as an inducible protective protein and as an antigen.

The heat shock (HS) response is a general homeostatic mechanism that protects cells and the entire organism from the deleterious effects of environmental stresses. It has been demonstrated that heat shock proteins (HSP) play major roles in many cellular processes, and have a unique role in several areas of cell biology, from chronic degenerative diseases to immunology, from cancer research to interaction between host and parasites. This review deals with the hsp70 gene family and with its protein product, hsp70, as an antigen when pathogens infect humans. Members of HSP have been shown to be major antigens of many pathogenic organisms when they experience a major temperature shift upwards at the onset of infection and become targets for host B and T cells.

Heat shock response in the liver: expression and regulation of the hsp70 gene family and early response genes after in vivo hyperthermia.

Heat shock response in cultured cells has been studied extensively; however few data are available on heat shock response in an intact organ of a living animal. In this study we analyzed the kinetics of expression of the heat shock protein 70 gene family (heat shock protein 70, heat shock cognate protein 73 and glucose-regulated protein 78) in the liver of the thermally stressed rat. New synthesis of heat shock protein 70 and heat shock cognate protein 73 was shown in liver slices pulse labeled in vitro with 35S-methionine. Accumulation of heat shock protein 70 and heat shock cognate protein 73 proteins was shown in total cellular extracts. 32P-labeled complementary DNA probes encoding heat shock protein 70, heat shock cognate protein 73 and glucose-regulated protein 78 were used to show that the levels of the corresponding messenger RNAs increase as a fraction of total RNA and in polysomes at different extents and with different kinetics. The induction of heat shock protein 70 and heat shock cognate protein 73 messenger RNAs reflected the increase in the synthesis of the corresponding proteins. Run-on transcription analysis indicated that the expression of heat shock protein 70 and heat shock cognate protein 73 genes was mainly regulated at the transcriptional level. On the contrary, both transcriptional and posttranscriptional regulatory mechanisms can explain the induction of the glucose-regulated protein 78 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Expression of heat shock factor 2 in mouse testis: potential role as a regulator of heat-shock protein gene expression during spermatogenesis.

We have examined the expression and function of heat shock transcription factor 2 (HSF2) in spermatogenic cells of mouse testis. The results of in situ RNA hybridization analysis, RNA filter hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) analysis indicate that HSF2 mRNA expression in testis is subject to developmental and cell type-dependent, as well as stage-dependent, regulation. Localized expression of HSF2 mRNA in testis first appears between Day 14 and Day 21 of postnatal development. In adult testis, HSF2 mRNA is found at highest levels in spermatocytes and round spermatids. Immunocytochemical staining and gel mobility shift analysis demonstrate that HSF2 protein is localized to the nuclei of spermatocytes and round spermatids and that this transcription factor exists in testis in a constitutively active DNA-binding state. We further demonstrate that the constitutive HSF2 DNA-binding activity present in testis is able to interact with promoter sequences of the hsp70.2 gene, a testis-specific member of the hsp70 gene family. Taken together, our results show that the expression and functional properties of HSF2 are regulated in spermatogenic cell types of the mouse testis, supporting a role for this transcription factor as a regulator of hsp gene expression during spermatogenesis.

Hsp70 genes are linked to the Xenopus major histocompatibility complex

Some of the inducible forms of the heat shock protein 70 (Hsp70) gene family are encoded in the class III region of the major histocompatibility complex (MHC) of mammals. This study was undertaken to determine whether Hsp70 genes are linked to the MHC of Xenopus, an amphibian last sharing a common ancestor with mammals 300-350 million years ago. Segregation analyses involving seven haplotypes demonstrated the linkage of two or three inducible Hsp70 genes to the frog MHC. Another Hsp70 gene is not closely linked to the MHC. We conclude that the physical association of MHC class I and class II genes with Hsp70 genes is ancient.

The Hsp70 heat-shock gene family of the mosquito Anopheles albimanus.

Four Hsp70 genes of the malaria vector Anopheles albimanus were isolated from a genomic DNA library as two non-overlapping clones each containing a pair of divergently transcribed genes having 75% DNA sequence similarity to the protein-coding regions of the Drosophila melanogaster Hsp70 genes. The clones were assigned to two loci on chromosome 2R by in situ hybridization. These clones hybridize strongly to heat-shock but only weakly to non-shocked mosquito RNA. The Hsp70 gene family of A. albimanus is undergoing concerted evolution probably by gene conversion. The general arrangement of the genes suggests that divergently transcribed pairs of genes at two loci is an ancient Dipteran arrangement predating the Nematocera/Cyclorrapha divergence.

Molecular cloning and cellular localization of a bip homologue in Trypanosoma brucei: Divergent er retention signals in a lower eukaryote

Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989) Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.

Genomic structure and sequence analysis of Drosophila melanogaster HSC70 genes

We report the identification of two new members of the Drosophila melanogaster HSP70 gene family, HSC3 and HSC5. DNA sequence analysis predicts that HSC3 encodes a 72-kDa protein with a hydrophobic leader sequence and a C-terminal retrieval tetrapeptide, KDEL, characteristics associated with luminal endoplasmic reticulum (ER) proteins. Sequence analysis predicts that HSC5 encodes a 74-kDa protein with a characteristic mitochondrial leader sequence. We report the deduced amino acid (aa) sequence for the previously identified gene, HSC1. HSC1 encodes a 70-kDa protein lacking a leader sequence and is presumed to have a cytoplasmic localization. A comparison of the deduced aa sequences of these and hsc70 proteins from different species indicates that hsc70 proteins residing in the same intracellular compartment in different organisms are more similar to each other than are hsc70s from the same organism, but different organelles.

Chromosomal Localization of Five Murine HSP70 Gene Family Members: Hsp70.1, Hsp70-2, Hsp70-3, Hsc70t, and Grp78

The 70,000-D heat shock protein (HSP70) gene family includes both heat-inducible and constitutively expressed genes. We have mapped five murine HSP70 genes to specific sites on three separate chromosomes. Southern blot analysis of Chinese hamster X mouse somatic cell DNAs was used to assign the gene for the 78,000-D glucose-regulated protein (Grp78) to Chromosome (Chr) 2, the mule germ cell-specific Hsp70-2 and Hsc70t genes to Chr 12 and Chr 17, respectively, and the heat-inducible Hsp70-3 gene also to Chr 17. Southern blot analysis of DNA from the progeny of two multilocus crosses confirmed the Grp78 location on Chr 2 and suggested the order: centromere-Vim-Abl-Grp78-Hc. Similar analysis also confirmed the initial Hsp70-2 assignment to Chr 12 with the order: Hsp70-2-Aat-Igh . The Hsp70-3 and Hsc70t genes on Chr 17, along with the heat-inducible Hsp70-1 gene, were further localized by Southern blot analysis of genomic clones to the H-2 histocompatibility region with the order: Hsp70-1-Hsp70-3-Hsc70t-Bat-6 (human G7a, valyl-tRNA synthetase).

The effect of age on the synthesis of two heat shock proteins in the hsp70 family.

Hepatocytes isolated from male F344 rats were exposed to elevated temperatures (40 degrees C to 45 degrees C), and the incorporation of [35S]-L-methionine into proteins was measured from fluorograms of two-dimensional polyacrylamide gels. The synthesis of two proteins was induced by temperatures of 40 degrees C to 42.5 degrees C; however, 45 degrees C inhibited the synthesis of all proteins. Based on their apparent molecular weights and pI values and their recognition by a monoclonal antibody to the HSP70 gene family, the two proteins induced by hyperthermia were found to be the highly heat-inducible hsp70 and the constitutive hsc70. Because a heat shock of 42.5 degrees C for 30 minutes had very little effect on cell viability and induced the synthesis of hsp70 and hsc70, this heat shock was used to study the effect of age on the ability of hepatocytes to respond to the stress of hyperthermia. Neither hepatocytes isolated from young adult (5-7 months) nor old (25-27 months) rats synthesized detectable amounts of hsp70 when incubated at 37 degrees C. However, heat shock induced the synthesis of both hsp70 and hsc70 in hepatocytes isolated from young adult and old rats. The induction of hsp70 synthesis was significantly lower (37%) for hepatocytes isolated from old rats compared to hepatocytes isolated from young adult rats. However, neither the basal level nor the induced level of hsc70 synthesis changed significantly with age. Thus, aging resulted in a decrease in the ability of hepatocytes to synthesize hsp70 in response to hyperthermia; this effect, however, was specific for hsp70.

Vascular heat shock protein expression in response to stress. Endocrine and autonomic regulation of this age-dependent response.

Adaptation to stress requires coordinated interactions between the vascular and endocrine systems. Previously we demonstrated that restraint stress induces the expression of the major heat shock protein, HSP70, in the adrenal cortex of the rat. Here we demonstrate that restraint also induces expression of HSP70 in the vasculature. We further demonstrate that the adrenal and vascular responses are differentially regulated: the adrenal response is adrenocorticotropin dependent, whereas the vascular response is under adrenergic control. In addition, the adrenal response is restricted to members of the HSP70 gene family, whereas in vascular tissue the low molecular weight HSP, HSP27, is also induced by restraint. Further characterization of the vascular response revealed that HSP70 induction occurred in both the thoracic and abdominal aortas as well as in the vena cava. However, no HSP70 induction was apparent in the heart or in a wide variety of other tissues examined. In situ hybridization showed that the vascular expression was localized to the aortic smooth muscle cells with minimal expression in the endothelium. Induction of HSP70 mRNA in both the adrenal cortex and aorta was followed by an elevation in HSP70 protein. Maximum HSP70 protein levels were seen within 3-12 h after restraint, but declined thereafter. Stress induced HSP70 expression was dramatically reduced with age, which may explain, in part, the diminished tolerance to stress seen in elderly individuals.

Regulation of hsc70 expression in the human histiocytic lymphoma cell line, U937.

The transcription of hsc70, a cognate member of the hsp70 gene family, is suppressed in the terminally differentiated human histiocytic lymphoma cell line, U937, and appears to be regulated in the absence of heat shock. We have examined the 5' upstream regulatory region of 450 bp which contains a putative regulatory sequence of 18 bp in addition to known cis-elements such as HSEs, CCAAT boxes, Sp1 binding sites and AP2 binding sites. The 18 bp cis-element formed larger complexes with nuclear localized transfactors when extracts were prepared from differentiated rather than from proliferating cells. These larger complexes were also detected in nuclear extracts of serum-deprived cells, suggestng that such complexes may be related to the suppression of hsc70 gene transcription in Go arrested cells. The level of HSC70 protein as determined using monoclonal antibodies, increased threefold in differentiated cells. The apparent discrepancy between the levels of hsc70 mRNA and HSC70 protein can be explained by a rapid turnover of HSC70 in proliferating cells.

Isolation of four HSP70 genes in the pig and localization on chromosomes 7 and 14.

For insight into the general organization of the swine leukocyte antigen (SLA) complex, the swine major histocompatibility complex (MHC), four sequences related to the heat-shock proteins HSP70 were characterized by screening of a pig genomic cosmid library with a swine cDNA HSP70 2.6-kb probe. This yielded three positive clones: HC2.2, HC3.2, and HC4.2. Restriction site maps revealed a large overlap of HC2.2 with HC3.2, whereas HC4.2 was independent. Southern blot hybridization with the 5' section, the central section, and the 3' section of the 2.6-kb probe and also with a swine 4.5-kb HSP70 genomic probe suggested the existence, within the overlapping clones, of three distinct HSP70 sequences encompassing a segment no longer than 22 kb. The HC4.2 clone, which hybridized with the same probes, displayed a single band of 7.3 kb, probably corresponding to one gene only. Fluorescent in situ hybridization on swine chromosome metaphases with the whole HC2.2 or HC4.2 cosmids allowed the assignment of HC2.2 to MHC region on Chromosome (Chr) 7 (Cen-p1.1), and of HC4.2 to Chr 14 (q2.4-2.5). Thus, as in humans, the swine MHC comprises three closely linked HSP70 loci. The presence of additional genes belonging to the same inducible HSP70 gene family can be expected from what is known in humans. The HSP70 gene found here on the pig Chr 14 may be one of these putative unidentified genes.

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.

Cloning and sequencing of an hsp70 gene of Schistosoma mansoni

Schistosomes have a complex life cycle (vertebrate and molluscan hosts as well as larvae living freely in water) in which they are exposed to different environments and temperatures (20°C37°C). Since heat shock genes are activated in response to stress and during development [1], it is of interest to study the hsp70 gene family in schistosome. To approach this issue we have isolated from Schistosoma mansoni a genomic clone containing the complete coding region of hsp70 and the 5′ flanking DNA with transcription regulatory elements including HSE (heat shock element) sequences.

Characterization of a new member of the sea urchin Paracentrotus lividus hsp70 gene family and its expression

We have sequenced a second gene of the hsp70 family derived from a genomic clone of the sea urchin, Paracentrotus lividus. The structure of this gene, named hsp70 IV gene, is interrupted by one intron and diners from the previously analyzed sea urchin hsp70II gene, which contains several introns. Two open reading frames of hsp70IV gene encode a predicted protein of 639 amino acids with an M r of 69 672. The 5′ flanking region of the gene contains a putative TATA element, three heat-shock elements made up of some arrays of the 5-bp units, NGAAN and NTTCN (N = A,C,G or T), a canonic conensus sequence for binding of the regulatory activating transcription factor (ATF), and a purine box. The 3′ flanking region contains four putative polyadenylation sites located at different sites downstream from the stop codon. Using Northern blot hybridization analysis, carried out using a probe corresponding to a 3′ noncoding fragment ( UTR) peculiar to hsp70IV gene, we found that this gene is transcribed only under heat shock (Hs) and that the transcript can be recovered from the polysomal pellet. The hsp70IV gene may be classified as a Hs gene 70 although it contains one intron.

Induction of HSP70 gene expression by the antiproliferative prostaglandin PGA2: a growth-dependent response mediated by activation of heat shock transcription factor.

Prostaglandins (PG) of the A series are potent inhibitors of cell proliferation. Recently, it was shown that PGA2-induced growth arrest was associated with the increased synthesis of stress proteins encoded by the HSP70 gene family. In this study, we have examined the molecular basis for this increases HSP70 expression. Northern (RNA) blot analysis and nuclear run-on assays demonstrated that induction of high levels of HSP70 mRNA results from an increase in the rate of transcription. High-level induction is specific to the HSP70 family of heat shock proteins and is rapid, reversible, dose dependent, and specific for PGs capable of growth-arresting HeLa cells. In addition, the response was found to be highly dependent on the growth state of the cells, as induction occurs in growing but not in confluent nongrowing cell populations. Induction is dependent on the activation of heat shock factor. Cycloheximide pretreatment, which inhibits the antiproliferative effects of PGA2, prevents activation of the heat shock factor and induction of HSP70 mRNA by PGA2. These results support a role for HSP70 in mediating the antiproliferative effects of PGA2.