Structure, organization, and expression of the 16-kDa heat shock gene family of Caenorhabditis elegans

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 690-697 ◽  
Author(s):  
E. P. M. Candido ◽  
D. Jones ◽  
D. K. Dixon ◽  
R. W. Graham ◽  
R. H. Russnak ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Normal Growth ◽  
Growth Conditions ◽  
Amino Acid Sequences ◽  
Heat Shock Gene ◽  
Coding Region ◽  
Heat Shock Element ◽  
Hypersensitive Sites ◽  
Intergenic Regions

Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in a mouse alpha-crystallin gene. The compact intergenic regions of both hsp16 loci contain a TATA element and a heat shock element (HSE) for each member of the pair, and are very similar in sequence overall. Expression studies, however, show that the level of transcripts from the hsp16-2/41 pair may be up to 14-fold higher on a per gene basis, as the level of RNA from the hsp16-1/48 pair, depending upon the induction conditions and developmental stage. This difference in message levels seems to be due to differences in the kinetics of inactivation of the genes rather than in transcription rates or rates of mRNA turnover. Distinct DNAseI hypersensitive sites are present upstream of each HSE in chromatin when the genes are inactive; these disappear and the whole intergenic region seems to become DNAse sensitive when the genes are maximally active.Key words: heat shock, 16-kDa polypeptides, gene structure, transcription, DNAseI hypersensitive sites, Caenorhabditis elegans.

HSF1 granules: a novel stress-induced nuclear compartment of human cells

1997 ◽  
Vol 110 (23) ◽  
pp. 2925-2934 ◽  
Author(s):  
J. Cotto ◽  
S. Fox ◽  
R. Morimoto
Keyword(s):  
Heat Shock ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Normal Growth ◽  
Growth Conditions ◽  
Heat Shock Gene ◽  
Heat Shock Factor 1 ◽  
Flag Epitope ◽  
Nuclear Structures ◽  
Stress Dependent

Heat shock factor 1 (HSF1) is the ubiquitous stress-responsive transcriptional activator which is essential for the inducible transcription of genes encoding heat shock proteins and molecular chaperones. HSF1 localizes within the nucleus of cells exposed to heat shock, heavy metals, and amino acid analogues, to form large, irregularly shaped, brightly staining granules which are not detected during attenuation of the heat shock response or when cells are returned to their normal growth conditions. The kinetics of detection of HSF1 granules parallels the transient induction of heat shock gene transcription. HSF1 granules are also detected using an HSF1-Flag epitope tagged protein or a chimeric HSF1-green fluorescent protein which reveals that these nuclear structures are stress-induced and can be detected in living cells. The spatial organization of HSF1 granules in nuclei of stressed cells reveals that they are novel nuclear structures which are stress-dependent and provides evidence that the nucleus undergoes dynamic reorganization in response to stress.

scholarly journals Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.

1989 ◽  
Vol 9 (6) ◽  
pp. 2615-2626 ◽  
Author(s):  
E Hickey ◽  
S E Brandon ◽  
G Smale ◽  
D Lloyd ◽  
L A Weber
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Normal Growth ◽  
Gene Families ◽  
Growth Conditions ◽  
Complete Sequence ◽  
Start Codon ◽  
Hybridization Probe ◽  
Reading Frame ◽  
Alpha Gene

Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

Modular recognition of 5-base-pair DNA sequence motifs by human heat shock transcription factor

1991 ◽  
Vol 11 (7) ◽  
pp. 3504-3514
Author(s):  
N F Cunniff ◽  
J Wagner ◽  
W D Morgan
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Gene Promoter ◽  
Heat Shock Transcription Factor ◽  
Heat Shock Gene ◽  
Gene Promoters ◽  
Sequence Motifs ◽  
Binding Modes ◽  
Heat Shock Element

We investigated the recognition of the conserved 5-bp repeated motif NGAAN, which occurs in heat shock gene promoters of Drosophila melanogaster and other eukaryotic organisms, by human heat shock transcription factor (HSF). Extended heat shock element mutants of the human HSP70 gene promoter, containing additional NGAAN blocks flanking the original element, showed significantly higher affinity than the wild-type promoter element for human HSF in vitro. Protein-DNA contact positions were identified by hydroxyl radical protection, diethyl pyrocarbonate interference, and DNase I footprinting. New contacts in the mutant HSE constructs corresponded to the locations of additional NGAAN motifs. The pattern of binding indicated the occurrence of multiple DNA binding modes for HSF with the various constructs and was consistent with an oligomeric, possibly trimeric, structure of the protein. In contrast to the improved binding, the extended heat shock element mutant constructs did not exhibit dramatically increased heat-inducible transcription in transient expression assays with HeLa cells.

Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells

1988 ◽  
Vol 8 (11) ◽  
pp. 4736-4744
Author(s):  
D D Mosser ◽  
N G Theodorakis ◽  
R I Morimoto
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Heat Shock ◽  
Gene Transcription ◽  
Elevated Temperatures ◽  
Binding Activity ◽  
Heat Shock Gene ◽  
Hsp70 Gene ◽  
Heat Shock Element ◽  
Amino Acid Analogs

Activation of human heat shock gene transcription by heat shock, heavy metal ions, and amino acid analogs required the heat shock element (HSE) in the HSP70 promoter. Both heat shock- and metal ion-induced HSP70 gene transcription occurred independently of protein synthesis, whereas induction by amino acid analogs required protein synthesis. We identified a HSE-binding activity from control cells which was easily distinguished by a gel mobility shift assay from the stress-induced HSE-binding activity which appeared following heat shock or chemically induced stress. The kinetics of HSP70 gene transcription paralleled the rapid appearance of stress-induced HSE-binding activity. During recovery from heat shock, both the rate of HSP70 gene transcription and stress-induced HSE-binding activity levels declined and the control HSE-binding activity reappeared. The DNA contacts of the control and stress-induced HSE-binding activities deduced by methylation interference were similar but not identical. While stable complexes with HSE were formed with extracts from both control and stressed cells in vitro at 25 degrees C, only the stress-induced complex was detected when binding reactions were performed at elevated temperatures.

Heat shock factor is required for growth at normal temperatures in the fission yeast Schizosaccharomyces pombe

1993 ◽  
Vol 13 (2) ◽  
pp. 749-761
Author(s):  
G J Gallo ◽  
H Prentice ◽  
R E Kingston
Keyword(s):  
Heat Shock ◽  
Schizosaccharomyces Pombe ◽  
Normal Growth ◽  
Growth Conditions ◽  
Heat Shock Factor ◽  
Binding Ability ◽  
Lower Growth ◽  
General Role ◽  
Functional Conservation ◽  
Cellular Processes

Schizosaccharomyces pombe is becoming an increasingly useful organism for the study of cellular processes, since in certain respects, such as the cell cycle and splicing, it is similar to metazoans. Previous biochemical studies have shown that the DNA binding ability of S. pombe heat shock factor (HSF) is fully induced only under stressed conditions, in a manner similar to that of Drosophila melanogaster and humans but differing from the constitutive binding by HSF in the budding yeasts. We report the isolation of the cDNA and gene for the HSF from S. pombe. S. pombe HSF has a domain structure that is more closely related to the structure of human and D. melanogaster HSFs than to the structure of the budding yeast HSFs, further arguing that regulation of HSF in S. pombe is likely to reflect regulation in metazoans. Surprisingly, the S. pombe HSF gene is required for growth at normal temperatures. We show that the S. pombe HSF gene can be replaced by the D. melanogaster HSF gene and that strains containing either of these genes behave similarly to transiently heat-shocked strains with respect to viability and the level of heat-induced transcripts from heat shock promoters. Strains containing the D. melanogaster HSF gene, however, have lower growth rates and show altered morphology at normal growth temperatures. These data demonstrate the functional conservation of domains of HSF that are required for response to heat shock. They further suggest a general role for HSF in growth of eukaryotic cells under normal (nonstressed) growth conditions.

Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae

1987 ◽  
Vol 7 (5) ◽  
pp. 1906-1916
Author(s):  
M R Slater ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Promoter Region ◽  
Inducible Expression ◽  
Proximal Promoter ◽  
Heat Shock Gene ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hybrid Promoter ◽  
Shock Gene

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.

The expression of heat-shock genes in higher plants

1986 ◽  
Vol 314 (1166) ◽  
pp. 453-468 ◽  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Higher Plants ◽  
Structural Features ◽  
Amino Acid Sequences ◽  
Heat Shock Gene ◽  
Regulatory Sequences ◽  
Heat Shock Genes ◽  
Control Of Gene Expression ◽  
Shock Proteins

High-temperature stress or heat shock induces the vigorous synthesis of heat-shock proteins in many organisms including the higher plants. This response has been implicated in the acquisition of thermotolerance. The biological importance of a group of low-molecular-mass proteins in the response of plants is indicated by the conservation of the corresponding genes. The steady-state levels of mRNAs for these proteins shift from undetectable levels at normal temperature to about 20 000 molecules per gene in the cell after heat shock. The analysis of ‘run-off’ transcripts from isolated soybean nuclei suggests a transcriptional control of gene expression. The DNA sequence analysis of soybean heat-shock genes revealed a conservation of promoter sequences and 5'-upstream elements. A comparison of the deduced amino acid sequences of polypeptides showed a conservation of structural features in heat-shock proteins between plants and animals. The implication of a common regulatory concept in the heat-shock response makes genes belonging to this family (15-18 kDa proteins) in soybean favourable candidates for investigating thermoregulation of transcription. We have exploited the natural gene transfer system of Agrobacterium tumefaciens to introduce a soybean heat-shock gene into the genomes of sunflower and tobacco. The gene is thermoinducibly transcribed and transcripts are faithfully initiated in transgenic plants. Experiments are in progress to define the regulatory sequences 5'-upstream from the gene. The expression of heat-shock genes in a heterologous genetic background also provides the basis for studying the function of the proteins and their possible role in thermoprotection.

scholarly journals Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally.

1986 ◽  
Vol 6 (9) ◽  
pp. 3134-3143 ◽  
Author(s):  
R J Kay ◽  
R J Boissy ◽  
R H Russnak ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Gene Pair ◽  
Mammalian Cells ◽  
Heat Shock Gene ◽  
Promoter Strength ◽  
Wild Type ◽  
Kinase Gene ◽  
Mouse Fibroblasts ◽  
Bidirectional Transcription

A divergently transcribed pair of Caenorhabditis elegans hsp16 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. The hsp16 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. In a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hsp16 transcripts in each cell after 2 h of heat shock treatment. The hsp16 transcript levels were more than 100 times higher than those of a gene with a herpes simplex virus thymidine kinase gene promoter carried on the same vector. A single heat shock promoter element (HSE) could activate bidirectional transcription of the two hsp16 genes when placed between the two TATA elements, but the transcriptional efficiency was reduced 10-fold relative to that of the wild-type gene pair. Four overlapping HSEs positioned between the two TATA elements resulted in inducible bidirectional transcription at greater than wild-type levels. The number of HSEs can therefore be a major determinant of the promoter strength of heat-inducible genes in mammalian cells. Partial disruption of an alternating purine-pyrimidine sequence between the two hsp16 genes had no significant effect on their transcriptional activity.

scholarly journals Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (5) ◽  
pp. 1906-1916 ◽  
Author(s):  
M R Slater ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Promoter Region ◽  
Inducible Expression ◽  
Proximal Promoter ◽  
Heat Shock Gene ◽  
Heat Shock Element ◽  
Yeast Saccharomyces Cerevisiae ◽  
Hybrid Promoter ◽  
Shock Gene

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.

Hemin-induced transcriptional activation of the HSP70 gene during erythroid maturation in K562 cells is due to a heat shock factor-mediated stress response

1989 ◽  
Vol 9 (8) ◽  
pp. 3166-3173
Author(s):  
N G Theodorakis ◽  
D J Zand ◽  
P T Kotzbauer ◽  
G T Williams ◽  
R I Morimoto
Keyword(s):  
Stress Response ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
K562 Cells ◽  
Heat Shock Gene ◽  
Specific Gene ◽  
Hsp70 Gene ◽  
Heat Shock Element ◽  
Molecular Events ◽  
Erythroid Maturation

Hemin-induced differentiation of the human erythroleukemia cell line K562 results in the expression and accumulation of erythroid-specific gene products such as embryonic and fetal hemoglobins and the elevated synthesis of the major heat shock protein HSP70. This activity was suggested to represent activation of a heat shock gene during erythroid maturation independent of stress induction. In this study, we demonstrate that hemin induces the transcription of two members of the human HSP70 gene family, HSP70 and GRP78 (BiP). However, the induction of HSP70 by hemin showed characteristics consistent with the molecular events associated with a heat shock or stress response. The increase in HSP70 gene transcription was accompanied by induction of the stress-induced form of the heat shock transcription factor. Moreover, a heat shock element was required for the hemin responsiveness of chimeric heat shock promoter-chloramphenicol acetyltransferase genes transiently expressed in transfected K562 cells.

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