Small Heat Shock Genes Research Articles

Abundant, diverse, and consequentialPelements segregate in promoters of small heat‐shock genes inDrosophilapopulations

The present study extends evidence that Drosophila heat-shock genes are distinctively evolvable because of insertion of transposable elements by examining the genotypic diversity and phenotypic consequences of naturally occurring P element insertions in the proximal promoter regions of two small heat-shock genes. Detailed scrutiny of two populations revealed 16 distinctive P transposable elements collectively segregating in proximal promoters of two small heat-shock genes, Hsp26 and Hsp27. These elements vary in size, orientation and insertion site. Frequencies of P element-containing alleles varied from 5% to 100% in these populations. Two Hsp26 elements chosen for detailed study, R(s)P(26) and D(2)P(m), reduced or abolished Hsp26 expression respectively. The R(s)P(26) element increased or did not affect inducible tolerance of high temperature, increased fecundity, but decreased developmental rate. On the other hand, the D(2)P(m) element decreased thermotolerance and fecundity. In lines subjected to experimental evolution, the allelic frequency of the R(s)P(26)P element varied considerably, and was at lower frequencies in lines selected for increased longevity and for accelerated development than in controls. Transposable element insertions into small Hsp genes in Drosophila populations can have dramatic fitness consequences, and therefore create variation on which selection can act.

RNA thermometers are common in α- and γ-proteobacteria

Expression of many rhizobial small heat-shock genes is controlled by the ROSE element, a thermoresponsive structure in the 5'-untranslated region of the corresponding mRNAs. Using a bioinformatics approach, we found more than 20 new potential ROSE-like RNA thermometers upstream of small heat-shock genes in a wide variety of alpha- and gamma-proteobacteria. Northern blot analyses revealed heat-inducible transcripts of the representative candidate Caulobacter crescentus CC2258, Escherichia coli ibpA and Salmonella typhimurium ibpA genes. Typical sigma(32)-type promoters were mapped upstream of the potential RNA thermometers by primer extension. Additional translational control was demonstrated in a lacZ reporter system and by site-directed mutagenesis. RNA secondary structure predictions strongly suggest that the Shine-Dalgarno sequence in the RNA thermometers is masked at low temperatures. Combining two regulatory modules, a sigma(32) promoter and a ROSE-type RNA thermometer, provides a novel stringent mechanism to control expression of small heat-shock genes.

Protein Kinase A Regulates Constitutive Expression of Small Heat-Shock Genes in an Msn2/4p-Independent and Hsf1p-Dependent Manner in Saccharomyces cerevisiae

Hsf1p, the heat-shock transcription factor from Saccharomyces cerevisiae, has a low level of constitutive transcriptional activity and is kept in this state through negative regulation. In an effort to understand this negative regulation, we developed a novel genetic selection that detects altered expression from the HSP26 promoter. Using this reporter strain, we identified mutations and dosage compensators in the Ras/cAMP signaling pathway that decrease cAMP levels and increase expression from the HSP26 promoter. In yeast, low cAMP levels reduce the catalytic activity of the cAMP-dependent kinase PKA. Previous studies had proposed that the stress response transcription factors Msn2p/4p, but not Hsf1p, are repressed by PKA. However, we found that reduction or elimination of PKA activity strongly derepresses transcription of the small heat-shock genes HSP26 and HSP12, even in the absence of MSN2/4. In a strain deleted for MSN2/4 and the PKA catalytic subunits, expression of HSP12 and HSP26 depends on HSF1 expression. Our findings indicate that Hsf1p functions downstream of PKA and suggest that PKA might be involved in negative regulation of Hsf1p activity. These results represent a major change in our understanding of how PKA signaling influences the heat-shock response and heat-shock protein expression.

Cloning and characterization of the hsp 18.55 gene, a new member of the small heat shock gene family isolated from wine Lactobacillus plantarum

Using a molecular approach based on PCR, RT-PCR and northern blot analysis, a new member of the small heat shock family of wine, Lactobacillus plantarum, was cloned and characterized. The protein sequence deduced from the isolated gene had a calculated molecular mass of 18.548 kDa and was therefore named HSP 18.55. The gene codes for a protein homologous to the previously characterized HSP 19.3 and HSP 18.5 and is co-transcribed with an upstream gene of unknown function. Analysis of the 5′ flanking region of the hsp 18.55 gene revealed the presence of putative cis elements able to bind alternative sigma factor σ B. Based on its structure, the gene was classified as belonging to class II of the heat shock genes according to Bacillus subtilis nomenclature for shock-responsive genes. Expression of the newly identified small heat shock gene, analyzed by RT-PCR and northern blot analysis, was induced by a wide range of abiotic stresses including heat, cold and ethanol, suggesting that the small family of heat shock genes is probably involved in the general stress response in wine L. plantarum. Moreover, the expression of hsp 18.5, hsp 18.55 and hsp 19.3 genes, analyzed over a complete culture cycle, revealed that early growing cells contained substantial amounts of hsp 18.5, hsp 18.55 and hsp 19.3 mRNAs, which rapidly declined upon entry into stationary phase.

Cloning, molecular characterization and expression analysis of two small heat shock genes isolated from wine Lactobacillus plantarum.

Understanding the molecular response to stress tolerance of wine Lactobacillus plantarum. Two genes codifying for heat shock proteins were cloned from wine L. plantarum. The coding regions of the two heat shock genes are 420 and 444 nucleotides long, and started with an ATG codon suggesting that they were translated. The protein sequences deduced from the isolated genes have a molecular mass of 18.483 and 19.282 kDa, respectively, and were therefore named hsp18.5 and hsp19.3. The expression of small heat shock genes was analysed by RT-PCR analysis. Moreover, the 5' and 3' noncoding regions were cloned and sequenced. The expression of the heat shock genes was strongly induced by heat, cold and ethanol stress. Analysis of the 5' and 3' flanking regions of hsp18.5 and hsp19.3 genes, revealed the presence of an inverted repeat sequence (TTAGCACTC-N(9)-GAGTGCTAA) homologue to the CIRCE elements found to the upstream regulatory region of heat shock operons, and an inverted sequence that could form a stem and loop structure that it is likely to function as a transcriptional terminator. Based on their structures, the genes were classified as belonging to Class I of heat shock genes according to the B. subtilis nomenclature of heat response genes. Small heat shock genes isolated from wine L. plantarum might have a role in preventing damage by cold stress.

NELF and DSIF cause promoter proximal pausing on the hsp70 promoter in Drosophila.

NELF and DSIF collaborate to inhibit elongation by RNA polymerase IIa in extracts from human cells. A multifaceted approach was taken to investigate the potential role of these factors in promoter proximal pausing on the hsp70 gene in Drosophila. Immunodepletion of DSIF from a Drosophila nuclear extract reduced the level of polymerase that paused in the promoter proximal region of hsp70. Depletion of one NELF subunit in salivary glands using RNA interference also reduced the level of paused polymerase. In vivo protein-DNA cross-linking showed that NELF and DSIF associate with the promoter region before heat shock. Immunofluorescence analysis of polytene chromosomes corroborated the cross-linking result and showed that NELF, DSIF, and RNA polymerase IIa colocalize at the hsp70 genes, small heat shock genes, and many other chromosomal locations. Finally, following heat shock induction, DSIF and polymerase but not NELF were strongly recruited to chromosomal puffs harboring the hsp70 genes. We propose that NELF and DSIF cause polymerase to pause in the promoter proximal region of hsp70. The transcriptional activator, HSF, might cause NELF to dissociate from the elongation complex. DSIF continues to associate with the elongation complex and could serve a positive role in elongation.

High-osmolarity signalling in Saccharomyces cerevisiae is modulated in a carbon-source-dependent fashion.

High-osmolarity-induced expression of the small heat-shock gene HSP12 is regulated by the HOG (high-osmolarity glycerol) pathway and PKA (protein kinase A). To analyse the regulatory input of both signal transduction pathways, high-salt-induced HSP12 expression in different genetic backgrounds on glucose-, ethanol- and glycerol-based culture media was examined. Upon exposure to high-osmolarity stress, the kinetics of induction of HSP12 in cells growing on the non-fermentable carbon sources are strikingly different from those on glucose. Derepression of HSP12 gene expression under non-stress conditions was observed in cells growing on non-fermentable carbon sources. High-salt challenge resulted in a lower induction of the HSP12 mRNA levels in ethanol-grown cells as compared to glucose-grown cells, whereas in glycerol-grown cells hardly any high-salt induction of HSP12 mRNA levels could be detected. Analysis of signalling through the HOG pathway suggested that glycerol may influence the activity of this signalling route, possible via negative feedback. Furthermore, the cellular level of PKA activity was found to have a great impact on stress-responsive gene transcription. On the basis of the data obtained it was concluded that modulation of PKA activity plays a major role in the stress response. A glucose-dependent, PKA-regulated cellular component is postulated to affect high-osmolarity-induced HSP12 expression.

Putative evolutionary origin of plasmids carrying the genes involved in leucine biosynthesis in Buchnera aphidicola (endosymbiont of aphids).

An 8.5-kb plasmid encoding genes (leuABCD) involved in leucine biosynthesis and a small plasmid of 1.74 kb of yet unknown function were found in the intracellular symbiont, Buchnera aphidicola, of two divergent aphid species, Thelaxes suberi and Tetraneura caerulescens, respectively. The leuABCD-carrying plasmid (pBTs1) was amplified from total aphid DNA by inverse long PCR, using outwardly oriented oligonucleotide primers specific to leuA. The resulting 8.2-kb PCR fragment as well as the 1.74-kb plasmid (pBTc1) were cloned and sequenced. pBTs1 differed from a previously described B. aphidicola plasmid (pRPE) of the aphid Rhopalosiphum padi by the presence of a small heat shock gene (ibp) and in the order of the leuABCD and repA genes. Comparison of both leucine plasmids to the small plasmid pBTc1 revealed extensive similarity with respect to putative replication functions as well as in the presence of a highly conserved open reading frame that was found to be homologous to Escherichia coli YqhA and Haemophilus influenzae HI0507 and which may encode an integral membrane protein. The three B. aphidicola plasmids most likely evolved from a common ancestral replicon, which in turn may be distantly related to IncFII plasmids. Phylogenetic affiliations of the B. aphidicola strains of the two aphid species were assessed by sequencing of their 16S rRNA genes. Evaluation of the distribution of the leuABCD-encoding plasmids within a phylogenetic framework suggests independent origins for pBTs1 and pRPE from an ancestral replicon resembling pBTc1. The implications for symbiotic essential amino acid biosynthesis and provisioning are discussed.

The role of ecdysone in the induction and maintenance of hsp27 transcripts during larval and prepupal development of Drosophila

The Drosophila melanogaster small heat-shock gene hsp27 carries the canonic ecdysone response element (EcRE) at -537. This EcRE has been used extensively both in cultured cells and in vitro for studies of the ecdysone response. We have characterised the developmental expression of hsp27 in wild-type larval and prepupal salivary glands in parallel with that of the E74B and E74A primary ecdysone response transcripts, which are induced in the mid and late third larval instar by a minor and major increase in ecdysone titre respectively. The induction of hsp27 occurs between these two events in larvae and in parallel with that of E74A in prepupae. Transcript levels are severely reduced in ecd1 and dor (22) (deep orange) mutant larvae but are only moderately reduced in larvae mutant for the Broad-Complex allele br (t435) . By culturing salivary glands of different ages with low (1.8×10(-8) M) or high (1.8×10(-6) M) concentrations of hormone, we show that the response of an EcRE varies during development and that the timing of the response cannot be predicted solely from its apparent strength in cell line analyses.

The Bradyrhizobium japonicum rpoH1 gene encoding a sigma 32-like protein is part of a unique heat shock gene cluster together with groESL1 and three small heat shock genes.

The heat shock response of Bradyrhizobium japonicum is controlled by a complex network involving two known regulatory systems. While some heat shock genes are controlled by a highly conserved inverted-repeat structure (CIRCE), others depend on a sigma 32-type heat shock sigma factor. Using Western blot (immunoblot) analysis, we confirmed the presence of a sigma 32-like protein in B. japonicum and defined its induction pattern after heat shock. A B. japonicum rpoH-like gene (rpoH1) was cloned by complementation of an Escherichia coli strain lacking sigma 32. A knockout mutation in rpoH1 did not abolish sigma 32 production in B. japonicum, and the rpoH1 mutant showed the wild-type growth phenotype, suggesting the presence of multiple rpoH homologs in this bacterium. Further characterization of the rpoH1 gene region revealed that the rpoH1 gene is located in a heat shock gene cluster together with the previously characterized groESL1 operon and three genes encoding small heat shock proteins in the following arrangement: groES1, groEL1, hspA, rpoH1, hspB, and hspC. Three heat-inducible promoters are responsible for transcription of the six genes as three bicistronic operons. A sigma 32-dependent promoter has previously been described upstream of the groESL1 operon. Although the hspA-rpoH1 and hspBC operons were clearly heat inducible, they were preceded by sigma 70-like promoters. Interestingly, a stretch of about 100 bp between the transcription start site and the start codon of the first gene in each of these two operons was nearly identical, making it a candidate for a regulatory element potentially allowing heat shock induction of sigma 70-dependent promoters.

Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter

We have isolated two sunflower genes, Ha hsp18.6 G2 and Ha hsp17.7 G4, that encode small heat shock proteins (sHSPs). RNAse A protection experiments, carried out with RNA probes transcribed from each gene and hybridized to sunflower total RNA, allowed us to distinguish their mRNA accumulation patterns. In sunflower, Ha hsp17.7 G4 mRNAs accumulated during zygotic embryogenesis at 25 degrees C. In vegetative tissues, these mRNAs accumulated in response to either heat shock (42 degrees C), abscisic acid (ABA), or mild water stress treatments. In all cases, the mRNAs were transcribed from the same initiation site. In contrast, Ha hsp 18.6 G2 mRNAs accumulated only in response to heat-shock. This result demonstrates differential regulation of these two sHSP genes. The complex regulation depicted by the Ha hsp 17.7 G4 promoter has been further analyzed in transgenic tobacco, using G4::GUS translational fusions. Developmental induction of Ha hsp 17.7 G4 during zygotic embryogenesis was faithfully reproduced in the transgenic plants. 5'-distal sequences (between -1132 and -395) were required to confer a preferential spatial expression of GUS activity in the cotyledons. More proximal sequences (from -83 to +163) conferred to the chimeric genes most of the developmental regulation, and the responses to ABA and heat shock characteristic of the Ha hsp17.7 G4 promoter. The water stress response of this gene was not reproduced in transgenic tobacco and, thus, could be uncoupled from its regulation during embryogenesis.

Short Transcripts of the Ternary Complex Provide Insight into RNA Polymerase II Elongational Pausing

Expression of thehsp70 gene ofDrosophila melanogasteris controlled at the level of transcript elongation. In the uninduced state,hsp70 possesses an RNA polymerase II complex, elongationally engaged, but paused early in the transcription unit. In this study, we have used a powerful new selection-amplification technique to analyze the RNA transcripts associated with such “paused polymerases” under non-heat shock and heat shock-induced conditions. They reveal a region of pausing on the uninduced genein vivospanning from +21 to +35. This region is interrupted by an area of low polymerase density, centered at about +26. Upon induction, an accumulation of short transcripts, similar in size to those associated with the paused complex, was seen. Models for polymerase pausing and release are discussed in light of these data. The increased sensitivity of our new technique also allowed us to investigate ternary complexes associated with genes with much lower levels of engaged RNA polymerase. These included two of the small heat shock genes (hsp26 andhsp27) and two metabolic genes (Gapdh-1andGapdh-2), where paused polymerase were thought to be present, plus two other genes (Mtn andyp1), where polymerases pausing has not been detected in the past. In both of the small heat shock genes, we found evidence of previously unknown sites of transcriptional termination, residing immediately upstream of the regions of polymerase pausing.

The expression of a small heat shock gene is activated during induction of tobacco pollen embryogenesis by starvation*

ABSTRACTA tobacco (Nicotiana tabacum L.) cDNA clone, Nthsp 18P, encoding a class I low‐molecular‐weight heat shock protein (LMW HSP), has been isolated from a mid‐biccllular pollen cDNA library by hcterologous hybridization with a low‐molecular‐weight heat shock cDNA clone of alfalfa. Northern analysis showed that Nthsp 18P expression is activated at normal temperature during the dehydration phase of in situ pollen development, just before anthesis. Induction of pollen embryogenesis by an in vitro starvation treatment of mid‐bicellular tobacco pollen was accompanied by a dramatic increase in the levels of the Nthsp 18P transcript, which accumulated in embryogenic pollen grains at 25°C; lower levels of this transcript were also detected in early pollen‐derived embryos. A similar pattern of expression was observed in transgcnic tobacco plants containing the promoter of Gmhsp 17.3‐B, a soybean homologue of the Nthsp 18P gene, fused to the GUS reporter gene. This is the first characterized gene shown to be transcriptionally activated during the induction of pollen embryogenesis.

An Evaluation of the Usefulness of the Small Heat Shock Genes for Phylogenetic Analysis in Plants

An Evaluation of the Usefulness of the Small Heat Shock Genes for Phylogenetic Analysis in Plants

Multiple interacting elements delineate an ecdysone-dependent regulatory region with secondary responsive character

Within the 2.2 kb region between hsp23 and gene 1 of the small heat shock gene locus 67B1 of Drosophila melanogaster, an approximately 1 kb perturbation of the chromatin architecture has previously been observed to occur in response to the steroid hormone ecdysone. Transient expression assays in hormonally-responsive Drosophila tissue culture cells utilizing hsp70-lacZ chimeric reporter constructs revealed the presence of ecdysone-dependent regulatory sequences in this hsp23-gene 1 intergenic region. The analysis delimited five functional segments: three core regions which were completely encompassed within the region of chromatin perturbation, and two gene-proximal regions which appear to be functionally equivalent under some circumstances. None of the delineated regions was capable of stimulating expression independently, while sub-maximal expression was obtained when combinations of two or three regions were monitored. This requirement for multiple DNA segments to drive maximal transcription suggested that cooperative interactions between the regions were essential for full hormonal responsiveness. Unexpectedly, no binding of the ecdysone receptor was detectable within any of the delineated regions, implying the involvement of multiple non-receptor factors in the observed hormonal responsiveness. The ecdysone-dependent activation of reporter constructs driven by these sequences showed a significant time lag and was coupled with a marked sensitivity to low concentrations of cycloheximide. The data obtained strongly suggest that the cis-acting elements delimited within the hsp23-gene 1 intergenic region respond to ecdysone in a secondary manner, presumably by requiring interaction with the product(s) of primary ecdysone-responsive genes.

In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.

The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp70 gene of Drosophila melanogaster, for example, an RNA polymerase II complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk of capping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.

Rapid changes in Drosophila transcription after an instantaneous heat shock.

Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes provides a framework for evaluating the mechanisms of activation and repression of transcription. Here, using two methods, we examined the changes in RNA polymerase II association on a set of Drosophila genes at 30-s intervals following an instantaneous heat shock. In the first method, Drosophila Schneider line 2 cells were quickly frozen to halt transcription, and polymerase distribution was analyzed by a nuclear run-on assay. RNA polymerase transcription at the 5' end of the hsp70 gene could be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase could already be detected near the 3' end of the gene. A similar rapid induction was found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene was found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. Similar results were obtained by an in vivo UV cross-linking assay. In this second method, cell samples removed at 30-s intervals were irradiated with 40-microseconds bursts of UV light from a Xenon flash lamp, and the distribution of polymerase was examined by precipitating UV cross-linked protein-DNA complexes with an antibody to RNA polymerase II. Both approaches also showed the in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene to be approximately 1.2 kb/min.

Rapid Changes in Drosophila Transcription After an Instantaneous Heat Shock

Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes provides a framework for evaluating the mechanisms of activation and repression of transcription. Here, using two methods, we examined the changes in RNA polymerase II association on a set of Drosophila genes at 30-s intervals following an instantaneous heat shock. In the first method, Drosophila Schneider line 2 cells were quickly frozen to halt transcription, and polymerase distribution was analyzed by a nuclear run-on assay. RNA polymerase transcription at the 5' end of the hsp70 gene could be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase could already be detected near the 3' end of the gene. A similar rapid induction was found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene was found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. Similar results were obtained by an in vivo UV cross-linking assay. In this second method, cell samples removed at 30-s intervals were irradiated with 40-microseconds bursts of UV light from a Xenon flash lamp, and the distribution of polymerase was examined by precipitating UV cross-linked protein-DNA complexes with an antibody to RNA polymerase II. Both approaches also showed the in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene to be approximately 1.2 kb/min.

Comparison of regulatory and structural regions of the Xenopus laevis small heat-shock protein-encoding gene family

We have isolated several unique Xenopus laevis hsp30 (encoding heat-shock protein 30) genomic clones, one of which contains two complete hsp30 genes ( hsp30C and hsp30D), as well as the promoter and N-terminal coding region of a third gene ( hsp30E). Nucleotide sequence and restriction enzyme analysis revealed that this gene cluster is different from a cluster isolated previously. The hsp30C and hsp30D genes encode proteins of approx. 24 kDa. In all, the hsp30 gene family contains a minimum of seven genes. The strand exchange and breakage of the duplication events which generated this gene family appear to have occurred within tracts of DNA which potentially can assume a Z-DNA conformation. Comparing the amino acid (aa) sequences of each known Hsp30 protein with bovine α-crystallin revealed a high degree of shared conservation of aa that constitute the major structural feature(s) of α-crystallin.

Response to heat shock of gene 1, a Drosophila melanogaster small heat shock gene, is developmentally regulated

The expression of gene 1, a member of the small heat shock gene family from the Drosophila melanogaster chromosomal locus 67B was studied. In contrast to the other heat shock genes, the response of gene 1 to stress was modulated during development. In the absence of stress, gene 1 was expressed at the beginning of pupation, and at a very low level in adult males. Expression of gene 1 was substantially increased by heat shock in pupae, but was one to two orders of magnitude lower in adults or in embryos. Under the same conditions, hsp70 or hsp26 were induced to similar levels in all stages. This developmental effect could be mimicked in cultured Drosophila cells: expression of gene 1 was stimulated by heat shock in the presence, but not in the absence, of the moulting hormone ecdysterone, while the level of expression of hsp26 and hsp70 in response to heat shock was independent of the presence of the hormone. Thus, the presence and activity of the heat shock transcription factor are not sufficient for the maximal response of gene 1 to stress. These results suggest that the heat shock activator protein requires additional factors, which are developmentally regulated, to activate transcription of gene 1. Furthermore, S1 nuclease mapping analysis revealed several gene 1 mRNA species, which are generated by the use of alternative polyadenylation sites and by the use of differentially regulated transcriptional initiation sites.