Hsp27 Gene Research Articles

Species‐specific RFLP pattern in the Heat Shock Protein26 gene (Hsp26): a single‐locus tool for species identification and experimental testing of habitat‐induced isolation in the New World Artemia species

The brine shrimp Artemia (Crustacea, Branchiopoda), a paradigmatic inhabitant of hypersaline lakes, has molecular features to survive under stressful conditions, such as the p26 heat shock protein. We report the RFLP fingerprinting pattern (four restriction enzymes) of a 217 bp fragment of exon2 of the Hsp26 gene in six Artemia franciscana and four Artemia persimilis populations, the most genetically divergent Artemia species co-occurring in latitudinal extremes of Chile. The species-specific RFLP pattern observed is a simple and cost-effective single-locus tool for species delimitation and experimental testing the habitat-induced isolation barrier between them.

SAGA and Rpd3 Chromatin Modification Complexes Dynamically Regulate Heat Shock Gene Structure and Expression

The chromatin structure of heat shock protein (HSP)-encoding genes undergoes dramatic alterations upon transcriptional induction, including, in extreme cases, domain-wide nucleosome disassembly. Here, we use a combination of gene knock-out, in situ mutagenesis, chromatin immunoprecipitation, and expression assays to investigate the role of histone modification complexes in regulating heat shock gene structure and expression in Saccharomyces cerevisiae. Two histone acetyltransferases, Gcn5 and Esa1, were found to stimulate HSP gene transcription. A detailed chromatin immunoprecipitation analysis of the Gcn5-containing SAGA complex (signified by Spt3) revealed its presence within the promoter of every heat shock factor 1-regulated gene examined. The occupancy of SAGA increased substantially upon heat shock, peaking at several HSP promoters within 30-45 s of temperature upshift. SAGA was also efficiently recruited to the coding regions of certain HSP genes (where its presence mirrored that of pol II), although not at others. Robust and rapid recruitment of repressive, Rpd3-containing histone deacetylase complexes was also seen and at all HSP genes examined. A detailed analysis of HSP82 revealed that both Rpd3(L) and Rpd3(S) complexes (signified by Sap30 and Rco1, respectively) were recruited to the gene promoter, yet only Rpd3(S) was recruited to its open reading frame. A consensus URS1 cis-element facilitated the recruitment of each Rpd3 complex to the HSP82 promoter, and this correlated with targeted deacetylation of promoter nucleosomes. Collectively, our observations reveal that SAGA and Rpd3 complexes are rapidly and synchronously recruited to heat shock factor 1-activated genes and suggest that their opposing activities modulate heat shock gene chromatin structure and fine-tune transcriptional output.

Proteasome inhibition induces hsp30 and hsp70 gene expression as well as the acquisition of thermotolerance in Xenopus laevis A6 cells

Previous studies have shown that inhibiting the activity of the proteasome leads to the accumulation of damaged or unfolded proteins within the cell. In this study, we report that proteasome inhibitors, lactacystin and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), induced the accumulation of ubiquitinated proteins as well as a dose- and time-dependent increase in the relative levels of heat shock protein (HSP)30 and HSP70 and their respective mRNAs in Xenopus laevis A6 kidney epithelial cells. In A6 cells recovering from MG132 exposure, HSP30 and HSP70 levels were still elevated after 24 h but decreased substantially after 48 h. The activation of heat shock factor 1 (HSF1) may be involved in MG132-induced hsp gene expression in A6 cells since KNK437, a HSF1 inhibitor, repressed the accumulation of HSP30 and HSP70. Exposing A6 cells to simultaneous MG132 and mild heat shock enhanced the accumulation of HSP30 and HSP70 to a much greater extent than with each stressor alone. Immunocytochemical studies determined that HSP30 was localized primarily in the cytoplasm of lactacystin- or MG132-treated cells. In some cells treated with higher concentrations of MG132 or lactacystin, we observed in the cortical cytoplasm (1) relatively large HSP30 staining structures, (2) colocalization of actin and HSP30, and (3) cytoplasmic areas that were devoid of HSP30. Lastly, MG132 treatment of A6 cells conferred a state of thermotolerance such that they were able to survive a subsequent thermal challenge.

Characterization of the shsp genes in Drosophila buzzatii and association between the frequency of Valine mutations in hsp23 and climatic variables along a longitudinal gradient in Australia

The small heat shock gene (shsp) cluster of Drosophila buzzatii was sequenced and the gene order and DNA sequence were compared with those of the shsps in Drosophila melanogaster. The D. buzzatii shsp cluster contains an inversion and a duplication of hsp26. A phylogenetic tree was constructed based on hsp26 genes from several Drosophila species of the Sophophora and Drosophila subgenera. The tree shows first a separation of the Sophophora and the Drosophila subgenera and then the Drosophila subgenus is divided into the Hawaiian Drosophila and the repleta/virilis groups. Only the latter contain a duplicated hsp26. Comparing the gene organisation of the shsp cluster shows that all the Drosophila subgenus species contain the inversion. Putative heat shock elements (HSE) were found in the promoters of all the shsp and putative regulator elements for tissue specific expression were found in the promoter of hsp23, hsp27 and one of the hsp26 genes. hsp23 was found to be polymorphic for four non-synonymous changes that all lead to exchange of a Valine. The duplicated hsp26 gene in D. buzzatii (phsp26) was polymorphic for two non-synonymous changes. The allele frequencies of these variants were determined in nine D. buzzatii populations covering most of its distribution in Australia using high-resolution melting curves. The allele frequencies of one of the hsp23 variants showed a significant linear regression with longitude and the pooled frequency of the four Valine changes of hsp23 in the nine populations showed a significant linear regression with longitude and with a composite measure of climatic variables.

Survival in Acute and Severe Low O2 Environment

Hypoxia whether present during physiologic states (e.g., embryogenesis) or during pathologic states (e.g., obstructive sleep apnea and sickle cell anemia), challenges the vertebrate or invertebrate organism. Clearly, hypoxia can lead to sublethal cell injury or death and consequently organ or systemic injury and failure, depending on severity. We discovered that the adult Drosophila melanogaster is tolerant to a low O(2) environment, withstanding approximately 3-4 hours of total O(2) deprivation or anoxia without showing any evidence of cell injury. This opened major avenues for us since the Drosophila has been used so effectively in so many relevant research areas. We investigated the changes in gene expression in D. melanogaster after severe (1% O(2)) intermittent or constant hypoxia treatment for 2.5 hours. Our microarray analysis has identified multiple gene families that are up- or downregulated in response to acute constant (CH) and intermittent hypoxia (IH). We observed that even for short-term the gene expression response to IH and CH varied not only in the number of genes but also type of gene families. Furthermore, by utilizing powerful Drosophila genetic tools we studied the role of single genes (up- or downregulated in arrays) in survival under either paradigm in adult flies. We observed significant increased adult survival (as compared to controls) of P-element lines for Hsp70 and Hsp23 genes during CH and Mdr49 and l (2)08717 genes during IH. This suggests that the increased transcript levels as observed in array data after either paradigm play an important role under severe hypoxia. Indeed, we found for example that over-expressing Hsp70 in vivo in specific fly organs (such as heart) significantly increased adult survival during CH as compared to controls. These data provide further clues about the mechanisms by which intermittent and constant hypoxia lead to cell injury and morbidity or adaptation and survival.

The histone demethylase Dmel\\Kdm4A controls genes required for life span and male-specific sex determination in Drosophila

Histone methylation plays an important role in regulating chromatin-mediated gene control and epigenetic-based memory systems that direct cell fate. Enzymes termed histone demethylases directly remove the methyl marks from histones, thus contributing to a dynamically regulated histone methylated genome; however, the biological functions of these newly identified enzymes remain unclear. The JMJD2A-D family belongs to the JmjC domain-containing family of histone demethylases (JHDMs). Here, we report the cloning and functional characterization of the Drosophila HDM gene Dmel\\Kdm4A that is a homolog of the human JMJD2 family. We show that homologs for three human JHDM families, JHDM1, JHDM2, and JMJD2, are present in Drosophila and that each is expressed during the Drosophila lifecycle. Disruption of Dmel\\Kdm4A results in a reduction of the male life span and a male-specific wing extension/twitching phenotype that occurs in response to other males and is reminiscent of an inter-male courtship phenotype involving the courtship song. Remarkably, certain genes associated with each of these phenotypes are significantly downregulated in response to Dmel\\Kdm4A loss, most notably the longevity associated Hsp22 gene and the male sex-determination fruitless gene. Our results have implications for the role of the epigenetic regulator Dmel\\Kdm4A in the control of genes involved in life span and male-specific sex determination in the fly.

イネ登熟種子由来リン酸化タンパク質 Hsp82 および NDPK1 の発現特性

イネ登熟種子由来リン酸化タンパク質 Hsp82 および NDPK1 の発現特性

Heat shock protein patterns in the bovine ovary and relation with cystic ovarian disease

The present study was performed to determine how the development of cystic ovarian disease (COD) affecting the ovarian expression of heat shock proteins (HSP) in cows were expressing extrous cycles. HSP27, HSP60, HSP70 and HSP90 were evaluated in different ovarian components by Western blot and semiquantitative immunohistochemical analysis. Greater expression of the HSP27 gene was detected in the granulosa and theca cells of primary, secondary, tertiary and cystic follicles, with decreasing amount in atretic follicles. HSP60, HSP70 and HSP90 showed a similar pattern of immunostaining, with moderate gene expression in primary and secondary follicles, increased expression in tertiary and atretic follicles with the greatest gene expression in cystic follicles. HSP were also localized in the corpus luteum, corpus albicans, interstitial tissue and tunica albuginea. The relative amount of protein in the follicular wall of small and large healthy follicles and cystic follicles as analysed by Western immunoblot was consistent with the immunohistochemical data. We speculate that altered expression of HSP genes decreases apoptosis in the follicular wall and leads to the delayed regression of cystic follicles. This study supports earlier observations suggesting that aberrant HSP gene expression, observed in cells of the cystic follicles, is probably associated with the intra-ovarian component of COD pathogenesis.

Heat Shock Protein 27 as a New Therapeutic Target for Radiation Sensitization of Head and Neck Squamous Cell Carcinoma

In a wide range of human cancers, increased levels of heat shock protein 27 (Hsp27) are closely associated with tumorigenesis, metastasis, resistance to anticancer therapeutics, and thus poor prognosis. In this study, we evaluate the radiosensitizing effects of Hsp27 gene silencing using OGX-427, a second-generation antisense oligonucleotide (ASO), on the radioresistant head and neck squamous cell carcinoma (HNSCC) SQ20B cells. In vitro, the downregulation of Hsp27 significantly enhanced radiation-induced apoptotic and clonogenic death, and promoted Akt inactivation. In vivo, combining OGX-427 with local tumor irradiation (5 x 2 Gy) led to a significant regression of SQ20B tumors related to a high rate of apoptosis and decreased levels of glutathione antioxidant defenses. Increasing the total radiation dose (15 x 2 Gy) significantly amplified the radiosensitizing effect of OGX-427. Treatment of tumors with OGX-427 plus radiation resulted in a decrease in angiogenesis associated with a reduced activation of the Akt pathway. Furthermore, the combined treatment enhanced the survival of SQ20B-bearing mice and showed no signs of acute and delayed toxicity. Our findings demonstrate for the first time that Hsp27 knockdown enhances the cytotoxic effects of radiotherapy in vivo and provide preclinical proof of principle for clinical trials using Hsp27 antisense technology in the treatment of patients with HNSCC radioresistant cancers.

PAMAM Dendrimers Mediate siRNA Delivery to Target Hsp27 and Produce Potent Antiproliferative Effects on Prostate Cancer Cells

RNA interference (RNAi) holds great promise for the treatment of inherited and acquired diseases, provided that safe and efficient delivery systems are available. Herein we report that structurally flexible triethanolamine (TEA) core PAMAM dendrimers are able to deliver an Hsp27 siRNA effectively into prostate cancer (PC-3) cells by forming stable nanoparticles with siRNA, protecting the siRNA nanoparticles from enzymatic degradation, and enhancing cellular uptake of siRNA. The Hsp27 siRNA resulted in potent and specific gene silencing of heat-shock protein 27, an attractive therapeutic target in castrate-resistant prostate cancer. Silencing of the hsp27 gene led to induction of caspase-3/7-dependent apoptosis and inhibition of PC-3 cell growth in vitro. In addition, the siRNA-dendrimer complexes are non-cytotoxic under the conditions used for siRNA delivery. Altogether, TEA core PAMAM dendrimer-mediated siRNA delivery, in combination with RNAi that specifically targets Hsp27, may constitute a promising approach for combating castrate-resistant prostate cancer, for which there is no efficacious treatment.

Down-regulation of HSP27 sensitizes TRAIL-resistant tumor cell to TRAIL-induced apoptosis

Tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL) has recently emerged as a cancer therapeutic agent because it preferentially induces apoptosis in human cancer over normal cells. Most tumor cells, including lung cancer cell line A549, unfortunately, are resistant to TRAIL treatment even at high dose. Recent studies indicated that TRAIL-resistant cancer cells could be sensitized to TRAIL by combination therapy. Stress and heat shock proteins such as HSP90, HSP70 and HSP27 are induced in response to a wide variety of physiological environmental insults including heat, reactive oxygen species or anticancer drugs. Their elevated expressions facilitate cells to survive in stress circumstances. The HSP27 expression is enhanced in many tumor cells, implying that it is involved in tumor progression and the development of treatment resistance in various tumors, including lung cancer. This fact suggests a novel strategy for the treatment of cancer via inhibiting the function of HSP27. In this study, we investigated the inhibitory effect of a small interfering (si) RNA on the expression of HSP27 gene in the TRAIL-resistant human lung adenocarcinoma cell line A549, and the effect of HSP27 siRNA on drug sensitization of A549 cells to TRAIL treatment. The results showed that treatment of A549 cells with HSP27 siRNA down-regulated HSP27 expression but did not induce significant apoptosis. However, combination of HSP27 siRNA with TRAIL-induced significant apoptosis in TRAIL-resistant A549 cells. In addition to inducing caspases activation and apoptosis, combined treatment with HSP27 siRNA and TRAIL also increased JNK and p53 expression and activity. Collectively, these findings provide a conclusion that siRNA targeting of the HSP27 gene specifically down-regulated HSP27 expression in A549 cells, and sensitized the cells to TRAIL-induced apoptosis.

Erratum

Insect Molecular BiologyVolume 18, Issue 3 p. 419-420 Free Access Erratum This article corrects the following: The hsp27 gene of the Mediterranean fruit fly, Ceratitis capitata: structural characterization, regulation and developmental expression G. Kokolakis, M. Tatari, A. Zacharopoulou, A. C. Mintzas, Volume 17Issue 6Insect Molecular Biology pages: 699-710 First Published online: November 6, 2008 First published: 15 May 2009 https://doi.org/10.1111/j.1365-2583.2009.00888.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Kokolakis et al., 2008, Insect Mol Biol. 17(6):699–710, errors were discovered. The following corrections have been made. Figure 2. The bars indicating the 5 HSEs, the arrow indicating transcription initiation and the arrows indicating the two primers (F and R) have been moved leftwards by 3 letters. b) The two lines corresponding to No 2731 and 2801 have been deleted because they were duplicated (see the two lines below). Table 1. The open box corresponding HSE-1 has been moved leftwards by 1 letter while the open box corresponding the HSE-4 has been moved rightwards by 1 letter. Table 1. Drosophila heat shock factor (DHSF) binding sites of Cchsp27 gene HSF binding sites Optimized matrix threshold Position Core similarity Matrix similarity Sequence* 1 0.77 2199–2223 1.000 0.796 2 0.77 1856–1880 0.810 0.780 3 0.77 1846–1870 1.000 0.823 4 0.77 1654–1678 0.891 0.790 * Capital letters indicate the core sequence. Open boxes indicate putative HSEs. The corrected figure and table can be found below. We apologize for this error. Reference Kokolakis, G., Tatari, M., Zacharopoulou, A. & Mintzas, A.C. (2008) The hsp27 gene of the Mediterranean fruit fly, Ceratitis capita: structural characterization, regulation and development expression. Insect Mol Biol 17(6): 699– 710. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume18, Issue3June 2009Pages 419-420 ReferencesRelatedInformation

Expression of hsp22 and hsp70 Transgenes Is Partially Predictive of Drosophila Survival Under Normal and Stress Conditions

Drosophila Hsp70 is a highly conserved molecular chaperone with numerous cytoplasmic targets. Hsp22 is an alpha-crystallin-related chaperone (small hsp) that localizes to the mitochondrial matrix. The hsp70 and hsp22 genes are induced in response to acute heat and oxidative stress and are also upregulated during normal aging. Here the hsp22 promoter (-314 to +10) and the hsp70 promoter (-194 to +10) were used to drive expression of the fluorescent reporter proteins green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRED) in transgenic flies. Multiple transgenic lines were analyzed under normal culture conditions and under oxidative stress and heat stress conditions that significantly shorten life span. Flies were individually housed, and GFP (or DsRED) was quantified at young-age time points using the fluorescence stereomicroscope and image analysis software. Expression of the hsp reporters in young flies was partially predictive of remaining life span: Young flies with high expression tended to die sooner under both control and stress conditions.

Simultaneous exposure of Xenopus A6 kidney epithelial cells to concurrent mild sodium arsenite and heat stress results in enhanced hsp30 and hsp70 gene expression and the acquisition of thermotolerance

In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1–10 µM sodium arsenite at a mild heat shock temperature of 30 °C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 µM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 °C with larger responses at 28 and 30 °C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 µM) and heat shock (30 °C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 °C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.

Hsp90 and p23 facilitate steroid hormone receptor signaling in yeast

Steroid hormone receptors (SHRs) are among the best‐characterized "client" proteins of Hsp90 complexes. However, effects of Hsp90 and its co‐chaperone on SHR signal transduction have not been examined in a common system. We used a yeast system engineered to express human androgen, estrogen αand ?, glucocorticoid, mineralocorticoid, or progesterone receptors along with relevant reporter genes and tested whether Hsp90 isoproteins and p23 affected SHR signaling. Deletion of the endogenous HSC82 (homolog of human Hsp90 ?) or SBA1 (homolog of human p23) genes in this system strongly inhibited ligand‐mediated signaling of all SHRs in reporter gene assays. HSC82 gene deletion had greater impact on SHR signaling than did SBA1 gene deletions. Deletion of the HSP82 gene (homolog of human Hsp90α) did not affect SHR signaling, suggesting that the Hsc82p‐Sba1p complex specifically supports SHR signaling in this system. Effects of a lack of Hsc82p or Sba1p were restricted to SHR signaling because both cell growth and signaling by an unrelated reporter construct were unaffected by chaperone status. Geldanamycin and radicicol (Hsp90 inhibitors) strongly reduced SHR signaling at doses that did not affect cell growth. Collectively these results indicate that all SHRs, including estrogen receptorβ, are dependent on the Hsp90 chaperone system for proper function. Funded by NCI 1R33CA101622‐01A2.

Two hsp23 genes in the Mediterranean fruit fly, Ceratitis capitata: structural characterization, heat shock regulation and developmental expression

In the present study, we characterized a 3320-bp genomic DNA fragment encoding two medfly (Ceratitis capitata) homologues of the Drosophila melanogaster heat shock protein 23 (hsp23) gene, named Cchsp23-alphaand -beta. The two medfly hsp23 genes are transcribed in opposite directions and encode two almost identical proteins. Furthermore, the two genes exhibit a very high degree of similarity in their 5' untranslated and proximal promoter regions. Phylogenetic analysis indicated that the CcHsp23 proteins are orthologous to Drosophila Hsp23 and Sarcophaga crassipalpis Hsp23. Structural analysis of the 5' flanking regions of the Cchsp23 genes revealed the presence of several putative heat shock elements. Both CcHsp23 genes are induced by heat in a similar manner. In addition to heat-induction, the Cchsp23 genes are expressed at several stages of normal development as well as in ovaries and testes. In general, the developmental expression patterns of the medfly genes are similar, suggesting that they are under similar regulatory mechanisms. However, the expression of the Cchsp23 genes differs significantly from the expression of the Drosophila hsp23 gene in certain embryonic and larval stages, suggesting differential regulation of the hsp23 genes in the two dipteran species. The expression of both Cchsp23 genes in adult flies is increased with age, especially in males.

Spt6 enhances the elongation rate of RNA polymerase II in vivo

Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.

Functional Rescue of Mutant Human Cystathionine β-Synthase by Manipulation of Hsp26 and Hsp70 Levels in Saccharomyces cerevisiae

Many human diseases are caused by missense substitutions that result in misfolded proteins that lack biological function. Here we express a mutant form of the human cystathionine beta-synthase protein, I278T, in Saccharomyces cerevisiae and show that it is possible to dramatically restore protein stability and enzymatic function by manipulation of the cellular chaperone environment. We demonstrate that Hsp70 and Hsp26 bind specifically to I278T but that these chaperones have opposite biological effects. Ethanol treatment induces Hsp70 and causes increased activity and steady-state levels of I278T. Deletion of the SSA2 gene, which encodes a cytoplasmic isoform of Hsp70, eliminates the ability of ethanol to restore function, indicating that Hsp70 plays a positive role in proper I278T folding. In contrast, deletion of HSP26 results in increased I278T protein and activity, whereas overexpression of Hsp26 results in reduced I278T protein. The Hsp26-I278T complex is degraded via a ubiquitin/proteosome-dependent mechanism. Based on these results we propose a novel model in which the ratio of Hsp70 and Hsp26 determines whether misfolded proteins will either be refolded or degraded.

Genetic manipulation of HSP26 and YHR087W stress genes may improve fermentative behaviour in wine yeasts under vinification conditions

Throughout wine production yeast cells are affected by a plethora of stress conditions that compromise their ability to carry out the whole process. In recent years important knowledge about the mechanisms involved in stress response in both laboratory and wine yeast strains has been obtained. Several studies have indicated that a correlation exists between stress resistance, expression of stress response genes and fermentative behaviour. In this work we introduce several genetic manipulations in two genes induced by several stress conditions: HSP26 (which encodes a heat shock protein) and YHR087W (encoding a protein of unknown function) in two different wine yeasts, ICV16 and ICV27. These manipulations include expression in multicopy and centromeric plasmids, and substitution of the promoter in one of the genomic copies of these genes for that of the SPI1 gene, encoding for a cell wall protein of unknown function, or the PGK1 gene, which encodes the phosphoglycerate kinase glycolytic enzyme. Our results indicate that some of these modifications result in strains with higher expression of these genes, better resistance to certain stress conditions, and even improved fermentative behaviour. The modifications of the YHR087W gene are particularly interesting, and suggest an important role of this gene in the vinification process.

Isolation and expression of heat shock protein 30 gene fromPenicillium marneffei

Penicillium marneffei is a dimorphic fungus that can cause disseminated mycosis, especially in AIDS patients. The role of heat shock proteins and stress response-related proteins in P. marneffei remains unknown. In this study, we isolated a cDNA encoding for heat shock protein 30 (Hsp30) of P. marneffei using an antibody screening method. The DNA sequence and deduced amino acid sequence analysis showed high homology to other fungal hsp30 genes. Expression of P. marneffei hsp30 in response to temperature increase was determined by Northern blot analysis. A high level of hsp30 transcript was detected in yeast cells grown at 37 degrees C, whereas a very low or undetectable transcript level was observed in mycelial cells at 25 degrees C. A recombinant Hsp30 protein was produced and tested preliminarily for its immunoreactivity with sera from P. marneffei-infected AIDS patients using Western blot analysis. The positive immunoblot result, with some serum samples, confirmed the antigenic property of the Hsp30. Collectively, the high response of hsp30 to temperature increase could indicate it may play a role in heat stress response and cell adaptation. This is the first report showing that this small heat shock protein could elicit the human immune response.