Heat-shock gene expression in animal embryonic systems

1986 ◽  
Vol 28 (6) ◽  
pp. 1093-1105 ◽  
Author(s):  
John J. Heikkila ◽  
L. W. Browder ◽  
L. Gedamu ◽  
R. W. Nickells ◽  
G. A. Schultz
Keyword(s):  
Heat Shock ◽  
Incubation Temperature ◽  
Embryo Cell ◽  
In Vitro Translation ◽  
Heat Shock Gene ◽  
Northern Hybridization ◽  
Transcriptional Level ◽  
Mrna Accumulation ◽  
Stress Genes

We have examined the expression of heat shock or stress genes in fish, echinoderm, amphibian, and mammalian embryonic systems. In a Chinook salmon embryo cell line, elevation of the incubation temperature or exposure to metal ions (e.g., cadmium and zinc) induced a set of heat-shock proteins HSPs. Transcriptional inhibitor, in vitro translation, and Northern hybridization studies suggest that fish HSP synthesis is regulated at the transcriptional level. The synthesis of HSPs during early development of Arbacia punctulata, Xenopus laevis, mouse, and rabbit is a stage-dependent phenomenon. In each of the developmental systems, HSP synthesis could not be induced until after cleavage stages. The ability of the embryo to undergo a heat-shock response (i.e., HSP synthesis) was correlated with the ability to detect HSP mRNA accumulation by either in vitro translation or Northern hybridization assays. Thus, the stage-dependent synthesis of HSPs appears to be controlled at the transcriptional level. Finally, in all of the organisms studied, the capacity to synthesize HSPs and accumulate HSP mRNA also coincides with acquisition of thermotolerance.Key words: heat shock, transcription, mRNA, actin, thermotolerance.

scholarly journals Sequence and expression of the mRNA encoding HSP22, the mitochondrial small heat-shock protein in pea leaves

1995 ◽  
Vol 311 (3) ◽  
pp. 805-813 ◽  
Author(s):  
C Lenne ◽  
M A Block ◽  
J Garin ◽  
R Douce
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Molecular Mass ◽  
Transit Peptide ◽  
Small Heat Shock Protein ◽  
Apparent Molecular Mass ◽  
In Vitro Translation ◽  
Transcriptional Level ◽  
Small Hsps

A 3 h treatment at 40 degrees C of pea (Pisum sativum var. Douce Provence) plants induces production and accumulation of a small heat-shock protein of 22 kDa apparent molecular mass, designated HSP22, in the matrix compartment of mitochondria [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261]. We show here that the HSP22 precursor (i.e. the mature protein plus the transit peptide) has an apparent molecular mass of 26 kDa after in vitro translation of mRNA extracted from heat-stressed pea plants and immunodetection. We have isolated, cloned and sequenced the full-length cDNA encoding the precursor of the mitochondrial HSP22. An analysis of the amino acid sequence of the mitochondrial HSP22 reveals that this protein is a representative member of the low-molecular-mass heat shock protein (HSP) superfamily, exhibiting the specific consensus regions that are typical of the small HSPs. Most importantly, comparison of the mitochondrial HSP22 sequence with that of chloroplast small HSPs indicates that HSP22 does not contain the typical chloroplast consensus region III. We have also analysed the kinetics of HSP22 induction, and report results on the temporal expression of HSP22 at the transcriptional level. HSP22 mRNA was detected as soon as 10 min after the temperature was raised to a high temperature of 40 degrees C. Then the amount of HSP22 mRNA declined considerably even though pea plants were still submitted to the heat treatment. These results are discussed in light of the translation data previously published [Lenne and Douce (1994) Plant Physiol. 105, 1255-1261], particularly concerning the physiological behaviour of mitochondria when plants are heat-stressed. Furthermore, we have studied the dependence of HSP22 accumulation with temperature and demonstrate that the pea mitochondrial heat-shock response is only developed under extreme environmental growth conditions.

Heat and sodium arsenite act synergistically on the induction of heat shock gene expression in Xenopus laevis A6 cells

1987 ◽  
Vol 65 (4) ◽  
pp. 310-316 ◽  
Author(s):  
J. J. Heikkila ◽  
S. P. Darasch ◽  
D. D. Mosser ◽  
N. C. Bols
Keyword(s):  
Heat Shock ◽  
Sodium Arsenite ◽  
Heat Shock Gene ◽  
Northern Hybridization ◽  
Epithelial Cell Line ◽  
Hsp 70 ◽  
Dot Blot ◽  
Mrna Accumulation ◽  
A6 Cells ◽  
Heat Shock Gene Expression

Heat shock protein (HSP) synthesis was studied in the Xenopus epithelial cell line A6 in response to heat and sodium arsenite, either singly or together. Temperatures of 33–35 °C consistently brought about the synthesis of HSPs at 87,73,70,54,31, and 30 kilodaltons (kDa), whereas sodium arsenite at 25–100 μM induced the synthesis of HSPs at 73 and 70 kDa. In cultures exposed to 10 μM sodium arsenite at 30 °C, HSP synthesis in the 68- to 73-kDa and 29- to 31-kDa regions was much greater than the HSP synthesis in response to each treatment individually. RNA dot blot analysis using homologous genomic subclones revealed that heat shock induced the accumulation of HSP 70 and 30 mRN As. The sizes of the HSP 70 and 30 mRN As determined by Northern hybridization were 2.7 and 1.5 kilobases, respectively. Sodium arsenite (10–100 μM) also induced the accumulation of both HSP 70 and 30 mRNAs. Finally, a mild heat shock (30 °C) plus a low concentration of sodium arsenite (10 μM) acted synergistically on HSP 70 and 30 mRN A accumulation in A6 cells. Thus sodium arsenite and heat act synergistically at the level of both HSP synthesis and HSP mRNA accumulation.

scholarly journals Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

2003 ◽  
Vol 23 (8) ◽  
pp. 2953-2968 ◽  
Author(s):  
Ville Hietakangas ◽  
Johanna K. Ahlskog ◽  
Annika M. Jakobsson ◽  
Maria Hellesuo ◽  
Niko M. Sahlberg ◽  
...  
Keyword(s):  
Heat Shock ◽  
Phosphorylation Site ◽  
Stress Granules ◽  
Heat Shock Factor ◽  
Transcriptional Level ◽  
Heat Shock Factor 1 ◽  
Protection Mechanism ◽  
Sumo Modification

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

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.

scholarly journals Stimuli that induce a yeast heat shock gene fused to beta-galactosidase.

1984 ◽  
Vol 4 (12) ◽  
pp. 2573-2579 ◽  
Author(s):  
C Brazzell ◽  
T D Ingolia
Keyword(s):  
Heat Shock ◽  
Heat Shock Gene ◽  
Enzyme Assays ◽  
Lacz Gene ◽  
Directed Synthesis ◽  
Shock Gene ◽  
Chemical Stimuli ◽  
Physical And Chemical ◽  
Beta Galactosidase

Saccharomyces cerevisiae contain a multigene family related to the Drosophila heat shock gene hsp70. Two members of this family, YG100 and YG101, have been previously characterized (Ingolia et al., Mol. Cell. Biol. 2:1388-1398, 1982), and only YG100 was found to have elevated levels of transcription after heat shock. The yeast hsp70 genes contained on YG100 and YG101 were truncated and fused to the Escherichia coli lacZ gene contained on pMC1587 (Casadaban et al., Methods Enzymol. 100:283-308, 1983). The resulting plasmids directed synthesis of the beta-galactosidase gene as measured by in vitro enzyme assays and by colorimetric assays on plates. The expression level from the YG101 gene was constant under all the conditions tested, whereas expression driven by the YG100 gene could be induced over 50-fold. Other stimuli besides heat, including recovery from anoxia and high cell density, were found to strongly induce YG100 gene expression. Most physical and chemical stimuli tested, including UV irradiation, zymolyase treatment, and ethanol, did not stimulate expression of this heat shock gene.

scholarly journals Isolation of cDNA clones for genes exhibiting reduced expression after differentiation of murine teratocarcinoma stem cells.

1984 ◽  
Vol 4 (10) ◽  
pp. 2142-2150 ◽  
Author(s):  
R A Levine ◽  
G J LaRosa ◽  
L J Gudas
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Cyclic Amp ◽  
Cdna Library ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Transcriptional Level ◽  
Cdna Clones ◽  
Dibutyryl Cyclic Amp

In the absence of retinoic acid, PSA-G teratocarcinoma stem cells spontaneously differentiate at a moderate frequency into fibroblast-like cells. In the presence of retinoic acid and dibutyryl cyclic AMP, PSA-G stem cells differentiate into parietal endoderm cells. We prepared a cDNA library from undifferentiated PSA-G teratocarcinoma stem cells; this cDNA library was then screened for gene sequences which exhibit a reduction in expression during the differentiation of these stem cells. From ca. 1,000 clones screened, eight independent sequences were isolated. The level of expression of these cloned genes decreases by 3.0-fold to more than 10-fold after differentiation of PSA-G cells into fibroblast-like cells. After treatment of either PSA-G or F9 teratocarcinoma cells with retinoic acid and dibutyryl cyclic AMP for 72 h, the expression of seven genes is inhibited by two- to fourfold. This decrease of clone-specific transcripts can be detected within 12 h after the addition of retinoic acid. Hybridization-selection and in vitro translation experiments identified the proteins encoded by three of the cloned genes: pST 6-23 codes for a 89,000-dalton protein, pST 7-105 codes for a 41,000-dalton protein, and pST 9-31 codes for a 34,000-dalton protein. The 89,000-dalton protein encoded by pST 6-23 is a heat shock protein. In vitro transcription experiments demonstrate that the retinoic acid-mediated decrease in pST 6-135- and pST 1-68-specific RNA occurs at the transcriptional level and that dibutyryl cyclic AMP acts posttranscriptionally to further depress the levels of these RNAs.

A family of related proteins is encoded by the major Drosophila heat shock gene family

1982 ◽  
Vol 2 (3) ◽  
pp. 286-292
Author(s):  
S C Wadsworth
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Heat Shock Protein ◽  
Sodium Dodecyl ◽  
Heat Shock Gene ◽  
Partial Digestion ◽  
Shock Gene ◽  
Shock Proteins

At least four proteins of 70,000 to 75,000 molecular weight (70-75K) were synthesized from mRNA which hybridized with a cloned heat shock gene previously shown to be localized to the 87A and 87C heat shock puff sites. These in vitro-synthesized proteins were indistinguishable from in vivo-synthesized heat shock-induced proteins when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of the pattern of this group of proteins synthesized in vivo during a 5-min pulse or during continuous labeling indicates that the 72-75K proteins are probably not kinetic precursors to the major 70K heat shock protein. Partial digestion products generated with V8 protease indicated that the 70-75K heat shock proteins are closely related, but that there are clear differences between them. The partial digestion patterns obtained from heat shock proteins from the Kc cell line and from the Oregon R strain of Drosophila melanogaster are very similar. Genetic analysis of the patterns of 70-75K heat shock protein synthesis indicated that the genes encoding at least two of the three 72-75K heat shock proteins are located outside of the major 87A and 87C puff sites.

scholarly journals Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

1992 ◽  
Vol 12 (8) ◽  
pp. 3490-3498 ◽  
Author(s):  
N Hosokawa ◽  
K Hirayoshi ◽  
H Kudo ◽  
H Takechi ◽  
A Aoike ◽  
...  
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Heat Shock Factor ◽  
Mobility Shift ◽  
Human Colon Cancer ◽  
Heat Shock Element ◽  
Mrna Accumulation ◽  
Cell Extracts

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

scholarly journals Three light-inducible heat shock genes of Chlamydomonas reinhardtii.

1989 ◽  
Vol 9 (9) ◽  
pp. 3911-3918 ◽  
Author(s):  
E D von Gromoff ◽  
U Treier ◽  
C F Beck
Keyword(s):  
Thermal Stress ◽  
Elevated Temperature ◽  
Heat Shock ◽  
Chlamydomonas Reinhardtii ◽  
Heat Shock Gene ◽  
Mrna Levels ◽  
Heat Shock Genes ◽  
Drastic Increase ◽  
Shock Gene

Genomic clones representing three Chlamydomonas reinhardtii genes homologous to the Drosophila hsp70 heat shock gene were isolated. The mRNAs of genes hsp68, hsp70, and hsp80 could be translated in vitro into proteins of Mr 68,000, 70,000, and 80,000, respectively. Transcription of these genes increased dramatically upon heat shock, and the corresponding mRNAs rapidly accumulated, reaching a peak at around 30 min after a shift to the elevated temperature. Light also induced the accumulation of the mRNAs encoded by these heat shock genes. A shift of dark-grown cells to light resulted in a drastic increase in mRNA levels, which reached a maximum at around 1 h after the shift. Thus, in Chlamydomonas, expression of hsp70-homologous heat shock genes appears to be regulated by thermal stress and light.

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