Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

1987 ◽  
Vol 65 (2) ◽  
pp. 87-94 ◽  
Author(s):  
J. J. Heikkila ◽  
N. Ovsenek ◽  
P. Krone
Keyword(s):  
Heat Shock ◽  
Xenopus Laevis ◽  
Heat Shock Protein ◽  
Incubation Temperature ◽  
Effect Of Temperature ◽  
Continuous Exposure ◽  
Hsp 70 ◽  
Mrna Accumulation ◽  
Mrna Induction ◽  
Xenopus Laevis Embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33–35 °C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27–37 °C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 °C, with relatively greater levels at 30–35 °C and lower levels at 37 °C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27–35 °C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33–35 °C induced maximum relative levels of hsp 70 mRNA within 1–1.5 h, while at 30 and 27 °C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively. Finally, placement of Xenopus neurulae at 22 °C after a 1-h heat shock at 33 °C produced an initial decrease in hsp 70 mRNA within 15–30 min, followed by a transient increase in hsp 70 mRNA at 1–2 h before decaying to background levels by 7 h.

Heat shock gene expression in Xenopus laevis A6 cells in response to heat shock and sodium arsenite treatments

1988 ◽  
Vol 66 (8) ◽  
pp. 862-870 ◽  
Author(s):  
S. Darasch ◽  
D. D. Mosser ◽  
N. C. Bols ◽  
J. J. Heikkila
Keyword(s):  
Heat Shock ◽  
Xenopus Laevis ◽  
Sodium Arsenite ◽  
Polyacrylamide Gel Electrophoresis ◽  
Heat Shock Gene ◽  
Epithelial Cell Line ◽  
Continuous Exposure ◽  
Mrna Levels ◽  
Hsp 70 ◽  
Mrna Accumulation

Continuous exposure of a Xenopus laevis kidney epithelial cell line, A6, to either heat shock (33 °C) or sodium arsenite (50 μM) resulted in transient but markedly different temporal patterns of heat-shock protein (HSP) synthesis and HSP 70 and 30 mRNA accumulation. Heat-shock-induced synthesis of HSPs was detectable within 1 h and reached maximum levels by 2–3 h. While sodium arsenite induced the synthesis of some HSPs within 1 h, maximal HSP synthesis did not occur until 12 h. The pattern of HSP 70 and 30 mRNA accumulation was similar to the response observed at the protein level. During recovery from heat shock, a coordinate decline in HSPs and HSP 70 and 30 mRNA was observed. During recovery from sodium arsenite, a similar phenomenon occurred during the initial stages. However, after 6 h of recovery, HSP 70 mRNA levels persisted in contrast to the declining HSP 30 mRNA levels. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of 5 HSPs in the HSP 70 family, of which two were constitutive, and 16 different stress-inducible proteins in the HSP 30 family. In conclusion, heat shock and sodium arsenite induce a similar set of HSPs but maximum synthesis of the HSP is temporally separated by 12–24 h.

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts

1986 ◽  
Vol 6 (4) ◽  
pp. 1088-1094
Author(s):  
R B Widelitz ◽  
B E Magun ◽  
E W Gerner
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mrna Levels ◽  
Hsp 70 ◽  
Mrna Accumulation ◽  
General Protein Synthesis ◽  
Rat Fibroblasts ◽  
General Protein ◽  
Rat Embryonic Fibroblasts

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.

Enhanced accumulation of constitutive heat shock protein mRNA is an initial response of eye tissue to mild hyperthermia in vivo in adult Xenopus laevis

2002 ◽  
Vol 80 (11) ◽  
pp. 1119-1123 ◽  
Author(s):  
Adnan Ali ◽  
John J Heikkila
Keyword(s):  
Heat Shock ◽  
Xenopus Laevis ◽  
Heat Shock Protein ◽  
Specific Interaction ◽  
Initial Response ◽  
Heat Shock Element ◽  
Mrna Accumulation ◽  
Mild Hyperthermia ◽  
Hsp Genes

We have examined the effect of mild hyperthermia in vivo on heat shock transcription factor (HSF) binding activity and heat shock protein (hsp) gene expression in eye tissue of adult Xenopus laevis. A specific interaction between HSF and a synthetic oligonucleotide corresponding to the proximal heat shock element of the Xenopus hsp70B gene was greatly enhanced in eyes from hyperthermic animals compared with controls. Given these results, we examined the effect of hyperthermia in vivo on the expression of five hsp genes (hsp70, hsc70, BiP, hsp90, and hsp30) in eye tissue. Interestingly, at 28°C constitutively expressed hsp genes hsc70, BiP, and hsp90 were strongly enhanced, with further accumulation at 30°C. However, hsp70 and hsp30 mRNA accumulation were not detectable at 28°C but were strongly induced at 30°C. No enhancement of the relative levels of cytoskeletal actin mRNA was observed in the eye tissue of hyperthermic animals. These results suggest that one of the primary responses of eye tissue to hyperthermia in vivo is in the elevation of mRNAs encoding a set of constitutively expressed molecular chaperones.Key words: Xenopus, mRNA, eye, heat shock, heat shock factor.

scholarly journals Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

1986 ◽  
Vol 6 (4) ◽  
pp. 1088-1094 ◽  
Author(s):  
R B Widelitz ◽  
B E Magun ◽  
E W Gerner
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mrna Levels ◽  
Hsp 70 ◽  
Mrna Accumulation ◽  
General Protein Synthesis ◽  
Rat Fibroblasts ◽  
General Protein ◽  
Rat Embryonic Fibroblasts

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.

Analysis of the temperature-dependent temporal pattern of heat-shock-protein synthesis in fish cells

1983 ◽  
Vol 3 (7) ◽  
pp. 647-658 ◽  
Author(s):  
Lashitew Gedamu ◽  
Beverly Culham ◽  
John J. Heikkila
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Temporal Pattern ◽  
Stress Proteins ◽  
Incubation Temperature ◽  
Stress Protein ◽  
In Vitro Translation ◽  
Continuous Exposure ◽  
Temperature Dependent

Continuous exposure of Chinook salmon embryo cells to an elevated incubation temperature of 24°C induces the transient expression of a set of heat-shock or stress proteins whereas maintenance of the cells at a higher incubation temperature of 28°C produces a continuous synthesis of these stress proteins. In vitro translation studies suggest that the temperature-dependent temporal pattern of stress-protein synthesis is correlated with the levels of stress-protein mRNA. This was verified using a recombinant-DNA probe complementary to the 70K heat-shock-protein mRNA. A transient increase in the level of the fish heat-shock 70K mRNA was observed in RNA samples isolated from cells continuously exposed at 24°C However, a constant increase in the level of this specific mRNA was found in RNA preparations obtained from cells maintained at 28°C Therefore, the temperature-dependent pattern of fish heat-shockprotein synthesis appears to be directly related to the level of heat-shock-protein mRNA.

Analysis of hsp 30, hsp 70 and ubiquitin gene expression in Xenopus laevis tadpoles

Development ◽  
1988 ◽  
Vol 103 (1) ◽  
pp. 59-67
Author(s):  
P.H. Krone ◽  
J.J. Heikkila
Keyword(s):  
Heat Shock ◽  
Xenopus Laevis ◽  
Temporal Pattern ◽  
Size Range ◽  
Continuous Exposure ◽  
Hsp 70 ◽  
Blastula Stage ◽  
Tailbud Stage ◽  
Actin Mrna ◽  
Continuous Heat

Heat-induced accumulation of hsp 30 mRNA (1.1 kb) during early development of Xenopus laevis was first detectable at the tailbud stage (stage 30–34). This contrasts with heat-induced accumulation of hsp 70 mRNA (2.7 kb) and ubiquitin mRNA (size range = 1.7–3.1 kb), which was first detectable at the mid- to late-blastula stage. Continuous exposure of tadpoles to a 33 degrees C heat shock resulted in a coordinate, transient accumulation of hsp 30, hsp 70 and ubiquitin mRNA. A coordinate, temporal pattern was also observed for the decay of hsp 30, hsp 70 and ubiquitin mRNA in tadpoles recovering at 22 degrees C following a 1 h heat shock at 33 degrees C. Thus, while hsp 30 genes are regulated differently during development compared with hsp 70 and ubiquitin genes, these genes all exhibit a coordinate heat-inducible pattern of expression at the tadpole stage. Levels of alpha-cardiac actin mRNA remained unchanged during continuous heat shock and recovery experiments.

scholarly journals A role for glyceraldehyde-3-phosphate dehydrogenase in the development of thermotolerance in Xenopus laevis embryos.

1988 ◽  
Vol 107 (5) ◽  
pp. 1901-1909 ◽  
Author(s):  
R W Nickells ◽  
L W Browder
Keyword(s):  
Enzyme Activity ◽  
Heat Shock ◽  
Xenopus Laevis ◽  
Heat Shock Protein ◽  
Specific Activity ◽  
Glycolytic Enzyme ◽  
Polyacrylamide Gels ◽  
Different Temperatures ◽  
Peptide Maps ◽  
Xenopus Laevis Embryos

During heat shock, Xenopus laevis embryos exhibit an increase in the rate of accumulation of lactate and a loss of ATP relative to non-heat-shocked control embryos. These results suggest that heat shock stimulates a shift in energy metabolism to anaerobic glycolysis while at the same time causing an increase in the demand for ATP. We have evidence indicating that the embryo may meet such demands placed on it by increasing the levels of some glycolytic enzymes. In this report, we show that heat shock stimulates increases in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase [( EC 1.2.1.12] GAPDH). The specific activity of GAPDH shows a significant increase after heat shock, which correlates with the accumulation of GAPDH in heat-shocked embryos as detected by immunoblotting. Increases in GAPDH-specific activity are variable, however, and are inversely proportional to the levels of specific activity in control embryos; i.e., constitutive enzyme activity. We further analyzed the heat-enhanced accumulation of GAPDH by electrophoretically separating GAPDH isozymes on nondenaturing polyacrylamide gels. Control embryos exhibit a single isozyme of GAPDH, whereas heat-shocked embryos exhibit two isozymes of GAPDH. When these isozymes are labeled with [35S]methionine, separated by nondenaturing gel electrophoresis, and analyzed by fluorography, a heat-shock protein is found to comigrate with the isozyme unique to the heat-shocked sample. Enzyme activity assays at different temperatures suggest that this isozyme has optimum enzymatic activity only at heat-shock temperatures. We have correlated a 35-kD heat-shock protein (hsp35) with GAPDH using the following evidence: this hsp comigrates with GAPDH on one-dimensional SDS polyacrylamide gels; heat-enhanced increases in GAPDH specific activity correlate with hsp35 synthesis; and hsp35 and GAPDH have similar peptide maps. This relationship also provides a compelling explanation for the restriction of hsp35 synthesis to the vegetal hemisphere cells of heat-shocked early gastrulae reported previously (Nickells, R. W., and L. W. Browder. 1985. Dev. Biol. 112:391-395).

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