The Arabidopsis HSP18.2 promoter/GUS gene fusion in transgenic Arabidopsis plants: a powerful tool for the isolation of regulatory mutants of the heat-shock response

A detailed study of the expression of the promoter of the HSP18.2 gene from Arabidopsis fused to the bacterial gene for β-glucuronidase (GUS) in transgenic Arabidopsis plants is described. High levels of GUS activity were induced in all organs of transformants except for seeds during heat shock. The optimum temperature for expression of GUS in Arabidopsis was 35°C regardless of the plant growth temperature. Heat shock of 40°C did not induce any detectable levels of GUS activity. Pre-incubation at 35°C was found to have a protective effect on the induction of GUS activity at 40°C. GUS activity was also increased in response to a gradual increase in temperature. Histochemical analysis revealed that basal levels of GUS activity were induced in the vascular tissue of leaves and sepals, as well as at the tips of carpels, at the normal growth temperature. Heat treatment of a limited part of the plant tissue did not appear to cause systemic induction of GUS activity. To extend the analysis of the plant heat-shock response, we attempted to screen mutations in genes involved in the regulation of the induction of heat-shock protein (HSP) genes, using the GUS gene as a selection marker in transgenic Arabidopsis plants, and the results of this analysis are described.     

Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus

Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28°C. When the infected cells were shifted from 28 to 37°C for 90 min (heat shock conditions), the synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28°C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2) the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells.     

Molecular characterization and gene evolution of the heat shock protein 70 gene in snakehead fish with different tolerances to temperature

Temperature tolerances (including lethal limits) and associated rates of thermal acclimation of fish are critical information in predicting fish responses to global climate changes. In this study, a partial sequence of the heat shock protein 70 gene (HSP70) from the fish species Channa striatus was isolated and characterized. Evolutionary process that led to the diversity of HSP70 specific to vertebrates was also analysed. Results revealed that HSP70 is highly homologous in other fish families. The conservation of the HSP 70 gene among fish families could be driven by forces of natural selection due to climatic change. We exposed C. striatus to heat shock (32 °C) and cold shock (16 °C) respectively, in order to examine the differences of temperatures in influencing the expression patterns of HSP70. We revealed that expression of HSP70 was higher at 32 °C than at 16 °C in most of the organs. Specifically, occurrence of chaperone activity of HSP70 was found at low temperature. Therefore, this fish was postulated that to seems to be able to survive at lower temperature compared to higher temperature indicating there is force of natural selection acting towards this HSP 70 gene. This will demonstrate the effect of global warming towards the fish survivability.     

Effect of salicylic acid on the development of induced Thermotolerance and induction of heat shock protein synthesis in the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> cell culture

Salicylic acid (SA) could be involved in the development of tolerance to abiotic stresses, to heat shock in particular. Under normal conditions (26°C), treatment with SA improved the tolerance of heterotrophic Arabidopsis thaliana (L.) Heynh culture to severe heat shock (50°C). Under mild heat shock (37°C) inducing the development of thermotolerance, the presence of SA, in contrast, reduced the capability of arabidopsis cells to tolerate high temperature (50°C) and simultaneously suppressed induction of HSP synthesis (Hsp101 and Hsp17.6) important for the development of induced thermotolerance. Since SA suppressed cell respiration and activated the alternative pathway of electron transport, SA is supposed, by modulating mitochondria functions, to be an endogenous regulator of plant stress gene expression.     

Production of medium-chain-length hydroxyalkanoic acids from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in pH stat

The production of β-glucuronidase (GUS) driven by the Arabidopsis small heat shock protein 18.2 promoter in liquid cultures of transgenic tobacco (Nicotiana tabacum) hairy roots is reported. Clone GD-3, showing high GUS heat induction and a moderate growth rate, was selected from 436 clones for study. Treatment of GD-3 with heat shock at 36–42°C for 2 h then recovery at 27°C resulted in an increase in GUS specific activity, while higher heat-shock temperatures led to a decline. These results were in accordance with the change in esterase activity, a measure of tissue viability. Using 2 h of 42°C heat shock and a recovery phase at 27°C, GUS specific activity increased rapidly and reached a maximum of 267.6 nmol 4-methylumbelliferyl β-D-glucuronic acid (MU) min⁻¹ mg⁻¹ protein at 24 h of recovery. When tissues were continuously heated at 42°C and tested without a recovery period, GUS mRNA was detectable at 2 h and peaked at 5 h, but GUS activity was not seen until 10 h and did not peak until 28 h; in addition, the maximum activity was lower than that seen after heat shock for only 30 min or 2 h, followed by recovery. This shows that recovery at normal temperature is crucial for the heat-inducible heterogeneous expression system of transgenic hairy roots. Multiple heat-shock treatments showed that this system was heat reinducible, although a gradual decline in GUS specific activity was seen in the second and third cycles.     

Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes’ expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.     

Induction of gene expression in bacteria at optimal growth temperatures

Traditional temperature-sensitive systems use either heat shock (40–42 °C) or cold shock (15–23 °C) to induce gene expression at temperatures that are not the optimal temperature for host cell growth (37 °C). This impacts the overall productivity and yield by disturbing cell growth and cellular metabolism. Here, we have developed a new system which controls gene expression in Escherichia coli at more permissive temperatures. The temperature-sensitive cI857-P _L system and the classic lacI-P _lacO system were connected in series to control the gene of interest. When the culture temperature was lowered, the thermolabile cI857 repressor was activated and blocked the expression of lacI from P _L. Subsequently, the decrease of LacI derepressed the expression of gene of interest from P _lacO . Using a green fluorescent protein marker, we demonstrated that (1) gene expression was tightly regulated at 42 °C and strongly induced by lowering temperature to 25–37 °C; (2) different levels of gene expression can be induced by varying culture temperature; and (3) gene expression after induction was sustained until the end of the log phase. We then applied this system in the biosynthesis of acetoin and demonstrated that high yield and production could be achieved using temperature induction. The ability to express proteins at optimal growth temperatures without chemical inducers is advantageous for large-scale and industrial fermentations.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

Effects of rearing and induction temperature on the temporal dynamics of heat shock protein 70 expression in a butterfly

The temporal dynamics of heat shock protein 70 (HSP70) expression in response to longer‐term acclimation and rapid hardening in the butterfly Lycaena tityrus is investigated. After a 1‐h exposure to 1 °C or 37 °C, HSP70 is quickly up‐regulated within 1 h and down‐regulated within 2 h. The fast dynamic of HSP70 expression is in contrast to the patterns found in organisms inhabiting more stable thermal environments, and is interpreted as an adaptation to the large and rapid temperature variation experienced by flying ectotherms. HSP70 expression is higher in males than in females, as well as in animals reared at 27 °C than at 20 °C, although it is very similar across the high and low induction temperatures. Animals reared at the higher temperature, however, respond less strongly to high‐temperature stress.