Abstract
Most of the members of the mammalian heat-shock protein (HSP) gene family have been studied and isolated from human and mouse cells. Few studies have concentrated on the HSPs of rat, a commonly used experimental animal. We have isolated and characterized a novel inducible rat HSP70 gene using an HSP70 cDNA sequence obtained from an ischaemic rat heart cDNA library. The isolated rat HSP70 gene was found to be a functional gene, as indicated by RNAase-protection and Northern-blot analysis. The deduced amino acid sequence of the inducible rat HSP70 exhibits a high degree of similarity to previously isolated mammalian inducible HSP70 gene products. Expression of the inducible HSP70 gene in rat myogenic cells (H9c2) is markedly increased after relatively short periods of hypoxia as well as by heat shock. Two heat-shock elements (HSE) are present in the rat HSP70 promoter. Transient transfection of rat HSP70 promoter/chloramphenicol acetyltransferase constructs into H9c2 cells shows that the presence of either of the two HSEs is sufficient for heat-shock inducibility. In contrast, induction of the rat HSP70/chloramphenicol acetyltransferase constructs by hypoxia is only detectable when both HSEs are present. This leads us to conclude that the induction of HSP70 by hypoxia and heat shock occurs through the same regulatory HSEs but the activation of the inducible HSP70 gene by heat shock is several-fold higher than by hypoxia.