Characterization of the heat shock proteins in cultured trout cells Mach II

• Main Author: Burgess, Elizabeth Anne
• Language: English
• Published: University of British Columbia 2010
• Online Access: http://hdl.handle.net/2429/25278

Despite considerable investigation, the function of the heat shock proteins (hsps) remains obscure. In this thesis, the heat shock proteins of Salmo gairdnerii RTG-2 cells were examined by two-dimensional gel electrophoresis, and the induction of the 70,000 dalton hsp (hsp70) was studied. As in Drosophila melanogaster each trout hsp detected by one dimensional analysis separated into several isoelectric variants following two-dimensional gel electrophoresis. Post-translational modification of a single polypeptide could have given rise to charge variants similar to those observed here, but no evidence for either the phosphorylation of the hsps or for changes in the phosphorylation of other proteins was obtained. Messenger RNA was isolated from heat shocked cells and translated in the in vitro rabbit reticulocyte system.. Most of the isoelectric variants were detected in the in vitro translation suggesting that either different transcripts encode the isoelectric variants or that post-translational modification is occurring in the in vitro system. Heat shock mRNA was isolated by hybridization to a cDNA clone of the 70K hsp and translated in vitro. Two-dimensional gel electrophoresis of the translation products revealed that all 70K isoelectric variants were present. The induction of hsp70 shortly after the addition of sodium arsenite was examined using an antibody to chick hsp70. Some cross-reaction was observed with a polypeptide of 70,000 in unshocked cells, which could indicate the presence of hsp70 in non-shocked cells. This is not conclusive since the antibody was polyclonal. It was still possible to detect the induction of hsp70 above this background. A sharp increase in the level of antibody binding was detected after 15 minutes. The relationship of histone acetylation to hsp induction was also examined using sodium butyrate under conditions which cause a high degree of histone hyperacetylation. Synthesis of the heat shock proteins was neither induced nor blocked. Therefore, the state of histone acetylation does not affect the heat shock response. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate