AbstractThe study focuses on the effects of heat stress on Serpula lacrymans and S. himantioides (which have been previously shown to be relatively thermosensitive and thermotolerant respectively) in relation to the heat shock response. A part of this response is the production of heat-shock proteins (hsps). Comparative studies of chick embryo fibroblasts and Saccharomyces cerevisiae (wine strain, L-2226) were used to demonstrate possible differences between these three very distinct eukaryotic systems.
The aims of the project were to determine whether S. lacrymans and S. himantioides undergo the heatshock response with respect primarily to induced thermotolerance and the production of hsps. The approach taken to establish the above was by systematic analysis of the different pathways involved in this response. Firstly, overall heat induced changes in protein and mRNA synthesis were analysed, in conjunction with the identification of specific proteins related to the heat-shock response, namely hsp60, hsp70 which have been shown in other eukaryotic systems to play key roles in this response. Secondly, immunological studies were also used to detect changes in the pattern of protein ubiquitination, which resulted from heat stress. Thirdly, changes in hsp70 mRNA were monitored by RT-PCR. The cDNA and genomic amplimer products generated by RT-PCR and PCR respectively were cloned and sequenced to identify a putative of hsp70 gene sequence. Fourthly, the AMP-activated protein kinase (AMPK) cascade has been demonstrated to be activated in response to stress and steps were made to detect and identify AMPK activity in cellular extracts o f both Serpula species. Finally, the effect of heat upon trehalose accumulation and mobilisation during the heat-shock response was studied as there is growing evidence to link increased thermotolerance to transient increase in trehalose levels, which is now considered to be a stress metabolite.
|Date of Award||May 1999|