Jones Dawn L, Petty June, Hoyle David C, Hayes Andrew, Oliver Stephen G, Riba-Garcia Isabel, Gaskell Simon J, Stateva Lubomira
Department of Biomolecular Sciences, UMIST, Manchester M60 1QD, UK.
Comp Funct Genomics. 2004;5(5):419-31. doi: 10.1002/cfg.415.
We have used DNA microarray technology and 2-D gel electrophoresis combined with mass spectrometry to investigate the effects of a drastic heat shock from 30 to 50 on a genome-wide scale. This experimental condition is used to differentiate between wild-type cells and those with a constitutively active cAMP-dependent pathway in Saccharomyces cerevisiae. Whilst more than 50% of the former survive this shock, almost all of the latter lose viability. We compared the transcriptomes of the wildtype and a mutant strain deleted for the gene PDE2, encoding the high-affinity cAMP phosphodiesterase before and after heat shock treatment. We also compared the two heat-shocked samples with one another, allowing us to determine the changes that occur in the pde2Delta mutant which cause such a dramatic loss of viability after heat shock. Several genes involved in ergosterol biosynthesis and carbon source utilization had altered expression levels, suggesting that these processes might be potential factors in heat shock survival. These predictions and also the effect of the different phases of the cell cycle were confirmed by biochemical and phenotypic analyses. 146 genes of previously unknown function were identified amongst the genes with altered expression levels and deletion mutants in 13 of these genes were found to be highly sensitive to heat shock. Differences in response to heat shock were also observed at the level of the proteome, with a higher level of protein degradation in the mutant, as revealed by comparing 2-D gels of wild-type and mutant heat-shocked samples and mass spectrometry analysis of the differentially produced proteins.
我们运用DNA微阵列技术以及二维凝胶电泳结合质谱分析法，在全基因组范围内研究了从30℃急剧升温至50℃的热休克效应。此实验条件用于区分酿酒酵母中的野生型细胞和那些具有组成型活性cAMP依赖性途径的细胞。虽然超过50%的野生型细胞能在这种热休克中存活，但几乎所有具有组成型活性cAMP依赖性途径的细胞都会失去活力。我们比较了热休克处理前后野生型和缺失编码高亲和力cAMP磷酸二酯酶的PDE2基因的突变株的转录组。我们还将两个热休克后的样本相互比较，从而确定pde2Δ突变体中发生的变化，这些变化导致其在热休克后活力急剧丧失。几个参与麦角固醇生物合成和碳源利用的基因表达水平发生了改变，这表明这些过程可能是热休克存活的潜在因素。这些预测以及细胞周期不同阶段的影响通过生化和表型分析得到了证实。在表达水平发生改变的基因中鉴定出了146个功能未知的基因，并且发现其中13个基因的缺失突变体对热休克高度敏感。在蛋白质组水平上也观察到了对热休克反应的差异，通过比较野生型和突变体热休克样本的二维凝胶以及对差异产生的蛋白质进行质谱分析发现，突变体中蛋白质降解水平更高。
