Analysis of gene filter, macroarray, data for ethanol-stressed cells in the

Although a growing number of studies have documented the changes in global gene expression in ethanol-stressed yeast cultures using global gene expression, there have been no such reports on the stimulatory effect of added acetaldehyde to ethanol-stressed

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cultures. The following presents the first such data from gene filter, macroarray experiments.

At the one-hour time point there were 214 ORFs MHE in the acetaldehyde-treated ethanol-stressed culture than in the untreated ethanol-stressed culture, and only 4 ORFs had reduced expression (Tables 3.3a and 3.3b, in Appendix III). At the five-hour time point 347 were MHE and 49 were LHE (Tables 3.4a and Table 3.4b, in Appendix III).

One of the most interesting aspects of the array data is that, when acetaldehyde was added to the ethanol-stressed yeast cells, there was increased expression of many genes. Perhaps the most striking feature of the MHE ORFs was the large number that are associated with synthesis of ribosomal proteins, DNA synthesis, transcription and cell cycle, and this was evident at both time points (Tables 5.1 and 5.2). This is consistent with growth curve data shown in Figures 4.3 and 4.4, in which it clearly showed that cells are rescued from lag considerably faster when acetaldehyde is present in ethanol- containing cultures, relative to cultures without acetaldehyde; when acetaldehyde is present the cells commence division more rapidly.

Another notable feature of this study was that stress response genes and genes associated with trehalose synthesis were not highly expressed during acetaldehyde stimulation of ethanol-stressed cultures relative to untreated ethanol-stressed cultures. This finding is interesting, because the well-documented increased expression of HSP

genes and trehalose synthesis genes in response to ethanol stress (Chandler et al., 2004; Alexandre et al., 2001; Piper et al., 1994 and Piper, 1995) has led to the proposal that stress response genes protect against the damaging effect of ethanol stress. This may be the case but results presented here suggest they are unlikely to have a role in acetaldehyde-induced tolerance to ethanol stress.

As described in Section 5.2.2.2 150 genes associated with transport were LHE in ethanol-stressed cultures relative to the unstressed control. In the presence of acetaldehyde 18 of these (along with two that were not affected by ethanol stress alone), displayed increased level of expression relative to ethanol stressed cells. This change in expression, brought about by the addition of acetaldehyde to ethanol stressed cultures, may represent a small fraction of the overall number of transport genes that were LHE

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under ethanol stress, but it suggests that the cell is attempting to activate transport processes that are otherwise compromised in ethanol-stress conditions. For example, several HXT hexose and PHO phosphate transport genes were LHE under ethanol stress, a finding that is consistent with the findings of Chandler et al. (2004) and Alexandre et al. (2001). However, when acetaldehyde was added to the ethanol- stressed culture, HXT3, PHO84, PHO3 and PHO88 showed increased expression levels at one hour time point, and HXT1 at five hour time point. It has been proposed that cells under ethanol stress are likely to be in a pseudo-starvation state where nutrients, such as glucose, are present in the growth medium but are not accessible to the cell (Chandler et al., 2004). The increased expression of these transport genes following addition of acetaldehyde to the ethanol-stressed culture may ameliorate ethanol-induced stress by facilitating the transport of nutrients for normal cell metabolic activities.

Another group of genes that are noteworthy here because they were MHE when acetaldehyde was added to the ethanol-stressed culture are genes associated with plasma membrane structure and function. These include the ergosterol synthesis genes, ERG3, ERG11, ERG25 and ERG26 at one-hour time point and ERG3 and ERG9 at five-hour time point. There was only one fatty acid metabolism gene, FEN1 (YCR034W) with increased expression across both time points.

As discussed in Section 2.4.1, ethanol tolerance in yeast is thought by some workers to correlate with increasing ergosterol levels. Thus, increased expression of ERG genes in an ethanol-stressed culture with added acetaldehyde might be a significant factor in promoting ethanol tolerance. However, when Walker-Caprioglio et al. (1985) performed fluorescence anisotropy measurement of the plasma membrane during ethanol stressin the presence of small amounts of added acetaldehyde no reversal of the ethanol-induced changes to plasma membrane was observed; i.e. acetaldehyde did not reverse the ethanol-induced changes to plasma membrane. Thus, while the cell may be attempting to alter its membrane structure to better tolerate ethanol stress, actual changes in membrane composition may not be achieved. Membrane lipids were not analysed for work described in this thesis, therefore it is not possible to ascertain whether increased expression of ERG genes had any impact on the membrane.

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Table 5.2: Summary of macroarray data: functional classes of genes with changed expression following five hours exposure to 7% (v/v) ethanol in the presence and absence of acetaldehyde.

Effect of ethanol stress Effect of acetaldehyde on

ethanol-stressed cells

Effect of acetaldehyde on unstressed cells

Functional class of genes No. of ORFs

MHE No. of ORFs LHE No. of ORFs MHE No. of ORFs LHE No. of ORFs MHE No. of ORFs LHE

Ribosomal Protein & Ribosomal subunit - 149 55 - - -

Stress response 7 18 8 - - -

Cell cycle & growth - 25 13 1 1 -

Protein metabolism 1 34 - - 1 1

Transport and translocation 3 91 30 4 - -

Transcription & translation factors 5 125 30 3 - -

Energy utilization 9 23 5 2 - -

Protein synthesis & folding - 28 24 - - -

Signal transduction 1 8 3 - - -

Cytoskeleton organization & maintenance - 35 5 - - -

Cell wall & membrane proteins 4 49 8 2 - -

Lipid metabolism 1 12 3 - - Nucleotide metabolism 9 109 13 5 - - Histone metabolism - 12 - - - - Miscellaneous 9 308 47 2 - - Unknown function 36 394 103 30 - - Total ORFs 85 1420 347 49 2 1

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5.2.2.4 Comparison of one- and five-hour time points using data from gene