Effect of temperature on the ability of genetically diverse S. cerevisiae strains to grow and ferment

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Gel electrophoresis

DNA products were separated by size and visualised using submerged agarose gels with 1-3 % agarose in TBE buffer (Table 2-1). Electrophoresis was at 100 V, 400 A, for 40 min. The samples loaded in each well contained 5 μL DNA, 1 μL water and 4 μL electrophoresis loading dye. Additionally, 10 μL 1 kb Plus DNA ladder (Invitrogen) was loaded in the leftmost lane of each gel as a size standard to measure the length of the DNA fragments. Gels were stained using 0.5 mg L-1 ethidium bromide in TBE for 15-30 min and DNA was visualised using UV light and a gel viewer (Molecular Imager Gel Documentation System, Biorad Laboratories). The Quantity One® software (Version 4.5.1) was used to optimise the brightness and contrast of gel images and to print and save data.

PCR product clean-up

PCR products were purified using the High Pure PCR Product Purification Kit (Roche Applied Science), according to the manufacturer’s instructions. DNA was concentrated by evaporation for 40 min, at room temperature, followed by 30 min at 30°C (reduction from 50 μL to 10 μL) using the Concentrator 5301 (Eppendorf). DNA concentrations of purified products were determined via NanoDrop® (Section 2.5.8).

RNA extraction

Yeast RNA was extracted from frozen cell samples harvested from fermentations (Section 2.4.6) using the standard phenol:chloroform method (COLLART and OLIVIERO 2001). Surfaces were wiped with ethanol to minimise contact with contaminating RNases, gloves were changed regularly and barrier tips were used. Water, buffers and solutions were pretreated with 0.1 % (v/v) DMDC for 12 h, 25°C, with shaking at 100 rpm, to deactivate contaminating RNases. RNA was stored at -80°C until required for microarrays (Section 2.5.9).

Nucleic acid quantification

The quantity and quality of RNA (Section 2.5.7) was determined using the NanoDrop® spectrophotometer ND-1000 (NanoDrop® Technologies Inc.) and the Agilent 2100 BioAnalyzer® (Agilent Technologies Inc.). Quantification was carried out according to the manufacturer’s instructions. An A260/A280 ratio for each sample was an indicator of RNA quality. Samples were electrophoresed using the BioAnalyzer® to determine the quality of the RNA using 28S:18S rRNA ratios and to confirm the concentrations obtained via the NanoDrop®.

Microarrays

RNA samples were given to L. Williams (Affymetrix Centre, Centre for Genomics and Proteomics, SBS, University of Auckland) for microarray processing, as described in the GeneChip® Expression Analysis Technical Manual (Affymetrix, Santa Clara, California). RNA samples were subjected to poly (A)+ RNA purification, cDNA synthesis, in vitro transcription (IVT) to produce biotin-labelled cRNA and cRNA fragmentation, using the MessageAmp III Premier RNA amplification kit (Ambion®), following manufacturer’s instructions. The cRNA samples were hybridised to Affymetrix GeneChip Yeast Genome 2.0 Arrays (Affymetrix, Santa Clara, California) and processed as described by the manufacturer (Eukaryotic Arrays GeneChip® Expression Analysis and Technical Manual, Affymetrix, Santa Clara, California).
Microarray analysis was performed using Bioconductor (http://www.bioconductor.org/), an open-source online tool for comprehensive analysis of large genomic datasets, which utilises R statistical programming language (GENTLEMAN et al. 2004). Free downloadable add-on packages are also available from the Comprehensive R Archive Network (CRAN) to extend functionality (http://cran.r-project.org/). Pre-processing of array data involved extracting probeset IDs which correspond to S. cerevisiae (5900), while masking Schizosaccharomyces pombe probesets (5028), using a code developed by GILLESPIE et al. (2010) . Microarray chip quality was analysed qualitatively, by visualising raw chip hybridisation intensity distributions to identify anomalies, and quantitatively via quality control (QC) metrics, following the methods of ALVORD et al. (2007). Differential expression analysis of the microarray data was performed using the limmaGUI package in Bioconductor (WETTENHALL and SMYTH 2004). Background correction was performed by subtracting the signal intensity of the area between spots, and robust multi-array averaging (RMA) was used to normalise
expression data, followed by data-fitting to a linear model. Box plots were used to visualise probe intensities across chips before and after normalisation.
Various contrasts were made between pairs of conditions in a fixed matrix to obtain a list of differentially expressed genes for each contrast. Plots to visualise post-RMA and post–model fitting QC parameters, including differences in log intensity for each probe (M-value) vs. their average across all arrays and channels (A-value) (MA plots), were also constructed using limmaGUI and TKRplot. Genes were considered to have significant differential expression if the M-values (log2-fold change calculated from the ratio between two intensities) were ≥ 1 or ≤ -1, and the P-values were < 0.05, after applying the Benjamini and Hochberg (BH) method to adjust for false discovery rates (FDR) (BENJAMINI and HOCHBERG 1995).
All probe sets were annotated and linked to their gene descriptions using the yeast2 annotation package (yeast2.db) and the SGD (http://www.yeastgenome.org). For further analysis, FunCatDB (http://mips.helmholtz-muenchen.de/genre/proj/yeast/Search/index.html) was used to group genes into biological pathways based on the Munich Information Centre for Protein Sequences (MIPS) classification (MEWES et al. 2000; ROBINSON et al. 2002).

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Yeast transformation

Yeast transformation was performed using a modification of the SCHIESTL and GIETZ (1989) lithium acetate protocol developed by H. Niederer (née Brown), this laboratory. Yeast cells were prepared two days prior to transformation, first by culturing them overnight in 10 mL YPD (with or without selection) at 28°C and 200 rpm, then scaling up to a series of 30 mL YPD cultures with varying inoculums from the previous culture (1, 2, 4, 8 and 10 μL). These dilutions were incubated overnight at 28°C, 200 rpm. On the day of transformation, the culture with an OD600 nm reading between 0.5-0.6 (exponential phase) was chosen, transferred to a 50-mL falcon tube and centrifuged at 1000g for 5 min. The pellet was suspended in 20 mL sterile water, centrifuged again, then resuspended with 20 mL water. After the previous spin, the pellet was resuspended in 10 mL S1 (Table 2-1), centrifuged for 1000g for 5 min and the supernatant was removed to leave ~500 μL. The culture was resuspended and transferred to a microcentrifuge tube and incubated for 15 min at 30°C. Meanwhile, 5 μL PCR product (to be transformed) and 20 μL freshly boiled co-TF DNA (Table 2-1) were pipetted into new tubes. A no-DNA negative control and plasmid DNA positive control (pFLR-A, a 2μ-based vector plasmid with nourseothricin resistance (NatR), K. Richards and M. Harsch, this laboratory) were also included. After incubation, 50 μL of the culture was transferred into the microcentrifuge tubes with the DNA and 285 μL S2 (Table 2-1) was added, mixed gently five times and incubated at room temperature for 5 min. The tubes were incubated at 30°C for a further 30 min, then 42°C for 25 min. Tubes were centrifuged at 2000g for 2 min and pellets resuspended in 1 mL YPD. Transformed cells were transferred to 14-mL culture tubes, another 1 mL YPD was added, and the cultures were incubated with shaking at 125 rpm for 3 h. Yeast were plated onto selection plates and incubated at 30°C for 2-5 d until colonies appeared.

Chapter 1. Introduction
1.1. General characteristics of S. cerevisiae
1.2. S. cerevisiae and alcoholic fermentation
1.3. Low temperature fermentation .
1.4. Aims and significance of the research
Chapter 2. Materials and methods .
2.1. Laboratory reagents .
2.2. Yeast strains .
2.3. Yeast culture and handling
2.4. Yeast growth and fermentation experiments
2.5. Molecular biology techniques
2.6. Quantification of aroma compounds in wine ..
Chapter 3. Effect of temperature on the ability of genetically diverse S. cerevisiae strains to grow and ferment
3.1. Introduction
3.2. Initial yeast screening before testing for variation in growth and fermentation
3.3. Screening commercial wine yeast for cold fermentation parameters
3.4. Variation among strains for growth and fermentation across a range of temperatures
3.5. Discussion
3.6. Conclusion
Chapter 4. Effect of low temperature fermentation on gene expression
4.1. Introduction .
4.2. Strain selection and fermentation for microarray sampling .
4.3. Quantity and quality of extracted total RNA .
4.4. Microarray pre-processing and analysis
4.5. Overview of microarray results
4.6. Comparison 1: Growth phase
4.7. Comparison 2: Temperature
4.8. Comparison 3: Strain
4.9. Discussion
4.10. Conclusion
Chapter 5. Effect of low temperature fermentation on wine aroma
Chapter 6. Identification of genes important for low temperature fermentation
Chapter 7. Final discussion