Analysis of the Meiotic Transcriptome in Genetically Distinct Budding Yeasts Using High Density Oligonucleotide Arrays

The last three years have witnessed a massive production of wholegenome expression data in the yeast field using two different types of gene arrays: commercially available high-density oligonucleotide arrays (GeneChips) and PCR-based gene arrays. In the case of GeneChips every yeast gene is represented by 20 oligonucleotides (each being a 25-mer) synthesized in situ onto a glass plate (which is then inserted into a cartridge for experimental manipulation; Fig. 1, panel b, top left image). Poly A+ RNA (e.g. prepared from cells at various stages of spore development; Fig. 1, panel a) is reverse transcribed into cDNA, labeled with a fluorophor and hybridized to the GeneChip. The fluorescence signal intensities of each set of 20 oligonucleotides are directly proportional to the mRNA concentration in the sample (Fig. 1, panel b, image of a GeneChip hybridization pattern). To eliminate the problem of cross hybridization a set of wild-type oligonucleotides (perfect match) are compared to a set of oligonucleotides containing a point mutation (mismatch) that destabilizes the DNA-DNA interaction. Two examples of a correct hybridization pattern (panel b, middle left) and a clear case of cross-hybridization (panel b, bottom left) are shown in Fig. 1. Open image in new window Fig. 1a–d A GeneChip-based analysis of the meiotic transcriptome in yeast. Panel (a) shows a Nomarski view (top half) and Hoechst-stained nuclei using UV-light microscopy (bottom half) of mature asci formed after 12 hours of spore development in the yeast strain SKI. Panel (b) summarizes the image of a GeneChip cartridge (top left), the result of a fluorescence scan (right) and individual hybridization results indicating correct ratios between perfect match and mismatch (middle left) and a case of clear cross-hybridization (bottom left). Panel (c) shows a bar diagram of 130 selected meiotic expression patterns as identified in SKI (ordered over initial time of induction and then clustered on the basis of overall similarity). Each column corresponds to a time-point (t=0, 1, 2, 3, 4, 6, 8 and 10 hours of sporulation), each line represents a gene. Red and blue indicate high and low levels of expression, respectively. Panel (d) displays the graphical view of 33 selected meiotically induced genes whose expression patterns are very similar (correlation coefficient 0.9). Fluorescence intensities that are directly proportional to mRNA concentrations are plotted versus hours of sporulation in SK1