Yeast microarrays for genome wide parallel genetic and gene expression analysis

Abstract

We have developed high-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96-well plates, and the resulting products were arrayed using an automated micro arraying device. Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of f luorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Applications of the microarrays are shown for genetic and gene expression analysis at the whole genome level.The genome sequencing projects have generated and will continue to generate enormous amounts of sequence data. The genomes of Saccharomyces cerevisiae, Haemophilus influenzae (1), Mycoplasma genitalium (2), and Methanococcus jannischii (3) have been completely sequenced. Other model organisms have had substantial portions of their genomes sequenced as well including the nematode Caenorhabditis elegans (4) and the small flowering plant Arabidopsis thaliana (5). Given this everincreasing amount of sequence information, new strategies are necessary to efficiently pursue the next phase of the genome projects-the elucidation of gene expression patterns and gene product function on a whole genome scale.One important use of genome sequence data is to attempt to identify the functions of predicted ORFs within the genome. Many of the ORFs identified in the yeast genome sequence were not identified in decades of genetic studies and have no significant homology to previously identified sequences in the database. In addition, even in cases where ORFs have significant homology to sequences in the database, or have known sequence motifs (e.g., protein kinase), this is not sufficient to determine the actual biological role of the gene product. Experimental analysis must be performed to thoroughly understand the biological function of a given ORF's product. Model organisms, such as S. cerevisiae, will be extremely important in improving our understanding of other more complex and less manipulable organisms.To examine in detail the functional role of individual ORFs and relationships between genes at the expression level, this work describes the use of genome sequence information to study large numbers of genes efficiently and systematically. The procedure was as follows. (i) Software scripts scanned annotated sequence information from public databases for predicted ORFs. (ii) The start and stop position of each identified ORF was extracted automatically, along with the sequence data of the ORF and 200 bases flanking either side. (iii) These data were used to automatically select PCR primers that would amplify the ORF. (iv) The primer sequences were automatically input into the automated multip...

Harnessing Natural Diversity to Probe Metabolic Pathways

“…Of the strains tested, only W303 and the related laboratory strain S288c were able to utilize Dasparagine in the PM assay (see Figure 2E). Genomic analyses utilizing microarrays have noted a lack of detectable ASP3 in Y55 [57] and a clinical isolate, YJM789 [3]. Neither Y55 nor our clinical isolate, YAT7, were capable of utilizing the putative Asp3p substrates (see Figure 2).…”

Remodeling of Yeast Genome Expression in Response to Environmental Changes

“…The 283 repressed genes are dominated by genes associated with translation and protein synthesis. Many of these have previously been observed to be repressed in response to specific stresses (Kim and Warner, 1983;DeRisi et al, 1997;Lashkari et al, 1997;Eisen et al, 1998;Jelinsky and Samson, 1999;Rep et al, 2000). The repressed set includes genes for cytoplasmic ribosomal proteins, polymerase I, II, and III transcription, tRNA synthetases, proteins required for processing rRNAs, and a subset of translation initiation factors.…”

Gene Expression Analysis

“…This cDNA array allows every gene of the S. cer6isiae genome to be simultaneously analyzed under different experimental conditions. In a series of experiments, altered expression of major genes associated with heat shock (36 genes), cold shock (29 genes), and change from glucose to galactose metabolism (40 genes) have been identified (20). Arrays that contain the entire set of genes expressed in the melanocyte (or a specific subset) could be used in the same way to follow the effect of different experimental conditions on the expression of these genes (2, 3).…”