Expression Data Set 43 details

Information Item	Value
Dataset Name	SAGE transcript profiles from yeast grown on two different carbon sources
Dataset Number	43
Short Description	SAGE analysis of wild type and mutant cells grown on oleate for comparison with SAGE analysis of cells grown on glucose
Source URL	http://www.molbiolcell.org/cgi/content/full/10/6/1859/DC1
Reference	Kal, AJ; et al (see LongDescrip for Complete Reference), Dynamics of Gene Expression Revealed by Comparison of Serial Analysis of Gene Expression Transcript Profiles from Yeast Grown on Two Different Carbon Sources, Mol. Biol. Cell 2000, 10(6):1859-1872
Strains	Wild type strain BJ1991 (MATalpha, leu2, trp1, ura3-52, pip4-3, prb1-1122; Jones, 1977), mutant strain BJ1991 pip2/oaf1 (MATalpha, leu2, trp1 ura3-52, pep4-3 prbl-1122, PIP2::KANMX4,OAF1::LEU2; Rottenstein et al, 1997)
Conditions	See LongDescrip
Date Added to ExpressDB	Sep 15 2000 3:07:47:103PM
Number of Measures on ExpressDB	10 (here to download dataset and view measure details)
Long Description	Abstract - We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE). Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source. A major fraction (>20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of ~6300 yeast genes. Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters). In oleate-grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal-oxidation enzymes required for degradation of fatty acids. The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes. We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2 and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins. Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment. We describe a statistical method for analysis of data obtained by SAGE. Data analysis methods Initial data analysis was performed using the SAGE Software package version 1.0 (Velculescu et al., 1995). The tag list from wild type cells and pip2/oaf1 cells contained 10,943 and 3847 tags, respectively, of which 577 and 234, respectively, were derived from linker sequences. These tags were excluded from the analysis. The resulting tag lists contained 10,366 total tags from wild type cells and 3613 tags from pip2/oaf1 cells. We compiled a database of all potential tags of the complete yeast genome (over 69,000 10-bp sequences) and linked each tag to the gene annotations in the MIPS database (as of 9th December 1998). Next, we merged this dataset with the tags found with SAGE. Tag numbers can be converted to number of mRNA transcripts per cell assuming a total of 15,000 mRNA molecules per cell (see below). Classification in Functional Categories was done according to the yeast protein functional catalogue (Goffeau, 1997; Mewes et al., 1997); also available via the World Wide Web at http://websvr.mips.biochem.mpg.de/proj/yeast). Usage of the SAGE data Determine expression levels To determine the expression level of a certain gene, follow the guidelines below. 1. Use the systematic name of the gene, e.g. YMR303C is the systematic name for the ADH2 gene. For searches, always use the systematic names. Not all synonyms are included in the descriptions, and sometimes the same acronym is used for different genes (e.g. CTP1 = citrate transport protein or copper transport protein). 2. Only consider tags that are within the 500 bp 3' of the ORF. If multiple tags match a gene within the ORF or within the 500 bp 3' of the ORF AND these tags have only one genome hit, tags can be considered to originate from the same gene and can be added. If tags match the genome at multiple places, all places should be checked. Sometime the 11th bp of the tag can be identified using the SAGE software, this sometimes resolves ambiguities. 3. Calculate the expression level (mRNA copies per cell) by dividing the number of tags by the total number of tags for that condition, and multiply the resulting number by 15,000 (total number of mRNA molecules per cell). E.g. 100 tags from a gene in the wild type oleate library equals an expression level of 100/10,366*15000=145 mRNA copies per cell. Note that a single tag can originate from multiple genes (e.g. tag GGTGAAAACG can originate from ADH1, ADH2 or DYN1 genes), that a single gene can give multiple tags and that tags that originate from chromosome localizations far away (>500 bp) from annotated ORF can originate from NORFs (Non-annotated ORFs). Complete Reference - Kal, AJ; van Zonneveld, AJ; Benes, V; van den Berg, M; Koerkamp, G; Albermann, K; Strack, N; Ruijter, J M; Richter, A; Dujon, B; Ansorge, W; Tabak, HF, Dynamics of Gene Expression Revealed by Comparison of Serial Analysis of Gene Expression Transcript Profiles from Yeast Grown on Two Different Carbon Sources, Mol. Biol. Cell 2000, 10(6):1859-1872 Conditions - Strains were precultured 24 h on minimal medium containing 0.3% glucose to obtain a derepressed culture. After a shift to medium containing 0.12% oleate, 0.2% Tween 40, 0.3% yeast extract, 0.5% bacto-peptone, and 0.5% potassium phosphate buffer, pH 6.0, the cells were cultured for 18 h at 280C. Cell growth was stopped by the addition of an equal volume of ethanol (800C), and RNA was extracted immediately. mRNA was isolated using the Poly-A-tract kit from Promega (Madison, WI) according to the manufacturer's protocol.

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