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note
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Isogenic strains were derived from Saccharomyces cerevisiae ∑1278b MATa ura3-52 leu2::hisG his3::hisG. Mating types were switched (for example, MATa to MATα, and MATa/α to MATa/a) by transient expression of the HO endonuclease from pGAL-HO. A brief exposure to galactose was followed by curing of pGAL-HO. Mating types were confirmed through mating and pheromone production assays. Matings produced the desired ploidy (for example, 2n MATa/a × 2n MATα/α → 4n MATa/a/α/α). Ploidies of these zygotes were confirmed by tetrad dissections and analysis of mating-type segregation.
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Total RNA was extracted and polyadenylated RNA was selected from cell samples. Using the methods of D. J. Lockhart et al. [Nature Biotechnol. 14, 1675 (1996)] and L. Wodicka et al., [ibid. 15, 1359 (1997)], we generated cDNA, produced biotin-labeled cRNA, hybridized fragmented cRNAs to high-density oligonucleotide arrays, stained with streptavidin-conjugated phycoerythrin, washed and scanned the arrays, and determined hybridization signal intensities, which are proportional to the target RNA concentration. Expression was measured as a trimmed average difference between 20 perfect-match and mismatch oligonucleotide probe pairs for each yeast gene. Scan-to-scan variations in intensity were corrected by a bulk scaling method analogous to adding equal amounts of total RNA to gel lanes in Northern blot expression analysis. For each scan, the sum of average differences was tallied. The median sum was found. The scaling factor for each scan (applied to each average difference in that scan) was the ratio of the median sum to the scan sum. All scaling factors were less than 2 and greater than 0.5. Scaled average differences ranged from ∼0 to ∼33,000; those less than 1 were set to 1.
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Lockhart, D.J.1
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Total RNA was extracted and polyadenylated RNA was selected from cell samples. Using the methods of D. J. Lockhart et al. [Nature Biotechnol. 14, 1675 (1996)] and L. Wodicka et al., [ibid. 15, 1359 (1997)], we generated cDNA, produced biotin-labeled cRNA, hybridized fragmented cRNAs to high-density oligonucleotide arrays, stained with streptavidin-conjugated phycoerythrin, washed and scanned the arrays, and determined hybridization signal intensities, which are proportional to the target RNA concentration. Expression was measured as a trimmed average difference between 20 perfect-match and mismatch oligonucleotide probe pairs for each yeast gene. Scan-to-scan variations in intensity were corrected by a bulk scaling method analogous to adding equal amounts of total RNA to gel lanes in Northern blot expression analysis. For each scan, the sum of average differences was tallied. The median sum was found. The scaling factor for each scan (applied to each average difference in that scan) was the ratio of the median sum to the scan sum. All scaling factors were less than 2 and greater than 0.5. Scaled average differences ranged from ∼0 to ∼33,000; those less than 1 were set to 1.
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All experimental cell samples were taken from asynchronous yeast cultures at late exponential phase (optical density at 600 nm = 1) growing in SC 2% glucose medium [F. Sherman, Methods Enzymol. 194, 3 (1991)] at 30°C and 250 rpm. Samples were tested for viability by comparison of cell count with colony formation on plates as well as by staining with methylene blue. The growth of haploids and diploids was nearly indistinguishable. Triploids and tetraploids showed a 10 to 15% decrease in growth rates and an increased growth lag. MATa/α cells had a slight growth rate advantage and a shorter lag than their MAT-homozygous counterparts.
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note
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Expression data for each gene, as well as each reference pattern, were normalized to have mean = 0 and SD = 1. Pearson correlation coefficients were then calculated for each gene against a reference pattern either proportional to ploidy (haploids = 1, diploids = 2, and so forth) or inversely proportional (haploids = 1, diploids = 1/2, and so forth). The first criterion was that the correlation exceeded a threshold (Fig. 1). The second criterion was that the maximum and minimum average difference values for a gene differed by at least 100 units and at least a factor of 3. Ten random data sets (within-gene permutations of the experimental data set) showed that these criteria resulted in less than one false positive on average.
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0344365293
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To download the data set, search the data, and view additional results, including an analysis of mating type-dependent expression, see http://staffa.wi.mit. edu/fink_public/ploidy/.
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16
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0344797095
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data not shown
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T. Galitski, A. J. Saldanha, C. A. Styles, E. S. Lander, C. R. Fink, data not shown.
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Galitski, T.1
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Lander, E.S.4
Fink, C.R.5
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0345228033
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note
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Using the same total RNA samples used for microarray expression analysis, equal amounts of total RNA were added to each gel lane for Northern blot analysis. Radioactively probed mRNAs were visualized and quantitated by autoradiography and phosphor-Imager analysis. Total RNA samples derived from independent experiments (not shown) confirmed ploidy-dependent expression patterns.
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We thank G. Acton, A. Dranginis, Q. Feng, T. Golub, M. Gordon, P. Hecht, F. Holstege, E. Jennings, F. Lewitter, H. Madhani, T. Ni, T. Orr-Weaver, J. Park, S. Rozen, D. Slonim, P. Tamayo, N. Watson, and R. Young for their contributions. Supported by Bristol-Meyers Squibb Company, Affymetrix Inc., and Millennium Pharmaceutical Inc., and by NIH grant GM35010. G.R.F. is an American Cancer Society Professor of Genetics. T.G. is a postdoctoral fellow of the Helen Hay Whitney Foundation.
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