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note
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Unless otherwise indicated, all experiments in this study were done in strains isogenic to W303. A single selection strain, DA2050A (a/a bar1/bar1 mec1-1/ mec1-1 CDC28VF::LEU2/CDC28-VF::LEU2 GAL1-MPS1/CAL1-MPS1), was engineered to identify inhibitors of the spindle checkpoint or of the pheromone response pathway. The bar1 mutation increases sensitivity to α factor by removing a protease that degrades the pheromone, and the CDC28-VF mutation blocks the ability of cells to adapt to overexpression of Mps1 from the GAL1 promoter. The mec1-1 mutation, which abolishes the DNA damage checkpoint, is irrelevant to the experiments reported here.
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For both selections, transformants were grown on uracil-deficient plates for 2 days. Cells were scraped from these plates, and aliquots were plated onto selection plates [lacking uracil and containing galactose (-uracil/+galactose) for the spindle checkpoint selection; lacking uracil and containing α factor (-uracil/+α factor) (1 μg/ml) for the pheromone pathway selection] and grown at 30°C for 2 or 3 days. Plasmid DNA was recovered from the entire population of colonies that grew on these plates, purified, amplified by transformation into E. coli, and transformed into DA2050A to enrich for peptamers that conferred resistance as opposed to genomic mutations that caused resistance. Cell populations harboring active peptamers typically grew as lawns on selection plates, and individual plasmids were isolated from these plates and retested to confirm that they gave plasmid-dependent resistance.
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note
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Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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A yeast ORF-Gal4 activation domain fusion array has been assembled that expresses about 85 to 90% of the predicted ORFs of 5. cerevisiae in strain pJ69-4a (P. James, J. Halladay, E. Craig Genetics 144, 1425 (1996)]. In this strain, GAL4 is absent and the reporters HIS3 and ADE2 are under Gal4 control. To probe the Gal4-AD fusion array for protein-protein interactions, we mated the array to a strain expressing a Gal-4-DNA binding domain fusion. After selecting for diploids, we identified two-hybrid positives by testing diploids on plates containing different concentrations of 3-aminotriazole.
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DN. Each comparison was done on four arrays composed of two pair of hybridizations done with reversal of the fluorophore labelings to eliminate biases of fluorophore incorporation. We used two different sources of mRNAs for labeled cDNA production, including a total RNA protocol (http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/) and twice-purified polyadenylated mRNAs [(M. J. Marton et al. Nature Med. 4, 1293 (1998)].
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0345227037
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note
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Supported by grants from NIH, Human Frontier Science Program, and Chiron (A.W.M.); an NIH senior fellowship (T.C.N.); an NIH grant and the Herbert W. Boyer Fund (S.M.O.); and NIH grant P41-RR11823 (B.L.D. and S.F.). S.F. is an Investigator of the Howard Hughes Medical Institute. We thank L. Hartwell, I. Herskowitz, L. Huang, and J. Rine for yeast strains and plasmids; T. Geyer, Alejandro Colman-Lerner, and R. Brent for communicating unpublished results; and D. Gottschling, I. Herskowitz, L. Pillus, J. Simon, and members of the Seattle Project and the Murray laboratory for valuable discussions.
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