Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles

Science

Volumn 287, Issue 5454, 2000, Pages 873-880

  Roberts, Christopher J ^a   Nelson, Bryce ^a   Marton, Matthew J ^a   Stoughton, Roland ^a   Meyer, Michael R ^a   Bennett, Holly A ^a   He, Yudong D ^a   Dai, Hongyue ^a   Walker, Wynn L ^a   Hughes, Timothy R ^a   Tyers, Mike ^a   Boone, Charles ^a   Friend, Stephen H ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

MITOGEN ACTIVATED PROTEIN KINASE;

ARTICLE; CELL CYCLE; ENZYME INDUCTION; GENE EXPRESSION REGULATION; GENOME; MORPHOGENESIS; NONHUMAN; PRIORITY JOURNAL; SIGNAL TRANSDUCTION; YEAST;

CELL CYCLE PROTEINS; FUNGAL PROTEINS; G1 PHASE; GENE EXPRESSION PROFILING; GENE EXPRESSION REGULATION, FUNGAL; GENOME, FUNGAL; LIPOPROTEINS; MAP KINASE SIGNALING SYSTEM; MITOGEN-ACTIVATED PROTEIN KINASES; MULTIGENE FAMILY; OLIGONUCLEOTIDE ARRAY SEQUENCE ANALYSIS; PEPTIDES; PROTEIN KINASE C; REPRESSOR PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TRANS-ACTIVATION (GENETICS); TRANSCRIPTION FACTORS;

EID: 0034603061     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5454.873     Document Type: Article

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59. Potential filamentation genes were induced in experiments 19, 22, 23, 31, 32, and 33, which includes the pheromone response profile of fus3Δ cells, the rst1Δ rst2Δ profile, and the high osmolarity cross talk of hog1Δ cells (9), and coordinately repressed in experiment 30, the pheromone response profile of rst1Δ rst2Δ cells. Genes encoding secreted proteins (PGU1 and PRY2), potential cell-surface proteins (YLR042c, YIL177c), proteins involved in glycosylation and chitin biosynthesis (KTR2, CHS7, and GFA1), and a regulatory protein implicated in invasive and filamentous growth (PHD1) were all induced by Kss1p signaling. PGU1 encodes a secreted form of a pectin-degrading enzyme, which might facilitate yeast invasion of fruits [P. Blanco, C. Sieiro, N. M. Reboredo, T. G. Villa, FEMS Microbiol. Lett. 164, 249 (1998); S. Gognies, A. Gainvors, M. Aigle, A. Belarbi, Yeast 15, 11 (1999)]. Consistent with our results, PGU1 has recently been shown to be induced by Ksslp signaling in Σ1278b cells [H. D. Madhani, T. Galitski, E. S. Lander, G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 96, 12530 (1999)]. It is also noteworthy that α-factor treatment of rst1Δ rst2Δ cells leads to further induction of several pheromone-responsive genes (11). Pheromone-induced gene expression in the absence of Rstlp and Rst2p indicates that Ste12p activation may be controlled by another negative regulatory mechanism, perhaps one involving Kss1p [J. G. Cook, L. Bardwell, J. Thorner, Nature 390, 85 (1997); L. Bardwell et al., Genes Dev. 12, 2887 (1998)]. Alternatively, Fus3p may phosphorylate and activate Ste12p directly [W. Hung, K. A. Olson, A. Breitkreutz, I. Sadowski, Eur. J. Biochem. 245, 241 (1997)].
60. Potential filamentation genes were induced in experiments 19, 22, 23, 31, 32, and 33, which includes the pheromone response profile of fus3Δ cells, the rst1Δ rst2Δ profile, and the high osmolarity cross talk of hog1Δ cells (9), and coordinately repressed in experiment 30, the pheromone response profile of rst1Δ rst2Δ cells. Genes encoding secreted proteins (PGU1 and PRY2), potential cell-surface proteins (YLR042c, YIL177c), proteins involved in glycosylation and chitin biosynthesis (KTR2, CHS7, and GFA1), and a regulatory protein implicated in invasive and filamentous growth (PHD1) were all induced by Kss1p signaling. PGU1 encodes a secreted form of a pectin-degrading enzyme, which might facilitate yeast invasion of fruits [P. Blanco, C. Sieiro, N. M. Reboredo, T. G. Villa, FEMS Microbiol. Lett. 164, 249 (1998); S. Gognies, A. Gainvors, M. Aigle, A. Belarbi, Yeast 15, 11 (1999)]. Consistent with our results, PGU1 has recently been shown to be induced by Ksslp signaling in Σ1278b cells [H. D. Madhani, T. Galitski, E. S. Lander, G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 96, 12530 (1999)]. It is also noteworthy that α-factor treatment of rst1Δ rst2Δ cells leads to further induction of several pheromone-responsive genes (11). Pheromone-induced gene expression in the absence of Rstlp and Rst2p indicates that Ste12p activation may be controlled by another negative regulatory mechanism, perhaps one involving Kss1p [J. G. Cook, L. Bardwell, J. Thorner, Nature 390, 85 (1997); L. Bardwell et al., Genes Dev. 12, 2887 (1998)]. Alternatively, Fus3p may phosphorylate and activate Ste12p directly [W. Hung, K. A. Olson, A. Breitkreutz, I. Sadowski, Eur. J. Biochem. 245, 241 (1997)].
61. Potential filamentation genes were induced in experiments 19, 22, 23, 31, 32, and 33, which includes the pheromone response profile of fus3Δ cells, the rst1Δ rst2Δ profile, and the high osmolarity cross talk of hog1Δ cells (9), and coordinately repressed in experiment 30, the pheromone response profile of rst1Δ rst2Δ cells. Genes encoding secreted proteins (PGU1 and PRY2), potential cell-surface proteins (YLR042c, YIL177c), proteins involved in glycosylation and chitin biosynthesis (KTR2, CHS7, and GFA1), and a regulatory protein implicated in invasive and filamentous growth (PHD1) were all induced by Kss1p signaling. PGU1 encodes a secreted form of a pectin-degrading enzyme, which might facilitate yeast invasion of fruits [P. Blanco, C. Sieiro, N. M. Reboredo, T. G. Villa, FEMS Microbiol. Lett. 164, 249 (1998); S. Gognies, A. Gainvors, M. Aigle, A. Belarbi, Yeast 15, 11 (1999)]. Consistent with our results, PGU1 has recently been shown to be induced by Ksslp signaling in Σ1278b cells [H. D. Madhani, T. Galitski, E. S. Lander, G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 96, 12530 (1999)]. It is also noteworthy that α-factor treatment of rst1Δ rst2Δ cells leads to further induction of several pheromone-responsive genes (11). Pheromone-induced gene expression in the absence of Rstlp and Rst2p indicates that Ste12p activation may be controlled by another negative regulatory mechanism, perhaps one involving Kss1p [J. G. Cook, L. Bardwell, J. Thorner, Nature 390, 85 (1997); L. Bardwell et al., Genes Dev. 12, 2887 (1998)]. Alternatively, Fus3p may phosphorylate and activate Ste12p directly [W. Hung, K. A. Olson, A. Breitkreutz, I. Sadowski, Eur. J. Biochem. 245, 241 (1997)].
62. Potential filamentation genes were induced in experiments 19, 22, 23, 31, 32, and 33, which includes the pheromone response profile of fus3Δ cells, the rst1Δ rst2Δ profile, and the high osmolarity cross talk of hog1Δ cells (9), and coordinately repressed in experiment 30, the pheromone response profile of rst1Δ rst2Δ cells. Genes encoding secreted proteins (PGU1 and PRY2), potential cell-surface proteins (YLR042c, YIL177c), proteins involved in glycosylation and chitin biosynthesis (KTR2, CHS7, and GFA1), and a regulatory protein implicated in invasive and filamentous growth (PHD1) were all induced by Kss1p signaling. PGU1 encodes a secreted form of a pectin-degrading enzyme, which might facilitate yeast invasion of fruits [P. Blanco, C. Sieiro, N. M. Reboredo, T. G. Villa, FEMS Microbiol. Lett. 164, 249 (1998); S. Gognies, A. Gainvors, M. Aigle, A. Belarbi, Yeast 15, 11 (1999)]. Consistent with our results, PGU1 has recently been shown to be induced by Ksslp signaling in Σ1278b cells [H. D. Madhani, T. Galitski, E. S. Lander, G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 96, 12530 (1999)]. It is also noteworthy that α-factor treatment of rst1Δ rst2Δ cells leads to further induction of several pheromone-responsive genes (11). Pheromone-induced gene expression in the absence of Rstlp and Rst2p indicates that Ste12p activation may be controlled by another negative regulatory mechanism, perhaps one involving Kss1p [J. G. Cook, L. Bardwell, J. Thorner, Nature 390, 85 (1997); L. Bardwell et al., Genes Dev. 12, 2887 (1998)]. Alternatively, Fus3p may phosphorylate and activate Ste12p directly [W. Hung, K. A. Olson, A. Breitkreutz, I. Sadowski, Eur. J. Biochem. 245, 241 (1997)].
63. Potential filamentation genes were induced in experiments 19, 22, 23, 31, 32, and 33, which includes the pheromone response profile of fus3Δ cells, the rst1Δ rst2Δ profile, and the high osmolarity cross talk of hog1Δ cells (9), and coordinately repressed in experiment 30, the pheromone response profile of rst1Δ rst2Δ cells. Genes encoding secreted proteins (PGU1 and PRY2), potential cell-surface proteins (YLR042c, YIL177c), proteins involved in glycosylation and chitin biosynthesis (KTR2, CHS7, and GFA1), and a regulatory protein implicated in invasive and filamentous growth (PHD1) were all induced by Kss1p signaling. PGU1 encodes a secreted form of a pectin-degrading enzyme, which might facilitate yeast invasion of fruits [P. Blanco, C. Sieiro, N. M. Reboredo, T. G. Villa, FEMS Microbiol. Lett. 164, 249 (1998); S. Gognies, A. Gainvors, M. Aigle, A. Belarbi, Yeast 15, 11 (1999)]. Consistent with our results, PGU1 has recently been shown to be induced by Ksslp signaling in Σ1278b cells [H. D. Madhani, T. Galitski, E. S. Lander, G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 96, 12530 (1999)]. It is also noteworthy that α-factor treatment of rst1Δ rst2Δ cells leads to further induction of several pheromone-responsive genes (11). Pheromone-induced gene expression in the absence of Rstlp and Rst2p indicates that Ste12p activation may be controlled by another negative regulatory mechanism, perhaps one involving Kss1p [J. G. Cook, L. Bardwell, J. Thorner, Nature 390, 85 (1997); L. Bardwell et al., Genes Dev. 12, 2887 (1998)]. Alternatively, Fus3p may phosphorylate and activate Ste12p directly [W. Hung, K. A. Olson, A. Breitkreutz, I. Sadowski, Eur. J. Biochem. 245, 241 (1997)].
64. Potential filamentation genes were induced in experiments 19, 22, 23, 31, 32, and 33, which includes the pheromone response profile of fus3Δ cells, the rst1Δ rst2Δ profile, and the high osmolarity cross talk of hog1Δ cells (9), and coordinately repressed in experiment 30, the pheromone response profile of rst1Δ rst2Δ cells. Genes encoding secreted proteins (PGU1 and PRY2), potential cell-surface proteins (YLR042c, YIL177c), proteins involved in glycosylation and chitin biosynthesis (KTR2, CHS7, and GFA1), and a regulatory protein implicated in invasive and filamentous growth (PHD1) were all induced by Kss1p signaling. PGU1 encodes a secreted form of a pectin-degrading enzyme, which might facilitate yeast invasion of fruits [P. Blanco, C. Sieiro, N. M. Reboredo, T. G. Villa, FEMS Microbiol. Lett. 164, 249 (1998); S. Gognies, A. Gainvors, M. Aigle, A. Belarbi, Yeast 15, 11 (1999)]. Consistent with our results, PGU1 has recently been shown to be induced by Ksslp signaling in Σ1278b cells [H. D. Madhani, T. Galitski, E. S. Lander, G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 96, 12530 (1999)]. It is also noteworthy that α-factor treatment of rst1Δ rst2Δ cells leads to further induction of several pheromone-responsive genes (11). Pheromone-induced gene expression in the absence of Rstlp and Rst2p indicates that Ste12p activation may be controlled by another negative regulatory mechanism, perhaps one involving Kss1p [J. G. Cook, L. Bardwell, J. Thorner, Nature 390, 85 (1997); L. Bardwell et al., Genes Dev. 12, 2887 (1998)]. Alternatively, Fus3p may phosphorylate and activate Ste12p directly [W. Hung, K. A. Olson, A. Breitkreutz, I. Sadowski, Eur. J. Biochem. 245, 241 (1997)].
65. All yeast strains were based on the 5288c genetic background and were derived from BY4741 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0) or BY4742 (MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0) [C. B. Brachmann et al., Yeast 14, 115 (1998)]. The bar1Δ (R276) and hog1Δ (R7326) deletions were constructed by gene replacement in BY4741 with the KanMX cassette [A. Wach, A. Brachat, R. Pohlmann, P. Philippsen, Yeast 10, 1793 (1994)]. R426, the control strain for mutant experiments, was created by integration of URA3 at the ura3Δ0 locus in R276. Genes were deleted from derivatives of R276 and replaced with URA3 sequences [J. S. Jones and L. Prakash, Yeast 6, 363 (1990)], creating the following strains: ste2Δ (R1269), ste4Δ (R410), ste5Δ (R413), ste7Δ (R415), ste11Δ (R416), ste12Δ (R418), ste18Δ (R419), ste20Δ (R421), fus3Δ (R500), kss1Δ (Y1399), bni1Δ (R994), sst2Δ (R1331), far1Δ (R496), and tec1Δ (Y1543). Y1787 (fus3Δ tec1Δ) was created by replacing the FUS3 gene of Y1543 with LEU2 sequences. Y1460 (MATa fus3Δ kss1Δ lys2Δ0) was isolated by sporulation of the diploid formed by crossing R500 and Y1438 (MATα kss1Δ::URA3 bar1Δ::KanMX his3Δ200 leu2Δ0 met15Δ0 lys2Δ0). Y1612 (MATa rst1Δ rst2Δ lys2Δ0) was isolated by sporulation of a diploid formed by crossing Y1458 (MATa rst1Δ::URA3 rst2Δ:.KanMX his3Δ200 leu2Δ0 lys2Δ0) and Y1400 (MATα rst1Δ::URA3 bar1Δ::KanMX his3Δ200). Y1469 (MATa bar1Δ::KanMX trp1-63 his3Δ200 leu2Δ0 ura3Δ0 met15Δ0) was isolated from sporulation of the diploid formed by crossing R276 and R154 (MATα lys2α0 ura3α0 trp1-63).
66. All yeast strains were based on the 5288c genetic background and were derived from BY4741 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0) or BY4742 (MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0) [C. B. Brachmann et al., Yeast 14, 115 (1998)]. The bar1Δ (R276) and hog1Δ (R7326) deletions were constructed by gene replacement in BY4741 with the KanMX cassette [A. Wach, A. Brachat, R. Pohlmann, P. Philippsen, Yeast 10, 1793 (1994)]. R426, the control strain for mutant experiments, was created by integration of URA3 at the ura3Δ0 locus in R276. Genes were deleted from derivatives of R276 and replaced with URA3 sequences [J. S. Jones and L. Prakash, Yeast 6, 363 (1990)], creating the following strains: ste2Δ (R1269), ste4Δ (R410), ste5Δ (R413), ste7Δ (R415), ste11Δ (R416), ste12Δ (R418), ste18Δ (R419), ste20Δ (R421), fus3Δ (R500), kss1Δ (Y1399), bni1Δ (R994), sst2Δ (R1331), far1Δ (R496), and tec1Δ (Y1543). Y1787 (fus3Δ tec1Δ) was created by replacing the FUS3 gene of Y1543 with LEU2 sequences. Y1460 (MATa fus3Δ kss1Δ lys2Δ0) was isolated by sporulation of the diploid formed by crossing R500 and Y1438 (MATα kss1Δ::URA3 bar1Δ::KanMX his3Δ200 leu2Δ0 met15Δ0 lys2Δ0). Y1612 (MATa rst1Δ rst2Δ lys2Δ0) was isolated by sporulation of a diploid formed by crossing Y1458 (MATa rst1Δ::URA3 rst2Δ:.KanMX his3Δ200 leu2Δ0 lys2Δ0) and Y1400 (MATα rst1Δ::URA3 bar1Δ::KanMX his3Δ200). Y1469 (MATa bar1Δ::KanMX trp1-63 his3Δ200 leu2Δ0 ura3Δ0 met15Δ0) was isolated from sporulation of the diploid formed by crossing R276 and R154 (MATα lys2α0 ura3α0 trp1-63).
67. All yeast strains were based on the 5288c genetic background and were derived from BY4741 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0) or BY4742 (MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0) [C. B. Brachmann et al., Yeast 14, 115 (1998)]. The bar1Δ (R276) and hog1Δ (R7326) deletions were constructed by gene replacement in BY4741 with the KanMX cassette [A. Wach, A. Brachat, R. Pohlmann, P. Philippsen, Yeast 10, 1793 (1994)]. R426, the control strain for mutant experiments, was created by integration of URA3 at the ura3Δ0 locus in R276. Genes were deleted from derivatives of R276 and replaced with URA3 sequences [J. S. Jones and L. Prakash, Yeast 6, 363 (1990)], creating the following strains: ste2Δ (R1269), ste4Δ (R410), ste5Δ (R413), ste7Δ (R415), ste11Δ (R416), ste12Δ (R418), ste18Δ (R419), ste20Δ (R421), fus3Δ (R500), kss1Δ (Y1399), bni1Δ (R994), sst2Δ (R1331), far1Δ (R496), and tec1Δ (Y1543). Y1787 (fus3Δ tec1Δ) was created by replacing the FUS3 gene of Y1543 with LEU2 sequences. Y1460 (MATa fus3Δ kss1Δ lys2Δ0) was isolated by sporulation of the diploid formed by crossing R500 and Y1438 (MATα kss1Δ::URA3 bar1Δ::KanMX his3Δ200 leu2Δ0 met15Δ0 lys2Δ0). Y1612 (MATa rst1Δ rst2Δ lys2Δ0) was isolated by sporulation of a diploid formed by crossing Y1458 (MATa rst1Δ::URA3 rst2Δ:.KanMX his3Δ200 leu2Δ0 lys2Δ0) and Y1400 (MATα rst1Δ::URA3 bar1Δ::KanMX his3Δ200). Y1469 (MATa bar1Δ::KanMX trp1-63 his3Δ200 leu2Δ0 ura3Δ0 met15Δ0) was isolated from sporulation of the diploid formed by crossing R276 and R154 (MATα lys2α0 ura3α0 trp1-63).
68. Plasmids described previously include the following: pRS314 (CEN TRP1), pRS315 (CEN LEU2), and pR5316 (CEN URA3) [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)]; YCplac111 (CEN LEU2) and YC- plac22 (CEN TRP1) [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)]; pDL242 carries the CAL1/10 promoter (pr) regulating PKC1-R398A [M. Watanabe, C. Y. Chen, D. E. Levin, J. Biol. Chem. 269, 16829 (1994)]; pL914 carries GAL1/10pr-RHO1-Q68H [H. Qadota et al., Science 272, 279 (1996)]; p2107 carries GAL1/10pr-STE4 in vector pRS315; pGFP-CS5- CTM carries the GAL1/10pr-GFP-STES-CTM [P. M. Pryciak and F. A. Huntress, Genes Dev. 12, 2684 (1998)]; p2087, CEN TRP1 plasmid, carries GAL1/ 10pr-STE11-4; pNC252, CEN URA3 plasmid, carries GAL1/10pr-STE12. p3S6 carries GAL1/10pr-BNI1ΔN (20) in vector pRS316; pJB290 (CEN LEU2) carries a FUS3 kinase dead aliele, fus3-K42R (8); and p3019 (CEN LEU2) carries FG:TY1-lacZ (8). V85, a YC- plac111-based plasmid containing the lacZ gene, was used for reporter construction. Promoter sequences were amplified by polymerase chain reaction (PCR) from W303-1A (MATa ura3-1 leu2-3, 112 his3-11, 15 trp1-1 ade2-1 can1-100) genomic DNA. The 3′ downstream primer included the start codon of the gene designed for an in-frame fusion with lacZ. The 5′ upstream primer determined the size of the promoter-containing PCR product: YLR042C, 600 base pairs (bp); PGU1, 743 bp; FUS3, 690 bp; KSS1, 791 bp; FUS1, 834 bp; FIG1, 800 bp; SVS1, 793 bp; YPL192C, 800 bp.
69. Plasmids described previously include the following: pRS314 (CEN TRP1), pRS315 (CEN LEU2), and pR5316 (CEN URA3) [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)]; YCplac111 (CEN LEU2) and YC-plac22 (CEN TRP1) [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)]; pDL242 carries the CAL1/10 promoter (pr) regulating PKC1-R398A [M. Watanabe, C. Y. Chen, D. E. Levin, J. Biol. Chem. 269, 16829 (1994)]; pL914 carries GAL1/10pr-RHO1-Q68H [H. Qadota et al., Science 272, 279 (1996)]; p2107 carries GAL1/10pr-STE4 in vector pRS315; pGFP-CS5- CTM carries the GAL1/10pr-GFP-STES-CTM [P. M. Pryciak and F. A. Huntress, Genes Dev. 12, 2684 (1998)]; p2087, CEN TRP1 plasmid, carries GAL1/ 10pr-STE11-4; pNC252, CEN URA3 plasmid, carries GAL1/10pr-STE12. p3S6 carries GAL1/10pr-BNI1ΔN (20) in vector pRS316; pJB290 (CEN LEU2) carries a FUS3 kinase dead aliele, fus3-K42R (8); and p3019 (CEN LEU2) carries FG:TY1-lacZ (8). V85, a YC- plac111-based plasmid containing the lacZ gene, was used for reporter construction. Promoter sequences were amplified by polymerase chain reaction (PCR) from W303-1A (MATa ura3-1 leu2-3, 112 his3-11, 15 trp1-1 ade2-1 can1-100) genomic DNA. The 3′ downstream primer included the start codon of the gene designed for an in-frame fusion with lacZ. The 5′ upstream primer determined the size of the promoter-containing PCR product: YLR042C, 600 base pairs (bp); PGU1, 743 bp; FUS3, 690 bp; KSS1, 791 bp; FUS1, 834 bp; FIG1, 800 bp; SVS1, 793 bp; YPL192C, 800 bp.
70. Plasmids described previously include the following: pRS314 (CEN TRP1), pRS315 (CEN LEU2), and pR5316 (CEN URA3) [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)]; YCplac111 (CEN LEU2) and YC- plac22 (CEN TRP1) [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)]; pDL242 carries the CAL1/10 promoter (pr) regulating PKC1-R398A [M. Watanabe, C. Y. Chen, D. E. Levin, J. Biol. Chem. 269, 16829 (1994)]; pL914 carries GAL1/10pr-RHO1-Q68H [H. Qadota et al., Science 272, 279 (1996)]; p2107 carries GAL1/10pr-STE4 in vector pRS315; pGFP-CS5- CTM carries the GAL1/10pr-GFP-STES-CTM [P. M. Pryciak and F. A. Huntress, Genes Dev. 12, 2684 (1998)]; p2087, CEN TRP1 plasmid, carries GAL1/ 10pr-STE11-4; pNC252, CEN URA3 plasmid, carries GAL1/10pr-STE12. p3S6 carries GAL1/10pr-BNI1ΔN (20) in vector pRS316; pJB290 (CEN LEU2) carries a FUS3 kinase dead aliele, fus3-K42R (8); and p3019 (CEN LEU2) carries FG:TY1-lacZ (8). V85, a YC- plac111-based plasmid containing the lacZ gene, was used for reporter construction. Promoter sequences were amplified by polymerase chain reaction (PCR) from W303-1A (MATa ura3-1 leu2-3, 112 his3-11, 15 trp1-1 ade2-1 can1-100) genomic DNA. The 3′ downstream primer included the start codon of the gene designed for an in-frame fusion with lacZ. The 5′ upstream primer determined the size of the promoter-containing PCR product: YLR042C, 600 base pairs (bp); PGU1, 743 bp; FUS3, 690 bp; KSS1, 791 bp; FUS1, 834 bp; FIG1, 800 bp; SVS1, 793 bp; YPL192C, 800 bp.
71. Plasmids described previously include the following: pRS314 (CEN TRP1), pRS315 (CEN LEU2), and pR5316 (CEN URA3) [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)]; YCplac111 (CEN LEU2) and YC- plac22 (CEN TRP1) [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)]; pDL242 carries the CAL1/10 promoter (pr) regulating PKC1-R398A [M. Watanabe, C. Y. Chen, D. E. Levin, J. Biol. Chem. 269, 16829 (1994)]; pL914 carries GAL1/10pr-RHO1-Q68H [H. Qadota et al., Science 272, 279 (1996)]; p2107 carries GAL1/10pr-STE4 in vector pRS315; pGFP-CS5- CTM carries the GAL1/10pr-GFP-STES-CTM [P. M. Pryciak and F. A. Huntress, Genes Dev. 12, 2684 (1998)]; p2087, CEN TRP1 plasmid, carries GAL1/ 10pr-STE11-4; pNC252, CEN URA3 plasmid, carries GAL1/10pr-STE12. p3S6 carries GAL1/10pr-BNI1ΔN (20) in vector pRS316; pJB290 (CEN LEU2) carries a FUS3 kinase dead aliele, fus3-K42R (8); and p3019 (CEN LEU2) carries FG:TY1-lacZ (8). V85, a YC- plac111-based plasmid containing the lacZ gene, was used for reporter construction. Promoter sequences were amplified by polymerase chain reaction (PCR) from W303-1A (MATa ura3-1 leu2-3, 112 his3-11, 15 trp1-1 ade2-1 can1-100) genomic DNA. The 3′ downstream primer included the start codon of the gene designed for an in-frame fusion with lacZ. The 5′ upstream primer determined the size of the promoter-containing PCR product: YLR042C, 600 base pairs (bp); PGU1, 743 bp; FUS3, 690 bp; KSS1, 791 bp; FUS1, 834 bp; FIG1, 800 bp; SVS1, 793 bp; YPL192C, 800 bp.
72. Plasmids described previously include the following: pRS314 (CEN TRP1), pRS315 (CEN LEU2), and pR5316 (CEN URA3) [R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989)]; YCplac111 (CEN LEU2) and YC- plac22 (CEN TRP1) [R. D. Gietz and A. Sugino, Gene 74, 527 (1988)]; pDL242 carries the CAL1/10 promoter (pr) regulating PKC1-R398A [M. Watanabe, C. Y. Chen, D. E. Levin, J. Biol. Chem. 269, 16829 (1994)]; pL914 carries GAL1/10pr-RHO1-Q68H [H. Qadota et al., Science 272, 279 (1996)]; p2107 carries GAL1/10pr-STE4 in vector pRS315; pGFP-CS5-CTM carries the GAL1/10pr-GFP-STES-CTM [P. M. Pryciak and F. A. Huntress, Genes Dev. 12, 2684 (1998)]; p2087, CEN TRP1 plasmid, carries GAL1/ 10pr-STE11-4; pNC252, CEN URA3 plasmid, carries GAL1/10pr-STE12. p3S6 carries GAL1/10pr-BNI1ΔN (20) in vector pRS316; pJB290 (CEN LEU2) carries a FUS3 kinase dead aliele, fus3-K42R (8); and p3019 (CEN LEU2) carries FG:TY1-lacZ (8). V85, a YC-plac111-based plasmid containing the lacZ gene, was used for reporter construction. Promoter sequences were amplified by polymerase chain reaction (PCR) from W303-1A (MATa ura3-1 leu2-3, 112 his3-11, 15 trp1-1 ade2-1 can1-100) genomic DNA. The 3′ downstream primer included the start codon of the gene designed for an in-frame fusion with lacZ. The 5′ upstream primer determined the size of the promoter-containing PCR product: YLR042C, 600 base pairs (bp); PGU1, 743 bp; FUS3, 690 bp; KSS1, 791 bp; FUS1, 834 bp; FIG1, 800 bp; SVS1, 793 bp; YPL192C, 800 bp.
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84. 10(expression ratio) as color, where red is up-regulation, green is down-regulation, and black represents no change, as illustrated in the color bar of Fig. 5A (gray denotes no reliable data). Each row in the display represents an experiment condition pair, and each column corresponds to a gene. Rows and columns are displayed in the order given by the clustering output trees in the two dimensions. The clustering operation and this rearrangement are independent and noninterfering between the two dimensions. Not all genes are retained in the clustering analysis, only those that had a minimum amplitude of response of threefold at a 99% confidence level (4, 11) in at least two experiments. This focuses attention on the most informative genes, but it does not bias the clustering result toward any a priori assumptions about the mechanism. The growth conditions, strains, and plasmids for the experiments included in Fig. 5 were as described in (10, 28, 29). The 46 experiments whose response profiles were clustered in Fig. 5 include those described in Figs. 2 and 3 as well as the experiments described below. Experiments 1 to 5, 9, 10, 26, and 27 involved various concentrations of α-factor, each for a 30-min treatment of R276 (wt) cells. Experiments 2, 5, 6, 7, 11 to 13, and 28 correspond to a kinetic analysis of α-factor response for R276 (wt) cells (Fig. 2F). Experiments 2 and 5 involved identical α-factor treatments for R276 (wt) cells, which provided the data for the "wt ± 50 nM αF, 30 min" experiment shown in the correlation plots of Figs. 2 and 3. Experiments 14, 15, and 16 (Fig. 2H) correspond to the α-factor response of bni1Δ cells. Experiments 17, 18, 24, 25, and 35 to 37 involved galactose induction of STE4, STE5-CTM (GFP-STE5-CTM), STE12, STE11-4, BNI1ΔN, PKC1-R398A, and RHO1-Q68H (Fig. 21) in Y1469 (wt) cells, respectively. Experiments corresponding to comparisons of R276 (wt) cells versus mutant cells are as follows: sst2Δ, 21; rst1Δ rst2Δ, 22; fus3Δ, 32; bni1Δ, 34; fus3Δ kss1Δ, 38; ste7Δ, 39; ste12Δ, 40; ste18Δ, 41; ste4Δ, 42; ste5Δ, 43; ste11Δ, 44. Experiments corresponding to treatments of mutants with 50 nM α-factor for 30 min are as follows: kss1Δ, 8; fus3Δ, 19; far1Δ, 20; ste20Δ, 29; rst1Δ rst2Δ, 30; fus3-K42R, 31. Direct comparisons of wt versus mutant strains, both of which were treated with 50 nM α-factor, identified genes induced or repressed specifically in the mutant strain, as follows: R276 (wt) versus fus3Δ (30 min), 33; R276 (wt) versus tec1Δ (30 min), 45; R276 (wt) versus tec1Δ (120 min), 46. Experiment 23 involved treating hog1Δ cells with 1 M sorbitol for 2 hours.
85. Supported by Rosetta lnpharmatics, and grants to C.B. from the Natural Sciences and Engineering Research Council of Canada and the National Cancer Institute of Canada. We thank G. Fink, L. Hartwell, I. Herskowitz, H. Madhani, P. Pryciak, and S. O'Rourke for plasmids and yeast strains; F. Naider for synthetic a-factor; T. R. Ward and S. Whelen for yeast strains; K. Kennedy and A. Tong for assistance with β-galactosidase experiments and figures; and M. Ashby, A. Breitkreutz, H. Bussey, N. Davis, L. Harrington, L. Hartwell, P. Pryciak, J. Rine, T. Roemer, and P. Young for comments on the manuscript.