Genetic Dissection of Transcriptional Regulation in Budding Yeast Rachel B. Brem, Gael Yvert, Rebecca Clinton, Leonid Kruglyak

Probe pairs that serve as good genetic markers were selected as follows. First, data from 3 hybridization experiments for each parent strain were filtered by removing probe pairs with saturated intensity (> 40,000 units) on any chip, and probe pairs with a low perfect match (pm) intensity (< 3,500 units) or a low difference between pm and mismatch (mm) intensities (<2,000 units) in any BY experiment. All further analyses were performed on log(pm/mm) values. To correct for global chip effect, each distribution was centered by subtracting its mode. Following Efron et al. (1) we formed two scores for each probe pair: Z = D / (A + S), and z = d / (a + s), where D is the average of the 3 differences BY_j - RM_j (j = 1 to 3), S is their standard deviation, and A is the 90^th percentile of all S values; d is the average of the four differences BY₂ - BY₁, BY₃ - BY₂, RM₂ - RM₁, RM₃ - RM₂, s is their standard variation, and a is the 90^th percentile of all s values. As expected, we observed that the Z distribution was skewed toward high values compared to the z distribution, and we selected 6444 probe pairs with Z>0.5 (only 75 probe pairs had a higher z). For these probe pairs, we analyzed all 49 DNA hybridization experiments (3 for each parent, 4 for a tetrad, and 39 for additional segregants). We removed probe pairs having missing values in more than 10 experiments, normalized log(pm/mm) values by dividing by the mode of their distribution, and for each probe pair, performed k-means clustering (k = 2) of the experiments. Probes were retained when this clustering separated the parental scores (all BY chips in the "high" cluster, all RM chips in the "low" cluster). 4702 probe pairs remained after this step. For each of these, a gaussian distribution was fitted to each of the 2 clusters and the probability of having a BY origin given a score x was computed as pBY = H(x) / ( L(x) + H(x) ), where L(x) and H(x) are the probabilities of drawing x from the gaussians fitted to the low and high clusters, respectively. Genotypes were called as follows: "BY" if pBY > 0.965, "RM" if pBY < 0.035, "ambiguous" otherwise. The list of markers was then filtered to remove probe pairs with more than 10 ambiguous genotypes, probe pairs with <2 instances of either genotype in the 43 segregants (which is highly unlikely under random segregation), and probe pairs corresponding to Ty elements (because their exact location is unknown in the strains used). Finally, 16 markers were removed because their genetic map location in the cross disagreed with their physical location. The genotypes used in linkage analysis were based on the resulting genetic map of 3312 markers. Genotypes were corrected when they were in disagreement with two flanking markers located within 20kb (~7cM).

Reference

1. Efron B, Tibshirani R, Goss V, Chu G. Microarrays and their use in a comparative experiment. Technical Report No. 213. Stanford University, 2000.

Genes in Groups 1-8

Group 1

YLR042C
YNL327W EGT2 Cell wall protein. Putatively involved in glucan metabolism
YNL208W
YOR264W DSE3 Daughter Specific Expression
YER124C DSE1 Daughter Specific Expression
YPR106W ISR1 protein kinase
YJL078C PRY3 Similar to plant PR-1 class of pathogen related proteins
YGR041W BUD9 involved in bud-site selection
YHR117W TOM71 71-kDa component of the protein translocase of the outer membrane of mitochondria
YDR119W Member of the 14-spanner drug:[H+] antiporter (DHA14) family of multidrug-resistance (MFS-MDR) proteins in the major facilitator superfamily (MFS)
YNL066W SUN4 Protein involved in the aging process
YGL028C SCW11 soluble cell wall protein
YJR147W HMS2 heat shock transcription factor homolog
YDL055C PSA1 mannose-1-phosphate guanyltransferase; mutants exhibit failure of cell separation and/or cytokinesis, impaired bud growth and bud-site selection
YOR263C
YNR067C DSE4 Daughter Specific Expression
YOR342C
YHR143W DSE2 Daughter Specific Expression

Group 2

YLR089C Putative mitochondrial alanine aminotransferase
YBR068C BAP2 probable amino acid permease for leucine, valine, and isoleucine
YGL114W Protein member of the oligopeptide transporter (OPT) family of membrane transporters
YMR096W SNZ1 encodes highly conserved 35 kDa protein that shows increased expression after entry into stationary phase
YNL104C LEU4 alpha-isopropylmalate synthase (2-Isopropylmalate Synthase)
YFR030W MET10 subunit of assimilatory sulfite reductase
YJR016C ILV3 dihydroxyacid dehydratase
YLR348C DIC1 mitochondrial dicarboxylate transport protein
YGL009C LEU1 isopropylmalate isomerase
YKR071C
YLR303W MET17 O-Acetylhomoserine-O-Acetylserine Sulfhydralase
YGL010W
YHR208W BAT1 branched-chain amino acid transaminase, highly similar to mammalian ECA39, which is regulated by the oncogene myc
YGL125W MET13 putative methylenetetrahydrofolate reductase (mthfr)
YMR108W ILV2 acetolactate synthase
YOR225W
YER073W ALD5 mitochondrial Aldehyde Dehydrogenase
YOR108W LEU9 Alpha-isopropylmalate synthase II
YGR146C
YKL120W OAC1 mitochondrial oxaloacetate transport protein
YOR226C ISU2 Iron-sulfur cluster nifU-like protein

Group 3

YLR041W
YIL015W BAR1 extracellular protease synthesized in a-cells that cleaves and inactivates alpha factor
YDR459C
YFL026W STE2 alpha-factor pheromone receptor; seven-transmembrane domain protein
YKL177W
YKL209C STE6 ABC transporter, glycoprotein, component of a-factor secretory pathway
YCR038C BUD5 GTP/GDP exchange factor for Rsr1 protein
YNL145W MFA2 mating a-factor pheromone precursor
YJL170C ASG7 An a-specific gene that is induced to a higher expression level by alpha factor
YFL027C GYP8 induced 28-fold by alpha mating pheromone
YNL036W NCE103 involved in non-classical protein export pathway
YCL065W
YJR004C SAG1 alpha-agglutinin
YLR040C in MAT expression cluster, Spellman et al.
YGL089C MF(alpha)2 alpha mating factor
YNL146W induced by mating pheromones, Erdman et al.
YDR461W MFA1 a-factor mating pheromone precursor
YKL178C STE3 a factor receptor
YGL032C AGA2 adhesion subunit of a-agglutinin
YCR097W-A
YCR097W a1 haploid specific gene repressor
YCL067C HMLAlpha2 Mating type protein alpha-2
YMR173W-A
YCR096C a2 Regulatory protein MATa2p (no known function); sequence is the same as the last 119 residues of MATalpha2p
YFL025C BST1 negative regulator of COPII vesicle formation
YGL090W LIF1 interacts with DNA ligase protein
YPL187W MF(alpha)1 mating factor alpha
YCL066W HMLalpha1 transcription factor involved in the regulation of alpha-specific genes

Group 4

YOL126C MDH2 cytosolic malate dehydrogenase
YJL130C URA2 carbamoyl-phophate synthetase, aspartate transcarbamylase, and glutamine amidotransferase
YEL052W AFG1 ATPase family gene
YKL216W URA1 dihydroorotate dehydrogenase
YLR420W URA4 dihydrooratase
YOL143C RIB4 6,7-dimethyl-8-ribityllumazine synthase (DMRL synthase)
YOL114C

Group 5

YMR015C ERG5 cytochrome P450 involved in C-22 denaturation of the ergosterol side-chain
YDR044W HEM13 Coproporphyrinogen III oxidase
YNL111C CYB5 cytochrome b5
YLR100W ERG27 3-keto sterol reductase
YJL048C
YLR205C HMX1 Homology to heMe oXygenases
YGR235C
YGR049W SCM4 suppressor of cdc4 mutations
YGR234W YHB1 Flavohemoglobin
YKR046C Protein possibly involved in respiration
YHR072W ERG7 2,3-oxidosqualene-lanosterol cyclase
YER044C ERG28 Protein involved in synthesis of ergosterol
YNR043W MVD1 mevalonate pyrophosphate decarboxylase
YNL156C
YJR048W CYC1 iso-1-cytochrome c
YEL058W PCM1 Phosphoacetylglucosamine Mutase
YDR502C SAM2 S-adenosylmethionine synthetase
YDL086W Putative carboxymethylenebutenolidase
YMR208W ERG12 mevalonate kinase
YEL039C CYC7 iso-2-cytochrome c
YMR009W
YLR101C
YML126C ERG13 3-hydroxy-3-methylglutaryl coenzyme A synthase
YLR153C ACS2 acetyl-coenzyme A synthetase
YEL034W HYP2 Translation initiation factor eIF-5A
YPL028W ERG10 acetoacetyl CoA thiolase
YMR134W Protein involved in iron metabolism
YJR047C ANB1 anaerobically expressed form of translation initiation factor eIF-5A

Group 6

YER190W YRF1-2 Y'-helicase protein 1
YBL113C member of the subtelomeric Y' family of putative helicases
YJL225C member of the subtelomeric Y' family of putative helicases
YNL339C YRF1-6 Y'-helicase protein 1
YLL066C member of the subtelomeric Y' family of putative helicases
YML133C member of the subtelomeric Y' family of putative helicases
YHL050C member of the subtelomeric Y' family of putative helicases
YDR545W YRF1-1 Y'-helicase protein 1
YIL177C member of the subtelomeric Y' family of putative helicases
YGR296W YRF1-3 Y'-helicase protein 1
YPL283C YRF1-7 Y'-helicase protein 1
YHR219W member of the subtelomeric Y' family of putative helicases
YLR467W YRF1-5 Y'-helicase protein 1
YLL067C member of the subtelomeric Y' family of putative helicases
YEL077C member of the subtelomeric Y' family of putative helicases
YFL068W member of the subtelomeric Y' family of putative helicases

Group 7

YKL127W PGM1 phosphoglucomutase, minor isoform
YER114C BOI2 involved in bud formation, has SH3 domain
YOR009W TIR4 Member of the seripauperin (PAU) family of possible cell wall mannoproteins
YER087W Putative prolyl-tRNA synthetase
YBR017C KAP104 karyopherin beta 2, yeast transportin
YBR127C VMA2 vacuolar ATPase V1 domain subunit B (60 kDa)
YKL152C GPM1 Phosphoglycerate mutase
YGL195W GCN1 translational activator of GCN4 through activation of GCN2 in response to starvation
YHR147C MRPL6 Mitochondrial ribosomal protein MRPL6 (YmL6)
YDL178W AIP2 D-Lactate Dehydrogenase (Cytochrome), mitochondrial
YML050W
YMR193W MRPL24 Mitochondrial ribosomal protein MRPL24 (YmL24)
YLR204W QRI5
YDR347W MRP1 37 kDa mitochondrial ribosomal protein
YLR253W
YHR091C MSR1 Arginyl-tRNA synthetase
YBL090W MRP21 Component of the small subunit of mitochondrial ribosomes
YKL138C MRPL31 15.5 kDa mitochondrial ribosomal protein YmL31
YDR511W ACN9
YGR192C TDH3 Glyceraldehyde-3-phosphate dehydrogenase 3
YPR099C
YBR129C OPY1 involved in mating pathway
YMR158W
YER058W PET117 cytochrome c oxidase assembly factor; active in mito.
YLR312W-A MRPL15 Mitochondrial ribosomal protein MRPL15 (YmL15)
YBR120C CBP6 Translational activator of COB mRNA; active in mito.
YKL170W MRPL38 mitochondrial ribosomal protein L14
YNL137C NAM9 putative mitochondrial S4 ribosomal protein
YGL143C MRF1 Mitochondrial polypeptide chain release factor
YPL215W CBP3 involved in cytochrome-c reductase assembly; active in mito.
YDR337W MRPS28 Mitochondrial ribosomal protein MRPS28 (E. coli S15)
YDR494W
YBL038W MRPL16 Mitochondrial ribosomal protein MRPL16
YKL053C-A
YPR100W
YOR200W
YGR165W falls in mitochondrial expression cluster, Hughes et al.
YJL046W
YHR024C MAS2 53 kDa subunit of the mitochondrial processing protease
YBR146W MRPS9 Probable mitochondrial ribosomal protein S9
YJR101W RSM26 protein of the small subunit of the mitochondrial ribosome
YMR157C falls in mitochondrial expression cluster, Hughes et al.
YBR122C MRPL36 Mitochondrial ribosomal protein MRPL36 (YmL36)
YOR150W MRPL23 mitochondrial ribosomal protein of the large subunit
YGR076C MRPL25 Mitochondrial ribosomal protein MRPL25 (YmL25)
YLR239C LIP2; active in mito.
YOL023W IFM1 mitochondrial initiation factor 2
YHR038W FIL1 Putative mitochondrial ribosome recycling factor
YNL252C MRPL17 mitochondrial ribosomal protein of the large subunit
YJL096W MRPL49 mitochondrial ribosomal protein of the large subunit
YCR003W MRPL32 Mitochondrial ribosomal protein MRPL32 (YmL32)
YHR059W FYV4
YKL169C
YEL050C RML2 mitochondrial ribosomal protein L2 of the large subunit
YNL005C MRP7 Mitochondrial ribosomal protein MRP7 (YmL2) (E. coli L27)
YDR261C EXG2 Exo-1,3-b-glucanase
YPL118W MRP51 Component of small subunit of the mitochondrial ribosome
YDR237W MRPL7 Mitochondrial ribosomal protein MRPL7 (YmL7)
YGR219W
YPL184C
YNR040W
YMR024W MRPL3 Mitochondrial ribosomal protein MRPL3 (YmL3)
YDR175C RSM24 protein of the small subunit of the mitochondrial ribosome
YDR041W RSM10 protein of the small subunit of the mitochondrial ribosome
YDR493W
YJR113C RSM7 protein of the small subunit of the mitochondrial ribosome
YGR220C MRPL9 Mitochondrial ribosomal protein MRPL9 (YmL9) (E. coli L3) (human MRL3)
YDR322W MRPL35 Mitochondrial ribosomal protein MRPL35 (YmL35)
YDR197W CBS2 cytochrome b translational activator; active in mito.
YFR009W GCN20 Member of ATP-binding cassette (ABC) family of proteins
YNL004W HRB1 hypothetical RNA-binding protein
YNR020C
YIL093C RSM25 protein of the small subunit of the mitochondrial ribosome
YER182W
YLR069C MEF1 mitochondrial elongation factor G-like protein
YCR024C Asn-tRNA synthetase, mitochondrial
YGL129C RSM23 protein of the small subunit of the mitochondrial ribosome
YNL177C falls in mitochondrial expression cluster, Hughes et al.
YNL284C MRPL10 Mitochondrial ribosomal protein MRPL10 (YmL10)
YGL107C
YJL180C ATP12 (putative) involved F1F0-ATPase complex assembly; active in mito.
YMR098C
YOL042W NGL1 Protein with possible DNAse/RNAse function
YDR375C BCS1 Mitochondrial ATPase (AAA family)
YBR185C MBA1 involved in assembly of mitochondrial respiratory complexes
YHR116W falls in mitochondrial expression cluster, Hughes et al.
YNL122C
YBR262C
YOR354C MSC6
YPL097W MSY1 Tyrosyl-tRNA synthetase, mitochondrial
YIL070C MAM33 Mitochondrial protein involved in respiration
YJL063C MRPL8 Mitochondrial ribosomal protein MRPL8 (YmL8) (E. coli L17)
YNL081C
YKL167C MRP49 16 kDa mitochondrial ribosomal large subunit protein

Group 8

YGR052W
YOL109W ZEO1 Overexpression causes resistance to Zeocin
YNL234W Hemoprotein with similarity to mammalian globins
YDR533C
YML128C MSC1 Meiotic Sister Chromatid recombination-elevated phenotype
YIR038C GTT1 Glutathione transferase
YDL204W
YGR043C may be involved in signal transduction, has strong similarity to Tal1p
YOR040W GLO4 Mitochondrial glyoxylase-II
YLR178C TFS1 CDC25-dependent nutrient- and ammonia-response cell cycle regulator
YKR049C
YJL116C NCA3 involved in regulating expression of F0F1 ATPase subunits
YDR453C Cytoplasmic thiol peroxidase
YPR184W GDB1 the enzyme that debranches the glycogen having a glucanotranferase + 1-6amyloglucosidase activity
YER150W SPI1 similar to Sed1; highly expressed in stationary phase
YOR173W
YDR070C
YDL223C
YDR406W PDR15 probable multidrug resistance transporter