FIGURE 24: tup1 Repressive Effects
D N A TECHNIQUES
Preparation of plasmid D N A
The m ajority of plasm id preparations were perform ed using a n A utogen plasm id preparation machine. Plasm ids used for in v itr o
tran scrip tio n /tran slatio n , were prepared using a Q uiagen m axiprep kit. For m anual m iniprep DNA a Tris / ED TA /N P40/SD S buffer was used to d isru p t transform ed bacteria. Plasmid DNA was precipitated by the addition of
am m o n iu m sulphate and cold ethanol followed by 10 m inutes of cetrifugation at lOK.
R estriction digestion
Quick digests to check constructs were perform ed in a 20pl v o lu m e in a m icrotitre using 5 units of restriction enzym e in the appropriate buffer (Boehringer) and w ith an incubation tim e of 1-2 hours. For digestion of vectors for use in subcloning, approxim ately Ipg of vector was digested for 3-16 hours w ith approxim ately 20 units of enzym e in a volum e of 50|uil. For digestion of the ends of FCR products, the PCR reaction was first gel pu rified .
P h o p h atasin g vectors
Ip l of calf intestinal alkaline phosphatase was added directly to th e restriction digest after the incubation period. The reaction was incubated for a further 2-16 hours.
Ligation reactions
All ligation reactions were perform ed in a voum e of 20\i\ using 10 un its of T4 DNA ligase (NEB). Sticky end ligations containing lOng vector DN A and 200ng foreign DNA in a 20|il reaction m ixture were incubated at room tem p eratu re for 1-3 hours in a ligation buffer (lOX Buffer: SOOmM Tris-HCl pH7.8, lOOmM MgCl2, lOmM ATP, 200mM D ithiothreitol). B lu n t end ligations w ere incubated longer, typically overnight at 16 °C.
D N A generation by PCR
For generating fragm ents for subcloning, high fidelity Vent or Pfu polym erases were used. Typically a lOOpl reaction was set up in buffer supplied w ith the enzyme. A standard protocol w as used for all fram ents of IKbp in length or less: 5mins@ 94°C, 30x(l m in at 94°C, 1 min@50°C, 1 min@72°C) and 5 m ins at 72°C. For generating DNA probes, Taq polym erase produced by the I.C.R.F. central services was used, in a buffer of lOmM Tris.Cl ph8.3, 50mM KCl, 1.5mM MgCl2, 0.1% gelatin.
D N A sequencing
All sequencing was perform ed using either an ABI or an ALF autom ated sequencer, which w ere operated by the I.C.R.F sequencing service.
A n n ealin g single stranded oligonucleotides
Ip g of each oligo dissolved in H2O was added to a m icrofuge tube, and placed in a beaker of boiling water. The w ater w as allowed to cool slow ly to room tem perature.
End labelling double stranded oligonucleotides for EMSA probes
5ng of double stranded oligo w ere mixed w ith 12 pi H2O, 2pl lOx PNK buffer (700mM Tris.Cl pH7.6, lOOmM MgCl2 5mM DTT), 3|tl g32P dATP an d ip l of polynucleotide kinase. The reaction was incubated at 37”C for 20 m inutes. U nincorporated nucleotides w ere rem oved using a Sephadex G-50 c o lu m n .
OLIGONUCLEOTIDES
YEAST EXPRESSION CONSTRUCTS: N d e l/N o tl ligations w /R ep81
N deA tfl GGCCGGCCATATGTCCCCGTCTCCCG N d e A tfllS GGCGTTCCATATGCCTTCAGGTGTGCCTCCTGCG N deA tfl43 GGCGTTCCATATGTTAAGTGGAGCTCCAGGTATTGC N deA tfl75 GGCGTTCCATATGTATGATGCTACTCTTCGTCCTG NdeAtf294 GGCGTTCCATATGTCCTCTGAAAACGATCAAGCAGC NdeAtf363 GGCGTTCCATATGCCATCTGTTTACGGCG NdeAtf443 GGCGTTCCATATGAATGGTAAGGCTTCTAGTGAATCTG Atf566Not CCGCGTTAGCGGCCGCGGTACCCTAAATTGATTC
GST CONSTRUCTS: N col /X hol ligations w/pGEX-KG
N co lA tflf CGGCCGCCATGTCCCCGTCTCCCG NcoAtf 81f CGGCCGCCATGGTAGGCTCCTTGAAGCTGGAT NcoAtf 87f CGGCCGCCATGGATTACGAACCCAACC NcoAtf 94f CGGCCGCCATGGAGCATTCATTTGGCTCTA N coA tfl21f CGGCCGCCATGGCTTCAGGTGTGCCTCCTG N coA tfl38f CGGCCGCCATGGCCATAGCATCACCTGATA N co lA tfl9 0 f GCCGGGATCCGATGCATCTGCAGCAGCTAGATG Atf566Xhol GTAGTTTAGCGGCCGCTAGTACCCTAAATTGAT A tf294Xhol GTAGTTTAGCGGCCGCGCTGAAGCTTTTGCTAC
Atfl48Sacl Endogenous S a d site used to m ake GST (N co l/S acl)
P aplN co CGGCCGCCATGTCCGGACAAACTGAGACGTT
PaplX ho CGCCTCGAGATCGTCTTTCATCGAG
N coPcrl GCCGCCATGGCTGCCAAAAAAAAAGAAGTTG
PcrlXho CCGCTCGAGATGGGCCCACCAAGGGA
LexA FUSION OLIGOS FOR LacZ ASSAY: N o tl /S p e ltB a m H li ligations
N otA tfl CGACGACCTAGCGGCCGCTATGTCCCCGTCTCCCGTCAATAC N otA tf 144 CGACGACCTAGCGGCCGCTTTAAGTGGAGCTCCAGGTATTGC N otA tfl54 CGACGACCTAGCGGCCGCTTTGGGCTATCCTGCTTGGTC N otA tfl75 CGACGACCTAGCGGCCGCTTATGATGCTACTCTTCGTCCTGAC NotAtf295 CGACGACCTAGCGGCCGCTTCCTCTGAAAACGATCAAGCAGCA NOT393 CGACGACCTAGCGGCCGCTTCCACAAATCTACCGÇAAAAAACTTC Atf295Bam CGGGGATCCGATGGGTTGCTGAAGCTTTTGC
Atf393 endogenous B am H l site used
Atf566Spe GGACTAGTGTACCCTAAATTGATTCTTTG EMSA PROBE OLIGOS
g p d l l GAACGCGTTTGCGACCAAATGG g p d lb CAAATCCTTGGGATCCATGTC flpflPLDf GGTAGTTTACGTAATACTTGGT aptlFLDb ACCAAGTATTACGTAAACTACC a p llK F li GAATAGGATTAGTCAGAAATGG ap flA P lb CCATTTCTGACTAATCCTATTC
E4 CRE/M E4 CRE/SV40 API from Takeda 1995 NORTHERN PROBE OLIGOS
g p d l f CGCGAATGACCCTTTTTTGTCAGG g p d lb GGTAAAAGAGAAGAGCGGG hsp9f ATGTCTGATCCCGCAAGAAAGTC hsp9b CTTGTCATGAGCCTCTTGAGAGGT hsp 104b C AGCAAATTGGCAGCGTCCATGCC ACCT hsplOâl ATGGCTGATTATCCTTTTACTGACA e n a lf ATGGTAACCATTAATATCTCGAATC ena lb GCAAATGTCAGTAACACCTCCTAAGG e n o l f ATGGCCATTCAGAAAGTCTTTGCTCG e n o lb TGGCGGTACGAAGACGCTTGACGTTG g lo K ATGGCTTCTACTACTGACATGA g lo lb TACTTCTGTTCAATGACCTCAAC
dakl f ATGG ATA AGC ACTTT ATC A ACG ATCCT
da klb CGATAGCTTCAACAGGTTGGTCAATC
gcyl f ATGTC A ATT A A AG AATTTGCTA AG A ACG
g cylb AGCATGAGTTTGTTTAGGCGTGCCA
f b p l i CTCGACGAAATCGACACTGACATTGTCAC
n t h l f CGTGGATACAGAAGCCATTTCAAATGATG n t h l b GGCCACCAGGAACGACAAATGGCACAC w is2f TCGACAACGTAGTTCCCAAAAGTGTC wis2b GCAGGATCGTTACCAGCCTTAGCA tp s K ATGTCGGATGCTCATGATACCA tp slb CAAGCTCATTGACAACGGCTCTAA tps2f TGCAGGGTCTGCGATACACCAGTC tps2b GAACAATTGGAACCGGCTGATTA t s l l l GGAGATTGACTCGACGAATGCACA fs/2b CGATGGAGATCGGAAGGGCAATCA pyp2f ATGCTCCATCTTCTGTCTAAAGAC pyp2b CATTCTGTGCATTGCTATCACGT g p x if ATGTCTCATTTCTACGACTTGGC g p x lb GACACTCTCGATATCGTTTTCG
REFERENCES
Aiba H, Yamada H, Ohmiya R, Mizuno T (1995) The osm o-inducible gp d l+ gene is a target of the signaling pathw ay involving W isl M A P-kinase kinase in fission yeast. FEBS Lett 376, 199-201
A khtar N, Blomberg A, A dler L (1997) O sm oregulation and p ro te in expression in a pbs2delta m u tan t of Saccharomyces cerevisiae d u rin g adaptation to hypersaline stress. FEBS Lett 403, 173-80
Albertyn J, H o h m an n S, Thevelein JM, Prior BA (1994) GPDl, w h ich encodes glycerol-3-phosphate dehydrogenase, is essential for grow th u n d e r osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osm olarity glycerol response pathw ay. Mol Cell Biol 14, 4135-44
A lepuz PM, C unningham KW, Estruch F (1997) Glucose repression affects ion hom eostasis in yeast through the regulation of the stress-activated E N A l gene Mol. Microbiol. 26, 91-8
Altschul, Stephen F., W arren Gish, Webb Miller, Eugene W. Myers, and David J. L ipm an (1990). Basic local alignm ent search tool. J. Mol. Biol.
215:403-10.
Bardwell L, Cook JG, Voora D, Baggott DM, M artinez AR, T horner J (1998) Repression of yeast Stel2 transcription factor by direct binding of u n p h osphorylated Kssl MAPK and its regulation by the Ste7 MEK. Genes Dev 12, 2887-98
Bardwell L, Cook JG, Chang EC, Cairns BR, Thorner J (1996) Signaling in the yeast pherom one response pathway: specific and high-affinity interaction of the m itogen activated protein (MAP) kinases Kssl and Fus3 w ith th e upstream MAP kinase kinase Ste7. Mol Cell Biol 16, 3637-50
Binetruy B, Smeal T, Karin M (1991) Ha-Ras augm ents c-Jun activity and stim ulates phosphorylation of its activation dom ain. N ature 351, 122-7 Blomberg A, A dler L (1992) Physiology of osm otolerance in fungi. A d v
Microb Physiol 33, 145-212
Bocco JL, Bahr A, Goetz J, Hauss C, Kallunki T, Kedinger C, Chatton B (1996) In vivo association of ATFa w ith JNK /SA P kinase activities. Oncogene 12, 1971-80
Bokemeyer D, Sorokin A, Yan M, A hn NG, Tem pleton DJ, D unn MJ (1996) Induction of m itogen-activated protein kinase phosphatase 1 by the stress- activated protein kinase signaling pathw ay but not by extracellular signal- regulated kinase in fibroblasts. J.Biol Chem 271, 639-42
Borsch-Haubold AG, K ram er RM, W atson SP (1997) Phosphorylation and activation of cytosolic phospholipase A2 by 38-kDa m itogen-activated protein kinase in collagen-stim ulated hum an platelets. Eur J B io chem 245, 751-9
Bos TJ, B ohm ann D, Tsuchie H, Tjian R, Vogt PK (1988) v-jun encodes a nuclear protein w ith enhancer binding properties of AP-1. Cell 52, 705-12
Boy-Marcotte E, Perrot M, Bussereau F, Boucherie H, Jacquet M (1998) M sn2p and Msn4p control a large num ber of genes induced at the diauxic transition w hich are repressed by cyclic AMP in Saccharomyces cerevisiae. /
Bacteriol 180, 1044-52
Brewster, J.L., de Valoir, T., Dwyer, N.D., W inter, E. & G ustin M.C. (1993) An osm osensing signal transduction pathw ay in yeast. Science 259, 1760-63
Brondello JM, Brunet A, Pouyssegur J, McKenzie FR (1997) The d u al specificity m itogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44M APK cascade. / Biol Chem 272,1368-76
Brown, J.L., Bussey, H. & Stewart, R.C. (1994) Yeast SknZp functions in a eukaryotic two-com ponent regulatory pathw ay. EMBO J. 13, 5186-94
Brunet A, Pouyssegur J (1996) Identification of MAP kinase dom ains by redirecting stress signals into grow th factor responses. Science 272, 1652-5
Buehrer BM, Errede B (1997) C oordination of the m ating and cell integrity m itogen-activated protein kinase pathw ays in Saccharomyces cerevisiae.
M ol Cell Biol 17, 6517-25
Cam ps M, N ichols A, Gillieron C, A ntonsson B, M uda M, Chabert C, Boschert U, A rkinstall S (1998) Catalytic activation of the phosphatase MKP- 3 by ERK2 m itogen-activated protein kinase. Science 280, 1262-5
Cam ps M, Chabert C, M uda M, Boschert U, Gillieron C, A rkinstall S (1998) Induction of the m itogen-activated protein kinase phosphatase MKP3 by nerve grow th factor in differentiating PC12. FEBS Lett 425, 271-6
Canagarajah BJ, Khokhlatchev A, Cobb MH, Goldsm ith EJ (1997) A ctiv atio n m echanism of the MAP kinase ERK2 by dual phosphorylation. Cell 90, 859- 69
Cavigelli, M., Dolfi, F., Claret, F.X. & Karin, M. (1995) Induction of cFos expression th ro u g h JNK-m ediated TCF/Elk-1 phosphorylation. EMBO J. 14, 5957-5964
Chang C, Kwok SF, Bleecker AB, Meyerowitz EM (1993) A rabidopsis ethylene-response gene FTRl: sim ilarity of product to tw o -co m p o n en t regulators. Science 262, 539-44
C hapm an RE, W alter P (1997) Translational attenuation m ediated by a n mRNA intron. Curr Biol 7, 850-9
C hatton B, Bocco JL, Caire M, Hauss C, R eim und B, Goetz J, Kedinger C (1993) Transcriptional activation by the adenovirus larger E la product is m ediated by m em bers of the cellular transcription factor ATE family w h ich can directly associate w ith Ela. Mol Cell Biol 13, 561-70
Chow CW, Rincon M, Cavanagh J, Dickens M, Davis RJ (1997) N u clear accum ulation of NFAT4 opposed by the JNK signal transduction pathw ay.
Science 278, 1638-41
Chu B, Soncin E, Price BD, Stevenson MA, Calderw ood SK (1996) Sequential phosphorylation by m itogen-activated protein kinase and glycogen synthase kinase 3 represses transcriptional activation by h eat shock factor-1. / Biol Chem 271, 30847-57
Clifton AD, Young PR, Cohen P (1996) A com parison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activ atio n by cytokines and cellular stress. FEBS Lett 392, 209-14
Cohen, P. (1997) The search for physiological substrates of MAP and SAP kinases in m am m alian cells. Trends Cell Biol. 7, 353-361.
C onrad KE, Oberwetter JM, V aillancourt R, Johnson GL, G utierrez- H artm ann A (1994) Identification of the functional com ponents of the Ras signaling pathw ay regulating pituitary cell-specific gene expression. M o l
Cell Biol 14, 1553-65
Cook JG, Bardwell L, Kron SJ, Thorner J (1996) Two novel targets of th e MAP kinase K ssl are negative regulators of invasive grow th in the yeast Saccharomyces cerevisiae. Genes Dev 10, 2831-48
Costigan C, G ehrung S, Snyder M (1992) A synthetic lethal screen identifies SLKl, a novel protein kinase hom olog im plicated in yeast cell m orphogenesis and cell growth. Mol Cell Biol 12, 1162-78
Cottarel G (1997) Mcs4, a tw o-com ponent system response regulator hom ologue, regulates the Schizosaccharomyces pom be cell cycle control.
Genetics 147, 1043-51
Courchesne WE, Kunisaw a R, Thorner J (1989) A putative protein kinase overcom es pherom one-induced arrest of cell cycling in S. cerevisiae. Cell
58,1107-19
Cox JS, W alter P (1996) A novel m echanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87, 391-404
D avenport KR, Sohaskey M, Kamada Y, Levin DE, G ustin MG (1995) A second osm osensing signal transduction pathw ay in yeast. Hypotonic shock activates the PKCl protein kinase-regulated cell integrity pathw ay. / B iol Chem 270, 30157-61
Deak M, Clifton AD, Lucocq LM, Alessi DR (1998) M itogen- and stress- activated protein kinase-1 (MSKl) is directly activated by MAPK and SAPK2/p38, and m ay m ediate activation of CREB. EMBO J 17, 4426-41
Degols, G., Shiozaki, K. & Russell, P. (1996) A ctivation and regulation of the Spcl stress- activated protein kinase in Schizosaccharomyces p o m b e.
Mol. Cell. Biol. 16, 2870-2877
Degols, G. & Russell, P. (1997) Discrete roles of the Spcl kinase and the A tfl transcription factor in the UV response of Schizosaccharomyces p o m b e.
Derijard, B., Hibi, M., W u, I.H., et al. (1994) JNKl: a protein kinase stim ulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation dom ain. Cell 76,1025-1037
DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680-6
De Virgilio C, Burckert N, Bell W, Jeno P, Boiler T, W iem ken A (1993) D isruption of TPS2, the gene encoding the 100-kDa subunit of the trehalose- 6-phosphate sy n thase/phosphatase complex in Saccharomyces cerevisiae, causes accum ulation of trehalose-6-phosphate and loss of trehalose-6- phosphate phosphatase activity. Eur J Biochem 212, 315-23
De Vit MJ, W addle JA, Johnston M (1997) Regulated nuclear translocation of the M igl glucose repressor. Mol Biol Cell 8, 1603-18
Dibrov P, Smith JJ, Young PC, Fliegel L (1997)
Identification and localization of the sod2 gene product in fission yeast.
FEBS Lett 405, 119-24
D odou E, T reism an R (1997) The Saccharomyces cerevisiae MADS-box transcription factor R lm l is a target for the M pkl m itogen-activated p ro tein kinase pathw ay. Mol Cell Biol 17, 1848-59
Doi K, G artner A, A m m erer G, Errede B, Shinkawa H, Sugim oto K, M atsum oto K (1994) MSG5, a novel protein phosphatase p ro m o tes adaptation to pherom one response in S. cerevisiae. EMBO J 13, 61-70
D udley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR (1995) A synthetic inhibitor of the m itogen-activated protein kinase cascade. Proc N atl A ca d
Egger, L.A., Park, H. & Inouye, M. (1997) Signal transduction via histidyl- aspartyl phosphorelay. Genes Cells 2, 167-184.
Elion, E.A. (1995) Ste5: a m eeting place for MAP kinases and their associates.
Trends Cell Biol. 5, 322-327.
Elion EA, Satterberg B, Kranz JE (1993) FUS3 phosphorylates m u ltip le com ponents of the m ating signal transduction cascade: evidence for STE12 and FARl. Mol Biol Cell 4, 495-510
Elliott B, H altiw anger RS, Futcher B (1996) Synergy betw een trehalose and H sp 104 for therm otolerance in Saccharomyces cerevisiae. Genetics 144, 923- 33
Ellis J (1987) Proteins as m olecular chaperones. Nature 328, 378-9
Engelberg D, Klein C, M artinetto H, Struhl K, Karin M (1994) The U V response involving the Ras signaling pathw ay and AP-1 tran scrip tio n factors is conserved betw een yeast and mammals. Cell 77, 381-90
Estruch, F. & Carlson, M. (1993) Two hom ologous zinc finger genes identified by m ulticopy suppression in a SNFl protein kinase m u tan t o f
Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 3872-3881.
Fernandes, L., Rodrigues-Pousada, C. & Struhl, K. (1997) Yap, a family of eight bZIP proteins in Saccharomyces cerevisiae w ith distinct biological functions. Mol. Cell. Biol. 17, 6982-6993.
Ferrando A, Kron SJ, Rios G, Fink GR, Serrano R (1995) Regulation of cation transport in Saccharomyces cerevisiae by the salt tolerance gene HAL3. Mol Cell Biol 15, 5470-81
Foury F (1997) H um an genetic diseases: a cross-talk betw een m an and yeast.
Gene 195, 1-10
Fukuda M, Gotoh Y, N ishida E (1997) Interaction of MAP kinase w ith M AP kinase kinase: its possible role in the control of nucleocytoplasmic tran sp o rt of MAP kinase. EMBO J 16,1901-8
Fukuda M, Gotoh I, Gotoh Y, N ishida E (1996) Cytoplasmic localization of m itogen-activated protein kinase kinase directed by its N H 2-term inal, leucine-rich short am ino acid sequence, w hich acts as a nuclear export signal. / Biol Chem 271, 20024-8
Gaits, F., Degols, G., Shiozaki, K. & Russell, P. (1998). Phosphorylation and association w ith the transcription factor A tfl regulate localization of th e Sty 1/S p c l stress-activated kinase in fission yeast. Genes Dev. 12, 1464-1473.
Galcheva-Gargova, Z., Derijard, B., W u I-H & Davis, R. (1994) A n osm osensing signal transduction pathw ay in m am m alian cells. Science 265, 806-808.
Gallo GJ, Schuetz TJ, Kingston RE (1991) Regulation of heat shock factor in Schizosaccharomyces pom bem ore closely resembles regulation in m am m als than in Saccharomyces cerevisiae. Mol Cell Biol 11, 281-8
Gaxiola R, de Larrinoa IF, Villalba JM, Serrano R (1992) A novel and conserved salt-induced protein is an im portant determ inant of salt tolerance in yeast. EMBO J 11, 3157-64
Gentz R, Rauscher FJ 3d, Abate C, C urran T (1989) Parallel association of Fos and Jun leucine zippers juxtaposes DNA binding dom ains. Science 243, 1695-9
G erondakis S, G rum ont R, Rourke I, G rossm ann M (1998) The reg u latio n and roles of R el/N F-kappa B transcription factors during lym phocyte activation. Curr Opin Immunol 10, 353-9
Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldm ann H, Galibert F, H oheisel JD, Jacq C, Johnston M, Louis EJ, Mewes H W , M urakam i Y, Philippsen P, Tettelin H, Oliver SG (1996) Life w ith 6000 genes. Science 274, 546, 563-7
Glover JN, H arrison SC (1995) Crystal structure of the heterodim eric bZIP transcription factor c-Fos-c-Jun bound to DNA. Nature 373, 257-61
Gorlich D, Mattaj I (1996) Nucleocytoplasmic transport. Science 271, 1513-8
C orner W, D urchschlag E, M artinez-Pastor MX, Estruch F, A m m erer G, H am ilton B, Ruis H, Schuller C (1998) Nuclear localization of the C2H2 zinc finger protein Msn2 is regulated by stress and protein kinase A activity.