10022891local text STA1 STA1Q03125P149222957under STA1 promoter function-inducing conditions and wasconsidered a candidate for the multicopy inhibitor of STA1
10022891local text MSS1 glucoamylaseInterestingly, the NRG1 gene has previously been identifiedas MSS1 in a genetic screen for a multicopy suppressor of elevated glucoamylase production caused by sns1 mutation (1).The fact that the same gene was isolated by two different approaches to identify negative factors for STA1 expressionimplies that the NRG1 gene may play an important role in the negative regulation process.Nrg1 is involved in glucose repression of STA1 gene expression.
10022891local text nrg STA1nrg1D alleviates glucose repression of STA1 transcription.
10022891local text Nrg1 Ssn6These two-hybrid results, together with the genetic interac-tion between Nrg1 and Ssn6-Tup1, suggested the possibility of direct interaction between Nrg1 and Ssn6.
10022891local text major nrg1Although we cannot exclude the possibility of the in-volvement of Mig1, Mig2, and/or some unknown repressors, we suggest that Nrg1 is the major repressor responsible for theglucose repression of the STA genes since nrg1
10022891local text Snf1 Mig1In theabsence of glucose, the Snf1 kinase inhibits the function of Mig1 protein directly or indirectly, leading to derepression ofglucose-repressed genes (3, 4).
10022891local text Nrg1 Ssn6hybrid and GST pull-down experiments demonstrate the phys-ical interaction between Nrg1 and Ssn6 both in vivo and in vitro.Nrg1 joins Mig1 and Mig2 as a DNA-binding repressor for glucose repression.
10022891local text STA1 MATRegulation of STA1 gene expression by MAT during thelife cycle of Saccharomyces cerevisiae.
10022891local text Nrg1 Ssn6Nrg1 interacts with Ssn6 both in vivo and in vitro.
10022891local text Nrg1 STA1STA-TATASTA-TPK2-integrated cells grown inglucose (data not shown), it is possible that Nrg1 mediates
10022891local text Nrg1 Ssn6Nrg1 interacts with Ssn6.
10022891local text Nrg1 Ssn6Two-hybrid and glutathione S-transferase pull-down experiments show an interaction of Nrg1 with Ssn6 both in vivo and in vitro.
10022891local text Nrg1 Ssn6The interaction of Nrg1 with Ssn6 was confirmed by another two-hybridassay (Fig. 7A).
10022891local text mig1 glucoamylasethis notion are our observations that the STA promoters do notcontain a consensus Mig1-binding sequence and mig1
10022891local text Nrg1 STA1These findings indicate that Nrg1 acts as a DNA-binding repressor and mediatesglucose repression of the STA1 gene expression by recruiting the Ssn6-Tup1 complex.
10022891local text Nrg1 STA1Nrg1 relieves glucose repression of STA1 gene expression.Northern blot analysis was performed to determine whether the increased glucoamylase activity in nrg1D cells correlateswith the transcription level of the STA1 gene.
10022891local text glucoamylase nrg1Second, Northern analyses show that the increased glucoamylase level iscorrelated with the increased level of STA1 transcript in nrg1
10022891local text nrg1 glucoamylaseIn contrast, nrg1
10022891local text Nrg1 STA1Taken together, the results of the glucoamylase assay and Northernanalysis indicate that Nrg1 is required for glucose repression of STA1 gene expression.Nrg1 binds to an upstream element of the STA1 promoter.
10022891local text Nrg1 Ssn6The results revealed that Nrg1 interacts with Ssn6 in vitro (Fig. 7B).
10022891local text Ssn6 a2The tetratricopeptiderepeats of Ssn6 interacts with the homeo domain of
10022891local text Glucoamylase nrg1Glucoamylase production in wild-type and nrg1D cells was monitored during growthin minimal medium containing glucose as the sole carbon source.
10022891local text Nrg1 Ssn6The results from the two-hybrid and GST pull-down analyses, therefore, indicatethe physical interaction of Nrg1 with Ssn6 both in vivo and in vitro.
10022891local text Nrg1 Ssn6In vivo interaction of Nrg1 with Ssn6 was tested with the plasmids expressingDBD-Nrg1 and AD-Ssn6 (Ssn6 residues 1 to 403 fused with the Gal4 AD) (48) and strain Y190 (12).
10022891local text glucoamylase Sta1STA-TATASTA-TPK2 (Fig. 1B) and significantly de-creased glucoamylase production to the Sta1 strain (Table 2)
10022891local text Nrg1 STA1DISCUSSION We provide here several lines of evidence for the identifi-cation of Nrg1 as a transcriptional repressor responsible for
10022891local text glucoamylase nrg1Nrg1 relieves glucose repression of STA1 gene expression.Northern blot analysis was performed to determine whether the increased glucoamylase activity in nrg1D cells correlateswith the transcription level of the STA1 gene.








10024454local text PSAT AMGSince Arg42 and Arg77 of PSAT both take
10024454local text PSAT AATP23721P237213913A comparison of substrate analogue complexes of PSAT and AAT
10024454local text PLP PLPHydrogen-bonding of PLP and PLP-AMG to PSAT
10024454local text nine AMGA third argi- nine binds the AMG side-chain indirectly through a solvent molecule but is expected to position itself during catalysis between the PLP phosphate group and the substrate side-chain.
10024454local text la desdans la deA^termination des structures cristallines.
10024454local text a3 PLPThe most rel- evant structural differences responsible for this are the already mentioned shift of a-helix 5 of AAT- AMA with respect to helix a3 of PSAT and the hydrogen bonding of Arg266 of AAT-AMA with the PLP phosphate group (Figures 4(a), 5(a) and 7(c); JaE` ger et al., 1994).
10024454local text PSAT AMGAnalysis of the active site of PSAT and of the complex with AMG demonstrates that the enzyme is well adapted for binding a highly negatively charged substrate side-chain.
10024454local text area PSATThe total accessible surface area of the PSAT dimer is 26,667 AE^ 2 when a probe
10024454local text PLP PLPOne solvent molecule mediates hydrogen bonds of Gly10 and Thr240 to PLP while a second mediates a hydrogen bond of Arg77 to PLP.
10024454local text PSAT PLPIn the unligated state of PSAT no hydrogen bond to atom O30 of PLP is made.
10024454local text ase pHkinetics of the reaction of aspartate aminotransfer- ase with aspartate at low pH reveals dual routes in the enzyme substrate association process.








10026184local text RNA polymerase II holoenzymeNonetheless, BRCA1 has been reported associated with certain chromatin structures in vivo and linked to the RNA polymerase II holoenzyme complex (15, 16).
10026184local text BARD1 BRCA1P38398P3839817831BARD1 homodimer and BRCA1/BARD1 heterodimer sample (1.0 ml)concentrations were adjusted to 1
10026184local text BRCA1 BARD1Mapping the Functional Domains of BRCA1 INTERACTION OF THE RING FINGER DOMAINS OF BRCA1 AND BARD1*
10026184local text BRCA1 BARD1Proteolysis mapping and fluorescence experiments provide general structural insights as a preliminary step toward building a picture of the BRCA1/BARD1 heterodimer.
10026184local text BRCA1 BARD1BRCA1 and BARD1 N-terminal complexes do not bindnucleic acids.








10026191local text II LiveTransforming Growth Factor-b Induces Formation of aDithiothreitol-resistant Type I/Type II Receptor Complex in Live Cells*
10026191local text step PAI-1P37173P3717310669DTT Pretreatment Prevents TGF-b-induced Activation of the Type I Receptor--In epithelial cells, an essential step in TGFb-mediated growth inhibition and PAI-1 promoter activation is activation of the ability of TbRI to rapidly phosphorylate Smad3 at its C-terminal SSVS motif (28).
10026191local text fold Smad3TGF-b Receptor Complexes 5719 20-fold increase in Smad3 phosphorylation; two-dimensional tryptic mapping indicated that this in vitro phophorylation occurred at the same site as in vivo (data not shown).








10026196local text Fc BAFTABLE IISummary of complex formation and BAF data for biotinylated ligands Biotinylatedligand Location ofmutation Ratio IL-11R-Fc binding Ratio complex formationa Ratio BAF stimulation
10026196local text II gp130-Q00560P4787310012This allowed us to examine the binding of these site II mutants to gp130-Fc (in the presence of soluble IL-11R), which we were unable to do by measuring competitive binding between bIL-11 and the mutants, as the mutant proteins were still able to compete for soluble IL-11R.
10026196local text gp130 pIGDot-blot analysis showed that both monoclonal antibodies preferentially recognized native mIL-11 and not de-natured mIL-11, indicating that their reactivity with mIL-11 is conformation-dependent.IL-11R-Fc and gp130-Fc Expression Constructs--The construction of the eukaryotic expression plasmids pIG/IL-11R-Fc and pIG/gp130-Fchas been described previously (17).
10026196local text Fc BAFTABLE ISummary of receptor binding and BAF assay data for mIL-11 mutants Wild type or mIL-11mutant Location ofmutationa Ratio IL-11R-Fc binding Ratio BAF stimulation
10026196local text IL-11 gp130IL-11 has been shown to function in a similar manner, and, as for IL-6, a ligand-specific IL-11 receptor (IL11R) (15, 16) functions to promote the formation of a high affinity complex between IL-11 and gp130 (17).
10026196local text IL-6 gp130Site III enables IL-6 to bind to a second gp130 molecule and form hexameric complexes (29), and site III of LIF enables it to bind to the LIF receptor (32).
10026196local text IL-11 gp130A complex of IL-11 and the IL-11R interacts with gp130 to induce this homodimerization.
10026196local text IL-11 IL-11A complex of IL-11 and the IL-11 receptor(IL-11R) has been shown to interact with gp130, with high affinity, and to induce gp130- dependent signaling.In this study, we have identified residues crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R andgp130 by examining the activities of mIL-11 mutants in receptor binding and cell proliferation assays.
10026196local text fold BAFThis biotinylated mutant bound to IL-11R-Fc with a 5-fold increase in affinity
10026196local text protease a proteinCleavage of the glutathione S-transferasemIL-11 fusion protein with 3C protease produces a protein consisting ofamino acids 1-178 of mIL-11 with an extra glycine at the NH
10026196local text Fc F3Activity of Site II Mutants--The activities of the site II mutants were assessed in both the IL-11R-Fc binding assay and the Ba/F3-mgp130/mIL-11R proliferation assay (Table I and
10026196local text Fc F3Activity of Site III Mutants--The activities of the site III mutants W147A and R151A were assessed in the IL-11R-Fc binding assay and the Ba/F3-mgp130-mIL-11R proliferation assay (Table I and Figs.








10026281local text plastocyanin cytochromeThe results are similar to the effects of ionic strength on the reduction of the positivelycharged cytochrome c and the negatively charged plastocyanin by the cytochrome bf complex.
10026281local text AMP ETFP38975P3897411018Close analysis of the structure reveals that the loop containing a^I63 isin part responsible for conferring the high specificity of AMP binding by the ETF protein.
10026281local text ETF ETF-QO7, 1999 1987 denitrificans and human ETF semiquinones as substrates ofpig ETF-QO in the disproportionation reaction catalyzed by
10026281local text ETF ETF-QOPreviously, a chimeric human-Paracoccus ETF protein was constructedin which the first 84 residues of the human a^-subunit were substituted by those of the P. denitrificans sequence (19).While this region still retains 64% sequence identity with the original human ETF protein, this chimeric ETF was foundto be markedly diminished in the reaction between ETF and ETF-QO, suggesting that residues within the N-terminalportion of the a^-subunit are necessary for interactions with ETF-QO (19).
10026281local text AMP theaAMP binds ETF exclusively within thea^-subunit, with residues in the binding site being contributed by those within strand 1, at the C-terminal end of strand 2,and from strands 4, 4a, and 4b (see Figure 3B).
10026281local text ETFs pigThe reaction between the ETFs and pig ETFQO was assayed by the catalyzed disproportionation ETFsemiquinone reaction (35).
10026281local text plastocyanin cytochromeHowever, in thereaction of plastocyanin and the cytochrome bf complex, the results are apparently due to local charges on the redoxproteins in the electron transfer complex and therefore shortrange forces (47).The original description of P. denitrificans ETF indicated that the bacterial ETF did not reduce mammalian (pig) ETF-QO when assayed as a ubiquinone reductase (3).
10026281local text MCAD ETFFigure 7 comparesthe ionic strength dependence of the reaction of MCAD with human ETF, P. denitrificans ETF, and a human-Paracoccuschimeric ETF (19); all reactions were conducted at 1 uM ETF at pH 8.0, the pH optimum for the dehydrogenase.








10048790local text Cdc25 adenylyl cyclaseP04821P086782663These studies suggest that a direct interaction between Cdc25 and adenylyl cyclase promotes efficient assembly of the adenylyl cyclase complex.
10048790local text Cdc25p cAMPP01120P086782664SDS-PAGE gels were transferred to Cdc25p Decrease the cAMP Response to Glucose nitrocellulose membranes or stained with Coomassie blue.
10048790local text Ras adenylyl cyclaseP04821P086782663Ras and SH3 do-mains cause synergistic activation of adenylyl cyclase activity.Precipitations were carried out by using soluble membrane ex-tracts prepared from strain JF36 (ras1, ras2) [32].
10048790local text map Cdc25pThese dataThe cAMP measurements suggested that Cdc25p SH3 suggest that Cdc25-SH3 binds directly to adenylyl cyclase.binds directly to other components of the Ras signalling sysTo further map the region bound by Cdc25p1-134, wetem.
10048790local text Cdc25 adenylyl cyclaseCdc25 may prefer non-PXXP binding partners.binding site is in the C-terminal region of adenylyl cyclase near the centre of the catalytic region, a region where there Several lines of evidence suggest that the interaction between Cdc25 and adenylyl cyclase is a bonafide SH3 inter-are no PXXP consensus binding-site motifs.
10048790local text adenylyl cyclase CapGrb2 also binds Cap, but Grb2 has two SH3 sequently does not associate with the membrane is nonfunctional when expressed from the chromosome but isdomains; perhaps one binds adenylyl cyclase and the other binds Cap.
10048790local text Cdc25p cAMPcerevisiae Cdc25p Binds Adenylyl Cyclase and Facilitates Ras Regulation of cAMP Signalling
10048790local text Protein GRFSH3SF9 Cell Protein Expression and Extract Preparation domain and Ras GRF domain are shown.
10048790local text Cyclase RasCyclase Activity by Ras and SH3 Domains
10048790local text Cdc25 CapFirst, Cdc25 SH3 binds to cyclase and not Cap,
10048790local text Cdc25p cAMPMutations in theSH3 domain of Cdc25p decrease the cAMP response to glucose.Glucose-starved yeast cells were given glucose to a final concen-tration of 25 mM.
10048790local text Grb2 GSTexpression of Grb2 as a fusion to GST in E. coli (GST-activate Ras [3].
10048790local text Grb2 CapGrb2 also binds Cap, but Grb2 has two SH3 sequently does not associate with the membrane is nonfunctional when expressed from the chromosome but isdomains; perhaps one binds adenylyl cyclase and the other binds Cap.
10048790local text Ras adenylyl cyclaseThe binding accelerated Ras activation of adenylyl cyclase in vitro.
10048790local text Ras GRFGTP-bound Ras binds Ras-GRF, which is activated by Ca21 [11].and activates downstream effectors such as Raf, a protein kiIn yeast, Cdc25p is the Ras GEF [12-15].
10048790local text Cdc42 cAMPFurthermore, PIX potentiates Cdc42 ac-several deletions in the N-terminus caused severe growth tivation of Pak and even enables a Cdc42 mutant that failsdefects and reduced the cAMP response to glucose feeding, to bind Pak to activate it [49].
10048790local text adenylyl cyclase manyWe suggest that the SH3 domains either cause hypermains have been identified through sequence comparisonsactivation of adenylyl cyclase or increase Ras binding to adand many ligand interactions have been studied by screen-enylyl cyclase.








10049744local text IFN IFNg and its receptor(31), it is unusual that IFN a and IFNb interact withthe same receptor chains but IFN
10049744local text IFNb a2P17181P0157410914Furthermore, the presence of IFNb1b inthe immunoprecipitated IFN receptor complex suggests that IFNb interacts and binds differently to thereceptor than IFN
10049744local text IFNb IFNAR2If dissociation of IFNb from the receptor complex didoccur then the IFNAR1/IFNAR2 complex would need to be held together largely by interactions between thereceptor chains themselves.
10049744local text IFNAR1 b1bIFN activity was detected in anti-IFNAR1 immunoprecipitates from Daudi cells stimulatedwith IFN
10049744local text IFN IFNAR1aand IFN b may have similar receptor affinities (4,11),but assumes IFN








10049915local text rad53-31 CDC7rad53-31 double mutants are viable because rad53-31onstrates a genetic interaction with CDC7.The rad53-11 ( 5mec2-1) is synthetically lethal with retains an intact checkpoint function.We reasoned that, because
10049915local text rad53- rad53-31P22216P323252669the level of DBF4 message was increased in the rad53- protein expression in the rad53-31 and rad53-11 strainswas similar to that seen for mRNA expression.
10049915local text RAD53 DBF4Second, RAD53 regu-lates the expression of DBF4 at the message and proteindominant but weak checkpoint signal from rad53-31.
10049915local text RNR1 RNR1Genes of RNR1 because high-copy expression of RNR1 can Dev.
10049915local text rad53 Dbf4pbypass rad53D::URA3.Genetic interactions between rad53 and various cell cycle Interaction of Rad53p and Dbf4p: Given that RAD53 mutations were tested by crossing individual strains together.The diploids were sporulated and dissected and the resulting interacts genetically with CDC7 and DBF4, we asked if
10049915local text DBF4 DBF4RAD53 regulates DBF4 expression at the mRNA andfirst complemented by wild-type DBF4 on a plasmid, protein levels: To understand how rad53-31 manifests
10049915local text URA3 Dbf4ption that allows repeated use of URA3 selection in the construc-With the dramatic reduction of Dbf4p protein, the
10049915local text rad53-11 cdc7The A. Interaction between rad53-11 and various cdc7 alleles results are complicated by the fact that strains bearingWild type (299) Viable (10 tetrads)
10049915local text rad53- rad53-11the level of DBF4 message was increased in the rad53- protein expression in the rad53-31 and rad53-11 strainswas similar to that seen for mRNA expression.
10049915local text Rad53p Dbf4pThe results shownificant deviation from the 1PD:4T:1NPD ratio was observed that Rad53p interacts weakly with Dbf4p, but not with
10049915local text replication replicationkinase complex is brought to origins of replication via
10049915local text Dbf4p Rad53pLonger exposures re-vealed a very low level of Dbf4p expression in the rad- tion of Rad53p, and the essential function of RAD53(Zheng et al. 1993).
10049915local text rad53-31 cdc8-1Interaction between rad53-31 and cdc8-1 in the diploid.
10049915local text Rad53p Dbf4pTwo-hybrid analysis indicates that the Rad53p protein binds to Dbf4p.
10049915local text DBF4 RAD53This work was supported by U.S. Public Health Service grant GM35078 awarded to R.A.S.In addition, the activation of DBF4 transcription and/
10049915local text bob1-1 rad53A. bob1-1 or second site suppressor suppresses rad53D::URA3 mutationbob1/ 1 1/rad53D::URA3 4:0 viable:inviable 0(299 3 P119) 3:1 viable:inviable 9a2:2 viable:inviable 16
10049915local text epsilon replicationepsilon links the DNA replication machinery to the S phase check-Donaldson, A. D., W. L. Fangman and B. J. Brewer, 1998 Cdc7 point.
10049915local text Rad53p Dbf4pIn addition, we show that Rad53p interacts with Dbf4p and controls the level of expression of DBF4.cell cycle and allow the completion of DNA repair orreplication (Sidorova and Breeden 1997).
10049915local text bob1-1 rad53The bob1-1 mutation cannot suppress a rad53D::URA3 (Figure 3A).
10049915local text cdc7-1 Dbf4pof reduced activity of the cdc7-1 gene product and a interact directly through Dbf4p.
10049915local text rad53-31 dbf4-1C. Interaction between rad53-31 and dbf4-1 suppressor was responsible for the suppression, notdbf4-1 (PDY029) Slow growth phenotype (31, 24 tetrads








10064605local text replication topoisomeraseP12956P1592740059USA, 95, 6049-6054.Tsao,Y.P., Russo,A., Nyamusa,G., Silber,R. and Liu,L.F. (1993) Interaction between replication forks and topoisomerase I-DNAcleavable complexes: studies in a cell-free SV40 DNA replication system.
10064605local text topoisomerase RPA2P27694P7852740061treated with topoisomerase inhibitors and ionizingradiations RPA2 phosphorylation was determined by Western blottingof cell lysates using specific monoclonal antibody.
10064605local text Rb p21P12956P15927400591 phase accumulation induced by UCN-01 isassociated with dephosphorylation of Rb and CDK2 proteins as well
10064605local text p53 p53P15927P7852740057Miller,S.D., Moses,K., Jayaraman,L. and Prives,C. (1997) Complexformation between p53 and replication protein A inhibits the sequencespecific DNA binding of p53 and is regulated by single-strandedDNA.
10064605local text RPA2 protein kinaseP12956P7852740058Inhibition of RPA2 phosphorylation and DNA-PKactivation by the protein kinase inhibitors wortmannin and UCN-01Next, we used wortmannin to obtain further evidence for a role of DNA-PK in RPA2 phosphorylation.
10064605local text p34 Cdc2, 57, 3386-3389.Niu,H., Erdjument-Bromage,H., Pan,Z.Q., Lee,S.H., Tempst,P. and Hurwitz,J. (1997) Mapping of amino acid residues in the p34 subunitof human single-stranded DNA-binding protein phosphorylated by DNA-dependent protein kinase and Cdc2 kinase in vitro.
10064605local text cdc2 replication, 4, 968-977.Dutta,A. and Stillman,B. (1992) cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA and activate DNA replication.EMBO J., 11, 2189-2199.