Title of Invention

PROMOTER FOR THE FUNCTIONAL CHARACTERIZATION OF G-PROTEIN COUPLED RECEPTORS IN THE YEAST SACCHAROMYCES CEREVISIAE

Abstract

A motorcycle in which an engine unit arranged below a main frame is suspended from a body frame having a main frame extending down backward from a head pipe, in which the main frame is covered at its two sides with a pair of left and right main frame side covers, and in which the main frame is covered at its upper portion with an upper cover. At least part of the front upper ends of the paired main frame side covers (31L, 31R) supported by the body frame (F) are joined at the back of the head pipe (12), and the front portion of the upper cover (34) is fastened to the joined portions of the two main frame side covers (31L, 31R). The invention relates to a promoter, DNA fragments containing said promoter and the use thereof, in particular in methods for the identification of substances displaying an activating or inhibiting effect on G-protein receptors.

Full Text

Promoter for the functional characterization of G protein-coupled receptors in the yeast Saccharomyces cerevisiae The present invention relates to a promoter, to DNA fragments containing said promoter and to the use thereof, in particular in methods for identifying substances exhibiting an activating or inhibiting action on G protein-coupled receptors. GPCRs (G protein-coupled receptors) form a gene family of structurally and functionally linked transmembrane proteins. GPCRs are target molecules of great importance for medical research and the development of pharmacological active substances and occupy a key position in a multiplicity of pathologies (Stadel et al., 1997). The ciassic example of the central position of GPCRs in the pharmacological treatment cf asthma is P2AR (P2-adrenergic receptor) or the involvement of a Kaposi sarcoma (KS)-associated herpesvirus GPCR in KS pathogenesis (Geras-Raaka et al., 1998). This ciass of receptors bind a broad spectrum of ligands such as, for example, protein hormones, chemokines. peptides or divalent cations. The identification of activators and inhibitors of G protein-coupled receptors is thus one of the most promising approaches toward a better knowledge and treatment of diseases (Wilson et al., 1998). Many components of the GPCR signal transduction pathway have orthologous components in the GPCR signal transduction pathway of bakers' yeast, Saccharomyces cerevisiae. Stimulation with mating factor (pheromone a or pheromone a) activates, for example, the pheromone-dependent mitogen-activated protein kinase cascade (denoted MARK cascade hereinbelow) (Frederickson, 1999). This property of yeast can be utilized for assaying mammalian GPCRs, in particular human GPCRs, in a yeast model, in order to search for activators or inhibitors of the relevant GPCR or the corresponding signal transduction pathway, in particular since GPCRs of foreign species can be functionally expressed in yeast, resulting via the yeast MARK cascade in a measurable cellular response when the introduced GPCR is activated by a specific ligand and this signal is passed on to the yeast MAPK cascade. The search for activators or inhibitors of GPCRs to which, up until now, no functions have been ascribed, the orphan GPCRs", is of particular interest. The pheromones a and a act in S. cerevisiae via the endogenous G protein-coupled receptors Ste2p und Ste3p (Gustin et al., 1998). In this connection, pheromone a acts directly on the pheromone-dependent MAPK cascade, thereby regulating the expression of particular S. cerevisiae genes. Whether a substance acts in an activating or inhibiting way on a GPCR heterologously expressed in S. cerevisiae, may be detected by expressing the S. cerevisiae genes which are regulated by the MAPK cascade (marker genes). The promoters of such marker genes may be used, for example in connection with a suitable reporter system, for identifying GPCR activators or inhibitors. Pheromone-regulated genes may be identified either by using transposon mutagenesis (Ross-Macdonald et al., 1999) or by using DNA microchips, which allows expression analysis of all mRNAs of a cell at a particular time (WodicKa et al., 1997). Up until now, functional assays for identifying inhibitors or activators of GPCRs have been carried out mainly in mammalian cells:(Wilson et al. (1998) British Journal of Pharmacology 125, 1387-1392; Geras-Raaka er al M998) J. Exp. Med. 188 No.2, 405-408). The use of promoters of the FUS1 and -US2 genes for functional assays in S. cerevisiae has been described (Cisnowsk; et al (1999) Nature 17, 878-883; Frederickson (1999) Nature Biotechnology A, 552-553). FUS1 is a gene whose expression in wild-type cells of the yeast Saccharomyces cerevisae is increased, after activation with pheromone a. In US 5,063,153. FUS1 and FUS2 promoters are used in order to express in large amounts a structural gene which codes for a protein of interest. It was an object of the present invention to identify a different S. cerevisiae promoter which can be activated by pheromone a and which can be used to achieve strong expression of the regulated gene. The invention relates to the promoter of the S. cerevisiae YNL279w gene. The invention relates to the promoter having the sequence SEQ ID NO. 4. The invention also relates to a recombinant DNA fragment containing the Saccharomyces cerevisiae YNL 279 w gene promoter which, when functionally linked to a structural gene, regulates transcription thereof and to a recombinant DNA fragment containing a promoter having the DNA sequence SEQ ID NO. 4. The invention relates to a recombinant DNA fragment which contains the YNL 279 w promoter and a structural gene and in which the promoter is functionally linked to the structural gene. The structural gene codes, for example, for a receptor gene or for a protein which is intended to be produced in large quantities in S. cerevisiae. The invention relates to a DNA vector and a recombinant S.cerevisiae cell, which contain such a recombinant DNA fragment. Such a recombinant S. cerevisiae cell preferably expresses no or only small quantities of functionally active endogenous receptors Ste2p and Ste3p. The invention also relates to methods for functional cnaracterization of GPCRs and for screening of GPCR inhibitors and/or activators and to methods for preparing proteins in S. cerevisiae. For example, methods for identifying activators and/cr inhibitors of G protein-coupled receptors, wherein a) a recombinant S. cerevisiae cell is produced, which contains a reporter gene under the control of the promoter of the YNL 279 w gene and which expresses a heterologous G protein-coupled receptor; b) the cell is incubated with a substance to be studied, and c) the change in reporter gene transcription is determined. Methods for identifying constitutively active mutants of G protein-coupled receptors, wherein a) a recombinant S. cerevisiae cell is produced, which contains one or more reporter genes under the control of the promoter of the YNL279w gene b) and which expresses a mutated heterologous G protein-coupled receptor, the modification resulting in a constitutively active G protein-coupled receptor, c) the cell is incubated wiith an activating or inhibiting substance, and d) the change in reporter gene transcription is determined. In the recombinant S. cerevisiae cell, the appropriate endogenous GPCRs, Step 2 and/or Step 3, are preferably not expressed, for example because these genes have been deleted. This deletion may be carried out as described, for example, in Broach Thorner (1996) Nature, 384, 14-16. Another embodiment relates to methods for preparing heterologous proteins in S. cerevisiae, wherein the structural gene of the heterologous protein to be expressed is functionally linked to the promoter of the YNL279w gene and is expressed under the ccnirol thereof. Detailed description of the invention: Pneromone-inducibie and -repressible ORFs (open reading frames) were identified by treating cultures of bakers' yeast Saccharomyces cerevisiae with or without pheromone a, in order to determine which known genes and, in particular, which as ye: uncharacterized genes are induced or repressed by pheromone a. DNA microchips were used, which can display in parallel the expression patterns of the more than 6000 identified yeast genes. Comparison of the gene expression patterns of two cells or cultures of cells which have been treated in different ways, for example with or without pheromone, makes it possible, with the aid of the DNA microarrays after appropriate preparation of the corresponding mRNAs, to determine which genes have been specifically activated or repressed. After addition of pheromone a, ORF YNL279w showed a strong time-dependent increase in its expression (figures 2 and 3), Since transcription and expression of genes are normally regulated via regulatory sequences 5' from the translational starting point, the "promoters", the promoters having such regulatory sequences are of particular interest. In order to identify activators or inhibitors of pharmacologically and medically interesting GPCRs, it is possible to clone said inducible or repressible promoters in the same way in front of genes or structural genes, which represent a suitable reporter system, in order to identify substances which, after addition, show activating or inhibiting action on the reporter system. Examples of suitable reporter systems are the genes of LacZ, iuciferase, aequorin, green fluorescent protein (GFP), DsRed, H1S3, URA3, TRP1 and LEU2, but also resistance genes against particular antibiotics, for example kanamycin. These "reporter genes" may be, for example, genes such as lacZ, for which an action of particular substances on the MARK cascade can be detected via color change of the cell colonies. The reporter genes may also be, for example, genes which make it possible for the cell to grow under particular selection conditions such as, for example, auxotrophic genes. Examples of DNA fragments (DNA constructs) containing the promoter of the YNL279W gene and reporter genes cicned behind said promoter are listed in table 1. Said DNA constructs may be used for example, for identifying GPCR activators (agonists) and inhibitors (antagonists)in functional assays. Table 1: DNA construct Detection system (read out) Promoter(YNL279w)-lacZ Colorimetric/chemiluminescence read out Promoter(YNL279w)-luciferase Chemiluminescence read out Promoter(YNL279w)-aeqourin Chemiluminescence read out Promoter(YNL279w)-GFP Fluorescent colonies (cells) Promoter(YNL279w)-EGFP Fluorescent colonies (cells) Promoter(YNL279w)-KAN Growth in selection medium Promoter(YNL279w)-HIS3 Growth in histidine-depleted medium Promoter(YNL279w)-URA3 Growth in uracil-depleted medium Promoter(YNL279w)-TRP1 Growth in tryptophan-depleted medium Promoter(YNL279w)-LEU2 Growth in leucine-depleted medium Promoter(YNL279w)-ADE2 Growth in adenine-depleted medium Promoter(YNL279w)-CAN1 Growth in canavanine-containing medium (when adding an inhibitor) The DNA which codes for such a receptor gene is fused 3' to the pheromone-inducible promoter of ORF YNL279w of the yeast Saccharomyces cerevisae and cloned, for example, into a high-copy vector or a low-ccpy vector. These vectors may then be used for transforming yeast cells. Likewise, it is possible for the DNA fused to the promoter to be stably integrated into the yeast genome. Yeast strains which are genetically manipulated with reporter genes belonging to the group of auxotrophic genes (e.g. HISS, URA3, TRP1, LEU2, ADE2 and LYS1) must be mutated, i.e. functionally inactivated, for the corresponding gene. Stimulation of yeast cells by pheromone leads to activation of the yeast promoter YNL 279 w and to increased expression of the reporter gene regulated by the promoter. When searching for GPCR agonists, the promoter of the gene YNL279w is preferably cloned in front of reporter genes such as, for example, HIS, ADE, TRP or LEU, since this combination allows a read out via cell growth in an appropriately depleted medium. Activation of a selected GPCR by a substance to be studied results in activation of the promoter YNL279w which is at the bottom of the MARK cascade and thus in expression of the reporter gene regulated by the YNL279w promoter. In the case of HIS, ADE, TRP or LEU, this leads to growth in the corresponding selection medium. An agonist can be identified by the corresponding S. cerevisiae cells growing in the selection medium. When using the genes for LacZ, GFP or EGFP, a read out may be carried out via colorimetric or luminometric measurements. Only those cells whose heterologous GPCR has been activated by an agonist show chemiluminescence. When the reporter gene used is CAN1, the cells grow in canavanine-containing medium. The CAN1 gene is expressed in the presence of activators (agonists) of a heterologously expressed GPCR so that the cells cease growing in the canavanine-containing medium. If an antagonist (inhibitor) is added, the cultures will grow in said selection medium. The screening methods may be carried out on microtiter plates seeded with yeast cells which have been transformed beforehand with the promoter-reporter gene construct (e.g. a DNA constnjct from table 1). In addition, said cells are transformed with a heterologous GPCR for which an agonist or antagonist is to be found. It is possible, for example, to use in said methods human GPCRs, for exampie adenosine receptors, somatostatin receptors, dopamine receptors, bradykinin receptors, lysolipid receptors, β-adrenergic receptors and muscarinic acetylcholine receptors. As a pheromone α-induced gene, the YNL279w promoter may also be used for identifying activators and inhibitors of the pheromone-dependent MARK cascede. Since the YNL279w promoter is, after pheromone a treatment, more sensitive to stimulation than, for example, the FUS1 promoter, using the YNL279w promoter as proposed herein, is advantageous compared with other screening methods using, for example, the FUS1 promoter. Figure 4 shows that stimulation of the YNL279vv promoter/aequorin construct (p415YNL279-AEQ) reacts substantially better than is the case for a promoter element corresponding to a region from the promoter of the FUS1 gene (denoted 4PRE-AEQ here). Figure 1a: Sequence of YNL279w (GenBank acc.No.:Z71555, SEQ ID NO, 3). The ATG startcodon is underlined. Sequence regions in bold type represent pheromone-responsive elements (PRE). Figure 1 b: Promoter region (5'-UTR) of YNL279w (SEQ ID NO. 4) Figure 1c: Pheromone-responsive elements (PRE) in the promoter region of YNL279w Figure 2: Expression profile of Saccharomyces cerevisiae ORF YNL279w after induction with pheromone a. The observed changes in gene expression are based on results of the studies using DNA microchips. Changes in gene expression of the FUS1 gene are shown for comparison. Figure 3: Northern blot analysis of the expression profile of the YNL279w gene after stimulating the yeast cultures with pheromone a in yeast mutant sst1. Figure 4: Induction of aecuorin expression (as relative light units) after stimulating the yeast cells with pheromone a at the concentrations stated (B). The values indicated in the diagram represent the averages of three independently carried out experiments, and the corresponding individual values are depicted in (A). In the case of pYNL279-AEQ, the aequorin reporter gene is under the control of the YNL279w promoter. In the case of 4PRE-AEQ, aequorin is under the control of a region (-271 to -1 before start) of the FUS1 gene. Each stimulation experiment was carried out three times. Figure 5: Diagrammatic representation of the promoter-reporter gene vector construct comprising the YNL279w promoter region and the aeqourin reporter gene in vectcr p415 (a derivative of vector pRS415: ATCC 87520). Examples Example 1: Identification of ORF YN279w Cell cultures of the Saccharomyces cerevisiae yeast strain having the genotype (MAT a sst1::LEU2; ade2;can1-100; his3-11,15; Ieu2-3,112; trp1-1; ura3-1) were stimulated with 1 (JM pheromone a (dissolved in 90% methanol); control cells were treated only with 90% methanol. 20 min, 90 min and 180 min after stimulation, an amount of cells corresponding to about 10 ODsoonm was removed. Total RNA from these cells was isolated according to standard methods (e.g. Sambrock et al. Molecular Chemistry Press, (Cold Spring Harbor Laboratory 1989), and in each case 30 pg of total RNA were use for first strand synthesis. The protocol for first and second strand synthesis and also for the purification steps of the double-stranded cDNA generated, the in-vitro transcription and preparation of cRNA for hybridization corresponds to the protocol from Affymetrix Inc. (Santa Clara, CA, USA), The YNL279W ORF was identified, via comparison of the expression profiles of pheromone a-induced and noninduced yeast cells, as one of the ORFs induced most strongly. The activity and usability of the promoter in a screening system with a suitable reporter was determined by amplifying tne YNL279w promoter region from genomic Saccharomyces cerevisiae DNA. Beispiel 2: Isolation of the YNL279w promoter ("promoter") The promoter region of the YNL279w gene was amplified using primers YNL279F1 (SEQ ID NO. 1; 5*-CCGAGTCCTACTCCTATGCTGTTTACAAGG-3') and YNL279R (SEQ ID NO. 2; 5'-TGCTCTAGA,ATCATCAACGTTCACAAATTCG-3'). The identity of the resulting ampiificate was determined by standard sequencing methods. The length of the promoter region amplified via YNL279F1/YNL279R is 473 bp without restriction cleavage sites; this fragment of the promoter region is denoted "promoter" below. Example 3: Determination of functionality The functionality of the proposed screening method was determined by cloning the amplified promoter region of the YNL279w gene into vector p415 (see figure 5; derivative of vector pRS415, ATCC 87520) in front of the aequorin-encoding gene. This construct was transformed into the Saccharomyces cerevisiae strain (Mat a far1::hisG sst2::ura3FOA fus1::HIS3) using the Li acetate method (Ito et aL, 1983). After stimulating the MAPK cascade in the Saccharomyces cerevisiae strain (Mat a far1:;hisG sst2::ura3FOa fus1::HlS3) with pheromone a, aequorin was detected as follows: 1. 25 µl of cell suspension (1.5x106 cells per 25 µl in selection medium, SC/glucose leucine) were added to a well of a 96-well plate; 2. 25 µl of a 2x concentrated stimulation mix containing mating factor a in serial dilution (10-6 M to 10-8 M) and 1 pM coelenterazine (final concentration 0.5 pM) were added to the cells; 3. the 96-well plate is carefully agitated; 4. the 96-well plate is incubated in a humid atmosphere at 30°C :n the dark for 1.5 hours; 5. for detection, 150 pi of calcium/lysis buffer are added; aequorin amits photons immediately after adding this mix; the resulting signal is integrated for 15 s; the signal is detected by means of a luminometer (Luminoskan, LABSYSTEM) Mating factor a stock solution: 100 µM in 90% methanol Coelenterazine stock solution: 1 mM in 100% methanol (MOLECLAR PROBES: ref C-2944) CaCl2 stock solution: 1 M in 1 M Tris-HCI solution (diluted 100 fold in lysis buffer prior to use) Lysis buffer: yeast protein extraction reagent; PIERCE, ref 78990 References Cismowski MJ, Takesono A, Ma C, Lizano JS, Xie X, Fuernkranz H, Lanier SM, Duzic E. (1999) Genetic screens in yeast to identify mammalian nonreceptor modulators of G-proteinsignaling. Nat Biotechnot 17:878-83 Erdman S, Lin L. Malczynski M, Snyder M. (1998) Pheromone-regulated genes required for yeast mating differentiation. J Cell Biol 140:461-83 Fink, OR, Trueheart, J, Ellon, EA. (1991) Pheromone-inducible yeast promoter. United States Patent 5.063.154 Frederickson RM. (1999) Budding actors in mammalian G-protein signaling. Nat Biotechnol 17:852-3 Geras-Raaka E. Varma A, Ho H, Clark-Lewis I, Gershengorn MC. (1998; human interferon-gamma-inducible protein 10 (IP-10) inhibits constitutive signaling of Kaposi's sarcoma-asscciated herpesvirus G protein-coupled receptor. J Exp Med 188:405-8 Gustin MC. Albertyn J. Alexander M, Davenport K. (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62:1264-300 Stadel JM, Wilson. S, Bergsma, DJ. (1997) Orpahn G protein-coupled receptors: a neglected opportunity for pioneer dnjg discovery. TIPS 18:430-437 Wilson S, Bergsma DJ, Chambers JK, Muir Ai, Fantom KG, Ellis C, Murdcck PR, Herrity NC, Stadel JM (1998) Orphan G-protein-coupled receptors: the next generation of drug targets? Br J Pharmacol 125:1387-92 Wodicka L, Dong H, Mittmann M, Ho MH, Lockhart DJ. (1997) Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat Biotechnol 15:1359-67 Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol Jan;153:163-8 Ross-Macdonald P, Coeiho PS, Roemer T, Agarwal S, Kumar A, Jansen R, Cheung KH, Sheehan A, Symoniatis D, Umansky L, Heidtman M, Nelson FK, Iwasaki H, Hager K, Gerstein M, Miller P, Roeder GS, Snyder M (1999) Large-scale analysis of the yeast genome by transposon tagging and gene disruption. Nature 402:413-8 Patent claims: 1. A recombinant DNA fragment comprising the Saccharomyces cerevisiae YNL 279 w gene promoter which, when functionally linked to a structural gene, regulates transcription thereof. 2. A recombinant DNA fragment comprising a promoter having the DNA sequence CCTTT TATTCTCTAC ACCGAATTTG TCTTTACTCC TATGCTGT7T AGAAGGTCTA TCTGATAAGC AATTGCGCAA GAAAATAGTA GAATGAAAA3 7GAT7ATT.AA AAAGAAJ^GGT AAA.CTCAAGC CTGACTTGAT GCTGAGACGG AGTAGGTGAA AAACGTCCGT TATGCAAAAC CCTTTATATG CACAAGCTTG AGACAATGGA AATTTCCGAT GATGCCTACA TACAAAAGAG GGAAAGGCGA TATAAATTTT TTTGACGGGA TTTTCGTTTA GGTGAAAATA AAATGAACGA CAGA.GCATGG AGAGTCCGGG TAATACATAT GTTTGAATAC TGTTTGAAAA AGTTTCAGA AGTGGGTCAC ATATTAATTT TAACTTATAA CTGGCCTG7T GGTGGCAAGA GGTATATATA TATGACGAAT GTGAGCAACA TAAGTCCTTA. AGATAATGCG GAAATATTTG GTTAGGATGA TTCCCTTTCG AATTTGTGAA CGTTGATGAT 3. The recombinant DNA fragment as claimed in either of claims 1 or 2, comprising the promoter and a structural gene, wherein the promoter is functionally linked to the structural gene and regulates expression thereof. 4. The recombinant DNA fragment as claimed in claim 3, wherein the structural gene codes for a reporter gene. 5. The recombinant DNA fragment as claimed in claim 4, wherein the reporter gene is selected from lacZ, luciferase, aequorin, GFP, dsRed, HIS 3, URA 3, TRP1 and LEU2. 6. A DNA vector comprising a recombinant DNA fragment as claimed in any of claims 1 to 5. 7. A recombinant S. cerevisiae cell comprising a recombinant DNA fragnhent as claimed in any of claims 1 to 5. 8- A methcd for identifying activators of G protein-coupled receptors, wherein c) a recombinant S. cerevisiae cell Is produced, which contains one or more reporter genes under the control of the promoter of the YNL 279 w gene and which expresses a heterologous G protein-coupled receptor; d) the ceil is incubated with a substance to be studied, and e) the change in reporter gene transcription is determined. 9. A method for identifying inhibitors of G protein-coupled receptors, wherein a) a recombinant S. cerevisiae cell is produced, which contains one or more reporter genes under the control of the promoter of the YNL 279 w gene and which expresses a heterologous G protein-coupled receptor; b) the cell is incubated with a substance to be studied, and c) the change in reporter gene transcription is determined. 10. A method for preparing heterologous proteins in S. cerevisiae, wherein the structural gene of the heterologous protein is functionally linked to the promoter of ihe YNL 279w gene and is expressed under the control thereof. 11. A method for identifying constitutively active mutants of G protein-coupled receptors, wherein a) a recombinant S. cerevisiae cell is produced, which contains one or more reporter genes under the control of the promoter of the YNL279w gene b) and which expresses a mutated heterologous G protein-coupled receptor, the modification resulting in a constitutively active G protein-coupled receptor, c) the ceil is incubated wiith an activating or inhibiting substance, and d) the change in reporter gene transcription is determined. 12. A recombinant DNA fragment substantially as herein described with reference to the accompanying drawings. 13. A method for identifying activators of G protein-coupled receptors substantially as herein described with reference to the accompanying drawings.

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