EP1084231A1 - Transformed cell lines which express heterologous g-protein-coupled receptors - Google Patents
Transformed cell lines which express heterologous g-protein-coupled receptorsInfo
- Publication number
- EP1084231A1 EP1084231A1 EP99923357A EP99923357A EP1084231A1 EP 1084231 A1 EP1084231 A1 EP 1084231A1 EP 99923357 A EP99923357 A EP 99923357A EP 99923357 A EP99923357 A EP 99923357A EP 1084231 A1 EP1084231 A1 EP 1084231A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- receptor
- gene
- gpc
- heterologous
- transformed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- 229960000553 somatostatin Drugs 0.000 description 1
- NRAUADCLPJTGSF-VLSXYIQESA-N streptothricin F Chemical compound NCCC[C@H](N)CC(=O)N[C@@H]1[C@H](O)[C@@H](OC(N)=O)[C@@H](CO)O[C@H]1\N=C/1N[C@H](C(=O)NC[C@H]2O)[C@@H]2N\1 NRAUADCLPJTGSF-VLSXYIQESA-N 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 102000027257 transmembrane receptors Human genes 0.000 description 1
- 108091008578 transmembrane receptors Proteins 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
Definitions
- the invention relates to transformed Zeil lines according to the preamble of the first independent patent claim, which Zeil lines express heterologous G protein-coupled receptors (GPC receptors) and are suitable for detecting interactions between the GPC receptors or through the GPC receptors control signal transmission systems and substances (ligands, modulators), or to find substances that interact with the receptors or the signal transmission systems.
- GPC receptors heterologous G protein-coupled receptors
- the invention further relates to vectors for the production of these Zeil lines and the use of these Zeil lines for the detection of said interactions and for the detection of substances acting on the receptors or signal transmission systems (screening assays).
- G protein-coupled receptors are receptors with seven transmembrane domains, which together with heterotrimeric, guanyl nucleotide-binding, regulatory proteins (G proteins) form signal transduction systems for the transmission of many extracellular signals [H.G. Dohlmann, J. Thorner, M. Caron, and R. J. Lefkowitz (1991) Annu. Rev. Biochem., 60, 653-688].
- G proteins G protein-coupled receptors
- Somatostatin receptor is a prototype of the GPC receptors in mammalian cells. Somatostatin has extensive modulatory effects in the central nervous system and peripheral tissues and acts on a number of receptor subtypes.
- the signal transmission through a signal transmission system with GPC receptor and G protein has the following general characteristics: Heterotrimeric G proteins function as signal transmitters between a transmembrane receptor molecule (GPC receptor) and an enzyme called effector, which produces a secondary messenger. Adenylate cyclase, phospholipaseC and ion channels are examples of well-studied effectors in mammalian systems.
- G proteins consist of a guanyl nucleotide-binding ⁇ subunit, a ⁇ subunit and a 7 subunit (G ⁇ , Gß, G ⁇ subunit)
- G proteins exist in two different forms, depending on whether GDP or GTP is bound to the ⁇ subunit.
- GDP When GDP is bound, the G protein occurs as a heterotrimeric ⁇ ß ⁇ complex.
- the ⁇ subunit dissociates, leaving a ⁇ 7 complex behind.
- Association of a G ⁇ ß complex with an activated GPC receptor in the cell membrane leads to an increase in the exchange rate of GTP for bound GDP.
- the rate of dissociation of the bound G ⁇ subunit from the Gß ⁇ complex increases.
- the free ⁇ subunit and the Gß ⁇ complex can relay signals to cellular effectors of various signaling routes.
- the GPC receptors are important target molecules for therapeutic compounds.
- the human genome probably contains about 5000 different GPC receptor genes, on which new therapeutic compounds could act as ligands. Screening test systems are used for the investigation of such interactions of ligands and receptors and also of modulators and corresponding signal transmission systems, in which biochemical ligand binding studies, reporter systems in mammalian cells, or reporter systems in yeast cells are used according to the state of the art.
- yeast cells used in such test systems are transformed such that they express heterologous GPC receptors.
- Such yeast cells are described in the publication WO-92/05244 (US-5739029). These contain a first heterologous DNA sequence that expresses a heterologous GPC receptor and a second heterologous DNA sequence that expresses an ⁇ subunit of a mammalian G protein.
- the endogenous GPC receptors in yeast cells mediate the recognition of different cell types via extracellular peptides, so-called pheromones.
- the pheromone-activated signal transmission path initiates a development program which leads to the fusion of haploid ⁇ and a cells and the formation of diploid ⁇ / a cells [M. Whiteway and B.
- GPC receptor-controlled signal transduction in mammals and in yeast Another fundamental difference between GPC receptor-controlled signal transduction in mammals and in yeast is that so far no effector enzyme has been discovered in yeast cells that generates a secondary messenger in response to receptor stimulation.
- the ß ⁇ complex probably passes the pheromone signal directly on to the Ste20 protein kinase, which in turn activates a protein kinase cascade consisting of Stell, Ste7, Fus3 and Kssl, and the "Gerüs protein Ste5 [I. Herskowitz (1995) Cell, 80, 187-197].
- the cellular consequences of pheromone stimulation in yeast include the transcriptional induction of a whole range of genes and the arrest of the cell cycle.
- pheromone-inducible genes code for products that are required for the biosynthesis of the pheromones (MFA1, MFA2, MFal, MFa2, STE6, STE13), for the production of the receptors STE2 and STE3, for the pheromone signal transmission (GPA1, FUS3) , for cell cycle standstill (FAR1, CLN2, CLN3), for morphological changes and cell fusion (FUS1, FUS2, CHS1) and for pheromone desensitization (SST2, BARI).
- Pheromone-controlled transcription is mediated by the sequence-specific DNA binding protein Stel2.
- the transcription of the STE12 gene cannot be induced by pheromone.
- the functionally redundant MAP kinase homologues Fus3 and Kssl of the pheromone signaling pathway activate the transcription factor Stel2 by specific phosphorylation.
- Pheromone-inducible genes have cis-acting DNA sequences in their promoter region, the so-called "pheromone response element" (PRE).
- PRE pheromone response element
- the occurrence of PRE sequences in the promoter region of a gene in yeast is not sufficient for the transcription of this gene to be pheromone-inducible.
- the STE12 gene has multiple PREs, but the expression of STE12 is not pheromone-inducible.
- the object of the invention is to create transformed cell lines (human, animal or plant cells and also fungal cells) which express heterologous GPC receptors. These transformed Zeil lines are said to are suitable for the detection of interactions between substances (ligands, modulators) and GPC receptors or GPZ receptor-controlled signal transmission systems or for finding substances acting on the receptors or the signal transmission systems in screening assays. In order to be suitable for this purpose, the transformed cells should show a high sensitivity to such an interaction.
- the Zeil lines according to the invention (human, animal or plant cells and also fungal cells) have a signal transmission system with GPC receptors that can be activated by a ligand, which signal transmission system shows positive feedback.
- the mechanism of positive feedback is that the transcription of the gene coding for the GPC receptor-activatable transcription factor can be induced by receptor stimulation.
- the Zeil lines according to the invention can naturally have such a GPC receptor signal transmission system with positive feedback or a positive feedback mechanism can be built into an existing GPC receptor signal transmission chain by means of recombinant DNA technology.
- the natural or correspondingly modified signal transduction system with a positive feedback mechanism has a sensitivity which is significantly higher than that of known, cellular detection systems and which can be used for the detection of the above-mentioned interactions.
- Zeil lines with such a positive feedback mechanism thus react more sensitively than the corresponding, known cellular detection systems for activation of the GPC receptors, regardless of whether these receptors are endogenous or heterologous receptors introduced by transformation.
- the maize brandy fungus Ustilago maydis is an example of a cellular test system for GPC receptors in which a positive feedback mechanism is naturally present in the GPC receptor-controlled signal transmission chain. This positive feedback in pheromone-activatable signal transduction is described for Ustilago maydis by H. A. Hartmann, R. Kahmann and M. Bölker [EMBO J., 15, 1632-1641 (1996)].
- the Basidiomycet Ustilago maydis is used as a eukaryotic model organism. In its pathogenic form, it causes the cornburn on its host plant maize and is therefore also used as a model system for studying pathogenic fungi. The genetic constitution of U. maydis can be changed relatively easily [F. Banuette (1995) Annu. Rev. Genet., 29, 179-208].
- Zeil lines are therefore used for the detection of interactions between certain GPC receptors or corresponding signal transmission systems and test substances, which in their natural or in a genetically modified state have a GPC receptor-activatable signal transmission system in which the GPC receptor -induced transcription of target genes through feedback is reinforced. Furthermore, they have an endogenous or heterologous reporter gene, the expression of which is controlled by a promoter which can be induced by activating the receptor, the expression of the reporter gene being measurable (for example essential growth enzyme which causes measurable cell growth or other enzymes), which lead to measurable effects in biochemical reactions).
- the heterologous GPC receptor can associate with endogenous G protein or with heterologous G protein, in particular with a heterologous ⁇ subunit of the G protein.
- the cell can additionally contain a mutation in the gene which inactivates the endogenous ⁇ subunit responsible for the GPC receptor-controlled signal transmission and thereby facilitates the interaction between the heterologous receptor and the heterologous G protein.
- FIG. 1 shows the comparison between a signal transmission system with positive feedback (left), as is the case according to the invention
- Zeil lines e.g. Ustilago maydis
- a signal transmission system without positive feedback such as yeast cells have.
- heterologous is used in the present description in relation to the respective Zeil lines and consequently relates to DNA sequences, proteins and other materials which are introduced into the respective Zeil line by other organisms, or to combinations, that do not occur naturally in the respective Zeil lines.
- upstream and downstream are used below in relation to the direction of transcription and translation.
- a sequence that is transcribed or translated before another sequence is said to be “upstream” from the other sequence.
- Ustilago maydis, the detection and fusion of compatible cell types (al and a2) is naturally made possible by a signal transmission system, as was also described at the beginning for the yeast. This signal transmission system is controlled by pheromones and the corresponding pheromone receptors [J. Kronstad and C. Stäben (1997) Annu. Rev. Genet., 31, 245-276].
- Ustilago maydis cells with the crossing type al secrete the peptide pheromone Mfal, which binds to the specific Mfal receptor (Pra2) in a2 cells.
- Junction type a2 cells secrete the Mfa2 pheromone, which only works in Al cells that have the Mfa2 receptor (Pral) express.
- the pheromone receptors Pral and Pra2 are GPC receptors.
- the cellular effector of Gpa3 appears to be an adenylate cyclase (Uacl), since mutations in the gpa3 gene on the one hand make pheromone signal transmission impossible and on the other hand lead to filamentous growth, which is reminiscent of the growth of adenylate cyclase-deficient mutants.
- the filamentous growth of gpa3 mutants can be reversed by adding cyclic AMP, the secondary messenger that is produced by the adenylate cyclase [R. Kahmann and C. Basse (1997) Trends in Plant Sei., 2, 366-368; S. Gold, G. Duncan, K. Barrett and J.
- Pheromone stimulation in Ustilago ultimately results in the transcriptional induction of all genes that are at the intersection loci a and b, ie mfal, mfa2, pral, pra2, lga2, rga2, bE, bW [M. Urban, R. Kahmann and M. Bölker (1996) Mol. Gen. Genet., 251, 31-37]. All of these genes have at least one cis-acting DNA sequence, the "pheromone response element" (PRE), in their associated gene regulatory regions.
- the PRE-Se sequences from Ustilago maydis are recognized by the sequence-specific DNA binding protein Prfl.
- Pheromone stimulation leads to the activation of Prfl, which mediates the pheromone-inducible transcription of these genes. Since the promoter of the prfl gene itself also has PREs, the transcription of the prfl gene is also activated by pheromone stimulation (HA Hartmann, R. Kahmann and M. Bölker (1996) EMBO J., 15, 1632-1641). Due to the pheromone-inducible transcription of the prfl gene, a positive feedback mechanism is inherent in the Ustilago pheromone signal transmission pathway.
- GPC receptor-controlled signal transmission systems with positive feedback can be recognized by the fact that the transcription of the gene which codes for the transcription factor which is activated by stimulation of the GPC receptor and thereby controls the GPC receptor-controlled transcription of target genes, itself is also induced by stimulation of the GPC receptor.
- the mechanism described above is shown schematically on the left in FIG. This mechanism leads to a significantly higher sensitivity with which, for example, binding of ligands to the receptor can be perceived.
- the corresponding yeast system and other known cellular detection systems lack such a positive feedback mechanism.
- the expression of the GPC receptor-activatable transcription factor (Stel2) is not induced by receptor stimulation (see Fig. 1).
- the promoter of the STE12 gene which codes for the transcription factor activated by pheromone stimulation, can be replaced by a promoter which is pheromone-inducible (eg FUSI promoter).
- Expression vectors are replicable DNA constructs that are used to express a heterologous DNA sequence in a host cell.
- the heterologous DNA sequence to be expressed must be equipped with suitable control sequences which are capable of controlling the expression in the intended host of a protein or protein subunit encoded by the heterologous DNA sequence.
- Control sequences include a transcriptional promoter, optional cis-acting DNA sequences to regulate transcription, suitable DNA sequences which mediate efficient initiation of translation, and DNA sequences which terminate transcription and polyadenylation control the mRNA.
- Suitable vectors for the production of Zeil lines according to the invention include plasmids, viruses and integrable DNA fragments, ie DNA fragments which can be integrated into the host genome via genetic recombination.
- Suitable vectors contain control sequences derived from species that are functional in the intended expression host.
- Ustilago vectors can contain an autonomously replicating sequence (UARS) which enables the plasmid to replicate in high copy number in the Ustilago cell, a promoter, heterologous DNA sequences which code for the heterologous proteins to be expressed, sequences for the polya - Denylation and a selectable marker gene.
- UARS autonomously replicating sequence
- plasmid is pJW42 [J. Wang, D.W. Holden and S.A Leong (1988) Proc. Natl. Acad. Be. USA 85, 865-869].
- This plasmid contains the hph gene from Escherichia coli [L. Gritz and J. Davies (1983) Gene, 25, 179-188], which confers resistance to the antibiotic hygromycin B and can thus be used as a selectable marker.
- Other applicable marker genes are, for example, the cbx gene from Ustilago maydis, which confers resistance to the fungicide carboxin [P.L.E. Broomfield and JA. Hargreaves (1992) Curr.
- Suitable promoter sequences include the promoters of the hsp70 gene [DW Holden, JW Kronstad and S.A Leong (1989) EMBO J., 8, 1927-1934], of the glyceraldehyde-3-phosphate dehydrogenase gene [TL Smith and S.A. Leong (1990) Gene, 93, 111-117] and the translation elongation factor gene [HA Hartmann, R. Kahmann and M. Bölker (1996) EMBO J., 15, 1632- 1641].
- Other promoters with the additional advantage of transcriptional control through the growth conditions are the promoter of the crgl gene [A. Bottin, J. Kämper and R. Kahmann (1996) Mol. Gen.
- the termination sequences associated with these genes can also be ligated into the expression vectors downstream of the heterologous sequences.
- novel expression vectors have been developed. These expression vectors contain the Ustilago maydis hsp70 promoter and terminator, which mediate the transcription of the cDNAs for GPC receptors. Additional restriction enzyme sites are introduced between the hsp70 promoter and terminator to clone DNA segments to be expressed, e.g. Simplify GPCR cDNAs.
- DNA sequences encoding Ustilago pheromone receptors eg the pral gene encoding the Mfa2 pheromone receptor and pra2 gene encoding the Mfal pheromone receptor
- Ustilago Genes encoding GPC receptors that can be used to construct such vectors.
- a second segment which is located downstream of the said first segment and is in the correct reading frame therewith, comprises a DNA sequence which codes for a heterologous GPC receptor. Such adjustments to the translation initiation site can increase expression of a heterologous protein.
- the first and second segments are operatively associated with a promoter, such as the constitutive one hsp70 promoter or the inducible crgl promoter, which are functional in Ustilago cells.
- Each GPC receptor and the corresponding DNA sequences which code for these receptors can be used to produce the Zeil lines according to the invention.
- Examples of such receptors are adrenergic receptors ( ⁇ or ⁇ ), adenosine receptors, angiotensin receptors, bradykinin receptors, cannabinoid receptors, chemokine receptors, dopamine receptors, glucagon receptors, neurokinin receptors, neurotensin receptors. gates, serotonin receptors, opiate receptors, muscarinic receptors, somatostatin receptors and vasopressin receptors.
- the term "receptor” used here also includes subtypes and their mutants and homologs and also the DNA sequences which code for them.
- G ⁇ subunits and the corresponding DNA sequences which code for these G ⁇ subunits can be used to produce the Zeil lines according to the invention.
- G ⁇ subunits are Gs subunits, Go subunits, Gq subunits, Gi subunits and Gz subunits.
- the term "G ⁇ subunit” used here includes subtypes as well as their mutants and homologs and also DNA sequences which code for them.
- the functional expression of such heterologous G ⁇ subunits in Ustilago can easily be checked, since a defect in the Ustilago G ⁇ subunit Gpa3 leads to a characteristic, visually observable filamentous growth, in contrast to the yeast-like growth form of Ustilago cells with an intact gpa3 -Gene.
- Heterologous G ⁇ subunits which take on the function of the endogenous G ⁇ subunit Gpa3 in the pheromone signal transmission chain, complement the filamentous growth defect of the gpa3 mutant cells to normal, yeast-like growth and can therefore be easily identified.
- Gß ⁇ subunit and the corresponding DNA sequences coding for these Gß ⁇ subunits can be used to produce the Zeil lines according to the invention.
- the term "Gß-subunit” used here includes subtypes as well as their mutants and homologs and also DNA sequences which code for them.
- a third DNA construct which comprises a promoter and a reporter gene.
- the promoter can be induced by activating the heterologous GPC receptor.
- the reporter gene is placed downstream of the GPC receptor-inducible promoter and is operatively associated with it. The expression of the reporter gene can be measured and reflects the activation of the heterologous GPC receptor by suitable ligands.
- the promoter of the mfal gene, the promoter of the mfa2 gene, the promoter of the pral gene, the promoter of the pra2 gene or the promoter of the prfl gene can be used as GPC receptor-inducible promoters .
- Different, endogenous or heterologous genes can also be used as reporter genes. Examples of reporter genes are the pyr6 gene [JW Kronstad, J. Wang, SF Covert, DW Holden, GL McKnight and SA Leong (1989) Gene, 79, 97-106], the pyr3 gene [A Spanos, N.
- GFP Green Fluorescent Protein
- the Ustilago expression vectors pDT78 and pDT99 were constructed.
- the 3.1 kb Hindlll fragment of the autonomously replicating Ustilago vector pCM54 [T. Tsukuda, S. Carleton, S. Fotheringham and WK Holloman (1988) Mol. Cell. Biol, 8, 3703-3709] by a 2 kb Hind fragment of the plasmid pDWHIO [J. Wang, DW Holden and SA Leong (1988) Proc. Natl. Acad. Be. USA, 85, 865-869].
- This 2 kb Hindlll fragment contains the promoter and the transcription terminator of the U. maydis hsplO gene, separated by a BgUl interface.
- plasmid pDT48 was cut with S ⁇ cl and Pstl, and a 1.5 kb Sacl-Pstl fragment isolated from plasmid pNATl (pDT65) was inserted which contains the natl gene from Streptomyces noursei, which is resistant to the antibiotic of the streptothricin family Nourseothricin mediates [H. Krügel, G. Fiedler, C. Smith and S. Baumberg (1993) Gene, 127, 127-131].
- the expression of the natl gene in U. maydis is determined by the Promoter of the U.
- GPDH glyceraldehyde-3-phosphate dehydrogenase
- the resulting plasmid pDT78 has a single interface for BgUl between the U. maydis hsp70 promoter and the U. maydis hsp70 terminator. This restriction enzyme interface can be used to insert a DNA sequence to be expressed in U.
- U. maydis for example a cDNA which codes for a heterologous GPC receptor.
- the transcription of the cDNA that specifies the heterologous GPC receptor is thus mediated by the transcription control sequences of the U. maydis hsp70 promoter.
- the pDT78 expression vector and its derivatives can be introduced into Ustilago maydis using published transformation methods [J. Wang, DW Holden and SA Leong (1988) Proc. Natl. Acad. Be. USA 85, 865-869] and by adding the antibiotic nourseothricin (40 ⁇ g / ml) into the growth medium, selection is made for the presence of this plasmid in U. maydis cells.
- pDT49 is identical to the pDT48 described above, except that the 2 kb HindIII fragment, which contains the promoter and the transcription terminator of the U. maydis hsp70 gene, was introduced in the reverse orientation, ie the E. coli lacZ promoter of the plasmid and the introduced hsp70 promoters mediate transcription in the opposite direction.
- pDT49 was digested with Sm ⁇ l and with Pstl, the 3 'overhang of the Pstl interface was removed with T4 DNA polymerase and the plasmid was religated.
- pDT90 contains a gene which mediates resistance to the fungicide carboxin in U. maydis [PLE Broomfield and JA. Hargreaves (1992) Curr. Genet., 22, 117-121].
- pDT99 can be introduced into U. maydis cells by means of transformation [J. Wang, DW Holden and SA Leong (1988) Proc. Natl. Acad. Be. USA, 85, 865-869] and by adding the fungicide carboxin (2 ⁇ g / ml) to the growth medium, the presence of this plasmid is selected in U. maydis cells.
- the DNA sequence of the 1.2 kb ⁇ mHI b2-AR PCR product thus cloned was verified.
- the 1.2 kb BamHI fragment of the resulting plasmid pDT87 was then inserted into the BglII site of the expression vector pDT78 in such a way that the ⁇ 2-AR sequence is transcribed in the correct orientation by the hsp70 promoter.
- the resulting ⁇ 2-AR expression plasmid pDT94 can now be published using published transformation methods [J. Wang, DW Holden and SA Leong (1988) Proc. Natl. Acad. Be. USA 85, 865-869] can be introduced into U. maydis cells.
- U. maydis The biochemical proof that the human ß2-AR is expressed in U. maydis can be provided by means of ligand binding studies.
- membrane fractions of U. maydis cells transformed with pDT94 can be carried out for binding studies with, for example, the radioactively labeled ⁇ 2-AR ligand 3- [ 12S I] - iodocyanopindolol according to published protocols [HK Dohlman, MG Caron, A. DeBlasi, T Frielle and RJ Lefkowitz (1990) Biochemistry, 29, 2335-2342].
- Evidence that the human ⁇ 2-AR expressed in U. maydis is functional and interacts with the pheromone signaling chain of U.
- the plasmid pMUl contains the bacterial uidA gene, which codes for the enzyme ⁇ -glucuronidase (GUS) [RA Jefferson, SM Burgess and D. Hirsh (1986) Proc. Natl. Acad. Be. USA 86, 8447-8451] and its expression is regulated by the strongly pheromone-inducible promoter of the mfal gene [M. Urban, R. Kahmann and M. Bölker (1996) Mol. Gen. Genet., 251, 31-37].
- GUS ⁇ -glucuronidase
- U. maydis transformants which contain pDT94 and pMUl, are stimulated with, for example, the ß-adrenergic agonist isoproterenol.
- the stimulation of the receptor can thus be demonstrated by a simple biochemical test for GUS activity, as described, for example, in A. Gururaj Rao and P. Flynn (1992) in GUS Protocols; SR Gallagher (ed.), Academic Press Inc., 89-99.
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Abstract
According to the invention, transformed cell lines, for example of Ustilago maydis, are used for detecting interactions between GPC-receptors (receptors with seven transmembrane sites, which interact with guanyl nucleotide-binding, regulatory proteins) or GPC-receptor-controlled signal transmission systems and test substances (ligands, modulators), or for searching for substances which are capable of interacting with receptors or signal transmission systems of this type. The transformed cell lines have a GPC-receptor-controlled signal transduction path with positive feedback and are transformed in such a way that they express a heterologous GPC- receptor. The transformed cell lines also contain a reporter gene, the expression of which can be detected using measuring techniques and which is controlled by a promotor. Said promoter can be induced by stimulating a GPC-receptor, and is endogenous. The reporter gene is endogenous or heterologous.
Description
TRANSFORMIERTE ZELL-LINIEN, DIE HETEROLOGE G-PROTEIN-GEKOPPELTE REZEPTOREN EXPRIMIEREN TRANSFORMED CELL LINES THAT EXPRESS HETEROLOGIST G-PROTEIN-COUPLED RECEPTORS
Gebiet der ErfindungField of the Invention
Die Erfindung betrifft transformierte Zeil-Linien nach dem Oberbegriff des ersten, unabhängigen Patentanspruchs, welche Zeil-Linien heterologe G-Pro- tein-gekoppelte Rezeptoren (GPC-Rezeptoren) exprimieren und sich eignen zum Nachweis von Wechselwirkungen zwischen den GPC-Rezeptoren oder den durch die GPC-Rezeptoren kontrollierten Signalübeπnittlungssystemen und Substanzen (Liganden, Modulatoren), bzw. zum Auffinden von Sub- stanzen, die mit den Rezeptoren oder den Signalübermittlungssystemen in Wechselwirkung treten. Die Erfindung betrifft ferner Vektoren zur Herstellung dieser Zeil-Linien und die Verwendung dieser Zeil-Linien zum Nachweis der genannten Wechselwirkungen und zum Auffinden von auf die Rezeptoren oder Signalübermittlungssysteme wirkenden Substanzen (Screening Assays).
Stand der TechnikThe invention relates to transformed Zeil lines according to the preamble of the first independent patent claim, which Zeil lines express heterologous G protein-coupled receptors (GPC receptors) and are suitable for detecting interactions between the GPC receptors or through the GPC receptors control signal transmission systems and substances (ligands, modulators), or to find substances that interact with the receptors or the signal transmission systems. The invention further relates to vectors for the production of these Zeil lines and the use of these Zeil lines for the detection of said interactions and for the detection of substances acting on the receptors or signal transmission systems (screening assays). State of the art
G-Protein-gekoppelte Rezeptoren (GPC-Rezeptoren) sind Rezeptoren mit sieben transmembranären Domänen, die zusammen mit heterotrimeren, Gua- nylnukleotid-bindenden, regulatorischen Proteinen (G-Proteine) Signaltrans- duktionssysteme zur Übermittlung vieler extrazellulärer Signale bilden [H.G. Dohlmann, J. Thorner, M. Caron, and R. J. Lefkowitz (1991) Annu. Rev. Biochem., 60, 653-688]. Derartige Signaltransduktionssysteme kommen in einem breiten Spektrum von Organismen vor, angefangen bei einfachen Pilzen bis zum Menschen.G protein-coupled receptors (GPC receptors) are receptors with seven transmembrane domains, which together with heterotrimeric, guanyl nucleotide-binding, regulatory proteins (G proteins) form signal transduction systems for the transmission of many extracellular signals [H.G. Dohlmann, J. Thorner, M. Caron, and R. J. Lefkowitz (1991) Annu. Rev. Biochem., 60, 653-688]. Such signal transduction systems occur in a wide range of organisms, from simple fungi to humans.
Ein Beispiel eines derartigen Rezeptors ist der Somatostatin-Rezeptor, der einen Prototyp der GPC-Rezeptoren in Säugerzellen darstellt. Somatostatin hat weitreichende modulatorische Wirkungen im zentralen Nervensystem und im peripheren Gewebe und wirkt auf eine Reihe von Rezeptor-Subtypen.An example of such a receptor is the somatostatin receptor, which is a prototype of the GPC receptors in mammalian cells. Somatostatin has extensive modulatory effects in the central nervous system and peripheral tissues and acts on a number of receptor subtypes.
Die Signalübermittlung durch ein Signalübermittlungssystem mit GPC-Rezeptor und G-Protein hat die folgenden generellen Merkmale: Heterotrimere G- Proteine funktionieren als Signalübermittler zwischen einem transmembranären Rezeptor Molekül (GPC-Rezeptor) und einem als Effektor bezeichneten Enzym, das einen sekundären Botenstoff produziert. Adenylatzyklase, Phos- pholipaseC und Ionenkanäle sind Beispiele gut untersuchter Effektoren in Säugetier-Systemen.The signal transmission through a signal transmission system with GPC receptor and G protein has the following general characteristics: Heterotrimeric G proteins function as signal transmitters between a transmembrane receptor molecule (GPC receptor) and an enzyme called effector, which produces a secondary messenger. Adenylate cyclase, phospholipaseC and ion channels are examples of well-studied effectors in mammalian systems.
G-Proteine bestehen aus einer Guanylnukleotid-bindenden α-Untereinheit, einer ß-Untereinheit und einer 7-Untereinheit (Gα-, Gß-, Gγ-Untereinheit)G proteins consist of a guanyl nucleotide-binding α subunit, a β subunit and a 7 subunit (Gα, Gß, Gγ subunit)
[M. I. Simon, M. P. Strathmann and N. Gautam (1991) Science, 252, 802-808].
G-Proteine existieren in zwei unterschiedlichen Formen, abhängig davon, ob GDP oder GTP an die α-Untereinheit gebunden ist. Wenn GDP gebunden ist, kommt das G-Protein als heterotrimerer αßγ-Komplex vor. Durch die Bindung von GTP an das G-Protein, dissoziiert die α-Untereinheit und lässt einen ß7-Komplex zurück. Assoziation eines Gαß -Komplexes mit einem aktivierten GPC-Rezeptor in der Zellmembran führt zu einer Erhöhung der Austauschrate von GTP für gebundenes GDP. Als Folge erhöht sich die Dissoziationsrate der gebundenen Gα-Untereinheit vom Gßγ-Komplex. Die freie α-Untereinheit und der Gßγ-Komplex können Signale an zelluläre Effektoren verschiedener Signalübermittlungswege weiterleiten.[MI Simon, MP Strathmann and N. Gautam (1991) Science, 252, 802-808]. G proteins exist in two different forms, depending on whether GDP or GTP is bound to the α subunit. When GDP is bound, the G protein occurs as a heterotrimeric αßγ complex. By binding GTP to the G protein, the α subunit dissociates, leaving a β7 complex behind. Association of a Gαß complex with an activated GPC receptor in the cell membrane leads to an increase in the exchange rate of GTP for bound GDP. As a result, the rate of dissociation of the bound Gα subunit from the Gßγ complex increases. The free α subunit and the Gßγ complex can relay signals to cellular effectors of various signaling routes.
Die GPC-Rezeptoren stellen wichtige Zielmoleküle für therapeutische Verbindungen dar. Das menschliche Genom enthält vermutlich etwa 5000 ver- schiedene GPC-Rezeptor-Gene, auf die neue therapeutische Verbindungen als Liganden wirken könnten. Für die Untersuchung derartiger Wechselwirkungen von Liganden und Rezeptoren und auch von Modulatoren und entsprechenden Siganübermittlungssystemen werden Screening Testsysteme verwendet, in denen gemass dem Stande der Technik biochemische Ligandenbindungs-Stu- dien, Reportersysteme in Säugetierzellen, oder Reportersysteme in Hefezellen zur Anwendung kommen.The GPC receptors are important target molecules for therapeutic compounds. The human genome probably contains about 5000 different GPC receptor genes, on which new therapeutic compounds could act as ligands. Screening test systems are used for the investigation of such interactions of ligands and receptors and also of modulators and corresponding signal transmission systems, in which biochemical ligand binding studies, reporter systems in mammalian cells, or reporter systems in yeast cells are used according to the state of the art.
In solchen Testsystemen verwendete Hefezellen sind derart transformiert, dass sie heterologe GPC-Rezeptoren exprimieren. In der Publikation WO- 92/05244 (US-5739029) werden derartige Hefezellen beschrieben. Diese enthalten eine erste heterologe DNA-Sequenz, die einen heterologen GPC-Rezeptor exprimiert, und eine zweite heterologe DNA-Sequenz, die eine α-Untereinheit eines Säuger-G-Proteins exprimiert.
Die endogenen GPC-Rezeptoren in Hefezellen vermitteln die Erkennung von unterschiedlichen Zelltypen via extrazelluläre Peptide, sogenannte Pheromo- ne. Der Pheromon-aktivierte Signalübermittlungsweg leitet ein Entwicklungsprogramm ein, welches zur Verschmelzung von haploiden α- und a-Zellen und zur Ausbildung diploider α/a-Zellen führt [M. Whiteway and B. Errede (1993) in: Signal Transduction, Prokaryotic and Simple Eukaryotic Systems, ed. J. Kurjan and B. L. Taylor, Academic Press, pp. 189-237]. Zellen des Kreuzungstyps α sekretieren α-Faktor, welcher in a-Zellen an den α-Faktor- Rezeptor (Ste2) bindet, und Zellen des Kreuzungstyps a sekretieren a-Faktor, welcher in α-Zellen an den spezifischen a-Faktor Rezeptor (Ste3) bindet. Sowohl Ste2 als auch Ste3 gehören zur Familie der GPC-Rezeptoren. Nach der Bindung des Pheromons an den entsprechenden Rezeptor ändert der Pheromon-Rezeptor wahrscheinlich seine Konformation. Dies führt zur Dissoziation der Gα-Untereinheit (Gpal) vom Gßγ-Komplex (Ste4, Stel8) und somit zur Aktivierung des G-Proteins. Interessanterweise - und im Gegensatz zur Funktion von G-Proteinen in Säugetier Systemen - übt die Gα-Untereinheit in Hefe einen negativen Einfluss auf die Signalübermittlung aus, während die Gßγ-Untereinheit das Pheromon-Signal weiterleitet.Yeast cells used in such test systems are transformed such that they express heterologous GPC receptors. Such yeast cells are described in the publication WO-92/05244 (US-5739029). These contain a first heterologous DNA sequence that expresses a heterologous GPC receptor and a second heterologous DNA sequence that expresses an α subunit of a mammalian G protein. The endogenous GPC receptors in yeast cells mediate the recognition of different cell types via extracellular peptides, so-called pheromones. The pheromone-activated signal transmission path initiates a development program which leads to the fusion of haploid α and a cells and the formation of diploid α / a cells [M. Whiteway and B. Errede (1993) in: Signal Transduction, Prokaryotic and Simple Eukaryotic Systems, ed. J. Kurjan and BL Taylor, Academic Press, pp. 189-237]. Cells of the crossing type α secrete α factor, which binds to the α factor receptor (Ste2) in a cells, and cells of the crossing type a secrete a factor, which binds to the specific a factor receptor (Ste3 ) binds. Both Ste2 and Ste3 belong to the family of GPC receptors. After the pheromone binds to the corresponding receptor, the pheromone receptor is likely to change its conformation. This leads to the dissociation of the Gα subunit (Gpal) from the Gßγ complex (Ste4, Stel8) and thus to the activation of the G protein. Interestingly, and in contrast to the function of G proteins in mammalian systems, the Gα subunit in yeast has a negative influence on signal transmission, while the Gßγ subunit passes on the pheromone signal.
Ein weiterer, grundlegender Unterschied zwischen GPC-Rezeptor-kontrollier- ter Signaltransduktion in Säugetieren und in Hefe besteht darin, dass bis jetzt in Hefezellen noch kein Effektor-Enzym entdeckt worden ist, welches einen sekundären Botenstoff generiert als Reaktion auf Rezeptorstimulation. Wahrscheinlich leitet der ßγ-Komplex das Pheromonsignal direkt weiter an die Ste20-Proteinkinase, welche wiederum eine Proteinkinasen-Kaskade aktiviert, bestehend aus Stell, Ste7, Fus3 und Kssl, und dem "Gerüs -Protein Ste5 [I. Herskowitz (1995) Cell, 80, 187-197].
Zu den zellulären Konsequenzen der Pheromon-Stimulation in Hefe gehören die transkriptionelle Induktion einer ganzen Reihe von Genen und der Stillstand des Zellzyklus. Diese Pheromon-induzierbaren Gene kodieren für Produkte, die für die Biosynthese der Pheromone benötigt werden (MFA1, MFA2, MFal, MFa2, STE6, STE13), für die Produktion der Rezeptoren STE2 und STE3, für die Pheromon-Signalübermittlung (GPA1, FUS3), für Zellzyklus-Stillstand (FAR1, CLN2, CLN3), für morphologische Veränderungen und Zellfusion (FUS1, FUS2, CHS1) und für Pheromon-Desensibilisie- rung (SST2, BARI).Another fundamental difference between GPC receptor-controlled signal transduction in mammals and in yeast is that so far no effector enzyme has been discovered in yeast cells that generates a secondary messenger in response to receptor stimulation. The ßγ complex probably passes the pheromone signal directly on to the Ste20 protein kinase, which in turn activates a protein kinase cascade consisting of Stell, Ste7, Fus3 and Kssl, and the "Gerüs protein Ste5 [I. Herskowitz (1995) Cell, 80, 187-197]. The cellular consequences of pheromone stimulation in yeast include the transcriptional induction of a whole range of genes and the arrest of the cell cycle. These pheromone-inducible genes code for products that are required for the biosynthesis of the pheromones (MFA1, MFA2, MFal, MFa2, STE6, STE13), for the production of the receptors STE2 and STE3, for the pheromone signal transmission (GPA1, FUS3) , for cell cycle standstill (FAR1, CLN2, CLN3), for morphological changes and cell fusion (FUS1, FUS2, CHS1) and for pheromone desensitization (SST2, BARI).
Pheromon-kontrollierte Transkription wird durch das sequenzspezifische DNA-Bindungsprotein Stel2 vermittelt. Die Transkription des STE12-Gens ist nicht durch Pheromon induzierbar. Es wird vermutet, dass die funktioneil redundanten MAP-Kinase-Homologe Fus3 und Kssl des Pheromon-Signal- übermittlungswegs den Transkriptionsfaktor Stel2 durch spezifische Phospho- rylation aktivieren. Pheromon-induzierbare Gene weisen cis-wirkende DNA- Sequenzen in ihrer Promotorregion auf, das sogenannte "Pheromon Response Element" (PRE). Das Vorkommen von PRE-Sequenzen in der Promotorre- gion eines Gens in Hefe genügt jedoch nicht, dass die Transkription dieses Gens Pheromon-induzierbar ist. So hat z.B. das STE12-Gen mehrere PREs, aber die Expression von STE12 ist nicht Pheromon-induzierbar.Pheromone-controlled transcription is mediated by the sequence-specific DNA binding protein Stel2. The transcription of the STE12 gene cannot be induced by pheromone. It is assumed that the functionally redundant MAP kinase homologues Fus3 and Kssl of the pheromone signaling pathway activate the transcription factor Stel2 by specific phosphorylation. Pheromone-inducible genes have cis-acting DNA sequences in their promoter region, the so-called "pheromone response element" (PRE). However, the occurrence of PRE sequences in the promoter region of a gene in yeast is not sufficient for the transcription of this gene to be pheromone-inducible. For example, the STE12 gene has multiple PREs, but the expression of STE12 is not pheromone-inducible.
Grundzüge der ErfindungBasics of the invention
Die Erfindung stellt sich die Aufgabe, transformierte Zeil-Linien (humane, tierische oder pflanzliche Zellen sowie auch Pilzzellen), die heterologe GPC- Rezeptoren exprimieren, zu schaffen. Diese transformierten Zeil-Linien sollen
sich eignen für den Nachweis von Wechselwirkungen zwischen Substanzen (Liganden, Modulatoren) und GPC-Rezeptoren bzw. GPZ-Rezeptor-kontrol- lierten Signalübermittlungssystemen bzw. zum Auffinden von auf die Rezeptoren bzw. die Signalübermittlungssysteme wirkenden Substanzen in Screening Assays. Um für diesen Zweck geeignet zu sein, sollen die transformierten Zellen eine hohe Sensitivität auf eine derartige Wechselwirkung zeigen.The object of the invention is to create transformed cell lines (human, animal or plant cells and also fungal cells) which express heterologous GPC receptors. These transformed Zeil lines are said to are suitable for the detection of interactions between substances (ligands, modulators) and GPC receptors or GPZ receptor-controlled signal transmission systems or for finding substances acting on the receptors or the signal transmission systems in screening assays. In order to be suitable for this purpose, the transformed cells should show a high sensitivity to such an interaction.
Diese Aufgabe wird gelöst durch die transformierten Zellen-Linien, wie sie in den Patentansprüchen definiert sind.This object is achieved by the transformed cell lines as defined in the patent claims.
Die erfindungsgemässen Zeil-Linien (humane, tierische oder pflanzliche Zellen sowie auch Pilzzellen) weisen ein durch einen Liganden aktivierbares Signalübermittlungssystem mit GPC-Rezeptoren auf, welches Signalübermittlungssystem eine positive Rückkoppelung zeigt. Der Mechanismus der positiven Rückkoppelung besteht darin, dass die Transkription desjenigen Gens, das für den GPC-Rezeptor-aktivierbaren Transkriptionsfaktor kodiert, selbst durch Rezeptor-Stimulation induzierbar ist.The Zeil lines according to the invention (human, animal or plant cells and also fungal cells) have a signal transmission system with GPC receptors that can be activated by a ligand, which signal transmission system shows positive feedback. The mechanism of positive feedback is that the transcription of the gene coding for the GPC receptor-activatable transcription factor can be induced by receptor stimulation.
Die erfindungsgemässen Zeil-Linien können natürlicherweise ein solches GPC-Rezeptor-Signalübermittlungssystem mit positiver Rückkoppelung aufweisen oder ein positiver Rückkoppelungsmechanismus kann mittels rekom- binanter DNA-Technologie in eine bestehende GPC-Rezeptor-Signalübermitt- lungskette eingebaut werden. Dadurch weist das natürliche oder entsprechend modifizierte Signaltransduktionssystem mit positivem Rückkoppelungsmechanismus eine gegenüber bekannten, zellulären Nachweissystemen bedeutend höhere Sensitivität auf, die für den Nachweis der genannten Wechselwirkun- gen ausgenützt werden kann. Zeil-Linien mit einem derartigen, positiven Rückkoppelungsmechanismus reagieren also sensitiver als die entsprechenden,
bekannten zellulären Nachweissysteme auf eine Aktivierung der GPC-Rezeptoren, ungeachtet, ob diese Rezeptoren die endogenen sind oder durch Transformation eingeführte heterologe Rezeptoren.The Zeil lines according to the invention can naturally have such a GPC receptor signal transmission system with positive feedback or a positive feedback mechanism can be built into an existing GPC receptor signal transmission chain by means of recombinant DNA technology. As a result, the natural or correspondingly modified signal transduction system with a positive feedback mechanism has a sensitivity which is significantly higher than that of known, cellular detection systems and which can be used for the detection of the above-mentioned interactions. Zeil lines with such a positive feedback mechanism thus react more sensitively than the corresponding, known cellular detection systems for activation of the GPC receptors, regardless of whether these receptors are endogenous or heterologous receptors introduced by transformation.
Der Maisbrand-Pilz Ustilago maydis ist ein Beispiel eines zellulären Testsystems für GPC-Rezeptoren, in dem ein positiver Rückkoppelungsmechanismus in der GPC-Rezeptor-kontrollierten Signalübermittlungskette natürlicherweise besteht. Diese positive Rückkoppelung in der Pheromon-aktivierbaren Signal- transduktion wird für Ustilago maydis beschrieben von H. A. Hartmann, R. Kahmann and M. Bölker [EMBO J., 15, 1632-1641 (1996)]. Der Basidiomycet Ustilago maydis wird als eukaryontischer Modellorganismus genutzt. Er verursacht in seiner pathogenen Form den Maisbrand auf seiner Wirtspflanze Mais und dient aus diesem Grunde auch als Modellsystem zum Studium von pathogenen- Pilzen. Die genetische Konstitution von U. maydis kann relativ leicht verändert werden [F. Banuette (1995) Annu. Rev. Genet., 29, 179-208].The maize brandy fungus Ustilago maydis is an example of a cellular test system for GPC receptors in which a positive feedback mechanism is naturally present in the GPC receptor-controlled signal transmission chain. This positive feedback in pheromone-activatable signal transduction is described for Ustilago maydis by H. A. Hartmann, R. Kahmann and M. Bölker [EMBO J., 15, 1632-1641 (1996)]. The Basidiomycet Ustilago maydis is used as a eukaryotic model organism. In its pathogenic form, it causes the cornburn on its host plant maize and is therefore also used as a model system for studying pathogenic fungi. The genetic constitution of U. maydis can be changed relatively easily [F. Banuette (1995) Annu. Rev. Genet., 29, 179-208].
Es ist anzunehmen, dass auch andere Zeil-Linien, insbesondere Pilze der Ustilago-Familie oder der Basidiomyceten überhaupt, derartige GPC-Rezep- tor-aktivierbare Signaltransduktionswege mit positiver Rückkoppelung aufweisen und sich dadurch wie Ustilago maydis natürlicherweise für die genannten Nachweisreaktionen anbieten.It can be assumed that other Zeil lines, in particular fungi from the Ustilago family or the Basidiomycetes in general, have such GPC receptor-activatable signal transduction pathways with positive feedback and, as a result, like Ustilago maydis, are naturally suitable for the detection reactions mentioned.
Gemass Erfindung werden also für den Nachweis von Wechselwirkungen zwischen bestimmten GPC-Rezeptoren bzw. entsprechenden Signalübermittlungssystemen und Testsubstanzen Zeil-Linien verwendet, die in ihrem natürlichen oder in einem gentechnisch modifizierten Zustand ein GPC-Rezeptor-aktivierbares Signalübermittlungssystem aufweisen, in dem die GPC-Rezeptor-induzierte Transkription von Zielgenen durch Rückkoppelung
verstärkt ist. Ferner weisen sie ein endogenes oder heterologes Reportergen auf, dessen Expression von einem Promotor gesteuert ist, der durch Aktivierung des Rezeptors induzierbar ist, wobei die Expression des Reportergens messtechnisch erfassbar ist (z.B. essentielles Wachstumsenzym, durch das messbares Zellwachstum bewirkt wird, oder andere Enzyme, die in biochemischen Reaktionen zu messbaren Wirkungen führen).According to the invention, Zeil lines are therefore used for the detection of interactions between certain GPC receptors or corresponding signal transmission systems and test substances, which in their natural or in a genetically modified state have a GPC receptor-activatable signal transmission system in which the GPC receptor -induced transcription of target genes through feedback is reinforced. Furthermore, they have an endogenous or heterologous reporter gene, the expression of which is controlled by a promoter which can be induced by activating the receptor, the expression of the reporter gene being measurable (for example essential growth enzyme which causes measurable cell growth or other enzymes), which lead to measurable effects in biochemical reactions).
In den Zellen der erfindungsgemässen Zeil-Linien kann der heterologe GPC- Rezeptor sich mit endogenem G-Protein assoziieren oder mit heterologem G- Protein, insbesondere mit einer heterologen α-Untereinheit des G-Proteins. Die Zelle kann zusätzlich eine Mutation in demjenigen Gen enthalten, welches die für die GPC-Rezeptor-kontrollierte Signalübermittlung verantwortliche endogene α-Untereinheit inaktiviert und dabei die Interaktion zwi- sehen dem heterologen Rezeptor und dem heterologen G-Protein erleichtert.In the cells of the Zeil lines according to the invention, the heterologous GPC receptor can associate with endogenous G protein or with heterologous G protein, in particular with a heterologous α subunit of the G protein. The cell can additionally contain a mutation in the gene which inactivates the endogenous α subunit responsible for the GPC receptor-controlled signal transmission and thereby facilitates the interaction between the heterologous receptor and the heterologous G protein.
Kurze Beschreibung der FigurenBrief description of the figures
Figur 1 zeigt den Vergleich zwischen einem Signalübermittlungssystem mit positiver Rückkoppelung (links), wie es die erfindungsgemässenFIG. 1 shows the comparison between a signal transmission system with positive feedback (left), as is the case according to the invention
Zeil-Linien aufweisen (z.B. Ustilago maydis), und einem Signal- Übermittlungssystem ohne positive Rückkoppelung (rechts), wie es beispielsweise Hefezellen aufweisen.Zeil lines (e.g. Ustilago maydis), and a signal transmission system without positive feedback (right), such as yeast cells have.
Detaillierte Beschreibung der Erfindung
Der Begriff "heterolog" wird in der vorliegenden Beschreibung in Bezug auf die jeweiligen Zeil-Linien benutzt und bezieht sich demzufolge auf DNA- Sequenzen, Proteine und andere Materialien, die von anderen Organismen in die jeweilige Zeil-Linie eingebracht werden, oder auf Kombinationen, die nicht natürlicherweise in den jeweiligen Zeil-Linien vorkommen.Detailed description of the invention The term "heterologous" is used in the present description in relation to the respective Zeil lines and consequently relates to DNA sequences, proteins and other materials which are introduced into the respective Zeil line by other organisms, or to combinations, that do not occur naturally in the respective Zeil lines.
Die Begriffe "stromaufwärts" und "stromabwärts" werden im Folgenden in Be- zug auf die Richtung der Transkription und der Translation benutzt. Eine Sequenz, die vor einer anderen Sequenz transkribiert oder translatiert wird, wird als "stromaufwärts" von der anderen Sequenz bezeichnet.The terms "upstream" and "downstream" are used below in relation to the direction of transcription and translation. A sequence that is transcribed or translated before another sequence is said to be "upstream" from the other sequence.
Methoden und Materialien, mit deren Hilfe die erfindungsgemässen Zeil- Linien hergestellt werden, werden für das Beispiel Ustilago maydis im Detail beschrieben. Dies soll die erfindungsgemässen Zeil-Linien aber in keiner Weise auf diese Spezies beschränken. Für andere Zeil-Linien sind die Methoden und Materialien entsprechend anzuwenden, was für den Fachmann ohne Probleme möglich ist.Methods and materials with which the Zeil lines according to the invention are produced are described in detail for the Ustilago maydis example. However, this should in no way limit the Zeil lines according to the invention to this species. For other Zeil lines, the methods and materials are to be used accordingly, which is possible without any problems for the person skilled in the art.
Für Ustilago maydis wird natürlicherweise die Erkennung und Fusion von kompatiblen Zelltypen (al und a2) durch ein Signalübermittlungssystem er- möglicht, wie dies eingangs auch für die Hefe beschrieben wurde. Dieses Signalübermittlungssystem wird durch Pheromone und die entsprechenden Pheromonrezeptoren kontrolliert [J. Kronstad and C. Stäben (1997) Annu. Rev. Genet., 31, 245-276]. Zellen von Ustilago maydis mit dem Kreuzungstyp al sekretieren das Peptid-Pheromon Mfal, welches in a2-Zellen an den spezi- fischen Mfal-Rezeptor (Pra2) bindet. Zellen des Kreuzungstyps a2 sekretieren das Mfa2-Pheromon, welches nur in al-Zellen wirkt, die den Mfa2-Rezeptor
(Pral) exprimieren. Bei den Pheromonrezeptoren Pral und Pra2 handelt es sich um GPC-Rezeptoren.For Ustilago maydis, the detection and fusion of compatible cell types (al and a2) is naturally made possible by a signal transmission system, as was also described at the beginning for the yeast. This signal transmission system is controlled by pheromones and the corresponding pheromone receptors [J. Kronstad and C. Stäben (1997) Annu. Rev. Genet., 31, 245-276]. Ustilago maydis cells with the crossing type al secrete the peptide pheromone Mfal, which binds to the specific Mfal receptor (Pra2) in a2 cells. Junction type a2 cells secrete the Mfa2 pheromone, which only works in Al cells that have the Mfa2 receptor (Pral) express. The pheromone receptors Pral and Pra2 are GPC receptors.
Aktivierung der Rezeptoren Pral und Pra2 durch Bindung des entsprechenden Pheromons führt wahrscheinlich zur Dissoziation eines heterotrimeren G- Proteins, von dem bis jetzt nur die α-Untereinheit Gpa3 bekannt ist [E. Regenfelder, T. Spellig, A. Hartmann, S. Lauenstein, M. Bölker and R. Kahmann (1997) EMBO J., 16, 1934-1942]. Gpa3 - im Gegensatz zum funktionel- len Homolog in Hefe (Gpal) - übt einen positiven Einfluss auf die Pheromon- Signalübermittlung aus. Interessanterweise scheint der zelluläre Effektor von Gpa3 eine Adenylatzyklase zu sein (Uacl), da Mutationen im gpa3-Gen einerseits die Pheromon-Signalübermittlung verunmöglichen und andererseits zu filamentösem Wachstum führen, welches an das Wachstum von Adenylatzy- klase-defizienten Mutanten erinnert. Des weiteren kann das filamentöse Wachstum von gpa3-Mutanten rückgängig gemacht werden durch Zugabe von cyclischem AMP, dem sekundären Botenstoff, der von der Adenylatzyklase produziert wird [R. Kahmann and C. Basse (1997) Trends in Plant Sei., 2, 366-368; S. Gold, G. Duncan, K. Barrett and J. Kronstad (1994) Genes Dev., 8, 2805-2816]. Demzufolge scheint die Pheromon-Signalübermittlung in Ustilago den entsprechenden Mechanismen in Säugetier-Systemen ähnlicher zu sein als dies der Fall ist für die Pheromon-kontrollierte Signalübermittlung in Hefe.Activation of the receptors Pral and Pra2 by binding the corresponding pheromone probably leads to the dissociation of a heterotrimeric G protein, of which only the α subunit Gpa3 is known to date [E. Regenfelder, T. Spellig, A. Hartmann, S. Lauenstein, M. Bölker and R. Kahmann (1997) EMBO J., 16, 1934-1942]. Gpa3 - in contrast to the functional homolog in yeast (Gpal) - has a positive influence on the pheromone signal transmission. Interestingly, the cellular effector of Gpa3 appears to be an adenylate cyclase (Uacl), since mutations in the gpa3 gene on the one hand make pheromone signal transmission impossible and on the other hand lead to filamentous growth, which is reminiscent of the growth of adenylate cyclase-deficient mutants. Furthermore, the filamentous growth of gpa3 mutants can be reversed by adding cyclic AMP, the secondary messenger that is produced by the adenylate cyclase [R. Kahmann and C. Basse (1997) Trends in Plant Sei., 2, 366-368; S. Gold, G. Duncan, K. Barrett and J. Kronstad (1994) Genes Dev., 8, 2805-2816]. As a result, pheromone signaling in Ustilago appears to be more similar to the corresponding mechanisms in mammalian systems than is the case for pheromone-controlled signaling in yeast.
Pheromon-Stimulation in Ustilago resultiert letztendlich in der transkriptionel- len Induktion aller Gene, die sich an den Kreuzungstypen-Loci a und b befinden, d.h. mfal, mfa2, pral, pra2, lga2, rga2, bE, bW [M. Urban, R. Kahmann and M. Bölker (1996) Mol. Gen. Genet., 251, 31-37]. All diese Gene besitzen in ihren assoziierten genregulatorischen Regionen mindestens eine cis-wirken- de DNA-Sequenz, das "Pheromon Response Element" (PRE). Die PRE-Se-
quenzen von Ustilago maydis werden vom sequenzspezifischen DNA-Bindungsprotein Prfl erkannt. Pheromon-Stimulation führt zur Aktivierung von Prfl, welches die Pheromon-induzierbare Transkription dieser Gene vermittelt. Da der Promotor des prfl-Gens selbst auch PREs aufweist, wird auch die Transkription des prfl-Gens durch Pheromon-Stimulation aktiviert (H. A. Hartmann, R. Kahmann and M. Bölker (1996) EMBO J., 15, 1632-1641). Durch die Pheromon-induzierbare Transkription des prfl-Gens ist dem Phero- mon-Signalübermittlungsweg von Ustilago ein positiver Rückkoppelungsmechanismus inhärent.Pheromone stimulation in Ustilago ultimately results in the transcriptional induction of all genes that are at the intersection loci a and b, ie mfal, mfa2, pral, pra2, lga2, rga2, bE, bW [M. Urban, R. Kahmann and M. Bölker (1996) Mol. Gen. Genet., 251, 31-37]. All of these genes have at least one cis-acting DNA sequence, the "pheromone response element" (PRE), in their associated gene regulatory regions. The PRE-Se sequences from Ustilago maydis are recognized by the sequence-specific DNA binding protein Prfl. Pheromone stimulation leads to the activation of Prfl, which mediates the pheromone-inducible transcription of these genes. Since the promoter of the prfl gene itself also has PREs, the transcription of the prfl gene is also activated by pheromone stimulation (HA Hartmann, R. Kahmann and M. Bölker (1996) EMBO J., 15, 1632-1641). Due to the pheromone-inducible transcription of the prfl gene, a positive feedback mechanism is inherent in the Ustilago pheromone signal transmission pathway.
GPC-Rezeptor-kontrollierte Signalübermittlungssysteme mit positiver Rückkoppelung sind daran zu erkennen, dass die Transkription des Gens, das für denjenigen Transkriptionsfaktor kodiert, der durch Stimulation des GPC-Re- zeptors aktiviert wird und dadurch die GPC-Rezeptor-kontrollierte Transkription von Zielgenen steuert, selbst auch induziert wird durch Stimulation des GPC-Rezeptors.GPC receptor-controlled signal transmission systems with positive feedback can be recognized by the fact that the transcription of the gene which codes for the transcription factor which is activated by stimulation of the GPC receptor and thereby controls the GPC receptor-controlled transcription of target genes, itself is also induced by stimulation of the GPC receptor.
Der oben beschriebene Mechanismus ist in der Figur 1 links schematisch dargestellt. Dieser Mechanismus führt zu einer bedeutend höheren Sensitivität, mit der beispielsweise ein Bindung von Liganden an den Rezeptor wahrgenommen werden kann. Wie bereits oben erwähnt und wie in der Figur 1 rechts als Vergleich dargestellt, fehlt dem entsprechenden Hefesystem und anderen bekannterweise angewendeten, zellulären Nachweissystemen ein derartiger positiver Rückkoppelungsmechanismus. Im entsprechenden Mechanismus der Hefe wird die Expression des GPC-Rezeptor-aktivierbaren Transkriptionsfaktors (Stel2) nicht induziert durch Rezeptor-Stimulation (siehe Fig.l).
Es ist jedoch durchaus denkbar, Hefestämme, die zum Nachweis von Wechselwirkungen zwischen heterologen Rezeptoren und Liganden benutzt werden, so zu modifizieren, dass sie einen positiven Rückkoppelungsmechanismus aufweisen. Um dies zu erreichen, kann der Promotor des STE12-Gens, welches für den durch Pheromon-Stimulation aktivierten Transkriptionsfaktor kodiert, durch einen Promotor ersetzt werden, der Pheromon-induzierbar ist (z.B. FUSl-Promotor).The mechanism described above is shown schematically on the left in FIG. This mechanism leads to a significantly higher sensitivity with which, for example, binding of ligands to the receptor can be perceived. As already mentioned above and as a comparison shown in FIG. 1 on the right, the corresponding yeast system and other known cellular detection systems lack such a positive feedback mechanism. In the corresponding mechanism of the yeast, the expression of the GPC receptor-activatable transcription factor (Stel2) is not induced by receptor stimulation (see Fig. 1). However, it is quite conceivable to modify yeast strains used to detect interactions between heterologous receptors and ligands so that they have a positive feedback mechanism. In order to achieve this, the promoter of the STE12 gene, which codes for the transcription factor activated by pheromone stimulation, can be replaced by a promoter which is pheromone-inducible (eg FUSI promoter).
Verschiedene Ustüago-Stämme und geeignete Expressionsvektoren für die Transformation von Ustilago sind bekannt. Expressionsvektoren sind replizierbare DNA-Konstrukte, die benutzt werden, um eine heterologe DNA-Sequenz in einer Wirtszelle zu exprimieren. Die zu exprimierende heterologe DNA- Sequenz muss mit geeigneten Kontrollsequenzen ausgestattet sein, welche fähig sind, die Expression eines durch die heterologe DNA-Sequenz kodierten Proteins oder Proteinuntereinheit im beabsichtigten Wirt zu steuern. Kontrollsequenzen umfassen einen transkriptioneilen Promotor, fakultative cis-wirken- de DNA-Sequenzen, um die Transkription zu regulieren, geeignete DNA- Sequenzen, welche eine effiziente Initiation der Translation vermitteln, und DNA-Sequenzen, welche die Termination der Transkription und die Polya- denylierung der mRNA steuern.Various Ustüago strains and suitable expression vectors for the transformation of Ustilago are known. Expression vectors are replicable DNA constructs that are used to express a heterologous DNA sequence in a host cell. The heterologous DNA sequence to be expressed must be equipped with suitable control sequences which are capable of controlling the expression in the intended host of a protein or protein subunit encoded by the heterologous DNA sequence. Control sequences include a transcriptional promoter, optional cis-acting DNA sequences to regulate transcription, suitable DNA sequences which mediate efficient initiation of translation, and DNA sequences which terminate transcription and polyadenylation control the mRNA.
Geeignete Vektoren, für die Herstellung von erfindungsgemässen Zeil-Linien umfassen Plasmide, Viren und integrierbare DNA-Fragmente, d.h. DNA-Fragmente, die in das Wirtsgenom integrierbar sind via genetische Rekombination. Geeignete Vektoren enthalten Kontrollsequenzen, welche von Spezies stammen, die im beabsichtigten Expressionswirt funktionell sind.
Ustilago-Vektoren können eine autonom replizierende Sequenz (UARS) enthalten, welche das Plasmid befähigt, in hoher Kopienzahl in der Ustilago- Zelle zu replizieren, einen Promotor, heterologe DNA-Sequenzen, die für die zu exprimierenden heterologen Proteine kodieren, Sequenzen für die Polya- denylierung und ein selektierbares Markergen.Suitable vectors for the production of Zeil lines according to the invention include plasmids, viruses and integrable DNA fragments, ie DNA fragments which can be integrated into the host genome via genetic recombination. Suitable vectors contain control sequences derived from species that are functional in the intended expression host. Ustilago vectors can contain an autonomously replicating sequence (UARS) which enables the plasmid to replicate in high copy number in the Ustilago cell, a promoter, heterologous DNA sequences which code for the heterologous proteins to be expressed, sequences for the polya - Denylation and a selectable marker gene.
Ein Beispiel eines solchen Plasmids ist pJW42 [J. Wang, D. W. Holden and S. A Leong (1988) Proc. Natl. Acad. Sei. USA 85, 865-869]. Dieses Plasmid enthält das hph-Gen von Escherichia coli [L. Gritz and J. Davies (1983) Gene, 25, 179-188], welches Resistenz gegen das Antibiotikum HygromycinB vermittelt und dadurch als selektierbarer Marker genutzt werden kann. Andere, anwendbare Markergene sind beispielsweise das cbx-Gen von Ustilago maydis, das Resistenz gegen das Fungizid Carboxin vermittelt [P.L.E. Broomfield and JA. Hargreaves (1992) Curr. Genet.,22, 117-121], oder das natl-Gen von Streptomyces noursei, das Resistenz gegen das Antibiotikum Nourseothricin vermittelt [H. Krüger, G. Fiedler, C. Smith and S. Baum- berg(1993) Gene, 127, 127-131].An example of such a plasmid is pJW42 [J. Wang, D.W. Holden and S.A Leong (1988) Proc. Natl. Acad. Be. USA 85, 865-869]. This plasmid contains the hph gene from Escherichia coli [L. Gritz and J. Davies (1983) Gene, 25, 179-188], which confers resistance to the antibiotic hygromycin B and can thus be used as a selectable marker. Other applicable marker genes are, for example, the cbx gene from Ustilago maydis, which confers resistance to the fungicide carboxin [P.L.E. Broomfield and JA. Hargreaves (1992) Curr. Genet., 22, 117-121], or the natl gene from Streptomyces noursei, which mediates resistance to the antibiotic nourseothricin [H. Krüger, G. Fiedler, C. Smith and S. Baumberg (1993) Gene, 127, 127-131].
Geeignete Promotor Sequenzen umfassen die Promotoren des hsp70-Gens [D. W. Holden, J. W. Kronstad and S. A Leong (1989) EMBO J., 8, 1927-1934], des Glyceraldehyd-3-Phosphat Dehydrogenase Gens [T. L. Smith and S. A Leong (1990) Gene, 93, 111-117] und des Translation-Elongationsfaktor-Gens [H. A. Hartmann, R. Kahmann and M. Bölker (1996) EMBO J., 15, 1632- 1641]. Andere Promotoren mit dem zusätzlichen Vorteil der transkriptionellen Kontrolle durch die Wachstumsbedingungen sind der Promotor des crgl-Gens [A. Bottin, J. Kämper and R. Kahmann (1996) Mol. Gen. Genet., 253, 342- 352], welcher durch Arabinose induziert und durch Glukose reprimiert wird, und der Promotor des sidl-Gens, welcher negativ reguliert ist durch die Eisen-Konzentration im Wachstumsmedium [Z. An, B. Mei, W. M. Yuan and S.
A. Leong (1997) EMBO J., 16, 1742-1750]. Um die Polyadenylierung und die Termination der mRNA zu gewährleisten, können auch die Teπninationsse- quenzen, die mit diesen Genen assoziiert sind, stromabwärts der heterologen Sequenzen in die Expressionsvektoren ligiert werden.Suitable promoter sequences include the promoters of the hsp70 gene [DW Holden, JW Kronstad and S.A Leong (1989) EMBO J., 8, 1927-1934], of the glyceraldehyde-3-phosphate dehydrogenase gene [TL Smith and S.A. Leong (1990) Gene, 93, 111-117] and the translation elongation factor gene [HA Hartmann, R. Kahmann and M. Bölker (1996) EMBO J., 15, 1632- 1641]. Other promoters with the additional advantage of transcriptional control through the growth conditions are the promoter of the crgl gene [A. Bottin, J. Kämper and R. Kahmann (1996) Mol. Gen. Genet., 253, 342-352], which is induced by arabinose and repressed by glucose, and the promoter of the sidl gene, which is negatively regulated by the iron concentration in the growth medium [Z. An, B. Mei, WM Yuan and S. A. Leong (1997) EMBO J., 16, 1742-1750]. To ensure polyadenylation and termination of the mRNA, the termination sequences associated with these genes can also be ligated into the expression vectors downstream of the heterologous sequences.
Um die effiziente Expression von heterologen GPC-Rezeptoren in Ustilago zu ermöglichen, wurden neuartige Expressionsvektoren entwickelt. Diese Expressionsvektoren enthalten Ustilago maydis hsp70-Promotor und -Terminator, welche die Transskription der cDNAs für GPC-Rezeptoren vermitteln. Zwischen dem hsp70-Promotor und -Terminator werden zusätzliche Schnittstellen für Restriktionsenzyme eingeführt, um die Klonierung von zu exprimierenden DNA-Segmenten, z.B. GPCR-cDNAs, zu vereinfachen.To enable efficient expression of heterologous GPC receptors in Ustilago, novel expression vectors have been developed. These expression vectors contain the Ustilago maydis hsp70 promoter and terminator, which mediate the transcription of the cDNAs for GPC receptors. Additional restriction enzyme sites are introduced between the hsp70 promoter and terminator to clone DNA segments to be expressed, e.g. Simplify GPCR cDNAs.
Um die intrazelluläre Lokalisierung der heterologen GPC-Rezeptoren zu der Plasmamembran in Ustilago zu optimieren, ist es auch möglich, Expressionsvektoren zu konstruieren, die ein erstes Segment enthalten, welches Ustilago- DNA-Sequenzen umfasst, die mindestens ein Segment der aminoterminalen kodierenden Sequenz eines Ustilago-GPC-Rezeptors beinhalten. DNA-Sequenzen, die für Pheromon-Rezeptoren von Ustilago kodieren (z.B. das pral- Gen, das für den Mfa2-Pheromon-Rezeptor kodiert, und das pra2-Gen, das für den Mfal-Pheromon-Rezeptor kodiert) sind Beispiele für Ustilago-Gene, die für GPC-Rezeptoren kodieren, welche benutzt werden können, um solche Vektoren zu konstruieren. Ein zweites Segment, das stromabwärts des genannten ersten Segments liegt und im korrekten Leseraster mit diesem ist, umfasst eine DNA-Sequenz, die für einen heterologen GPC-Rezeptor kodiert. Solche Anpassungen der Translationsinitiationsstelle können die Expression eines heterologen Proteins erhöhen. Die ersten und zweiten Segmente sind funktionsfähig assoziiert mit einem Promotor, wie z.B. dem konstitutiven
hsp70-Promotor oder dem induzierbaren crgl-Promotor, welche funktionsfähig sind in Ustilago-Zellen.In order to optimize the intracellular localization of the heterologous GPC receptors to the plasma membrane in Ustilago, it is also possible to construct expression vectors which contain a first segment which comprises Ustilago DNA sequences which comprise at least one segment of the amino-terminal coding sequence of a Ustilago -GPC receptor include. DNA sequences encoding Ustilago pheromone receptors (eg the pral gene encoding the Mfa2 pheromone receptor and pra2 gene encoding the Mfal pheromone receptor) are examples of Ustilago Genes encoding GPC receptors that can be used to construct such vectors. A second segment, which is located downstream of the said first segment and is in the correct reading frame therewith, comprises a DNA sequence which codes for a heterologous GPC receptor. Such adjustments to the translation initiation site can increase expression of a heterologous protein. The first and second segments are operatively associated with a promoter, such as the constitutive one hsp70 promoter or the inducible crgl promoter, which are functional in Ustilago cells.
Jeder GPC-Rezeptor und die entsprechenden DNA-Sequenzen, die für diese Rezeptoren kodieren, können benutzt werden, um die erfindungsgemässen Zeil-Linien herzustellen. Beispiele solcher Rezeptoren sind adrenerge Rezeptoren (α oder ß), Adenosin-Rezeptoren, Angiotensin-Rezeptoren, Bradykinin- Rezeptoren, Cannabinoid-Rezeptoren, Chemokin-Rezeptoren, Dopamin-Re- zeptoren, Glukagon-Rezeptoren, Neurokinin-Rezeptoren, Neurotensin-Rezep- toren, Serotonin-Rezeptoren, Opiat-Rezeptoren, muskarinische Rezeptoren, Somatostatin-Rezeptoren und Vasopressin-Rezeptoren. Der hier benutzte Begriff "Rezeptor" schliesst auch Subtypen sowie deren Mutanten und Homologe ein und auch die DNA-Sequenzen, die für diese kodieren.Each GPC receptor and the corresponding DNA sequences which code for these receptors can be used to produce the Zeil lines according to the invention. Examples of such receptors are adrenergic receptors (α or β), adenosine receptors, angiotensin receptors, bradykinin receptors, cannabinoid receptors, chemokine receptors, dopamine receptors, glucagon receptors, neurokinin receptors, neurotensin receptors. gates, serotonin receptors, opiate receptors, muscarinic receptors, somatostatin receptors and vasopressin receptors. The term "receptor" used here also includes subtypes and their mutants and homologs and also the DNA sequences which code for them.
Jede Gα-Untereinheit und die entsprechenden DNA-Sequenzen, die für diese Gα-Untereinheiten kodieren, können benutzt werden, um die erfindungsgemässen Zeil-Linien herzustellen. Beispiele solcher Gα-Untereinheiten sind Gs-Untereinheiten, Go-Untereinheiten, Gq-Untereinheiten, Gi-Untereinhei- ten und Gz-Untereinheiten. Der hier benutzte Begriff "Gα-Untereinheit" schliesst Subtypen sowie deren Mutanten und Homologe ein und auch DNA- Sequenzen, die für diese kodieren. Die funktionelle Expression solcher hete- rologer Gα-Untereinheiten in Ustilago kann leicht überprüft werden, da ein Defekt in der Ustilago Gα-Untereinheit Gpa3 zu einem charakteristischen, visuell beobachtbaren filamentösen Wachstum führt, im Gegensatz zur hefeartigen Wachstumsform von Ustilago-Zellen mit einem intakten gpa3-Gen. Heterologe Gα-Untereinheiten, welche die Funktion der endogenen Gα-Untereinheit Gpa3 in der Pheromon-Signalübermittlungskette übernehmen, kom- plementieren den filamentösen Wachstumsdefekt der gpa3 mutanten Zellen
zu normalem, hefeartigem Wachstum und können somit leicht identifiziert werden.Each Gα subunit and the corresponding DNA sequences which code for these Gα subunits can be used to produce the Zeil lines according to the invention. Examples of such Gα subunits are Gs subunits, Go subunits, Gq subunits, Gi subunits and Gz subunits. The term "Gα subunit" used here includes subtypes as well as their mutants and homologs and also DNA sequences which code for them. The functional expression of such heterologous Gα subunits in Ustilago can easily be checked, since a defect in the Ustilago Gα subunit Gpa3 leads to a characteristic, visually observable filamentous growth, in contrast to the yeast-like growth form of Ustilago cells with an intact gpa3 -Gene. Heterologous Gα subunits, which take on the function of the endogenous Gα subunit Gpa3 in the pheromone signal transmission chain, complement the filamentous growth defect of the gpa3 mutant cells to normal, yeast-like growth and can therefore be easily identified.
Jede Gßγ-Untereinheit und die entsprechenden DNA-Sequenzen, die für diese Gßγ-Untereinheiten kodieren, können benutzt werden, um die erfindungsgemässen Zeil-Linien herzustellen. Der hier benutzte Begriff "Gßγ-Untereinheit" schliesst Subtypen sowie deren Mutanten und Homologe ein und auch DNA-Sequenzen, die für diese kodieren.Each Gßγ subunit and the corresponding DNA sequences coding for these Gßγ subunits can be used to produce the Zeil lines according to the invention. The term "Gß-subunit" used here includes subtypes as well as their mutants and homologs and also DNA sequences which code for them.
Um die Bindung eines Liganden an einen heterologen GPC-Rezeptor oder allgemein die Wechselwirkung zwischen einem Modulator und dem GPC- Rezeptor-kontrollierten Signalübermittlungssystem in erfindungsgemässen Zeil- Linien- nachzuweisen, ist es besonders zweckdienlich, die Zellen mit einem dritten DNA-Konstrukt auszustatten, welches einen Promotor und ein Reportergen umfasst. Der Promotor ist induzierbar durch die Aktivierung des heterologen GPC-Rezeptors. Das Reportergen ist stromabwärts vom GPC- Rezeptor-induzierbaren Promotor plaziert und funktionsfähig mit diesem assoziiert. Die Expression des Reportergens ist messtechnisch erfassbar und wiederspiegelt die Aktivierung des heterologen GPC-Rezeptors durch geeignete Liganden. Im beispielhaften Ustilago maydis System können z.B. der Promotor des mfal-Gens, der Promotor des mfa2-Gens, der Promotor des pral-Gens, der Promotor des pra2-Gens oder der Promotor des prfl-Gens als GPC-Rezeptor-induzierbare Promotoren benutzt werden. Auch können verschiedene, endogene oder heterologe Gene als Reportergene benutzt werden. Beispiele für Reportergene sind das pyr6-Gen [J. W. Kronstad, J. Wang, S. F. Covert, D. W. Holden, G. L. McKnight and S. A. Leong (1989) Gene, 79, 97- 106], das pyr3-Gen [A Spanos, N. Kanuga, D. W. Holden and G. R. Banks (1992) Gene, 117, 73-79], das lacZ-Gen, das hph-Gen (Hygromycin-Resistenz) [L. Gritz and J. Davies (1983) Gene, 25, 179-188], das ble-Gen (Phleomycin-
Resistenz) [D. Drocourt, T. Calmels, J. P. Reynes, M. Baron and G. Tiraby (1990) Nucl. Acids Res., 18, 4009], ein GFP-Gen (Green Fluorescent Protein) [T. Spellig, A Bottin and R. Kahmann (1996) Mol. Gen. Genet., 225, 503-509] oder das uidA (GUS) Gen [R. A. Jefferson, S. M. Burgess and D. Hirsh (1986) Proc. Natl. Acad. Sei. USA 86, 8447-8451].In order to detect the binding of a ligand to a heterologous GPC receptor or generally the interaction between a modulator and the GPC receptor-controlled signal transmission system in Zeil lines according to the invention, it is particularly useful to equip the cells with a third DNA construct, which comprises a promoter and a reporter gene. The promoter can be induced by activating the heterologous GPC receptor. The reporter gene is placed downstream of the GPC receptor-inducible promoter and is operatively associated with it. The expression of the reporter gene can be measured and reflects the activation of the heterologous GPC receptor by suitable ligands. In the exemplary Ustilago maydis system, for example, the promoter of the mfal gene, the promoter of the mfa2 gene, the promoter of the pral gene, the promoter of the pra2 gene or the promoter of the prfl gene can be used as GPC receptor-inducible promoters . Different, endogenous or heterologous genes can also be used as reporter genes. Examples of reporter genes are the pyr6 gene [JW Kronstad, J. Wang, SF Covert, DW Holden, GL McKnight and SA Leong (1989) Gene, 79, 97-106], the pyr3 gene [A Spanos, N. Kanuga , DW Holden and GR Banks (1992) Gene, 117, 73-79], the lacZ gene, the hph gene (hygromycin resistance) [L. Gritz and J. Davies (1983) Gene, 25, 179-188], the ble gene (phleomycin Resistance) [D. Drocourt, T. Calmels, JP Reynes, M. Baron and G. Tiraby (1990) Nucl. Acids Res., 18, 4009], a GFP (Green Fluorescent Protein) gene [T. Spellig, A Bottin and R. Kahmann (1996) Mol. Gen. Genet., 225, 503-509] or the uidA (GUS) gene [RA Jefferson, SM Burgess and D. Hirsh (1986) Proc. Natl. Acad. Be. USA 86, 8447-8451].
Beispiel 1:Example 1:
Herstellung von Ustilago Expressionsvektoren (pDT78 und pDT99)Generation of Ustilago expression vectors (pDT78 and pDT99)
Um die Expression von heterologen GPC-Rezeptoren in U. maydis zu ermög- liehen, wurden die Ustilago Expressionsvektoren pDT78 und pDT99 konstruiert.In order to enable the expression of heterologous GPC receptors in U. maydis, the Ustilago expression vectors pDT78 and pDT99 were constructed.
Für den Expressionsvektor pDT78 wurde das 3.1 kb Hindlll Fragment des autonom replizierenden Ustilago Vektors pCM54 [T. Tsukuda, S. Carleton, S. Fotheringham and W. K. Holloman (1988) Mol. Cell. Biol, 8, 3703-3709] durch ein 2 kb Hindin Fragment des Plasmids pDWHIO [J. Wang, D.W. Holden and S. A Leong (1988) Proc. Natl. Acad. Sei. USA, 85, 865-869] ersetzt. Dieses 2 kb Hindlll Fragment enthält den Promotor und den Transkrip- tionsterminator des U. maydis hsplO Gens, getrennt durch eine BgUl Schnittstelle. Das resultierende Plasmid, pDT48, wurde mit Sαcl und Pstl geschnitten, und ein 1.5 kb Sacl-Pstl Fragment, isoliert vom Plasmid pNATl (pDT65), wurde eingefügt, welches das natl Gen von Streptomyces noursei enthält, das Resistenz gegen das Antibiotikum der Streptothricin Familie Nourseothricin vermittelt [H. Krügel, G. Fiedler, C. Smith and S. Baumberg (1993) Gene, 127, 127-131]. Die Expression des natl Gens in U. maydis wird durch den
Promotor des U. maydis Glyceraldehyd-3-Phosphat Dehydrogenase (GAPDH) Gens [T.L. Smith and S.A. Leong (1990) Gene, 93, 111-117] und den Transkriptions-Terminator des cycl Gens von Saccharomyces cerevisiae [D. Dro- court, T. Calmels, J.P. Reynes, M. Baron and G. Tiraby (1990) Nucleic Acids Res. 18, 4009] gesteuert. Das daraus resultierende Plasmid pDT78 besitzt eine einzige Schnittstelle für BgUl zwischen dem U. maydis hsp70 Promotor und dem U. maydis hsp70 Terminator. Diese Restriktionsenzym-Schnittstelle kann dazu benützt werden um eine in U. maydis zu exprimierende DNA Sequenz einzufügen, z.B. eine cDNA welche für einen heterologen GPC-Rezeptor kodiert. Die Transkription der cDNA die den heterologen GPC-Rezeptor spezifiziert, wird somit durch die Transkriptions-Kontrollsequenzen des U. maydis hsp70 Promotors vermittelt. Der pDT78 Expressionsvektor und dessen Derivate können mittels publizierten Transformations-Methoden in Ustilago maydis eingeführt werden [J. Wang, D.W. Holden and S. A. Leong (1988) Proc. Natl. Acad. Sei. USA 85, 865-869] und mittels Beifügen des Antibiotikums Nourseothricin (40 μg/ml) ins Wachstumsmedium wird für die Präsenz dieses Plasmids in U. maydis Zellen selektioniert.For the expression vector pDT78, the 3.1 kb Hindlll fragment of the autonomously replicating Ustilago vector pCM54 [T. Tsukuda, S. Carleton, S. Fotheringham and WK Holloman (1988) Mol. Cell. Biol, 8, 3703-3709] by a 2 kb Hind fragment of the plasmid pDWHIO [J. Wang, DW Holden and SA Leong (1988) Proc. Natl. Acad. Be. USA, 85, 865-869]. This 2 kb Hindlll fragment contains the promoter and the transcription terminator of the U. maydis hsplO gene, separated by a BgUl interface. The resulting plasmid, pDT48, was cut with Sαcl and Pstl, and a 1.5 kb Sacl-Pstl fragment isolated from plasmid pNATl (pDT65) was inserted which contains the natl gene from Streptomyces noursei, which is resistant to the antibiotic of the streptothricin family Nourseothricin mediates [H. Krügel, G. Fiedler, C. Smith and S. Baumberg (1993) Gene, 127, 127-131]. The expression of the natl gene in U. maydis is determined by the Promoter of the U. maydis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene [TL Smith and SA Leong (1990) Gene, 93, 111-117] and the transcription terminator of the cycl gene from Saccharomyces cerevisiae [D. Dro-court, T. Calmels, JP Reynes, M. Baron and G. Tiraby (1990) Nucleic Acids Res. 18, 4009]. The resulting plasmid pDT78 has a single interface for BgUl between the U. maydis hsp70 promoter and the U. maydis hsp70 terminator. This restriction enzyme interface can be used to insert a DNA sequence to be expressed in U. maydis, for example a cDNA which codes for a heterologous GPC receptor. The transcription of the cDNA that specifies the heterologous GPC receptor is thus mediated by the transcription control sequences of the U. maydis hsp70 promoter. The pDT78 expression vector and its derivatives can be introduced into Ustilago maydis using published transformation methods [J. Wang, DW Holden and SA Leong (1988) Proc. Natl. Acad. Be. USA 85, 865-869] and by adding the antibiotic nourseothricin (40 μg / ml) into the growth medium, selection is made for the presence of this plasmid in U. maydis cells.
Um die Klonierung von in Ustilago zu exprimierenden DNA Sequenzen zu vereinfachen, wurden weitere Expressionsplasmide mit dem hsp70 Pomotor konstruiert, welche aber zwischen dem hsp70 Promotor und Terminator statt nur einer Restriktionsschnittstelle, wie z.B. pDT78, Schnittstellen für mehrere verschiedene Restriktionsenzyme besitzen. Dies wird hier am Beispiel von pDT99 veranschaulicht.In order to simplify the cloning of DNA sequences to be expressed in Ustilago, further expression plasmids were constructed with the hsp70 promoter, which, however, between the hsp70 promoter and terminator instead of only one restriction site, e.g. pDT78, have interfaces for several different restriction enzymes. This is illustrated here using the example of pDT99.
pDT49 ist identisch mit dem oben beschriebenen pDT48, ausser dass das 2 kb Hindlll Fragment, welches den Promotor und den Transkriptions-Terminator des U. maydis hsp70 Gens enthält, in der umgekehrten Orientierung eingeführt wurde, d.h. der E. coli lacZ Promotor des Plasmids und der eingeführte
hsp70 Promotor vermitteln die Transkription in entgegengesetzter Richtung. Um die Smal, BamHl, Xbal, Saϊl und Pstl Schnittstellen von pDT49 zu eliminieren, wurde pDT49 mit Smαl und mit Pstl verdaut, der 3'-Überhang der Pstl Schnittstelle wurde mit T4 DNA Polymerase entfernt und das Plasmid wurde religiert. In die BgUl Schnittstelle zwischen dem hsp70 Promotor und Terminator des resultierenden Plasmids pDT85 wurde ein doppelsträngiges Oligo- nukleotid, welches Schnittstellen für die Restriktionsenzyme Kpnl, EcoRl, Notl, Ncol, Mlul, Stul, Sphl, BamHl und SαcII (in dieser Reihenfolge) enthält, so ein-ligiert, dass die SαcII Schnittstelle näher beim hsp70 Promotor zu lie- gen kam als die Kpnl Schnittstelle. In das resultierende Plasmid pDT90 wurde in die S cl Schnittstelle ein 2.3 kb Sαcl Fragment des Plasmids pjahCbxδ eingebracht, welches ein Gen enthält, das in U. maydis Resistenz gegen das Fungizid Carboxin vermittelt [P.L.E. Broomfield and JA. Hargreaves (1992) Curr. Genet., 22, 117-121]. pDT99 kann mittels Transformation in U. maydis Zellen eingebracht werden [J. Wang, D.W. Holden and S. A. Leong (1988) Proc. Natl. Acad. Sei. USA, 85, 865-869] und mittels Beifügen des Fungizids Carboxin (2 μg/ml) ins Wachstumsmedium wird für die Anwesenheit dieses Plasmids in U. maydis Zellen selektioniert.pDT49 is identical to the pDT48 described above, except that the 2 kb HindIII fragment, which contains the promoter and the transcription terminator of the U. maydis hsp70 gene, was introduced in the reverse orientation, ie the E. coli lacZ promoter of the plasmid and the introduced hsp70 promoters mediate transcription in the opposite direction. In order to eliminate the Smal, BamHl, Xbal, Saϊl and Pstl interfaces of pDT49, pDT49 was digested with Smαl and with Pstl, the 3 'overhang of the Pstl interface was removed with T4 DNA polymerase and the plasmid was religated. In the BgUl interface between the hsp70 promoter and terminator of the resulting plasmid pDT85 was inserted a double-stranded oligonucleotide which contains interfaces for the restriction enzymes Kpnl, EcoRl, Notl, Ncol, Mlul, Stul, Sphl, BamHl and SαcII (in this order) ligated in such a way that the SαcII interface came closer to the hsp70 promoter than the Kpnl interface. A 2.3 kb Sαcl fragment of the plasmid pjahCbxδ was introduced into the resulting plasmid pDT90, which contains a gene which mediates resistance to the fungicide carboxin in U. maydis [PLE Broomfield and JA. Hargreaves (1992) Curr. Genet., 22, 117-121]. pDT99 can be introduced into U. maydis cells by means of transformation [J. Wang, DW Holden and SA Leong (1988) Proc. Natl. Acad. Be. USA, 85, 865-869] and by adding the fungicide carboxin (2 μg / ml) to the growth medium, the presence of this plasmid is selected in U. maydis cells.
Beispiel 2:Example 2:
Expression des humanen ß2-adrenergen Rezeptors in U. maydis (pDT94)Expression of the human β2-adrenergic receptor in U. maydis (pDT94)
Um den humanen ß2-adrenergen Rezeptor (ß2-AR) in U. maydis zu exprimieren, wurden ca. 0.1 μg DNA des Plasmids pTF3 [B.K. Kobilka, R.AF. Dixon, T. Frielle, H.G. Dohlman, MA. Bolanowski, S. Sigal, T.L. Yang-Feng, U. Francke, M.G. Caron, R.J. Lefkowitz (1987) Proc. Natl. Acad. Sei. USA 84, 46-50] welches eine cDNA des humanen ß2-adrenergen Rezeptors enthält, mit den Primern 5'-CGGGATCCACAATGACCCAACCCGGCAACGGCAGCG-
3' und 5'-CGGGATCCTCAGAGCAGCGAGTCATTTGTGCTACA-3' (wobei A = Adenosin; C = Cytosin; G = Guanin; T = Thymidin) mittels der Polymerase-Kettenreaktion (PCR) amplifiziert. Im Vergleich zur humanen DNA Sequenz des ß2-adrenergen Rezeptors, wurde insbesondere die Umge- bung des Translations-Initiations ATG Kodons so verändert, dass sie der entsprechenden Konsensus-Sequenz für filamentöse Pilze entspricht [D. J. Bailance (1991) in Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi; S : Leong and R.M. Berka (eds.), Dekker, New York, pp 1-29]. Das resultierende 1.2 kb PCR Produkt wurde mit BamHl ver- daut und in die BamHl Schnittstelle des mit BamHl linearisierten und de- phosphorylierten Vektors pBLUESCRIPTII KS+ (Stratagene Inc.) kloniert. Die DNA Sequenz des somit klonierten 1.2 kb ßαmHI b2-AR PCR Produkts wurde verifiziert. Das 1.2 kb BamHl Fragment des resultierenden Plasmids pDT87 wurde dann in die Bglll Schnittstelle des Expressionsvektors pDT78 so eingefügt, dass die ß2-AR Sequenz in der richtigen Orientierung vom hsp70 Promotor transkribiert wird. Das resultierende ß2-AR-Expressionsplasmid pDT94 kann nun mittels publizierten Transformations-Methoden [J. Wang, D.W. Holden and S. A Leong (1988) Proc. Natl. Acad. Sei. USA 85, 865-869] in U. maydis Zellen eingeführt werden.In order to express the human β2-adrenergic receptor (β2-AR) in U. maydis, approx. 0.1 μg DNA of the plasmid pTF3 [BK Kobilka, R.AF. Dixon, T. Frielle, HG Dohlman, MA. Bolanowski, S. Sigal, TL Yang-Feng, U. Francke, MG Caron, RJ Lefkowitz (1987) Proc. Natl. Acad. Be. USA 84, 46-50] which contains a cDNA of the human β2-adrenergic receptor with the primers 5'-CGGGATCCACAATGACCCAACCCGGCAACGGCAGCG- 3 'and 5'-CGGGATCCTCAGAGCAGCGAGTCATTTGTGCTACA-3' (where A = adenosine; C = cytosine; G = guanine; T = thymidine) amplified by means of the polymerase chain reaction (PCR). In comparison to the human DNA sequence of the β2-adrenergic receptor, the environment of the translation initiation ATG codon in particular has been changed so that it corresponds to the corresponding consensus sequence for filamentous fungi [DJ Bailance (1991) in Molecular Industrial Mycology: Systems and Applications for Filamentous Fungi; S: Leong and RM Berka (eds.), Dekker, New York, pp 1-29]. The resulting 1.2 kb PCR product was digested with BamHl and cloned into the BamHl site of the BamHl linearized and dephosphorylated vector pBLUESCRIPTII KS + (Stratagene Inc.). The DNA sequence of the 1.2 kb βαmHI b2-AR PCR product thus cloned was verified. The 1.2 kb BamHI fragment of the resulting plasmid pDT87 was then inserted into the BglII site of the expression vector pDT78 in such a way that the β2-AR sequence is transcribed in the correct orientation by the hsp70 promoter. The resulting β2-AR expression plasmid pDT94 can now be published using published transformation methods [J. Wang, DW Holden and SA Leong (1988) Proc. Natl. Acad. Be. USA 85, 865-869] can be introduced into U. maydis cells.
Der biochemische Nachweis, dass der humane ß2-AR in U. maydis exprimiert wird kann mittels Liganden-Bindungsstudien erbracht werden. So können Membran-Fraktionen von mit pDT94 transformierten U. maydis Zellen für Bindungsstudien mit z.B. dem radioaktiv markierten ß2-AR-Liganden 3-[12SI]- Iodocyanopindolol durchgeführt werden gemass publizierten Protokollen [H.K. Dohlman, M.G. Caron, A. DeBlasi, T. Frielle and R.J. Lefkowitz (1990) Bio- chemistry, 29, 2335-2342].
Der Nachweis, dass der in U. maydis exprimierte humane ß2-AR funktionell ist und mit der Pheromon-Signalübermittlungskette von U. maydis interagiert, kann nun erbracht werden, indem ein Pheromon-induzierbares Reportergen in die mit pDT94 transformierten U. maydis Zellen eingebracht wird. Das Plas- mid pMUl enthält das bakterielle uidA Gen, welches für das Enzym ß-Gluku- ronidase (GUS) kodiert [R. A. Jefferson, S. M. Burgess and D. Hirsh (1986) Proc. Natl. Acad. Sei. USA 86, 8447-8451] und dessen Expression durch den stark Pheromon-induzierbaren Promotor des mfal Gens reguliert wird [M. Urban, R. Kahmann and M. Bölker (1996) Mol. Gen. Genet., 251, 31-37]. Demzufolge kann die Bindung eines ß2-AR Agonisten an den in U. maydis exprimierten ß2-AR nachgewiesen werden, indem U. maydis Transformanden, welche pDT94 und pMUl enthalten, mit z.B. dem ß-adrenergen Agonisten Isoproterenol stimuliert werden. Die Stimulation des Rezeptors kann somit durch einem einfachen biochemischen Test für GUS-Aktivität nachgewiesen werden, wie z.B. beschrieben in A. Gururaj Rao and P. Flynn (1992) in GUS Protocols; S.R. Gallagher (ed.), Academic Press Inc., 89-99.
The biochemical proof that the human ß2-AR is expressed in U. maydis can be provided by means of ligand binding studies. Thus, membrane fractions of U. maydis cells transformed with pDT94 can be carried out for binding studies with, for example, the radioactively labeled β2-AR ligand 3- [ 12S I] - iodocyanopindolol according to published protocols [HK Dohlman, MG Caron, A. DeBlasi, T Frielle and RJ Lefkowitz (1990) Biochemistry, 29, 2335-2342]. Evidence that the human β2-AR expressed in U. maydis is functional and interacts with the pheromone signaling chain of U. maydis can now be provided by inserting a pheromone-inducible reporter gene into the U. maydis cells transformed with pDT94 . The plasmid pMUl contains the bacterial uidA gene, which codes for the enzyme β-glucuronidase (GUS) [RA Jefferson, SM Burgess and D. Hirsh (1986) Proc. Natl. Acad. Be. USA 86, 8447-8451] and its expression is regulated by the strongly pheromone-inducible promoter of the mfal gene [M. Urban, R. Kahmann and M. Bölker (1996) Mol. Gen. Genet., 251, 31-37]. Accordingly, the binding of a ß2-AR agonist to the ß2-AR expressed in U. maydis can be demonstrated by U. maydis transformants, which contain pDT94 and pMUl, are stimulated with, for example, the ß-adrenergic agonist isoproterenol. The stimulation of the receptor can thus be demonstrated by a simple biochemical test for GUS activity, as described, for example, in A. Gururaj Rao and P. Flynn (1992) in GUS Protocols; SR Gallagher (ed.), Academic Press Inc., 89-99.
Claims
1. Transformierte Zeil-Linie für den Nachweis von Wechselwirkungen mit GPC-Rezeptoren oder mit einem GPC-Rezeptor-kontrollierten Signalübermittlungssystem, wobei die Zellen der transformierten Zeil-Linie einen heterologen GPC-Rezeptor exprimieren sowie einen durch Stimula- tion des heterologen GPC-Rezeptors induzierbaren Promotor und ein durch den Promotor gesteuertes Reportergen aufweisen, dadurch gekennzeichnet, dass die Zellen ein endogenes oder gentechnisch eingeführtes GPC-Rezeptor-kontrolliertes Signalübermittlungssystem mit positiver Rückkoppelung aufweisen.1. Transformed Zeil line for the detection of interactions with GPC receptors or with a GPC receptor-controlled signal transmission system, the cells of the transformed Zeil line expressing a heterologous GPC receptor and one by stimulating the heterologous GPC receptor Inducible promoter and a reporter gene controlled by the promoter, characterized in that the cells have an endogenous or genetically introduced GPC-receptor-controlled signal transmission system with positive feedback.
2. Transformierte Zeil-Linie nach Anspruch 1, dadurch gekennzeichnet, dass die Zellen das GPC-Rezeptor-kontrollierte Signalübermittlungssystem mit positiver Rückkoppelung natürlicherweise aufweisen.2. Transformed Zeil line according to claim 1, characterized in that the cells naturally have the GPC receptor-controlled signal transmission system with positive feedback.
3. Transformierte Zeil-Linie nach Anspruch 2, dadurch gekennzeichnet, dass sie eine Linie von Ustilago maydis ist.3. Transformed Zeil line according to claim 2, characterized in that it is a line from Ustilago maydis.
4. Transformierte Zeil-Linie nach Anspruch 1, dadurch gekennzeichnet, dass die positive Rückkoppelung im GPC-Rezeptor-kontrollierten Signalübermittlungssystem mittels genetischer Rekombination erzeugt ist.
4. Transformed Zeil line according to claim 1, characterized in that the positive feedback in the GPC receptor-controlled signal transmission system is generated by means of genetic recombination.
5. Transformierte Zeil-Linie nach einem der Ansprüche 1 bis 4, dadurch gekennzeichnet, dass der heterologe GPC-Rezeptor ein α-adrenerger Rezeptor, ein ß-adrenerger Rezeptor, ein Adenosin-Rezeptor, ein Angio- tensin-Rezeptor, ein Bradykinin-Rezeptor, ein Cannabinoid-Rezeptor, ein Chemokin-Rezeptor, ein Dopamin-Rezeptor, ein Glukagon-Rezeptor, ein Neurokinin-Rezeptor, ein Neurotensin-Rezeptor, ein Serotonin-Rezeptor, ein Opiat-Rezeptor, ein muskarinischer Rezeptor, ein Somatostatin-Re- zeptor oder ein Vasopressin-Rezeptor ist.5. Transformed Zeil line according to one of claims 1 to 4, characterized in that the heterologous GPC receptor is an α-adrenergic receptor, a β-adrenergic receptor, an adenosine receptor, an angiosensin receptor, a bradykinin Receptor, a cannabinoid receptor, a chemokine receptor, a dopamine receptor, a glucagon receptor, a neurokinin receptor, a neurotensin receptor, a serotonin receptor, an opiate receptor, a muscarinic receptor, a somatostatin-Re - is a zeptor or a vasopressin receptor.
6. Transformierte Zeil-Linie nach einem der Ansprüche 1 bis 5, dadurch gekennzeichnet, dass die Zellen zusätzlich heterologe Untereinheiten von G-Proteinen exprimieren.6. Transformed Zeil line according to one of claims 1 to 5, characterized in that the cells additionally express heterologous subunits of G proteins.
7. Transformierte Zeil-Linie nach Anspruch 6, dadurch gekennzeichnet, dass die Zellen heterologe Gα-Untereinheiten exprimieren, die Gs-, Go-, Gq-, Gi- oder Gz-Untereinheiten sind.7. Transformed Zeil line according to claim 6, characterized in that the cells express heterologous Gα subunits that are Gs, Go, Gq, Gi or Gz subunits.
8. Transformierte Zeil-Linie nach Anspruch 6, dadurch gekennzeichnet, dass die Zellen heterologe Gßγ-Untereinheiten exprimieren.8. Transformed Zeil line according to claim 6, characterized in that the cells express heterologous Gßγ subunits.
9. Transformierte Zeil-Linie nach Anspruch 3, dadurch gekennzeichnet, dass der durch Stimulation des heterologen GPC-Rezeptors induzierbare Promotor der Promotor des Ustilago maydis Gens mfal, mfa2, pral, pra2
9. Transformed cell line according to claim 3, characterized in that the promoter inducible by stimulation of the heterologous GPC receptor is the promoter of the Ustilago maydis gene mfal, mfa2, pral, pra2
10. Transformierte Zell-Linie nach einem der Ansprüche 1 bis 9, dadurch gekennzeichnet, dass das durch den Promotor gesteuerte Reportergen das lacZ-Gen, das hph-Gen, das ble-Gen, ein GFP-Gen oder das uidA-Gen ist.10. Transformed cell line according to one of claims 1 to 9, characterized in that the reporter gene controlled by the promoter is the lacZ gene, the hph gene, the ble gene, a GFP gene or the uidA gene.
11. Transformierte Zell-Linie nach Anspruch 3, dadurch gekennzeichnet, dass das durch den Promotor gesteuerte Reportergen das pyr6-Gen von Ustilago maydis oder das pyr3-Gen von Ustilago maydis ist.11. Transformed cell line according to claim 3, characterized in that the reporter gene controlled by the promoter is the pyr6 gene from Ustilago maydis or the pyr3 gene from Ustilago maydis.
12. Vektor zur Herstellung der transformierten Zell-Linie gemass einem der12. Vector for producing the transformed cell line according to one of the
Ansprüche 1 bis 11, dadurch gekennzeichnet, dass der Vektor zwischen einem Promotor und einem Terminator, die in einer zu transformieren- den Zelle funktionell sind, eine heterologe, für einen GPC-Rezeptor kodierende DNA-Sequenz aufweist und ein Markergen.Claims 1 to 11, characterized in that the vector between a promoter and a terminator, which are functional in a cell to be transformed, has a heterologous DNA sequence coding for a GPC receptor and a marker gene.
13. Vektor nach Anspruch 12, dadurch geennzeichnet, dass der Promotor und der Terminator Gene von Ustilago maydis Genen sind.13. Vector according to claim 12, characterized in that the promoter and the terminator are genes of Ustilago maydis genes.
14. Vektor nach Anspruch 13, dadurch gekennzeichnet, dass der Promotor/ - Terminator vom Ustilago maydis hsp70-Gen sind oder dass der Promotor der Arabinose-induzierbare Promotor des crgl-Gens von Ustüago maydis ist.14. Vector according to claim 13, characterized in that the promoter / terminator of the Ustilago maydis hsp70 gene or that the promoter is the arabinose-inducible promoter of the crgl gene from Ustüago maydis.
15. Vektor nach einem der Ansprüche 12 bis 14, dadurch gekennzeichnet, dass das Markergen das cbx-Gen von Ustilago maydis, das natl-Gen von15. Vector according to one of claims 12 to 14, characterized in that the marker gene is the cbx gene from Ustilago maydis, the natl gene from
Streptomyces noursei oder das hph-Gen von Escherichia coli ist.
Streptomyces noursei or the hph gene from Escherichia coli.
16. Vektor nach einem der Ansprüche 13 bis 15, dadurch gekennzeichnet, dass die für einen heterologen GPC-Rezeptor kodierende DNA-Sequenz ein Translations-Initiations Kondon mit einer Konsensus-Sequenz für filamentöse Pilze aufweist.16. Vector according to one of claims 13 to 15, characterized in that the DNA sequence coding for a heterologous GPC receptor has a translation initiation condon with a consensus sequence for filamentous fungi.
17. Vektor nach einem der Ansprüche 12 bis 16, dadurch gekennzeichnet, dass der Vektor eine DNA-Sequenz die mindestens ein Segment der aminoterminalen kodierenden Sequenz eines GPC-Rezeptors der zu transformierenden Zelle beinhaltet und stromabwärts von dieser DNA- Sequenz im korrekten Leseraster die für den heterologen GPC-Rezeptor kodierende DNA-Sequenz aufweist.17. Vector according to one of claims 12 to 16, characterized in that the vector contains a DNA sequence which comprises at least one segment of the amino terminal coding sequence of a GPC receptor of the cell to be transformed and downstream of this DNA sequence in the correct reading frame the for DNA sequence encoding the heterologous GPC receptor.
18. Vektor nach einem der Ansprüche 12 bis 17, dadurch gekennzeichnet, dass die DNA-Sequenz, die für einen heterologen GPC-Rezeptor kodiert, für einen α-adrenergen Rezeptor, einen ß-adrenergen Rezeptor, einen Adenosin-Rezeptor, einen Angiotensin-Rezeptor, einen Bradykinin-Re- zeptor, einen Cannabinoid-Rezeptor, einen Chemokin-Rezeptor, einen18. Vector according to one of claims 12 to 17, characterized in that the DNA sequence which codes for a heterologous GPC receptor for an α-adrenergic receptor, a β-adrenergic receptor, an adenosine receptor, an angiotensin Receptor, a bradykinin receptor, a cannabinoid receptor, a chemokine receptor, one
Dopamin-Rezeptor, einen Glukagon-Rezeptor, einen Neurokinin-Rezep- tor, einen Neurotensin-Rezeptor, einen Serotonin-Rezeptor, einen Opiat- Rezeptor, einen muskarinischen Rezeptor, einen Somatostatin-Rezeptor oder einen Vasopressin-Rezeptor kodiert.Coded dopamine receptor, a glucagon receptor, a neurokinin receptor, a neurotensin receptor, a serotonin receptor, an opiate receptor, a muscarinic receptor, a somatostatin receptor or a vasopressin receptor.
19. Verwendung der transformierten Zell-Linie nach einem der Ansprüche 1 bis 11 zum Nachweis von Wechselwirkungen von Testsubstanzen mit dem heterologen GPC-Rezeptor oder mit dem GPC-Rezeptor-kontrollierten Signalübermittlungssystem, wobei Testsubstanz und Zellen in Interaktion gebracht und die Expression des Reportergens messtechnisch erfasst wird.
19. Use of the transformed cell line according to one of claims 1 to 11 for the detection of interactions of test substances with the heterologous GPC receptor or with the GPC receptor-controlled signal transmission system, whereby the test substance and cells are brought into interaction and the expression of the reporter gene is measured is recorded.
20. Verwendung nach Anspruch 19, dadurch gekennzeichnet, dass das Reportergen ein essentielles Wachstumsenzym exprimiert und dass das Zellwachstum durch Trübungsmessung erfasst wird.20. Use according to claim 19, characterized in that the reporter gene expresses an essential growth enzyme and that the cell growth is detected by turbidity measurement.
21. Verwendung nach Anspruch 19, dadurch gekennzeichnet, dass die Expression des Reportergens einer biochemischen Reaktion unterzogen wird und das Reaktionsprodukt messtechnisch erfasst wird.21. Use according to claim 19, characterized in that the expression of the reporter gene is subjected to a biochemical reaction and the reaction product is measured.
22. Verwendung nach Anspruch 21, dadurch gekennzeichnet, dass das Reportergen das uidA-Gen ist und für ß-Glukuronidase kodiert und dass die exprimierte ß-Glukuronidase mit einem geeigneten biochemischen Test erfasst wird.
22. Use according to claim 21, characterized in that the reporter gene is the uidA gene and codes for β-glucuronidase and that the expressed β-glucuronidase is detected using a suitable biochemical test.
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| PCT/CH1999/000246 WO1999064567A1 (en) | 1998-06-05 | 1999-06-04 | Transformed cell lines which express heterologous g-protein-coupled receptors |
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|---|---|---|---|
| EP99923357A Withdrawn EP1084231A1 (en) | 1998-06-05 | 1999-06-04 | Transformed cell lines which express heterologous g-protein-coupled receptors |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1084231A1 (en) |
| JP (1) | JP2002517228A (en) |
| AU (1) | AU4028199A (en) |
| CA (1) | CA2334215A1 (en) |
| WO (1) | WO1999064567A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002533095A (en) * | 1998-12-22 | 2002-10-08 | アイシス・イノベーション・リミテッド | Sequence identification method |
| MY166780A (en) | 2011-06-10 | 2018-07-23 | Temasek Life Sciences Laboratory Ltd | Genetic manipulation and expression system for pucciniomycotina and ustilaginomycotina subphyla |
| CN113444724B (en) * | 2021-05-10 | 2023-03-17 | 西南大学 | Promoter, recombinant vector and application |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2628750B1 (en) * | 1988-03-21 | 1991-11-08 | Pasteur Institut | SEQUENCE MODIFIED VECTOR ENCODING AMINO ACID SEQUENCE CONTAINED IN MAMMALIAN PROTEIN HAVING BIOLOGICAL MEMBRANE RECEPTOR ACTIVITY |
| US5462856A (en) * | 1990-07-19 | 1995-10-31 | Bunsen Rush Laboratories, Inc. | Methods for identifying chemicals that act as agonists or antagonists for receptors and other proteins involved in signal transduction via pathways that utilize G-proteins |
| EP1029915A3 (en) * | 1990-09-13 | 2004-11-17 | Duke University | Expression of G protein coupled receptors in yeast |
| JP2001508292A (en) * | 1996-12-27 | 2001-06-26 | メルク エンド カンパニー インコーポレーテッド | Galanin receptor GALR2 and nucleotides encoding the receptor |
-
1999
- 1999-06-04 WO PCT/CH1999/000246 patent/WO1999064567A1/en not_active Application Discontinuation
- 1999-06-04 JP JP2000553557A patent/JP2002517228A/en not_active Withdrawn
- 1999-06-04 EP EP99923357A patent/EP1084231A1/en not_active Withdrawn
- 1999-06-04 CA CA002334215A patent/CA2334215A1/en not_active Abandoned
- 1999-06-04 AU AU40281/99A patent/AU4028199A/en not_active Abandoned
Non-Patent Citations (1)
| Title |
|---|
| See references of WO9964567A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1999064567A1 (en) | 1999-12-16 |
| JP2002517228A (en) | 2002-06-18 |
| AU4028199A (en) | 1999-12-30 |
| CA2334215A1 (en) | 1999-12-16 |
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