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Definitions

• Phosphatases and kinases are enzymes that are generally involved in the regulation of enzyme activity by adding or removing of phosphate groups to enzymes.
• the activity of these enzymes is often regulated by the binding of another protein and/or by an autoinhibitory domain. This binding of the autoinhibitory domain again is regulated, e.g., by a peptidic or non-peptidic compound that binds to the autoinhibitory domain.
• a protein phosphatase of the 2B subfamily one of four classes of phosphoserine/ phospho- threonine-specific phosphoprotein phosphatases that have been distinguished on the basis of their sensitivities to inhibitors and divalent cations, is calcineurin.
• Calcineurin is the only known phosphatase that is regulated specifically by Ca 2+ and calmodulin.
• the calcineurin holoenzyme purified from mammalian cells (Guerini et al., Proc. Natl. Acad. Sci. USA (1989), 86, 9183-9187) is a heterodimer consisting of a large catalytic subunit (51-61kD) and a small regulatory subunit ( ⁇ 19kD).
• the catalytic subunit binds calmodulin, and the regulatory subunit attaches up to four molecules of Ca 2+ .
• the catalytic activity of the calcineurin holoenzyme is completely inhibited by a synthetic peptide corresponding to a region in the C-terminus of the catalytic subunit in the presence of Ca + -calmodu!in. This suggests that the catalytic subunit contains an autoinhibitory domain (AID) (Hashimoto et al., J. Biol. Chem. (1990), 265, 1924-1927).
• the budding yeast Saccharomyces cerevisiae contains proteins with properties similar to the mammalian catalytic and regulatory subunits of calcineurin.
• yeast cells possess two genes, CNA1 and CNA2, which encode for the mammalian catalytic subunit (Cyert et al., Proc. Natl. Acad. Sci. USA (1991), 88, 7376-7380).
• CNA1 and CNA2 proteins contain regions which have similarity to the putative AID sequence of the mammalian enzyme (Hashimoto etai, J. Biol. Chem. (1990), 265, 1924-1927).
• Cyclosporin A is an undecapeptide (Schreiber, Science (1991 ), 251 , 283-287) and acts as an inhibitor of T-cell activation. It is currently the favored therapeutic agent for prevention of graft rejection after organ and bone marrow transplantation. CsA binds to family of conserved proteins, named cyclophilins, having high affinity for CsA (Schreiber, Science (1991), 251 , 283-287).
• FK506 a macrolide; Liu, Immunol. Today (1993), 14, 290-295) has been discovered that also inhibits T-cell activation.
• FKBPs FK-binding proteins
• PKA cAMP dependent protein kinase
• This enzyme is a member of the large group of eukaryotic protein kinases that play critical roles in regulation of cell growth and comprises a catalytic and a regulatory subunit.
• PKA in its inactive state consists of a tetramer consisting of two identical catalytic and two identical regulatory subunits.
• This enzyme is activated, like the other protein kinases, by dissociation of the regulatory and the catalytic subunit to form a dimer of active catalytic subunits. This dissociation is mediated, e.g., by cAMP.
• p70S6 a single chain enzyme involved in the regulation of protein synthesis.
• the target for p70S6 is the 40S ribosomal protein S6. It is supposed that phosphorylation of S6 either triggers or facilitates the activation of protein synthesis.
• p70S6 is supposed to be regulated by an autoinhibitory domain at the C-terminus (Flotow et al., J. Biol. Chem., (1992), 267, 3074-3078) and through serine/threonine phosphorylation by insulin/mitogen-activated protein kinases.
• a new approach for the detection of protein-protein interaction is the two-hybrid system (Fields et al., Nature (1989), 340, 245-246).
• This system provides a convenient method in which the possible interactions between a peptide and a protein can be examined in a cellular environment.
• the system depends on the reconstitution of a transcription factor from its two essential components, the DNA-binding domain and its activating sequence.
• two fusion proteins are generated, each comprising one of the two domains of the transcription factor and one of the two proteins that are being tested for their ability to bind to each other. Only when the proteins to be tested interact, a functional transcription reconstitutes.
• the activation of gene transcription varies. To monitor the formation of a functional transcription factor, the expression of the gene triggered by said reconstituted transcription factor is measured.
• this dual-hybrid approach is capable of determining the interaction between a phosphatase or a kinase and its autoinhibitory domain (AID).
• AID autoinhibitory domain
• inventive approach it is su ⁇ risingly possible to measure the influence of antagonists and agonists and, hence, to find new regulatory polypeptides and to design regulatory polypeptides with increased regulatory properties. It has been su ⁇ risingly found, for example, that cyclosporin A and FK506 despite their different structure inhibit the formation of active-center-AID-complex.
• inventive method it is su ⁇ risingly possible to identify new agonists or antagonists of a phosphatase or a kinase. Additionally, it has been found su ⁇ risingly that autoinhibitory domains exhibit better binding properties when present in several copies.
• the current invention concems a transcription system for measuring the interaction between a phosphatase or kinase, or a mutein or fragment thereof that binds an autoinhibitory domain, with said autoinhibitory domain, comprising a) the DNA binding domain of a transcription factor and the transcription activation domain of a transcription factor that are separated, wherein one of the two transcription factor domains is linked to a polypeptide comprising a phosphatase or kinase or a fragment thereof that binds an autoinhibitory domain; and the other of the two transcription factor domains is linked to a polypeptide comprising an autoinhibitory domain that is capable of binding to the polypeptide linked to the first transcription factor domain, and b) a DNA that is transcribed when the DNA binding domain of said transcription factor and the transcription activation domain of said transcription factor are connected via the polypeptides linked thereto.
• a transcription system for measuring the interaction between a phosphatase or kinase, or a mutein
• a preferred phosphatase or kinase comprises an autoinhibitory domain (AID) that can be located on the catalytic or a regulatory subunit.
• a phosphatase or kinase are phosphatase A1 and A2B, cyclic GMP dependent kinase (cGPK), myosin light chain kinase (MLCK), myosin heavy chain kinase, dsRNA-dependent kinase, dsDNA-dependent kinase, protease activated kinase I and II, cell cycle kinase cdc-28 MPF, growth factor regulated kinase, mammary gland casein kinase, glycogen synthase kinase-3, AMP activated protein kinase, pyruvate dehydrogenase kinase, branched chain ⁇ -ketoacid dehydogenase kinase, end
• a form of said phosphatase or kinase or a mutein or fragment of said phosphatase or kinase that has a free AID binding domain In this form the AID that is part of the original enzyme is not bound to the catalytic domain. This form can be achieved via phosphorylation, lack of certain deactivating factors, addition of activation factors or via a mutein that does not comprise a functional AID.
• the AID can be inactivated using several approaches. It is, for example, possible to delete the AID on DNA-level, to alter the AID by mutagenesis to be inactive or to use only a fragment of the protein that does not comprise the AID.
• the mutein is not changed to such an extent that the autoinhibitory domain used in the dual hybrid test cannot bind anymore. It is, for example, possible to use a fragment of calcineurin that lacks the C-terminal AID, especially the C-terminal 40-60 amino acids.
• Autoinhibitory domains that interact with or bind to a phosphatase or kinase are well known in the art.
• the interaction of these autoinhibitory domains and the corresponding enzymes by formation of a complex is sensitive to various parameters like compounds that compete with the autoinhibitory domain for the same attachment site, compounds that change the affinity of the autoinhibitory domain for the attachment site or compounds that change the affinity of the attachment site for the autoinhibitory domain.
• These compounds can be, for example, peptides, chemical compounds, metal salts or compounds comprising peptidic and non-peptidic parts.
• Examples for known regulatory compounds are DNA, RNA, steroids, macrolides, cAMP, cGMP, calmodulin, phosphatidyl serine, diacyl glycerol, insulin, epidermal growth factor, interleukins, cytokines, growth factors like IGF and GMCSF, and Ca 2 ⁇ Preferred examples for such autoinhibitory domains are the inhibitory or auto ⁇ inhibitory domains of the catalytic or regulatory subunit of a phosphatase or kinase (Kemp et al., Trends in Biochem. Sci. (1990), 15, 342-346), e.g.
• the autoinhibitory domain and the fragment that binds an autoinhibitory domain originate from the same enzyme komplex.
• the autoinhibitory domain and the fragment that binds an autoinhibitory domain originate from the same enzyme.
• artificial substrates or pseudosubstrates of the phosphatase or kinase to be tested are also included.
• Pseudosubstrates are, for example, created by replacing amino acids of natural occurring autoinhibitory domains with amino acids that promote binding to the enzyme. Examples are the replacement of one or more serine, threonine and/or thyrosine by another amino acid like alanine (Hardie, Nature (1988), 335, 592-593).
• a preferred polypeptide that binds to the phosphatase or kinase as described above is a polypeptide comprising the regulatory subunit of PKA, the autoinhibitory domain of calcineurin and or p70S6 kinase, as well as fragments thereof capable of binding to enzyme in the test.
• the regulatory subunit of PKA a fragment of calcineurin comprising the C-terminal 40 to 60 amino acid and a fragment of p70S6 kinase comprising amino acid 332 to 502 or 399 to 502.
• the autoinhibitory domain can be linked to the transcription factor domain as a single copy or in several copies which is preferred, e.g., 1 to 5 copies of the AID or 2 to 4 copies which is preferred. It is also possible to link different autoinhibitory domains to the same transcription factor to test, e.g., different influences.
• the different autoinhibitory domains or the copies of the same autoinhibitory domain may be connected directly or with spacer groups. Spacer groups are, for example, ⁇ -helix breakers like short peptides of repeated amino acids like Gly 3 , Gly 4 or Gly 5 .
• Transcription factors are proteins that bind to a certain region of DNA (promoter) and initiate the transcription of a DNA sequence that belongs to it. These transcription factors usually comprise a domain that is responsible for the DNA recognition or binding and a domain that initiates transcription. DNA recognition and transcription initiation domains are known for several transcription factors such as Ace1 , Gal4, VP16, p53 and lexA.
• both domains necessary for transcription initiation of a certain gene can originate from two different transcription factors or from the same transcription factor.
• both domains may originate form the CUP1 or Gal4 transcription factor or the DNA binding domain originates from the lexA transcription factor and the transcription activating domain originates from the VP16 or p53 transcription factor; or any other combination thereof.
• the DNA binding domain is located in the region comprising N-terminal amino acids, preferred are the N-terminal 122 amino acids, and the transcription activation domain is located in the region comprising C-terminal amino acids, wherein the C-terminal 100 amino acids are preferred, (Cizewski et al., Proc. Natl. Acad. Sci. USA (1988), 86, 8377-8381).
• one of the two transcription factor domains is linked to a polypeptide comprising a phosphatase or kinase or a fragment thereof that binds an autoinhibitory domain, as described above.
• the other of the two transcription factor domains is linked to a polypeptide comprising an autoinhibitory domain that is capable of binding to the phosphatase or kinase linked to the first transcription factor domain, as described above.
• a further aspect of the invention is an expression cassette for the expression of a fusion protein comprising the DNA binding domain or the transcription activation domain of a transcription factor and a polypeptide comprising a phosphatase or kinase or a fragment thereof that binds an autoinhibitory domain; or a polypeptide comprising said autoinhibitory domain or an active fragment thereof that is capable of binding to said phosphatase or kinase or a fragment thereof.
• Another aspect of the current invention is a DNA that is transcribed when the DNA binding domain of a transcription factor and the transcription activation domain of a transcription factor are joined via the polypeptides linked thereto, as described above, if this DNA is not already part of the natural genome of the host that is used for the test.
• This DNA is usually comprised in an expression cassette comprising a promoter that is operably linked to said DNA and to a sequence containing a transcription termination signal, as described above.
• said DNA is coding for a polypeptide.
• the promoter of the expression cassette is chosen in accordance with the transcription factor fragment used.
• suitable transcription factor/promoter combinations are Ace1/CUP1p or Gal4/Gal1-Gal10p (Chien et al., Proc. Natl. Acad. Sci. USA (1991), 88, 9578-9582; Yang et al., Science (1993), 257, 680-682).
• the promoter is the CUP1 promoter.
• a suitable DNA that is transcribed under the control of this promoter usually causes an effect that can be measured easily during or after transcription or translation.
• transcription or translation products that can be measured easily e.g. via the measurement of the amount of protein, mRNA or DNA produced; or that cause an effect that can be measured easily, e.g. cell growth, an enzymatic reaction or color.
• the transcribed DNA codes, for example, for a protein that is produced in an amount that is related to the amount of activation of said promoter and that is, e.g., not produced elsewhere in the chosen microbiological host under the applied conditions.
• suitable proteins are the metallothionein that is encoded by the yeast CUP1 gene, ⁇ -galactosidase or luciferase. Preferred is metallothionein and ⁇ -galactosidase.
• An expression cassette for said suitable DNA may be already present in the microbiological host.
• the corresponding original transcription factor is inactivated or replaced by the inventive transcription factor fusion constructs.
• the reconstitution of the promoter will induce the transcription of a genuine gene, that is, e.g., necessary for survival of the microbiological host under the condition applied.
• An example is the replacement of the original Ace1 domain(s) with the separated Ace1 domain(s) modified according to the invention, resulting in a yeast whose sensitivity to Cu + depends on the reconstitution of the functional Ace1.
• a microbiological host transformed with this inventive system will, for example, grow in Cu 2+ containing media only if the two transcription factor domains are linked via the associated phosphatase or kinase, as defined above, and the associated autoinhibitory domain of this phosphatase or kinase, as defined above.
• the promoter is operably linked to the encoded protein. Additional sequences, such as pro- or spacer-sequences which may or may not carry specific processing signals can also be included in the constructions to facilitate accurate processing of precursor molecules. Altematively fused proteins can be generated containing internal processing signals which allow proper maturation in vivo or in vitro.
• the expression cassettes according to the present invention comprise also the 3'-flanking sequence of a gene which contains the proper signals for transcription termination and polyadenylation. Suitable 3'-flanking sequences are for example those of the gene naturally linked to the promoter used, or 3'-flanking sequences of other yeast genes, e.g., of the PHO5 or the SUC2 gene.
• An expression cassette comprising the DNA that is transcribed under the control of the inventive transcription factor construct may, for example, contain additionally a DNA sequence encoding a signal peptide.
• the expression cassettes for the transcription factor fusion proteins and the DNA that is transcribed by the reconstituted transcription factor may be present on different plasmids or some or all together on one plasmid.
• These plasmids may be integrated into the genome of the microbiological host or may retain as separate units. It is for example possible to integrate the expression cassettes for the modified transcription factor domains into the genome of the microbiological host, e.g. at the position of the original transcription factor domains and inactivating therewith the original factor, as described in (Munder & F ⁇ rst, Mol. Cell. Biol. (1992), 12, 2091-2099).
• the expression cassette expressing the polypeptide to be monitored may be inserted, if it is not already part of the genome of the microbiological host, in form of a stable plasmid, e.g., in Saccharomyces cerevisiae in form of a two-micron based plasmid as described in EP-341215.
• hybrid vectors according to the invention are selected from the group consisting of a hybrid plasmid and a linear DNA vector and are further selected depending on the host organism envisaged for transformation.
• the invention relates also to hybrid vectors comprising the expression cassettes described above.
• Hybrid plasmids that retain as separate units carry the expression cassettes according to the invention and as additional DNA sequences a yeast replication origin and a selective genetic marker for yeast.
• Hybrid plasmids containing a yeast replication origin e.g. an autonomously replicating segment (Ars)
• a yeast replication origin e.g. an autonomously replicating segment (Ars)
• a yeast replication origin e.g. an autonomously replicating segment (Ars)
• Hybrid plasmids containing sequences homologous to yeast 2 ⁇ plasmid DNA can be used as well. These hybrid plasmids will get integrated by recombination into 2 ⁇ plasmids already present within the cell or will replicate autonomously. 2 ⁇ sequences are especially suitable for high- frequency transformation plasmids and give rise to high copy numbers.
• the preferred hybrid plasmids according to the invention may include a DNA sequence as part of a gene present in the host yeast chromosome (e.g. the gene for the transcription factor domain or its promoter linked to the transcription factor constructs).
• a DNA sequence as part of a gene present in the host yeast chromosome (e.g. the gene for the transcription factor domain or its promoter linked to the transcription factor constructs).
• the homologous sequence which should amount to a stretch of at least 20 to 100 deoxynucleotides, the whole plasmid or linear fragments thereof can be stably inco ⁇ orated into the host chromosome by recombination.
• the progeny cells will retain the introduced genetic material even without selective pressure.
• any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker.
• Suitable markers for yeast are particularly those expressing antibiotic resistance or, in the case of auxotrophic yeast mutants, genes which complement host lesions. Corresponding genes confer, for example, resistance to the antibiotic cycloheximide or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2. HIS3 or TRP1 gene. It is also possible to employ as markers structural genes which are associated with an autonomously replicating segment providing that the host to be transformed is auxotrophic for the product expressed by the marker.
• the additional DNA sequences which are present in the hybrid plasmids according to the invention also include a replication origin and a selective genetic marker for a bacterial host, especially Escherichia coli.
• a replication origin and a selective genetic marker for a bacterial host, especially Escherichia coli.
• an E. coli replication origin and an E. coli marker in a yeast hybrid plasmid.
• large amounts of hybrid plasmid DNA can be obtained by growth and amplification in E. coli and, secondly, the construction of hybrid plasmids is conveniently done in E. coli making use of the whole repertoire of cloning technology based on E. coli.
• E. coli plasmids, such as pBR322 and the like contain both E. coli replication origin and E. coli genetic markers conferring resistance to antibiotics, for example tetracycline and ampiciilin, and are advantageously employed as part of the yeast hybrid vectors.
• vector DNA which together with the yeast promoter and the coding region is forming a hybrid plasmid according to the invention.
• vector DNA which together with the yeast promoter and the coding region is forming a hybrid plasmid according to the invention.
• vector DNA which together with the yeast promoter and the coding region is forming a hybrid plasmid according to the invention.
• the preferred hybrid vectors of the present invention are prepared by methods known in the art, for example by linking a suitable promoter, a coding region, the 3' flanking sequence of a yeast gene and optionally vector DNA.
• vector DNA for example bacterial plasmid DNA or the like (see above), having at least one restriction site, preferably two or more restriction sites, can be employed.
• the vector DNA already contains replication origins and gene markers for yeast and/or a bacterial host.
• the vector DNA is cleaved using an appropriate restriction endonuclease.
• the restricted DNA is ligated to the DNA fragment containing the yeast promoter and to the DNA segment coding for the desired protein.
• restriction and ligation conditions are to be chosen in such a manner that there is no interference with the essential functions of the vector DNA and of the promoter.
• the hybrid vector may be built up sequentially or by ligating two DNA segments comprising all sequences of interest.
• Blunt ends (fully base- paired DNA duplexes) produced by certain restriction endonucleases may be directly ligated with T4 DNA ligase. More usually, DNA segments are linked through their single-stranded cohesive ends and covalently closed by a DNA ligase, e.g. T4 DNA ligase.
• T4 DNA ligase e.g. T4 DNA ligase.
• Such single- stranded "cohesive termini” may be formed by cleaving DNA with another class of endonucleases which produce staggered ends (the two strands of the DNA duplex are cleaved at different points at a distance of a few nucieotides).
• Single strands can also be formed by the addition of nucieotides to blunt ends or staggered ends using terminal transferase ("homopolymeric tailing") or by simply chewing back one strand of a blunt- ended DNA segment with a suitable exonuclease, such as I exonuclease.
• a further approach to the production of staggered ends consists in ligating to the blunt-ended DNA segment a chemically synthesized linker DNA which contains a recognition site for a staggered-end forming endonuclease and digesting the resulting DNA with the respective endonuclease.
• the components of the hybrid vector according to the invention such as the yeast promoter, the structural gene optionally including a signal sequence, transcription terminator, the replication system etc., are linked together in a predetermined order to assure proper function.
• the components are linked through common restriction sites or by means of synthetic linker molecules to assure proper orientation and order of the components.
• a linear DNA vector is made by methods known in the art, e.g. linking the above yeast promoter to the coding region in such a manner that the coding region is controlled by said promoter, and providing the resulting DNA with a DNA segment containing transcription termination signals derived from a yeast gene.
• Joining of the DNA segments may be done as detailed above, viz. by blunt end ligation, through cohesive termini or through chemically synthesized linker DNAs.
• the DNA fragments coding for fragments or domains of polypeptides can be isolated by methods known in the art. It is for example possible to isolate the desired fragment from a gene bank using specific hybridization probes, to synthesize the desired fragment in a DNA synthesizer, to amplify the specific fragment via PCR techniques or to use any combination thereof.
• Excision of a portion of a DNA coding for a protein-domain like the transcription activation or the DNA binding domain of a transcription factor, or a phosphatase or kinase lacking the autoinhibitory domain may be effected by using restriction enzymes.
• a prerequisite of this method is the availability of appropriate restriction sites in the vicinity of the ends of the DNA coding for the desired domain.
• the latter is advantageously contained in a greater DNA segment provided with appropriate linkers which allow insertion and cloning of the segment in a cloning vector.
• a preferred microbiological host for the constructs described above is a yeast or bacterial cell, wherein yeast is more preferred and Saccharomyces cerevisiae is especially preferred.
• the expression cassettes or hybrid vectors described above can be integrated into the microbiological host by standard methods in genetic engineering.
• the microbiological host originating therefrom comprises one or more expression cassettes for the two modified transcription factors and one ore more expression cassettes expressing the polypeptide to be monitored.
• the transcription system according to the invention or the host transformed therewith can be used for the identification of an agonist or antagonist of a phosphatase or kinase as described above.
• the inventive transcription system can be used to identify an antagonist of the phosphatase or kinase as described above.
• a further embodiment of the invention is a method for the identification of a phosphatase or kinase agonist and antagonist comprising a) treating a transformed microbiological host as defined above with a test compound, and b) measuring the amount of transcription activation caused by the two modified transcription factor fragments, e.g. by measuring the amount of a produced mRNA, DNA, protein or by measuring an effect caused by the induced transcription or translation product.
• the agonist and antagonist identified using the inventive system have valuable pharmacological properties an can be used in a method of treatment. Indications for these agonists and antagonists are, for example, cancer, arthritis, psoriasis, graft rejection, autoimmune diseases, allergy, Alzheimer's disease and regulation of cell proliferation. It is, for example, possible to use the calcineurin antagonists in a method of treatment that is characterized by inhibiting T-cell activation, especially in a method of treatment or prevention of graft rejection. Also enclosed by the present invention is a pharmaceutical composition comprising one or more of the compounds identified by the inventive system or pharmacologically acceptable salt thereof, optionally together with pharmacologically suitable carrier.
• Fig. 1 is a schematic illustration of plasmid pRH1.
• Fig. 2 is a schematic illustration of plasmid pRH14C.
• Fig. 3 is a schematic illustration of plasmid YipCL.
• Fig. 4 is a schematic illustration of plasmid pMH7.
• Fig. 5 is a schematic illustration of plasmid pMH10.
• the enzyme employed for all PCR reactions is Vent polymerase (New England Bio-Labs)
• the Primers are synthesized on a DNA synthesizer
• Example 1 Isolation of vCNA1 and vCNA2 yCNA1 and yCNA2 are the complete genes from the yeast genome encoding the two isozymes for the catalytic subunit of yeast calcineurin.
• PCR polymerase chain reaction
• yeast genomic DNA obtained from the wild type strain S288C
• primers SEQ ID NOs 1 , 2 and 3, 4.
• the primers correspond to the published sequences of yeast CNA1 and CNA2 (Cyert et al., Proc. Natl. Acad.
• the yeast cDNA is amplified in 30 cycles of PCR.
• the Bglll-Hindlll fragments containing the coding regions of yCNA1 and yCNA2 are ligated to the plasmid pUC19Bgl, completely digested with Bglll/Hindlll.
• An aliquot of the ligation mixture is added to calcium- treated, transformation-competent E. coli HB101 cells, ⁇ ampicillin resistant E. coli transformants from each of the two transformations are grown in the presence of 100 mg l ampicillin.
• Plasmid DNA is prepared and analyzed by digestion with Bglll/Hindlll, Bglll/BstEII and Hindlll/BstEII.
• the two correct clones with the expected fragments are named pUC19Bgl/Bglll-Hindlll/yCNA1 and pUC19Bgl/Bglll-Hindlll/yCNA2, respectively.
• the DNA inserts in the two plasmids are confirmed by sequencing (using the Applied Biosystems DNA sequencer 370A).
• the vector pUC19Bgl used for the above ligations, is a modified pUC19 (Boehringer Mannheim GmbH, Germany).
• pUC19 is digested with the restriction enzyme BamHI and the sticky ends are flushed with the large fragment of Klenow polymerase.
• the blunt ended DNA is ligated with Bglll linkers (double-stranded octamers, the sense strand being 'CAGATCTG'; Boehringer), digested with Bglll and religated.
• An aliquot of the ligation mixture is transformed in HB101 cells. Plasmid DNA is prepared from 6 HB101 transformants and are analyzed by digestion with Scal/Bglll and Scal/BamHI.
• pUC19Bgl One correct clone with the expected fragments is named pUC19Bgl.
• the BamHI and Bglll restriction sites are both present in the vector pUC19Bgl.
• the plasmid pUC18Bgl is prepared in an identical manner from pUC18.
• Example 2 Construction of vCNAI ⁇ and vCNA2 ⁇ , C-terminal truncated versions of the genes encoding the isozymes (i.e. vCNA1 and vCNA2) for the catalytic subunit of yeast calcineurin
• PCR is performed on the complete yeast CNA1 and CNA2 (pUC19Bgl/ Bglll-Hindlll/ yCNA1 and pUC19Bgl/Bglll-Hindlll/yCNA2, as templates; see Example 1 ), using two pairs of deoxyoligoribonucleotides as primers (see SEQ ID NOs 1 , 5 and 2, 6).
• the yeast cDNA is amplified as described in Example 1. Two 1670 bp and 1530 bp Bglll-Hindlll fragments, encoding yCNAI ⁇ and yCNA2 ⁇ (i.e.
• yCNA1 and yCNA2 lacking their putative autoinhibitory domains are isolated. After digestion with Bglll/Hindlll, the fragments are ligated to the plasmid pUC19Bgl (see Example 1). DNA obtained from transformants are analyzed as described in Example 1). The two correct clones with the expected fragments are named pUC19Bgl/Bglll-Hindlll/yCNA1 ⁇ and pUC19Bgl/Bglll- Hindlll/yCNA2 ⁇ , respectively. The truncated gene fragments are confirmed by sequencing, as in Example 1.
• Example 3 Construction of vKAID) and v2(AID), the gene fragments encoding the autoinhibitory peptides of yeast calcineurin vCNA1 and vCNA2
• the Bglll-Xbal autoinhibitory sequences y1 (AID) and y2(AID) (Cyert et al., Proc. Natl. Acad. Sci. USA (1991), 88, 7376-7380), are obtained by PCR using pUC19Bgl/Bglll-Hindlll/yCNA1 and pUC19Bgl/Bglll-Hindlll/yCNA2, as templates (see Example 1).
• the primers employed are depicted in SEQ ID NOs 7, 8 and 9, 10.
• the Bglll-Xbal fragments are subcloned in pUC19Bgl.
• the two clones with correct inserts are named pUC19Bgl/Bglll-Xbal/y1 (AID) and pUC19Bgl/Bglll-Xbal/y2(AID), respectively.
• the SUC2 gene (Taussig et al., Nucleic Acids Res. (1983), 11 , 1943-1954) terminator is isolated as four unique fragments (-300 bp) by PCR, using yeast genomic DNA as template: (i) an EcoRI-Sacl fragment with primers in SEQ ID NOs 11 and 12; (ii) an Xbal- Sacl fragment with primers in SEQ ID NOs 13 and 12; (iii) a Bglll-Sacl fragment with primers in SEQ ID NOs 14 and 12; (iv) an EcoRI-Kpnl fragment with primers in SEQ ID NOs 11 and 15. All the three fragments are subcloned in pUC19 or pUC19Bgl and the DNA inserts are confirmed by DNA sequencing (see Example 1). The resulting plasmids are:
• a 393 bp truncated version of the constitutive glyceraldehyde dehydrogenase promoter (GAPCLp) is cloned as a -670 bp Sall-EcoRI fragment (Meyhack et al., in Hershberger, Queener, Hegeman (ed.), Genetics and molecular biology of industrial microorganisms, 1989, p. 311-321. American Society for Microbiology, Washington, DC) into pBluescriptllKS- vector (Stratagene), yielding the plasmid pRH2.
• the -670 bp Sall-EcoRI insert in pRH2 contains a 276 bp Sall-BamHI fragment from pBR322 upstream of the GAPCLp.
• a 80 bp EcoRI-Spel double-stranded DNA linker (see SEQ ID NO 16), encoding the nuclear localization signal (NLS) from the simian virus 40 Tantigen (Kalderon et al., Cell (1984), 39, 499-509; and Nelson et al., Mol. Cell. Biol. (1989), 9, 384-389), is subcloned in pRH2 and the DNA insert is confirmed by sequencing (see Example 1 ).
• One correct clone is referred to as pRH3.
• the -304 bp Xbal-Bglll ACE1C transcriptional activation domain from the yeast transcriptional activator ACE1 (Munder et al., Mol. Cell. Biol. (1992), 12, 2091-2099) is isolated as a PCR product using two primers (see SEQ ID NOs 17 and 18) with the plasmid pTM4 as template (Munder et al., Mol. Cell. Biol. (1992), 12, 2091-2099).
• the Xbal-Bglll fragment is subcloned in pUC19Bgl.
• the insert is confirmed by sequencing (see Example 1 ) and it contains two extra nucieotides after the last complete codon at the 3' end of the gene fragment (see SEQ ID NO 18).
• the plasmid with the correct insert is named pUC19Bgl/Xbal-Bglll/ACEc_3.
• the -670 bp Sall-EcoRI fragment from pRH2 and the -304 bp Xbal-Bglll fragment from pUC19Bgl Xbal-Bglll/ACEc_3 are subcloned in pUC19Bgl completely digested with Sail and Bglll. Correct clones are confirmed by appropriate restriction enzyme digests. One such clone is referred to as pRHIOC.
• Example 6 Construction of yeast expression vectors for the expression of ACE1 C- vKAID) and ACE1C-v2(AID): y1 (AID) and y2(AID) encode the autoinhibitory peptides of the yeast calcineurin catalytic subunits yCNA1 and yCNA2.
• the -1075 bp Sall-Bglll fragment from pRHIOC (see Example 5), the Bglll-Xbal fragment from either pUC19Bgl/Bglll-Xbal/y1(AID) or pUC19Bgl/Bglll-Xbal/y2(AID) (see Example 3) and the -300 bp Xbal-Sacl fragment from ⁇ UC19/Xbal-Sacl/SUC2t (see Example 4) are ligated to the vector pDP34 which has been completely digested with Sail and Sad.
• the plasmid pDP34 is an E. coli-S. cerevisiae shuttle vector, which contains the complete S.
• cerevisiae 2-micron plasmid and encodes the S. cerevisiae URA3 and dLEU2 genes as yeast selection markers (Meyhack et al., in Hershberger-CL; Queener-SW; Hegeman-G (ed.), Genetics and molecular biology of industrial microorganisms, 1989, p. 311-321. American Society for Microbiology, Washington, DC).
• yeast selection markers Meyhack et al., in Hershberger-CL; Queener-SW; Hegeman-G (ed.), Genetics and molecular biology of industrial microorganisms, 1989, p. 311-321. American Society for Microbiology, Washington, DC.
• DNA is prepared from 12 transformants. Analyses with Sall/Sacl and BamHI-Sacl confirm the correct clones. Two such clones are referred to as pMH10 [encoding y1 (AID)] and pCS100 [encoding y2(AID)
• a control plasmid which contains only NLS-ACE1C under the control of the GAPCLp, is prepared by ligating the -1075 bp Sall-Bglll fragment from pRHIOC (see Example 5) and the -300 bp Bglll-Sacl fragment from pUC19/Bglll-Sacl/SUC2t (see Example 4) to the vector pDP34 which has been completely digested with Sail and Sad.
• the plasmid is named pRH C (Fig. 1).
• Example 7 Construction of a yeast expression vector for the expression of ACE1C-- vKAIDHGIv..rVl (AID) and ACE1C-v2.AIDHGIv),.-v2(AID), encoding a fusion between the transcription-activation domain ACE1 C and duplicate fragments of the autoinhibitory domains of yCNA1 and vCNA2
• the -1075 bp Sall-Bglll fragment from pRHIOC (see Example 5) and the Bglll-Xbal fragments from either pUC19Bgl Bglll-Xbal/y1 (AID) or pUC19Bgl/Bglll-Xbal/y2(AID) (see Example 3) are ligated to pUC19 which has been completely digested with Sail and Xbal.
• the two correct clones are named pUC19/Sall-Xbal/GAPFLp-NLS-ACE1C-y1(AID) or pUC19/Sall-Xbal/G APFLp-NLS-ACEI C-y2(AID).
• Unphosphorylated linkers (encoding a stretch of four glycines) with SEQ ID NO 19 are ligated to Xbal digested pUC19/Sall-Xbal/GAPFLp-NLS-ACE1C-y1(AID) or pUC19/Sall- Xbal/GAPFLp-NLS-ACE1C-y2(AID).
• the ligated molecules are isolated on a 1% agarose/Tris-acetate gel.
• the fragments are isolated, purified by GeneClean (Bio 101 , CA, USA) and re-annealed by incubating at 95°C followed by slow cooling to room temperature.
• the DNA obtained from HB101 transformants are analyzed on a 2% agarose gel.
• the clones which show a distinct increase in the molecular size of the Bglll-Xbal fragments from the ones obtained from the starting plasmids are named pUC19/Sall-Xbal/GAPFLp-NLS-ACE1C-y1(AID)-(Gly) 4 and pUC19/Sall-Xbal/ GAPFLp-NLS- ACE1C-y2(AID)-(Gly) 4 .
• a second copy of y1(AID) is isolated as a Spel-Hindlll fragment by PCR, using the primers in SEQ ID NOs 20 and 21 and the plasmid pUC19/Sall-Xbal/ GAPFLp-NLS-ACE1C-y1(AID) as template. This is subcloned along with a Hindlll-Sacl yeast PH05 transcriptional terminator fragment in pBluescriptKS+. One correct clone is referred to as pBluescriptKS+/Spel-Sacl/y1(AID)-PH05t.
• a second copy of y2(AID) is isolated as a Spel-Sacl fragment by PCR, using the primers in SEQ ID NOs 22 and 12 and the plasmid pCS100 (see Example 6) as template. This is subcloned in pBluescriptKS+.
• One correct clone is referred to as pBluescriptKS+/Spel- Sacl/y2(AID)-SUC2t.
• the -1100 bp Sall-Xbal fragment from pUC19/Sall-Xbal/GAPFLp-NLS-ACE1C-y1(AID) ⁇ (Gly) 4 and the Spel-Sacl fragment from pBluescriptKS+/Spel-Sacl/y1(AID)-PH05t are ligated to the vector pDP34 which has been completely digested with Sail and Sad. After transformation in E. coli HB101 , DNA is prepared from 12 transformants. Analyses with Sall/SacI and BamHI-Sacl confirm the correct clones. One such clone is referred to as pMH11 (encoding two copies of y1 (AID) linked by a glycine linker).
• the -1100 bp Sall-Xbal fragment from pUC19/Sall-Xbal/GAPFLp-NLS-ACE1C-y2(AID)- (Gly) 4 and the Spel-Sacl fragment from pBluescriptKS+/Spel-Sacl/y2(AID)-SUC2t are ligated to the vector pDP34 which has been completely digested with Sail and Sacl.
• analyses with Sall/SacI and BamHI-Sacl confirm the correct clones.
• One such clone is referred to as pCS101 (encoding two copies of y2(AID) linked by a glycine linker).
• Example 8 Construction of hCNRA2 and hCNRA2 ⁇ . the complete and truncated genes encoding the catalytic subunit of human calcineurin
• the 3' end ( ⁇ 1280bp) of the human gene (Guerini et al., Proc. Natl. Acad. Sci. USA (1989) 86, 9183-9187) is isolated as two fragments (a -563 bp Ncol-Sacl fragment and a -713 bp Sacl-EcoRI fragment) from human fetal brain cDNA library (Stratagene) by PCR, using two pairs of primers with SEQ ID NOs 23, 24 and 25, 26.
• the -300 bp 5' end (a Bglll-Ncol fragment) of hCNRA2 is constructed with two overlapping deoxyoligoribonucleotides (which are chemically synthesized using yeast-biased codons; see SEQ ID NOs 27 and 28).
• primer extension is performed which is followed by a PCR with primers (see SEQ ID NOs 29 and 30; which code for the sense and the anti- sense strand of the 5' and 3' ends of the -300 bp fragment).
• the Bglll-Ncol fragment is subcloned in pUC19bg_nc (see above; this Example).
• Six individual clones with -300 bp inserts are sequenced (using the Applied Biosystems DNA sequencer 370A).
• One correct clone is named pUC19bg_nc/Bglll-Ncol/5'hCNRA2.
• the complete hCNRA2 gene is assembled from the chemically synthesized -300 bp Bglll- Ncol fragment and the -1280 bp Ncol-EcoRI fragment by subcloning in pUC18Bgl (constructed in the way pUC19Bgl is made; Example 1).
• Six individual clones are analyzed by restriction enzyme digests. One correct cloned is named pK010.
• the complete hCNRA2 gene (but lacking the DNA encoding the 2 amino acids at the N-terminus, Met 1 , Ala 2 ) is isolated as a Bglll-EcoRI fragment by PCR, using primers with SEQ ID NOs 31 and 32 and pKO10 as template.
• the gene encoding the C-terminal truncated version of hCNRA2 i.e. hCNRA2 ⁇
• hCNRA2 ⁇ which lacks the autoinhibitory domain (and also the DNA encoding the N-terminal residues, Met 1 , Ala 2 ) is isolated as a Bglll-EcoRI fragment by PCR, using primers with SEQ ID NOs 31 and 33 and pK010 as template.
• Example 9 Construction of two yeast expression vectors for the expression of ACE1C- h(AID) and ACE1C-h(AIDHGIv) 4 -h(AIDV.
• h(AID) encodes the autoinhibitory peptide of the human brain calcineurin catalytic subunit hCNRA2
• the second copy of h(AID) is isolated as a Spel-Sacl fragment using the primers with SEQ ID NOs 36 and 12 and pCS102 as template (similar to Example 7).
• the expression plasmid for ACE1C-h(AID)-(Gly) 4 -h(AID) in pDP34 is constructed in an identical manner to that of pCS101 (see Example 7).
• the plasmid is named pCS103.
• Example 10 Construction of pRH1, the vector for expression of fusion genes between the DNA-binding domain of ACE1 (i.e. ACE1 N) and a gene of interest
• the plasmid pTM9 (Munder et al., Mol. Cell. Biol. (1992), 12, 2091-2099) is digested with EcoRI and Kpnl.
• the -4400 bp fragment is isolated on a 1% agarose gel and purified by GeneClean ® .
• a -300 bp EcoRI-Kpnl fragment from pUC19/EcoRI-Kpnl/SUC2t (see Example 4) is ligated to the -4400 bp linearized fragment and transformed in E. coli. Analysis of transformants with EcoRI and Kpnl yielded a plasmid where the CYC1 terminator in pTM9 is replaced by the SUC2 terminator fragment.
• This plasmid is named pRH1 (Fig. 2) and is a vector which can be used for integration of any gene which is fused to ACE1 N (the DNA-binding domain of the yeast transcription factor ACE1) into the yeast chromosome.
• Example 11 Construction of veast expression vectors for the expression of ACE1N- vCNA1-PHQ5t. ACE1 N-vCNA2-PHQ5t, ACE1 N-vCNA1 ⁇ -PHQ5t. ACE1 N- yCNA2 ⁇ -PHQ5t, ACE1 N-hCNRA2-SUC2t and ACE1 N-hCNRA2 ⁇ -SUC2t in PRH1
• ACE1N-yCNA1-PH05t The subcloning of ACE1N-yCNA1-PH05t, ACE1N-yCNA2-PH05t, ACE1N-yCNA1 ⁇ -PH05t and ACE1 N-yCNA2 ⁇ -PH05t in pRH1 is performed in an identical manner.
• a Bglll-Hindlll fragment containing the complete yCNA1 or yCNA2 genes (or their truncated versions; Examples 1 and 2) and the Hindlll-Kpnl fragment of the yeast PH05 transcription terminator are ligated to pRH1 (see Example 10) which is completely digested with Bglll and Kpnl. Restriction enzyme analysis of DNA, obtained from E.
• the plasmids are named pMH6, pMH7 (encoding ACE1N-yCNA1 and ACE1N- yCNAI ⁇ , respectively) and pMH21, pMH22 (encoding ACE1N-yCNA2 and ACE1 N-yCNA2 ⁇ , respectively)
• the Bglll-EcoRI fragments containing the hCNRA2 and hCNRA2 ⁇ genes are subcloned directly in Bglll-EcoRI digested pRH1.
• the plasmids are named pK011 , pK012 (encoding the ACE1 N-hCNRA2 and ACE1 N-hCNRA2 ⁇ fusions, respectively).
• Example 12 Construction of a veast vector for expression of vTPK1. the complete gene encoding the catalytic subunit of veast cAMP-dependent protein kinase
• a -1200 bp Bglll-EcoRI fragment of the yTPK1 gene (the complete coding sequence; Toda et al., Cell (1987), 50, 277-287) is isolated from the yeast genome by PCR using the primers with SEQ ID NOs 37 and 38.
• the -1200 bp Bglll-EcoRI fragment of yTPK1 is subcloned in pRH1 (see Example 10).
• One clone, which contains the correct insert (confirmed by restriction enzyme analysis and DNA sequencing) is named pSK1 A.
• Example 13 Construction of a veast expression vector for expression of the regulatory subunit of yTPK1 , i.e. yBCYI : the complete gene which also encodes the inhibitory peptide of yTPK1
• a -1260 bp BamHI-EcoRI fragment of the yeast BCY1 gene (the complete coding sequence, Toda et al., Mol. Cell. Biol. (1987), 7, 1371-1377) is isolated from the yeast genome by PCR, using the primers with SEQ ID NOs 39 and 40.
• yBCYI As a ACE1C-yBCY1 fusion, the -1075 bp Sall-Bglll fragment from pRHIOC (see Example 5), the -1260 bp BamHI-EcoRI fragment of the yeast BCY1 gene and the -300 bp EcoRI-Sacl fragment from pUC19/EcoRI-Sacl/SUC2t (see Example 4) are ligated to the vector pDP34 which has been completely digested with Sail and Sad.
• pSK5 One clone, which contains the correct insert (confirmed by restriction enzyme analysis and DNA sequencing) is named pSK5.
• Example 14 Construction of veast expression vectors for the expression of ACE1C- BCYKwt ID) ACE1 C-BCY1 (Mut1 ID) and ACE1 C-BCY1 (Mut2 ID) which encode a single copy of the gene fragments encoding the inhibitory peptide (ID) of veast BCY1 (wild type, pseudosubstrate and a mutated pseudosubstrate)
• the resulting correct clones are named pUC19/Sall-Xbal/GAPCLp- ACE1C-BCY1 (wt-ID), pUC19/Sall-Xbal/GAPCLp-ACE1C-BCY1 (Mut1 -ID) and pUC19/Sall- Xbal/GAPCLp-ACE1 C-BCY1 (Mut2-ID).
• the individual Sall-Xbal fragments from the above plasmids and a Xbal-Sacl fragment from pUC19/Xbal-Sacl/SUC2t are subcloned in pDP34 (as described in Example 6).
• the expression plasmids are named pSK26, pSK21 and pSK22 [encoding ACE1C- BCYI(wt-ID)], pSK21 [encoding ACE1C-BCY1(Mut1-ID)] and pSK22 [encoding ACE1C- BCY1 (Mut2-ID)].
• Example 15 Construction of veast expression vectors for the expression of ACE1 C- BCYKMut/l ID)-(Glv) 4 -BCY1 (Mut1 ID) and ACE1C-BCY1 (Mut2 ID)-(Glv) 4 - BCY1(Mut2 ID), the gene fragments encoding the inhibitory peptides of veast BCY1 (pseudosubstrate and a mutated pseudosubstrate): two copies linked by qlycine linkers
• BCYI(wt-ID), BCY1(Mut1-ID) and BCY1 (Mut2-ID) are isolated by PCR using the primers (SEQ ID NOs 44 and 45), the templates being pSK26, pSK21 and pSK22, respectively.
• Expression plasmids in pDP34 are made as described in Example 7.
• Example 16 Isolation of the three fragments from human p70 s6 kinase (p70s6k). the complete gene, a C-terminal truncated version of p70s6k (p70s6k ⁇ C) and an N-terminal truncated version of p70s6k (p70s6k ⁇ N), for expression as fusion genes with ACE1 N
• PCR is performed on the cDNA encoding the mitogen-activated S6 kinase (i.e. p70s6k) obtained from the rat liver (Kozma et al., Proc. Natl. Acad. Sci. USA (1990), 87, 7365-7369).
• the rat gene encodes a polypeptide identical to the human protein.
• Three pairs of deoxyoligoribo ⁇ nucleotides are used as primers (see SEQ ID NOs 45 and 46; 45 and 47 and 48 and 46).
• the genes, obtained as BamHI-Xbal/ BamHI-EcoRI fragments, are subcloned in pRH1 (see Example 10).
• the resulting plasmids are named pSK18 (encoding wild type (wt) p70s6k), pSK6 (encoding p70s6k ⁇ C) and pSK27 (encoding p70s6k ⁇ N), respectively.
• Example 17 Construction of veast expression vedors for the expression of ACE1C-s6k(wt AID), ACE1C-s6k(Mut1-AID) and ACE1C-s6k(Mut2-AID): single copies of the gene fragments encoding the autoinhibitory peptides of human p70 s6 kinase (wild type, pseudosubstrate and a mutated pseudosubstrate)
• the Bglll-EcoRI -312 bp fragments containing the wild type or mutated autoinhibitory peptide sequences (Banerjee, et al., Proc. Natl. Acad. Sci. USA (1990), 87, 8550-8554) are subcloned in the context of the activation domain of the yeast transcriptional activator ACE1 in the expression vector pDP34 (as in Example 6).
• the pair of deoxyoligoribonucleotides with SEQ ID NOs 49 and 46 are used to isolate the wild type (wt) and the two mutated sequences as Bglll-EcoRI fragments.
• the resulting plasmids are named pSK11 (wt), pSK13 (Mut1 ) and pSK12 (Mut2).
• a Bglll-EcoRI -513 bp fragment (comprising the above -312 bp fragment and a 5' extension of -200 bp) is subcloned by PCR from the wt gene and the mutated sequences, which encode the Mut1-AID and Mut2-AID mutations.
• the pair of deoxyoligoribonucleotides with SEQ ID NOs 50 and 46 are used to isolate the wt and the two mutated sequences as Bglll- EcoRI fragments. They are cloned as ACE1C fusions in pDP34, as detailed in Example 6. They are referred to as pSK8 (wt), pSK10 (Mut1) and pSK9 (Mut2).
• Example 18 Construction of plasmids for the expression of p70s6k. p70s6k ⁇ C and p70s6k ⁇ N from veast multi-copy vectors
• a -670 bp Sall-Bglll fragment (containing the GAPCL promoter) is isolated from pRH2 (see Example 5) by PCR using the primers with the SEQ ID NOs 51 and 52.
• the above Sall-Bglll fragment, the BamHI-Xbal/ BamHI-EcoRI fragments (encoding the sequences of p70s6k, p70s6k ⁇ C and p70s6k ⁇ N; see Example 16) and a Xbal-Sacl/ EcoRI- Sacl fragment of the SUC2 terminator (see Example 4) are subcloned in pDP34 digested completely with Sail and Sad.
• the correct clones are named pSK16 (encoding p70s6k), pSK18 (encoding p70s6k ⁇ C) and pSK28 (encoding p70s6k ⁇ N).
• yeast transformants of pSK16 yield an active protein. No protein is seen in cells harboring pSK18 and pSK28. However, the amount of mRNA obtained from the latter is the same as in cells expressing full length p70S6k.
• Example 19 Chromosomal integration of ACE1 N fusion genes in the veast strain TFY2
• the Bio-Rad gene pulser is employed for transformation of yeast cells (see Table 1) by electroporation (Methods. Enzymol. (1991), 194, 182-187).
• the strain TFY2 (Mat ⁇ his ura3- 52 tro1-285 acel LEU2::YipCL CUP1) (Munder et al., Mol. Cell. Biol. (1992), 12, 2091-2099) is used for all integrations of ACE1N-gene fusions into the yeast chromosome. Correct gene integration and gene replacement events are verified by PCR with primers flanking the insertion sites. Subsequently, the amplified fragments are analyzed by agarose gel electrophoresis.
• Example 20 Yeast transformations with plasmids. in strains harboring gene fusions with ACE1N
• Yeast strains harboring ACE1N gene fusions are transformed with plasmids bearing the different ACE1C fusions as described in Example 19. The transformants are depicted in Table 2.
• Example 21 Assay for copper resistance: induction of metallothionein expression
• Equal numbers of cells are spotted onto SD-agar plates (2 % glucose, 0.67 % yeast nitrogen base without amino acids, 2 % agar) containing (a) no CuS0 4) (b) 100 ⁇ M CuS0 4 and (c) 200 ⁇ M CuS0 4 (Munder et al., Mol. Cell. Biol. (1992), 12, 2091-2099).
• the results are portrayed in Table 2.
• Yeast cells (Example 19) are grown in YPD medium (1 % bacto-yeast extract, 2 % bacto- peptone, 2 % glucose) for 17 h at 30°C. Cells are first washed in SD medium (0.67 % yeast nitrogen base without amino acids, 2 % glucose) and then the transcription of the reporter gene is induced. Induction and permeabilization of cells are performed as described previously (F ⁇ rst et al., Cell (1988), 55, 705-717). ⁇ -galactosidase activity, measured as o-nitrophenyl- ⁇ -D-galacto-pyranoside hydrolysis at 420 nm, is normalized to cell culture density and expressed as arbitrary units. The activity in the control strains, harboring the plasmid pRH14C, is set to the value 1. The results are portrayed in Table 2.
• Cells are initially grown in YPD, as in Example 22. After washing in SD, cells are preincubated with small organic compounds (dissolved in dimethyl sulphoxide and 10-20 ⁇ molar in final concentration) for 90 min at 30°C. Later, ⁇ -galactosidase activity is measured, exactly as in Example 22.
• Example 24 Disruption of protein-peptide interaction in CNY15 with the immunosuppressants cyclosporin A and FK506
• Example 22 ⁇ -galactosidase activity is measured, as in Example 22.
• three individual transformants from CNY16 see Table 2 are used.
• the results, an average of three transformants (in duplicate), are depicted in Table 3.
• the units shown are arbitrary (see Example 22).
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• 1995
  • 1995-07-12 EP EP95925863A patent/EP0775206A1/de not_active Withdrawn
  • 1995-07-12 JP JP8505403A patent/JPH11506301A/ja active Pending
  • 1995-07-12 AU AU29832/95A patent/AU2983295A/en not_active Abandoned
  • 1995-07-12 WO PCT/EP1995/002724 patent/WO1996003501A1/en not_active Application Discontinuation
• 1997
  • 1997-01-17 FI FI970205A patent/FI970205A/fi not_active Application Discontinuation
  • 1997-01-21 NO NO970258A patent/NO970258L/no unknown

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