WO1991016456A1 - Techniques de triage de composes inhibant la liaison de c-myc a l'adn - Google Patents

Techniques de triage de composes inhibant la liaison de c-myc a l'adn Download PDF

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WO1991016456A1
WO1991016456A1 PCT/US1991/001962 US9101962W WO9116456A1 WO 1991016456 A1 WO1991016456 A1 WO 1991016456A1 US 9101962 W US9101962 W US 9101962W WO 9116456 A1 WO9116456 A1 WO 9116456A1
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myc
expression
host
dna
protein
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PCT/US1991/001962
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Robert E. Kingston
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The General Hospital Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • This invention is directed to methods for identifying compounds which inhibit c-myc transcriptional activity, and specifically compounds which inhibit c-myc heterodimer complex binding to specific DNA sequences.
  • the cellular c-myc gene encodes the c-myc protein.
  • Expression of large amounts of c-myc in a variety of cell types allows the cells to grow indefinitely in cell culture (reviewed in Bishop, J.M., Ce77 42:23-38 (1985); and Weinberg, R.A., Science 230:770-776 (1985)).
  • C-myc expression has been implicated as a factor in at least 10% of all human cancers.
  • overexpression of c-myc in normal rat fibroblasts together with expression of an activated ras oncogene product, transforms the fibroblasts and endows them with the ability to form tumors in living animals (Land, H. et al . , Nature 304:595-601 (1983); Ruley, H.E., Nature 304:602-
  • c-myc protein The function of the c-myc protein remains unknown despite evidence suggesting possible roles in transcriptional regulation, RNA processing, and replication. Recent studies suggest that oncoproteins such as c-myc alter gene expression and immortalize cells by regulating the promoter activity of specific target genes and thus activating or repressing transcription of those target genes (see, for example, Var us, H.E., Science 238:1337-1339 (1987)); Springfield, R.E. et al., Cell 41:3-5 (1985); Bishop, J.M., Ce77 42:23-38 (1985);
  • myc proteins are sequence specific DNA binding proteins. However, only nonspecific interactions with DNA have been reported for c-myc and for N- myc. (Donner, P. et al. , Nature 295:262-265 (1982); Persson,
  • E motifs generally are variants of the 5'-CAGGTGGC-3' consensus sequence.
  • the ⁇ El motif is GTCAAGATGGC
  • ⁇ E2 motif is AGCAGCTGGC
  • E3 is GTCATGTGG
  • iE5 is TGGCAGGTGT (Murre, C. et al . , Cel l 55:777-783 (1989).
  • One report found no binding of N-myc to any the E box sequences (Murre, C. et al . , Cel l 55:777-783 (1989)).
  • the invention provides a reliable and accurate method for objectively classifying compounds, including human pharmaceuticals, as an inhibitor of c-myc activity.
  • the invention further provides a method for identifying and classifying the mechanism of action of a bioactive c-myc- inhibiting compound.
  • the invention further provides an assay for the monitoring of the isolation and/or purification of an c-myc- inhibiting compound or mixture of such compounds from a crude preparation.
  • the transcription of a codi ' ng sequence which is operably-1inked to a promoter element is induced by factors which "activate” the promoter's activity; transcription of a coding sequence which is operably-1inked to a promoter element is inhibited by factors which "repress" the promoter's activity.
  • a promoter region would be operably-1inked to the coding sequence of a protein if transcription of the coding sequence activity was influenced by the activity of the promoter.
  • response is intended to refer to a change in any parameter which can be used to measure, indicate or otherwise describe c-myc heterodimer binding to the ⁇ E2 DNA sequence.
  • the response may be revealed as a physical change (such as a change in phenotype) or, it may be revealed as a molecular change (such as a change in a reaction rate or affinity constant). Detection of the response may be performed by any means appropriate.
  • Compound is intended to refer to a chemical entity, whether in the solid, liquid, or gaseous phase.
  • the term should be read to include synthetic compounds, natural products and macromolecular entities such as polypep- tides, polynucleotides, or lipids, and also small entities such as neurotrans itters, ligands, hormones or elemental compounds.
  • Bioactive Compound is intended to refer to any compound which induces a detectable or measurable response in the methods of the invention.
  • promoter is a DNA sequence located proximal to the start of transcription at the 5' end of the transcribed sequence.
  • the promoter may contain multiple regulatory elements which interact in modulating transcription of the operably-1inked gene.
  • Expression is the process by which the information encoded within a gene is revealed. If the gene encodes a protein, expression involves transcription of the DNA into mRNA, the processing of mRNA (if necessary) into a mature mRNA product, and translation of the mature mRNA into protein.
  • a nucleic acid molecule, such as a DNA or gene is said to be "capable of expressing" a polypeptide if the DNA contains the coding sequences for the polypeptide and expression control sequences which, in the appropriate host environment, provide the ability to transcribe, process and translate the genetic information contained in the DNA into a protein product, and if such expression control sequences are operably-1inked to the nucleotide sequence which encodes the polypeptide.
  • Cloning vehicle is any molecular entity which is capable of delivering a nucleic acid sequence into a host cell for cloning purposes.
  • Examples of cloning vehicles include plasmids or phage genomes.
  • a plasmid which can replicate autonomously in the host cell is especially desired.
  • a nucleic acid molecule which can insert into the host cell's chromosomal DNA is especially useful.
  • Cloning vehicles are often characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be spliced in order to bring about its replication and cloning.
  • the cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are tetracycline resistance or ampicillin resistance. The word “vector” is sometimes used for "cloning vehicle.”
  • Expression vehicle is a vehicle or vector similar to a cloning vehicle but is especially designed to provide sequences capable of expressing the cloned gene after transformation into a host.
  • the gene to be cloned is usually operably-1inked to certain control sequences such as promoter sequences.
  • Expression control sequences will vary depending on whether the vector is designed to express the operably-1inked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • host any organism that is the recipient of a cloning or expression vehicle.
  • at least two genetic constructs are utilized. First, a reco binant construct capable of expressing c-myc; and second, a reporter gene whose expression is operably linked to c-myc heterodimer binding to the ⁇ E2 sequence. If desired, a recombinant construct capable of expressing a c-myc partner protein may also be used.
  • Constructs which are capable of expressing c-myc protein may be constructed utilizing the guidelines as described below or purchased commercially.
  • the ⁇ E2 motif sequence may be operably linked to any gene which confers a selectable marker in yeast.
  • a marker gene which allows phenotypic selection in Saccharo yces cerevisiae is used.
  • Yeast which have been co-transformed with both an expressible c-myc gene and with the ⁇ E2 binding sequence may be used to (1) identify the presence or absence of endogenous host proteins which interact with c-myc in a manner which permits it to bind to the ⁇ E2 sequence; (2) classify a protein as a c-myc partner protein; and (3) identify and classify compounds as agents which disrupt 'heterodimer formation between c-myc and its partner proteins.
  • Hosts which have been co-transformed with both an expressible c-myc gene and with the ⁇ E2 binding sequence may be used to identify the presence or absence of endogenous host proteins which interact with c-myc in a manner which permits c-myc to bind to the ⁇ E2 sequence if c-myc expression in such host is sufficiently low that the moles of c-myc which are expressed do not overwhelm the moles of the partner protein which is endogenously in the cell.
  • such partner protein may be isolated using techniques known in the art such as gel mobility shift analysis, cDNA expresison cloning vectors such as, for example, ⁇ gtlO and ⁇ gtll, or other cloning systems specifically designed for high- efficiency cloning and expression of full-length cDNA in yeast such as, for example, pGl and pTRP56, all of which are commerically available (Clontech, Palo Alto, California).
  • c-myc partner proteins it is not necessary that the host be completely deficient in c-myc partner proteins to be useful in the method of the invention. As described below, if c-myc is expressed at levels much greater than those found in the host, reporter gene transcription from endogenous partner proteins may be negligible, or of such low amount that it does not otherwise prevent the utility of the methods of the invention. If the c-myc expression is transcribed with a strong promoter, and/or if the c-myc expression cassette is supplied on a high copy number vector, the levels of c-myc will be high enough to overcome a low level background and such c-myc constructs may be used to analyze the ability of cloned c-myc partners to influence c-myc DNA binding.
  • One of ordinary skill in the art can adapt the expression system to the level of expression desired using methods known in the art.
  • the partner protein if supplied as a recombinant construct to the host cell, should be capable of expressing at levels comparable to that of the c-myc protein.
  • C-myc partner proteins can be identified by utilizing a phage plaque assay, as described in the inventor's copending U.S. patent application entitled “Protein Partner Screening Assays and Uses Thereof," Serial No. 07/510,254 f filed the same day as this application, April 19, 1990 and incorporated herein by reference. Proteins identified by the screening assay can be subcloned into eukaryotic expression vectors known in the art and commerically available so as to provide a recombinant source of partner protein gene expression.
  • the genetic constructs of the invention may be placed on different plasmids, or combined on one plas id.
  • a construct may also be inserted into the genome of a host cell.
  • the construct coding for the c-myc protein and the construct coding for the partner protein are provided to the host on two different plasmids.
  • the ⁇ E2 motif may be located at any site in the transcription cassette of the reporter gene which allows for the transcription of that gene to be operably-1inked to c- yc-partner complex binding.
  • such motif may be located 5' to the transcriptional start site or 3' to the transcriptional start site, for example, in an intron, similar to its location relative to the promoter region in the immunoglobulin genes.
  • the reporter gene whose expression is operably linked to c-myc heterodimer binding to the ⁇ E2 sequence may be any gene whose expression can be monitored. Any detectable phenotype change may serve as the basis for the methods of the invention.
  • the reporter gene is a gene not normally expressed by the host, or a gene which replaces the host's endogenous gene. Any reporter gene which is capable of being operably-1inked to a promoter capable of responding to the binding of the the c-myc-partner protein heterodi ers to ⁇ E2 may be used.
  • genes which endow the host with an ability to grow on a selective medium are useful.
  • yeast use of the yeast LEU2 gene as a reporter gene in strains which normally lack LEU2 allows such yeast to grow on leucine as a sole carbon source. Expression the reporter gene is monitored by merely observing whether the host retains the ability to grow on leucine.
  • suc2 gene as a reporter gene would allow growth of the a suc2" yeast host on sucrose to be used as the detection method.
  • growth on the indicated substrate indicates ⁇ E2 DNA binding of the c-myc-partner protein heterodimer and lack of such growth indicates lack of binding or lack of heterodimer formation.
  • a construct (and host) which is gall + gall ⁇ would respond to galactose in the medium; a construct (and host) which is lac2 + gall + would be lactose sensitive.
  • Other reporter genes include ⁇ /s3, ura3 and trp5.
  • Reporter constructs in which the ⁇ E2a motif and the 7acZ reporter gene are operably linked will express ⁇ - galactosidase in response to binding of a c-myc-partner protein complex. Such expression can be easily scored by monitoring the ability of the host to produce ⁇ - galactosidase (Maniatis, T. et al.. Molecular Cloning (A Laboratory Manual). 2nd edition, Cold Spring Harbor Laboratory, 1989). The production of 0-galactosidase may be visually monitored by detecting its activity to reduce the chromophoric dye, X-gal (commercially available from International Biotechnologiers, Inc., New Haven, CT). ⁇ - galactosidase reduces X-gal to a form which possesses a blue color.
  • the coding sequence of chlora phenicol acetyltransferase (CAT) is used as the reporter gene.
  • any detection method which can identify expression of the reporter gene may be used.
  • levels of the product of the reporter gene may be directly assayed with an immuno- assay.
  • immunoassays include those wherein the antibody is in a liquid phase or bound to a solid phase carrier.
  • the reporter gene can be detectably labeled in various ways for use in immunoassays.
  • the preferred immuno- assays for detecting a reporter protein using the include radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), or other assays known in the art, such as immunofluorescent assays, chemiluminescent assays, or bioluminescent assays.
  • yeast strains which express such heterodimers and which contain the ⁇ E2 binding site may be plated and grown as lawns and the compound to be tested may be applied to the plates on a filter paper disk that is impregnated with such compound.
  • the compound may be incorporated into the media within which the host cells are growing.
  • the methods of the invention can be used to screen compounds in their pure form, at a variety of concentrations, and also in their impure form.
  • the methods of the invention can also be used to identify the presence of such inhibitors in crude extracts, and to follow the purification of the inhibitors therefrom.
  • the methods of the invention are also useful in the evaluation of the stability of the inhibitors identified as above, to evaluate the efficacy of various preparations.
  • the permeability of cells to various compounds can be enhanced, if necessary, by use of a mutant cell strain which possess an enhanced permeability or by using compounds which are known to increase permeability.
  • a mutant cell strain which possess an enhanced permeability or by using compounds which are known to increase permeability.
  • compounds which are known to increase permeability for example, in yeast compounds such as polymyxin B nonapeptide may be used to increase the yeast's permeability to small organic compounds.
  • DMSO dimethyl sulfoxide
  • Analogs of such compounds which are more permeable across yeast membranes may also be used. For example, dibutyryl derivatives often display an enhanced permeability.
  • the genetic constructs and the methods for using them are utilized in eukaryotic hosts, and especially in yeast, insect and mammalian cells.
  • the introduced sequence is incorporated into a plasmid or vector- capable of either autonomous replication or integrative activity.
  • the DNA sequence of the fusion protein and/or target gene may be chemically constructed if it is not desired to utilize a clone of the genome or mRNA as the source of the genetic information.
  • Methods of chemically synthesizing DNA are well known in the art ⁇ Oligonucleotide Synthesis, A Practical Approach, M.J. Gail, ed., IRL Press, Washington, D.C., 1094; Synthesis and Applications of DNA and RNA, S.A.
  • a cloned protein encoding DNA sequence obtained through the methods described above, (preferably in a double-stranded form), may be operably- 1inked to sequences controlling transcriptional expression in an expression vector, and introduced, for example by transformation, into a host cell to produce recombinant proteins useful in the methods of the invention, or functional derivatives thereof.
  • Such techniques are well known in the art ⁇ Recombinant DNA Methodology, Wu, R. et al . , eds., Academic Press, (1989); Maniatis, T. et al .. Molecular Cloning (A Laboratory Manual), second * edition, Cold Spring Harbor Laboratory, 1989).
  • Transcriptional initiation regulatory signals can be selected which allow for repression or activation of the expression of the c-myc construct or the partner protein construct or both, so that expression of such constructs can be modulated, if desired.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, for example, by a metabolite, salt, or substrate added to the growth medium.
  • sequences functional in the host cell may be substituted.
  • constructs of the invention may result in different post-translational modifications which may alter the properties of the proteins expressed by these constructs. It is necessary to express the proteins in a host wherein the ability of the protein to retain its biological function is not hindered. Expression of proteins in yeast hosts is preferably achieved using yeast regulatory signals.
  • the vectors of the invention may contain operably-1inked regulatory elements such as upstream activator sequences in yeast, or DNA elements which confer species, tissue or cell-type specific expression on an operably-1inked gene.
  • expression vectors containing transcriptional regulatory sequences are used in connection with a host. These sequences facilitate the efficient transcription of the gene fragment operably-1inked to them.
  • expression vectors also typically contain discrete DNA elements such as, for example, (a) an origin of replication which allows for autonomous replication of the vector, or, elements which promote insertion of the vector into the host's chromosome in a stable manner, and (b) specific genes which are capable of providing phenotypic selection in transformed cells.
  • Eukaryotic expression vectors may also contain elements which allow it to be maintained in prokaryo- tic hosts; such vector are known as shuttle vectors.
  • yeast are used as the host cells.
  • the elements necessary for transcriptional expression of a gene in yeast have been recently reviewed (Struhl, K. Ann. Rev. Biochem. 58:1051-1077 (1989)).
  • UAS upstream activator sequence
  • TATA TATA
  • I initiation
  • Some promoters also contain operator elements.
  • mammalian cells are used as the host cells.
  • a wide variety of transcriptional and translational regulatory signals can be derived for expression of proteins in mammalian cells and especially from the genomic sequences of viruses which infect eukaryotic cells.
  • the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, for example by transformation. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector- containing cells. Expression of the cloned gene sequence(s) results in the production of the protein. This expression can take place in a continuous manner in the transformed cells, or in a controlled manner.
  • Genetically stable transformants may be constructed with episomal vector systems, or with integrated vector systems whereby the fusion protein DNA is integrated into the host chromosome. Such integration may occur de novo within the cell or be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with retroviral vectors, transposons or other DNA elements which promote integration of DNA sequences in chromosomes.
  • Cells which have been transformed with the DNA vectors of the invention are selected by also introducing one or more markers which allow for selection of host cells which contain the vector, for example, the marker may provide biocide resis- tance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the marker may provide biocide resis- tance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like.
  • the transformed host cell can be fermented or cultured according to means known in the art to achieve optimal cell growth, and also to achieve optimal expression of the cloned fusion protein sequence fragments.
  • a high level of fusion protein expression for the cloned sequences coding for the proteins can be achieved according to a preferred procedure of this invention.
  • the following examples further describe the materials and methods used in carrying out the invention. The examples are not intended to limit the invention in any manner.
  • Example 1 Screening a cDNA expression library for proteins able to interact with the helix-loop-helix domain of c-Mvc.
  • This expression unit was subcloned into pACYC177, a low copy number plasmid (10-15 copies/cell) with a kanamycin R gene which is commercially available. Cells transformed with this construct were shown to be resistant to lambda phage infection by a dot plaque assay.
  • the . coli strain Y1090 transformed with pYC192cIHLH was used to screen a human tonsil/B cell ⁇ gtll expression library. 5 x 10 6 pfu were sceened in duplicate on the above transformed strain, as well as a Y1090:: ⁇ lysogen strain. On each of test plates 800 plaques formed. On the control plates there were no plaques were plaque-purified once and a single plaque was picked and suspended in suspension medium (SM). The 160 purified plaques were grouped according to plaque size: 20 small, 70 medium, 70 large. (Four of the "small" group did not form plaques in the plaque purification step.) Each of these was then screened by a dot plaque assay on an . . coli .
  • yeast host cells are transformed with plasmids carrying c-myc (host 'a'); or c-myc and a 46,000 dalton partner protein identified as above (host 'b').
  • all yeast strains are cotransformed with a plasmid which contains the coding sequence for ⁇ -galactosidase operably-1inked to the ⁇ E2 sequence as described above.
  • a lawn of each of the transformed yeast strains is spread on agar plates containing X-gal in the medium and small filter disks containing compound W, X, Y, or Z are placed on the lawns.
  • the yeast are allowed to grow and the plates are monitored for colony growth and colony color by visual observation. Typical results from such an experiment are shown in Table 1.
  • Compound Y does not prevent induction of ⁇ -galactosidase activity in the 'b' host cells. Therefore, compound Y is not an inhibitor of heterodimer formation.
  • Compound Z shows an interesting effect of inducing ⁇ - galactosidase activity in the 'a' host cells which does not contain the c-myc partner protein used in the 'b' hosts, rather than preventing heterodimer formation. This suggests that compound Z may induce synthesis of a partner protein which is not otherwise present in the yeast host cells or that it may be (or mimic) such a protein.
  • compound W would be identified as an inhibitor of c-myc heterodimer fomration and thus of c-myc transcriptional activity in vivo.

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Abstract

Techniques de triage et de classification de composés utilisés comme inhibiteurs de l'action de la protéine c-myc, permettant de détecter la capacité d'un composé d'inhiber ou d'empêcher la formation d'un hétérodimère de c-myc.
PCT/US1991/001962 1990-04-19 1991-03-22 Techniques de triage de composes inhibant la liaison de c-myc a l'adn WO1991016456A1 (fr)

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US510,253 1990-04-19

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0528827A4 (fr) * 1990-04-19 1992-11-16 Gen Hospital Corp Techniques de triage de partenaires de proteines et leurs utilisations.
EP0550592A1 (fr) * 1990-09-24 1993-07-14 Roger Brent Methodes de triage.
WO1993019176A1 (fr) * 1992-03-23 1993-09-30 Imperial Cancer Research Technology Limited Sequences de leucine type zipper
WO1994009133A1 (fr) * 1992-10-14 1994-04-28 The General Hospital Corporation Essai de depistage de proteines associees et ses utilisations
WO1995020652A1 (fr) * 1994-01-28 1995-08-03 Medigene Gmbh Procede pour determiner l'activite d'un facteur regulateur et application de ce procede
WO1995029254A1 (fr) * 1994-04-20 1995-11-02 Gene Shears Pty. Ltd. SYSTEME D'EXPRESSION DE GENES $i(IN VIVO)
WO1996017960A2 (fr) * 1994-12-07 1996-06-13 Scriptgen Pharmaceuticals, Inc. Procedes d'inhibition de l'activite amplificatrice de transcription de la proteine x du virus de l'hepatite b
WO1997040379A2 (fr) * 1996-04-23 1997-10-30 Cold Spring Harbor Laboratory EPREUVES ET REACTIFS PERMETTANT L'IDENTIFICATION DE MODULATEURS D'ACTIVATION DE LA MITOSE INDUITE PAR Cdc25
US5747338A (en) * 1996-08-15 1998-05-05 Chiron Corporation Method and construct for screening for inhibitors of transcriptional activation
WO1998048274A1 (fr) * 1997-04-22 1998-10-29 Smithkline Beecham Corporation Test par fluorescence homogene pour mesurer l'effet des composes sur l'expression d'un gene
WO2020128015A3 (fr) * 2018-12-20 2020-08-06 The University Of Bath Essai de criblage

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, Volume 174, (1), issued 1988, DANG, "Detection and Use of Recombinant Staphylococcus Protein a Fusion Proteins", pages 313-317. *
GENES. DEV., Volume 3, (12B), issued 1989, SMEAL et al., "Different Requirements for Formation of Jun Jun and Jun Jos Complexes", pages 2091-2100. *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0528827A1 (fr) * 1990-04-19 1993-03-03 Gen Hospital Corp Techniques de triage de partenaires de proteines et leurs utilisations.
US5322801A (en) * 1990-04-19 1994-06-21 The General Hospital Corporation Protein partner screening assays and uses thereof
EP0528827A4 (fr) * 1990-04-19 1992-11-16 Gen Hospital Corp Techniques de triage de partenaires de proteines et leurs utilisations.
US5580721A (en) * 1990-09-24 1996-12-03 The General Hospital Corporation Assays for inhibitors of myc oncoprotein
EP0550592A1 (fr) * 1990-09-24 1993-07-14 Roger Brent Methodes de triage.
EP0550592A4 (en) * 1990-09-24 1994-12-07 Roger Brent Screening assays
WO1993019176A1 (fr) * 1992-03-23 1993-09-30 Imperial Cancer Research Technology Limited Sequences de leucine type zipper
WO1994009133A1 (fr) * 1992-10-14 1994-04-28 The General Hospital Corporation Essai de depistage de proteines associees et ses utilisations
WO1995020652A1 (fr) * 1994-01-28 1995-08-03 Medigene Gmbh Procede pour determiner l'activite d'un facteur regulateur et application de ce procede
WO1995029254A1 (fr) * 1994-04-20 1995-11-02 Gene Shears Pty. Ltd. SYSTEME D'EXPRESSION DE GENES $i(IN VIVO)
WO1996017960A3 (fr) * 1994-12-07 1996-08-29 Scriptgen Pharm Inc Procedes d'inhibition de l'activite amplificatrice de transcription de la proteine x du virus de l'hepatite b
WO1996017960A2 (fr) * 1994-12-07 1996-06-13 Scriptgen Pharmaceuticals, Inc. Procedes d'inhibition de l'activite amplificatrice de transcription de la proteine x du virus de l'hepatite b
US6051373A (en) * 1994-12-07 2000-04-18 Scriptgen Pharmaceuticals, Inc. Methods for screening for inhibitors of the transcription-enhancing activity of the X protein of hepatitis B virus
WO1997040379A2 (fr) * 1996-04-23 1997-10-30 Cold Spring Harbor Laboratory EPREUVES ET REACTIFS PERMETTANT L'IDENTIFICATION DE MODULATEURS D'ACTIVATION DE LA MITOSE INDUITE PAR Cdc25
WO1997040379A3 (fr) * 1996-04-23 1998-01-29 Cold Spring Harbor Lab EPREUVES ET REACTIFS PERMETTANT L'IDENTIFICATION DE MODULATEURS D'ACTIVATION DE LA MITOSE INDUITE PAR Cdc25
US5747338A (en) * 1996-08-15 1998-05-05 Chiron Corporation Method and construct for screening for inhibitors of transcriptional activation
WO1998048274A1 (fr) * 1997-04-22 1998-10-29 Smithkline Beecham Corporation Test par fluorescence homogene pour mesurer l'effet des composes sur l'expression d'un gene
WO2020128015A3 (fr) * 2018-12-20 2020-08-06 The University Of Bath Essai de criblage

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YU66491A (sh) 1994-06-24
IL97702A0 (en) 1992-06-21
ZA912302B (en) 1991-12-24

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