EP1062368A1 - Detection de l'interaction de proteines et piegeage du facteur de transcription - Google Patents

Detection de l'interaction de proteines et piegeage du facteur de transcription

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Publication number
EP1062368A1
EP1062368A1 EP99936098A EP99936098A EP1062368A1 EP 1062368 A1 EP1062368 A1 EP 1062368A1 EP 99936098 A EP99936098 A EP 99936098A EP 99936098 A EP99936098 A EP 99936098A EP 1062368 A1 EP1062368 A1 EP 1062368A1
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Prior art keywords
dna
protein
cell
dna sequence
transcriptional regulatory
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EP99936098A
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German (de)
English (en)
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Christopher J. Ong
Frank R. Jirik
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University of British Columbia
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University of British Columbia
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Publication of EP1062368A1 publication Critical patent/EP1062368A1/fr
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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/71—Fusion polypeptide containing domain for protein-protein interaction containing domain for transcriptional activaation, e.g. VP16
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/40—Systems of functionally co-operating vectors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/60—Vectors containing traps for, e.g. exons, promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • This invention relates to the use of gene trapping methods for the identification of genes and two-hybrid methodology for the identification of protein-protein interactions .
  • Virtually all cellular responses, including growth and differentiation, are stringently controlled by physiological signals in the form of growth factors, hormones, nutrients, and contact with neighbouring cells. These various signals are processed and interpreted by signal transduction mechanisms which ultimately induce the cell to mount an appropriate response. Signalling pathways stimulated by physiological signals involves a network of specific protein-protein interactions which function to transmit the signal to downstream effector molecules that execute the response. Thus, specific interactions between proteins are critical for signal transduction mechanisms as well as regulation of cellular architecture and responses to physiological signals. Given that specific protein-protein interactions are involved in execution of virtually all cellular functions, technologies which simplify and facilitate detection and analysis of specific protein-protein interactions will be valuable for the discovery, design and testing of drugs that target highly specific biological processes.
  • Eukaryotic gene expression is regulated by a class of proteins variously known as transcriptional activators, or enhancer binding proteins and are referred to herein as "transcriptional regulatory proteins". These molecules, bind to specific sequences on DNA within the promoters of genes they regulate, and function by recruiting the general transcriptional initiation complex to the site where - 2 -
  • transcription of DNA into messenger RNA begins.
  • the general eukaryotic transcriptional initiation complex may consist of two large protein complexes represented by transcription factor IID (TFIID) , which contains the TATA-element binding protein that functions to position the general initiation complex at a precise location on the promoter, and the RNA polymerase II holoenzyme, which contains the catalytic function necessary to unwind the double stranded DNA and transcribe a copy of the DNA template into mRNA.
  • transcription factor IID transcription factor IID
  • RNA polymerase II holoenzyme which contains the catalytic function necessary to unwind the double stranded DNA and transcribe a copy of the DNA template into mRNA.
  • Known transcriptional activators are understood to function by forming direct protein-protein interactions with parts of TFIID and/or the RNA polymerase holoenzyme, and catalysing their assembly into an initiation complex at TATA-element of the promoter.
  • Transcriptional regulatory proteins typically possess two functional elements, a site-specific DNA-binding domain and a transcriptional activation domain which can interact with either TFIID or the RNA polymerase holoenzyme.
  • Eukaryotic transcriptional regulatory proteins are typified by the Saccharomyces yeast GAL4 protein, which was one of the first eukaryotic transcriptional activators on which these functional elements were characterized.
  • GAL4 is responsible for regulation of genes which are necessary for utilization of the six carbon sugar galactose. Galactose must be converted into glucose prior to catabolism; in Saccharomyces this process typically involves four reactions which are catalysed by five different enzymes.
  • Each enzyme is encoded by a GAL gene (GAL 1, 2, 5, 7, and 10) which is regulated by the transactivator GAL4 in response to the presence of galactose.
  • GAL gene has a cis-element within the promoter, termed the upstream activating sequence for galactose (UAS G ) , which contains 17 base-pair sequences to which GAL4 specifically binds.
  • UAS G upstream activating sequence for galactose
  • the GAL genes are repressed when galactose is absent, but are strongly and rapidly induced by the presence of - 3 -
  • GAL4 is prevented from activating transcription when galactose is absent by a regulatory protein GAL80.
  • GAL80 binds directly to GAL4 and likely functions preventing interaction between GAL4's activation domains and the general transcriptional initiation factors.
  • yeast are given galactose, transcription of the GAL genes is induced.
  • Galactose causes a change in the interaction between GAL4 and GAL80 such that GAL4 ' s activation domains become exposed to allow contact with the general transcription factors represented by TFIID and the RNA polymerase II holoenzyme and catalyse their assembly at the TATA-element which results in transcription of the GAL genes.
  • GAL4 The functional regions of GAL4 have been defined by a combination of biochemical and molecular genetic strategies. GAL4 binds as a dimer to its specific cis-element within the UAS G of the GAL genes. The ability to form tight dimers and bind specifically to DNA is conferred by an N-terminal DNA-binding domain. This fragment of GAL4 (amino acids 1-147) can bind efficiently and specifically to DNA but cannot activate transcription. Two parts of the GAL4 protein are necessary for activation of transcription, called activating region 1 and activating region 2. The activating regions are thought to function by interacting with the general transcription factors. The large central portion of GAL4 between the two activating regions is required for inhibition of GAL4 in response to the presence of glucose. The C-terminal amino acids of GAL4 bind the negative regulatory protein GAL80; deletion of this segment causes constitutive induction of GAL transcription.
  • VP16 Herpes viral protein 16
  • GAL80 a transcriptional activator protein
  • GAL80-B17 fusion protein when co-expressed with GAL4 , was found to cause activation of a GAL4-dependent reporter gene to a greater extent than GAL4 alone.
  • the standard two-hybrid assay relies upon the fact that many eukaryotic transcriptional regulatory systems consist of the separate domains discussed above: the DNA-binding domain (DNA-BD) that binds to a promoter or other cis-transcriptional regulatory element; and, the activation domain (AD) that directs RNA polymerase II to transcribe a gene downstream from the site on the DNA where the DNA-BD is bound.
  • the DNA binding domain and the activation domain may be separate proteins but will function to activate transcription as long as the AD is in proximity to a DNA-BD bound to the transcriptional regulatory element. Where each of the AD and the DNA-BD is fused to members of a pair of interacting proteins, the AD will function via the link to the DNA-BD created by the interacting proteins.
  • the two-hybrid assay may be used to investigate whether interaction occurs between two proteins (termed “bait” and “prey”) expressed as fusion products with DNA-BD and AD peptides, respectively.
  • a positive event is identified by activation of a reporter gene having an upstream promoter to which the DNA-BD binds .
  • the two-hybrid assay may be carried out in a variety of eukaryotic cells including yeast (see: Fields, S. and Song. 0. 1989 A Novel Genetic System to Detect
  • a variant of the two-hybrid method employs the principle of using separate fusions with DNA-binding and transactivation domains, except that the bait is fused to LexA, which is a sequence-specific DNA binding protein from E. coli , and an artificial transactivation domain known as B42 (31) is used for the "prey" fusions. Interaction between the bait and prey fusions is detected by expressed of a LexA-responsive reporter gene .
  • Reverse Two-Hybrid A modification of the standard two-hybrid system known as "Reverse Two-Hybrid" (Erickson et al . U.S. Pat No. 5,535,490; Vidal et al . International Application Number PCT/US96/04995) has been described which is intended for use in identifying specific inhibitors of a standard two-hybrid protein-protein interaction.
  • the reverse two- hybrid system operates by driving the expression of relay gene, such as the GAL80 gene, that encodes a protein that bind to and masks the activation domain of a transcriptional activator such as GAL4. Expression of the reporter gene is made dependent upon the functioning of the activation domain of the transcriptional activator. Only when the level of the masking protein is reduced because a compound interferes with the two-hybrid interaction will the activation domain of the transcriptional activator be unmasked and allowed to function.
  • cDNA expression libraries possess some intrinsic disadvantages. For example, cDNA libraries produce a bias toward cloning of highly expressed genes and rare gene transcripts are unlikely to be discovered.
  • the source of the mRNA for the generation of the cDNA library is critical since many tissue restricted genes and developmentally or temporally regulated genes are not represented by a particular cDNA library.
  • Gene trap vectors target the prevalent introns of the eukaryotic genome. These vectors may consist of either a splice-acceptor (SA) site upstream of a reporter sequence, or an unpaired splice-donor (SD) site downstream from a reporter sequence. Preferably, on the latter vector comprising a SD, the reporter sequence is driven by an appropriate transcriptional regulatory element (eg. promoter) . Integration of the above-described gene trap vectors into an intron results in production of m-RNA in which a transcript of the vector is joined to an transcript of an adjacent exon. (see:* Skarnes, W.C. et al . 1992.
  • tagging may also be accomplished by using a vector comprising a peptide encoding segment and both an upstream SA and a downstream SD (see United States Patent No. 5,652,128 of Jarvik) .
  • gene trapping can provide information about coding regions of most genes that is independent of their transcription status
  • gene trapping is independent of the source of mRNA (therefore, rare as well as tissue specific genes and developmental temporally regulated genes may be trapped) .
  • Gene trap methodologies provide a repertoire of protein domains encoded by exon sequences found within the genome. Two-hybrid techniques permit identification of protein-protein interactions. This invention makes use of a combination of gene trap and two-hybrid methodologies for the identification and characterization of genes according to protein-protein interactions of the gene product or for the identification of genes encoding transcriptional activator domains (AD) . Interaction of an exon-encoded protein domain with a given protein, or functioning of the exon-encoded domain as an AD, is detected by reconstituting the activity of a transcriptional activator.
  • AD transcriptional activator domains
  • This invention also provides gene trap vectors adapted for use in a two-hybrid assay and methodologies for identification of genes encoding proteins capable of interacting with a selected protein. This invention also provides gene trap vectors and methodologies for the selective identification of genes encoding transcription activator domains.
  • This invention provides a DNA construct comprising a DNA sequence encoding a transcriptional regulatory protein moiety selected from the group consisting of a DNA-BD and a AD; and, a m-RNA splice site.
  • m-RNA splice site is defined herein as being a splice acceptor sequence
  • SA an unpaired splice donor sequence
  • SD an unpaired splice donor sequence
  • This invention also provides a DNA construct comprising a DNA sequence encoding a transcriptional regulatory protein moiety selected from the group consisting of a DNA-BD and an AD; and, a downstream SD .
  • This DNA construct preferably contains no nucleic acid sequence which would encode a protein that will interact - 9 -
  • the only protein encoded by the construct or the portion of the construct between the 5' end of the sequence encoding the transcriptional regulatory protein moiety and the 3 ' end of the SD is the transcriptional regulatory protein moiety itself.
  • this construct will have a transcriptional regulatory element (eg. a promoter) operably linked to the sequence encoding the transcriptional regulatory protein moiety.
  • This invention also provides a DNA construct comprising a DNA sequence encoding a transcription regulatory protein moiety selected from the group consisting of a DNA-BD and an AD, together with an upstream SA and a downstream SD.
  • this DNA construct may comprise an SA upstream of a transcriptional regulatory protein moiety selected from the group consisting of a DNA-BD and an AD; and, a downstream poly-adenylation signal.
  • these DNA constructs will not encode any protein which will interact with a test protein as used on this invention.
  • the only protein encoded by the construct or the portion of the construct between the SA and the SD or the SA and the poly-adenylation signal will be the transcriptional regulatory protein moiety.
  • This invention also provides a method of making the DNA constructs of this invention comprising the step of joining a DNA sequence encoding a transcriptional regulatory protein moiety as defined above with one or both of a SA and a SD . Preferably, at least three such DNA constructs are made in three different reading frames.
  • This invention also provides cells comprising the DNA constructs of this invention obtainable by the method of transforming eucaryotic cells with one or more DNA constructs of this invention. - 10 -
  • kits that comprise the above-described DNA construct of this invention.
  • the DNA constructs may be in the form of plasmids.
  • the kits may also comprise host cells, two-hybrid vectors or reporter gene constructs as described herein.
  • the two-hybrid vectors of the kit may be plasmids constructed
  • kits may also comprise materials and reagents useful for DNA insertions, reporter gene activity assays, or sequencing of inserts
  • This invention also provides host cells whose genome optionally comprises a reporter gene as described herein and wherein the cell expresses a two-hybrid vector as described herein.
  • the two-hybrid vector may include a sequence encoding a test protein.
  • This invention also provides a method for detecting interaction between an endogenous protein of a cell and a test protein, wherein said cell contains a first DNA sequence encoding a reporter under transcriptional control of a transcriptional regulatory element, and a second DNA sequence that is expressed by the cell and which encodes a first hybrid protein comprising:
  • a first transcriptional regulatory protein moiety selected from the group consisting of: a DNA-BD that recognizes a binding site on the transcriptional regulatory element controlling the first DNA sequence and, a AD functional in the cell;
  • test protein (b) a test protein
  • the DNA construct comprising a third DNA sequence described in the method above may be selected from the following group, in which it is preferable that the only protein encoded by the construct or the portion of the construct described above, be the transcriptional regulatory moiety itself :
  • a gene trap vector comprising the third DNA sequence to reconstitute a transcriptional regulatory protein, followed by a SD; and preferably, a transcriptional regulatory element operably linked to the third DNA sequence;
  • the third DNA sequence to reconstitute a transcriptional regulatory protein; and preferably, the third DNA sequence is followed by a poly-adenylation signal;
  • (III) a gene trap vector comprising the third DNA sequence to reconstitute a transcriptional regulatory protein, with an upstream SA and a downstream SD.
  • the DNA construct comprising the third DNA sequence will encode an AD.
  • the DNA construct will comprise a DNA-BD capable of binding to the transcriptional regulatory element controlling the reporter.
  • the third DNA sequence will preferably encode an AD, not a DNA-BD. This may minimize false positives resulting from reconstitution of a transcriptional regulatory protein when the third DNA sequence is expressed with an exon that encodes an endogenous protein that itself is capable of functioning as an AD. - 13 -
  • This invention also provides a method for detecting an endogenous transcription activator domain (AD) of a cell, wherein the cell contains a first DNA sequence encoding a reporter under transcriptional control of a transcriptional regulatory element, wherein the method comprises the steps of:
  • the DNA construct comprising a second DNA sequence as used in the above-described method for detecting an endogenous transcription activator domain may be selected from the following group in which it is preferred that the only protein encoded by the construct or the portion of the construct described above, be the transcriptional regulatory moiety itself :
  • a gene trap vector comprising the second DNA sequence, followed by a SD; and preferably, a transcriptional regulatory element is operably linked to the second DNA sequence;
  • V a gene trap vector without a transcriptional regulatory element and comprising a SA upstream of the second DNA sequence; and preferably, the - 14 -
  • second DNA sequence is followed by a poly-adenylation signal
  • VI a gene trap vector comprising the second DNA sequence, with an upstream SA and a downstream
  • a cell as used in the method of this invention is a eukaryotic cell .
  • a "DNA construct” is a deoxynucleic acid (DNA) molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of DNA combined and juxtaposed in an arrangement not existing in nature.
  • reporter may refer to a polynucleotide sequence (structural sequence) encoding a reporter protein or the term may refer to the reporter protein itself, depending upon the context.
  • operably linked is intended to mean that a
  • DNA sequence is linked to a regulatory sequence in a manner which allows expression of the DNA sequence.
  • a regulatory sequence includes promoters, enhancers and other expression control elements.
  • polypeptide refers to a polymer of amino acid residues.
  • endogenous refers to that which is produced or arises from within a cell or organism.
  • Plasmid refers to a circular, double stranded, extrachromosomal bacterial DNA into which additional DNA segments may be ligated and which replicates automatically. Methodologies for selection and construction of vectors, plasmids and DNA constructs may be found, for example in: Molecular Cloning: A Laboratory Manual ; (2d), Sambrook et al . 1989, Cold Spring Harbor Laboratory Press. Suitable host cells are discussed further in Goeddel; "Gene Expression Technology” in: Methods in Enzymology 185, Academic Press, San Diego, California (1990) .
  • DNA constructs are introduced into a host cell and expressed in the host cell in sufficient quantities for a reporter gene to be activated.
  • the host cell may be any eukaryotic cell, including yeast, zebrafish, C. eleqans , Drosophila and mammalian cells having a genome one would like to screen for interactive protein encoding exons or AD encoding exons .
  • the host cell is constructed to contain and ultimately express a reporter gene having a transcription regulatory element known to include a binding site for the DNA-BD to be employed.
  • the reporter gene product produces a detectable signal when the reporter gene is transcriptionally activated.
  • a reporter is a moiety whose transcription is detectable, or which expresses a detectable protein or a protein the expression of which may otherwise be determined by monitoring an effect of - 16 -
  • reporter gene products that are readily detectable are well-known and include: /3-galactosidase, green fluorescent protein, luciferase, alkaline phosphatase, and chloramphenicol acetyl transferase (CAT) as well as other enzymes and proteins that are also known as selectable markers.
  • detectable signals include cell surface markers such as CD4.
  • the reporter gene used is the pac gene which encodes the puromycin resistance marker.
  • the reporter gene may be homologous the yeast URA3 gene, the yeast CAN1 gene, the yeast GAL1 gene, the yeast HIS3 gene, or the E. coli LacZ gene.
  • the reporter gene may be homologous to the CAT gene, the LacZ gene, the SEAP gene, the Luciferase gene, the GFP gene, the BFP gene, the CD2 gene, the Flu HA gene, or the tPA gene.
  • the reporter gene in the host cell will be driven by a transcriptional regulatory element (including promoters and enhancers) that is capable of binding the DNA-BD employed in the assay and is functional in the host cell .
  • a transcriptional regulatory element including promoters and enhancers
  • suitable regulatory elements are well- known, particularly, promoters including those described below.
  • the assay may make use of host cells in which the reporter gene has been previously incorporated, or a construct containing the reporter gene may be introduced to the cell at the same time as other vectors used in the assay.
  • vectors used in the assay include a gene trap vector and a two-hybrid vector.
  • the gene-trap vector is employed for random insertion of a transcriptional - 17 -
  • regulatory protein moiety into the genome of the host cell may comprise DNA encoding either a AD or a DNA-BD and either: an upstream splice acceptor (SA) ; or, an upstream transcriptional regulatory element (eg. a promoter) capable of functioning in the host cell for transcription of the downstream AD or DNA-BD which in turn is followed by an unpaired splice donor sequence (SD) .
  • SA upstream splice acceptor
  • SD unpaired splice donor sequence
  • the gene trap vector has both an upstream SA and a downstream SD.
  • incorporación of the gene trap vector within an intron will permit processing of a chimeric message comprising a transcript of a flanking endogenous exon joined to the transcript for the DNA-BD or AD.
  • Use of a gene trap vector having a downstream SD and an upstream promoter is preferred since transcription of the chimeric message will not be dependent upon endogenous expression of the host cell gene.
  • a splice donor is defined as a nucleotide moiety having an ability to effect m-RNA splicing to a splice acceptor site.
  • a splice acceptor is defined by its ability to effect mRNA splicing to a splice donor site.
  • an unpaired splice donor includes the 3' end of an exon and the 5' end of an intron
  • a splice acceptor includes the 3' end of an intron and the 5' end of an exon (eg. as defined by Alberts, B. et al . , at page 373 of Molecular Biology of the Cell (1994) , (3d) Garland Publishing, N.Y. Sequences that may be used as splice acceptors and donors are known and include the examples of SA and SD sequences as set out in the Examples herein.
  • the two-hybrid vector will comprise an upstream transcriptional regulatory element (eg. a promoter) capable of a functioning in the host cell and driving transcription - 18 -
  • an upstream transcriptional regulatory element eg. a promoter
  • the two-hybrid vector will express either a DNA-BD or a AD as the case may be, depending upon the makeup of the gene trap vector.
  • the two-hybrid vector will express DNA-BD.
  • the two-hybrid vector also contains a nucleotide sequence which is under the control of the regulatory element and which encodes a selected protein (including a peptide or a polypeptide) of interest (test protein) in respect of which protein-protein interactions are to be determined.
  • Expression of the two-hybrid vector in the host cell results in the translation of a chimeric protein comprising the transcriptional regulatory protein moiety (eg. DNA-BD) fused with the test protein.
  • a chimeric protein comprising the transcriptional regulatory protein moiety (eg. DNA-BD) fused with the test protein.
  • Incorporation of the gene trap vector into a gene encoding a protein capable of interaction with the selected protein will result in production in the cell of a reconstituted transcription regulatory protein via interaction of the test protein and the protein product of the trapped gene.
  • Activation of the reporter gene occurs as a result of binding of the DNA-BD to the reporter gene promoter.
  • interaction of proteins (such as an endogenous protein with a test protein) means any interaction whereby proteins tend to be associated in proximity. Such interaction includes any known form of chemical bonding occurring between proteins that are found to be interacting.
  • the gene trap vector comprising a DNA-BD is used without a two-hybrid vector.
  • the gene trap vector integrates into a gene containing an exon that encodes a protein capable of - 19 -
  • the resulting gene product is a chimeric protein that joins both the DNA-BD coded for by the vector DNA and the AD coded for by the endogenous exon.
  • a transcriptional regulatory protein is constituted, capable of activating the reporter gene in the cell.
  • a DNA-BD and a AD employed in DNA constructs for use in this invention may be derived from a single known transcriptional regulatory protein having separate DNA-binding and transcriptional activation domains (for example, the yeast GAL4 and GEN4 proteins) .
  • the DNA-BD and AD moieties may be derived from separate known sources.
  • the DNA-BD may be derived from LexA in E. coli .
  • the DNA-BD may be from DNA binding proteins other than activators (eg. repressers) .
  • the AD could be derived from amino acids 147-238 of GAL4.
  • the moieties may also be synthetic, such as the B42 activation domain.
  • the DNA-BD and the AD are from different proteins.
  • the DNA-BD should not be capable of functioning significantly as an activator domain on its own and the AD should not be capable of binding to the promoter of the reporter gene .
  • the DNA-binding domain is derived from the N-terminal region of the yeast GAL4 protein (eg. amino acids 1-147) and the transcriptional activation domain is derived from the transcriptional activator of Herpes Simplex Virus VP16 (eg. amino acids 411-455 of VP16) which is known not bind to DNA but will function as a transcriptional activator.
  • Herpes Simplex Virus VP16 eg. amino acids 411-455 of VP16
  • the reporter gene may be present in the genome of the host cell at the time of introduction of the first and/or second DNA constructs.
  • a construct comprising the reporter gene may be introduced into the - 20 -
  • host cell genome at the same time as the first and/or second DNA constructs.
  • further DNA constructs to be used in this invention may be introduced to the cell and made part of the host cell genome before further constructs are introduced, or such constructs may be introduced at the same time.
  • DNA constructs, plasmids and the like, as used in this invention can be delivered or placed in cells in vivo using methods known in the art and the methods referred to in the Examples herein. Such methods include direct injection of DNA, receptor-mediated DNA uptake or viral-mediated transfection. Direct injection has been used to introduce named DNA into cells in vivo (see eg. Acsadi et al . (1991) Nature 332:815-818; Wolff et al . (1990) Science 247:1465- 1468) .
  • a delivery apparatus eg. a "gene gun" for injecting DNA into cells in vivo can be used. Such an apparatus is commercially available (eg. from BioRad) .
  • Naked DNA can also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G. and Wu, CH. (1998) J. Biol. Chem. 263:14621; Wilson et al . (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320). Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis.
  • a cation such as polylysine
  • DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al . (1991) Proc. Natl.
  • Endogenous genes into which the gene trap vector has integrated may be cloned and sequenced, for example by the
  • undifferentiated embryonic stem (ES) cells can be further used to generate mice mutated from the endogenous gene .
  • Heterologous DNA can be inserted into the site of the endogenous gene by known methods including homologous recombination and site directed to recombination .
  • RACE 5' Rapid PCR amplification of cDNA ends
  • ES cell lines which may be used in this invention are: porcine (eg. U.S. Patent 5523226 Transgenic Swine Compositions and Methods); murine (eg. D3 , Rl, CGR8, AB1 ES cell lines) ; primate (eg. rhesus monkey) ; rodent; marmoset; avian (eg. chicken); bovine; rabbit; sheep; and horse .
  • Murine Rl ES cells from A. Nagy [Proc. Nat. Acad. Sci. U.S.A. (1993) 90, 8424-8428] may be grown on Primary Embryonic Fibroblast feeder layers or on gelatinized dishes in the presence of 1000 U/ml murine leukemia inhibitory factor (LIF) , ESGROTM (GIBCO BRL) . Selection conditions can be: 150 ⁇ g/ml G418, 1.0 ⁇ g/ml puromycin, 110 ⁇ g/ml Hygromycin B.
  • Rl cells eg. 2 x 10 7 cells
  • Rl cells may be electroporated with, for example, 100 ⁇ g linearized DNA in 0.8 ml PBS at 500 ⁇ F and 240 V with a BioRad Gene PulserTM at room temperature.
  • Mammalian MatchmakerTM two-hybrid assay kit is modified and supplemented to provide a reporter gene as a selectable marker (pac) for puromycin resistance; a DNA-BD from GAL4
  • the first DNA construct (two-hybrid vector) comprises a sequence encoding a GAL4 DNA-BD which recognizes a binding site on the reporter gene and further comprises a sequence encoding p53 protein (Clontech, pM-53 plasmid) .
  • the second DNA construct (gene trap vector) is novel and comprises a promoter capable of operation in the host cell, driving a VP16 AD upstream of a splice donor sequence.
  • the novel gene trap vector does not contain a promoter and has a splice acceptor sequence upstream of the VP16 AD followed by a poly-adenylation signal.
  • a transcriptional regulatory protein comprising GAL4 BD and the VP16 AD is constituted.
  • Expression of the reporter gene in a host cell as a result of binding by the DNA-BD is detected by culturing the transformed cells in the presence of puromycin. Cells in which the reporter gene has been activated will survive.
  • the reporter used in the assay could remain as CAT and determination of - 23 -
  • reporter gene activity may be carried out according to standard assay procedures, for example as taught in the Clontech kit instructions.
  • Host cells are transformed by any of the well-known methods, selected as being suitable for the particular cell type. Electroporation or calcium phosphate mediated transfection are suitable for mammalian cells . Transfection procedures as taught in the Clontech kit instructions may be used. A preferred method known for ES cells is electroporation.
  • the reporter is a modified version of the GAL4 responsive CAT reporter construct from the Clontech MatchmakerTM kit (pG5CAT) .
  • the CAT reporter gene is replaced by the selectable marker pac, generating a reporter construct containing the puromycin resistance gene under the control of the adenovirus Elb minimal promoter used in the Clontech plasmid.
  • Upstream are five copies of the 17 nucleotide consensus GAL4 binding site (galactose upstream activating sequence : UAS G ) .
  • the second plasmid is the pM-53 vector from the MatchmakerTM kit which is an expression plasmid containing the SV40 promoter driving a GAL4 DNA-BD.
  • the commercial construct encodes p53 protein, but the multiple cloning site downstream from the DNA-BD may be used to insert different bait proteins. This functions as the two-hybrid vector.
  • a gene trap vector plasmid is constructed by inserting an oligomer sequence encoding a consensus SD sequence in frame into a Sall/BspMI digested pVP16 plasmid (Clontech) simultaneously deleting the stop codons and - 24 -
  • a gene trap vector comprising an SV40 promoter driving expression of the AD.
  • Three versions of this vector were created resulting in splicing in all three potential reading frames .
  • AGGTAAGT SEQ ID N0:1
  • AGGTGAGT SEQ ID NO: 2
  • An alternate gene trap vector plasmid may be constructed containing the VP16 AD downstream of a SA sequence. Three constructs should be generated, each resulting in splicing in each of three possible reading frames .
  • SA sequences comprise a polypyri idine tract followed by a nucleotide, T or C, AG, and at least G or A. Examples are the murine En-2 splice acceptor and the splice acceptors from human 3-globin and rabbit b-globulin.
  • VP16 gene trap vectors The following methods may be used for construction of VP16 gene trap vectors :
  • Pair #1 5' tcgacaggtaagt 3' (SEQ ID NO : 3 ) 5' tcatacttacctg 3' (SEQ ID NO: 4)
  • Pair #2 5' tcgaccaggtaagt 3' (SEQ ID NO: 5) 5' tcatacttacctgg 3' (SEQ ID NO: 6)
  • Pair #3 5' tcgacccaggtaagt 3' (SEQ IDNO:7)
  • the three forms of the gene trap vector representing all three potential reading frames are placed in a head to tail tandem array allowing the use of alternate promoters to generate three hybrid mRNAs fusing the VP16 domain in all three possible reading frames to a adjacent exon upon integration into a gene within the host cell genome.
  • ES reporter murine embryonic stem
  • reporter cell lines Characterize the reporter cell lines for its ability to detect protein-protein interactions by electroporating with pVP16T (Clontech) as a positive control and pVP16-CP - 27 -
  • pVP16T expresses a fusion of the VP16 activation domain to the SV40 large T antigen, which is known to interact with p53.
  • the pVP16-CP negative control plasmid expresses a fusion of the VP16 activation domain to a viral coat protein, which does not interact with p53.
  • the methods employed in the preceding example are used in an assay employing the ES host cell, the same reporter gene construct (pG5CAT) employed in the preceding example, and a gene trap vector plasmid designed to trap genes expressing endogenous protein capable of functioning as a transcriptional activator domain (AD) in conjunction with the DNA-BD expressed by the gene trap - 28 -
  • pG5CAT reporter gene construct
  • AD transcriptional activator domain
  • DNA-BD fused to an endogenous protein capable of functioning as a AD will result in activation of the reporter gene which comprises a binding site for the DNA-BD.
  • the gene trap vector plasmid is constructed by inserting an oligomer sequence encoding the consensus SD sequence in frame into the Sall/BspMI digested pM plasmid (Clontech) resulting a vector comprising of the SV40 promoter driving the GAL4 DNA-binding domain linked to a SD sequence. Three versions of this vector are created resulting in splicing in each of the three potential reading frames, respectively.
  • a consensus splice donor sequence domain contains the following:
  • Pair #3 5' tcgacccaggtaagt 3' (SEQ ID NO: 7) 5' tcatacttacctggg 3' (SEQ ID NO: 8)
  • the three forms of the gene trap are then placed in a head-to-tail tandem array allowing the use of alternative promoters to generate three hybrid mRNAs fusing the GAL4 DNA domain in all three possible reading frames to the next endogenous exon upon integration into a gene within the genome .
  • ES reporter murine embryonic stem
  • pM3-VP16 (Clontech) as a positive control
  • pM-53 (Clontech) as a negative control for transcriptional activator domains.
  • pM3-VP16 expresses a fusion of the VP16 activation domain to the GAL4 DNA binding domain which is known transactivate the GAL4 responsive promoter in pG5Puro.
  • the pm-53 negative control plasmid expresses a fusion of the VP16 activation domain to p53, which does not transactivate the GAL4 responsive promoter in pG5Puro.

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Abstract

L'invention concerne des procédés dans lesquels on utilise une combinaison de méthodes, méthode de piégeage de gène et méthode à deux hybrides, pour identifier et caractériser des gènes inconnus en fonction des interactions protéines/protéines du produit génique, ou pour identifier et caractériser des gènes inconnus codant des domaines activateurs de transcription. L'interaction entre un domaine de protéine codé par un exon et une protéine connue, ou le fonctionnement du domaine codé par exon en tant que domaine activateur de transcription, sont détectés par reconstitution de l'activité d'un activateur de transcription. L'invention concerne également des vecteurs appropriés de piégeage de gènes.
EP99936098A 1998-02-25 1999-02-25 Detection de l'interaction de proteines et piegeage du facteur de transcription Withdrawn EP1062368A1 (fr)

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CA 2224475 CA2224475A1 (fr) 1998-02-25 1998-02-25 Collecteur des facteurs de transcription et d'interaction de proteine
PCT/CA1999/000173 WO1999043848A1 (fr) 1998-02-25 1999-02-25 Detection de l'interaction de proteines et piegeage du facteur de transcription

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US7807447B1 (en) 2000-08-25 2010-10-05 Merck Sharp & Dohme Corp. Compositions and methods for exon profiling
US6713257B2 (en) 2000-08-25 2004-03-30 Rosetta Inpharmatics Llc Gene discovery using microarrays
CA2446207A1 (fr) 2001-05-04 2002-11-14 Health Research, Inc. Dosage complet a haut rendement pour l'identification d'agents modificateurs d'expression genetique

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US5525490A (en) * 1994-03-29 1996-06-11 Onyx Pharmaceuticals, Inc. Reverse two-hybrid method
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