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WO1998046789A1 - Nouveaux vecteurs-reporter pour systemes a un hybride et a deux hybrides - Google Patents

Nouveaux vecteurs-reporter pour systemes a un hybride et a deux hybrides Download PDF

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Publication number
WO1998046789A1
WO1998046789A1 PCT/EP1998/002194 EP9802194W WO9846789A1 WO 1998046789 A1 WO1998046789 A1 WO 1998046789A1 EP 9802194 W EP9802194 W EP 9802194W WO 9846789 A1 WO9846789 A1 WO 9846789A1
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Prior art keywords
reporter
nucleic acid
acid sequence
cell
gene
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PCT/EP1998/002194
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German (de)
English (en)
Inventor
Robert Cormack
Imre Somssich
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO1998046789A1 publication Critical patent/WO1998046789A1/fr

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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

Definitions

  • the invention relates to a reporter vector which comprises a regulatory nucleic acid sequence which contains an operator and a promoter linked to a reporter gene which codes for a photoprotein, the expression of which can be detected by irradiation or treatment.
  • the invention further relates to a method for the detection of molecular interactions, transiently or stably transformed cells, and the use of these cells for the investigation of molecular interactions.
  • Reporter vectors are plasmids which, in addition to the components which are generally customary for a plasmid (origin of replication, selection markers, etc.) carry a reporter gene under the control of a promoter. Strong, constitutive promoters such as the CMV promoter (cytomegalovirus) or the Ca V 35S (cauliflower mosaic virus) are generally used as promoters for reporter vectors. Reporter genes should be easily detectable. For this reason, genes are used whose expression product is easily detectable by a chemical reaction that is easy to carry out, such as a color reaction. Examples of such reporter genes are the LacZ gene or the CAT gene (chloroamphenicol acetyltransferase). Another group of reporter genes are photoproteins, e.g. B. the GFP isolated from Aequorea victoria (Green Fluorescent
  • Such reporter vectors are used, for example, to check the transfection effectiveness in the transfection of cells.
  • _5 Reporter vectors are also used in other examination methods. They are adapted to the respective process by appropriate modifications.
  • molecular interactions are understood to mean an interaction between polypeptides and nucleic acids or the interaction of two or more polypeptides with one another. Either these interactions are investigated per se or one wants to identify molecules that can interact specifically with a certain nucleic acid sequence or with a certain polypeptide.
  • a reporter vector with the LacZ reporter gene described above is used and transformed into a yeast cell _5.
  • Two further expression vectors are transformed into the same cell, an interaction of the two expression products of these vectors leads to activation of the expression of the reporter gene of the reporter vector.
  • the expression is then by adding a chemical inducer, e.g. B. IPTG, made visible.
  • a chemical inducer e.g. B. IPTG
  • the detection of expression can also be verified using the filter assay.
  • An impression of the colonies is taken from the culture plate and the color reaction is carried out using an X-Gal solution (5-bromo-5 4-chloro-3-indolyl / 3-D-galactoside).
  • the colonies in question must be picked and inoculated in culture medium for quantitative detection.
  • the expression can only be quantified in a further process step after an appropriate culture time. 0
  • the determination of the expression level is therefore time and cost intensive. In addition, the number of colonies that can be tested in this way is limited.
  • the reporter vector according to the invention which comprises a regulatory nucleic acid sequence, o which contains an operator and a promoter, operatively linked to a reporter gene, which is characterized in that the reporter gene codes for a photoprotein, the expression of which can be detected by radiation.
  • the reporter vector according to the invention makes it possible to detect positive clones by irradiation, the background of the system surprisingly being very low in vivo and having a good signal / background ratio.
  • the great advantage lies in the fact that for the detection of expression of the reporter gene neither filter impressions have to be made nor that a chemical reaction has to be carried out in a further working step.
  • the colonies can now be applied more densely to the culture plates, so that fewer culture plates are necessary and 5 a larger number of colonies can be screened simultaneously in one work step.
  • Another advantage is that the reporter vector according to the invention the use of expensive chemicals for the detection of positive clones is eliminated. This saves materials and expensive working hours. Above all, however, the working time required to identify positive clones is reduced.
  • the reporter vector according to the invention can in principle be any vector, e.g. B. an extrachromosomal or chromosomal vector.
  • the vector can be a viral vector, a plasmid or an artificial chromosome.
  • the reporter vector is preferably a plasmid. It expediently contains the components required for propagation in the intended host cell. They include an origin of replication e.g. B. a bacterial origin of replication such as Col E 1 or p5A or a eukaryotic origin of replication such as the yeast 2 micron origin of replication.
  • the reporter vector can contain one or more selection markers such as ampicillin, penicillin, tetracycline for selection in bacterial cells and / or selection markers which enable selection in yeast cells, such as the gene for Ura3, Leu2, Lys2, Trpl, Cys3 or Ade2 . These selection markers are used in conjunction with corresponding auxotrophic yeast strains and suitable yeast culture media. The cultural media are also referred to as "dropout" media. By using them, the number of 5 false positive clones can be reduced.
  • the reporter vector contains a Transkriptionstermiationssequenz such as a ADHL sequence. It was surprising that the reporter vector according to the invention showed no basal expression of the reporter gene after transfection in the cell. This advantageously leads to a very good signal / background ratio when used. In addition, the signal strength upon irradiation correlates with the expression level and thus also allows conclusions to be drawn about the strength of the molecular interaction. 5
  • Any suitable promoter that is operatively linked to the reporter gene can be used as the promoter in the reporter vector. is knotting.
  • the reporter vector preferably contains the Gall, 10 minimal promoter.
  • the operator can contain further different sequence motifs, preferably comprising at least one Gal4 binding motif and / or at least one LexA binding motif.
  • the photoprotein is preferably a GFP (Green Fluorescent Protein) from the organism Aequorea victoria or a modified GFP (see, for example, WO 95/07463; WO 95/21191; WO 96/23810; WO 96/23910 or WO 96 / 23898).
  • GFP Green Fluorescent Protein
  • the reporter vector may also have unique cloning sites.
  • singular cloning site is meant that a restriction enzyme can only cut at one point in the reporter vector.
  • the singular cloning site is preferably located 5 'to the side of the ATG start codon of the reporter gene.
  • the distance between the ATG and the singular cloning site is preferably 50 to 4,000 bp.
  • the singular cloning site can be an interface for any restriction enzyme, preferably the singular cloning site contains 5 sites for restriction enzymes with a 6 to 8 bp recognition sequence, e.g. B. Notl, Nrul and / or Spei.
  • a heterologous nucleic acid fragment is inserted in the singular cloning site. This can vary in size, preferably it has a size of 20 bp to 4 kbp.
  • Another object of the invention is a cell that is transiently or stably transformed with a reporter vector as described above.
  • Yet another object of the invention is a method for the detection of molecular interactions between a nucleic acid and a polypeptide, wherein a reporter vector into which a first heterologous nucleic acid sequence is inserted is transformed into a cell, at least a second vector is transformed into this cell, which contains a second heterologous nucleic acid sequence under the control of a promoter, the transformed cell is cultivated under conditions such that expression of the second nucleic acid sequence can take place and the expression of the reporter gene is examined in the cell, the increase in expression of the reporter gene resulting in An interaction between the expression product of the second nucleic acid sequence and the first nucleic acid sequence can be detected on the reporter vector.
  • the vectors used in the process which is referred to as a one-hybrid process, are transformed into the cells by processes known to those skilled in the art.
  • a selection with suitable selection methods is carried out if necessary.
  • auxotrophic yeast strains in connection with suitable media and corresponding genes, which are contained in the transformed vectors.
  • the reporter vector and the second vector can be transformed into the cell simultaneously or in succession.
  • the vectors can be transiently or stably transformed into the cell. It is also possible to first transform the reporter vector into the cell and to produce stably transformed cells. In a second transformation, these cells are then transformed transiently or stably with the second vector to investigate the molecular interaction.
  • first heterologous nucleic acid sequence in the reporter vector, whose interaction with poly- peptides to be examined.
  • the method can be used to isolate nucleic acid sequences which code for polypeptides which can interact with a first given heterologous nucleic acid sequence which is inserted in the reporter vector.
  • the second heterologous nucleotide sequence from a sequence library which contains a large number of different, potentially interacting sequences, e.g. B. from a genomic library or a cDNA library.
  • the first heterologous nucleic acid sequence preferably comes from a genomic or cDNA library.
  • the present method offers the advantage that a large number of clones and different gene banks can be examined for the presence of expression products which interact with a specific nucleic acid sequence.
  • nucleic acid sequences that can interact with the given polypeptide can also be identified quickly and easily for a given expression product.
  • the invention relates to a cell that is transiently or stably transformed with a reporter vector as shown above and / or at least one second vector as shown above.
  • Cell 5 is preferably a yeast cell.
  • Another object of the invention is a method for the detection of molecular interactions between two polypeptides, wherein a reporter vector, which optionally contains an o heterologous nucleic acid sequence, is transformed into a cell, at least a second vector is transformed into this cell, which contains a fusion gene from a first heterologous nucleic acid sequence and a nucleic acid sequence which codes for a binding domain of the operator on the reporter vector 5, under the control of a promoter, at least one third vector is transformed into this cell, which contains a fusion gene from a second heterologous nucleic acid sequence and a nucleic acid sequence which is suitable for encodes a transcription activator of the reporter gene, contains under control o a promoter, the transformed cell is cultivated under such conditions that expression of the heterologous nucleic acid sequences can take place and expression of the reporter gene is examined in the cell, the occurrence of an interaction between the expression product of the first nucleic acid sequence and the expression product of the second nucleic acid sequence being detectable by an increase
  • the first 5 or second heterologous nucleic acid sequence preferably comes from a gene bank.
  • the method is carried out in cells compatible with the vectors used.
  • a yeast cell is preferably used. le.
  • yeast strains and selection markers and suitable culture media PL Bartel et al. , (Cellular Interactions in Development-A Practical Approach, Ed. DA Hartley IRL Press, 1993), 5 which is hereby incorporated into this application.
  • the second vector contains a fusion gene that contains a binding domain.
  • This binding domain can bind to a sequence of the operator of the reporter vector.
  • the binding domain preferably comprises a LexA sequence.
  • the third vector used in this method contains a fusion gene that encodes a transcriptional activator of the reporter gene.
  • This activator can be any suitable activator, preferably it comprises a B42, GAL4 sequence and / or a VP16 (Herpex-Simplex) sequence.
  • one or both interaction partners can be composed of two or more subunits.
  • the individual subunits can be encoded by different vectors or their coding sequences can be present together on one vector.
  • the cells are irradiated, which leads to a color signal.
  • the radiation preferably comprises a wavelength of 280 nm to 400 nm. Most preferably it comprises 350 nm to 375 nm. 0
  • the invention relates to a cell which is transiently or stably transformed with a reporter vector as described above and / or with at least one second vector as described above or / and 5 and with at least a third vector as described above.
  • Another object of the invention is the use of a transiently or stably transformed cell as described above, for the investigation of molecular interactions.
  • FIG. 1 The examples together with FIG. 1 describe the invention in more detail.
  • a BamHI fragment containing the LTA gene from pTDI was subcloned in the same orientation into the unique restriction site BamHI from pJG4-5.
  • the recombinant plasmid was linearized with EcoRI, 5 then filled in with T4 DNA polymerase and recircularized with T4 DNA ligase to put the LTA gene "in-frame”.
  • the reporter vectors pGNGl and pGNG2 are shown in FIG. 1.
  • the minimal Gall, 10 promoter sequence in pSH18-34 (2) contains 4 copies of the LexA binding site. It was amplified using the polymerase chain reaction (4).
  • the first primer was positioned 2 base pairs 5 'on the 5' side of the LexA operator 5 and provided with a HindIII restriction site at its 5 'end.
  • the second primer was complementary to the Gall, 10 sequence up to the ATG start codon for the LacZ gene, without containing it and it contained an Xbal restriction site at its 5 'end.
  • the PCR product was subcloned into the plasmid pGFPuv (Clontech, Palo Alto, California, 5 USA) between the unique restriction sites HindiII and Xbal. From this construct, a HindiII-EcoRI DNA fragment containing the minimal promoter-GFP fusion gene was subcloned into the yeast shuttle vector YEp357R (5) between the HindiII and EcoRI restriction sites of the "Multiple Cloning Site" to give the vector pPGNGl .
  • the complementary deoxyribonucleotides MCSl (5 '-AGCTTGCGGCCGCTCGCGAC-TAGT and MCS2 (5' -AGCTACTAGTCGCGAGCGGCGCA) with compatible Hindlll ends were hybridized and then into the unique restriction site HindiII from pPGNG25 bp EcoRI - 5 XmnI fragment, which contains the ADH1 terminator from pGAD424 (6), cloned into the 6 kbp EcoRI -PvuII fragment of pPGNGl or pPGNG2 in order to give the reprotector vectors pGNGl or pGNG2.
  • the S.cerevisiae strain EGY48 was transformed with the lithium acetate method (7) with pGNGl and on synthetic drop-out uracil (SD-Ura) plates containing 2% (w / v) galactose and 1% (w / v) raffinose contained, selected.
  • the resulting strain 5 was then transformed with different "bait" and pJG4-5-based "prey" plasmids based on pEG202 and selected on a suitable SD medium containing galactose and raffinose. Culture plates containing yeast transformants were examined under a UV lamp (366 nm).
  • a reporter vector for the two-hybrid system (interaction trap), pGNGl (FIG. 1) was produced, which contains the GFP gene (Green Fluorescent Protein).
  • the vector backbone was derived from YEp357R, which contains the URA3 selection marker and a 2 micron origin of replication.
  • the GFPuv gene which was used in the vector, is a "cycle3" variant of the GFP, which is 18 times brighter than the wild-type protein (8).
  • the reporter vector for the one hybrid system pGNG2 (Fig. 1), was constructed. This contains approximately 450 bp on the 5 'side of the ATG start codon of the GFPuv gene, a "multiple cloning site".
  • the reporter vector pGNG2 contains the unique cloning sites Notl, Nrul and Spei. A "bait" DNA sequence can be inserted here for examination in the one-hybrid system.
  • yeast cells containing pGNGl and "bait" vectors showed that capable were able to activate the LacZ reporter gene encoded by pESH18-34, also the ability to activate the GFPuv gene.
  • yeast cells that expressed "Baitl” fluoresced significantly more under UV light than those that expressed "Bait2". This indicates that the transactivation of the "Baitl” was stronger than that of the "Bait2".
  • Yeast cells were also co-transformed with vectors containing a p53-LexA binding domain hybrid protein and the Large-T antigen
  • the reporter vector pGNGl shows no detectable basal expression of the GFP in the absence of the "Bait” and "Prey” vectors. However, in the presence of molecular interactions kung or transactivating "bait" proteins expressed enough GFP in the yeast cells to allow their detection.
  • the sensitivity of the reporter vector according to the invention is lower than that of the known LacZ reporter vectors, 5 which are regulated by the same minimal promoter.
  • the interaction between p53 and the Large-T antigen and the other three protein pairs (Bait and Prey) already detected with other reporter genes was also detected with the aid of the reporter vector according to the invention.
  • the lower sensitivity compared to the known reporter vectors represents an advantage, since it can reduce the number of false positive clones.
  • the variability in the intensity of the fluorescence for each pair of the proteins tested represents a qualitative measurement of the strength of the 5 interactions. This was observed in the same way with the other reporter genes.
  • a practical advantage of the reporter vector according to the invention over the known reporter vectors in the process of the one- and two-hybrid system is the fact that the colonies obtained can be held directly under a UV lamp (366 nm) and the positive clones immediately can be made visible without cultivating them again. The steps of picking the colonies, testing the activity or cultivating on culture plates containing X-Gal, or cultivating again for quantitative detection, are thus no longer necessary in the previously used reporter vectors.
  • the reporter vector pGNG2 for the one-hybrid system also showed no basal expression of GFP in yeast cells. Reporter genes co-transformed with different "bait” and "prey” vectors in S.cerevisiae EGY48

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Abstract

La présente invention porte sur un vecteur-reporter comprenant une séquence régulatrice d'acide nucléique, laquelle contient un opérateur et un promoteur en liaison fonctionnelle avec un gène-reporter codant pour une photoprotéine dont l'expression peut être mise en évidence par irradiation. L'invention concerne en outre un procédé de mise en évidence des interactions moléculaires, des cellules transformées de manière transitoire ou stable, ainsi que l'utilisation de ces cellules aux fins de l'examen desdites interactions moléculaires.
PCT/EP1998/002194 1997-04-15 1998-04-15 Nouveaux vecteurs-reporter pour systemes a un hybride et a deux hybrides WO1998046789A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19715683A DE19715683C2 (de) 1997-04-15 1997-04-15 Neue Reportervektoren für One-Hybrid und Two-Hybrid Systeme
DE19715683.5 1997-04-15

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WO1998046789A1 true WO1998046789A1 (fr) 1998-10-22

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283173A (en) * 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
WO1995007463A1 (fr) * 1993-09-10 1995-03-16 The Trustees Of Columbia University In The City Of New York Methodes d'utilisation de proteines fluorescentes vertes
WO1995021191A1 (fr) * 1994-02-04 1995-08-10 William Ward Indicateur bioluminescent fonde sur l'expression d'un gene codant pour une proteine modifiee a fluorescence verte
WO1995034646A1 (fr) * 1994-06-14 1995-12-21 American Cyanamid Company Nouveaux systemes cellulaires ayant une interaction specifique des paires de liaisons de peptides
WO1996023810A1 (fr) * 1994-11-10 1996-08-08 The Regents Of The University Of California Proteines fluorescentes vertes modifiees

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5283173A (en) * 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
WO1995007463A1 (fr) * 1993-09-10 1995-03-16 The Trustees Of Columbia University In The City Of New York Methodes d'utilisation de proteines fluorescentes vertes
WO1995021191A1 (fr) * 1994-02-04 1995-08-10 William Ward Indicateur bioluminescent fonde sur l'expression d'un gene codant pour une proteine modifiee a fluorescence verte
WO1995034646A1 (fr) * 1994-06-14 1995-12-21 American Cyanamid Company Nouveaux systemes cellulaires ayant une interaction specifique des paires de liaisons de peptides
WO1996023810A1 (fr) * 1994-11-10 1996-08-08 The Regents Of The University Of California Proteines fluorescentes vertes modifiees

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALLEN J B ET AL: "FINDING PROSPECTIVE PARTNERS IN THE LIBRARY: THE TWO-HYBRID SYSTEM AND PHAGE DISPLAY FIND A MATCH", TIBS TRENDS IN BIOCHEMICAL SCIENCES, vol. 20, December 1995 (1995-12-01), pages 511 - 516, XP000198723 *
CHALFIE M ET AL: "GREEN FLUORESCENT PROTEIN AS A MARKER FOR GENE EXPRESSION", SCIENCE, vol. 263, 11 February 1994 (1994-02-11), pages 802 - 805, XP002003599 *
CORMACK ET AL.: "ISOLATION OF PUTATIVE PLANT TRANSCRIPTIONAL COACTIVATORS USING A MODIFIED TWO-HYBRID SYSTEM INCORPORATING A GFP REPORTER GENE", PLANT, vol. 14, no. 6, 1998, pages 685 - 692, XP002076526 *
HAWLEY-NELSON P ET AL: "RAPID, HIGH LEVEL DETECTION OF MAMMALIAN CELL TRANSFECTION WITH PGREEN LATERN-1, A HUMANIZED MUTANT GFP REPORTER VECTOR", FASEB JOURNAL, vol. 10, no. 6, 2 June 1996 (1996-06-02), XP002030425 *
KAIN S R ET AL: "GREEN FLUORESCENT PROTEIN AS A REPORTER OF GENE EXPRESSION AND PROTEIN LOCALIZATION", BIOTECHNIQUES, vol. 19, no. 4, October 1995 (1995-10-01), pages 650 - 655, XP002033687 *

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DE19715683C2 (de) 1999-11-11
DE19715683A1 (de) 1998-12-24

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