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WO2002031165A1 - Procede de construction par recombinaison d'une masse d'adn dans laquelle a ete transferee une mutation par deletion et procede d'identification d'une region de liaison de proteines - Google Patents

Procede de construction par recombinaison d'une masse d'adn dans laquelle a ete transferee une mutation par deletion et procede d'identification d'une region de liaison de proteines Download PDF

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WO2002031165A1
WO2002031165A1 PCT/JP2001/007285 JP0107285W WO0231165A1 WO 2002031165 A1 WO2002031165 A1 WO 2002031165A1 JP 0107285 W JP0107285 W JP 0107285W WO 0231165 A1 WO0231165 A1 WO 0231165A1
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dna
yeast
protein
producing
deletion mutation
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Japanese (ja)
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Takashi Ito
Kazuhisa Ota
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Todai TLO Ltd
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Todai TLO Ltd
Center for Advanced Science and Technology Incubation Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids

Definitions

  • the present invention relates to a method for preparing a DNA population into which a deletion mutation has been introduced stepwise from the 5 ′ end or 3 ′ end, a DNA population produced by the method, a yeast for use in the method, and The present invention relates to a method for identifying a binding region of a protein to another protein.
  • Protein-protein interactions are fundamental to various life phenomena, and their analysis is an important tool for understanding mechanism. It is important to identify the small area involved in the interaction as a mochi loaf j jet in order to perform higher order mochi loaf later.
  • the binding ability of each 0EF product fragmented by restriction enzyme digestion or PCR is examined.
  • the selection of the cutting point is arbitrary and the number thereof is practically limited, and accurate identification of the minimum binding region by this method is inefficient. And unrealistic.
  • protein folding often has a large effect even with a single breakpoint residue, so a method that can easily create many breakpoints is desired in molecular biology and especially in structural biology research. I have.
  • comprehensive analysis of protein-protein interactions has begun. However, streamlining is difficult with the above methods, which require individual strategies to be studied for each target protein.
  • An object of the present invention is to provide a means for preparing a DNA population into which a deletion mutation has been introduced stepwise from one direction, and to improve the efficiency of protein functional analysis.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, have found that the use of homologous recombination makes it possible to easily prepare a DNA population into which a deletion mutation has been introduced stepwise from one direction.
  • the invention has been completed.
  • the present invention relates to a method for producing a DN aggregate into which a deletion mutation has been introduced stepwise from the 5 ′ end, which comprises the following steps: .
  • the present invention also provides a method for preparing a DNA population into which a deletion mutation has been introduced stepwise from the 3 ′ end, comprising the following steps.
  • the present invention is a method for producing a DN ⁇ group into which a deletion mutation has been introduced stepwise from the 5 'end, which comprises the following steps.
  • 3) a step of preparing a mixture of a DNA fragment consisting of a sequence homologous to the other terminal region of the linearized vector and an arbitrary sequence located on the 3 ′ side of the sequence, 4) a step of introducing a mixture of the linear vector obtained in step 1), the DNA fragment obtained in step 2), and the DNA fragment obtained in step 3) into a microorganism to cause homologous recombination. .
  • the present invention relates to a method for preparing a DNA population into which a deletion mutation has been introduced stepwise from the 3 ′ end, which comprises the following steps.
  • step 4) a step of introducing a mixture of the linear vector obtained in step 1), the DNA fragment obtained in step 2), and the DNA fragment obtained in step 3) into a microorganism to cause homologous recombination.
  • the present invention also provides a DNA assembly prepared by using any one of the above-described methods for producing a DNA group.
  • the present invention provides a yeast for use in any one of the above-described methods for producing a DNA population, which comprises a region involved in self-ligation of linearized DNA in a genome of the yeast.
  • the above-described yeast wherein a region involved in auxotrophy of the yeast has been deleted.
  • the present invention is a method for identifying a binding region of a certain protein to another protein, the method comprising the following steps:
  • the DNA used in the present invention is a double-stranded double strand, and since the respective DNAs constituting the strand are symmetrical to each other, which end is the 5 ′ end or the 3 ′ end)
  • the person skilled in the art will appropriately determine: However, in the present specification, the terms “5 ′ end” or “3, end” are used for convenience in describing the method of the present invention more clearly. Please be careful. However, single-stranded DNA (primer sequence) is used in the orientation (5 'end or 3' end) as described in this specification.
  • the method for producing a fiber group of the present invention comprises the following steps 1) to 3). Is what you do.
  • Stepwise means that the lengths of the regions deleted by deletion mutation in each DNA in the DNA population differ from each other by at least one base.
  • a linearized vector containing a DNA into which a deletion mutation is to be introduced and having a portion near the 5 ′ end of the DNA truncated is prepared.
  • the DNA into which the deletion mutation is to be introduced is not particularly limited, and may be a DNA encoding a protein or a DNA not encoding a protein.
  • DNA encoding proteins that interact with other proteins DNA encoding enzymes, promoters, enhancers, silencers, terminators, Insley Yuichi, centromeres, telomeres, replication origins, etc.
  • the DNA into which the deletion mutation is to be introduced encodes a protein
  • the protein may be expressed as a fusion protein with another protein.
  • the cleavage site in the vector is not particularly limited as long as homologous recombination described below can occur. No, but it is preferable that the DNA to introduce the deletion mutation is located at the 5 'end of the DNA (! ⁇ 50 bases upstream.
  • the vector can be cut using a restriction enzyme that recognizes rare cut ter sites. .
  • step 2) a mixture of a DNA fragment consisting of a sequence homologous to the upstream region of the cleavage site of the linearized vector and an arbitrary sequence located on the 3 ′ side of the sequence is prepared.
  • the length of the sequence homologous to the upstream region of the cleavage site of the linearization vector is not particularly limited as long as it is within a range where homologous recombination described below can occur, but is preferably about 40 to 1000 bases.
  • Nx any sequence can be denoted as "Nx".
  • N represents any one of bases selected from A, G, C or T
  • X represents any natural number. Therefore, it indicates that X bases represented by Nx ⁇ N are arranged in a sequence. However, when X is 2 or more (ie, N is 2 or more), even if the bases indicated by each N are the same as each other, It may be different.
  • the length of this arbitrary sequence (that is, X) is not particularly limited as long as the homologous recombination can occur, but is preferably about 1 to 25 bases, and more preferably about 8 to 25 bases.
  • DNA fragment as used herein comprises two sequences: a sequence homologous to the upstream region of the cleavage site of the linear primer, and an arbitrary sequence located on the 3 'side of the homologous sequence. Means fragment.
  • mixture refers to a mixture of the DNA fragments. Since any sequence in the DNA fragment is a sequence (Nx) that can have various base compositions, the mixture includes DNA fragments having various base sequences.
  • the number of DNA fragments constituting the mixture is most preferably 4 ⁇ (X is the number of bases in an arbitrary sequence), but if a DNA population into which deletion mutations are introduced stepwise can be prepared, It may be less.
  • step 3 the mixture of the linearized vector and the DNA fragment is introduced into a microorganism to cause homologous recombination.
  • the introduction of the mixture of the linearized vector and the DNA fragment into a microorganism may be performed by a general method used for DNA introduction, for example, according to a lithium acetate method, a spheroplast method, an electoral portation method, or the like. be able to.
  • yeast is preferably used because of its high homologous recombination ability.
  • yeast include bacteria such as Saccharomyces cerevisiae and Schizosaccharomyces pombe.
  • Strains for example, PJ69-2A strain, PJ69-4A strain, AH109 strain, Y190 strain, CG1945 strain, HF7c strain, L40 strain can be used.
  • a yeast in which a region involved in self-ligation of linearized DNA and a region involved in auxotrophy of the yeast are deleted from the yeast genome is used.
  • the region involved in the self-ligation of the linearized DNA that is, the linearized vector
  • the full-length DNA DNA having no deletion mutation
  • the possibility that the requirement of the yeast is restored independently of the vector introduced into the yeast can be reduced.
  • yeast strains from which such a region has been deleted include yeast strain PJ69-2 - ⁇ 1 ig4 ⁇ leu2 prepared by the present inventors.
  • the yeast was prepared by adding 114 4 (3 ⁇ 4005 (: 337343 to 334509 region of chromosome 15 and leu2: YCL018W: 90915 to 92518 region of chromosome 3) from the yeast PJ69-2A genome to Ulrich Guldener et al. Nucleic Acids Research, 1996 Vol. 24 No. 13: 2519-2524), which was prepared by sequential deletion using the method of efficient gene disruption cassette for repeated use in budding yeast.
  • microorganisms other than yeast as described above can be used as long as they can cause homologous recombination between the introduced linearized vector and the mixture of DNA fragments.
  • E. coli or the like may be used.
  • it is preferable to perform a treatment such as supplying an excess amount of a recombinase to improve the recombining ability.
  • a DN group into which a deletion mutation has been introduced stepwise from the 5 'end side can be prepared.
  • Such a DN fiber group can be used to identify the original DNA (lean in which no deletion mutation has been introduced) N A) or the functional region of the protein encoded by it.
  • step 1) the cleavage site of the linear vector is changed from near the 5 'end to near the 3' end (more specifically, at a position about 0 to 50 bases downstream from the 3 'end).
  • step 2) a sequence homologous to the upstream region at the cut f ⁇ position of the linearized vector is changed to a sequence homologous to the downstream region at the cut 15 position.
  • the length of the sequence homologous to the linearization vector This is the same as the method for preparing a DNA population described above.
  • step 2) an arbitrary sequence is placed on the 5 'side, not on the 3' side, of a sequence homologous to the linear vector.
  • the length of the arbitrary sequence is the same as the above-described method for preparing a DNA population.
  • Gal4 AD Gal4 transversion H-activation domain
  • 0RF the DNA into which the deletion mutation is to be introduced
  • «ADH1 terminator is located downstream. Yuichi is arranged (“ ⁇ ” in the figure), and these three DNA regions are contained in one vector (top of Figure 1).
  • the vector is cut near the boundary between 0RF and Gal4 AD, and consists of a sequence homologous to Gal4 AD and an arbitrary sequence ("Nx" in the figure).
  • Nx an arbitrary sequence
  • the vector is cut near the boundary between 0RF and Gal4 AD, and consists of a sequence homologous to Gal4 AD and an arbitrary sequence ("Nx" in the figure).
  • homologous recombination occurs not only between Gal4 ADs but also between the NRF and the 5'-terminal region of 0RF.
  • Each sequence constituting Nx causes homologous recombination at a sequence portion having a high affinity in the region on the 5 'end side of the 0RF. Therefore, homologous recombination occurs at various positions at the 5 'end of 0EF, and 0EF causes deletions of different lengths in each yeast (FIG. 1, lower left).
  • the vector when a deletion mutation is to be introduced at the 3 'end, the vector is cleaved near the boundary between 0EF and T and consists of a sequence homologous to the T-dish and an arbitrary sequence ("Nx" in the figure). Make a mixture of DNA fragments ( Figure 1, right center).
  • Figure 1, right center When the mixture of the linear vector and the DNA fragment is introduced into yeast, homologous recombination occurs in the same manner as described above, and 0RF is deleted in different lengths for each yeast (FIG. 1, lower right).
  • the yeast for use in the method for producing a DNA population according to the present invention includes a region involved in self-ligation of linearized DNA and a region involved in auxotrophy of the yeast in the genome of the yeast. It is characterized by being deleted.
  • yeast strain PJ69-2AAlg4Aleu2 produced by the present inventors.
  • the method for producing the yeast strain and the effect of using the yeast are as described in the method for producing a DNA population. 3. DNA population preparation method (2)
  • Another method for preparing the DNA population of the present invention that is, another method (2) for preparing a DN fiber into which a deletion mutation has been introduced stepwise from the 5 ′ end side, is as follows (1) to 4) It is characterized by including the steps of:
  • a linear vector is prepared.
  • the vector to be linearized is not particularly limited as long as it is a vector generally used in the two-hybrid method.
  • a pGAD series pGAD-C1 or the like
  • This vector was reported by James et al. In Genetics, 144, 1425-1436. The region around the leu2 gene described above was determined with reference to the sequence contained in this vector.
  • Linearization of the vector can be performed by cutting the vector using a restriction enzyme known to those skilled in the art.
  • the restriction enzyme is not particularly limited as long as it has a recognition site in the multicloning site of the vector to be used.
  • 3 ⁇ 4coRI and Pstl can be used.
  • These restriction enzymes may be used alone or in combination of two or more.
  • two or more types of restriction enzymes for example, TEcoRI and: Ps11
  • two or more types of restriction enzymes for example, TEcoRI and: Ps11
  • step 2) a DNA fragment is prepared in which a sequence homologous to one end of the linearized vector is added to the 3 ′ side of the DNA into which a deletion mutation is to be introduced.
  • DNAJ into which a deletion mutation is to be introduced and“ homologous ” have the same meaning as described in the method for producing a DNA population (1) of the present invention.
  • the length of the sequence homologous to the one terminal region of the linearized vector is not particularly limited as long as it is within a range in which homologous recombination can occur, but is preferably about 40 to 100 bases.
  • the DNA fragment is prepared by converting the DNA into which the deletion mutation is to be introduced into a type II, using a DNA-specific primer usually used for the DNA, and one end of the linearized vector. This can be carried out by performing PCR using a combination of a primer containing a sequence homologous to the DNA and a specific sequence with the DNA. In the latter primer, a sequence homologous to a region at one end of the linearized vector is used. Is to introduce a deletion mutation into the DNA from the 5 'end, and vice versa if introducing a deletion mutation into the DNA from the 3' end. Place at the end.
  • the sequence of the former (DNA-specific primer) is STE5-N: 5'-ATGATGGAAACTCCTACAGAC-3 '(SEQ ID NO: 3),
  • the latter sequence is ST E5-C + 40: 5'-GGGGTTTTTCAGTATCTACGATTCATAGATCTCTGCAGGT + CTATATATAATCCATATGGAG-3 '
  • step 3 a mixture of a DNA fragment consisting of a sequence homologous to the other terminal region of the linearized vector and an arbitrary sequence located on the 3 ′ side of the sequence is prepared.
  • the length of the sequence homologous to the other terminal region of the linearized vector is not particularly limited as long as it is within a range where homologous recombination described below can occur, but is preferably about 40 to 100 bases.
  • the number of DNA fragments constituting the mixture is most preferably (X is the number of bases in an arbitrary sequence), but if a DNA population into which deletion mutations are introduced stepwise can be prepared, It may be less.
  • step 4 a mixture of the linearized vector obtained in step 1), the DNA fragment obtained in step 2), and the DNA fragment obtained in step 3) is introduced into a microorganism. causes homologous recombination. ⁇
  • a DN group into which a deletion mutation has been introduced stepwise from the 5 'end side can be prepared.
  • Such a D group can be used for specifying the original DNA (NA without deletion mutation) or the functional region of the protein encoded by it.
  • the method (2) for producing a DNA population of the present invention has the following advantages as compared with the above-mentioned method (1) for DN fiber.
  • the method for preparing the DN fiber group (1) it is necessary to subcut the DNA into which the deletion mutation is to be introduced in a dedicated vector beforehand. At this time, operations such as ⁇ PCR, restriction enzyme treatment, ligation, E. coli transformation, selection of positive clones, and sequence confirmation by sequence are required.
  • the fragment containing the DNA into which the deletion mutation is to be introduced and the linearized vector can be directly used for recombination. Can be omitted.
  • a DNA population into which a deletion mutation has been introduced stepwise from the 3 ′ end side can be prepared.
  • step 2) the sequence homologous to the region at one end of the linearization vector is changed to a sequence homologous to the region at the other end of the linearization vector.
  • the length of the sequence homologous to the other terminal region of the linearized vector is the same as in the above-described method for preparing a DNA population (2).
  • step 3 the position of an arbitrary sequence is set to the 5 'side of the sequence homologous to the other terminal region of the linearized vector, not to the 3' side.
  • the length of the arbitrary sequence is the same as in the above-described method (2) for preparing a DM population.
  • FIG. 5 shows a schematic diagram of the method (2) for preparing a DNA population of the present invention.
  • a template for nucleotide sequence determination can be prepared.
  • the yeast for use in the method for producing a DNA population according to item 3 of the present invention includes a region involved in the self-ligation of linearized DNA in the genome of the yeast and an auxotrophy of the yeast. It is characterized in that the region has been deleted.
  • examples of such yeast include yeast strain PJ69-2AAlg4Aleu2 produced by the present inventors.
  • the method for producing the yeast strain and the effect of using the yeast are as described in the method for producing a DNA population.
  • the method for identifying a binding region of a protein of the present invention comprises the following steps 1) to 6).
  • step 1) a group of expression vectors containing a DNA encoding the protein A and a DNA comprising the DNA encoding the protein in which the deletion mutation has been introduced stepwise from the 3 ′ end or the 5 ′ end. Is prepared.
  • Protein A is a protein that activates gene transcription by forming a complex with the key protein B.
  • a protein generally used in a two-hybrid method can be used. Specifically, one of the Gal4 transmutation domain and the Gal4 DNA binding domain, a protein used in the MATCHMAKER LexA Two-Hybrid System (Clontech), and a protein used in the CytoTrap two-hybrid system (Stratagene) And the like.
  • the stepwise deletion mutation can be introduced according to the method for preparing a DNA population described above, but may be performed by another method. Encode protein A in the vector! ) In addition to M and the DNA encoding the relevant protein, insert a promoter or terminator for expressing them as necessary.
  • an expression vector containing a DNA consisting of a DNA encoding the protein B and a DNA encoding the other protein is prepared.
  • Protein B is a protein that activates gene transcription by forming a complex with protein A described above.
  • a protein generally used in a two-hybrid method can be used.
  • a promoter for expression of these and an evening initiator, if necessary, may be used. Insert it.
  • a yeast having a repo overnight gene whose transcription is activated by a complex of protein A and protein B was prepared in one of the expression vector groups prepared in 1) and in 2). Introduce an expression vector.
  • yeast those commonly used in the two-hybrid method may be used.
  • PJ69-2A strain, PJ69-4A strain, AH109 strain, Y190 strain, CG1945 strain, HF7c strain, L40 strain, etc. may be used.
  • ADE2, HIS3, U3, LEU2 and the like can be used as the reporter gene.
  • the transcriptional activation of the reporter gene by the complex of protein A and protein B can be performed by activating the complex activation sequence upstream of the reporter gene (for example, by placing AS if the? -Complex is Gal4).
  • the vector can be introduced, for example, according to the lithium acetate method, the spheroplast method, the elect-portation method, and the like. Wear.
  • a yeast in which expression of the repo overnight gene has been confirmed is selected from the above yeasts.
  • the repo is expressed only when a complex of protein A and protein B is formed. Also, a complex of protein A and protein B is formed only when the ability of one protein to bind to another protein is maintained. Therefore, the yeast population selected above is referred to as a “yeast population containing a protein that has not lost binding ability”.
  • a DNA fragment in which the deletion mutation has been introduced in a stepwise manner contained in the expression vector of 1) is isolated from the yeast population selected in 4). Isolation of a DNA fragment can be performed by PCR using primers corresponding to a region adjacent to the DNA fragment.
  • the shortest fragment among the isolated DNA fragments is detected, and the binding region of the protein is identified from the length of the fragment.
  • the shortest DNA fragment can be detected by electrophoresing the PCE ⁇ in step 5).
  • the DNA encoding this protein is 5'-terminal or 3 ' Prepare a DNA population into which a deletion mutation has been introduced from the terminal side.
  • DNA is selected from this DNA population using the expression of the repo overnight gene as an index, it is possible to select DNA whose binding region has not been broken even by the introduction of a deletion mutation.
  • the DNAs of the selected and non-selected populations are amplified by PCR and then developed by electrophoresis, the fragments are detected with a ladder-like spread in the non-selected population. Since it is not included, fragments shorter than a certain length are not detected.
  • the length of the shortest detected DNA corresponds to the position from the N-terminus or C-terminus of the binding region on the protein.
  • the third DNA from the top is the shortest, but the length of this DNA (arrow a in the figure) varies from the C-terminus of the protein to the binding region.
  • the ninth DNA from the top is the shortest, but the length of this DNA (arrow b in the figure) is from the N-terminal of the protein to the C-terminal of the binding region. Against distance.
  • the position and range of the binding region can be identified from the length of NA.
  • the above-described method for identifying a binding region of a protein can be performed by selecting various combinations of a certain protein and another protein, thereby mapping various regions, for example, mapping the expression control region of a promoter fragment, and promoting transcription factors on DNA. This method can be applied to mapping of binding regions, mapping of ligated proteins on the thigh, and mapping of active regions such as enzymes, liposomes, and abdomers.
  • This description includes part or all of the contents as disclosed in the description and Z or the drawings of Japanese Patent Application No. 2009/039836, which is a priority document of the present application.
  • FIG. 1 is a diagram showing the principle of the method (1) for producing a DNA population of the present invention.
  • FIG. 2 is a diagram showing the principle of the method for identifying a protein binding region of the present invention.
  • FIG. 3 shows the results of electrophoresis of 24 yeast strains into which linearized pGAD-RC-Ste5 and GAD-Nx were introduced.
  • FIG. 4 is a diagram showing the results of electrophoresis of a DNA amplification product encoding Ste5 (left), and the binding state of Ste7 and Stell to Ste5 (right).
  • FIG. 5 is a schematic diagram of the method (2) for producing a DNA population of the present invention.
  • pGAD-RC-Ste5 A plasmid that expresses a fusion protein consisting of the Gal4 transduction domain and Ste5 was prepared by inserting a DNA fragment encoding the plasmid (hereinafter, this plasmid is referred to as “pGAD-RC-Ste5”). Since stop codons corresponding to the three readings are inserted downstream of the DNA encoding Ste5 of pGAD-RC-Ste5, the amino acids attached to Ste5 are deleted even if the stop codon of Ste5 is deleted.
  • the linearized pGAD-RC-Ste5 and GAD-Nx prepared as described above were introduced into yeast strain PJ69-4A lig4 leu2 to cause homologous recombination.
  • DNA was introduced by the lithium acetate method.
  • PGAD_RC-Ste5 was extracted from the transformed yeast, and it was examined whether or not a deletion mutation was introduced into the inserted DNA encoding Ste5. Deletion mutations were detected using pGAD-RC-Ste5 as a type I, a primer corresponding to the sequence near the 3 'end of the DNA encoding the Gal4 transcription activation domain, and ADH 1 min.
  • 'PCR was performed using a primer corresponding to the sequence near the end, and the amplified product was subjected to electrophoresis.
  • the sequence of the primer used for PCR is as follows.
  • GAD1 5'-CGCGTTTGGAATCACTACAG-3 '(SEQ ID NO: 1)
  • GAD2 5'-GGGGTTTTTCAGTATCTACG-3 '(SEQ ID NO: 2)
  • FIG. 3 shows the results of electrophoresis of 24 yeast strains into which linearized pGAD-RC_Ste5 and GAD-Nx were introduced.
  • the DNA region encoding Ste5 varies in length for each yeast. This indicates that the oligonucleotide mixture Nx caused homologous recombination at various sites on the DNA region encoding Ste5, and deletion mutations were introduced stepwise from the 5 'end of the DNA encoding Ste5. Indicates that a DNA population was obtained.
  • Nx -ADH1 An oligonucleotide mixture consisting of 8 bases (this mixture contains ⁇ kinds of oligonucleotides) was added to the 5 'end of ADH1 every day and every minute (hereinafter, this mixture of DNA fragments was called “Nx -ADH1 ”).
  • Example 3 In order to cause homologous recombination between Nx-ADHl and the linearized pGAD-RC-Ste5 prepared in Example 1, the strain was introduced into yeast strain P; i69-4Allig4 leu2. PGAD-RC-Ste5 was extracted from the transformed yeast, and it was examined whether a deletion mutation was introduced into the inserted DNA encoding Ste5. The deletion mutation was detected in the same manner as in Example 1. As a result of electrophoresis, amplified fragments having different lengths were obtained for each yeast, as in FIG. This means that for the DNA encoding Ste5, 3 'It indicates that a DM population into which a deletion mutation has been introduced stepwise from the terminal side can be obtained. (Example 3)
  • Example 2 a vector group containing DNA encoding a GaM transcriptionally active domain and DNA into which a deletion mutation was introduced stepwise from the 3 ′ end of DNA encoding Ste5 was prepared.
  • One of this vector group and a vector expressing a fusion protein consisting of Gal4 DNA binding domain and Ste7 were introduced into yeast strain PJ69-ig4 leu2. Since the repo overnight genes ADE2 and HIS3 are located downstream of the Gal upstream activation sequence in this yeast strain, if there is an interaction between Ste5 and Ste7, whose C-terminal part has been partially deleted, Gal4 A transcriptional activation domain and a DNA binding domain form a complex, and the repo all-over-one gene is expressed.
  • Lane 1 in the figure is a sample of the amplification product obtained from the unselected population expressing Ste7
  • Lane 2 is a sample of the amplification product obtained from the selected population expressing Ste7.
  • ladder-like DNA fragments were detected in the entire region of the amplification products from the unselected population, whereas the amplification products obtained from the selected i * group expressing Ste7 and Stell were Did not detect DNA fragments shorter than 3000 bp and 200 Obp, respectively.
  • the present invention provides a novel method for producing a DNA population and a novel method for identifying a binding region of protein. By using these methods, it becomes possible to efficiently perform the function of proteins.

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Abstract

Procédé de construction par recombinaison d'une masse d'ADN dans laquelle une mutation par délétion a été transférée par étapes à partir du côté de la terminaison 5' ou 3' à l'aide de la recombinaison homologue, masse d'ADN ainsi obtenue, levure utilisée dans ledit procédé et procédé d'identification d'une région de liaison de protéines à l'aide du procédé susmentionné et du procédé des deux hybrides.
PCT/JP2001/007285 2000-10-10 2001-08-24 Procede de construction par recombinaison d'une masse d'adn dans laquelle a ete transferee une mutation par deletion et procede d'identification d'une region de liaison de proteines Ceased WO2002031165A1 (fr)

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WO2009027495A1 (fr) * 2007-08-30 2009-03-05 Procomcure Biotech Gmbh Procédé de fabrication d'un peptide modifié

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JP2001269181A (ja) * 2000-03-28 2001-10-02 Hokkaido Technology Licence Office Co Ltd 変異を検出する方法

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Ito T, et.al., "Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins.", Proc. Natl. Acad. Sci. U.S.A. (February 2000), Vol.97, No.3, pp.1143-1147 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009027495A1 (fr) * 2007-08-30 2009-03-05 Procomcure Biotech Gmbh Procédé de fabrication d'un peptide modifié
EP2034020A1 (fr) * 2007-08-30 2009-03-11 Procomcure Biotech GmbH Procédé pour la fabrication d'un peptide modifié

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