JP2003079400A - Method for evaluation of significance of mutation in gene cording transcription factor protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for deciding whether a mutation in a gene of a transcription factor protein is the mutation affecting transcriptive activity or not without examining many clinical specimens. SOLUTION: This method for deciding whether a mutation in a gene of transcription factor protein is the mutation affecting transcriptive activity or not comprises preparing a transcription factor protein coded by a gene having mutation and coding the transcription factor protein bonding with DNA or fragment concerning to bond of the DNA, bonding the resultant with the bonding DNA fragment and comparing the bondability with that of the DNA with the normal transcription factor protein or its fragment.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for determining the significance of mutation of a gene encoding a transcription factor protein.

[0002]

2. Description of the Related Art In recent years, various kinds of so-called gene diagnosis have been carried out in which diagnosis of diseases is based on the nucleotide sequences of genes. Genetic diagnosis is performed by examining the presence or absence of gene mutations known to cause disease. However, not all mutations of a gene cause a disease, and even if the mutation is located in a structural gene and causes a mutation of an amino acid, no particular disease may appear. Therefore, when there is a mutation in a gene, it is unknown whether the mutation causes a disease, and in order to investigate this, it is necessary to examine a large number of clinical specimens and perform statistical treatment. However, this is a labor-intensive task, and in the first place, it is not easy to obtain a large number of specimens of patients having a specific disease for research. In particular, in the case of diseases caused by genes, there are often mutations in multiple parts of the gene, and there may be multiple groups with different combinations of mutations. It is often difficult to determine if they are affected.

On the other hand, various transcription regulatory factors are known, and it is also known that their abnormalities contribute to cancer, diabetes, hypertension and the like.

[0004]

The object of the present invention is to determine whether a mutation in a gene encoding a transcriptional regulatory factor protein is a mutation affecting transcriptional activity, without examining a large number of clinical specimens. It is to provide a determination method.

[0005]

As a result of earnest research, the present inventors have prepared a transcriptional regulatory factor protein encoded by a gene having a mutation, and designated the transcriptional regulatory factor protein as a DNA fragment to which it binds. It is possible to determine whether or not the mutation is a mutation affecting transcriptional activity by binding and comparing the binding property with the binding property between the normal transcriptional regulator protein and the DNA. The present invention has been completed and the present invention has been completed.

[0006] That is, the present invention relates to a gene encoding a transcriptional regulator protein that binds to DNA, which is a transcriptional regulator protein encoded by a gene having a mutation, or a fragment thereof involved in binding to the above DNA. And preparing the transcriptional regulator protein or fragment thereof,
The mutation of the gene encoding the transcriptional regulatory protein includes binding to the binding DNA fragment and comparing the binding property with the binding between the normal transcriptional regulatory factor protein or the fragment and the DNA. A method for determining whether or not the mutation affects activity is provided.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, in the method of the present invention, first, a transcriptional regulator protein encoded by a gene having a mutation or a fragment involved in binding to the above DNA is prepared. Since the method of the present invention is for determining whether or not the mutation of interest is a mutation that affects transcriptional activity, the mutation has been identified before carrying out the method of the present invention. It is intended for. Therefore, the sequence of the transcriptional regulator of interest and the transcriptional regulator protein encoded thereby is known prior to performing the method of the invention. In order to prepare a transcriptional regulatory factor protein having a mutation, first, the cDNA encoding the transcriptional regulatory factor protein contained in the cDNA library is amplified by a nucleic acid amplification method such as PCR, or reverse transcription PCR ( RT-PCR), starting from the mRNA of the transcriptional regulator protein to give cDNA
A is amplified and the resulting amplified cDNA is cloned. Next, a desired point mutation is introduced into the cloned cDNA. Introduction of point mutations can be easily performed by site-directed mutagenesis. The site-directed mutagenesis method itself is well known, and a kit therefor is commercially available (eg, QuikChange Site-Direct manufactured by STRATAGENE).
ed Mutagenesis kit). Site-directed mutagenesis can be easily performed using such a commercially available kit. If there are multiple abrupt displacements, multiple abrupt displacements can be introduced by performing site-directed mutagenesis multiple times. Then, the gene having the mutation introduced therein is expressed by a conventional method to prepare a transcriptional regulatory factor protein or a fragment thereof to be tested.

The transcriptional regulator protein may be prepared in its full length, or may be prepared as a fragment of its region involved in DNA binding. When the region involved in binding to DNA is known, a partial fragment consisting of the known region can be prepared. When the region involved in binding to DNA is unknown, the region involved in binding to DNA is determined by analyzing the binding state between normal transcription regulatory factor protein and DNA by a known X-ray analysis method. You can also The X-ray analysis method itself for investigating the binding state between protein and DNA is known, and is disclosed in, for example, JP 2001-204A.
A preferred example is described in 469. However, DN
When the region involved in the binding with A is unknown and such analysis is troublesome, the full length is prepared.

When the target transcriptional regulatory factor protein is composed of a plurality of subunits, each subunit is prepared. In this case, a mutation may be introduced into only one of the subunits, or a mutation may be introduced into each of a plurality of subunits.

On the other hand, a DNA fragment that binds to the transcription factor protein is prepared. When the DNA region that binds to the target transcriptional regulatory factor is known, DNA containing the known region may be synthesized. When the region is unknown, the DNA may be bound by the above-mentioned X-ray analysis method or the like. Check the area to be used. The DNA fragment may be a fragment containing one or more bases upstream and downstream of the region that binds to the target transcriptional regulatory factor protein. However, longer fragments may be synthesized. The DNA fragment can be easily chemically synthesized using a commercially available DNA synthesizer.

Next, the synthesized transcription regulatory factor protein or fragment thereof is bound to the DNA fragment. The binding can be performed by simply bringing them into contact with each other in a buffer solution. In this case, the concentration of the transcriptional regulatory factor protein or its fragment and the DNA fragment is not particularly limited and can be set appropriately, but usually, it is 1 to 10 mM and 10 n, respectively.
M to about 1 pM (for EMSA), or 1 to 1 for both
It is on the order of 0 mM (in the case of X-ray crystal structure analysis and NMR). The salt concentration of the buffer solution is not particularly limited,
It can be appropriately selected, but it is usually 0 to 300 mM in terms of NaCl,
It is preferably about 50 to 200 mM. The mixing temperature is not particularly limited and is preferably room temperature to 37 ° C., and room temperature is the most convenient. The mixing time is not particularly limited and is usually several minutes to ten and several minutes.

Next, the binding properties of the two will be investigated. This includes, for example, a transcriptional regulator protein or fragment thereof and the DNA described above.
It can also be carried out by measuring the dissociation constant of the bound product with the fragment, or by comparing the mobility of the bound product with that of the normal form by electrophoresis (for example, by electromobility shift assay (EMSA)). Can also be done. The former is preferred for quantitative and detailed analysis. The dissociation constant of the bound substance itself can be measured by a known analysis method, for example, surface plasmon resonance method or isothermal calorimetric titration (i.
It can be measured by sothermal titration calorimetry (ITC). Apparatuses for performing these methods are commercially available, and can be easily performed by a conventional method using commercially available apparatuses.

The measurement result is compared with the measurement result of the binding product of the normal transcriptional regulatory factor protein or its fragment and the DNA fragment. If the result obtained for the mutant type is not much different from the result obtained for the normal type, for example, if the dissociation constant is within 2 times, especially within 1.5 times that of the normal type, the mutation The body can bind to DNA, and usually the mutation has little effect on the transcriptional activity, and when there is a large difference, for example, when the dissociation constant is 3 times or more, particularly 5 times or more of the normal type. In particular, the mutant is unable to bind well to DNA and therefore the mutation has a large effect on transcriptional activity and is likely to cause some disease or deficiency. If the magnification is intermediate between these, the influence on the transcriptional activity is also intermediate, and there is a possibility that some kind of disease or deficiency is caused.

By the method of the present invention, once it is determined whether or not the mutant of the gene encoding the transcriptional regulator protein is a mutation that affects transcriptional activity, the base sequence of the target gene is known in advance. Therefore, in order to detect a mutation determined to affect transcriptional activity, the region containing the mutation is directly sequenced,
By detecting the mutation by a conventional method such as PCR-RFLP, it is possible to know whether or not the transcriptional regulatory factor contained in each sample has a mutation that affects transcriptional activity.
Alternatively, even in the case of searching for a mutation causing a disease by comparing a gene mutation with a clinical result, the mutation affecting the transcriptional activity is generally known by the determination method of the present invention. The task will be much easier because you will only have to search for mutations. In particular, when there are multiple mutations in one disease and it is unknown which mutation causes the disease, the determination method of the present invention can be used to analyze the causative mutation. It will be significantly easier.

[0015]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Reference Example 1 (1) Preparation of X-ray Diffraction Core Binding Factor (CBF) is a transcriptional regulatory factor consisting of α subunit (CBFα) and β subunit (CBFβ) And plays an important role in hematopoiesis.
CBF in patients with acute myelogenous leukemia and clavicle dysplasia
Some have been found to have mutations in.

A partial fragment of mouse CBFα (Gly60-Lys182, AM
Cd encoded by L / Runx-1 gene)
NA was amplified from a mouse gene library using the RTPCR method and obtained by introducing a start codon before Gly60 and a stop codon after Lys182. Also, mouse CB
A cDNA encoding Fβ (Met1-Gln141) was obtained from a mouse gene library by amplification using the RTPCR method, and Met1 was used as a start codon, and a stop codon was introduced after Gln141. The base sequences of these cDNAs are
AML1 / Runx-1 is Proc. Natl. Acad. Sci. USA, 90, 6859-
6863 (1993), GenBank Accession No. D14636, CBFβ
Virology, 194, 314-331 (1993), GenBank Accession N
o. See D14572. Each cDNA was amplified by PCR by a conventional method, the amplified product was digested with restriction enzymes EcoRI and BamHI, subcloned into pBS-KS vector (STRATAGENE), and the nucleotide sequence was determined. The restriction enzyme NdeI
And digested with BamHI, and the NdeI-BamHI fragment was inserted into the pAR2156 vector (Gene, 56, 125-135 (1987)). The recombinant pAR2156 vector into which the CBFα fragment gene or CBFβ gene had been inserted was introduced into BL21 (DE3) cells (manufactured by PROMEGA) and overproduced. The produced protein was recovered from cells and purified to obtain CBFα fragment and CBFβ.

A crystal of a complex comprising the obtained CBFα fragment, CBFβ and a DNA fragment to which the CBFα fragment binds is disclosed in JP-A-2001.
-204469. The DNA fragment (double-stranded) used here is shown in FIG.
Has the sequence shown on the right side of.

X-ray analysis was performed using the obtained crystals.
The production of crystals and X-ray analysis were specifically carried out as follows. CBFα-β-C / EBPβ-DNA, CBFα-C / EBP
In order to prepare each complex of β-DNA and CBFα-DNA, each component contained 10-60 mM DTT and pH 7.4 without salt.
Mix equimolar amounts in the above solution and add 5-10% excess DNA.
Was added to prepare a complex solution having a concentration of 9-11 mg / ml. Crystallization was performed at a temperature of 297 K using the sitting-drop vapor diffusion method. Crystallization conditions are CBFαβC / EBP
For the βDNA complex, 200 mM KCl, 10 mM MgCl ₂ , 20 mM DTT, 4.
5% w / v PEG8000,1% v / v glycerol and 50 mM MES buffer containing 1% v / v MPD (pH5.6 ), 5mM MgSO 4 in CBFαC / EBPβDNA complex, 3% w / v PEG4000 and 2 50 mM MES buffer (pH 5.6) containing% v / v dioxane, 200 for CBFα DNA complex
mM ammonium acetate, 150 mM magnesium acetate and 5% w / v
50 mM HEPES sodium buffer containing PEG4000 (pH 7.0)
And In order to obtain larger crystals, the macro seeding method was performed as needed.

CBFαβC / EBPβDNA was used to perform an X-ray diffraction experiment by cooling the crystal to a temperature of about 100 K with liquid nitrogen.
In the complex crystal, 25 mM MES buffer (pH 5.MPM) containing 200 mM KCl, 4.5% w / v PEG8000, 2% v / v glycerol and 25% v / v MPD.
6), CBFαC / EBPβ DNA complex crystals contained 25 mM MES buffer (pH 5.6) containing 3% w / v PEG4000 and 30% v / v PEG400, CBF
In the αDNA complex crystals, 50 mM HEPES sodium buffer (pH 7.0) containing 200 mM ammonium acetate, 150 mM magnesium acetate, and 30% v / v PEG400 was used as a cryoprotectant, and after immersing the crystal in the cryoprotectant, Crystals were frozen in liquid nitrogen and X-ray diffraction images were collected using the RIKEN beamline at SPring-8, a synchrotron radiation facility.

(2) Structural determination CBFα-β-C / EBPβ-DNA was prepared by the MAD method (Sci) using a gold derivative.
ence, 254, 51-58 (1991)). One major gold binding site is identified by the Bijuboe difference Patterson map (Bijvo
et difference Patterson Map). The difference anomaly Fourier map calculated using the initial MAD phase with one gold atom revealed two other minor sites to which the gold atom binds. These were added and refined for final MAD phasing at 31 nm resolution.
Phase extension from 30-40 nm resolution, combined with density modification, yielded a high quality electron density map with clear tracing of the backbone and most side chains of the protein were well matched. 4, 5, or 6 deoxythymidine,
D substituted with 5-iododeoxyuracil at the corresponding position
The orientation of the DNA atoms was confirmed using a complex containing the three heavy atoms of the NA fragment. TURBO-FRODO software (Rouss
el, A., and Cambillau, C .; turbo@lccmb.cnrs-mrs.f
r) to build the model, and use CNA version 0.9a (Brung
er AT et al., Acta Crystallogr. D54, 905-921). CBFα-C / EBPβ-
The structures of DNA and CBFα-DNA were analyzed by the molecular replacement method using the corresponding fragment of the refined structure of CBFα-β-C / EBPβ-DNA as a search model, and then refined using CNS. The crystals of the latter two complexes contained two complex molecules in the asymmetric unit. The statistics used to evaluate the quality of the final refined and refined structures are shown in Table 1.

[0022]

[Table 1]

The structure determined as described above is shown in FIG.
It shows in FIG. FIG. 1 shows the structure of the CBFα-β-C / EBPβ-DNA quaternary complex viewed from three directions. A is a front view, B is a side view, and C is a plan view. As is clear from the side view of B and the plan view of C, only CBFα is bound to DNA and CBFβ is bound to CBFα.

On the left side of FIG. 2, CBFα used for the structure determination.
The amino acid sequences of the fragment and CBFβ are shown along with the secondary structure (loops and regions). The 26 bp sequence in the upper row of the nucleotide sequence shown on the right side of FIG. 2 is the DNA used for the structure determination of CBFα-β-C / EPBβ-DNA and CBFα-C / EBPβ-DNA complex.
This is the base sequence of the fragment, and the lower 16 bp sequence is CBFα-D.
It is the base sequence of the DNA fragment used for the structure determination of NA complex.

FIG. 3 shows CBFα (left side) and CBFβ (right side).
Shows a topology diagram of the structure. The first and last amino acid numbers of each secondary structure are listed. R80, R142, R171, R1 in the structure of CBFα (left side)
74 and R177 are amino acid residues involved in specific recognition of DNA bases, R135, R139, G143, K167 and V170 are amino acid residues involved in nonspecific interaction with the DNA main chain, T84,
K83 and D171 are amino acid residues involved in water-mediated interactions. In the structure of CBFβ (right side), the electron density in the region of loop 3 (L3) (amino acid residues 71-81) was not observed (dashed line).

FIG. 4 is a diagram schematically showing DNA recognition by CBFα. The solid line shows intermolecular hydrogen bonds and the dotted line shows van der Waals contacts. DNA bases that interact directly with amino acids are G18, G20, C20 ', G21, C21', A23 '.
And C24 '. The DNA base involved in water-mediated base recognition is A22 '. DNA recognition by the amide of the peptide backbone was marked NH. Recognition of the DNA minor groove is boxed in the lower right of FIG.

FIG. 5 shows sterically the specific interaction between CBFα and DNA. The thick tube is the backbone of the CBFα peptide. Dotted lines indicate intermolecular hydrogen bonds and small balls indicate water molecules.

6 to 9 show a stabilizing network between CBFα, CBFβ and DNA. FIG. 6 shows a CBFα-CBFβ heterodimer structure complexed with DNA. CBFα and CB
Two major hydrophilic interaction regions between Fβ (Area I and Ar
ea II) is shown by the dotted salt. FIG. 7 schematically shows a stabilizing network between CBFα, CBFβ and DNA.

FIG. 8 shows the results in the CBFα-β-C / EBPβ-DNA complex.
FIG. 9 shows a three-dimensional structure focusing on the hydrogen bond network between CBFα, CBFβ and the DNA portion in Area II.
Is the CB at Area I in the CBF α-β-C / EBP β-DNA complex.
The three-dimensional structure focusing on the hydrogen bond network between Fα, CBFβ and the DNA portion is shown. In each figure, hydrogen bonds are indicated by dotted lines.

Example 1 EMSA (1) Preparation of CBFα and CBFβ mutants In the method for preparing the CBFα fragment described in Reference Example 1 (1),
After preparing the recombinant pAR2516 vector and before introducing it into BL21 (DE3) cells, QuikChange Site-Directed Mutagenesis
Using a kit (manufactured by STRATAGENE), site-directed mutagenesis was carried out by the method described in the package insert of the product, except that a mutation was introduced into the DNA encoding the CBFα fragment or CBFβ. Reference Example 1 (1) Various CBFα fragment mutants were prepared by the same procedure as in. The mutant is obtained by mutating various amino acid residues that are presumed to play an important role, based on the structures shown in FIGS. 1 to 9 determined in Reference Example 1. In the description of the mutations in the present specification and the drawings, for example, “D66A”
Indicates that aspartic acid (D), which is the 66th amino acid (the amino acid numbering is shown in FIG. 2), is mutated to alanine (A). The same applies to other displays. In addition, for example, "D110A / E111A" indicates that the 110th aspartic acid is mutated to alanine,
In addition, it shows that the glutamic acid at position 111 was mutated to alanine. In addition, "WT" shows a wild type.

(2) EMSA EMSA was carried out using the various mutant CBFα fragments and CBFβ prepared in (1) and the DNA fragment having the 16 bp sequence shown in the lower right column of FIG. In addition, both cases with and without addition of CBFβ were investigated. CBFα fragment, CBFβ and DNA fragment tripartite or CBFα fragment and DNA fragment bipartite binding reaction is <2.9 pM DNA, 2.5 nM CBFα fragment (wild type or mutant type), and in the case of tripartite binding, 3 nM
Alternatively, it was carried out at 20 ° C for 20 to 30 minutes in a buffer solution in which 100 nM CBFβ fragment (wild type or mutant type) was dissolved. The buffer solution is 25 mM HEPES (pH 7.4), 50 mM KCl, 5 mM MgCl ₂ , 0.5.
It contained mM EDTA, 5% Ficol, 10 mM DTT (dithiothreitol) and 0.2 mg / ml BSA (bovine serum albumin). The resulting binding reaction was subjected to polyacrylamide gel (15%) electrophoresis (120V) at 20 ° C. for 90 minutes.

The results are shown in FIG. FIG. 10 shows the relevant portions of the obtained electrophoretic pattern taken out. The position indicated by "CBFα-CBFβ-DNA →" indicates the position at which a band of a conjugate of a wild-type CBFα fragment and a wild-type CBFβ-DNA fragment is formed, and "CBFα-DN
“A →” indicates the position where a band of a conjugate of a wild-type CBFα fragment and a DNA fragment is formed, and “DNA →” indicates DNA.
The position where a band is formed when alone is shown. Mutations in CBFα and CBFβ have been described by the expression method described above. In addition, "-" in the row of "CBFβ" means that CBFβ was not added, and "+" means that it was added. Figure 10
A and C are B when the concentration of wild type CBFβ is 100 nM.
Shows the results when the concentration of wild-type CBFβ was 3 nM, D shows the results when the concentration of mutant CBFβ was 3 nM, and E shows the results when the concentration of mutant CBFβ was 100 nM.

As shown in FIG. 10, wild-type CBFα
When only one of the various amino acids of the fragment or CBFβ is mutated, it is divided into those that form a band at the positions of “CBFα-CBFβ-DNA →” and “CBFα-DNA →” and those that do not, and in many cases, It is very clear whether or not to form the bands at these positions. Those in which no band was formed at these positions were those in which binding of CBFα-DNA or CBFα-CBFβ-DNA did not occur, and a transcriptional regulatory factor can bind to DNA even with a mutation of only 1 amino acid. It is clear that there are cases where it is possible and cases where it is not. Whether or not a transcriptional regulatory factor having a mutation can bind to DNA
It was revealed that the above-mentioned EMSA using the fragments can be examined.

Example 2 Surface plasmon resonance method BIAcore 2000 biosensor system (PHARMACIA BIOSE
21-bp DNA fragment (5'-CAAACTCTG derived from the CSF-1R promoter and CBFα using NCER, Upsala)
Interaction with TGGTTGCCTTGC-3 ') was measured. Biotinylated DNA was bound as a ligand via streptavidin that was previously immobilized to dextran on the surface of biosensor chip SA. The ligand was 300 mM NaCl and 0.0
10 mM HEPES buffer containing 05% Tween 20® (pH
It was diluted to 0.2 μM in 7.5) and added to the sensor chip at a rate of 5 ml / min until the resonance unit (RU) was about 400. 180 mM NaCl and 0.005% Tween were used as running buffer.
A 10 mM HEPES buffer solution (pH 7.4) containing 20 (registered trademark) was used. The CBFα fragment prepared in Reference Example 1 (1) or its mutant prepared in Example 1 which is an analyte was diluted to various concentrations with the same buffer solution (provided that R142A and K144M are 100% each).
Diluted to a concentration of mM and 140 mM), injected over immobilized ligand at a rate of 15 μl / min until the injection volume reached 250 μl. In an experiment using CBFβ, 500 nM CBFβ or its mutant (produced in Reference Example 1 (1) or Example 1) was used as CBFα.
Added to the solution. The dissociation of the analyte was then measured by injecting only running buffer. All experiments were performed at 25 ° C. Data is BIAevaluation version
Using 2.1 (PHARMACIA BIOSENCER), analysis was carried out by the nonlinear least squares method, and the association rate constant k _a and the dissociation rate constant k _d were obtained. The equilibrium dissociation constant K _D was calculated by the formula K _D = k _d / k _a or by Scatchard analysis. The results are shown in Table 2 below.

[0035]

[Table 2] Table 2 Kinetics and dissociation constants of CBFα or its variant binding to DNA in the presence or absence of CBFβ or its variant The increase in a RU was not monophasic or too small to be measured from the sensorgram. b These values were obtained in the presence of 100 mM NaCl instead of 180 mM NaCl. c This value was calculated using Scatchard analysis. d These values were obtained in the presence of 140 mM NaCl instead of 180 mM NaCl.

As shown in Table 2, CBFα fragment and CBF
The K _D for both β types of wild type (WT) is 7.56 ± 0.47. Those less than or equal to this value, for example, C66
For A, D110A, E111A, etc., the EMSA shown in FIG.
A band is clearly formed at the position indicated by "BFβ-DNA →". On the other hand, if K _D is much larger than this (that is too large to measure), for example CBF
For N109A of α and N104A of CBFβ, EMSA shown in FIG.
No band is formed at the position indicated by α-CBFβ-DNA → ”. Therefore, by measuring the equilibrium dissociation constant of the mutant and comparing it with that of the wild type, it is possible to know whether or not the mutant of the transcriptional regulator can bind to DNA.

[0037]

INDUSTRIAL APPLICABILITY The present invention provides for the first time a method for determining whether or not a mutation in a gene encoding a transcription factor protein is a mutation affecting transcriptional activity, without examining a large number of clinical specimens. Was done. Once it is determined by the method of the present invention whether or not the mutant of the gene encoding the transcriptional regulator protein is a mutation that affects transcriptional activity, the base sequence of the target gene is known in advance. By detecting and detecting the mutation determined to affect the transcriptional activity by a conventional method, it is possible to know whether or not the transcriptional regulatory factor contained in each sample has a mutation that affects the transcriptional activity. . Further, even when confirmation is performed using a clinical sample, since the mutation that affects the transcriptional activity is generally known by the determination method of the present invention, it suffices to search by paying attention to the mutation. Therefore, the work is greatly facilitated.

[0038]

[Sequence list] <110> The Kanagawa Academy of Science and Technology Foundation <120> Method for evaluating significance of mutations of genes encoding translational regulatory factor proteins <160> 5

[0039] <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <223> DNA fragment used for binding with CBFα <400> 1 caaactctgt ggttgccttg c 21

[0040] <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <223> DNA fragment used for determining the structures of the CBF α-β-C / EBPβ-DNA and CBFα-C / EBPβ-DNA complexes <400> 2 gaagatttcc aaactctgtg gttgcg 26

[0041] <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <223> DNA fragment used for determining the structures of the CBFα-DNA complex <400> 3 gaactctgtg gttgcg 16

[0042] <210> 4 <211> 123 <212> Protein <213> Homo sapiens <400> 4 Gly Glu Leu Val Arg Thr Asp Ser Pro Asn Phe Leu Cys Ser Val Leu 1 5 10 15 Pro Thr His Trp Arg Cys Asn Lys Thr Leu Pro Ile Ala Phe Lys Val             20 25 30 Val Ala Leu Gly Asp Val Pro Asp Gly Thr Leu Val Thr Val Met Ala         35 40 45 Gly Asn Asp Glu Asn Tyr Ser Ala Glu Leu Arg Asn Ala Thr Ala Ala     50 55 60 Met Lys Asn Gln Val Ala Arg Phe Asn Asp Leu Arg Phe Val Gly Arg 65 70 75 80 Ser Gly Arg Gly Lys Ser Phe Thr Leu Thr Ile Thr Val Phe Thr Asn                 85 90 95 Pro Pro Gln Val Ala Thr Tyr His Arg Ala Ile Lys Ile Thr Val Asp             100 105 110 Gly Pro Arg Glu Pro Arg Arg His Arg Gln Lys         115 120

[0043] <210> 5 <211> 141 <212> Protein <213> Homo sapiens <400> 5 sMet Pro Arg Val Val Pro Asp Gln Arg Ser Lys Phe Glu Asn Glu Glu   1 5 10 15 Phe Phe Arg Lys Leu Ser Arg Glu Cys Glu Ile Lys Tyr Thr Gly Phe             20 25 30 Arg Asp Arg Pro His Glu Glu Arg Gln Thr Arg Phe Gln Asn Ala Cys         35 40 45 Arg Asp Gly Arg Ser Glu Ile Ala Phe Val Ala Thr Gly Thr Asn Leu     50 55 60 Ser Leu Gln Phe Phe Pro Ala Ser Trp Gln Gly Glu Gln Arg Gln Thr 65 70 75 80 Pro Ser Arg Glu Tyr Val Asp Leu Glu Arg Glu Ala Gly Lys Val Tyr                 85 90 95 Leu Lys Ala Pro Met Ile Leu Asn Gly Val Cys Val Ile Trp Lys Gly             100 105 110 Trp Ile Asp Leu His Arg Leu Asp Gly Met Gly Cys Leu Glu Phe Asp         115 120 125 Glu Glu Arg Ala Gln Gln Glu Asp Ala Leu Ala Gln Gln     130 135 140

[Brief description of drawings]

FIG. 1 is a diagram showing the structure of a CBFα-β-C / EBPβ-DNA quaternary complex prepared in Reference Example as viewed from three directions.

FIG. 2 is a diagram showing the amino acid sequences of the CBFα fragment and CBFβ prepared in Reference Example together with the secondary structure (loop and region) (left side) and the nucleotide sequences of the DNA fragments used for the binding of CBFα and CBFβ or CBFα alone. FIG.

FIG. 3 CBFα fragment (left side) and CBFβ prepared in Reference Example
A topological diagram of the structure (right) is shown.

FIG. 4 is a diagram schematically showing DNA recognition by CBFα prepared in Reference Example.

FIG. 5 is a diagram showing three-dimensionally the specific interaction between CBFα and DNA prepared in Reference Example.

FIG. 6: CBFα-CB complexed with DNA prepared in Reference Example
It is a figure which shows Fβ heterodimer structure.

FIG. 7 is a schematic view showing a stabilizing network between CBFα, CBFβ and DNA prepared in Reference Example.

FIG. 8 shows a three-dimensional structure focusing on the hydrogen bond network between CBFα, CBFβ and the DNA portion in Area II in the CBFα-β-C / EBPβ-DNA complex prepared in Reference Example.

FIG. 9 shows a three-dimensional structure focusing on the hydrogen bond network between CBFα, CBFβ and the DNA portion in Area I in the CBFα-β-C / EBPβ-DNA complex prepared in Reference Example.

FIG. 10 is a diagram showing an electrophoretic pattern by EMSA performed in Example 1.

Claims (3)

[Claims] 1. A gene encoding a transcriptional regulator protein that binds to DNA, wherein the transcriptional regulator protein encoded by a gene having a mutation or a fragment thereof involved in binding to the DNA is prepared, DNA that binds a transcription factor protein or a fragment thereof
Mutation of a gene encoding a transcriptional regulator protein affects transcriptional activity, including binding to a fragment and comparing the binding property with the binding between a normal transcriptional regulatory protein or a fragment thereof and the DNA A method of determining whether or not a mutation. 2. The method according to claim 1, wherein the binding property is measured by measuring a dissociation constant of a binding product of the transcriptional regulator protein or its fragment and the DNA fragment. 3. The method according to claim 2, wherein the dissociation constant is measured by a surface plasmon resonance method.