JP2003024069A - NEW TdT-BINDING PROTEIN AND GENE ENCODING THE SAME PROTEIN - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein and a gene thereof useful for treatment and diagnosis of acute leukemia. SOLUTION: A new protein directly promoting a DNA polymerase activity of TdT by binding to the C-terminal region of a terminal deoxynucleotidyl- transferase(TdT), a gene encoding the protein and a vector expressing the same are provided. These substances become medicines enhancing a therapeutic effect caused by administration of a hormone on acute leukemia.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein having a binding property to DNA polymerase TdT and a gene encoding the protein.

[0002]

2. Description of the Related Art Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase discovered by Bollum1 ⁾ , which does not require a template strand for the reaction and polymerizes dNTP as a random sequence at the 3'end of single-stranded DNA. It is characterized by High levels of TdT are detected in thymus and bone marrow cells, whereas it is rarely expressed in other organs. However, since it is specifically expressed in certain leukemias such as acute lymphocytic leukemia (ALL) and acute undifferentiated leukemia (AUL), it is used as a tumor marker.

As a treatment for acute leukemia, hormone therapy in which synthetic hormones such as steroid hormones prednisolone and dexamethasone are administered has been carried out. In order to monitor the therapeutic effect, the DNA polymerase activity of TdT was measured. There is. However, regarding the mechanism by which the expression of TdT disappears due to the administration of hormones,
No clear conclusion has been reached.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel protein and its gene useful for treating and diagnosing acute leukemia.

[0005]

[Means for Solving the Problems] In the treatment of acute leukemia, the present inventors continued their earnest research as a result of the prediction that the protein on which steroid hormones act may be different from TdT. We found a new protein, TdIFl, consisting of 329 amino acids, and cloned the gene encoding the protein.

[0006] TdlFl binds to the C-terminal region of TdT and directly interacts with TdT to enhance the DNA polymerase activity of TdT. Furthermore, it was confirmed that the amino acid sequence of TdlFl shows high identity with 65 kD oncofetal protein (p65) (Fig. 3) and HMGI-C.

P65 is a member of the nuclear receptor superfamily with two C4 type Zn finger motifs, isolated from the human breast cancer cell line MCF7. p65 is an effective tumor marker and is induced at a very early stage in carcinogenesis ^{2), 3)} . In particular, p65 is highly expressed in acute lymphocytic leukemia, acute myeloma leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia ⁴⁾ , and the highest level of expression is observed in acute lymphocytic leukemia and acute myelogenous leukemia cells. To be observed. TdT is also highly expressed in acute lymphocytic leukemia and acute myeloma leukemia cells
^{5) -7)} , the expression pattern of TdT in leukemia cells is p
Consistent with 65. Furthermore, TdlFl is expressed in normal rat thymus, bone marrow and spleen cells. Thus, TdIF1 is specifically expressed in lymphocyte-related tissues, suggesting that it is directly involved in lymphocyte-specific functions.

Proteins belonging to the nuclear receptor superfamily are transcriptional regulators composed of A / B (AF-1), DNA binding and ligand binding (AF-2) domains. The transcriptional activity of the nuclear receptor depends on the C-terminal ligand-binding domain (AF-2) and the N-terminal A / B domain, and AF-1 shows ligand-independent transcriptional activity. Indicates ligand-dependent transcriptional activity. Due to the lack of the AF-1 and Zn finger motifs, TdlFl does not possess the typical basic structure of the nuclear receptor superfamily, but has regions of high homology with AF-2 of p65 ( (Figure 3). From this, it is expected that TdlFl binds to a ligand similar to p65, which has not been identified yet, and expresses ligand-dependent transcriptional activity ⁸⁾ .
That is, TdIF1 is expected to function in vivo as a transcription factor capable of interacting with TdT.

[0009] Recent studies, cleavage of tissue and differentiation stage-specific V (D) J, was shown to be controlled by a change in approach easiness of recombinant device (recombinase) for recombination sites ^{9 )} . For example, specific deletion of Ig and TcR enhancers significantly inhibits recombination of gene fragments especially in mutated aryls. ^{10
) -15)} . In addition to V (D) J cleavage, insertion of the N region between the coding ends is also regulated in a tissue- and differentiation-stage specific manner. Therefore, it is suggested that TdlFl may control the synthesis of N region by activating V (D) J recombination-related gene while enhancing TdT activity. Although a factor that binds to an enhancer and / or a promoter and controls V (D) J recombination has not been identified yet, TdlFl
May be the first transcription factor controlling all processes of V (D) J recombination.

High mobility group IC (HMGI-C) protein is a protein that is abundant in highly proliferating undifferentiated cells. High level expression of this protein is characteristic in early embryonic tissues and various tumors. By acting as a construction factor, HMGI-C promotes the activity of NF-κB, which is a transcription factor for the β interferon (βIFN) promoter. Here, HMGI-C has three DNA-binding domains, AT-hook (Fig. 4), and binds to NRDI and PRDII factors of βIFN promoter at the first and second AT-hooks, respectively ¹⁶⁾ .

TdIF1 also has three AT-hooks and can bind to both single-stranded and double-stranded DNA. In the HMGI-DNA complex, the tripeptide Arg-Gly-Arg (RGR) is AT-hoo.
It has been revealed by NMR analysis that it exists in the central part of k. This tripeptide RGR is the second AT-of TdIFl.
In the hook, the first arginine is changed to lysine, but in the first AT-hook it is completely conserved.
These are AT-hook where TdIF1 is a DNA binding domain.
It strongly suggests that it is bound to DNA via.

It is considered that the HMG protein causes structural changes in DNA and chromatin and functions as a structural element that creates a three-dimensional structure that activates or represses various DNA transcriptions. Therefore, it is considered that TdIF1 exerts a function similar to that of HMG in V (D) J recombination. TdIFl binds to DNA after cleavage of the hairpin structure at the end of the coding region and
Is fixed at the end of the coding region and TdT is
Expected to synthesize regions.

Recent studies have reported that many proteins, including TdT, synthesize the N region of the Ig and TCR genes. The present inventor isolated a cDNA clone encoding a TdT binding protein other than TdIF1 by the two-hybrid screening method. Those proteins included PCNA, a known protein that is an auxiliary protein that promotes the activity of DNA polymerase δ. Notably, TdIF
Unlike l promoting TdT activity, PCNA reduced TdT activity by 17% in the Tris-Mg assay system. PCN in TdT
The binding region of A is in the C-terminal region, which is the binding region in common with TdIFl. That is, PCNA and TdIFl competitively compete with C of TdT.
It is thought that it can bind to the terminal region. Experiments using the yeast two-hybrid system confirmed that TdIFl does not interact directly with PCNA. Furthermore, TdIF
l binds to single-stranded and double-stranded DNA and promotes TdT activity, whereas PCNA does not bind to single-stranded DNA and does not suppress TdT activity.
These findings indicate that the synthesis of N region by TdT is PCNA and TdIFl.
Suggests that they are negatively and positively controlled, respectively. PCNA when inserting N area
It is considered that the switch from the negative control due to TdIFl to the positive control due to TdIFl occurs.

The present inventor has further isolated a cDNA clone of a protein that interacts with TdIF1 by screening a human thymus cDNA library using the two-hybrid system. As a result, lymphoid cell-specific cDNA was found in the isolated clones, suggesting that TdIFl functions widely as a regulator of DNA metabolism.

TdlFl is A of the nuclear receptor superfamily
It has a region very similar to F-2, which means Tdl
It strongly suggests that Fl is a steroid hormone-dependent transcription factor. Hormone administration is used for the treatment of acute leukemia, but TdT activity disappears with the remission of leukemia.
T activity measurement is used.

Further, it was revealed by the present invention that TdIF1 enhances TdT activity as described above. This suggests that the TdT-TdIF1 interaction is involved in the reduction of leukocyte TdT activity by administration of steroid hormones.

On the other hand, although the mechanism of treatment of acute leukemia by hormone administration is currently unknown, it is possible that such therapeutic effect is mediated by TdIF1 as well as TdT activity regulation because TdT activity decreases with the therapeutic effect. Is high. From this viewpoint, TdIF1, which is expected to mediate the action effects of hormones
It is expected that the therapeutic effect on acute leukemia due to hormone administration can be enhanced by administering or excessively expressing the drug.

[0018]TdIF1 gene The present invention provides a gene encoding the TdIF1 protein.
It This gene expresses human TdT cDNA and interacts with this protein.
Cloning a cDNA encoding an interacting protein
Can be obtained by
Based on the sequence
Cloning and phosphoamida using genetic engineering techniques
DNA prepared by chemical synthetic methods such as the Ito method
You may. Its form is cDNA or genomic DNA.
Alternatively, chemically synthesized DNA is included, but there is no particular limitation.

The DNA of the present invention may be single-stranded or may bind to DNA or RNA having a sequence complementary thereto to form a double-stranded chain or triple-stranded chain. The DNA may be labeled with an enzyme such as horseradish peroxidase (HRPO), a radioisotope, a fluorescent substance, a chemiluminescent substance, or the like.

If the nucleotide sequence of the TdIF1 gene is provided, the RNA sequence derived therefrom and the complementary DNA and RNA sequences are uniquely determined. It should be understood that RNA corresponding to DNA or DNA and RNA having a sequence complementary to the DNA of the present invention is also provided.

Further, the DNA of the present invention also includes a DNA which hybridizes with the DNA having the nucleotide sequence of SEQ ID NO: 2 under stringent conditions.

Therefore, a DNA which hybridizes with the DNA sequence shown in SEQ ID NO: 2 and which has a function substantially equivalent to the TdIF1 protein, that is, a DNA encoding a protein which interacts with the TdT protein, is within the scope of the present invention. Should be understood to be included in. Also, due to the presence of so-called codon degeneracy, there may be a nucleic acid that encodes the same protein as the TdIF1 protein and has a sequence different from the sequence shown in SEQ ID NO: 2, but this is also within the scope of the present invention. Needless to say.

In other words, the DNA sequence is partially changed by various artificial treatments such as site-directed mutagenesis, random mutation by treatment with a mutagen, mutation / deletion / ligation of DNA fragment by restriction enzyme cleavage, etc. However, these DNAs hybridize with the DNA of SEQ ID NO: 2 under stringent conditions and interact with the TdIF1 protein or a protein having substantially the same function as the protein, that is, TdT protein. It is to be understood that any DNA encoding the protein described above is included within the scope of the present invention regardless of the difference from the DNA sequence shown in SEQ ID NO: 2.

The degree of mutation of the above DNA is within an allowable range as long as it has 70% or more, preferably 80% or more, more preferably 90% or more homology with the DNA sequence of SEQ ID NO: 2. is there. Further, as the degree of hybridization, under normal conditions, for example, when the probe is labeled with a DIG DNA Labeling kit (Cat No. 1175033 manufactured by Boehringer Mannheim), a 32 ° C DIG Easy Hyb solution (manufactured by Boehringer Mannheim) is used. (Cat No. 1603558), and the membrane is washed in 0.5 x SSC solution (containing 0.1% (w / v) SDS) at 50 ° C (1 x SSC is 0.15 MN).
Southern hybridization with aCl, 0.015 M sodium citrate) is sufficient as long as it hybridizes to the nucleic acid shown in SEQ ID NO: 2.

The transformed cell in the present invention can be prepared by applying a technique known to those skilled in the art, for example, various vectors that are commercially available or generally available to those skilled in the art are used, and suitable cells can be prepared. It is possible to integrate the DNA of the invention into a host cell. At that time, the expression of the TdIF1 gene in the host cell can be arbitrarily controlled by placing the TdIF1 gene under the influence of an expression control gene represented by a promoter or an enhancer. This technique is preferably used in the production of TdIF1 protein using transformed host cells.

Furthermore, a transgenic animal can be prepared based on a mouse or other appropriate animal by combining the DNA of the present invention with a known method. Using such a transgenic animal, it becomes possible to analyze the onset mechanism of acute leukemia.

[0027]TdIF1 protein Deduced amino acid of TdIF1 protein encoded by TdIF1 gene
The sequence is as shown in SEQ ID NO: 1.

The side chains of amino acid residues, which are constituent elements of proteins, differ in hydrophobicity, charge, size, etc., but they do not substantially affect the three-dimensional structure (also called a three-dimensional structure) of the whole protein. In the sense, some relationships are known that are highly conservative. For example, for substitution of amino acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Le)
u) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), T
Examples include hr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg), and the like. In addition, many mutations are known to those skilled in the art, even when the above-mentioned conservativeness is impaired, the essential function of the protein is not lost. further,
There are many cases in which the same type of protein, which is conserved among different species of organisms, retains its essential function even if some amino acids are deleted or inserted in a concentrated or dispersed manner.

Therefore, even in the case of the mutant protein having the substitution, insertion, deletion, and / or addition on the amino acid sequence shown in SEQ ID NO: 1, the mutation is functionally equivalent to the TdIF1 protein, that is, the mutant protein is compatible with the TdT protein. These can be said to be within the scope of the present invention as long as they are proteins that act.

Such amino acid changes are recognized in nature such as sequence diversity found in different organisms or so-called genetic polymorphisms, and methods known to those skilled in the art, for example, mutagenesis such as NTG. It can be artificially generated by utilizing a mutagenesis method using an agent or a site-directed mutagenesis method using various recombinant gene techniques.
The mutation site and number of amino acids are not particularly limited as long as the mutant protein retains the transcriptional regulatory activity, but the number of mutations is usually within tens of amino acids, preferably within 10 amino acids.

[0031]

DETAILED DESCRIPTION OF THE INVENTIONNucleic acid The DNA of the present invention expresses human TdT cDNA and
Using the lid system (CLONTECH), etc.,
Cloning a cDNA encoding an interacting protein
Can be obtained by See also SEQ ID NO: 2.
Probes and anti-TdIF1 antibodies prepared based on the nucleotide sequences
Can also be obtained from a DNA library.
As an example of obtaining the DNA of the present invention from a DNA library,
The appropriate genomic DNA library or cDNA library is
Screening method by hybridization, antibody
Screening by immunoscreening method using
And grow a clone containing the desired DNA,
There is a method of cutting out with a restriction enzyme or the like. Hybrid
The screening by the zation method is described in SEQ ID NO: 2.
DNA containing the above nucleotide sequence or part of it is
Label it as a probe and pair it with any cDNA library.
Then, it can be carried out by a known method. Immuno screen
The antibody used in the ning method is the antibody of the present invention described below.
can do. The novel DNA of the present invention also includes genomic DNA.
PC using the library or cDNA library as a template
It can also be obtained by R (Polymerase Chain Reaction)
It PCR is based on the nucleotide sequence shown in SEQ ID NO: 2 and
And prepare antisense primer and antisense primer.
For the desired DNA library,
It is also possible to obtain the DNA of the invention. D used in the above various methods
NA library is a DNA library containing the DNA of the present invention.
Select and use. The DNA library is
Any library with clear DNA
Can also be used, and a commercially available DNA library was used.
, Lymphocytes and other cells having the DNA of the present invention to cDN
Select cells suitable for preparing A library
A cDNA library can be prepared and used according to the method.
You can

The recombinant vector having the DNA of the present invention is
It may be in any form such as cyclic or linear. Such a recombinant vector may have other base sequences in addition to the DNA of the present invention, if necessary. With other base sequences, enhancer sequence, promoter sequence, ribosome binding sequence, base sequence used for the purpose of amplification of copy number, base sequence encoding signal peptide, base sequence encoding other polypeptide, It is a poly A addition sequence, a splicing sequence, an origin of replication, a base sequence of a gene serving as a selection marker, and the like.

For gene recombination, an appropriate synthetic DN
It is also possible to add a translation initiation codon or a translation termination codon to the DNA of the present invention using an A adaptor, or to newly generate or delete an appropriate restriction enzyme cleavage sequence in the base sequence. These are within the scope of operations normally performed by those skilled in the art, and can be arbitrarily and easily processed based on the DNA of the present invention.

Further, the vector carrying the DNA of the present invention is
An appropriate vector may be selected and used according to the host to be used, and it is also possible to use various viruses such as bacteriophage, baculovirus, retrovirus, vaccinia virus in addition to the plasmid, and there is no particular limitation. .

Methods for introducing DNA into a vector are known. That is, the DNA and the vector may be digested with appropriate restriction enzymes, and the obtained fragments may be ligated with DNA ligase.

[0036]protein The protein of the present invention is produced by a genetic engineering method.
be able to. Transformation using the recombinant vector of the previous section
There is no particular limitation on the host cell to be used, and E. coli, B. subtil
For genetic engineering methods represented by is or S. cerevisiae
Available in lower cells, insect cells, COS7 cells, CHO
Cells, many cells such as animal cells represented by Hela cells
However, it can also be used for the present invention.

As a method for introducing the recombinant vector into a host cell, known methods such as electroporation method, protoplast method, alkali metal method, calcium phosphate precipitation method, DEAE dextran method, microinjection method, and method using virus particles can be used. However, any method may be used.

To produce the protein by genetic engineering, the above-mentioned transformant is cultured to recover a culture mixture,
The protein is purified.

As a method for purifying the protein of the present invention from the culture mixture, an appropriate method can be appropriately selected from the methods usually used for protein purification.
That is, salting out method, ultrafiltration method, isoelectric precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, affinity chromatography such as hydrophobic chromatography and antibody chromatography, chromatofocusing method, adsorption method. Appropriate methods may be appropriately selected from commonly used methods such as chromatography and reverse phase chromatography, and if necessary, purification may be performed in an appropriate order using an HPLC system or the like.

As described above, the protein of the present invention can be converted into various forms. For example, processing by various methods known to those skilled in the art such as various chemical modifications of proteins, binding with polymers such as polyethylene glycol, binding to insoluble carriers, and the like can be considered. Further, depending on the host used, the presence or absence of addition of sugar chains or the degree thereof is different. Even in such a case, as long as it is a protein functionally equivalent to TdIF1 protein,
It should be under the spirit of the present invention.

The present invention will be described in more detail with reference to the following examples, but the present invention should not be limited to these examples.

[0041]Example 1 Plasmid Decoy used for enzyme two-hybrid screening
To construct a plasmid (pAS2-1-TdT (full length))
In addition, human TdT cDNA (H14) was cloned into pAS2-1 vector
Introduced). The full-length TdT cDNA clone H14 is a MOLT3 clone.
Screened pcD library constructed from vesicle mRNA
Isolated by¹⁷⁾.

The TdIFl cDNA was subcloned into pGEX4T-2 (Amersham Pharmacia Biotech) for expression of the GST fusion protein in E. coli. In addition, pEGFP C2 (Clontech) subcloned between the EcoRl site and the Xhol site to identify the localization of the gene product was prepared.

Furthermore, three expression vectors pGEX-TdIFl (full length), pGEX-TdIFl (EX) and pGEX-TdIFl (XX) were constructed.
These are TdIFl residues 1-329, 1-218, and 218, respectively.
It encodes ~ 329 residues. To identify the binding site of TdIFl to TdT, a deletion mutant of TdT was prepared and subcloned, and then inserted into pAS2-1 vector. The constructed plasmid is shown in FIG.

[0044]Example 2 Yeast two-hylide system
Cloning of TdIF1 gene Recommended by the manufacturer for isolation of the desired clone
Under the condition The MATCHMAKER GAL4 System (Clonetech)
Was used. Decoy plasmid pAS2-1-TdT (full length)
Transfecting yeast strain Y190 and then Trp Au
Select cells transfected with totrophy
It was Then GAL4 activation domain fusion cDNA library
Transfect bait cells with (Clontech)
It was Not a candidate expressing a protein that interacts with TdT
Clones on Trp / His / Leu deficient plates
Isolate by selection and filter β-galactosyl
Β-galactosidase reporter
The activation of the gene was tested. Positive clones are reporters
Cotransfection with yeast (strain Y190) together with decoy plasmid
And confirmed the interaction. Transfection
Performed by standard lithium acetate method. Autotrophy
And positive selected by β-galactosidase assay
The clone was transfected into E. coli HB101 strain,
A clone with leucine autotrophy was selected. 7 x 1
0^FiveScreen clones on Trp / His / Leu deficient plates
And 68 candidate clones were isolated. Isolated cDNA
Clone DNA sequence dideoxy termination method
¹⁸⁾Determined by As a result, 13 out of 68 clones
The DNA sequences of the clones were confirmed to be identical. Figure 1
The translated amino acid sequence is shown. TdlFl has 329 amino acids
The molecular weight was estimated to be 37 kD. Determined DNA distribution
The columns are NCBI type using BLASTN and BI-ASTP algorithms.
Compared by a similarity search tool¹⁹⁾. as a result,
TdlFl is a 65kD oncofetal protein (p65) (Fig. 3) and HMGI-C
It was confirmed that it showed high identity with (Fig. 4). TdIFl
Is a protein of the N-terminal and nuclear receptor superfamily
It does not possess the characteristic C4 type Zn fingers, but p6
It has 60% homology with the amino acid sequence up to 278th C-terminal of 5.
It was confirmed to do. Homologous region of TdlFl and p65 is C4 type Zn
It extended from the rear half of the finger to the C-terminal (Fig.
3).

[0045]Example 3 Chromosomal mapping of TdIFl Chromosome mapping of TdIFl using NCBI human genome BLAST
Carried out. As a result, the TdlFl gene has a q on chromosome 20.
It is located in 12-q13 and is confirmed to consist of 13 exons.
Accepted (Fig. 2).

The mouse TdIF1 cDNA sequence is the human TdIF1 cDNA sequence.
Was confirmed by in silico cloning.
In addition, using the mouse EST database, TdIF1 mice
The cDNA sequence was deduced (SEQ ID NO: 4). To cover the full length of TdIFl cDNA, 20 mouse EST clones were picked up, and the gene analysis software DNASIS (Hitach Software Engi
neering Co., Ltd. Yokohama Japan). Figure 1 shows the translated amino acid sequence of mouse TdIF1. The mouse sequence of TdlFl showed 96.4% homology with the human sequence.

[0047]Example 4 Determination of TdIFl binding region in TdT To determine the TdIFl binding region of TdT, the yeast two hybrid
A dead assay was performed. Deletion of human TdT cDNA
The mutant (Fig. 6) was subcloned into the pAS2-1 vector.
Was constructed to construct a TdT-deficient plasmid. Mutant
Plasmid together with pACT2-TdIFl (full length) plasmid.
SD / -Leu / -Trp play co-transfected into mother cells
Cultured in Fermentation of protein-protein interactions
Detected by mother-to-hybrid system. As shown in Figure 6.
As described above, TdlFl and TdT-de12 containing the C-terminal 360 amino acids of TdT
TdT-de contains N-terminal 150 amino acids while interacting
Did not interact with 11. TdT-de12, -de13, -de14, -d
Other raids including e15, -de17, -de18, -de19 and -del10
Deliation Mutant does not interact with TdlFl
It was. Therefore, the interaction between TdT and TdlFl is the C-terminal 360 in TdT.
It was concluded that all amino acids are required.

[0048]Example 5 Interaction between TdT and TdlFl 10 of MOLT-4 cells⁷Collect the pieces, wash with PBS, and then
Fur (PBS, 1% TritonX-100, 5 mM dithiothreitol,
1 mM PMSF, 1 μM pepstatin, 1 μM leupepsin,
5 μM benzamidine, 5% glycerol)
It was homogenized with a non-type homogenizer. Homo
Treat the dinate with ethidium bromide at 4 ℃, 16,000
Centrifuge at g for 30 minutes. Ethidium bromide is pull dow
-Independent protein-tamper in protein assay
Without having a clear impact on the quality interaction
Selective DNA-dependent protein-protein interactions
Inhibit²⁰⁾. Rabbit antibody raised against bovine TdT
MOLT4 cells using Western blotting by
The expression of TdT was confirmed.

The TdIFl GST fusion protein was expressed in E. coli using the pGEX4T-2 vector. GST-TdIFl (full length)
The fusion protein was non-covalently linked to glutathione-Sepharose 4B. GST-TdIFl-conjugated glutathione beads were mixed with MOLT4 cell lysate and incubated at room temperature for 4 hours.
Incubated for 0 minutes. The beads were washed three times with 100 μM B buffer (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1 mM dithiothreitol (DTT) and 10% glycerol), and the protein bound to the beads was washed with 10 mM. Elution was performed with PBS containing glutathione. The eluate was analyzed by Western blot. Glutathione Sepharose 4B was purchased from Amersham-Pharmacia Biotech. Bovine thymus (1.5 g) was prepared with 7.5 mL of C buffer (40 mM Tris-HCl (pH 7.
4), 40 mM KCl, 5 mM MgCl ₂ , 0.1% Nonidet P40, ₂ m
Homogenize with M DTT, 10% glycerol, 12,000 r
It was centrifuged at pm for 20 minutes, treated with ethidium bromide, and then centrifuged again at 4 ° C. and 13,000 rpm for 30 minutes. Supernatant and anti-TdT rabbit antibody conjugated to protein A sepharose 4B and 4
The reaction was carried out at ℃ for 3 hours. Wash the beads with C buffer,
The protein bound to the beads was eluted with PBS containing 2% SDS. The eluate was analyzed by Western blotting.
As shown in Fig. 5, TdT is GST-TdIF1 bound to beads.
It was sedimented by (full length) (lane 4) but not by GST-conjugated beads (lane 3).

Proteins belonging to the nuclear receptor superfamily have a dimerization domain in AF-1. Therefore, it was confirmed using a yeast two-hybrid system whether TdlFl directly interacts with itself. The result is a blue signal that indicates a direct protein-protein interaction,
This indicates that TdlFl binds to and dimerizes with other TdlFl in yeast (data not shown).

[0051]Example 6 Measurement of TdT activity The effect of TdIFl on TdT activity is (dT)₁₆Oligonucleo
Standard TdT activity assay using Tide as primer
Tested by system. The reaction mixture was 40 mM Tris-HCl (pH 8.
0), 40 mM KCl, 1 mM 2-mercaptoethanol, 6 mM Mg
Cl₂, 200 μg / ml BSA, 0.66 μM of each protein (GST-TdI
Fl (full length) fusion protein or GST), 7.5 unit TdT
(Gibco), 10 μg / ml (dT)₁₆Oligonucleotides and 0.
100 μM of [having a specific activity of 611 Ci / mmol³H] dTTP included
Mu. The total volume in each assay is 24 μl. TdT-TdIF
30 minutes on ice to form l protein complex
Between TdT and [^ThreePreincubate the reaction mixture excluding [H] dTTP.
And then TdT and [^ThreeAdd [H] dTTP to the mixture and add 37 ° C.
And incubated for 60 minutes. Whatman DE- reaction mixture
Spot on 81 discs, 5% disodium hydrogen phosphate
After washing 5 times with purified water and 2 times with purified water, dehydrate in ethanol.
It was Dried discs are scintillation counter
(Beckman). As a result, when TdlFl was added to the reaction mixture, TdlFl was not present.
The activity of TdT was promoted 4-fold as compared with that in the case (Fig. 7).

[0052]Example 7 Confirmation of localization of TdIF1 in cells Use pEGFPC2-TdIFl according to the manufacturer's instructions for ribofectin (given
Cos) into COS7 cells. Trang
36 hours after the infection, the localization of Gyp-TdIF1 (full length) was detected.
It was observed with a light microscope. MOL, a human T-cell leukemia cell line
T4 was cultured at 37 ° C. in RPMI1640 medium supplemented with 10% FCS. Service
COS7, a kidney cell line from Leval, is Dulbecco's with 10% FCS.
The modified Eagle medium was cultured at 37 ° C.

As shown in FIG. 8, the localization of EGFP-TdlFl in the nucleus was confirmed. In addition, the N-terminal region of TdlFl and EGFP
It was confirmed that the fusion protein with and is also localized in the nucleus. The localization of TdIF1 in the nucleus was also confirmed by immunostaining of MOLT4 cells with a rabbit polyclonal antibody against TdIF1 (data not shown).

[0054]Example 8 DNA cellulose column chromatography
Graph Single-stranded or double-stranded calf thymus DNA cellulose (sig
Column) and perform TdIF1 DNA binding.
Total activity was tested. Expressing GST-TdIFl or GST
E. coli lysate was suspended in B buffer at 4 ℃, 16,000
 Centrifuge at g for 30 minutes. Replace each supernatant with single-stranded or double-stranded DNA
Added to the cellulose column. Column has 13 times the volume of B buffer
And washed with a gradient of 0-2 M NaCl. Fractionated
Separate eluate by SDS-PAGE and perform Western blot
did. Specific to GST or TdIFl for immune response
Rabbit antibody was used. Figure 9 (a) shows DNA-cellulose color
The elution pattern of column chromatography is shown. Of each fraction
The eluate is subjected to SDS-PAGE and Western blotting
Was performed (FIG. 9 (b)). GST-TdlFl elutes at 0.6 M NaCl
(25th fraction in FIG. 9). Double-stranded DNA-cell
Similar results were obtained by sloat column chromatography
(No data).

[0055]Example 9 SDS-PAGE and Western block
Ting From glutathione beads or DNA cellulose columns
The eluate was denatured and then separated by 10% SDS-PAGE.
Transfer of polyacrylamide gel to nitrocellulose membrane
Transfer of proteins to semi-dry electrophoresis blotting
So I went. Membrane is 5% skim milk (Difco)
-Soak in PBST (PBS, 0.05% Tween 20) for 12 hours at room temperature,
The protein on the membrane is the primary antibody (bovine TdT
Rabbit polyclonal antibody) for 90 minutes. Next
The cells were allowed to react with the POD-labeled secondary antibody in PBST for 90 minutes. antibody
Protein bound to ECL Western blotting
It was detected with the detection reagent.

[0056]

EFFECTS OF THE INVENTION The gene is a gene encoding a novel protein TdIF1 having an amino acid sequence similar to that of a nuclear receptor and interacting with TdT. The protein is a drug which enhances the effect of steroid administration on acute leukemia. Can be. Further, the nucleic acid of the gene or the vector expressing the nucleic acid can also be a therapeutic agent for acute leukemia.

[0057]

[References] 1. Bollum FJ .: Terminal deoxynucleoti
dyltransferase. The Enzymes 10, 145-71 (1974). 2. Hanausek M, Szemraj J, Adams AK, Walaszek Z .:
The oncofetal proteinp65 a new member of the st
eroid / thyroid receptor superfamily.Cancer Detect
Prev. 20, 94-102 (1996). 3. Niewiadomska H, Mirowski M, Stempien M, Olbors
ki B, Blonski JZ, Hanausek M, Wierzbicki R .: 65 kD
a oncofetal protein (p65), proliferating cell nucl
ear antigen (PCNA) and Ki67 expression in breast c
ancer patients.Neoplasma 45, 216-22 (1998). 4. Hanausek M, Wang SC, Blonski JZ, Kulesza EP, W
alaszek Z, Mirowski M .: Expression of an oncofetal
65-kDa phosphoprotein in lymphocytic and granuloc
ytic leukemias. Int. J .Hematol. 63, 193-203 (199
6). 5. McCaffrey R, Harrison TA, Parkman R, Baltimore
D .: Terminal deoxynucleotidyl transferase activi
ty in human leukemic cells and in normal human thy
mocytes. N .Engl. J .Med. 292, 775-80 (1975). 6. Coleman MS, Hutton JJ, De Simone P, Bollum F
J .: Terminal deoxyribonucleotidyl transferase in h
uman leukemia. Proc. Natl. Acad. Sci. USA .71, 4
404-8 (1974). 7. Sahai Srivastava BI, Khan SA, Henderson ES .: H
igh terminal deoxynucleotidyl transferase activity
in acute myelogenous leukemia. Cancer Res. 36, 384
7-50 (1976). 8. Niewiadomska H, Mirowski M, Bartkowiak J, Stem
pien M, Blonski JZ, Walaszek Z, Hanausek M, Wierzb
icki R .: Oestrogen / progesterone receptor anda 65 k
D tumour-associated phosphoprotein (p65) expressio
n in breast cancer patients.Biomedical Letter. 5
6, 91-99 (1997). 9. Sikes ML, Suarez CC, Oltz EM .: Regulation of V
(D) J recombination by transcriptional promoters. M
ol. Cell. Biol. 19, 2773-81 (1999). 10. Takeda S, Zou YR, Bluethmann H, Kitamura D, M
uller U, Rajewsky K .: Deletion of the immunoglobuli
n kappa chain intron enhancer abolishes kappa chai
n gene rearrangement in cis but not lambda chain g
ene rearrangement in trans.EMBO J. 12, 2329-36 (1
993). 11. Chen J, Young F, Bottaro A, Stewart V, Smith
RK, Alt FW .: Mutationsof the intronic IgH enhancer
and its flanking sequences differentially affect a
ccessibility of the JH locus. EMBO J. 12, 4635-45
(1993). 12. Sleckman BP, Gorman JR, Alt FW .: Accessibilit
y control of antigen-receptor variable-region gene
assembly: role of cis-acting elements. Annu. Rev.
Immunol. 14, 459-81 (1996). 13. Bories JC, Demengeot J, Davidson L, Alt FW .:
Gene-targeted deletion and replacement mutations
of the T-cell receptor beta-chain enhancer: the ro
le of enhancer elements in controlling V (D) J recom
bination accessibility. Proc. Natl. Acad. Sci. US
A. 93, 7871-6 (1996). 14. Bouvier G, Watrin F, Naspetti M, Verthuy C, N
aquet P, Ferrier P .: Deletion of the mouse T-cell r
eceptor beta gene enhancer blocks alphabeta T-cell
development. Proc. Natl. Acad. Sci. USA .93, 7
877-81 (1996). 15. Zhong XP, Carabana J, Krangel MS .: Flanking n
uclear matrix attachment regions synergize with th
e T cell receptor delta enhancer to promoteV (D) J r
ecombination. Proc. Natl. Acad. Sci. USA .96,11
970-5 (1999). 16. Schwanbeck R, Manfioletti G, Wisniewski JR .:
Architecture of high mobility group protein IC.
DNA complex and its perturbation upon phosphorylat
ion by Cdc2 kinase. J. Biol. Chem. 275, 1793-801
(2000). 17. Koiwai O, Morita A .: Isolation of putative pr
omoter region for human terminal deoxynucleotidylt
ransferase gene.Biochem.Biophys.Res.Commun. 15
4 (1), 91-100 (1988). 18. Sanger F, Nicklen S, Coulson AR .: DNA sequen
cing with chain-terminating inhibitors. Proc. Nat
l. Acad. Sci. US A. 74, 5463-7 (1977). 19. Altschul SF, Madden TL, Schaffer AA, Zhang J,
Zhang Z, Miller W, Lipman DJ .: Gapped BLAST and P
SI-BLAST: a new generation of protein database sea
rch programs.Nucleic Acids Res. 25, 3389-402 (19
97). 20. Lai JS, Herr W .: Ethidium bromide provides a
simple tool for identifying genuine DNA-independen
t protein associations. Proc. Natl. Acad. Sci. US
A. 89, 6958-62 (1992).

[0058]

[Sequence list]                       SEQUENCE LISTING    <110> Osamu, Koiwai; Medical & Biological Laboratories, Co., Ltd. <120> A novel TdT binding protein and a gene coding for the protein <130> <160> 4 <170> PatentIn Ver. 2.1    <210> 1 <211> 329 <212> PRT <213> homo sapiens <400> 1 Met Gly Ala Thr Gly Asp Ala Glu Gln Pro Arg Gly Pro Ser Gly Ala   1 5 10 15 Glu Arg Gly Gly Leu Glu Leu Gly Asp Ala Gly Ala Ala Gly Gln Leu              20 25 3 Val Leu Thr Asn Pro Trp Asn Ile Met Ile Lys His Arg Gln Val Gln          35 40 45 Arg Arg Gly Arg Arg Ser Gln Met Thr Thr Ser Phe Thr Asp Pro Ala      50 55 60 Ile Ser Met Asp Leu Leu Arg Ala Val Leu Gln Pro Ser Ile Asn Glu  65 70 75 80 Glu Ile Gln Thr Val Phe Asn Lys Tyr Met Lys Phe Phe Gln Lys Ala                  85 90 95 Ala Leu Asn Val Arg Asp Asn Val Gly Glu Glu Val Asp Ala Glu Gln             100 105 110 Leu Ile Gln Glu Ala Cys Arg Ser Cys Leu Glu Gln Ala Lys Leu Leu         115 120 125 Phe Ser Asp Gly Glu Lys Val Ile Pro Arg Leu Thr His Glu Leu Pro     130 135 140 Gly Ile Lys Arg Gly Arg Gln Ala Glu Glu Glu Cys Ala His Arg Gly 145 150 155 160 Ser Pro Leu Pro Lys Lys Arg Lys Gly Arg Pro Pro Gly His Ile Leu                 165 170 175 Ser Ser Asp Arg Ala Ala Ala Gly Met Val Trp Lys Pro Lys Ser Cys             180 185 190 Glu Pro Ile Arg Arg Glu Gly Pro Lys Trp Asp Pro Ala Arg Leu Asn         195 200 205 Glu Ser Thr Thr Phe Val Leu Gly Ser Arg Ala Asn Lys Ala Leu Gly     210 215 220 Met Gly Gly Thr Arg Gly Arg Ile Tyr Ile Lys His Pro His Leu Phe 225 230 235 240 Lys Tyr Ala Ala Asp Pro Gln Asp Lys His Trp Leu Ala Glu Gln His                 245 250 255 His Met Arg Ala Thr Gly Gly Lys Met Ala Tyr Leu Leu Ile Glu Glu             260 265 270 Asp Ile Arg Asp Leu Ala Ala Ser Asp Asp Tyr Arg Gly Cys Leu Asp         275 280 285 Leu Lys Leu Glu Glu Leu Lys Ser Phe Val Leu Pro Ser Trp Met Val     290 295 300 Glu Lys Met Arg Lys Tyr Met Glu Thr Leu Arg Thr Glu Asn Glu His 305 310 315 320 Arg Ala Val Glu Ala Pro Pro Gln Thr                 325    <210> 2 <211> 1296 <212> DNA <213> Homo sapiens <400> 2 ggcacgaggg ccactttccg gccggtgaca gagtccagcg gagttgtggg ggccggaggc 60 gccatgggag ccactggcga cgccgagcag ccgcggggac ccagcggggc cgagaggggc 120 ggcttggagc tgggggatgc gggcgcagcg gggcagctgg ttcttacgaa cccttggaac 180 ataatgataa agcaccggca ggtgcagcgg aggggccgcc gctcacagat gacaacaagt 240 ttcacagatc ctgccatctc catggatctc ctccgagctg tcctgcagcc cagcatcaac 300 gaggagatcc agactgtctt caacaagtac atgaagttct tccagaaggc agcactgaac 360 gtgcgagaca atgttgggga ggaggtggac gcagagcagc tgatccagga agcctgtcgg 420 agctgcctgg agcaggctaa actgctcttt tcagatggag aaaaagtaat acccagattg 480 acccatgagc ttccaggaat aaagcgtggc cgtcaggcag aagaagaatg tgcccatcga 540 ggaagccccc ttcctaaaaa gaggaaagga cggcctcctg gacacatcct gtcaagcgac 600 cgggcagccg ccggcatggt atggaaacca aaatcctgtg aaccaattcg ccgggaaggc 660 cccaagtggg acccagctcg cctgaatgaa tctaccacct ttgtgttggg atctcgagcc 720 aacaaagccc tggggatggg gggcaccaga ggaagaatct acatcaagca cccacacctc 780 tttaagtatg cagctgaccc ccaggataag cactggctgg ctgagcagca tcacatgcgg 840 gcaacagggg gcaagatggc ctacctcctc atcgaggagg acatccggga ccttgcggcc 900 agtgatgatt acagaggatg cctggatctg aagctagagg aattgaaatc ctttgtccta 960 ccctcctgga tggtggagaa gatgagaaag tatatggaga cactacggac agagaatgag 1020 catcgtgctg ttgaagcacc tccacagacc tgaggccggg tcccctggcc acacttggca 1080 gccctcctcc aaagccctct tcctcacgtg gctgaggcca ccgctgggac tgctcctaga 1140 tggatctcag cggcattaag ctgtgcctga gcgagtttgt agtgactcac tgcacagcac 1200 ccccagacta gcatgtggtt ctatatttgt aaagttattg ggataagaaa caattaaaca 1260 gtttgtagta aacacagaaa aaaaaaaaaa aaaaaa 1296    <210> 3 <211> 328 <212> PRT <213> Mouse <400> 3 Met Gly Ala Thr Gly Asp Thr Glu Gln Pro Arg Gly Pro Gly Gly Ala   1 5 10 15 Glu Arg Gly Gly Leu Glu Leu Gly Asp Ala Gly Ala Ala Gly Gln Pro              20 25 30 Val Leu Thr Asn Pro Trp Asn Ile Met Ile Lys His Arg Gln Val Gln          35 40 45 Arg Arg Gly Arg Arg Ser Gln Met Thr Thr Ser Phe Thr Asp Pro Ala      50 55 60 Ile Ser Met Asp Leu Leu Arg Ala Val Leu Gln Pro Ser Ile Asn Glu  65 70 75 80 Glu Ile Gln Gly Val Phe Asn Lys Tyr Met Lys Phe Phe Gln Lys Ala                  85 90 95 Ala Leu Asn Val Arg Asp Asn Val Gly Glu Glu Val Asp Ala Glu Gln             100 105 110 Leu Ile Gln Glu Ala Cys Arg Ser Cys Leu Glu Gln Ala Lys Leu Leu         115 120 125 Phe Ser Asp Gly Glu Lys Val Ile Pro Arg Leu Ala His Glu Leu Pro     130 135 140 Gly Ile Lys Arg Gly Arg Gln Ala Glu Glu Glu Ser His Arg Gly Ser 145 150 155 160 Pro Ile Pro Lys Lys Arg Lys Gly Arg Pro Pro Gly His Val Leu Ser                 165 170 175 Asn Asp Arg Ala Ala Ala Gly Met Val Trp Lys Gln Lys Ser Cys Glu             180 185 190 Pro Ile Arg Arg Glu Gly Pro Lys Trp Asp Pro Ala Arg Leu Asn Glu         195 200 205 Ser Thr Thr Phe Val Leu Gly Ser Arg Ala Asn Lys Ala Leu Gly Met     210 215 220 Gly Gly Thr Arg Gly Arg Ile Tyr Ile Lys His Pro His Leu Phe Lys 225 230 235 240 Tyr Ala Ala Asp Pro Gln Asp Lys His Trp Leu Ala Glu Gln His His                 245 250 255 Met Arg Ala Thr Gly Gly Lys Met Ala Tyr Leu Leu Ile Glu Glu Asp             260 265 270 Ile Arg Asp Leu Ala Ala Ser Asp Asp Tyr Arg Gly Cys Leu Asp Leu         275 280 285 Lys Leu Glu Glu Leu Lys Ser Phe Val Leu Pro Ser Trp Met Val Glu     290 295 300 Lys Met Arg Lys Tyr Met Glu Thr Leu Arg Thr Glu Asn Glu His Arg 305 310 315 320 Ala Ala Glu Ala Pro Pro Gln Thr                 325    <210> 4 <211> 1235 <212> DNA <213> Mouse <400> 4 tcgagctgag gttgttgtgg gcaggggggg ccatgggggc gactggcgac accgagcagc 60 cgcggggccc cggcggggcg gagcgaggtg gcctggagct gggcgacgcg ggcgcggcgg 120 gccagccggt tctcacgaac ccttggaaca taatgataaa acatcggcag gtgcagcgaa 180 ggggccgccg atctcagatg accacaagtt tcacagaccc agccatctct atggatctcc 240 tccgtgctgt cctgcagcct agcatcaatg aggagatcca gggtgtcttc aacaagtaca 300 tgaagttctt ccagaaggca gcgctgaatg tgcgagacaa tgttggggaa gaagtggacg 360 cagagcagtt gattcaggag gcctgccgca gctgcctgga gcaggcaaag ctgctctttt 420 ctgatggaga gaaagtgata cccagattgg cccatgagct tccagggatc aagcggggcc 480 ggcaagcaga agaggagtcc caccgaggaa gccccattcc caaaaagagg aaaggtcggc 540 ctcctggaca cgtcctgtca aatgaccgcg cagctgctgg catggtatgg aaacaaaaat 600 cctgtgaacc aattcgccga gaaggcccca agtgggaccc agctcggctg aatgaatcta 660 ccacctttgt tttggggtct cgagccaaca aggccttggg gatgggaggc accagaggga 720 gaatctacat caagcaccca cacctcttta agtatgcagc agatcctcag gacaagcact 780 ggctggctga gcagcatcac atgcgggcaa caggcggaaa gatggcgtac cttctcattg 840 aagaagacat ccgggacttg gctgccagcg atgactacag aggatgcttg gacctgaagt 900 tggaggagct gaaatccttt gttctgccat cctggatggt tgagaagatg cggaaataca 960 tggagacact gcggacagaa aatgagcacc gcgctgcgga agcgcctccc cagacctgag 1020 ccgggtgtcc tggctactac acttggcggc ctgcctccaa gaccctcttt ccccacccgg 1080 ctgaggccat catggggatt tggtctagtt gactcttaac agcattaagc tgtacatgag 1140 ctagtttgta gtgactcact gcagagcacc ccagactggc atgtggttct gtttctaaag 1200 ttattggaat aagaagcaat taaacagttt gtaat 1235

[Brief description of drawings]

FIG. 1 shows human and mouse TdF1 deduced amino acid sequences.

FIG. 2 shows TdF1 genomic DNA and spliced mRNA.

FIG. 3 shows a comparison of the amino acid sequences of TdIF1 and p65.

FIG. 4 shows the amino acid sequences of the AT-hook region of TdIF1 and HMGI-C.

FIG. 5 shows Western blotting in which the TdTF1-TdIF1 complex was detected with an anti-TdT antibody. Lane 3 is a reaction solution of GST-bound beads and TdT, and lane 4 is a reaction solution of GST-TdIF1-bound beads and TdT.

FIG. 6 shows a TdT deletion plasmid.

FIG. 7 shows a comparison of TdT enzyme activity in the presence (left column) and absence (middle column) of TdIF1.

FIG. 8 shows the results of immunofluorescence microscopy showing that the expressed protein is localized in the nucleus in COS7 cells transfected with the EGFP-TdIF1 fusion protein expression plasmid.

FIG. 9 (a) shows an elution pattern of DNA-cellulose column chromatography. Anti-TdIF1 (b) and anti-GST
The Western blotting of each fraction using (c) is shown.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 15/00 ZNAA 1/21 5/00 A 5/10 A61K 37/02 (72) Inventor Osamu Osamu Osamu Osamu 2641 Yamazaki, Noda, Chiba Prefecture Tokyo University of Science Faculty of Science and Technology F-term (reference) 4B024 AA01 AA11 CA04 4B065 AB01 BA02 CA24 CA44 CA46 4C084 AA07 AA13 BA01 BA08 BA22 BA23 CA53 NA14 ZB27 4C087 AA01 BC83 CA12 ZB27 4H045 AA10 AA11 CA40 EA28 EA51

Claims (6)

[Claims] 1. A DNA of the following (a) or (b); (a) a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2; (b) a hybridized with the DNA of SEQ ID NO: 2 under stringent conditions, A DNA that encodes a protein that has the ability to bind to TdT. 2. A protein encoded by the DNA according to claim 1. 3. A protein of (a) or (b) below: (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) a deletion, substitution or addition of an amino acid in the amino acid sequence of SEQ ID NO: 1. Consists of the amino acid sequence
A protein having the binding property to TdT. 4. A recombinant vector containing the DNA according to claim 1. 5. A transformed cell transformed with the recombinant vector according to claim 4. 6. An antibody against the protein according to claim 2 or claim 3 or a partial peptide thereof.