JPH09191884A - Dna capable of coding human neurod protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA, capable of coding a human neuroD protein, having a specific amino acid sequence and expressible in a neuroblastomatous cell and a brain or a mutant of the human neuroD protein and useful as a probe or a primer, etc., for identifying the neuroblastomatous cell and the brain, etc. SOLUTION: This DNA is a new one, capable of coding a human neuroD protein, having an amino acid sequence represented by the formula and specifically expressible in a neuroblastomatous cell and a brain or a protein, prepared by adding, deleting or substituting one or plural amino acid residues in the amino acid sequence and having activities for differentiating a human nonneuronal ectoblast into a neuron and useful as a probe or a primer, etc., for identifying the neuroblastomatous cell, the brain and the neuron in the initial development of a fetus. The DNA is readily obtained by screening a cDNA library derived from an mRNA in a human neuroblastomatous cell or a human brain according to a plaque hybridization method, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to human neuroD
Relating to DNA.

[0002]

2. Description of the Related Art neuroD was introduced in 1995 by a two-hybrid system (Nature 340, 2).
45 (1989)), a gene encoding 358 amino acids isolated and identified from a mouse fetal cDNA library as a basic helix-loop helix-type transcription factor that interacts with the transcription factor E2A. It is considered to be transiently expressed in neurons of the central nervous system and peripheral nervous system (Science)
268: 836 (1995)). The neuroD cDNA has been isolated also in Xenopus laevis, and it has been shown to have a function of differentiating non-neural ectoderm and neuroblast into neurons in the Xenopus laevis system (Science 268: 836 ( 1995)).

As described above, neuroD plays an important role in the differentiation process of nerve cells in the central nervous system and peripheral nervous system. Cloning of the human neuroD gene and clarifying its structure are very important for studying the development and differentiation of the brain and nervous system.

Furthermore, if the obtained gene shows a tissue- or cancer cell-specific expression pattern, the gene fragment can recognize a tissue or a cell, or can be a gene marker effective for cancer diagnosis. .

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a gene encoding a human neuroD protein which is transiently expressed in neurons of the central nervous system and peripheral nervous system of the fetal period. The present invention further provides a human neuroD protein encoded by the gene.

Further, the present invention provides a DNA fragment useful as a probe or a primer for identifying nerve cells in the early stage of fetal development.

[0007]

[Means for Solving the Problems] As a result of intensive studies, the present inventors have found that a cDNA encoding human neuroD.
Was isolated, its base sequence was determined, and its amino acid sequence was deduced. As a result, it was revealed that the human neuroD protein has the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing described later. In this specification, “
neuroD "means a basic helix-loop-helix type transcription factor that interacts with the transcription factor E2A.

Hereinafter, the present invention will be described in detail.

NeuroD gene The neuroD gene of the present invention has the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or one or more amino acids added, deleted or substituted in the amino acid sequence,
It is also a DNA encoding a protein having the biological activity of human neuroD. The DNA of the present invention is human ne
The fact that it encodes a protein having the biological activity of uroD was confirmed by having 95% homology with mouse neuroD reported in Science 268: 836 (1995).

Human neuroD determined by the present invention
The nucleotide sequence of the gene encoding is shown in SEQ ID NO: 2 together with the deduced amino acid sequence encoded by it. As is clear from this sequence, this cDNA has base number 1
To 1068 total 1068 base pairs. Base numbers 1 to 3 correspond to the translation initiation genetic code, and base number 106
6 to 1068 correspond to the translation-stopped genetic code. The sequence of base numbers 1066 to 1068 (***) is T
It may be GA, TAA or TAG. SEQ ID NO: 1 shows the amino acid deduced from the base sequence shown in SEQ ID NO: 2.

Therefore, the gene of the present invention can be obtained from a cDNA library derived from human neuroblastoma cell or human brain mRNA, for example, by using the plaque hybridization method, as described in the following Examples. However, based on the nucleotide sequence of DNA determined by the present invention, mRNA of human neuroblastoma cells or human brain
It can also be easily prepared by a PCR method using a cDNA library derived from the template, or an RT-PCR method using an mRNA of human neuroblastoma cells or human brain as a starting material. Human neuroD c thus prepared
A mutant can also be prepared using DNA by the method described below.

Since there are a plurality of codons coding for one amino acid, DNA having any base sequence is included in the scope of the present invention as long as the coded amino acid sequences are the same. Therefore, any DNA encoding the amino acid sequence represented by SEQ ID NO: 1 is included in the scope of the present invention.

Furthermore, in general, when the amino acid sequence of a peptide having physiological activity is slightly changed, that is, one or more amino acids in the amino acid sequence are substituted or deleted, or one or more amino acids are added. It is a well-known fact that the physiological activity of the peptide may be maintained even when the treatment is performed. Therefore, such a modification is added to the amino acid sequence shown in SEQ ID NO: 1,
And human neuro having biological activity of human neuroD
Variants of the oD protein are also within the scope of this invention. Further, a DNA encoding the mutant protein is also included in the scope of the present invention.

Mutants by addition, deletion or substitution of amino acids can be obtained by, for example, site-directed mutagenesis (eg, Nucleic Acid) which is well known in the art, for example, in the DNA encoding the same.
Research, Vol.10, No.20, p.6487-6500, 1982). As used herein, the term "one or more amino acids" means a number of amino acids that can be added, deleted or substituted by site-directed mutagenesis.

The site-directed mutagenesis method is carried out as follows, for example, using a synthetic oligonucleotide primer complementary to the single-stranded phage DNA to be mutated except for a specific mismatch which is a desired mutation. be able to. That is,
A double-stranded DNA obtained by synthesizing a strand complementary to a phage using the above-mentioned synthetic oligonucleotide as a primer
Transform the host cells with A. A culture of transformed bacteria is plated on agar and plaques are formed from single cells containing phage. Then, theoretically, 50% of the new colonies contain the phage having the mutation as a single strand, and the remaining 50% have the original sequence. The obtained plaque is hybridized with a kinase-synthesized synthetic probe at a temperature at which it hybridizes with a DNA having the desired mutation but does not hybridize with a DNA having the original strand. Let Next, a plaque that hybridizes with the probe is picked up, cultured, and the DNA is recovered.

In addition to the above-mentioned site-directed mutagenesis, other methods for making substitutions, deletions or insertions of one or more amino acids in the amino acid sequence of a biologically active peptide such as an enzyme without losing its activity are There is also a method of treating a gene with a mutagen and a method of selectively cleaving the gene, then removing, adding or substituting a selected nucleotide, and then ligating. Both ends of the DNA of the present invention, that is, the translation start code and the translation stop code, are arbitrary D
It can bind to NA fragments. The size of the nucleotide sequence of this DNA fragment is not important, and any DNA fragment suitable for ligation with an extranuclear gene possessed by bacteria or the like may be used. For example, in the following Examples, the translation initiation code has a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 3, and the translation termination code has a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 4.
Each is associated with a fragment.

Variants may include conservatively substituted sequences, which means that a particular amino acid residue may be replaced by a residue having similar physicochemical characteristics. ing. Non-limiting examples of conservative substitutions include substitutions between aliphatic group-containing amino acid residues such as Ile, Val, Leu or Ala mutual substitutions, or polar group-containing amino acid residues such as between Lys and Arg. Intervening substitutions are included.

Nucleotide sequences that fall within the scope of the present invention include human neuroD that hybridizes to the human neuroD nucleotide sequences disclosed herein under mild or harsh stringency conditions and that is biologically active. Isolated DNA and RNA encoding Conditions for mild stringency hybridization are described, for example, in Sambrook et al., Molecular Cloning: A Labor.
atory Manual, 2 ed. Vol.1, pp.1. 101-104, Cold Spr
ing Harbor Laboratory Press, (1989). Mild stringency conditions, as defined by Sambrook et al., Include 5 × SS.
This includes the use of a prewash solution of C, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions at about 55 ° C., 5 × SSC, overnight. Harsh stringency conditions include higher temperature hybridization and washing. At that time, the temperature and the salt concentration of the washing solution are appropriately adjusted according to various factors such as the length of the probe.

Study on differentiation and development of human neuroD gene
Use as a research reagent The DNA of the human neuroD gene of the present invention can be used for comparing differences in expression patterns in human organs and cells. This promotes research on human organs and cell differentiation and development. For example, the present inventors show that human neuroD is expressed in human liver carcinoma cell line, human cervical carcinoma cell line, human adult pancreas, kidney, skeletal muscle, liver, lung, placenta, and heart based on the results of Northern analysis. Notably, it was found to be expressed only in human neuroblastoma cells and human adult brain.

As a probe for the human neuroD gene
Use of the present invention is based on the finding that human neuroD is expressed only in human neuroblastoma cells and adult human brain as described above, and thus a DNA fragment that specifically hybridizes to the sequence of human neuroD gene was obtained. Techniques are provided that are labeled with appropriate labels to be used as probes to identify human neuroblastoma cells and human adult brain.

The probe may be designed based on any of the sequences of SEQ ID NO: 2, 3 or 4. The length is preferably at least 15 continuous bases or more,
The length may be any length up to the total length of each SEQ ID NO: or the total length of SEQ ID NO: 3, 2 and 4 as long as it is 15 bases or more. For example, one that can be used as a probe has the sequence of SEQ ID NO: 5. The probe may be single-stranded or double-stranded, but will be single-stranded at least when used. Furthermore, as described above, the nucleotide sequences 2, 3
Alternatively, the DNA fragment probe selected from 4 is modified by addition, deletion, alteration, etc. of bases so as not to lose the property of specifically hybridizing to the human neuroD gene.

Without limitation, the probe is an isolated ne obtained by the method disclosed herein.
It can be prepared by cleaving the cDNA fragment of the uroD gene with an appropriate restriction enzyme. Or neur
The probe can also be prepared by performing a PCR reaction using a sample containing the oD gene. Or,
Single-stranded DN to be used as a probe by a method commonly used with a commercially available DNA synthesizer (for example, Perkinermer)
A can also be synthesized.

The probe can be labeled by a conventional method, for example, a radioisotope, a detectable enzyme or the like. For example, when ³² P is used, it is convenient to label it with a random priming label when using a cDNA fragment of the neuroD gene, and to label the 5 ′ end with a phosphorylating enzyme when using a synthetic primer. Hybridization between the sample expected to contain neuroD and the probe of the present invention can be carried out by a commonly used method. Generally, it is performed at moderate hybridization strength (hybridization at 42 ° C to 50 ° C and washing with 0.1xSSC).

For example, mRN extracted from the bone marrow fluid of a patient
When A is electrophoresed and the hybridization between the substance transferred to a nylon membrane and the probe is detected by an autoradiograph or the like, the sample is diagnosed as containing neuroblastoma cells. Neuroblastoma cells are cancers that develop in children's adrenal medulla and systemic sympathetic ganglia, and are characterized by a tendency to metastasize to bone. The examination for childhood cancer has been performed so far by detecting the neuroblasts contained in the urine, but in some cases, the neuroblasts are not excreted in the urine. Therefore, by examining whether or not neuroblastoma cells are contained in the bone marrow fluid, it is possible to improve the accuracy of pediatric cancer detection.

PCR primer for human neuroD gene
Use as a primer A DNA fragment that hybridizes with the sequence of the human neuroD gene or a part of its complementary strand can also be used as a PCR primer. PCR is carried out using such a set of DNA fragments as primers to determine the mRNA in the sample.
The presence of human neuroblastoma cells or brain cells can be examined by examining whether or not the cDNA prepared from is amplified.

Two of the primers of the present invention can be selected from SEQ ID NO: 2, 3 or 4 so as to satisfy the following conditions.

1) The length of the primer is 15-30 bases,
20 to 25 bases are preferable; 2) G (guanine) + C (cytosine) in the primer
Is 40-60%, preferably 45-55%, more preferably about 50%; 3) A (adenine), T contained in the primer sequence.
Since the distribution of (thymine), G (guanine), and C (cytosine) is not partially biased, for example, a region where GT is repeatedly distributed is considered to have low specificity, it cannot be used as a primer.

4) The distance on the nucleotide sequence of the human neuroD gene corresponding to the two selected primers is 100.
-1000 bases, preferably. 150-500 bases, more preferably 15-300 bases; and 5) there is no complementary sequence portion within each primer itself or between the two primers.

Examples of preferable primers are as follows.

5'side primer: 5'-CACATCA
TGAGCGAGTCATG-3 ′ (base numbers 1019 to 1138 of SEQ ID NO: 2) 3 ′ side primer: 5′-TAGGCTCAGATTT
ATTACAT-3 ′ (base number 1036 of SEQ ID NO: 4
Complementary sequence of -1055) These primers may be used in combination of two,
Each may be used in combination with another suitable DNA fragment. In addition, the preferred primer described above is added to neuroD
In order not to lose the property of specifically hybridizing to
Sequences modified by adding, deleting or changing bases are also PCR
It can be used as a primer.

If the primer sequences are selected, commercially available D
Single-stranded DNA as a primer can be synthesized by a method commonly used with an NA synthesizer (for example, Perkin Elmer). The primer may be single-stranded or double-stranded, but will be single-stranded when used.

Furthermore, the base sequence selected from SEQ ID NO: 2, 3 or 4 so as to satisfy the above selection condition is
A sequence modified by addition, deletion, change, etc. of bases is also included in the scope of the present invention so as not to lose the property of specifically hybridizing to uroD.

The polymerase used in the DNA strand synthesis reaction of PCR is preferably a thermotolerant polymerase (typically Taq polymerase). Polymerase for PCR reaction, deoxyribonucleic acid (dATP, dCTP, d
GTP, dTTP), reaction solution and the like are commercially available as a kit if necessary.

The PCR reaction has one cycle of a series of replication reactions consisting of three steps of annealing of a cDNA prepared from mRNA in a sample and a primer, a polymerase reaction and a heat denaturation. Although not necessarily limited to the following conditions, in the present invention, the annealing is performed at 35 to 70 ° C., preferably 45 to 55 ° C., more preferably about 50 ° C. for 20 seconds to 2 minutes, preferably about 1 Do minutes.
The polymerase reaction is carried out at 60-80 ° C, preferably 70-
At 75 ° C. for 1 to 3 minutes. Heat denaturation at 90-95 ° C for 3
Perform 0 seconds to 1 minute. The number of cycles is preferably 25 to 35 times. The PCR reaction can be performed using a commercially available automatic device for PCR (for example, manufactured by Perkin Elmer).

As a result of the PCR reaction, for example, when the neuroD gene is present in the cDNA prepared from the mRNA of the bone marrow fluid of the patient, the region between the template DNAs annealed by the two kinds of primers used is amplified. When the solution after the reaction is electrophoresed in a medium such as agarose or polyacrylamide, a band having a length expected from the used primer can be detected. If bands are detected in this way, the presence of neuroblastoma cells in the sample is diagnosed.

Production of Recombinant NeuroD Polypeptide Further, the present invention provides that expression vectors containing human neuroD cDNA and host cells containing these expression vectors are cultured under conditions suitable for expression of neuroD, and the expressed neuroD is expressed. Also provided is a method of producing a recombinant neuroD polypeptide by recovering

In order to produce the recombinant neuroD polypeptide of the present invention, a suitable expression vector selected depending on the host cell used is derived from a mammalian, microbial, viral, or insect gene. Insert a human neuroD DNA sequence linked to transcriptional or translational regulatory nucleotide sequences. Examples of regulatory sequences include transcription promoters, operators or enhancers, mRNA ribosome binding sites, and appropriate sequences controlling initiation and termination of transcription and translation. In addition, human neuro
A sequence encoding an appropriate signal peptide that is not naturally present in the D gene may be incorporated into the expression vector.

Suitable host cells for the expression of human neuroD polypeptide include prokaryotic cells, yeast or higher eukaryotic cells. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manu.
al, Elsevier, New York, (1985).

Prokaryotes include Gram-negative or Gram-positive bacteria such as E. coli or Bacillus subtilis. In prokaryotes such as E. coli, the neuroD polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in prokaryotes. This N-terminal Met can be cleaved from the recombinant neuroD polypeptide after expression.

The expression vector used in the prokaryotic host cell is
Generally, it contains one or more phenotype selectable marker genes. A phenotype selectable marker gene is, for example, a gene that confers antibiotic resistance or auxotrophy. Examples of expression vectors suitable for prokaryotic host cells include commercially available plasmids such as pBR322 (ATCC37017) or those derived therefrom. Since pBR322 contains genes for ampicillin and tetracycline resistance, it is easy to identify transformed cells. Suitable promoter and n
The euroD DNA sequence is inserted into this pBR322 vector. Other commercially available vectors include, for example, p
KK223-3 (Pharmacia F, Uppsala, Sweden)
ine Chemicals) and pGEM1 (Promega Biotec, Madison, Wisconsin, USA).

Promoter sequences commonly used in expression vectors for prokaryotic host cells include β-lactamase (penicillinase), the lactose promoter (Chang et al., N.
ature 275: 615, 1978; and Goeddel et al., Nature 281: 54.
4, 1979) etc. are included. A particularly useful prokaryotic host cell expression system is the phage λP _L promoter and cI857ts
A thermostable repressor sequence is used. Plasmid vectors available from the American Type Culture Collection that incorporate derivatives of the λP _L promoter include plasmid pHUB2 (E. coli strain JMB9 (A
TCC37092)) and pPLc28 (present in E. coli RP1 (ATCC53082)).

Human neuroD may also be expressed in yeast host cells. Preferably, the genus Saccharomyces (eg, S. cerevisiae) is used, but Pichia
Alternatively, other yeast genera such as Kluyveromyces may be used. The yeast vector is a sequence of origin of replication from 2μ yeast plasmid, an autonomously replicating sequence (ARS),
It often contains a promoter region, sequences for polyadenylation, sequences for termination of transcription, and a selectable marker gene. Using the yeast alpha factor leader sequence,
Secretion of human neuroD polypeptide can also be effected. Other leader sequences suitable for promoting secretion of a recombinant polypeptide from a yeast host are also known. Methods for transforming yeast are described, for example, in Hinnen et al., Proc. Nat.
USA Acad. Sci. USA 75: 1929, 1978.

Mammalian or insect host cell culture systems can also be used to express recombinant human neuroD polypeptide. Cell lines of mammalian origin can also be used. Transcription and translation control sequences for mammalian host cell expression vectors can be obtained from the viral genome. Commonly used promoter and enhancer sequences are derived from polyomavirus, adenovirus 2 and the like. SV40 viral genome, eg SV
40 origin, early and late promoters, enhancers,
DNA sequences derived from splice sites and polyadenylation sites may be used to provide other genetic elements for expression of the structural gene sequences in mammalian host cells. Expression vectors for use in mammalian host cells are described, for example, by Okayama and Berg (Mol. Cell. Biol. 3:28.
0, 1983).

One method for producing the human neuroD protein of the present invention is D encoding human neuroD.
Culturing a host cell transformed with an expression vector containing the NA sequence under conditions in which human neuroD is expressed. Then, depending on the expression system used, human neu
The roD protein is recovered from the culture medium or cell extract. The procedure for purifying recombinant human neuroD will be appropriately selected according to factors such as the type of host cell used and whether human neuroD is secreted into the culture medium.

The human neuroD protein of the present invention is
It is expected to be a valuable reagent for studying the tissue differentiation process in the human brain and for studying the signal transduction mechanism in the human nervous system. Furthermore, as a result of such studies human n
If the action of the euroD protein is clarified, human n
It is expected that the euroD protein itself or its inhibitor can be used as a therapeutic drug for nervous system diseases.

[0046]

The present invention will be described below with reference to examples. In the following examples, in the nucleic acid sequence of SEQ ID NO: 2, the nucleic acid having the sequence represented by SEQ ID NO: 3 at the base of base number 1 is:
D having a base sequence in which the nucleic acid having the sequence represented by SEQ ID NO: 4 is bound to the base of base number 1071
A method for manufacturing NA will be described. However, it is obvious that the present invention is not limited to the following examples. Unless otherwise specified, each operation in the following examples was performed by Maniatis et al.
Cloning Laboratory Manual Second Edition (198
9), according to the method described in Cold Spring Harbor. In addition, as the restriction enzyme, a commercially available one was used, and the specific usage thereof was in accordance with the instructions for the commercially available product. Furthermore, the neuroblastoma cells CHP134, LA-N- used in the examples.
1, LA-N-2, LA-N-5, MC-NB-1, N
B69 and human hepatoma-derived cell line HepG2 are available from RIKEN Cell Development Bank, HeLa,
IMR32 is Japan of Cancer Research Promotion Foundation
ese Cancer Research Resou
Available from rcers Bank.

(1) m of human neuroblastoma cell CHP134
RNA preparation In approximately 10 ⁷ human neuroblastoma cells CHP134 (derived or available) 18 ml of D solution (4M guanidine thiocyanate, 25 mM sodium citrate, 0.
5% sarcosyl, 0.1M 2-mercaptoethanol) was added, and the cells were peeled and lysed with a rubber policeman, followed by rapid homogenization. The obtained homogenate was transferred to a 50 ml centrifuge tube, and 2 ml of 2M sodium acetate (pH 4) was added and mixed. Next, 20 ml of water-saturated phenol was added and mixed. Finally, 5 ml of chloroform was added and mixed, and then ice-cooled for 15 minutes. Then, it was centrifuged at 4 ° C. at 4000 × g for 1 hour. After centrifugation, the aqueous layer was transferred to another centrifuge tube, and 32 ml of ethanol was added to the aqueous layer and mixed. RNA was precipitated by placing it at -20 ° C for 1 hour. RNA recovered by centrifugation
Was washed with 80% ethanol, vacuum dried, and dissolved in sterile distilled water. Next, the obtained RNA aqueous solution was treated according to the following procedure to separate mRNA having poly (A).
That is, 2 × extraction buffer (20 mM Tris-HCl, 2 mM EDTA, 0.1 mM) was added to RNA dissolved in sterile distilled water.
2% SDS) and O sold by Takara Shuzo Co., Ltd.
Ligotex-dT30 solution was added in an amount of 2 times and stirred. After stirring, heat treatment was performed at 65 ° C. for 5 minutes and ice cooling for 3 minutes. After cooling with ice, 1/10 volume of 5M NaCl solution was added,
Oligotex-dT30 bound to mRNA was recovered by centrifugation at 37 ° C. for 10 minutes. The recovered Oligotex-dT30 was washed with a washing buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1
% SDS, 0.1 M NaCl). After washing, add sterilized distilled water, heat treatment at 65 ℃ for 5 minutes,
The mRNA was eluted. MRN eluted by centrifugation
A was isolated and recovered by ethanol precipitation. The collected mRNA is washed with 80% ethanol, vacuum dried,
It was dissolved in sterile distilled water. Using this mRNA, the following c
DNA synthesis was performed.

(2) 3'directed cDNA
Preparation of library By the method of Okubo et al. (Dna Seq. 2 (1991) 137-144), 3'direct using pUC119 as a vector, using the mRNA of the human neuroblastoma cell CHP134.
An ed cDNA library was prepared. By this method, a plasmid library in which the sequence at the 3'end of the cDNA was inserted between the BamHI site and the PstI site of pUC119 was prepared.

Approximately 1300 randomly selected from this library
The cDNA nucleotide sequence of the clone was analyzed. From among the analyzed clones, a clone considered to be specifically expressed in human neuroblastoma cell CHP134 was selected. The selection was made in the laboratory of Professor Kenichi Matsubara of the Center for Cell Biotechnology, Osaka University (Gene 135
(1993) 265-274) Gene Expression Information Data Bank (Body
expression map of human genes) was used.

(3) Confirmation of Expression of GS007656 Gene in Human Cell Lines and Human Organs As one of clones which are considered to be specifically expressed in human neuroblastoma cell CHP134 and brain and nervous system, GS007656 gene Was selected. The nucleotide sequence of the GS007656 gene is shown as SEQ ID NO: 5 in the sequence listing below. The expression of GS007656 gene in human cell lines and human organs was analyzed using the Northern analysis method. As an analysis of human cell lines, human neuroblastoma cell line CHP13
4, LA-N-1, LA-N-2, LA-N-5, MC
-NB-1, NB69, IMR32, human hepatocellular carcinoma HepG2, human cervical cancer cell line HeLa-derived total RN
After electrophoresing 30 μg of A on agarose, a transferred nylon membrane was prepared. In addition, mRN derived from human brain, pancreas, kidney, skeletal muscle, liver, lung, placenta, and heart
After electrophoresing 2 μg of each A on an agarose gel, a transferred nylon membrane (manufactured by Clontech) was purchased and used for analysis. These nylon membranes were placed in a HYBRIPAK and 10 ml of hybridization buffer (5 × SSPE, 50% formamide, 5%
× Denhardt's, 0.5% sodium dodecyl sulfate, 40
μg / ml salmon sperm DNA) was added and reacted at 42 ° C. for 1 hour.

The ³² P label of the probe is the B of pUC119.
GS0076 inserted between the amHI and PstI sites
It was performed using a PCR fragment of 56 genes as a template and a multi-primer kit (manufactured by Amersham). This ³² P-labeled probe was added to the above reaction solution, and a hybridization reaction was further performed for 14 hours or more. Then filter the filter with 0.1% sodium dodecyl sulfate 500
Wash with 2 ml of 2 × SSC at 50 ° C. twice for 20 minutes each, and further add 0.1% sodium dodecyl sulfate 50
It was washed with 0 ml of 0.1 × SSC twice at 50 ° C. for 20 minutes each. Then, autoradiography was performed at -70 ° C for 2 days using an intensifying screen. The results are shown in FIG. The base sequences of the primers used in the above PCR are as follows.

5'-TGTAAAACGACGGCCAGT-3 '5'-ACCATGATTACGCCAAGCTTG-3' GS007656 gene is a human liver cancer cell line, human cervical cancer cell line, human adult pancreas, kidney, skeletal muscle, liver, lung, placenta, It is found that it is not expressed in the heart, but is expressed only in human neuroblastoma cells and human adult brain. GS
[0076] The gene may be used as a gene marker for human neuroblastoma cells and human adult brain.

(4) m of human neuroblastoma cell IMR32
Preparation of RNA To obtain the full-length cDNA of GS007656 gene,
A library was prepared. First, human neuroblastoma cells I
MR32 mRNA was prepared. To about 10 ⁷ human neuroblastoma cells IMR32, 18 ml of D solution (4M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1M 2-mercaptoethanol) was added, and the cells were peeled with a rubber policeman. After dissolving, it was quickly homogenized. The obtained homogenate was transferred to a 50 ml centrifuge tube, and 2 ml of 2M sodium acetate (pH 4) was added and mixed. Next, 20 ml of water-saturated phenol was added and mixed. Finally, 5 ml of chloroform was added and mixed, and then ice-cooled for 15 minutes. Then, it was centrifuged at 4 ° C. at 4000 × g for 1 hour. After centrifugation, the aqueous layer was transferred to another centrifuge tube, and 32 ml of ethanol was added to the aqueous layer and mixed. RNA was precipitated by placing it at -20 ° C for 1 hour. RNA recovered by centrifugation
Was washed with 80% ethanol, vacuum dried, and dissolved in sterile distilled water. Next, the obtained RNA aqueous solution was treated according to the following procedure to separate mRNA having poly (A).
That is, RNA dissolved in sterile distilled water has an equal amount of 2 × extraction buffer (20 mM Tris-HCl, 2 mM EDT).
A, 0.2% SDS) and Oligotex-dT30 solution commercially available from Takara Shuzo Co., Ltd.
Stirred. After stirring, heat treatment was performed at 65 ° C. for 5 minutes and ice cooling for 3 minutes. After cooling with ice, 1/10 volume of 5M NaCl solution was added, and the mixture was allowed to stand at 37 ° C for 10 minutes, and then centrifuged for mRN.
Oligotex-dT30 bound to A was recovered. The recovered Oligotex-dT30 was washed with a washing buffer (10 mM Tris-HCl, 1 mM EDTA,
Wash with 0.1% SDS, 0.1M NaCl). After washing, sterile distilled water was added, and heat treatment was performed at 65 ° C. for 5 minutes to elute mRNA. M eluted by centrifugation
RNA was isolated and recovered by ethanol precipitation. The recovered mRNA was washed with 80% ethanol, vacuum dried, and dissolved in sterile distilled water. The following cDNA synthesis was performed using this mRNA. (5) Preparation of cDNA library GIBCO was used to synthesize cDNA from the prepared mRNA.
Reverse transcriptase SUPER S commercially available from BRL
It was performed using CRIPT II according to the protocol attached to the product. After synthesizing cDNA complementary to mRNA by reverse transcriptase, ribonuclease H
Then, by inserting a nick and a gap in the RNA chain, and repair-synthesizing this with E. coli DNA ligase, RN
The A chain was replaced with cDNA. Finally, both ends of the cDNA chain were blunted with T4 DNA polymerase. Next, λMOSS lox commercially available from Amersham
A cDNA library was created using the library system. All methods were performed according to the protocol attached to the product. That is, first, EcoRI adapters were added to both ends of the synthetic cDNA, and this was previously
It was connected to a λMOSS lox arm that had been cut with oRI. Then, in vitro packaging was performed and the Escherichia coli ER strain (attached to the product) was infected to prepare a cDNA library.

(6) Full length cDNA of GS007656 gene
Selection of clones Of the cDNA library prepared according to the above (5), about 100,000 phage groups were absorbed by Escherichia coli ER strain, plated on an agar plate, and cultured at 37 ° C for 12 hours. Place the nylon membrane filter on the agar and let it stand for 1 minute to allow the phage to adsorb to the filter, then remove the filter from the agar, and denature the solution (0.5 M NaOH,
A denaturation treatment was carried out for 5 minutes in (1.5 M NaCl). After sufficiently removing the denaturing solution on a paper towel, perform a neutralization reaction in a neutralizing solution (3M sodium acetate pH 5.5) for 5 minutes and irradiate with ultraviolet light, and then 2 × SSC (1 × SSC is
It was washed for 30 minutes in 15 mM sodium citrate-hydrochloric acid buffer containing 0.15 M sodium chloride, pH 7). This filter was immersed in a hybridization solution (7% polyethylene glycol 6000, 10% sodium dodecyl sulfate) and reacted at 65 ° C for 1 hour.

³² P labeling of the probe was carried out using a multi-primer kit (manufactured by Amersham) using the PCR fragment of the GS007656 gene prepared in (3) above as a template. This ³² P-labeled probe was added to the above reaction solution, and a hybridization reaction was further performed for 14 hours or more. Thereafter, the filter was washed twice with 500 ml of 2 × SSC containing 0.1% sodium dodecyl sulfate at 65 ° C. for 20 minutes each time, and further washed with 500 ml of 0.1 × SSC containing 0.1% sodium dodecyl sulfate.
It was washed twice at 5 ° C. for 20 minutes each. Then, autoradiography was performed at -70 ° C for 2 days using an intensifying screen.

A DNA probe labeled with ³² P binds to the phage DNA in which the cDNA containing the sequence of the GS007656 gene used as a probe is inserted. Therefore, the portion corresponding to the plaque of the phage DNA adsorbed on the nylon membrane filter produces black dots on the X-ray film. The plaque phage that produced this sunspot were isolated from the agar plate, and the cDNA of about 2.5 kbp long was isolated.
A clone with A inserted was obtained.

Escherichia coli BM from the obtained phage clone
The plasmid containing the insert fragment was recovered by a method using a strain (attached to the product of Amersham). All the detailed methods at this time followed the protocol of Amersham.

(7) Determination of Total Nucleotide Sequence of cDNA Insert Fragment The entire nucleotide sequence of the cDNA insert fragment was determined by the primer walking method using a dideoxy terminator kit (manufactured by ABI). The determined entire base sequences are shown as SEQ ID NOS: 2, 3 and 4. Here, the sequence represented by SEQ ID NO: 3 is bound to the base of base number 1 of the sequence represented by SEQ ID NO: 2, and the sequence represented by SEQ ID NO: 4 is the base of sequence represented by SEQ ID NO: 2. It is bound to the base number 1068. The clone pHneuroD contains 2520 base pairs as an inserted DNA fragment and has the translation initiation genetic code ATG.
To the translation-arrested genetic code TAG had 1068 base pairs as the largest continuous open reading frame. The amino acid sequence derived from the DNA base sequence represented by SEQ ID NO: 2 is shown in the sequence listing as SEQ ID NO: 1.

D inserted in the clone pHneuroD
The amino acid sequence deduced from the base sequence of the NA fragment showed homology with the amino acid sequence of mouse neuroD.

[0060]

INDUSTRIAL APPLICABILITY As described above, the present invention provides a gene encoding human neuroD. This gene can be used as a probe or primer for identifying human neuroblastoma cells or human adult brain, i.e. as a probe or primer for identifying human neuroblastoma or human adult brain according to the present invention. Possible DNA fragments were provided.

[0061]

[Sequence list]

SEQ ID NO: 1 Sequence length: 356 amino acid residues Sequence type: Amino acid Number of chains: Single chain Sequence type: Protein sequence Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro 1 5 10 15 Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu 20 25 30 Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala 35 40 45 Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu 50 55 60 Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gln Lys 65 70 75 80 Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu 85 90 95 Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110 Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120 125 Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg 130 135 140 Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly 145 150 155 160 Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly Leu 165 170 175 Ser Gln Pro Thr Thr Asn Leu Val Gly Gly Cys Leu Gln Leu Asn Pro 180 185 190 Arg Thr Phe Leu Pro Glu Gln Asn Gln Asp Met Pro Pro His Leu Pro 195 200 205 Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro 210 215 220 Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe 225 230 235 240 His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe 245 250 255 Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro 260 265 270 Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu 275 280 285 Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro 290 295 300 Ala Ala Thr Leu Ala Gly Ala Gln Ser His Gly Ser Ile Phe Ser Gly 305 310 315 320 Thr Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe 325 330 335 Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gln Leu Asn Ala 340 345 350 Ile Phe His Asp 355

SEQ ID NO: 2 Sequence length: 1071 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA sequence ATG ACC AAA TCG TAC AGC GAG AGT GGG CTG ATG GGC GAG CCT CAG CCC 48 Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro 1 5 10 15 CAA GGT CCT CCA AGC TGG ACA GAC GAG TGT CTC AGT TCT CAG GAC GAG 96 Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu 20 25 30 GAG CAC GAG GCA GAC AAG AAG GAG GAC GAC CTC GAA GCC ATG AAC GCA 144 Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala 35 40 45 GAG GAG GAC TCA CTG AGG AAC GGG GGA GAG GAG GAG GAC GAA GAT GAG 192 Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu 50 55 60 GAC CTG GAA GAG GAG GAA GAA GAG GAA GAG GAG GAT GAC GAT CAA AAG 240 Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gln Lys 65 70 75 80 CCC AAG AGA CGC GGC CCC AAA AAG AAG AAG ATG ACT AAG GCT CGC CTG 288 Pro Lys Arg Arg Gly Pro Lys Lys Lys Ly s Met Thr Lys Ala Arg Leu 85 90 95 GAG CGT TTT AAA TTG AGA CGC ATG AAG GCT AAC GCC CGG GAG CGG AAC 336 Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110 CGC ATG CAC GGA CTG AAC GCG GCG CTA GAC AAC CTG CGC AAG GTG GTG 384 Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120 125 CCT TGC TAT TCT AAG ACG CAG AAG CTG TCC AAA ATC GAG ACT CTG CGC 432 Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg 130 135 140 TTG GCC AAG AAC TAC ATC TGG GCT CTG TCG GAG ATC CTG CGC TCA GGC 480 Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly 145 150 155 160 AAA AGC CCA GAC CTG GTC TCC TTC GTT CAG ACG CTT TGC AAG GGC TTA 528 Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly Leu 165 170 175 TCC CAA CCC ACC ACC AAC CTG GTT GGG GGC TGC CTG CAA CTC AAT CCT 576 Ser Gln Pro Thr Thr Asn Leu Val Gly Gly Cys Leu Gln Leu Asn Pro 180 185 190 CGG ACT TTT CTG CCT GAG CAG AAC CAG GAC ATG CCC CCC CAC CTG CCG 624 Arg Thr Phe Leu Pro Glu Gln A sn Gln Asp Met Pro Pro His Leu Pro 195 200 205 ACG GCC AGC GCT TCC TTC CCT GTA CAC CCC TAC TCC TAC CAG TCG CCT 672 Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro 210 215 220 GGG CTG CCC AGT CCG CCT TAC GGT ACC ATG GAC AGC TCC CAT GTC TTC 720 Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe 225 230 235 240 CAC GTT AAG CCT CCG CCG CAC GCC TAC AGC GCA GCG CTG GAG CCC TTC 768 His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe 245 250 255 TTT GAA AGC CCT CTG ACT GAT TGC ACC AGC CCT TCC TTT GAT GGA CCC 816 Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Ser Phe Asp Gly Pro 260 265 270 CTC AGC CCG CCG CTC AGC ATC AAT GGC AAC TTC TCT TTC AAA CAC GAA 864 Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu 275 280 285 CCG TCC GCC GAG TTT GAG AAA AAT TAT GCC TTT ACC ATG CAC TAT CCT 912 Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro 290 295 300 GCA GCG ACA CTG GCA GGG GCC CAA AGC CAC GGA TCA ATC TTC TCA GGC 960 Ala Ala Thr Leu A la Gly Ala Gln Ser His Gly Ser Ile Phe Ser Gly 305 310 315 320 ACC GCT GCC CCT CGC TGC GAG ATC CCC ATA GAC AAT ATT ATG TCC TTC 1008 Thr Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe 325 330 335 GAT AGC CAT TCA CAT CAT GAG CGA GTC ATG AGT GCC CAG CTC AAT GCC 1056 Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gln Leu Asn Ala 340 345 350 ATA TTT CAT GAT TAG 1071 Ile Phe His Asp ** * 355

SEQ ID NO: 3 Sequence length: 109 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA sequence CGGCCACGAC ACGAGGAATT CGCCCACGCA GGAGGCACGG CGTCCGGAGG CCCCAGGGTT 60 ATGAGACTAT CACTGCTCAG GACCTACTAAT CAACAAAG 109

SEQ ID NO: 4 Sequence length: 1340 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA sequence AGGCACGCCA GTTTCACCAT TTCCGGGAAA CGAACCCACT GTGCTTACAG TGACTGTCGT 60 GTTTACAAAA GGCAGCCCTT TGGGAGTACTAC TGCTGCAA CCAAGCTTCA 120 AGTGATATAT GTATTTATTG TCATTACTGC CTTTGGAAGA AACAGGGGAT CAAAGTTCCT 180 GTTCACCTTA TGTATTATTT TCTATAGCTC TTCTATTTAA AAAATAAAAA AATACAGTAA 240 AGTTTAAAAA ATACACCACG AATTTGGTGT GGCTGTATTC AGATCGTATT AATTATCTGA 300 TCGGGATAAC AAAATCACAA GCAATAATTA GGATCTATGC AATTTTTAAA CTAGTAATGG 360 GCCAATTAAA ATATATATAA ATATATATTT TTCAACCAGC ATTTTACTAC TTGTTACCTT 420 TCCCATGCTG AATTATTTTG TTGTGATTTT GTACAGAATT TTTAATGACT TTTTATAATG 480 TGGATTTCCT ATTTTAAAAC CATGCAGCTT CATCAATTTT TATACATATC AGAAAAGTAG 540 AATTATATCT AATTTATACA AAATAATTTA ACTAATTTAA ACCAGCAGAA AAGTGCTTAG 600 AAAGTTATTG TGTTGCCTTA GCACTTCTTT CCTCTCCAAT TGTAAAAAAA AAAAAAAAAA 660 AAAAAAAAAAAAAAAAATTG CACAATTTGA GCAATTCATT TCACTTTAAA GTCTTTCCGT 720 CTCCCTAAAA TAAAAACCAG AATCATAATT TTCAAGAGGA GAAAAAATTA AGAGATACAT 780 TCCCTATCAC AACATATCAA TTCAACACAT TACTTGCACA AGCTTGTATA TACATATTAT 840 AAATAGATGC CAACATACCC TTCTTTAAAT CACAAGCTGC TTGACTATCA CATACAATTT 900 GCACTGTTAC TTTTTAGTCT TTTACTCCTT TGCATTCCAT GATTTTACAG AGAATCTGAA 960 GCTATTGATG TTTCCAGAAA ATATAAATGC ATGATTTTAT ACATAGTCAC CCCCATGGTG 1020 GGTTGTCATA TATTCATGTA ATAAATCTGA GCCTAAATCT AATCAGGTTG TTAATGTTGG 1080 GAGTTATATC TATAGTAGTC AATTAGTACA GTAGCTTAAA TAAATTCCCC CCATTTAATT 1140 CATAATTAGA ACAATAGCTA TTGCATGTAA AATGCAGTCC AGAATAAGTG CTGTTTGAGA 1200 TGTGATGCTG GTACCACTGG AATCGATCTG TACTGTAATT TTGTTTGTAA TCCTGTATAT 1260 TATGGTGTAA TGCACAATAATA AGAAAACATT CATCGAATTAAAAAGT

SEQ ID NO: 5 Sequence length: 116 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA sequence GATCTGTACT GTAATTTTGT TTGTAATCCT GTATATTATG GTGTAATGCA CAATTTAGAA 60 AACATTCATC CAGTTGCAAT AAAATAGTAT TGAAAGTGAA AAAAAAA AAAAAA 116

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of Northern analysis for examining the expression of GS007656 gene in human cell lines and human organs.

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[Procedure amendment]

[Submission date] April 24, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1 is an electrophoretic photograph showing the results of Northern analysis for examining the expression of the GS007656 gene in human cell lines and human organs.

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

Front page continuation (72) Inventor Kosaku Okubo 1-3 Yamadaoka, Suita City, Osaka Prefecture Osaka Biotechnology Center

Claims (7)

[Claims] 1. A protein having the amino acid sequence set forth in SEQ ID NO: 1, or 1 in the above amino acid sequence.
Alternatively, a DNA encoding a protein in which a plurality of amino acid residues have been added, deleted, or substituted, and which has an activity of differentiating human non-neural ectoderm into nerve cells. 2. The DN according to claim 1, which encodes a protein having the amino acid sequence set forth in SEQ ID NO: 1.
A. 3. The DNA according to claim 2, which has the base sequence shown in SEQ ID NO: 2. 4. A probe for diagnosing neuroblastoma, which comprises a DNA fragment having a sequence of at least 15 consecutive bases in the base sequence set forth in SEQ ID NO: 2, 3 or 4 or a base sequence complementary thereto. . 5. The probe according to claim 4, which comprises a DNA fragment having the base sequence shown in SEQ ID NO: 5. 6. A primer used in a replication chain reaction (PCR) for identifying neuroblastoma cells and brain cells, which is the neuron set forth in SEQ ID NO: 2, 3 or 4.
From the D base sequence, select two regions satisfying the following conditions: 1) the length of each region is 15-30 bases; 2) the ratio of G + C in each region is 40-60%; 3) The distribution of A, T, G, C in each region is not partially biased; 4) The distance between each region is 100-1000 bases; 5) Each region itself or two regions There is no complementary sequence portion between them; and a single-stranded D having the same base sequence as the above region or a base sequence complementary to the above region
The above primer, which is produced by a method comprising producing NA and, if necessary, producing the above single-stranded DNA modified so as not to lose the binding property to the above neuroD nucleotide sequence. 7. The following nucleotide sequence: 5'-CACATCAT.
GAGCGAGTCATG-3 ′ (base numbers 1019-1138 of SEQ ID NO: 2) and 5′-TAGGCTCA
The primer according to claim 6, which comprises a set of DNA fragments having GATTTATTACAT-3 ′ (complementary sequence of base numbers 1036-1055 of SEQ ID NO: 4).