JP3075309B2 - Gene AML1 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a chromosome 21 which is useful as a marker for understanding the state of human acute myeloid leukemia (AML) and is found in AML patients;
The present invention relates to a novel gene AML1 existing at the 21 chromosome translocation site.

[0002]

2. Description of the Related Art Chromosome translocation t (8; 21) (q22; q
22) is frequently found in M2 cases of AML patients (Fourth International Workshop on Chromosomes in
LeukemiaCancer Genet Cytogenet 11,284-287 (198
Four)). However, cloning of this translocation site has not been resolved due to the lack of suitable DNA markers around it.

Recently, the site of the translocation breakpoint of (15; 17) with acute promyelocytic leukemia (APL) has been
Detailed analysis was performed using the A marker, chromosome 17qNotI linking clone and pulsed-field gel electrophoresis (PFGE) (Borrow et al. Science 249, 15).
77-1580 (1990); de The et al. Nature 347,558-561 (1990); L
emons et al. Genes Chrom Cancer 2,79-87 (1990)). The translocation breakpoint is concentrated in the first intron of the retinoic acid receptor and contains two CpG islands.
CpG islands are present in the 5 'untranslated region of certain genes (Bird. Trend Genet. 3,342-347 (198
7)). Retinoic acid receptor α is APLt (15; 1
(7) It is thought that it is involved in leukemia pathogenesis, and it is thought that binding of trans retinoic acid to all of its receptors leads to reduction of symptoms in APL (Huang et al.). ．Blood 72,567-572
(1988)).

The present inventor has reported that the LL263 clone, one of NotI linking clones specific to human chromosome 21, has a DNA of an AML patient having a t (8:21) translocation.
The translocation can be detected, and the detailed analysis of the restriction enzyme map shows that the translocation site is 13 to 13 LL from the LL263NotI site.
Proved to be 100 kilobases (Shimizu et al. .G
enes, Chromosomes & Cancer 3,163-167 (1991)).

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide an AM
The chromosomal translocation site frequently observed in the M2 line of L patients was analyzed at the molecular level, and human 2 previously developed by the present inventors was analyzed.
Using the NotI linking library of chromosome 1 (Saito et al., Experimental Medicine Vo18, No. 8, 991-996 (1990)), the gene AML1 was identified by cloning a novel gene AML1 existing at the translocation site. is there.

[0006]

Means for Solving the Problems Cloning of a chromosomal translocation site requires an appropriate DNA marker around it. The present inventor made a restriction map of human chromosome 21 using a NotI linking library,
Based on the knowledge obtained therefrom, a NotI linking clone located near the translocation site was obtained, and the gene AML1 present at the translocation site found in AML patients was successfully identified.

Hereinafter, the present invention will be described in detail. The present inventors performed chromosome walking from LL263 and isolated three types of genomic clones λE3, λE4, and λE12. The genomic clone λE4 was used for cloning from a human bone marrow cDNA library and identified cDNA clone C6. This was used to obtain a full-length AML1 cDNA having a single long open reading frame encoding 250 amino acids shown in the sequence listing below. This amino acid sequence did not show significant homology to known proteins in the Swiss-Prot database. Therefore, this new gene was named AML1. The AML1 cDNA contains proline and arginine as high as 9.6% and 9.2%, respectively, and the binding site of ATP or GTP was found in the amino acid sequence 138 to 145 using the PCGENE PROSITE program.

[0008] From the study of the detailed restriction enzyme maps of human genomics and cDNA, as shown in FIG.
The NA contained at least seven exons, and the translocation breakpoints of the three AML patients examined were located in the same intron of the gene AML1. Further, the chromosome translocation t (8; 2)
Analysis of the other six patients with 1) by Southern blotting revealed that rearrangements were found in the BamH1 fragment. From the knowledge obtained so far,
The translocation breakpoint on human chromosome 21 of an AML patient with the t (8; 21) translocation was found to cluster in the same intron of the AML1 gene.
This cluster also suggests that the AML1 gene is involved in cancerous conversion in AML.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples unless it exceeds the gist.

Example 1 (Preparation of NotI linking clone of human chromosome 21) 1) Preparation of marker gene SupF (1) Plasmid πvx (Maniatis, T. et al. Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, New
York, Cold Spring Harbor Laboratory, 1982) was prepared in large amounts using Escherichia coli W3110r ⁻ m ⁺ (p3). (2) πvx was digested with EcoRI, electrophoresed on a polyacrylamide gel, and a 203 bp SupF band was cut out. (3) SupF DNA was eluted from the cut gel,
Purified. (4) SupF DNA was blunt-ended by an end-filling reaction using Klenow enzyme. (5) This was ligated with an excessive amount of NotI linker, then digested with NotI, and electrophoresed on a polyacrylamide gel. (6) SupF DNA with NotI linker attached
The (215 bp) band is cut out and D
NA was eluted and purified. (7) The above DNA was incorporated into a plasmid having a NotI site and cloned. (8) The SupF fragment was excised from the plasmid by NotI digestion, purified, and then dephosphorylated at the 5 ′ end with bovine intestinal alkaline phosphatase.

2) Selection of NotI Linking Clones The principle of selecting NotI linking clones is as follows.
By incorporating a specific marker gene only into clones having I site and growing in a specific E. coli,
That is what you choose. The Sharon 21A vector has two ambar mutations (introducing the stop codon UAG) in the structural gene and can grow only in E. coli (su ⁺ ) having a suppressor tRNA for reading the codon. Lambda DNA is a linear molecule, but in a solution, it anneals to each other at 12 bases called cos sites at both ends to form a partially circular molecule or multimer. After dissociating them by heat to form a monomer, they are once self-cyclized by a low-concentration ligation reaction as shown in FIG. Further, while the suppressor tRNA gene (SupF) having NotI linkers attached to both ends is incorporated into the molecule linearized by NotI digestion, the molecule is again circularized. After packaged in vitro packaging, su ^-
Grow in E. coli and select only clones containing SupF, ie NotI linking clones. That is,

(1) amplifying the genomic library,
Lambda DNA was prepared. (2) Using 1.5 ml eppendorf tube, add 10 ml of distilled water to 10 μg / ml of lambda DNA.
X ligation reaction buffer [67 mM Tris-HCl (pH 7.
5), 67 mM-MgCl ₂ ]. (3) The mixture was heated at 68 ° C. for 5 minutes to dissociate the binding by the cos portion of λDNA. (4) 0.1 M ATP, 1 M DTT and T4DN
A ligase was added to final concentrations of 1 mM, 10 mM and 40 U / ml, respectively. 500-6 total reaction volumes
The volume was 00 μl. (5) The ligation reaction was carried out at 15 ° C. overnight. (6) Heating was performed at 68 ° C. for 10 minutes to inactivate T4 DNA ligase. (7) NotI and 10 × digestion buffer [1.5 M Na
Cl, 60 mM Tris-HCl (pH 7.5), 60 mM
MgCl ₂ , 60 mM β-mercaptoethanol, 1 mg / m
lBSA] was added to each to give a final concentration of 300 U / ml, 1x, and reacted at 37 ° C overnight. (8) Extracted with phenol / chloroform. The extract was transferred to a dialysis tube, and TE [10 mM Tris-HCl (p
H8.0), 1 mM EDTA]. After dialysis, it was collected in an Eppendorf tube. (9) Selectable marker gene Su purified according to 1) above
pF and lambda DNA were mixed at a molar ratio of 1: 1 and ligated at 15 ° C. overnight. The concentration of lambda DNA itself was the same as in the first ligation reaction. (10) Extract with phenol / chloroform, add 1/10 volume of 3M sodium acetate (pH 5.2) and 2.5 volume of ethanol, place at -20 ° C overnight, centrifuge and DNA
Was precipitated. (11) The precipitate was dried and dissolved with a small amount (about 10 μl) of TE. (12) A part of the solution was replaced with ethidium bromide (C ₂₁ H ₂₀ B
rN ₃ ), and the DNA concentration of the solution was measured by comparing the degree of fluorescence under ultraviolet light with a DNA solution of known concentration. (13) Packaging was performed in vitro using a commercially available packaging kit. (14) Packaging lysate is treated with su ⁺ bacteria (for example, V
CS257) and the packaging efficiency was calculated. (15) The packaging lysate was infected with su ^- bacteria. This includes CA274 (HfrClac _am125 trp
_am ). Clones that formed blue plaques on plates containing IPTG and X-Gal were selected as NotI linking clones. (16) Lambda DNA was purified from individual plaques.

Example 2 (Genomic Clones and cDNA Cloning) The partial restriction enzyme map of BamH1 in FIG. 2 was prepared by Southern blotting analysis of genomic DNA and genomic clones using various restriction enzyme fragments of cDNA as probes. The AML1 cDNA was mapped onto six genomic BamH1 fragments, one of which was covered by λE4 and contained at least two exons. Thus, the AML1 cDNA contains at least seven exons. ΛD11 and λD13 shown in FIG.
Each contains one exon. The cutting point is P1,
Indicated by arrows P2 and P3. Dotted line is genomic DNA
The approximate location of the exon in the figure is shown.

The genomic clones λE3 and λ of FIG.
E4 is a human leukocyte genomic library (Clontech, EMBL-3) using LL263L obtained by digesting linking clone LL263 with NotI as a probe.
From. λE12 is λE4 from the same library
Was isolated using. Ie

(1) Escherichia coli (for example, LE392) is cultured in an L-medium at 37 ° C. overnight, and 1 ml of the culture is added to an L-medium
The cells were seeded in 0 ml, cultured with shaking at 37 ° C. for 3 hours, centrifuged to collect the cells, and suspended in 10 mM MgSO _{4 so} that OD = 0.5. (2) 100 μl of a diluted human leukocyte genomic phage library was added to 100 μl, and 1
Incubate for 5 minutes at 37 ° C. (3) To this, add 3 ml of soft agar kept at 50 ° C.
Plated and incubated at 37 ° C. for 7-8 hours. (4) Carefully place the nylon thin film on agar, let stand for 1 minute, gently lift the thin film and place it in the denaturing solution (0.
5 N NaOH, 1.5 M NaCl) on a filter paper soaked in soft agar for 5 minutes with the surface in contact with soft agar facing up,
A was bonded to the thin film. Next, neutralize solution (0.5M Tris-
The solution was transferred onto a filter paper soaked with HCl (pH 7.5, 1.5 M NaCl) and allowed to stand for 5 minutes to neutralize the thin film. 2 x it
Wash with SSC (1 × SSC: 0.15 M NaCl, 15 mM sodium citrate), air dry, DNA side down and irradiate the DNA with a transilluminator for 2-5 min. Fixed to. (5) 6 × SSC, 1 × Denhardt's solution (0.1% BS
A, 0.1% Ficoll, 0.1% polyvinylpyrrolidone), prehybridization was performed with a solution containing 1% SDS and 50% formamide. (6) 1 mg of heterologous DNA (eg, salmon sperm DNA)
0.5 ml of the / ml solution was denatured by heating at 100 ° C for 5 minutes. This is added to the pre-hybridization solution
It was adjusted to 0 μg / ml and incubated with the thin film at 42 ° C. for 1 hour. (7) DNA probe (LL263L) labeled using Amersham's multi-prime labeling kit
Was denatured by heating at 100 ° C. for 5 minutes. (8) This probe was added to the pre-hybridization solution and incubated at 42 ° C. for at least 12 hours. (9) The filters were washed with 2 × SSC and 0.1% SDS. (10) Replace the solution with 0.1 × SSC, 0.1% SDS,
Incubated at 65 ° C. for 10 minutes. This operation was repeated as needed. (11) Take out the filter, wrap it in Saran wrap,
I took an autoradiograph.

The cDNA clones C6 and B1-3
Is a human bone marrow cDNA library (Clonetech, λgt
From 10), λE4 and HindIII-EcoR of C6
The I fragment C6E6H2 was isolated in the same manner as described above, using each as a DNA probe. P6-1
Has a poly (A) chain of 90 bases or more. Several other overlapping cDNA clones shown in FIG. 2 were obtained from a human peripheral blood leukocyte cDNA library (Clonetech, λgt).
Similarly isolated from 10). The nucleotide sequences of these cDNA clones were determined using the method described below.

Genomic clones λD11 and λD
13 is a human lymphocyte genomic library (Strata
gene, λDASH) using C6E6H2 as a probe.

Example 3 (Patient Samples and Somatic Cell Hybrids) Peripheral blood and bone marrow samples were collected from t (8; 21) AML patients at the onset of leukemia and at remission. Since the patient's white blood cells are only available in very small amounts and these cells are extremely difficult to proliferate outside the body, to obtain large amounts of DNA, somatic cell hybrids were used in mouse myeloma X63 (HPRT).
^- ) Prepared by fusion of cells and patient white blood cells.

(DNA sequencing and Southern blotting analysis) cDNA integrated into phage strain
Is EcoRI of pBluescript II KS ⁺ (Staratagene)
Recloned to the site. For DNA strands on both sides of the cDNA clone, Sanger dideoxy chain termination method (Sanger et al. Proc. Natn. A) using Sequenase version 2.0 (USB).
cad. Sci. 74, 5463-5467 (1977)). The nucleotide sequence was determined according to the USB protocol.

(1) pBl obtained by recloning cNDA
uescript II KS ⁺ double-stranded DNA (3-5 μg)
Single dissociation was performed in M NaOH, 0.2 mM EDTA, and ethanol precipitation was performed. After drying the precipitate, it was dissolved in 7 μl of sterile distilled water. (2) Primer 1 μl, 5-fold concentrated reaction solution (200 mM
Tris-HCl (pH 7.5), 100 mM MgCl ₂ ,
After adding 2 μl of 250 mM NaCl), the mixture was heated at 65 ° C. for 2 minutes, and then slowly cooled to room temperature. Keep on ice until labeling reaction. (3) 1 μl of 0.1 M DTT, 2 μl of a 5-fold diluted labeling mixture, [α- ³⁵ S] dCTP (1000 C
(i / mmol) 0.5 μl and 2 μl of a 9-fold diluted sequence enzyme were added, and the mixture was allowed to stand at room temperature for 2 to 5 minutes. (4) ddGTP, ddATP, ddTTP, ddCT
Each of the four types of termination mixed solutions of P
Each μl was dispensed into four Eppendorf tubes and kept at 37 ° C. for at least 1 minute. (5) The labeled reaction solution was dispensed in 3.5 μl aliquots into the tube of (4) and kept at 37 ° C. for 3 to 5 minutes. (6) 4 μl of the reaction stop solution was added and mixed. (7) After heating at 75 ° C. to 80 ° C. for 2 minutes, 2-3 μl
Each was overlaid on a sequence gel, and electrophoresis was performed. (8) After the electrophoresis, the gel is dried, and autoradiography is performed at room temperature using Kodak Ortho M film.
The nucleotide sequence of the DNA was determined.

For Southern blotting analysis, 10
μg of genomic DNA was digested with an appropriate restriction enzyme, separated by agarose gel electrophoresis, and transferred to Hybond-N (Amersham). That is, 20 × S
Place agarose gel on filter paper wet with SC, and put on it Nylon membrane filter (HiBond-N) soaked in 2 × SSC, 3 filter papers soaked in 2 × SSC, 7 dry filter papers, dry paper towel A 5 cm-long glass plate was placed in this order, and an appropriate weight of weight was placed on the glass plate, and the plate was allowed to stand overnight. To ensure that the bottom filter paper is always wet with 20 × SSC, add 20 × SSC to the buffer tank. When the blotting is completed, remove the thin film and use 2 × SSC
Wash the agarose with UV light (312nm)
The DNA was fixed to the thin film by irradiating for 5 minutes. For hybridization with a probe labeled with a random primer, a mixture of 6 × SSC, 10% dextran sulfate, 1% SDS, 1 × Denhardt's solution, 50% formamide and denatured salmon sperm DNA (100 μg / ml) was used at 42 ° C. Used in For the last wash, 0.1 × SSC,
Performed at 65 ° C. using a 0.1% SDS solution. Autoradiography is bio image analyzer, FUJ
IX BAS2000 was used.

[0022]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1912 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence type: cDNA Origin: Human bone marrow or peripheral blood, white blood cell Sequence characteristics Amino acid coding site: 768-1518 ATP or GTP Binding site: 1179-1203 poly (A) signal: 1880-1885

[0023] SEQ ATTGAGAGAA CAGAGAAGAT GACAAGTACT TCTAGCTCAG CACTGCTCCA ACTACTGAAG 60 CTGATTTTCA AGGCTACTTA AAAAAATCTG CAGCGTACAT TAATGGATTT CTGTTGTGTT 120 TAAATTCTCC ACAGATTGTA TTGTAAATAT TTTATGAAGT AGAGCATATG TATATATTTA 180 TATATACGTG CACATACATT AGTAGCACTA CCTTTGGAAG TCTCAGCTCT TGCTTTTCGG 240 GACTGAAGCC AGTTTTGCAT GATAAAAGTG GCCTTGTTAC GGGAGATAAT TGTGTTCTGT 300 TGGGACTTTA GACAAAACTC ACCTGCAAAA AACTGACAGG CATTAACTAC TGGAACTTCC 360 AAATAATGTG TTTGCTGATC GTTTTACTCT TCGCATAAAT ATTTTAGGAA GTGTATGAGA 420 ATTTTGCCTT CAGGAACTTT TCTAACAGCC AAAGACAGAA CTTAACCTCT GCAAGCAAGA 480 TTCGTGGAAG ATAGTCTCCA CTTTTTAATG CACTAAGCAA TCGGTTGCTA GGAGCCCATC 540 CTGGGTCAGA GGCCGATCCG CAGAACCAGA ACGTTTTCCC CTCCTGGACT GTTAGTAACT 600 TAGTCTCCCT CCTCCCCTAA CCACCCCCGC CCCCCCCCAC CCCCCGCAGT AATAAAGGCC 660 CCTGAACGTG TATGTTGGTC TCCCGGGAGC TGCTTGCTGA AGATCCGCGC CCCTGTCGCC 720 GTCTGGTAGG AGCTGTTTGC AGGGTCCTAA CTCAATCGGC TTGTTGTG ATG CGT ATC 777 Met Arg Ile CCC GTA GAT GCC AGC ACG AGC CGC CGC TTC ACG CCG CCT TC C ACC GCG 825 Pro Val Asp Ala Ser Thr Ser Arg Arg Phe Thr Pro Pro Ser Thr Ala 5 10 15 CTG AGC CCA GGC AAG ATG AGC GAG GCG TTG CCG CTG GGC GCC CCG GAC 873 Leu Ser Pro Gly Lys Met Ser Glu Ala Leu Pro Leu Gly Ala Pro Asp 20 25 30 35 GCC GGC GCT GCC CTG GCC GGC AAG CTG AGG AGC GGC GAC CGC AGC ATG 921 Ala Gly Ala Ala Leu Ala Gly Lys Leu Arg Ser Gly Asp Arg Ser Met 40 45 50 GTG GAG GTG CTG GCC GAC CAC CCG GGC GAG CTG GTG CGC ACC GAC AGC 969 Val Glu Val Leu Ala Asp His Pro Gly Glu Leu Val Arg Thr Asp Ser 55 60 65 CCC AAC TTC CTC TGC TCC GTG CTG CCT ACG CAC TGG CGC TGC AAC AAG 1017 Pro Asn Phe Leu Cys Ser Val Leu Pro Thr His Trp Arg Cys Asn Lys 70 75 80 ACC CTG CCC ATC GCT TTC AAG GTG GTG GCC CTA GGG GAT GTT CCA GAT 1065 Thr Leu Pro Ile Ala Phe Lys Val Val Ala Leu Gly Asp Val Pro Asp 85 90 95 GGC ACT CTG GTC ACT GTG ATG GCT GGC AAT GAT GAA AAC TAC TCG GCT 1113 Gly Thr Leu Val Thr Val Met Ala Gly Asn Asp Glu Asn Tyr Ser Ala 100 105 110 115 GAG CTG AGA AAT GCT ACC GCA GCC ATG AAG AAC CAG GTT GCA AGA TTT 1161 Glu Leu Arg Asn Ala Thr Ala Ala Met Lys Asn Gln Val Ala Arg Phe 120 125 130 AAT GAC CTC AGG TTT GTC GGT CGA AGT GGA AGA GGG AAA AGC TTC ACT 1209 Asn Asp Leu Arg Phe Val Gly Arg Ser Gly Arg Gly Lys Ser Phe Thr 135 140 145 CTG ACC ATC ACT GTC TTC ACA AAC CCA CCG CAA GTC GCC ACC TAC CAC 1257 Leu Thr Ile Thr Val Phe Thr Asn Pro Pro Gln Val Ala Thr Tyr His 150 155 160 AGA GCC ATC AAA ATC ACA GTG GAT GGG CCC CGA GAA CCT CGA AGA CAT 1305 Arg Ala Ile Lys Ile Thr Val Asp Gly Pro Arg Glu Pro Arg Arg His 165 170 175 CGG CAG AAA CTA GAT GAT CAG ACC AAG CCC GGG AGC TTG TCC TTT TCC 1353 Arg Gln Lys Leu Asp Asp Gln Thr Lys Pro Gly Ser Leu Ser Phe Ser 180 185 190 195 GAG CGG CTC AGT GAA CTG GAG CAG CTG CGG CGC ACA GCC ATG AGG GTC 1401 Glu Arg Leu Ser Glu Leu Glu Gln Leu Arg Arg Thr Ala Met Arg Val 200 205 210 AGC CCA CAC CAC CCA GCC CCC ACG CCC AAC CCT CGT GCC TCC CTG AAC 1449 Ser Pro His His Pro Ala Pro Thr Pro Asn Pro Arg Ala Ser Leu Asn 215 220 225 CAC TCC ACT GCC TTT AAC CCT CAG CCT CAG AGT CAG ATG CAG GAG GAA 1497 His Ser Thr Ala Phe Asn Pro Gln Pro Gln Ser Gln Met Gln Glu Glu 230 235 240 GAC ACA GCA CCC TGG AGA TGT TA AGGCAGAAGT CAGTTCTTCT GTCCATCCCT 1550 Asp Thr Ala Pro Trp Arg Cys-245 250 CCCCAG AGGATAGAGC TATCTTTTCC ATCTCATCCT CAGAAGAGAC TCAGAAGAAA 1610 GATGACAGCC CTCAGAATGC ACGTTATGAG GAAGGCAGAA TGTGGGTCTG TAATTCCTCC 1670 GTGTCCCTTC TCCCCCTCTG CAAACCGTCG TAACAATAAT AGTTCCTAAC ACATGGGACA 1730 ATTGTGAGGA TTAAATGAGT TAGCCTGCAG AAATCACTTG ATGCACAGCA CATGGGAAGC 1790 ATTGTGTGTA TTTATTAATC CTTCACAAAG TCTTTGAGAT ATATTTTTAT CAAATATTTA 1850 GCATGGATCC CGGTACACTT TCAATACTTA ATAAATGGTC AATGTTATTC TTTTTCACTA 1910 TT (A) n 1912

[Brief description of the drawings]

FIG. 1 shows a method for selecting a NotI linking clone of human chromosome 21.

FIG. 2 shows a restriction enzyme map of the t (8; 21) translocation site of human chromosome 21 and AML1 cDNA.

Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) MEDLINE (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A gene AML1 wherein the AML1 cDNA has the nucleotide sequence and amino acid sequence of SEQ ID NO: 1. 2. An amino acid consensus sequence for a binding site of ATP or GTP, Gly-Arg-Ser-Gly-
The gene AML1 according to claim 1, wherein Arg-Gly-Lys-Ser is present.