JPS61227784A - Probe useful for detection of chromosme translocation - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a single-stranded nucleic acid molecule useful as a zorope for detecting chromosomal translocations, which nucleic acid molecule resides within an exon of a eukaryotic chromosomal gene and An exon contains a nucleic acid sequence containing at least one sequence that is complementary to the encoded mRNA, and the gene is characterized by a chromosomal breakpoint within which another nucleotide sequence can be translocated. ) There is at least one site. In a particular embodiment, the translocation is a Phyladelphia translocation, the eukaryotic chromosomal gene is on human chromosome 22, and the translocated nucleotid q-abl oncogene (onc
ogene).

The present invention also relates to purified hybrid ° (hybrid) mRNA molecules useful as indicators of chromosomal translocations. This mRNA is an abnormality (aberr) caused as a result of chromosomal translocation.
ant) is a transcript of a hybrid DNA sequence present on a chromosome. Furthermore, the present invention is directed to this hybrid l-'
It concerns hybrid polybutides encoded by mRNA molecules and antibodies directed against antigenic determinants on these hybrid polybutides.

Using the single-stranded nucleic acid molecules of the present invention, polyzotiP, and antibodies, chromosomal translocations can be detected and diseases related thereto can be diagnosed.

BACKGROUND OF THE INVENTION Throughout this specification, various publications are referenced by numbers in parentheses. All of these publications are listed together as "References" at the end of this specification.

To indicate the state of the art to which this invention pertains, the disclosures of these publications are incorporated herein in their entirety.

Several chromosomal translocations are known to be associated with human cancer. The chromosomal rearrangements that occur as a result of these translocations can activate the translocated human cellular oncogenes.

Chronic myeloid leukemia (CML) is a multipotent stem cell disease characterized by the presence of the Philadelphia (ph') chromosome in leukemic cells in 96% of all CML patients. p
The h' chromosome results from a translocation between chromosomes 22 and 9 (1). h) The c-all oncogene is located on the long arm (q) of chromosome 9 on the chromosome map (2
). From the analysis of somatic cell hybrid 0, the present inventors showed that this oncogene is located on the Ph' positive 0 MALPh' (22q) chromosome. This means c-
It has been shown that abl is involved in the 9;22 translocation (3). It was shown by isolating DNA fragments from chromosome 9q+ of CML patients that the c-abl oncogene is located adjacent to a translocation breakpoint in CML. That is, this fragment is the ninth
Chromosome and W, 22 different'! I9q+ chromosome was generated (
4). No. 22 isolated from this hybrid DNA fragment.
It has become possible to sequence chromosomes. Cutting point cluster region (
break-point cluster region
n, bcr) /lY1g Identified on chromosome 22←
Ta. The DNA of all Ph'-adjacent CML patients tested to date (more than 30) has breakpoints in this region up to 5.8 kb, strongly suggesting a role for BCR in chronic myeloid leukemia. (5) We were able to identify patients with Ph'h' using bcr-specific genomic probes (our pending U.S. Patent Application No. 571, filed January 18, 1984; , the contents of which are incorporated herein by reference), but were experimentally difficult and often required the use of several different probes.

In 1975, Lozzio and Lozzio reported the isolation of cell line K-562 from the fresh water of an adult CML patient (6). This cell line is an erythroid lineage (eh
ythroid lineage) and exhibit induced and spontaneous globin synthesis (7)
. The present inventors and other researchers (4, 8, 9) have demonstrated that c-a
bl oncogene and immunoglobulin light chain constant region (C)
was shown to be amplified at least 4-fold in this cell line. In contrast, ζζ, normally located on chromosome 22, but translocated (moved) to chromosome 9 in Ph′h′
Another human oncogene, C-1, is not amplified (4). These data indicate that -562 contains a Ph'h' form that is amplified at least 4-fold. However, this is not confirmed by cytogenetic data.

That is, in our study, the ph77 chromosome was not detected in various pedigree of this cell line, and other researchers (6) suggested the existence of a single ph' chromosome. There is. Because of these findings, the question remains unresolved whether the amplification of the c-abl oncogene in K-562 is associated with the presence of amplified Ph'h'.

If such a relationship exists, K-562 would be
It will clearly be shown that it is derived from the patient's Ph' positive leukemia cells. The inventors have a DN of -562.
It was shown that a Ph' chromosomal breakpoint exists in A. This cleavage occurs in bcr, and the research results of bcr et al. show that the progenitor cells of K-562 contain a ph' translocation, and that C and c
It is established that -abl are located on the same amplification unit.

The research results of the present inventors have shown that -562 in the CML17iE cell line is present in all CML patients tested to date.
Similar to the h'-positive material, it is shown that the breakpoint is within the breakpoint cluster region on chromosome 22. However, in contrast to DNA isolated from patient material, K-562
The DNA consists of the remaining region of the Ph'-chromosome (rem
nants), a portion of bcr and an amplified portion of c-abl. These amplified regions are present on a single acrocentric marker chromosome rather than on a multicopy complete ph'-chromosome Is, *phase #t7 (9). This territory is the highest. By restriction-μ analysis using various restriction enzymes, all copies of the citrus breakpoint region contain breakpoint fragments of the same size.

There is no evidence that the leukemic cells of the patient from which K-562 was established contained more than one Ph'-chromosome. It is more likely that amplification occurred after the establishment of the cell line, resulting in the selection of cells favorable for proliferation.

In addition, -5628 cell has chromosome 99 in the sachin method (5628).
They also differ from other CML cells in that they cannot be detected by external blot analysis. This is consistent with the cytogenetic analysis result (9) that it was not observed in f'19(l chromosome Kani-562).

The absence of chromosome 99+ means that chromosome 22Q
- Reinforces the hypothesis that chromosomes are critical to the malignant growth of these leukemic cells.

In K-562, amplification of the human c-al ■ DNA sequence is coupled with increased expression of the abnormally sized 8.5 kb c-all mRNA (12.19).

This suggests that transcription from the translocated and amplified c-all oncogene is active. Recently, it has been shown that the altered human C-ab protein is distinct from normal human tannosoriplasm both in size and in the presence of associated proteinase activity (21). However, these previous studies on altered c-abl protein and mRNA
+, was not determined to be a molecular S hybrid 9 extracellular child. The present inventors have actually developed Hypritz ljmR
We show that NA and the produced tanno-Z-protein exist as a result of the Philadelphia translocation, and based on the hybrid nature of these aberrant gene products, we present a novel diagnostic test for CML and other diseases associated with similar translocations. invented the law.

The present invention relates to the construction and use of DNA probes to detect aberrant chromosomes resulting from translocations, such as the Philadelphia translocation, and to assay mRNA and protein products obtained from these aberrant chromosomes. The present invention relates to a method for detecting these + translocations by:

SUMMARY OF THE INVENTION Single-stranded nucleic acid molecules provided by the present invention are useful as probes for detecting chromosomal translocations, and are useful as probes for detecting chromosomal translocations that are located within and encoded by exons of eukaryotic chromosomal genes. It has a nucleotide sequence that includes at least one sequence that is complementary to RNA. This eukaryotic chromosomal gene is characterized by the presence within it of at least one chromosomal break site into which another nucleotide sequence can be translocated.

In one embodiment of the invention, said sequence is located within an exon located 3' to the chromosomal break site into which the oncogene may be translocated. In one presently preferred embodiment of the invention,
The translocation is a Philadelphia translocation, and the eukaryotic chromosomal gene is located on human chromosome 22 and is characterized by the presence of a breakpoint cluster region within it, and the translocated sequence is a c-abl IIl tumor. At least part of the gene.

Single-stranded nucleic acid molecules may be DNA or RNA, preferably from about 20 to about 8,500 base pairs in length, and may be labeled with detectable moieties, such as radioisotopes. These labeled probe molecules can be used to detect chromosomal translocations, such as the Philadelphia translocation.

The invention also provides purified mRNA molecules useful as indicators of chromosomal translocations. This mRNA is a transcript of a DNA sequence that includes at least a portion of an exon of a eukaryotic chromosomal gene and at least a portion of another nucleotide sequence that is translocated into this gene.
In one embodiment of the invention, the nucleotide sequence of the exon is within an exon located 5' of the translocation sequence. In one currently preferred embodiment of the invention, the nucleotide sequence is located in the breakpoint cluster region (bcr) of human chromosome 22, and the translocated nucleotide sequence is at least part of the c;-abl oncogene.

Furthermore, the present invention provides a polybezotide encoded by a purified mRNA molecule, e.g.
'bcr/c-abl encoded by RNA molecules
Poly-butylene is provided. Antibodies, such as monoclonal antibodies, can be generated against antigenic determinants on this polypeptide. These antibodies are polybutides present as a result of chromosomal translocations, such as the Philadelphia translocation, such as bcr/c-ab! It can be used to detect polypeptides and diagnose related diseases such as chronic myeloid leukemia, acute lymphocytic leukemia or acute myeloid leukemia.

(Margin below)

[Brief explanation of the drawing]

Human is a restriction enzyme map of human chromosome 22 sequence, 5
．． 8kb DANGL II-BJ徂HI (erect construction) area is 0
．． Contains a 6HB probe and a 1.2HBg probe. D is a restriction enzyme map of the ph' chromosome in Ni-562, with the ph' chromosome breakpoints indicated by arrows. B and C are detailed restriction enzyme maps of parts of A and D, respectively. The thick bar represents the sequence of chromosome 22. Open bars indicate sequences derived from chromosome 9. Probe used for research? Shown above the person and below D. (A-Aval, B-BamHI, Bg-11■, E
mEcoRl, Hmdanind [[, K -4I *P
-Ps t l, R-88t ■, X h-mX
ho () Figure 2 Restriction enzyme map of plasmid ■-3 cDNA insert Isolation of the ■-30 DNA plasmid will be described in detail later. PolyG indicates the 5' end of the cDNA, and polyA
Tail indicates 3' end. The break point in K562 is within the intron, and the M chison indicated by the arrow remains on chromosome 22 and forms part of the hybrid mRIJA. Probes A and B are subcloned into the p8P vector and are the 5' and 3' fragments of cDNA. (
Poly A tail-3'. pv-upper”II-Bg-L 1 [, Fl-) 11ndl
, Ps-shitan1. A-2■) Figure 3 The DNA sequence and translated amino acid residues of the ■-3 sequence are indicated by the 1c1 letter symbol below the sequence. The exact positions of the exons designated 1-6 in the -3bcr cDNA are indicated by their respective numbers above the sequence.The DNA sequence was determined from M1.
The restriction enzyme fragment of CDNA subcloned into phage 3 was subjected to the dideoxy chain terminator method. The entire region of both strands was sequenced. Figure 4 shows the details of the restriction enzyme map of bar cDNA ■-3 W-3. The thick black bar indicates the sequence of the open reading frame, and the thin line indicates the untranslated sequence. The 3' end containing the tail was designated as An (A-
MU I s Ap"= ALLI, B (Ba-Bat, gll) Figure 5: DNA sequence analysis of the K-562 breakpoint. The upper part is the normal DNA sequence of chromosome 22, and the lower part is the corresponding one.
This is the DNA sequence of the breakpoint region within 62. K-! 562
0.3 kb and II-shi4-RX fragments (Fig. 1C) and 0.37 kb of normal chromosome 22 Ava ■/P
The at I fragment (Fig. 1B) was sequenced by the dideoxy method described in Habo 22. The region shown in the figure is A.
It is located 241 bp st side from the va 1 site. During ph translocation, cleavage occurs within the intron circle of the bar gene on chromosome 22 and within or 5' of the c-abl tumor gene on chromosome 9. These DN person sequences t-
Head to tail on the zph chromosome, bCr is the centromere (c@ntromer) c-ab
1 are physically connected as telomarics. By this arrangement. Transcription of a hybrid mRNA consisting of the 5' exon fused to the c-abL coding sequence is enabled. This mEINA is translated into a hybrid polypeptide in which the ball is the amino terminal and the c-abl is the carboxy terminal. FIG. 7 Genomic structure of the bar gene Restriction enzyme map of chromosome 22 sequence containing bar@ is shown in YA. The exons are shown as black boxes below the restriction enzyme map.The positions of the exons marked with numbers were determined to be ■-3 cDNA&c by engineering and hybridization. A restriction enzyme map of the breakpoint cluster region is shown in B with the exons indicated. Below this map are various CML t)NA
The approximate location of the cutting point is indicated by a horizontal or vertical arrow. Lc) (AmAva l, BmB, KHI, Bg
Once o'. EmEcoRI%ifm once 1ndl [, p-yo stj
. 8 = 1 Ujs 8s = iij, , I , X-Xho
The breakpoint of chromosome 22q-derived from ( ) DNAO319129 was isolated from 9, 5 as previously described (37).
Charon as kb Egl If fragment
) was cloned for 30 yen. 7.2 kb Bam
HI fragment and 7.7 kb F! The 99 fragment was isolated by cloning the co RI fragment into ShaC1730 and Lambda gtvras, respectively. Figure 8 DNA breakpoint sequence 031 of two CML patients
The sequence of 9129 DNA is shown in '4tA. Arrangement is 5'-3
’ is oriented. Normal chromosome 9 West 2nd Fil
r meeting f11i11) and middle 1'%KT+f%+m1%rs
%? 1l-=-2-1hM Sq+ and 22q- sequences (2nd and 3rd columns) are patient DN 0319129
A-derived, normal chromosome 22 sequence (column 4) +
It is derived from DNA that is not zcML. Arrows (1) indicate breakpoints on chromosome 9 and chromosome 22. Nucleotide C, which is found at the breakpoint in both the 9q+ and 22q- sequences, is framed. Limited regions of homology with the sequence of chromosome 22 are underlined. BK shows the breakpoint sequence of 021201850NA, and the normal sequence of chromosomes 9 and 22 (each pair) (columns 1 and 3) were derived from non-CML DNA.The sequence of chromosome 9q+ shown in the second column was derived from normal chromosome 9 or chromosome 22, whose sequence was determined by the inventors' experiments. Contains a boxed area to indicate that it is not derived from a chromosome.The points above the sequence of chromosome 9 are break points based on the results of comparison between the envelope map and the chromosome 22 map. We selected a small restriction enzyme fragment containing exon-intron structure A with 9th @ bcrY
The restriction enzyme map of the 5.8 kb breakpoint cluster region is shown on the map. In this area, Son's location is shown roughly (not precisely drawn) below the map. The numbered exons will be discussed later. Restriction enzyme map of B bar cDNA, cDIJA
The numbered and shaded regions correspond to similar markings in A. The citrus cDNA probes used in the present study are shown below the map, (A-Δval. Bg-shizumi n11 Bs-j!AiE
II, E-Eco RX, H-Hlndll, PmPs
t I, "66Pv # Pvu l. 3' of cDNA
AAAN at the end indicates a polyA tail) DETAILED DESCRIPTION OF THE INVENTION The present invention relates to single-stranded nucleic acid molecules useful as probes for detecting chromosomal translocations. This molecule exists within one exon of a eukaryotic chromosomal gene and contains a small sequence (both 1 and 1) complementary to the mRNA encoded by this exon.
It has an array containing . A feature of this eukaryotic chromosomal gene is the presence of at least one chromosomal break site into which another nucleotide sequence can be translocated. The invention also relates to nucleic acid molecules that are substantially complementary to these single-stranded nucleic acid molecules. These complementary nucleic acid molecules can also be used as probes for nucleic acid hybridization analysis of chromosomal breakpoints. Is the single-stranded nucleic acid molecule of the present invention a DNA molecule or an RNA molecule? It may consist only or mostly of these sequences (or both exons and introns). It is preferable that at least one of the sequences present in one exon is located 3' to the chromosomal break site(s). In this case, sequences residing within exons, located 3' to the chromosomal break site(s), are used. This single-stranded nucleic acid molecule is used in a nucleic acid hybridization experiment. DNA molecules of various sizes can be prepared by methods known to those skilled in the art, such as restriction enzyme cleavage or oligonucleotide synthesis, preferably from about 20 base pairs to about 8,500 base pairs. Preferably, the eukaryotic chromosomal gene in which the exonic nucleotide sequence is located is a human chromosome characterized by the presence of a breakpoint cluster region (bar) within it. may be located within either an intron or an exo/intron. In one aspect of the invention, the gene is located on human chromosome 22 and the exon nucleotide sequence is within the BCR group (fami17).
nucleotide sequences corresponding to one or more of the following nucleotide sequences. In a particular embodiment of the invention, the gene is located within the genus that is specifically involved in the Philadelphia translocation in patients with chronic myeloid disease (CML). In more than 90% of patients with CML and in a smaller or larger proportion of patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), C -abl @-, usually located on chromosome 9, is present. At least part of the nucleotide sequence of the 14 genes was transferred to the 22nd gene due to the Philadelphia translocation.
The construction of a single-stranded DNA molecule of the present invention translocated to a chromosome is described in the "Experimental" section of this specification. Several different probe molecules can be constructed from this DNA molecule by methods such as restriction enzyme cleavage and oligonucleotide synthesis. For example, using plasmid ■-3, one can construct several different probes (as shown in Figure 2). This molecule has been sequenced, and the sequence is shown in Figure 3. Other single-stranded DNA molecules useful as locus probes can be constructed using the methods of the invention as described in the Experimental section. Therefore, it is possible to construct a vector molecule containing the DNA molecule of the present invention.That is, it is possible to construct a vector molecule containing the DNA molecule of the present invention.That is, it is possible to construct a vector molecule containing the DNA molecule of the present invention. These vector molecules can be incorporated as inserts into vectors such as standard recombinant DNA molecules known to those skilled in the art.
It can be prepared by A technique. Alternatively, this molecule can be subcloned into a plasmid vector, such as the 8P6 vector system (26, 27), with a complementary RN suitable for use as a probe in hybridization methods.
A molecule may also be created. The single-stranded DNA molecule of the present invention contains a detectable substance (moie).
It can also be labeled with tiθS). These labeled molecules can be used in a variety of nucleic acid hybridization methods known to those skilled in the art. The molecule may be a fluorescent marker, a prime marker or a radioactive marker (e.g. 32PQ).
can be labeled with any type of detectable substance, such as nick translation). These labeled molecules can be used to detect chromosomal translocations on a given eukaryotic chromosome. This combined DNA is processed to obtain r4A cleft fragments or single-stranded DNA molecules, and appropriate conditions are established to enable hybridization between complementary single-stranded molecules. The probe is then contacted with a labeled single-stranded nucleic acid probe molecule of the present invention, and the presence of the hybridized molecule is detected. Thus, by analyzing the hybridization results, the presence of a chromosomal translocation is detected. Furthermore, the present invention provides a method for detecting chromosomal translocations,
In this method, a suitable restriction enzyme is used. The chromosomal DNA is cleaved using the above methods, and the resulting fragments are treated by gel electrophoresis and denaturation to obtain single-stranded DNA molecules, which are recovered from the gel and placed on a suitable solid support. body. Fix it on a nitrocellulose filter. The immobilized single-stranded DNA fragments are then contacted with labeled single-stranded nucleic acid probe molecules of the present invention under suitable conditions that allow hybridization between complementary single-stranded molecules, thus causing hybridization. The soybean molecules are identified, for example by autoradiography, to identify abnormalities in the restriction pattern of chromosomal DNA caused by chromosomal translocations. One particular embodiment of the invention relates to a method for detecting a Philadelphia translocation in a human subject, in which DNA fragments are detected using one or more suitable restriction substances under suitable conditions such that DNA fragments are generated. The entire chromosomal DNA of the examiner is cleaved and the resulting fragments are then processed to obtain single-stranded DNA molecules, allowing hybridization between complementary molecules of the single-stranded DNA molecules thus obtained. Under the right conditions the second
A single-stranded nucleic acid molecule complementary to the exon nucleotide of the bar region of chromosome 2 is contacted, the presence of hybridized molecules is detected, and chromosomal translocations are thus detected. One example of such a method involves the detection of a Philadelphian translocation in a human subject, in which the entire chromosomal DNA of the subject is extracted using a suitable restriction enzyme over 1'a under suitable conditions to generate DNA fragments. cleavage, and the fragment thus obtained is single-stranded D
NA molecules were processed by gel electrophoresis and denaturation, and the single-stranded DNA molecules thus obtained were recovered from the gel (ρ) and fixed on a suitable solid support, e.g. a nitrocellulose filter, for primary immobilization. Under appropriate conditions that allow hybridization between complementary single-stranded molecules such as single-stranded DNA molecules, μ of chromosome 22 can be combined with labeled single-stranded nucleic acid molecules complementary to the exons of the region, e.g. Contact the insert fragment of plasmid M-3 as probe A in Figure 2. The molecules thus hybridized are identified, for example by autoradiography. In this way, abnormalities in the restriction pattern of chromosomal DNA caused by the Philadelphia translocation are detected. Methods of using the probe molecules of the invention are not limited to those described herein. The probe of the invention also includes: Other nucleic acids known to those skilled in the art! redization method,
For example, it can also be used for in situ hybridization. The invention also relates to purified hybrid mRNA molecules useful as indicators of chromosomal translocations. This m
An RNA molecule is a transcript of a DNA sequence that contains a portion of an exon nucleotide sequence of a eukaryotic chromosomal gene and a portion of another nucleotide sequence translocated within this gene. In some embodiments, the exon nucleotide sequence of the gene is located 5' to the translocated sequence; in other embodiments, the translocated nucleotide sequence is at least part of an oncogene. In one embodiment, the S exon nucleotide sequence of the translocated sequence is from the h positive region of chromosome 22, or in a third embodiment, the purified mRNA molecule is from about 6.0 to about &
A bcr/c-abl mFINA molecule of Okb length, which is the result of a Philadelphia translocation in which the c-abl oncogene is translocated from chromosome 9 to chromosome 22. The identification and isolation of this molecule is described in the "Experimental" section of this specification. The mRNA molecules of the invention can be purified by any method known to those skilled in the art, such as gel electrophoresis, column chromatography, density gradient centrifugation, or nucleic acid hybridization methods. Single-stranded c-DNA molecules such as
Cellulose beads containing c-T)NA molecules complementary to 'mRNA may be bound to a nitrocellulose filter. After hybridization of complementary strands, unbound nucleic acid molecules can be washed away. The nucleic acid duplex can then be denatured, m
BNA molecules can be obtained in purified form. Purified hybrid mRNA molecules of the invention can also be used to prepare DNA or cRNA probes. Once the fflRNA molecule has been purified, it is possible to produce cDNA or cRNA naturally;
For example, cDNA can be produced using reverse transcriptase. These cDNA molecules can then be cloned into an appropriate vector system by standard recombinant DNA techniques known to those skilled in the art. Various probe molecules can then be constructed from the cloned cDNA. Alternatively, purified mRNA or cRNAt' can be labeled with a detectable substance and used as a probe. For example, it may be enzymatically labeled with 33P by RNA & polynucleotide kinase. This mRNA probe can then be utilized to identify and isolate the translocated gene present on the abnormal chromosome by using standard molecular biology techniques known to those skilled in the art. The invention also relates to polypeptides encoded by purified hybrid mRNA molecules. These polypeptides contain polypeptide sequences originally encoded by two different genes. One of the two polypeptides is b
bar/ encoded by cr/c-abl mRNA
c-abl protein, and this polypeptide is the second
It contains a portion encoded by the exon nucleotide sequence of the bar region of chromosome 2 and a portion encoded by the oncogene c-abl. Antibodies to these polypeptides can also be prepared, and these antibodies are directed against antigenic determinants on this polypeptide,
For example, a determinant specifically created by the fusion of two genes, such as a determinant that is present in the hybrid protein but not present in either of the separate proteins of the fusion gene, can be opposed. Such antibodies can be used in a variety of diagnostic and therapeutic methods. In another aspect of the invention, more than one antibody can be prepared against the polypeptide, each antibody being directed against an antigenic determinant present on a different portion of the polypeptide. One example of such an antibody is a portion of a polypeptide encoded by a gene located on a chromosome to which another sequence has been translocated, such as the bar/c-abl polypeptide on chromosome 22. Other antibodies may be directed against antigenic determinants present in the portion of the polypeptide encoded by the exon nucleotide sequence encoding the polypeptide. These antibodies may be used to raise specific protein products. Diseases associated with chromosomal translocations that result, such as the Philadelphia translocation and CML in which the presence of the bcr/c-abl polypeptide can be diagnosed. 2 for parts
Two different antibodies were tested in an immunoassay (immunomatri).
c aaaays ) or a sandwich-type assay. In a preferred embodiment of the present invention, one or both of these antibodies are monoclonal antibodies.The antibody of the present invention also contains a radioactive substance, such as 1! ! It can be labeled with a detectable substance such as a fluorescent substance, such as fluorescein, or an enzymatic substance, such as peroxidase. Conditions suitable for the formation of antibody-antigen complexes are approximately 6
．． Solution phase and emulsion reaction systems maintained at a pH of 0 to about 8.0 and a temperature of about 45°C to about 45°C, preferably aqueous. Polyclonal antibodies and monoclonal antibodies of the present invention can be prepared by methods known to those skilled in the art. Monoclonal antibodies are described by Kohler et al., Nature, vol. 256, 995-497T4 (
(1975) can be prepared by techniques such as those disclosed by K. This process involves injecting mice or other animals with an immunogen. This mouse was then killed, cells were removed from its pancreas, and fused with myeloma cells to produce norubibrid. generate. A population of hybridomas generated in vitro is screened to isolate individual clones, each secreting a single antibody specific for the antigen. The antibodies produced by this I hybridoma are then screened to identify those with the highest affinity for the immunogen in question.To produce monoclonal antibodies directed against various parts of the hybrid polypeptide, the entire polypeptide must be isolated. Used directly as an immunogen, the primary generated hybridomas can be screened for antibodies directed against antigenic determinants on various portions of the polypeptide. Alternatively, fragments of various portions of hybrid polypeptides. The bar/c-abl polypeptide on chromosome 22 (
7) The portion encoded by the Ekifun nucleotide sequence present on the bar region can be used as an immunogen. For each purpose, one can use similar synthetic peptides as immunogens using standard prokaryotic and eukaryotic expression systems known to those skilled in the art, using the restriction data disclosed herein; For example bar/c-abL
A synthetic polypeptide is used that resembles the antigenic determinant present in the bcrlls portion of the polypeptide. One aspect of the invention is to determine the presence of a polypeptide, e.g., bcr/c-abl, encoded by an mRNA resulting from a chromosomal translocation, e.g. It relates to a method of detecting. In this method, a sample containing a polypeptide is labeled with an antibody-antigen complex substance)
contact with. This antibody may be polyclonal or monoclonal. The unbound labeled antibody' is then separated from the labeled antibody-antigen humbrex, and the presence of the labeled antibody-antigen complex is then detected by suitable means, such as autoradiography or fluorescence detection. Antibodies of the invention, whether polyclonal or monoclonal, have two antibodies directed against antigenic determinants on different parts of a polypeptide.
Staining with two different antibodies allows the sample containing the polypeptide to be isolated from the ason nucleotide sequence of the gene in which another nucleotide sequence has been translocated under suitable conditions such that an antibody-antigen complex is formed. is contacted with a measured amount of a first antibody directed against an antigenic site on the portion of the polypeptide encoded by the antibody. This first antibody is sm'd with a detectable substance. The labeled antibody-antigen complex is then transferred to a nucleotide sequence that has been translocated into the chromosome, such as a portion of a polypeptide encoded by an oncogene, under suitable conditions that allow the formation of a labeled antibody-antigen-antibody complex. A second antibody directed against the above antigenic site is contacted. This second antibody is bound to a suitable solid support, such as a microtiter well, cellulose beads, or a nitrocellulose filter. The unbound labeled antibody is then bound to the solid support. Labeled antibody-antigen-antibody complex-h) Chi4+
-Releasing enzyme 11 peptide 0 is heated and either the amount of bound labeled antibody-antigen-antibody complex or the amount of unreacted labeled antibody is measured. Alternatively, the above procedure can be carried out using a method in which the first antibody is unlabeled and bound to a solid support and the second antibody is labeled with a detectable substance. One particular embodiment of the invention relates to a method of detecting the presence of a bar/c-abl polypeptide and a Philadelphia translocation. In this method, a sample obtained from a subject is exposed to antigenic sites on the portion of the polypeptide encoded by the c-abl pain gene under suitable conditions that allow the formation of antibody-antigen complexes. 1. Contact with a known amount of antibody No. 1. This first antibody is labeled with a detectable substance. The complex thus formed is contacted with a second antibody directed against an antigenic site on the portion of the labeled antibody-antigen-antibody combination encoded by the exunion nucleotide sequence of the gene on chromosome 22. This second antibody is bound to a suitable solid support. The unbound labeled antibody is separated from the labeled antibody-antigen-antibody complex bound to the solid support. To detect the presence of the polypeptide, either the amount of labeled antibody-antigen-antibody complex bound to the solid support or the unbound labeled antibody is measured. Alternatively, the above procedure may be performed using a method in which the first antibody is unlabeled and bound to a solid support, and the second antibody is labeled with a detectable substance. Another method for detecting chromosomal translocations on a given eukaryotic chromosome, such as the Philadelphia translocation, involves isolating the polyA mRNA encoded by the gene on the chromosome. For example, polyA mRNA is isolated by poly under appropriate conditions that allow for the formation of Tux on the ph chromosome. The separated RNA molecules are immobilized on a suitable solid support, for example in a dry gel or on a ditrocellulose filter, and labeled probes of the invention are immobilized under suitable conditions that allow hybridization between complementary molecules. RNA
contact with molecules. The presence of hybridized molecules is detected, thus detecting the presence of chromosomal translocations. Another aspect of the invention is to detect CML in a human subject by detecting the presence of a Philadelphia translocation. The present invention relates to a method for diagnosing AML or ALL. (Left below) Details of the mRNA experiment containing the mRNA encoded by the gene According to tests to date, there is a break point on chromosome 22 in the DNA of most Ph'-positive CML patients. This can be demonstrated using the HBg probe (Figures 5 and 1). For example, a representative group of OML patients 0212
DNA of 0185.0319129 and 0311068
There's an abnormal womb inside! Restriction enzyme fragments were clearly present. Molecular cloning of a restriction enzyme fragment having these cut points (495) revealed that it was 9q'Ift#sheepvl. In a cell line isolated from the pleural fluid of an adult suffering from CML, testing after hybridization to the 12 HBg probe revealed that the abnormal DNA fragments were present in the pleural fluid or some others. It could not be detected with each of the restriction enzymes. Similarly, several more Bh
It was not possible to prove Bh' positivity using this probe with respect to the DNA obtained from 'positive OML patient 1. This may indicate that ph'-positive leukemia species such as K-562 do not have internal breakpoints on chromosome 22, or that the use of genomic DNA probes such as L2HBg may detect these breakpoints. Indicates that it is not suitable for point detection. To address this more fully, a probe closer to 5' within the frame (α6HB, see Figure 1A) was prepared in A, digested with bulk enzymes, and hybridized to -562 DNA. 5). This probe detects not only the normal &Okb divil fragment but also the abnormal 3l fragment in 1562 DNA. Furthermore, one of these abnormal fragments is amplified more than four times. The amplified abnormal restriction enzyme fragment in K-562 could also be detected using another enzyme. In order to analyze in more detail that the 0.6 HB probe detects the 22q-fragment in the DNA of many OML patients, it was partially digested with Mbo I and gl (-562DN human) using the known operating procedure (b). A cosmy library was constructed from 100,000 recombinants. Numerous colonies of approximately 100,000 recombinants hybridized with the o, a Hi probe. Three such positive colonies containing overlapping parts of the same region were used for subsequent restriction enzyme analysis. was selected for analysis (Figure 1D).Comparison of detailed restriction enzyme maps between the normal chromosome 22 sequence (Figure 1B) and that of a -562DN person (Figure 1C) revealed that the homology between the two was 5. It is clear that the cut ends at 3' of the cut point closest to '.
The og probe (Figure 1D) hybridizes to DNA isolated from a somatic cell hybrid containing human chromosome 9 without human chromosome 22, but not from a hybrid containing chromosome 22. Does not hybridize to DNA. One region indicating that the breakpoint is included is amplified more than 4 times. A sequence specific to chromosome 9 is also amplified. This means that the LOE probe amplifies the sequence of the control DN chromosome, It begins at the point where the breakpoint occurs on chromosome 9 at the locus and extends in the direction of the telomere, which contains the C-L oncogene. Since the distance between the nearby V-υ Meng homologous exofungus is at least 64 kb, the amplification region is relatively wide. These results indicate that -562 contains a break point within the cell line. However, it was found that this cutting point could not be detected with a probe originating from the 3' region of the crotch. We obtained a glove capable of detecting the Ph' breakpoint in sample material from all patients, and conducted an experiment to isolate a specific cDNA in order to test whether the Ph' breakpoint is part of the gene. Start friend. α6 of several genomic DNA probes tested
The HB probe (Figure 1) is a Northern probe (Nor).
(thern blot) showed that it hybridized to RN and therefore contained one or more epifunes. Therefore, the α6HB probe was used to screen a human cDNA library. 10μ of GM 637 cDN-containing plasmid was obtained from Hiroto Okayama (Hiroto Okayama). This library is a cDNA library of the human fibroblast cell line GM637 transformed with 8V-40. The construction of Ripe 2S is described by Okayama and Berg (2). It will be apparent to those skilled in the art that any of the two representative human cDN λ types may be used. It will also be clear to those skilled in the art that equivalent cDNA libraries can be prepared from the human) 0M637 cell line. The cell line is N40M8 H+aman Genetic Mut Cell Repository (N40M8 H+aman Genetic Mut
anta Ce1l Reposltory, Camden. 20-0 Jersey Power 1
It can be obtained from Using standard recombinant DNA procedures known to those skilled in the art, lOA
E. coli (
E.coli) DH1t-transformed. The transformed E. coli culture was plated out into 1 kilo large plate. Replicas of these plates were manufactured and bacterial t
-Scrape and culture. Ten large-scale plasmid preparations were prepared from these recombinant bacteria to construct an 8V-cDN human library. The preparations were named 8V-1-8V-10. 100 μL of SaI containing 42 units of restriction enzyme Sal
10 μq of each plasmid g product was digested with t1 restriction enzyme buffer. Digestion lasted for 4 hours with appropriate buffer and temperature conditions known to those skilled in the art. After digestion, each DN sample was phenol extracted and ethanol precipitated. The samples were then subjected to electrophoresis at 5-18 volts for 4 days on an alpha 6% agate gel.
r) and joule (5 chuell)). 0,6 nick-translated with HB probe 'k''P and hybridized to cDNA samples immobilized on nitrocellulose filters using operating procedures αη known to those skilled in the art. After completion of hybridization α3XS80,
Filters were washed under high stringency conditions at 65°C. Autoradiograms of the washed filters show that all samples except the lil sample 5v-c+ hybridized to a DNA band of approximately 5 kb. The entire 8V-cDN human library was then filtered through a large nitrocellulose filter (Mifjlpore).
HATF). Replicas of these filters were made and hybridized to the α61(B probe). Positive colonies were replated onto small nitrocellulose filters at a density suitable for isolation of single colonies. These filters were replicated and hybridized to the α6HB probe. The colonies that hybridized to the globe were picked and grown on a scale suitable for large-scale DNA isolation. Two positive colonies consisting of plasmids VI-3 and -1 were further analyzed. Plasmid VI
-3 contained the largest insert of approximately 2.2 kb and was further analyzed. The second step is to create a detailed restriction enzyme map for this insert.
Figure), the 5'-3' orientation of the cDNA was determined. Next, using the cDNA insert of plasmid v1-3 as a probe, human
A hybridization experiment was conducted on genomic DNA of two chromosomes. This region of chromosome 22 is included in cosmid clones 0a22-1 and 0a22-2. Cosmid clone preparation, restriction digestion, electrophoresis, plotting, and hybridization procedures were performed using standard methods known to those skilled in the art. Numerous restriction enzyme fragments in adjacent order in the Gram DN human hybridized to the VI-3 probe. This means that this cDNA is an mR encoded by a gene that includes addition in its coding region.
This clearly shows that it is a reverse transcription copy of the NA portion. The W-3 cDNA insert was sequenced (Figure 3). The dideoxy method was used for sequencing. According to the hybridization data of 0a22-2 and 0a22-1 and VI-3, W-3eDNA (
7) It was estimated that the sequence had at least 13 exofuns and that these exofuns were dispersed in a region of about 45 kb of the genomic DNA. These 13 exono 6←string remaining exfuns exist at 5' and 3' of the phase region. Therefore, a relatively small region of the VI-3CDN human insert, e.g. 500 base pairs, can be isolated and used as a probe.
It is possible to detect chromosomes 9q+ and 22q- in ML patients. A large number of such globes can be constructed from this cDNA insert. Such a globe preferably includes a 5'-most G-l restriction site and a 5'-most G-l restriction site. To detect all chromosomal aberrations in the region t:β of the internal M-3 cDNA insert with the marked restriction site (Figure 2), the region containing the breakpoint fragment in the K-562 cell line was detected. Probably. The -3 cDNA sequence is useful as a detection probe for Philadelphia translocation. The probe may be prepared as described herein, or the probe or substantially equivalent polynucleotide molecule may be synthesized using any polynucleotide synthesis method known to those skilled in the art, such as enzymatic or chemical. May be prepared by polynucleotide synthesis. Probes synthesized using this method cost $22
Through the use of such probes that detect chromosomal DNA sequences (which can be used on cDNA libraries or genomic DNA), one skilled in the art will be able to isolate chromosome 22 sequences from any representative human cDNA library or genomic DNA. In order to accurately determine the position of the cutting point of K-562,
The DNA sequence of the -562 breakpoint region was compared with the DNA sequence of chromosome 22 of the normal genome. The two arrays are the fifth
They are completely equal up to one point in the approximate center of the area shown in the figure. On the 3' side of this point, the sequence of chromosome 9 joins the sequence of chromosome 22. No sequence homology is found between the DNA sequences of chromosome 9 and chromosome 22. The lack of homology between the sequences involved in the OML 9;22 recombination event suggests that Burkira)
I) Consistent with the results obtained from sequence analysis of the 8;14 translocation in lymphoma. In the latter case, no obvious homology between the recombinant sequences was detected (to date). Furthermore, these sequence results clearly indicate that the breakpoint within K-562 occurred within the intron of the coward gene. A hybridization test was performed using a DNA probe prepared from the 'fl-3L cDNA human, and it was determined that the cleavage was present in the region corresponding to the Ka1/Baho fragment of the 17-3 cDNA human (see arrow in Figure 2). ). The exon 5' of the breakpoint shown remains on chromosome 22 at -562. As in the case of K-562, the chromosomal breakpoints of the four separate DNAs isolated from separate patient samples were located within the introns of the vulgar region. Therefore, the Philadelphia translocation creates a break within the gene that has a portion of the gene. In this translocation, c-a
The sequence of bL tumor tumorigenic gene Kinyo 9th chromosome is spliced into the gene containing Oshi. To examine the effect of translocation of the human c-sbtm oncogenic gene on expression, K-562 and control) L
Polyhuman RNA was isolated from EL human cells. Similar to the results obtained by other researchers (6), C-
Meng-specific PCR) detects &0 and 7. Ok b mRNAs in both LELA cells and -562''. In addition, &0 and 7. Ok b mRNAs are detected in both LELA cells and -562''. This new c-rM homology m
RN individuals were also detected in sample material from OML patients (1
9.20). Since the exact location of the breakpoint mentioned above for K-562 is known, the aberrant &5 kb mRNA found in K-562 in the OML cell line can be found in the bcr/
Further hybridization experiments were performed to determine whether it was c-abA mRNA. The experiment was carried out for -562c-abA mRNA plots as described above.
It contained hybridization of A. Hybridization analysis was performed using three different probes. The three probes were: (a) α6 kb of genomic DNA obtained from the 5′ region of the c-atμ gene containing Ekifun and R1/B;
amH1 fragment. (b) VI-3 cDNA 5' of the K-562 break point
Pvu I/K1 fragment (probe person in Figure 2). (c) VI-3 cDNA 3' side of K-562 break point
(probe B in Figure 2). The c-abA globe detected &5 kb amplified mRNA as in the previous experiment. Globe people are also abnormal & 5kb m
RNA was detected, but Proop B detects this abnormal m RNA.
could not be detected. These results showed that there were 1,562 pH' positive cell lines! It can be concluded that there are 201 mRNA individuals. Furthermore, we detected an abnormally sized &5 kb mRNA hybridizing to the probe human in ANA isolated from bone marrow cells of five CML patients. This &5kb
The mRNA is &5k out of 1562 in that it hybridizes to probe A but not to probe B.
It is similar to bmRNA. Therefore, this mRNA is Qui2
It may be unique to OML patients who exhibit Delphi translocations. Therefore, this mRNA (D-LZ knee 1 difference protein product also exists, and it is likely that this protein is involved in the cause and/or progression of OML. 0M63
These probes hybridized to the expected restriction fragments when used in Southern blot hybridization analysis of DNA derived from seven cell lines. These results confirm that probe A detects the abnormal restriction pattern associated with Philadelphia translocation and is therefore an excellent secondary hybridization probe and is effective in diagnosing OML. It is believed that the portion of the VI-3 sequence described above is not present in a single copy of the human genome. When hybridized to whole human DNA digested with the same restriction enzyme as the 5'Pvul restriction enzyme, under stringent washing conditions only fragments corresponding to the corresponding genomic DNA in this region are found. VI-
All proteins in the 3 cDNA detect large numbers of hybridized fragments in the DNA even after extremely stringent washes. This is at least VI-3 cDNA
It means that one + part of ``gives'' appear in one gene group. A single epifun containing a probe isolated from the 3' region of 0a22-2 will hybridize to at least four separate fragments in whole human DNA cleaved with separate restriction enzymes; A gene group has a minimum of four members. Two members of this group are those described herein.
Molecularly cloned from a 62 cosmid library. Overlapping cosmids spanning an 80 kb region were isolated. It contains a region of high homology with the VI-33' sequence. These cosmids, which are called cosmid VKII-11-prototype, are located on the 5' side of the 'fjc1 region (also located on chromosome 22).
(closer to the dynamic body). The second member was isolated in one nismid spanning a 40 kb region. This cosmid VKII-3 also has the above 0.46
Contains sequences that hybridize to the kbPs fragment. At least three members of this gene group are present on chromosome 22, indicating that somatic hybrid P G
Indicated by its presence in Me25-NuO DNA (3). PG Me25-Nu contains human chromosome 22 as the only human chromosome complement. The three F# crystals of this gene on chromosome 22 are the corresponding sequences attached to cosmid VKII-1, (
b) Sequences present in the bcr region, and (cl
Cosmid VKII-3 [9 Hiroshi, # 3' of corresponding area]
The arrangement on the side (end small grain side) is the same as the one on the side (terminus small grain side).
Although it is not amplified in chromosome 62, it is presumed to be translocated from chromosome 22 by ph' translocation. Since the region of chromosome 22 is always specifically involved in translocations to form the Ph' chromosome, there are known specific translocations that occur between chromosome 22 and another chromosome, such as Ewing's sarcoma. It is reasonable to assume that other members of this group are also involved in genetic disorders such as overgrowth or Day George syndrome. Therefore, the methods and probes of the present invention may also be useful in diagnosing other diseases associated with specific chromosomal translocations. By restriction enzyme mapping (Figure 4) and sequence analysis,
Maximum cDNA containing Z2kb insert. VI-3t - Determined in detail. The DNA sequence and translation are shown below (see Figure 3). Amino acid residues are indicated by single letter symbols below the sequence. The exact positions of the next Ekifun (figure 7) labeled 1 to 6 within the VI-3bsx cDNA are indicated by numbers above the sequence. cDNA sack quartered in M137 medium
DNA sequencing of restriction fragments of A was performed using the dideoxy termination method. The entire region was sequenced on both strands. The cDNA contains one long reading frame starting from the 5' polyG tail and continuing to nucleotide 1770 until the stop codon is reached. All reading frames other than this one have multiple stop codons within the entire region. This long reading frame i;j has the capacity to encode 589 amino acids9, which has a molecular weight of approximately 65,
Corresponds to 000 proteins. At the 3' end, there is a polyadenylation signal at nucleotide 2182 and base 220
A polyA tail starting at 8 follows. This is cD
The NA is shown to contain the complete 3' end of the gene. Although the translation initiation sequence is present on the 5' shoulder, it is unlikely that this cDNA contains a complete copy of the mRNA, as Northern blot analysis shows the presence of bcr mRNA with a size of approximately 40 and a5 kb. Unthinkable. Computer searches of newly isolated protein sequences, either derived directly from the protein or deduced from cDNA human nucleotide sequences, often identify proteins with partial homology. Such information is useful, and it is possible to infer in advance the functions of tannokuri, which are generally unknown. Therefore - Bill Fastp (PIRpAsTp) program was used to search for homologous proteins. No proteins with significant homology were detected. This indicates that the cellular function exhibited by the protein is currently unidentified. 1 Ire that determines the orientation of the Nagi gene on chromosome 22
” u re 'I'^IPl++go<-W,'l
The Ph It was confirmed that the 3' end of the 3' gene was retained after translocation.
;22) translocates to chromosome 9. c DNA is the second
It hybridizes to restriction enzyme fragments distributed over a 45 kbj region of bichromosomal DNA (Figure 7A). There are at least 13 exhausts within this area. To determine the exact location and number of exophyles within the breakpoint cluster region, all hybridized regions of the groin were sequenced and compared with W-acDNA. As shown in FIG. 7B, there are four relatively small ex-funs designated 1-4 in the upper interior, and their sizes differ from each other in the range of 76 to 105 bp. In the cDNA, these exosomes correspond to nucleotides 483-836. Since the eyelid is defined as the region where the Ph' breakpoint of chromosome 22 is detected, it can be concluded that the breakpoint occurred within the gene. After determining the location of the Ekifun within the vassal, the next problem is:
The purpose of this study was to know whether the break point occurs in the exfunctor region or the intron region. For OML DNA such as OML patient 0481's DNA, this question can be easily answered. As mentioned above (
5), certain OML D such as patient 0481's DNA
In NA, a break point was detected inside the L2 kb H/Bg casing fragment. Since there is no coding sequence within this region (Figure 7), patients such as 0481 must have a chromosomal breakpoint in the intron between Ekifun3 and Ekifun4. In the case of the DNA of patients 031106g and 77010, the situation is even more complicated. However, these DNA
By cloning the 9q cleavage point fragment from Ekifun 2 and sequentially performing restriction enzyme analysis and Southern hybridization, we succeeded in detecting the cleavage point between Ekifun 2 and Ekifun 3 (Figure 7). Cloned as previously described -1 (4°5) patients 0319129 and 0212018
The 99 breakpoint fragment with 5 was analyzed by DNA sequencing. Additionally, the 22q breakpoint fragment tube of 562m was cloned into patient 0319129 and cell line. By combining the DNA sequence analysis of the break point regions of these fragments and the sequence analysis of the corresponding region of chromosome 22, we were able to determine the translocation points of these DNAs (Fig. 7). None of the nine breakpoints analyzed here were detected within the exon. This means that the Ph' translocation breakpoint is bcr
It is shown that this occurs within the intronic region of . In four of the six analyzed DNAs, as a result of the translocation, mRNAs containing l equinones 1 and 2 (Figure 7)'f: were transcribed and 2
There is no substantial increase in OML DNA. The DNA sequence of the translocation junction should give more information about the mechanism of chromosomal translocation.
(sover paint) f: Shows the sequence of the containing region and compares it with the normal DNA sequences of chromosome 22 and chromosome 9. In the DNA of 0319129, the chromosomal break occurred briefly and ``exactly'', resulting in normal chromosomes 22 and 9.
22q- and 9q accurately reflect the DNA sequence of the chromosome
However, it is noteworthy that although nucleotide 0 is present in both the 9q+ and 22q- sequences at the breakpoint, the chromosome 22 sequence contains G at the same location. Both have a limited range of homology in the vicinity of the breakpoint (Fig. 8A, underlined area). ODN DNA analysis revealed that this region is homologous to a non-human repeat sequence. A similar open homologous sequence between chromosome 9 and JII122 is not seen at the breakpoint in the DNA of patient 021015 (Figure 8B) or in the -562DN individual ((. This is highly likely to suggest that a secondary event occurred in the normal chromosome 9 between the break points of chromosome 9 and chromosome 22. In this case, the arrow indicating the break point of chromosome 9 Figure 8B) probably indicates the deletion point. Furthermore, the chromosome 9 sequence of the 9q fragment has 13 nucleotide changes within a range of 81 bp compared to the normal chromosome 9 sequence. (' 4U→U These changes may reflect individual differences (the control chromosome 9 sequence was isolated from the human lung cancer layer cosmid library 'lJ). 4-), #4 written by 藋＾Special t5 臥 Ii nomfu) Tsuu. Furthermore, the chromosome 22 portion of the 9q10 fragment does not contain any nucleotide changes within either the intronic or echinonic sequences.
It is demonstrated that DNN damage repair is ineffective upon deletion of sequences on chromosome 9. These results demonstrate that the deletion is the part of the gene with the 5' end that directs the dynamics of chromosome 22. It has been found. In the Philadelphia translocation, the cleavage occurs intrauterinely, and all Ph'-positive subjects (5, 38) and the joining of the c-abL sequence are associated with a complex translocation and cytogenetically associated Ph' chromosome. It is highly KOML-specific since it is an arrangement (coaf muguratloa) that was also observed in the leukemia cells of one OML patient with the defect. Chromosome 9 c-a
Since the orientation of bL is also a dynamic body 5'-3'-terminal small grain,
Ta and C-0μ are extensions of each other (he
ad-to-tail). most mar 0
1 The distance from the highly homologous echinone to the physical cut point is 1
Although the difference ranges from 4 kb (in the case of patient 0319129) to more than 100 kb (in the case of K-sag), the citrus gene and c
The effect of translocation on expression with -7μ is very similar in different patients. -1562 and ph'-positive L-1562 (28, 30). Conclusive evidence for the existence of hybrid mRNA humans is
A hybrid cDN from K-562 cells was obtained by human molecular cloning. Hybrid mRNA individuals must be the result of transcription initiated by the auditory gene promoter. Depending on the precise position detection of the break point, the transcripts include - echinons 1 and 2 or echinons 1, 2 and 3 plus 6'
It will include all the engineering codes. Transcription occurs between the most 6' mer01 homologous exon and phosphorusin (phosphoto).
yroaine) continues within the C-abt oncogenic gene, including at least the 3' equinone sequence containing the acceptor site. It remains unclear whether inclusion of echinone-3 in hybrid mRNA individuals has any effect on disease progression. Chromosomal aberrations may result from specific events involving recombination of prone DN human sequences, or such recombination events may occur more or less randomly. In all cases, a very small number of translocations result in genetic alterations that disrupt normal proliferation and differentiation. It was found that in Ph' translocation, the breakpoint of chromosome 9 extends to a region of 100 kb. The breakpoint of chromosome 22 occurs within a small region of about &Okb. However, when comparing the breakpoint region or the coding region of c-abL and the pathogenic gene of various OML patients, no sequence homology was detected. Therefore, the process that underpins Ph' translocation is likely to occur when random recombinant recombination occurs and hybridize LZJ
Genomic configuration (conf) that enables transcription of μmRNA
The abnormally sized molecule fk21 and 000 c-tL4 proteins were detected in 0K-562 cells, which caused malignant proliferation of cell-specific cells. The potential for humans to be translated into proteins is extremely large. In contrast to the normal C-υL protein, P210 has synkinase activity. (Left below) Materials and Methods Restriction enzymes were Niney England Biolaps (N
ew England Biolab) or Bethesda Research 2PO2Tries (Bethesda
Re5search I, laboratories. (BRL)) and used them according to their respective specifications. The DNA was digested with restriction enzymes, subjected to 0.75% agarose gel electrophoresis, and purified using nitrocellulose (Schleier) as described by Southern (25).
her) and s--le (3chuell), pH
7,9) IC was transferred. Probe nick translation and filter hybridization were performed as known (23, 24). Specific activity of probe is 2-5
x 10” epm/μf. After hybridization, DuPont Lightning
Dupont Lightning P1
u+s) XAR-2 film (Kodak) for up to 5 days at -70°C using an intensifying screen
K exposure. Digest the DNA with an appropriate restriction enzyme and transfer it to low melting point agarose (B
RL) to prepare a DNA probe. The desired band was excised from the gel and dissolved by heating at 65° C. for 30 minutes. 0.3M Na0Ac. Extracted twice with phenol equilibrated at pH 5.0 and extracted with phenol/chloroform/isoamyl alcohol (25:2).
4:1) to remove the agarose. As a carrier, 20 pf/at dextrin (Dex
tran) T-500 (Pharmacia
acia)) in the presence of ethanol and 0.2M Na0.
DNA was precipitated using Ac, pH 4.8. c-abl mRNA in 1562 using the following procedure
Northern blot analysis was performed. K-562 and H
20 μt of polyA RNA from ELA cells was subjected to 1.2% agarose gel electrophoresis in the presence of formaldehyde (13). After plotting, use the nitrocellulose probe or probe A or B (see text and Figure 2).
hybridized to. Total RNA was isolated using the Li(J/urea method (16). Ori-r dT cellulose KRNA
was passed once to obtain po17A RNA. As a molecular weight standard, denatured and kinased
-H1nd III was used. Cosmid library construction and manipulations for screening, isolation, and recombinant propagation were performed using known procedures (11, 25). -562 and 0M637 to the cell line MU line were NigeAX (NI
GMS). Human Genetic Mutan Cell Repository
ts Ce1l Repository), Institute for Medical Research rTnHikitit
auto fnr Medical RAqatr+
= h): I-Deutz- and Davies Street (Copewood and Davies 3tre)
ets), Camden: / (Camden), 2z-・'
Obtained from New Jersey, 08103. Methods Tested Patients Five patients with Ph'-positive CML were tested. Four patients were found to have normal translocations and one patient was found to have complex translocations9. Patient 2128 is a 37-year-old East Indian male, 19
My friend was diagnosed with CML on June 17, 1982. The patient is complex
(9; 12), which had a t(12n 22 ) translocation (38). According to the chromosome test, 46, XY, P
It was determined that the karyotype was h'. The patient is still in the chronic phase. Peripheral blood is normal. Patient 2252 is a 45 year old Caucasian male. 1983 1
On April 26th, he presented with CML symptoms and had a standard t(9;22) translocation. The karyotype (46,XY, Ph') was unchanged, and peripheral blood was normal. Patient 2397 is a 43-year-old female with a 46. XX, t(9
;22). It has a Ph' karyotype. The first person diagnosed with CML was 19 years old.
It was October 17th, 1983. Periodic examination reveals no changes in the karyotype. Peripheral blood is normal. Patient 1708 is a Caucasian female diagnosed with KCML on July 24, 1980. All 40 test cells were 46.
XX, t(9;22), Ph' nine with nuclear deafness. The last bone marrow aspiration was performed on July 9, 1984. Similar cytogenetic findings were seen in 40 tested cells. The patient is chronic. For the past 6 years, I have been conducting continuous cytogenetic testing. Peripheral blood cells stimulated with PHA are normal. Patient 2172 is a 52 year old Caucasian male. CML was first diagnosed on September 22, 1982. From now on 3
I track it every month. The karyotype is 46°XY, t(9;2
2), maintained at Ph/. Peripheral blood stimulated with PEA is normal. Cytogenetic Manipulation First, 20 metaphase cells were banded (stained) using G-banding, one sample per time. Next, the R-band method (R
-banding) 20 cells were banded by K. Metaphase cells were photographed in color and printed in monochrome. Color slides were projected and cytogenetically examined. LiCA! Total RNA was isolated according to the /urea method (18). 25- of 3M LiCl on ice with a fresh bone marrow sample (1-2.5 m)! ! and 6M urea and homogenized using a Polytron homogenizer for 2×30 seconds. The homogenate was kept on ice for 3 hours. The precipitate was centrifuged at 10,001 l for 1 h and
Sux-MC1, pH 7, 5, 5mM EDTA. A 1:1 mixture of 0.2% 50S) and phenol/chloroform/isoamyl alcohol (25:24:1) was dissolved at 3.2 dK. The aqueous phase was precipitated with phenol/lyoaform/isoamyl alcohol (25:24:1) overnight. Wash the RNA three times with 70% ethanol, -70
Stored at °C. Pol RNA was obtained after passing the RNA once through oligo-dT cellulose. d5 of 10Pf! jA
RNA was subjected to 1tIb agarose gel electrophoresis (16) in the presence of formaldehyde. (16) Nitrocellulose blotting (Schleicher and Schue)
ll) and pf(7,9) were performed. A RNA probe was generated. 1-5X 10” cpm of P2 health RNA for each hybridization
It was used. This is insin)DNAIμf is 0.
5-2.5 x 10" cpm equivalent to Ic. XAR-2 film A (Koda
k) Ic sodium phosphate buffer, pH 6,5 and 5
X5 SSC and 1mM EDTA and O, 1% SDS and 2
．． 5 x Denhard (DenhardLs) and 25071
97- salmon sperm DNA and 250 μt/- calf liver R
The plots were first prehybridized for 4 hours at 55°C in NA and 4vh of ioμ2/-. 35-hybridization mix (4 parts prehybridization mix and 1 part 50% dextran sulfate)
Hybridization was carried out overnight at CP55°C. Filters were washed 2 x 20 min at 55°C in 7 pl of 20 mM sodium phosphate buffer (6.5 tmM EDTA (!: 0.1% SDS and 2.5X SSC) at 55°C. 20mM Sodium Phosphate pH 6.5 and 1mM EDTA and 0.1% SDS
．． The cells were washed 2×20 minutes at 65° C. with 3×SSC, and then washed with similar solutions containing 0, 1×5SC, and 0.03×SSC sequentially at 65°C. Results bar/c-abl mR in bone marrow cells of CML patients
Proving the presence of NA required a large number of probes for rounding to perform maser/plot analysis. It was found that most of all Ph' breakpoints occur in the intronic sequence between exons ■ and ■ or in the intron between exons and ■ (Fig. 9A) (5). This indicates that in Ph/translocation, the eDNA portion corresponding to Zorope AK (Figure 9B) resides at 3% on chromosome 22, and the eDNA sequence corresponding to probe B is translocated to chromosome 9. Both probes were inserted into the pSP vector in a reverse orientation so that 32P-labeled RNA complementary to the normal bcr mRNA (in the opposite orientation) could be isolated. This vector construct was used to generate labeled probes. This is because previous experiments have shown that such probes produce a 5 times or more enhanced signal in Northern hybridization compared to conventional nick-translated DNA probes. Similarly, the human c-abl fragment encompassing the most 5' v-abl homologous exfun (ploop C) was transformed into pS
P vector (17). If bcr/abl mRNA is present in leukemic cells of CML patients, one would expect to detect abnormally sized abl transcripts. This abnormally sized mRNA is 5' b
Hybridizes with the ar probe (probe A) but 3
' It should not hybridize with the bar probe (probe B). This is because the genomic sequence corresponding to this probe is translocated to chromosome 9 at t(9:22). All patients have an 8.5 kb transcript that hybridizes with both the c-ak and the 5'-specific probes. 3
The 'specific -bar probe does not -hybridize with this transcript. This transcript is therefore excluded from the normal fib+■ metsenger. The 8.5 kb Methsenjar is not detected in normal control cells or in cells of other types of white mening disease (19,29) and therefore appears to be unique to Ph'-positive cells. Both the 5' and 3' bar probes exhibit approximately 7.5°. 7.5. 7.0
and 4.5 kb transcripts in different human cell lines and tissues,
For example, several other myeloid and linomimyoid cell lines,
It is also present in the red cell lineage progenitor cells, skin, intestines, kidneys and tiles. Among the test samples, 〒Q q し h k M %-
The bone marrow cells of all the patients were 6.0 and 7. It is assumed that both c-abl of Okb start from the same promoter but terminate separately at the 3' end of the abl oncogene. All five patients have both the 8.5 kb hybrid and the normal protein 1 transcript, but the hybrid transcript is expressed at higher levels. (Left below) The cell line contains an abnormal c-abl % driving transcript of approximately 8.5 kb (19, 20, 29). The present inventors demonstrated that this abnormal mRNA was found in the RNA of each of five ph'-positive CML patients.
A, and this is bcr/c-ab1
It was demonstrated that the results correspond to the transcripts. The specific presence of this new transcript in leukemic cells of CML patients suggests that both c-abl and μd are involved in CML and that the fusion of bcr c-abl sequences is crucial for this disease. Re that is the event? 1t' be recognized. In a t(9;12) CML patient lacking a cytogenetically detectable Ph' chromosome, an internal breakpoint on chromosome 12 was recently demonstrated (40) and on chromosome 12. The fact that the genome 1 sequence and the c-abl sequence were localized may also support the above event. Furthermore, this result highlights the limitations of cytogenetic detection of the Ph' chromosome as a prognostic and diagnostic marker. Approximately 15 patients with acute lymphoblastic leukemia (ALL)
The Ph' chromosome is detected in leukemic cells in ~25 days. The inability to obtain high quality metaphase chromosome distributions (apreads) from the leukemic cells of these patients is an obstacle to accurate diagnosis of this type of ALL. bcr DNA probes, such as those used in the present invention, are an alternative diagnostic tool for detecting Ph' translocations in ph'-positive ALLO, liI and certain cytogenetically Ph'-negative CML patients. I get it. Furthermore, some types of AL
It is also possible that hybrid transcripts are detected simultaneously in L patients. The v-alternative probe was used to detect abnormally sized c-abl RNA in the human ALL cell line 5M5-8B (41). As found in five CML patients, hybrid mRNA
Translation of A will result in a bar/abl fusion tanbore. The presence of such proteins indicates that similar bc
The r-c-ab1 transcript was detected and demonstrated in the CMLO cell line, in which an unusual size c-abl protein (p210) with a molecular weight of 210 was detected. This and I indicate that this tan/Q is a translation product of bcr/abl mRNA. Although the mm-forming potential of this tannoq gene is unknown, v-abl, which is the result of recombination between the murine leukemia virus and the mouse c-abl oncogenic gene (4), rapidly eradicates B-cell lymphoma in mice. I can't invite you. It is currently unknown how the abnormal bcr-c-abl hybrid tongue/Q rise contributes to possible tumorigenic effects. However, the CML cell line c-ab
In contrast to tannoQ (14,47), it has tyrosine kinase activity. Therefore, this molecule's 1 rainbow
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230-238゜Brief explanation of the drawings Figure 1 is a restriction enzyme map of the breakpoint region on chromosome 22 of Hani-562. Figure 2 is a restriction enzyme map of the cDNA insert of plasmid VI-3. Figure 3 shows the DNA and amino acid sequences of the VI-3 sequence, Figure 4 shows a detailed restriction enzyme map of bcr cDNA VI-3, and Figure 5 shows the DNA sequence of the VI-3 sequence. Figure 6 is a diagram showing the results of DNA sequence analysis; Figure 6 is a schematic explanatory diagram showing the results of ph translocation; Figure 7 is a diagram showing the genome structure of the Shiku gene; Figure 8 is a diagram showing the results of ph translocation. FIG. 9 is a diagram showing the sequence of DNA breakpoints of a CML patient. FIG. 9 is a diagram showing an exon-intron structure having FLq, 3A Fig, 3B FLq, 3C MIIA MAAAA AAA AAA AAA AA
A AAA AAA AJIA AAAυ LectureFig, 4

Claims (76)

[Claims] (1) It has a nucleotide sequence that is present in an exon of a eukaryotic chromosomal gene and includes at least one sequence that is complementary to the mRNA encoded by this exon, and A single-stranded nucleic acid molecule useful as a probe for detecting chromosomal translocation, characterized by the presence of at least one chromosomal cleavage site into which a nucleotide sequence can be translocated. (2) A nucleic acid molecule complementary to the nucleic acid molecule according to claim 1. (3) The DNA molecule according to claim 1. (4) RNA molecule according to claim 1. (5) The nucleic acid molecule according to claim 1, which consists of only a nucleotide sequence existing within an exon. (6) The nucleic acid molecule according to claim 1, which comprises a nucleotide sequence existing within an exon and a sequence existing within an intron. (7) The nucleic acid molecule according to claim 6, characterized in that it consists essentially of a nucleotide sequence present within an exon. (8) The nucleic acid molecule according to claim 1, which contains one sequence present in an exon located 3' of the chromosomal break site. (9) Claim 1, wherein at least one exon sequence includes a plurality of sequences existing within a plurality of exons located 3' of a chromosomal break site.
Nucleic acid molecules described in Section. (10) The nucleic acid molecule according to claim 1, wherein the molecule has a length of about 20 to about 8,500 base pairs. (11) The nucleic acid molecule according to claim 1, wherein the cleavage site is located within an intron of a gene. (12) The nucleic acid molecule according to claim 1, wherein the cleavage site is located within an exon of a gene. (13) The nucleic acid molecule according to claim 1, wherein a breakpoint cluster region is present in a eukaryotic chromosomal gene. (14) The nucleic acid molecule according to claim 1, wherein the eukaryotic chromosome is a human chromosome. (15) The chromosomal translocation is a Philadelphia translocation, a breakpoint cluster region exists in a eukaryotic chromosomal gene, and this gene is located on human chromosome 22,
An exon is located 3' to the cleavage site located within the cluster region, and other nucleotide sequences are c-\a
The nucleic acid molecule according to claim 1, which is at least a part of a bl\ oncogene. (16) The nucleic acid molecule according to claim 15, which comprises at least a part of the sequence of VI-3 cDNA shown in the attached FIG. 3. (17) A double-stranded DNA molecule comprising the single-stranded DNA molecule according to claim 3. (18) A vector comprising the DNA molecule according to claim 3. (19) The plasmid according to claim 18. (20) The cosmid according to claim 18. (21) The phage according to claim 18. (22) The nucleic acid molecule according to claim 1, which is labeled with a detectable substance. (23) The DNA molecule according to claim 22. (24) The RNA molecule according to claim 22. (25) The nucleic acid molecule according to claim 22, wherein the substance is radioactive. (26) The nucleic acid molecule according to claim 15, which is labeled with a detectable substance. (27) The DNA molecule according to claim 26. (28) The RNA molecule according to claim 26. (29) Chromosomal DNA derived from eukaryotic chromosomes is cleaved using one or more appropriate restriction enzymes under conditions suitable for generating DNA fragments, and the resulting fragments are treated with 1
A double-stranded DNA molecule is obtained, and the single-stranded DNA molecule thus obtained is subjected to suitable conditions that allow hybridization between complementary single-stranded molecules to form a double-stranded DNA molecule according to claim 22. A method for detecting chromosomal translocations on eukaryotic chromosomes, comprising contacting a stranded nucleic acid molecule, detecting the presence of a hybridized molecule, and thus detecting a chromosomal translocation. (30) Chromosomal DNA is cleaved using one or more appropriate restriction enzymes under appropriate conditions to generate DNA fragments, and the resulting fragments are subjected to gel electrophoresis and denaturation treatment.
A double-stranded DNA molecule is obtained, the single-stranded DNA molecule thus obtained is recovered, it is immobilized on a suitable solid support, and the immobilized single-stranded DNA molecule is subjected to hybridization between complementary single-stranded molecules. contacting with a single-stranded nucleic acid molecule according to claim 22 under suitable conditions allowing for dissolution and detecting the presence of hybridized molecules and thus detecting chromosomal translocations. A method for detecting chromosomal translocations. (31) A method for detecting Philadelphia translocation in a human subject, which involves cleaving the entire chromosomal DNA of the subject using one or more appropriate restriction enzymes under conditions suitable for generating DNA fragments. The resulting fragment was processed to produce single-stranded DNA.
A molecule is obtained and the single-stranded DNA molecule thus obtained is subjected to the single-stranded nucleic acid according to claim 26 under appropriate conditions that allow hybridization between complementary single-stranded molecules. A method consisting of contacting a molecule and detecting the presence of a hybridized molecule, thus detecting a Philadelphia translocation. (32) A method for detecting Philadelphia translocation in a human subject, which involves extracting the entire chromosomal DNA of the subject using one or more appropriate restriction enzymes under conditions suitable for generating DNA fragments. cleavage, gel electrophoresis and denaturation of the resulting fragments to obtain single-stranded DNA molecules, recovery of the single-stranded DNA molecules thus obtained, immobilization on a suitable solid support, and immobilization. Complementary 1
contact with a single-stranded nucleic acid molecule according to claim 26 under suitable conditions that allow hybridization between the double-stranded molecules, detecting the presence of hybridized molecules, and thus translating philadelphia. A method consisting of detecting loci. (33) having a nucleotide sequence comprising a sequence encoded by at least a portion of an exon of a eukaryotic chromosomal gene and a sequence encoded by at least a portion of another nucleotide sequence translocated into the gene; Purified hybrid RNA molecules useful as indicators of chromosomal translocations. (34) The RNA molecule according to claim 33, wherein the sequence encoded by the exon is located 5' of the sequence encoded by the translocated nucleotide sequence. (35) Claim 3, wherein the translocated nucleotide sequence includes at least a part of an oncogene.
RNA molecule according to item 4. (36) The chromosomal translocation is a Philadelphia translocation, a breakpoint cluster region exists in a eukaryotic chromosomal gene, and this gene is located on human chromosome 22;
Claim 35, characterized in that the exon is located 5' of the translocated nucleotide sequence, and the translocated nucleotide sequence comprises at least a portion of c-abl.
The RNA molecule described in Section. (37) having an amino acid sequence comprising a sequence encoded by at least a portion of an exon of a eukaryotic chromosomal gene and a sequence encoded by at least a portion of another nucleotide sequence translocated into the gene; Purified hybrid polypeptides useful as indicators of chromosomal translocations. (38) A polypeptide encoded by the RNA molecule according to claim 35. (39) A polypeptide encoded by the RNA molecule according to claim 36. (40) An antibody against an antigenic determinant unique to the polypeptide according to claim 37. (41) The monoclonal antibody according to claim 40. (42) The antigenic determinant is present in a portion encoded by an exon nucleotide sequence located 5' of the translocated nucleotide of the polypeptide. antibody. (43) The antibody according to claim 40, wherein the antigenic determinant is present in a portion of the polypeptide encoded by the translocated nucleotide sequence. (44) An antibody against an antigenic determinant unique to the polypeptide according to claim 38. (45) The monoclonal antibody according to claim 44. (46) Claim 44, characterized in that the antigenic determinant is present in a portion of the polypeptide encoded by an exon nucleotide sequence located 5' of the translocated tumor gene. antibodies. (47) The antibody according to claim 44, wherein the antigenic determinant is present in a portion of the polypeptide encoded by the nucleotide sequence of the translocated tumor gene. (48) An antibody against an antigenic determinant unique to the polypeptide according to claim 39. (49) The monoclonal antibody according to claim 48. (50) A claim characterized in that the antigenic determinant is present in a portion of the polypeptide encoded by an exon nucleotide sequence located 5' of the translocated tumor gene ¥c-abl¥ Antibody according to paragraph 48. (51) The antigenic determinant is present in a portion of the polypeptide encoded by the nucleotide sequence of the translocated tumor gene ¥c-abl¥. antibody. (52) The antibody according to claim 40, which is labeled with a detectable substance. (53) The antibody according to claim 44, which is labeled with a detectable substance. (54) The antibody according to claim 48, which is labeled with a detectable substance. (55) A single-stranded DNA molecule complementary to the RNA molecule according to claim 33. (56) The DNA molecule according to claim 55, which is labeled with a detectable substance. (57) A method for detecting a chromosomal translocation by determining the presence of the polypeptide according to claim 37, which comprises treating a sample containing the polypeptide with an antibody.
Under appropriate conditions that allow the formation of an antigen complex, this polypeptide is contacted with an antibody directed against an antigen site on the polypeptide that is labeled with a detectable substance, and the unbound labeled antibody is combined with the labeled antibody-antigen. A method consisting of separating from the complex and detecting the presence of a labeled antibody-antigen complex. (58) A method for detecting chromosomal translocation by determining the presence of the polypeptide according to claim 38, wherein a sample containing this polypeptide is used to form an antibody-antigen complex. The polypeptide is contacted with an antibody directed against an antigenic site on the polypeptide that is labeled with a detectable substance under appropriate conditions that allow for unbound labeled antibody to be separated from the labeled antibody-antigen complex. , a method comprising detecting the presence of a labeled antibody-antigen complex. (59) A method for detecting a Philadelphia translocation by determining the presence of the polypeptide according to claim 39, the method comprising: contact with an antibody directed against an antigenic site on the polypeptide and labeled with a detectable substance under appropriate conditions that allow the unbound labeled antibody to be separated from the labeled antibody-antigen complex; A method consisting of detecting the presence of a labeled antibody-antigen complex. (60) A method for detecting chromosomal translocation by detecting the polypeptide according to claim 37, the method comprising: a first antibody against an antigenic site encoded by an exonic nucleotide sequence of a gene in which another nucleotide sequence is translocated within said polypeptide under appropriate conditions, and is detectable. nucleotide sequences translocated within said polypeptide under suitable conditions to allow the formation of a labeled antibody-antigen-antibody complex. a labeled antibody-antigen-antibody bound to the solid support; The presence of the polypeptide is determined by separating unbound labeled antibody from the complex and measuring either the amount of labeled antibody complex bound to the solid support or the amount of unreacted labeled antibody. A method consisting of things. (61) Claim 60, characterized in that the first antibody is unlabeled and bound to a suitable solid support, and the second antibody is labeled with a detectable substance. The method described in. (62) The method according to claim 60, wherein the antibody is a monoclonal antibody. (63) A method for detecting chromosomal translocation by detecting the polypeptide according to claim 38, wherein a sample containing this polypeptide is treated in such a manner that an antibody-antigen complex is formed. a first antibody against an antigenic site encoded by an exonic nucleotide sequence of a gene in which another nucleotide sequence is translocated within said polypeptide under appropriate conditions, and is detectable. nucleotide sequences translocated within said polypeptide under suitable conditions to allow the formation of a labeled antibody-antigen-antibody complex. a labeled antibody-antigen-antibody bound to the solid support; The presence of the polypeptide is determined by separating unbound labeled antibody from the complex and measuring either the amount of labeled antibody complex bound to the solid support or the amount of unreacted labeled antibody. A method consisting of things. (64) Claim 63, characterized in that the first antibody is unlabeled and bound to a suitable solid support, and the second antibody is labeled with a detectable substance. The method described in. (65) The method according to claim 63, wherein the antibody is a monoclonal antibody. (66) A method for detecting Philadelphia translocation in a human subject by detecting the presence of the polypeptide according to claim 39, the method comprising: a first antibody directed against an antigenic site at the portion encoded by the c-abl oncogene within said polypeptide under appropriate conditions to allow complex formation and labeled with a detectable substance; The complex thus formed is then contacted with a known amount of the first antibody that has been labeled, and the complex thus formed is exposed to a gene on chromosome 22 within said polypeptide under suitable conditions that allow the formation of a labeled antibody-antigen-antibody complex. a labeled antibody bound to the solid support, which is contacted with a second antibody directed against an antigenic site located in the portion encoded by the exonic nucleotide sequence and bound to a suitable solid support; The unbound labeled antibody is separated from the antigen-antibody complex, and either the amount of the labeled antibody-antigen-antibody complex bound to the solid support or the unbound labeled antibody is measured to determine the polypeptide. A method consisting of detecting the presence of. (67) The method according to claim 66, wherein the first antibody is unlabeled and bound to a solid support, and the second antibody is labeled. (68) The method according to claim 66, wherein the antibody is a monoclonal antibody. (69) A method for detecting chromosomal translocations on a given eukaryotic chromosome, the method comprising: isolating a polyA mRNA molecule encoded by a gene on this chromosome; immobilized on a suitable solid support and contacting the immobilized RNA molecule with an RNA molecule according to claim 24 under suitable conditions that allow hybridization between complementary molecules; A method consisting of detecting the presence of hybridized molecules and thus detecting chromosomal translocations. (70) A method for detecting Philadelphia translocation in a human subject, the method comprising: isolating polyA mRNA molecules of the human subject; separating the thus obtained mRNA molecules by gel electrophoresis; The RNA of claim 28 is immobilized on a suitable solid support and the immobilized RNA molecule is subjected to suitable conditions that allow hybridization between complementary single-stranded molecules. A method comprising contacting a molecule, detecting the presence of a hybridized molecule, and thus detecting an abnormality in the mRNA of a subject caused by a Philadelphia translocation. (71) A method for detecting chromosomal translocations comprising isolating total mRNA from eukaryotic cells, immobilizing such molecules on a solid support, and translating the immobilized molecules between complementary single-stranded molecules. contacting a single-stranded DNA molecule according to claim 26 under conditions that allow hybridization of the molecule, detecting the presence of the hybridized molecule, and thus detecting a chromosomal translocation. How to become. (72) A method for diagnosing a disease by detecting the presence of the polypeptide according to claim 37 associated with the disease. (73) Human chronic myeloid leukemia, acute myeloid leukemia,
or how to diagnose acute lymphoblastic leukemia. (74) A method for diagnosing chronic myeloid leukemia, acute myeloid leukemia, or acute lymphocytic leukemia by detecting the presence of a Philadelphia translocation using the method according to claim 31. . (75) A method for diagnosing chronic myeloid leukemia, acute myeloid leukemia, or acute lymphocytic leukemia by detecting the presence of a Philadelphia translocation using the method according to claim 59. . (76) A method for diagnosing chronic myeloid leukemia, acute myeloid leukemia, or acute lymphocytic leukemia by detecting the presence of a Philadelphia translocation using the method according to claim 70. .