WO1991013172A1 - Facteurs de transcription jouant un role pathogene dans des neoplasmes humains - Google Patents

Facteurs de transcription jouant un role pathogene dans des neoplasmes humains Download PDF

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WO1991013172A1
WO1991013172A1 PCT/US1991/001168 US9101168W WO9113172A1 WO 1991013172 A1 WO1991013172 A1 WO 1991013172A1 US 9101168 W US9101168 W US 9101168W WO 9113172 A1 WO9113172 A1 WO 9113172A1
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nucleic acid
dna
gene
fusion
acid sequence
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PCT/US1991/001168
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Michael L. Cleary
Julia D. Mellentin
David Baltimore
Cornelis Murre
Patrick S. Mccaw
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The Board Of Trustees Of The Leland Stanford Junior University
Whitehead Institute For Biomedical Research
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Publication of WO1991013172A1 publication Critical patent/WO1991013172A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3061—Blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • ALL acute lymphoblastic leukemias
  • chromosome banding remains unknown.
  • the present invention relates to a method of detecting and/or quantitating either or both of the genes localized to the breakpoint of a consistently recurring chromosomal translocation present in a human neoplasm, the fusion DNA which crosses the breakpoint, or their encoded products, as well as to reagents useful in the method.
  • it relates to a method of detecting either or both of the genes localized to the t(1;19) breakpoint on chromosome 19 and chromosome 1 in human acute lymphoblastic leukemias, the fusion DNA which crosses the breakpoint or their encoded products.
  • the two genes localized at the t(1;19) breakpoint are the E2A gene, which is chromosome 19 derived, encodes a ubiquitous transcription factor and is structurally altered by most t(1;19) chromosomal translocations and the pr1 gene, which is chromosome 1 derived (1q23) and encodes a previously undescribed homeoprotein designated Pr1.
  • the altered E2A gene which is a component of the fusion DNA which crosses the t(1;19) breakpoint includes the E2A effector domain, but lacks the region which
  • the gene(s) localized to the breakpoint or the fusion DNA is de tected and , if des ired , quantitate d through the us e o f a selected nucleic acid sequence or probe which is capable of hybridizing to one or both of the translocated genes (i.e., the genes present at the
  • the probe is complementary to all or a portion of the E2A gene and, particularly, to a portion which includes the
  • a nucleic acid probe which is complementary to all or a portion of the prl gene or to all or a portion of the fusion DNA
  • a nucleic acid probe can be used to detect and/or quantitate the fusion RNA transcript shown to be produced from the fusion DNA. It is also possible to detect the presence of and/or quanti tate the product of the altered E2A gene, the prl gene or the fusion DNA or mRNA . This is done through the use of antibodies, either polyclonal or monoclonal, capable of recognizing (binding to) cells which produce the fusion protein produced by cells containing the t(1;19) translocation.
  • Detection of the altered E2A gene, the prl gene, the fusion mRNA or DNA or gene products is useful in the initial diagnosis of acute lymphoblastic leukemia in an individual and in confirmation of a diagnosis of acute lymphoblastic leukemia made by other means. It is also useful in monitoring the effects of therapy instituted once a diagnosis has been made.
  • neoplasia such as acute lymphoblastic leukemia, in which the t(1;19) chromosomal translocation has occurred
  • the present method to inhibit (reduce or eliminate) the effect or activity of the altered translocated gene.
  • a nucleotide sequence capable of hybridizing with all or a portion of the translocated sequence can be introduced into cells in which the translocation has occurred. Hybridization of the introduced (anti- sense) sequence to the translocated sequence renders the latter unavailable for transcription and, thus, prevents production of the mRNA transcript and the encoded protein.
  • the introduced nucleotide sequence can be one sufficiently complementary to all or a portion of the altered E2A or of the prl gene now known to be localized at the t(1;19) breakpoint that it hybridizes to the gene or gene portion under the conditions used.
  • the introduced nucleotide sequence can be one sufficiently complementary to the fusion DNA.
  • the effect of the trans- located gene can be inhibited by introducing into cells an anti-sense construct whi ch hybridiz e s with the fus ion mRNA trans cr ip t produced from the fusion DNA present at the translocation.
  • translocated genes or to the fusion DNA and nucleotide sequences capable of hybridizing to the mRNA transcript of the translocated gene can also be used together to produce inhibition.
  • nucleotide sequences may be the same (i.e., one sequence which hybridizes to both the translocated gene and the mRNA transcript) or may be two or more different sequences.
  • the protein encoded by the translocated genes is targeted and its activity
  • Figure 1 shows the nucleotide sequence
  • Figure 1A is the
  • FIG. 1A is a histogram showing the distribution of silver grains over the human chromosome complement.
  • Figure 1C is a blot showing hybridization of E2A cDNA subclone pE47M to human x rodent hybrid cell line DNAs. Lane 1, total human DNA; lanes 2-5, somatic cell hybrids CF100-1, G38F3, G24A9 and CF104-22, respectively.
  • Figure 1D is the nucleotide sequence of the cDNAs E12 and E47, present in subclones pE12P and pE47P, respectively.
  • Figure 2 shows results of Southern blot analysis of various cell line and leukemia DNAs using the E2A cDNA subclone pE47M as a hybridization probe.
  • Figure 3 is a diagrammatic representation of the t (1;19) (q23;p13) and its unbalanced variant.
  • Figure 4 shows results of Northern blot analysis of E2A mRNA in various lymphoid cell lines.
  • Lane 1 t(1;19)-carrying cell line SUP-B27; lanes 2-4, B lineage lymphoid cell lines FL18; SU-DUL-5; SU-DHL-4; lanes 5 and 6, T lineage ALL cell lines SUP-T7 and SUP-T8, respectively.
  • Figure 5 shows the results of RNAse pro te ction analysis of total RNA isolated from t(1;19)-carrying cell lines (SUB-B27; 697 and RCH-ACV) and cell lines without cytogenetic abnormalities of chromosome 19 (RCH-ADD and FL18), which demonstrate E2A mRNA
  • Figure 6 shows physical maps of E2A cDNAs isolated from the t (1;19)-carrying cell line SUP-B27.
  • Figure 6A is a comparison of restriction maps of normal E2A cDNA and of E2A cDNAs isolated from t (1;19)-carrying cell line SUP-B27. Restriction enzymes represented: EcoRI (R), Sad (S), TaqI (T), Xhol (X), Hindlll (H) and StuI (St).
  • Figure 6B is a diagrammatic representation of normal and fusion E2A cDNAs based on nucleotide
  • Figure 7 shows results of Southern blot analysis demonstrating that sequences linked to E2A cDNA derive from chromosome 1. Arrows indicate the positions of hybridizing bands present in both total human DNA and the chromosome 1 hybrid cell line. All other bands correspond to cross-hybridizing rodent genes.
  • Figure 8 is the nucleotide sequence of fusion cDNAs isolated from t(1;19)-carrying cell line SUP-B27.
  • Figure 9 shows the similarity of predicted fusion protein with various other homeodomains.
  • Figure 10 shows the results of Northern blot analyses of E2A and Prl-homologous RNAs in various lymphoid cell lines using a probe specific for E2A (A) or for the chromosome lq23 gene Prl (B). Migration of normal E2A and Prl mRNAs are shown by dashes. Fusion transcripts are indicated by arrows.
  • Figure 11 shows the results of analysis of total cellular RNA from t (1;19)-carrying cell lines SUP-B27, 697, and RCH-ACV, lymphoblastoid cell line RCH-ADD and B-cell lymphoma cell line FL18, which demonstrate (by PCR) presence of identical E2A/Prl mRNA fusions in the t(1;19)-carrying cell lines. Arrow denotes position of amplified fusion products. Migrations of size markers are shown in base pairs.
  • Figure 12 shows the results of in vitro analys is of E2A and E2A fusion proteins.
  • Figure 13 shows clustering of t(1;19) chromosomal breakpoints in a single intron of the E2A gene.
  • Figure 14 is the nucleotide sequence of the cDNAs representing the normal E2A.E12 transcript and the E2A/prl fusion transcript, obtained from t(1;19) carrying cell line 697.
  • Figure 14A is the nucleotide sequence of cDNA containing the putative coding and 3' untranslated region of E2A.E12.
  • Figure 14B is the nucleotide sequence of the 3' portion of a cDNA representing a fusion transcript between the E2A and prl sequence.
  • the present invention relates to a method of detecting transcription factors which have a pathogenetic role in human neoplasias.
  • a gene encoding a transcription factor has a role in a cancer-associated genetic rearrangement.
  • the E2A gene which codes for immunoglobulin enhancer-binding factors E12 and E47, is consistently located at the breakpoint of the t(1;19) translocation which is a feature of many acute
  • the fusion mRNA codes for a fusion protein in which the DNA-binding and dimerization motif of the transcription factor-encoding gene have been replaced by sequences of the second chromosome which encode a protein which contains a homeodomain (a homeoprotein).
  • the work described herein demonstrates that a recurrent chromosomal translocation in acute leukemias results in synthesis of a fusion protein with features characteristic of a chimeric transcription factor. It also suggests that homeoproteins and transcription factors in general play an important role in the pathogenesis of human neoplasias.
  • the genes localized to the t(1;19) breakpoint site on chromosomes 1 and 19 in human leukemia-associated chromosomal rearrangements have been identified and shown to be present at the translocation in the form of a fusion DNA which includes an altered transcription factor-encoding gene (an altered E2A gene) and DNA which encodes a homeoprotein.
  • E2A/Prl fusion protein contributes importantly to the malignant state of the cells and that the E2A/prl gene is an oncogene.
  • the altered E2A gene has been shown to be a component of fusion DNA which crosses the t(1;19) breakpoint. This is the first time that such an oncogene, formed by fusing the coding regions of two DNA-binding proteins, has been identified.
  • the altered E2A gene includes the putative E2A effector domain, but lacks the region which normally (in the unaltered E2A gene) encodes a helix-loop-helix DNA binding and dimerization motif, and is linked to DNA from chromosome 1 (1q23) shown to encode a homeoprotein, designated Prl (for Pre-B cell leukemia).
  • Prl for Pre-B cell leukemia
  • the t(1;19) breakpoints within the E2A gene are associated with a novel fusion RNA transcript and an encoded fusion protein which has the features of a chimeric transcription factor and may be pathogenetically important in leukemias carrying a t(1;19).
  • detecting and/or quantitating the individual genes or the fusion DNA present at the translocation in DNA obtained from an individual in whose cells a translocation is present is also available.
  • Reagents e.g., DNA probes, specific antibodies
  • the encoded fusion protein can be targeted; its effect can be reduced by introducing into cells a drug which inhibits the activity of the fusion product by inactivating or destroying it.
  • the following is a description of the method by which the identities of the genes at the t(1;19) break- point site on chromosomes 1 and 19 in human acute lymphoblastic leukemias were determined; of the discovery that the t(1;19) breakpoints are associated with a novel fusion mRNA transcript; and of the characterization of the encoded altered E2A protein and Prl protein. These are described in greater detail in the Examples.
  • the following also describes use of the present method to detect other transcription factors, homeoproteins or chimeric transcription factors at a translocation and diagnostic and therapeutic
  • E-box motif found in both the heavy chain and kappa light chain enhancers (Church et al., Cell 313 : 798 (1985); R. Sen and D. Baltimore, Cell 46:705 (1986); Lenardo et al., Science: 236 : 1573 (1987)).
  • E-box elements have been identified in the enhancer region which are required for optimal transcription and bind distinct proteins in vitro (R. Sen and D. Baltimore, Cell 46:705 (1986);
  • E47 and E12 Two cDNAs, designated E47 and E12, that encode kE2-binding proteins have been isolated using an oligonucleotide screening procedure (See Example 1). They derive from one gene, which has been called the E2A gene.
  • the nucleotide sequence of E2A is shown in Figure 1A; the nucleotide sequences E12 and E47 are shown in Figure 10.
  • the cDNAs code for nearly identical proteins containing a region that appears to represent a novel helix-loop-helix DNA-binding and dimerization motif.
  • predicted protein must also be structurally altered following some, if not all, t(1;19) translocations.
  • E2A gene is specifically altered by chromosomal translocation t(1;19), resulting in synthesis of a fusion mRNA that crosses the breakpoint between chromosomes 1 and 19.
  • the fusion mRNA codes for a protein in which the DNA- binding and dimerization motif of E2A has been replaced by sequences from chromosome lq23 containing a homeo- domain.
  • these data demonstrate that a specific, recurrent chromosomal translocation in acute leukemias consistently results in synthesis of a fusion protein with the features of a chimeric transcription factor.
  • the following is a description of analysis of the fusion RNA in the SUP-B27 cell line and in the 697 cell line.
  • RNAse protection assay was employed to investigate the nature of E2A mRNA abnormalities in three cell lines carrying the t(1;19) chromosomal translocation.
  • the templates for synthesis of antisense RNA probes consisted of subcloned E2A cDNA fragments of 1.75 kb (probe A) and 600 bp (probe B), whose positions in the E2A cDNA are shown in Figure 5.
  • RNAse protection studies with both probes showed bands corresponding to fully protected probe in all RNA samples analyzed
  • a differential screening procedure was employed to isolate E2A cDNAs lacking the helix-loop-helix DNA-binding motif and representing candidate fusion cDNAs that potentially crossed the t(1;19) breakpoint. (See Example 3).
  • a cDNA library was prepared from SUP-B27, a cell line that carries a t(1;19) and shown, as described above, to express a novel 8.3 kb E2A transcript.
  • Initial screening was performed with an E2A cDNA fragment (probe B, Figure 5) identical to that used for the RNAse protections.
  • B-lineage cell line SU-DHL-4 ( Figure 6A). However, all of the cDNAs diverged from E2A in their 3' portions, as would be expected based on the screening strategy for their isolation and consistent with the RNAse protection data showing divergence at about the midpoint of the E2A mRNA.
  • SUP-B27 might cross the t(1;19) breakpoint, the chromosomal origin of the 3' portion of KJ6 was determined.
  • a 1.2 kb StuI-EcoRI fragment from the 3' end of this cDNA (probe D, Figure 6B) was used as a hybridization probe on DNA isolated from human sperm; the
  • the chromosome lq23-derived polypeptide contained a limited but significant similarity to several proteins, including the Drosophila engrailed, ultrabithorax, and an tennapedia proteins, the yeast MAT ⁇ 1, MAT ⁇ 2 and MAT ⁇ 2 proteins, and others, all of which have in common the fact that they are homeoproteins (Scott, et al., Ann Rev. Biochem., in press (1990)). Furthermore, the peptide sequence in the fusion protein (amino acids #621-684, underlined in Figure 8) identified by the similarity search overlapped with a region in these proteins that corresponded to their homeodomains.
  • Amino acid identities of selected homeodomains with the chromosome lq23-derived Prl sequence are indicated by dashes.
  • residues conserved through the entire homeodomain family are shown in single-letter code.
  • Asterisks denote four invariant residues in non-yeast homeodomains; eight additional highly conserved residues described previously and amino acids that form the proposed helix-turn-helix motif are indicated.
  • the alignment indicates that this portion of the fusion protein represents a homeodomain, since it contains the four invariant amino acids present in all non-yeast homeodomains and is identical or has cons ervative matches at e ight other h ighly conserved positions (asterisks, colons and periods in Figure 9).
  • Leukemia should be considered a new member of the homeoprotein family.
  • E2A/Prl fusion mRNAs are a consistent feature of t(1;19)
  • Prl, E2A and the fusion mRNAs were examined by Northern blot analysis of RNA isolated from several different lymphoid cell lines. Using a probe from the 3' end of the fusion cDNA (probe D, Figure 6) that was spec if ic for the chromosome 1 portion, 3.0 and 8.3 kb mRNAs were detected in all three t(1;19)- carrying cell lines ( Figure 10; the 8.3 kb RNA in
  • RNA is a fusion product that crosses the t(1;19) breakpoint and, thus, it may represent an imcompletely or alternatively processed form of the 3.0 kb fusion mRNA.
  • the Northern data suggested that fusion mRNAs of similar or identical size were present in all cell lines with a t(1;19).
  • the precise point of E.2A/Prl mRNA fusion in the cell lines SUP-B27, 697 and RCH-ACV was determined using a reverse PCR analysis. (See Example 3).
  • cDNA synthesis was specifically primed with an oligonucleotide complementary to Prl sequences 3' of the fusion point.
  • the resulting cDNAs were then amplified using a primer homologous to E2A sequences 5' of the fusion site in combination with the oligonucleotide used to prime cDNA synthesis.
  • RNA fusion products that spanned the fusion site were detected in the three t (1;19)-carrying cell lines, but not in s everal cell lines lacking the t(1;19) ( Figure 11). Furthermore, the size of the amplified product was identical in all three t(1;19)-carrying cell lines, confirming that the fusion of chromosome 19 and chromosome 1-derived RNA sequences was identical. Similar results have been observed in RNA preparations from all t(1;19)-carrying leukemia cells examined to date, further suggesting that the RNA fusion products described here are a consistent feature of all t(1;19) breakpoints.
  • the reciprocal fusion product is not transcriptionally active, as shown by the fact that a 5'Prl-3'E2A fusion transcript could not be detected by sensitive RNAse protection studies ( Figure 5).
  • the fusion transcript and predicted protein appear to be a
  • the fusion proteins appear to be encoded by a fusion gene whose precursor RNA is processed into the mature mRNA described here. It has also been shown that at least for the E2A gene, t(1;19) breakpo ints tightly cluster in a single E2A intron and cells with the unbalanced t(1;19) containing only the 19p+ chromosome no longer retain exons containing the 3' end of E2A.
  • a pre-B ALL cell line called 697 which harbors the t(1;19) translocation, was examined to determine whether the translocation produces a fused mRNA and protein and to study the gene on chromosome 1 that participates in the translocation.
  • 697 a pre-B ALL cell line that harbors the t(1;19) translocation
  • 697 a novel 90 kDa protein was identified in 697 cells that is likely to be the product of such a fusion transcript.
  • mRNA from 697 cells was used to
  • E2A/Prl fusion protein to leukemia pathogenesis is suggested by the role of helix-loop-helix and homeoproteins in control of developmental and tissue-specific gene expression.
  • the E2A gene codes for two proteins, E12 and E47, which bind the Ig kappa E-box motif kE2 in vitro.
  • the E12 and E47 proteins are nearly Identical; they differ only in a 60 amino acid region that corresponds to a putative helix-loop-helix- DNA-binding and dimeriz ation motif.
  • E2A-encoded proteins may be more generalized than Ig kappa gene expression, as suggested by the fact that they are expressed in many non-lymphoid tissues and the kE2 site is a constituent of other enhancers, including those in the Ig heavy chain, insulin and creatine kinase genes.
  • Prl also has features to suggest that it might under normal circumstances play a role in regulation of gene expression. Sequence analyses indicated that the portion of Prl encoded by the fusion cDNA contains a homeodomain. Homeodomains were first described as highly conserved features of Drosophila homeotic proteins which are important in controlling the
  • the mammalian transcription factors Pit1, Oct1, and 0ct2 contain homeodomains, which for 0ct2 is required for its specific binding to the octamer DNA motif (Ko, et al., Cell, 55:135 (1988)).
  • Prl homeodomain is quite divergent from those described previously; it shows only 30% amino acid identity with any of the other 80 plus homeodomain sequences and requires a 3 amino acid insertion to obtain an optimal alignment. However, Prl still maintains all of the highly conserved residues found in homeodomains and the observed three amino acid insertion is also a feature of the homeodomain for one other protein, i.e., yeast MATa2. Thus, Prl appears to be a new member of this family of proteins that apparently function as sequence-specific DNA-binding proteins. Some, but not all of these proteins have been shown to play important roles in development and it is not unreasonable to predict that Prl plays an important role in early development of the lymphoid system.
  • t(1;19) results in activation of Prl. This may in part be accomplished by transfer of the E2A effector domain to Prl, resulting in a chimeric transcription factor consisting of the Prl DNA-binding site fused to the E2A effector domain.
  • Chimeric transcription factors have been produced experimentally to demonstrate that the bipartite structure of some transcriptional activators is composed of discrete DNA- binding and effector domains (Keegan et al., 1986).
  • the C1 homeotic mutation in Drosophila joins the homeobox exon of the ubx gene to the non-homeobox exons of abd-A to produce a functional fusion protein. It is therefore likely that the E2A/Prl chimeric protein retains its ability to bind to sites normally recognized by Prl although it is possible that its binding specificity is altered.
  • t(1;19) also appears to result in a transcriptional deregulation of the Prl gene.
  • extensive studies of prl expression have not been performed, using Northern blot analysis, expression of the non-translocated prl gene was not observed in a number of lymphoid cell lines ( Figure 10 and unpublished observations).
  • Most significantly, no prl-homologous mRNAs were observed in pre-B cell leukemia cell lines lacking the t(1;19), suggesting that prl is not transcriptionally active at this stage of B cell differentiation.
  • the E2A gene appears to be constitutively expressed in a wide variety of tissues, both lymphoid and non-lymphoid.
  • t(1;19) fusion of the 5' end of the E2A gene to the 3' end of the Prl gene results in a chimeric gene expressed at levels expected for E2A.
  • t(1;19) in part results in transcriptional deregulation of Prl, which normally appears to be quite restricted in its expression or expressed at levels too low to be detected by Northern blot analysis.
  • Prl may be inactivated by t(1;19) due to interruption of its effector domain. This s eems less likely because the fragment of E2A translocated to Prl in at least three cases is identical and the nontranslocated Prl gene remains intact in t(1;19) cells, as judged by Southern analysis. Thus, a recessive mechanism requiring inactivation of Prl cannot be completely ruled out but appears less likely.
  • Activation of Prl by E2A implies an important function for the amino terminal two-thirds of the E2A protein. Published studies of E2A proteins E12/E47 have focused on the HLH motif involved in DNA-binding and dimerization. Murre, C. et al., Cell , 56:777-783 (1989); Murre, C. et al., Cell, 58:537-544 (1989).
  • E2A effector domains include a proline-rich region near its amino terminus or a region slightly upstream of the fusion point whose presence in a GAL4 fusion protein is required for activating transcription off of a GAL4 responsive promoter (Hewthorn, P., et al., Science , in press
  • the method of the present invention is useful to detect and/or quantitate in a sample obtained from an individual, all or a portion of a gene or genes
  • leukemias with a pre-B cell phenotype in which a t (1;19) (q23;p13.3) chromosomal translocation occurs.
  • a sample such as a tumor or blood sample
  • the sample is processed in a manner appropriate for rendering DNA and/or RNA present in cells available for hybridization with a complementary nucleotide sequence. For example, it is treated to disrupt cells, to release nucleic acids (DNA and/or RNA), which are subsequently treated to render them single stranded (e.g., by heating).
  • the treated sample and the nucleic acid probe which is preferably
  • nucleic acid probe is a DNA probe whose sequence is all or a portion of the nucleotide sequence now known to occur at the t(1;19) translocation. For example, it can be all or a portion of the nucleotide sequence represented in
  • the probe used will be sufficiently long to hybridize to sample DNA and remain hybridized under the conditions used; it will generally be at least eight nucieotides long, although under certain conditions, may be shorter. After sufficient time for hybridization of complementary sequences to occur, hybridization is detected, using known techniques.
  • antibodies which bind specifically to a protein of polypeptide encoded by all or a portion of the DNA or mRNA which crosses the breakpoint between chromosome 1 and 19 can be used to detect the expression product.
  • a protein of polypeptide encoded by all or a portion of the DNA or mRNA which crosses the breakpoint between chromosome 1 and 19 can be used to detect the expression product.
  • a protein of polypeptide encoded by all or a portion of the DNA or mRNA which crosses the breakpoint between chromosome 1 and 19 can be used to detect the expression product.
  • a protein of polypeptide encoded by all or a portion of the DNA or mRNA which crosses the breakpoint between chromosome 1 and 19 can be used to detect the expression product.
  • monoclonal antibody specific for the product encoded by the altered E2A gene, by the prl gene, which encodes Prl or by the fusion DNA or mRNA can be produced, using known techniques, and used to analyze a sample (e.g., blood or tumor sample) for the occurrence of the encoded product.
  • An assay which detects and/or quantitates a gene or gene product can be used not only in the diagnosis of acute lymphoblastic leukemia, but also in monitoring treatment of individuals for the condition. Evidence of effective treatment would be available from such an assay carried out, for example, periodically because results would indicate whether levels of the gene or gene product have changed during the treatment (i.e., if treatment is effective,
  • drugs useful in treating acute lymphoblastic leukemia can be any drugs useful in treating acute lymphoblastic leukemia.
  • a nucleotide sequence which hybridizes to all or a portion of the gene localized to the t(1;19) breakpoint can be introduced into cells, in which it will hybridize to the gene or gene portion, rendering it unavailable for transcription, thus reducing or eliminating expression of the encoded product.
  • an oligonucleotide comple- mentary to the altered E2A gene is introduced into cells; hybridization of the complimentary oligonucleotide to the altered E2A gene results in inactivation of the gene.
  • an oligonucleotide complementary to the pril gene is introduced into cells;
  • the presence of a gene encoding a transcription factor at the breakpoint of a different chromosomal translocation can be determined in a similar manner as that described with specific reference to detection of the gene localized to the t(1;19) breakpoint.
  • the methods and probes used for determining that an altered E2A gene and the prl gene are present at the t(1:19) breakpoint can be used to determine whether either (or both) is present at a different chromosomal translocation associated with human
  • E2A-encoded proteins are also expressed in many non-lymphoid tissues and the KE2 site is a constituent of other enhancers, including those in the immunoglobulin heavy chain, insulin and creatine kinase genes. Thus, it is reasonable to expect that the E2A gene or an altered E2A gene is present at other translocation breakpoints.
  • Prl has features which suggest that under normal circumstances, it might play a role in regulation of gene expression. For example, sequence analyses
  • Homeodomains were first described as highly conserved features of Drosophila homeotic proteins which are important in controlling the development fate of embryonic cells. Homeodomains contain a number of highly conserved basic amino acids and a predicted helix-turn-helix secondary structure similar to those found in bacterial DNA-binding
  • homeotic proteins function as sequence-specific DNA-binding proteins and, in many cases, regulate the expression of developmentally important genes. More recently, the mammalian transcription factors Pit1, Oct1, and 0ct2 were shown to contain homeodomains and several lines of evidence suggest that the homeodomain mediates the sequence-specific DNA-binding activities of these proteins.
  • Prl homeodomain is quite divergent from tho s e described previously in that it shows less than 36% amino acid identity with any of the other 89 plus homeodomain sequences and requires a 3 amino acid insertion to obtain an optimal alignment. However, Prl still maintains all of the highly conserved
  • yeast MATa2 homeodomain for one othe protein, the yeast MATa2.
  • Prl appears to be a new member of this family of proteins that apparently function as sequence-specific DNA-binding proteins, some of which have been shown to play important roles in development. Alteration of Prl by chromosomal translocations during early lymphoid differentiation may induce subsequent maturation arrest at the pre-B cell stage, contributing to the development of an acute leukemia. Recent observations that constitutive expression of the Hox2.4 gene is caused by nearby integration of an intracisternal A particle in the murine WEHI-3 myelomonocytic leukemia cell line provide additional support for the potential importance of homeoproteins in leukemogenesis. It is possible that Prl or a similar homeodomain protein plays a role in the pathogenesis of human neoplasias other than acute lymphoblastic leukemias.
  • nucleotide s equence s described herein for localization or detection of the altered E2A gene and/or the prl gene can be used to detect the occurrence of similar sequences present at other chromosomal translocations.
  • a general protocol for determining whether a transcription factor other than E2A, a homeoprotein other than Prl or a chimeric transcription factor which includes both is present is as follows:
  • control cells cells in which the same chromosomal translocation is not present (control cells);
  • Step 5 localization of the cellular nucleotide sequence is at the breakpoint of the chromosomal translocation of interest and compare with its occurrence in control cells;
  • Step 6 If the cellular nucleotide sequence is shown in Step 6 to localize to the chromosomal trans location breakpoint, determine whether it is a component of a chimeric transcription factor or fusion protein which is consistently associated with the chromosomal translocation and consistently absent in control cells.
  • chromosomes was carried out using 3 H-labeled subclone pE47M, which contains a 847 bp insert fragment that codes for 283 carboxy-terminal amino acids, including the helix-loop-helix DNA-binding motif and the
  • DNAs that encode KE2 binding proteins and plasmids containing the DNAs were produced as follows: To isolate a cDNA encoding a specific KE2 binding factor, an oligo (dT)-primed ⁇ gtll library derived from
  • poly (A)-containing RNA of the human B cell lymphoma cell line BJAB was constructed.
  • the unamplified library contained 500,000 recombinants.
  • the probe for the library screen was constructed by cloning three copies of the oligonucleotide tcgaAAGGCAGGTGGCCCAagct
  • TTCCGTCCACCGGGT into the Sail site of PUC 18 and radiolabeling the EcoRI-Hindlll fragment.
  • the library was screened with the trimer probe as described previously (Singh et al. , Cell, 52:415-423 (1988); Staudt et al., Science,
  • nitrocellulose membranes (Schleicher and Schuell) were soaked in 10mM IPTG and air dried at room temperature. LB plates containing phage plaques that were incubated for 3 hr at 42°C were overlaid with one filter for a 6 hr incubation. Next the filters were incubated for 60 min in 50 mM Tris (pH 7.5), 50 mM NaCI, ImM EDTA, 5% nonfat dry milk.
  • the filters were then incubated in 10 mM Tris (pH 7.5), 50 mM NaCI, 1 mM EDTA, 1 mM DTT, 5 ⁇ g/ml sonicated, denatured calf thymus DNA, and approximately 2 x 10 6 cpm/ml of 32 P-labeled probe.
  • the filters were washed three times with the same buffer for 30 min, air dried, and exposed overnight.
  • E12 From the primary screen of 500,000 phage plaques, one recombinant, designated E12, was isolated and used to rescreen the BJAB cDNA library. Several more recombinants were identified by hybridization to the
  • E12 cDNA One of these, E47, containing a 1.8 Kb insert, was chosen for further analysis. Recombinants
  • E12 and E47 were subcloned into PUC18, resulting in plasmids pE12 and pE47.
  • the 1.3 kb EcoRI insert of plasmid pE12 was blunt-ended using Klenow fragment and subcloned, in frame, in the Smal site of pBS-ATG.
  • pBS-ATG was kindly provided by H. Singh (MIT) and was constructed by ligating a 27 bp oligonucleotide
  • Bluescript plasmid (S tratagene). It contains an ATG, Kozak sequences, and a recognition site for T3 RNA polymerase. Clerc et al., Genes, Dev.2,, 1570-1582 (1988). A truncated E47 gene was constructed by cloning a 280 bp Smal-EcoRI fragment into pBS-ATG, resulting in plasmid pE47S. Deletions of pE12 were made using Bal31 exonuclease. The termination sites of the deletion derivatives, designated Bal7, Bal13, and Bal14, were sequenced using the dideoxy chain
  • Truncated versions of pE12 and pE47 were obtained by cloning a Pvull fragment
  • pE47M was produced using the same method.
  • the pE47M subclone was radiolabeled to a specific activity of 7.9 x 10 8 cpm/ microgram by hexamer priming, as described by Feinberg and Vogelstein.
  • Hybridization was carried out at 44oC to chromosome preparations from a chromosomally normal female at a final probe
  • Southern blot analys is of human x rodent hybrid cell line DNAs whose
  • chromosome 19 localizes to chromosome 19 is denoted by an arrow; an additional cross-hybridizing band not present on chromosome 19 and observed only in lane 1 is denoted by a dash. Faint bands at approximately 16 and 18 kb represent cross-hybridizing hamster and mouse bands, respectively. The right hand bracket indicates the smallest region of chromosome 19 containing detectable pE47M-hybridizing DNA. (Lane 1, total human DNA; lanes 2-5, somatic cell hybrids CF100-1, G38F3, G24A9 and CF104-22, respectively.)
  • Results confirmed the chromosomal assignment of the E2A gene detected by the pE47M clone in human DNA. Analysis of EcoRI digested DNA showed two bands
  • the 11 kb EcoRI band observed in total human DNA (lane 1) does not map to chromosome 19 since it is not s e en in lane s 2 - 5 .
  • the s e data indicate the pres ence of an additional pE47M-homologous human sequence that maps to a site other than chromosome 19. This secondary site may not have been observed in the chromosomal in situ hybridization experiments due to a higher
  • DNA samples 2 through 9 were prepared from leukemia cells obtained from children with acute lymphoblastic leukemia whose karyotyp i c evaluation showed a balanced or unbalanced t(1;19) chromosomal translocation.
  • Sample 1 contained a chromosome 19 abnormality that was not due to a translocation with chromosome 1.
  • Cell lines 697 and SUP-B27 were established from t(1;19)-carrying acute lymphoblastic leukemias that phenotypically typed as pre - B cells as described elsewhere (Findley et al., Blood 60:1305 (1982);
  • Lane 1 t(1;19) carrying cell line SUP-B27; lanes 2-4, B lineage lymphoma cell lines FL18, SU-DUL-5, SU-DHL-4; lanes 5 and 6, T lineage ALL cell lines SUP-T7 and SUP-T8, respectively.
  • Size standards consisted of a ladder of phage lambda-homologous marker RNAs (BRL, Gaithersburg, MD).
  • SUP-B27 was established under conditions similar to those described earlier (Smith et al., Blood 71:395-402 (1988); Smith et al., Blood 73:2182-2187 (1989)) from a patient with pre-B cell acute lymphoblastic leukemia. Karyotype studies showed that SUP-B27 was
  • RCH-ADD is an EBC-immortalized lymphoblas told line from the same patient from which RCH-ACV was established (Jack, et al., Cancer, 19:261 (1986)). FL18 was established from a t (14;18)-carrying B cell lymphoma as described earlier (Doi et al., Blood 70 : 1619 - 1623
  • the chromosome 19 and chromosome 1-containing rodent/human hybrid cell lines (5HL9 and MCH 6M2B2 , respectively) were established as described previously (Tro, et al., Proc. Natl . A cad. Sci. USA, 84 : 4180
  • t(1;19)-carrying cell line SUP-B27 were converted to double-stranded cDNA essentially as described (Cleary et al., J. Exp. Med. 167 : 682 - 687 (1988)) with minor modifications. Following EcoRI linker addition, the cDNA was size fractionated by electrophoresis through a 0.8% agarose gel and products of 2-4.5 kb were purified and ligated into lambda gt10.
  • cDNA inserts were subcloned into M13mp18 and M13mp19 phage for nucleotide sequence analyses (Norrander, et al., Gene, 26:101 (1983); Sanger, et al., Proc. Natl. Acad. Sci., U SA 74:5463-5467 (1977)).
  • cDNA clones of the normal E2A mRNA were isolated from a library constructed from the B cell lymphoma cell line SU-DHL-4 described earlier (Cleary, et al . , Cel l
  • Anti-sense E2A RNA probes were transcribed in vitro from pSP64 plasmids containing fragments of the
  • E2A cDNA (probe A or probe B, Figure 5) using SP6 polymerase in the presence of [alpha- 32 P]CTP under conditions provided by Riboprobe System (Promega).
  • RNAse digestion buffer (10 mM Tris pH7.5, 5 mM EDTA, 0.3 M NaCI) containg 40 ⁇ g/ml of RNAse A and 2 ⁇ g/ml of RNAse T1 were added.
  • RNAses were inactivated with SDS and
  • reaction products (shown in Figure 11) were subjected to agarose gel electrophoresis, transfer to nylon membranes, and hybridization with an internal oligonucleotide
  • DNA and RNA were isolated from cultured cell lines using methods described previously (Cleary et al. , Cell 47:19-28 (1986)). Southern and Northern blotting techniques and hybridizations were performed as described (Cleary et al., Cell 47:19-28 (1986)). Gel isolated DNA fragments used as hybridization probes were radiolabeled by random hexanucleotide priming using the Klenow fragment of DNA polymerase and
  • nuclease-treated rabbit reticulocyte lysate (Promega) under conditions recommended by the supplier.
  • the in vitro translation products were analyzed by 10%
  • transcripts initiated within the E2A promoter should extend through E2A-specific sequences and into those from chromosome 1.
  • the translated fusion protein would contain the amino-terminal effector domain of E2A and the carboxyl terminus of a protein encoded by a gene on chromosome 1, and should be reactive with anti-E2A sera.
  • antiserum against the 440 carboxyl-terminal amino acids of the E2A product (clone E12, described herein) was used to immunoprecipitate normal
  • E2A-related proteins from the human pre-B cell line, Nalm-6, as well as potential products of a chimeric E2A transcript in 697 cells, which contain the
  • polypeptides a protein of approximately 90 kDa. This 90 kDa protein was not immunoprecipitated from these cells by control sera, and has not been found in a variety of other cell lines, indicating that it
  • oligo (dT)-primed, size-selected cDNA library (>2.0 kbp) was created using mRNA from the mature human B-cell line, BJAB. Twenty- two positive clones were characterized and the largest, containing an insert of 4.4 kbp, was selected for sequence
  • the cDNA contained a single open reading frame encoding a polypeptide of 654 amino acids and 2.4 kbp of 3' untranslated mRNA ( Figure 14A).
  • Figure 14A is the nucleotide sequence of a cDNA containing the entire coding and 3' untranslated region of E2A.E12.
  • the amino acid sequence is numbered at the left, and the nucleic acid sequence is numbered at the right.
  • the in-frame termination codon both upstream of the E2A.E12 that is excluded from the E2A/prl cDNA is underlined, occurring just after the codon for Serine 483.
  • the broken line designates those amino acids that exhibit the capability to both bind DNA and from dimers. Two probable polyadenylation signals,
  • nucieotides 2512 and 4374 are underlined.
  • the polyadenylation signal at 4374 was not followed by a sequence of adenylate residues.
  • the sequence of four of 22 cDNA clones terminated approximately 300 bases from an Eco RI site (italicized) located at nucleotide 4140, which would place them 60 bases from this
  • the size of this cDNA would suggest that it corresponds to the larger, 4.8 kb mRNA species, and such in interpretation is supported by the observation that if the internal polyadenylation signal located at nucleotide 2512 were used, the size of the transcript would be reduce 1900 bases to 2.9 kb, which is the size of the smaller mRNA.
  • the seventh codon 5' to the first methionine codon was a termination codon, indicating that the sequence would not be translated. There are two possible initiating methionine codons. The first contains a consensus purine (A) at position -3, but lacks a guanosine at position 4, which creates an even more favorable environment for the initiation of translation.
  • a second inframe ATG resides six codons downstream from the first within a sequence having adenosine at -3 and guanosine at +4.
  • nucieotides 678 to 2015 of this clone were identical in sequence to the nucieotides 9 to 1343 of the E12 isolate of the E2A gene ( Figure 1D) and
  • E2A ⁇ The calculated molecular mass of the protein encoded by E2A.E12, designated E2A ⁇ , is 67.6 kDa, which would correspond to one of the intermediate sizes of polypeptides that react with antibodies to E2A.
  • Five of the 22 cDNA clones contained internal fragments not represented in the sequence of E2A.E12, and these cDNA's may code for some of the additional proteins that react with the antisera to E2A ⁇ .
  • a comparision of amino acids 1-470 of E2A with proteins in the computer base did not reveal a significant degree of homology with any other
  • mRNA was isolated from the cytosol of BJAB and 697 cells and purified twice by adsorption to oligo(dT)
  • Sepharose (Pharmacia). Synthesis of the first strand was accomplished using Moloney reverse transcriptase (Promega) after denaturation with methyl mercuric hydroxyde. Second strand synthesis was accomplished using DNA pol I, RNase H, and E.coli DNA ligase. The 3' overhangs were blunted using T4 DNA polymerase and Eco RI/Not I adaptors were ligated to the DNA
  • the 2143-2741 Pvu II fragment was radiolabeled and used to detect homologous transcripts by Northern analysis. Strong hybridization was observed to two RNA species of 3.0 and 8.0 kb, and these transcripts were found in mRNA from 697 cells, but not in the mature T cell lines HSB2 or Jurkat, the mature B cell lines BJAB or LY65, or the pre-B cell line Nalm-6, which does not contain the t(1;19)(q23;p13) translocation. Each of the two transcripts also contained E2A sequences, since both bound an E2A-specific probe as well. To determine the primary structure of these putative chimeric transcripts, a clone designated
  • cDNA clones were prepared for sequence analysis by cloning into the vectors, pBSK+ and pBSK- (Stratagene).
  • JM109 were transformed with the deletion mutants and superinfected with helper phage VCS-M13 at a multiplicity of
  • transcript sequences from the SUP-B27 pre-B ALL cell line The sequence of the larger of the four clones was analyzed in this region and found to contain this same 113 bp insert.
  • the internal 113 nucleotide sequence is flanked by GT at its 5' end and AG at its 3' end (underlined in Figure 14B), consensus sequences that typify an intron.
  • the larger transcript and its potention protein product are designated "type I” and the shorter transcript and corresponding protein are designated "type II”.
  • the gene that contributes the unique 3' sequence has been designated prl, for pre-B cell lymphoblastic leukemia.
  • the fusion transcript is called E2A/prl, because it replaces the E12-specific portions of the E2A.E12 transcript with the prl sequence.
  • the prl-specific portion of the E2A/prl fusion transcript was used as a probe to identify homologous sequences in mouse DNA.
  • Transcripts encode proteins that lack the carboxyl terminal 171 amino acids comprising the
  • E2A/prl fusion transcript exhibits two
  • the type I transcript contains an insertion of 113 nucieotides whose boundaries matched 5'-GT....AG-3' splice donor and acceptor sites, indicating that the larger species is likely the unspliced precursor of the smaller one.
  • the type I transcript was observed in only one of four clones obtained from the 697 cell line found in six of seven clones obtained from SUP-27 (see above). It is not yet known whether these two related proteins have related or different functions.
  • An interesting parallel can be drawn again to the CTF family of transcriptional activators.
  • the CTF-I transcript contains a translated 3' sequence that is spliced out in the CTF-II transcript.
  • the sequence GGGSAAAAAAAAASGG Between the proline-rich domain and the homeobox of the E2A/Prl fusion protein is located the sequence GGGSAAAAAAAAASGG, the underlined portion of which contains the most alphahelical character in the entire protein as determined using an alogrithm designed by Chou and Fasman (Chou and Fasman, 1978).
  • the serine residues that flank this probable helix are helix-breaking residues and both serine and glycine permit maximal flexibility in the polypeptide chain.
  • This sequence may introduce a flexible linkage between the transcription and DNA-binding domains.
  • the longest proteins contained in the datebase the longest
  • sequences of repeated alanines are 11 and 14 residues, contained in the proteins encoded by the eve and en homeotic genes of Drosophilia.
  • the alanines also separate the homeodomain from a proline-rich domain containing 34 proline residue in 300 amino acids (Frasch et al; 1987).
  • the 14 alanines are located 65 amino acids from the amino terminus whereare the homeobox resides at residues 450-510, near the carboxylterminus of this 552 amino acid protein (Poole et al.; 1985). Whether or not such sequences of alanine residues separate functional domains is not yet clear; however, it is curious that such polyalanine sequences are contained in a number of proteins that contain a homeobox.
  • the Prl protein has a remarkable 19 of 27 amino acid identify with the predicted second and third
  • the yeast ⁇ 2 gene product functions as a repressor of a-specific genes by binding as a dimer to a 5' regulatory sequence 31 bp in length (Sauer et al., 1988), and the a1 protein alters the DNA-binding specificity of the ⁇ 2 protein.
  • Prl exhibits no sequence identity with yeast al in the amino-terminal half of its homeobox, but it exhibits maximal sequence identity in this region with the homeobox of the yeast ⁇ 2 protein (8/30, Astell et al., 1981). Two of these sequence identities are phenylalanine residues,
  • ATGTGAATGAATACAT the a1/ ⁇ 2 binding motif in yeast (Miller et al., 1985).
  • hypotheses should be considered, each based on the facts that normally prl is not expressed in pre-B cells, and that the t(1;19) translocation
  • Prl constitutively expresses E2A/Prl proteins that can be expected to alter the transcription of Prl-responsive genes.
  • Prl may be a transcriptional activator that simply becomes expressed by a translocation involving E2A and whose transcriptional activity is not altered by fusion with E2A.
  • Prl could be a tissue-specific transcriptional activator that requires other tissue-specific factors to be functional;
  • Prl would overcome this biological restrication and now be capable of stimulating transcription through ubiquitous factors.
  • Prl might normally function as a transcriptional repressor that would be converted to an activator by replacement of its aminoterminal domain with that of E2A.
  • fibroblasts and bone marrow cells as well as that of its ability to initiate disease in mice, might clarify its role in human leukemia.
  • Translation of the type I transcript terminates 342 amino acids from the
  • the type IIE2A/Prl protein terminates 14 amino acids from the boundary of the spliced intron, contains 259 residues on the carboxy-terminal side of the breakpoint, and has a calculated molecular mass of 77.2 kDa.
  • Prl Encodes a Homebox
  • a computer-generated comparison with known protein sequences revealted significant homology between amino acids 631-691 of Prl and the 60 amino acids that comprise the homeoboxes encoded by other distinct genes. At least three criteria are used to judge whether or not a sequence encodes a DNA-binding protein of the homeobox family.
  • the hallmark of the homeobox is a strict conservation of tryptophan, phenylalanine, asparagine, and arginine residues at positions
  • Prl contains all of these residues.
  • the residues that do not satisfy such conservation, found at positions 5, 12, 17, and 20 are, curiously, locations that are also divergent in the homeobox of the yeast a2 gene, and both genes encode phenylalanine residues at amino acids 5 and 17, which is residue that is not observed at these positions in any of the other homeoboxes.
  • the homeodomain should have a sequence compatible with a helix 2-turn-helix 3 secondary structure, encompassed by amino acids 27 to 51, which is strongly implicated in forming an essential portion of the DNA-binding surface the homeobox.
  • Analysis by the Chou and Fasman algoritum suggests strongly that amino acids 27-37
  • Prl satisfies a majority of the criteria that typify most homeoboxes.
  • yeast al homeobox contained the most sequence identify with the Prl homeobox, having 19 of 27 identities in its carboxyl-terminal half. No significant homologies with other proteins were identified in regions outside the homeobox.

Abstract

Un procédé et des réactifs permettent de détecter et/ou de quantitifer l'un ou les deux gènes situés dans le point de rupture d'une translocation chromosomique récurrente dans un néoplasme humain, l'ADN de fusion qui traverse le point de rupture, ou leurs produits codés. Notamment, un procédé permet de détecter l'un ou les deux gènes situés dans le point de rupture t(1;19) du chromosome 19 et du chromosome 1 dans des leucémies lymphoblastiques aiguës d'êtres humains, l'ADN de fusion qui traverse le point de rupture ou leurs produits codés.
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Publication number Priority date Publication date Assignee Title
WO1993023549A2 (fr) * 1992-05-20 1993-11-25 Centre National De La Recherche Scientifique (Cnrs) Acide nucleique correspondant a un gene du chromosome 22 implique dans les translocations chromosomiques recurrentes associees au developpement de tumeurs cancereuses
DE19610255A1 (de) * 1996-03-15 1997-09-18 Univ Heidelberg Nukleinsäuresequenzen und Verfahren zum Nachweis von Translokationen zwischen Chromosomen
WO1998024928A2 (fr) * 1996-12-06 1998-06-11 Niels Pallisgaard Detection d'anomalies chromosomiques
US5858682A (en) * 1996-08-02 1999-01-12 Pharmingen E2A/pbx1 fusion protein specific monoclonal antibodies

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EP0189628A1 (fr) * 1984-11-15 1986-08-06 The Wistar Institute Détection de néoplasme des B-cellules
EP0252685A2 (fr) * 1986-07-09 1988-01-13 The Wistar Institute Procédés diagnostiques pour la détection de lymphomas chez les humains
EP0338713A1 (fr) * 1988-04-18 1989-10-25 City Of Hope Diagnostic de leucémie chronique de myélogène

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EP0252685A2 (fr) * 1986-07-09 1988-01-13 The Wistar Institute Procédés diagnostiques pour la détection de lymphomas chez les humains
EP0338713A1 (fr) * 1988-04-18 1989-10-25 City Of Hope Diagnostic de leucémie chronique de myélogène

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Leukemia, Vol. 4, No. 12, December 1990 Philippe Jonveaux et al.: "Specific In Vitro Amplified Probe Detects the E2A Gene Rearrangement in the t(1;19) Acute Lymphoblastic Leukemia ", *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993023549A2 (fr) * 1992-05-20 1993-11-25 Centre National De La Recherche Scientifique (Cnrs) Acide nucleique correspondant a un gene du chromosome 22 implique dans les translocations chromosomiques recurrentes associees au developpement de tumeurs cancereuses
FR2691475A1 (fr) * 1992-05-20 1993-11-26 Centre Nat Rech Scient Séquence d'ADN des produits de fusion résultant de la translocation chromosomique récurrente t(11;22) (q24;q12) associée au développement d'un groupe de tumeurs cancéreuses.
WO1993023549A3 (fr) * 1992-05-20 1994-03-31 Centre Nat Rech Scient Acide nucleique correspondant a un gene du chromosome 22 implique dans les translocations chromosomiques recurrentes associees au developpement de tumeurs cancereuses
US5968734A (en) * 1992-05-20 1999-10-19 (Cnrs Centre National De La Recherche Scientifique Nucleic acid corresponding to a gene of chromosome 22 involved in recurrent chromosomal translocations associated with the development of cancerous tumors, and nucleic acids of fusion resulting from said translocations
DE19610255A1 (de) * 1996-03-15 1997-09-18 Univ Heidelberg Nukleinsäuresequenzen und Verfahren zum Nachweis von Translokationen zwischen Chromosomen
DE19610255B4 (de) * 1996-03-15 2004-11-04 Universität Heidelberg Verfahren zur Herstellung von Nukleinsäuresequenzen und Verfahren zum Nachweis von Translokationen zwischen Chromosomen
US5858682A (en) * 1996-08-02 1999-01-12 Pharmingen E2A/pbx1 fusion protein specific monoclonal antibodies
WO1998024928A2 (fr) * 1996-12-06 1998-06-11 Niels Pallisgaard Detection d'anomalies chromosomiques
WO1998024928A3 (fr) * 1996-12-06 1999-03-25 Niels Pallisgaard Detection d'anomalies chromosomiques

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