TRANSCRIPTION FACTORS HAVING A PATHOGENETIC ROLE IN HUMAN NEOPLASIAS

Abstract: 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 reagents useful in the method. In particular, 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.

Description: TRANSCRIPTION FACTORS HAVING A PATHOGENETIC ROLE IN HUMAN NEOPLASIAS Description Background of the Invention Chromosomal translocations are implicated as important events in the pathogenesis of tumors of the hematopoietic lineage. The vast majority of acute lymphoblastic leukemias (ALL) have been shown to carry such karyotypic abnormalities, although more than 15 different consistently recurring rearrangements have been described (F. Mitelman, Cancer (1988) ; Heim, et al. , Cancer (1987). One of the most frequently re¬ ported cytogenetic changes in ALL is the t(l;19) (g23;pl3.3) chromosomal translocation, first reported in 1984 and subsequently observed in up to 6% of pediatric acute leukemias and in approximately 30% of leukemias with a pre-B cell phenotype (Carroll et al. , Blood :721 (1984); Michael et al. , Cancer 12;333 (1984); Williams et al. , Cell 36:101 (1984); Michael et al. , Cancer 17:79 (1985)). The association of this translocation with a particular leukemia phenotype has pro pted several investigations into the identity of genes that may map to the breakpoint loci on chromo¬ somes 1 and 19 (Yang-Fong et_al., Science 22j$:728 (1985); Kodoru et_al. , Cell 43:181 (1986)). However, in spite of suggestive chromosomal localizations, neither the insulin receptor gene at 19pl3.2-pl3.3 nor the proto-oncogene c-ski at lq22-24 has been shown to be directly involved by the t(l;19). The identity of the genes which map to the breakpoint loci on chromosome banding remains unknown. Disclosure of the Invention 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. In particular, it relates to a method of detecting either or both of the genes local- ized to the t(l;19) breakpoint on chromosome 19 and chromosome 1 in human acute lymphoblastic leukemias, the fusion DNA which crosses the breakpoint or their encoded products. In the case of acute lymphoblastic leukemias, it has been shown that the two genes localized at the t(l;19) breakpoint are the E2A gene, which is chromosome 19 derived, encodes a ubiquitous transcription factor and is structurally altered by most t(l;19) chromosomal translocations and the prl gene, which is chromosome 1 derived (lq23) and encodes a previously undescribed homeoprotein designated Prl. The altered E2A gene which is a component of the fusion DNA which crosses the t(l;19) breakpoint, includes the E2A effector domain, but lacks the region which normally (in the unaltered E2A gene) encodes a helix- loop-helix DNA binding and dimerization motif. According to the present method, the gene(s) localized to the breakpoint or the fusion DNA is detected and, if desired, quantitated through the use of a selected nucleic acid sequence or probe which is capable of hybridizing to one or both of the trans¬ located genes (i.e. , the genes present at the breakpoint). In the case of the gene localized to the t(l;19) breakpoint on chromosome 19, the probe is complementary to all or a portion of the E2A gene and, particularly, to a portion which includes the structural alteration shown to be caused by most t(l;19) chromosomal translocations . 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 (i.e. , the altered E2A gene from chromosome 19 linked to the prl gene from chromosome 1 (lq23)) can also be used. Alternatively, 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 quantitate 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(l;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. After diagnosis of human neoplasia, such as acute lymphoblastic leukemia, in which the t(l;19) chromo- so al translocation has occurred, it is possible to use the present method to inhibit (reduce or eliminate) the effect or activity of the altered translocated gene. For example, a nucleotide sequence capable of hybridiz¬ ing 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. In the case of acute lymphoblastic leukemias in which the t(l;19) chromosomal trans¬ location occurs, 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(l;19) breakpoint that it hybridizes to the gene or gene portion under the conditions used. Alternatively, the introduced nucleotide sequence can be one sufficiently complementary to the fusion DNA. In another embodiment, the effect of the trans- located gene can be inhibited by introducing into cells an anti-sense construct which hybridizes with the fusion mRNA transcript produced from the fusion DNA present at the translocation. Nucleotide sequences capable of hybridizing to either or both of the 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. Such 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. In another embodiment, the protein encoded by the translocated genes is targeted and its activity inhibited, either by destruction of the protein or by rendering it unable to have the effect it would have if not inhibited. This can be carried out by introducing into cells one or more drugs which will inhibit the activity of the fusion protein encoded by the fusion DNA, such as by destroying the protein and/or preventing it from carrying out its usual or unusual function (e.g. , by binding to the protein, by pre¬ venting it from interacting with other cellular con¬ stituents by blocking its ability to interact) . This can be carried out using existing drugs or drugs designed for the particular purpose; such drugs can be targeted to affected cells, if desired, by attaching or linking them to a substance (e.g. , a monoclonal anti¬ body) which is specific for cells in which the protein is produced. Brief Description of the Drawings Figure 1 shows the nucleotide sequence and localization of the E2A gene. Figure IA is the nucleotide sequence of the E2A gene. Figure IB 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 CFlOO-1, G38F3, G24A9 and CF104-22, respectively. Figure ID 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 ;pl3) and its unbalanced variant. Figure 4 shows results of Northern blot analysis of E2A mRNA in various lymphoid cell lines. Lane 1, t(l;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 protection analysis of total RNA isolated from t(l; 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 abnormalities in t(1 ; 19) -carrying cell lines. 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 sequence data shown in Figure 8. Open areas , E2A coding sequences; cross-hatched box, potential leucine zipper motif; checkered box, helix-loop-helix motif; stippled areas, non-E2A sequences; filled box, homeobox motif; solid lines, 5' and 3' untranslated sequences. 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 RNAs 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(l;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 analysis of E2A and E2A fusion proteins. Figure 13 shows clustering of t(l;19) chromosomal breakpoints in a single intron of the E2A gene. Physical maps of a portion of the E2A gene and cloned t(l;19) breakpoints (BP3, BP6 , 697, KJ and rKJ) are shown in a 5' to 3' orientation. Filled boxes with numbers denote exons of the E2A gene. Thin horizontal lines denote chromosome 19 DNA; thick horizontal lines denote chromosome 1 DNA. The intron in which t(l;19) breakpoints cluster is indicated by bracket. Restriction enzyme cutting sites as follows: B, BamHI; H, Hindlll, S, Sfil; X, Xhol; Bg, Bglll. Figure 14 is the nucleotide sequence of the cDNAs representing the normal E2A.E12 transcript and the E2A/prl fusion transcript, obtained from t(l;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. Detailed Description of the Invention The present invention relates to a method of detecting transcription factors which have a pathogenetic role in human neoplasias. As described herein, for the first time it has been demonstrated that a gene encoding a transcription factor has a role in a cancer-associated genetic rearrangement. It has now been shown that the E2A gene, which codes for immunoglobulin enhancer-binding factors E12 and E47 , is consistently located at the breakpoint of the t(l;19) . translocation which is a feature of many acute leukemias showing a pre-B cell phenotype. Thus, for the first time, the' potential role of transcription factors in neoplasias has been extended to human leukemias . Demonstration of the involvement of E2A in a cancer-associated chromosomal rearrangement establishes an important precedent. This is particularly true because, in light of the large number of potential genes which code for transcription factors, it is likely that many cytogenetic abnormalities in human neoplasia involve similar types of genes. Furthermore, as is also described herein, it has been shown that the gene which encodes the transcription factor is altered by the chromosomal translocation, resulting in synthesis of a fusion mRNA which crosses the breakpoint between the two chromosomes involved in the trans- location. 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) . Thus, 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. As described herein, the genes localized to the t(l;19) breakpoint site on chromosomes 1 and 19 in human leukemia-associ¬ ated chromosomal rearrangements have been identified and shown to be present at the translocation in the form of a fusion DNA which includes an altered trans¬ cription factor-encoding gene (an altered E2A gene) and DNA which encodes a homeoprotein. In particular, as described below, it has been demonstrated that the E2A gene, which encodes a ubiquitous transcription factor, is localized to the breakpoint of a consistently recurring chromosomal translocation present in many acute leukemias and is structurally altered by most t(l;19) chromosomal translocations . The high percentage of t(l;19) -positive pre-B ALL'S suggest that the 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(l;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. In the fusion DNA, 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 (lq23) shown to encode a homeoprotein, designated Prl (for Pre-B cell leukemia). As is also described, particularly in Examples 3 and 4, the t(l;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(l;19). As a result, a method is now available for 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 trans¬ location is present. Also available is a method for detecting and/or quantitating the fusion mRNA which crosses the translocation and the encoded fusion protein. Reagents (e.g. , DNA probes, specific anti¬ bodies) useful in the method are also now available. In addition, it is now possible to determine whether submicroscopic lesions of the E2A gene or a gene on chromosome 1 are associated with leukemias which lack the t(l;19) chromosomal translocation. Further, based on the work described herein, it is possible to identi¬ fy other genes associated with chromosomal transloca- tions which, in turn, are associated with human neo- plasias. Also as a result of these discoveries, methods for inhibiting (reducing or eliminating) the effects of transcription factors on cells in which they occur are available. For example, it is possible to select or design drugs useful for inhibiting the activity of the transcription factor or of the encoded fusion protein in cells in which the characteristic chromosomal translocation occurs. As described below, anti-sense technology can be used to inhibit transcription factors. Alternatively, 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(l;19) break- point site on chromosomes 1 and 19 in human acute lymphoblastic leukemias were determined; of the disco¬ very that the t(l;19) breakpoints are associated with a novel fusion mRNA transcript; and of the characteriza¬ tion of the encoded altered E2A protein and Prl pro- tein. 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 application of the present invention. Chromosomal Localization of__the E2A Gene Extensive studies of the immunoglobulin genes have resulted in the description of various trans-acting factors and sequence motifs required for their tissue- specific expression (Hatzopoulos et_ 1. , in Eukaryotic -^^_.i-Z2.£ --L.£ϊ.§._--2^_ -E£££^ Biology Series, pp. 43 (1988)). One class of such sequences is the so-called n E-box motif" found in both the heavy chain and kappa light chain enhancers (Church e , t__al . , Cell 313 : 798 (1985); R. Sen and D. Baltimore, Cell 46_:705 (1986); Lenardo et al . , £cience i_2_ _: 1573 (1987)). Several studies have identified trans-acting factors that interact with the E-box sites both i.n vi.vo and in vitro (Church et_al. , Cell 313:798 (1985); R. 05 Sen and D. Baltimore, Cell 46:705 (1986)). For the kappa light chain gene, E-box elements have been identified in the enhancer region which are required for optimal transcription and bind distinct proteins i ^ n lill. . ( R - Sen and D - Baltimore, Cell 46:705 (1986); 0 Lenardo et_al. , j3c:Le ce^2 6 : 1573 (1987)). Two cDNAs , designated E47 and E12, that encode kE2-binding pro¬ teins have been isolated using an oligonucleotide screening procedure (See Example 1) . They derive from one gene, which has been called the E2A gene. The 5 nucleotide sequence of E2A is shown in Figure IA; 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. o This motif shares significant similarity with domains in several proteins involved in the control of dif¬ ferentiation and proliferation in various cellular lineages (Davis et al . , EMB0_J^ 2 :2585 (1988); Caudy et -=----• » Cell 5_5:1061 (1988); Lagouy et_a1. , EMBO J . :3359 (1987); Stanton et_a1. , Proc... Natl ._Aca.d. Sci^ ,_USA 3:1772 (1986); Thisse et_al. , EMB0_J^ 7 : 2175 (1988); Villares et_al. , Cell 5_0:415 (1987); Wright et_al. , Cell jj6_:607 (1989)). Because several members of this group (e.g. , the Myc proteins and Lyl-1), are associ¬ ated with genetic rearrangements and abnormalities of growth control in various neoplasms (Alt £;_ 1. , Qu a Biol. LI 931 (1986)), the potential role of E2A in similar processes was examined. To determine the chromosomal localization of the E2A gene, in .si ^ tu hybridization to human chromosomes (T.A. Donlon, et al . , Am. J. Hum. Genet. , 1 :1097 3 (1984)) was carried out with a H-labeled subclone (pE47M) containing a 847 bp insert fragment that codes for 283 carboxy-terminal amino acids, including the helix-loop-helix DNA-binding motif and the dimerization domain of immunoglobulin enhancer-binding factor E2A (Murre et_al. , Cell 5_6:777 (1989)). As described in Example 1, of 34 grains observed on chromosome 19, 25 (74 percent) were located on bands 19pl3.2-pl3.3 , with a peak on the distal subband 19pl3.3 (See Figure IB). In addition, out of 206 chromosomal grains observed in 100 metaphase cells, 12.1 percent were found over 19pl3.2-pl3.3 and no other sites were labeled above background. The chromosomal assignment of the gene detected by the pE47M subclone was confirmed by Southern blot analysis of rodent x human somatic cell hybrids carrying fragments of human chromosome 19 whose break¬ points have been -well-characterized as described elsewhere. (See Example 1). (Brook £t_al-. , Human 5 Genetics 8:282 (1984); Lusis et_al . , Natl. Acad. Sci ? USA 8_3_:3939 (1986).) These breakpoints are shown in Figure 1C. Analysis of EcoRI digested DNA showed two bands homologous to the pE47M probe in human genomic DNA corresponding to sizes of 23 and 11 kb (lane 1) . Q In the somatic cell hybrids, the 23 kb band was present in lanes 2 and 3 , each containing DNA from human chromosome 19ql3.2-19pter . This band was not observed in lanes 4 and 5 containing DNA from hybrid cells carrying human chromosome 19pl3.2-19pter and 19q, respectively. These results are consistent with ±τi £i u hybridization data which localized pE47M- homologous sequences in 19pl3.2-pl3.3. The 11 kb EcoRl band observed in total human DNA (lane 1) does not map to chromosome 19, as shown by the fact that it is not seen in lanes 2-5. These data indicate the presence of an additional pE47M-homologous human sequence that maps to a site other than chromo¬ some 19. This secondary site may not have been ob¬ served in the chromosomal in situ hybridization experi¬ ments due to a higher stringency of hybridization. The data in Figure 1 also indicate that the E2A gene is highly conserved in rodents, since the pE47M probe cross-hybridizes with a single band of approximately 16 or 18 kb which is present in the somatic cell hybrid DNAs (lanes 2-5) but not in human DNA. Investigation of E2A Involvement in t(1; 19) (q23 ; !3) Chromosomal Translocations The localization of an E2A gene to 19pl3.2-pl3.3 prompted examination of the possible involvement of E2A in t(1 ; 19) (q23 ;pl3) chromosomal translocations . As described in Example 2, for this purpose, Southern blots were carried out on DNA obtained from nine leukemia specimens and two cell lines that carry t(l;19) cytogenetic abnormalities (Findley <≥t_a1. , Blood 60:1305 (1982); Green e _a1. , Cancer 1_ : 251 (1982); Barker et_al. , Cancer 25:379 (1987)) . Using the pE47M subclone as a probe, DNA rearrangements were observed in all but one of these DNAs (Patient #1) , following digestion with EcoRl, BamHI or Hindlll (Figure 2) . Germline DNA from these patients was not available. However, the inability to detect non- germline bands in an equal number of acute leukemia DNAs lacking cytogenetic alterations of chromosome 19 strongly suggests that the observed rearrangements were not inherited polymorphisms but, rather, resulted from translocation breakpoints within or near the NF-E2 gene in each of the leukemias. In most cases, only a single non-germline pE47M- homologous fragment was observed. However, DNA pre¬ pared from the leukemic cells of Patient #3 and the cell line SUP-B27 was found to have two rearranged fragments. As shown in the Table (Example 2), these two cases were the only ones that carried balanced t(l;19) translocations . All other analyzed samples (except #1, which had no E2A rearrangements and did not carry a translocation involving chromosome 1) carried the more common form of t(l:19), shown in Figure 3, whereby the derivative chromosome 1 had been lost by the leukemia cells and only the derivative 19 chromosome was retained (F. Mitelman, Cancer (1988); S. Heim and F. Mitelman, Cancer (1987)). Thus, there was complete correlation between the Southern data and the karyotype data, indicating thatboth products of the translocation were detected as rearranged DNA fragments for the two cases in which they were present. The fact that the pE47M DNA subclone hybridized to both derivative chromosome 1 and 19 translocation products in cases with a balanced t(l;19) Indicates that the breakpoint occurred within the portion of the E2A gene detected by this probe, thereby resulting in a structural interruption of the gene. Furthermore, becausethe p#47M probe consisted of a cDNA fragment coding for approximately 260 C-terminal amino acids and no untranslated portions of the E2A mRNA (Murre e_t_al . , C_ell 5_6_:777 (1989)), the data indicate that the predicted protein must also be structurally altered following some, if not all, t(l;19) translocations . A.ss_ me_nt:_ f_E2^_Tran £riEt:ion^ Products To further investigate the possibility that there is structural alteration of the gene and of the encoded protein, the transcriptional activity and products of the E2A gene were studied in several different cell lines, which included lymphoid cell lines and t(l ; 19) -carrying cell lines. (See Examples 3 and 4). Northern blot analysis of RNAs hybridized with the pE47M cDNA subclone showed two major E2A transcripts of 2.9 and 4.4 kb in all lymphoid cell lines examined (Figure 4). However, in the t(l ; 19) -carrying cell line SUP-B27, an additional, novel RNA of approximately 8.3 kb was also observed (See Example 3) . The presence of an abnormal-sized RNA in SUP-B27 is consistent with the Southern blot data indicating that the E2A transcriptional unit is disrupted by the translocation. In the t(l;19) -carrying cell line 697, a NovLl RNA of approximately 8-0 Kb was observed (See Example 4). The fact that this novel RNA is significantly larger than the normal-sized E2A mRNAs observed in other lymphoid cell lines suggested that it may cross the t(l;19) breakpoint to produce a fusion transcript. Further assessment demonstrated that the E2A gene is specifically altered by chromosomal translocation t(l;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. As described below, 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 trans¬ cription factor. The following is a description of analysis of the fusion RNA in the SUP-B27 cell line and in the 697 cell line. £UP^B27 Truncation , of the E2A , .mR.NA_.by t(l; 19)_ . An RNAse protection assay was employed to investi- gate the nature of E2A mRNA abnormalities in three cell lines carrying the t(l;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 (Figure 5), consistent with earlier Northern blot studies showing expression of full-length, unaltered E2A mRNA in these cell lines. In addition, a partially protected product (arrows in Figure 5) was observed with both probes in RNA preparations from the t(l;19)- carrying cell lines, but not in those from cell lines lacking this cytogenetic abnormality. The sizes of the partially-protected fragments indicated the presence of very similar transcripts in each of the t(1 ; 19)-carry- ing cell lines which diverged from the probe at a position near the midpoint of the E2A mRNA (arrow in Figure 5, lower) between the sequences coding for the previously described leucine zipper and helix-loop- helix motifs. These data also suggested that the divergent RNAs lacked the sequences encoding the E2A DNA-binding motif, consistent with other observations that the t(l;19) results in truncation of the E2A gene with loss of the exons for the DNA-binding domain from the derivative 19 chromosome. i £iati£n_£f_E2^_fu i.on_cDNAs^ Based on these observations a differential screen¬ ing 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(l;19) breakpoint. (See Example 3). A cDNA library was prepared from SUP-B27, a cell line that carries a t(l;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. Phages hybridizing with this probe but not with a 3' E2A cDNA fragment (probe C, Figure 5) were purified for further analyses . Mapping analyses for several restriction enzyme cutting sites showed that cDNAs isolated by the above procedures (KJ3 , KJ6-9) were identical in their 5' halves with the normal E2A cDNA isolated from the 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 pro¬ tection data showing divergence at about the midpoint of the E2A mRNA. To determine whether the cDNAs isolated from SUP-B27 might cross the t(l;19) breakpoint, the chromo¬ somal 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 rodent/human somatic cell hybrid 5HL9 containing human chromosome 19 to which the E2A gene maps, and the CHO cell line MCH 6M2B2 containing a neomycin plasmid- tagged human chromosome 1 as the only non-rodent chromosome (Tao, et_al. , Proc . Natl. ; Acad___ ci ^ ^USA, _J4:4180 (1987)). Results are shown in Figure 7. Five hybridizing bands were observed in total human DNA (H) with this probe, all of which were detected in the chromosome 1 cell line, but not in the chromosome 19-containing cell line. By comparison, Southern analysis of these same DNAs with an E2A probe showed hybridizing bands in total human DNA and DNA from the somatic hybrid containing human chromosome 19, but not in MCH 6M2B2 DNA. Several additional bands observed in the rodent cell line DNAs corresponded to cross-hybridizing rodent sequences, indicated by the fact that they were not observed in total human DNA. The Southern data demonstrated that the cDNAs isolated from SUP-B27 crossed the t(l;19) breakpoint and consisted of 5' sequences of E2A (chromosome 19) fused to 3' sequences from chromosome 1. Fu ^on_of_E2 ^ A_t _a_chr£m£ £me__jL__e_n£C)ded The complete nucleotide sequences of the SUP-B27 fusion cDNAs were determined and a composite sequence is shown in Figure 8. In addition, the nucleotide sequence of cDNAs coding for the normal 3.0 kb E2A mRNA was determined. Clone KJ9 differed from the other four fusion cDNAs in that it had a small deletion (113 bp) of chromosome 1-derived DNA, as shown in Figures 6 and 8. Translation of the fusion cDNA nucleotide sequence showed one large open reading frame that initiated near the 5' end and terminated at nucleotide 2547. In-frame ATG codons were found at nucieotides 70 and 87, with the latter (underlined in Figure 8) appearing most favorable for translation initiation according to the criteria of Kozak, Cell 44:283-292 (1986). The reading frame could not initiate 5' of nucleotide 70 because an in-frame stop codon was found slightly upstream. Comparison of the fusion cDNA nucleotide sequence with that for E2A showed that they were identical up to nucleotide #1519. This portion of the two sequences overlapped and matched that of partial E2A cDNAs (E12/E47) described earlier (Murre et_al . , Cell 56:777-783 (1989), except for a 3 base insertion at nucieotides #1236-38. After nucleotide #1519 the two sequences were completely divergent, apparently repre¬ senting the fusion of chromosome 19 and chromosome 1-derived sequences. The primary structure of the fusion site appeared to be a consistent feature since seven fusion cDNAs independently isolated from SUP-B27 showed an identical nucleotide sequence at the point of divergence from E2A. The open reading frame of fusion cDNAs KJ3 , 6, 7 and 9 continued 1028 nucieotides beyond the fusion point to an in-frame stop codon that was followed by 0.4 kb of 3' untranslated sequence. Schematic representations of the E2A and fusion cDNAs based on their nucleotide sequences are shown in Figure 6B. Results of Southern blot analysis (Figure 7) demonstrated that sequences linked to E2A cDNA derive from chromosome 1. Translation of the nucleotide data showed that all but one of the fusion cDNAs coded for a predicted 85 kdal protein, whereas the deleted variant KJ9 predicted an 80 kdal product (compared to the E2A predicted product of 68 kdal) . Although the predicted fusion proteins contained the amino two-thirds of E2A, they no longer retained the helix-loop-helix DNA-binding and dimerization motif, which was replaced by a polypeptide encoded by DNA from chromosome 1. This was used as a query sequence in a similarity search of the Protein Identification Resource (National Biomedical Research Foundation) . Search results showed that the chromosome lq23-derived polypeptide contained a limited but significant similarity to several proteins, including the Drosophila ngrai.l d, ultra i hor x, and -Q-££S- .££di.a proteins, the yeast MATβl, MATo2 and MATα2 proteins, and others, all of which have in common the fact that they are homeoproteins (Scott, __t_al. , Ann , 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 . A partial alignment of the fusion protein with several selected homeodomains is shown in Figure 9. Amino acid identities of selected homeodomains with the chromosome lq23-derived Prl sequence are indicated by dashes. In the consensus sequence, 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- urn-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 conservative matches at eight other highly conserved positions (asterisks, colons and periods in Figure 9) . To achieve the alignment with the homeodomains shown in Figure 9, it was necessary to introduce a 3 amino acid insertion in all but the yeast MATa2 sequence. This was needed because the homeo¬ domain of the lq23 gene consists of 64 amino acids similar to that of MATa2 , as opposed to all other previously described homeodomains, which are only 61 amino acids in length. This appears to be a dis¬ tinguishing feature of the homeodomain for the fusion protein, which is at most 30% identical to any other homeodomain (Figure 9 and data not shown) . The similarity to other homeoproteins does not extend beyond the sequences shown in Figure 9. These data indicate that the chromosome lq23-encoded portion of the fusion protein, designated Prl (P_re-B cell Leukemia) , should be considered a new member of the homeoprotein family. E2_A Prl_fu i£n__mRNA _are_a_££n i. t£nt_feature_of Expression of Prl, E2A and the fusion mRNAs was 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 specific for the chromosome 1 portion, 3.0 and 8.3 kb RNAs were detected in all three t(l;19)- carrying cell lines (Figure 10; the 8.3 kb RNA in RCH-ACV was evident on longer exposures only) . When an E2A-specific probe was used on these same RNA prepar¬ ations, E2A transcripts of 3, 4.4 and 8.3 kb were observed In the t(l;19) -carrying cell lines (Figure 10); E2A RNAs of only 3 and 4.4 kb were observed in cell lines lacking the t(l;19). These data are con¬ sistent with the cDNA analyses and confirmed that the 3 kb fusion RNA and a 3 kb RNA normally transcribed from the E2A gene comigrated with each other in t(l; 19)- carrying cell lines. Complementary DNA representative of the full-length 8.3 kb RNA was not cloned and therefore its complete composition is not clear. However, the Northern blotting data clearly demon¬ strated that this RNA is a fusion product that crosses the t(l;19) breakpoint and, thus, it may represent an imcompletely or alternatively processed form of the 3.0 kb fusion mRNA. Although E2A trancripts were observed in RNA preparations from all cell lines examined, p_rl. trans¬ cripts were observed only in the t(l; 19) -carrying cell lines and corresponded to fusion RNAs. No p_r].-homo- logous RNAs were detected in various lymphoid cell lines, including pre-B leukemia cell lines lacking the t(l;19). This indicates that the j>rl. gene may not normally be transcriptionally active at these stages of lymphoid differentiation. The Northern data suggested that fusion mRNAs of similar or identical size were present in all cell lines with a t(l;19). To further investigate this, 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). Using RNA preparations from various cell lines, cDNA synthesis was specifi¬ cally 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. Using a kinased internal oligonucleotide, amplification products that spanned the fusion site were detected in the three t(1 ; 19) -carrying cell lines, but not in several cell lines lacking the t(l;19) (Figure 11) . Furthermore, the size of the amplified product was identical in all three t(l; 19) -carrying cell lines, confirming that the fusion of chromosome 19 and chromo¬ some 1-derived RNA sequences was identical. Similar results have been observed in RNA preparations from all t(l; 19) -carrying leukemia cells examined to date, further suggesting that the RNA fusion products des¬ cribed here are a consistent feature of all t(l;19) breakpoints. Thus, results showed that the t(l;19) chromosomal translocation results in synthesis of a novel transcript that crosses the t(l;19) breakpoint and codes for a predicted fusion protein with the properties of a chimeric transcription factor. As shown by others, this translocation has been convinc- ingly associated with a specific subset of childhood acute lymphoblastic leukemias that have a pre-B cell phenotype. (A. J. Carroll e.t_al. , Bl.££d, 6_3_:721 (1984); P.M. Michael et al . , Cancer Genet. Cytogenet. , 12:333 (1984), P.M. Michael et^al^, Cancer Genet. Cytogenet. , L7:79 (1985); D.L. Williams et_al., Cell , 3_6_:101 (1984). The evidence presented herein suggests that the translocation-induced fusion products are important in the pathogenesis of t(1; 19) -carrying leukemias for several reasons: First, there appears to be selective retention of a characteristic E2A/Prl fusion gene by these cells. The 19p+ chromosome which is present in all leukemias carrying the t(l;19) encodes the E2A/Prl fusion gene, since it is present in the 697 cell line which carries an unbalanced form of the t(l;19). This is consistent with cytogenetic studies suggesting that the critical genetic rearrangement is the translocation of material from chromosome 1 to chromosome 19. This is supported by the fact that the t(l;19) is frequently unbalanced with loss of the lq- chromosome. (Mitelman, F. , Cancer Cyto enetics , A.R. Liss, New York (1987)). Furthermore, in cell lines carrying a balanced t(l;19) with retention of both translocated chromosomes, 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). Second, the fusion transcript and predicted protein appear to be a consistent feature of all t(1; 19) -carrying cells that were examined. The point of fusion for E2A and Prl mRNA sequences was shown to be the same in three t(l ; 19) -carrying leukemia cell lines, suggesting a selective pressure during leukemogenesis for fusion of specific portions of the E2A and Prl proteins. 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(l;19) breakpoints tightly cluster in a single E2A intron and cells with the unbalanced t(l;19) containing only the 19p+ chromo- some no longer retain exons containing the 3' end of E2A. Figure 13 and Mellentin, et al . , S_£uan£ , 4_> :379-382 (1989)). These observations allow a deduction of the orientation for both genes on their respective chromosomes which is 5' to 3' , centromere to telomere; a fusion gene results from the exchange of the telomeric portions of each chromosome and gene. 69_7 As described in Example 4, a pre-B ALL cell line called 697, which harbors the t(l;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. Through use of an antiserum that recognizes the E2A products, 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 synthesize, isolate, and characterize cDNA's representing possible chimeric mRNA's transcribed across the breakpoint. Four chimeric cDNA's were characterized. Each contained sequences encoding the aminoterminal 484 amino acides of E2A. After this point, at the 3' edge of an exon, the DNA sequence diverged from that of E2A, to sequence encoding either 259 or 342 amino acids containing a heretofore underscribed homeobox. Homeobox fusion mRNA The contribution of an E2A/Prl fusion protein to leukemia pathogenesis is suggested by the role of helix-loop-helix and ho eoproteins 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 puta¬ tive helix-loop-helix- DNA-binding and dimerization motif. Since cDNAs for E12/E47 were originally isolated based on their ability to bind kE2 , they are implicated in Ig kappa light chain gene expression, but this has not yet been demonstrated in vivo. The role of 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. G.M. Church et_al . Cell, 3_1 :798 (1985); A. Ephrussi et_al. , Science , 2^7:134 (1985); L.G. Moss et_al. , M l ___Ce11___Bi£l__, 8 : 2620-2627 (1988); J.N. Buskin and S.D. Hauschka, Mo^Cell^iol^, in press (1989). Sequence comparisons (Murre, C. t_a^., <3eJL.l, 56_:777-783 (1989)) suggest that the E12/E47 proteins may be the mammalian counterparts of the Drosophila Daughterless protein, which plays a role in neural and sex determination as defined by several mutations. Caudy, M. et_al . , Genes _Dev.. , 2 : 843-852 (1988); Cline, T.W. , Genetics, 84:723-742 (1976); Cronmiller, C. and T.W. Cline, Cell, 4_J:479-487 (1987). Recent studies show a functional similarity of the E12/E47 and Daughterless proteins in that they form heterodimers with Achaete-scute and MyoD proteins, resulting in complexes that bind in vitro with enhanced avidity to the kE2 site (Murre, C. et al. , Cell, 58:537-544 (1989)). The prevailing thought is that the E2A- encoded proteins are ubiquitous components of an enhancer-binding regulatory system whose specificity is determined at least in part by heterodimerization with tissue-specific proteins, resulting in high-affinity sequence-specific transcription factors. 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 developmental fate of embryonic cells. Scott, M.P. t al. , Ann. R£v_. Biochem. , in press (1990) . 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 pro¬ teins. There is increasing evidence that homeotic proteins function as sequence-specific DNA-binding proteins and, in many cases, regulate the expression of developmentally important genes (Scott, M.P. , et_al. , nn_Rev__Bi L £ h£m_^, in press (1990)). The mammalian transcription factors Pitl, Octl, and 0ct2 contain homeodomains, which for 0ct2 is required for its specific binding to the octamer DNA motif (Ko , et al . , C£li» 55:135 (1988)). The 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 in¬ sertion 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. Alteration of Prl by chromosomal transloca- tions during early lymphoid differentiation could result in subsequent maturation arrest at the pre-B cell stage contributing to the development of an acute leukemia. The potential importance of homeoproteins in leukemogenesis is also suggested by the constitutive expression of the Hox2.4 gene caused by nearby inte¬ gration of an intracisternal A particle in the murine WEHI-3 myelomonocytic leukemia cell line Blatt et al, EMB0_J__, 7:4283-4290 (1988). ϊiiϊ5£.EA£_t£an£5 ri : £t^£ _factor_i.n_acute_leukemia Based on the work described herein, it appears most likely that t(l;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 trans¬ cription factor consisting of the Prl DNA-binding site fused to the E2A effector domain. Chimeric transcrip¬ tion factors have been produced experimentally to demonstrate that the bipartite structure of some trans - criptional activators is composed of discrete DNA- binding and effector domains (Keegan et al . , 1986). In addition, the Cl 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(l;19) also appears to result in a transcrip- tional deregulation of the Prl gene. Although ex¬ tensive studies of £rl expression have not been per¬ formed, using Northern blot analysis, expression of the non-translocated £rl gene was not observed in a number of lymphoid cell lines (Figure 10 and unpublished observations). Most significantly, no £rl L -homologous mRNAs were observed in pre-B cell leukemia cell lines lacking the t(l;19) , suggesting that £rl is not trans¬ criptionally active at this stage of B cell differenti¬ ation. The E2A gene, on the other hand, appears to be constitutively expressed in a wide variety of tissues, both lymphoid and non-lymphoid. Following t(l;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. Thus, t(l;19) in part results n 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. Alternatively, Prl may be inactivated by t(l;19) due to interruption of its effector domain. This seems 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(l;19) cells, as judged by Southern analysis. Thus, a reces- sive 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_a , Cell, 5^:537-544 (1989). However, this motif is specifically and consistently deleted by t(l;19), indicating that the "activating" properties are independent of the HLH motif. Initial studies suggested that the E2A-encoded proteins E12 and E47 contained a domain with regularly spaced leucines representing a potential leucine zipper. The location of this domain in E2A would result in its retention in the fusion protein following t(l;19). The leucine zipper has been found in several transcription factors and functions as a helical dimerization site to determine the specificity of protein-protein hetero- dimer formation, presumably via a coiled coil struc¬ ture. Landschultz et_al. (1988); O'Shea et_al. (1989). However, contrary to original suggestions, sequence analyses show that E2A does not contain a heptad leucine repeat with a helical secondary structure, suggesting that other upstream portions may be im¬ portant for activation of Prl. Other candidates for 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. , t_a1.. , S_£jLe_n£e , in press (1990)). Consistent retention of the latter domain with concurrent deletion of the HLH motif in a fusion transcript correlates with the tightly clustered occurrence of t(l;19) breakpoints in a single intron of the E2A gene. It is possible that the translocated segment of E2A allows Prl to interact with components of the transcription apparatus with which it normally does not interact or does so less effectively. The effector domain of E2A may be particularly potent, given its probable involvement in the expression of Ig heavy and light chain genes which are transcribed at high levels in B lymphocytes. ^ERii£ ti£n_£f_th _M th£d_and_ a j _ent _£f_ 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 localized to the t(l;19) breakpoint on chromosomes 1 and 19 in acute lymphoblastic leukemias or a product encoded by one of the genes. This method is useful, for example, in the diagnosis of acute lymphoblastic leukemia, such as pediatric acute leukemias and leukemias with a pre-B cell phenotype, in which a t(1; 19) (q23 ;pl3.3) chromosomal translocation occurs. In the present method, a sample, such as a tumor or blood sample, is obtained from an individual, using known techniques. In the embodiment in which a nucleic acid probe is used, 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 labelled for subsequent detection, are combined under conditions appropriate for hybridization of comple¬ mentary nucleotide sequences to occur. The nucleotide sequence used is one sufficiently complementary to all or a portion of: 1) the altered E2A gene; 2) the prl gene; 3) the DNA present at the chromosomal transloca¬ tion (referred to as the fusion DNA, which includes the altered E2A gene and the prl gene) or 4) the fusion mRNA to hybridize and remain hybridized under the conditions used. In one embodiment, the 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(l;19) translocation. For example, it can be all or a portion of the nucleotide sequence represented in Figure 8. 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 hybridiza¬ tion of complementary sequences to occur, hybridization is detected, using known techniques. In another embodiment, 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. For example, a 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, as described above, 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, decreased levels will be observed) . As a result of the present invention, drugs useful in treating acute lymphoblastic leukemia can be designed. For example, a nucleotide sequence which hybridizes to all or a portion of the gene localized to the t(l;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. In one embodiment, an oligonucleotide co ple- mentary to the altered E2A gene is introduced into cells; hybridization of the complimentary oligonucleo¬ tide to the altered E2A gene results in inactivation of the gene. Alternatively, an oligonucleotide comple¬ mentary to the pril gene is introduced into cells; hybridization results in reduction or elimination of Prl protein production. Detecti£n_£f_th£_E2A_A^tered_Tran £ri£ti.£n_Fa £Ξ_th _Ch^m ric_E2. Prl_Tr n £r_L££^ C h£r_Chr£m£ omajL_Tran l£ t^£n_Br ak££i.nt 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(l;19) breakpoint. For example, the methods and probes used for determining that an altered E2A gene and the prl gene are present at the t(l:19) breakpoint can be used to determine whether either (or both) is present at a different chromosomal translocation associated with human neoplasia. Other data suggest that the role of E2A- encoded proteins is more generalized than simply immunoglobulin kappa gene expression. 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 indicated that the portion of Prl encoded by the fusion DNA encodes a homeodomain. 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- urn-helix secondary structure similar to those found in bacterial DNA-binding proteins. There is increasing evidence that 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 Pitl, Octl, 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 . The Prl homeodomain is quite divergent from those 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 residues found in homeodomains and the observed three amino acid insertion is also a feature of the homeodomain for one othe protein, the 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 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 develop¬ ment 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 . Using the method of the present invention, it is possible to assess the role of an altered E2A gene, the prl gene, the fusion DNA which includes both or a functional equivalent of the genes or fusion DNA in other human neoplasias associated with a chromosomal translocation. For example, nucleotide sequences 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. If stringency conditions used are lowered to increase hybridization between nucleotide sequences present in a sample containing a suspected translocation and probes which are sequences presented herein for detecting the altered E2A gene and the prl gene, the likelihood of detection will be increased. D t Ϊ£n_£f_0_:h r_Tran £ri^£i£n_Fa t;£r ∑ - £i _£r_Chim£ri. _T£an £ri££^£n_ a£_: r _Pr£ nt_a_: _Chr£m£ £mal._Tra 2.££a_;i£n_Br a.k££l.nt Alternatively, it is possible to identify a gene encoding a transcription factor other than E2A or a gene encoding a homeoprotein other than Prl present at the breakpoint of a chromosomal translocation associated with a human neoplasia, by modifying the method described above. 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 : 1. Obtain cells in which a chromosomal translocation associated with human neoplasia is present and cells in which the same chromosomal translocation Is not present (control cells) ; 2. Treat the cells to render nucleic acids present available for hybridization with nucleic acid probes complementary in sequence ; 3. Assay the cells with a nucleic acid probe(s) capable of detecting (complementary to the nucleotide sequence of) a selected (known) transcription factor(s) or a selected homeoprotein; 4. Detect hybridization of complementary sequences, if any; 5. Determine the chromosomal localization of the cellular nucleotide sequence to which the nucleotide probe(s) hybridized; 6. Determine whether the chromosomal localization of the cellular nucleotide sequence (Step 5) is at the breakpoint of the chromosomal translocation of interest and compare with its occurrence in control cells; 7. If the cellular nucleotide sequence is shown in Step 6 to localize to the chromosomal translocation 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. The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way. EXAMPLE_1. Chromosomal. Localizatj L on_of_the_E^A_G£ng In order to determine the chromosomal localization of the E2A gene, i.n_siju hybridization to human 3 chromosomes was carried out using 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 dimerization domain of immunoglobulin enhancer-binding factor E2A. The nucleotide sequence of pE47M is represented in Figure IA. 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 λgtll library derived from poly(A) -containing RNA of the human B cell lymphoma cell line BJAB was constructed. The unamplified library contained 500,000 recombi- nants . The probe for the library screen was con¬ structed 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 £_ _.. , Cell, .52:415-423 (1988); Staudt et al. , Science, 241:577-578 (1988)). Briefly, nitrocellulose membranes (Schleicher and Schuell) were soaked in lOmM 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 M Tris (pH 7.5), 50 mM NaCI, 1 mM EDTA, 1 mM DTT, 5 μg/ml sonicated, denatured calf thymus DNA, and approximately 2 x 10 32 cpm/ml of P-labeled probe. The filters were washed three times with the same buffer for 30 min, air dried, and exposed overnight. 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 TGCACACCATGGCCATCGATATCGATC into the Pstl site of pBS Bluescript plasmid (Stratagene) . It contains an ATG, Kozak sequences, and a recognition site for T3 RNA polymerase. Clerc et al , , G £ , Djs ^.^, 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 , Ball3, and Ball4, were sequenced using the dideoxy chain termination technique. Truncated versions of pE12 and pE47 were obtained by cloning a Pvull fragment, encoding approximately 350 amino acids of both proteins, in frame into the Smal site of pBG-ATG, resulting in pE12P and pE47P. pE47M was produced using the same method. For in situ hybridizations, the pE47M subclone was g radiolabeled to a specific activity of 7.9 x 10 cpm/ microgram by hexamer priming, as described by Feinberg and Vogelstein. Feinberg, A. P. and B. Vogelstein, A a_t^._Biochem^, 13.1-6 (1984). Hybridization was carried out at 44 C to chromosome preparations from a chromosomally normal female at a final probe concentration of 10 pg/microliter, using methods described by Harper and Saunders . Harper, M.E. and G.F. Saunders, Chromosome , ^:431 (1981). The preparations were exposed for five days, developed and analyzed. T.A. Donlon ££_al._ L , 3^:1097 (1984). Additional studies using less stringent hybridization conditions showed a primary hybridization peak at chromosome bands 19pl3.2-pl3.3 and a possible secondary site on chromosome 9p . Visualization was carried out using a light microscope and results are shown in Figure IB, which is a histogram showing the distribution of silver grains over the human chromosome complement. Of 34 grains observed on chromosome 19, 25 (74 percent) were localized on bands 19pl3.2-pl3.3 with a peak on the distal subband 19pl3.3 (Figure IB). Of 206 grains observed in 100 metaphase cells, 25 (12.1%) were localized to 19pl3.2-pl3.3. To confirm the chromosomal assignment of the gene detected by the pE47M subclone, Southern blot analysis of human x rodent hybrid cell line DNAs whose breakpoints have been well-characterized were carried out using the same probe (pE47M) . J.D. Brook e _a ., Human_ Genetics , 6_8_:282 (1984) and A.J. Lusis et al . , Proc. Natl. Acad. Sci. , USA, 83:3929 (1986). Ten micrograms of EcoRI digested DNA was applied to all lanes and Southern blots hybridized with the pE47M subclone as a probe .using previously described methods. M.L. Cleary et_al_ , Cell, 47:19 (1986). The results of these analyses are shown in Figure IC. The human chromosome 19 content of each cell line Is indicated by brackets below the autoradiogram. The pE47M-homologous band in the autoradiogram that 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 CFlOO-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 homologous to the pE47M probe in human genomic DNA corresponding to sizes of 23 and 11 kb (lane 1). In the somatic cell hybrids, the 23 kb band was present in lanes 2 and 3, each containing DNA from human chromosome 19ql3.2-19pter . This bands was not observed in lanes 4 and 5 containing DNA from hybrid cells carrying human chromosome 19pl3.2-19qter and 19q, respectively. These results are consistent with the m i.t hybridization data which localized pE47M- homologous sequences to 19pl3.2-pl3.3. The 11 kb EcoRI band observed in total human DNA (lane 1) does not map to chromosome 19 since it is not seen in lanes 2-5. These data indicate the presence 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 stringency of hybridization. A. P. Feinberg and B. Vogelstein, Anal___Biochem_ ^ , 112:6 (1984); M.E. Harper and G.F. Saunders, Chromosome , i{_ : -* 31 (1981); T.A. Donlon et_a l , Am _ _Hum Λ _Geιιet^, ^35:1097 (1984) . The data in Figure 1 also indicate that the E2A gene is highly conserved in rodents since the pE47M probe cross-hybridizes with a single band of approximately 16 or 18 kb present in the somatic cell hybrid DNAs (lanes 2-5) but not in human DNA. 5^1i;I_2. Hybridization Analysis of_Various_CeIl_Line and Leukemia DNAs To evaluate the possible role of the E2A gene in t(1; 19) (q23;pl3) chromosomal translocations, DNA from nine leukemia specimens and two cell lines which carry the t(l;19) cytogenetic abnormalities was analyzed by Southern blots, using the pE47M subclone as a probe. Results are shown in the Table. TABLE P r£i.a^__kar2£ 2£e£_a d_ outh£rn_bJL£ _re ul ^ _:£_on ∑ar^£U _£ell_l^ne _and_acut _leukemi.a_ £eci.men Patient or Cel^_li.ne art^a3 : _kar_r£t:__2 1 -19,+der(19) 2 -19,+der(19) t(1 ;19) (q23;p!3) 3 t(l;19)(q23; P 13) 4 -19,+der(19) t(1; 19) (q23 ;pl3) 5 -19,+der(19) t(1;19) (q23 ; P 13) 6 -19,+der(19) (1;19) (q23;pl3) 7 -19,+der(19) t(l ;19) (q23 ;pl3) 8 -19,+der(19) t(l;19) (q23 ;pl3) 9 -19,+der(19) t(l;19) (q23;pl3) 697 -19,+der(19) t(1;19) (q23 ;pl3) SUP-B27 t(l;19)(q23;pl3) DNA samples 2 through 9 were prepared from leukemia cells obtained from children with acute lymphoblastic leukemia whose karyotypic evaluation showed a balanced or unbalanced t(l;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 _>0:1305 (1982); Green £t_al., Cancer 2 :257 (1982); Barker et_a1. , Cancer 2.5:379 (1987); Smith et_al. , Bl££d 21:395 (1988); Smith £t_al. , ibid__, in press) . X^ lϊ ; _3. A £ ment_£f_Tran £ri££i£nal_A£ti.vi.ty_£f ^l£_-§-?.A_C£n To investigate the possibility that the breakpoint occurred within the portion of the E2A gene detected by the pE47M probe, thereby resulting in structural interruption of the gene and that the predicted protein must also be structurally altered following some, if not all, t(l;19) translocations, the transcriptional activity and products of the E2A gene were studied in several different cell lines. Poly(A)+mRNAs were size- fractionated in formaldehyde-agarose gel, transferred and hybridized with a cDNA probe (pE47M) using a previously described method. M.L. Cleary £t_al._ , C≥ll, 47_-19 (1986). Results are shown in Figure 2. A novel 8.3 kb mRNA present in cell ine SUP-B27 is indicated with an arrow. Lane 1, t(l;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. Derivation and characterization of cell lines have been described previously. S.D. Smith et_al . , Bl££d, 71:395 (1988); A. Epstein et al . , Cancer, 42 ^ :2379 (1978); S.D. Smith et_al_. , ibid, in press; S.H. Doi _st_a_ _, Bl££d, 2-2:1619 (1987) . Size standards consisted of a ladder of phage lambda-homologous marker RNAs (BRL, Gaithersburg, MD) . Assessment of E2A mRNA Abnormalities and Alterations__in M the_Enc£ded_E2A_Pr£duct The establishment and characterization of t(l;19) -carrying cell lines 697 and RCH-ACV have been described previously (Findley £__al . , Blood _i0:1305-1309 (1982); Barker £t_al. , Cancer 25:379-380 (1987); Jack et_al. , Cancer 19:261-269 (1986)). SUP-B27 was established under conditions similar to those described earlier (Smith _t_al. , Bl££d 2I : 395 -402 (1988); Smith et_al. , B_-££d 22:2182-2187 (1989)) from a patient with pre-B cell acute lymphoblastic leukemia. Karyotype studies showed that SUP-B27 was representative of the patient's leukemia since both carried a reciprocal t(l;19) and trisomy chromosome 8 as the only detectable cytogenetic abnormalities. RCH-ADD is an EBC-immortalized lymphoblastold line from the same patient from which RCH-ACV was established (Jack, _t_al. , Cancer, L9:261 (1986)). FL18 was established from a t(14 ; 18)-carrying B cell lymphoma as described earlier (Doi et al. , Bl£_d 10 ^ : i i 9 - 1623> (1987)). The chromosome 19 and chromosome 1-containing rodent/human hybrid cell lines (5HL9 and MCH 6M2B2 , respectively) were established as described previously (Tro , et_a1. , Pr££__Na11 _____cad__Sci__USA , 8_ : 4180 (1987); Thompson, L.M. , ___al . , G_£n£mi_£ , in press (1989)) . £DNA_C^£ning_and_Chara£_:eri.£ati.£n Three micrograms of poly (A) RNA from the t(l; 19) -carrying cell line SUP-B27 were converted to double-stranded cDNA essentially as described (Cleary £-£_ !• > 16_2: 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 gtlO. Approximately 2 million recombinant phage were subjected to a differential screening procedure consisting of an initial screen with a hybridization probe that spanned the E2A fusion point (probe B, Figure 5), followed by hybridization of the same library filters with a 3' fragment of the E2A cDNA (probe C, Figure 5) consisting mostly of 3' untranslated sequences. Approximately 50% of the plaques that hybridized to probe B did not hybridize to probe C and ten of these were further purified and analyzed for locations of restriction enzyme cutting sites. The cDNA inserts were subcloned into M13mpl8 and M13mpl9 phage for nucleotide sequence analyses (Norrander, et al . , Gene, 2^:101 (1983); Sanger, et St - . 24:5463-5467 (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. , Cell 47:19-28 (1986)). Nucleotide sequences of E2A and fusion cDNAs were determined on both strands of two and five independent isolates, respectively. RNAj» _Pr£te tΪ£n_A _ 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 32 polymerase in the presence of [alpha- P]CTP under conditions provided by Riboprobe System (Promega) . Test RNA samples and anti-sense RNA probes were denatured in 30 microliters of hybridization buffer (80% formamide, 40 M PIPES pH 6.7, 0.4 M NaCI, 1 mM EDTA) at 85"C for 5 minutes and hybridized at 48°C overnight. Following hybridization, 0.3 ml of RNAse digestion buffer (10 mM Tris pH7.5 , 5 mM EDTA, 0.3 M NaCI) containg 40 μg/ml of RNAse A and 2 μg/ml of RNAse Tl were added. Following incubation at 30 β C for 60 minutes, RNAses were inactivated with SDS and proteinase K, and samples were extracted, precipitated and analyzed in 4% or 6% polyacrylamide/urea gels. PCR Analyses of mRNA fusions 1.5 μg of total or 1.0 μg of poly(A) test RNA was converted to cDNA using an oligonucleotide primer (5* -CCACGCCTTCCGCTAACAGC-3' ) complementary to chromosome 1 sequences 3' of the fusion point (nucieotides 1587 to 1606 of fusion cDNA in Figure 8) and MLV reverse transcriptase (200 units) in PCR buffer containing dNTPs and 20 units of RNAsin (20 μl reaction volume) at 42 β CX for 45 minutes. Reaction volume was increased to 100 μl, Taq polymerase and a second primer corresponding to the sense strand of E2A mRNA (5' -GCACAACCACGCGGCCC-3' , nucieotides 1446 to 1462 in Figure 8) added, and amplifications carried out for 36 cycles in a Perkin-Elmer Thermocyler (1 minute denaturation at 94 β C, 1 minute annealing at 55°C, 1.5 minute elongation at 72 β C). Reaction products (shown in Figure 11) were subjected to agarose gel electro- phoresis, transfer to nylon membranes, and hybridi¬ zation with an internal oligonucleotide (5' -GGACCAGGAACCCACAG-3' , nucieotides 1542 to 1558 of fusion cDNA in Figure 8). S∑^-£i^i≥^- Ϊ£--l-_------:^;i∑-§. -£_£^_B:^^—^S^— --!—--- DNA and RNA were isolated from cultured cell lines using methods described previously (Cleary _t_al. _ C_ ^L1 4-2:19-28 (1986)). Southern and Northern blotting techniques and hybridizations were performed as des¬ cribed (Cleary et_al. , Cell 42-19-28 (1986)). Gel isolated DNA fragments used as hybridization probes were radiolabeled by random hexanucleotide priming using the Klenow fragment of DNA polymerase and 32 [[aallpphhaa-- 32 PP]]ddCCTTPP ((FFeeiinnbbee:rg and Vogelstein, Anal^_Bi£ chem. 132:6-13 (1983)). In Vitro Translations Translations were performed using RNA transcribed in vitro from two different cDNA constructs consisting of a 2.3 kb EcoRl-Hindlll fragment of the E2A cDNA (Figure 6A) and a 2.7 kb EcoRI fragment containing the complete fusion cDNA (KJ7 in Figure 6A) subcloned Into pSP65 (Promega) . Plasmids were linearized with Hindlll and transcribed into RNA using SP6 polymerase (Promega) . RNA was translated in vitro using a nuclease-treated rabbit reticulocyte lysate (Promega) under conditions recommended by the supplier. The in vitro translation products were analyzed by 10% SDS-polyacrylamide gel electrophoresis. The gels were fixed, treated with Amplify (Amersham) and vacuum dried prior to autoradiography at room temperature. C_£m£uter_Anal ^ _ £ The University of Wisconsin Genetics Computer Group software package was used on a Digital Equipment Corp. VAX 8550 computer. Peptide sequences predicted from nucleotide sequence analyses of fusion cDNAs were analyzed using the search algorithm FASTP (Lipman and Pearson, Science 2JT7 : 1435- 1441 (1985)). Exam£].£_4 A sessment_of_Occurren e_of__2._.ι 5£B£^iBiS£_ZH_i£B_J__2 £ :E-t_i ___Z_---£-L-L--L__B--- Altgga ion.s_..in_ the Encoded..Product As a result of the t(1 ; 19) (q23 ;pl3.3) translocation, 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. To examine this question, 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 t(1 ; 19) (q23 ;pl3.3) translocation. In Nalm-6, anti-E2A sera reacted with a group of proteins whose apparent molecular masses ranged from 65 to 85 kDa. The 697 cells contained, in addition to these normal 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 represents a potential chimeric protein, synthesized from a fusion transcript between the E2A gene and chromosome 1-derived sequences. This fusion protein was also found in numerous patient samples having the t(l;19) (q23 ;pl3.3) translocation. The structure of the entire normal E2A transcripts was first assessed before fusion transcripts resulting from the t(l;19) translocation in 697 cells were identified. In all cell lines examined, E2A mRNA migrates as two heterogeneous populations averaging 2.9 and 4.8 kb in denaturing gels. An 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 analysis. 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, beginning at 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 polyadenylation signal. 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. With the exception of a single in-frame insertion of 3 bases, 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 ID) and consequently this cDNA has been designated E2A.E12. The inserted triplet results in the addition of glutamine 390. 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 E2Aα. A comparision of amino acids 1-470 of E2A with proteins in the computer base (see below) did not reveal a significant degree of homology with any other transcription or replication factors. A distinctive feature of the amino-terminal domain of E2Aα, however, is its high content of proline and glycine. To investigate whether an E2A-containing fusion transcript was produced in 697 cells, a cDNA library was constructed using mRNA from 697 cells, and 12 clones that hybridized to E2A.E12 cDNA were examined for the presence of unique 3' sequences. cDNA Synthesis 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 (Invitrogen) . After the addition of linkers, the dephosphorylated cDNA was fractionate by electrophoresis through a 1.0% agarose gel and the population of cDNA larger than 2.0 kbp was selected for ligation into Eco Rl-cut lambda gtlO (Stratagene) . The ligated cDNA was packaged (gigapack, Stratagene), and plated on an hfl-strain of C600. Recombinant clones containing E2A sequences were identified by hybridization with an E2A.E12 probe. Four clones contained E2A sequences up to the Bgl I at nucleotide 1435 but lacked the diagnostic Not 1 site centered at nucleotide 1507. Instead, these clones contained either 1350 bp or 1450 bp of unique sequences 3' to the Bgl I site at 1435. A fragment of DNA from the 3' end of the E2A gene that encodes the DNA-binding and proposed helix-loop-helix dimerization domain was radiolabeled and found not to hybridize to any of the four variant clones, affirming that the novel cDNA's did not contain sequences encoding the natural DNA-binding portion of E2A. Likewise, a DNA probe isolated from sequences between the Pvu II sites at 2143 and 2741 of the unique sequences (Figure 14B) hybridized to each of the variant clones but did not bind any of the normal cDNA's. To confirm that these cDNAs representing putative fusion transcripts were not cloning artifacts, but rather a reflection of a subpopulation of mRNA's in 697 cells, 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 ;pl3) 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 697-4, was selected for sequence analysis (Figure 14B) . ■£ ££ _Q £_A£i l-Z Ϊ _£- _ϊyϊ-^ cDNA clones were prepared for sequence analysis by cloning into the vectors, pBSK+ and pBSK- (Stratagene) . Nested sets of deletion mutants were made using unidirection deletion by Exo III (Promega) . JM109 were transformed with the deletion mutants and superinfected with helper phage VCS-M13 at a multiplicity of approximately 20. Single stranded viral DNA was prepared 8 hours later. The single stranded DNA's were sequenced using Sequenase (United States Biochemicals) . The 5' end of this partial clone began at nucleotide 852 of E2A.E12, and its nucleotide sequence matched that of the E2A.E12 clone until nucleotide 1478. After this point, the sequence diverged. The nucleotide within the cDNA where the sequence diverges corresponds exactly to the junction between the fourth intron from the 3' end of the cDNA and its upstream exon, the boundary of which has the sequence GTG/GT, where GT begins the intron. The cDNA sequence was complete at its 3' end, having a polyadenylation signal (underlined) 17 bases 5' from an 18-base, poly(A)tail. The sequence of this E2A fusion cDNA was 113 bases shorter in its 3' coding region than the major 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. A probe containing DNA from nucieotides 1527 (PvuII) to 1684 (Dpn 1), which is GC-rich, was not useful because it bound nonspecifically to many fragments on Southern blots and to 28S RNA on Northern blots. By contrast, a Pvu II to Dpn I fragment encompassing the homeodomain sequences from nucleotide 1888 to 2143, as well as a Pvu II fragment from 2143 to 2741, hybridized strongly to one, and weakly to two, fragments of mouse genomic DNA, digested with Eco. These two probes also hybridized specifically to the two fusion mRNA's on Northern blots. The predicted amino acid sequence encoded by the E2A/prl transcripts is illustrated above their respective cDNA sequences in Figure 14. Both Transcripts encode proteins that lack the carboxyl terminal 171 amino acids comprising the DNA-binding/dimerization domain of E2A. The E2A/prl fusion transcript exhibits two alternative 3' termini that result in the production of two different proteins, both with the same homeodomain. 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. Although 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. This event also leads to the utilization of two different reading frames at the 3' junction of the splice site (Santoro et al. , 1988; Mermod et al , 1989). In spite of these differences, both proteins function as transcriptional activators and both will induce the initiation of DNA replication from the Adenovirus replication origin. The type I and II proteins encoded by E2A/prl likewise exhibit similar biological activities. 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 Fas an (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. Among the 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. In Eve, the alanines also separate the homeodomain from a proline-rich domain containing 34 proline residue in 300 amino acids (Frasch et al; 1987). However, in Engrailed, 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. Tne P^l protein has a remarkable 19 of 27 amino acid identify with the predicted second and third DNA-binding helices of the yeast al mating type protein, which is the same structural motif that homeodomains share with bacterial and bacteriophage DNA-binding proteins that modulate transcription (Figure 5B, Matthews et al. , 1982; Sauer et al . , 1982; Mondragon et al. , 1989; Pabo and Lewis, 1982; Anderson et al., 1987; Anderson et al. , 1981; McKay and Steitz, 1981; Steitz et al. , 1982; Schevitz et al . , 1985). The yeast α2 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 al protein alters the DNA-binding specificity of the α2 protein. The two have been proposed to form a heterodimeric complex that binds a consensus element 5' of haploid-specific genes and represses transcription, thereby permitting sporulation (Miller et al., 1985; Nasmyth and Shore, 1987). Whether or not al itself binds DNA is the absence of α2 has not yet been reported. 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 α2 protein (8/30, Astell et al . , 1981). Two of these sequence identities are phenylalanine residues, occurring at the conserved positions 5 and 17, which contain charged amino acids in most homeoboxes. The replacement of these residues by phenylalanine in both α2 and Prl suggests a possible structural similarity and/or evolutionary conservation between Prl and α2. One explanation, albeit speculative, is that yeast al and α2 have diverged independently after duplicatin of an ancestral gene, which has been retained as a single gene in mammals, and is prl. Consequently, it is possible tha Prl binds a sequence very similar to ATGTGAATGAATACAT , the al/α2 binding motif in yeast (Miller et al. , 1985) . Recently, Treisman et al. (1989) have demonstrated that changing the third aminio acid in the highly conserved WF_N sequence of one homeobox to that found in a second homeobox will convert the DNA-binding specificity of the first homeodomain to that of the second. Most homeodomains containing the same amino acid at this location exhibit measurable affinities for the same binding mofifs. Although specific binding sequences have been determined for homeoboxes containing glutamine, lysine, cystine, and serine residues at this position (Lonai et al . , 1987) , and its target sequence is not know. How might the E2A/Prl fusion protein be involved in the generation of pre-B ALL? At least three hypotheses should be considered, each based on the facts that normally prl is not expressed in pre-B cells, and that the t(l;19) translocation constitutively expresses E2A/Prl proteins that can be expected to alter the transcription of Prl-responsive genes. First, 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. Second, Prl could be a tissue-specific transcriptional activator that requires other tissue-specific factors to be functional; however, by exchanging domains with that of E2A, Prl would overcome this biological restrication and now be capable of stimulating transcription through ubiquitous factors. Third, 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. Although mutations within the coding sequence of homeobox genes have not been observed outside of this study, both activation and deletion of homeobox genes have. In the mouse myeloid leukemia cell line, WEHI-3B, the Hox-2.4 homeobox gene is constitutively activated due to the insertion of an intracisternal A particle (Blatt et al. , 1988). In addition, 55 of 59 mouse myeloid leukemias contain a deletion in chromosome 2 that encompasses the Hox-4.1 gene (Blatt and Sachs, 1988). The importance of these events, however, is not clear, since neither study demonstrated that either activation Hox-2.4 or deletion of Hox-4.1 was clearly involved in the evolution of these leukemias. Our results, likewise, reflect guilt by association, and the biological activity of the E2A/Prl needs to be demonstrated. A determination of the biochemical effects of the E2A/Prl protein in 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 breakpoint, and the resulting polypeptide has a calculated molecular mass of 85.4 kDa. Because the reading frame is altered by the elimination of the 113 bp intron in the type II transcript, 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_En££de _a_H£m££££X 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. First, with the exception of the yeast genes, the hallmark of the homeobox is a strict conservation of tryptophan, phenylalanine, asparagine, and arginine residues at positions 49,50,52, and 54, respectively; Prl contains all of these residues. Second, twelve additional positions are highly conserved, and 8 of the 12 highly conserved residues are also encoded by prl, located at positions 16, 25, 35, 40, 45, 52, 57, and 59. 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 . Third, according to the precedent established by the ant gene and a number of bacteriophage repressor proteins, 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 (357-367 in the complete sequence) encoded by prl form helix 2 of this structure; however, the subsequent amino acids do not exhibit a strong alpha helical tendency. Consequently, Prl satisfies a majority of the criteria that typify most homeoboxes. Prl exhibited amino acid identities ranging from 10-18 of 60 with the sequence of 22 distinct families of homeoboxes. The )east 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.

Claims: CLAIMS 1. A method of detecting, in cells obtained from an individual in whom a t(l;19) chromosomal translocation is suspected to be present, a gene localized to the t(l;19) breakpoint in the chromosomal translocation, comprising the steps of: a) treating cells obtained from the individual in such a manner that nucleic acids present in the cells are rendered available for hybridization with complementary nucleic acid sequences ; b) contacting the product of step (a) with a nucleic acid probe selected from the group consisting of a nucleic acid sequence comple¬ mentary to all or a portion of the altered E2A; a nucleic acid sequence complementary to all or a portion of the prl gene; and a nucleic acid' sequence complementary to all or portion of the fusion mRNA, under condi¬ tions appropriate for hybridization of complementary nucleic acid sequences to occur; and c) detecting hybridization of complementary nucleic acid sequences. 2. The method of Claim 1 wherein the nucleic acid probe is a labeled nucleic acid probe and hybridization of complementary nucleic acid sequences in step (b) produces complexes comprising labeled nucleic acid probe hybridized with at least one gene localized to the t(l;19) breakpoint in the chromosomal translocation. 3. The method of Claim 2 further comprising quantita- ting in cells obtained from an individual in whom a t(l;19) chromosomal translocation is suspected to be present, a gene localized to the t(l;19) breakpoint in the chromosomal translocation, by detecting the extent to which complexes comprising labeled nucleic acid probe hybridized with a gene localized to the t(l;19) breakpoint in the chromosomal translocation are formed. 4. A method of detecting, in DNA obtained from an individual, a t(l;19) chromosomal translocation associated with a human neoplasia comprising the steps of: a) contacting DNA from the individual with a nucleic acid sequence selected from the group consisting of: nucleic acid sequences which hybridize to all or a portion of the altered E2A gene; nucleic acid sequences which hybridize to all or a portion of the prl gene; and nucleic acid sequences which hybridize to the fusion DNA, under conditions appropriate for hybridization of comple¬ mentary nucleic acid sequences to occur; and b) detecting hybridization of complementary nucleic acid sequences. 5. The method of Claim 4 wherein the nucleic acid sequence which hybridizes to all or a portion of the altered E2A gene is selected from the group consisting of the subclone pE47M and the subclone pE47P. 6. The method of Claim 4 further comprising quantita¬ ting, in DNA obtained from an individual, the t(l;19) chromosomal translocation by determining the extent to which hybridization of complementary nucleic acid sequences occurs. 7. A method of diagnosing in an individual acute lymphoblastic leukemia in which a t(l;19) chromo¬ somal occurs, comprising the steps of: a) obtaining from the individual a sample selected from the group consisting of blood samples and tumor samples; b) treating the sample obtained in step (a) to render nucleic acids present available for hybridization with a complementary nucleic acid sequence; c) contacting the product of step (b) with a nucleic acid probe which is complementary to a nucleic acid sequence selected from the group consisting of: the nucleic acid sequence of the altered E2A gene; the nucleic acid sequence of the prl gene; and the nucleic acid sequence of the fusion mRNA, under conditions appropriate for hybridization of complementary nucleic acid sequences to occur; and d) detecting hybridization of complementary nucleic acid sequences. 8. A method of detecting, in cells obtained from an individual, the fusion protein encoded by the fusion DNA or by the fusion mRNA present in cells containing a t(l;19) chromosomal translocation, comprising contacting cells obtained from the individual with an antibody specific for the fusion protein or a porton thereof and detecting binding of the antibody to the fusion protein or a portion thereof. 9. Isolated DNA consisting essentially of the nucleic acid sequence of the E2A gene. 10. Isolated DNA of Claim 9 consisting essentially of the nucleic acid sequence of Figure 1, or a portion thereof. 11. Isolated DNA consisting essentially of the nucleic acid sequence of the prl gene. 12. Isolated DNA consisting essentially of the nucleic acid sequence complementary to the fusion cDNA of Figure 8 , or a portion thereof. 13. Isolated DNA having a nucleotide sequence selected from the group consisting of the nucleotide sequence of E12 as represented in Figure ID and the nucleotide sequence of E47 as represented in Figure ID. 14. Isolated fusion DNA consisting essentially of the nucleotide sequence of the altered E2A derived from chromosome 19 and a chromosome 1-derived nucleotide sequence. 15. Isolated DNA of Claim 14 wherein the chromosome 1-derived nucleic acid sequence is a prl gene. 16. Isolated fusion DNA of Claim 15 which encodes a fusion protein which is a chimeric transcription factor . 17. An isolated nucleic acid sequence consisting essentially of a nucleic acid sequence comple¬ mentary to the nucleic acid sequence represented in Figure 8. 18. Isolated mRNA encoding the fusion cDNA of Figure 8. 19. A nucleic acid probe consisting essentially of the nucleotide sequence of Figure 8, or a portion thereof. 20. An isolated nucleic acid sequence which hybridizes to the nucleic acid probe of Claim 19. 21. A nucleic acid probe consisting essentially of a nucleic acid sequence which hybridizes to all or a portion of at least one gene localized to the breakpoint in a t(l;19) chromosomal translocation. 22. Isolated polypeptide consisting essentially of an amino acid sequence selected from amino acid sequences encoded by the fusion DNA present in leukemia cells in which a t(l;19) chromosomal translocation is present and the fusion RNA present in leukemia cells in which a t(l;19) chromosomal translocation is present. 23. Isolated polypeptide of Claim 15 consisting essentially of the amino acid sequence represented in Figure 8. 24. Isolated polypeptide encoded by the isolated fusion DNA of Claim 16. 25. An antibody specific for a fusion protein which is encoded by the fusion DNA present in leukemia cells containing a t(l;19) chromosomal transloca- tion, said fusion DNA consisting essentially of the altered E2A gene derived from chromosome 19 and the prl gene derived from chromosome 1. 26. An antibody of Claim 25 specific for a fusion protein having the amino acid sequence represented in Figure 8. 27. An antibody specific for the product encoded by the altered E2A gene present in cells containing a t(l;19) chromosomal translocation. 28. An antibody specific for the product encoded by the prl gene which is derived from chromosome 1 and present in cells containing a t(l;19) chromo¬ somal translocation. 29. The cell line SUP-B27. 30. The fusion cDNA present in SUP-B27. 31. RNA, approximately 8.3 Kb in size, consisting essentially of a transcript of the fusion cDNA present in SUP-B27. 32. A method of inhibiting expression of DNA which is localized to the breakpoint of the t(l;19) chromo- somal translocation and present in human leukemia cells, comprising introducing into the human leukemia cells a nucleic acid sequence which hybridizes to all or a portion of DNA localized to the breakpoint, the nucleic acid sequence selected from the group consisting of: a) nucleic acid sequences complementary to the DNA of the altered E2A gene derived from chromosome 19; b) nucleic acid sequence complementary to the DNA of the prl gene derived from chromosome l; c) nucleic acid sequences complementary to fusion DNA consisting essentially of the DNA of the altered E2A gene derived from chromo¬ some 19 and the DNA of the prl gene derived from chromosome 1; and d) equivalents thereof, said equivalents being nucleic acid sequences which are sufficiently similar in sequence to the complementary nucleic acid sequences to be capable of hybridizing with all or a portion of DNA localized to the breakpoint and inhibiting its expression. 33. Use of a substance for the manufacture of a medicament for treating, in a human, acute lympho¬ blastic leukemia in which a t(l;19) chromosomal translocation is present 34. Use of Claim 33 wherein the substance inhibits expression of fusion DNA present at the breakpoint of the t(l;19) chromosomal translocation or inhibits the activity of a fusion product encoded by the fusion DNA present at the breakpoint of the t(l;19) chromosomal translocation. 35. Use of Claim 34 wherein the fusion DNA consists essentially of an altered E2A gene derived from chromosome 19 and a prl gene derived from chromosome 1.

Inventors: BALTIMORE DAVID, CLEARY MICHAEL L, MCCAW PATRICK S, MELLENTIN JULIA D, MURRE CORNELIS, BALTIMORE, DAVID, CLEARY, MICHAEL, L., MCCAW, PATRICK, S., MELLENTIN, JULIA, D., MURRE, CORNELIS
Applicants: UNIV LELAND STANFORD JUNIOR, WHITEHEAD BIOMEDICAL INST, THE BOARD OF TRUSTEES OF THE LELAND STANFORD, JR., WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH
Application year: 1991
Main classification: C12Q1
IPC codes: C07K16, C12Q1