The oncogenic nucleoporin CAN/Nup214 is essential in vertebrate cells. Its depletion results in defective nuclear protein import, inhibition of messenger RNA export and cell cycle arrest. We recently found that CAN associates with proteins of 88 and 112 kDa, which we have now cloned and characterized. The 88 kDa protein is a novel nuclear pore complex (NPC) component, which we have named Nup88. Depletion of CAN from the NPC results in concomitant loss of Nup88, indicating that the localization of Nup88 to the NPC is dependent on CAN binding. The 112 kDa protein is the human homologue of yeast CRM1, a protein known to be required for maintenance of correct chromosome structure. This human CRM1 (hCRM1) localized to the NPC as well as to the nucleoplasm. Nuclear overexpression of the FG-repeat region of CAN, containing its hCRM1-interaction domain, resulted in depletion of hCRM1 from the NPC. In CAN-/- mouse embryos lacking CAN, hCRM1 remained in the nuclear envelope, suggesting that this protein can also bind to other repeat-containing nucleoporins. Lastly, hCRM1 shares a domain of significant homology with importin-beta, a cytoplasmic transport factor that interacts with nucleoporin repeat regions. We propose that hCRM1 is a soluble nuclear transport factor that interacts with the NPC.

The translocation (6;9)(p23;q34) in acute nonlymphocytic leukemia results in the formation of a highly consistent dek-can fusion gene. Translocation breakpoints invariably occur in single introns of dek and can, which were named icb-6 and icb-9, respectively. In a case of acute undifferentiated leukemia, a breakpoint was detected in icb-9 of can, whereas no breakpoint could be detected in dek. Genomic and cDNA cloning showed that instead of dek, a different gene was fused to can, which was named set. set encodes transcripts of 2.0 and 2.7 kb that result from the use of alternative polyadenylation sites. Translocations are the best-studied nonrandom chromosomal aberrations associated with specific subtypes of leukemia. As a result of a translocation, an oncogene can be activated through alterations in regulatory DNA sequences that leave the encoded protein intact (e.g., myc) or through formation of a fusion gene, encoding a chimeric protein (e.g., bcr-abl). The t(9;22) associated with chronic myeloid leukemia, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (29) results in the expression of a chimeric BCR-ABL protein with enhanced tyrosine kinase activity (16,19,27,38,45). Pendergast et al. showed that defined sequences encoded by the first exon of bcr interact with the SH2 domain of ABL (33). This interaction is essential for the activation of the ABL tyrosine kinase activity and for the transforming capacity of BCR-ABL. More recently, other fusion genes have been isolated. t(1;19), occurring in childhood pre-B-cell acute leukemia, fuses the E2a gene, encoding transcription factors E12 and E47, to a novel homeobox gene, PBX1 (26, 32). t(15;17), strongly associated with acute promyelocytic leukemia, fuses part of the retinoic acid receptor type a gene (RARt) to a novel gene on chromosome 15 named PML, which is predicted to be a transcription factor (9, 25). bcr-abl, E2A-pbx, andpml-RARao seem to be highly consistent partners.Previously we reported the cloning of t(6;9) breakpoints (43). t(6;9) is the hallmark of a specific subtype of AML characterized by a poor prognosis and a young age of onset. It is classified in the French-American-British system mostly as M2/M4 and rarely as Ml or refractive anemia with excess of blast cells (RAEB) (2,36,39). On chromosome 9, break-* Corresponding author.points take place in a specific intron, icb-9, of a large gene (>140 kb) named Cain (can) (43). On chromosome 6, breakpoints also occur in a single intron, icb-6, of a gene named dek (42). The result of t(6;9) is the formation of a dek-can fusion gene on chromosome 6p-, which is transcribed into an invariable, 5.5-kb, leukemia-specific dek-can mRNA (39). The fusion transcript encodes a 165-kDa chimeric protein, which derives from the in-frame fusion of dek and can open reading frames (ORFs). Sequence comparison of DEK and CAN with entries in the EMBL data base shows no homology to any known protein sequences. CAN contains several putative dimerization motifs, and the C-terminal part may function as an ancillar...

The translocation (6;9) is associated with a specific subtype of acute myeloid leukemia (AML). Previously, it was found that breakpoints on chromosome 9 are clustered in one of the introns of a large gene named Cain (can). cDNA probes derived from the 3' part of can detect an aberrant, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. cDNA cloning of this mRNA revealed that it is a fusion of sequences encoded on chromosome 6 and 3' can. A novel gene on chromosome 6 which was named dek was isolated. In dek the t(6;9) breakpoints also occur in one intron. As a result the dek-can fusion gene, present Defined karyotypic aberrations are associated with specific subtypes of leukemia. Detailed molecular characterization of these aberrations may identify genes involved in leukemogenesis and in the precise regulation of proliferation and differentiation in the hematopoietic system. Translocations are the best-studied chromosomal abnormalities. As the result of a translocation, the function or activity of oncogenes located at or near the translocation breakpoint is altered. In myeloid leukemia three translocation breakpoints have been cloned and analyzed at the molecular level.The two best studied, t(9;22) in chronic myeloid leukemia (27, 43) and t(15;17) in acute promyelocytic leukemia (2,8,12), result in the formation of chimeric genes that encode fusion proteins. In chronic myeloid leukemia this is a BCR-ABL protein that has an enhanced tyrosine kinase activity (34, 49) directly responsible for its in vivo tumorigenic potential (14,25). In acute promyelocytic leukemia a PMLRARa fusion protein that represents an altered transcription factor (16, 33) is found.The third translocation is the t(6;9) (p23;q34), found in a specific subtype of acute myeloid leukemia (AML) (1,39,41). This leukemia is characterized by a poor prognosis, affects young adults, and is classified mostly as M2 or M4 and rarely as Ml (according to the French-American-British classification of AML). A region on chromosome 9 situated 360 kb downstream of the c-abl gene was cloned and analyzed. It was found that breakpoints were clustered in a region of 8 kb in five patients, four with t(6;9) AML and one with acute undifferentiated leukemia (AUL) (47). Through cDNA cloning this region could be identified as one of the introns of a large gene (>100 kb) encoding a 7-kb transcript. This intron was named icb-9; the intron containing the breakpoints on chromosome 9 and situated in the middle of * Corresponding author. a gene named Cain (can). The 3' part of can is translocated to the 6p-chromosome, and only 3' can probes detect an additional, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. No additional transcripts were detected with 5' can probes. The breakpoint region on chromosome 6p23 was isolated from a genomic XEMBL3 library constructed of bone marrow DNA from one of the t(6;9) patients. An area of 40 kb of chromosome 6 DNA was cloned in overlapping phages. Southern blot analysis sh...

The Philadelphia (Ph) chromosome, the product of t(9:22), is the cytogenetic hallmark of chronic myelogenous leukemia. The c-abl oncogene on chromosome 9 is translocated to the Ph chromosome and linked to a breakpoint cluster region (bcr), which is part of a large bcr gene. This results in the formation of a bcr-c-abl fusion gene, which is transcribed into an 8.5 kb chimeric mRNA encoding a 210 kd bcr-c-abl fusion protein. The Ph chromosome is also found in acute lymphoblastic leukemia (Ph+ ALL). Although the c-abl is translocated and a new 190 kd c-abl protein has been identified, no breakpoints are observed in the bcr (Ph+bcr- ALL). Here we show that in Ph+bcr- ALL, breakpoints in chromosome 22 occur within the same bcr gene, but more 5' of the bcr. Cloning of a chimeric bcr-c-abl cDNA demonstrates that the fusion gene is transcribed into a 7 kb mRNA, encoding a novel fusion protein.

Communicated by Stylianos E. AntonarakisHbVar (http://globin.bx.psu.edu/hbvar) is a locus-specific database (LSDB) developed in 2001 by a multi-center academic effort to provide timely information on the genomic sequence changes leading to hemoglobin variants and all types of thalassemia and hemoglobinopathies. Database records include extensive phenotypic descriptions, biochemical and hematological effects, associated pathology, and ethnic occurrence, accompanied by mutation frequencies and references. In addition to the regular updates to entries, we report significant advances and updates, which can be useful not only for HbVar users but also for other LSDB development and curation in general. The query page provides more functionality but in a simpler, more user-friendly format and known single nucleotide polymorphisms in the human α-and β-globin loci are provided automatically. Populationspecific β-thalassemia mutation frequencies for 31 population groups have been added and/or modified and the previously reported δ-and α-thalassemia mutation frequency data from 10 population groups have also been incorporated. In addition, an independent flat-file database, named XPRbase (http://www.goldenhelix.org/xprbase), has been developed and linked to the main HbVar web page to provide a succinct listing of 51 experimental protocols available for globin gene mutation screening. These updates significantly augment the database profile and quality of information provided, which should increase the already high impact of the HbVar database, while its combination with the UCSC powerful genome browser and the ITHANET web portal paves the way for drawing connections of clinical importance, that is from genome to function to phenotype.

The t(6;9) associated with a subtype of acute myeloid leukemia (AML) was shown to generate a fusion between the 3' part of the CAN gene on chromosome 9 and the 5' part of the DEK gene on chromosome 6. The same part of the CAN gene appeared to be involved in a case of acute undifferentiated leukemia (AUL) as well, where it was fused to the SET gene. Genomic sequences around the translocation breakpoint were determined in two t(6;9) samples and in the case of the SET-CAN fusion. Although coexpression of myeloid markers and terminal deoxynucleotidyl transferase was shown to be one of the characteristics of t(6;9) AML, no addition of random nucleotides at the translocation breakpoint could be found. In addition, the breakpoint regions did not reveal heptamer-nonamer sequences, purine-pyrimidine tracts, a chi-octamer motif, or Alu repeats. The sequence in which the translocation breakpoints occurred was enriched in A/T. Notably, the specific introns in which clustering of breakpoints occurs in DEK and CAN both contain a LINE-I element. As LINE-I elements occur with a moderate frequency in the human genome, the presence of such an element in both breakpoint regions may be more than coincidental and may play a role in the translocation process.

The Tel gene (or ETV6) is the target of the translocation (12;22)(p13;q11) in myeloid leukemia. TEL is a member of the ETS family of transcription factors and contains the pointed protein interaction (PNT) domain and an ETS DNA binding domain (DBD). By contrast to other chimeric proteins that contain TEL's PNT domain, such as TEL-platelet-derived growth factor ␤ receptor in t(5;12)(q33;p13), MN1-TEL contains the DBD of TEL. The N-terminal MN1 moiety is rich in proline residues and contains two polyglutamine stretches, suggesting that MN1-TEL may act as a deregulated transcription factor. We now show that MN1-TEL type I, unlike TEL and MN1, transforms NIH 3T3 cells. The transforming potential depends on both N-terminal MN1 sequences and a functional TEL DBD. Furthermore, we demonstrate that MN1 has transcription activity and that MN1-TEL acts as a chimeric transcription factor on the Moloney sarcoma virus long terminal repeat and a synthetic promoter containing TEL binding sites. The transactivating capacity of MN1-TEL depended on both the DBD of TEL and sequences in MN1. MN1-TEL contributes to leukemogenesis by a mechanism distinct from that of other chimeric proteins containing TEL.The Tel gene (or ETV6) encodes a member of the ETS family of transcription factors and is located on chromosome 12 band p13. Tel was discovered as part of a fusion gene created by a translocation (5;12) in a case of chronic myelomonocytic leukemia (20). Extensive analysis of other leukemia cases by fluorescent in situ hybridization analysis revealed that Tel is a frequent target of translocations. Over 30 different rearrangements involving Tel have been described, and over 10 of these have been cloned (45). Tel encodes two proteins, one starting at the first AUG (methionine 1) and one at the second AUG (methionine 43). Both isoforms contain an N-terminal pointed (PNT) domain involved in protein-protein interactions and a C-terminal ETS domain that binds DNA. In transient-transfection experiments, TEL appears to function as a transcriptional repressor by recruitment of histone deacetylases via the transcriptional corepressors mSin3A, SMRT,13,34). In addition, the central region of TEL also contains two autonomous repression domains (34), which depend on TEL self-association.In most cases, Tel translocations encode fusion proteins that contain the PNT domain fused to phosphotyrosine kinase (PTK) domains, such as those of platelet-derived growth factor ␤ receptor, ABL, JAK2, NTRK3, and ARG (7,12,20,21,26,29,40,41). In these fusion proteins, the PNT domain provides the oligomerization interface (24) needed for activation of the fused PTK moieties (5, 21, 47). The activated PTKs are directly responsible for the in vitro and in vivo transforming activities of these proteins (6,21,33,47,50). The PNT domain is also present in several fusions with transcription factors such as AML1, MDS1 (or EVI1), and CDX2 (9,19,41,44). It is unknown at present how addition of the PNT domain alters the functions of these transcription factors or how this wou...

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