Search

The localization of the adenovirus E1B-55K-E4orf6 protein complex is critical for its function. Prior studies demonstrated that E4orf6 directs the nuclear localization of E1B-55K in human cells and in rodent cells that contain part of human chromosome 21. We show here that the relevant activity on chromosome 21 maps to RUNX1. RUNX1 proteins are transcription factors that serve as scaffolds for the assembly of proteins that regulate transcription and RNA processing. After transfection, the RUNX1a, RUNX1b, and RUNX1-DeltaN variants allowed E4orf6-directed E1B-55K nuclear localization. The failure of RUNX1c to allow nuclear colocalization was relieved by the deletion of amino-terminal residues of this protein. In the adenovirus-infected mouse cell, RUNX1 proteins were localized to discrete structures about the periphery of viral replication centers. These sites are enriched in viral RNA and RNA-processing factors. RUNX1b and RUNX1a proteins displaced E4orf6 from these sites. The association of E1B-55K at viral replication centers was enhanced by the RUNX1a and RUNX1b proteins, but only in the absence of E4orf6. In the presence of E4orf6, E1B-55K occurred in a perinuclear cytoplasmic body resembling the aggresome and was excluded from the nucleus of the infected mouse cell. We interpret these findings to mean that a dynamic relationship exists between the E4orf6, E1B-55K, and RUNX1 proteins. In cooperation with E4orf6, RUNX1 proteins are able to modulate the localization of E1B-55K and even remodel virus-specific structures that form at late times of infection. Subsequent studies will need to determine a functional consequence of the interaction between E4orf6, E1B-55K, and RUNX1.

We report a case of acute myeloid leukemia (AML), M2 subtype according to the French-American-British (FAB) classification, with extramedullary myeloblastoma of the uterus and a masked type of variant translocation of t(8;21)(q22;q22). A 45-year-old Japanese woman presented with metrorrhagia, and AML (M2) with uterine invasion was diagnosed. The patient received an allogeneic peripheral blood stem cell transplantation after remission, and her pelvis was irradiated locally. Cytogenetic study at first showed t(8;17)(q22;p13) by G-banding. Spectral karyotyping (SKY) analysis modified this interpretation to a 3-way translocation involving chromosomes 8,17, and 21 and identified a masked type of variant t(8;21)(q22;q22) translocation. Results of fluorescence in situ hybridization using the AML1/ETO probe, and of detection of the AML1/ETO fusion transcript by reverse transcriptase-polymerase chain reaction were consistent with the karyotyping result. SKY analysis is useful to compensate for the limitations of cytogenetic studies.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

To detect a translocation (8;21)(q22;q22) in interphase cells by fluorescence in situ hybridization (FISH), we investigated three probe combinations: single-color hybridization with two cosmid probes (cY8 and cY3), single-color hybridization with four cosmid probes (cY8, cY3, cY107, and cYR4), and dual-color hybridization with two cosmid probes (cY107 and cYR4) from the AML1 gene flanking or overlapping the breakpoint region. Over 95% of nuclei gave sufficient signals in all three probe combinations, and the detection rates were not statistically different among them. Among 18 patients examined at diagnosis, 12 with t(8;21) were also monitored for the number of residual leukemic cells after chemotherapy or bone marrow transplantation (BMT). There were some discrepancies between morphology and genetic (especially FISH) results at partial, or even complete remission. As leukemic cells with t(8;21) can maturate, morphological assessment alone is insufficient to evaluate the residual leukemic cells. Interphase FISH provided more precise information about the clinical status of patients with an 8;21 translocation after treatment.

Fluorescence in situ hybridization (FISH) analysis was applied to detect t(12;21) using two yeast artificial chromosome probes and cosmid probes covering the TEL(ETV6) and the AML1 gene to clarify the incidence of abnormality of t(12;21) in Japanese childhood acute lymphoblastic leukemia (ALL). We detected seven TEL/AML1 fusion positive patients (9.5%), all of whom were diagnosed as B-lineage ALL, among 74 childhood ALL. On the other hand, no TEL/AML1 fusion positive patients were found among 37 adult ALL. The incidence among Japanese seemed to be lower than that among other nations. Of the seven patients with the TEL/AML1 fusion, five exhibited normal karyotype, one was t(8;12)(q11;p13), i(21q) and the remaining one exhibited a near-triploid karyotype in conventional G-banding. The FISH method clearly demonstrated that all patients with the TEL/AML1 fusion had subpopulations of leukemic cells with deletion of the normal TEL allele, which is significant for understanding the progression of leukemia with t(12;21).

The t(16;21)(q24;q22) translocation is a rare but recurrent chromosomal abnormality associated with therapy-related myeloid malignancies and a variant of the t(8;21) translocation in which the AML1 gene on chromosome 21 is rearranged. Here we report the molecular definition of this chromosomal aberration in four patients. We cloned cDNAs from the leukemic cells of a patient carrying t(16;21) by the reverse transcription polymerase chain reaction using an AML1-specific primer. The structural analysis of the cDNAs showed that AML1 was fused to a novel gene named MTG16 (Myeloid Translocation Gene on chromosome 16) which shows high homology to MTG8 (ETO/CDR) and MTGR1. Northern blot analysis using MTG16 probes mainly detected 4.5 kb and 4.2 kb RNAs, along with several other minor RNAs in various human tissues. As in t(8;21), the t(16;21) breakpoints occurred between the exons 5 and 6 of AML1, and between the exons 1 and 2 or the exons 3 and 4 of MTG16. The two genes are fused in-frame, resulting in the characteristic chimeric transcripts of this translocation. Although the reciprocal chimeric product, MTG16-AML1, was also detected in one of the t(16;21) patients, its protein product was predicted to be truncated. Thus, the AML1-MTG16 gene fusion in t(16;21) leukemia results in the production of a protein that is very similar to the AML1-MTG8 chimeric protein.

The frequent occurrence of 21q deletions in human non-small cell lung carcinoma (NSCLC) indicates the presence of a tumor suppressor gene on this chromosome arm. Since the ANA (Abundant in Neuroepithelium Area) gene, a member of an antiproliferative gene family, was mapped to 21q11.2-q21.1, we searched for genetic alterations of the ANA gene in human lung cancers. The gene was homozygously deleted in a human NSCLC cell line, Ma17. The gene was mapped in the 0.33 Mb Not1 fragment at 21q21.1 of the Not1 restriction map for 21q. Loss of heterozygosity (LOH) at this locus was detected in 24/47 (51.1%) of NSCLC, and the frequency of LOH in brain metastases was significantly higher than that in stage I-II primary tumors (P = 0.018). These results suggested that the homozygously deleted region harbors a novel tumor suppressor gene involved in NSCLC progression. Since mutation of the ANA gene was not detected in other lung cancer cell lines and fresh lung tumors with LOH at this locus, it is unlikely that the ANA gene is a target gene inactivated by two mutational events in this chromosomal region. Physical mapping of the homozygously deleted region showed that the deletion had occurred interstitially at 21q11.1-q21.1 and the size of the deletion was estimated as being more than 3 Mb. Our mapping results will facilitate further efforts to identify a tumor suppressor gene on 21q.

We have investigated a case of acute myelocytic leukaemia derived from myelodysplastic syndrome (MDS-AML) with an 8;21 translocation. In this case the AML1/MTG8 (ETO) fusion transcript was not detected by reverse transcriptase-polymerase chain reaction (RT-PCR), and the rearrangement of the AML1 gene locus was not detected by Southern blot nor pulse field gel electrophoresis (PFGE) analyses using specific probes for the AML1 gene. Fluorescence in-situ hybridization (FISH) study using cosmid probes for 21q22 revealed that the breakpoint of 21q22 was telomeric to the AML1 gene locus and centromeric from D21S259, 351, 3421 loci. This is the first report concerning the t(8;21)(q22;q22) carrying AMLs (de novo AML, MDS-AML and therapy-related AML) to show that the breakpoint at 21q22 is located outside the AML1 gene locus. It is also noteworthy that the cell-surface antigen expression pattern of the bone marrow (BM) blasts was changed from CD7+ CD2+ CD13+ CD33+ CD19- CD11b+ CD14+ CD36+ to CD7- CD2- CD13+ CD19+ CD11b- CD14- CD33+ CD34+ CD36- CD56+ during leukaemic progression, and the pattern in leukaemic phase was similar to the characteristic phenotype of de novo AML cases with t(8;21), when the AML1/MTG8 fusion transcripts are always detected by RT-PCR.

16;21 translocation is a recurrent primary abnormality in acute myeloid leukemia (AML). The genes involved in this translocation are ERG on chromosome 21 and TLS/FUS on chromosome 16. The rearrangement of the two chromosomes forms the TLS/FUS-ERG fusion gene and produces a consistent chimeric transcript on the der (21) chromosome. In this study, we analyzed the clinical characteristics of 19 patients with t(16;21)-AML, including 2 patients who evolved from myelodysplastic syndrome, and detected the chimeric transcripts of the TLS/FUS-ERG fusion gene in the patients during various clinical stages by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. We found that the patients with t(16;21) are characterized by a relatively younger age (median age, 22 years old), involvement of various subtypes of French-American-British classification and a poor prognosis: 18 of the 19 patients died of the disease (median survival was 16 months). Four types of TLS/FUS-ERG chimeric transcripts including a novel type were noted in the RT-PCR analysis. The novel transcript contained an additional 138 nucleotides consisting of TLS/FUS exon 8 and ERG exons 7 and 8 and had an in-frame fusion. These chimeric transcripts were consistently detectable in the samples obtained not only at diagnosis and relapse but also in short and long complete remission, suggesting that t(16;21)-AML is resistant to conventional chemotherapy. Thus, we recommend that t(16;21) should be monitored by RT-PCR even in clinical remission and the patients should be treated by other more powerful modality like stem-cell transplantation in the first remission.

The translocation t(8;21)(q22;q22) is found in 40% of cases of acute myeloid leukemia (AML) designated as the subtype M2 in the French-American-British (FAB) classification. The 8;21 translocation is clinically of interest because patients with this subtype have a good prognosis. We describe a masked type of the translocation, t(8;12;21)(q22.1;q24.1;q22.1). The translocation was first interpreted as t(8;12)(q22;q24) based on cytogenetics, but was reevaluated as a result of Southern blot and fluorescence in situ hybridization (FISH) analyses.

The Down syndrome (DS) region has been defined by analyses of partial trisomy 21. The 2.5-Mb region between D21S17 and ERG is reportedly responsible for the main features of DS. Within this 2.5-Mb region, we focused previously on a distal 1.6-Mb region from an analysis of Japanese DS patients with partial trisomy 21. Previously we also performed exon-trapping and direct cDNA library screening of a fetal brain cDNA library and identified a novel gene TPRD. Further screening of a fetal heart cDNA library was performed and a total of 44 possible exons and 97 cDNA clones were obtained and mapped on a BamH1 map. By rescreening other cDNA libraries and a RACE reaction, we isolated nearly full-length cDNAs of three additional genes [holocarboxylase synthetase (HCS), G protein-coupled inward rectifier potassium channel 2 (GIRK2), and a human homolog of Drosophila minibrain gene (MNB)] and a coding sequence of a novel inward rectifier potassium channel-like gene (IRKK). The gene distribution and direction of transcription were determined by mapping both ends of the cDNA sequences. We found that these genes, except IRKK, are expressed ubiquitously and are relatively large, extending from 100 kb to 300 kb on the genome. These nearly full-length cDNA sequences should facilitate understanding of the detailed genome structure of the DS region and help to elucidate their role in the etiology of DS.

The t(8;21) chromosome translocation frequently occurs in the AML, acute myeloid leukemia, M2 sub-type. This translocation juxtaposes the AML1 gene on chromosome 21 with the MTG8(ETO) gene on chromosome 8, resulting in the expression of the AML1-MTG8(ETO) fusion transcript. The fusion product is thought to play a critical role in the abnormal proliferation and differentiation of myeloid leukemia cells. We investigated the effects of various differentiation inducers of myeloid leukemia cells on the growth and differentiation of Kasumi-1 and SKNO-1 cells, AML cell lines with t(8;21). These cells resisted differentiation into mature granulocytes and macrophages in response to various inducers of myelomonocytic differentiation, such as dimethyl sulfoxide, retinoic acid, butyrate, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1alpha,25-dihydroxyvitamin D3. On the other hand, dexamethasone can induce apoptosis in these cells at low concentrations, whereas other myelomonocytic leukemia cell lines tested were resistant to glucocorticoid-induced apoptosis. The levels of glucocorticoid receptor gene expression were high in Kasumi-1 and SKNO-1 cells. Expression of the AML1-MTG8(ETO), bcl-2, and c-myc genes was unchanged following exposure to dexamethasone. Glucocorticoids might induce the apoptosis of some types of AML cells, just like that of some lymphoid leukemia cells.

The Down syndrome (DS) region on chromosome 21, which is responsible for the DS main features, has been defined by analysis of DS patients with partial trisomy 21. Within the DS region, we constructed a 1.6-Mb P1 contig map previously. To isolate gene fragments from the 1.6-Mb region, we performed direct cDNA library screening and exon trapping using the P1 clones and a human fetal brain cDNA library, and obtained 67 cDNA fragments and 52 possible exons. Among them, 23 cDNA fragments and 4 exons were interpreted to be derived from a single gene by localization on P1 clones and by Northern analysis. To obtain the full-length cDNA sequence, longer cDNA clones were further screened from another human cDNA library which was enriched with longer cDNA species. These clones were sequenced and assembled to a sequence of 9045 bp. This transcribed sequence encodes a novel 2025 amino-acid protein containing tetratricopeptide repeat (TPR) motifs and therefore the gene was designated as TPRD (a gene containing the TPR motifs on the Down syndrome region). The TPR domain has been found in a certain protein phosphatase and in other proteins involved in the regulation of RNA synthesis or mitosis. The TPRD gene, the novel gene which was proved to be in the 1.6-Mb region and to have the interesting features described above, is a candidate for genes responsible for the DS phenotypes.

The gene encoding human transcription factor E4TF1-60 was previously mapped to chromosome 21q21. We analyzed the localization of the E4TF1-60 gene in more detail by genomic Southern hybridization and determined the sequence of the exons and the regions surrounding the intron boundaries. We report here that E4TF1-60 locates in the long arm of chromosome 21 at q21.2-q21.3 and contains a total of ten exons.

Fifty-three cDNA fragments previously obtained by hybridization selection from random clones in the chromosome 21 cosmid library LL21CNO2 failed to identify clones in the chromosome 21 YAC contig described by Chumakov et al. (1992, Nature 359: 380-387). Using an expanded panel of somatic cell hybrids, we have verified that the majority of these cDNAs map to chromosome 21 and that in particular a very high proportion, approximately 85%, localize to a 5-Mb region of distal 21q22.3. Pulsed-field analysis coupled with information from the NotI restriction map of the region further indicate that 17 cDNA fragments map within 650 kb of the PFKL gene and thus may be candidates for genetic diseases linked to this gene. This work helps to characterize a region poorly represented in the CEPH YAC contig and adds to the number of cDNAs useful in analysis of chromosome 21-associated diseases.

We have undertaken a detailed analysis of several hundred YACs from widely available YAC libraries which map to human chromosome 21 with the goal of improving the physical map of chromosome 21 and determining the feasibility of producing a minimal tiling path of well characterized, stable, non-chimeric YACs spanning the long arm of the chromosome (21q). We report information on over 500 YACs known to contain STS from 21q including information on size, stability, chimerism, marker content, and NotI restriction sites. YACs derive from the CEPH and St. Louis YAC libraries, and STSs include the set of 198 markers originally used do assemble a YAC contig of 21q, as well as additional anonymous probes and gene markers. This information has assisted in refinements of STS order, has defined a region of general instability in 2lq22.3, has identified an increased number of NotI restriction sites, and has defined cryptic gaps, particularly in 2lq2l, for which few or no markers are available. These results have allowed us to develop and assess a minimal tiling path of overlapping YACs consisting of 59 YACs (and two PI clones), largely non chimeric, stable, and of verified STS content. They total 30 mb of non-overlapping DNA, and contain all chromosome 21 specific STSs originally used to define the 810 YAC 21q YAC contig. When integrated with the analysis of a somatic cell hybrid mapping panel of chromosome 21 reported in the accompanying manuscript, a greatly enhanced understanding of the physical map of chromosome 21 is obtained.

The human CBFA2T1 (also known as MTG8) gene, on chromosome 8, has been identified through its involvement in the t(8;21) chromosomal translocation, frequently found in acute myeloid leukemia. We report here the isolation and characterization of the mouse homologue of the CBFA2T1 gene, Cbfa2t1h. Nucleotide sequence analysis of Cbfa2t1h cDNA clones revealed an open reading frame encoding a protein of 577 amino acids with an extremely high degree of amino acid identity (99.3%) to the human protein. The nucleotide sequence is also highly conserved between mouse and human in the 5'- and 3'-untranslated regions (87.0, 92.0, and 93.7% identities for 5'-untranslated, coding, 3'-untranslated regions, respectively). The 3'-untranslated region of Cbfa2t1h contains a (CA)n dinucleotide repeat, and the polymerase chain reaction amplification of the (CA)n repeat region revealed fragment length polymorphism among mouse strains. Using this polymorphism, we have mapped Cbfa2t1h to mouse chromosome 4 close to the centromere using SMXA recombinant inbred strains and 106 intersubspecific backcross progenies of the (DBA/2 x Mae) x Mae cross. The chromosomal location was also confirmed by fluorescence in situ hybridization.

The t(16;21)(p11;q22) translocation is a recurrent chromosomal abnormality found in several types of myeloid leukemia. We have previously demonstrated that the breakpoints of this translocation are clustered in a specific intron of the ERG gene on chromosome 21, which has recently been reported to be involved in Ewing's sarcoma. We show here that the TLS/FUS gene on chromosome 16 is fused with the ERG gene to produce the TLS/FUS-ERG chimeric transcript by this translocation. The TLS/FUS gene has been identified as a translocated gene in myxoid liposarcoma by the t(12;16)(q13;p11) translocation and encodes an RNA-binding protein that is highly homologous to the product of the EWS gene involved in Ewing's sarcoma. Thus, the TLS/FUS-ERG gene fusion in t(16;21) leukemia is predicted to produce a protein that is very similar to the EWS-ERG chimeric protein responsible for Ewing's sarcoma.

The t(3;21)(q26;q22) translocation, which is one of the consistent chromosomal abnormalities found in blastic crisis of chronic myelocytic leukemia (CML), is thought to play an important role in the leukemic progression of CML to an acute blastic crisis phase. The AML1 gene, which is located at the translocation breakpoint of the t(8;21)(q22;q22) translocation found in acute myelocytic leukemia, was also rearranged by the t(3;21)(q26;q22) translocation. Screening of a cDNA library of the t(3;21)-carrying leukemic cell line cells (SKH1) resulted in the isolation of two potentially complete AML1-EVI-1 chimeric cDNAs of 6 kb. Two species of AML1-EVI-1 fusion transcripts of 8.2 and 7.0 kb were detected in SKH1 cells. These cells expressed the 180 kDa AML1-EVI-1 fusion protein containing an N-terminal half of AML1 including a runt homology domain which is fused to the entire zinc finger EVI-1 protein. The AML1-EVI-1 fusion transcript was consistent in all three cases of the t(3;21)-carrying leukemia examined by RNA-based PCR. These findings strongly suggest that the t(3;21) translocation results in the formation of a new class of chimeric transcription factor which could contribute to the leukemic progression of CML through interference with cell growth and differentiation.

From human chromosome 21-specific libraries, 22 SfiI linking clones and 38 P1 clones were isolated and regionally mapped on the chromosome. The terminal sequences of these clones were determined and pairs of PCR primers were generated which could specifically amplify the sequenced regions. These sequence-tagged sites (STSs) should be useful for constructing a high resolution map of human chromosome 21.

The t(8;21) translocation is one of the most frequent chromosomal abnormalities in acute myeloid leukaemia and results in gene fusion between AML1 on chromosome 21 and MTG8 (= ETO or CDR) on chromosome 8. AML1 contains a region of sequence homology to the Drosophila runt gene and the mouse polyomavirus enhancer binding protein PEBP2 alpha gene. The rearrangement occurs within a specific intron of the AML1 gene and results in the formation of a chimaeric protein with the consistent feature that the region of sequence homology of AML1 is fused with almost the entire MTG8 protein. MTG8 (ETO, CDR) is predicted to be a transcription activation factor from its sequence with zinc-finger motifs and proline-rich domains. Thus the rearrangement is a fusion between two probable transcription activation factors.

The t(16;21)(p11;q22) translocation is a nonrandom chromosomal abnormality found in several types of myeloid leukemia, which show variable cytomorphological features. We constructed rodent-human somatic cell hybrids containing the der(16) chromosome from leukemic cells of a patient with t(16;21). Using these hybrids, we mapped the translocation breakpoint on the Not I restriction map of chromosome 21 which we had previously constructed. The result showed the proximity of the breakpoint to the ERG gene, a member of the ets oncogene superfamily. Polymerase chain reaction and Southern blot analyses of genomic DNA from the hybrids and from peripheral blood cells and bone marrow cells of patients with t(16;21) showed that the breakpoints were clustered within a single intron in the coding region of the ERG gene. This finding and the results obtained by Northern blot analysis suggested the formation of a chimeric product(s) by fusion of the ERG gene and an unknown counterpart gene on chromosome 16.

The chromosomal translocation, t(8;21), is found frequently in acute myeloid leukemia (AML) with maturation (FAB-M2). We have previously mapped the translocation breakpoints of t(8;21) in a specific intron of the AML1 gene on chromosome 21. In this study, we cloned cDNAs synthesized from a cell line carrying t(8;21) by reverse transcription polymerase chain reaction (RT-PCR) using an AML1-specific primer. The analysis of the cDNAs structure has led to the identification of the fusion of AML1 with a gene named MTG8 on chromosome 8, which seems to be identical to ETO. Northern analysis using MTG8 (ETO) probes detected 7.8-kb and 6.2-kb RNAs and several minor RNAs in the cell line with t(8;21), but failed to detect any transcripts in a cell line without t(8;21). A set of primers were designed to detect the AML1/MTG8(ETO) fusion by PCR. The PCR amplified identical products in all 6 patients and one cell line with t(8;21), suggesting that the AML1/MTG8(ETO) fusion is a constant feature associated with t(8;21) and the junctions of the AML1/MTG8(ETO) fusion are restricted in a unique site. Because the PCR detection of the AML1/MTG8(ETO) fusion at the RNA level is highly sensitive, it can be used as a sensitive method for diagnosis and detection of minimal residual disease in t(8;21) leukemia.

A variety of maps of the human genome have been constructed, including cloned DNA maps. We have isolated 40 of the 42 NotI sites that exist on the long arm of human chromosome 21, as NotI linking clones and constructed a complete NotI restriction map spanning the entire region. This map, which provides the most reliable ordering and distance estimation in the region from a pericentromeric locus to the terminus, demonstrates the usefulness of linking clone mapping for analysing human chromosomes.

We isolated and analyzed 19 NotI site-containing clones specific for human chromosome 21. The overall process consisted of selective isolation of NotI site-containing clones from flow-sorted chromosome 21 libraries, selection of independent clones by their restriction patterns and nucleotide sequences, and assignment of the clones to chromosome 21. Sequence analysis showed that the regions around the NotI sites had features typical of CpG islands, such as extremely high GC content, high-frequency appearance of CpG dinucleotide sequences, and lack of methylation of these CpGs. PCR conditions for these extremely G + C-rich templates were optimized to establish these NotI sites as sequence-tagged sites.

The t(8;21) translocation is one of the most frequent chromosome abnormalities in acute myeloid leukemia. It has been shown that the t(8;21) breakpoints on chromosome 21 cluster within a single specific intron of the AML1 gene, which is highly homologous to the Drosophila segmentation gene runt. Here we report that this translocation juxtaposes the AML1 gene with a novel gene, named MTG8, on chromosome 8, resulting in the synthesis of an AML1-MTG8 fusion transcript. The fusion protein predicted by the AML1-MTG8 transcript consists of the runt homology region of AML1 and the most part of MTG8, which contains putative zinc finger DNA binding motifs and proline-rich regions constituting a characteristic feature of transcription factors. The MTG8 gene is not expressed in normal hematopoietic cells, whereas AML1 is expressed at high levels. Our results indicate that the production of chimeric AML1-MTG8 protein, probably a chimeric transcription factor, may contribute to myeloid leukemogenesis.

The AML1 gene was rearranged in leukemic cells with t(8;21)(q22;q22) or its variant, complex t(8;V;21) translocations from 33 acute myeloid leukemia (AML) patients. The AML1 rearrangement was also detected in three AML patients without t(8;21); two had a normal diploid karyotype, and one had a karyotype of 45,X, - X. The AML1 rearrangement in the t(8;21) breakpoint cluster region was not detected in leukemic cells with cytogenetic abnormalities other than t(8;21), or with normal diploidy obtained from 23 AML patients. Because leukemic cells of the five patients with complex t(8;V;21) translocations had a der(8)t(8;21) chromosome with a break in band 8q22 in common, the juxtaposition of the 5' side of AML1 to a predicted counterpart gene located in the breakpoint region of 8q22 may be an essential step in the leukemogenesis of AML with t(8;21). Our findings show that the 8;21 translocation, its variants, and the masked t(8;21) may all be detectable by the Southern hybridization method using the AML1 probes.

The AML1 gene on chromosome 21 was rearranged by the t(8;21) chromosomal translocation in acute myeloid leukemia (AML). Southern blot analysis of 21 AML patients with t(8;21), including three with complex translocations, t(8;V;21), demonstrated that all the breakpoints occurred at random within a single intron between two coding exons of AML1. Clustering of the breakpoints in the restricted intron suggests the formation of a unique fusion gene between the AML1 gene and a presumable counterpart gene on chromosome 8. Nucleotide sequencing of the breakpoint region revealed that the translocation event was accompanied by deletion of a short stretch of nucleotides.

The t(8;21)(q22;q22) translocation is a non-random chromosomal abnormality frequently found in patients with acute myeloid leukemia (AML) with maturation (M2 subtype). We report here the cloning of a gene, named AML1, on chromosome 21 that was found to be rearranged in the leukemic cell DNAs from t(8;21) AML patients. The breakpoints in 16 out of 21 patients were clustered within a limited region of AML1, and detailed analysis in 3 patients revealed that the breakpoints occurred in the same intron of the gene. Sequencing of cDNA clones identified a long open reading frame encoding a 250-amino acid protein. Northern blot analysis detected four constant mRNA species in t(8;21) leukemic and normal cells; the largest species was more abundant in the leukemic cells than in normal cells. In addition, two mRNA species limited to the leukemic cells were found. These findings indicate that the AML1 gene may be involved in neoplastic transformation of AML with the t(8;21) translocation.

Effective procedures have been developed for constructing NotI linking libraries starting from chromosome-specific genomic libraries. Fifteen different single copy and two rDNA NotI linking clones from human chromosome 21 were identified in two libraries. Their chromosomal origin was confirmed, and regional location established using hybrid cell panels. Hybridization experiments with these probes revealed pairs of genomic NotI fragments, each ranging in size from less than 0.05 to 4.0 Mb. Many fragments displayed cell type variation. The total size of the NotI fragments detected in a human fibroblast cell line (GM6167) and mouse hybrid cell containing chromosome 21 as its only human component (WAV17) were approximately 32 and 34 Mb, respectively. If these fragments were all non-overlapping, this would correspond to about 70% of the 50-Mb content estimated for the whole chromosome. The linking clones will be enormously useful in the subsequent construction of a NotI restriction map of this chromosome. Characterization of these clones indicates the presence of numerous additional sites for other enzymes that recognize sequences containing CpG. Thus most NotI linking clones appear to derive from CpG islands and probably identify the 5' end of genes.

An 8;21 translocation is a common chromosome abnormality associated with acute myeloblastic leukemia with maturation (M2 of French-American-British (FAB) classification). We have isolated chromosome 21 Notl linking clones; pulsed field gel electrophoresis analysis with one clone (LL263) detected an altered fragment of Notl-digested leukemic cell DNA carrying t(8;21). The altered fragment was shown to be produced by the 8;21 translocation. The breakpoint in chromosome 21 was located about 13 kb to 100 kb proximal to the LL263 Notl site. Because the LL263 clone has a CpG island and is a short distance from the breakpoint, the clone itself may be considered as a candidate for part of the t(8;21) associated gene.