From www.bloodjournal.org by guest on January 12, 2015. For personal use only. RAPID COMMUNICATION Molecular Cloning of Complex Chromosomal Translocation t(8; 14; 12)(q24.1; q32.3; q24.1) in a Burkitt Lymphoma Cell Line Defines a New Gene (BCL7A) With Homology to Caldesmon By V.J. Zani, N. Asou, D. Jadayel, J.M. Heward, J. Shipley, E. Nacheva, K. Takasuki, D. Catovsky, and M.J.S. Dyer Chromosome 12q24.1 is a recurrent breakpoint in high-grade B-cell non-Hodgkin lymphoma (B-NHL). To identify the genes involved at 12q24.1, molecular cloning of a three-way translocation t(8; 14;12)(@4.1;q32.3;q24.1) in a Burkitt lymphoma cell line (Wien 133) was performed; all four translocation breakpoints were cloned and sequenced. Analysis of clones encompassing the der(12)(12;14)(q24.1;q32.3) breakpoint showed a CpG island from chromosome 12q24.1 juxtaposed in a tail-to-tail configuration with a productively rearranged Ig VA-DHJH5 gene. A total of 4.5 kb of genomic DNA including the CpG island was sequenced and analyzed using gene-identificationprograms; all three programs identified a potential 92-bp exon within the centromeric boundary of the CpG island. Using this as a probe, an RNA transcript of 3.8 kb, expressed at low levels in a wide variety of normal tissues, was detected. Overlapping cDNA clones were isolated and sequenced. The longest open-reading frame predicted a serine-rich protein of 231 amino acids. This protein, termed BCL7A, exhibited no recognizable protein motifs but showed homology with the actin-binding protein, caldesmon. In Wien 133, the BCL7A breakpoint occurred within the first intron and resulted in a MYC-BCL7A fusion transcript, with exon I of BCL7A being replacedby MYCexon 1. The normal, untranslocated allele of BCL7A was also expressed without mutation. One of the 11 other 8-NHL cell lines examined with 12q24.1 cytogenetic abnormalities, a mediastinal B-NHL cell line (Karpas 1106). showed biallelic rearrangement within the first intron of BCL7A, which was adjacent to the breakpoint observed in Wien 133. Disruption of the amino-terminus of BCL7A defines a new mechanism in the pathogenesis of a subset of high-grade B-NHL. 0 1996 by The American Society of Hematology. R Specific histologic subgroups of B-NHL may be associated with specific translocations juxtaposing the Ig heavy chain (IGH) locus at 14q32.3 with various oncogenes. Thus, low-grade follicular B-NHL is associated with the t( 14; 18) (q32.3;q21.3) involving the BCL2 gene at 18q21.3, mantlecell B-NHL with the t(l1; 14)(q13;q32.3) involving BCLU CCNDl at 1 lq13, and Burkitt’s lymphoma with the t(8; 14) (q24.1;q32.3) involving the MYC oncogene at 8q24.1.*,I ’ Translocation of the oncogene to the IGH locus results in deregulated expression, presumably in part due to the presence of potent B-cell enhancers within IGH.” Until recently, no specific cytogenetic abnormality had been associated with high-grade diffuse large-cell B-NHL. The cloning of the BCL6 gene allowed the identification of biallelic abnormalities of this gene by both translocation and mutation in up to 30% of cases.”.” A feature of BCM translocations was the marked promiscuity of translocation partners, with the B C U gene being involved with multiple other loci apart from IGH. 1 3 ~ ’ 4 ~ ’However, 6 further molecular analysis of these aggressive malignancies has been hampered by their cytogenetic complexity. In an attempt to overcome some of these difficulties, we have established a large number of spontaneously growing Epstein-Barr virus-negative B-NHL cell lines.”-z0 High-resolution cytogenetic analysis supplemented by fluorescent in situ hybridization showed that chromosome 12q24.1 was the site of recurrent abnormalities in these and other2‘cell lines; the cytogenetic abnormalities included both translocations and interstitial deletions (Nacheva et ai, manuscript in preparation). The nature of the genes involved in the various 12q24.1 abnormalities was unknown. We report here the complete molecular cloning of a complex three-way translocation from one of our Burkitt lymphoma cell lines, Wien 133,” in which MYC and IGH had became juxtaposed with unknown sequences on 12q24.1 and the subsequent isolation of a new gene of unknown functions (BCL7Aj from chromosome 12q24.1. Disruption of the amino-terminal portion of this ECURRENT CHROMOSOMAL abnormalities have been recognized in all the lymphoid malignancies. Some, but not all, are of prognostic significance, indicating their central importance both to the pathogenesis and subsequent biologic behavior of these diseases.I4 In B-cell precursor acute lymphoblastic leukemias (BCP-ALL), a single chromosomal translocation, generally involving the fusion of transcription factors controlling cell differentiation and development, may be sufficient to drive the neoplastic c10ne.l.~In contrast, B-cell non-Hodgkin lymphomas (BNHL) are characterized by marked cytogenetic complexity with multiple genetic events including concurrent activation of dominant oncogenes and loss of tumor suppressor gene functions, necessary to produce the full neoplastic phenotype! For example, concurrent activation of BCL2 and MYC produces marked synergistic effects: whereas p53 mutation and biallelic deletion of p16/p15 have been associated with high-grade transformation.’.’’ From the Academic Department of Haemarology and Cytogenetics and the Department of Experimental Pathology, Institute of Cancer Research-Royal Marsden Hospital, Sutton, Surrey, UK; the Department of Haematology, University of Cambridge, Cambridge, UK; and the Second Department of Internal Medicine, Kumamoto University School of Medicine, Kumamoto, Japan. Submitted April 25, 1995; accepted December 18, 1995. Supported by the Kay Kendall Leukaemia Trust, a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and a grant from the Okukubo Memorial Fund. Address reprint requests to M.J.S. Dyer, MD, DPhil, Academic Department of Haematology and Cytogenetics, Institute of Cancer Research-Royal Marsden Hospital, Haddow Laboratories, 15 Cotswold Rd, Sutton, Surrey, SM2 51vG UK. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. OW6-4971/96/8708-0$3.00/0 3124 Blood, Vol 87,No 8 (April 15), 1996: pp 3124-3134 From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 3125 BCL7A GENE IN LYMPHOMA gene defines a new mechanism in the pathogenesis of a subset of high-grade B-NHL. MATERIALS AND METHODS Cell Culture, Cytogenetics, and Fluorescent In Situ Hybridization (FISH) Analysis The derivation of most cell lines used in this study has been reported previously; references may be obtained on request to M.J.S.D. Cell lines were obtained either directly from their originators or from the German Tissue Culture Repository (Braunschweig, Germany). Cell lines were grown in either RPMI or Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) under standard conditions. Cytogenetic analysis was performed as described” using a computer-based image analysis system (Smart Capture; Digital Scientific, Cambridge, UK). The ISCN 1991 nomenclature was used to describe abnormal chromosomes. FISH was performed with whole chromosome paints (Cambio, Cambridge, UK), a-satellite centromeric probes (Oncor, Gaithersburg, MD), cosmid, and P1 clones using previously described Images were captured by a CCD camera (Photometrics, Tucson, AZ) aided by dedicated software (Digital Scientific). DNA Blotting Genomic Cloning and DNA Sequencing DNA blotting was performed as de~cribed.~’ High molecular weight DNA from all cell lines was digested with all the following restriction enzymes to assess for rearrangement of the BCL7A gene: EcoRI, HindIII, BamHI, Xba I, Bgl 11, and Sac I (Promega, Madison, WI). All probes were used as gel-purified inserts and were labeled with ’2P-deoxycytidine triphosphate to a specific activity of 2 ~ 2X IO9 d p d p g DNA by the method of oligo-priming. The IGH probes MYC probes included a used in this study have been de~cribed.2~ full-length MYC cDNA probe” and exon I- and exon 11-specific MYC genomic probes.25 Molecular cloning of IGH and MYC rearrangements in cell line Wien 133 was performed by ligating unfractionated EcoRI- and BamHI-digested DNA into bacteriophage EMBL4 and EMBL3 arms, respectively (Stratagene, La Jolla, CA). Recombinant phage were screened with IGH and MYC probes and positive clones hybridizing to one or more probes were plaque purified by two successive replatingsz6 Cloned phage were mapped and the regions of interest subcloned into either the phagemid vector pBSII (Stratagene) or into pSLl180 (Pharmacia, Uppsala, Sweden) for the production of nested deletions. Phage clone AIn, a 6.0-kb EcoRI fragment, was subcloned into EcoRI-digested pSLl180 in both orientations and a series of nested deletions produced by Mung-bean exonuclease digestion (Promega). This strategy allowed most of AI11 to be sequenced in both orientations. Gaps were filled by using primer extension. DNA sequencing was performed on either single-stranded phagemid DNA or on double-stranded DNA by cycle-sequencing (Amersham, Amersham, UK). DNA sequence compressions were sequenced using deaza-GTP. The DNA sequence of clone AIU was analyzed using three geneidentification programs, ie, GRAIL,” GENE-ID:* and Staden,” packages, via the computing facilities provided by the UK Medical Research Council, Human Genome Mapping Resource Centre (Hinxton Hall, Cambridge, UK). Northern Blots, Isolation of cDNA Clones, and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Experiments Northern blots using 2 pg poly (A’) of RNA extracted from cell lines with 12q24.1 abnormalities were performed as described.I8 Multiple tissue Northern blots containing 2 pg poly (Af) RNA from a variety of normal tissues were purchased from Clontech (Palo Alto, CA). All Northern blots were probed with either actin or GAPDH probes to confirm RNA loading. cDNA clones were isolated from a normal fetal brain library (Stratagene) and a Raji Burkitt lymphoma cell line library (Clontech) using conventional methods.2h Poly (A+) RNA from normal skeletal muscle and thymus (Clontech) was reverse-transcribed using Superscript reverse-transcriptase (GIBCO-BRL, Gaithersburg, MD) in the presence of oligo (dT). The BCL7A open-reading frame (OW) was amplified by PCR using the primers W125: 5’ ATGTCGGGCAGGTCGG’IT 3’ and W126: 5‘ ATCGCTGGGGCAAAGTTG 3’. The former primer contains the initiation ATG codon of the BCL7A cDNA, whereas the latter is found in the 3’ untranslated region (UTR). The 711-bp products were cloned into the pCRII-TA vector (Invitrogen, San Diego, CA) and sequenced. In an attempt to amplify the presumptive MYC-BCL7A fusion “A, poly (A+) RNA from Wien 133 and skeletal muscle was reverse transcribed in the presence of a BCL7A gene-specific primer, W 128 (5’ AACTGTTCTCCGGAGTGGTCAC 3’). and then PCR amplified using a nested BCL7A primer (W129) and a MYC exon I primer (HCI): W129: 5‘ CGGAGTGGTCACCTCTGA 3‘ and HC1: 5’ ATGCGAGGGTCTGGACGGCTG 3’. A 686-bp product was observed in Wien 133 cDNA samples. This was cloned into pCRII vector (Invitrogen) and sequenced in both directions to confirm the chimeric nature of the product. Genomic PCR Experiments: Isolation of BCL7A PI and Cosrnid Clones The potential 92-bp exon identified by gene-identification programs was PCR amplified from genomic DNA using the primers W103: 5‘ TGTCGGGCAGGTCGG’ITC 3’ and W77: 5’ CCATITGCGCACTTTCTCG 3‘. The product was cloned into the pGEM-T vector (Promega) and sequenced. BCL7A P1 clones were isolated by PCR screening of a commercial P1 library (Genome Systems, St Louis, MO) using primers from the 3‘ BCL7A UTR: W122: 5‘ CTGCACTGGAGTTCTGACTC 3’ and W123: 5’ CCTCACCCTCAGAAACTC’IT3’. COSmids from a normal human placental library (Clontech) were obtained by conventional screening methods using genomic 5’ BCL7A probes. RESULTS DNA Blotting of Cell Line Wien 133 The cell line Wien 133, derived spontaneously from a child with chemotherapy-resistant Burkitt’s lymphoma, exhibited an apparently unique, three-way translocation, (8; 14; 12)(q24.1;q32.3;q24.1).22These derivative chromosomes were confirmed by three-color FISH with chromosome paints for chromosomes 8, 12, and 14 (data not shown) and raised the possibility that either MYC (at 8q24.1) and/ or IGH (at 14q32.3) sequences might be juxtaposed with novel sequences from 12q24.1. DNA from the cell line was therefore digested with multiple restriction enzymes, blotted, and probed with various MYC and IGH probes. Representative results are shown in Fig I. Several features were of note. First, despite only having two copies of chromosome 14, three rearranged JH fragments of differing intensity were noted in all restriction digests. One allele of C p was retained and the other was deleted; as the cell line expressed IgM, this was therefore the functional allele. One allele of C a l was rearranged; the From www.bloodjournal.org by guest on January 12, 2015. For personal use only. ZANl ET AL 3126 A B 111 D C I E F G H 1 J EcoRI BamHI h R l BamHl rr; W J 6.6 > 4.4 > 23 > 2.0 5 DICEST: h R I PROBE; JH BamHI RamHI JH 9 EwRI BamHI MYC exon 11 14 8 EeoRI MYC exon I BLG4lSH A l l 1 Xho0.5 12 Fig 1. Southern blot data of cell line Wien 133 and normal human leukocyte DNAs digested with EcoRl or BamHl and probed with IGH MYC and BCL7A probes. Rearranged fragments in Wien 133 are denoted by arrowheads. The same filters were stripped and reprobed successively with IGH, MYC, and BCL7A probes. Three rearranged JHfragments were observed in Wien 133 in all enzyme digests; there was one strongly hybridizing band and two weakly hybridizing bands. One C p allele (the productive IGH allele) was retained; this fragment did not comigrate with any of the three JH rearrangements in BamHl digests. Different-sizedrearranged fragments were seen in Wien 133 with MYC exon I and II probes (compare ID] and IF]),indicative of a breakpoint in MYC intron 1. (G) and (J) show the patterns of hybridizationwith the derived BCL7A genomic clones BLG4lSH and All1 XhoO.5. Both probes detected germline fragments of the same size, indicatinga reciprocal BCL7A translocation. Rearranged BCL7A fragments in Wien 133 comigrated with the JH rearrangements. other C a l allele and both alleles of Ca2 were retained in germline configuration (data not shown). The same filters were probed with MYC exon I and exon I1 probes. Different sized rearranged fragments were observed with both probes, indicating that the MYC translocation breakpoint occurred between these two exons. The MYC exon I1 rearrangement comigrated with the Ca1 rearrangement in EcoRI digests. The MYC exon I rearrangement comigrated with the 14.7kb EcoRI J H rearrangement, this being one of the two faint J H bands. Thus, one J H rearrangement represented the productive IGH allele, the 14.7-kb EcoRI J H fragment comigrated with exon I of MYC, whereas the third fragment could not readily be accounted for. Both the breakpoint within MYC between exons I and I1 and the juxtaposition of exon I1 to C a l are typical of sporadic Burkitt's lymphoma.'"." Molecular Cloning of JH and MYC Rearrangement in Wien 133 To determine the precise structure of these rearrangements and in an attempt to isolate novel sequences from I2q24. I , complete EcoRI and BamHI genomic libraries of Wien 133 were prepared and screened with J H and MYC exon I and I1 probes. Clones containing all three J H fragments and both 5' and 3' MYC rearrangements were obtained and all breakpoints were sequenced. Restriction maps of the three clones containing DNA from chromosome 12q24.1 are shown in Fig 2. The structure of these three clones is discussed below. Clone BA64RI. This 14.7-kb EcoRI clone was derived from the der(@@;14; 12)(q24.1;q32.3;q24.1) and hybridized both with MYC exon I and J H probes. DNA sequencing showed a MYC-Sp breakpoint, as anticipated. However, sequencing of the JH' 2.7-kb Hind111 subclone showed loss of homology with IGH sequences within the 23-bp spacer between the nonamer and heptamer recombination signal sequences (RSS) upstream of JH6 (Fig 3A): beyond that point, the sequences showed no homology with any other sequences on the databases. The 0.8-kb Sma I-Hind111 fragment probe (BLG4lSH in Fig 2) was a single copy and evolutionarily conserved (data not shown). Clone AIM This 6.0-kb EcoRI JH' fragment was derived from the der( 12)(12; 14)(q24.1;q32.3) and contained a productively rearranged and mutated VH4 gene that had rearranged to the JH5 segment. Identity with IGH continued up to immediately before the JH6 segment, where all homology to IC sequences was lost (Fig 3B). This sequence also had no homology on the databases. Both 0.6- and 0.5-kb Xho I fragments from AI11 were conserved single-copy probes, and with probe BLG4ISH from BA64RI, gave rearranged fragments of the same size From www.bloodjournal.org by guest on January 12, 2015. For personal use only. BCL7A GENE IN LYMPHOMA 3127 CENTROMERE CLONE BA64RI der(8)1(8; 14;I 2)(q24.l;q32.3;q24. I) ' E TELOMERE Bg Bg H X S H I I I I Bg M H S In1 I I E x x x CLONE AI I I der( I2)1( 12;I4)(q24. I ;32.3) E I JH5 D VH4 . . . CLONE WB54 I kb wlZppO.4 - Xho 0.5 BLG41SH ~ w52 w63 Xho 0.65 Fig 2. Restriction maps of bacteriophage clones from Wien 133 containing 12q24.1 sequences. Clones BA64RI and All1 represent the der(8)t(8;14;12) and the der(12)(12;14), respectively. WB54 represent the germline 12q24.1 BamHl clone. Single-copy and evolutionarily conserved probes are indicated by heavy horizontal bars. None of these probes detected RNA transcripts by Northern blot in either normal or Wien 133 RNA samples. as the JH fragments on Southern blot, indicating that no major alterations or artefacts had occurred during cloning. Furthermore, all three probes detected the same germline bands in EcoRI and BamHI restriction digests, suggesting a reciprocal translocation (Fig 1). This was confirmed by cloning the 14-kb BamHI germline chromosome 12 region (clone WB54) from the EMBL3 Wien 133 library using the probe BLG4ISH. Clone WB54. This clone represented the germline allele of chromosome 12q24.1 and contained both Xho I fragments as well as BLG4ISH. indicating that a reciprocal translocation had indeed occurred. DNA sequencing of the germline region corresponding to the translocation breakpoint showed that 30 bp of chromosome 12 had been deleted during the translocation, whereas 27 bp of IGH had been duplicated (Fig 3C and D). To confirm that the new sequences genuinely arose from 12q24.1, a normal human placental DNA cosmid library was screened with the two Xho I fragments from clone AI11 and a positive cosmid mapped onto normal human metaphases by FISH along with a chromosome 12 centromeric paint. All signal was observed at 12q24.1, with no detectable crosshybridization to other sites (data not shown). Isolation and Sequence of BCL7A cDNA Clones None of the single copy probes shown in Fig 2 detected RNA transcripts in Northern blot experiments. However, restriction analysis of clone AI11 showed an extremely high density of rare-cutting restriction sites, including 3 Xho I, 2 Not I, 3 Sfi I, 6 BssHII, and 9 Eag I sites, indicating the presence of a CpG island.32Furthermore, by using Not ]/Sac 1 and other double digests, it was possible to show that the CpG island was not methylated in any B-cell NHL lines examined by the presence of the 0.6-kb Not I fragment observed in plasmid DNA in all cases (data not shown). Unmethylated CpG islands are intimately associated with genes.33We therefore sequenced the entire 6.0 kb of clone AI11 by producing a series of nested deletions and analyzed the sequence using three gene-identification programs. The complete sequence of this clone has been deposited in the EMBL database (accession no. X 90000). Features were a 2.0-kb CpG island with Alu, (CGC&, and (CA),, repeats immediately centromeric of the CpG island. There was no significant homology of this sequence with any other genomic sequences on the databases. Use of the gene-identification programs failed to detect any potential conding sequences within the two conserved Xho I fragments. However, all three programs did identify a 92-bp potential exon zt the centromeric boundary of the CpG island, which was bounded by potential Kozak translation initiation and a RNA splice donor sites. No other comparable potential coding sequences were identified in AI11 using these programs. This 92-bp fragment was amplified by PCR and used as a probe in Northern blot experiments and showed low-level expression of a 3.8-kb RNA transcript in nearly all normal tissues (data not shown); the highest levels of expression were found in the thymus. Transcripts ranging from 1.8 to 6.0 kb in size were seen in skeletal muscle. Using this fragment as a probe, cDNA clones were isolated from oligo (dT)-primed normal fetal brain and Burkitt lymphoma (Raji) cell line libraries. Overlapping cDNA clones spanning 4.5 kb were isolated and sequenced. Part of this sequence along with the predicted protein is shown in Fig 4. This gene has been termed BCL7A. Although the 5' end of the BCL7A mRNA has not been precisely defined, it is likely that the predicted ORF is correct (1) because this From www.bloodjournal.org by guest on January 12, 2015. For personal use only. ZANl ET AL 3128 A I-6 i4q32.3 AGAAGGTCTCGGGTGGACTGGGT~TGTGGGGTGAGGATGGACATTCTGCCUFZTGA~TACTACTACTACTACG] BA64R1 TGAA~GGAAGCTGGACCATGAGATCCCAAAGGTGAGGATGGACATTCTGC~ATTACTACTACTA~ACG 12q24.1 TGAACTGGAAGCTGGACCATGAGATCCCAAAGCCTGAGGTCTTCAGAGGACACGATGTGTTG~CCCCAAACCCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIJIIIIIIIIII IlIIIIIlIIIIIIIIlIlIIIIllIIIIl/I B I-6 i4q32.3 GGGTGGACTWQTTTTETGGGGTGAGGATGGACATTCTGCC&TWEA~TACTACTACTACTACGGTATGGACGTCTI IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Alll GGGTGGACT~TGGGGTGAGGATGGACATTCTGC~TTTCCCAAACCCAGGGAGGCGCAGAGGTA IIIIIIIllIIIIIIIIlllllIIIIIlII 12q24.1 C Alll GGGTGGACT~TGGGGTGAGGATGGACATTCTGCCXL7SEGTTTCCCAAACCCAGGGAGGCGCAGAGGTA IIIIIIlIIllIIIIIIllIlIIIIIllII lZq24.1 G G A C C A T G A G A T C C C A A A G C C T G A G G T C T T C A G A G G A C A C IIIIIIIIIIIIIIIIIII BA64R1 GGACCATGAGATCCCAAAGGTGAGGATGGACATTCTGCCUFZTGA~ACTACTACTACTACTGCGCTATGGACGT~~ IGJRb D Alll I I I I I I I I"I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I 14q32.3 GGGTGGACT~TGGGGTGAGGATGGACATTCTGC~T~TTGTGA~TACTACTACTACTACGGTATGGACGTCT~ I I I I I I I I I I I I I I I I I I I I I -1 BA64R1 - 1 GCTGGACCATGAGATCCCTGAGGATGGACA~CTGCCUFZTGA~TACTACTACTACTACGGTATGGACGTCT~ IGJX6 Fig 3. DNA sequence of some of the translocation breakpoints in cell line W e n 133. All the translocation breakpoints within the cell line W e n 133 were cloned in phage, subcloned into plasmid, and sequenced (Asou et al, unpublished observations). Shown here are the breakpoints involved with chromosome 12q24.1. DNA sequences from the hybrid, translocated clones are shown in comparison with germline IGHl14q32.3) and BCL7A (12q24.1). Underlined sequences denote heptamer.nonamer recombination sites associated with JH6;shaded sequences denote JH6 sequences. (A) Clone BA64R1 [der(E)t(8;14;12)1 showing breakpoint between JH6nonamer and heptamer sites. IB) Clone A l l l [derl121t(12;14)1 showing breakpoint immediately 5' of JH6 with duplication of 27 bp of chromosome 14 material. IC) Germline BCL7A sequences from clone WB54 compared with both A l l l and BA64R1 sequences showing deletion of 30 bp from germline BCL7A occurring as a consequence of the translocation. (D) Comparison of A l l l and BA64R1 t o show duplication of 27 bp of IGH. was the longest O W within the entire cDNA, (2) because there were termination codons in all possible ORFs 5' of the ORF shown, and (3) because of amino-terminal protein identity (but not nucleotide sequence identity) with other human genes (BCL7B, C, and D) and with a C elegans EST (Dyer et al, manuscript in preparation). The initiating methionine of BCL7A marked the ATG codon at the start of the 92-bp fragment identified by the gene-identification programs and therefore denoted the first coding exon of BCL7A. cDNA clones containing the 5' end of the BCL7A gene were difficult to isolate despite the relatively small size of the gene, and only one clone (from a Raji cDNA library) contained these sequences. Other groups have obtained sequence from the 3' end of BCL7A but not the 5' end; six clones were identical to the BCL7A sequence. The reason for the lack of representation of BCL7A 5' sequences in many cDNA libraries is not known. Two of these clones (NIB 1857 and clyh04) were obtained from Dr J. Sikela (Department of Pharmacology, University of Colorado, Boulder, CO) and from GCnCthon (Evry, France), respectively, and were sequenced in their entirety. These sequences were identical with the sequence of our own clones. A BCL7A PI clone isolated using primers designed from the 3' end of the BCL7A sequence also contained the BCL7A CpG island and was mapped by FISH back to chromosome 12q24.1 (data not shown). To confirm the sequence of the BCL7A ORF in normal tissues and in B-NHL cell lines, mRNA from normal skeletal muscle and thymus and B-NHL cell lines Wien 133 and DoHH2 was reverse-transcribed and the BCL7A ORF was amplified by PCR, cloned, and sequenced. No differences in the BCL7A ORF sequence was observed in any of the clones apart from a T to A transition at nucleotide 430 and a C to T transition at nucleotide 629. Neither change would produce any change in the predicted amino acid sequence and are polymorphisms. RT-PCR also showed the presence of two types of clone with and without a 63-bp exon, as indicated in Fig 4. Analysis of the predicted BCL7A protein showed no major homologies with other proteins on the databases and no recognizable protein motifs. However, caldesmon, a ubiquitously expressed actin-binding protein, which is thought to control the interaction of actin and myosin in smooth muscle cells;M exhibited two interesting homologies with BCL7A. First, the amino-terminus of BCL7A showed significant homology with the extended a-helical repeat of the smooth- From www.bloodjournal.org by guest on January 12, 2015. For personal use only. BCL7A GENE IN LYMPHOMA 3129 A ORF BCL7A S A 1 1 I pRI I FBI.1 FR 4.6 C-29AIO NIR 1857 FB 7.2 FR 7 3 FB I 3 C-IFEOI C-IFEI?. C-IYH04 B -253 -203 -153 -103 -53 - 03 48 98 GGCGGCGCGGCTCCCCCTGCTCTGTGCAGCTGCCGCCCGGGCTTGCGCTG GGCCAGGCGCGCGGCGGCCCCGGGCTTTGTGTGTGTGTATGTGTGTGTGT GTGTGTGTGTGTGTGTGTGAGAGTGTGTGCGTGTGAGAGTGCGAGTGTCT GTGCGCGAGTGAGTGAGCGGCGGGCGGGCGCGAGTGTGGCCGGCCGGAGC GCGAGCATGACCCGGCGGGCGCGCTCCCCAGCCTCCGTCTCCCCGCCGGA M S G R S V R A E T R S R A K D ACCATGTCGGGCAGGTCGGTTCGAGCCGAGACGAGGAGCCGGGCCAAAGA D I K R V M A A I E K V R K W E TGATATCAAGAGGGTCATGGCGGCGATCGAGAAAGTGCGCAAATGGGAGA K K W V T V G D T S L R I Y K W V AGAAATGGGTGACCGTTGGTGACACATCCCTACGAATCTACAAATGGGTC P V T E P K V D D K N K N K K K O 148 CCTGTGACGGAGCCCAAGGTTGATGACAAAAACAAGAATAAGWGG 198 CAAGGACGAGAAGTGTGGCTCAGAGGTGACCACTCCGGAGAACAGTTCCT 248 CCCCAGGGATGATGGACATGCATGACGATAACAGCAACCAGAGCTCCATC K S Fig 4. (A) Overlapping BCL7A cDNA clones. pR11 and pR123 were cloned from Raji cDNA library and FB1.l, 1.3,4.6,7.2, and 7.3 were cloned from a normal fetal brain library. NIB1857 was an homologous EST cloned and kindly provided by J. Sikela (Department Pharmacology, University of Colorado) and c-29A10, c-lfe01, c-lfel2, and c-lyh04 ESTs were cloned and kindly supplied by Genethon. Point S indicates the position of the alternatively sliced exon. Point A indicates a potential alternative poly-adenylation site. (6) Partial cDNA (nucleotides -253 t o 1047) and predicted protein sequences of the BCL7A gene. The complete BCL7A sequence may be found under EMBL accession no. X89984. The first exon of BCL7A of 92 bp identified by gene recognition programs starts at nucleotide 0. An alternatively spliced 3' exon, identified from RT-PCR experiments on normal skeletal muscle, is shown by the shaded area. Polymorphic nucleotides are underlined. 430 was T in 2 fetal brain cDNA and 2 skeletal muscle RT-PCR clones and was an A in 2 Raji cDNA and 2 Wien 133 RT-PCR clones. 629 underwent a C t o a T transition in 4 of 7 skeletal muscle RT-PCR clones. Neither affected the predicted ORF. 298 348 398 448 498 548 598 648 698 748 798 848 898 94 8 998 D P E G K M C M G D S M E H V D T D T N P S E N N Q S S S S I A D A S P I K Q E N S S N S S P A GCAGATGCCTCCCCCATCAAACAGGAGAACAGCAGCAACTCCAGCCCCGC P E P N S A V P S D G T E A K V TCCAGAGCCCAACTCGGCTGTGCCCAGCGACGGCACCGAGGCCAAGGTGG D E A Q A D G K E H P G A E D A S ATGAGGCCCAGGCTGATGGGAAGGAGCACCCTGGAGCTGAAGATGCTTCT D E Q N S Q S S M E H S M N S S E GATGAGCAGAATTCACAGTCCTCGATGGAACATTCGATGAACAGCTCAGA K V D R Q P S G D S O L A A E T GAAAGTAGATCGGCAGCCGTCTGGAGACTCGGGTCTGGCCGCAGAGACGT S A I S Q V P R S R S Q R G S Q I CTGCAATCTCTCAGGTACCTCGCTCGAGGTCTCAGAGGGGCAGCCAGATC G Q R P I G L S G D L E G V P P S GGCCGGGAGCCCATTGGGTTGTCGGGGGATCTGGAAGGAGTGCCACCCTC K K M K L E A S Q Q N S E E M . TAAAAAGATGAAACTGGAGGCCTCTCAACAAAACTCCGAA CGATGCTTTAAGCCTCCGATAACTGTTCCATGGAAGGTACATCAGCAATT AATTCTAGAGCAACTTTGCCCCAGCGATTCCTCTTGGGTGCGAACAGAAC TACTAACGTTTCAAGTTTACCAAGTGCAAATCCAAGAAGACCCAGACGGC GTCACTTCTCAGACACTGAAGAACTCTGCTGTGAAGCAI#ACACTCAAAC CTTTAAGGGACTGTCCTTGGGGAGGCAGGCGGGGCTGACAGCTCAGGAGT GTCTGCACACTGTCTCGGAAGCCAGGATTCCATTTGTGTT TTCCCCCCACTTCTCTATGTAACGATATAAGCTATCGGAGGGTGGTACCG From www.bloodjournal.org by guest on January 12, 2015. For personal use only. ZANl ET AL 3130 A BCL7A : AET RSR AKD DIK RVM AA1 EKV RKW EKK Caldesmon : EK: AEE RQR IKE EEK RAA EER QRI KEE EKR K: : R. .. ::: : . B BCL7A 'I2 Caldesmon 757 NSS PAP EPN SAV PSD N.S PAP .P. P:D NKS PAP KPS DLR PGD muscle isoform of caldesmon (Fig 5A). The function of this domain of caldesmon is not known and, unlike other forms of the protein, is expressed only in smooth muscle." A second region of homology was identified in the carboxy-terminal domain of caldesmon, within the region implicated in actin binding (Fig 5B and Redwood et a13"). This region contains the serine 759, which is the major MAP kinase phosphorylation site of caldesmon. The MAP kinase site found in caldesmon is maintained in BCL7A. Phosphorylation or mutagenesis of this serine residue completely abolishes the tropomysin-dependent high-affinity binding of caldesmon to actin.'6 Homology of BCL7A to caldesmon in this region suggests that BCL7A is an actin-binding protein (Dyer et al, manuscript in preparation). 34 Fig 5. (A) Comparison of BCL7A amino-terminal region with a-helical repeat of smooth-muscle isoof caldesform of caldesmon. The a-helical region mon is an extended a-helix of 55 turns, resulting from the region shown being repeated up t o 10 times.",'5 BCL7A therefore has multiDle homologies with this region of caldesmon. The homology shown has 33.3% identity and 59.3% similarity. (B)Comparison of the actin-binding domain of caldesmon with BCL7A. The phosphorylated serine of caldesmon is shown in bold; this is a MAP kinase site in both caldesmon and BCL7A. Apart from this, BCL7A exhibits multiple other potential phosphorylation sites: casein kinase 2 sites residues 37,76,98, and 156; protein kinase C residues 2, 5, 42, 165, 193, and 216; MAP kinase 77,83,103, and 114; and CAMPat resides 10, 37. 73. and 173. were derived from various subtypes of high-grade or transformed follicular B-NHL (Table I). Conventional DNA blot using W63 and clyh04 to detect 5' and 3' BCL7A rearrangements, respectively, showed 5' rearrangements in only one additional cell line, Karpas 1106, derived from a patient with systemic relapse of mediastinal B-NHL." This cell line in fact showed two rearrangements within the first intron of BCL7A affecting both BCL7A alleles. The breakpoints within the BCL7A intron in Karpas 1106 were within 500 bp of that observed in Wien 133 (Fig 8 and Zani et al, manuscript in preparation). No full-length BCL7A ORF could be amplified from Karpas 1106 cDNA, confirming the biallelic Consequences of the t(8;14; 12) and BCL7A Expression in Cell Line Wien 133 Northern blotting of poly (A') RNA from malignant hematopoietic cell lines with and without 32q24.1 chromosome abnormalities showed low levels of apparently normal-sized BCL7A transcript. Higher levels of expression were observed in the Jurkat T-cell NHL cell line, whereas lowest levels were observed in cell lines of the myeloid lineage (Fig 6). However, the t(8; 14; 12) translocation in the cell line Wien 133 produced a break within the first intron of the BCL7A gene and juxtaposed the first (noncoding) exon of MYC in a head-to-tail arrangement. Unlike other MYC translocations in other B-cell malignancies,'" this therefore raised the possibility that a fusion product consisting of MYC exon I fused to BCL7A exon I1 might be a pathologic consequence of the translocation. That this was indeed the case was shown in RT-PCR experiments using 5' MYC and 3' BCL7A primers. In Wien 133, but not in normal skeletal muscle, a hybrid MYC-BCL7A PCR product was obtained. Cloning and sequencing of this product showed fusion between exon I of MYC and exon I1 of BCL7A (Fig 7). Frequency of BCL7A Rearrangements in Other B-NHL Cell Lines With 12924. I Abnormalities A total of 15 malignant lymphoid cell lines with cytogenetic abnormalities of 12q24.1 have been identified (Nacheva and Dyer, manuscript in preparation). Of these, 1 1 A -28s -18s B Fig 6. Expression of BCL7A in malignant hematopoietic cell lines. Approximately 2 p g of poly (A') RNA was blotted and probed with (AI BCL7A exon 1 probe (upper panel) or (B) GAPDH probe (lower panel). Exposure times are 10 days and overnight, respectively. Note the low level of expression of BCL7A in all cell lines, and particularly so in the cell lines of the myeloid lineage (HL60, K562, and Kasumi11. The highest levels of expression were observed in the Jurkat TNHL cell line. Chromosome 12q24.1 abnormalities were observed in cell lines Wien 133, DoHH2, SSK41, and Granta 452, but all exhibited expression of BCL7A of apparently normal size. From www.bloodjournal.org by guest on January 12, 2015. For personal use only. 3131 BCL7A GENE IN LYMPHOMA Fig 7. Sequence of the chimeric MYC-BCL7A mRNA in Wien 133. Primers used for amplification and sequencing are indicated. BCL7A sequences are denoted in bold italics. Numbering of nucleotides is from the germline MYCDNA sequence (EMBL accession no. X003641. 2302 HC-1 TAATGCGAGG GTCTGGACGG CTGAGGACCC CCGAGCTGTG CTGCTCGCGG 2352 CCGCCACCGC CGGGCCCCGG CCGTCCCTGG CTCCCCTCCT GCCTCGAGAA 2402 GGGCAGGGCT TCTCAGAGGC TTGGCGGGAA AAAGAACGGA GGGAGGGATC 2452 GCGCTGAGTA TAAAAGCCGG TTTTCGGGGC TTTATCTAAC TCGCTGTAGT 2502 AATTCCAGCG AGAGGCAGAG GGAGCGAGCG GGCGGCCGGC TAGGGTGGAA 2552 GAGCCGGGCG AGCAGAGCTG CGCTGCGGGC GTCCTGGGAA GGGAGATCCG 2602 GAGCGAATAG GGGGCTTCGC CTCTGGCCCA GCCCTCCCGC TGATCCCCCA 2652 GCCAGCGGTC CGCAACCCTT GCCGCATCCA CGAAACTTTG CCCATAGCAG 2702 CGGGCGGGCA CTTTGCACTG GAACTTACAA CACCCGAGCA AGGACGCGAC 2752 TCTCCCGACG CGGGGAGGCT ATTCTGCCCA TTTGGGGACA CTTCCCCGCC 2802 GCTGCCAGGA CCCGCTTCTC TGAMGGCTC TCCTTGCAGC TGCTTAGACG 2852 CTGGATTTTT TTCGGGTAGT GGAAAACCAG GGAGAAGAAA TGGGTGACCG TTGGTGACAC ATCCCTACGA ATCTACAAAT GGGTCCCTGT GACGGAGCCC AAGGTTGATG ACAAAAACAA GAATAAGAAA AAAGGCAAGG ACGAGAAGTG W129 W128 TGGCTCAGAG GTGACCACTC CGGAGAACAG T T BCL7A rearrangement within the first intron; whether hybrid BCL7A fusion transcripts are expressed in this cell line remains to be determined. DISCUSSION Chromosomal translocations in BCP-ALL frequently result in the fusion of transcription factors controlling cell differentiation. In contrast, translocations in B-NHL generally involve the ZGH locus at 14q32.3 with genes of other functions, including BCLIKCNDI, which is involved in the regulation of the cell cycle, and BCL2, which is involved in the control of apoptosis. We report here the isolation of a new gene of unknown functions by molecular cloning of a complex three-way Table 1. BCL7A Rearrangementsin B-NHL Cell Lines With 12q24.1 Rearrangements Cell Line 1 Wien 133 2 Karpas 1106 3 BL58 4 NAMALWA IPNl45 5 VAL 6 PRI 7 SSK41 8 DoHH2 9 DS 10 GRANTA 452 11 GRANTA 519 Derivation 12q24.1 Abnormality Other Translocations BCL7A (W63) GIR RIR GIG GIG GIG Burkitt's Mediastinal B-NHL Burkitt's Burkitt's B-ALL t(8;14; 12)(q24.1;q32.3;q24.1) ins(12)(q13.1;q24.1;q24.3) der( 12)t(l2; 14;8)(q24.1;q24q32.3;q24.1) add(12Hq24.1) 1 der(12M12; 14;8)(q24.1;q24q32.3;q24.1) add(12)(q24.1) int del(12)(q24.lq24.3) t~l;6~~qll;qll~ t(X; 13; 18)(q28;q12.l;q21.3) t(8; 14)(q24.1;q32.3) t(8; 14;)(q24.1;q32.3) t(8; 14; 18)(q24.l;q32.3;q21.3) t(14; 18)(q32.3;q21.3) t(8;22)(q24.l;qll) t(8; 14; 18) inv(12)(p13q24.1) int de1(12)(q24.lq24.3) int de1(12)(q24.lq24.3) t(8;14) plus t(14;18) t(8;22)(q24.l;qll) t(l1;14)(q13;q32.3) Transformed follicular B-NHL B-ALL B-ALL Transformed Mantle-cell der(l2)(1;12)(q24;q24.1) GIG GIG GIG GIG GIG GIG DNA from all cell lines shown was digested with BamHI, EcoRI, Hindlll, Xba I, Bg/ 11, and Sac I and probed with the BCL7A 5' probes W63 (Fig 2) and with the 3' BCL7A cDNA probe clyoh4 (Fig 4). No rearrangements were seen with the latter probe. However, rearrangements were detected in both Wien 133 and Karpas 1106 in all enzyme digests. Both alleles of Karpas 1106 were seen to be rearranged with the W63 probe and other probes from the 5' end of BCL7A; however, an additional rearranged fragment was also seen indicative of a third telomeric rearrangement, thus indicating a complex rearrangement. G denotes germline with probe W63; R denotes rearranged. Reference for the derivation of all cell lines used in this study may be obtained on direct request to M.J.S.D. Note that both translocations der(12)t(12;14;8)(q24.l;q24q32.3;q24.1) (cell lines BL58 and PRI) and interstitial deletion (12)(q24.1;q24.3) (cell lines DoHH2, Granta 452, and Granta 519) were recurrent events (Nacheva et al, manuscript in prepartion). From www.bloodjournal.org by guest on January 12, 2015. For personal use only. ZANl ET AL 3132 N N K K N n d 1 4 . 0 k b 6.2kb - -a DIGEST Hindm PROBE AIlIXho 0.5 BamFII W63 translocation t(8; 14; I2)(q24.1 ;q32.3;q24.1). This translocation resulted from the formation of a regular t(8; 14)(q24.1; q32.3) followed by a second reciprocal translocation in which the BCL7A gene was disrupted by translocation to the der@)@;14)(q24.1;q32.3) via a peculiar break within the JH segments (Fig 9). Thus, the one IGH allele was the target for both M Y C and BCL7A translocations. Similar double rearrangements result in the formation of t(8; 14; 18). in which both MYC and BCL2 become translocated to the same IGH allele.” The translocation involving the BCL7A gene occurred after the MYC translocation and was therefore a secondary event. From our own and others’ cytogenetic analyses of 12q24.1 translocations, it seems likely that most 12q24.1 translocations are secondary events3’ (Nacheva et al, manuscript in preparation). In Wien 133, the 12q24.3 breakpoint fell within the first intron of the BCL7A gene immediately telomeric to the BCL7A CpG island. The resulting derivative chromosomes comprised der(8)(8; 14; 12) (q24.l;q32.3;q24.1) and der(12)(12; 14)(q24.1;q32.3). The latter clone comprised exon I of BCL7A in tail-to-tail configuration with a productively rearranged V&-DH-JH5gene and was therefore unlikely to be transcriptionally active. On the other hand. the der(8) comprised MYC exon I-(IGH intronic enhancer)-BCL7A exon I1 in the correct orientation to allow the formation of a MYC-BCL7A hybrid mRNA, with fusion of MYC exon I and BCL7A exon 11. This was shown by RT-PCR experiments. The pathologic consequences of this fusion gene product in Wien 133 remain to be determined. However, the first Fig 8. 6CL7A rearrangements in mediastinal BNHL cell line Karpas 1106. Karpas 1106 exhibitedtwo copies of chromosome 12, one of which was considered to have ins(12;?l(q13.lql3.3) on the basis of high-resolutioncytogenetics alone.’” Lack of involvement of any other chromosome was shown by FISH using chromosome 12 paints (data not shown). However, all restriction digests showed at least two rearranged fragments with either All1 or W63 probes, indicating biallelic rearrangement within the first exon of 6CL7A; in 6amHl digests, it was clear that all W63 fragments were in fact of abnormal size. These data indicate complex internal rearrangements within both the alleles of the 6CL7A gene in this cell line. K, DNA from Karpas 1106 cell line; N, DNA from a normal individual. exon of MYC is normally noncoding and the fusion product might therefore initiate translation at the first available methionine (residue 86 of the BCL7A ORF). Apart from removing the amino-terminus of the BCL7A gene, this product would also lack the cysteine residue present in intact BCL7A at residue 71 and a potential nuclear targeting sequence (KKKGK: residues 63-67). However, it should be noted that, in Wien 133, the remaining BCL7A allele appears to be expressed normally, without mutation. This significance of this expression is that BCL7A loss or inactivation does not appear to be the mechanism of oncogenesis. The direct involvement of the BCL7A gene in 2 of the 15 cell lines examined with clustering of breakpoints to the first intron and the creation of a MYC-BCL7A fusion gene in Wien 133 suggest a pathogenic role for BCL7A in a subset of high-grade B-NHL. The incidence of BCL7A rearrangements in a large series of fresh B-NHL is currently being assessed. However, the lack of rearrangements detected in the remaining cases also indicated the presence of other pathogenic genes within 12q24.3. From preliminary pulsedfield data, the region of 12q24.1 around BCL7A contains a high density of CpG islands, with other CpG islands being detected less than 50 kb both telomeric and centromeric of the BCL7A gene (Jadayel et al, unpublished observations). Whether the genes adjacent to BCL7A are also involved in translocation is currently being determined. ACKNOWLEDGMENT We thank Dr Abraham Karpas (University of Cambridge, Cambridge, UK) for providing many of the cell lines used in this study; From www.bloodjournal.org by guest on January 12, 2015. For personal use only. BCL7A GENE IN 3133 LYMPHOMA Cal SalSp JD V IGH 14q32.3 Cal sa1sp Fig 9. Summary of genomic rearrangements in Burkitt cell line Wien 133. IGHsequencesare shown in red, MYC in blue, and BCL7A in green. Although the cell line expressedIgM, the other IGH allele had undergone classswitching t o C d before the MYC translocation occurred, as shown by the presence of a hybrid SallSp region in the der(l4)t(8;14) (Asou et al, unpublished observations). The der(E)t(8;14) typical of Burkitt lymphoma then became the target for a second reciprocal translocation via the residual JH segment, rewlting in der(8)tiE;14; 121 and der(12W112;14). MYC exon I therefore became juxtaposed with exon II of BCL7A to allow the formation of a MYC exon IBCL7A hybrid mRNA. Despite the proximity of the IGH enhancer, there was no demonstrable overexpression of this fusion mRNA (Fig 61. Transcriptional orientation is denoted by horizontal arrows. II III MYC der(14)t(8;14)(q24.l;q32.3) MYCI JDV der(8)t(8;14)(q24.1;q32.3) MYC I t - J BCL7AII -- BCL7AI JD V der(8)t(8;14;12)(q24.1;q32.3;q24.1) der(12)t(12;14)(qU.l;q32.3) Rebecca Berry, T.J. Stevens, and Dr J. Sikela (Department of Pharmacology, University of Colorado, Boulder, CO) for kindly providing clone NIB1857; the Genexpress cDNA program, Laboratoire Gknkthon (Evry, France) for kindly providing clones clfel2, c29a10, clyhO4, and clfeol; and the UK MRC Human Genome Mapping Resource Centre (Hinxton Hall, Cambridge, UK) for computing facilities. We thank Dr S.B. Marston (National Heart Lung Institute, London, UK) for helpful discussions and Maurizio Valeri for his help in growing the cell lines. REFERENCES 1. Rabbitts TH: Chromosomal translocations in human cancer. Nature 372:143, 1994 2. Offit K Chromosome analysis in the management of patients with non-Hodgkin’s lymphoma. Leuk Lymphoma 7:275, 1992 3. Juliusson G, Oscier DG, Fitchett M, Ross FM, Stockdill G, Mackie MJ, Parker AC, Castoldi GL, Cuneo A, Knuutila S, Elonen E, Gharton G: Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities. N Engl J Med 323:720, 1990 4. 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Saglio G, Borello MG, Guerrasio A, Sozzi G, S e r a A, di Celle PF, Foa R, Ferrarini M, Roncella S, Pignatti CB, Marradi P, lzzo P, Soler J, Pierotti M: Preferential clustering of chromosomal breakpoints in Burkitt’s lymphomas and L3 type acute lymphoblastic leukemias with a t(8; 14) translocation. Genes Chromosom Cancer X:l, 1993 32. Bird AP: CpG-rich islands and the function of DNA methylation. Nature 321:209, 1986 33. Bird AP: CpG islands as gene markers in the vertebrate nucleus. Trends Genet 12:342, 1987 34. Matsumara F, Yamashiro S: Caldesmon. Cum Opin Cell Biol 5:70, 1993 35. Hayashi K, Yano H, Hashida T, Takeuchi R, Takeda 0,Asada K, Takayashi E, Kat0 I, Sobue K: Genomic structure of the human caldesmon gene. Proc Natl Acad Sci USA 89: 121222, 1992 36. Redwood CS, Marston SB, Gusev N K The functional effects of mutations Thr 673 + A S P and Ser 702 ASP at the pro-directed kinase phosphorylation sites in the C-terminus of chicken gizzard caldesmon. FEBS Lett 327235, 1993 37. 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For personal use only. 1996 87: 3124-3134 Molecular cloning of complex chromosomal translocation t(8;14;12)(q24.1;q32.3;q24.1) in a Burkitt lymphoma cell line defines a new gene (BCL7A) with homology to caldesmon VJ Zani, N Asou, D Jadayel, JM Heward, J Shipley, E Nacheva, K Takasuki, D Catovsky and MJ Dyer Updated information and services can be found at: http://www.bloodjournal.org/content/87/8/3124.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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