Molecular Cloning of Complex Chromosomal

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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
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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
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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
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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
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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-
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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.
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+
From www.bloodjournal.org by guest on January 12, 2015. 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
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