Download Structural analysis of both products of a reciprocal translocation

volume 13 Number 6 1985
Nucleic Acids Research
Structural analysis of both products of a reciprocal translocation between c-myc and
inununoglobulin lod in Burkitt lympboma
Christopher Moulding, Aaron Rapoport, Paula Goldman, Jim Battey, Gilbert M.Lenoir1 and Philip
Leder
Department of Genetics, Harvard Medical School, 45 Shattuclc Street, Boston, MA 02115, USA, and
'International Agency for Research on Cancer, 150 Cours Albert-Thomas, 69372 Lyon, France
Received 27 December 1984; Accepted 5 February 1985
ABSTRACT
The balanced transiocations that occur between the c-myc and Iranunoglobulin l o c i In B u r k i t t lymphoraa provide an unusual opportunity to analyze
both products of a reciprocal recombination.
Accordingly, we have determined t h e ' s t r u c t u r e of the two reciprocal products of a translocation t h a t
j o i n s the 5' portion of the c-myc gene on chromosome 8 to the Immunologulin
(i switch recombination signal on chromosome 14. By determining the nucleot i d e sequences at the translocation crossover points of both product chromosomes, we precisely locate these points with respect to nearby genes.
This determination allows us to conclude that translocation Involves nonhomologous recombination, Is highly conservative of c-myc sequences ( d e l e t ing only 16 bp at the crossover p o i n t ) , but deletes over 2 Kb of Immunoglobulin sequences from the \i switch s i g n a l . The p constant and c-rayc genes
are joined head-to-head about 3 Kb apart, while the IgH enhancer and an
aberrantly rearranged D/J region are linked to sequences 51 of c-rayc on the
reciprocal product.
INTRODUCTION
The occurrence of reciprocal translocations between the c-myc gene and
the Imraunoglobulin loci In Burkitt lymphoma provides a rare opportunity to
analyze both the substrates and the products of a reciprocal recombination
that occurs In a human cell (1-3). Such analyses are Important because
they r e f l e c t the mechanisms by which these translocations occur and allow
us to explore the structural and functional consequences of juxtaposing
genes normally located on different chromosomes. Several reciprocal translocations have been analyzed 1n mouse piasmacytomas where recombination
generally Involves the Immunoglobulin a switch signal (1,4-10), a sequence
thought to be Involved In specific recombinational events that occur during
maturation of B lymphocytes and formation of antibody genes. In human Burk i t t c e l l s , these translocations generally—but not always—Involve a d i f ferent recombination signal, the (i switch, a sequence that occurs near the
region encoding the Immunoglobulin v heavy chain (1,11-13).
A less coranon class of translocation Involves the c-myc gene and the X
© IRLPres* Limited, Oxford, England.
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l i g h t chain locus (14).
One example has been exmained In p a r t i c u l a r d e t a i l
and has been found to Involve
that j o i n s a region near the \
a highly
conservative
recombination
constant region genes to a point
thousand bases downstream (3') from the c-myc gene (15).
event
several
In t h i s case no
switch region signal Is found on either segment Involved 1n the translocation.
Because the basis of these recombination events 1s s t i l l
not clear,
we have examined both reciprocal products of a translocatlon that has occurred between the c-myc gene and the 1mmunoglobul1n \i switch region in the
B u r k i t t lymphoma BL22.
In contrast to the example cited above, the recip-
rocal exchange 1s r e l a t i v e l y conservative of c-myc sequences, but not of \L
switch region 1n which a 2 Kb segment 1s l o s t
translocatlon.
from the products of
the
Furthermore, the translocatlon has not associated the IgH
enhancer (16-18) w i t h the c-nyc gene, rather the enhancer Is found on the
product reciprocal
to the n constant-c-myc linkage.
The segment carrying
the rearranged c-rayc gene renains t r a n s c r i p t i o n a l l y a c t i v e , though deregulated (19.20).
MATERIALS AND METHODS
B u r k i t t Lywphoma Cell Line, BL22
The B u r k i t t lyraphoma cell l i n e BL22 was established from a pleural effusion of a 19 year old Congolese female (14).
The malignant c e l l s were
characterized by the presence of the EBV genome, a t(8;14) and a II,K phenotype.
Genomic DNA Preparation and Southern B l o t t i n g
DMA samples from normal human peripheral white blood c e l l s and the
B u r k i t t lyraphoma c e l l l i n e BL22 were prepared as described previously (21).
AHquots of genomic DNA were digested with appropriate r e s t r i c t i o n enzymes
(New England Biolabs), electrophoresed, blotted to n i t r o c e l l u l o s e , and hybridized to nick-translated DHA r e s t r i c t i o n fragment probes.
tivity
of
probes was t y p i c a l l y
100-300 cpra/pg.
Specific ac-
Hybridized f i l t e r s
were
washed at 52°C 1n 15 niM NaCl, 1.5 mM sodium c i t r a t e and 0.11 NaDodS04.
Genomic Cloning
The human germline c-myc genomic clone and the c-myc containing recorabinant clone frora BL22 were obtained as described previously (19).
ciprocal
The r e -
recombined (17 Kb EcoRl) fragment was obtained by screening ap-
proximately 2 x 10
recombinant phage \ CH4A (22) clones containing
size
p u r i f i e d 17 Kb EcoRl r e s t r i c t i o n fragments frora BL22 genomic DNA. The hyb r i d i z a t i o n probe was a 1 Kb C l a - H i n d l l l
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the germline c-myc clone.
The gennline innunoglobuHn heavy chain clone 1s
described elsewhere ( 2 3 ) .
DMA Sequencing
DNA sequencing was performed using the H13 "dideoxy" chain termination
method (24) essentially as described In the Bethesda Research Laboratories
sequencing manual (1981).
RESULTS
I d e n t i f i c a t i o n and Cloning of c-myc Reciprocal Translocation Fragments
The two products of the reciprocal
translocation that occurred 1n the
Burkitt l i n e BL22 were detected by making use of mapping data which I n d i cated that the translocation breakpoint occurred about 1 Kb 5' to the c-myc
promoter ( 1 9 ) .
A probe was made from a normal c-myc fragment located Im-
mediately 5 1 to the BL22 recombination s i t e (5 1 break, F1g. 1 ) .
When this
probe 1s hybridized to EcoRl-digested BL22 and control DNA's, I t detects an
unrearranged 12.5 Kb c-myc DNA EcoRI fragment 1n the control sample and two
fragments In BL22 genomic DNA.
One of
the BL22 fragments corresponds to
the unrearranged 12.5 Kb EcoRI fragment; the other,
EcoRI r e s t r i c t i o n
fragment.
to a rearranged 17 Kb
When a segment of the c-myc gene I t s e l f
Is
used as a probe (F1g. 1 ) , the unrearranged c-myc gene Is again detected 1n
both DNA samples, but a rearranged fragment 9 Kb 1n length, the other product of the reciprocal translocation, Is detected In the tumor c e l l s .
The 17 Kb EcoRI fragment represents one of the two reciprocally
ranged fragments
generated by the BL22
translocation.
rear-
The 9 Kb EcoRI
c-rayc-containing fragment represents the other (F1g. 1) and has I t s Iraraunoglobulin recombination site In the middle of the p. switch signal, extending
Into sequences 3 1 to this region ( 1 6 ) .
The reciprocal
of
this
fragment
would therefore be expected to contain sequences 5' to \i switch. Including
the heavy chain J region cluster.
A J region probe also detects a co-mi-
grating, rearranged 17 Kb EcoRI fragment In BL22 genomic DNA, as expected
I f this fragment represents the reciprocal fragment (data not shown).
This
17 Kb fragment was cloned Into a phage \ vector from s i z e - p u r i f i e d , EcoRI
cleaved BL22 genomic DNA using the 5'
break fragment ( F i g . 1) as a probe.
Comparative r e s t r i c t i o n mapping and b l o t hybridization experiments showed
that this fragment contains Immunoglobulin heavy chain sequences extending
from J5 to the 5'
border of the u switch.
In a d d i t i o n ,
this
reciprocal
fragment contains v i r t u a l l y a l l c-myc sequences 5' to the previously determined site of translocation ( F i g . 1 ) .
The 17 Kb reciprocal
fragment also
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A
17Kb
12.5Kb—,
DNA
Control
BL22
I
Probe:
c-myc Recombinont
(9 Kb)
BL22
I
L__
MYC
5' Break
14
L .fr
Reciprocal
(17 Kb)
IgH
Control
1
H
C
14
X H
14
F1g. 1 . Structure of germline and recorabinant DNA segments around the
c-rayc-IgH translocation s i t e s . A) Genoraic DNA from normal human polymorphonuclear c e l l s (control) and the B u r k i t t lymphona cell l i n e BL22 was d i gested with EcoRl, b l o t t e d , and hybridized with either a 5' break or c-myc
probe. The map positions of these two probes on the gerraline c-myc f r a g ment are designated below In p a r t B. Both probes Identify an unrearranged
12.5 Kb EcoRl fragment 1n control and BL22 DNA. The myc probe detects an
additional 9.0 Kb EcoRl fragment 1n BL22 which Is the c-myc recorabinant.
The 5 1 breakpoint probe hybridizes to the 17 Kb reciprocal recorabined
fragment 1n BL22.
B) The structure of the human germline c-myc locus and the germline
IgH locus are compared to the c-rayc containing recorabinant and reciprocal
recotnbined fragment. All c-myc derived DNA (chromosome 8) Is represented
as a stippled bar and Inmunoglobulin heavy chain DNA (chromosome 14) Is
shown as an open bar. A thin l i n e designates rearranged DNA of uncertain
origin flanking J5 on the reciprocal recombined fragment. Translated exons
are f i l l e d boxes above the bars; untranslated exons are unshaded boxes.
The 16 bp deleted from germline c-myc and the 2 Kb deleted from IgH by r e -
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combination are Indicated by t r i a n g l e s . A hatched box Indicates Iromunoglobulin rau switch sequences. The directions of transcription of c-myc and
IgH genes are shown by arrows. The structure of the gerraline IgH locus 1s
as described by Ravetch e t a i . ( 2 3 ) . Abbreviations used: Cn = (i constant
region, E « Iraraunoglobulin enhancer element, Sji = |i switch sequences, J H »
heavy chain J segment cluster. Restriction s i t e s : R - EcoRl, H =• H i n d l l l ,
C = C l a l , X - Xbal, B - BamHl.
contains approximately 8 Kb of DMA adjacent to J5 that 1s not homologous to
either c-myc or IgH Iramunoglobulin J sequences ( F i g . 1 and see below).
Sixteen Basepairs are Deleted from Germline c-myc at the Site of Translocation.
DNA sequences a t the translocation crossover point from both the c-myc
containing recorabinant (9.0 Kb Rl) and the reciprocal recombinant fragment
(17 Kb) were compared to their
unrearranged counterparts In the gerraline
c-myc region (12.5 Kb Rl) ( F i g . 2 ) . With the exception of a 16 nucleotlde
segment of DNA derived from the gerraline c-myc locus, a l l
sequences are found In either
gerraline c-rayc
the c-myc containing recombinant or I t s
re-
ciprocal (F1g. 2 ) .
About Two Kilobases of n Switch DNA are Deleted from the Translocated Heavy
Chain Locus.
The c-myc containing recorabinant In BL22 contains about 1 Kb of
switch sequence adjacent to I t s c-myc recombination s i t e ( 1 6 ) .
germline \i switch region extends over 3 Kb ( 2 3 ) .
u
The e n t i r e
Restriction mapping and
genoraic blotting experiments comparing the germline IgH locus with the r e ciprocal recorabined fragment showed that the reciprocal
no switch sequences (data not shown).
fragment contained
This result Indicates that at l e a s t
2 Kb of IgH |i switch sequences are deleted at the translocation s i t e . DNA
sequence comparison of
the translocation
site
In
the reciprocal
fragment
and the germline IgH locus precisely locates the recombination s i t e 50 bp
5'
to the n switch region ( F i g . 1 and 2 ) , as predicted from mapping and
blotting experiments.
The possibility that this 2 Kb deletion occurred as a consequence of
the cloning
procedure,
could be ruled
out by comparing
fragment length of cloned and genomic DNA.
the
restriction
BL22 genomic DNA and the r e -
ciprocal clone were digested with H i n d i I I , blotted and probed with the 5'
break probe (Figs. 1 and 3A).
This probe detects a 2 Kb H i n d l i l
fragment
In the cloned DNA which co-migrates with I t s 2 Kb rearranged H i n d l i l count e r p a r t In BL22 genomic DNA ( F i g . 3A).
This 2 Kb H i n d l i l
fragment spans
the translocatlon crossover point In which the switch deletion has occurred
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F1g. 2 . Hucleotide sequences a t the translocation s i t e . A) The DNA
sequences a t the transiocat.1 on s i t e of the two reciprocal fragments are
compared to their germiine c-nyc counterparts. The regions sequenced are
designated by bars above the three structural representations. The 16 nucleotides deleted from germline c-myc a t the recombination site are boxed.
B) The nucleotlde sequence of the non c-myc containing fragment ( r e ciprocal) compared to IgH sequences bordering the n switch. The region
sequenced 1s designated by bars 0—>) above structural representations of
these DNA segments.
(Fig.
1 ) , since the fragments
are of Identical
length,
the deletion has
occurred In the Burkitt c e l l . Similar comparisons of the c-myc recombinant
clone and BL22 genomic r e s t r i c t i o n fragments confirmed that no major deletions occurred during cloning o f this genomic fragment (data not shown).
The J Cluster on the Reciprocal Fragment has Undergone an Aberrant Immunogiobulin J-Type Recombination Event.
Restriction mapping and Southern b l o t comparison between a germline
IgH clone
(23) and the cloned reciprocal
fragment
from BL22
showed an
abrupt break In homology between these two cloned DMAs within the J cluster
(F1g. 1 ) . In order to define precisely the location of this break 1n homology, DMA sequence from the reciprocal J region was determined and compared
to that
previously
determined genaline
for the IgH nucleotlde
sequence
( 2 3 ) . The two sequences are homologous through the 3' end of the J c l u s t e r ,
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but sharply diverge three nucieotides
Into
the J5 segment (see F i g .
4).
These results are consistent with a recombination event a t J5.
VDJ joining 1s mediated by characteristic recombination signal sequences that flank germiine human Immunoglobuiin D segments on their 5 1 and 3'
sides ( 2 3 , 2 5 ) .
The consensus recombination signal sequence usually found
on the 5' side of gerraline D segments 1s GGTTTTTGT—12 bp—CACTGTG ( 2 3 , 2 5 ) .
I f the DJ-type recombination event had joined a germline 0 to J5, the 5 1 D
recombination signal sequence should be found 10-30 nucleotides 51 to the
J5 sequence In the reciprocal recotnbined fragment.
No such signal sequence
1s found within several hundred nucleotides of the J5 segment ( F i g . 4 ) .
It
Is clear that a normal germline D segment has not participated 1n a DJ recombination at J5 1n the reciprocal, unless a V-D joining
has also taken
place.
I f the recombination at J5 In the reciprocal Is a VDJ j o i n i n g , a l l recombination signal sequences associated with the joined D segment would be
deleted.
Instead, DNA sequence encoding a highly conserved ami no a d d se-
quence In the framework three region (amino a d d s 90-92) found In a l l known
human heavy chain variable regions should be observed within 30-50 bases 5'
to J5 (see Fig. 4) ( 2 6 ) .
No such ami no acid sequence could be translated
In any frame from DNA sequences within several hundred bases 5' to J5, r u l ing out a functional
or aberrant V-region joining 1n the BL22 reciprocal
recombined fragment ( F i g . 4 ) .
The DNA Fragment Joined to J5 on the Reciprocal Recombinant is Homologous
to a Family of D-Bear1ng Fragments
In order to better understand the nature of the sequences rearranged
5' to J5, a probe was prepared from this region and hybridized to P v u I I cleaved genomic DNA from a normal Individual
and BL22.
The probe hybri-
dizes to at least four fragments In normal genomic DNA (F1g. 3 8 ) .
Genomic
DNA from BL22 shares one of these homologous fragments, and also contains
two homologous fragments of d i f f e r i n g sizes. The smallest homologous f r a g ment In BL22 genomic DNA comigrates with a 748 bp PvuII-PvuII
fragment containing
the J5 breakpoint from the reciprocal
PvuII fragment Is J5 break probe)
(Fig. 3 ) .
rearranged
clone
(748 bp
In separate experiments
(not
shown) we find that this segment strongly crosshybridizes to a family of
cloned DNA fragments known to encode multiple human D segments (U. Sieben11st, personal communication).
The Nature of the Re combinational Event
Comparison of the germline and translocated DNA segments from a Bur-
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c
o
o
20 —
10 —
B
CM
CVJ
c
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CD
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o
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BB
m•
10
5
2
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Enzyme:
Hind IE
Probe: 5' Break
Pvu H
J5 Break
5' Break
c-myc
5' Break
J5 Break
Reciprocal
F1g. 3. Genomic representation of the reciprocally translocated junction fragment and the aberrantly rearranged J5 segment. A) Ten pg of genomic DKA from normal human polymorphonuclear cells (control) and the Burk i t t lynphoma cell Une BL22 are digested with HindiII and compared to 20
picograras DMA digested with the reciprocal rearranged clone 1n a genomic
blot hybridization. The probe (5' break) hybridizes to an approximately 15
Kb unrearranged genomic fragment in control and BL22 genomic DNA. A 2 Kb
H i n d l l l fragment Is rearranged by translocation In BL22 and hybridizes to
the probe. The b l o t shows that the same 2 Kb H i n d l l l fragment Is In the
reciprocal clone and coraigrates with I t s genomic counterpart In BL22 DNA.
B) Ten ug of genoraic DNA from control and Burkitt lymphoma BL22 are
digested with PvuII and compared to 20 picograms of PvuII-cut DNA from the
reciprocal clone by Southern blot analysis. The probe u t i l i z e d 1n the hybridization (J5 break) Is a 748 bp PvuII fragment which spans the J5 rearrangement site from the reciprocal rearranged clone. This probe hybridizes
to a fatally of at least four genoraic fragments 1n normal DNA. All but one
of these fragments 1s deleted or rearranged 1n BL22 genomic DNA. One of the
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rearranged fragments In BL22 genomic DNA comigrates with the 748 bp PvuII
fragment 1n the cloned DNA that spans the J5 rearrangement s i t e , clearly
demonstrating that the rearrangement found In cloned BL22 DNA 1s not a
cloning a r t i f a c t .
A schematic diagram of the reciprocal rearranged fragment and germline
c-myc DNA 1s shown below the autoradiogram. The position of the two probes
used (5' Break and J5 Break) Is shown in the two clones. Stippled bars denote c-myc derived sequences and open bars indicate IgH derived sequences.
The BL22 translocation site 1s designated by a triangle on gennline c-myc.
The three exon structure of the c-myc gene and the direction of I t s transcription Is Indicated. The J segments and enhancer region (E) are shown
on the reciprocal.
k1tt lymphoma (BL22) Indicates that this translocation event is highly conservative with respect to c-myc sequences 1n the Immediate v i c i n i t y of the
translocation breakpoint. This result 1s similar to the 8;22 variant translocation 1n the Burkitt lyraphoma BL37, where no nucleotfdes are lost from
c-myc sequences during recombination (8). Analysis of an analogous mouse
plasmacytoroa (Bl-8 \i) c-rayc translocation revealed loss of only 7 bp of
c-myc sequences—also highly conservative (9).
Approximately two kilobases of ti switch sequences are lost from the
translocation site In this Burkitt lymphoma.
I t Is Impossible to know
whether this DNA was deleted before, during or after translocation, or
whether the sequences were lost from either the c-myc containing recotnbinant or Its reciprocal recombined fragment. This represents a larger deletion than has been previously observed at c-myc trans locations, and may
r e f l e c t the recombinational I n s t a b i l i t y of this highly repeated sequence.
In addition, i t Is Important to note that the changes occurring In the
Immediate v i c i n i t y of the translocation breakpoint are not the only a l t e r ations detected In the structure of the c-tnyc gene. Detailed sequence analysis of the untranslated leader exon of BL22 c-rayc gene, a region about 1.5
Kb from the crossover point described here, contains a number of point mutations and small Insertions and deletions (20).
The structural basis of the recombination event Is not obvious from
Inspection of various sequences surrounding the translocational recombination site (4-10,13,15). Clearly there 1s no region of evident homoiogy 1n
the sequences presented here. Furthermore, from a variant translocation
Involving the X l i g h t chain locus, 1t is clear that switch region sequences
are not a necessary component of the recombination site (15). What Is then
responsible for this recurrent non-homologous recombination event? I t Is
s t i l l possible that some subtle feature of the primary structure of the DNA
surrounding the various sites of translocation w i l l be found (e.g. note the
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YYC
F1g. 4 . Hudeotide sequence comparison of gerraiine IgH J region and
the reciprocal recorabined fragment a t the J recombination s i t e . The two
sequences are compared and show evidence for a J-type rearrangement three
nucleotides from the 5' end of J5 1n the reciprocal. The boundaries of J5
are Indicated, and the J5 translation shown above I t s germline sequence.
The DNA sequence from the reciprocal around the J5 rearrangement site 1s
translated In three forward frames below the sequence. Asterisks designate
stop codons. Neither a consensus 5' D signal nor the Invariant IgH V amlno
adds 90-92 are found within several hundred nucleotides of the J5 rearrangement s i t e In the reciprocal sequence. The consensus 5' D signal sequence (GGTTTTTGT— 12 bp— CACTGTG) and Invariant araino a d d s 90-92 (YYC)
are shown a t the bottom of the f i g u r e . Phenylalanine (F) can occasionally
substitute for tyrosine (Y) at amlno a d d s 90 and 9 1 , but the cysteine (C)
at amlno a d d 92 1s absolutely I n v a r i a n t . Schematic diagrams of the two
DNA segments (germline IgH and reciprocal) are shown below the sequence.
Bars designate the regions In wrrich sequences are compared In the figure.
CCAAACCC found near the breakpoint In the c-myc gene ( F i g . 2A) and also
found 1n this form or as I t s complement near the recombination point 1n several Burkitt translocations (14,15) and mouse myelomas ( 7 , 9 , 1 0 ) ) .
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other hand, since no general and consistent sequence has yet emerged, I t Is
more useful to think of the ImmunoglobulIn locus as one that physiologica l l y must undergo recombination In pre-B cells and, therefore, usually exists 1n a chromosomal configuration suitable for recombination.
Making
this assumption contributes l i t t l e to our understanding of why the c-myc
locus should serve as a partner In this process unless we assume I t 1s not
the only fragment drawn Into the Imraunoglobulin locus by translocation, but
one of the few that leads to the dramatic transforming events that result
In Its detection.
The Translocated IgH Enhancer
Transcription enhancer sequences In immunoglobuHn genes have been
shown to markedly Increase transcription In B-cells from IgH V region and
other promoter sequences joined 1n close proximity to these enhancer elements (16,17). The human homologue to the known mouse IgH enhancer sequence (18) has been located between the J cluster and the \L switch region
by DNA sequence analysis (F1g. 1).
This sequence resides not with the
translocated c-rayc gene, but with I t s reciprocal In this Burkitt lyraphoma.
This c1s-act1ng sequence therefore cannot be responsible for altering c-myc
gene expression In this lymphoma or, 1n fact, In any other c-rayc translocation occurring In this orientation at an Imraunoglobulin switch region. The
translocation event has moved this enhancer near DNA sequences normally l o cated > 1 Kb 5' to c-myc. These 5' sequences are not normally transcribed
1n B lymphoblastoid c e l l s . RNA blot experiments using probes from the region 5' to c-rayc show no novel transcripts In BL22 (A.R., unpublished observations). I t thus appears that this enhancer does not activate new transcripts within five kilobases of the DNA fragment normally flanking the 5'
portion of the c-rayc gene. Furthermore, the fact that no transcripts are
found corresponding to the IgH portion of the reciprocal fragment reflects
the fact that the Ig V region and I t s promoter sequence has not yet been
joined to this region.
ACKNOWLEDGMENTS
We would l i k e to thank U. S i e b e n l i s t f o r numerous h e l p f u l
and h i s generous g i f t o f T c e l l genooic DNAs.
discussions
We are g r a t e f u l t o R. Taub
f o r her generous g i f t o f B c e l l genomic DNAs and BL22 RNA and we would a l s o
l i k e t o thank L. Hennighausen f o r h i s h e l p f u l comments. We g r a t e f u l l y a c knowledge the support o f grants from the American Business Cancer Research
Foundation and E . I . DuPont deNenours and Co. f o r p o r t i o n s o f t h i s work.
2151
Nucleic Acids Research
J.B. was supported In p a r t by a postdoctoral
f e l l o w s h i p from the American
Cancer S o c i e t y .
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