Download Localization of human variable and constant

volume 10 Number 24 1982
Nucleic Acids Research
Localization of human variable and constant region immunoglobulin heavy chain genes on
subtdomeric band q32 of chromosome 14
O.Wesley McBride1, James Battey3, Gregory F.Hollis3, David C. Swan2, Ulrich Siebenlist3 and
Philip Leder3
'Laboratories of Biochemistry and Cellular and Molecular Biology, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20205, and department of Genetics, Harvard Medical
School, Boston, MA 02115, USA
Received 18 August 1982; Revised and Accepted 15 November 1982
ABSTRACT
Analysis of a group of human/rtfaent somatic cell hybrids with nucleic
acid probes prepared from cloned human variable region (VJJ) , junctional (Jy) ,
and constant region (Ce) heavy chain immunoglobulin genes indicates that all of
these IgH genes are localized on the subtelomeric (q32) band of chromosome 14.
Somatic cell hybrids were isolated in selective medium after fusing human
fibroblasts with hprt~ Chinese hamster cells. The human parental cells contained
two translocation chromosomes representing a reciprocal translocation between
chromosomes X and 14. Only those hybrid cell lines retaining a complete human
autosome 14 or the X/14 translocation chromosome (i.e. containing band 14q32)
retained the human IgH genes. Retention of these genes did not correlate with
the presence of the other translocation chromosome, 14/X. These results indicate
that all human IgH genes (VH, Jg, and C H ) map to the same chromosomal band (14q32)
which is commonly involved in reciprocal translocations with human chromosome
8 (8q24) in B-cell neoplasms.
INTRODUCTION
Three specific human chromosome rearrangements have been reported in
Burkitt's lymphoma and other B-cell neoplasms and each involves a reciprocal
translocation between a specific region of chromosome 8 (8q24) and specific
break points on human chromosomes 2, 22 or 14 (1-4).
These same three chromo-
somes bear the kappa, lambda, and heavy chain human immunoglobulin genes,
respectively (5-9).
Analogous translocations involving mouse chromosome 15
with chromosomes bearing kappa (chr. 6) and heavy chain (chr. 12) immunoglobulin
genes have also been described in mouse plasmacytomas (10). These observations
led Klein (2) and Rowley (3) to propose that B-cell neoplasms may arise by
translocation of part of an active immunoglobulin locus to the vicinity of a
cellular oncogene thereby activating the one gene. Regional chromosome assignments of the human Ig genes are required to evaluate this proposal.
Although the molecular organization of both human and rodent immunoglobulin genes have been studied intensively, the distance between variable
region and constant region Ig genes in germline cells is still unknown.
) I R L Press Limited, Oxford, England.
0305-1048/82/1024-8155S 2.00/0
Hence,
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regional chromosomal mapping of Ig genes might also provide information
relating to the distance between these two regions which undergo activation
by somatic rearrangement in lymphocytes.
The approach we have used to regionally localize the variable region,
junctional, and constant region heavy chain genes on human chromosome 14 was
to analyze DNA isolated from human/rodent hybrid cell lines containing a
well characterized translocation of this chromosome with labeled DNA probes
prepared from cloned Ig sequences.
Our results indicate that all of these
genes are located on a single subtelomeric band (14q32) of this chromosome.
These findings confirm and extend recent reports (11, 12, 13) localizing some
heavy chain Ig genes to this same chromosome segment.
MATERIALS AND METHODS
Somatic Cell Hybridization.
The parental human fibroblast line (GM0073)
which was used for hybrid cell isolation contained a balanced translocation
between the X chromosome and chromosome 14 (Fig. 1 ) . The karyotype of this
line is 46X, t(X; 14) (Xpter > Xql3::14q32 > 14qter; 14pter > 14q32:: Xql3
> Xqter).
These GMOO73 human cells and hprt~ Chinese hamster flbroblasts
(CHTG49) were cocultivated (1:1 mixture) In plastic petri dishes (6 cm
diameter) for 24 hrs until confluent before induction of cell fusion with
52.5% polyethylene glycol 1000 (14). The culture medium was replaced with
selective HAT medium (100 \H hypoxanthine, 1 \H amethopterin, 16 \H thymidine, 100 viM glycine) 24 hrs after cell fusion and this selective medium
was replaced twice weekly.
Ouabain (50 yM) was included during the initial
3 or 4 days of selection to prevent survival of nonhybridized human cells.
.2.
p
2
2
1
q
t
1
4
X/14
14
MPflT
G6PO
14/X
Figure 1. Diagram of the reciprocal balanced translocation in GMOO73 flbroblasts . These cells contain one normal X chromosome and one normal autosome
14. Broken lines extending from these two chromsomes indicate the translocation breakpoint in this cell line. The translocation fragments in both
rearranged chromosomes are separated by solid arrows. The chromosomal location
of some genes is shown alongside each chromosome.
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Independent colonies were cloned after 10 to 14 days.
Each hybrid cell clone was expanded in HAT medium for about 2 weeks
(15-20 cell generations) to a total of approximately 10? cells.
Frozen
stocks were prepared and aliquots of each cell line were seeded into petri
dishes at cloning densities in HAT medium.
Independent colonies were again
cloned after 10 to 14 days and expanded to 10^ to 10? cells (15-20 cell generations) over an interval of about 2 weeks in HAT medium.
Aliquots of cells from 9 subclones, each originating from different
hybrid clones, were propagated for 1 week (7-14 generations) in nonselective
(1:1 Ham's F12: Dulbecco) medium (15) containing 5% (vol/vol) fetal bovine
serum.
Each cell line was then subcultured and half of the cells were
expanded in HAT medium and the other half in medium containing 6-thioguanine
(50 pM). The media containing HAT or 6-thioguanine permit survival of those
cells which have retained or lost the 14/X translocation chromosome, respectively.
Isoenzyme Analysis.
Washed cell pellets were simultaneously prepared for DNA
isolation and isoenzyme analyses from hybrid cell lines which had been expanded to 3-10 x 108 cells in the appropriate HAT or thioguanine selective media.
Cell pellets were stored at -80°C until used.
The human chromosomes present
in each hybrid cell line were identified by starch gel electrophoretic
analysis (16, 17) of isoenzyme markers which have been previously assigned
to each of the human chromosomes (18). Purine nucleoside phosphorylase
(NP; EC2.4.2.1) and brain type creatine kinase (CKBB; EC2.7.3.2) were the
isoenzymes used to identify human chromosome 14; CKBB has not previously
been regionally localized on chromosome 14 and NP is located proximal to
the break point with respect to the centromere on chromosome 14 in GM0073
cells.
Hypoxanthine phosphoribosyltransferase (HPRT; EC2.4.2.8), glucose-
6-phosphate dehydrogenase (G6PD; EC1.1.1.49), and phospoglycerate kinase
(PGK; EC2.7.2.3) were employed as markers for the X chromosome and all
3 markers are located distal to the break point with respect to the centromere of the X chromosome in GMOO73 cells.
Hence, the 14/X translocation
chromosome in these cells is positive for human NP, PGK, HPRT, and G6PD
enzymes.
Isoenzyme markers used for detection of the other human chromo-
somes have been described (7).
Nucleic Acid Hybridization Analysis of Cell Hybrids.
DNA was isolated from
hybrid cell lines (19), digested with BamHI, size fractionated by 0.75%
agarose gel electrophoresis, transferred to nitrocellulose paper, and
hybridized with cloned, nick translated, 32p_iabeled DNA probes as described
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JH
-H4H-4MHHH
PROBES
I
1
LR3SV
I
-ih
1
J 2 - J5
I
1
EPSILON
Figure 2. Diagram of the human heavy chain imraunoglobulin gene region
illustrating the relative locations of cloned sequences used as Ig gene
probes. Distances are not drawn to scale and some genes have been omitted.
previously (7). The DNA probes (Fig. 2) were: IgE probe, a 2.6Kb BamHI
fragment containing the entire epsilon constant region gene and flanking
sequences (20); JJJ probe, a 1.2Kb Sau3A fragment extending from within the
J2 coding sequence to a region between the J4 and J5 heavy chain junction
sequences (21); and Vy, a 363 BP BamHI fragment including about 100 BP of
flanking sequences 51 to this gene (J. Ravetch, unpublished results) subcloned from a human IgM cDNA clone designated LR-35 (21).
RESULTS
Isolation and Characterization of Hybrid Cell Lines.
Regional localization
of the human IgH genes required isolation of hybrid cell lines which retained
only a specific well-characterized portion of human chromosome 14 in the
absence of the normal homologue.
This problem was simplified by isolating
hybrid cells after fusing Chinese hamster hprt~ mutant fibroblasts with a
human parental cell line (GMOO73) which contained the selectable hprt gene
attached to a large translocation fragment of human chromosome 14. Since
the normal X chromosome is preferentially inactivated in somatic cells containing a balanced X-autosomal translocation (13), hybrid cells could be
selected for retention or loss of the human 14/X translocation chromosome by
growth in medium containing HAT or thioguanine, respectively.
The possibility
for both forward and reverse selection was important in analyzing these
hybrids.
In contrast, other human chromosomes, including the normal 14
homologue, were slowly lost from the human/rodent hybrid cells by chromosome
segregation during cell growth in either medium.
Based on these facts, a strategy was used for isolating hybrid cell lines
which permitted us to distinguish by isoenzyme analysis between lines containing
a 14/X translocation chromosome alone and those retaining a normal human
chromosome 14 as well.
Each hybrid cell line was subcloned after chromosome
segregation during growth in HAT for 15-20 cell generations to obtain hybrids
retaining a limited number of specific human chromosomes including t 14/X.
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These subcloned hybrid segregants were expanded in HAT and then cultured for
one week in non-selective medium to permit survival of hybrid cell progeny
which lost the t 14/X chromosome during continuing chromosome segregation.
Other human chromosomes were also lost from individual hybrid progeny during
the interval following subcloning but it is unlikely that any of these other
chromosomes would have been lost from an entire population of hybrid cells.
In the absence of additional subcloning, it could be anticipated that only
the 14/X translocation chromosome would be selectively retained or lost from
mass cell cultures in the HAT and thioguanine media, respectively.
All
other human chromosomes, including the normal chromosome 14, which were
present when the hybrid cells were subdivided would be retained in both cell
populations despite heterogeneity in specific human chromosome content among
Individual hybrid cells.
Therefore, analysis for human nucleoside phosphory-
lase in cell extracts prepared from each hybrid line after forward and reverse
selection permitted identification of those hybrid lines which retained the
normal human chromosome 14. All cell extracts prepared from the hybrids grown
in HAT medium contain human NP activity since these cells all selectively
retain t 14/X irrespective of the presence or absence of the normal 14
homologue.
However, only those hybrid cell lines which retain the normal
chromosome 14 also express human NP after reverse selection in thioguanine
medium for cells having lost the 14/X translocation chromosome.
Human Chromosome Content of Hybrid Cell Lines.
The specific human chromosomes
present in each hybrid cell line were determined by isoenzyme analysis (Fig. 3 ) .
Each human chromosome except chromosome 10 was present in at least one hybrid
cell line, and only chromosome 6 was present in all lines.
chromosome could not be identified by isoenzyme analyses.
The inactive X
Human glucose-6-
phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and hypoxanthine
phosphoribosyltransferase (HPRT) isoenzyme activities were present in the
extracts from each line cultured in HAT and absent in the parallel lines
cultured in thioguanine with one exception.
Human G6PD was not detected in
hybrid 40H or 40T and this suggests the presence of a chromosome break or
translocation involving the distal portion of the translocated X chromosome
long arm in this line.
Human nucleoside phosphorylase activity was detected
in extracts of all hybrids cultured in HAT medium and it was absent from hybrid
lines 33, 37 and 40 after growth in thioguanine medium (Fig. 4 ) .
Thus, we concluded that the normal human chromosome 14 was absent from these
3 hybrid lines and present in the other 6 lines.
This interpretation was
greatly strengthened by the unexpected observation that brain type creatine
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33H
34H
35H
36H
5 37H
£
T
I 38H
T
39H
40H
41H
1
4
8
12
16
HUMAN CHROMOSOME
20 22X *
"
H
Figure 3. Distribution of specific human chromosomes in each human/hamster
hybrid cell line. Individual hybrid cell lines are represented on the
ordinate and H or T indicates growth of that cell line in HAT or 6-thioguanine
medium, respectively. Specific human chromosomes are represented on the
abscissa. Solid boxes indicate the presence of a particular human chromosome
in a hybrid line, and open boxes indicate absence of the chromosome. The
presence of Vy, D^, JJJ and C e sequences in hybrid cell lines is shown
by hatched boxes in the last column. The presence of the 14/X human translocation chromosome in cell lines was inferred by the detection of human
NP, G6PD, PGK, and HPRT. The presence of a normal human chromosome 14 was
deduced by the presence of human NP and CKBB activity in cell extracts of a
hybrid line after parallel growth in both thioguanine and HAT media. Hybrid
lines exhibiting human CKBB activity during growth in both selective media
but human NP activity only during culture in HAT were judged to contain the
X/14 translocation chromosome; its presence could not be determined in hybrids
retaining the normal autosome 14.
kinase (CKBB) did not segregate concordantly with NP in these hybrids.
Human CKBB was detected in extracts of all hybrids except lines 33 and 37,
and the presence of this enzyme was unrelated to previous growth of the
cells in either HAT or thioguanine selective medium (Fig. 5 ) .
Since CKBB has been previously assigned to human chromosome 14, it must be
located distal to the breakpoint on this chromosome in GM0073 cells.
Thus,
CKBB is a marker for the chromosome 14q32 band and this enzyme would be
detected in hybrids containing either a normal chromosome 14 or the t X/14
chromosome.
The retention of human CKBB despite the loss of human NP after
thioguanine selection of hybrid line 40, Indicates that the t X/14 chromosome
is present in this hybrid.
The presence or absence of the t X/14 chromosome
could not be determined in those hybrid lines containing a normal human
chromosome 14, but this fact was irrelevant to our analysis.
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I x i X I I l l 2
2 i- I- I-
-ORIGIN
-HUMAN
-HAMSTER
Figure 4. Nucleoside phosphorylase (NP) isoenzyme separation by vertical (12%)
starch gel electrophoresis in Tris-EDTA-borate buffer (16) at pH 8.6. The
anodally migrating NP activity was detected in situ as dark formazan bands by
standard procedures (17). Two heteropolymeric bands can be seen between the
trimeric homopolymers of human and Chinese hamster NP activity. The light
staining bands represent superoxide dismutase activity.
that, except for t 14/X, the same human chromosomes would be present in a
hybrid cell population irrespective of its final growth in HAT or thioguanine
was generally confirmed.
A single deviation from that pattern was found in
three hybrid lines (33, 35 and 37) and three exceptions were observed in a
fourth line (39). These probably all represented instances in which the
fraction of cells containing a particular human chromosome corresponded
closely to the proportion of cells required for detection of the isoenzyme
marker.
Analysis of Hybrid Cell Lines with Human Immunoglobulln Probes.
DNA was
isolated from each hybrid line after growth in either HAT or thioguanine,
and the DNA was analyzed for the presence of specific human IgH genes.
Hybridization of a cloned 2.6Kb epsilon constant region probe with DNA blots
after BamHI digestion revealed intensely hybridizing bands of 2.6Kb and 6Kb
size and weak hybridization with a 9Kb band (Fig. 6 ) . These 3 respective
bands represent the active human epsilon gene and the two pseudogenes, tyel
and \)ie2 (20, 22). Both the human e and t|>el sequences were detected in
all hybrid lines except 33H, 33T, 37H and 37T.
In contrast, there is a
different distribution of hybrid lines which contain the processed human
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oft
jj
at
co c o c o w e o
co co m
i
o
«*
n
» w ^ O p ) f ) j
ORIGtJ
GKBB
Figure 5. Creatlne kinase, brain type (CKBB) lsoenzyme separation by (12%)
starch gel electrophoresls in citrate-phosphate buffer at pH 6.8 (16). Creatine
kinase activity was detected by a standard procedure (17) using creatine phosphate substrate and a tetrazollum-linked stain. Hamster type CKBB is not
expressed in CHV79 partental cells or hybrids of these cells. Human type
CKBB is expressed in GMOO73 parental cells and GM0073/hamster hybrids retaining
human autosome 14. The less anodally migrating bands above CKBB represent
adenylate kinases and these bands persisted when creatine phosphate was omitted
from the reaction mixture.
epsilon pseudogene ( tjie2) as previously reported (23).
Southern analysis of the BamHI digested hybrid cell DNA samples with a
1.2Kb J H region probe (Fig. 7) showed an intensely hybridizing 17 Kb band in
both human placental DNA and DNA from hybrid cell lines containing human
epsilon sequences.
A much smaller, faintly hybridizing band was also detected
in all hybrid lines as well as Chinese hamster DNA, and this band presumably
represented homologous hamster J region sequences.
Hybridization of the blots with a cloned heavy chain variable region probe
(Fig.
8) revealed a predominant 12Kb band as well as only partially resolved
larger bands and several faint smaller bands in human placental DNA.
A similar
pattern of hybridization was observed with all hybrid cell lines except 33H,
33T,
37H and 37T.
Lines 33 and 37 showed only a faint smear of hybridization
with very large DNA fragments which was indistinguishable whether the cells
had been grown in HAT or thioguanine.
This suggests that VJJ pseudogenes or
some other human sequences with weak homology to the VJJ probe may be located
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Kb
2.6
Figure 6. Hybridization of the 32p_iabeie<j human epsilon Ig probe with
hybrid cell DNAs. DNA preparations isolated from controls (Chinese hamster
liver and human placenta) and somatic cell hybrid lines were digested with
BamHI, fractionated by 0.75% agarose gel electrophoresis (30ug/lane),
transferred to nitrocellulose, hybridized with a C e probe, and visualized
by autoradiography. The hybridizing 2.6Kb band represents the functional e
gene and the 6 and 9Kb bands represent pseudogenes. Somatic cell hybrid
lines used for DNA isolation are shown above the lanes ; T and H indicate
the same cell line cultured in 6-thioguanine or HAT, respectively. Sizes of
DNA in hybridizing bands is shown on the left. Lane A1H contained a smaller
quantity of DNA (as indicated by ethidium bromide staining) and the hybridizing
bands are poorly visualized in this reproduction.
on other human chromosomes than autosome 14.
The distribution of hybrid
cell lines hybridizing with a cloned diversity region probe was identical to
the pattern with VJJ, Vj, and C £ probes (not shown).
These results clearly indicate that all classes of heavy chain immunoglobulin genes can be assigned to a region of human chromosome 14 distal to
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E
X
c
a
E
3
X
4 1
H
T
40
H T
T
H
T
H
T
H
T
H
33
34
35
36
37
38
39
H
T
H
T
H
T
Kb
17
Figure 7. Hybridization of size fractionated BamHI DNA restriction fragments
from somatic cell hybrid lines with a 1.2Kb ^2P-labeled human J H probe.
Cell lines used for DNA isolation are shown above the lanes; H and T represent
growth in HAT and 6-thioguanine, respectively. The conditions and procedures
are described in Fig. 6.
the break point in the subtelomeric band of this chromosome in GM0073 cells.
These genes segregated discordantly with all other human chromosomes including
the 14/X translocation chromosome which contains the entire chromosome 14
with the exception of the most distal band (i.e. I4q32).
Our basic observa-
tion is that human IgH genes are detected in hybrids expressing either human
CKBB alone or CKBB and human NP while these IgH genes are not found in hybrids
expressing only human NP.
Localization of the IgH genes to 14q32 is thus based
primarily upon the absence of these genes from hybrid lines 33H and 37H.
Even if these two lines had survived due to the presence of an active normal
X chromosome, the presence of human NP in these lines indicates that the
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a
m
c
a
-
:
5
41
H
T
40
H
T
30
H
T
38
H
T
37
H
T
36
H
T
35
H
T
34
H
T
33
H
T
23.7
Figure 8. Hybridization of a human -"P-labeled v H probe designated LR35V
with BamHI digested, hybrid cell DNAs. The size markers along the ordinate
indicate the positions of HinD3 cut lambda bacteriophage DNA fragments.
Cell lines used for DNA isolation are shown above the lanes, H and T
represent growth in HAT and 6-thioguanine, respectively. The conditions of
DNA digestion, electrophoresis, transfer, and hybridization were identical
to those described in Fig. 6.
proximal portion of chromosome 14 was also present and that the 14/X translocation chromosome segregates discordantly with the human IgH genes in these
lines.
The alternate possibility that both of these human NP+ CKBB" hybrids
(33H and 37H) retained an active normal human X chromosome, or X-chromosomal
long arm, in combination with a proximal portion of chromosome 14 resulting
from spontaneous breakage of this human chromosome during culture is considered
unlikely.
The additional requirement that this putative proximal fragment
of chromosome 14 would have been retained non-selectively during culture in
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HAT while it was lost from parallel mass cultures of the same lines (33T and
37T) during growth in thioguanine is exceedingly improbable, but detailed
karyotypic analysis will be required to exclude this very remote possibility.
Moreover, the presence of IgH genes in hybrid 40T in the absence of human NP
indicates that the IgH genes are located on the distal portion of chromosome
14.
The finding that CKBB segregates concordantly with the IgH genes and
discordantly with NP means that CKBB is also located on 14q32.
The use of CKBB as a marker for human chromosome 14q32 requires consideration of several additional facts. Many established mouse and Chinese
hamster fibroblast lines, and some human lymphoblastoid lines, do not express
CKBB (24, 25). Human and rodent CKBB are expressed independently of each
other in human/rodent somatic cell hybrids; i.e., expression of CKBB from the
inactive parental cell is not reactivated and CKBB expression from the active
parent is not suppressed in these hybrids (24). Two groups (25, 26) have
assigned the CKBB locus to human chromosome 14 while two other groups (24, 27)
concluded that the structural locus for CKBB is probably on this chromosome but,
the presence of human chromosome 17, or other chromosomes, may also be required
for human CKBB expression.
Our results (and our unpublished studies) support
all these conclusions except we find no evidence that expression of human
CKBB requires the presence of any specific human chromosome in addition to
no. 14.
It is suggested that these conflicting interpretations may arise
from the location of the CKBB locus on the telomeric end of chromosome 14.
DISCUSSION
Human heavy chain variable region, junctional, and constant region Ig
genes have all been regionally localized to a single subtelomeric band (14q32)
on chromosome 14 by analysis of a group of human/hamster somatic cell hybrids
with cloned human Ig probes.
The analysis was simplified by fusing human
parental fibroblasts containing a reciprocal X; 14 chromosome translocation with
hprt~ hamster cells thereby permitting selection of hybrid cells which either
specifically retained or lost the 14/X translocation chromosome.
While the
normal human X chromosome is always inactivated in lymphocytes obtained from
females with an X/autosomal balanced translocation, this is not invariably
true in fibroblasts from these patients (28). The presence of an active normal
human X chromosome in some of our hybrids would have prevented selection for
retention of the t 14/X chromosome in those lines, but it would not affect our
interpretations.
There have been several recent reports (11-13) indicating that some human
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IgH genes are located on 14q32.
Cox et al. (12) studied a patient who has a
ring chromosome 14. Based upon karyotyping and GM allotyping, they concluded
that this Y chain genetic marker could be localized to the most terminal portion
of I4q at band q32.3.
Kirsh et al. (11) mapped a heavy chain Ig gene to
chromosome band 14q32 by in situ hybridization using a cloned heavy chain y4
gene probe.
Balazs et al. (13) used a cloned DNA probe to map a site for
restriction fragment length polymorphism (D14S1) to chromosome band 14q32
based upon analysis of human/mouse somatic cell hybrids containing the human
GMOO73 translocation chromosome.
They showed linkage (3% recombination
fraction) between the D14S1 site and the GM locus by examining a family
segregating for GM-variants at the Y-l locus.
These reports all strengthen
our assignment of the IgH genes to band 14q32.
However, our results directly
map both the variable and constant region IgH genes to this chromosome segment
and thereby eliminate the possibility that only some of the sequences required
for IgH expression are localized to this region.
Since both Vfj and C H genes are localized on the same band, their
orientation with respect to the centromere cannot be determined.
CJJ gene cluster is probably located on band 14q32.
The entire
Either the functional
epsilon gene or \\iz-l is the penultimate gene in this cluster (20) and either
sequence would be detected with our e probe.
Moreover, the i)iel and e gene
can be detected in EcoRI restriction fragments of about 25 and 30 Kbp length,
respectively, containing the human a genes.
Hence, the epsilon probe would
have detected a sequence located less than 25-30 Kbp from the 3'-terminus of
the C H gene cluster.
In contrast, other families of V K region genes may be
located 5' to the sequences detected with our VJJ probe.
These sequences could
possibly extend proximal to the breakpoint on chromosome 14 in GMOO73 cells
if the orientation of IgH genes on this chromosome is centromere + VJI + C^ *
telomere.
It can be estimated by several methods that the DNA content of band 14q32
is about 10^> Kbp (11, 13). Hence, this represents the maximum separation which
could exist between Vy and CJJ genes in germ line cells.
The heavy chain immuno-
globulin genes, excluding the interval between the VJJ and CJJ regions, probably
occupy about 10% of this entire chromosome band.
Results obtained by cloning
human IgH constant region genes in several laboratories indicate that the
general arrangement and total length of the human C H gene cluster is similar
to that found in the mouse or about 200 Kbp length (29). The human J H sequences
are separated from Cy by only 6 Kbp and occupy an additional 3 Kbp (21).
Matthyssens and Rabbitts (30) reported that one human V H family contained about
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23 genes spaced at 12-16 Kbp Intervals, so that the entire V^ region containing a minimum of three V H families probably comprises at least 500-1000 Kbp.
The location and length of DNA occupied by diversity region genes is not clear
but one of these genes is located within the human J^ cluster between i|^
and Ji (21).
The most common chromosome rearrangement associated with B-cell neoplasms
is a reciprocal translocation between 8q24 and 14q32.
Thus, assignment of all
IgH genes to band 14q32 is primarily important as it may relate to these neoplasms.
Currently, chromosomal breakpoints cannot be determined with greater
precision than about 1000 Kbp even using high resolution banding techniques
(31), and the usual precision is about 10-fold lower.
This restricts the
ability to precisely relate a chromosome breakpoint in lymphomas to the localization of Ig genes.
Considering the length of DNA occupied by the IgH genes,
there is about 10% probability that a break anywhere in the 14q32 band would
be located close to an IgH gene (i.e. within about 20-50 Kbp). A more precise
correlation between the 14q32 breakpoint in lymphomas and IgH gene locations
will probably require application of recombinant DNA techniques to neoplastic
cells containing the 8:14 translocation.
A recent report by Lenoir et al. (32) supports the hypothesis that the
association between reciprocal translocations Involving human chromosome 8
with Ig-bearing chromosomes and B-cell malignancies may not be coincidental.
They found complete concordance between the class of secreted Ig light chains
in lymphomas and the specific chromosome translocation (i.e. 2:8 or 8:22
translocations are associated with < or X light chain secretion, respectively).
Mechanisms have been suggested with might promote translocations involving
Ig-bearing chromosomes and thereby produce B-cell malignancies (11). Klein
(2) and Rowley (3) have proposed methods by which these translocations might
induce the malignant state and one method involves activation of a cellular
one gene on chromosome 8 by an immunoglobulin gene.
It may be relevant that
two human one gene analogues, human c-mos (33) and human c-myc (D. Swan, W.
McBride, S. Tronick and S. Aaronson, in preparation), have recently been
assigned to chromosome 8.
The possibilities that specific Ig-bearing chromo-
some translocations and/or cellular one genes are causually related to lymphoid
neoplaslas is now open to experimental testing by a variety of methods.
Since preparation of this manuscript, there has been a report (34) that
the chromosome 14 break point occurs within the V H region in a Burkitt
lymphoma cell line containing a reciprocal 8; 14 chromosome translocation.
The results also indicate that the orientation of IgH genes on chromosome 14
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is centromere • CJJ + Vpj + telomere.
ACKNOWLEDGEMENTS
We are grateful to M.C. Otey, A. Kerr, and D. Keithley for expert
technical assistance, to C. Mock for preparing photographs, and to Gail Taff
for expert help in preparing the manuscript.
The GM0073 cell line was obtained
from the Institute for Medical Research (Caraden, NJ).
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