Proc. NatL Acad. Sci. USA
`Vol. 79, pp. 4957-4961, August 1982
`Biochemistry
`
`Localization of human immunoglobulin K light chain variable
`region genes to the short arm of chromosome 2 by
`in situ hybridization
`(human K genes/gene mapping/chromosome translocation/Burkdtt lymphoma)
`S. MALCOLM*, P. BARTON*, C. MURPHYt, M. A. FERGUSON-SMITHt, D. L. BENTLEYt, AND T. H. RABBITTS*
`*Department of Biochemistry, Queen Elizabeth College, Campden Hill Road, London W8, England; Institute of Medical Genetics, Royal Hospital for Sick Children,
`Yorkhill, Glasgow, Scotland; and *Laboratory of Molecular Biology, Ile MRC Centre, Hills Road, Cambridge CB2 2QH, England
`Communicated by C. Milstein, May 26, 1982
`
`The genes for human immunoglobulin K light
`ABSTRACT
`chains have been localized in normal lymphocyte and fibroblast
`chromosomes by in situ hybridization ofprobes from cloned DNA
`fragments of the K variable region locus. The localization was
`achieved by counting grains (after autoradiography) over chro-
`mosomes in a number of karyotypes.. The variable region gene
`probes hybridized in a cluster on a region of the chromosome 2
`short arm close to the centromere (2cenvp12). This location was
`confirmed in lymphocytes from a balanced translocation carrier
`46XXt (2; 16) (q13; q22). Our results show that human K light chain
`genes are located in the region ofthe break point observed in spe-
`cific chromosomal translocations associated with Burkitt lymphoma.
`The three sets ofunlinked genes code for antibody proteins: the
`heavy chain, the K light chain, and the A light chain genes.
`Attempts to map these genes in man have been hindered by the
`lack of variants suitable for family studies and the loss of im-
`munoglobulin expression in somatic cell hybrids. Despite these
`problems, chromosome assignments for some human immu-
`noglobulin genes have been made by using panels of mouse-
`human hybrid cell lines with different human chromosome
`complements. Correlation between immunoglobulin expres-
`sion and the presence of specific human chromosomes in hy-
`brids of mouse myeloma cells and human B cells has led to as-
`signment of A light chains to chromosome 22 (1) and of heavy
`chains to chromosome 14 (2). Human heavy chain genes have
`also been assigned to chromosome 14 on the basis of filter hy-
`bridizations between cloned heavy chain genes and DNA from
`a panel ofmouse-human hybrid cell lines with different human
`chromosome complements (3).
`A completely different technique-with the advantage of
`using normal cells rather than a panel of hybrid cell lines-is
`that of in situ hybridization to fixed metaphase chromosomes.
`This technique previously has been used to confirm the location
`of human a- and /3-globin genes to their respective chromo-
`somes (4, 5). In this paper we describe the localization of human
`K light chain genes by in situ hybridization. Two cloned genomic
`DNA fragments, each containing a single K variable region (VK)
`gene, were used as hybridization probes. The results of the in
`situ hybridization with these VK probes localize the K chain
`genes to chromosome 2 in man.
`
`MATERIALS AND METHODS
`Chromosome Preparation. Translocation carrier and normal
`human metaphase chromosome spreads prepared from phyto-
`hemagglutinin-stimulated peripheral blood cultures (6) were
`
`The publication costs ofthis article were defrayed in part by page charge
`payment. This article must therefore be hereby marked "advertise-
`ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
`
`banded by mild treatment with 1% lipsol detergent (LIP Equip-
`ment and Services, Shipley, Yorkshire, United Kingdom),
`stained with Giemsa, and photographed. Chromosomes were
`prepared from fibroblast cultures as described by de Grouchy
`et aL (7) except that the culturing medium used contained Harris
`F-10 medium with 10% newborn calf serum (GIBCO, Paisley,
`Scotland), 10% fetal calf serum (GIBCO), penicillin (Glaxo,
`Middlesex, United Kingdom; 1,000 units/ml), and strepto-
`mycin (Glaxo; 25 mg/ml).
`In Situ Hybridization. Prior to hybridization, chromosomes
`were denatured in 60% (vol/vol) formamide/0.1 mM EDTA/
`5 mM Hepes, pH 7, at 550C for 15 min, washed in 0.30 M NaCl/
`0.03 M sodium citrate, and dehydrated through a series of al-
`cohols. The [3H]cRNA used as a probe was prepared as de-
`scribed (5) under conditions that gave random initiation and
`produced equal transcription of vector and inserted DNA.
`[3H]cRNA (specific activity, 1.7 X 108 dpm/,ug) in 50% form-
`amide/0.6 M NaCI/5 mM Hepes, pH 7.6/0.1 mM EDTA was
`hybridized to denatured chromosomes at 430C for 20 hr (ap-
`proximately 20 ng of cDNA in 5 1.l per slide). Unhybridized
`cRNA was removed by mild RNase treatment and extensive
`washes in 0.30 M NaCl/0.03 M sodium citrate; the slides were
`then dipped in Ilford (Essex, United Kingdom) K2 dipping
`emulsion and exposed for 23-26 days. Previously photographed
`spreads were relocated and scored for silver grains occurring
`over chromosomes. The method of prehybridization banding
`allows unequivocal identification ofchromosomes, which is oth-
`erwise hampered by changes in morphology during denatura-
`tion, and also prevents observer bias in choice of spreads.
`Alternatively, HK101 was labeled by nick-translation with
`"I-labeled dCTP to a specific activity of about 5 x 108 cpm/
`/ig. This material was hybridized to metaphase chromosomes
`by using 50 ng of heat-denatured "2I-labeled HK101 per slide
`in 20 Al as above except for the addition of 10% dextran sulfate.
`Exposure time was 6 days.
`
`RESULTS
`Characteristics of Cloned Hybridization Probes. Two frag-
`ments ofhuman fetal liver DNA, cloned in A phage Charon 4A
`(8), were used for our experiments. Clone HK101 contains a
`single VK gene of subgroup 1 in 18.6 kilobases ofchromosomal
`DNA and has been fully described elsewhere (9, 10). Clone
`HK122 was isolated from the same library of fetal liver DNA
`as HK1O1 and contains a 10.5-kilobase-long insert including a
`single VK gene also ofsubgroup 1. These two VK genes are about
`87% homologous in their coding sequences (unpublished data).
`No highly repeated sequences were detected in either HK101
`
`Abbreviation: VK, K variable region.
`
`4957
`
`Genzyme Ex. 1014, pg 302
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`

`
`4958
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`Biochemistry: Malcolm et aL
`
`or HK122 by hybridization with nick-translated total human
`DNA (11). Cloned segments of flanking region sequences from
`HK101 exhibit a low copy number in human DNA when hy-
`bridized under conditions of high stringency (10). Clones
`HK101 and HK122 do not cross-hybridize to any significant
`degree except within the VK coding segments themselves.
`Hybridization of HKIOI Probe to Normal Stimulated Lym-
`phocyte Preparations. [3H]cRNA made from clone HK101 was
`hybridized to metaphase chromosome spreads from peripheral
`blood lymphocytes. A total of 106 prephotographed spreads
`from seven slides were analyzed for the presence ofsilver grains
`in the region of the individual chromosomes. Data from each
`slide were treated separately and from all slides as a whole. The
`grain distribution over the chromosomes was analyzed in two
`ways. First, the number ofgrains per unit length for each chro-
`
`Proc. Nad Acad. Sci. USA 79 (1982)
`mosome was calculated by dividing the total number of grains
`found on each chromosome by the percentage of the genome
`contained in that chromosome. This procedure was adopted to
`allow for the difference in background found on different length
`chromosomes. Chromosome 2 was more heavily labeled than
`any other chromosome, with a grains/unit length value of 93.5
`compared to a median of 68.4 and falling 2.75 SD away from
`the median. Because this method of calculation will produce a
`bias against the longer chromosomes (because the site-specific
`grains will be averaged over the whole chromosome), a second
`method of analysis was used.
`In the second method the number ofgrains expected on each
`chromosome, if all grains were randomly distributed along
`equal lengths of the genome, was calculated and the deviation
`from this random value was calculated (Fig. 1A). The excess
`
`A
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`
`CHROMOSOME NUMBER
`
`FIG. 1. In situ hybridization of HK101 [3H]cRNA to human chromosomes: distribution of grains over the genome. (A) Hybridization to lym-
`phocyte chromosomes from 106 karyotyped cells hybridized with HK101 [3H~cRNA (18 ng/5 1l; 1.67 x 10i dpm/,ug). Autoradiographs were exposed
`for 23 days. Expected values are derived from a proportional distribution of the 6,999 grains counted according to chromosome lengths. (B) Hy-
`bridization to fibroblast chromosomes from nine karyotyped cells hybridized with HK101 [3H]cRNA (21 ng/5 j.l; 1.6 x 108 dpm/,ug). Autoradiograph
`was exposed for 26 days. Expected values are calculated from a proportional distribution of the 1,525 grains counted.
`
`Genzyme Ex. 1014, pg 303
`
`

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`Biochemistry: Malcolm et al.
`grains on chromosome 2 are extremely striking (+ 194) and fall
`outside (3.94 SD from the mean) the range ofdeviation, positive
`and negative, found on the other chromosomes. In situ hybrid-
`ization of human peripheral blood lymphocytes was also carried
`out with iodinated HK101 labeled by nick-translation using
`"NI-labeled dCTP (12). It is clear from the karyotype analysis
`(Fig. 2) that both chromosomes 2 show a localization of silver
`grains whereas no other pairs ofchromosomes display this con-
`sistent labeling. In four other pairs of chromosome 2 (Fig. 2
`Lower), all members show labeling at the same approximate
`position. Therefore, the results obtained with [3H]cRNA and
`'25I-labeled DNA were identical in demonstrating hybridization
`of the human K light chain probes to chromosome 2 in lym-
`phocytes.
`Hybridization of HK1O1 to Metaphase Chromosomes Pre-
`pared from Fibroblast Cell Cultures and a Cell Line with a
`Balanced Translocation of Chromosome 2. Although we used
`phytohemagglutinin-stimulated lymphocytes [consisting mainly
`of T cells which generally show no rearrangement of their K
`genes (13)], we controlled against any possible rearrangement
`ofthe immunoglobulin genes in the stimulated lymphocytes by
`in situ hybridization of [3H]cRNA probes to cultured fibro-
`
`Proc. Natl. Acad. Sci. USA 79 (1982)
`
`4959
`
`blasts. Nine spreads from two slides were analyzed. The chro-
`mosomal distribution ofgrains, shown in Fig. 1B, indicates that
`chromosome 2 was significantly labeled, on the basis of grains
`per unit length, compared with the other chromosomes. There-
`fore, these data show that the K light chain probe hybridizes to
`chromosome 2 in fibroblasts as it did in peripheral blood lym-
`phocytes.
`As a further check on the specificity of hybridization and to
`define the position ofthe gene with respect to a cytogenetically
`defined break point, we carried out in situ hybridization of
`HK101 [3H]cRNA to chromosomes from a carrier of a balanced
`translocation between chromosomes 2 and 16 [46XXt (2; 16)
`(q13; q22)]. The break point in chromosome 2 is at q13-i.e.,
`in the long arm just below the centromere. Cells from this in-
`dividual contain one copy each of the normal chromosomes 2
`and 16, an abnormal 2/16 (2pter.2ql3; 16q22.16qter), and an
`abnormal 16/2 chromosome (16pter416q22; ql342qter). Re-
`sults ofan analysis ofgrain distribution obtained from 22 karyo-
`types are shown in Fig. 3. Both the normal 2 and the abnormal
`2/16 chromosomes show significant hybridization with the K
`probe, thus confirming the location of the VK gene to chro-
`mosome 2 and locating the position above the break point q13.
`
`K ARYO TV PE
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`FIG. 2. In situ hybridization of '251-labeled HK101 to human chromosomes. Chromosome spreads from blood lymphocytes were hybridized with
`HK101 '25I-labeled by nick-translation (12): 50 ng of heat-denatured '251-labeled HK101 DNA hybridized in 20 ,ul for 20 hr. Autoradiography was
`for 6 days. (Upper) Karyotype of lymphocyte chromosomes with associated grains after hybridization. (Lower) Pairs of chromosomes 2 taken from
`four other hybridized chromosome spreads.
`
`Genzyme Ex. 1014, pg 304
`
`

`
`4960
`
`Biochemistry: Malcolm et al.
`
`Proc. Nad Acad. Sci. USA 79 (1982)
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`CHROMOSOME NUMBER
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`FIG. 3. In situ hybridization of HK101 [3H]cRNA (18 ng/5 01; 1.67 x 10' dpm/,gg) to translocated (2; 16) chromosomes: distribution of grains
`over each chromosome from 22 karyotyped cells. Autoradiographs were exposed for 26 days. Expected grain values are derived from a proportional
`distribution of the total grain count according to chromosome length and number of chromosomes counted.
`
`similar analysis of the grains over the same number of chro-
`mosomes 7 or chromosomes 20 was carried out [both of which
`showed small positive values in grain distribution (Fig. 1)], a
`random distribution was observed, consistent with a nonspecific
`origin of these grains.
`The localization of the K genes near the centromere on the
`short arm ofchromosome 2 was confirmed with an independent
`K probe, HK122. Because this probe only cross-hybridizes with
`the VK coding region of HK101, it acts as a control against pos-
`sible in situ hybridization of the flanking (i.e., noncoding) re-
`gions which might be unrelated to the VK segment. With this
`VK probe, chromosome 2 was also found to show specific hy-
`bridization (data not shown) and the grain distribution along
`chromosome 2 (Fig. 4B) indicated that the hybridization was
`localized to the same section (2cen+1p12) as that observed with
`clone HK101.
`
`DISCUSSION
`The results described here were obtained by using two inde-
`pendently derived K chain probes. There are two main lines of
`evidence for the specificity of hybridization of these two cloned
`K probes. (i) Chromosome 2 is consistently more heavily labeled
`than any other chromosome in both lymphocytes and fibro-
`blasts. (ii) The excess grains are restricted to the short arm of
`chromosome 2 in both normal and translocated chromosomes
`and, furthermore, they are clustered in one region encompass-
`ing 10% of this chromosome.
`The data presented in this paper show that the human VK
`locus occurs on the short arm of chromosome 2 near the cen-
`tromere (2pl2). We assume that the K constant region gene is
`closely linked to the VK genes and therefore also is on chro-
`mosome 2. Thus, the three immunoglobulin loci in man have
`now been mapped to separate autosomes, K chains on chro-
`mosome 2 (this paper), A chains on chromosome 22 (1), and
`heavy chains on chromosome 14 (2, 3). A corollary of mapping
`human K light chain genes to chromosome 2 concerns the Kidd
`blood group genes. This blood group locus appears to be linked
`to the K chain genes (14), so this locus would also seem to reside
`on chromosome 2.
`The position of the K chain genes within chromosome 2 is of
`interest because it is in the same area (2pl2) as the specific break
`point in 8;2 translocations, which have been described in some
`Burkitt lymphomas (15, 16). Any possible relationship between
`this specific translocation and the transformation process is ob-
`
`Location of K Light Chain Genes Within Chromosome 2. In
`order to locate the K gene locus more precisely, each photo-
`graph (data from Fig. 1A) ofchromosome 2 (arm ratio q: p, 1.57)
`was divided into 10 units. Because chromosome 2 contains
`7.76% of the total genome length, each unit corresponds to
`0.776% of the genome or 2.3 x 107 base pairs. Fig. 4A shows
`the result ofdetermining the number ofgrains within each sec-
`tion of chromosome 2 from 106 karyotyped lymphocytes.
`Clearly, the grains were localized to the region ofchromosome
`2 closest to the centromere on the short arm, which would be
`defined as 2cen+p12 by classical banding techniques. When a
`
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`Distribution of grains along chromosome 2 after in situ
`FIG. 4.
`hybridization of [3H]cRNA transcribed from VK probes. p and q, short
`and long arms; vertical arrow, position of centromere; broken line,
`number of grains expected from a proportional distribution of the total
`number of grains on all chromosomes. (A) Distribution after hybrid-
`ization to HK101 (data from Fig. 1A). (B) Distribution after hybrid-
`ization to HK122 (data from 18 karyotypes).
`
`Genzyme Ex. 1014, pg 305
`
`

`
`Biochemistry: Malcolm et aL
`scure at present, but it has also been observed that other Burkitt
`lymphomas show a translocation of a similar piece of chromo-
`some 8 to either chromosome 14 (which carries the immuno-
`globulin heavy chain genes) or chromosome 22 (which carries
`the immunoglobulin A light chain genes). In connection with
`the translocations, it is interesting that the immunoglobulin
`genes undergo extensive DNA rearrangement during the ac-
`tivation of human lymphocytes: this can involve variable region
`rearrangement to joining segments near the constant regions
`or switching between heavy chain constant region genes. It is
`possible, therefore, that these rearrangements facilitate the
`specific translocations that occur between immunoglobulin-en-
`coding chromosomes and chromosome 8 in the Burkitt lym-
`phomas and that these specific chromosome translocations rep-
`resent the neoplastic transformation event.
`We thank Dr. M. Baker forproviding the cell line with the 2/16 trans-
`location. This work was supported by the Medical. Research Council;
`D.L.B. is a recipient of a Beit Memorial Fellowship.
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`Genzyme Ex. 1014, pg 306