Proc. Natl. Acad. Sci. USA Vol. 75, No. 9, pp 4494-4498, September 1978 Immunology
Assignment of genes for immunoglobulin K and heavy chains to chromosomes 6 and 12 in mouse (somatic cell hybrids/metacentric chromosomes)
HANS HENGARTNER, TOMMASO MEO, AND EDITH MULLER* Basel Institute for Immunology, Grenzacherstrasse 487, Postfach, 4005 Basel 5, Switzerland
Communicated by N. K. Jerne, July 3, 1975
ABSTRACT Using somatic cell hybrids from fusions of lymphocytes of two different mouse stocks with the myeloma cell line X63-Ag8, we have assigned genes for the immunoglobulin heavy and K-type light chains to chromosomes 12 and 6, respectively. The two mouse stocks exhibit karyotypes consisting of nine pairs of metacentric chromosomes as a result of centric fusions of acrocentric chromosomes in different combinations. In the hybrid cells these metacentric chromosomes can be distinguished from the acrocentric chromosomes of myeloma origin, permitting correlation of Ig chain expression with mitotic loss of individual metacentric chromosomes.
thine/aminopterin/thymidine (HAT) and secretes an IgG1 immunoglobulin (MOPC 21). The cells were grown in suspension in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 15% heat-inactivated horse serum, glutamine, antibiotics, and 50 ,uM 2-mercaptoethanol. Fusion and Cloning. The fusion method was basically as described by Kohler and Milstein (3), using 2000 hemagglutination units of ,3-propionelactone-inactivated Sendai virus to fuse 108 mouse spleen cells with 107 X63-Ag8 cells. The hybrids were selected by HAT medium (4) in 48 separate wells (Costar no. 3524) containing 2 ml of medium and 105 peritoneal exudate cells of BALB/c origin (5). Cell hybrids were cloned in soft agar as described (6). For some clones it was absolutely necessary to include 106 peritoneal exudate cells of BALB/c origin in the 10 ml of feeder layer to get any clones growing. Analysis of Products of Cell Hybrids. Supernatants of [14C]leucine-labeled cell cultures (6) were analyzed by sodium dodecyl sulfate (NaDodSO4)/10% polyacrylamide gel electrophoresis under reducing conditions (7) and by isoelectric focusing (8). Radioactive intracellular Ig was obtained by
Previous attempts to map the mouse Ig loci by using conventional linkage-testing stocks and recombinant inbred mouse lines have been unsuccessful, with the exception of the finding that markers of the K light chain variable region (VK) are linked to the Ly 2,3 region on chromosome 6 (1). We have approached this problem by using somatic cell hybrids from fusions of lymphocytes with a myeloma cell line in order to study the correlation of Ig chain expression with mitotic loss of individual chromosomes. The cytogenetic analysis was facilitated by taking advantage of two newly described feral mouse populations, CB and CD (2), which, as a result of extensive centric fusions (translocations), exhibit karyotypes consisting of nine metacentric chromosomes. In a hybrid cell these metacentric chromosomes can be easily distinguished from the homologous acrocentric chromosomes. With one exception, the metacentric chromosomes of CB and CD mice represent different combinations of the original murine acrocentric chromosomes. The involvement of the same chromosomes in different translocations allows us to assign genes to the original acrocentric chromosomes. Here we report results that identify one chromosome controlling the synthesis of K and another controlling the synthesis of ,u Ig chains. MATERIALS AND METHODS Animals and Cells. Spleen cells used for fusion, derived from CD and CB female mice (2), were kindly provided by E. Capanna, University of Rome. These mice were homozygous for 9 metacentric chromosomes derived by Robertsonian translocation (i.e., centric fusion) between 18 acrocentric autosomes. With one exception (16/9), the metacentric chromosomes of CB and CD mice represent different combinations of acrocentric chromosomes. In both stocks, the sex chromosomes and chromosome 19 remain acrocentric. For fusion we used the myeloma cell line X63-Ag8 (3), which is sensitive to hypoxan-
anti-immunoglobulin precipitation of a Nonidet P-40 lysate of cells that had been labeled for 2 hr in a medium containing [14C]leucine as described (9). The light chains of spleen cell origin were shown to be of the K type by Ouchterlony gel diffusion. Cytogenetic Analysis. Mitosis was arrested by adding Colcemid at a final concentration of 1 ,ug/ml for 2 hr. After centrifugation the cells were suspended in 75 mM KCl for 15 min and fixed with methanol/acetic acid (3:1 vol/vol), using three changes of this fixative. The metaphases were then spread on a cold wet slide, and the preparations were carefully dried by flaming. After 1 week of aging at room temperature, the chromosome preparations were stained by a trypsin-G banding technique as described by Buckland et al. (10). Karyograms of the cell hybrids of XCB-23-4-D5, XCB-23-5-D5, XCD-9-C5C2, and XCD-9-C5-A2 were made from photographs of randomly selected metaphases. For the other clones, chromosomes were counted by direct observation; karyograms of the metacentric chromosomes were prepared from selected metaphases containing the modal or higher number of metacentric chromosomes. Chromosome analysis was done "blind." That is, the person carrying out this analysis had no prior knowledge of the origin of the subclone or of the gel electrophoresis and isoelectric focusing data.
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. §1734 solely to indicate this fact.
Abbreviations: HAT, hypoxanthine/aminopterin/thymidine; HLGK, heavy (,) and light (K) chains of spleen cell and the gamma and kappa chains of myeloma origin; NaDodSO4, sodium dodecyl sulfate; VK, K light chain variable region. * Present address: Naturhistorisches Museum, Basel, Switzerland.
4494
Immunology: Hengartner et al.
C~
Proc. Natl. Acad. Sci. USA 75 (1978) XCB23
b
XCD9
random selection (20 clones)
4495
random selection (20 clones)
XCD-C5 (HLGK)
XCB23-4
recloning, 43 random clones
-B5
-
-C2
XCD9-C5:
-A2
-A4
-B2
\1XCD9-C5:
recloning, 108 random clones
XCB23-4-D5 (LGK) C
(LGK)
-C4
-Dl
-Cl
(HLGK)
recloning. 62 random clones
and XCB23-5 (HLGK)
XCB23-5-D5 (LGK) XCB1
(HLGK)
cloning, 49 random clones
-A2-16
-A2-8
-A4-5
-A4- 10
-CI-4
(LG)
(LG)
(LGK)
(LG)
(GK)
-Cl-1 1 (GK)
XCB1-41
XCB1-66
XCB1-33
XCB1-61
(HLGK)
(HLGK)
(LGK)
(LGK)
FIG. 1. Derivation of subclones from hybrids XCD9 (a), XCB23 (b), and XCB1 (c).
RESULTS
trophoresis and in some cases by isoelectric focusing. Those clones showing chain losses were saved for cytogenetic analysis. The relationship of the numerous subclones to each other is shown in Fig. 1. In order to describe lines secreting various sets of Ig chains, we use a special notation (3). The set of four possible secreted chains is designated as HLGK [i.e., the heavy (A) and light (K) chains of spleen cell origin and the gamma and kappa chains of myeloma origin]. Loss of production of one or more of these chains leads to deficient sets such as LGK, LG, or GK (e.g., LGK has lost the heavy chain of spleen origin).
The fusion of 108 CB spleen cells with 107 X63-Ag8 cells resulted in cell growth in 23 out of 48 wells (XCB hybrids). The fusion of 108 CD spleen cells with 107 X63-Ag8 cells produced 27 positive wells out of 48 (XCD hybrids). The low frequency of positive wells suggests that in many wells there was only a single clone growing. The supernatant of all positive wells were analyzed by NaDodSO4/polyacrylamide gel electrophoresis. All contained, in addition to myeloma X63-Ag8 IgG1 (K), a new heavy and a new light chain. In most cases, the new heavy chain was g; in each of the two hybridizations one 7y-producing line was observed. Growth of all hybrids was highly dependent on the presence of peritoneal exudate cells as feeders. On the basis of good cell growth and of the distinguishability of the Ig chains, hybrids from three wells were cloned and used for further analysis. The three lines (XCD9, XCB23, and XCB1) were recloned. At each stage of the cloning, culture supernatants were analyzed by NaDodSO4/polyacrylamide gel elec-
Derivation of Ig chain loss variants Six out of 43 randomly picked subelones of XC9-C5 (HLGK) lost the ,. chain so as to secrete the set of chains LGK. Analysis of 48 subelones of XCD9-C5-A2 and XCD9-C5-A4 (LGK type, Fig. la) showed that three clones had lost the myeloma K-chain expression. All 14 subclones of XCD9-C5-C1 showed loss of L-chain expression (Figs. la, 2, and 3).
a
b a_ _
_
_
_
....
_
YJ~ ~
_
_
K-E= N,
Assignment of genes for immunoglobulin K and heavy chains to chromosomes 6 and 12 in mouse (somatic cell hybrids/metacentric chromosomes)
HANS HENGARTNER, TOMMASO MEO, AND EDITH MULLER* Basel Institute for Immunology, Grenzacherstrasse 487, Postfach, 4005 Basel 5, Switzerland
Communicated by N. K. Jerne, July 3, 1975
ABSTRACT Using somatic cell hybrids from fusions of lymphocytes of two different mouse stocks with the myeloma cell line X63-Ag8, we have assigned genes for the immunoglobulin heavy and K-type light chains to chromosomes 12 and 6, respectively. The two mouse stocks exhibit karyotypes consisting of nine pairs of metacentric chromosomes as a result of centric fusions of acrocentric chromosomes in different combinations. In the hybrid cells these metacentric chromosomes can be distinguished from the acrocentric chromosomes of myeloma origin, permitting correlation of Ig chain expression with mitotic loss of individual metacentric chromosomes.
thine/aminopterin/thymidine (HAT) and secretes an IgG1 immunoglobulin (MOPC 21). The cells were grown in suspension in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 15% heat-inactivated horse serum, glutamine, antibiotics, and 50 ,uM 2-mercaptoethanol. Fusion and Cloning. The fusion method was basically as described by Kohler and Milstein (3), using 2000 hemagglutination units of ,3-propionelactone-inactivated Sendai virus to fuse 108 mouse spleen cells with 107 X63-Ag8 cells. The hybrids were selected by HAT medium (4) in 48 separate wells (Costar no. 3524) containing 2 ml of medium and 105 peritoneal exudate cells of BALB/c origin (5). Cell hybrids were cloned in soft agar as described (6). For some clones it was absolutely necessary to include 106 peritoneal exudate cells of BALB/c origin in the 10 ml of feeder layer to get any clones growing. Analysis of Products of Cell Hybrids. Supernatants of [14C]leucine-labeled cell cultures (6) were analyzed by sodium dodecyl sulfate (NaDodSO4)/10% polyacrylamide gel electrophoresis under reducing conditions (7) and by isoelectric focusing (8). Radioactive intracellular Ig was obtained by
Previous attempts to map the mouse Ig loci by using conventional linkage-testing stocks and recombinant inbred mouse lines have been unsuccessful, with the exception of the finding that markers of the K light chain variable region (VK) are linked to the Ly 2,3 region on chromosome 6 (1). We have approached this problem by using somatic cell hybrids from fusions of lymphocytes with a myeloma cell line in order to study the correlation of Ig chain expression with mitotic loss of individual chromosomes. The cytogenetic analysis was facilitated by taking advantage of two newly described feral mouse populations, CB and CD (2), which, as a result of extensive centric fusions (translocations), exhibit karyotypes consisting of nine metacentric chromosomes. In a hybrid cell these metacentric chromosomes can be easily distinguished from the homologous acrocentric chromosomes. With one exception, the metacentric chromosomes of CB and CD mice represent different combinations of the original murine acrocentric chromosomes. The involvement of the same chromosomes in different translocations allows us to assign genes to the original acrocentric chromosomes. Here we report results that identify one chromosome controlling the synthesis of K and another controlling the synthesis of ,u Ig chains. MATERIALS AND METHODS Animals and Cells. Spleen cells used for fusion, derived from CD and CB female mice (2), were kindly provided by E. Capanna, University of Rome. These mice were homozygous for 9 metacentric chromosomes derived by Robertsonian translocation (i.e., centric fusion) between 18 acrocentric autosomes. With one exception (16/9), the metacentric chromosomes of CB and CD mice represent different combinations of acrocentric chromosomes. In both stocks, the sex chromosomes and chromosome 19 remain acrocentric. For fusion we used the myeloma cell line X63-Ag8 (3), which is sensitive to hypoxan-
anti-immunoglobulin precipitation of a Nonidet P-40 lysate of cells that had been labeled for 2 hr in a medium containing [14C]leucine as described (9). The light chains of spleen cell origin were shown to be of the K type by Ouchterlony gel diffusion. Cytogenetic Analysis. Mitosis was arrested by adding Colcemid at a final concentration of 1 ,ug/ml for 2 hr. After centrifugation the cells were suspended in 75 mM KCl for 15 min and fixed with methanol/acetic acid (3:1 vol/vol), using three changes of this fixative. The metaphases were then spread on a cold wet slide, and the preparations were carefully dried by flaming. After 1 week of aging at room temperature, the chromosome preparations were stained by a trypsin-G banding technique as described by Buckland et al. (10). Karyograms of the cell hybrids of XCB-23-4-D5, XCB-23-5-D5, XCD-9-C5C2, and XCD-9-C5-A2 were made from photographs of randomly selected metaphases. For the other clones, chromosomes were counted by direct observation; karyograms of the metacentric chromosomes were prepared from selected metaphases containing the modal or higher number of metacentric chromosomes. Chromosome analysis was done "blind." That is, the person carrying out this analysis had no prior knowledge of the origin of the subclone or of the gel electrophoresis and isoelectric focusing data.
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. §1734 solely to indicate this fact.
Abbreviations: HAT, hypoxanthine/aminopterin/thymidine; HLGK, heavy (,) and light (K) chains of spleen cell and the gamma and kappa chains of myeloma origin; NaDodSO4, sodium dodecyl sulfate; VK, K light chain variable region. * Present address: Naturhistorisches Museum, Basel, Switzerland.
4494
Immunology: Hengartner et al.
C~
Proc. Natl. Acad. Sci. USA 75 (1978) XCB23
b
XCD9
random selection (20 clones)
4495
random selection (20 clones)
XCD-C5 (HLGK)
XCB23-4
recloning, 43 random clones
-B5
-
-C2
XCD9-C5:
-A2
-A4
-B2
\1XCD9-C5:
recloning, 108 random clones
XCB23-4-D5 (LGK) C
(LGK)
-C4
-Dl
-Cl
(HLGK)
recloning. 62 random clones
and XCB23-5 (HLGK)
XCB23-5-D5 (LGK) XCB1
(HLGK)
cloning, 49 random clones
-A2-16
-A2-8
-A4-5
-A4- 10
-CI-4
(LG)
(LG)
(LGK)
(LG)
(GK)
-Cl-1 1 (GK)
XCB1-41
XCB1-66
XCB1-33
XCB1-61
(HLGK)
(HLGK)
(LGK)
(LGK)
FIG. 1. Derivation of subclones from hybrids XCD9 (a), XCB23 (b), and XCB1 (c).
RESULTS
trophoresis and in some cases by isoelectric focusing. Those clones showing chain losses were saved for cytogenetic analysis. The relationship of the numerous subclones to each other is shown in Fig. 1. In order to describe lines secreting various sets of Ig chains, we use a special notation (3). The set of four possible secreted chains is designated as HLGK [i.e., the heavy (A) and light (K) chains of spleen cell origin and the gamma and kappa chains of myeloma origin]. Loss of production of one or more of these chains leads to deficient sets such as LGK, LG, or GK (e.g., LGK has lost the heavy chain of spleen origin).
The fusion of 108 CB spleen cells with 107 X63-Ag8 cells resulted in cell growth in 23 out of 48 wells (XCB hybrids). The fusion of 108 CD spleen cells with 107 X63-Ag8 cells produced 27 positive wells out of 48 (XCD hybrids). The low frequency of positive wells suggests that in many wells there was only a single clone growing. The supernatant of all positive wells were analyzed by NaDodSO4/polyacrylamide gel electrophoresis. All contained, in addition to myeloma X63-Ag8 IgG1 (K), a new heavy and a new light chain. In most cases, the new heavy chain was g; in each of the two hybridizations one 7y-producing line was observed. Growth of all hybrids was highly dependent on the presence of peritoneal exudate cells as feeders. On the basis of good cell growth and of the distinguishability of the Ig chains, hybrids from three wells were cloned and used for further analysis. The three lines (XCD9, XCB23, and XCB1) were recloned. At each stage of the cloning, culture supernatants were analyzed by NaDodSO4/polyacrylamide gel elec-
Derivation of Ig chain loss variants Six out of 43 randomly picked subelones of XC9-C5 (HLGK) lost the ,. chain so as to secrete the set of chains LGK. Analysis of 48 subelones of XCD9-C5-A2 and XCD9-C5-A4 (LGK type, Fig. la) showed that three clones had lost the myeloma K-chain expression. All 14 subclones of XCD9-C5-C1 showed loss of L-chain expression (Figs. la, 2, and 3).
a
b a_ _
_
_
_
....
_
YJ~ ~
_
_
K-E= N,
