Proc. Natl. Acad. Sci. USA Vol. 75, No. 6, pp. 2883-2887, June 1978

Immunology

Chromosomal locations of mouse immunoglobulin genes (chromosomes/molecular hybridization/mouse-human hybrid cells)

OSCAR VALBUENA*, KENNETH B. MARCU*, CARLO M. CROCEt, KAY HUEBNERt, MARTIN WEIGERT*, AND ROBERT P. PERRY* * The Institute for Cancer Research, Fox Chase Center, and t The Wistar Institute, Philadelphia, Pennsylvania Contributed by Robert P. Perry, February 24, 1978

The chromosomal locations of the structural ABSTRACT genes coding for the constant portions of mouse heavy (H) and light chain immunoglobulins were studied by molecular hybridization techniques. Complementary DNA probes containing the constant-region sequences of K and AI light chain and a, 72b, and u heavy chain mRNAs were annealed to a large excess of DNA from a series of eight mouse-human hybrid cell lines that are deficient for various mouse chromosomes. The lines were scored as positive when a high proportion of a probe annealed and negative when an insignificant proportion annealed. Some lines were clearly negative for H and A and clearly positive for K. Others were positive or intermediate for A, psitive for K and negative for H. Still others, including a line that was selected for the absence of the mouse X chromosome, were positive for all immunoglobulin species. These results demonstrate that the CA, C,,, and CH genes are located on different autosomes in the mouse. In contrast, the three heavy-chain families exhibited consistently uniform hybridization results, suggesting that the genes for C., Cy, and CA are located on the same chromosome. A comparison of karyotypic data with hybridization data has limited the possible locations of the Ig genes to only a few chromosomes.

In the mouse, as in other mammals, the chromosomal locations of the genes coding for antibody heavy and light chain structures have not been clearly identified. From genetic studies it has been inferred that these genes are located on different autosomes (1), and it has been suggested on the basis of a variable region marker that the mouse K locus may be on chromosome 6 (2). However, owing to the lack of a demonstrable linkage between heavy or light chain constant-region genes and other loci and to the possible existence of regulatory loci affecting the expression of the immunoglobulin structural genes (3, 4), a more definitive study is called for. We have therefore attempted to solve this problem by use of the molecular hybridization techniques. In these experiments, cDNA probes representing the constant regions of mouse K and AI light chain mRNAs and a, 72h and M heavy chain mRNAs were annealed to a large excess of DNA from a series of mouse-human hybrid cell lines that are deficient for various mouse chromosomes (5). A similar approach using hybrids segregating human chromosomes has shown that the structural gene for human a-globin is on chromosome 16 (6). The results of our studies demonstrate clearly that genes CA, CK, and CH are autosomal and not linked. Moreover, a comparison of karyotypic and hybridization data has limited the possible locations of these genes to only a few chromosomes. Interestingly, our results indicate that the chromosome containing the CK gene is probably not chromosome 6. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

MATERIALS AND METHODS Cell Lines. The methods of formation of hybrid cells and the conditions of cultivation of the various cell lines used in this study have been described (5). For simplicity we have replaced the original complete codes for the hybrid cell lines with Roman numerals. The correspondence is: I = 55-14 F1, C129; II = 55-14 F1, C119; III = 55-14 F7, C154; IV =55-14 F1, C126; V = 55-91 F2 C14; VI = 55-54 F2 (d); VII = 55-14 F7 (early passage); VIII = 55-84 F8; and IX = 55-84 F8 6TG. The hybrid lines 55-14, 55-54, and 55-91 were derived from three independent fusions of HT-1080-6TG human fibrosarcoma cells with BALB/c mouse peritoneal macrophages; the hybrid line 55-84 was derived from a fusion of HT-1080-6TG cells with OTT6050 mouse teratocarcinoma cells (5). The F numbers refer to different flasks in which hybrids were formed following the initial fusion event, and Cl numbers refer to different clonal isolates from the initial hybrid cell cultures. Line VII is an early sample of the 55-14 F7 culture that was kept frozen until its cultivation for the present series of experiments. Karyotype analyses were carried out by using the trypsin-Giemsa banding technique

(5).

DNAcDNA Annealing Experiments. The cDNA probes were synthesized from highly purified heavy and light chain mRNAs isolated from various mouse plasmacytomas. The tumors used as sources of the particular mRNAs were: AI, H2020 (obtained from Melvin Cohn, Salk Institute); k, M321 (obtained from Michael Potter, National Institutes of Health); a, H2020; 7Y2b MPC 11 (obtained as a cell culture line from Matthew Scharff, Einstein College of Medicine);,u, PCNZB 3741 (produced at the Institute for Cancer Research). All have been maintained as solid subcutaneous tumors in either BALB/c or (BALB/c X NZB) F1 progeny. The methods for mRNA isolation and purification by dT-cellulose chromatography, sucrose

gradient sedimentation, and 98% (vol/vol) formamide/polyacrylamide gel electrophoresis are detailed elsewhere (7). Synthesis of cDNA probes (7) was carried out in 13-,l reaction volumes containing 100 ,M unlabeled deoxynucleotide triphosphates, avian myoblastosis virus reverse transcriptase, and 30 uM 32P-labeled dGTP or dCTP (200-350 Ci/mmol). The ratio of transcriptase to mRNA template was 3.3 and 6.6 units/pmol for heavy and light chain mRNAs, respectively. Purification of the cDNAs by hybridization to their templates and subsequent S1 nuclease treatment (7) was used in some cases. Cellular DNAs were isolated with sodium dodecyl sulfate/chloroform/phenol (8) from frozen pellets of hybrid cells, parental human fibrosarcoma cells, 10- to 14-day NZB mouse Abbreviations: Crt, initial concentration of total RNA (moles of nucleotide/liter) X time (seconds); Cot, initial concentration of total DNA (moles of deoxynucleotide/liter) X time (seconds).

2883

2884

Immunology: Valbuena et al. 1.

5 0 i

Proc. Nati. Acad. Sci. USA 75 (1978)

1 0.5

-ID

>=

jj

(D

iiJC-iiA -2t®®

1.0

..........._ S _ _ Ti}

data have a statistical uncertainty due to the limited number of metaphase figures analyzed for each cell line. This uncertainty is assessed by assigning to each value for chromosome l~~~~~~~~~~~~~~.0 frequency a 90% one-sided confidence limit that is based on a sample size equivalent to two copies of a particular chromosome for each metaphase group scored. Another source of error in these analyses is the ambiguity in identifying individual mouse when less than a full complement is present per chromosomes ~~~~~~~~~~~~~~.0 0.5 cell. On average there were 1.6 unidentifiable chromosomes 05 or chromosome fragments per cell for lines I-VL This represents about 15% of the chromosomes scored in these cells. To help remove such ambiguity, the identification of chromosomes was confirmed by isozyme analyses in those cases for which suitable isozyme markers are available-i.e., for chromosomes 1, 2, 3, 4, 5, 7, 8, 9, 11, 12, 14, 15,17, 18, 19, and X (5).

+ _ 1.5 i

_

1.5 0 0

o

0.5

1.5

2.0

1.5 1.0

05

2 4 6 8 102141618X

2 4 6 81012141618 X

Mouse chromosome number

FIG. 1. Results of the mouse karyotype analyses of hybrid cell lines. For individual mouse chromosomes the frequency (average number per cell) is based on an analysis of the following numbers of metaphase figures for lines I-VIII: 23, 27, 15, 14, 18, 11, 10, and 12. The vertical bars represent the 90% one-sided confidence limits calculated with a cumulative binomial distribution computer (10). The average number of unidentifiable chromosome fragments per cell were 2.2, 2, 0.6, 3, 1.5, and 0.5 for lines I-VI. The dashed lines at 0.5 and 0.25 chromosome per cell represent estimated limits for detection of positive and negative hybridization according to the calibration curve -of Fig. 4.

embryos, and human placenta (kindly supplied by Anna O'Connell). The conditions of mRNA-cDNA and DNA-cDNA annealing and the analysis of hybrids are described elsewhere (7). Other details are given in the legends of figures and tables.

RESULTS

Chromosomal composition of hybrid cell lines The hybrid cells used in these experiments contain an average of 76-97 human chromosomes (9) and 9-29 mouse chromosomes. One line designated VIII, which contained at least one copy of each mouse chromosome in almost every cell, served as a control. Another line, designated IX, was derived from line VIII by selection in media containing 6-thioguanine at 30 This line lacks the mouse X chromosome and does not Ag/ml. express two X-linked isozymes, hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The results of mouse karyotype analysis of eight hybrid cell lines used as sources of DNA are diagrammed in Fig. 1. For most of the cell lines, the karyotype analyses were performed on the same cultures used for DNA extraction or on cultures sampled within one to five doublings of those used for DNA extraction. In these cases, we may presume that the DNA is derived from a distribution of chromosomes that is faithfully represented by the recorded karyotype. For cell lines III and VI, the chromosome analysis was made several passages prior to the source culture for DNA isolation and, therefore, for these lines the karyotype might be less representative. The karyotype

Properties of cDNA probes Because, for our purposes, it was desirable to minimize hybridization of the cDNA probes to human Ig sequences, and because constant-region amino acid sequences have less interspecies homology than do variable-region sequences (11), we chose conditions for the reverse transcriptase reaction that would yield probes representing predominately the 3' untranslated and constant regions of the mRNA molecules. Using a protocol with an adequate ratio of enzyme to template and with relatively low concentrations (30 MM) of labeled deoxynucleotide triphosphates, we produced probes of about 50-600 nucleotides in length, as determined by sedimentation in alkaline sucrose gradients. These probes had high specific activities (200-550 cpm/pg) which enabled us to carry out hybridization reactions at very high ratios of cellular DNA to cDNA without expending large amounts of cellular DNA (tide infra).

The purity of the mRNAs used as templates was judged by several criteria to be about 90% and 60% for light and heavy chain mRNAs, respectively (7). In our initial experiments the cDNAs were purified by hybridization to their respective mRNAs at a Crt of 5 X 10-2 mol liter'1 sec and sequential treatments with S1 nuclease and alkali. This purification step was later omitted for light chain probes, but it was routinely used for the heavy chain probes, resulting in the elimination of a relatively unreactive fraction amounting to about 30-40%. The general quality of the probes was evaluated by kinetic complexity analysis using an excess of the various template mRNAs as drivers (Fig. 2). A high purity is indicated by the substantial proportions of probe annealing at relatively low Crt values. In contrast, negligible amounts of hybrid were formed when the probes were annealed with mRNAs of the other Ig classes, thus attesting to the specificity of each type of probe. Under our annealing conditions the XI probe should hybridize effectively with XI, sequences (12). For annealing of the probes to cellular DNA, we first determined conditions that would maximize the amount of hybrid formed with mouse embryo DNA. The highest practical ratio of DNA to cDNA was used so that we would have maximum sensitivity of detecting Ig genes in the mouse-human hybrid cells. At a DNA/cDNA ratio of 4 X 107 and a Cot value of 5 X 104, 75-90% of a cDNA probe can be drawn into S1 nucleaseresistant hybrid by mouse DNA (Fig. 3). In the absence of DNA about 15% of the cDNA self-annealed into S1 nuclease-resistant duplexes after similar incubations, and when the probes were incubated with human DNA from either HT1080 fibrosarcoma cells or human placenta, the amount of hybrid formed was only 15-30% (i.e., 0-15% above the self-annealing background). Thus, there is a spread of approximately 60% between the

Immunology: Valbuena et al.

Proc. Natl. Acad. Sci. USA 75 (1978)

2885

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0

C

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N >0

n 40 I

-°50 Ahi

.5

10-3

0o-2 Crt, mol Iiter-'

10-1 sec

FIG. 2. Specificity of cDNA probes. The cDNA probes containing light chain and heavy chain sequences were annealed at 68° to their respective mRNA templates or to other Ig mRNAs, and the amount of hybrid resistant to digestion by S1 nuclease was measured. Each point is derived from a 50-Ml reaction mixture containing 10 ng of mRNA, 0.42 ng of [3H]cDNA (-2000 cpm) (7), 0.6 M NaCl, 40 mM Tris-HCl (pH 7.2), and 2 mM EDTA. The abscissa is plotted as Crt multiplied by a factor of 5 to normalize to standard concentrations of monovalent cation. The data are corrected for probe self-annealing which amounted to about 2% after the longest incubation periods.

of annealing observed with mouse and with human DNA, which readily allows the detection of mouse Ig genes in the presence of human DNA. In order to estimate the relative content of mouse Ig genes in hybrid cells, a calibration curve was constructed as follows. Mouse embryo and human fibrosarcoma DNAs were mixed in various proportions and annealed with cDNA probes at a Cot of 5 X 104 and an overall DNA/cDNA ratio of 4 X 107. A "relative hybridization" parameter, R, was defined as 100 (X - h)/(m - h) in which X, m, and h are the percentages of probe hybridized by the mixture, pure mouse, and pure human DNA, respectively. The values of R were then plotted against the fraction of mouse DNA in the mixture (Fig. 4). These fractions are experimentally equivalent to the fractional dilution of the chromosomes bearing a particular Ig gene in the hybrid cells. Thus, for example, in a hybrid cell containing one copy of the mouse chromosome carrying the CA gene, 19 other mouse chromosomes, and 80 human chromosomes, 1 of 100 chromosomes would bear the CX gene, whereas in a diploid mouse cell, amount

.°0 _ ~~~~~~~~~~~ N

FIG. 3.

c 25

-}

Annealing of cDNAs to genomic DNA. Mouse embryo or

68° for various periods tEach point is derived from a 40-Ml reaction mixture containing 400 fisg of genomic DNA, 10 pg of P32P~cDNA, and the salts human fibrosarcoma DNA was annealed at

of time to K-cDNA.

and buffer used in the experiments of Fig. 2. For the other species of cDNA the percentage hybridizing with mouse embryo DNA at the highest Cot value varied from 75 to 90%. The data points are not corrected for probe self-annealing which amounted to about 15% at

the longest incubation times. DNA/cDNA =t4 x 10. 0, mouse DNA; 0, A, human DNA with a, s, and AlcDNA probes, respectively.

0

I I 0.4 0.6 0.8 Fraction of mouse DNA

0.2

1.0

FIG. 4. Relative hybridization of Ig cDNA probes with mixtures of mouse and human genomic DNAs. Mouse embryo DNA and human fibrosarcoma DNA were mixed in various proportions and annealed with [32PJcDNA as in Fig. 3. The relative hybridization, R (see text), is plotted against the proportion of mouse DNA in the mixture. For this series of experiments, the hybridization with pure mouse and pure human DNA was 80 + 10% and 20 ± 5%, respectively. DNA/cDNA =4 X 107. *, K cDNA; A, a cDNA; v, A cDNA.

2 of 40 chromosomes bear the CA gene. The fractional dilution would be (1/iOO) + (2/40) = 0.2, assuming that the DNA contents of the resident human and mouse genomes are roughly equivalent. The data of Fig. 4 indicate that we would detect significant hybridization (R > 25%) with our X cDNA probe, even at a fractional dilution of 0.05, which is equivalent to an average of 0.25 chromosome bearing CA genes per hybrid cell. Thus, an R value 0.5 CA-bearing chromosome per cell. For 0.25-0.5 CA chromosome per cell, an R value of 25-50% might be expected. Hybridization of cDNAs to the DNA of mouse-human

hybrid cells

The cDNAs were annealed at a Cot of 6 X 104 to the genomic DNA of different hybrid cell lines and, for each experimental series, to mouse embryo DNA and HT1080 human fibrosarcoma DNA. The ratio of genomic DNA to cDNA was 4 X 107. With one exception, the relative hybridizations were either clearly positive (>58%) or clearly negative (0.5 and discordant with frequencies S0.25. Conversely, we considered negative hybridizations to be concordant with frequencies 0.5. If the 90% confidence limits of a particular chromosome frequency fell entirely within a positive or negative zone (cf. Fig. 1) the correlation was considered to be fully concordant or discordant, whereas if one of the limits fell into the intermediate zone, the correlation was ranked as a half concordance or discordance. Intermediate region frequencies with 90% limits in both positive and negative zones were omitted from the correlation analysis, except for the annealing of the X probe with the DNA of cell line III, for which the hybridization value was, itself, intermediate. In order to diminish reliance on the karyotype designation of any one cell line we required more than one full discordancy or two half-discordancies before eliminating a chromosome as a possible bearer of the gene locus. Using this basis for correlation we can eliminate with reasonable certainty all chromosomes except 9, 13, 14, 15, and 16

Table 2. Hybridization of k cDNA to genomic DNAs at varying DNA/cDNA ratios

DNA/cDNA X 10-6 40 20 10 8 5 4 2.5

M

% of probe hybridized I III IV V

76 75 63 64

73 66 54

57 51 51

71 73

62 58 54.

-

-

54

-

54 49

46

43

-

-

32

38

39 28

% of maximum*

H

M

I

III:

IV

V

24 25 31

100 98 79 80 64 56 -

100 86 61 45

100 82 82

100 104

20

-

34

19

-

100 89 79

-

64

-

58

-

-

-

16

42

9 -

39 26

All hybridizations were carried out to a Cot of 6 X 104. M and H represent mouse embryo and HT1080 human fibrosarcoma DNAs, respectively. The Roman numerals refer to cell line numbers. * Calculated by subtracting a 15% self-annealing correction from mouse values or a 24% correction from hybrid values for the average human plus self-annealing background, and then expressing the result as a percentage of the value for the highest DNA/cDNA ratio. -, Not tested.

Proc. Natl. Acad. Sci. USA 75 (1978)

Immunology: Valbuena et al. Table 3. Probable and possible chromosomal locations of mouse constant region genes Gene

Chromosomes More probable Possible*

CH CMI

15

9, 13, 14, 16 9, 13, 16 C, 1,2,3,or8 4,5,7,9,10,17,18,19 * Gene assignments must be mutually exclusive based on the results of Table 1 which indicate that the CH, Cxj, and C, genes reside on different chromosomes.

duction of 5-fold or more in order to elicit a similar decrease. The results of such experiments with four hybrid lines (Table 2) indicate that a relatively large diminution in DNA/cDNA ratio is required in order to cause a marked decrease in the proportion of K probe hybridized. This suggests that the CK_ bearing chromosome is one of the most abundant chromosomes common to these four lines. According to the karyotypes of Fig. 1, this would be chromosome 1, 2, 3, or 8. DISCUSSION The results of the foregoing experiments show clearly that the CH, C,, and CAI genes of the mouse must reside on different chromosomes. For example, cell line I DNA anneals efficiently with CK DNA but does not significantly hybridize with either the Cx or the CH cDNAs. Cell line VI DNA anneals with both CX and C, cDNAs but not CH cDNAs. This finding is in agreement with genetic data in other species (1) but, due to lack of allelic markers for Cx and C, in the mouse, a genetic test of C-region linkage has not yet been carried out. Our result is supported by certain genetic analyses that may be indirect tests for CH and CL linkage. These include the findings that CH allotypes and certain VK markers or CH allotypes and the locus controlling CXI level (which may be linked to or within the X structural gene) segregate independently (2). Our formal proof that the genes coding for the C regions of the different antibody subunits are on separate chromosomes has important implications for the regulation of antibody synthesis. Induction of antibody synthesis must involve gene activation on at least two different chromosomes, one carrying a light chain gene and another carrying the heavy chain genes. Furthermore, allelic exclusion must occur at sites on each of the three different autosomal chromosomes carrying the CK, Cx, and CH genes. Models that postulate allelic exclusion to be a consequence of the inactivation of an entire chromosome now become unlikely, and it is reasonable to assume that the mechanism is restricted to the antibody genes. In all of the lines studied, hybridization with the cDNAs of three different CH regions, CA, Ca, and C-y2b, were concordant. This result is not surprising in view of the extensive genetic data in man and mouse showing close linkage of CH genes. In mouse, for example, no recombinants between heavy chain loci have been observed in over 3000 backcross mice examined (1, 13). A comparison of the hybridization and karyotypic data permit us to exclude certain chromosomes as sites for C-region genes and thereby identify particular chromosomes as possible sites of constant-region genes (Table 3). At least one gene on each mouse chromosome has previously been tested for linkage for CH and Pre, a gene 10-12 centimorgans from CH, and so

2887

far, close linkage has not been observed (13-15). These genetic data do not exclude CH's being on any of these chromosomes but suggest that the CH genes are not located near the tested markers or that they are located near a recombinational "hot spot." Although there are no genetic linkage data for CK and Cx, the VK genes have been presumed to be on chromosome 6 on the basis of a uniform segregation of VK markers with Ly 3 (no recombinants between Ly 3 and the VK markers have been observed) (2). If the VK locus on chromosome 6 does indeed code for V. structural genes, our results would mean that VK and CK are unlinked. If VK and CK are linked, as is the case for the V and C genes of the heavy chain locus (1, 16), our finding that CK is not on chromosome 6 provides evidence that the expression of certain VK genes is regulated by the Ly locus or a locus closely linked to Ly. Although we have not unequivocally localized C region genes to individual chromosomes, we have narrowed the possibilities to a relatively few candidates. Given this limited number of possibilities, it now becomes feasible to use certain experimental protocols to test these candidates, and thus one may anticipate a precise localization of these genes in the near future. We thank Irene Kieba and Patricia McBreen for excellent technical assistance. This work was supported by grants from the National Science Foundation, National Institutes of Health, American Cancer Society, and National Foundation-March of Dimes and an appropriation from the Commonwealth of Pennsylvania. C.M.C. is a recipient of a Research Career Development Award, National Cancer Institute; K.B.M. is a National Institutes of Health postdoctoral fellow; 0. V. acknowledges a fellowship from the Venezuelan government. 1. Mage, R., Lieberman, R., Potter, M. & Terry, W. D. (1973) in The Antigens, ed. Sela, M. (Academic, New York), pp. 299-376. 2. Gottlieb, P. D. (1974) J. Exp. Med. 140, 1432-1437. 3. Bosma, M. J. & Bosma, G. C. (1974) J. Exp. Med. 139, 512527. 4. Strosberg, A. D. (1977) Immunogenetics 4,499-513. 5. Croce, C. M. (1976) Proc. Natl. Acad. Sci. USA 73, 32483252. 6. Deisseroth, A., Nienhuis, A., Turner, P., Velez, W. F., Anderson, F., Ruddle, F., Lawrence, J., Creagan, R. & Kucherlapati, R. (1977) Cell 12,205-218. 7. Marcu, K. B., Valbuena, 0. & Perry, R. P. Biochemistry, in press. 8. Varmus, H. E., Heasley, S. & Bishop, J. M. (1970) J. Virol. 14, 895-903. 9. Miller, 0. J., Miller, D. A., Vaithilingim, G. D., Tantravati, R. & Croce, C. M. (1976) Proc. Nati. Acad. Sci. USA 73, 45314535. 10. Muench, J. 0. (1964) A cummulative binomial distribution computer, Monograph SC-R-64-1348 (Sandia Corporation, Al-

buquerque, NM). 11. Dayhoff, M. 0. (1972) in Atlas of Protein Sequence and Structure (National Biomedical Research Foundation Washington, D.C.), Vol. 5. 12. Honjo, T;, Packman, S., Swan, D. & Leder, P. (1976) Biochemistry

15,2780-2785. 13. Herzenberg, L. A., McDevitt, H. 0. & Herzenberg, L. A. (1968) Annu. Rev. Genet. 2,209-244. 14. Taylor, B. A., Bailey, D. W., Cherry, M., Riblet, R. & Weigert, M. (1975) Nature 256,644-646. 15. Taylor, B. A. & Eicher, E. M. (1977) Immunogenetics 5, 511. 16. Blomberg, B., Geckeler, W. R. & Weigert, M. (1972), Science 177,

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Chromosomal locations of mouse immunoglobulin genes.

Proc. Natl. Acad. Sci. USA Vol. 75, No. 6, pp. 2883-2887, June 1978 Immunology Chromosomal locations of mouse immunoglobulin genes (chromosomes/mole...
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