GENOMICS 1,277-2%-l (1987)

SHORT COMMUNICATION

Localization of the Murine X5 Gene on Chromosome 16 AKIRA KUDO,* DIMITRINA PRAvrcHEvA,t NOBUO SAKAGUCHI, *J FRANK H. RuDcxE,t AND FRITZ MELCHERS*

*Base/ Institute for Immunology, Base/, Switzerland, and Wepartment of Biology, Yale University, New Haven, Connecticut

Received May 7, 1987; revised September 11, 1987

The chromosomal location of the murine X6 gene was analyzed by Southern hybridization using re- striction enzyme-digested DNA from a panel of 16 mouse x hamster somatic cell hybrids. Sequences ho- mologous with those of X, DNA were detected in DNA of 6 hybrids. In all 5 hybrids X6 was contained in re- striction fragments of equal sizes, the lengths of which indicated that the germline coniiguration of X6 with three exons and the restriction sites expected from its genomic structure were present. Southern hybridization with the murine X1 gene as a probe de- tected the same 5 hybrids as positive. The only mouse chromosome present on all of the positive hybrids, and absent from negative ones, was number 16. We conclude that b is situated on the same chromosome as X1, i.e., on the murine chromosome 16. Q 1987

Academic Pra, Inc.

X,, originally called pZ183, is a single-copy gene which is selectively expressed during B-cell differen- tiation in pre-B cells, but not in mature cells of the B lineage, nor in any other eukaryotic cell tested so far (Sakaguchi et al., 1986). The primary transcript of the gene encoded in three exons within a stretch of 4 kb of genomic DNA is spliced to form a 1.2-kb mRNA cod- ing for a presumed protein of 179 amino acids (Saka- guchi and Melchers, 1986; Kudo et al., 1987). Parts of the second exon show strong sequence homologies to those encoding the J segments of X light (L) chains of immunoglobulins, while the third exon has similarly strong homologies to sequences encoding the constant (C) region domains of XL chain, a finding which has prompted the name X5 for the pZ183 gene. The 5’-10- cated first exon shows no detectable strong homolo- gies to any known nucleotide sequences.

The gene segments encoding the variable (V), J, and C parts of XL chains have been localized on chro- mosome 16 of the mouse (D’Eustachio et aZ., 1981). This was accomplished through the use of DNA prep- arations of 11 mouse X hamster hybrids with different assortments of mouse and hamster chromosomes and

1 Present address: Department of Immunology, Saga Medical School, Nabeshima, Saga, Japan.

analysis by Southern hybridization with radiolabeled XL chain gene probes. Here we report the chromo- somal localization of the X6 gene using DNAs from the mouse X hamster hybrids. By controlling the string- ency of hybridization, we have learned to distinguish X6 gene sequences from XL chain gene sequences (Sa- kaguchi and Melchers, 1986). We identify the genomic form of X5 by the characteristic length of the restric- tion enzyme-digested fragments detected with radio- labeled Xs probes containing the 5’ and the 3’ parts of the gene.

Somatic cell hybrids were formed between Chinese hamster cell line E36 and peritoneal macrophages from A/HeJ mice, fibroblasts from Balb/c fetal mice, cells from a tissue culture-adapted subline of the MethA murine fibrosarcoma, or cells from the murine cell line CTllc or CllDH3. The mouse parental cells for some of the hybrids (clones 2 and 4 in Table 1) were derived from the in vitro-maintained Balb/c sar- coma line CMS4.

Hybrid cell lines were grown in monolayer culture in antibiotic-free Dulbecco’s modified essential me- dium, high-glucose formation (Grand Island Biologi- cal Co., Grand Island, NY), supplemented with 10% fetal bovine serum and HAT (lo-’ M hypoxanthine, 1.6 X 10m5 M aminopterine, 1.6 X lo-’ M thymidine). Murine pre-B lymphoma cells 7OZ/3 obtained from Dr. Christopher Paige (Base1 Institute for Immunol- ogy) were grown in Dulbecco’s modified medium con- taining 2 mM glutamine, 10% fetal calf serum (Gibco). DNAs were extracted using a standard method. Each lo-pg DNA sample was digested with Hind111 and EcoRI restriction enzymes (P. H. Stehelin 8z Cie AG, Basel). Digested DNAs were fractionated by agarose gels and transferred to Zeta probe membranes (Bio- Rad) using 0.4 M NaOH as transfer buffer (Reed and Mann, 1985). After transfer, filters were rinsed twice in 2X SSC, dried at 80°C under vacuum, and hybrid- ized with oligolabeled probes as described previously (Feinberg and Vogelstein, 1984). Hybridization was performed with 1.5X SSPE, 1.0% SDS, 0.5% nonfat powdered milk, 0.5 mg/ml salmon sperm DNA. The

277 o&w7543187 $3.00 Copyright Q 1987 by Academic Press, Inc.

All rights of reproduction in any form reserved.

278 SHORT COMMUNICATION

4

14.5kb

W

I 2 3 4 5 6 7 8 9 10 11 12 13 141516 17 18 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18

-9.6 kb

1 2 4 5 6 8 9 11 12 13 15 16 1718 702 I 2 4 5 6 8 911 121315 161718702

FIG. 1. DNAs from mouse X hamster hybrids (numbers l-4,6, and 3-17 in Table 1) from the Balb/c sarcoma line CMS4 (number 7), from the hamster cell line E36 (numbera 5 and la), and from the mouse pre-B cell line 70213 (70213) were digested with HindI (a) and EcoRI (b). After electrophoresis DNAs were transferred to a nylon membrane filter. Hybridization was carried out using the 3’ DNA of the X, genomic gene (a, left), the 5’ DNA of the X6 genomic gene (b, left), and CX1 DNA (a, b, right) as probes.

5’-side DNA of the X6 genomic gene (7pB12 PstI-KpnI fragment, 760 bp), the 3’-side DNA of the X5 genomic gene (7pB12 EcoRI fragment, 360 bp) (Kudo et al., 1987), and the CX, DNA (Bothwell et al., 1981) (MOPC-104E XhoI-Hind111 fragment, 460 bp) were used as radiolabeled probes.

After hybridization, filters were washed in 0.2~ SSC, 0.1% SDS at 65°C for 30 min and subjected to autoradiography at -70°C using intensifying screens.

Zeta probe membranes were used successively after regeneration in 0.1X SSC, 0.1 SDS at 95°C.

DNA extracted from mouse X hamster hybrids, from the mouse Balb/c sarcoma line CMS4, and from the Chinese hamster cell line E36 (D’Eustachio et al., 1981) was digested with restriction enzyme Hind111 or EcoRI, and the resulting DNA fragments were ana- lyzed for the presence of X5 coding sequences and XL chain coding sequences, as well as for the size of frag- ments containing such coding sequences, by Southern blot analyses.

It is evident from the results in Fig. 1 that DNA from mouse X hamster hybrids contains Xs and XL chain coding sequences. It is also apparent that DNA from the same hybrids contains 5’- and 3’-X5 as well as hL chain coding sequences. The lengths of fragments hybridizing to 5’- and to 3’-X6 DNA sequences are the same (data not shown). They, as well as those mea- sured by detection with the XL chain probe, are those expected from the known restriction maps of Xs and

XIL chain genes (Blomberg and Tonegawa, 1982; Miller et al., 1981). In HindIII-digested DNA, Xs probes detect a 14.5-kb-long fragment, and the XIL chain probe a 9.6-kb-long fragment. In EcoRI-di- gested DNA, X5 probes detect a 10.2-kb-long frag- ment, and the XIL chain probe an 8.5-kb-long frag- ment. Furthermore, mouse chromosome 16 was the only one present in all of the positive hybrids and absent from all of the negative ones, allowing the gene to be assigned to this chromosome (Table 1).

Fragments crosshybridizing with hamster DNA were detected with a X6 probe under mild washing conditions. We conclude from these results that one copy of the X5 gene is present within the murine ge- nome on chromosome 16, in linkage with the genes for XL chains. fn situ hybridization of X, on murine chro- mosomes and analyses by chromosomal walking, to be done in future experiments, should tell us how close this linkage is.

Our results add one further complexity to the al- ready complex genetic organization of the XL chain locus, which in mice has been shown to be differently complex in different strains (Scott et al., 1982) and which in man appears even more complex than in mice (Taub et al., 1983). They prove again how power- ful, i.e., how sensitive and rapid, the method of as- signment of the location of a gene to a given chromo- some can be with the aid of the mouse X hamster hybrids.

SHORT COMMUNICATION 279

TABLE 1

Mouse x Hamster Hybrid Cell Lines Tested for the Presence of the Murine A6 Gene

Clone number” Mouse chromosome number

1 14,* 15 2 5,6,* 8,’ 12, 13, 14, 16, 17, 19, X 3 1, 2,3, 4, 6, 7,8,9, 10, 12, 13, 15, 17, 18, 19, X 4 2, 5,6,* 8,* 10, 12, 13, 14, 15, 16, 17, 18, 19, X 5 Chinese hamster cell line E36 6 X, 6 7 Balb/c sarcoma line CMS4 8 2, 7, 12, 15, 19 9 x, 12

10 1, 2, 3, 4, 7, 9, 12, 15, 19, x* 11 1, 2, 3, 4,5, 6, 7,8,** 9, lo,** 12, 13, 15, 16, 17, 19, X 12 1, 2, 3, 6, 7, 8, 9, 12, 13, 15, 17, 19, X 13 1, 2, 3, 4, 7, 8, 9, 10, 12, 13, 15, 16, 17, 18, 19, X 14 17,18, plus an additional unidentified

chromosomal segment 15 X, 16 16 x, 14 17 x, 1

a These clones (1-17) were from the same cell lines as the sources of DNA (l-17) in Fig. 1.

* Rearranged chromosome. ** Chromosomes are present in 15% of the cells.

The product of the X6 gene is an 18-kDa protein which we have tentatively identified on Western blots by peptide-specific antibodies. Since no conforma- tion-specific antibodies now exist for the A6 protein, the site of expression of the protein in or on cells of the B lineage is not yet known. Through its penulti- mate cysteine at the carboxy-terminal end, it could form homodimers (As/As) or heterodimers. H chains could be partners in this heterodimer formation as soon as they are expressed in pre-B cells, and this may allow H chains to appear on the surface of pre-B cells,

as previous experiments have indicated (Melchers et al., 1977). Such a surface deposition of expressed H chains together with A5 may function in recognition processes which could govern pre-B cell development.

ACKNOWLEDGMENTS

We thank Dr. Steven Bauer for critical reading of our manu- script. The Base1 Institute for Immunology was founded and is supported by F. Hoffmann-La Roche, Ltd. Co., Baeel, Switzerland. D.P. and F.H.R. were supported in parts by grants.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

REFERENCES

BLOMBERG, B., AND TONEGAWA, S. (1982). Proc. Natl. Acad. Sci. USA 79: 530-533.

BOTHWELL, A. L. M., PASKIND, M., SCHWARTZ, R. C., SON- NENSHEIN, G. E., GEFIXR, M. L., AND BALTIMORE, D. (1981). Nature (London) 290: 65-67.

D’EUSTACHIO, P., BOTHWELL, A. L. M., TAKARO, T. K., BAL- TIMORE, D., AND RUDDLE, F. H. (1981). J. Exp. Med. 163: 793-800. FEINBERG, A. P., AND VOGELSTEIN, B. (1984). Anal. Biochem. 137: 266-267. KUDO, A., SAKAGUCHI, N., AND MELCHERS, F. (1987). EMEO J. 6: 103-107. MELCHERS, F., ANDER~~ON, J., AND PHILLIPS, R. A. (1977). Cold Spring Harbor Symp. Quunt. Biol. 41: 147-158. MILLER, J., BOTHWELL, A., AND STORB, U. (1981). Proc. N&l. Acad. Sci. USA 78: 3829-3833.

REED, K. C., AND MANN, D. A. (1985). Nucleic Acids Bee. 13: 7201-7221. SAKAGUCHI, N., BERGER, C. H., AND MELCHERS, F. (1988). EMBO J. 5: 2139-2147. SAKAGUCHI, N., AND MELCHERS, F. (1988). Nature Ohdon) 324: 579-582. SCOTT, C. L., MUSHINSKI, J. F., HO~PI, K., WEIGERT, M., AND POUR, M. (1982). Nature (London) 300: 757-760.

TAIJB, R. A., HOLLIS, G. F., HIETER, P. A., KORSMEYER, S., WALDMANN, T. A., AND LEDER, P. (1983). Nature (London) 304: 172-174.