The mouse immune interferon receptor gene is located on chromosome 10.

When mouse L cells are incubated with 32P-labeled recombinant murine immune interferon ( [32P]Mu-IFN-gamma) and subsequently cross-linked with disuccinimidyl suberate, a major complex with an apparent molecular mass of 95,000-125,000 daltons can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The complex was not formed when the binding was performed in the presence of excess unlabeled Mu-IFN-gamma or when Chinese hamster ovary cells were used. This complex therefore represents the Mu-IFN-gamma receptor (or its interferon-binding subunit). The chromosomal location of the Mu-IFN-gamma receptor (or the binding subunit of the receptor) gene, termed Ifgr, was identified by performing the binding and cross-linking reactions on a series of mouse-hamster somatic cell hybrids with different subsets of mouse chromosomes. The presence of mouse chromosome 10 was shown to be necessary and sufficient for the formation of the cross-linked complex. Thus, the gene coding for the binding subunit of the Mu-IFN-gamma receptor was localized to mouse chromosome 10. The presence of this chromosome in the hybrid cells was not sufficient, however, to confer antiviral resistance to the hybrids when they were treated with Mu-IFN-gamma and challenged with encephalomyocarditis virus.

The interferons (IFNs)' are a family of proteins which display antiviral, antiproliferative, and i m m u n o r e~l a t o~ activities. To date, three classes of interferons have been isolated and characterized (reviewed in Refs. [1][2][3][4][5]. These are the leukocyte (IFN-a), fibroblast (IFN-B), and immune (IFN- 7) interferons. In order to elicit a response, the interferons must first bind to specific cell surface receptors (6-9). The preparation of radiolabeled interferons has stimulated many studies of the interferon receptors (reviewed in Refs. 9 and 10). So far, it seems that IFN-a and IFN-8 share a common receptor which is distinct from the IFN-7 receptor.
Most of the IFN receptor studies have utilized "51-labeled interferons as ligands. Recently, recombinant human (11,lZ) and murine (13)   have been radiolabeled to high specific radioactivities with [y3'P]ATP and the catalytic subunit of * 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. Section 1734 solely to indicate this fact.
$ Present address: Dept. of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635.
.___ bovine heart muscle CAMP-dependent protein kinase. The 32P-labeled recombinant human (Hu) IFN-7 was initially used to measure equilibrium and kinetic parameters for the interaction between the ['*P]Hu-IFN-y and its receptor (12). Subsequently, human-mouse and human-hamster somatic cell hybrids were cross-linked with [32P]Hu-lFN-y in order to localize the gene for the Hu-IFN-7 receptor to human chromosome segment 6q (14).
In the present study, mouse-hams~r somatic cell hybrids were cross-linked with [32P]Mu-IFN-7 to determine the chromosome location of the Mu-IFN-7 receptor gene.

EXPERIMENTAL PROCEDURES
Interferon, Radiolabeling, and Interferon Assay--Recombinant murine IFN-? with a specific activity of 1.02 X lo7 units/mg was isolated from ~s c h e~h~ coli as described previously (15) and kindly supplied by Dr. H. M. Shepard (Genentech). The protein concentration of the interferon was determined by amino acid analysis.
Mu-IFN--y was phosphorylated according to procedures developed for the phosphorylation of Hu-IFN-7. Briefly, 0.9 pg of Mu-IFN-y was incubated at 37 "C for 15 min with 0.5 mCi [y-32P]ATP (>5000 Ci/mmol; Amersham Corp.) and 5 units of the catalytic subunit of CAMP-dependent protein kinase from beef heart (Sigma) in a 3 0 -~1 reaction volume containing 20 mM Tris.HC1, pH 7.4, 100 mM NaCI, 12 mM MgCI,, and 1 mM dithiothreitol. The reaction was terminated by cooling on ice and adding 0.4 ml of 5 mg/ml bovine serum aibumin in 10 mM sodium pyrophosphate (NaPPi), pH 6.7. After extensive dialysis at 4 "C against 10 mM NaPPi, pH 6.7, aliquots were stored in liquid nitrogen.
The antiviral activity of the Mu-IFN-7 was routinely measured on mouse L cells by a cytopathic effect inhibition assay (16) with encephalomyocarditis virus. The antiviral effect of MU-IFN-7 on the mouse-hamster hybrid cells was measured in the same manner. Somatic Cell Hybrkfs-Somatic cell hybrids were made by fusing cells of the Chinese hamster line E36 and peritoneal or spleen cells of BALBIC, A/J, or NFSAkv-2 congenic mice. Th& production and characterization of these hybrids has been described previously (17-
Binding and Covalent Cross-linking of p2P]Mu-IFN-r to Cells-" Cells were rinsed twice with 5 ml of Dulbecco's phosphate-buffered saline lacking Mg2+ and Ca2+ and then with 2.5 ml of trypsin-EDTA solution (Ix in phosphate-buffered saline; Gibco). Trypsinization was performed at 37 "C until the cells were released from the tissue culture flask. The cells were resuspended in the appropriate medium and counted. After centrifugation at 500 X g at 4 "C for 10 min, the cells were resuspended a t 5 X lo6 cetls/ml. The binding and cross-linking reactions were done as described previously (14) with slight modification. About lo6 cpm of [32P]Mu-IFN--y (-516-2,110 Ci/mmol), with or without 1 pg of unlabeled Mu-

Mouse Immune
Interferon Receptor Gene 5813 IFN-7 as a competitor, were added to 0.3 ml of cells in a 1.5-ml polypropylene tube. Binding was allowed to proceed at 22 "C for 60 min with gentle resuspension every 15 min. The reactions were chilled on ice, and the cells were pelleted for 20 s at 15,000 X g in a Brinkman microfuge. The cells were washed once with and resuspended in 500 pl of cold phosphate-buffered saline. A 5 0 -m~ solution of disuccinimidyl suberate (Pierce Chemical Co.), freshly prepared in dimethylsulfoxide, was added to a final concentration of 0.5 mM. After 20 min on ice, the cross-linking reaction was quenched by the addition of 10 pl of 1 M Tris-HC1, pH 7.5. After 5 min on ice, the cells were pelleted as described above and extracted with 100 gl of 0.5% Triton X-100 in phosphate-buffered saline containing 5 mM EDTA a t 4 "C for 20 min. Insoluble material was sedimented at 15,000 X g a t 4 "C for 10 min. The supernatants were then analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis on 1.5-mm thick slab gels containing 8% acrylamide (20). Gels were dried under vacuum and subjected to autoradiography at -70 "C with Kodak XRP-1 film and Cronex Lightning Plus intensifying screens (DuPont).

RESULTS
The y which was bound to the receptor but was not cross-linked. When mouse-hamster somatic cell hybrids were examined in this manner, the 110,000-dalton band characteristic of the mouse L cells was noted with several of the hybrids (e.g. hybrid 2A5E5 in Fig. 1). In all hybrid cells where this band was present, it was completely eliminated when the binding reaction contained excess unlabeled IFN-y.
The presence of the major cross-linked complex was correlated with the mouse chromosome complements of the mouse-hamster hybrids ( Table I) Formation of this complex is completely inhibited by excess unlabeled Mu-IFN-y (Fig. 1). If this represents a 1:l complex between Mu-IFN-y and its receptor, then the binding subunit of the murine IFN-y receptor has a molecular mass of 80,000-110,000 daltons as was recently reported elsewhere (13,21). Although several hybrid lines expressed the binding subunit of the Mu-IFN-7 receptor, the cells were not protected from encephalomyocarditis virus killing by prior treatment with Mu-IFN-7. This failure to establish an antiviral state was also observed in human-mouse and human-hamster hybrid cells which expressed the Hu-IFN-y receptor-binding subunit (14). Several explanations for this phenomenon may be offered. First, the number of IFN-y receptors expressed on the surface of the hybrid cells may not be sufficient to elicit an antiviral effect when the cells are treated with IFN-y. In the case of the Hu-IFN-y receptor, hybrid cells seemed to express the binding subunit at one-fifth to one-half the level expressed by human A431 cells (14). However, in the present case, two of the positive hybrid lines seemed to express the murine receptor-binding subunit at the same level as mouse L cells (data not shown). Another possibility is that the binding subunit of the IFN-7 receptor is not fully functional in somatic cell hybrids either because of some modification in the protein itself (e.g. post-translational modifications affecting glycosylation or proteolytic cleavage) or because the binding subunit fails to couple efficiently with other host cell-encoded factors involved in the antiviral response. Perhaps this coupling has a high degree of species specificity, and the mouse binding subunit cannot function in a hamster cell.
It should be noted that the gene coding for Mu-IFN-y has also been localized to chromosome 10 (22). This contrasts with results for Hu-IFN-y where the Hu-IFN-y gene resides on chromosome 12 (23), and the gene coding for the binding subunit of the receptor is on the long arm of chromosome 6 (14).
A comparison of the human and mouse gene maps indicates that human chromosome 6 has some homology to mouse chromosome 10. The human and murine homologs for the avian myeloblastosis virus (myb) proto-oncogene have also been localized to the same chromosomes as the genes encoding the binding subunit of the IFN-y receptor (24,25). All of the mouse-hamster hybrids used in the present study were tested for the presence of the murine myb homolog and perfect correlation with mouse chromosome 10 and Ifgr was observed (data not shown). Since a comparison of all the other known genes on these two chromosomes indicates extensive gene rearrangement, it is likely that the myb proto-oncogene and the IFN-y receptor gene are genetically closely linked. This interesting observation may provide a means for the isolation of the IFN-y receptor gene and the eventual structural characterization of the receptor itself.
A c k~~~~~n~-W e thank Kathy Cairoli and Louise Brenner for assistance in the preparation of this manuscript.