type could be rescued by fusion to an antigenically distinct cell ... Blots were hybridized with the nick-translated probes shown in panel D: probe A (used in panel ...
The EMBO Journal vol.5 no.1 pp. 103- 111, 1986
A mutational hot-spot within f2-microglobulin gene
an
intron of the
Jane R.Parnes, Kurt C.Sizer, J.G.Seidmant, Valerie Stallings2 and Robert Hyman2 Department of Medicine, Division of Immunology, Stanford University, Stanford, CA 94305, 'Department of Genetics, Harvard Medical School, Boston, MA 02115, and 2Cancer Biology Laboratory, The Salk Institute, PO Box 85800, San Diego, CA 92138, USA Communicated by R.Flavell
02-Microglobulin is the smaller, relatively non-polymorphic chain of class I major histocompatibility complex proteins. We have previously described a mutant mouse cell line which had been selected for loss of the class I thymus leukemia (TL) antigen and had concomitantly lost surface expression of H-2k antigens. Expression of class I antigens on the cell surface was restored by fusion to an antigenically distinct mouse lymphoma line, and the defect in the mutant was shown to be the loss of a functional 02-microglobulin gene. We now describe three additional mutants with the same phenotype, all selected for loss of TL but after different types of mutagenesis. All of these mutants have genomic rearrangements resulting in the absence of a functional (2-microglobulin gene. These data provide strong evidence for the requirement of 032-microglobulin for cell surface expression of the heavy chain of class I major histocompatibility complex proteins. We further show that the defects in at least one (32-microglobulin gene in each mutant cell line map to the same small DNA segment within the first intron. The breakpoints of these mutations define a hypermutable site within the mouse 32-microglobulin gene. Key words: 02-microglobulin/hypermutable/MHC/B1 repeat/ transplantation antigen Introduction Class I major histocompatibility complex (MHC) proteins are heterodimeric structures expressed on the surface of most mammalian cells and serve as restriction elements for the recognition of virally infected, chemically altered or tumor cells by cytotoxic T lymphocytes (Klein, 1975, 1979; Snell et al., 1976; Vitetta and Capra, 1978; Ploegh et al., 1981; Zinkernagel and Doherty, 1980). In the mouse these proteins include the classical transplantation antigens (H-2K, D and L), the thymus leukemia antigen (TL), Qa antigens and perhaps other as yet unidentified, proteins which are encoded by a large multi-gene family on chromosome 17. The class I proteins consist of a polymorphic, MHC-encoded transmembrane glycoprotein of -45 kd which is tightly but non-covalently associated with the relatively invariant 12-kd polypeptide jO2-microglobulin. 032-Microglobulin, which is not encoded within the MHC, does not itself span the plasma membrane, but rather is found on the cell surface only by virtue of its association with class I heavy chains (reviewed in Klein, 1975; Snell et al., 1976; Vitetta and Capra, 1978; Ploegh et al., 1981). Data from both human and mouse systems have suggested that © IRL Press Limited, Oxford, England
mouse
class I MHC molecules are not expressed on the cell surface in the absence of (32-microglobulin, despite the fact that both chains have their own signal peptides (Dobberstein et al., 1979; Steinmetz et al., 1981; Parnes and Seidman, 1982). The human Daudi cell line (established from a Burkitt lymphoma) has a single defective (32-microglobulin gene with a point mutation which creates an early termination codon, thereby preventing translation of 02-microglobulin mRNA (Rosa et al., 1983; de Preval and Mach, 1983). This line is surface HLA-A and -B negative, but HLA heavy chains are present within the cell, and fusions with (32-microglobulin-positive human or mouse cells result in rescue of surface expression of the Daudi HLA-A and -B antigens (Arce-Gomez et al., 1977; Ploegh et al., 1979). In the mouse system we found that a mutant cell line selected in vitro for loss of expression of TL after chemical mutagenesis had lost surface expression not only of this class I antigen, but also of H-2 antigens (Hyman and Stallings, 1976). As in the case of the Daudi cell, surface expression of class I antigens of the parental type could be rescued by fusion to an antigenically distinct cell Table I. Cell surface expression of class I antigens by mutants and somatic cell hybrids Cell line
Relative DNA content/cell
Relative antigen expression/cell TL
Experiment 1 RI. I
H-2k
ND 1.00 1.07
1.0
