Transfectants (TK+) were selected and maintained in medium containing hypoxanthine, aminopterin, and thymidine (10). Metabolic Labeling of Transfectants.
Proc. Nati. Acad. Sci. USA Vol. 82, pp. 5920-5924, September 1985 Immunology
Structure and expression of genes encoding murine Qa-2 class I antigens (major histocompatibility complex)
ANDREW L. MELLOR*, JANE ANTONIOU*, AND PETER J. ROBINSONt *Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, United Kingdom; and tDepartment of Immunology, German Cancer Centre, Deutsches Krebsforschungszentrum, Postfach 101949, 6900 Heidelberg 1, Federal Republic of Germany
Communicated by Peter Medawar, May 20, 1985
ABSTRACT DNA structural analysis of the Qa region in two BALB/c mouse substrains with different Qa-2 phenotypes reveals that a deletion of DNA has occurred in BALB/cBy (Qa-2-) mice relative to BALB/c (Qa-2+) mice. We propose that this deletion arises from unequal crossing-over and recombination between adjacent BALB/c class I genes and results in the generation of a hybrid class I gene in BALB/cBy mice. Furthermore, we suggest that this is a direct cause of the change in Qa-2 phenotype. Further support for this model was obtained from transfection experiments in which cloned genes from the equivalent part of the Qa region in C57BL/10 mice were introduced into L cells. Four C57BL/10 genes, arranged in two almost identical pairs, encode polypeptides that are precipitated from lysates of transfectants with anti-Qa-2/3 antiserum. Although loss of one pair of these genes in BALB/c mice has no qualitative effect on Qa-2 phenotype, the loss of both pairs of genes via gene fusion leads to the loss of the Qa-2+ phenotype in BALB/cBy mice.
into mouse L cells and tested transfectants for the expression of Qa-2-reactive polypeptides. MATERIALS AND METHODS Inbred Mouse Strains. C57BL/10 (hereafter referred to as B10) and BALB/c mice of Qa-2+ phenotype were obtained from stocks maintained at the Clinical Research Centre. BALB/cBy mice were a recent gift (1983) of D. Bailey (The Jackson Laboratory) (7). Cells and Antisera. Mouse L cells (TK-, thymidine kinaseminus) were obtained from F. Grosveld (National Institute for Medical Research, Mill Hill, UK) and grown in minimal essential medium alpha supplemented with 5% fetal calf serum (GIBCO). Antiserum B6.K1 anti-B6 (anti-Qa-2/3) was a gift from L. Flaherty (New York State Dept. of Health,
Albany, NY) (8). Preparation and Analysis of DNA. Mouse DNA was prepared from spleen and digested with restriction enzymes (Anglian Biotechnology, Colchester, UK) according to the supplier's specifications. Cosmids containing cloned B10 (3) and BALB/c (2) class I genes have been described previously. Digested mouse spleen DNA (5 ,ug) or cosmid DNA (1 ,ug) was electrophoresed in a 0.7% agarose gel, transferred to nitrocellulose (Schleicher & Schuell) filters (9), and hybridized to nick-translated 32P-labeled DNA probes (108 cpm/,ug) as described (3). Deoxynucleoside [a-32P]triphosphates for nick-translation were obtained from Amersham. Transfection of L Cells. L-cells (TK-) were transfected with cloned cosmid DNA (3) by the Ca3(PO4)2/DNA-mediated gene-transfer method (10). Transfectants (TK+) were selected and maintained in medium containing hypoxanthine, aminopterin, and thymidine (10). Metabolic Labeling of Transfectants. L-cell transfectants were grown in 75-cm2 flasks (GIBCO) until semiconfluent. Cells were removed mechanically and washed three times in medium without methionine. Radiolabeling (108 cells per ml) was for 15 min at 37°C, using [35S]methionine (Amersham) at 1 mCi/ml (1 Ci = 37 GBq). Labeling was arrested in an ice-water bath, and the cells were pelleted and then solubilized in Triton X-100-containing buffer as described (11). Immunoprecipitation (11) and two-dimensional gel electrophoresis (12) are described elsewhere. Gels were processed with dimethyl sulfoxide/2,5-diphenyloxazole and fluorographed for 8 weeks at -70°C.
The class I gene family within the mouse major histocompatibility complex consists of about 30 highly homologous genes (1-3) which are distributed among four genetic subregions, H-2K, H-2D,L; Qa and TL (4, 5). Genes located in the H-2K and H-2D,L subregions encode H-2 class I cell surface polypeptides which function as guiding molecules for cytotoxic T cells during cell-mediated immune responses. However, most of the class I genes map to the Qa and TL subregions and, although serologically defined Qa and TLregion gene products are related biochemically to the H-2K, -D, and -L antigens, there are several differences distinguishing them (5). Unlike the H-2K, -D, and -L antigens, Qa and TL antigens (0) are expressed mainly in cells of haematopoietic lineage, (ii) exhibit only low levels of genetic polymorphism, and (iii) are not recognized as guiding molecules by cytotoxic T cells. Consequently, the biological function(s) of the Qa and TL class I antigens is unknown. The Qa subregion contains about 10 class I genes in both C57BL/10 (B10) (3) and BALB/c (2) mice and controls the expression of the Qa-2 antigen and possibly other class I polypeptides (5). The Qa-2 antigen exhibits little, if any, structural polymorphism (6), and mouse inbred strains type serologically as either Qa-2+ (e.g., B10 and most BALB/c strains) or Qa-2- (e.g., AKR; see ref. 7). We have used two complementary experimental approaches to identify the class I gene(s) that encode the Qa-2 antigen. In the first approach, we have made use of cloned, low copy number, B10 DNA probes (3) to compare the organization of Qa region class I genes in the BALB/c (Qa-2+) mouse and in the BALB/cBy (Qa-2-) substrain. In our second approach, we have introduced cloned class I genes from the Qa region of B10 mice
RESULTS DNA Probes Used for Structural Analysis of the Qa Region. Fig. 1 shows an Xba I restriction enzyme-site map for part of the B10 Qa region, as well as the published BamHI map (3). B10 DNA probes 1-3, derived from the B10 H-2K region and which cross-hybridize to DNA in the Q6-Q9 gene segment
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: B10, C57BL/10; kb, kilobase(s). 5920
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Immunology: Mellor et al. 190 1
Proc. Natl. Acad. Sci. USA 82 (1985) 210
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Balb/cBy FIG. 1. Map of part of the murine Qa region between map positions 190 and 270 kilobases (kb) (top scale) of the B10 Qa region (3). Four B10 class I genes (Q6 to Q9) and Xba I (X) and BamHI (B) restriction enzyme sites are indicated (+ indicates additional, unmapped Xba I sites). Identical Xba I and BamHI restriction enzyme sites are present in cloned BALB/c DNA spanning the Q6-Q7 region (2). Probes 1, 2, and 3 (see text) hybridize to B10 DNA as shown. Bottom two lines show the structure of equivalent regions in BALB/c (3) and BALB/cBy mice (see Results). Arrows indicate gene orientation (5- )3'). Broken lines represent DNA deletions in BALB/c substrains relative to B10.
(3), were used in Southern blot (9) hybridization analysis of the corresponding region in BALB/cBy. DNA probe 1 (the 5' flanking K-region probe described in ref. 3) hybridizes to DNA flanking all four genes shown in Fig. 1, as well as to DNA flanking two more Qa region genes (Q5 and QJO, not shown) and the two H-2K-region class I genes (K) and H-2Kb) in B10 mice (3). DNA probes 2 and 3 are subclones of the H-2Kb gene and hybridize to the 5' and 3' ends, respectively, of all four genes shown in Fig. 1 as well as to all known complete class I genes in the B10 mouse genome. Previous comparisons (3) of the Q6-Q9 B10 DNA segment and equivalent regions in the genome of Qa-2+ BALB/c mice (2) indicate that this DNA segment is almost identical in both strains, except for a deletion of DNA between genes Q8 and Q9 in BALB/c (see Fig. 1). This deletion has resulted in the generation of a fusion gene in BALB/c mice that has a
b
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13
characteristics of both the B10 Q8 (5' end) and the B10 Q9 (3' end) genes. The Xba I restriction-site map between genes Q6 and Q7 is identical in both Qa-2+ strains as shown by Southern blot analysis of BALB/c cosmids that span this DNA segment (unpublished results). Structural Analysis of the Qa Region in BALB/cBy Mice. Hybridization of DNA probes 1-3 to Southern blots of restriction enzyme digests of B10 and BALB/cJ (both Qa-2+) and of BALB/cBy (Qa-2-) spleen DNA reveals structural differences in the Qa region of all three mouse strains. Initially, we hybridized probe 1 to BamHI digests of spleen DNA (Fig. 2a). This probe hybridizes to several BamHI fragments including a 0.64-kb BamHI fragment and a 0.68-kb BamHI fragment that is underrepresented in BALB/cJ spleen DNA with respect to B10 spleen DNA. The 0.68-kb BamHI fragment is absent in digests of BALB/cBy spleen
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5922
Immunology: Mellor et al.
DNA. Furthermore, no additional bands are present in BALB/cBy compared to BALB/cJ DNA digests, implying that DNA in BALB/cJ has been deleted in BALB/cBy. In B10 mice, the 0.68-kb BamHI fragment is present in three copies (3), corresponding to the 5' flanking regions of the KJ, Q7, and Q9 genes (Fig. 1). In BALB/c (Qa-2+) mice the H-2K region does not contribute a 0.68-kb BamHI fragment (unpublished results) and the fusion of genes Q8 and Q9 eliminates the DNA segment 5' of Q9 which would otherwise contribute a 0.68-kb BamHI fragment. Thus, only one copy of this fragment is present in BALB/c mice and is situated in the 5' flanking region of Q7. Analysis of BALB/c cosmids derived from this DNA segment confirms the presence of such a fragment at this location in BALB/c Qa-2' mice (data not shown). Loss of this band in BALB/cBy strongly suggests that the deletion involves DNA between the BALB/c equivalents of the Q6 and Q7 genes. In contrast, no change to the 0.64-kb BamHI fragment flanking the B10 Q6, Q8, and H-2Kb genes (3) is detected in the blots hybridized with probe 1. Similar results reported recently by Flaherty et al. (13) prompted us to investigate the extent of this DNA deletion more precisely by using class I gene probes derived from the H-2Kb gene (probes 2 and 3). Of several different restriction enzyme digests of BALB/cJ and BALB/cBy spleen DNA, those using Xba I were most informative when probed with DNA probes 2 and 3 (5' and 3' gene probes, respectively). Each probe detects a single difference between BALB/c substrains. Thus, use of probe 2 (Fig. 2b) reveals the absence of a weakly hybridizing 3.5-kb Xba I fragment in BALB/cBy DNA, whereas probe 3 reveals the absence of a 3.7-kb Xba I fragment. A 3.7-kb Xba I fragment, which also hybridizes to a 3' class I gene probe and which is present only in DNA derived from Qa-2+ mouse strains, was described by Flaherty et al. (13). Similarly, use ofprobe 1 reveals the loss of a 3.5-kb and a weakly hybridizing 4.8-kb Xba I fragment in BALB/cBy DNA (Fig. 2b). As with probes 2 and 3, no additional hybridizing bands are detected in BALB/cBy DNA digests, implying that most, if not all, of these Xba I fragments are deleted in BALB/cBy DNA. Identification of the DNA Segment Deleted in BALB/cBy. Because probes 2 and 3 hybridize to class I gene sequences, it is likely that the deletion in BALB/cBy involves one or more class I genes. Furthermore, this gene(s) must have a 5' end that is present on a 3.5-kb Xba I fragment and a 3' end that is present on a 3.7-kb Xba I fragment. To identify genes with these hybridization characteristics, Xba I digests of all 10 cloned B10 Qa region class I genes (3) were prepared and hybridized to probes 2 and 3 (Fig. 3 a and b, respectively). Digests of three genes (QS, Q7, and Q9) contain Xba I fragments of about 3.5 kb that hybridize to probe 2 (5' probe), whereas digests of three different genes (Q2, Q6, and Q8) contain Xba I fragments of about 3.7 kb that hybridize to probe 3 (3' probe). Experiments in which cloned B10 DNA and BALB/cBy spleen DNA were mixed prior to digestion with Xba I and hybridization to probe 2 or 3 reveal that genes Q7 and Q9 or Q6 and Q8, respectively, give rise to bands that correspond to the 3.5-kb or 3.7-kb bands absent in BALB/cBy DNA (data not shown). Moreover, a parallel analysis of overlapping cosmid clones spanning the BALB/c Qa region reveals that only two genes, equivalent to Q6 and Q7, give rise to 3.7-kb (3') and 3.5-kb (5') Xba I fragments, respectively. For example, cosmid 38.1, which contains two genes equivalent to the Q6 and Q7 genes (2, 3) gives rise to a 7-kb and a 3.7-kb Xba I fragment revealed by hybridization to probe 3 (Fig. 3c). Since clone 27.1, which contains only Q7 (2), gives rise to the 7-kb Xba I fragment, we conclude that Q6 gives rise to the 3.7-kb Xba I fragment. Taken together, these restriction mapping and hybridization data are consistent with the interpretation that a deletion
Proc. Natl. Acad. Sci. USA 82 (1985) Probe 2 07 08 09 02 04 Q1 03 05 06 07 Q8 09 010010
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FIG. 3. Autoradiograms of Southern blots of Xba I-digested, cloned B10 DNA (3) hybridized with probes 2 (a) and 3 (b) or cloned BALB/c DNA (2) hybridized to probe 3 (c). B10 class I gene(s) present in each DNA clone is indicated above each lane. In c, names of BALB/c clones containing Qa region class I genes are given above each lane. Detailed maps of these clones and of the genes present in each clone are given in ref. 2. Clones 27.1, 38.1 and 46.1 contain BALB/c genes Q7, Q6 plus Q7, and Q8/9, respectively (2, 3). Arrowheads indicate bands referred to in the text, and arrows indicate positions of marker DNA fragments.
of DNA has occurred between the Q6 and Q7 genes in the BALB/cBy substrain (Fig. 1). The deletion includes all three Xba I fragments (3.7 kb, 4.8 kb, and 3.5 kb), which span the
Immunology: Mellor et al.
Proc. Natl. Acad. Sci. USA 82 (1985)
region between these two class I genes, and probably results in the loss of most, if not all, of this 12-kb DNA segment since no extra bands are present in the BALB/cBy digests. Since most of the DNA in the 3.7-kb Xba I fragment lies within the Q6 gene, it is likely that the deletion removes part of the coding sequence of this gene. However, inspection of the detailed restriction enzyme-site map of the BALB/c Q7 gene (the 27.1 gene in ref. 14) reveals that the 3.5-kb Xba I fragment ends about 0.4 kb 5' of the first exon of this gene. This fragment is only detected because probe 2 contains DNA homologous to the 5' flanking regions of the Q6-Q9 genes (Fig. 1). We have attempted to ascertain whether the deletion extends into the 1.8-kb Xba I fragment that contains the 5' exons of the Q7 gene (ref. 14 and Fig. 1). Unfortunately, this proved to be difficult due to the fact that most genes in the Qa region give rise to a fragment of this size (Figs. 1 and 3a). This problem persists even when other restriction enzymes are used in attempts to discriminate between Qa region class I genes. Expression of Qa-2/3-Reactive Polypeptides in Transfected L Cells. All Qa region class I genes (QI to Q10, inclusive) cloned from the B10 mouse strain (3) were introduced into L cells (TK-) by use of the Ca3(PO4)2/DNA-mediated genetransfer method (10). Uncloned populations of TK' (hypoxanthine/aminopterin/thymidine-resistant) transfectant cells were radiolabeled with [35S]methionine, and lysates of these cells were immunoprecipitated with an anti-Qa-2/3 antiserum (8). Labeled polypeptides were analyzed by twodimensional gel electrophoresis (12). L cells transfected with B10 genes Q6, Q7, Q8, or Q9 contain Qa-2/3 reactive polypeptides (Fig. 4 a-d), whereas no such polypeptides precipitated specifically by anti-Qa-2/3 antiserum are detectable in extracts prepared from L cells transfected with individual genes QJ-Q5 and Q10 (e.g., Fig. 4e). The Qa-2/3reactive polypeptides present in L cells transfected with the Q6 gene alone (Fig. 4a) have a molecular weight of about 39,000 and are larger than those polypeptides (Mr -35,000) present in Q7 transfectants (Fig. 4b). Only Mr 39,000, Qa-2/3-reactive polypeptides are found in cells transfected with a cosmid clone (B3.3 in ref. 3) containing both Q7 and
Q8 (Fig. 4c). Since the Mr 35,000, Q7-associated polypeptides are absent, we assume that this cosmid clone does not contain a complete active Q7 gene and that the Mr 39,000 polypeptides detected are encoded by Q8. Similar Mr 39,000 polypeptides, as well as Qa-2/3-reactive Mr 35,000 molecules, are present in L cells transfected with a cosmid clone (B4.8 in ref. 3) containing Q8 and Q9 (Fig. 4d), indicating that both genes are expressed. Although the Q6 and Q8 products are indistinguishable by two-dimensional gel analysis, the Mr 35,000 polypeptides encoded by Q7 and Q9 are clearly different in charge, the Q7 products being more basic than the Q9 products. Taken together, the results suggest that the Q6 and Q8 gene products are closely related, as are the Q7 and Q9 gene products. Similar Qa-2/3-reactive polypeptides are also precipitated from C57BL/6 lymphoid cells (Fig. 4f).
DISCUSSION Our restriction mapping data show that the BALB/cBy (Qa-2-) mouse substrain differs from Qa-2+ BALB/c strains in that it has lost a segment of DNA located between two class I genes equivalent to the Q6 and Q7 genes (3). It is likely that this structural difference is the direct cause of the change in Qa-2 phenotype from Qa-2+ to Qa-2- in BALB/cBy mice (13). A comparison between class I genes present in the Qa region of B10 and of Qa-2+ BALB/c (2) mouse strains reveals that a gene fusion probably occurred between a pair of ancestral BALB/c genes equivalent to B10 genes Q8 and Q9 (3). This gene fusion probably took place via an unequal crossing-over between the homologous genes and subsequent recombination that resulted in loss of DNA between the genes and in the generation of a hybrid class I gene retaining some structural characteristics of both parental genes (i.e., 5' Q8/3' Q9). It is possible that the deletion we observe between genes Q6 and Q7 in BALB/cBy mice is exactly analogous to this gene fusion event, since it spans at least 12 kb, includes part of the Q6 gene, and extends to within 0.4 kb of the Q7 gene. Attempts to demonstrate that the 5' end of the Q7 gene is also involved in the deletion are hampered by the fact that the Q4-Q10 genes have very similar
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5923
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FIG. 4. Fluorograms of two-dimensional [isoelectric focusing (LEF) followed by NaDodSO4/PAGE] polyacrylamide gels of [35S]methioninelabeled Qa-2-reactive polypeptides synthesized by transfected L cells (a-e) or by mouse lymphocytes (f). L cells were transfected with plasmids or cosmids containing cloned B10 class I genes Q6 (a), Q7 (b), Q7 and Q8 (c), Q8 and Q9 (d), or Q4 and Q5 (e). (f) Qa-2-reactive polypeptides synthesized in C57BL/6 mouse lymphocytes. Spots present in all fluorograms at M, -40,000 are precipitated by nonspecific antisera and are probably actin. Open triangles indicate Mr 39,000 products; closed triangles indicate M, 35,000 products. Numbers at right indicate Mr X iO'. Fluorograms were exposed for 8 weeks.
5924
Immunology: Mellor et al.
restriction enzyme-site maps, making it difficult to discriminate between genes on the basis of hybridization analysis alone. However, no new bands are detected in BALB/cBy DNA digested with several different restriction enzymes and hybridized to probe 2, a surprising result if the deletion ends so close to the 5' end of the Q7 gene. We consider it more likely that the deletion extends between analogous regions within the Q6 and Q7 genes and results in the formation of a hybrid class I gene that yields no new restriction fragments because it retains the same overall structure as other genes in the Q4-QJO region. This possibility can be tested by resorting either to the use of more specific DNA probes that discriminate between each gene or to molecular cloning of the regions adjacent to the deleted segment in BALB/cBy DNA. The functional significance of the observation of these structural differences between two closely related BALB/c mouse substrains arises from the fact that the two strains have different Qa-2 phenotypes. This, combined with our conclusions from structural studies, implies that either gene Q6 or Q7 or both are structural genes that encode the murine Qa-2 antigen in mice. However, a more direct test of this is provided by our transfection experiments, in which we detected Qa-2-reactive polypeptides in L-cell transfectants containing B10 genes Q6-Q9. Moreover, the Mr 39,000, Qa-2-reactive polypeptides encoded by genes Q6 and Q8 differ biochemically from the Mr 35,000, Qa-2-reactive polypeptides encoded by each of the genes Q7 and Q9. Qa-2-reactive polypeptides with similar characteristics in two-dimensional gel electrophoresis are present in extracts of C57BL/6 lymphocytes, suggesting that the L-cell products are not artifacts of the transfection system. It is unclear whether the multiple spots detected in transfectants containing single class I genes (e.g., Fig. 4 B and D) are due to carbohydrate processing or to differential splicing. Use of longer metabolic labeling times gives rise to further heterogeneity of Qa-2-reactive products, making interpretation of the gels difficult. It is also unclear whether fibroblasts are capable of processing these molecules to mature cell surface products. So far, attempts to demonstrate Qa-2 antigen on the surface of L cells transfected with genes Q6-Q9 have failed. The reason for this is not known but it is clearly not due to a requirement for the presence of two nonidentical Qa genes (e.g., Q6 plus Q7 or Q8 plus Q9), as L cells transfected with cosmids containing pairs of such genes also fail to express surface Qa-2 antigen. L cells may have defective mechanisms for processing surface antigens, although this seems unlikely given the relative ease with which various H-2 class I antigens can be detected on the surface of transfected L cells (reviewed in ref. 1). Our results with transfected L cells demonstrate clearly that four genes in the B10 Qa region can encode class I polypeptides that are immunoprecipitable by use of anti-Qa-2/3 antibodies. Together with the observation that progressive loss of these genes in two successive gene fusion events in BALB/cJ (Qa-2+) and BALB/cBy (Qa-2-) leads ultimately to the loss of the Qa-2+ phenotype, these data constitute firm evidence that these genes encode the murine Qa-2 antigen. Mice with Qa-2+ phenotypes fall into two groups based on the level of detectable Qa-2 antigen present in lymphocytes. Thus, B10 is a high Qa-2+ strain and BALB/cJ is a low Qa-2+ strain (8). Since probe 1 is a useful probe for the Q6-Q9 gene region, we have used this probe on
Proc. Nati. Acad. Sci. USA 82 (1985) DNA samples extracted from a variety of inbred mice. We find a perfect correlation between the number and/or intensity of bands detected by probe 1 in mouse DNA and the Qa-2 phenotype (high or low Qa-2' and Qa-2-) of a particular inbred strain (see, for example, figure 4c in ref. 3). This provides additional support for our conclusion that gehes Q6-Q9 control the Qa-2 phenotype. One implication of this conclusion is that cells of BALB/cBy origin should not express Qa-2 antigen. This conflicts with reports that certain tumors derived from BALB/cBy mice appear to express Qa-2 antigen, since mice in which such tumor lines are grown produce anti-Qa-2 antisera (15). Perhaps structural analysis of the Qa region in these tumor lines will resolve this apparent discrepancy. In a previous study, Goodenow et al. (16) have demonstrated that a BALB/c class I gene, outside the Q6-Q9 region and which has no counterpart in the B10 mouse genome (3), gives rise to Qa-2 antigen on the surface of transfected L cells. We cannot exclude, from our studies, that additional genes in the BALB/c mouse strain encode Qa-2 antigens and this may explain the apparent discrepancy between the results. We thank Lorraine Flaherty for antisera, Don Bailey for BALB/ cBy mice, and Michael Steinmetz for BALB/c cosmid clones. We also thank Ivan Lefkovits, Pat Young, and Lotte Kuhn for gel analyses; Bruce Loveland, Elizabeth Simps6n, and Kirsten Fischer Lindahl for helpful discussions; and Pat McFarlane for typing the manuscript. 1. Hood, L., Steinmetz, M. & Malissen, B. (1983) Ann. Rev. Immunol. 1, 529-568. 2. Steinmetz, M., Winoto, A., Minard, K. & Hood, L. (1982) Cell 28, 489-498. 3. Weiss, E., Golden, L., Fahrner, K., Mellor, A., Devlin, J., Bullman, H., Tiddens, H., Bud, H. & Flavell, R. (1984) Nature (London) 310, 650-655. 4. Klein, J. (1975) Biology of the Mouse Histocompatibility Complex (Springer, Berlin). 5. Flaherty, L. (1981) in Role of the Major Histocompatibility Complex in Immunology, ed. Dorf, M. (Garland, New York), pp. 35-57. 6. Michaelson, J., Boyse, E., Chorney, M., Flaherty, L., Fleissner, E., Hammerling, U., Reinisch, C., Rosensori, R. & Shen, F.-W. (1983) Transplant. Proc. 15, 2033-2038. 7. Flaherty, L. (1978) in Origins of Inbred Strains, ed. Morse, H. (Academic, New York), pp. 409-422. 8. Michaelson, J., Flaherty, L., Bushkin, Y. & Yudkowitz, H. (1981) Immogenetics 14, 129-140. 9. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 10. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, T. & Axel, R. (1977) Cell 11, 223-232. 11. Dobberstein, B., Garoff, H., Warren, G. & Robinson, P. (1979) Cell 17, 759-769. 12. O'Farrell, P. (1975) J. Biol. Chem. 250, 4007-4021. 13. Flaherty, L., DiBiase, K., Lynes, M. A., Seidman, J. G., Weinberger, 0. & Rinchik, E. M. (1985) Proc. Natl. Acad. Sci. USA 82, 1503-1507. 14. Steinmetz, M., Moore, K., Frelinger, J., Taylor-Sher, B., Shen, F.-W., Boyse, E. & Hood, L. (1981) Cell 25, 683-692. 15. Rosenson, R. S., Flaherty, L., Levine, H. & Reinisch, C. (1982) J. Immunol. 129, 382-387. 16. Goodenow, R., McMillan, M., Nicolson, M., Taylor-Sher, B., Eakle, K., Davidson, N. & Hood, L. (1982) Nature (London) 300, 231-237.
