Proc. Nati Acad. Sci. USAVol. 79, pp. 1994-1998, March 1982Immunology

Structure and expression of a mouse major histocompatibilityantigen gene, H 2Ld

(gene cloning/transformation/nucleotide sequence)

GLEN A. EVANS*, DAVID H. MARGULIES*, R. DANIEL CAMERINI-OTEROt, KEIKO OZATOt, ANDJ. G. SEIDMAN**Laboratory of Molecular Genetics and WPregnancy Research Branch, National Institute of Child Health and Human Development, and tGenetics and BiochemistryBranch, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20205

Communicated by Igor B. Dawid, December 10, 1981

ABSTRACT A genomic clone encoding H-2Ld, a mouse majortransplantation antigen, has been identified and the structure ofthe H-2Ld gene has been partially determined. We isolated 35genomic clones from a BALB/c (H-2d) genomic library by hy-bridization to mouse or human probes. One of these clones en-codes H-2Ld as determined by two criteria. First, the gene en-codes a protein that is identical at the 76 known amino acidpositions for H-2Ld. Second, when introduced into L cells by DNA-mediated gene transfer, a new H-2 antigen is expressed that isrecognized by anti-H-2Ld monoclonal antibodies. The sequenceof the H-2Ld protein predicted by the DNA sequences shows morethan 80% homology to known H-2 antigens. H-2L-like sequencesare found in mutant H-2Kb molecules, suggesting that gene con-version or reciprocal recombination may play a role in the devel-opment of H-2 polymorphism.

Transplantation antigens are polymorphic cell-surface glyco-proteins that are involved in T-cell recognition phenomena suchas allograft rejection (see refs. 1 and 2 for a general review). Eachantigen consists of a nonpolymorphic 12,000-dalton light chain(,82-microglobulin) and a polymorphic 44,000-dalton heavychain (H-2 in the mouse). The complex family of H-2 genes isencoded on mouse chromosome 17 in the major histocompati-bility complex (MHC), but the single 82-microglobulin gene isencoded on another chromosome. Classical genetics suggeststhat the major transplantation antigens are encoded in two ge-netic loci within the MHC, H-2D and H-2K, which are sepa-rated by a recombination distance of 0.33 centimorgan (cM).More recently, the D region of certain haplotypes has beenshown to contain at least two transplantation antigen genes, H-2D and H-2L (3, 4).One approach to understanding the organization and function

of genes of the MHC is the use of cloned H-2 gene fragments.Several investigators have reported the isolation and charac-terization of human HLA and mouse H-2 cDNA clones (5-8).Initial studies with these cloned probes suggest that the ge-nomic organization of H-2 genes may be more complex thanpredicted by classical genetics. Hybridization of cloned probesto mouse genomic Southern blots demonstrates 10 to 20 H-2or H-2-like genes in the mouse genome and most of these mapto chromosome 17 (9). In addition, by hybridization ofthe DNAfrom recombinant and congeneic mice, at least three H-2-likegenes map to the Tla region, located 1.5 cM to the telomericside ofH-2D (9). An H-2-like pseudogene has been cloned andsequenced and maps to the Qa2,3 region (10). It is clear thatthere are many more H-2 or H-2-like genes present in themouse genome than are necessary to encode H-2D, -K, and-L antigens.

We and others (10) have been able to isolate large numbersof bacteriophage clones that contain H-2-like genes. In this re-port we present the analysis of an H-2 genomic clone that en-codes H-2Ld. The structure of an H-2Ld cDNA clone is alsopresented. The nucleotide sequence of this gene suggests thatit could encode a protein that is identical to H-2L in the 76positions for which amino acid sequence is available (11). Whenintroduced into mouse L cells by DNA-mediated gene transfer,this gene directs the synthesis ofa new cell-surface antigen thathas the antigenic specificities of H-2Ld. The structure of the H-2Ld gene confirms that this major transplantation antigen isclosely related to the classical transplantation antigens H-2Dand H-2K.

MATERIALS AND METHODS

Preparation of Mouse 1-2 cDNA Clones. A mouse cDNAlibrary was prepared from BALB/c lymphoma (LSTRA) mRNA(12). Poly(A)-rich mRNA was converted to double-strandedcDNA clones as described (13). H-2 cDNA clones were selectedby using an HLA cDNA probe kindly provided by S. Weissmanand his collaborators (8).

Isolation ofH-2 Genomic Clones. Thirty-five H-2 or H-2-likegenomic clones have been isolated by using various HLA or H-2 probes and several mouse genomic libraries. The clone usedin these studies was obtained by cross-hybridization with a hu-man HLA genomic probe. An HLA cDNA clone kindly pro-vided by J. L. Strominger, H. L. Ploegh, and H. T. Orr (7)consisting of the region coding for the carboxy-terminal 46amino acids and 375 base pairs (bp) of 3' untranslated mRNAsequence was used to screen a human B-cell library (14) fromwhich a single human clone was obtained. This recombinantphage contains a 14-kilobase pair (kb) insert in Charon 4A (15).A 5-kb EcoRI fragment containing the coding region was sub-cloned in pBR322, isolated from purified plasmid DNA byEcoRI digestion and electrophoresis on low-melting-point agar-ose gels, and labeled with 32p to a specific activity of 200-400cpm/pg by nick-translation (16). A mouse genomic library fromthe BALB/c plasmacytoma MOPC 41 (17) was screened bycross-hybridization with the human probe by using hybridiza-tion and washing conditions as described (14). A number ofcross-hybridizing clones were obtained and plaque purified,and phage DNA was prepared by double-banding in CsCl,phenol extraction, and ethanol precipitation. Clones weremapped by restriction endonuclease digestion and Southernblotting to human or mouse cDNA fragments containing 3' or5' coding regions. This genomic clone was named CH4A-H2Ldwhen it was shown that this clone encodes the H-2Ld gene.

Abbreviations: MHC, major histocompatibility complex; cM, centi-morgan; bp, base pair(s); kb, kilobase pair(s); TK, thymidine kinase.

1994

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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DNA Sequence Analysis. Nucleotide sequences were de-termined by using the dideoxy approach on DNA fragmentssubeloned in the single-stranded phage M13 (18, 19). Frag-ments for sequence determination were prepared by "shotgun"cloning into one of several M13 vectors with total phage DNAor DNA from purified restriction fragments. Sau3A fragmentswere cloned into the BamHI site of M13 mp7 (19), BamHI orBgl II fragments into the BamHI site of M13 mp8 or mp9 (Be-thesda Research Laboratories), and Pst I fragments into the PstI site of M13 mp2(Pst) (20), mp8, or mp9. Dideoxy reactionswere carried out by using a 26-bp double-stranded primer asdescribed (19), and sequences were analyzed on 80-cm 8% poly-acrylamide sequencing gels (21). On 0.4-mm thin gels, reso-lution of bands would allow the sequence to be read up to 430bp from the primer site. Our experience with 80-cm gels sug-gests that sequences determined in a single direction have asomewhat higher error rate than sequences determined in bothorientations. All fragments have been subjected to sequenceanalysis at least twice.Computer Analysis. DNA sequences were aligned to the se-

quences of several published H-2 cDNA sequences (5, 6) andto sequences we have determined (unpublished data) by usingthe SEQ sequence analysis program written by Brutlag et aL(22). Homology was analyzed using the dot-matrix method orfiltered-dot matrix program written by J. V. Maizel, Jr. (23).

DNA-Mediated Gene Transfer. The H-2Ld clone was intro-duced into the thymidine kinase (TK)-deficient, L-cell lineDAP-3 by cotransformation with the herpes virus thymidinekinase gene as described (24, 25). A calcium phosphate precip-itate containing 5 ,ug of CH4A-H2Ld DNA, 50 ng of pBRTKDNA, and 30 jig of DAP-3 carrier DNA was added to 1 X 106L cells, and transformants were selected by growth on hypo-xanthine/aminopterin/thymidine medium. From an initialpool ofTKV cells, a cloned cell line T1. 1.1 was obtained. Cellswere allowed to react with monoclonal antibodies that recognizeH-2 antigens (26) and fluorescein-conjugated goat F(ab')2 anti-mouse Fc as described (27). H-2 expression in transformed andparent cells was determined by flow cytofluorimetry with afluorescence-activated cell sorter (FACS II, Becton Dickinson).

A

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RESULTS AND DISCUSSION

Isolation of Two Mouse H-2 cDNA Clones from a BALB/c Lymphoma. The structure of the H-2 genes and the relation-ship to their protein products will be better understood whenthe nucleotide sequences of these genes and their mRNA prod-ucts are available. To this end, two groups (5, 6) have isolatedH-2 cDNA clones and determined their nucleotide sequences.Steinmetz et aL (5) isolated three H-2 cDNA clones by using ahuman HLA cDNA probe. We took a similar approach to isolatetwo cDNA clones, pMHC-1 and pMHC-2, from a cDNA librarymade from mRNA isolated from the BALB/c lymphoma LSTRA(unpublished data). The nucleotide sequence of most of the in-sert in pMHC-1 was determined by using the strategy shownin Fig. 1. The nucleotide sequence would encode an H-2-likeprotein from amino acids 66 to 286. Comparison ofthe predictedamino acid sequence with available H-2 protein sequences sug-gested that this mRNA could not encode H-2Kd or H-2Dd butcould encode H-2Ld in the 50 amino acid positions determinedin this region (11).

Isolation and Sequence of the H-2Ld Gene. The nucleotidesequence homologies between human and mouse gene se-quences permitted us to use a human DNA probe to identifymouse sequences. Hieter et al. (14) showed that the immuno-globulin K light chain sequences ofmouse and human are moreconserved in the portions of the gene encoding protein se-quence than in the 3' untranslated portion. Thus, portions ofthe mouse gene that encoded light chain constant region se-quences could be used as probes to isolate the human gene. Ourattempts to isolate a mouse genomic clone with the humancDNA probe (7) that corresponds to the 3' untranslated portionofthe HLA mRNA were unsuccessful. Therefore, a longer DNAprobe encoding all or most of an HLA gene was prepared.

Initial screening with this DNA probe resulted in sevenmouse genomic clones. Subsequent screening with this andother DNA probes has resulted in the isolation of 35 mousegenomic clones. One clone was selected for further structuralanalysis by DNA sequencing. This clone contains a 17-kb insertof mouse genomic DNA; the H-2 gene on this clone is about

mE =

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H2Ld Genomic 5' im - --L- M VA OFIl-I I a a I I I r r

91 91 183

N C1

I ** 1F *IIZ I

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183 275 275 315 315 326

C2 M 11-

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kb

FIG. 1. Restriction endonuclease analysis and sequence determination strategy for H-2Ld genomic and cDNA clones. (A) The H-2Ld genomicDNA was cut with BamHI; the 1.9- and 2.4-kb fragments containing H-2 encoding regions were mapped by restriction endonuclease cleavage andSouthern blotting with 5' and 3' cDNA probes. Coding blocks are labeled by analogy to the globular domains of the H-2Kb molecule (12): N, C1,C2, L (leader sequence), M (transmembrane portion), I, (internal cytoplasmic portion). Amino acid numbers represent the codon in which the splicesite occurs. The locations of these splice sites are inferred by comparison with the H-2Ld cDNA clone and the sequence of the H-2Kb protein. Thesequence from an H-2-like pseudogene (10) suggests that additional protein encoding blocks are present at the 3' end. These hypothetical exons areindicatedby hatched areas. (B) The sequence of the H-2Ld cDNA (pMHC-1) was determined by cloningSau3A-cleaved plasmidDNA into M13 vectorsand selecting subclones containing inserted sequences by hybridization to nick-translated insert DNA. Amino acid numbers indicate the extent ofthe coding region.

AA-21 1 1Domain L

B

H-2Ld cDNA

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7 kb from one end of the inserted DNA. Restriction enzymeanalysis and hybridization to cDNA probes (see below) dem-onstrated that the entire coding region was contained on twoBamHI fragments of 1.9 and 2.4 kb. These fragments were or-

dered and further digested with Sau3A. The Sau3A fragmentswere cloned into M13 vectors for nucleotide sequence analysis.The restriction map of this portion of CH4A-H2Ld is shown inFig. 1. The strategy for determining the nucleotide sequence

of about 2500 bp encoding most of the H-2 gene on this cloneis indicated in Fig. 1. The nucleotide sequence is shown in Fig.2.The location of the intervening sequences and coding seg-

ments of the H-2-like gene encoded on CH4A-H2Ld were de-termined by comparison of the gene's nucleotide sequence tothe nucleotide sequence of published H-2 cDNAs, pMHC-1,and an H-2-like pseudogene (5, 6, 10). For example, the nu-

cleotide sequence of the cDNA clone pMHC-1 correspondsprecisely to a portion ofthe nucleotide sequence ofthis H-2-likegene (Fig. 2) and defines two intervening sequences-betweenthe N and C1 domains and between the C1 and C2 domains.Further comparisons of the nucleotide sequence of this genewith other cDNA sequences (6, 7) permitted the identificationof two other intervening sequences (between C2 and M and

between M and I,, shown in Fig. 2). By translation of the nu-cleotide sequence 5' to N, an area with properties of a leadersequence was located. This sequence encodes a 21-amino acidhydrophobic sequence with an amino-terminal methionine res-idue and splice site. The nucleotide sequence of this gene wasalso compared to the nucleotide sequence ofan H-2 pseudogene(10). The two genes were >75% homologous for the portionsthat were compared and retained homology in both interveningsequences and coding blocks (data not shown).To determine which H-2 molecule is encoded by CH4A-

H2Ld, the amino acid sequence encoded by the HM2Ld gene wascompared directly to the amino acid sequences of various H-2molecules (Fig. 3). However, the amino acid sequence of onlyone H-2 molecule, H-2Kb, has been completely determined(11). Comparison of the H-2Kb sequence with the amino acidsequence encoded by this H-2-like gene indicates that this H-2-like gene is homologous to but not identical to H-2Kb. Com-parison of the predicted amino acid sequence with other partialH-2 sequences suggested that this gene could not encode H-2Dd or H-2Kd. However, this gene could encode a protein con-taining all 76 ofthe amino acid residues ofH-2Ld that have beendetermined (Fig. 3; unpublished data). The similarity betweenthe amino acid sequence encoded by the H-2-like gene on

10 20 30 40 50 60 70 80 90

0 GGATCCCAGATGGGGGCGATGGCTCCGCGCACGCTGCTCCTGCTGCTGGCGGCCGCCTGGCCCGACTCAGACCCGCGGGGTGAGTGGGGTCGGAGAGGGAMetAlaProArgThrLeuLeuLeuLeuLeuAlaAlaAlaTrpProAspSerAspProArgG

100 AACGGCCTCTGCCGGGGAGAGGGGCGGCAGGGGAAGCGTCCGGGTCGCCAACCGGACCCTCCGCCCCTTGTCCACCTGAAGCCCCCTGCCCAGATCCCGC

200 TCGCCTGGCCAGCCGCCCGGGTTTGTAAGTCGGGTCTCACGCGCGCGCGCCCCCCAGGCCCACACTCGATGCGGTATTTCGAGACCGCGGTGTCGCGGCClyProHisSerMetArgTyrPheGluThrAlaValSerArgPr

300 GGGCCTCGGGGAGCCCCGGTACATCTCTGTCGGCTATGTGGACAACAAGGAGTTCGTGCGCTTCGACAGCGACGCGGAGAATCCGAGATATGAGCCGCAGoGlyLeuGlyGluProArgTyrIleSerValGlyTyrValAspAsnLysGluPheValArgPheAspSerAspAlaGluAsnProArgTyrGluProGln

400 GCGCCGTGGATGGAGCAGGAGGGGCCGGAGTATTGGGAGCGGATCACGCAGATCGCCAAGGGCCAGGAGCAGTGGTTCCGAGTGAACCTGAGGACCCTGCAlaProTrpMetGluGlnGluGlyProGluTyrTrpGluArgIleThrGlnIleAlaLysGlyGlnGluGlnTrpPheArgValAsnLeuArgThrLeuL

500 TCGGCTACTACAACCAGAGCGCGGGCGGTGAGTGACCCCGCGTCGGAGGTCAGCCCCTCCATTTCCCGACACAGGGACGTGACGTGCGTCCCAAGTCCGAeuGlyTyrTyrAsnGlnSerAlaGlyG___________________________

600 GGTTCGGAACAGAACGGACCCGGAACCGTTTCCCTTTCAGTTTGGAGGAGTCGCGGCCGGGCGGCCGGGGCCGGGGCCGGGTACCGGGGCTGACCGGGGT

700 CCCGCAGGCACTCACACACTCCAGTGGATGTACGGCTGTGACGTGGGGTCGGACGGGCGCCTCCTCCGCGGGTACGAGCAGTTCGCCTACGACGGCCGTGlyThrHisThrLeuGlnTrpMetTyrGlyCysAspValGlySerAspGlyArgLeuLeuArgGlyTyrGluGlnPheAlaTyrAspGlyArgA_____________________________________________________________________________________________

800 ATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACATGGCGGCGCAGATCACCCGACGCAAGTGGGAGCAGGCTGGTGCTGCAGAGTAspTyrIleAlaLeuAsnGluAspLeuLysThrTrpThrAlaAlaAspMetAlaAlaGlnIleThrArgArgLysTrpGluGlnAlaGlyAlaAlaGluTy____________________________________________________________________________________________________

900 TTACAGGGCCTACCTGGAGGGCGAGTGCGTGGAGTGGCTCCACAGATACCTGAAGAACGGGAACGCGACGCTGCTGCGCACAGGTGCAGGGGCCGCGGGCrTyrArgAlaTyrLeuGluGlyGluCysValGluTrpLeuHisArgTyrLeuLysAsnGlyAsnAlaThrLeuLeuArgThrA___________________________________________________________________________________

1000 AGCCTCCTCCCTCTCGCCTCGGGCTGGGGGCTCAGTCCTGGGGAAGAAGAAACCCTCAGCTGGGGTGATGCCCTGTCTCAGAGGGAGAGAGTGACCCTGGt100 GTCTCCTGATGCCTCAGCACAGTGACTGCACTGACTCTCCAGGCTCATCTCCCTGGACATGGCCCAGCCTGTCTAGAGGAAGGAGAGAATTTCCCTGAGG1200 TAACAACAGCTGCTCCCTTCAGTTCCCCTGTAGCCTCTGTCAGCCAGTCCTCTCCCAGGCCGGGTTCTCTGCCCACGCCACTGTCGGTGACACTGACTCC1300 TGTCCTGGCTGAGTGTGTCAGCCCTTACACCTCAGGACCGGAAGTCGCCTTACCTGATTGGAAACATGGACTCCTATACACTAGCCTGGTTGCCCCAGCT1400 TCTAGAACTTTCCAGAGAATACATTCTCCCAGATC .................... GGATCCTGTGTGACACACCTGTGCCTTGTCCTCCAGAGTCAGGGG1500 CAGGGAATTTTCTCTGGCTACAGACTTTGTGATGGCTGTTCACTCGGACTGACAGTTAACGTTGGTCAGCAAGATGACCACAGTGGTTGAGTCTCAGTTG1600 GTGGGACCCTTCCAGTAGCATATGCCCCAATTTTGATATGAACTCCAAACAGATATTTAAATTACTTATTTTCCATTCCTATTTCATTCTTGGACACCCT1700 ACGCATGAACTCACATAAGATGCATGTCACCACTGCTACTAGTGATTCCCTCTTAGCTTCTTGTCCCAAAAGAAAATGTGCAGTCCTGTGCTGAGGGGAC1800 AGCTCTGCTTTTGGTCACTAGTGCAATGACAGTGTAGTGTCAAATAGACACATAGTTCACTCTATCATTGATTTAACTGAGTCTTGTGTAGATTTCAGTT

1900 TGTCTTGTTAATTGTGGAATTTCTTAAATCTTCCACACAGATTCCCCAAAGGCACATGTGACCCATCACCCCAGATCTAAAGGTGAAGTCACCCTGAGGTspSerProLysAlaHisValThrHisHisProArgSerLysGlyGluValThrLeuArgC

2000 GCTGGGCCCTGGGCTTTTACCCTGCTGACATCACCCTGACCTGGCAGTTGAATGGGGAGGAGCTGACCCAGGACATGGAGCTTGTGGAGACCAGGCCTGCysTrpAlaLeuGlyPheTyrProAlaAspIleThrLeuThrTrpGlnLeuAsnGlyGluGluLeuThrGlnAspMetGluLeuValGluThrArgProAI____________________________________________________________________________________________________

2100 AGGGGATGGAACCTTCCAGAAGTGGGCATCTGTGGTGGTGCCTCTTGGGAAGGAGCAGAATTACACATGCCGTGTGTACCATGAGGGGCTGCCCCATCCCaGlyAspGlyThrPheGlnLysTrpAlaSerValValValProLeuGlyLysGluGlnAsnTyrThrCysArgValTyrHisGluGlyLeuProHisPro____________________________________________________________________________________________________

2200 CTCACCCTGAGATGGGGTAAGGAGGGTGTGGGTGCAGAGCTGGGGTCAGGGAAAGCTGGAGGCCTTCTGCAGACCCTGAGGCTGGTCAGGGATGAGAGCTLeuThrLeuArgTrpG

2300 GGGGTCATAACCCTCACCTTCATTTCCTGTACCTGTCCTTCCCAGAGCCTCCTCCGTCCACTGACTCTTACATGGTGATC..........

luProProProSerThrAspSerTyrMetValIl1Q2400 .................GATCTTCCTAGAATTATTCACACTTTTCTTTTTCACAGGTGGAAAAGGAGGGGACTATGCTCTGGCTCCAGGTTAGTGTGGGGACAGG

lyGlyLysGlyGlyAspTyrAlaLeuAlaProG

2500 ATTGTCTGGGGGACATTGGAGTGAAGTTGGAGATGATGGGAGCTCTGGGAATTCCATAATAGCTCAGAGA.....

FIG. 2. Partial nucleotide sequence of the H-2Ld gene. The complete nucleotide sequence of the four external domains (exons 1-4) as well asa portion of the transmembrane and internal coding blocks is shown. Underlined regions represent sequences that are also present in a H-2LdcDNAclone (pMHC-1).

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LdKbQa2, 3Db

I 10 20 30 40 50 60 70 80 90 100+ + + + + + + + + + +

* **xd ** * N*6XX M*w M ** *** * **** M MGPHSMRYFETAVSRPGLGEPRYISVGYVDNKEFVRFDSDAENPRYEPQAPWMEQEGPEYWERITQIAKGQEQWFRVNLRTLLGYYNQSAGGTHTLQWMYG

L V ME DT R R E K N S D K S I VISQ LQ H WF DTQ M R R E M H S GS AQS K S

E R E K S N S Q --

FIG. 3. Comparison of H-2 protein sequences. The protein sequence predicted by the H-2Ld gene is compared with the first 100 amino acid res-idues of H-2K" (11) and the first 98 amino acid residues of H-2D (28). The H-2Ld sequence is also compared to the protein sequence inferred fromthe sequence of an H-2-like pseudogene that maps to the Qa2,3 region (10) and is indicated here as Qa2,3. -, unidentified residues; blanks, residueswhich are identical with the H-2Ld sequence; *, amino acid residue identified in H-2Ld. All 76 such residues are identical with the sequence predictedby the H-2Ld gene, but only those at positions 1-100 are shown here (4). The single-letter amino acid code is used (29) and amino acid numberingis by analogy to H-Kb (11).

CH4A-H2Ld and H-2Ld suggested that this cloned gene mightencode H-2Ld.

Expression of the Cloned H-2-like Gene in Mouse L Cells.The nucleotide sequence of the H-2-like gene on CH4A-H2Ldsuggests, but does not prove, that this gene encodes H-2Ld.Because only partial protein sequence data are available, it ispossible that this represents a gene that is highly homologousbut not identical to H-2Ld. Conceivably, this gene might cor-respond to an H-2 pseudogene, although the nucleotide se-quence does not indicate any serious defects. Alternatively,more than one H-2Ld gene could exist in the BALB/c mousegenome. To correlate sequence data with serological data, weintroduced this gene into mouse L cells by cotransformationwith the herpes virus TK gene and examined the transformedcells for their ability to bind monoclonal antibodies that rec-ognize H-2 antigens.The introduction of DNA sequences into L cells by cotrans-

formation with a viral TK gene is now a well-established pro-cedure (25). However, many introduced genes are not ex-pressed or are expressed at low levels (30). DNA frombacteriophage CH4A-H2Ld was introduced into mouse L cells,and these cells were examined for the expression of a new H-2 antigen. The parent mouse L cells are fibroblasts derived frommice of the k haplotype and therefore do not express H-2Ld.Monoclonal antibodies against H-2Ld showed minimal bindingto parent L cells as determined by flow cytofluorimetry (Table1). However, monoclonal antibodies specific for H-2Ld reactedwith the transformed L cell. Monoclonal antibodies against Ddand Ia gene products did not bind to the transformed cells. All

Table 1. Expression of H-2Ld on the transformed cell line

Mean fluorescencetDAP T1.1.1

Specificity Antibody* (parent) (transformed)Kk 36.7.5 S 620.5 162.8I-Ab, Iad 25.9.17 S (31) 118.8 104.2Ld/Rd 28.14.8 S (26) 118.3 459.6Ld 30.5.7 S (26) 125.0 492.2Dd 34.5.8 S 129.8 107.7

No Ab 110.0 98.0

Mouse L cells DAP-3 and a cell line transformed with theH-2Ld gene(T1.1.1) were analyzed for the expression of H-2 antigens. The cells(106) were incubated with hybridoma culture supernatant containingmonoclonal antibodies specific for H-2 antigens (26). After reactionwith fluorescein-conjugated goat F(ab')2 anti-mouse Fc, cells werefractionated by flow cytofluorimetry (27). Mean relative fluorescenceat constant gain was determined for 105 viable DAP-3 (L-cell parent)or T1.1.1 cells. The SD varied from 37 to 60 fluorescence units. Fouradditional experiments with this transformed cell line gave similarresults. In addition to clone T1.1.1, three additional transformationexperiments with this gene yielded transformed cells that produce H-2Ld determinants.* Only the specificity relevant to this study is shown. Reference num-bers are in parentheses for three entries.

t Menfluorescence normalized at gain 8.

cells of the transformed cloned cell line T1. 1.1 showed signif-icant binding with a clear single peak in the fluorescence pro-file. The relative intensity of anti-Ld approximated that of anti-Kk in the parent, implying that the amounts of H-2Ld producedare roughly equivalent to those ofthe endogenous H-2 antigens.Although we have no direct evidence that H-2Ld transcriptionis occurring from an H-2 promoter, it is likely that this is thecase, given the size of the 5' flanking region in this clone.

Relationship of H-2Ld to Other H-2 Molecules. From theanalysis of mouse DNA by Southern blotting there is evidencefor a large number of H-2-like genes in the mouse genome (5,9). At present, there is genetic evidence for at least two D-regiongenes, H-2Ld and H-2Dd, in H-2d (BALB/c) mice but only onegene, H-2D", in C57BL/6 mice. Fig. 3 shows a comparison ofthe amino acid sequence of the first 100 residues of H-2Kb and-Db and the sequence derived from a Qa2,3 pseudogene withthe sequence of H-2Ld derived from this gene. H-2Ld is 21%,24%, 25%, and 29% different from H2-Kb, H-2Dd, the H-2-likepseudogene, and H-2Kd, respectively, over the amino-terminal100 residues (10, 11, 28). However, H-2Ld is only 10% differentfrom H-2Db over the identified regions ofthe H-2Db molecule.From the closer homology of H-2Ld to H-2Db we suggest thattheir genes have derived from a common ancestral gene morerecently than H-2Ld separated from H-2Dd.Gene Conversion and the Evolution of H-2 Genes. Large

families of homologous genes may require mechanisms for themaintenance ofsequence homology and, in the H-2 gene family,the generation ofpolymorphism. Among many possible modelsfor the generation of diversity in H-2 genes are multiple single-point mutations, recombination between nonallelic genes, andgene conversion. Gene conversion implies the exchange ofDNA sequences between closely related genes with or withoutrecombination ofoutside markers during the course ofevolution(32). Evidence for the role ofgene conversion in the generationand evolution of H-2 genes may involve the demonstration thatH-2 gene sequences cannot be ordered into a normal evolu-tionary pathway based on single-point mutations. Additional H-2 gene sequence data are required to support or refute thishypothesis.

The H-2Ld sequence provides indirect evidence that geneticexchange between closely related H-2 genes can occur. Overthe past 20 years, a number ofmutant strains ofmice have beenisolated on the basis of altered graft rejection properties. Inparticular, a number of mutations in the H-2K" gene have beenisolated and the amino acid alterations in these genes have beendetermined (33). Surprisingly, some of these mutations involvemultiple amino acid changes. For example, H-2Kbml has ty-rosine residues at positions 155 and 156 replacing an arginineand a leucine residue found in the H-2Kb protein. Not only aretwo amino acid residues altered but each of these alterationsrequires two nucleotide changes. Nairn et al. (33) have sug-gested that these mutations may result from RNA splicing er-rors. However, the H-2Ld gene does not have intron-exon junc-tions at these positions, making this explanation unlikely.

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Interestingly, H-2Ld has tyrosine residues at positions 155 and156, as does the H-2Kbml molecule. Similarly, H-2Ld has thesame residue at each of the altered sites found in all of the mu-tant forms of the H-2Kb protein except the H-2Kbml molecule(33, 34). The finding that mutant H-2Kb molecules contain se-quences that could be derived from another H-2L-like gene(perhaps H-2Db) strongly suggests that gene conversion or re-ciprocal recombination has altered the sequences of theseH-2-like genes. Events similar to those that created the Kb mu-tants may play an important role in the generation of H-2polymorphism.Note Added in Proof. Since submission of this manuscript, the DNAsequence ofa H-2Ldgene has been published (35). This sequence differsin 65 of 2,100 nucleotide positions and in 6 of 286 amino acid residuesand demonstrates at least two restriction site differences from the se-quence presented here. These differences are unlikely to representsequence analysis or cloning artifacts and may indicate allelic variationin the H-2Ld gene or represent another nonallelic H-2Ld gene. Thus,there may be at least two H-2Ld genes that are expressed after intro-duction into mouse L cells. Both of these antigens are recognized bythe same monoclonal antibodies (36).We thank Dr. J. Coligan for discussion and making available unpub-

lished data, Drs. J. Strominger and S. Weissman for HLA cDNA clones,Drs. J. Messing and D. L. Bentley for M13 cloning vectors, Dr. J. V.Maizel for making his computer programs available to us, Ms. SusanSharrow and Mr. David Stephany for the FACS II analysis, and Ms.Terri Broderick for her editorial expertise.

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