2 mouse liver

Cell, Vol. 41, 469-478, June 1985,Copyright © 1985 by MIT

0092-8674/85/060469-10$02.00/0

Expression of Class I Genes in the Major Histocompatibility Complex: Identification of Eight Distinct mRNAs in DBA/2 Mouse Liver Jean-Louis Lalanne*, Catherine Transy, Sylvain Guerin,t Sylvie Darche, Pierre Meulien,$ and Philippe Kourilsky Unite de Biologie Moleculaire du Gene U. 277 I.N.S.E.R.M. associee au C.N.R.S. (U.A. 535) Institut Pasteur 25 rue du Dr. Roux 75724 Paris Cedex 15 France

Summary The mouse H-2 multigene family includes the genes coding for the major transplantation antigens and for genes located in the Qa-Tla region. We have studied a collection of class I cDNA clones made from liver mRNA of DBA/2 mice (H-2 d haplotype) and found that at least six distinct class I genes are transcribed, including three genes of the Qa-Tla region. Two of these six genes each yield two distinct mRNAs, resulting from alternate splicing. Altogether, liver cells may express at least eight distinct class I poiypeptides, of which three might be secreted, while one may be a new presumptive nonpolymorphic surface antigen. Introduction The major histocompatibility antigens are 45,000 dalton, polymorphic cell-surface glycoproteins, present on most somatic cells. They serve as targets for both allospecific and virus-restricted cytotoxic T lymphocytes. In the mouse, these molecules are encoded by genes located in the H-2K and in the H-2D regions of chromosome 17 (Klein, 1975). In the H-2 d haplotype, three surface antigens (H-2Kd, H-2D d, and H-2Ld) have been identified, but serological analyses have suggested the existence of additional class I molecules coded in the H-2K and H-2D regions (Demant et al., 1981; Hansen et al., 1983). In addition, some other class I molecules (i.e., antigens sharing structural similarities with the major transplantation antigens) encoded in the Tla region have been serologically defined. In contrast to the transplantation antigens, their expression is limited to lymphoid cells, especially T cells. Each of the defined antigens of the Qa-Tla series has a particular pattern of tissue distribution, being somewhat characteristic of certain differentiation stages in the lymphoid lineage (Flaherty, 1980). Although these data suggest a possible role in the differentiation pathway of lymphocytes, no precise function has so far been assigned to these antigens. When cDNA probes became available (Kvist et al., * Present address: RousseI-UCLAF,RecherchesBiotechnologiques, B.R n° 9, 93230 Romainville,France. tPresent address: UniversiteLaval, Faculte de Medecine,Departement de Microbiologie,Quebec,CanadaG1K 7P4. :l:Presentaddress:TransgeneS.A., 11 rue de Molsheim,67000Strasbourg, France.

1981; Steinmetz et ai., 1981a), it was shown that H-2 genes belonged to a relatively large multigene family (Cami et al., 1981; Steinmetz et al., 1981a). In BALB/c mice (H-2 d haplotype), this family comprises on the order of 36 genes (Steinmetz et al., 1982), five of which are located in the H-2K and H-2D regions, while the remainder lie in the QaTla region adjacent to H-2D on chromosome 17 (Winoto et al., 1983). Similar studies in B10 mice (H-2 b haplotype) have led to similar results, although the size of the H-2 family may be somewhat smaller (Weiss et al., 1984). A number of H-2 genes have been identified, mainly by the use of serological reagents after transfection of genes into mammalian cells in culture (Goodenow et al., 1982a; Evans et al., 1982; Mellor et al., 1982; Daniel-Vedele et al., 1984). It has also been shown that several genes mapping in the Qa-Tla region code for surface antigens (Goodenow et al., 1982b), but many genes so far have no identified product. On the basis of nucleotide sequence analysis, Steinmetz et al. (1981b) inferred that the 27-1 gene, isolated from BALB/c mice and mapped in the Qa region, was a pseudogene because it could not code for a membraneanchored protein. On the other hand, Cosman et al. (1982a; 1982b) identified a cDNA specifically expressed in liver, which was later shown to direct the synthesis of an H-2 related polypeptide secreted in the serum (Kress et al., 1983a; Maloy et al., 1984) and was shown to be encoded by a nonpolymorphic gene of the Qa region (Mellor et al., 1984). A detailed analysis of the products of H-2 genes is hampered by the lack of serological reagents against so far unknown polypeptides (especially if the latter are not polymorphic, in which case only xenogenic reagents can be used). An alternative approach is to make cDNA copies from mRNAs of a given tissue, to isolate cDNA clones, and to determine their nucleotide sequence. This information can then be used to synthesize peptides, or the cDNA can be engineered to produce a polypeptide in order to raise the appropriate serological reagents or to analyze the product directly. As a first step in this approach, we have undertaken a detailed analysis of H-2 and H-2-related transcripts in the liver of DBA/2 mice (H-2 d haplotype), cDNAs reacting with H-2 probes were isolated and were shown to belong to eight distinct types. From these data, we infer that mouse liver probably expresses at least eight distinct polypeptides, including H-2K d, Dd, Ld, and the liver-specific molecule described by Cosman et al. (1982b). The four other putative molecules include a molecule closely related to H-2Kd, produced from the same gene as the result of an alternative splicing process (Lalanne et al., 1983b; Transy et al., 1984); a molecule (presumably secreted) closely related to the molecule of Cosman et al. (1982a; 1982b), produced from the same gene as the result of an alternative splicing event; the product of the 27-1 pseudogene (Steinmetz et al., 1981b); and a presumptive new nonpolymorphic surface antigen, which may be expressed in liver and in several other tissues.

Cell 470

Table 1. Classification of Class I cDNA Clones Based on Their Hybridization to Specific Probes Probes Group I

II

III

IV

Clone Number 8 9 16" 17 22 23 24* 25 33

7 12 36* 38 39 44 14° 27° 30 ° 41 ° 46 32 37 18 19 31 4O 34 35

A B (exon 2) (exon 3) N C 2 ~

+

4444-

NC2/~

NC2y

NC25

2

Genetic Origin

5

Kd

4+ 444-

+ + Dd

Ld

4437 + Q10 +

+ +

+ +

27-1

Each NC2 probe defines a group of cDNA clones, cDNA clones that do not hybridize with any of the NC2 probes were ctassified by comparing their restriction maps with prototypes of the different groups (*) and/or (group II of cDNA) by hybridizing with oligonucleotidesspecific for D~ or Ld (o). Probes A and B were derived from the H-2K~ gene (Kvist et al., 1983). (Probe A) 327 bp Sma I-Sma I restriction fragment encompassing exon 2; (Probe B) 228 bp Sac II-Sac II restriction fragment encompassing exon 3; (Probe NC2,~) 170 bp Pst I-Hinf I restriction fragment from pH-2d-4 cDNA clone (Lalanne et al., 1982); (Probe NC2p) 190 bp Pst I-Pst I restriction fragment from pH-2d-1 cDNA clone (Lalanne et al., 1982); (Probe NC2y) 250 bp Pst I-Pst I restriction fragment from pH-2d-19 cDNA clone; and (probe NC26) 190 bp Sac I-Pst I restriction fragment from pH-2d-35 cDNA clone. Oligonucleotides 2, 5 (pH-2d-1 derived), and 3, 4 (pH-2d-3 derived) are described by Abastado et al. (1984). Used as probes with in situ hybridization experiments on the H-2 cosmid library constructed by Steinmetz et al. (1982), oligonucleotide 5 detects only the Dd gene containing clone, while oligonucleotides 3 and 4 are specific for the Ld gene. The genetic origin proposed for the different cDNA clones is discussed in the text.

Results Isolation and Classification of the cDNA Clones We have previously reported the construction of the cDNA library from liver m R N A of DBA/2 mice, the isolation of 40 independent cDNA clones by use of a hybridization probe encompassing the well-conserved third domain coding region (probe H: 600 bp Hinf I fragment of pH-2d-4, Lalanne et al., 1982) and the nucleotide sequence of two of these cDNA clones (Lalanne et al., 1983a, 1983b). The latter were shown to represent alternatively spliced transcripts of the H-2K d gene (Lalanne et al., 1983b; Transy et al., 1984). To characterize further the other cDNA clones, we used probes specific for the 5' and for the 3' moieties of H-2 mRNAs; probes A and B were isolated from the cloned H-2K d gene (Kvist et al., 1983), and they encompass exons 2 and 3 (first and second domains), respectively.

H-2 mRNAs differ in their 3' moiety (Lalanne et al., 1982; Cosman et al., 1982a), certain H-2 genes (including H-2L d) having a repeated sequence of the B2 type at their 3' ends (Steinmetz et al., 1981a; Cami et al., 1981; Moore et al., 1982). On this basis, we have previously distinguished, in the 3' noncoding (NC) region of H-2 cDNAs, two subregions; NC1 and NC2. NC1 is well conserved among different H-2 mRNAs, in contrast to NC2 (Lalanne et al., 1982), which exists either as the already mentioned repeated sequence (NC2/~) or as another sequence, NC2~, showing specificity for H-2K d (Xin et al., 1982). Probes corresponding to NC2~ and NC2p were isolated from cDNA clones of a previous series, pH-2d-1 (for NC2/~) and - 4 (for NC2a) (Kvist et al., 1981; Lalanne et al., 1982). Clones hybridizing with these various probes were further analyzed by restriction mapping and, where appropriate, by nucleotide sequencing. As their characterization

Liver Transcriptsof Class I Genes 471

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Figure 1. RestrictionMaps of cDNA Clones, Prototypesof the Different Groups, and SequencingStrategy Restriction enzymes used are as follow: Bal I (B), Bgl I (Bgll), Bgl II (Bglll), Hinf I (H) Pst I (P), Pvu II (Pv), Rsa I (R), Sac I (SI), and Sac II (SII). For pH-2d-18,-19,-35, and -37,which have been sequencedaccordingto the Maxam-Gilbertprocedure(1980),the sequenceanalysis strategy is indicatedas follows:open circles referto labelingof the 5' ends by y-32P-ATPexchange,and closedcircles referto labelingat the 3' ends, usingterminaltransferaseand o~-32P-ddATEFor pH-2d-19,restriction sites Hha I (Hh)and Hpa II (Hp) locatedin the vectorare used for end-labeling.

progressed, we found new classes that could be identified by their NC2 sequence. They are a transcript of the Q10 gene (Mellor et al., 1984), including a specific 3' nontranslated region (Cosman et al., 1982b) that we called NC2y, and another set defined by a sequence that was designated NC2& We finally arrived at the classifications summarized in Table 1. The nucleotide sequences of several cDNA inserts were determined (Figure 1, pH-2d-33 and -24 in our previous work, Lalanne et al., 1983a, 1983b, pH-2d-37, Figure 2, pH-2d-12, -18, -19, and -35, not shown). Three groups of clones could be assigned to Kd, Dd, and Ld, respectively, by hybridization with oligonucleotides of known specificity (Abastado et al., 1984; Table 1), and by sequence comparisons. We focus below on the transcripts of the other classes.

A Class I mRNA Showing a Distinct Second Domain Coding Sequence Two clones, pH-2d-32 and pH-2d-37, contained a repeated

sequence, but their restriction maps did not fit those of the H-2D d and H-2Ld transcripts (Figure 1), and they did not hybridize with the oligonucleotide probes specific for those genes (Table 1). Since pH-2d-32 appeared to be a shorter version of pH2d-37, the latter was sequenced. The insert is 1316 bp long (not including the poly(A) and poly(GC) tails). The sequence displays a 1071 bp open reading frame, starting from the first ATG encountered in the sequence (Figure 2). The deduced amino acid sequence can be aligned with that of known H-2 class I antigens and shares their characteristic features, the first 20 residues at the NH2 terminus are hydrophobic, as expected for a signal peptide. A distribution into three domains of 91 amino acids each can then be drawn by analogy with other class I molecules, with the second and third domains displaying the possibility for a disulfide bridge at the expected residues (Cys 101-Cys 164, Cys 203-Cys 259). After the third domain, a stretch of 20 residues, many hydrophobic, followed by four positively charged residues, has the expected traits of a membrane-spanning region. It is followed by a short sequence at the COOH terminus similar in size to the cytoplasmic tail of class I antigens. In addition, the sequence displays two possible N-glycosylation sites at positions 86 and 176. The 5' noncoding region in pH-2d-37 is 33 nucleotides long, compared with 25 in the H-2K d mRNA (Lalanne et al., 1983a; Kvist et al., 1983), suggesting that the insert is a full-length or nearly full-length copy of the mRNA. The 3' untranslated region is shorter than in other cDNAs so far analyzed. It comprises almost exclusively the B2 repeated element starting just a few nucleotides after the TGA stop codon. In other words, the "37" mRNA lacks the 300 nucleotide long NC1 sequence present in other H-2 mRNAs and must, therefore, be significantly shorter than the other H-2 mRNAs so far analyzed. The homology between the "37" nucleotide and amino acid sequences and those of other H-2 mRNAs and polypeptides can be scored, using the putative division into protein domains mentioned above. At the nucleotide level, the first NH2-terminal domain coding sequence is 85% homologous to that of other H-2 sequences, showing the least homology at its 3' end, while the second domain coding sequence is 70% homologous. This result is in contrast with the 90% homology observed in this region for all H-2 sequences described so far. The divergence between pH-2d-37 and other H-2 sequences is also reflected at the amino acid level. The third domain appears to be highly homologous, while the membranespanning region is more divergent, as with other class I antigens. The cytoplasmic region appears to be highly divergent, especially in the 3' moiety (corresponding to exon 7 of H-2Kd). The nucleotide sequence of pH-2d-37 predicts the existence of a thus far undescribed class I surface molecule, encoded by an mRNA 300 bp shorter than other class I mRNAs, with a distinctive second domain significantly different from that of other known class I antigens. It was likely, therefore, that the pH-2d-37 sequence cod-

Cell 472

PH2d37

T

M L L F A H L L Q L L V S A T V P T Q S S P H S L R Y F T G ATTCA G GTTCCTCA CAGAC C C A G G G A G T G A G G A T G T T G C T T T T T G C C C A C T T G C T T C A G C T G C T G G T C A G C G CCA CAGTC C CGAC CCAGAGTAGC CCACACTCGCTGCGGTAT T TCA C C 10 20 30 40 50 60 70 80 90 100 11 0 120 T A V S R P G L G E P R F I I V G Y V D D T O F V R F D S D A E N P R M E P R A AC C GC CGTGTCC C GGC CCGCCCTCGGGGAGC C C C G G T T C A T C A T T G T C G G C T A C G T G G A C G A C A C G C A G T T C G T G C G C T T C G A C A G C G A C G C G G A A A A T CC GAGGATGGAGC CTCGGGCG 130 140 150 160 170 180 190 200 210 220 230 240 @ R W I E Q E G P E Y W E R E T W K A R D M G R N F R V N L R T L L G Y Y N Q S N C G G T G G A T T G A G C A GGAGGGGC C G G A G T A T T G G G A G C G G G A G A C T T G G A A A G C CA G G G A C A T G G G G A G G A A C T T C A G A G T A A A C CTGAGGAC CCTGCTCG G CTACTA CA ATCA GAGTAAC 250 260 270 280 290 300 310 320 330 340 350 360 D E S H T L Q W M Y G ~ D V G P D G R L L R G Y C Q E A Y D G Q D Y I S L N E D GAC GAAT CT CA CA C GCTGCA G TGGATGTAC G GCTGCGACGTGGGGC CCGATGGGCGC CTGCTC C G C G G G T A T T G T C A G G A G G C CT ACGATGGCCAGGATTACATCTC C CTGAACGAGGAC 370 380 390 400 410 420 430 440 450 460 470 480 L R S W T A N D I A S Q I S K H K S E A V D E A H Q Q R A Y L Q G P C V E W L H CTGCGTT•CTGGACCGCGAATGACATAGCCTCACAGATCTCTAAGCACAAGTCAGAGGCAGTCGATGAGGCCCACCAACAGAGGGCATACCTGCAAGGTCCTTGCGTGGAGTGGCTCCAT 490 500 510 520 530 540 550 560 570 580 590 600

•

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R Y L R L G N E T L Q R S D P P K A H V T H H P R S E D E V T L R C W A L G F Y AGATACCTACGGCTGGGAAATGAGACACTGCAGCGCTCAGA•CCTCCAAAGGCACATGTGA•CCATCACCCTAGATCTGAAGATGAAGTCACCCTGAGGTGCTGGGCCCTGGGCTTCTAC 610 620 630 640 650 660 670 680 690 700 710 720

P A D I T L T W Q L N G E E L T Q D M E L V E T R P A G D G T F Q K W A A V V V C CT G CTGACATCAC C CTGAC CT G G C A G T T A A A T G G G G A G G A G C T G A C C C A G G A C A T G G A G C T T G T G G A G A C C AGGC CTGCAGGGGATGGAAC CTTC CAGA A G T G G G C A G C T G T C G T G G T G 730 740 750 760 770 780 790 800 81,3 820 830 840

P L G K E Q Y Y T C H V Y H E G L P E P L T L R W E P P P S T V S N M V I I A V C CT CTTGGGAAGGAG CA G T A T T A C A C A T G C C A T G T G T A C C A T G A G G G G C T G C C T G A G C C CCTCAC C CTGAGATGGGAGC CTC C TC CA TC CACTGTCTC C AACATGGTAATCAT AGCTGTT 850 860 870 880 890 900 910 920 930 940 950 960

T

L V V L G A V I I L G A V V A F V R K R R R H I G V K G C Y A H V L G S K $ F Q C T G G T T G T C C T T G G A G C T G T G A T C A T C C T T G G A G C T G T G G T G G C T T T T G T G A T G A A G A G G A G G A G A C A C A T A G G T G T A A A A G G A T G C T A T G C T C A T G T T C T A G G C A G C AA GAGC T TC CAG 970 980 990 1000 1010 1020 1030 1040 1050 1060 1070 1080

T S D W P Q K A * A C CT CT G A C T G G C C T CA GAAGGCA T GAAAATC C CT A GGGGG GCT GGTGAGA TGGCTCA GTGGGT A AGAGCACT G ACT G CT C T TCT G AAGGT C CA GAG T T CA A A TC C CA G C A A C C A C A T GG

1090

1100

1110

1120

1130

1140

1150

1160

1170

1180

1190

1200

T GGCTCA C AACCATC CGTAAC G AGATCTGACTC C C T C T T C T G G A G T G T • T G A A G A • A G C T A C A A T G T A • T T A C A T A T A A T A A A T A A A T A A A T A A A T A A A T A A A T A A A T A A A T A A A T A A

1210

1220

1230

1240

1250

1260

1270

1280

1290

1300

1310

Figure 2. Nucleotide Sequence of the pH-2-d-37 cDNA Clone and Deduced Amino Acid Sequence of the Putative "37" Protein Possible N-glycosylation sites are indicated by a closed circle above the corresponding Asparagine residues. Conserved Cystein residues implicated in disulfide bonds in known H-2 antigens are indicated by boldfaced type. Arrows indicate boundaries between exons that could be deduced for the first five exons by comparison with known H-2 genes.

ing for the second domain could serve as a specific, or low copy probe, in hybridization experiments. Two conveniently located Pst I sites bracket a 250 bp fragment that was used as probe (probe C) (Figure 3). DNAs from B10 (H-2b), BALB/c (H-2d), AKR (H-2k), DBA/1 (H-2q), and SJL/J (H-2 s) mouse strains were analyzed in Southern blot experiments with probe C. The four enzymes used gave similar patterns (Figure 3). Two bands are detected in b, d, k, and q haplotypes. One band is invariant (band I). The second one (band II), common to b, k, and q haplotypes, has a different position in the d haplotype. SJL/J DNA displays a unique band corresponding to a fragment 500 bp longer than in band I, with whichever enzyme was used. To map the genes corresponding to the two bands observed in the H-2 d haplotype, we used probe C to screen a set of 21 cosmid clones that contain the 36 class I genes isolated from BALB/c DNA, by Steinmetz et al. (1982). Two overlapping clones, 12.2 and 48.1, which contain genes 3-6 in cluster III Tla region, were found positive. Further analysis of these clones showed that gene 6 in cluster III is the only one to hybridize with probe C, but it corre-

sponds to band II in BALB/c genomic DNA, not to band I, which is characteristic of the "37" gene (Figure 3). This is shown in two ways. Gene 6 is complete in cosmid 12.2, as judged by hybridization with exon-specific probes (not shown). Probe C is a 250 bp Pst I fragment included in the second domain coding sequence of pH-2d-37. It is thus expected to be present in the corresponding exon of the "37" gene. However, when digested with Pst I, cosmids 12.2 and 48.1 yield a 550 bp Pst I fragment hybridizing with probe C. Furthermore, probe C has an inner Bgl II site located at 140 bp and 110 bp from its ends. When BALB/c DNA is digested with Bgl II, probe C detects three bands (10 kb, 1.5 kb, and 0.95 kb) in the genomic blots. In other mouse strains, the 1.5 kb and 0.95 kb fragments are invariant, while the larger fragment shows polymorphism (data not shown). Gene 6 in cosmid 12.2 contains no Bgl II site, and its mapping in the cosmid is compatible with its assignment to the larger polymorphic Bgl II band. In summary, gene 6 is related to, but not identical with, the "37" gene, and the latter is absent from the library of Steinmetz et al. (1982). The "37" low copy probe was also used to detect mRNA

Liver Transcripts of Class I Genes 473

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Figure 4. Expression of mRNA Related to pH-2(J-37cDNA Clone in Various Tissues of DBA/2 Mouse (A) Northern blot hybridized with probe C. Lane a, kidney poly(A)÷ RNA; lane b, liver poly(A)÷ RNA; and lane c, spleen poly(A)+ RNA. (B) The same blot as in (A) was rehybridized with probe H (detecting all H-2 mRNAs). Lane d, size markers (length indicated in kilobases).

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Figure 3. Southern Blot Analysis of Genes Hybridizing to pH-2d-37 Probe C The mouse strain from which DNA was extracted is indicated above each lane. 12-2 is an H-2 cosmid from the BALB/c genomic library constructed by Steinmetz et al. (1982). Probe C is a 250 bp Pst I-Pst I fragment derived from the second domain coding region of pH-2d-37 insert. Sizes of fragment are indicated in kilobases on the left and on the right. in various tissues. In Northern blot experiments, with mRNA from liver, spleen, and kidney, mRNA of about 1400 nucleotides was detected. This is, as expected from the pH-2d-37 sequence, 300 nucleotides shorter than other H-2 mRNAs (Figure 4). "37" mRNA has also been detected in total thymus RNA by dot blot experiments (data not shown). Thus, the "37" gene may be transcribed in all four tissues.

Alternative Spliced Transcripts of the Q10 Gene (Group III) The Q10 gene has been so named in the H-2 b haplotype

and has been mapped in the Qa region by Mellor et al. (1984). It is transcribed into the liver-specific mRNA first identified by Cosman et ai. (1982b) in mouse H-2q haplotype (a cloned copy of which is pH-16; Kress et al., 1983a) and encodes a polypeptide product secreted into the serum (Maloy et al., 1984). Comparison of sequences in the H-2 b and H-2q haplotypes (Mellor et al., 1984) indicates that the Q10 gene is not polymorphic, a conclusion strengthened by our sequence data, which show that it is not polymorphic in the H-2 d haplotype either (see below). We, therefore, retain the same nomenclature. One clone in our cDNA library, pH-2d-18, had a restriction map entirely compatible with that of the pH-16 insert of Cosman et al. (1982b), and another clone, pH-2d-19, was similar. Both were sequenced, and the sequences could be matched almost perfectly with those of the pH-16 cDNA or the Q10 gene (6 transitions G ~ A , compared with the Q10 gene sequence, were all located in the 3' noncoding sequence). However, while the pH-2d-18 sequence is colinear with that of pH-16, the sequence of pH-2d-19 lacks precisely the 276 nucleotides of exon 3, such that exon 2 is connected to exon 4, predicting the existence of a polypeptide product lacking a second domain. The transcript that gave rise to pH-2d-19 could have been generated from the Q10 gene by alternate splicing. We first verified that the Q10 gene is unique in the DBA/2 genome by Southern hybridization with the probe derived from the 3' untranslated region of pH-2d-19 according to Cosman et al. (1982), data not shown. Then, to demonstrate the existence of a Q10 transcript lacking the third exon, we hybridized RNA from liver or spleen to probes containing the 3' coding sequence of pH-2d-18 and pH-2 d19 and assayed protection against $1 nuclease digestion.

Cell 474

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In such experiments, the size of the protected DNA fragment is precisely predicted (Figure 5A). Probes were 5' labeled at the Bal I site located in exon 5. This site was chosen because upstream from it, a short sequence of 13 nucleotides is missing, when compared with all other known H-2 cDNAs. Since the Q10 gene is expressed specifically in liver, we used spleen DBA/2 RNA as a negative control. As depicted in Figure 5A, using the probe derived from pH-2d-18, fragments of 450 and 330 nucleotides should be protected by "18"-like and "19"-like mRNAs, respectively. With the probe derived from pH-2d-19, fragments of 329 nucleotides and 453 nucleotides should be protected by "18"-like and "19"-like mRNAs, respectively. Results in Figure 5B show that fragments of the expected size are found with both probes (Figure 5B, lanes b and e). Furthermore, liver RNA from C3H mice (H-2 k haplotype) gives the same pattern of fragments (Figure 5B, lanes c and f), suggesting that alternate splicing does not occur exclusively in the d haplotype. The ratio of 19 mRNA/18 mRNA is higher when the 19 probe, rather than the 18 probe, is used. When the 19 probe is hybridized with 18 mRNA, it creates an exon-3-containing-loop in the mRNA molecule, which, upon removal by $1 nuclease, yields a nicked RNA molecule. The latter may still protect the 19 probe, which may not be entirely cleaved. This would not happen when the 19 mRNA is hybridized with the 18 probe. We thus feel that, in this case, the signals should be more quantitative, suggesting that mRNA represents 5%-10% of the 18 mRNA. The 19 mRNA is detected directly in Northern blots, using the NC27 probe, as a faint band with the expected size, in agreement with this estimate (data not shown). Finally, there is no signal with spleen RNA either in experiments using $1 nuclease (Figure 5B, lanes a and b) or in Northern blots (not shown), confirming that Q10 transcription is liver-specific.

The 27-1 Pseudogene Is Transcribed in Liver

240

O

217

Figure 5. Detectionof pH-2d-18and pH-2d-19mRNAs by $1 Mapping (A) Experimentalstrategy. Exonsare shown as boxesand are numbered accordingto the usual intron-exonorganizationof H-2 genes. Wavy lines represent pBR327 sequences. The pH-2d-18-derived probe(1200bp Bal I-EcoRI fragment,5' labeledat the Bal I site)should give protectedfragmentsof 450 and 330 nucleotides,when hybridized to "18"and "19"mRNAs,respectively.The pH-2d-19derivedprobe(604 bp Bal I-Bgl I fragment,5'labeledat the Bal I site)shouldgiveprotected fragmentsof 453 and 330 nucleotides,whenhybridizedto "19"and "18" mRNAs, respectively.(B) $1 mappingresults using pH-2d-18-derived probe(lanesa to c) and pH-2d-19derivedprobes(lanesd to f). Lanes a and d, total DBA/2spleenRNA;lanesb and e, total DBA/2liver RNA; lanes c and f, total C3H liver RNA; lane g, Hpa II digestedpBR322as size marker.The lengthin nucleotidesof the protectedfragmentsis indicated on the left.

Two clones, pH-2d-34 and pH-2d-35, with identical restriction maps, were distinct from all the others. The nucleotide sequence of the 1000 bp insert of pH-2d-35 was determined. With the exception of four nucleotide substitutions and one nucleotide deletion, it fits exactly the nucleotide sequence of the "27-1" gene isolated from BALB/c DNA by Steinmetz et al. (1981b), data not shown. Alignment of the pH-2d-35 sequence with that of the gene shows the usual exon-intron distribution. However, exon 7 is lacking in the cDNA, which is expected since, according to the 27-1 gene sequence, it is bordered by an abnormal (AC instead of AG) 3' acceptor splice site (Steinmetz et al., 1981b). As shown in Table 1, the 190 bp Sac I-Pst I fragment (probe NC25) derived from the 3' noncoding region of pH2d-35 does not cross-hybridize with cDNA clones of the other groups. When assayed in Northern blot experiments with poly(A)÷ RNA from DBA/2 mice, this probe detects a 1700 nucleotide RNA species in the three tissues tested, that is, spleen, liver, and kidney (Figure 6A). However, probe NC25 detects four overlapping cosmids (46.1, 52.3, 47.2, and 65.1) in the library of Steinmetz et al. (1982b). These cosmids are included in cluster I located in the Qa

Liver Transcriptsof Class I Genes 475

a

b

in exon 5 and extending through the beginning of the insert (i.e., through the middle of exon 3) to the Eco RI site of the vector. The specificity of the experiment is increased by the fact that exon 4 in pH-2d-35 is 9 nucleotides longer than that of other class I mRNAs (except Dd). After hybridization of this probe with liver mRNA of DBA/2 mice and digestion with $1 nuclease, two major protected fragments are observed (Figure 6B). The longest one (460 nucleotides) has the size expected for protection by an mRNA homologous to pH-2d-35. The shortest fragment (350 nucleotides) could result from protection by an mRNA of a related gene. Alternatively, an mRNA homologous to pH-2d-35, but devoid of exon 3, should protect a fragment of exactly this size. A similar pattern was observed with spleen mRNA (data not shown), suggesting that this mRNA class, in contrast to Q10 mRNA, is not liver-specific.

c

4.5~ 2.9

1.4f

abc

Discussion

622 I

527

460-.-~ 404 350'.-,~-

309

240

217 Figure 6. Detection of mRNA from DBA/2 Mice Related to the pH-2 ~35 cDNA Sequence

(A) Northernblotanalysisusing NC2~as probeof poly(A)÷RNA.Probe NC25is describedin Table1. Lanea, kidneypoly(A)*RNA; laneb, liver poly(A)*RNA; lanec, spleenpoly(A)+RNA;sizeof markersin kilobases are indicatedon the left. (B) $1 mappinganalysis.The probeusedwas the 1200bp Bal I-EcoRI restrictionfragmentfrom pH-2d-35,5' labeled at the Bal I site. It contains 454 bp of the cDNA insert flanked by pBR327sequence.Lanea, probehybridizedwith liver DBA/2 mRNA; lane b, probehybridizedwith carrier RNA only; lanec, Hpa II digested pBR322 as size marker; lengthsof protectedfragmentsare indicated in nucleotideson the left. region, and only two of them (47.2 and 52,3) contain the 27-1 gene. Therefore, in addition to the 27.1 gene transcript, probe NC25 could potentially detect the transcripts of a few other genes. $1 mapping experiments were undertaken to test for the presence of "35" mRNA. As a probe, we used a 1200 bp fragment of pH-2d-35, 5' labeled at the Bal I site located

To explore the range of class I molecules expressed in a given tissue, we have analyzed, in detail, a cDNA library made from liver mRNA of DBA/2 mice (H-2 d haplotype). The summary of these experiments is given in Table 1. We find eight different transcripts originating from at least six distinct genes. In two instances, two transcripts originate from the same gene (H-2Kd and Q10) by an alternative use of splicing sites. All eight transcripts contain open reading frames potentially coding for eight polypeptides, three of which (the two produced from Q10 and the 27-1 product) have the potential to be secreted. Thus, our experiments suggest that the liver of DBA/2 mice makes at least eight class I or class I-related polypeptides, more than was previously suspected.

Classification of cDNA Clones and Probes Analysis of genes and transcripts of the H-2 multigene family is made difficult by the number of genes and by the scarcity of specific probes. In this respect, it is useful to review classifications of cDNA clones and of available specific probes, in light of the above results. Previous classifications of H-2 transcripts have been based on the analysis of their 3' noncoding regions. Three groups were distinguished by Cosman et al. (1982a). Now, this has to be extended to five groups built with five noncoding (NC) sequences (NC1, NC2m NC2/3, NC27, and NC25). NC1 is widely distributed in H-2 genes. NC2/3 is the repeated B2-1ike element, found in the family, particularly in H-2D d and in H-2L d in association with NC1, and in the "37" gene but without NCI. NC2a is the H-2Kd-specific probe. NC2), is a single-copy probe for the Q10 gene, and NC2(~ is a low copy probe for the 27-1 gene and for three others in the same cluster. These classifications, when refined, may become useful for tracing lineages between members of the family. In this respect, we have noted that our pH-2d-12 cDNA clone corresponds to a transcript of the H-2Dd gene in which the polyadenylation site located in the B2 repeat was not used. The region between the B2 repeat and the polyadenylation site downstream (128 bp long) is thus called NC2~. It is interesting that NC2~ dis-

Cell 476

plays significant sequence homology with NC2~, NC2y, and NC26 (not shown). This suggests that the B2 element was inserted after gene duplication. Additional probes characterized in this work include oligonucleotides 5 (H2Dd-specific), 3, 4 (H-2Ld-specific) (Abastado et al., 1984)i and the second domain probe of pH-2d-37, which hybridizes only with the "37" gene and a related gene. Quantitation of H-2 Transcripts in the Liver Forty H-2 clones were isolated from a cDNA library of 30,000 recombinants (0.15%). The characterized H-2 clones distributed as 3 0 0 , 2 5 % , and 20% for transcripts of H-2K d, D d, and Ld, and distributed as 25% for the other three genes. Whether such figures are significant is, of course, questionable because both sample size and cDNA cloning may have induced a bias in the representation of the mRNAs. It should be remembered that liver is not a homogeneous tissue and that rare mRNAs (or cDNAs) might, in fact, be more abundant in a subset of cells. Alternate Splicing Alternate splicing in H-2 genes has long been suspected on the basis of nucleotide sequence comparisons between related cDNAs (Steinmetz et al., 1981b). Stronger evidence has been provided by Kress et al. (1983b) in the 3' region of the H-2 d gene, and by us (Lalanne et a! 1983b) in the 5' region of the same gene, where a complex pattern of double alternate splicing has been found. Proof, based on $1 mapping experiments, has been provided by Transy et al. (1984). Here we report evidence for alternate splicing in the Q10 gene, which generates a transcript devoid of the third exon. In the two instances we have studied (H-2K d and Q10), the noncanonical transcript is relatively rare (5%-10%) with respect to its canonical counterpart. Thus, cDNA clones from alternatively spliced transcripts could have easily been missed in the present survey. As an example, cDNAs representing H-2K d transcripts alternatively spliced in 3'as shown by Kress et al. (1983b) have not been identified here. Data in Figure 6B lead us to suspect that alternative splicing takes place in the 27.1 gene. Altogether, it is possible that other alternatively spliced transcripts of H-2K d and Q10, or of other genes exist but have not been isolated as cDNA clones. Could these alternatively spliced mRNAs represent mere splicing errors? If this were the case, the rate of error (5%-10%) would be rather high. Also, the double alternative splicing at the 5' end of the H-2K d gene is a complex event that maintains an open reading frame and involves consensus splicing sequences, which are well conserved among H-2 and HLA genes (Transy et al., 1984). It seems more likely, therefore, that alternate splicing serves to increase the diversity of H-2 and H-2-related polypeptides. Predicted Membrane Bound Proteins: A New Surface Antigen? The H-2K d, D d, and Ld surface antigens are expressed in liver. Accordingly, three classes of cDNA clones have been found. It may be noted that pH-2d-12, which has been assigned to H-2D d (as has the previously character-

ized clone, pH-2d-1) is almost identical in sequence with another plasmid, pAG64, isolated by Brickell et al. (1983). These authors, who found an increased level of mRNA hybridizing to pAG64 probe in a variety of tumor cells, proposed that pAG64 encodes an antigen of the Qa-Tla series. This hypothesis should be revised, in light of the assignment of pH-2d-12 to H-2D d, and their results might indicatean elevation of H-2D d expression in these tumor cells. The isolation of pH-2d-37 leads to the prediction that liver expresses a surface molecule with all the traits of a bona fide class I antigen but with a sequence significantly divergent in the second domain. Using a low copy probe derived from that region, we could show that the corresponding gene (temporarily called the "37" gene) displays no polymorphism of restriction sites. The genomic blots with SJL/J DNA show a displaced "37" band. Since neither the direction nor the magnitude of the displacement depend on the restriction enzyme used, we conclude that SLJ/J mice carry an insertion of about 500 bp, in or near the vicinity of the "37" gene. With this exception, the absence of restriction site polymorphism makes it impossible to map the "37" gene by Southern probing of the DNA of the usual recombinant mouse strains. In addition, the "37" gene is absent from the cosmid library of Steinmetz et al. (1982). The only gene that cross-hybridizes with the "37" probe is located in the Tla region. Because the organization of the Qa-Tla region shows tandem and inverted duplications in the H-2 b and in the H-2d haplotypes (Weiss et al., 1984; Steinmetz et al., 1982) it is plausible that the "37" gene lies next to its cross-hybridizing gene in the Tla region. TL antigens encoded in this region are more distantly related to H-2K, D, L, and Qa-2 antigens than they are to each other, as judged from peptide maps and from available sequences (Solosky et al., 1982); so would be the "37" product, as judged from its nucleotide and deduced amino acid sequence. The "37" probe detects mRNA with the expected size in liver, spleen, and kidney. Dot spot hybridization shows mRNA in thymus. Because the probe cross-hybridizes with a second gene, we cannot ascertain that the "37" gene is expressed in all four tissues. At this stage, however, our results raise the interesting hypothesis that a nonpolymorphic class I molecule is present at the surface of a variety of cells, perhaps as ubiquitously in the animal as are H-2 antigens themselves. In this case, the "37" polypeptide should not correspond to any of the previously identified Qa or Tla antigens, since the expression of the latter has been shown to be limited to certain tissues (Flaherty, 1980). Predicted Secreted Molecules The nonpolymorphic Q10 gene is specifically transcribed in the liver and produces a polypeptide that can be found in the serum (Cosman et al., 1982b; Mellor et al., 1984; Maloy et al., 1984). Our results confirm that Q10 is not polymorphic and is specifically transcribed in the liver. In addition, the isolation of plasmid pH-2d-19 predicts the existence of a molecule carrying a first domain directly connected to a third domain, somewhat similar in strut-

Liver Transcripts of Class I Genes 477

ture to the extracellular part of class II molecules. If no determinant important for secretion is located in the second domain, this molecule is likely to be secreted, like the canonical Q10 product, but with a smaller molecular weight. Inspection of immunoprecipitates with the antiserum raised against a synthetic peptide corresponding to the NH2-terminal part of the Q10 product shows only one band (Maloy et al., 1984). However, since the noncanonical mRNA is present in lower amounts, the shortened polypeptide may have escaped detection. The 27.1 gene isolated by Steinmetz et al. (1981b) was believed to be a p s e u d o g e n e on the basis of two structural criteria, the occurrence of a stop codon in exon 5 (usually coding for the transmembrane region), and the existence of an abnormal splice junction bordering exon 7. However, the Q10 gene example shows that such a structure can encode a protein product that is secreted. Indeed, a functional m R N A is produced by splicing out the sequence between exons 5 and 8. Isolation of pH-2d-35 cDNA, apparently the transcript of the 27.1 gene, suggests that this gone, as well as Q10, could in fact code for a secreted protein, The similarity between the Q10 and the 27.1 genes, which are both located in the same cluster (Weiss et al., 1984), might even be greater if the existence of a 27.1 transcript lacking exon 3, suggested by our $1 mapping experiments, was demonstrated. Since the mRNA class defined by pH-2d-35 is not liver-specific, the two genes do not, however, appear to be coordinately regulated.

What Functions for Predicted Molecules? In the H-2 multigene family, there are presently more genes than there are identified protein structures. It is noteworthy that our studies, as well as those showing the existence of a secreted product of the Q10 gene, point to the existence of several other nonpolymorphic class I products. The only well documented function of class I transplantation antigen is their involvement in cell-cell recognition processes. This involvement could be demonstrated because of their polymorphism, which led to reveal the phenomenon of H-2 restriction and allowed, in practice, specific serological reagents to be raised. Nonpolymorphic class I products, membrane-bound or secreted, could also be involved in interactions between cells (by direct contact or via diffusible [secreted] products) and would not have been detected. This raises the hypothesis that class I products in general could participate in a broad set of cellular interactions of which only a minor set has been recognized to date, by virtue of the polymorphic traits of a few class I products.

Experimental Procedures Screening by In Situ Hybridization The cDNA clones were screened by in situ colony hybridization as described earlier (Lalanne et al., 1983a). The cosmid clones of Steinmetz et al. (1982) were screened by in situ colony hybridization in the same way. On occasion, the oligonucleotide probes described in Abastado etal. (1984) were also used under the conditions described by these authors.

Preparation of DNA and RNA Plasmid and cosmid purifications were performed according to the procedure of Birnboim and Doly (1979) modified for large scale purifications. Genomic DNA was isolated from mouse livers as described by Blin and Stafford (1976).All mice used were bred at the Pasteur Institute. Total RNA from tissues was prepared using the LiCI procedure described by Auffray and Rougeen (1980), Poly(A)*mRNA was purified by chromatography on oligo(dT) cellulose. Southern Blot Analysis Restricted DNA (genomic DNA or cosmid DNA) was separated by electrophoresis on 0.7% agarose gels in Tris acetate buffer, and was then transferred to nitrocellulose filters as described by Southern (1975). DNA probes were labeled by nick translation to a specific activity of 1-5 x 108 cpm/~g DNA. The filters were hybridized for 20 hr at 68°C in 5x Denhardt's (0.1% each of bovine serum albumine, Ficoll type 500, and polyvinyl pyrrolidone), 6x SSC (0.9% NaCI, 0.9 M sodium citrate), 1 mM EDTA,0.5% SDS, and 100/~g/m! denatured salmon sperm DNA. Washes were made in 0.1 SSC and 0.5% SDS for 2 hr at 68°C. Northern Blot Analysis Poly(A)÷ RNA (3/~g) denatured with glyoxal was separated by eiectrophoresis on 1% agarose gels in 10 mM phosphate buffer, pH 7. Transfer onto nitrocellulose filters was then performed as described by Thomas (1980). The filters were hybridized for 24 hr at 42°C in l x Denhardt's, 50% deionized fermamide, 5x SSC, 0.1% SDS, 50 mM phosphate buffer (pH 6.5), and 250/~g/ml denatured salmon sperm DNA. Two stringent washes were made in 0.1 SSC and 0.1% SDS at 50°C for 15 min. $1 Mapping Analysis Aliquots of the probes 5'-end*labeled by y exchange reaction (Maxam and Gilbert, t980) with polynucleotide kinase were coprecipitated with 3/~g of the poly(A)+ mRNA (or 50-100 ), of total RNA) to be analyzed. The pellet was resuspended in 40/~1of hybridization buffer containing 0.4 M NaCI, 40 mM PIPES (pH 6.4), 1 mM EDTA, and 80% formamide. After heating 10 min at 85°C, the samples were incubated 16 hr at 60°C for hybridization. $1 nuclease digestion was performed fer 2 hr at 37°C with 3000 units of enzyme in 300 #1 of buffer containing 0.2 M NaCI, 30 mM NaAc (pH 4.5), and 3 mM ZnAc (Favaloro et al., 1980). After phenol extraction and ethanol precipitation, the samples were resuspended in 2/~1 of formamide and were subjected to electrophoresis in an 8% acrylamide-urea gel. DNA Sequencing Nucleotide sequences of pH-2d-12 Pst i fragments were determined by the dideoxy sequencing technique (Sanger et al., 1980), with M13 mp701 as cloning vector, constructed by Dr. D. R. Bentley (personal communication). All other sequences were determined by the Maxam and Gilbert (1980) procedure. Enzymes and Reagents All enzymes were purchased from Boehringer Mannheim (ER.G.) or from Biolabs (Bishops, Starford, U.K.). Labeled nucleotides were from Amersham (U.K.). Nitrocellulose filters were obtained from Schleicher and SchLill (B85) (Dassel, F.R.G.). Agarose was from Sigma Chemical Co. (St. Louis, Missouri, U.S.A.). Oligo(dT) cellulose (type 3) was from Collaborative Research Inc. Acknowledgments We are grateful to many colleagues in the laboratory for helpful advice and for stimulating discussions, particularly Dr. G. Gachelin, Dr. M. Cochet, and Dr. A. Israel. We are indebted to Dr. M. Steinmetz, who kindly provided the cosmid clones carrying mouse class I genes, and to Dr. S. Kvist, who gave us the H-2Kd gene. We also thank Dr. R Candido and Dr. P. Baldacci for correcting the manuscript and Mrs. V. Caput for her invaluable help in preparing it. J.-L. L. was supported by the Elf Bie-recherches Co., and C. T., by a fellowship of the "Programme Mobilisateur, Essor des Biotechnologies~' S. G. was supported on a France-Quebec exchange program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

Cell 478

marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received November 12, 1984; revised March 11, 1985 References

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