]11111111110-
lmmunogenetics 37." 49-56, 1992
geneOs
© Springer-Verlag 1992
Transmembrane and cytoplasmic domain sequences demonstrate at least two expressed bovine MHC class I loci Shirley A. Ellis, Karen A. Braem, and W. Ivan Morrison Institute for Animal Health, Compton, Nr Newbury, Berks. RG16 ONN, UK Received February 3, 1992; revised version received March 30, 1992
Abstract. We have used the polymerase chain reaction to amplify cDNA from expressed bovine major histocompatibility complex class I genes. Sequences obtained from transmembrane and cytoplasmic domains were used to identify the number of expressed alleles. Data from three animals suggest that there are four major expressed alleles, representing the products of two (or more) loci. We have also demonstrated the presence of an alternatively spliced mRNA, which has been observed in five animals. The alternative splicing removes exon 7 (the major site of class I phosphorylation), which predicts a truncated molecule with a cytoplasmic portion 16 amino acids shorter than usual. This phenomenon was detected for only a single class I allele within each individual.
Introduction Class I major histocompatibility complex (MHC) antigens are highly polymorphic, membrane bound glycoproteins whose main function is to present peptides derived from foreign or self proteins to cytotoxic T lymphocytes (CTL; Yewdell et al. 1988). That expressed MHC molecules exhibit a high degree of protein polymorphism is central to their role in presenting a diverse range of peptides, thereby providing protection (at the population level) against evolving pathogens. This sequence polymorphism results in differences in the detailed shape and charge distribution in the antigen binding site, but does not significantly alter the overall structure, which appears to be well conserved between mammalian species. The selective advantage of polymorphism at the class I loci is clear, but the increase in antigen presenting capacity may be offset by a reduction in T-cell repertoire (Matzinger et al. 1984). This may account for the small number of functional class I loci (usually only two or Correspondence to: S.A. Ellis.
three) seen in all species studied Robinson and Kindt 1989, although there may be additional genes coding for less ubiquitously expressed nonclassical MHC molecules, e. g., mouse Qa, human HLA E, G (Stroynowski 1990). In cattle, the Bota complex is presently defined serologically as a singlle allelic series coding for 32 internationally defined allo-specificities (Bull 1989). Information regarding the genetic basis for these specificities was limited, until the recent publication of five bovine class I cDNA sequences (Ennis et al. 1988; Brown et al. 1989; Bensaid et al. 1991). There is evidence from 3' sequence comparisons and transfection experiments that these are encoded by at least two loci (Ennis et al. 1988; Toye et al. 1990; Bensaid et al. 1991); however there has been no systematic attempt to confirm the number of expressed products. The aim of this study was to establish how many expressed MHC class I alleles were present in individual cattle, and to estimate the number of MHC loci responsible, by using polymerase chain reaction (PCR) to amplify a region encompassing the transmembrane domain (TM), the cytoplasmic domain (C), and part of the 3' untranslated (UT) region. This region was chosen because it was possible to design appropriate primers based on conserved regions (in exon 4 and 3'UT region), maximizing the chance of amplifying all alleles present. Also, these regions have been shown in other species to possess locusspecific characteristics (Gussow et al. 1987) which would aid in the analysis of results.
Materials and methods Animals, MHCphenot~ping, and cell lines: All cattle used in this study were Friesian Holsteins. Peripheral blood mononuclear cells (PBMC) were Bota typed using altoantisera, in a microlymphocytotoxicity assay (Kemp et al. 1988), performed by Roger Spooner (IAPGR Edinburgh). PBMC were immortalized with the intracellular protozoan parasite Theileria annulata as described previously (Glass and Spooner 1990). The resulting cell lines were maintained in RPMI 1640 (Imperial,
50
S.A. Ellis et al. : Two expressed bovine MHC class I loci
Andover, UK) containing 10% fetal calf serum (FCS; Gibco Paisley, Scotland). The expression of MHC class I did not alter over periods of prolonged culture, as determined by isoelectric focusing (IEF), and as demonstrated previously (Spooner and Brown 1980).
MHC class I cDNA sequences, and were made using an Applied Biosystems 381 synthesizer (sequences shown in Fig. 1). These were used to amplify the TM an C class I domains present in eDNA from individual animals. PCR amplification was performed using a Programmable Thermal Controller (MJ Research, Boston, MA) and was carried out in a buffer containing 10 mM Tris HCI, 4 m M MgC1, 0.5 mM of each of dGTP, dATP, dTTP, dCTP, 1 IxM of each oligonucleotide primer, and 2.5 units of Taq DNA polymerase (Promega, Southampton, UK). An annealing temperature of 50 °C was used, followed by 35 cycles with the following conditions: 94 °C (1 rain), 50 °C (1 rain), 72 °C (3 min), with a last extension reaction of 72 °C for 10 min. In some experiments the eDNA was predigested with the restriction enzyme Ban II for 2 h prior to PCR; conditions were otherwise identical.
Preparation of eDNA. Messenger RNA (mRNA) was prepared directly from T. annulata-infected cell lines, by the method of Badley and coworkers (1988). eDNA was then prepared using a DNA synthesis system (BRL, Gaithersburg, USA).
Amplification by PCR and sequence analysis of TM and C domains. Oligonucleotide primers (18 mer) were designed, using published bovine a
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t Awl0 BL3-6 BL3-7 KN104
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10 times), compared with the published sequences A w l 0 (Bensaid et al. 1991), BL3-6 and BL3-7 (Ennis et al. 1988). In the TM region the sequences fall into two groups based on size, the A w l 0 / B L 3 - 6 group (37 amino acids) and the BL3-7 group (35 amino acids). The cytoplasmic region is 28 amino acids in all cases. The D19 sequences are all similar to the published sequences (-D19.2 is identical to A w l 0 in both TM and C domains). It is noticeable that variations between alleles are localized to particular positions, leaving other areas completely conserved. These sequences, and others obtained from five additional animals (data not shown), appear to belong to two loci. This is considered further, together with the KN104 sequence (Bensaid et al. 1991), in the Discussion. In addition to the four sequences shown (1319.1D19.4) we obtained a small number of aberrant sequences, some presumed to be generated by the PCR and others which required further investigation.
Analysis of amplified products. Amplificationproducts were examined on a 2% agarose gel, and DNA for further analysis was eluted from a low meltingpoint agarose gel. The DNA was the kinased and blunt end ligated into M13 mp18 (Maniatiset al. 1982). The dideoxy method of Sanger and co-workers (1977) was used to obtain the nucleotidesequence of a random selectionof clones, using a sequenasekit from US Biochemicals (Cleveland, OH).
Preparation of genomic DNA. GenomicDNA was prepared from T. annulata-infected cell lines as described by Maniatis and co-workers (1982). Amplificationof genomic DNA was carried out as for cDNA, using oligonucleotideprimers as shown in Figure 1, and described in Results.
Biosynthetic labelingof cells, immuonprecipitation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), T. annulatainfectedcell lines were incubatedat 37 °C for 30 min in methionine-free RPMI (Gibco) plus 10% FCS (Gibco), then for 4-6 h with 3Ss methionine(NEN) at 100 gCi to 5-10 x 106 cells. Fresh PBMCs were labeled for 16 h. To label cell products with 32p, cells were washed in phosphate-free medium (Eagles MEM, Flow), then resuspended at 2 x 106 cells per ml in the same mediumcontaining 10% FCS, and incubated at 37 °C for 45 min. 32p orthophosphate (NEN) was then added to a concentrationof 40 gCi/ml, and incubationcontinuedfor 3-6 h at 37 °C. After labeling,cells were lysed in 1 ml of buffer containing 50 mM Tris, 5 mM MgC12, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 0.5 % Nonidet P-40. Lysates were precleared using formalin fixed Staphylococcusaureus (Novabiochem, Nottingham, UK). MHC class I molecules were immunoprecipitated with the monoclonal antibody (mAb) ILA19 (Bensaidet al. 1989) which recognizesbovine class I a chain together with/32m. Immunoprecipitatedproducts were examined by SDS-PAGEas described by Laemmli (1970). Gels were dried and autoradiographed.
Identification o f an unusual pre-mRNA splicing pattern. In addition to the sequences shown in Figure 2, two (identical) D19 clones showed a truncated sequence which missed 48 base pairs (bp) from near the 3' end (Fig. 3). Because the sequence resumed with the last two bp before the TGA stop codon, and in other species class I exon 8 often consists of two bp, it seemed probable that this represented an alternatively spliced form of the gene, rather than a PCR artifact. This phenomenon was also observed in class I sequences derived from early fetal material (data not shown). Since no bovine class I genomic sequences are available, oligonucleotide primers were devised which could be used to amplify the regions of genomic DNA thought to contain the relevant introns (Fig. 1, primer pairs 4 and 3; 4' and 5).
Results PCR amplification o f bovine M H C class I exons 5-8. A T. annulata-infected cell line derived from animal D19 was used to prepare cDNA. The animal was serologically MHC class I typed as w l 1; w14 and family studies showed that the w l l and w14 specificities were inherited from the dam and sire respectively. MHC class I exons 5-8 were amplified by PCR, using oligonucleotide primers
Transmembrane region Awl0 BL3-6 D19.1 D19.2
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Fig. 2. The deduced amino acid sequence of Awl0 is shown (in the TM and C domains), and compared with BL3-6, BL3-7, KN104, and four DI9 clones, D19.1-D19.4. Dashes indicate identity, asterisks are deletions introduced to maximise homology.
52
S.A. Ellis et al.: Two expressed bovine MHC class I loci
a) D19.3 Exon 6 GGT GAA AAA GGA
GCC
CAG
CGG
ATC TAC ACC CAG GCT GCA
GGC TCT GAT GTG TCT CTC ACG GTT Ban II
CCT
Exon 7 AGT GAC AGT
A IGC
Exon 8 G] TT T G A
AAA
3'UT
Fig. 3. a The nucleotide sequence of clone D19.3 is shown. Indicate intron exon boundaries. The Ban II restriction site is indicated in exon 7; b shows the truncated form of D19.3.
b) D19.3 Short Exon 6 GGT GAA
Exon 8 AAA
GC~
CGG
ATC
TAC
ACC
CAG
GCT
GCA
Genomic DNA was prepared from a T. annulatatransformed cell line, from animal D 18. This is a half sibling of D19, and is MHC class I identical by serology and 1D-IEF. PCR with Oligos 4' and 5 resulted in a product of approximately 160 bp, and with Oligos 3 and 4 a product of approximately 200 bp. DNA was extracted and sequenced as before. Ten clones were sequenced, and each demonstrated the presence of an intron, with the characteristic nucleotide pairs GT and AG at the beginning and end. The positions of the introns are as shown in Figure 1, and the sizes are 136 bp and 164 bp respectively. Since they occur in the positions predicted by examination of MHC genomic sequences from other species, we assumed that the splice sites would be conserved in all alleles, and did not attempt to sequence these introns from every class I gene present. This result confirmed our observation as alternative splicing.
Identification of alternatively spliced class I pre-mRNAs using PCR. Having established that alternative splicing was occurring in at least one allele in animal D19, we wanted to find out if it was a general phenomenon, occurring in other alleles, in all individuals. Because the alternatively spliced sequence was only isolated twice in more than sixty clones, it seemed likely that the frequency would be low. It might therefore be difficult to detect, without sequencing a very large number of clones from each individual. In all of the available bovine class I sequences there is a Ban II restriction site in exon 7 (Fig. 3). This site does not occur anywhere else in the region which we are amplifying, i.e., exons 5-8. Therefore, digestion of cDNA with Ban II before PCR should eliminate most of the normally spliced product, but leave the alternatively spliced version intact, resulting in a relative increase in the amplification of truncated sequences. The experiment was carried out using cDNA from T. annulata lines from four animals: D18, D19, D595, and D602. A gel showing PCR products following Ban II digestion is shown in Figure 4. All four animals showed the predicted smaller band (approximately 200 bp), cor-
A ITT T G A
3' U T
D18 a
D19 b
.a
b
D595
D602
a
a
b
b
-250bp
--
~"
--~-----200bp
Fig. 4. A 2 % agarose gel showing PCR products from cDNA amplified with Oligos 2 and 3. In each case track a shows the product from undigested cDNA, track b the product from cDNA following Ban II digestion.
responding to a truncated product. DNA was extracted from this band in each case, and was subcloned into M13 for sequencing. The results showed that the DNA present in the 200 bp bands consisted exclusively of truncated sequences (Fig. 5). In each individual there appears to be one predominant allele which is present in the alternatively spliced form. In D19, D18, and D602 the sequences are identical, and correspond to the D19.3 allele. In animal D595 it is a different allele which is truncated [although it appears to belong to the same locus ('B') as Cytoplasmic domain
D19.3
Exon 6 GEKGRIYTQAA
Exon 7 SSDSAQGSDVSLTVPK Phosphorylation site
GEKGRIYTQAA GEKGRIYTQAA GEKRQTYTQAA GEKGRIYTQAA
I I I I
a
D19.3 DI8 D595 D602
Short Short Short Short
__) Exon 8 V
b Fig. 5. a Deduced amino acid sequence of the cytoplasmic domain of clone D 19.3. Gaps indicate exon boundaries. The expected phosphorylation site is indicated in exon 7; b shows the truncated clone found in each animal, D19, D18, D595, and D602.
S.A. Ellis et al.: Two expressed bovine MHC class I loci
53
the D19.3 allele]. The removal of exon 7 also results, in each case, in the substitution of an isoleucine residue for the usual valine, as the terminal amino acid.
to 35S-methionine labeled cells, confirming that a range of heavy chains is reaching the surface (data not shown).
Alternative class I transcripts are synthesized, and can be distinguished by differences in size and phosphorylation. In order to determine whether the alternatively spliced transcripts generate proteins, cells [T. annulata lines or fresh peripheral blood lymphocytes (PBL)] were labeled with either 35S-methionine or 32P-orthophospate (NEN), and class I heavy chains were immunoprecipitated using the mAb ILA19. Analysis of the products by SDS-PAGE demonstrated that 35S-methionine labeled products appeared as a cluster of bands, ranging in size fi'om Mr 41 000-47 000 (Fig. 6). This was seen most clearly using an 8 % gel (Fig. 6b). No 3:p was detected in the minor low M r product (41000). This result suggests that there is synthesis of a range of class I heavy chains, and that those with lower relative mass are not phosphorylated. It is thought that MHC class I molecules become phosphorylated at or near the cell surface, so the 32p-labeled products are likely to represent the phoshporylated molecules seen at the surface rather than intracellularly. Phosphorylation itself can affect the mobility of molecules during SDS-PAGE, and the existence of a mixture of phosphorylated and unphosphorylated molecules of the same molecular relative mass could therefore give rise to a range of bands. However this phenomenon alone seems unlikely to account for the wide size range which we consistently observed. Surface labeling of cells with 125I, followed by immunoprecipitation with ILA19, gave an identical pattern
Discussion
Fig. 6. a ImmunoprecipitatedMHC class I heavychains from D18 and D602 T. annulatainfected cells, labeled with 35S methionine(track 1), or 32porthophosphate(track2), run on a 10% SDS polyacrylamidegel. Relative molecular mass is indicated, b As a except an 8% gel.
Bovine MHC class I cDNAs have been cloned and sequenced (Ennis et al. 1988; Brown et al. 1989; Bensaid et al. 1991) but as yet the total number of expressed genes has not been established. The limited data available suggest that there are at least two loci (Ennis et ah 1988; Toye et al. 1990; Bensaid et al. 1991). However, conventional cDNA library screening would not necessarily reveal additional genes which may be expressed at low levels, without extensive and repetitive sequencing. For this reason we decided to utilize the PCR technique as an initial approach, and to look only at the TM and C domains, using a small number of animals. These regions have been shown in other species to demonstrate locus-specific characteristics (Gussow et al. 1987; Parham et ah 1988), and since together they consist of less than 300 bp, it is feasible to sequence rapidly large numbers of clones. One disadvantage of using this technique to amplify a mixture of very similar sequences is the occurrence of 'PCR artifacts', described in detail elsewhere (Ennis et al. 1990; Lawlor et al. 1991). However, because we sequenced large numbers of clones, it was relatively easy to identify the few sequences which demonstrated crossing-over between one clone and another. The results we obtained support the published data, in that we found evidence for two expressed loci, i.e., four alleles ( > 10 clones of each). Allowing for allelic variation, it was still easily possible to assign sequences to an 'A' or 'B' locus in each of the individuals we examined. We investigated three animals in detail ( > 40 clones), and another three in less detail ( < 20 clones). We always found approximately equal numbers of clones from each of the four alleles, suggesting that the two loci give rise to products which are expressed at similar levels (Fig. 2). The ' A ' locus sequences, which include those of the previously published cDNAs, Awl0 (Bensaid et al. 1991) and BL3-6 (Ennis et al. 1988), have a TM region consisting of 37 amino acids. The 'B' locus sequences, which include BL3-7 (Ennis et al. 1988), have a TM region of 35 amino acids, with the 'missing' residues always in the same position. Although there does appear to be ' A ' and 'B'-specific sequences in both the TM and C domains, the TM length will almost certainly prove to be the best diagnostic feature. We also found several aberrant sequences in each animal (occurring one or two times each) which contained nucleotide differences leading to amino acid substitutions. These were not easy to assign to PCR artifact, and could represent products of additional loci, expressed at much lower levels (comparable, for example, to HLA C). The
54 number of bands seen in IEF analysis of bovine class I does not exclude the possibility of a third expressed locus (data not shown). We are currently using PCR to look for these sequences in class I clones isolated from cDNA libraries made from these individuals. The cDNA sequence KN104 (Bensaid et al. 1991, and Fig. 1) complicates our straightforward interpretation. Bensaid and co-workers, when discussing the previously published class I sequences, suggest that Awl0, KN104, and BL3-7 may represent the products of three different loci. Awl0 and KN104 are both abundantly expressed class I molecules which function in presentation of antigen to CD8 ÷ T lymphocytes (Toye et al. 1990). KN104 has a TM region of 36 amino acids, with the 'missing' one in a different position to those seen in the putative 'B' locus sequences shown in Fig. 2. There are several possible explanations for this, one being that the 'B' locus is not as clearly defined as we have suggested, and that if we examined more animals we would find 'B' locus sequences that look like KN104. Another is that KN104 represents a third, 'C' locus, which we have yet to identify in our animals, as suggested by Bensaid and co-workers (1991). This seems unlikely given the number of clones we have sequenced, and the high levels of expression of KN104 (Kemp et al. 1990). The third explanation concerns the fact that both Awl0 and KN104 were cloned from an African Bos indicus animal, whereas all the others are from Bos taurus animals. These two types of cattle, which are distinguished by the presence of a hump in the Bos indicus, are considered as separate subspecies, although they readily interbreed. Bos indicus cattle appeared relatively recently (judged by archaeological evidence) and may have been selectively bred for agricultural purposes. They initially occurred in Asia (4-5000 years BC), and were not introduced by humans into Africa until approximately 2000 years ago (Epstein 1971). Since the Bos indicus cattle are presumed to be descended from the Bos taurus line (very recently in evolutionary terms) they would not be expected to have markedly different MHC genes; however, it is possible that selection pressure by particular pathogens has led to a high incidence of some class I alleles in African breeds of cattle. It should be possible to resolve some of these points by investigating further Bos indicus animals. Bensaid and co-workers (1988) demonstrated size heterogeneity in bovine class I heavy chains, following SDS-PAGE and western blotting. They suggested that at least some of these differences may be due to post transcriptional modifications. Our results, both SDS-PAGE (Fig. 6) and 1D-IEF (data not shown) also indicate the presence of multiple class I heavy chain isoforms. We have demonstrated that alternative splicing, resulting in removal of exon 7, occurred in some alleles (at a single locus), and that this may account for some of the heterogeneity seen.
s.A. Ellis et ah: Two expressedbovine MHC class I loci Several instances of class I alternative splicing have been described involving, in the mouse for example, H-2K b, H-2K q, H-2K k, H-2D d, and H-2L ° (Lew et al. 1987; Vogel et al. 1989), in human HLA A24, HLA A2 (Krangel 1986), and possibly HLA C (Mizuno et al. 1989), and in a sheep class I gene (Grossberger 1990). Most of these examples involve exons 7 and/or 8, and lead to truncated molecules, although there are a few examples involving the 5' end of the gene (Lalanne et al. 1985; Transy et al. 1984), and in the sheep the alternative splicing results in a longer molecule. Many of the nonclassical class I genes also demonstrate alternative splicing of exons 5-8 (Lew et al. 1987; Ulker et al. 1990). One reason why alternative splicing seems to occur quite frequently around exons 7 and 8 may be that they are small (2-48 bp). It has been suggested (Dominski et al. 1991) that the length of internal exons plays an important role in splice site selection. Internal exons less than 50 bp are rare ( < 4 % ) , and are more likely to be involved in 'exon skipping'. However, other factors, such as the length of the upstream polypyrimidine [poly(Y)] tract are also involved. This could explain why exon 6, which is even shorter (33 bp) is rarely spliced out. Examination of intron sequences may help to explain why particular alleles seem more susceptible to alternative splicing. In most cases described the alternatively spliced transcript is rare, which makes detection difficult. Despite this it could be a fairly widespread phenomenon, resulting in limited expression of altered molecules. Also, it may occur more frequently in certain cell types as in the case of H-2K u (Lew et al. 1986). Speculation as to the functional significance of these molecules is difficult since the role of the intracytoplasmic portion of full-length class I molecules is poorly understood. Comparison across species has shown that exon 7 is particularly well conserved, and the region indicated in Figure 5 (Ser-Asp-Val-Ser-Leu) is the usual site(s) ofphosphorylation (Guild et al. 1984). Phosphorylation-dependent signaling by class I molecules could be important during particular stages of development, or in particular tissues. Transfection studies with alternatively spliced class I molecules (Zuniga and Hood 1986) indicate that structural variations in the cytoplasmic domain can lead to use of distinct pathways of intracellular processing, and can effect the way the molecules interact with the cytoskeleton. Since class I aggregation in the membrane may be important in T-cell recognition, the presence of significant levels of a truncated class I allele could result in failure to elicit good in vivo CTL responses to certain pathogens. Although some studies suggest that the occurrence of alternative splicing of class I mRNAs is random (Vogel et al. 1989), indicating no specific function, other examples lead to the opposite interpretation. In the case of
S.A. Ellis et al.: Two expressed bovine MHC class I loci s o m e n o n c l a s s i c a l class I g e n e s t h e o c c u r r e n c e o f altern a t i v e s p l i c i n g is r e l a t e d to t h e a c t i v a t i o n state o f t h e cell, for e x a m p l e Q a - 2 w h e r e a c t i v a t i o n c a n r e s u l t in a n inc r e a s e in a s e c r e t e d f o r m o f t h e m o l e c u l e ( U l k e r et al. 1990). I n the h u m a n n o n c l a s s i c a l class I g e n e , HLA G, t h e r e is a stop c o d o n in e x o n 6, a n d e x o n 7 is spliced out, g i v i n g rise to a t r u n c a t e d m o l e c u l e (Ellis et al. 1990). T h e p r e s e n c e o f b o t h m e c h a n i s m s s u g g e s t that t h e t r u n c a t e d c y t o p l a s m i c tail m a y b e f u n c t i o n a l l y i m p o r t a n t . We have shown that there are four major expressed M H C class I alleles in h e t e r o z y g o u s B o s t a u r u s cattle, w h i c h a r e p r e s u m e d to r e p r e s e n t the p r o d u c t s o f t w o loci. I n a d d i t i o n w e h a v e d e m o n s t r a t e d a l t e r n a t i v e splicing inv o l v i n g e x o n 7 in at l e a s t t w o alleles. B o t h alleles a p p e a r to b e l o n g to t h e s a m e l o c u s - t h e s i g n i f i c a n c e o f this is at p r e s e n t u n k n o w n . F u r t h e r studies a r e u n d e r w a y o n o t h e r b r e e d s o f B o s t a u r u s , a n d B o s i n d i c u s cattle, to e s t a b l i s h w h e t h e r t h e r e are a l w a y s t w o e x p r e s s e d class I loci, a n d w h e t h e r in s o m e cases t h e r e are t h r e e , as m i g h t b e i n d i c a t e d b y t h e K N 1 0 4 sequence. Acknowledgments'. We would like to thank Dr. Roger Spooner for
serological typing of the cattle used in this study, Dr. Duncan Brown for provision of 7". sporozoites to establish cell lines, and Dr. John Young for helpful discussion and critical reading of the manuscript. This work was supported by the Agricultural and Food Research Council of Great Britain.
References Badley, J.E., Bishop, G.A., St. John, T., and Frelinger, J.A.: A simple, rapid method for the purification of Poly A + RNA. Biotechniques 6:114-116, 1988 Bensaid, A., Naessons, J., Kemp, S.J., Black, S.J., Shapiro, S.Z., and Teale, A.J.: An immunochemical analysis of class I (BoLa) molecules on the surface of bovine cells. Immunogenetics 27: 139-144, 1988 Bensaid, A., Kaushal, A., MacHugh, N. D., Shapiro, S. Z., and Teale, A. J.: Biochemical characterization of activation-associated bovine class I MHC antigens. Anim Genet 20: 241-255, 1989 Bensaid, A., Kaushal, A., Baldwin, C. L., Clevers, H., Young, J. R., Kemp, S. J., MacHugh, N. D., Toye, P. G., and Teale, A. J. : Identification of expressed bovine class I MHC genes at two loci and demonstration of physical linkage. Immunogenetics 33: 247-254, 1991 Brown, P., Spooner, R. L., and Clark, A. J.: Cloning and characterization of a BoLa class I cDNA clone, lmmunogenetics 29: 58-60, 1989 Bull, R.W.: Joint Report of the Third International bovine Antigen (BoLa) Workshop, Helsinki, 1986. Anita Genet 20: 109-132, 1989 Dominski, Z. and Kole, R.: Selection of splice sites in pre-mRNAs with short internal exons. Mol Cell Biol 11: 6075-6083, 1991 Ellis, S. A., Palmer, M. S., and McMichael, A. J.: Human trophoblast and the choriocarcinoma cell line BeWo express a truncated HLA class I molecule. J Immunol 144: 731-735, 1990 Ennis, P.D., Jackson, A.P., and Parham, P.: Molecular cloning of bovine class I MHC cDNA. J lmmunol 141: 642-651, 1988 Ennis, P. D., Zemmour, J., Salter, R, D., and Parham, P.: Rapid cloning of HLA-A, B cDNA by using the polymerase chain reaction:
55 frequency an nature of errors produced in amplification. Proc Natl Acad Sci USA 87: 2833-2837, 1990
Epstein, H.: Origin of the domestic animals of Africa. Africana Publishing Corporation, NY, 1971 Glass, E.J. and Spooner, R. L.: Parasite-accessory cell interactions in Theileriosis. Eur J lmmunol 20: 2491-2497, 1990 Grossberger, D., Hein, W., and Marcuz, A.: Class I MHC cDNA clones from sheep thymus: alternative splicing could make a longer cytoplasmic tail. Immunogenetics 32: 77-87, 1990 Guild, B.C. and Strominger, J.L.: Human and murine class I MHC antigens share conserved serrine 335, the site of HLA phosphorylation in vivo. J Biol Chem 259: 9235-9240, 1984 Gussow, D., Rein, R. S., Meijer, I., de Hoog, W., Seemann, G. H. A., Hochstenbach, F. M., and Ploegh, H. L.: Isolation, expression and the primary structure of HLA-Cwl and HLA-Cw2 genes: evolutionary aspects. Immunogenetics 25: 313-322, 1987 Kemp, S. J., Spooner, R. L., and Teale, A. J.: A comparative study of MHC antigens in East African and European cattle breeds. Anita Genet 19: 17-29, 1988 Kemp, S. J., Tucker, E. M., and Teale, A. J.: A bovine monoclonal antibody detecting a class I BoLA antigen. Anim Genet 21: 153-160, 1990 Krangel, M. S.: Secretion of HLA-A and -B antigens via an alternative RNA splicing pathway. J Exp Med 163: 1173-1190, 1986 Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970 Lalanne, J.-L., Trausy, C., Guerin, S., Darche, S., Meuhen, P., and Kouritsky, P. : Expression of class I genes in the mouse MHC: identification of eight distinct mRNAs in DBA/2 mouse liver. Cell 41: 469-478, 1985 Lawlor, D.A., Dichel, C.D., Hauswirth, W.W., and Parham, P.: Ancient HLA genes from 7500 year old archeological remains. Nature 349: 785-787, 1991 Lew, A.M., Margulies, D.H., Maloy, D.H., Lillehoj, W.L., McCluskey, J., and Coligan, J.E.: Alternative protein products with carboxyl termini from a single class I gene, H-2I(°. Proc Natl Acad Sci USA 83: 6084-6088, 1986 Lew, A.M., McCluskey, J., Maloy, W.L., Margulies, D.H., and Coligan, J.E.: Multiple class I molecules generated from single genes by alternative splicing of pre-mRNAs, lmmunol Res 6: 117-132, 1987 Maniatis, T., Fritsch, E. F., and Sambrook, J.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982 Matzinger, P., Zamoyska, R., and Waldmann, H. : Self tolerance is H-2 restricted. Nature 308: 738-741, 1984 Mizuno, S., Ng, J., Dupont, B., and Yang, S. Y.: The cloning, exon shuffling, and expression of the HLA Cwl gene. In B. Dupont (ed.): Immunobiology of HLA, vol 2. pp. 138-140, Springer-Verlag, New York, 1989 Parham, P., Lomen, C.E., Lawlor, D.A., Way, J.P., Holmes, N., Coppen, H.L., Salter, R.D., Wan, A.M., and Ennis, P.D.: Nature of polymorphism in HLA-A, -B and -C molecules. Proc Natl Acad Sci USA 85: 4005-4009, 1988 Robinson, M. A. and Kindt, T. J. : Major histocompatibility complex antigens and genes. In W. E. Paul (ed.): Fundamental Immunology. pp. 489-539, Raven Press, New York, 1989 Sanger, F., Nicklen, S., and Coulson, A.R.: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467, 1977 Spooner, R.L. and Brown, C.G.D.: Bovine lymphocyte antigens (BoLA) of bovine lymphocytes and derived lymphoblastoid lines transformed by Theileria parva and Theileria anulata. Parasite lmmunol 2: 163-174, 1980 Stroynowski, I. : Molecules related to class I MHC antigens. Annu Rev Immunol 8: 501-530, 1990
56 Toye, P.G., MacHugh, N.D., Bensaid, A . M . , Alberti, S., Teale, A. J., and Morrison, W. I.: Transfection into mouse L cells of genes encoding 2 serologically and functionally distinct bovine class I MHC molecules from a MHC-homozygous animal: evidence for a second class I locus in cattle. Immunology 70: 20-26, 1990 Transy, C., Lalanne, J.-L., and Kourilsky, P.: Alternative splicing in the 5' moiety of the H-2K d gene transcript. EMBO J 3: 2383-2386, 1984 Ulker, N., Lewis, K. D., Hood, L. E., and Stroynowski, I.: Activated T cells transcribe an alternatively spliced mRNA encoding a soluble form of Qa-2 antigen. EMBO J 9: 3839-3847, 1990
S.A. Ellis et al.: Two expressed bovine MHC class I loci Vogel, J. M., Morse, R. Y., and Goodenow, R. S. : A novel H-2K splice form: predictions for other alternative H-2 splicing events, lmmunogenetics 29." 33-43, 1989 Yewdell, J., Bennink, J., and Hosaka, Y.: Cells process exogenous proteins for recognition by cytotoxic T lymphocytes. Science 239: 637-640, 1988 Zuniga, M. C. and Hood, L. E. : Clonal variation in cell surface display of an H-2 protein lacking a cytoplasmic tail. J Cell Biol 102: 1-10, 1986
lmmunogenetics 37." 49-56, 1992
geneOs
© Springer-Verlag 1992
Transmembrane and cytoplasmic domain sequences demonstrate at least two expressed bovine MHC class I loci Shirley A. Ellis, Karen A. Braem, and W. Ivan Morrison Institute for Animal Health, Compton, Nr Newbury, Berks. RG16 ONN, UK Received February 3, 1992; revised version received March 30, 1992
Abstract. We have used the polymerase chain reaction to amplify cDNA from expressed bovine major histocompatibility complex class I genes. Sequences obtained from transmembrane and cytoplasmic domains were used to identify the number of expressed alleles. Data from three animals suggest that there are four major expressed alleles, representing the products of two (or more) loci. We have also demonstrated the presence of an alternatively spliced mRNA, which has been observed in five animals. The alternative splicing removes exon 7 (the major site of class I phosphorylation), which predicts a truncated molecule with a cytoplasmic portion 16 amino acids shorter than usual. This phenomenon was detected for only a single class I allele within each individual.
Introduction Class I major histocompatibility complex (MHC) antigens are highly polymorphic, membrane bound glycoproteins whose main function is to present peptides derived from foreign or self proteins to cytotoxic T lymphocytes (CTL; Yewdell et al. 1988). That expressed MHC molecules exhibit a high degree of protein polymorphism is central to their role in presenting a diverse range of peptides, thereby providing protection (at the population level) against evolving pathogens. This sequence polymorphism results in differences in the detailed shape and charge distribution in the antigen binding site, but does not significantly alter the overall structure, which appears to be well conserved between mammalian species. The selective advantage of polymorphism at the class I loci is clear, but the increase in antigen presenting capacity may be offset by a reduction in T-cell repertoire (Matzinger et al. 1984). This may account for the small number of functional class I loci (usually only two or Correspondence to: S.A. Ellis.
three) seen in all species studied Robinson and Kindt 1989, although there may be additional genes coding for less ubiquitously expressed nonclassical MHC molecules, e. g., mouse Qa, human HLA E, G (Stroynowski 1990). In cattle, the Bota complex is presently defined serologically as a singlle allelic series coding for 32 internationally defined allo-specificities (Bull 1989). Information regarding the genetic basis for these specificities was limited, until the recent publication of five bovine class I cDNA sequences (Ennis et al. 1988; Brown et al. 1989; Bensaid et al. 1991). There is evidence from 3' sequence comparisons and transfection experiments that these are encoded by at least two loci (Ennis et al. 1988; Toye et al. 1990; Bensaid et al. 1991); however there has been no systematic attempt to confirm the number of expressed products. The aim of this study was to establish how many expressed MHC class I alleles were present in individual cattle, and to estimate the number of MHC loci responsible, by using polymerase chain reaction (PCR) to amplify a region encompassing the transmembrane domain (TM), the cytoplasmic domain (C), and part of the 3' untranslated (UT) region. This region was chosen because it was possible to design appropriate primers based on conserved regions (in exon 4 and 3'UT region), maximizing the chance of amplifying all alleles present. Also, these regions have been shown in other species to possess locusspecific characteristics (Gussow et al. 1987) which would aid in the analysis of results.
Materials and methods Animals, MHCphenot~ping, and cell lines: All cattle used in this study were Friesian Holsteins. Peripheral blood mononuclear cells (PBMC) were Bota typed using altoantisera, in a microlymphocytotoxicity assay (Kemp et al. 1988), performed by Roger Spooner (IAPGR Edinburgh). PBMC were immortalized with the intracellular protozoan parasite Theileria annulata as described previously (Glass and Spooner 1990). The resulting cell lines were maintained in RPMI 1640 (Imperial,
50
S.A. Ellis et al. : Two expressed bovine MHC class I loci
Andover, UK) containing 10% fetal calf serum (FCS; Gibco Paisley, Scotland). The expression of MHC class I did not alter over periods of prolonged culture, as determined by isoelectric focusing (IEF), and as demonstrated previously (Spooner and Brown 1980).
MHC class I cDNA sequences, and were made using an Applied Biosystems 381 synthesizer (sequences shown in Fig. 1). These were used to amplify the TM an C class I domains present in eDNA from individual animals. PCR amplification was performed using a Programmable Thermal Controller (MJ Research, Boston, MA) and was carried out in a buffer containing 10 mM Tris HCI, 4 m M MgC1, 0.5 mM of each of dGTP, dATP, dTTP, dCTP, 1 IxM of each oligonucleotide primer, and 2.5 units of Taq DNA polymerase (Promega, Southampton, UK). An annealing temperature of 50 °C was used, followed by 35 cycles with the following conditions: 94 °C (1 rain), 50 °C (1 rain), 72 °C (3 min), with a last extension reaction of 72 °C for 10 min. In some experiments the eDNA was predigested with the restriction enzyme Ban II for 2 h prior to PCR; conditions were otherwise identical.
Preparation of eDNA. Messenger RNA (mRNA) was prepared directly from T. annulata-infected cell lines, by the method of Badley and coworkers (1988). eDNA was then prepared using a DNA synthesis system (BRL, Gaithersburg, USA).
Amplification by PCR and sequence analysis of TM and C domains. Oligonucleotide primers (18 mer) were designed, using published bovine a
OLIGO 2
)
Exon 4
Awl0 BL3-6 BL3-7 KN104
CCC
Awl0 BL3-6 BL3-7 KN104
GGC
CTC
.....
Ace
A
CTC, A A A
.......
.............
CTG
GTT
CTC
TC,G
G
~ii A A
CCT
CCT
CAG
CCC
TCC
TTC
~ii . . . . . . . . . . .
A ............
{ili . . . . . . . . . . .
A .........................
GTG
GTC
.................
A **
..*
*.T
ACT
GGA
GCT
GTG
GTG
GCT
***
GGA
GTT
G
** . . . .
AAG
AAG
.
.
.
.
.
.
.
CGC
.
.
C ............
TCA
.
.
.
. . . . . . . . . . . . . . .
GAA
AAA
C~
GGG
ii~i
. . . . . . . . .
Q . . . .
~
. . . . . . . . .
G
iili
. . . . . . . . .
Q . . . . . .
I I
ACT
GTG
,
ATC
C .........
G
T ..........
G
C
TAT
ATC
CAG
GCT
TCA
A
I I
I TC
I I C
I C
. . . . . .
iili i::i::
G I I
A . . . . . . . . . . . . . . . . .
I
~::~::
i::i::
)
) Exon 7
AGT
. . . . . .
GAC
...
TGC
.A . . . . . . . . . . . . . . . . .
c
OLIGO 4, 4'
( GC
GTT
Exon 6 i~iiG G T
OLIGO 5
Awl0 BL3-6 BL3-7 KN104
ATT
OLIGO 1
. . . . . . . . . . . . . . . I G .
ATC
............
Exon 5 ATG
GGC
T ................
........................
t Awl0 BL3-6 BL3-7 KN104
ATG
G .....
CTC
..........
ACC
G .....
......................................... ..T
CTC
AGT
Gee
c . . . . . . . .
CAG
GGC
TCT
GAT
GTG
TCT
CTC
ACG
GTT
CCT
G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10 times), compared with the published sequences A w l 0 (Bensaid et al. 1991), BL3-6 and BL3-7 (Ennis et al. 1988). In the TM region the sequences fall into two groups based on size, the A w l 0 / B L 3 - 6 group (37 amino acids) and the BL3-7 group (35 amino acids). The cytoplasmic region is 28 amino acids in all cases. The D19 sequences are all similar to the published sequences (-D19.2 is identical to A w l 0 in both TM and C domains). It is noticeable that variations between alleles are localized to particular positions, leaving other areas completely conserved. These sequences, and others obtained from five additional animals (data not shown), appear to belong to two loci. This is considered further, together with the KN104 sequence (Bensaid et al. 1991), in the Discussion. In addition to the four sequences shown (1319.1D19.4) we obtained a small number of aberrant sequences, some presumed to be generated by the PCR and others which required further investigation.
Analysis of amplified products. Amplificationproducts were examined on a 2% agarose gel, and DNA for further analysis was eluted from a low meltingpoint agarose gel. The DNA was the kinased and blunt end ligated into M13 mp18 (Maniatiset al. 1982). The dideoxy method of Sanger and co-workers (1977) was used to obtain the nucleotidesequence of a random selectionof clones, using a sequenasekit from US Biochemicals (Cleveland, OH).
Preparation of genomic DNA. GenomicDNA was prepared from T. annulata-infected cell lines as described by Maniatis and co-workers (1982). Amplificationof genomic DNA was carried out as for cDNA, using oligonucleotideprimers as shown in Figure 1, and described in Results.
Biosynthetic labelingof cells, immuonprecipitation, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), T. annulatainfectedcell lines were incubatedat 37 °C for 30 min in methionine-free RPMI (Gibco) plus 10% FCS (Gibco), then for 4-6 h with 3Ss methionine(NEN) at 100 gCi to 5-10 x 106 cells. Fresh PBMCs were labeled for 16 h. To label cell products with 32p, cells were washed in phosphate-free medium (Eagles MEM, Flow), then resuspended at 2 x 106 cells per ml in the same mediumcontaining 10% FCS, and incubated at 37 °C for 45 min. 32p orthophosphate (NEN) was then added to a concentrationof 40 gCi/ml, and incubationcontinuedfor 3-6 h at 37 °C. After labeling,cells were lysed in 1 ml of buffer containing 50 mM Tris, 5 mM MgC12, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 0.5 % Nonidet P-40. Lysates were precleared using formalin fixed Staphylococcusaureus (Novabiochem, Nottingham, UK). MHC class I molecules were immunoprecipitated with the monoclonal antibody (mAb) ILA19 (Bensaidet al. 1989) which recognizesbovine class I a chain together with/32m. Immunoprecipitatedproducts were examined by SDS-PAGEas described by Laemmli (1970). Gels were dried and autoradiographed.
Identification o f an unusual pre-mRNA splicing pattern. In addition to the sequences shown in Figure 2, two (identical) D19 clones showed a truncated sequence which missed 48 base pairs (bp) from near the 3' end (Fig. 3). Because the sequence resumed with the last two bp before the TGA stop codon, and in other species class I exon 8 often consists of two bp, it seemed probable that this represented an alternatively spliced form of the gene, rather than a PCR artifact. This phenomenon was also observed in class I sequences derived from early fetal material (data not shown). Since no bovine class I genomic sequences are available, oligonucleotide primers were devised which could be used to amplify the regions of genomic DNA thought to contain the relevant introns (Fig. 1, primer pairs 4 and 3; 4' and 5).
Results PCR amplification o f bovine M H C class I exons 5-8. A T. annulata-infected cell line derived from animal D19 was used to prepare cDNA. The animal was serologically MHC class I typed as w l 1; w14 and family studies showed that the w l l and w14 specificities were inherited from the dam and sire respectively. MHC class I exons 5-8 were amplified by PCR, using oligonucleotide primers
Transmembrane region Awl0 BL3-6 D19.1 D19.2
EP PQP • ...
-
.
T .
I IVGLVLLVVTGAVVAGVVI
...............
.... -
S FLTMG
Cytoplasmic domain
.
. . . . . . . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
CMKKRS
• ........
.
.
.
.
.
.
.
.
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.
A.
.... .
.
.
GEKRGTYIQAS
.........
.
.
.
.
.W. .
.R .....
.
S SDSAQGSDVSLTVPKV
...G.N......................
.
G.N .
.
.
.
.
.
. . . . . . . . . . . . . . . . . . . . . . o
°
.
°
.
.
. .
° ° o
.
.
.
.
.
.
°
. . .
BL3-7 D19.3 D19.4
....
T...I
**.L...A..WR
.......
GRI.T..A
. . . . . . . . . . . . . . . . .
....
T ................
**
.....
A..WR
.......
GRI.T..A
.................
....
T...I
**
.....
A..WR
........
KNI04
....
T
F . . W
. . . . . . . .
............
............
. . . . . . . . . . . . . .
* ........
Q..T..A.G...D
G.N
......
A . . . R
...........
. . . . . . . . . . .
Fig. 2. The deduced amino acid sequence of Awl0 is shown (in the TM and C domains), and compared with BL3-6, BL3-7, KN104, and four DI9 clones, D19.1-D19.4. Dashes indicate identity, asterisks are deletions introduced to maximise homology.
52
S.A. Ellis et al.: Two expressed bovine MHC class I loci
a) D19.3 Exon 6 GGT GAA AAA GGA
GCC
CAG
CGG
ATC TAC ACC CAG GCT GCA
GGC TCT GAT GTG TCT CTC ACG GTT Ban II
CCT
Exon 7 AGT GAC AGT
A IGC
Exon 8 G] TT T G A
AAA
3'UT
Fig. 3. a The nucleotide sequence of clone D19.3 is shown. Indicate intron exon boundaries. The Ban II restriction site is indicated in exon 7; b shows the truncated form of D19.3.
b) D19.3 Short Exon 6 GGT GAA
Exon 8 AAA
GC~
CGG
ATC
TAC
ACC
CAG
GCT
GCA
Genomic DNA was prepared from a T. annulatatransformed cell line, from animal D 18. This is a half sibling of D19, and is MHC class I identical by serology and 1D-IEF. PCR with Oligos 4' and 5 resulted in a product of approximately 160 bp, and with Oligos 3 and 4 a product of approximately 200 bp. DNA was extracted and sequenced as before. Ten clones were sequenced, and each demonstrated the presence of an intron, with the characteristic nucleotide pairs GT and AG at the beginning and end. The positions of the introns are as shown in Figure 1, and the sizes are 136 bp and 164 bp respectively. Since they occur in the positions predicted by examination of MHC genomic sequences from other species, we assumed that the splice sites would be conserved in all alleles, and did not attempt to sequence these introns from every class I gene present. This result confirmed our observation as alternative splicing.
Identification of alternatively spliced class I pre-mRNAs using PCR. Having established that alternative splicing was occurring in at least one allele in animal D19, we wanted to find out if it was a general phenomenon, occurring in other alleles, in all individuals. Because the alternatively spliced sequence was only isolated twice in more than sixty clones, it seemed likely that the frequency would be low. It might therefore be difficult to detect, without sequencing a very large number of clones from each individual. In all of the available bovine class I sequences there is a Ban II restriction site in exon 7 (Fig. 3). This site does not occur anywhere else in the region which we are amplifying, i.e., exons 5-8. Therefore, digestion of cDNA with Ban II before PCR should eliminate most of the normally spliced product, but leave the alternatively spliced version intact, resulting in a relative increase in the amplification of truncated sequences. The experiment was carried out using cDNA from T. annulata lines from four animals: D18, D19, D595, and D602. A gel showing PCR products following Ban II digestion is shown in Figure 4. All four animals showed the predicted smaller band (approximately 200 bp), cor-
A ITT T G A
3' U T
D18 a
D19 b
.a
b
D595
D602
a
a
b
b
-250bp
--
~"
--~-----200bp
Fig. 4. A 2 % agarose gel showing PCR products from cDNA amplified with Oligos 2 and 3. In each case track a shows the product from undigested cDNA, track b the product from cDNA following Ban II digestion.
responding to a truncated product. DNA was extracted from this band in each case, and was subcloned into M13 for sequencing. The results showed that the DNA present in the 200 bp bands consisted exclusively of truncated sequences (Fig. 5). In each individual there appears to be one predominant allele which is present in the alternatively spliced form. In D19, D18, and D602 the sequences are identical, and correspond to the D19.3 allele. In animal D595 it is a different allele which is truncated [although it appears to belong to the same locus ('B') as Cytoplasmic domain
D19.3
Exon 6 GEKGRIYTQAA
Exon 7 SSDSAQGSDVSLTVPK Phosphorylation site
GEKGRIYTQAA GEKGRIYTQAA GEKRQTYTQAA GEKGRIYTQAA
I I I I
a
D19.3 DI8 D595 D602
Short Short Short Short
__) Exon 8 V
b Fig. 5. a Deduced amino acid sequence of the cytoplasmic domain of clone D 19.3. Gaps indicate exon boundaries. The expected phosphorylation site is indicated in exon 7; b shows the truncated clone found in each animal, D19, D18, D595, and D602.
S.A. Ellis et al.: Two expressed bovine MHC class I loci
53
the D19.3 allele]. The removal of exon 7 also results, in each case, in the substitution of an isoleucine residue for the usual valine, as the terminal amino acid.
to 35S-methionine labeled cells, confirming that a range of heavy chains is reaching the surface (data not shown).
Alternative class I transcripts are synthesized, and can be distinguished by differences in size and phosphorylation. In order to determine whether the alternatively spliced transcripts generate proteins, cells [T. annulata lines or fresh peripheral blood lymphocytes (PBL)] were labeled with either 35S-methionine or 32P-orthophospate (NEN), and class I heavy chains were immunoprecipitated using the mAb ILA19. Analysis of the products by SDS-PAGE demonstrated that 35S-methionine labeled products appeared as a cluster of bands, ranging in size fi'om Mr 41 000-47 000 (Fig. 6). This was seen most clearly using an 8 % gel (Fig. 6b). No 3:p was detected in the minor low M r product (41000). This result suggests that there is synthesis of a range of class I heavy chains, and that those with lower relative mass are not phosphorylated. It is thought that MHC class I molecules become phosphorylated at or near the cell surface, so the 32p-labeled products are likely to represent the phoshporylated molecules seen at the surface rather than intracellularly. Phosphorylation itself can affect the mobility of molecules during SDS-PAGE, and the existence of a mixture of phosphorylated and unphosphorylated molecules of the same molecular relative mass could therefore give rise to a range of bands. However this phenomenon alone seems unlikely to account for the wide size range which we consistently observed. Surface labeling of cells with 125I, followed by immunoprecipitation with ILA19, gave an identical pattern
Discussion
Fig. 6. a ImmunoprecipitatedMHC class I heavychains from D18 and D602 T. annulatainfected cells, labeled with 35S methionine(track 1), or 32porthophosphate(track2), run on a 10% SDS polyacrylamidegel. Relative molecular mass is indicated, b As a except an 8% gel.
Bovine MHC class I cDNAs have been cloned and sequenced (Ennis et al. 1988; Brown et al. 1989; Bensaid et al. 1991) but as yet the total number of expressed genes has not been established. The limited data available suggest that there are at least two loci (Ennis et ah 1988; Toye et al. 1990; Bensaid et al. 1991). However, conventional cDNA library screening would not necessarily reveal additional genes which may be expressed at low levels, without extensive and repetitive sequencing. For this reason we decided to utilize the PCR technique as an initial approach, and to look only at the TM and C domains, using a small number of animals. These regions have been shown in other species to demonstrate locus-specific characteristics (Gussow et al. 1987; Parham et ah 1988), and since together they consist of less than 300 bp, it is feasible to sequence rapidly large numbers of clones. One disadvantage of using this technique to amplify a mixture of very similar sequences is the occurrence of 'PCR artifacts', described in detail elsewhere (Ennis et al. 1990; Lawlor et al. 1991). However, because we sequenced large numbers of clones, it was relatively easy to identify the few sequences which demonstrated crossing-over between one clone and another. The results we obtained support the published data, in that we found evidence for two expressed loci, i.e., four alleles ( > 10 clones of each). Allowing for allelic variation, it was still easily possible to assign sequences to an 'A' or 'B' locus in each of the individuals we examined. We investigated three animals in detail ( > 40 clones), and another three in less detail ( < 20 clones). We always found approximately equal numbers of clones from each of the four alleles, suggesting that the two loci give rise to products which are expressed at similar levels (Fig. 2). The ' A ' locus sequences, which include those of the previously published cDNAs, Awl0 (Bensaid et al. 1991) and BL3-6 (Ennis et al. 1988), have a TM region consisting of 37 amino acids. The 'B' locus sequences, which include BL3-7 (Ennis et al. 1988), have a TM region of 35 amino acids, with the 'missing' residues always in the same position. Although there does appear to be ' A ' and 'B'-specific sequences in both the TM and C domains, the TM length will almost certainly prove to be the best diagnostic feature. We also found several aberrant sequences in each animal (occurring one or two times each) which contained nucleotide differences leading to amino acid substitutions. These were not easy to assign to PCR artifact, and could represent products of additional loci, expressed at much lower levels (comparable, for example, to HLA C). The
54 number of bands seen in IEF analysis of bovine class I does not exclude the possibility of a third expressed locus (data not shown). We are currently using PCR to look for these sequences in class I clones isolated from cDNA libraries made from these individuals. The cDNA sequence KN104 (Bensaid et al. 1991, and Fig. 1) complicates our straightforward interpretation. Bensaid and co-workers, when discussing the previously published class I sequences, suggest that Awl0, KN104, and BL3-7 may represent the products of three different loci. Awl0 and KN104 are both abundantly expressed class I molecules which function in presentation of antigen to CD8 ÷ T lymphocytes (Toye et al. 1990). KN104 has a TM region of 36 amino acids, with the 'missing' one in a different position to those seen in the putative 'B' locus sequences shown in Fig. 2. There are several possible explanations for this, one being that the 'B' locus is not as clearly defined as we have suggested, and that if we examined more animals we would find 'B' locus sequences that look like KN104. Another is that KN104 represents a third, 'C' locus, which we have yet to identify in our animals, as suggested by Bensaid and co-workers (1991). This seems unlikely given the number of clones we have sequenced, and the high levels of expression of KN104 (Kemp et al. 1990). The third explanation concerns the fact that both Awl0 and KN104 were cloned from an African Bos indicus animal, whereas all the others are from Bos taurus animals. These two types of cattle, which are distinguished by the presence of a hump in the Bos indicus, are considered as separate subspecies, although they readily interbreed. Bos indicus cattle appeared relatively recently (judged by archaeological evidence) and may have been selectively bred for agricultural purposes. They initially occurred in Asia (4-5000 years BC), and were not introduced by humans into Africa until approximately 2000 years ago (Epstein 1971). Since the Bos indicus cattle are presumed to be descended from the Bos taurus line (very recently in evolutionary terms) they would not be expected to have markedly different MHC genes; however, it is possible that selection pressure by particular pathogens has led to a high incidence of some class I alleles in African breeds of cattle. It should be possible to resolve some of these points by investigating further Bos indicus animals. Bensaid and co-workers (1988) demonstrated size heterogeneity in bovine class I heavy chains, following SDS-PAGE and western blotting. They suggested that at least some of these differences may be due to post transcriptional modifications. Our results, both SDS-PAGE (Fig. 6) and 1D-IEF (data not shown) also indicate the presence of multiple class I heavy chain isoforms. We have demonstrated that alternative splicing, resulting in removal of exon 7, occurred in some alleles (at a single locus), and that this may account for some of the heterogeneity seen.
s.A. Ellis et ah: Two expressedbovine MHC class I loci Several instances of class I alternative splicing have been described involving, in the mouse for example, H-2K b, H-2K q, H-2K k, H-2D d, and H-2L ° (Lew et al. 1987; Vogel et al. 1989), in human HLA A24, HLA A2 (Krangel 1986), and possibly HLA C (Mizuno et al. 1989), and in a sheep class I gene (Grossberger 1990). Most of these examples involve exons 7 and/or 8, and lead to truncated molecules, although there are a few examples involving the 5' end of the gene (Lalanne et al. 1985; Transy et al. 1984), and in the sheep the alternative splicing results in a longer molecule. Many of the nonclassical class I genes also demonstrate alternative splicing of exons 5-8 (Lew et al. 1987; Ulker et al. 1990). One reason why alternative splicing seems to occur quite frequently around exons 7 and 8 may be that they are small (2-48 bp). It has been suggested (Dominski et al. 1991) that the length of internal exons plays an important role in splice site selection. Internal exons less than 50 bp are rare ( < 4 % ) , and are more likely to be involved in 'exon skipping'. However, other factors, such as the length of the upstream polypyrimidine [poly(Y)] tract are also involved. This could explain why exon 6, which is even shorter (33 bp) is rarely spliced out. Examination of intron sequences may help to explain why particular alleles seem more susceptible to alternative splicing. In most cases described the alternatively spliced transcript is rare, which makes detection difficult. Despite this it could be a fairly widespread phenomenon, resulting in limited expression of altered molecules. Also, it may occur more frequently in certain cell types as in the case of H-2K u (Lew et al. 1986). Speculation as to the functional significance of these molecules is difficult since the role of the intracytoplasmic portion of full-length class I molecules is poorly understood. Comparison across species has shown that exon 7 is particularly well conserved, and the region indicated in Figure 5 (Ser-Asp-Val-Ser-Leu) is the usual site(s) ofphosphorylation (Guild et al. 1984). Phosphorylation-dependent signaling by class I molecules could be important during particular stages of development, or in particular tissues. Transfection studies with alternatively spliced class I molecules (Zuniga and Hood 1986) indicate that structural variations in the cytoplasmic domain can lead to use of distinct pathways of intracellular processing, and can effect the way the molecules interact with the cytoskeleton. Since class I aggregation in the membrane may be important in T-cell recognition, the presence of significant levels of a truncated class I allele could result in failure to elicit good in vivo CTL responses to certain pathogens. Although some studies suggest that the occurrence of alternative splicing of class I mRNAs is random (Vogel et al. 1989), indicating no specific function, other examples lead to the opposite interpretation. In the case of
S.A. Ellis et al.: Two expressed bovine MHC class I loci s o m e n o n c l a s s i c a l class I g e n e s t h e o c c u r r e n c e o f altern a t i v e s p l i c i n g is r e l a t e d to t h e a c t i v a t i o n state o f t h e cell, for e x a m p l e Q a - 2 w h e r e a c t i v a t i o n c a n r e s u l t in a n inc r e a s e in a s e c r e t e d f o r m o f t h e m o l e c u l e ( U l k e r et al. 1990). I n the h u m a n n o n c l a s s i c a l class I g e n e , HLA G, t h e r e is a stop c o d o n in e x o n 6, a n d e x o n 7 is spliced out, g i v i n g rise to a t r u n c a t e d m o l e c u l e (Ellis et al. 1990). T h e p r e s e n c e o f b o t h m e c h a n i s m s s u g g e s t that t h e t r u n c a t e d c y t o p l a s m i c tail m a y b e f u n c t i o n a l l y i m p o r t a n t . We have shown that there are four major expressed M H C class I alleles in h e t e r o z y g o u s B o s t a u r u s cattle, w h i c h a r e p r e s u m e d to r e p r e s e n t the p r o d u c t s o f t w o loci. I n a d d i t i o n w e h a v e d e m o n s t r a t e d a l t e r n a t i v e splicing inv o l v i n g e x o n 7 in at l e a s t t w o alleles. B o t h alleles a p p e a r to b e l o n g to t h e s a m e l o c u s - t h e s i g n i f i c a n c e o f this is at p r e s e n t u n k n o w n . F u r t h e r studies a r e u n d e r w a y o n o t h e r b r e e d s o f B o s t a u r u s , a n d B o s i n d i c u s cattle, to e s t a b l i s h w h e t h e r t h e r e are a l w a y s t w o e x p r e s s e d class I loci, a n d w h e t h e r in s o m e cases t h e r e are t h r e e , as m i g h t b e i n d i c a t e d b y t h e K N 1 0 4 sequence. Acknowledgments'. We would like to thank Dr. Roger Spooner for
serological typing of the cattle used in this study, Dr. Duncan Brown for provision of 7". sporozoites to establish cell lines, and Dr. John Young for helpful discussion and critical reading of the manuscript. This work was supported by the Agricultural and Food Research Council of Great Britain.
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