Molecular Immunology, Vol.24,No. 2,pp.197-200, 1987
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km1 MUTATION: NUCLEOTIDE SEQUENCE THE H-2K AND COMPARATIVE ANALYSIS
John M. Martinko*+, Joyce C. Solheim* and Jan GeliebtetC
’ *Department of Microbiology, Southern Illinois University, Carbondale, Illinois 62901, USA. #Department of Cell Biology, Albert Einstein College of Medicine 1300 Morris Park Avenue, Bronx, New York 10461, USA. (Received 17 September 1986; accepted 23 September 1986)
ABSTRACT Nucleotide sequence analysis of mRNA from the class I murine MHC mutant H-ZKkml has est.ablisheda site of mutation to be at the codon for amino acid position 152. Complete sequence information for the nucleotides coding for amino acids 136-163 demonstrates an A -> C alteration at the codon for amino acid 152, changing Asp (GAT) in Kk to Ala (GCT) in Kkml. Several other murine and human class I MHC variants have sirnil= alterations at amino acid position 152, resulting in altered biological activity. Finally, the pH-2111 pseudogene of the H-2k haplotype has a GCT codon at amino acid position 152, suggesting that the GCT codon occuxg in Kkml is the result of a gene conversion event. INTRODUCTION The class I MHC glycoproteins have been implicated in a number of immune recognition phenomena (Klein, 1978). Mutations in several murine class I glycoproteins are known to cause tissue graft rejection between mutant and standard animals and alter the immune recognition patterns of the mutant animals (Klein, 1978; Kohn etg., 1978; Sherman, 1980). Analysis of a number of mutant glycoproteins and genes from the H-2Kb locus indicates that each of the known mutations occurs as a single discrete cluster of nucleotide/aminoacid substitutions (Nathensonetg., 1986). In the analogous HLA system, several CTL-selected variants also have clusters of sequence differences (Lew et &., 1986). Both the Kb and HLA variants are postulated to have arisen by a gene conversion-like event ?%olving other class I genes. A spontaneously occurring, histogenically active mutation in the H-2k haplotype has previously been mapped to the K locus of the CBA.M523 mouse strain (Blandova et al 1975). This report identifies a single nucleotide alteration in the standardykgide which results in the Kkml mutation: an A -> C substitution at the codon for amino acid 152 changes Asp (GAT)n -Kk to Ala (GCT) in Kkml. MATERIALS AND METHODS Mice. CBA.M523 mice, having the Kkml mutation, were obtained as breeding pairs from Dr. J. Forman (SouthwesternMedical School, Dallas, TX) and were maintained in the vivarium at Southern Illinois University. CBA/J mice having the standard Kk genotype were obtained from Jackson Laboratories, Bar Harbor, ME. Cell Line. RDM-4, a mouse lymphoma cell line which overproducesH-2Kk (Mescher et al., 1979) was kindly provided by John E. Coligan (Laboratory of Immunogenetics,NIH,Bethesda, MD). Preparation and 32P-labelingof Oligomers. The single-strandeddeoxynucleotide oligomers used as sequencing primers were made either manually (Matteuci and Caruthers, 1981) or as described in the manual for the Applied Biosyssqms oligonucleotidesynthesizer. A O.lug sample of oligomer was labeled with 70~ Ci of [v- P] ATP (4500 Ci/mmole; ICN Radiochemicals, Irvine, CA) and 10 units of polynucleotidekinase (New England Biolabs, Beverly, MA) for 30 min. at 37'C (Geliebteret&., 1986). Preparation of polyadenylatedRNA. Total cellular RNA was prepared from fresh livers of mice as described by Auffray and Rougeon (1980). The mRNA content of total RNA was enriched by isolating polyadenylatedRNA using oligo(dT)-cellulosechromatography (CollaborativeResearch, Inc., Lexington, MA) as described by Aviv and Leder (1972). RNA sequencing. Kk-specific mRNA sequencing was done according to previously described procedures gr oligonucleotide-directedcDNA synthesis and dideoxynucleotide sequencing of the resultant product (Geliebters&.,1986). Briefly, 5 ng of 32 P-labeled oligonucleotideand 12 pg of polyadenylatedRNA were denatured at 80°C and allowed to anneal for 1 hour in 12 ~1 of annealing buffer (250 mM KCl/lOmM Tris hydrochloride, pH 8.3/l mM EDTA). Two ~1 of the RNA-primer annealing solution was then added to 3.3 ~1 of transcription buffer [24 mM Tris hydrochloride, pH 8.3/16 mM MgC12/8 mM dithiothreitol/0.4mM dATP/0.4 u&ldCTP/0.4 mM dTTP/0.8 mM dGTP/actinomycinD (100 pg/ml)l containing 3-4 units of a'vianmyeloblastosisvirus reverse transciptase (Life + To whom reprint requests should be directed. 197
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JOHN M. MARTINKO et al
Sciences, St. Petersburg, FL) and one dideoxynucleoside triphosphate (final concentration 0.16 mM ddATP, 0.08 mM ddCTP, 0.16 mM ddGTP and 0.16 mM ddTTP). The reaction was incubated at 47'C for one hour, stopped with 2 ~1 of gel loading buffer (0.3% bromphenol blue, 0.3% xylene cyanol, 10 mM EDTA in formamide), boiled for 3 minutes and loaded onto an 8% polyacrylamide sequencing gel for electrophoresis. RESULTS Since virtually all immunological recognition of MHC molecules involves determinants on the three external domains of class I H-2 antigens (N, Cl and C2), we have undertaken the sequence analysis of this portion of the H-2Kkml mRNA. We have sequenced nucleotides coding for amino acid positions 68 to 291 and identified one site of mutation in this area. Oligomers used as sequencing primers were spaced 100 to 200 nucleotides apart, resulting in overlapping extended cDNA copies of the Kkml or Kk mRNA. Figure 1 depicts the Kkml mRNA in diagrammatic form, using the exon organization for Kk as described by Arnold -et al. (1984). The position of hybridization of each oligzucleotide used to initiate sequencing reactions and the length of sequences obtained after priming by each oligonucleotide are also shown. Use of these sequence primers has allowed us to assign 612 nucleotide positions over a total length of 670 nucleotides for the Kkml mRNA.
5’
L
,
N
I
Cl ClMl
c2 C2N
, I c2c
-CYT,, , I
TM
13’
TM
-ClM2 -ClN
Figure 1. Sequencing strategy for mRNA sequence analysis of H-2Kkm1, The top line represents the exon organization of H-2Kk as deduced from Arnold etg., 1984. L is the exon coding for the leader domain, N codes for the amino terminal domain, Cl and C2 code for the two disulfide-linked domains, TM codes for the transmembrane domain and Sequence-priming CYT codes for the 3 cytoplasmic domains. oligonucleotides are shown below their position of hybridization to Lines extending from the 5' ends of the oligonucleotides the mRNA. represent the maximum length of sequence obtained using each oligonucleotide. All oligonucleotides shown are specific for Kk with the exception of ClMl, which shares specificity with pH-ZIII.The only difference observed between the reported sequence for Kk (Arnold -et al 1984) and the sequence obtained for the Kkml transcript is at the cozn for amino acid 152. Figure 2 shows an autoradiogram of a nucleotide sequence of mRNA derived from Kkml and the corresponding region from Kk in mRNA extracted from the RDM-4 cell line. Kkml differs from Kk by an A -> C substitutzn in the codon for amino acid 152: GAT -> GCT, or, at Gino acid level, Asp -> Ala at position 152. Sequence data from CBA/J derived Kk mRNA showed the same results as the RDM-4 cell line: the codon at position 152 is the standard GAT (data not shown). Analogous studies of several spontaneous mutations in H-2Kb indicate that they arose from gene conversion events with other class I genes within the H-2b haplotype (Nathenson et &., 1986). The _Kkml mutation discovered in the H-2k haplotype may have also arisen from a gene conversion event. Figure 3 compares the nucleotide sequences of Kk (Arnold et al., 1984), an H-2k haplotype pseudogene designated pH-2111 (Steinmets et&.,1981), and Kkml from amino acids 136-163. The data establish that the coding sequence for Kkml is identical in this region to Kk at all positions with the exception of the codon for amino acid position 152. The reported sequence of pH-2111 is identical to Kkml at codon position 152 and, therefore, may have been a donor gene in a gene conversion event with Kk. The presence and transcription of the pH-2111 pseudogene in CBA.M523 mouse liver wasestablished using the cross-hybridizing oligomer ClMl (see Figure 1) as a sequencing primer. Two nucleotides were identified at several individual nucleotide positions where pH-2111 and Kk are not identical. Each double nucleotide assignment was homologous to both Kk and pH-2IE (data not shown). DISCUSSION The Kkml mutation in CBA.M523 has previously been investigated at the amino acid level using peptide map analysis. Ewald __. et al (1979) showed the presence of at least one amino acid difference between Kk and Kkml but did not identify specific positions of amino acid difference between these Glecules. This study defines a single base difference between the nucleotide sequence of Kkml from the CBA.M523 mutant mouse and the nucleotide sequence of the standard -Kk gene. This difference results in a single amino acid substitution between the mutant and standard histocompatibility molecules. The nucleotide sequence difference in codon 152, an A -> C substitution which results in an Asp -> Ala interchange at the amino acid level, can be explained in at least two ways. First, other murine class I mutations, notably several -Kb mutants,
199
H-2K'"'mutation: nucleotide sequence and analysis
Kk
Kkml A
C
G
T
AC
A
GTA A CC 147
*C K
AT152
AG 152 Figure 2. Autoradiograms of RNA sequencing gels. Polyadenylated CBA.M523 mRNA (Kkml) or RDM-4 mRNA (Kk) was sequenced as described (Geliebter et al.,86. Sequencing was initiated with an oligonucleotideprimer complementary to Kk mRNA at codons 193-199. The sequence, when read bottom to top, is complementary to the sense strand in the 3'-->5' orientation. Sequences shown correspond to codons 152-147 (bottom to top). The asterisks mark the non-identical base at codon 152 in each sequence.
H-2Kk pH2-III H-2Kkml
136 151 GCCGACATGGCGGCGCTGATCACCAAACACAAGTGGGAGCAGGCTGGT --G____________-__-__-__-____-____________-_---______-_-___-____--_~~~~_~~-~~~~-~~~~~~__~~--_
H-2Kk pH2-III H-2Kkml
152 167 GATGCAGAGAGAGACCGGGCCTACCTGGAGGGCACGTGCGTGGAGTGG -C____________--_______--__ -C_____-__-____-__________________________-__--
Figure 3. Nucleotide sequence comparison of H-2Kk, pH-2111 and H-2Kkml. The numbering system for H-2Kk is according to Arnold et a&, 1984. Information for pH-2.111is from Steinmetz et&., 1981. Dashes represent homology to Kk. The codon at amino acid position 152 differs from Kk in the Em1 sequence and codes for Ala instead of the standard Asp- The pH-2111 pseudogene from the H-2k haplotype is shown as a potential donor gene.
are postulated to result from a gene conversion-likemechanism (Nathensonet&., 1986). The fact that the H-2k pseudogene, pH-ZIII, contains the identical sequence found in Kkml at codon 152 suggests that Kkml was generated by interaction between Kk and pH-2111. Thr comparative data shown ingure 3 establish that the 5' boundary z a potential gene donation from pH-2111 is 3' to codon 136, where Kkml and pH-2111 differ. The 3' boundary of the potential gene donation cannot be accurately defined because the sequence of pH-2111 is identical to K.k_ at every codon 3' to the mutation site through codon 160, beyond which the pH-2111 sequence is unknown (SteinmetsCal., 1981). A second potential explanation for the GAT -> GCT substitution in Kkml is the occurrence of a random point mutation, Although this possibility cannot be ruled out, it seems unlikely because of the occurrence Kbm5 and Kbm16 mutations also of the GCT codon at position 152 in pH-2111. Furthermore, the -appear to be single nucleotide/amino acid substitutions at position 116[TAC (Tyr) -> TTC (Phe)] which exist In the H-Zb-derived Q4 gene, a potential donor gene. (Nathenson et a&,1986). Thus, all of theingle nucleotide substitutions observed in H-2 mutantshave potential donor genes and are probably not the result of random point mutation events. The existence of several other variations in expressed class I antigens at position 152 in the mouse and in man has been discovered, suggesting that this area of the class I MHC
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et al.
antigen is particularly susceptible to alteration. For example, the murine Kbml mutation has a Glu -> Ala interchange at position 152 in addition to other changes at amino acids 156 and 157 (Weiss sg., 1983). In man, serologically identical subtypes of HLA-A2 and A3 have been identified using CTL typing methods and analyzed using peptide mapping procedures. For a pair of HLA-A2 subtypes, a Glu -> Val interchange appears at position 152 in addition to other amino acid changes (Krangel et&., 1983). In HLA-A3 variants, the same Glu -> Val interchange occurs (van Schravendijk et&., 1985). The exchange of an uncharged, non-polar residue for a charged, acidic residue at position 152 creates a T cell-recognition site on the MHC class I molecule. The Kkml mutation clearly shows that a single amino acid substitution is sufficient to form a T cell-recognized antigenic determinant leading to the rejection of grafted tissue. Finally, we cannot strictly rule out other variations in w, since the entire transcript has not been sequenced. However, the sequence data obtained for Kkml around codon 152 include 93 contiguous nucleotides, 45 of which are 3' and 47 of which are 5' to the site of the mutation. All multiple substitutions in similar murine class I mutants occur in closely associated clusters within a span of 38 nucleotides or less (Nathenson et &., 1986). Thus, the mutation at codon 152 most probably represents the only alteration in Kkml and is responsible for the altered biological activity of the gene product. Acknowledgements. We thank Dr. J. Parker and Mr. J. Precup for providing us with the manual oligonucleotide synthesis methods and equipment, Dr. K. Girgis, Mr. W. Hildebrand and Ms. E. Palmieri-Lehner (of the Albert Einstein College of Medicine Oligonucleotide Synthesis Facility) for technical assistance, Ms. A. Gower for secretarial assistance and Drs. M. Lev and M. Madigan for reviewing the manuscript. These studies were supported in part by grants to Stanley G. Nathenson from the National Institutes of Health (AI 10702, AI 07289 and NC1 P30-CA13330), the American Cancer Society (IM-236) and the Irvington House Institute for Immunological Research and to J.M.M. from the National Institutes of Health (AI 21738). J.G. is supported by a training grant from the National Institute of Health (CA09173). REFERENCES Arnold, B., Burgert, H.G., Archipld, A.L. and Kvist, S. (1984). Complete nucleotide sequence of the murine H-2K gene. Comparison of three H-2K locus alleles. Nucleic Acids Res. 24:9473-9487. Auffray, C. and Rougeonc980). Purification of mouse immunoglobulin heavy chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 10:303-314. Purification of biological5 active globin messenger RNA by Aviv, H. and Leder, P. (1972). chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA -69 :1408-1412. Blandova, Z., Mnatsakanyan, Y.A. and Egorov, I.K. (1975). Study of H-2 mutations in mice. Immunogenetics 2:291-295. VI. M523, a new K end mutant. Ewald, S.J., Klein, J. and Hood, L.E. (1979). Peptide map analysis of mutant transplanation antigens. Immunogenetics x:551-559. Geliebter, J., Zeff, R.A., Melvold, R.W. and Nathenson, S.G. (1986). Mitotic recombination in germ cells generates two major hisbg$ompati$&ity complex mutant genes shown to be identical by RNA sequence analysis: K and K . Proc. Natl. Acad. Sci. USA 83:3371-3375. Herrmann, S.H. and Mescher, M.F. (1979). Purification of the H-2Kk molecule of the murine major histocompatibility complex. J. Biol. Chem. 254:8713-8716. Klein, J. (1978). H-2 mutations. Their genetics and effect onimmune function. Adv Immunol. 26:55-146. Kohn, H.I., Klxn, J., Melvold, R.W., Nathenson, S.G., Pious, D. and Shreffler, D.C. (1978). First H-2 mutant workshop. Immunogenetics 1:279-284. Krangel, M.S., Biddison, W.E., and Strominger, J.L. (1983). Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic lymphocytes II. Variant DKI: Evidence for a discrete CTL recognition region. J. Immunol. 130:1856-1862. Lew, A.M., Lillehoj, E.P., Cowan, E.P. Maloy, W.P., van SchravendiK M.R. and Coligan, Class I genes and molecules: an update. Immunology 57:3-18. J.E. (1986). Mateucci, M.D. and Caruthers, M.H. (1981). Synthesis of deoxyoligonuclztides on a polymer support. J. Am. Chem. Sot. 103:3185-3191. Nathenson, S.G., Geliebter, J., PfaTfenbach, G.M. and Zeff, R.A. (1986). Murine major histocompatibility complex class-1 mutants: molecular analysis and structure-function implications. Ann. Rev. Immunol. 2:471-502. Sherman, L. (1980). Dissection of the BlO.D2 anti-H-2Kb cytolytic lymphocyte receptor repertoire. J. Exp. Med. 151:1386-1397. Steinmetz, M., Frelinger, J.G.,xsher, D., Hunkapiller, T., Pereira, D., Weissman, S.W., Uehara, H., Nathenson, S.G. and Hood, L. (1981). Three cDNA clones encoding mouse transplantation antigens: homology to immunoglobulin genes. Cell 24:125-134. van Schravendijk, M.R., Biddison, W.E., Berger, A.E. and Coligan, J.E. n985). Comparative structural analysis of HLA-A3 antigens distinguishable by cytotoxic T lymphocytes: variant El. J. Immunol. 134:410-416. Weiss, E.H., Mellor, A., GolderL., Fahrner, K., Simpson, E., Hurst, J. and Flavell, R.A. The structure of a mutant H-2 gene suggests that the generation of (1983). polymorphism in H-2 genes may occur by gene conversion-like events. Nature -24: 671-674.