Genomics and Diversity of the Common Marmoset Monkey NK Complex1,2
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The Journal of Immunology Genomics and Diversity of the Common Marmoset Monkey NK Complex1,2 Anne Averdam,* Heiner Kuhl,‡ Mario Sontag,‡ Tamara Becker,† Austin L. Hughes,§ Richard Reinhardt,3‡ and Lutz Walter3,4* The common marmoset monkey (Callithrix jacchus) is a New World primate that is increasingly used in biomedical research as a model organism. Due to the occurrence of natural bone marrow chimerism, it represents a particularly useful primate model in immunological research. In this study, we describe the genomic organization of the CD94, NKG2, and LY49L genes in the NK complex (NKC) of the common marmoset based on complete sequencing of a bacterial artificial chromosome clonal contig. This region of the marmoset NKC is 1.5 times smaller than its human counterpart, but the genes are colinear and orthologous. One exception is the activating NKG2CE gene, which is probably an ancestral form of the NKG2C- and NKG2E-activating receptor genes of humans and great apes. The two completely sequenced marmoset bacterial artificial chromosome clones are derived from distinct haplotypes, which differ by 200 sites in the overlapping sequence. Analyses of NKC genes in nine additional marmoset individuals revealed a moderate degree of polymorphism of the CD94, NKG2A, NKG2CE, and NKG2D genes. Furthermore, expression analyses identified several alternatively spliced transcripts, particularly of the CD94 gene. Several products of alter- native splicing of NKC genes are highly conserved among primates. Alternative transcriptional start sites were found, but these probably do not lead to a change of the translational start site or result in longer or shorter cytoplasmic regions of these type II membrane receptors. The Journal of Immunology, 2007, 178: 7151–7161. atural killer cells are large granular lymphocytes that are mapping to the NKC include CD94 and the families of Ly49 and part of the innate immune system. Upon triggering, NK NK group (NKG) 2 genes (4, 11). Whereas LY49 and NKG2D N cells are able to release cytokines and to kill target cells, molecules form homodimers, CD94 and NKG2 molecules are ex- usually virally-infected cells or tumor cells (1–3). Expressing a pressed as heterodimers at the cell surface. The Ly49 gene family diverse array of activating and inhibitory receptors on their cell is expanded and diverse in rodents (12) and includes activating and surface (4, 5), NK cells scan target cells for the presence of inter- inhibitory receptors that are characterized by presence of a posi- acting ligands (6), which are in most cases members of the family tively charged residue in the transmembrane region and ITIM in of MHC class I molecules. The integration of such activating and the cytoplasmic region, respectively (13). The ITIM-containing inhibitory signals determines the functional outcome of the NK inhibitory receptor NKG2A and the activating receptor NKG2C cell (7). form heterodimers with the CD94 molecule, which exhibits neither Almost all NK cell receptors are encoded in two gene clusters, an activating nor an inhibitory motif. The CD94/NKG2A and the the leukocyte receptor complex (LRC)5 and the NK complex (NKC), which map to human chromosomes 19q13.4 (8) and 12p13 CD94/NKG2C molecules interact with the nonclassical MHC (9), respectively. LRC-encoded receptors contain Ig-like domains, class I ligand HLA-E (14). Recently, binding of CD94 and the whereas the NKC encodes C-type lectin-like receptors (10). Genes activating receptor NKG2E has been demonstrated (15), while the function of NKG2F, which has a truncated extracellular lectin-like domain and binds intracellularly to DAP12, is still unknown (16). *Department of Primate Genetics and †Primate Husbandry, German Primate Center, Although similarly named, the NKG2D molecule is only distantly ‡ Go¨ttingen, Germany; Max Planck Institute for Molecular Genetics, Berlin, Germany; related to the other members of the NKG2 family. NKG2D ho- and §Department of Biological Sciences, University of South Carolina, Columbia, SC 29208 modimers interact with stress-inducible MHC class I ligands Received for publication December 8, 2006. Accepted for publication March 7, 2007. MICA, MICB, ULBP1, ULBP2, ULBP3, and ULBP4 in hu- The costs of publication of this article were defrayed in part by the payment of page mans (17–19) and RAET1A, RAET1B, RAET1G, RAET1E, charges. This article must therefore be hereby marked advertisement in accordance H60, and MULT1 in mice (20, 21). NKG2D signaling involves with 18 U.S.C. Section 1734 solely to indicate this fact. the ITAM-containing adaptor molecules DAP10 and DAP12. 1 This work was supported by the German Primate Center (to A.A., T.B., and L.W.), the Max Planck Society (to H.K., M.S., and R.R.), and the German National Genome Notably, distinct splice products of NKG2D associate with Research Net. DAP10 and DAP12 in mice (22, 23), but not in humans, where 2 Sequences have been submitted to DDBJ/GenBank/European Molecular Biological the NKG2D gene is not differentially spliced and its product Laboratory database and have been assigned accession numbers EF050432–EF050450. associates with DAP10 only (24). 3 R.R. and L.W. share senior authorship. The CD94 gene is monomorphic and the NKG2 genes are mod- 4 Address correspondence and reprint requests to Dr. Lutz Walter, Forschergruppe erately polymorphic in humans and common chimpanzees, and all Primatengenetik, Deutsches Primatenzentrum, Kellnerweg 4, Go¨ttingen, Germany. E-mail address: [email protected] genes are subject to alternative splicing (25, 26). In this study, we 5 Abbreviations used in this paper: LRC, leukocyte receptor complex; BAC, bacterial show the complete sequence and organization of the CD94-LY49L artificial chromosome; NKC, NK complex; NKG, NK group; SNP, single nucleotide genomic interval in a New World monkey species, the common polymorphism; CRD, carbohydrate recognition domain. marmoset (Callithrix jacchus). The NKC of the common marmoset Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 monkey encodes only a single activating CD94/NKG2 receptor, www.jimmunol.org 7152 MARMOSET MONKEY NKC Table I. Primers used for RT-PCR analysis and 5ЈRACE Annealing Primer Sequence Temperature (°C) cjCD94-fw TCTCTACATTGCTCTTGGAAC 54 cjCD94-rev TCTACTCTCCACCTTCTCTG 54 cjNKG2A-fw ACTAACCTGGCCTCTCCACTA 58 cjNKG2A-rev ATGTGCTGCCAACTCAGATGC 58 cjNKG2CE-fw CAGTTATCACAGAGCACAGTC 56 cjNKG2CE-rev TGTCAGACTGCAAACTCAAATG 56 cjNKG2F-fw AGTCCCTGACATCACACAG 50 cjNKG2F-rev TCAGAATTCTTCAAAGCACAGG 50 cjNKG2E-rev CTACATGAGCACTGGAGCAC 56 cjNKG2D-fw GTGGATTGAAGACTTCAGATTC 54 cjNKG2D-rev CAGTGTTTCCGCTGGTATAG 54 Caja-CD94-5ЈRACE-rev TGTTGGGTCCTGGAGTAAATGCTGAC 63 Caja-NKG2A-5ЈRACE-rev CTTAGGTGTTCGTTGCTGCCTCTTTG 63 Caja-NKG2D-5ЈRACE-rev TGCCATCGTGTCGAAAAGTCACCT 61 Caja-NKG2CE-5ЈRACE-rev TTAGGTCTCCTTTGCTGCCTCTTTGG 63 Ly49-ex2fwd1 TGATCAGGGAGAGATTTATTCAAC 56 Caja-Ly49-ex3rev CACCAACATTGTGACTATCATCAG 56 which most likely represents an ancestral form of primate-activat- chemistry and analyzed in Applied Biosystems 3730 sequencers (Applied ing receptors CD94/NKG2C and CD94/NKG2E. Compared with Biosystems). Raw sequences were processed by Phred (available from Phil humans, the NKG2 and CD94 genes of the marmoset are more Green, University of Washington (www.phrap.org)) and assembled into a contiguous sequence by Phrap (available from Phil Green, University of variable. Washington (www.phrap.org)) (29). BLAST and FGENESH-2 algorithms (http://www.ncbi.nlm.nih.gov/BLAST/; http://www.softberry.com/all.htm) Materials and Methods identified exons and introns of genes in finished BAC clone sequences and Blood samples and RNA preparation annotations of genes were done manually. All BAC clone sequences have “finished sequencing” quality (1 putative mistake/100,000 bases) and the Blood samples (1 ml each) were obtained from nine healthy unrelated data have Phred values of Ͼ90. No gaps are present in the sequence. common marmoset monkeys (C. jacchus) that are maintained in the Ger- man Primate Centre breeding colony. Samples were brought to 15 ml with RT-PCR analysis and 5ЈRACE erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA) and were incubated at room temperature for 20 min. The remaining Two micrograms of total RNA was reverse transcribed for1hat42°C ϫ using oligo(dT) primer and 200 U of Moloney mouse leukemia virus re- blood cells were centrifuged for 10 min at 200 g and 7°C, washed in 15 ml of lysis buffer, and subjected to preparation of total RNA according to verse transcriptase (Promega) in a total volume of 25 l. For PCR, 1 lof standard methods (27). cDNA was used in standard PCR according to the different primers (Table I) and expected lengths of the amplificates. PCR products were separated Bacterial artificial chromosome (BAC) library screening and in 1% agarose gels and visualized with ethidium bromide staining. Bands probes were excised under UV light and DNA was recovered using standard silica adsorption. Isolated DNA was sequenced with respective PCR primers and High-density colony filters of BAC library CHORI-259 were obtained Applied Biosystems BigDye terminator chemistry. Alternatively, RT-PCR from Children’s Hospital Oakland Research Institute (http://bacpac.chori. products were cloned in a pGEM-T Easy PCR cloning vector according to org/home.htm). Filters were hybridized with probes of the human CD94, the supplier’s manual (Promega) and single clones were sequenced. NKG2A, and LY49L genes according to recommendations in filter-accom- The 5Ј ends of cDNAs were