Hydractinia Allodeterminant Alr1 Resides in an Immunoglobulin Superfamily-Like Gene Complex
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology 20, 1122–1127, June 22, 2010 ª2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.04.050 Report Hydractinia Allodeterminant alr1 Resides in an Immunoglobulin Superfamily-like Gene Complex Sabrina F.P. Rosa,1,4,5 Anahid E. Powell,1,7 Hydractinia colonies fuse if they share at least one allele at Rafael D. Rosengarten,2 Matthew L. Nicotra,5 both alr1 and alr2. The colonies reject if no alleles are shared Maria A. Moreno,2 Jane Grimwood,6 Fadi G. Lakkis,5 at either locus. The transitory fusion (TF) response, character- Stephen L. Dellaporta,2 and Leo W. Buss1,3,* ized by an initial fusion followed by rejection [10], occurs as 1Department of Ecology and Evolutionary Biology a dosage effect. In laboratory lines, transitory fusion arises 2Department of Molecular, Cellular and Developmental when colonies share one or more alleles at one locus but no Biology alleles at the other. 3Department of Geology and Geophysics Yale University, New Haven, CT 06511, USA Positional Cloning and Identification of an alr1 Candidate 4Institute for Molecular Biology and Medicine, Universite´ Libre A chromosome walk was initiated at a marker tightly linked to de Bruxelles, 6041 Gosselies, Belgium alr1 (marker 194, Figure 1A; [10]), using a bacterial artificial 5Thomas E. Starzl Transplantation Institute, Department chromosome (BAC) library constructed with DNA from the of Surgery and Department of Immunology, University of laboratory line homozygous for the f allele [7]. The alr1-con- Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA taining interval was circumscribed using a large pool of re- 6HudsonAlpha Genome Sequencing Center, HudsonAlpha combinants (Xs in Figure 1B) available between the markers Institute for Biotechnology, Huntsville, AL 35806, USA flanking alr1 (markers 194 and 18, Figure 1A) [9, 10]. Detailed recombination breakpoint mapping defined a minimal alr1-f interval of 300.8 kb. The syntenic alr1-r interval was obtained Summary from a BAC library constructed from a colony homozygous for the r allele [7] and found to span a region of at least Allorecognition, the ability to discriminate between self and 458.2 kb. The minimum tiling paths of both haplotypes were nonself, is ubiquitous among colonial metazoans and wide- sequenced (Figure 1A). spread among aclonal taxa [1–3]. Genetic models for the Allorecognition determinants are expected to be mem- study of allorecognition have been developed in the jawed brane-bound proteins bearing polymorphic extracellular vertebrates [4], invertebrate chordate Botryllus [5, 6], and recognition domains. alr2, the linked allodeterminant previ- cnidarian Hydractinia [7]. In Botryllus, two genes contribute ously identified from Hydractinia, is prototypical. alr2 was to the histocompatibility response, FuHC [5, 8] and fester [6]. determined to be an immunoglobulin superfamily (IgSF)- In the cnidarian Hydractinia, one of the two known allorecog- like molecule bearing three extracellular domains similar to nition loci, alr2, has been isolated [7], and a second linked Ig-like domains, a transmembrane domain, and a cytoplasmic locus, alr1, has been mapped to the same chromosomal tail bearing an ITIM-like signaling domain. The alr1-containing region, called the allorecognition complex (ARC) [9, 10]. interval was analyzed for gene content, and putative coding Here we isolate alr1 by positional cloning and report it to sequences (pCDSs) were identified (Figure 1C; see also Table encode a transmembrane receptor protein with two hyper- S1 available online). Three pCDSs (pCDS1, 4, and 7) raised variable extracellular regions similar to immunoglobulin immediate interest because they displayed domains similar (Ig)-like domains. Variation in the extracellular domain to Ig-like domains. For clarity in nomenclature, we hereafter largely predicts fusibility within and between laboratory refer to these domains as noncanonical Ig-like domains and strains and wild-type isolates. alr1 was found embedded in provide details of their differences from canonical Ig-like a family of immunoglobulin superfamily (IgSF)-like genes, domains in the Supplemental Results and Discussion (Fig- thus establishing that the ARC histocompatibility complex ure S2) where necessary. Moreover, pCDS1 and pCDS4 bore is an invertebrate IgSF-like gene complex. sequence similarity to the alr2 allorecognition molecule. The full gene complement of the interval is listed in the Supple- mental Results and Discussion and Table S1. Results and Discussion Polymorphism between our two inbred histoincompatible lines f and r was determined for the three pCDSs containing Hydractinia symbiolongicarpus is a colonial hydrozoan that noncanonical Ig-like domains (Table S1). Only pCDS1 and grows as a surface encrustation, often on hermit crab shells. pCDS4 were polymorphic, eliminating pCDS7 as an alr1 candi- Colonies encounter one another by growing into contact, date. The pCDS1 allele displayed structural variation between whereupon they display one of three responses: fusion, rejec- the pCDS1-f and pCDS1-r alleles. Specifically, the pCDS1-f tion, or transitory fusion [10–14]. The genetic basis of these allele was found to contain a partial gene duplication, and phenotypes was first determined by classical breeding exper- the pCDS1-r homolog sequence was missing the first exon iments [9, 15]. Near-congeneic lines segregating for two allor- of the predicted gene (Figure S1). Thus, only pCDS4 fulfilled ecognition haplotypes (f and r) were established, and two loci all of the required criteria to be a plausible allorecognition (alr1 and alr2) were mapped to a single chromosomal interval, receptor. the allorecognition complex (ARC). Within the congeneic lines, Full-length cDNA sequences were generated from homozy- gous f and r colonies, and the genomic organization of CDS4 *Correspondence: [email protected] was defined (Figure 1D). CDS4 covered a 22.4 kb interval 7Present address: Institute of Biology/Applied Genetics, Freie Universita¨ t and contained eight exons. The CDS4 predicted protein was a Berlin, 14195 Berlin, Germany 537 aa type I transmembrane protein and contained a 21 aa Allorecognition in Hydractinia 1123 A Figure 1. Positional Cloning of alr1 194 alr1 18 28 alr2 174 29 (A) Genetic map of the Hydractinia allorecogni- tion complex (ARC). The alr1 minimum tiling <0.1 1.1 0.2 0.1 0.1 0.1 cM path was distributed over four bacterial artificial chromosome (BAC) clones (heavy gray lines). (B) Physical map of the genomic alr1 interval (ARC-f haplotype). Breakpoints are designated YU018B13 YU031K01 as Xs. BAC end markers are in red, and markers YU025C05 YU001F08 designed to map the recombination breakpoints are in black. Markers 194s1 and 194c28 define the 300.8 kb minimum alr1 interval. B (C) Predicted coding sequences in the alr1-f interval (see also Table S1 and Figure S1). Boxes 194c27194i5 194i6 194s1 194i4 194c25 above and below the line represent predicted genes on the plus and minus strand, respectively. Boxes numbered 1, 4, and 7 represent coding 194c14 194c6 194c7 194c28 194i3 194c26 194c24 194c23 194c22 194c21 194c8 194c11 194c15 194c12194c16 194c9 194c10 sequences bearing sequence similarity to Ig-like xxx x xxx domains (see text and Figure S2). (D) Genomic organization of CDS4. White boxes, black boxes, and bent lines represent untrans- alr1 lated regions, exons, and introns, respectively. 300.8 kb (E) Alignment of the CDS4 ARC-f and -r amino acid sequences. Polymorphic residues are shown in red. Domain organization of the pre- C dicted CDS4 protein is shown within colored 1 boxes. The following labels are used: SP, signal peptide; domains I and II, noncanonical Ig-like domains; TM, transmembrane domain; SH2, 7 4 ITAM-like, and Src-SH3 sites, potential signal transduction motifs. The pale green box indicates D the segment of exon 4 missing in alternatively spliced variants. Exon organization is shown below the alignment. < and > indicate amino acids encoded by the last and first complete 1 kb codon of the exon, respectively, and ^ indicates a codon spanning two exons. E SP CDS4-f MGMDPLT FLC I T LCMY S HVQ S LNVKPV LPT I ST TFNATVELQWQVTLEPSEA I A SMT V R E 60 CDS4-r MGMD LFT FLC I T LCMYR HVQ S LNVKPV LPT I NA TFNATVELQWQVTLEPSEK I V SMT V R E < < >><< Domain I CDS4-f LP SLI T I LT G A V D G F NVAQG G R K LFGDRVSGI FNNRNN IVTLTLQNIQYNETLTFQLLVG 120 an immunoreceptor tyrosine-based CDS4-r LPRFI N I MA G T V N G L NVD KEG KA LFGDRVSGI FNNRNK IVTLTLQNIQYNETLTFQLLVV activation motif (ITAM)-like motif (Y-x-I- Domain II x10-Y-xx-I). The CDS4 ITAM-like motif CDS4-f SS- TL VKA AN I SIV E I SGSPRVCGRK LK SNYTVNEGDFRN I TQDI CGYPKPKV SWTLGQE 180 CDS4-r SSKLV VKT ANV SIE EISGSPRVCGRKLKSNYTVNEGDFRNITQDICGYPKPKVSWTLGQE contained all of the conserved ITAM resi- < > < dues (Figure 1E). Polymorphism across CDS4-f NAGS ST S FAVNNAT RQY EYHYKT RP FNR SDCGSN I A F I AKNT LGS I NGNAWI DVDFVP SG 240 CDS4 was assessed by aligning the CDS4-r NAGS ST S FAVNNAT RQY EYHYKT RP FNR SDCGSN I A F I AKNT LGS I NGNAWI DVDFVP SG CDS4-f and -r peptides (Figure 1E). A < >< total of 29 aa substitutions were found CDS4-f V S I V S I YNNNT NC I NVTWNKQ ET GSCN I KYHLR LNGK AT I YNT LDRHFT FC I SMKFENVT 300 between the two alleles, 24 of which CDS4-r V S I V S I YNNNT NC I NVTWNKQ ET GSCN I KYHLR LNGN AT I YNT LDRHFT FC I SMKFENVT alternative splicing were located in the 118 aa region span- CDS4-f VWASYKGKNGKMVSSSDVLTTPSTTTTTTTTTTSTRKSKTTTNKSGGQVIT NNGNGTNIG 360 ning domain I. CDS4-r VWASYKGKNGKMVSSSDVLTTPSTTTTTTTTT- STRKSKTTTNKSGGQVITNNGNGTNIG < BLASTP searches with CDS4 using TM >< CDS4-f L IVGIVGGILFVI I I I I IVVCVLKHKKKNGTGNHSGYSMRVTGGENNYVIDPTRSNGRPP 420 the NCBI nonredundant protein data- CDS4-r L IVGIVGGILFVI I I I I IVVCVLKHKKKNGTGNHSGYSMRVTGGENNYVIDPTRSNGRPP < base identified diverse IgSF proteins.