In fact, the genome of the related ascidian, contains a number of genes highly-homologous to those involved in natural killing in the vertebrates33

In fact, the genome of the related ascidian, contains a number of genes highly-homologous to those involved in natural killing in the vertebrates33. phase, these chordate structures are lost when the tadpole settles and metamorphs into a sessile, invertebrate body plan, called an oozooid. In addition, is usually a colonial organism, and this initial metamorphosis is usually followed by a recurring, highly coordinated budding process which eventually gives rise to a large colony of asexually derived, genetically identical individuals, called zooids, united by a common vascular network (Physique 1a, panel 1). Open in a separate window Physique 1 Histocompatibility and positional cloning of the FuHC locus in colony (panel 1) consists of asexually-derived individuals called zooids (Phospholipase A2-like, retinoblastoma binding protein-like D. Predicted structure of the FuHC protein, ss-signal sequence, EGF-epidermal growth factor repeat, Ig-immunoglobulin domain, TM-transmembrane domain. Black arrows indicate the location of two option splices, one which creates a secreted form of the protein, and another which shortens the cytoplasmic tail (see text). At the periphery of the colony, the vasculature stops in small protrusions called ampullae (Fig. 1a, panel 1). The ampullae are the site of a naturally occurring histocompatibility reaction initiated when two colonies asexually expand Triclosan into close proximity. Once juxtaposed, ampullae of each colony will reach out and begin an conversation (Fig. 1a, panel 2). This will result in either fusion of the two ampullae (forming a single chimeric colony sharing a common blood supply, panel 3), or a rejection reaction during which the interacting ampullae are destroyed, thus preventing vascular fusion (panel 4). Fusion or rejection is usually governed by a single, highly polymorphic locus called Bmp3 the FuHC 3C5. When two colonies share one or both FuHC alleles, they will fuse, Triclosan whereas rejection occurs if no alleles are in common. The FuHC is usually highly polymorphic, with most populations made up of tens to hundreds of alleles3C5. Thus this primitive chordate undergoes a histocompatibility reaction analogous to the vertebrate MHC-based reaction, and we wished to uncover the molecular mechanisms underlying this process. Positional Cloning of the FuHC locus We have created conditions to raise and breed in captivity and developed partially inbred lines homozygous for different FuHC alleles, allowing us to take a forward genetic approach to identify the FuHC locus6C8. As described previously8, mapping populations were created using FuHC defined individuals as parents, the FuHC locus was mapped using a combination of AFLPs and bulk segregant analysis (Physique 1b), and a genomic walk was initiated from the linked markers using both bacterial artificial chromosome (BAC) and Fosmid genomic libraries. The physical map now consists of 3 contigs spanning 1.3 Mbp, with one chromosomal breakpoint crossed (not shown). Over 1 Mbp of the minimal tiling path have been sequenced. The strategy for identifying candidate FuHC gene(s) is based on the remarkable polymorphism of the locus. Predicted genes from genomic sequence are analyzed for expression using RT-PCR, then compared to those from the genomic clones. The Triclosan genomic (BAC and Fosmid) and cDNA libraries are made from FuHC defined, but non-histocompatible individuals, thus we can concurrently survey for both expression and polymorphism of these cDNAs. The FuHC gene(s) should have polymorphisms with the following three characteristics: 1) absolute correlation with defined histocompatibility alleles in a fusion assay; 2) co-segregation with the same alleles in a defined cross and; 3) remarkable polymorphisms in colonies isolated from the wild. Finally, expression patterns should correlate spatially with the functional aspects of histocompatibility. Isolation and characterization of a candidate FuHC (cFuHC) gene A Genscan9 analysis of a sequenced contig consisting of two overlapping fosmid clones (531d19, 557i23; both segregated without recombination with the FuHC, Physique 1c) predicted a gene model encoding a transmembrane protein with an extracellular immunoglobulin (Ig) domain name. A full-length cDNA was isolated via RACE and was 3.2 kb in length, predicting an open reading frame of 1007 residues, and was highly polymorphic. The cFuHC is usually a type I transmembrane protein with the majority of the protein (852 residues) extracellular, followed by a transmembrane domain name and an intracellular tail of 128 residues. The domain name structure of the cFuHC is usually shown in Physique 1d. The N-terminus begins with a signal sequence, followed by an extracellular EGF repeat, then two tandem Ig domains, followed by the transmembrane domain name and an intracellular tail. BLAST searches show that this EGF repeat has homology to notch and tenascin at E values of 5e-05; the region encompassing the two Ig domains is usually homologous to Immunoglobulin Superfamily Member 4D/nectin-like 3 from a variety of vertebrate species (E = 7e-10), the highest homology is usually to chicken. No direct homolog was identified in.