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Identification of a Colonial Chordate Histocompatibility Gene

Science  26 Jul 2013:
Vol. 341, Issue 6144, pp. 384-387
DOI: 10.1126/science.1238036

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  1. Fig. 1 Genomic characterization of the cFuHC locus in B. schlosseri reveals two tightly linked genes.

    The cFuHC locus encodes two gene products, sFuHC (a secreted form) and mFuHC (a membrane-bound form). Sequences aligned, from bottom to top: (i) Genomic contigs from B. schlosseri draft assembly; (ii) fosmid clone used to characterize cFuHC (12) (table S5); (iii) predicted exon structures, with genomic coordinates indicated below in red (contigs with identical interexon distances to the fosmid are colored gray); (iv) B. schlosseri expressed sequence tags (ESTs) obtained from NCBI; (v) Sanger-sequenced PCR products resulting from selected cFuHC amplicons (table S1); (vi) representative RNA-Seq reads (100 bp × 2) from 17 colonies (table S4); (vii) translated primary sequences with predicted functional domains (14). All alignments were performed with megablast (mismatch penalty = –2, ≥90% identity, no query filtering, and otherwise default parameters). EGF, epidermal growth factor; IG, immunoglobulin domain; SP, signal peptide; TM, transmembrane domain.

  2. Fig. 2 Genome-wide analysis for candidate Fu/HCs reveals a single gene that exhibits perfect alignment with fusibility outcomes and defined Fu/HC genotypes.

    The ability to stratify known fusion or rejection outcomes was tested for all predicted genes from the draft assembly having transcriptome data covering ≥6 fusion and ≥6 rejection pairs, ≥20 common sites sequenced per pair, and at least 1 amino acid polymorphism (after filtering, n = 7523 genes) (table S5). (A) Classification errors across the genome are depicted as a boxplot showing the median (horizontal line), 25th to 75th percentiles (within the box), and 1st to 99th percentiles (whiskers). Although sFuHC is in the top 1% of best-performing genes, novel Fu/HC candidates with equal or better performance were also identified and are indicated in pink beneath the boxplot. Classification errors <0.2 (dotted line) have a P value of <0.001, as determined by 1 million random permutations of known fusibility outcomes for each gene analyzed in the assembly (table S6). (B) A B. schlosseri gene that exhibits perfect sequence concordance with fusion or rejection outcomes and defined genotypes, termed BHF. (C) BHF genomic and message sequence architecture (table S7), representative RNA-Seq coverage, and amino acid polymorphisms across all 17 colonies from the exploratory cohort (table S4).

  3. Fig. 3 BHF accurately predicts new fusibility outcomes and has expression patterns and function consistent with a Botryllus allorecognition determinant.

    (A) Known and predicted fusion or rejection outcomes among all 23 B. schlosseri colonies analyzed (table S4), including exploratory (n = 17) and validation cohorts (n = 6). All “blind” predictions were confirmed (6 of 6). (B) Expression analysis of BHF, sFuHC, and mFuHC under the conditions preceding fusion or rejection (“challenged”; n = 6) compared with unchallenged control colonies (“naïve”; n = 4) (*P = 0.009, two-tailed unequal variance t test; NS, not significant). Values are presented as means ± SEM. (C) BHF expression patterns assessed by whole-mount in situ hybridization, compared with control (sense probe). amp, ampullae. Scale bars, 50 μm. (D) Analysis of morpholino-induced knockdown of BHF. (Top) Unreactive ampullae from apposing colonies under BHF-knockdown conditions (left) and at lower magnification (right). (Bottom) Fused blood vessels between colonies injected with morpholino control (left) and at lower magnification (right). amp, ampullae. Scale bars, 1 mm.