title: Molecular evolution and in vitro characterization of Botryllus histocompatibility factor.
abstract: Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhc(sec). Finally, we observed BHF's localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of California-Santa Barbara, Santa Barbara, CA, 93106, USA.
authors.forename: Daryl A
authors.initials: DA
authors.lastname: Taketa
authors.affiliation: Division of Science and Mathematics, Centre College, Danville, KY, 40422, USA.
authors.forename: Marie L
authors.initials: ML
authors.lastname: Nydam
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of California-Santa Barbara, Santa Barbara, CA, 93106, USA.
authors.forename: Adam D
authors.initials: AD
authors.lastname: Langenbacher
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of California-Santa Barbara, Santa Barbara, CA, 93106, USA.
authors.forename: Delany
authors.initials: D
authors.lastname: Rodriguez
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of California-Santa Barbara, Santa Barbara, CA, 93106, USA.|Department of Developmental Biology, Stanford University, Stanford, CA, 94505, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Sanders
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, University of California-Santa Barbara, Santa Barbara, CA, 93106, USA. anthony.detomaso@lifesci.ucsb.edu.
authors.forename: Anthony W
authors.initials: AW
authors.lastname: De Tomaso
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019143:Evolution, Molecular; D020869:Gene Expression Profiling; D014644:Genetic Variation; D057809:HEK293 Cells; D006239:Haplotypes; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008285:Major Histocompatibility Complex / Q000235:genetics*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D014561:Urochordata / Q000235:genetics* / Q000276:immunology*; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D020033:Protein Isoforms; D000097573:Red Fluorescent Protein
keywords: Allorecognition; Botryllus histocompatibility factor (BHF); Botryllus schlosseri; Cellular localization; Selection