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Molecular evolution and in vitro characterization of Botryllus histocompatibility factor

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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.

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Acknowledgments

The authors thank Michael B. Caun for his expert care of the De Tomaso laboratory mariculture facility. They also thank Dr. Kathy Foltz and Dr. Jeffrey Bailey for their critical comments on this manuscript. They also thank Liviu Cengher and Michael A. Trebino for their assistance generating several constructs. They acknowledge the use of the NRI-MCDB Microscopy Facility and the Spectral Laser Scanning Confocal supported by the Office of The Director, National Institutes of Health of the NIH under Award # 1 S10 OD010610-01A1. This work was funded by the following grants through the National Institutes of Health: AI041588 (to AWD) and F32 GM108227 (to ADL). This work was also funded by California Institute for Regenerative Medicine: T3-00009 (to DR).

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Taketa, D.A., Nydam, M.L., Langenbacher, A.D. et al. Molecular evolution and in vitro characterization of Botryllus histocompatibility factor . Immunogenetics 67, 605–623 (2015). https://doi.org/10.1007/s00251-015-0870-1

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