Isolation and Characterization of Uncle Fester, an Allorecognition Molecule in the Primitive Chordate, Botryllus Schlosseri PDF Download

Author: Tanya Renee McKitrick
Publisher: Stanford University
ISBN:
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Languages : en
Pages : 150

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transmembrane helices and a short intracellular tail. Sequences from over 70 individuals collected from populations along the West Coast of North America and two populations from the North East coast revealed that uncle fester is remarkably non-polymorphic, with only two single nucleotide polymorphisms and one amino acid deletion found to be segregating within these populations. I first determined uncle fester expression by RT-PCR, and found that expression begins in the early stages of embryonic development, and continues throughout the life cycle of the individual. These studies also revealed that uncle fester is alternatively spliced in the embryo, tadpole and adult stages of development. Each adult examined thus far expresses three alternative splice variants; however, embryos and tadpole larvae express an additional set of splice variants. The roles of these splice variants remain unknown. I then performed in situ hybridization on tadpole larvae, juveniles and adult colonies using an uncle fester specific riboprobe, to determine where the uncle fester mRNA is localized. The results indicate that uncle fester mRNA is found on the larval adhesive papillae, the epithelia of the ampullae and on a subset of blood cells, all tissues known to be involved in the allorecognition response. I also created two monoclonal antibodies to the uncle fester protein, and results from whole-mount immunohistochemistry are concordant with the mRNA in situ hybridization data. The methods and characterization of the uncle fester antibodies can be found in Chapter 4. Previous studies have shown that fester is also expressed along the epithelia of the ampullae. To determine if both proteins are co-expressed on the same populations of cells, I performed double-labeled FACS and IF using directly conjugated fester and uncle fester mAbs. Results indicated that all cells that express uncle fester also express fester, and in addition there is a population of fester+/uncle fester- cells found migrating within the tunic. To determine if uncle fester was functionally involved in the allorecognition response, I used two distinct approaches. First, I delivered custom siRNA to individuals while surgically removing the ampullar tissue, essentially forcing the animals to regenerate new ampullae under the effects of the siRNA and eliminating the uncle fester protein. When incompatible colonies were paired while under the effects of siRNA, the allorecognition response was never initiated and the two colonies eventually grew over the top of each other. However, the application of uncle fester siRNA had no effect on compatible colonies, suggesting that the activation of a rejection reaction is independent of a fusion response. The second approach was to stimulate function using the uncle fester specific monoclonal antibodies. By conjugating the mAb to magnetic beads, I was able to localize the antibody to the epithelia of ampullae, which resulted in the formation of strong points of rejection on a single colony. In summary, I found that uncle fester is a non-polymorphic type I transmembrane protein that expresses a limited repertoire of alternative splice variants. It is co-localized with both the fuhc and fester on all tissues important to the allorecognition response. Functionally, uncle fester is required to activate a rejection response, but plays no role in compatible interactions. These results have transformed our working hypothesis from allorecognition consisting of a single activating pathway, to one consisting of two independent activating and inhibitory pathways that control histocompatibility outcomes.