Department Chair

Daniel L. Potts, Ph.D.

Date of Award

5-2020

Access Control

Open Access

Degree Name

Biology, M.A.

Department

Biology Department

Advisor

I. Martha Skerrett, Ph.D.

Department Home page

https://biology.buffalostate.edu/

First Reader

I. Martha Skerrett, Ph.D.

Second Reader

Daniel L. Potts, Ph.D.

Third Reader

Derek L. Beahm, Ph.D.

Abstract

In invertebrates gap junctions are formed by the innexin family of proteins. Remarkably, the genome of Hydra magnipapillata contains 17 innexin genes. This study focused on Hydra innexin-2 (h-Inx2) which is expressed in nerve cells and plays a role in contraction of the body column. The gene sequence of H-Inx2 was obtained from the National Center for Biotechnology Information (NCBI), the gene was synthesized externally and transferred to a vector suitable for expression in Xenopus oocytes (pcDNA3.1 CT-GFP TOPO). The TOPO CT-GFP vector includes a priming site for RNA polymerase which allows in vitro preparation of RNA. Another advantage is the optional GFP tag on the c-terminal tail, which could be useful for localization studies or protein purification. Also, the vector has been successfully used for expression of Drosphila innexins in oocytes. RNA encoding H-Inx-2 with the C-terminal GFP tag was transcribed in vitro and injected into Xenopus oocytes. The oocytes were then paired to allow formation of gap junctions, and the following day, coupling levels we assessed using electrophysiology. Injection of h-Inx2 RNA did not facilitate the formation of gap junctions. Positive controls included gap junctions formed by mouse Cx50, Drosophila innexin ShakBN16, and Hydra Inx3. Hydra innexins have not previously been expressed exogenously and to our knowledge this is the first attempt to express a GFP-tagged innexin in oocytes. Future work will involve insertion of a STOP codon to remove the GFP tag from H-Inx2 followed by studies of other Hydra innexins.

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