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Date of Award

Spring 2011

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biology

First Advisor

Dr. April Hill

Abstract

A Pax/Six/Eya/Dac (PSED) network is known to be involved in the development of eyes as well as various other organs in animals. This network of four transcription factors is animal-specific and highly conserved from insects to humans. To elucidate the early evolution of this network and basal functions of the network component genes, here we looked to sponges, the most ancient and basal animal phyla still present on the Earth today. The majority of our studies focused on Ephydatia muelleri, a globally distributed demosponge, but we frequently utilized the draft genomic sequence of Amphimedon queenslandica, another demosponge species, as a valuable resource. We found one copy of the Pax gene belonging to the PaxB subfamily and one copy of the Six gene belonging to the Six1/2 subfamily, but no Eya or Dac representatives, in the sponge genomes. When studying the functions of these two genes in E. muelleri, preliminary in situ hybridization and RNAi-qPCR data suggested that there is a regulatory relationship between the two genes in this basal animal. To determine whether or not it is the case that a Pax/Six (PS) network existed in E. muelleria, before the onset of the more complex PSED gene regulatory network, we overexpressed and purified the Em PaxB paired domain protein (EmPaxBprd) and tested whether it binds to putative cis-regulatory sequences near the Six1/2 gene in E. muelleri and A. queenslandica using EMSA. We found that for both E. muelleri and A. queenslandica, EmPaxBprd binds to three regulatory sites, two in the upstream region and one in the conserved intron of the Six1/2 gene. Thus our hypothesis that there is a regulatory relationship between the sponge PaxB and Six1/2 has been demonstrated by showing that PaxB protein directly regulates Six1/2 transcription by binding to its cis- regulatory elements. This study contributed to the understanding of the early evolutionary history of this important PSED network, which in turn provides a baseline to examine how the network has evolved over time. Additionally, we are pursuing a ChIP-Seq assay to survey additional targets of PaxB in the E. muelleri genome. Early ChIP assay results analyzed by a ChIP-Display method reveal a possible novel role of PaxB in sponge metabolism.

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