Kelsey Bitting

University Honors with Honors in Biology

Major: Biology

Supervisor: Thomas Shafer, Biology and Marine Biology

Sequence and Expression Pattern of Unusual Microsatellite containing Callinectes sapidus Gene

In its lifetime, the Callinectes sapidus (blue crab) goes through many molts where the cuticle is shed and a new one deposited and mineralized, making it a good model system to study the synthesis of the cuticle in crustaceans. In order to study the synthesis of the cuticle, gene expression patterns can be determined at different molt cycle stages in both calcifying and non-calcifying cuticle. Once gene expression patterns are determined, the proteins put into each layer and tissue type can be ascertained and the differences compared. A transcript believed to contain a Rebers-Riddiford sequence was selected from a Callinectes sapidus EST (expressed sequence tag) database and sequenced. A Rebers-Riddiford sequence indicates that a protein binds chitin. When the transcript was sequenced, it was revealed that the transcript contained an open reading frame and a TC microsatellite in the 3’UTR (untranslated region). The open reading frame of the molecule was virtually translated using Vector NTI, and the amino acid sequence was analyzed using PFAM and BLAST. PFAM discovered a signal sequence as well as confirmed the presence of an R-R sequence. The BLAST results indicated that the most closely matching proteins were from insects with three out of five being mosquito proteins. After further investigation, it was determined that this transcript most likely contains a RR-2 sequence. The expression pattern was determined using quantitative PCR (qPCR). The qPCR results indicated that the gene is expressed in calcifying and non-calcifying tissue during pre-molt, molting, and post-molt.