Mandy Levine
Professor Peter Brodfuehrer

Creating expression vectors containing accessory proteins to leech glutamate receptors

The leech is an excellent model organism, widely utilized in the field of neuroscience because it exhibits a variety of distinct behaviors, including swimming, that are easily observed. Previous research has shown that glutamate receptors mediate all of the excitatory synaptic connections in the swim network. One approach to characterizing the role of glutamate receptors in swimming is through expression in Xenopus oocytes. In order to correctly express these glutamate receptors, however, several accessory proteins, including SOL-1 and STG, must also be expressed. The leech transcriptome database was searched for putative coding regions and primers were designed for these regions. Using PCR, postulated protein encoding sequences were amplified from an existing leech cDNA library. These regions were cloned into pGEM-T Easy for sequencing and compared to existing databases. The sequences were then cloned into expression vectors to be expressed in Xenopus oocytes so that glutamate receptors could be accurately studied. Sequences for STG1, STG2, and SOL-1B were isolated, sequenced, and cloned into expression vectors for later use. Based on BLAST searches, it is likely that these sequences do in fact code for the accessory proteins necessary for proper glutamate receptor activity and therefore, once cloned into expression vectors, will be useful in studying glutamate receptors in Xenopus oocytes.