Print

Jacobs, Cassandra Lynn

Biology

2007

MS

The unicellular eulcaryote Puramecium tetraurelia is known to be attracted to the chemical stimulus glutamate. Recently, 14 putative genes showing homology to NMDA receptorlke proteins have been identified in the Paramecium genome. The gene that has the most identity to NMDA receptor-like proteins found in other organisms was termed pGluR1. Using an RNA interference (RNAi) construct, pGluR1 was down regulated yielding a phenotype indicating signtficantly dirmnished chemoresponse to glutamate. From these data, it is believed that pGluR1 is the main functional glutamate receptor expressed that is responsible for the organisms attractant response to Glutamate. Using a vector containing the gene codmg for Green Fluorescent Protein (GFP), a fusion protein was made with pGlul and GFP. Antibodies were used against GFP in immunostaining procedures and the staining patterns were observed using deconvolution microscopy. GFP staining shows that pGlul is trafficked to the surface of the cell and is also found in the cilia. Using western blot analysis, the GFP-pGluR1 can be found in the pellicle and cilia isolated portions of the cell. Transmembrane prediction programs used to determine how many transmembrane regions are in pGluR1 indicate that there are seven predicted domains. This characteristic has also been found in other species NMDA receptor-like proteins also. Therefore, RNAi was performed against the expression of G-beta. The down regulation of G-beta does not affect glutamate response in paramecium, indicating no involvement of G-proteins in the signal transduction pathway leading to the hyperpolarization of the cell and response to glutamate.