UVM Theses and Dissertations
Format:
Print
Author:
Czapla, Heather Marie
Dept./Program:
Biology
Year:
2012
Degree:
MS
Abstract:
Paramecium tetraurelia is attracted to the chemical stimulus, cyclic AMP. Over the past several years there has been a search to identify and locate the cyclic AMP receptors of P. tetraurelia. In the previous research it was found that cyclic AMP must bind to the cell surface for chemoattraction to occur. Through a BLAST (basic local alignment search tool) several putative receptors were found based on homology to the cyclic AMP receptors (cARs) of Dictyostelium discoideum. Previous work down regulating and testing chemoresponse to cyclic AMP narrowed down the potential receptors to three. The messenger RNA for potential receptors pCAR1, pCAR3.1 and pCAR3.2 were down regulated and as a result the chemoattraction to cyclic AMP was decreased in each case.
In this research, RNA interference (RNAi) was again used to confirm the results of down regulation of pCAR1 and pCAR3 separately (pCAR3.1 and pCAR3.2 are down regulated together with the same RNAi construct and are referred to as pCAR3). Upon down regulation of pCARl and pCAR3 separately, the chemoattraction to cyclic AMP was decreased. Reverse Transcriptase PCR (RT-PCR) conftrmed decreased levels of pCAR1 or pCAR3 mRNA. When pCARl expression was down regulated, pCAR3 expression was found to be unchanged, as well as the reciprocal.
Antisense expression vectors were used to down regulate pCAR1 and pCAR3 simultaneously. The antisense plasmids were microinjected into P. tetraurelia and the chemoresponse to cyclic AMP was tested. Down regulation of both pCARl and pCAR3 resulted in a loss ofchemoresponse to cyclic AMP. RT-PCR conftrmed that mRNA for both pCAR1 and pCAR3 decreased by about 70 to 80 percent.
Using a c-terminal FLAG epitope tagged to pCAR1 and an antibody made against pCAR3, the location of these receptors was studied. pCAR1 and pCAR3 were found to co-precipitate from pellicle membrane. Also, pCAR1 was found to be localized in the ciliary membrane through Western blot and immunostaining. Finally, it was inconclusive whether pCAR3 was located in the cilia.
In this research, RNA interference (RNAi) was again used to confirm the results of down regulation of pCAR1 and pCAR3 separately (pCAR3.1 and pCAR3.2 are down regulated together with the same RNAi construct and are referred to as pCAR3). Upon down regulation of pCARl and pCAR3 separately, the chemoattraction to cyclic AMP was decreased. Reverse Transcriptase PCR (RT-PCR) conftrmed decreased levels of pCAR1 or pCAR3 mRNA. When pCARl expression was down regulated, pCAR3 expression was found to be unchanged, as well as the reciprocal.
Antisense expression vectors were used to down regulate pCAR1 and pCAR3 simultaneously. The antisense plasmids were microinjected into P. tetraurelia and the chemoresponse to cyclic AMP was tested. Down regulation of both pCARl and pCAR3 resulted in a loss ofchemoresponse to cyclic AMP. RT-PCR conftrmed that mRNA for both pCAR1 and pCAR3 decreased by about 70 to 80 percent.
Using a c-terminal FLAG epitope tagged to pCAR1 and an antibody made against pCAR3, the location of these receptors was studied. pCAR1 and pCAR3 were found to co-precipitate from pellicle membrane. Also, pCAR1 was found to be localized in the ciliary membrane through Western blot and immunostaining. Finally, it was inconclusive whether pCAR3 was located in the cilia.