Meeting Abstract
P2.80 Friday, Jan. 4 REVERSE TRANSCRIPTION PCR OF A MOLLUSCAN AQUAPORIN KAPPER, M.A.*; HOOPENGARDNER, B.; Central Connecticut State University; Central Connecticut State University kapper@ccsu.edu
It is well known that during adaptation to altered salinities osmoconforming intertidal invertebrates adjust the total solute concentration of their intracellular fluids to match that of the environment. This is typically done by regulating the concentration of intracellular free amino acids. The metabolic pathways for amino acid metabolism are reasonably well worked out. Less well known are whether there are any adjustments to the water permeability of cell membranes during salinity adaptation. We hypothesize that during adaptation to increased salinity, the water permeability of cell membranes in gills of Geukensia demissa is modulated by adjusting the functionality of aquaporin water channel proteins. This can be accomplished by either regulating the amount of aquaporin or by regulating the cellular location or functioning of the protein. Preliminary experiments using western blot analysis of gill tissues from 15o/ooS adapted mussels transferred to 35o/ooS do not indicate any change in the amount of aquaporin-2 protein within 3 hours of transfer. Since 3 hours may not be enough time to complete new translation and transcription of proteins, we are attempting to quantify transcription of the aquaporin message. Total RNA was isolated from G. demissa gills and used as a template for reverse transcription. Degenerate PCR primers for aquaporin were designed by aligning amino acid sequences from the aquaporins from a number of spcies in the NCIB database and looking for areas of consensus.40 PCR cycles (95°/56°/65°) were run with analysis on 1% agarose gels. The aquaporin message was quantified relative to actin amplified from the same reverse transcription preparation. Actin primers were based on sequence from
