Term of Award

Summer 2010

Degree Name

Master of Science in Biology (M.S.)

Document Type and Release Option

Thesis (open access)

Department

Department of Biology

Committee Chair

James B. Claiborne

Committee Member 1

Jonathan Copeland

Committee Member 2

Oscar Pung

Abstract

Fish use their gills to excrete ammonia in order to eliminate nitrogenous waste. We hypothesize that this mechanism is accomplished by one or more transport proteins in the Rh glycoprotein (RhxG) family. Longhorn sculpin (Myoxocephalus octodecemspinosus) cDNA was amplified using polymerase chain reaction (PCR) and then the PCR products were visualized on an agarose gel. The cDNA from the gel bands was then sequenced and the gene sequence fragments were assembled and completed by rapid amplification of the cDNA ends (RACE). By this process we have obtained large portions of the gene sequences of the four known paralogues located in the sculpin gill (RhA, RhB, RhC1, and RhC2). Also, in vivo ammonia-loading experiments were done to determine the effect of increased internal ammonia on protein and mRNA expression. Treatment groups were exposed to a single ammonium bicarbonate, distilled water, or ammonium chloride (5 mM kg-1) infusion; then gill tissue was collected 4 hr postinfusion and analyzed using quantitative PCR to test changes in mRNA levels and dot blots for changes in RhxG protein levels. Preliminary QPCR data showed a trend of increase in response to ammonia loading. A second infusion test, with a chronic (8 hr) double load of ammonium bicarbonate, was completed with QPCR and dot blot analysis done on the gill tissue. Ambient water samples were also collected to determine in vivo ammonia efflux. In conclusion, from this data we have found protein expression changes in response to increased internal ammonia.

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