Date of Award:

5-1-2000

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Committee Chair(s)

Gregory Podgorski

Committee

Gregory Podgorski

Committee

Daryll DeWald

Committee

Noelle Cockett

Abstract

SNF1/AMPK protein kinases are a conserved group of eukaryotic serine/threonine protein kinases that mediate a variety of stress responses, including the response to nutrient deprivation. This report describes the isolation of snfA, the Dictyostelium discoideum SNF1/AMPK ortholog, and an examination of snfA protein function. snfA is a single copy gene that encodes a predicted 80.5 kD polypeptide. A single 2.8 kb snfA transcript was present at nearly constant levels during vegetative growth and all stages of development. The predicted snfA protein consists of three domains: an amino terminal kinase domain that is highly conserved in other SNF1/AMPK protein kinases, a less-well conserved C-terminal domain that in budding yeast regulates kinase activity, and a unique aspargine-rich central domain encoded by a series of AAT trinucleotide repeats. A SNF1/AMPK activity (snfA activity), assayed by phosphorylation of a specific peptide substrate, the SAMS peptide, was detected in Dictyostelium cell lysates. Attempts to create an snfA null mutant were unsuccessful, suggesting that snfA may be essential for growth. snfA activity was reduced to 35% of wild-type levels in transformants that expressed snfA antisense RNA. Since Dictyostelium development is a response to nutrient deprivation, the potential role of snfA in regulating the entry into development was investigated in antisense RNA expressing transformants. No evidence was obtained for alterations in the ability to enter or progress through development in cells with reduced snfA activity.

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