Cloning and characterization of glycogen synthase from Dictyostelium discoideum

Abstract

In Dictyostelium, glycogen metabolism plays a major role in development. Undifferentiated cells contain stores of glycogen that are broken down and converted to structural components in differentiated cells. The enzyme that synthesizes this developmentally important pool of glycogen is glycogen synthase. I have cloned the entire coding and 1.3 kb of upstream noncoding region, of glycogen synthase, using PCR amplification and genomic library screening. In order to clone the 3’ portion of the gene it was necessary to develop a new technique, enrichment-PCR, that relies on the base composition of the Dictyostelium genome. Due to the high A+T content of the Dictyostelium genome, a polyT primer and a gene specific primer were used to amplify an unknown DNA fragment, flanking a known sequence. Analysis of the complete coding region showed that glycogen synthase possesses three introns that contain the consensus splice sites for Dictyostelium.

The luciferase reporter gene was used to study the transcriptional regulation of glycogen synthase. I defined the cis-acting elements that are required for proper transcriptional regulation of glycogen synthase by using promoter/luciferase fusions of varying sizes. Using the luciferase reporter system a putative promoter element was identified. Additional luciferase constructs were made to identify the specific nucleotide involved in transcription of the glycogen synthase gene. Small defined deletions are often necessary for reporter gene analysis. We have developed a deletion cassette that can expand the functionality of any commonly-used vector. The deletion cassette confers the ability to make small specific sequential deletions of the DNA flanking the cassette. We have shown that this altered vector (pDNBL) now has the ability to create 2, 4, 5 or 9 bp deletions.

A number of experimental approaches were taken to study the regulation of glycogen synthase. Homologous recombination was used to try to generate a glycogen synthase (-) cell line. In addition, I have constructed a green fluorescent protein (GFP) vector (pNV) based on the pVTL2 vector. This reporter gene is useful for monitoring the expression of a particular gene in vivo.