Title page for ETD etd-10102005-131553
|Type of Document
||Molecular analysis of glycogen phosphorylase-1 gene expression during the development of dictyostelium discoideum
|Rutherford, Charles L.
|Claus, G. William
|Larson, Timothy J.
|Rogers, Patricia V.
- Dictyostelium discoideum Molecular aspects
|Date of Defense
The cellular slime mold, Dictyostelium discoideum, has two
developmentally regulated forms of the enzyme glycogen phosphorylase,
which are encoded by two distinct, but related genes (Rutherford, et. aI., 1991).
A complementary DNA (cDNA) encoding glycogen phosphorylase-}, gp-l, was
isolated from a 𝛌gtll expression library made from amoebae stage mRNA. The
5· upstream region of the gp-l gene was cloned by inverted polymerase chain
reaction (IPCR) and partial genomic DNA library screening. The gp-l gene
was found as one copy or low copy number gene in the Dictyostelium
genome, and an adjacent 22 kilobase pair region was physically mapped. The
deduced amino acid sequencing analysis revealed that there were 862 amino
acid residues encoded by the gp-1 mRNA of 2729 nucleotides. It was also found
that most regulatory and catalytic domains were similar to those in other
glycogen phosphorylases. One intron of 139 bp was verified beginning after
the 40th amino acid codon. The transcriptional start site was determined at 134
nucleotides upstream of the ATG initiation codon. Gel retardation assays
demonstrated that there were at least two nuclear DNA binding proteins from
vegetative amoebae (V 1 and V2 factors) and two from developing cells (D 1 and D2 factors). Experiments with a luciferase reporter gene suggested that a basal
expression of the gp-l gene can be conferred by the 5' region containing 363
bp upstream of the ATG codon and the entire regulatory region is located at 157
to 700 bp upstream of the ATG site. It was also demonstrated that the 363 bp
deletion fragment did not support cyclic AMP (cAMP) responsiveness of the
gp-l gene. DNase I footprinting mapped two regions that were protected by
nuclear DNA binding proteins and one of them was a palindromic sequence:
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