Molecular analysis of glycogen phosphorylase-1 gene expression during the development of dictyostelium discoideum
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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 ð gtll expression library made from amoebae stage mRNA. The 5Â· 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: CAAGTCGCTIG.
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