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TRANSCRIPTIONAL REGULATION OF THE GLYCOGEN PHOSPHORYLASE-2 GENE IN DICTYOSTELIUM DISCOIDEUM
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The expression of the glycogen phosphorylase- 2 gene (gp2) is initiated during early development and regulated by the extracellular morphogens cAMP and Differentiation Inducing Factor (DIF-1) [1-3]. Glycogen phosphorylase- 2 catalyzes the breakdown of glycogen reserves in developing cells to generate glucose precursors required for the synthesis of the end products of differentiation [4-6]. Thus, the expression of gp2 is a significant event for cellular differentiation. The sequence of the gp2 promoter, like other Dictyostelium promoters, has an AT-rich bias (88%) . Previous deletional analyses of the promoter provided a map of the regions that contained transcriptional regulatory elements. The regions thus identified contained either "TAAAAATGGA" or C-rich repeat sequences . These regions were dissected further by site-directed mutagenesis (SDM) to better define the physical boundaries of the regulatory elements. It was shown that the mutation of either one of the C-rich repeats resulted in a dramatic drop of about 95% in reporter gene levels. These data strongly suggested that both the C-rich repeats of gp2 functioned as transcriptional regulatory elements. I have identified and purified a factor called TF2 that demonstrates a high specificity for a C-rich transcriptional regulatory element, the 5' C box. TF2 was first detected with electrophoretic mobility shift assays of DEAE chromatographic fractions of cell-free extracts. The specificity of TF2 for the 5' C box was tested by competition analysis using six other oligonucleotides. Purification of TF2 was achieved by ion-exchange chromatography, DNA affinity chromatography, gel filtration chromatography, and preparative SDS-PAGE. SDS-PAGE analysis indicated an apparent subunit molecular weight of 28 kDa. The apparent molecular weight of the native protein as estimated by gel filtration was about 53 kDa. This suggested that TF2 binds gp2 as a homodimer. A cDNA clone of the tf2 gene was provided by the Japanese Dictyostelium cDNA project. This allowed me to synthesize probes for Southern and Northern blot analyses. Southern blot analysis indicated that there is only one form of the tf2 gene. Northern analysis showed little or no expression of tf2 in undifferentiated cells. During development tf2 expression increases up to a maximum at 8 h, then decreases in later stages. Attempts to disrupt the gene suggest that tf2 mutation may be lethal.
- Doctoral Dissertations