Title page for ETD etd-06112009-063539

Type of Document Master's Thesis
Author Khampang, Pawjai
URN etd-06112009-063539
Title Purification of the proteins that bind to the gp-2 promoter in Dictyostelium discoideum
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Rutherford, Charles L. Committee Chair
Esen, Asim Committee Member
Winkel, Brenda S. J. Committee Member
  • protein
Date of Defense 1995-12-15
Availability restricted

During Dictyostelium development, glycogen degradation is a crucial event that provides glucose monomers that are utilized to synthesize the essential structural components for cellular differentiation. The degradation is catalyzed by the product of the glycogen phosphorylase-2 gene. Cloning and sequencing of the gp-2 gene revealed several repeated sequences in the promoter region that are putative regulatory sites. I present here the purification of a DNA binding protein that binds to the 3' "C" box sequence in the gp-2 promoter using a DEAE Sephacel resin and specific "C" DNA affinity column chromatography. With undifferentiated amoebae cell extract, a DNA binding protein migrated at 0.40 Rf and with 17 hr differentiated cell extract, the protein migrated at 0.32 Rf. Both the 0.32 and 0.40 Rf proteins were purified to homogeneity and showed to consist of three subunits of 18 kD, 35 kD and 62 kD (for 0.40 Rf) or 81 kD (for 0.32 Rf). Amino acid sequence analysis showed identity between a region of the 62 and 81 kD subunits. I conclude that the difference in the shifted 0.40 Rf and 0.32 Rf bands in EMSA is due to the 62 and 81 k.D subunits. A southwestern blot analysis of the 17 hr cell extract demonstrated that the DNA binding activity resides in the 81 kD polypeptide. The effect of pH and phosphatase inhibitors on a "conversion" between the 0.32 and 0.40 Rf bands was examined. The results suggest that phosphorylation may be involved in the "conversion" reaction. This study suggests that the purified protein may be a trans-acting factor that is involved in gp-2 regulation.

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