Purification of the proteins that bind to the gp-2 promoter in Dictyostelium discoideum

During <i>Dictyostelium</i> 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 <i>gp</i>-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 <i>gp</i>-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 <i>gp</i>-2 regulation.

Name thesis:degree_name

Master of Science

Level thesis:degree_level

masters

Discipline thesis:degree_discipline

Biology

Department dc:contributor.department

Biology

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

1995

Author dc:creator

• Khampang, Pawjai

Chair dc:contributor.committeechair

• Rutherford, Charles L.

Committee members dc:contributor.committeemember

• Winkel, Brenda S. J.
• Esen, Asim

• protein

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Dc Identifier Other

etd-06112009-063539

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/43166