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

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dc.contributor.authorKhampang, Pawjaien
dc.contributor.committeechairRutherford, Charles L.en
dc.contributor.committeememberWinkel, Brenda S. J.en
dc.contributor.committeememberEsen, Asimen
dc.contributor.departmentBiologyen
dc.date.accessioned2014-03-14T21:37:58Zen
dc.date.adate2009-06-11en
dc.date.available2014-03-14T21:37:58Zen
dc.date.issued1995-12-15en
dc.date.rdate2009-06-11en
dc.date.sdate2009-06-11en
dc.description.abstractDuring <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.en
dc.description.degreeMaster of Scienceen
dc.format.extentix, 53 leavesen
dc.format.mediumBTDen
dc.format.mimetypeapplication/pdfen
dc.identifier.otheretd-06112009-063539en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-06112009-063539/en
dc.identifier.urihttp://hdl.handle.net/10919/43166en
dc.language.isoenen
dc.publisherVirginia Techen
dc.relation.haspartLD5655.V855_1995.K462.pdfen
dc.relation.isformatofOCLC# 34313365en
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectproteinen
dc.subject.lccLD5655.V855 1995.K462en
dc.titlePurification of the proteins that bind to the gp-2 promoter in Dictyostelium discoideumen
dc.typeThesisen
dc.type.dcmitypeTexten
thesis.degree.disciplineBiologyen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.levelmastersen
thesis.degree.nameMaster of Scienceen

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