Communications Project

Document Type:Master's Thesis
Name:Bekir Col
Degree:Master of Science
Department:Biology (Molecular&Cellular Biology Division)
Committee Chair: Dr. Charles Lee Rutherford
Committee Members:Dr. Reyna Favis
Dr. Asim Esen
Dr. Ann Stevens
Keywords:DNase I footprint analysis, transcription, gene expression, AT-rich, looping, large footprints, replication protein A, cell differentiation, Dictyostelium discoideum
Date of defense:December 12, 1997
Availability:Release the entire work for Virginia Tech access only.
After one year release worldwide only with written permission of the student and the advisory committee chair.


Glycogen phosphorylase 2 (gp-2) is a key enzyme during the development of Dictyostelium discoideum. The gp-2 enzyme breaks down glycogen into glucose monomers that are subsequently used to synthesize the terminal end products of cellular differentiation. This gene is an ideal candidate for studying the process of selective gene expression because its product figures so prominently in the development of this organism, implying a dependable control mechanism responsible for its developmentally regulated expression. I present in this thesis the identification of several putative cis-acting elements of gp-2 as revealed through footprint analysis. Due to the extreme AT-bias characteristic of Dictyostelium promoters, footprinting conditions required intensive optimization with respect to template, nonspecific competitor, source of protein extract and DNase I digestion. Using an endlabeled fragment containing seven repeated sequences (3 TA boxes [TAATTATA], 2 TAG boxes [TAAAAATGGT] and 2 C boxes [ACCCACT]), purified replication protein A and several developmental nuclear extracts were tested for DNA binding activity. Small footprints were observed on the TAG and C boxes of the promoter for both protein sources. However, using a more sensitive footprinting strategy involving multiple rounds of primer extension, larger footprints spanning the same promoter regions were detected. In both cases, the appearance of the footprints coincided with the documented transcriptional activity of the gene. It can be concluded from the data obtained that the TAG and C boxes are very likely cis-acting elements involved in the regulation of gp-2 expression.

List of Attached Files

Figure_1.2.pdf acknowledgement_table_of_contents.pdf figure1.4.pdf
figure2.1.pdf figure_1.1.pdf figure_1.2.pdf
figure_1.3.pdf figure_2.2.pdf figure_3.1___3.16.pdf
figure_4.1_and_4.2.pdf sections.pdf table_A_B.pdf

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