Date of Award:

5-1-1997

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Dennis L. Welker

Committee

Dennis L. Welker

Committee

Gregory J. Podgorski

Committee

Nabil N. Youssef

Committee

Noelle E. Cockett

Committee

Jon Y. Takemoto

Abstract

Dictyostelium discoideum plasmid Ddp6 from the wild strain NC47.2 is a 5257 bp nuclear plasmid with a copy number of 200-300 per cell. Analysis of the nucleotide sequence and structural organization of the Ddp6 plasmid has identified it as a new member of the Ddp2 plasmid family. Ddp6 contains a single gene that is homologous to the rep gene of Ddp2. As with Ddp2, a single constitutively expressed transcript of 3.3 kb was detected from the rep gene. The Ddp6 Rep open reading frame of 2.8 kb encodes a protein of 932 amino acids. A set of 10 conserved peptide sequences of unknown function was identified among the Rep proteins in the Ddp2 family. The distribution of these peptides throughout the Rep protein sequence suggests that the Rep proteins retain similar tertiary structure and functions. Ddp6 has a pair of 0.6 kb inverted repeat elements located upstream of the rep gene. These are similar in size and location to the inverted repeats found in all other members of the Ddp2 plasmid family. Deletion experiments revealed that the intact Ddp6 rep gene and sequences within the inverted repeat are essential for long-term stable maintenance of the plasmid in Dictyostelium cells. The yeast two-hybrid system was used to show that the Rep proteins of three members of the Ddp2 plasmid family (Ddp2, Ddp5, and Ddp6) are able to form homomultimers but heteromultimers. This analysis also revealed that multiple sites of the Rep proteins are likely to be required for homomultimerization to occur. One of these sites may lie near the carboxy terminus of the protein. The chromatin structure of the Ddp2 and Ddp6 plasmids in their inverted repeat and promoter regions was analyzed. Digestion experiments with micrococcal nuclease and restriction endonucleases showed that the Rep protein is a DNA-binding protein and is responsible for nucleosome positioning in the Ddp6 promoter region. On the other hand, nucleosome positioning in the inverted repeat regions appeared to be independent of the presence of Rep protein. This suggests the involvement of cellular factors, most likely the Dictyostelium origin recognition complex, although nucleosome positioning due to features of the DNA sequence cannot be excluded. Finally, the DNA-binding activity of the heterologously expressed Ddp2 Rep protein was tested in electrophoretic mobility shift assays. The binding activity of the purified in vitro expressed Ddp2 Rep protein towards plasmid-specific and nonspecific DNA was similar. This result confirms that Rep protein can bind double-stranded DNA and suggests that the presumed specificity of its binding requires the action of cellular factors, either for the binding itself or for post-translational modification of the protein.

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