Date of Award:

5-1-1993

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Dennis Welker

Committee

Dennis Welker

Committee

Gayle Knapp

Committee

Gregory J. Podgorski

Abstract

While nuclear-associated, eukaryotic plasmids are rare, cellular slime molds in the Dictyostelium genus harbor many of them. The Ddp5 plasmid is one carried by Dictyostelium discoideum strain WS2162. I have produced a restriction site map of Ddp5; Kpn I, Sal I, and Bgl II have unique sites. Subcloned Ddp5 sequences were stable in E. coli except that deletion derivatives missing about 2.5 or 3 kilobase pairs were observed with p34d5, a recombinant plasmid containing a region of Ddp5 and the cloning vector pGEM3Z, and with full length lambda clones. By cotransforming two of the subcloned DNA plasmids into the plasmid-free D. discoideum strain AX4, I isolated a stable, extrachromosomally replicating plasmid, p94d5, that contained about two-thirds of the entire Ddp5 sequence. Plasmids smaller than p94d5 were not stably maintained. Five RNA transcripts were detected from Ddp5 using poly (A) + RNA from HUD896, an AX4 transformant containing both Ddp5 and Ddp2, or from WS2162 cells. A 3.2 kilobase transcript was expressed at a low level in vegetative and aggregated cells, then expression decreased in culminating cells. A 2.6 kilobase transcript was expressed at low levels in vegetative and aggregated cells, and increased in abundance in culminating cells. A 4.1 kilobase transcript was not detected in vegetative or aggregated cells, and was weakly expressed in culminating cells. A transcript of 0.6 kilobases, absent from vegetative cells, was expressed weakly in aggregated cells and more strongly in culminating cells. A 1.0 kilobase transcript was constitutively transcribed in vegetative, aggregated, and culminating cells. Four putative RNA transcripts of 2.0, 1.7, 1.4, and 0.9 kilobases were also detected in a few experiments. The coding regions of the 3.2, 2.6, 4.1, 0.6, and 1.0 kilobase transcripts have been localized on the restriction site map of Ddp5. The Ddp5 origin of replication probably lies in a 1.7 kilobase pair region to which no transcripts appeared to map. In HUD940 cells (harboring p94d5) the 3.2, 0.6, and 1.0 kilobase transcripts were expressed. Expression of the 3.2 kilobase RNA in HUD940 cells was reduced, the 0.6 kilobase transcript was expressed as in HUD896 cells, and expression of the 1.0 kilobase transcript was also normal. A model for gene expression of Ddp5 has been proposed.

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