Date of Award:

5-1-1990

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Department name when degree awarded

Biology/Molecular Biology & Biochemistry

Committee Chair(s)

Dennis L. Welker

Committee

Dennis L. Welker

Committee

Jeffery Kondo

Committee

Gregory Podgorski

Abstract

While nuclear-associated DNA plasmids are very rare in eukaryotic organisms, cellular slime molds of the genus Dicytostelium harbor many of them. The Dgp1 plasmid is one carried by Dictyostelium giganteum strain DG6.1. A restriction endonuclease site map of Dgp1 has been produced. The GC content of the restriction site sequences was found to be 42%, which closely matches the typical GC content of Dictyostelium coding sequences. Micrococcal nuclease digestion demonstrated that this plasmid has a typical Dictyostelium chromatin structure indicating its nuclear location. The stability of the Dgp1 DNA sequence when cloned in several Escherichia coli vectors as well as its small size (4.4-kb) suggests that Dgp1 may be a good candidate for the construction of shuttle vectors between Dictyostelium and Escherichia coli. Thus, a Dictyostelium transformation vector was constructed based on the Dgp1 plasmid. Transformation of Dictyostelium discoideum with the plasmid Dgp1 and its recombinant derivatives revealed that the transforming DNA species were integrated into chromosomes in tandem arrays. Despite several attempts, it was not possible to transform D. giganteum cells. A single 3.3-kb transcript from the plasmid Dgp1 was found in DG6.1 cells. This transcript was replaced by several different Dgp1-homologous transcripts in the D. discoideum transformants. These transcripts were larger than the unit length of Dgp1. This different expression might explain why the Dgp1 sequence could not be maintained as an extra chromosomal element in the transformants and instead integrated into the chromosomes. The production of the endogenous 3.3-kb transcript appeared to be developmentally regulated. Expression seemed maximal at growth phase, dropped down at the onset of development through aggregation and early culmination, then increased at culmination and fruiting body formation. The transcripts in the D. discoideum transformants were also developmentally regulated, but the timing was different from that of the endogenous 3.3-kb Dgp1 transcript. The expression was at a lower level at growth phase, increased to a maximum at aggregation, then dropped down at culmination through fruiting body formation. The region coding the 3.3-kb transcript has been localized on the restriction map of Dgp1. Two repeated regions of several hundred base pairs have also been detected in Dgp1. Interestingly, the repeats seem to lie outside the region encoding the 3.3-kb transcript. This, compared to the sequence analyses of Dictyostelium plasmids pDG1 and Ddp2, suggests that the organization of Dgp1 includes a single long open reading frame surrounded by a pair of inverted repeats containing the plasmid origin of replication.

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