Thesis

Localization of a 3.7 KB plasmid of Polytomella Parva using photobiotin as a non-radioactive probe

The electrophoresis of whole cellular DNA from the non-photosynthetic alga Polytomella parva revealed a distinct 3.7 kb band. This band of DNA was isolated from the agarose gel and used to construct a non-radioactive probe utilizing Photobiotin. Cellular fractions were then isolated by sucrose gradient centrifugation and the mitochondrial fraction identified by succinic dehydrogenase activity. DNA was then isolated from the fraction that showed maximum enzymatic activity and used as a target for the 3. 7 kb Photobiotin probe in a Southern Transfer procedure. A successful annealing of the Photobiotin probe with a similar 3.7 kb segment of DNA is mitochondrial in origin. Lack of annealing with the nuclear and mitochondrial chromosomal DNA indicates that its sequence is not present in those DNA fractions. The Photobiotin probe was used to visualize the plasmid by attaching colloidal gold labeled avidin to the probe. The electron-micrographs indicate that the plasmid may be linear in structure.

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