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UBC Theses and Dissertations

Investigation of a kalilo plasmid in Louisiana Neurospora tetrasperma Kuehn, Monica M.


Senescence in Neurospora is caused by the insertion of a linear plasmid, kalilo, into the host mitochondrial genome. Kalilo-related senescence has been studied extensively in Hawaiian field-isolated strains of Neurospora intermedia. This work, however, investigated the presence of a kalilo-like plasmid, named kalilo', within Louisiana-isolated strains of Neurospora tetrasperma. Comparison of the physical characteristics of the kalilo andkalilo' plasmids revealed that the plasmids have identical restriction maps. In addition, digestion of the kalilo' plasmid with 5' and 3' exonucleases showed a 5' resistance to enzyme digestion and a 3' sensitivity to digestion. This suggested that kalilo' may have a protein bound to its 5' termini, as is the case with kalilo. Biological properties of the Louisiana plasmid were also examined. Investigation of inheritance patterns through crosses demonstrated a maternal inheritance, thereby suggesting that kalilo' was located within the mitochondria. Additionally, transmission of the plasmid by asexual culturing was relatively stable. In order to determine if the plasmid inserted into the host mitochondrial genome, DNA from the mitochondria was cut with the restriction endonuclease Pst I (an enzyme that does not cut the kalilo plasmid DNA). Unlike the Hawaiian kalilo, kalilo' did not appear to insert into the mitochondrial DNA. Finally, given that insertion did not occur, the relationship between kalilo' and senescence was investigated. Various cultures with and without kalilo' were sub cultured (serial vegetative transfers) and surveyed for senescence. Senescence failed to occur within the forty subcultures observed. While senescence did occur within growth tubes, the relationship between senescence and kalilo' are not tightly correlated. On consideration of these results, several questions arose. What was the evolutionary relationship between kalilo and kalilo'? If the kalilo' plasmid was identical or very similar to kalilo, why did integration not occur? What was the catalyst for senescence in N. tetrasperma? These matters have been addressed within the text, but definitive answers are still forthcoming.

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