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A functional analysis of the kalDNA plasmid from senescent strains of Neurospora intermedia Vickery, Daniel Barry


The 8.6 kb kalilo linear mitochondrial plasmid of Neurospora intermedia was found to give rise to multiple transcripts of 8.6, 4.4, 4.0, 1.3, 1.2, and 0.9 kb. A transcription map has been generated which shows similarities to other linear plasmids. These transcripts are all transcribed from a single, unique promoter sequence reiterated near the ends of the terminal inverted repeats of the linear plasmid. The transcripts are not processed, but instead utilize optional transcription stop sites. An analysis of sub-cellular RNA fractions has confirmed the mitochondrial location of kalilo transcription. The strong association of kalilo-specific RNA with rRNA to yield RNA artifacts is reported. Kalilo-specific RNA appears to be selectively unstable in affected strains of N. intermedia; this may be a general consequence of linear plasmid RNA. The 5' RNA start site was determined by primer extension and RNA sequencing. The sequence in this region does not show homology to any known mitochondrial, plasmid, nor nuclear promoter, and may constitute a novel element. The transcription start site shares homology with the terminus of the linear plasmid, and marks the end of a long series of direct repeats; therefore, the plasmid RNA polymerase may be bifunctional, it may recognize sequences at the ends of the plasmid as well as at the promoter. The analysis of the insertional behaviour of the linear mitochondrial plasmid was studied in parallel subculture series of the organism. It was determined that insertion, per se is not the event required to kill the organism. Generation of inserts of kalilo in the mtDNA is necessary, but not sufficient, for death to occur in all cases. An analysis of insertion sites has found one new site and good agreement with previously published locations. Insertion does not always appear to be random, so cultures may inherit undetectable amounts of mtlS-kalDNA. The analysis of insertion sites in one strain has suggested a novel possible structure for the mtDNA.

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