1. Retention or loss of mitochondrial markers CR321, OR1, PR454, TR (gene loci RIB1, OLI1, PAR1, TSM1 respectively has been analysed in a large number of ethidium bromide induced primary rho-clones. Retention of one or more of the four markers with a single clone was observed frequently, only 20 to 25% of clones were found to be (TOCOOOPO). Primary clones retaining two or more of the four markers were found to be mixed, i.e. the primary rho- cell contained a heterogeneous population of variously deleted mitDNA molecules which segregated into different cell lines in the corresponding primary clone. 2. A representative sample of the population of ethidium bromide induced rho- mutants has been analysed by a first subcloning performed after some 30 cell generations of vegetative multiplication in the abscence of the drug. At this level the heterogeneous population of mitDNA molecules, generated by the mutagenic treatment in the primary cell, has been sorted out. The cells forming secondary clones are thus essentially homoplasmic. In contrast to primary clones, genotypes of secondary clones therefore could be determined unambiguously, and the frequency of cell types can be regarded as a faithful representation of the frequency of mitDNA molecules. Retention of markers was low, in less than 2% of secondary clones one or several markers have been found. This observation has been interpreted as indicating that induction of rho-mutants by ethidium bromide is accompanied by deletion of very large sequences of mitDNA in a very large fraction of mitDNA molecules. 3. Five individual rho-clones retaining the four markers TRCRORPR have been isolated and analysed for spontaneous deletion of one or several of these markers during successive subclonings (pedigree analysis). High genetic stability (98-99.5% per cell generation) has been observed in these clones. 4. A method has been developed allowing an unambiguous determination of the order of the four markers on a circular map. It is based on the concomitant loss of two markers and retention of the other two markers (double loss/double retention analysis). The results of four out of five pedigrees of individual rho-clones analysed (spontaneous deletion) and the results of the analysis of populations of secondary rho-clones (ethidium bromide induced deletion) were in full agreement and the order of genes has been determined as being P-T-C-O-P. In the fifth pedigree results suggest an inversion of the T and C markers. 5. Relative distances between pairs of markers have been derived from the frequencies of separation of markers by deletion and were found to be C-T less than C-O less than T-O less than T-P less than C-P less than O-P. Linkage of the four markers could be established, and distances calculated are additive. 6. The general relevance of this approach of mapping by deletion and the methods used for the determination of order and distances of mitochondrial genes has been discussed. (ABSTRACT TRUNCATED)

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Journal Article

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Schweyen RJ, Steyrer U, Kaudewitz F

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