Figure 14.2 Cytochrome content in wild type and senescent nd strains of Neurospora crassa. The cytochromes were identified by recording the difference in absorbance between dithionite-reduced and ferricyanide-oxidized mitochondria at wavelengths of light ranging from 500 to 650 nm. The absorbance maxima at 550, 560, and 608 nm correspond to cytochrome c, b, and cytochrome oxidase aa3, respectively. The senescent strains nd9 and nd6 are deficient in cytochromes aa3 and c. The senescent strain maintained in a heter-okaryon (ndH) has spectra similar to wild-type. (From Seidel-Rogol et al. (1989). American Society of Microbiologists. With permission.)
The nuclei and mitochondria in the fused cell multiply and mix. To force heterokaryotic growth, auxotrophic markers, e.g., pantothenic acid or leucine, are incorporated into the fusing strains by prior crossing. Only the mixed cytoplasmic growth, i.e., heterokaryon (nonsenescent pan + senescent leu) grows on minimal media due to the complementation of nuclei from the original strains. The heterokaryotic nonsenescent mycelium can be propagated indefinitely. The genetically marked senescing and nonsenescing nuclear types from the heterokayon can be separated by plating conidia formed by heterokaryotic mycelium on appropriate media. For example, if the pan cultures senesce on pantothenate-supplemented medium, the senescent determinant is in the cytoplasm (mitochondria).
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