Characterization of variant Neurospora crassa mitochondrial DNAs which contain tandem reiterations.

Two variant mtDNA types ((types IIa and HI-10) have been identified in individual subcultures of the extra-nuclear poky] mutant of Neurospora crassa. Eco RI digests of type IIa mtDNA are characterized by an extra band, alpha, Mr = 1.4 Mdal, which arises from tandemly inserted reiterations of a 1.4 Mdal sequence. Restriction enzyme analysis and Southern hybridization experiments show: that the 1.4 Mdal repeats are located at the junction of Eco RI-4 and -6, that the repeats contain sequences ordinarily present in Eco RI-4 and -6, that the repeats are oriented head-to-tail and that the number of repeats per molecule (n) varies from n = 0 to n = 8, with about half of the molecules containing no repeats. The 1.4 Mdal repeats appear to be actively mained in type IIa mtDNA populations as a result of a specific alteration in mtDNA. Data are presented which suggest that this alteration may be located near small deletions and/or sequence changes in Eco RI-3 and -10, fragments almost exactly opposite the site of the repeats on the genome. The second variant, HI-10 mtDNA, arose in a heteroplasmic strain in which type IIa mtDNA was one component. The most striking feature of HI-10 mtDNA is the up to 5-fold amplification of an 18 Mdal segment extending from Eco RI-4 (the site of the 1.4 Mdal repeats) through the rRNA genes. Eco RI digests show that HI-10 possesses characteristic features of type IIa mtDNA, including the 1.4 Mdal repeats and the alteration in Eco RI-10. HI-10 mtDNA also shows a novel Eco RI fragment, beta, Mr = 2.9 Mdal. The variant Neurospora mtDNAs may be generated by mechanisms analogous to those which give rise to defective mtDNAs of yeast petite mutants. The possible consequences of defective mtDNAs in obligately aerobic organisms are discussed.