INACTIVATION OF THE GENE CODING FOR THE 30.4-KDA SUBUNIT OF RESPIRATORY-CHAIN NADH DEHYDROGENASE - IS THE ENZYME ESSENTIAL FOR NEUROSPORA

We have isolated and characterised the nuclear gene that codes for the 30.4-kDa subunit of the peripheral arm of complex I from Neurospora c rassa. The single-copy gene was localised on chromosome VI of the fung al genome by restriction fragment length polymorphism mapping. An extr a copy of the gene was introduced into a strain of N. crassa by transf ormation. This strain was crossed with another strain in order to inac tivate, by repeat-induced point mutations, both copies of the duplicat ion carried by the parental transformant. Ascospore progeny from the c ross were analysed and a mutant strain lacking the 30.4-kDa protein, n uo30.4, was isolated and further characterised. The mutant appears to assemble the membrane arm of complex I, while formation of the periphe ral arm is prevented. Nevertheless, the mutant grows reasonably well - indicating that this well conserved protein is not essential for vege tative growth - and is able to mate with other strains both as male or female. Strains with multiple mutations are readily obtained from het erozygous crosses between different complex I mutants of N. crassa. On the other hand, homozygous crosses between several mutants, including nuo30.4, fail to produce ascospores. These results suggest that compl ex I plays an essential role during the sexual phase of the life cycle of the fungus.