Inactivation of the Neurospora crassa Gene Encoding the Mitochondrial Protein Import Receptor MOM19 by the Technique of ''Sheltered RIP''

We have used a technique referred to as ``sheltered RIP'' (repeat
induced point mutation) to create mutants of the mom-19 gene of
Neurospora crassa, which encodes an import receptor for nuclear
encoded mitochondrial precursor proteins. Sheltered RIP permits the
isolation of a mutant gene in one nucleus, even if that gene is
essential for the survival of the organism, by sheltering the
nucleus carrying the mutant gene in a heterokaryon with an
unaffected nucleus. Furthermore, the nucleus harboring the RIPed
gene contains a selectable marker so that it is possible to shift
nuclear ratios in the heterokaryons to a state in which the nucleus
containing the RIPed gene predominates in cultures grown under
selective conditions. This results in a condition where the target
gene product should be present at very suboptimal levels and allows
the study of the mutant phenotype. One allele of mom-19 generated
by this method contains 44 transitions resulting in 18 amino acid
substitutions. When the heterokaryon containing this allele was
grown under conditions favoring the RIPed nucleus, no MOM19 protein
was detectable in the mitochondria of the strain. Homokaryotic
strains containing the RIPed allele exhibit a complex and extremely
slow growth phenotype suggesting that the product of the mom-19
gene is important in N. crassa.