Mild RIP - An alternative method for in vivo mutagenesis of the albino-3 gene in Neurospora crassa

We have used a biological phenomenon that occurs in Neurospora crassa, termed Repeat-Induced Point mutation (RIP), to create partially functional mutant alleles of the albino-3 (al-3) gene encoding geranylgeranyl pyrophosphate synthase, an enzyme involved in the biosynthesis of carotenoids and diverse prenylated compounds. A total of 70 RIP-induced al-3 mutants were identified by their pale albino phenotype, resulting from inactivation of carotenoid biosynthesis. Nucleotide sequence analysis of the al-3 gene in five of the RIP-induced mutants revealed that in each case RIP had introduced no more than six point mutations. The low frequency of RIP mutants (0.42%) and the isolation of only leaky mutants with very few mutations suggest that ascospores containing a heavily mutated al-3 gene do not survive. These results are evidence that the RIP phenomenon, used to inactivate and silence duplicated genes in N. crassa, may be exploited in its mild version as a method of sequence-specific in vivo mutagenesis to obtain functional mutant alleles of Neurospora genes. This mild form of mutagenesis may be particularly advantageous in selecting for leaky mutations in essential Neurospora genes.