A Method for the Selection of Deletion Mutations in the L-Proline Catabolism Gene Cluster ofAspergillus nidulans

Abstract

SUMMARY: Interest in the selection of mutations affecting L-proline catabolism inAspergillus nidulansis heightened by the involvement of one of the very few examples of a cluster of functionally related genes in an eukaryote and by an increasing awareness of the biological phenomena in which proline and proline catabolism participate. ThesasA-60 (semialdehyde sensitive) mutation inA. nidulansresults in toxicity of catabolic precursors of L-glutamic γ-semialdehyde (or its internal Schiff base L-Δ¹-pyrroline-5-carboxylate) and succinic semialdehyde, apparently without affecting the catabolic pathways concerned. AssasA-60 is unlinked to theprngene cluster, specifying the gene products necessary for L-proline catabolism and as L-proline, a precursor of L-glutamic γ-semialdehyde, is highly toxic tosasA-60 strains, this forms the basis of a powerful positive selection technique for obtaining a number of types ofprnmutations. Many of theseprnmutations can be directly classified according to the gene product(s) affected on the basis of growth phenotype with respect to L-arginine and L-ornithine utilization, proline-dependent resistance to certain toxic amino acid analogues and effect on supplementation of proline auxotrophies. The availability of both a positive selection technique and an extensive nutritional screening system has enabled the identification of fourteen spontaneous deletion mutations, recognized as extending into theprnB gene, specifying the principal L-proline permease, and into at least one otherprngene. These deletion mutations have been partially characterized both genetically and biochemically. In particular their use has greatly facilitated fine-structure mapping of theprncluster and aided studies of the regulation ofprngene expression.