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

SUMMARY Interest in the selection of mutations affecting L-proline catabolism in Aspergillus nidulans is 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. The sas A-60 (semialdehyde sensitive) mutation in A. nidulans results in toxicity of catabolic precursors of L-glutamic γ-semialdehyde (or its internal Schiff base L-Δ 1 -pyrroline-5-carboxylate) and succinic semialdehyde, apparently without affecting the catabolic pathways concerned. As sas A-60 is unlinked to the prn gene cluster, specifying the gene products necessary for L-proline catabolism and as L-proline, a precursor of L-glutamic γ-semialdehyde, is highly toxic to sas A-60 strains, this forms the basis of a powerful positive selection technique for obtaining a number of types of prn mutations. Many of these prn mutations 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 the prn B gene, specifying the principal L-proline permease, and into at least one other prn gene. These deletion mutations have been partially characterized both genetically and biochemically. In particular their use has greatly facilitated fine-structure mapping of the prn cluster and aided studies of the regulation of prn gene expression.