Orientation of enzymic domains in tryptophan synthase of Neurospora crassa: an immunoblot analysis of TRP3 mutant products.
Extracts of 52 TRP3 mutants of Neurospora crassa were tested for the presence of serologically cross-reacting material by the method of electrophoretic blot analysis. The test antigen was obtained by excision of lightly stained bands of denatured pure tryptophan synthase after SDS-polyacrylamide gel electrophoresis. Rabbit antisera raised against this antigen neutralized and precipitated native tryptophan synthase. Of the 52 strains, 19 exhibited banding patterns similar to wild type on electrophoretic blots, 25 strains gave no apparent bands, and 8 strains showed unique banding patterns. This evidence and related genetic fine structure mapping data indicate that strains exhibiting banding patterns similar to wild type carry missense mutations. Strains which did not exhibit any obvious bands may have resulted from certain kinds of missense or nonsense mutations or from frameshift mutations or extended deletions. Strains exhibiting unique banding patterns on electrophoretic blots were interpreted as carrying chain-terminating mutations or deletions. Genetic fine structure mapping data place the mutant lesions of these strains in a linear order corresponding to the apparent molecular weights of the crossreacting protein fragments which they exhibit. The direction of transcription and translation of the TRP3 locus in Neurospora was inferred from these relationships. The apparent organization of the Neurospora TRP3 gene is consistent with that ascribed to the Saccharomyces TRP5 system and suggests that the N-terminal portion of the protein corresponds to the "A" protein of the Escherichia coli system and that the C-terminal portion of the protein corresponds to the "B" protein of the bacterial system.
D003433:Crosses, Genetic; D005796:Genes; D005800:Genes, Fungal; D009154:Mutation; D009491:Neurospora / Q000235:genetics*; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics*; D013489:Suppression, Genetic; D014367:Tryptophan Synthase / Q000235:genetics*
D014367:Tryptophan Synthase