“One gene, one polypeptide” analysis of a Neurospora crassa auxotroph. Beadle and Tatum identified mutant Neurospora that were unable to synthesize the amino acid arginine. To investigate the metabolic pathway of arginine synthesis, they analyzed arginine auxotrophs for growth on minimal medium plus ornithine or citrulline, both precursors of arginine (or on minimal medium plus arginine, to be sure that the mutant grew when supplied with arginine). They found that class I mutants grow when supplied with any of the three compounds, so these mutants lack an enzyme that is upstream of these three compounds (i.e., an enzyme catalyzing an earlier reaction) in the synthetic pathway. Class II mutants do not grow on ornithine, and thus lack an enzyme downstream of this intermediate but upstream of citrulline. Class III mutants grow only on arginine and therefore lack an enzyme involved in the conversion of citrulline to arginine.