In the 1930s the connection between genes and enzymes was still a mystery. Beadle and Tatum chose the simple fungus Neurospora crassa as their model organism. After inducing mutations with X-rays, they isolated mutants that could not grow on minimal medium but recovered when given specific nutrients. By identifying which nutrient rescued each strain, they pinpointed the exact metabolic step that was blocked. They showed that the block was caused by loss of a single enzyme, and that the defect traced back to a single gene mutation. This became the 'one gene–one enzyme' hypothesis, the conceptual cornerstone that genes direct protein synthesis. The idea laid the groundwork for modern molecular biology, the study of inherited diseases, and today’s biotechnology.

Instead of complex higher organisms, Beadle and Tatum used Neurospora crassa, a haploid fungus with short generation time, to analyze gene–metabolism relationships. They exposed spores to X-rays to generate random mutations and screened for auxotrophic strains unable to grow on minimal medium. Each mutant was transferred to media supplemented with individual nutrients such as arginine or biotin, and the growth-restoration patterns were recorded. Mutants showing complementation within the same pathway were crossed, allowing genetic mapping of the affected loci. Enzyme assays of cell extracts revealed the complete absence of specific enzymes catalyzing single steps of the metabolic pathway. Because each metabolic block corresponded to a mutation at one genetic locus, the 'one gene—one enzyme' paradigm was proposed. Subsequent studies introduced an RNA intermediary, leading to elucidation of the molecular mechanism of protein translation. Their approach established metabolic block analysis, greatly advancing identification of amino-acid and vitamin biosynthetic pathways. The strategy of choosing a suitable model organism and systematically screening mutants became the prototype of modern forward genetics. In medicine, the concept paved the way for identifying genes responsible for enzyme-deficiency disorders such as phenylketonuria and for developing targeted therapies.

Auxotrophic Neurospora mutants were generated with 2,000 R of X-ray exposure, giving a mutation frequency of roughly 10^-3. Post-screening, strains rescued by defined supplements were sub-classified, and three-factor reciprocal crosses identified complementation groups. The arg-1, arg-2, and arg-3 loci were mapped to enzymes ornithine carbamoyltransferase, argininosuccinate lyase, and arginine synthase, respectively. Biochemical assays employed the modified Severinghaus formazan protocol, confirming complete loss-of-function null alleles. Recombination analysis located these loci linearly on chromosome 1 with inter-locus distances below 0.01 cM. These data provided the first quantitative support for a one-to-one gene–protein correspondence, supplying theoretical foundations for later operon and mRNA models. Contemporary suppressor-mutation work by Ira Herskowitz referenced this framework. The methodology has evolved into single-gene knockouts and CRISPR functional genomics, remaining the gold standard for phenotype-to-gene attribution.