Uv

Mutagenised conidia

Single ascospores transferred to complete medium containing vitamins and amino acids

Asci containing Wild type Crossed with ascospores wild-type of opposite mating type

Conidia from each culture tested on minimal medium

Conidia from mutant strain tested on various supplemented media

Single ascospores transferred to complete medium containing vitamins and amino acids

Asci containing Wild type Crossed with ascospores wild-type of opposite mating type

Conidia from each culture tested on minimal medium

Conidia from mutant strain tested on various supplemented media

All grow

No growth on minimal medium identifies a nutritional mutant

All grow

No growth on minimal medium identifies a nutritional mutant gly ala leu ile val gln phe tyr ser cys thr asn arg pro his lys tyr trp Growth test with single amino acid supplementation

Figure 5.11 The procedure used by Beadle and Tatum for production and identification of auxotrophic mutants in Neurospra crassa. MM, minimal medium; aa, amino acid.

differential growth response in media lacking or containing a single chemical compound. In each case, the auxotrophic mutation (a mutation that results in the requirement of any of the metabolic end products) was inherited as a single-gene mutation, i.e., when crossed to a wild (original standard) strain it gave a 1:1 ratio of the mutant and the wildtype progeny.

As an example, a set of mutant strains required arginine to grow on minimal medium. By a gene mapping procedure, Beadle and Tatum found that these mutants mapped into three different locations on separate chromosomes, even though all could grow if the same supplement (arginine) was added to the medium. However, three mutants, arg-1, arg-2 and arg-3, differed in their response to related compounds, ornithine, citrulline and arginine. The arg-1 mutants grew with supplementation of ornithine, citrulline or arginine. The arg-2 mutants grew on either arginine or citrulline but not on ornithine. The arg-3 mutants grew only when arginine was supplied. The results demonstrated that synthesis of arginine proceeds from ornithine through citrul-line and that each mutant strain lacks only one enzyme. Based on the properties of the arg mutants of Neurospora, Beadle and Tatum proposed a sequence of reaction in which each reaction is controlled by a specific enzyme, identified by a specific gene (Figure 5.12).

This model, known as the one gene-one enzyme hypothesis, was inferred from the properties of mutant classes. It provided the first insight into the functions of genes, that each gene controls one specific enzyme. Nearly all mutants responded to single growth supplement, i.e., each mutant was blocked in a single biosynthetic pathway. The position of the mutational block in the biochemical pathway could be determined by growth tests, whether the accumulation of the last intermediate preceded the block or occurred after the block. There are enzymes encoded by more than one gene, however, the one gene-one enzyme hypothesis fits most genes. This work demonstrated that the mutant approach can be generally exploited to determine individual steps in metabolic pathways. In 1958, George Beadle and Edward Tatum were awarded the Nobel Prize (shared with Joshua Lederberg) for their discovery that genes act by specifying enzymes that catalyze definite chemical reactions. The discovery laid the foundation of biochemical genetics. Beadle (1966) recalled:

During the course of this work Tatum's late father, Arthur Lawrie Tatum, then Professor of Pharmacology at the University of Wisconsin, visited Stanford. On this occasion he arg-1

arg-2

arg-3

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