Original Paper: Gross, S. R. 1965. The regulation of synthesis of leucine biosynthetic enzymes in Neurospora. Proceedings of the National Academy of Sciences USA 54:1538–1546.

The fungus Neurospora crassa first drew researchers’ attention as a common contaminant in French bakeries, and it quickly proved to be an exceptional model for research. Its nutritional requirements are simple, it grows quickly and easily in the lab, and it follows the rules of Mendelian genetics.

In one study, researchers experimented with Neurospora to identify the steps in the biosynthesis of the amino acid leucine. The figure below shows a partial pathway that was proposed early in this research.

Neurospora is haploid for most of its life cycle. During sexual reproduction, haploid cells of two different mating types fuse, producing a diploid cell that carries both sets of parental genes. Neurospora grows on minimal medium without added amino acids because it can synthesize all of the amino acids and other compounds it needs to carry out its metabolic functions.

With these properties in mind, the researchers isolated two mutant Neurospora strains and tested them for growth on various media. Fused cells produced from mating between the two Neurospora mutants were isolated and tested in the same way. The results are shown in the table, where “+” indicates growth and “–” indicates no growth.

Questions

Question 1

Leu-1 is a mutation in the protein catalyzing step C. The product of this step (α-ketoisocaproate) is the first in the pathway able to overcome blockage in this mutant; therefore, Leu-1 must be a mutation in the enzyme at step C. Leu-2 is a mutation in the protein catalyzing step B. The product of this step (3-isopropylmalate) is the first in the pathway able to overcome blockage in this mutant; therefore, Leu-2 must be a mutation in the enzyme at step B.

Question 2

The mutant cells carrying the Leu-1 mutation are deficient in enzyme C but carry the wild type enzyme B. The mutant cells carrying the Leu-2 mutation are deficient in enzyme B but carry the wild type enzyme C. Therefore, when the two cells fuse, the diploid cell contains one copy of wild type enzyme B and one copy of wild type enzyme C, which restores the wild type phenotype.

Question 3

Question 4

One mutant strain is affected in the gene encoding the α subunit and the other mutant strain is affected in the gene encoding the β subunit. Thus, haploid cells cannot produce a functioning α₂β₂ enzyme because these cells have only one copy of each gene, one of which is normal and one of which is not. Diploid cells, however, have two copies of each gene with one normal gene for α supplied by one of the mating pair and one normal gene for β, supplied by the other of the mating pair. The presence of a copy of a normal gene for each subunit allows the diploid cells to produce normal polypeptide chains of α and β, which associate to form a functional α₂β₂ enzyme.