MUTANTS OF NEUROSPORA CRASSA PERMEABLE TO HISTIDINOL

Abstract

IN Neurospora, L-histidinol is the immediate precursor of L-histidine, as shown by the isolation of L-histidinol dehydrogenase and the study of its catalytic functions (AMES 1957; CREASER, BENNETT and DRYSDALE 1965). The failure of histidine mutants, with the histidinol dehydrogenase function intact, to grow on histidinol, suggests that histidinol cannot enter the cell. However, CATCHESIDE ( 1960) and WEBBER, CASE and GILES (quoted in WEBBER 1960) have independently obtained variants of histidine mutants that can grow on histidinol. CATCHESIDE (1960) suggested that the mutation might involve a change in the system (permease) by which histidine is transported. The transport of histidine is connected with that of several amino acids. Growth of histidine mutants is inhibited by combinations of a neutral (especially aromatic) amino acid with arginine or lysine (HAAS, MITCHELL, AMES and MITCHELL 1952) and this was shown by MATHIESON and CATCHESIDE (1955) to be due to the prevention of histidine uptake. The present work sought to determine which genes were able to mutate to allow entry to histidinol and, by comparative physiological experiments, to discover the functions of the normal alleles of these genes. Such work should contribute to an understanding of these mutations and the mechanism of inhibition of uptake of histidine. MATERIALS AND METHODS The strains used were the wild type Emerson a and the mutants 30300 (arg-3) argininerequiring; K4-58 (his-3) histidine-requiring; AB9 (lys-4) lysine-requiring; 65001 (nt) nicotinictryptophan-requiring and E151 72 (sfo) sulfonamide-requiring. Strains permeable to histidinol will be described later. The minimal media of VOGEL (1964) and WESTERGAARD and MrrcHmL (1947), with appropriate supplements, were used for vegetative growth and crosses, respectively. VOGEL’S medium plus 0.5% L-sorbose and 0.1% sucrose (SS) with suitable supplements was used for scoring progeny. VOGFL’S medium plus 0.5% L-sorbose, 0.0125% D-glucose and 0.025% D-fructose (SGF) with suitable supplements was used for the isolation of mutants and germination of ascospores. The quantities of supplements (mg per 100 ml medium) used routinely for crosses, maintenance and scoring of cultures were as follows: 20 L-arginine HCI, L-citrulline, L-histidine HCl.H,O, L-lysine HCl; 60, L-histidinol2HCl; 1, nicotinamide. Cultures were tested for ability to grow on histidinol by inoculating conidia on solid VOGEL’S medium supplemented with histidinol; scoring was usually possible after 3 days of incubation at 25°C. It may be noted that K458 (his-3) also grows very poorly on solid medium containing histidinol but much less as compared to strains permeable to histidinol. Tests for sjo and nt were also done on solid VOGEL’S medium, sjo being scored after 1-2 days of incubation at 34°C.