Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains of Neurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater efficiency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase.

Mutants defective in polyol metabolism and/or in protoperithecial development were selected in Neurospora tetrasperma, a species in which protoperithecial development occurs at nonpermissively high temperature if certain polyols are used in lieu of sucrose as carbon source. Mutants selected for nonutilization of one of the four polyols tested, glycerol, mannitol, sorbitol, or xylitol, were usually found to be nonutilizers of the other three polyols as well. Mutants blocked at various stages of protoperithecial development complemented pairwise to produce more advanced developmental stages, usually mature protoperithecia and, when of opposite mating type, mature perithecia. About one-third of the mutants manifested both polyol auxotrophy and defective protoperithecial development upon initial isolation, but protoperithecial defectiveness in such mutants usually showed erratic segregation in crosses and/or instability to repeated vegetative transfer, whereas polyol auxotrophy usually did not and was, therefore, studied further. Two glycerol nonutilizing strains were introgressed into N. crassa to facilitate genetic analysis. One, glp-4, lacked both inducible and constitutive glycerol kinase and mapped to linkage group VI, between ad-1 and rib-1; the other, glp-5, lacked glyceraldehyde kinase and mapped to linkage group I, proximal to ad-9. Another mutant, gly-u(234), has been reported by other investigators to lack inducible glycerol kinase but to map to linkage group I, distal to ad-9.

The properties of NADP+-linked glycerol dehydrogenase (EC 1 . 1 . 1 .72) from Neurospora crassa were studied following 505-fold purification, with 54% yield, by gel filtration, ion exchange chromatography and affinity chromatography. Specific staining for the purified enzyme after disc gel electrophoresis revealed a single band and after isoelectric focusing, five bands. No enzymically inactive bands were detected by general protein staining in control gels. Total molecular weight was estimated to be about 160000, with a subunit molecular weight of about 43 000. The forward reaction from glycerol to D-glyceraldehyde had a pH optimum of 9.5 and was specific for glycerol as substrate, since no activity was detected with other polyols tested. The reverse reaction had a pH optimum of 6.5 and was maximal with D-glyceraldehyde as substrate. K , values for glycerol and D-glyceraldehyde were 1.43 x 10-1 M and 1.15 x M, respectively. Dihydroxyacetone also served as substrate in the reverse reaction. The enzyme was NADP+-specific.

Protoperithecial differentiation in Neurospora tetrasperma proceeds in four clearly discernible stages: ascogonial bud, ascogonial hook, ascogonial coil, and protoperithecium. This morphogenetic sequence was completely but transitorily blocked at 37°C. Under low oxygen tensions or in the absence of carbon dioxide, only ascogonial coils were differentiated. Glycolytic inhibitors such as 2-deoxy-D-glucose interfered with the ascogonial differentiation. Citric acid cycle inhibitors such as fiuoroacetate selectively blocked the further development of ascogonial coils into protoperithecia. Differentiating cultures at 25'^C responded positively to external sucrose with respiratory quotients above 1.0, whereas non-differentiating cultures at 37°C, although respiring at high rates, never reached RQ 1.0. These results indicate that carbohydrate metabolism via fermentative glycolysis functions normally in ascogonia differentiating (25 C) cultures only.Comparative chemical analysis showed delayed RNA synthesis, sharp disturbances in protein biosynthesis and metabolism, and lower levels of carbohydrate and bound lipids in young cultures at 37"C.Polyols, especially ribitol and xylitol utilized as sole source of carbon, induced abundant protoperithecial morphogenesis at 37'C. This alleviating effect of polyols was negated by the addition of sugars at 37°C and shown to be a specific metabolic effect.

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