ENZYME ACTIVITY AND CELL DIFFERENTIATION IN NEUROSPORA

ZALOKAR, MARKO. (Yale U., New Haven, Conn.) Enzyme activity and cell differentiation in Neurospora. Amer. Jour. Bot, 46(7): 555-559. Illus. 1959.-Morphological differences were observed in vegetative cells of Neurospora of different ages and in different parts of the mycelium. The surface layer of mycelium grown in standing cultures could be separated from the deep layer. The first contained most of the growing hyphae rich in protoplasm, while the second containecl heavily vacuolated hyphae laden with fat droplets. Specific activities of several enzymes were studied in conidia, young hyphae, and the surface and deep layers of mature mycelium. Succinic dehydrogenase was low in conidia and about 10 times more active in mature mycelium. The surface layer had twice the activity of the deep layer. Aldolase increased about 3 times after the germination of conidia; it was slightly lower in the surface than in the deep layer of mycelium. Tryptophan synthetase exhibited only small differences between conidia and mycelium and was slightly lower in the surface than in the deep layer. f-galactosidase was formed in appreciable amounts only after prolonged growth and had a much higher specific activity in the deep layer. The results were discussed in connection with cell differentiation and aging. IT HAS BEEN generally observed that there are progressive changes in the activity of enzymes in developing cultures of microorganisms, but such changes have seldom been correlated with concurrent changes in cell morphology. In Neurospora, B8-galactosidase has been found to increase with the age of the culture (Landman, 1951), while tryptophan synthetase decreases (Yanofsky, 1952a). It was not clear whether these changes were due to the aging of the entire culture or to the differentiation of cells which are endowed with different levels of enzyme activities. The latter assumption was suggested by the fact that the proportion of growing hyphal tips to old hyphae decreased with the age of the mycelium. The purpose of the present study was to investigate whether morphologically distinct types of hyphae in Neurospora mycelia show differences in enzymatic activities. Four enzymes, chosen to represent a variety of functions in metabolism, have been studied: (1) succinic dehydrogenase as a key enzyme in the citric acid cycle and for its localization in mitochondria; (2) aldolase as an essential enzyme in glycolysis; (3) 8-galactosidase as an induced enzyme; and (4) tryptophan synthetase as a constitutive enzyme under direct control of a particular gene (Yanofsky, 1952a). Enzymes which require either DPN or TPN as a co-factor could not be considered, since they could not be studied in conidial extracts which are rich in diphosphopyridine nucleotidase (Zalokar and Cochrane, 1956). MATERIAL AND METHODS.-Wild-type Neurospora crassa, E 5297, was used for all experiments. In order to obtain large quantities of conidia, Neurospora was grown on 500 ml. of nitrate medium (Zalokar and Cochrane, 1956) with 2% agar in 'Received for publication March 2, 1959. Fernbach flasks. It was found that conidiation was stimulated if cultures were kept for 3 days at 30?C. and subsequently moved to room temperature (20?-25?C.) for the next 4 days. Conidiation was further stimulated by replacing cotton plugs with several layers of gauze in order to increase aeration. The yield was as great as 5 g. fresh wt. of conidia per flask. Uniform growth of short, separate young hyphae was obtained on 200 ml. of minimal medium (Beadle and Tatum 1945) in 500-ml. Erlenmeyer flasks, vigorously shaken on a reciprocating shaker at 30?C. The cultures were inoculated with a heavy suspension of conidia (ca. 10 mg. fresh wt. per 100 ml. of medium) to provide enough material for chemical determinations. Older mycelia, free of conidia, were grown on a Fries minimal medium containing Tween 80 (Zalokar, 1954), in standing cultures. Large mycelial mats were grown in Roux' flasks in 200 ml. of media by incubation at 30?C. for 4 days. Layers of old and young parts of the mycelium could be separated in these mats after filtration on a Buchner funnel, as described below. It would, of course, have been desirable to work with material of various ages grown under similar conditions. This was not possible, because standing cultures were not appropriate for obtaining young hyphae in large amounts and shaken cultures were not suitable for obtaining older mycelia of uniform quality. In old, shaken cultures, hyphae coalesce into masses of mycelium of different sizes and unequal structure. The size of such masses limits the amount of tissue which has free access to the surrounding medium. This ambiguity could be avoided in standing cultures where the exposure of all the surface layer to the air and nutrients is uniform. Standing and shaking probably result in