The major part of the wall of Schizosaccharomyces pombe consists of (1-+3)-a-glucan and (1+3)-P-glucan with some (l&)-P-linkages. Although in hydrolysed samples only a minute amount of glucosamine could be detected, this amino sugar may play an essential role as an integral part of a glucosaminoglycan/glucan complex. Treatment of the wall with either nitrous acid or chitinase changed the solubility properties of the p-glucan, which suggests that the glucosaminoglycan/glucan complex is essentially similar to that found in walls of other fungi. An enzyme with properties similar to that of chitin synthase of other fungi, and probably responsible for the synthesis of the glucosaminoglycan, was detected in a mixed-membrane fraction.

Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae CHS2 gene product were used to identify and localize chitin synthase in the filamentous ascomycete Neumspora crassa. A single band of approximately 11 0 kDa was observed in Western blots of total protein extracts of N. crassa, probed with these antibodies. However, several additional bands were labelled when membrane fraction proteins (microsomes) were probed. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles (chitosomes), which are abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labelling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips.

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S U M M A R YProtoplasts of a strain of Pythiuii? (~~~2 1 4 2 ) could be obtained by incubating the mycelium with a combination of helicase and cellulase in the presence of an osmotic stabilizer. This process was inhibited when organic compounds were used as osmotic stabilizers but stimulated by pretreatment of the mycelium with a detergent. The most effective detergent was Triton X 100. Regeneration of protoplasts could be demonstrated in liquid or in solid media, but less than 30:/; of the protoplasts were able to grow into new mycelium. In contrast to protoplast formation, regeneration was inhibited by the inorganic salts used as osmotic stabilizer. The results suggest that the hyphal wall of Pythium is covered with a protective layer of lipid material. I N T R O D U C T I O NThe regeneration of fungal protoplasts is of current interest because of the importance of cell-wall formation in morphogenesis. To study this process in Pythiurn P R L~I Q a convenient method is needed to make a large number of protoplasts.It is possible to make protoplasts of Pj)thium P R L~I~~ with an enzyme system produced by a Streptomyces species, containing cellulase, exo-and endolaminaranase activity (Sietsma, Eveleigh & Haskins, 1969). The disadvantages of this method are that it takes a long incubation time (24 h) to get a sizeable number of protoplasts and that the preparation and purification of the enzyme complex is very tedious and laborious. A method has been found which makes large quantities of protopIasts of Pythium within a period of 4 h by using a combination of commercially available helicase and cellulase. The regeneration of these has been studied. M E T H O D SOrganism and growth conditions. Pythiuin PRL 2142 described by Haskins (1963) was grown in shake culture at 28 "C in a medium containing (g/l): KH,P04, 0.5; K2HP04, 0.5 ; K,S04, 0.5 ; MgC1,. 6H,O, 0.6 ; glucose, I o ; asparagine, I -2 ; and thiamine, 0-00 I .Before sterilization the pH of the medium was adjusted to 6.2, andfcholesterol (0.1 g) added as a 10% (w/v) solution in ethanol. After the culture had grown for a certain time, specified under results, the mycelium was collected by filtration, washed twice with distilled water, and then with an incubation medium containing an osmotic stabilizer dissolved in 0.005 M-acetate buffer, pH 6.2. The following substances were used as osmotic stabilizers: KCI, 0.4 M; NaCl, 0.4 M; NH,Cl, 0.4 M; MgS04, 0.65 M; sorbitol, 0.65 M; mannitol, 0.65 M ; sucrose, 0.65 M. The mycelium was finally suspended in an incubation medium at a concentration of 30 mg organismfml, helicase ( I -2 mglml) and cellulase (2 mgfml) added, and incubated at 30 "C. In some experiments the mycelium was either pretreated or incubated with SH-compounds, chelating agents or detergents as described below.