A modified Vogel's medium for crossings, mating-type tests and the isolation of female-sterile mutants of Neurospora crassa.

A modified Vogel's medium for crossing, mating-type tests and the isolation of female-sterile mutants of Neurospora crassa. This regular paper is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol32/iss1/4 T A B L E I TABLE I I Derepression of the effect of inositol upon the synthesis of MIPS in wild-type N. crassa Effect of various inhibitors upon the synthesis of MIPS in wild-type Neurospora crassa Growth* Enzyme Antigen “Specific ainiral activity content activity’ medium (h) U/mg μg/mg U/ μg protein protein antigen 0 16.3 8.7 1.9 0.5 18.4 7.1 2.6 1.0 21.5 9.2 2.3 2.0 4.0 33.5 12.8 2.6 75.5 29.3 2.6 22.0 8.0 77.1 31.3 2.5 72.4 27.8 2.6 *Cultures of strain RL-3-8A were pregrown in Vogel’s medium containing 50 μg inositol/ml, at 27° C for 22 h. Enzyme Antigen “Specific activity content activity” u/mg μg/mg U/μg protein protein antigen control° 16.3 8.7 1.9 at 0 h controI* 91.5 35.2 2.6 + cycloheximide* 2.5 μg/ml 5.0 13.6 0.4 + edein* 23.5 25.3 0.9 50 μg/ml °: strain RL-3-8A was grown at 27°C for 22 h in Vogel’s . medium containing 50 μg/ml inositol *: the cultures were further grown at 27°C for 5 h in Vogel’s medium with and without inhibitors, as shown We also examined how the MIPS derepression was affected by inhibitors of translation. Cycloheximide and edein were used as inhibitors of translation. The wild-type strain was cultured at 27°C for 22 h with 50 μg/ml inositol and then the growth was continued for 5 h in Vogel's minimal medium without inositol with the addition of the inhibitor. The cultures were then harvested' and enzyme activity and enzyme protein content were measured (Table II). It was found that enzyme production was significantly diminished in the presence of cycloheximide and edein. Actinomycin D and proflavin, as potential inhibitors of transcription, in concentrations (10, μg/ml) that completely inhibited the growth of our strain did not decrease enzyme synthesis. We conclude that: a> enzyme production is derepressed when inositol is washed from the culture medium; and b) MIPS production is regulated at the posttranscriptional level. The same type of regulation was observed in MIPS production of yeast (S. Henry et al., 1984' 12th International Conference on Yeast Genetics and Molecular Biology, Edinburgh). 1Department of Biochemistry and 2Biology, University Medical School of Debrecen, H-4012, Hungary. Russo, V.E. A. , T. Sommer and J.A. A. Chambers Our laboratory has been studying the photoinduction of protoperithecia A modified Vogel's medium for crossings, for several years. Originally all work mating-type tests and the isolation of was done with Westergaard and Mitchell female-sterile mutants of Neurospora medium, but this medium was found to be crassa. extremely inconvenient because it is difficult to prepare more concentrated than a 2x stock. Vogel's medium, although it can be prepared as a 50x stock, has a high nitrogen content which favours the production of conidia but not of protoperithecia. The convenience of a 50x stock prompted us to try to modify Vogel's medium for use as a crossing medium. We found that if the nitrogen concentration of the 50x stock was reduced from 100 gm to 10 gm NH4NO3 per liter then the medium promotes the production of enough protoperithecia for crossing and mating type testing. For colonial growth we replace the sucrose with 1.1% sorbose and 0.1% glucose. To test for protoperithecia production in this medium it was necessary to reduce the nitrogen concentration in the 50x stock to 1 gm of NH4NO3 per liter. Sorbose and sucrose are added from a sterile stock of 20% sorbose, 2% glucose that is steamed (9O°C, 30 min) twice with a 24h interval. On these plates protoperithecia appear after 4 days in the dark at 23°C. Typically a colony of 1 cm diameter has about 400 protoperithecia. Illumination accelerates the production of protoperithecia. TABLE I Ascospore yield end viability from different media (arg-10 , A x wc-l, a) Medium Ascospores %Germination+ Vogel's modifieda 3.6 x 104 60 " 2.2 x 104 60 " 4.3 x 104 82 Westergaard's 4.0 x 104 81 4.0 x 104 47 " 4.5 x 104 56 + After 24h germination at 34°C. a The 50x stock contained 10 gm NH4NO3/liter. Data from three crosses on each medium. Plates for testing mating type with a non-fluffy tester are incubated at 23°C for 3 days and conidia cleaned from the edges of the plate with an ethanol-soaked tissue on the third and fourth days. Mycelia can be fertilized between the 4th and 7th days. For crosses we use an essentially similar procedure in tubes with fertilization between the 5th and 7th days. Ascospores appear after about two weeks. If the female parent is an auxotroph for a potential nitrogen source we use'the minimal concentration compatible with good growth, e.g. 0.3 mg/ml-1 arginine for arg-1, -6, -10 and 0.7 mg/ml-1 glutamate for am132. We have been using this system for more than a year without any problems. We have compared the fertility of crosses on nitrogen-poor Vogel's and Westergaard and Mitchell's nineteen days after fertilization. Ejected ascospores were collected, counted and the efficiency of germination determined. We found the two to be comparable (Table I). Detailed experiments on the role of nitrogen in differentiation will be published later: We wish to acknowledge excellent technical support from Monika Götz and Uta Marehfelder and financial support from the Deutsche Forschungsgemeinschaft. MaxPlanck-Institut fur molekulare Genetik, Abt. Trautner, Ihnestrasse 63/73, B-1000 Berlin 33, West Germany.