Effect of various inhibitors on the production of myo-inositol-1-phosphate synthase in Neurospora crassa wild-type strain

Effect of various inhibitors on the production of myo-inositol-1-phosphate synthase in Neurospora crassa wild-type strain Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol32/iss1/3 These results suggest that an integration event resulted in a tandem arrangement of inl sequences leaving the expression of both inl+ and inlgenes intact. In Southern hybridization experiments, bands characteristic for covalently closed circular plasmids were detected in the DNA isolated from the F1 progeny of the transformants using the vector as a hybridization probe. (The principle of the detection was that the supercoiled plasmid DNA migrates faster than uncleaved chromomsomal DNA in an agarose gel-electrophoresis.) The copy-number of the transforming cosmid sequences was estimated to be 20-25/genome right after transformation and l-2/genome after two months of vegetative propagation on agar slants with periodic transfers. Eleven recombinant plasmids (pNCs) with different restriction patterns were recovered from the transformants by means of E. coli transformation. Some of these plasmids proved to be rearranged in N. crassa, e . g . deletions within the vector sequences could be detected. None of the pNCs transformed Neurospora to inositol prototrophy. There are a number of explanations for the presence of different plasmids in the F1 progeny of integrative-type transformants: (1) The plasmids were maintained during meiosis by autonomous replication. (2) They are the result of excision from the chromosome. (3) chromosomal DNA fragments carrying vector sequences were taken up and circularized by E. coli. Biochemistry*, Departments of Biology and University Medical School, H.-4012 Debrecen, Hungary. +Present Address: Department of Biology, University of Rochester, NY 14627. 1Kiss, A., 1A, Zsindely, The synthesis of myo-inositol-l-phosphate synthase (MIPS, 2M. Schablik and 2G. Szabo E.C.5.5.1.4.) in wild-type Neurospora crassa strains is almost completely repressed by inositol at a concentration Effect of various inhibitors of 50 μg/ml (Zsindely et al., 1983 on the production of myoBiochim. Biophys. Acta 741:273). inositol-l-phosphate synWe studied whether the enzyme was derepressed thase in Neurospora crassa after removing inositol from the medium. wild type strain. Wild-type Neurospora crassa strain RL-3-8A was grown at 27° C for 22 h in Vogel's culture medium containing 50 μg/ml inositol. Following harvest, the mycelium was washed, suspended in Vogel's minimal medium and growth was continued for 22 h during which samples were taken at various times. Enzyme activity and the amount of enzyme protein were determined in the 100,000 g supernatant after disintegration of the mycelium (Table I). Enzyme 'activity was determined according to Barnett et al. (1970, Biochem. J. 119: 183), as described earlier (Zsindely et al., 1977, Acta Biol. Acad. Sci. Hung. 28:281). One unit of activity is 1 nmol Pi released during 1 h incubation. The amount of protein reacting with monovalent immune sera produced against highly purified enzyme was determined by rocket immunoelectrophoresis according to Laurel.1 (1966, Anal. Biochem. 15: 45) in a 1% agrose gel containing 1% immune serum. Table I shows that MIPS becomes derepressed after removing inositol from the culture medium. Four h later the enzyme activity and the antigen content become similar to those measured in the crude extracts of the wild-type strain cultivated without inositol. No further change in enzyme activity or antigen content was observed up to 22h of cultivation. 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.


inositol-l-phosphate syn-
We studied whether the enzyme was derepressed thase in Neurospora crassa after removing inositol from the medium.wild type strain.

Wild-type
Neurospora crassa strain RL-3-8A was grown at 27° C for 22 h in Vogel's culture medium containing 50 µg/ml inositol.Following harvest, the mycelium was washed, suspended in Vogel's minimal medium and growth was tinued for 22 h during which samples were taken at various times.Enzyme activity a amount of enzyme protein were determined in the 100,000 g supernatant after disintegration of the mycelium (Table I).Enzyme 'activity was determined according to Barnett et al. (1970, Biochem. J. 119: 183), as described earlier (Zsindely et al., 1977, Acta Biol. Acad. Sci. Hung. 28:281).
One unit of activity is 1 nmol Pi released during 1 h incubation.
The amount of protein reacting with monovalent immune sera produced against highly purified enzyme was determined by rocket immunoelectrophoresis according to Laurel.1 (1966, Anal. Biochem. 15: 45) in a 1% agrose gel containing 1% immune serum.
Table I shows that MIPS becomes derepressed after removing inositol from the culture medium.Four h later the enzyme activity and the antigen content become similar to those measured in the crude extracts of the wild-type strain cultivated without inositol.No further change in enzyme activity or antigen content was observed up to 22h of cultivation.
T A B L E I °: 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).

TABLE II
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 *Cultures of strain RL-3-8A were pregrown in Vogel's medium containing 50 µg inositol/ml, at 27° C for 22 h.