Calcium effects in Neurospora crassa

Calcium effects in N. crassa 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/vol34/iss1/6 The resulting homogenate was shaken for 10 min and then centrifuged at 3,000 rpm for 15 min at 15° C. The aqueous phase was taken and an equal volume of phenol:chloroform: isoamyl alcohol; 49:49:2 was added. After shaking for 10 min, the mixture was centrifuged as above and the aqueous phase taken. Phenol was removed by four extractions with ether. To the RNA solution 2.5 volumes of cold absolute ethanol (-20° C) was added and the mixture was placed in a freezer at -80° C for at least 30 min. The RNA precipitate was collected by centrifugation, washed with cold absolute ethanol and then dried under a flow of N2 gas. The precipitate was dissolved in 5 ml of buffer containing 0.1 M sodium acetate (pH 5.0)/1 mM EDTA/1% SDS (buffer A). The crude RNA extract was subjected to gel filtration through a Sephadex G-100 column (2x32 cm) equilibrated with buffer A and fractionated into 2 ml fractions. RNA fractions 6 to 11 were pooled and the solution was diluted to 4-fold with buffer A. About 450 times as much RNA was recovered using method II compared with that recovered with method I. The gel filtration is useful to remove small RNAs and to remove free radioisotope when labeling of RNA was performed. To the RNA solution 2.5 volumes of cold absolute ethanol was added and the resulting mixture was stored at -80° C for at least 30 min. The ethanol precipitate was dissolved in 10 ml of 0.2 M NaCl and 25 ml of cold absolute ethanol added. After collecting the precipitate by centrifugation, it was dissolved in 10 ml of 10 mM Tris (pH 7.5)/0.5 M KCl. The RNA solution was loaded to an oligo (dT)-cellulose (Pharmacia, type 7) column. RNAs were eluted stepwise with buffers containing 10 mM Tris (pH 7.5)/0.5 M KCl, 10 mM Tris (pH 7.5)/0.1 M KCl and 10 mM Tris (pH 7.5). The polyadenylated mRNA fraction eluted with 10 mM Tris (pH 7.5) was pooled and loaded to an oligo (dT)-cellulose column. Polyadenylated mRNA was purified by 2 or 3 cycles of oligo (dT)-cellulose chromatography. Total amounts of RNA isolated by method II were 670-fold larger than those by method I, and polyadenylated mRNAs isolated by method II were 150-fold greater in amounts than those by method I. The ratio of polyadenylated mRNA to total RNA-was 1.0%; this value is very similar to those reported previously (M.C. Lucas et al. 1977, J. Bacteriol. 130:1192-1198. We are grateful to Mrs. M. Yazawa and Miss T. Imaizumi for excellent technical assistance. This research was supported by a Grant-in-Aid for Special Project Research from the Ministry of Education, Science and Culture of Japan (No. 60105002). National Inst. for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki 444 Japan Ghelani, S., B.G. Nair and H.S. Chhatpar Reissig and Kinney reported the role of Ca^+2 in the induction of apical branching in Calcium effects in Neurospora crassa N. crassa STL74A (Reissig and Kinney 1983 J. Bact. 154:1397-1402). Slayman et al. (1976 Biochim. Biophys. Acta 426:732-744) had suggested that spontaneous localized depolarization events could lead to localized Ca^+2 entry and hence branching. In the present study, attempts were made to determine the influence of calcium (Ca^+2) on carbohydrate metabolism and carotenogenesis in N. crassa. N. crassa (wild type, carotenogenic) obtained from the Division of Mycology and Plant Pathology, Indian Agricultural Research Institute, New Delhi, India was grown as described earlier (Nair and Chhatpar 1983 Neurospora Newsletter 30:11). Ca^+2 was added to the synthetic medium devoid of Ca^+2 as CaCl2 (anhydrous) at the desired concentration. Calcium deficient, calcium optimal and calcium supraoptimal conditions indicate no addition, addition of 10 ug/ml and 100 ug/ml or above to the growth medium, respectively. Methods for the preparation of cell-free extract, assay of FDP aldolase, isocitrate lyase, G6P dehydrogenase and protein were the same as described earlier (Savant et al. 1982 Experientia 38:310-311). Amylase was assayed according to the method of Bernfeld (Bernfeld, P. Meth. Enzymol. 1:149). Thin layer chromatography was carried out on 0.25 mm silica gel G plates (Ranboxy Co.). The solvent system used was 20% ethyl acetate in methylene chloride. Carotenoids were estimated according to the method of Davies (B.H. Davies, In:Chemistry and Biochemistry of Plant Pigments (T.W. Goodwin, Ed.) Academic Press. pg. 389) N. crassa grown under calcium deficient condition showed lower activity of extracellular amylase as compared to calcium optimal and supraoptimal conditions (Table 1). Calcium deficient cultures however showed higher activities of FDP aldolase and isocitrate lyase as compared to calcium optimal and supraoptimal conditions. No significant change was observed in the activity of FDP aldolase from calcium optimal to supraoptimal conditions. However, in the case of isocitrate lyase, the activity was found to be decreased in supraoptimal as compared to optimal conditions. Table 1: Effect of Ca^+2 on the activities of extracellular amylase, FDP aldolase, isocitrate lyase and G6P dehydrogenase in N. crassa. Growth conditions Extracellular FDP Isocitrate G6P amylase aldolese lyase dehydrogenase (U/50 ml) (U/mg protein) Calcium deficient Calcium optimal (10 ug/ml) Calcium supraoptimal (100 ug/ml) 1 5 0 1 9 3 7 9 9 3 9 7 2 8 0 1 2 0 4 4 0 3 2 2 2 6 1 1 2 6 3 8 1 3 4 8 Calcium supraoptimal 339 129 327 359 (1000 ug/ml) The mechanism of the effect of calcium on enzymes is not known. Whether calcium influences the activity of enzyme(s) or changes the rate of synthesis or changes the level of cyclic AMP which subsequently alters the level or activity of enzyme(s) is not known. Calcium mediated activation of amylase has been reported by Takegi et al. (1971 In:The Enzymes (P.D. Boyer, Ed.) Academic Press 5:235). In other studies in a number of instances, antagonistic regulatory roles of Ca^+2 and cyclic AMP have been suggested (M.J. Berridge 1975 Adv. Cyclic Nucleotide Res. 6:1-98; Rasmussen and Goodman 1977 Physiol. Rev. 57:421-509). Ealier, Flavell and Woodward had demonstrated that isocitrate lyase is subjected to catabolite repression (Flavell and Woodward 1971 J. Bacteriol. 105:200). In the present studies, if calcium changes the level of cyclic AMP at all, then the possibility exists it can affect the synthesis of isocitrate lyase. Calcium did not affect the activity of G6P dehydrogenase when added in the growth medium. G6P dehydrogenase is known to provide reducing power for the biosynthesis of lipids. The level of G6P dehydrogenase was not found to be affected whereas the level of carotenoids was significantly reduced under supraoptimal as compared to calcium deficient and calcium optimal conditions (Table 2.). This suggests that the effect of calcium may be on some of the enzymes(s) of a carotenogenic pathway. In order to ascertain if there was accumulation of any intermediate(s) in calcium supraoptimal conditions, thin layer chromatography of extracts of carotene from the Ca^+2 supraoptimal and calcium deficient cultures was carried out and the plates were then checked for fluorescence under UV light. The extract of calcium supraoptimal grown culture showed a distinct band which was not so prominent in the calcium deficient culture. Table 2: Effect of Ca^+2 on carotene production in N. crassa. Growth conditions Carotenoids (ug/g mat wet weight) Calcium deficient 25 Calcium optimal 34 (10 ug/ml) Calcium supraoptimal 6 (100 ug/ml Data obtained in this study indicate the influence of Ca^+2 in the growth medium on some enzymes of carbohydrate metabolism and the production of carotenoids in N. crassa. These studies on the regulatory effects of calcium on biochemical changes could be useful in boosting primary metabolism which can then trigger secondary metabolism. The desired products of secondary metabolism can potentially be increased by maintaining a suitable concentration of Ca^+2 in the growth medium. Dept. of Microbiology, Faculty of Science, M.S. University of Baroda, Baroda 390002, India.

In the present study, attempts were made to determine the influence of calcium (Ca^+2) on carbohydrate metabolism and carotenogenesis in N. crassa.
N. crassa (wild type, carotenogenic) obtained from the Division of Mycology and Plant Pathology, Indian Agricultural Research Institute, New Delhi, India was grown as described earlier (Nair and Chhatpar 1983 Neurospora Newsletter 30:11). Ca^+2 was added to the synthetic medium devoid of Ca^+2 as CaCl2 (anhydrous) at the desired concentration. Calcium deficient, calcium optimal and calcium supraoptimal conditions indicate no addition, addition of 10 ug/ml and 100 ug/ml or above to the growth medium, respectively. Methods for the preparation of cell-free extract, assay of FDP aldolase, isocitrate lyase, G6P dehydrogenase and protein were the same as described earlier (Savant et al. 1982 Experientia 38:310-311). Amylase was assayed according to the method of Bernfeld (Bernfeld, P. Meth. Enzymol. 1:149). Thin layer chromatography was carried out on 0.25 mm silica gel G plates (Ranboxy Co.). The solvent system used was 20% ethyl acetate in methylene chloride. Carotenoids were estimated according to the method of Davies (B.H. Davies, In:Chemistry and Biochemistry of Plant Pigments (T.W. Goodwin, Ed.) Academic Press. pg. 389) N. crassa grown under calcium deficient condition showed lower activity of extracellular amylase as compared to calcium optimal and supraoptimal conditions (Table 1). Calcium deficient cultures however showed higher activities of FDP aldolase and isocitrate lyase as compared to calcium optimal and supraoptimal conditions. No significant change was observed in the activity of FDP aldolase from calcium optimal to supraoptimal conditions. However, in the case of isocitrate lyase, the activity was found to be decreased in supraoptimal as compared to optimal conditions. Calcium deficient Calcium optimal (10 ug/ml) Calcium supraoptimal (100 ug/ml) 1 5 0 1 9 3 7 9 9 3 9 7 2 8 0 1 2 0 4 4 0 3 2 2 2 6 1 1 2 6 3 8 1 3 4 8 Calcium supraoptimal 339 129 327 359 (1000 ug/ml) The mechanism of the effect of calcium on enzymes is not known.
Whether calcium influences the activity of enzyme(s) or changes the rate of synthesis or changes the level of cyclic AMP which subsequently alters the level or activity of enzyme(s) is not known. Calcium mediated activation of amylase has been reported by Takegi  Ealier, Flavell and Woodward had demonstrated that isocitrate lyase is subjected to catabolite repression (Flavell and Woodward 1971 J. Bacteriol. 105:200).
In the present studies, if calcium changes the level of cyclic AMP at all, then the possibility exists it can affect the synthesis of isocitrate lyase.
Calcium did not affect the activity of G6P dehydrogenase when added in the growth medium.
G6P dehydrogenase is known to provide reducing power for the biosynthesis of lipids. The level of G6P dehydrogenase was not found to be affected whereas the level of carotenoids was significantly reduced under supraoptimal as compared to calcium deficient and calcium optimal conditions (Table 2.). This suggests that the effect of calcium may be on some of the enzymes(s) of a carotenogenic pathway. In order to ascertain if there was accumulation of any intermediate(s) in calcium supraoptimal conditions, thin layer chromatography of extracts of carotene from the Ca^+2 supraoptimal and calcium deficient cultures was carried out and the plates were then checked for fluorescence under UV light.
The extract of calcium supraoptimal grown culture showed a distinct band which was not so prominent in the calcium deficient culture.

Growth conditions
Carotenoids (ug/g mat wet weight) Calcium deficient 25 Calcium optimal 34 (10 ug/ml) Calcium supraoptimal 6 (100 ug/ml Data obtained in this study indicate the influence of Ca^+2 in the growth medium on some enzymes of carbohydrate metabolism and the production of carotenoids in N. crassa. These studies on the regulatory effects of calcium on biochemical changes could be useful in boosting primary metabolism which can then trigger secondary metabolism. The desired products of secondary metabolism can potentially be increased by maintaining a suitable concentration of Ca^+2 in the growth medium. ---Dept. of Microbiology, Faculty of Science, M.S. University of Baroda, Baroda 390002, India.