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Volume 118, Issue 2

A Method for Monitoring Macromolecular Synthesis in Filamentous Fungi and its Use to Study Control of Nucleic Acid Synthesis in Free

Abstract

A method is described for measuring the syntheses of macromolecules in the filamentous fungus It involves growth of mycelium on small filter papers in the presence of radioactively labelled -leucine to monitor total protein synthesis, and hence growth, and following the incorporation of differently labelled -uridine (to measure nucleic acid synthesis) or -leucine (to measure protein synthesis) under various conditions such as starvation for a metabolite or the presence of an inhibitor. Comparison of H/C or C/H labelling ratios (depending on the labelling combination used) shows the effects of the treatments on nucleic acid or protein synthesis. The method allows large numbers of measurements to be made and enables economical use of radioisotopes. Results are presented to show that, under the labelling conditions used, uridine is incorporated mainly into stable RNA. The method is used to demonstrate probable stringent control of stable RNA synthesis in and that inhibitors of protein synthesis such as cycloheximide and anisomycin also inhibit stable RNA synthesis. In contrast, starvation for -inositol affects nucleic acid synthesis much more strongly than it affects protein synthesis.

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© Society for General Microbiology 1980
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1980-06-01
2025-05-13
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References

  1. Arst H. N. JR 1968; Genetic analysis of the first steps of sulphate metabolism in Aspergillus nidulans . Nature; London: 219268–270
     [Google Scholar]
  2. Arst H. N. JR 1971; Mutants of Aspergillus nidulans unable to use choline-O-sulphate. Genetical Research 17:273–277
     [Google Scholar]
  3. Arst H. N. JR Page M. M. 1973; Mutants of Aspergillus nidulans altered in the transport of methylammonium and ammonium. Molecular and General Genetics 121:239–245
     [Google Scholar]
  4. Arst H. N., Scazzocchio C. 1971; RNA synthesis in Aspergillus nidulans . Heredity 26:346
     [Google Scholar]
  5. Arst H. N., Scazzocchio C. 1972a; Control of nucleic acid synthesis in Aspergillus nidulans . Biochemical Journal 127:18P
     [Google Scholar]
  6. Arst H. N., Scazzocchio C. 1972b; Control of nucleic acid synthesis in Aspergillus nidulans . Heredity 29:131
     [Google Scholar]
  7. Arst H. N. JR Scazzocchio C. 1975; Initiator constitutive mutation with an ‘up-promoter’ effect in Aspergillus nidulans . Nature; London: 25431–34
     [Google Scholar]
  8. Bailey C., Arst H. N. JR 1975; Carbon catabolite repression in Aspergillus nidulans . European Journal of Biochemistry 51:573–577
     [Google Scholar]
  9. Battaner E., Kumar B. V. 1974; Rifampin: inhibition of ribonucleic acid synthesis after potentiation by amphotericin B in Saccharomyces cerevisiae . Antimicrobial Agents and Chemotherapy 5:371–376
     [Google Scholar]
  10. Buckel P., Böck A. 1973; Lack of accumulat ion of unusual guanosine nucleotides upon amino acid starvation of two eukaryotic organisms. Biochimica et biophysica acta 324:184–187
     [Google Scholar]
  11. Clutterbuck A. J. 1974; Aspergillus nidulans. In Handbook of Genetics I pp. 447–510 Edited by King R. C. New York: Plenum Press;
     [Google Scholar]
  12. Cove D. J. 1966; The induction and repression of nitrate reductase in the fungus Aspergillus nidulans . Biochimica et biophysica acta 113:51–56
     [Google Scholar]
  13. Cybis J., Weglenski P. 1972; Arginase induction in Aspergillus nidulans. The appearance and decay of the coding capacity of messenger. European Journal of Biochemistry 30:262–268
     [Google Scholar]
  14. Darlington A. J., Scazzocchio C. 1967; Use of analogues and the substrate-sensitivity of mutants in analysis of purine uptake and breakdown in Aspergillus nidulans . Journal of Bacteriology 93:937–940
     [Google Scholar]
  15. Dutta S. K., Richman N., Woodward V. W., Mandel M. 1967; Relatedness among species of fungi and higher plants measured by DNA hybridization and base ratios. Genetics 57:719–727
     [Google Scholar]
  16. Edlin G., Broda P. 1968; Physiology and genetics of the ‘ribonucleic acid control’ locus in Escherichia coli . Bacteriological Reviews 32:206–226
     [Google Scholar]
  17. Foley J. M., Giles N. H., Roberts C. F. 1965; Complementation at the adenylosuccinase locus in Aspergillus nidulans . Genetics 52:1247–1263
     [Google Scholar]
  18. Golombek J., Wolf W., Wintersberger E. 1974; DNA synthesis and DNA-polymerase activity in synchronized yeast cells. Molecular and General Genetics 132:137–145
     [Google Scholar]
  19. Gravel R. A. 1976; Choline-O-sulphate utilization in Aspergillus nidulans . Genetical Research 28:261–276
     [Google Scholar]
  20. Gressel J., Galun E. 1967; Morphogenesis in Trichoderma: photoinduction and RNA. Developmental Biology 15:575–598
     [Google Scholar]
  21. Grivell A. R., Jackson J. F. 1968; Thymidine kinase: evidence for its absence from Neurospora crassa and some other micro-organisms, and the relevance of this to the specific labelling of deoxyribonucleic acid. Journal of General Microbiology 54:307–317
     [Google Scholar]
  22. Grollman A. P., Huang M. T. 1973; Inhibitors of protein synthesis in eukaryotes: tools in cell research. Federation Proceedings 32:1673–1678
     [Google Scholar]
  23. Gross K. J., Pogo A. O. 1974; Control mechanism of ribonucleic acid synthesis in eukaryotes. The effect of amino acid and glucose starvation and cycloheximide on yeast deoxyribonucleic acid-dependent ribonucleic acid polymerases. Journal of Biological Chemistry 249:568–576
     [Google Scholar]
  24. Grossman L. I., Goldring E. S., Marmur J. 1969; Preferential synthesis of yeast mitochondrial DNA in the absence of protein synthesis. Journal of Molecular Biology 46:367–376
     [Google Scholar]
  25. Hanson B., Brody S. 1979; Lipid and cell wall changes in an inositol-requiring mutant of Neurospora crassa . Journal of Bacteriology 138:461–466
     [Google Scholar]
  26. Henry S. A., Atkinson K. D., Kolat A. I., Culbertson M. R. 1977; Growth and metabolism of inositol-starved Saccharomyces cerevisiae . Journal of Bacteriology 130:472–484
     [Google Scholar]
  27. Keith A. D., Pollard E. C., Snipes W., Henry S. A., Culbertson M. R. 1977; Inositol-less death in yeast results in simultaneous increase in intracellular viscosity. Biophysical Journal 17:205–212
     [Google Scholar]
  28. Kessel M., Rosenberger R. F. 1968; Regulation and timing of deoxyribonucleic acid synthesis in hyphae of Aspergillus nidulans . Journal of Bacteriology 95:2275–2281
     [Google Scholar]
  29. Kobayashi G. S., Medofe G., Schlessinger D., Kwan C. N., Musser W. E. 1972; Amphotericin B potentiation of rifampicin as an antifungal agent against the yeast phase of Histoplasma capsulatum . Science 177:709–710
     [Google Scholar]
  30. Kudrna R., Edlin G. 1975; Nucleotide pools and regulation of ribonucleic acid synthesis in yeast. Journal of Bacteriology 121:740–742
     [Google Scholar]
  31. Lewis N. J., Hurt P., Sealy-Lewis H. M., Scazzocchio C. 1978; The genetic control of the molybdoflavoproteins in Aspergillus nidulans. IV. A comparison between purine hydroxylase I and II. European Journal of Biochemistry 91:311–316
     [Google Scholar]
  32. Markham P., Bainbridge B. W. 1979; Characterization of a new choline locus in Aspergillus nidulans and its significance for choline metabolism. Genetical Research 32:303–310
     [Google Scholar]
  33. Medoff G., Kobayashi G. S., Kwan C. N., Schlessinger D., Venkov P. 1972; Potentiation of rifampicin and 5-fluorocytosine as antifungal antibiotics by amphotericin B. Proceedings of the National Academy of Sciences of the United States of America 69:196–199
     [Google Scholar]
  34. O’Donovan G. A., Neuhard J. 1970; Pyrimidine metabolism in micro-organisms. Bacteriological Reviews 34:278–343
     [Google Scholar]
  35. Oliver S. G., Mclaughlin C. S. 1977; The regulation of RNA synthesis in yeast. I. Starvation experiments. Molecular and General Genetics 154:145–153
     [Google Scholar]
  36. Orr E., Rosenberger R. F. 1976; Initial characterization of Aspergillus nidulans mutants blocked in the nuclear replication cycle. Journal of Bacteriology 126:895–902
     [Google Scholar]
  37. Palmer L. M., Cove D. J. 1975; Pyrimidine biosynthesis in Aspergillus nidulans. Isolation and preliminary characterization of auxotrophic mutants. Molecular and General Genetics 138:243–255
     [Google Scholar]
  38. Pontecorvo G., Roper J. A., Hemmons L. A., Macdonald K. D., Bufton A. W. J. 1953; The genetics of Aspergillus nidulans . Advances in Genetics 5:141–238
     [Google Scholar]
  39. Ray D. B., Butow R. A. 1979a; Regulation of mitochondrial ribosomal RNA synthesis in yeast. I. In search of a relaxation of stringency. Molecular and General Genetics 173:227–238
     [Google Scholar]
  40. Ray D. B., Butow R. A. 1979b; Regulation of mitochondrial ribosomal RNA synthesis in yeast. II. Effects of temperature sensitive mutants defective in cytoplasmic protein synthesis. Molecular and General Genetics 173:239–247
     [Google Scholar]
  41. Roth R. M., Dampier C. 1972; Dependence of ribonucleic acid synthesis on continuous protein synthesis in yeast. Journal of Bacteriology 109:773–779
     [Google Scholar]
  42. Scazzocchio C., Arst H. N. JR 1978; The nature of an initiator constitutive mutation in Aspergillus nidulans . Nature; London: 274177–179
     [Google Scholar]
  43. Scazzocchio C., Darlington A. J. 1968; The induction and repression of the enzymes of purine breakdown in Aspergillus nidulans . Biochimica et biophysica acta 166:557–568
     [Google Scholar]
  44. Scazzocchio C., Holl F. B., Foguelman A. I. 1973; The genetic control of molybdoflavoproteins in Aspergillus nidulans. Allopurinol-resistant mutants constitutive for xanthine dehydrogenase. European Journal of Biochemistry 36:428–445
     [Google Scholar]
  45. Shatkin A. J., Tatum E. L. 1961; The relationship of m-inositol to morphology in Neurospora crassa . American Journal of Botany 48:760–771
     [Google Scholar]
  46. Shulman R. W., Sripati C. E., Warner J. R. 1977; Noncoordinated transcription in the absence of protein synthesis in yeast. Journal of Biological Chemistry 252:1344–1349
     [Google Scholar]
  47. Smith J. E., Pateman J. A. editors 1977 Genetics and Physiology of Aspergillus London: Academic Press;
     [Google Scholar]
  48. Somberg E. W., Salmon B. J., Davis F. F. 1966; Some effects of methionine starvation on a methionine auxotroph of Neurospora crassa . Biochimica et biophysica acta 129:419–421
     [Google Scholar]
  49. Stevens L. 1975; Ornithine decarboxylase in germinating conidia of Aspergillus nidulans . FEBS Letters 59:80–82
     [Google Scholar]
  50. Stevens L., Mckinnon I. M., Winther M. 1976; Polyamine and ornithine metabolism during the germination of conidia of Aspergillus nidulans . Biochemical Journal 158:235–241
     [Google Scholar]
  51. Storck R., Alexopoulos C. J. 1970; Deoxyribonucleic acid of fungi. Bacteriological Reviews 34:126–154
     [Google Scholar]
  52. Strauss B. S. 1958; Cell death and ‘unbalanced growth’ in Neurospora. Journal of General Microbiology 18:658–669
     [Google Scholar]
  53. Stunnenberg H. G., Wennekes L. M. J., van den Broek H. W. J. 1979; RNA polymerase from the fungus Aspergillus nidulans. Large-scale purification of DNA-dependent RNA polymerase I (or A). European Journal of Biochemistry 98:107–119
     [Google Scholar]
  54. Sullivan J. L., Debusk A. G. 1973; Inositolless death in Neurospora and cellular ageing. Nature New Biology 243:72–74
     [Google Scholar]
  55. Tisdale J. H., Debusk A. G. 1972; Permeability problems encountered when treating conidia of Neurospora crassa with RNA synthesis inhibitors. Biochemical and Biophysical Research Communications 48:816–822
     [Google Scholar]
  56. Verma I. M., Edelman M., Littauer U. Z. 1971; A comparison of nucleotide sequences from mitochondrial and cytoplasmic ribosomal RNA of Aspergillus nidulans . European Journal of Biochemistry 19:124–129
     [Google Scholar]
  57. Waldron C., Roberts C. F. 1974a; Cold-sensitive mutants in Aspergillus nidulans. I. Isolation and general characterisation. Molecular and General Genetics 134:99–113
     [Google Scholar]
  58. Waldron C., Roberts C. F. 1974b; Cold-sensitive mutants in Aspergillus nidulans. II. Mutations affecting ribosome production. Molecular and General Genetics 134:115–132
     [Google Scholar]
  59. Warner J. R., Gorenstein C. 1978; Yeast has a true stringent response. Nature; London: 275338–339
     [Google Scholar]
  60. Wehr C. T., Parks L. W. 1969; Macromolecular synthesis in Saccharomyces cerevisiae in different growth media. Journal of Bacteriology 98:458–466
     [Google Scholar]
  61. Wejksnora P. J., Haber J. E. 1974; Methionine-dependent synthesis of ribosomal ribonucleic acid during sporulation and vegetative growth of Saccharomyces cerevisiae . Journal of Bacteriology 120:1344–1355
     [Google Scholar]
  62. Williamson D. H. 1973; Replication of the nuclear genome in yeast does not require concomitant protein synthesis. Biochemical and Biophysical Research Communications 52:731–740
     [Google Scholar]
  63. Winther M., Stevens L. 1978; Polyamine stimulation of in vivo rates of macromolecular synthesis in a putrescine auxotroph of Aspergillus nidulans . FEBS Letters 85:229–232
     [Google Scholar]
  64. Zinchenko Y. B., Groshev B. V., Kameneva S. V. 1978; Use of a pyrimidine-dependent mutant for effective labelling of the DNA of Aspergillus nidulans . Microbiology, USSR (English translation) 47:851–854
     [Google Scholar]
/content/journal/micro/10.1099/00221287-118-2-443
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A Method for Monitoring Macromolecular Synthesis in Filamentous Fungi and its Use to Study Control of Nucleic Acid Synthesis in Aspergillus nidulans
Microbiology 118, 443 (1980); https://doi.org/10.1099/00221287-118-2-443
/content/journal/micro/10.1099/00221287-118-2-443
/content/journal/micro/10.1099/00221287-118-2-443
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