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

Abstract

A method is described for measuring the syntheses of macromolecules in the filamentous fungus Aspergillus nidulans. It involves growth of mycelium on small filter papers in the presence of radioactively labelled L-leucine to monitor total protein synthesis, and hence growth, and following the incorporation of differently labelled D-uridine (to measure nucleic acid synthesis) or L-leucine (to measure protein synthesis) under various conditions such as starvation for a metabolite or the presence of an inhibitor. Comparison of 3H/14C or 14C/3H 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 A. niduldns and that inhibitors of protein synthesis such as cycloheximide and anisomycin also inhibit stable RNA synthesis. In contrast, starvation for meso-inositol affects nucleic acid synthesis much more strongly than it affects protein synthesis.

Toward A Method For Measuring Instantaneous Fungal Growth Rates In Field Samples

“…For sediment slurries, low micromolar concentrations may be required (e.g., Fallon et al 1983, Fallon and Boylen 1990, van Duyl and Kop 1990). Note that other investigators have used 10-1-101 mmol/L added isotope-labeled precursors in order to obtain acceptably high activity in sterols or macromolecules in short (minutes to hours) incubations of fungi in culture or fungal enzyme systems (Pierce et al 1979, Arst and Scazzocchio 1980, Polak-Wyss et al 1985, Peacock and Goosey 1989, Henry 1990). One explanation for this is that fungal incorporation of 3H-thymidine into DNA-fraction-contaminating materials was rapidly rising with increased concentration of added thymidine, since fungal rate-saturation level might be expected to lie in the millimoles-per-litre range (Newell 1984; note that fungal mass is usually >98% of total microbial standing crop of standing-dead cordgrass: ).…”

“…It may be that millimolar concentrations of added labeled precursors are generally required to maximize assessment of fungal-synthesis rates in natural samples. Note that other investigators have used 10-'-10' mmol!L added isotope-labeled precursors in order to obtain acceptably high activity in sterols or macromolecules in short (minutes to hours) incubations of fungi in culture or fungal enzyme systems (Pierce et al 1979, Arst and Scazzocchio 1980, Polak-Wyss et al 1985, Peacock and Goosey 1989, Henry 1990).…”

“…In contrast, there has been only one method published for measurement of total fungal production in naturally decaying litter, and values for instantaneous rate of production were not obtained (Frankland 1975). Isotope-labeling techniques akin to those used to measure bacterial and microalgal productivity have been used in studies of fungal syntheses (e.g., Jack and Anekwe 1972, Taylor and Parks 1978, Pierce et al 1979, Arst and Scazzocchio 1980, Soderhall and Cerenius 1983, Seo et al 1986, Morita et al 1989, Peacock and Goosey 1989, Henry 1990), but in none of these cases was there any attempt to apply radioisotopic techniques to measurement of total fungal productivity.…”