Level and turnover of polyadenylate-containing ribonucleic acid in Neurospora crassa in different steady states of growth.

Mycelia of Neurospora crassa in a steady state of growth in different media have a ribosomal content proportional to the rate of growth. Moreover, both the percentage of polysomes and the average ribosomal activity are about the same at all different growth rates. The content of polyadenylated RNA was determined in three different conditions of exponential growth, which allowed growth rates that ranged from 0.26 to 0.51 duplications/h, and was found to constitute about the same fraction of total RNA (4.5--5.2%). Using a kinetic approach, an equation was derived which allowed determination of the average half-lives of polyadenylated RNA: in each medium the cultures were labeled from the moment of the inoculation with [32P]orthophosphate and were then given a 10-min pulse with [5-3H]uridine when they were in the exponential phase. It was found that the determined half-lives of polyadenylated RNA vary, depending on the growth medium, between 30 and 60 min, but with no direct correlation with the growth rate. Moreover, the rate of synthesis of polyadenylated RNA relative to that of stable RNA decreased with the growth rate. On the basis of previous data on the rates of synthesis of stable RNA, it was possible to make an evaluation of the absolute rate of synthesis of polyadenylated RNA. Whereas the rate of synthesis of stable ribosomal RNA increases as a function of the square of the number of duplications per hour, the increase in the rate of synthesis of polyadenylated RNA with the growth rate is much less consistent. It is concluded that in Neurospora the growth rate does not depend on the rate of synthesis of mRNA but rather on the rate of synthesis of rRNA, which sets both the ribosomal level and the steady-state level of mRNA.     

Picolinic acid, a catabolite of tryptophan, as the second signal in the activation of IFN-gamma-primed macrophages.

We have studied the effects of picolinic acid, a product of tryptophan degradation, on the activation of mouse peritoneal macrophages (M phi). Picolinic acid acts synergistically with IFN-gamma in activating M phi from C57BL/6 mice. Moreover, M phi from C3H/HeJ mice and C3H/HeN that do not become cytotoxic in response to IFN-gamma alone could be fully activated by exposure to picolinate plus IFN-gamma. These results indicate that picolinic acid is a potent costimulator of M phi activation that functions as a second signal. Inasmuch as we have previously demonstrated that the activation of cytotoxic M phi correlates with specific changes in ribosomal RNA (rRNA), we investigated whether picolinic acid could modify M phi RNA metabolism. Picolinic acid inhibited the synthesis of total M phi RNA, the accumulation of newly synthesized 28S rRNA, and augmented the steady state levels of rRNA precursors (pre-rRNA). These changes in RNA metabolism were similar to those previously described in murine M phi activated in vitro or in vivo to express tumoricidal activity. These results demonstrate that picolinic acid is a potent, biologic M phi second signal, suggest that the changes in rRNA are causally connected with the expression of tumoricidal activity, and suggest the existance of an autocrine effect mediated by picolinic acid.     

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.     

Inhibition of Growth, Synthesis, and Permeability in Neurospora crassa by Phenethyl Alcohol.

Lester, Gabriel (Reed College, Portland, Ore.). Inhibition of growth, synthesis, and permeability in Neurospora crassa by phenethyl alcohol. J. Bacteriol. 90: 29-37. 1965.-Inhibition of the growth of Neurospora crassa in still culture was detected at 0.05% and was complete at a level of 0.2% phenethyl alcohol (PEA). Benzyl alcohol was less inhibitory, and 3-phenyl-1-propanol and phenol were more inhibitory, than PEA; benzylamine and phenethylamine were less inhibitory than the analogous hydroxylated compounds. Inhibition by PEA was not reversed by synthetic mixtures of purines and pyrimidines or vitamins, or by casein digests, yeast extract, or nutrient broth. The germination of conidia was inhibited by PEA, but after an exposure of 8.5 hr no loss of viability was observed. The addition of PEA to growing shake cultures caused a simultaneous inhibition of growth and of the syntheses of ribonucleic and deoxyribonucleic acids and protein; the relationships of these compounds to mycelial dry weight and to one another were constant in growing mycelia, and PEA did not significantly affect these relationships. PEA partially inhibited the uptake of glucose, but severely restricted the accumulation of l-leucine, l-tryptophan, or alpha-aminoisobutyric acid in germinated conidia. The efflux of alpha-aminoisobutyric acid from germinated conidia was somewhat enhanced by PEA, but this effect was not so pronounced as the (complete) inhibition of alpha-aminoisobutyric acid accumulation by PEA. It is suggested that PEA affects primarily the initial influx of alpha-aminoisobutyric acid rather than the subsequent retention of alpha-aminoisobutyric acid.     

Nuclear division cycle in Neurospora crassa hyphae under different growth conditions.

Treatment with picolinic acid blocked Neurospora crassa nuclei in G1, and recovery from the treatment allowed a synchronous wave of deoxyribonucleic acid synthesis to occur. Nuclei, which appeared as compact globular bodies during the period of blockage, assumed a ring shape during the following S phase, which was also maintained in the G2 phase. The proportion of compact globular nuclei was much higher in hyphae growing at lower rates, whereas that of ring nuclei increased when the hyphae were growing at higher rates. Horseshoe nuclei (probably mitotic nuclei) and double ring nuclei were also observed in growing hyphae, but their frequencies were low and fairly independent of the rate of growth. The length of the S phase of the Neurospora nuclear division cycle was determined to be about 30 min. From the frequencies of the phase-specific nuclear shapes, the durations of the G1 phase and the combined S plus G2 phases were calculated. The results showed that variations in the growth rates of the mycelia were mainly coupled with variations in the G1 phase of the nuclear division cycle. For mycelia growing in minimal sucrose, the lengths of all of the phases of the nuclear division cycle were estimated.     

End-Product Regulation of the Tryptophan-Nicotinic Acid Pathway in Neurospora crassa

The regulation of the tryptophan-nicotinic acid pathway in Neurospora crassa was examined with mutants (nic-2, nic-3) which require nicotinamide for growth. The accumulation of N-acetylkynurenin and 3-hydroxyanthranilic acid by these mutants served to estimate the level of function of the early reactions in the pathway. In still cultures, maximal accumulation occurred with media containing growth-limiting amounts of nicotinamide; the accumulation of intermediates was almost negligible with nicotinamide in excess. Only nicotinamide and closely related compounds which also supported the growth of these mutants inhibited the accumulation of intermediates. The site of inhibition was assessed to be between tryptophan and kynurenin (or N-acetylkynurenin). The synthesis of N-acetylkynurenin was examined in washed germinated conidia suspended in buffer; the level of N-acetylkynurenin-synthesizing activity was inversely related to the concentration of nicotinamide in the germination medium. The addition of large amounts of nicotinamide to suspensions of germinated conidia did not affect their N-acetylkynurenin-synthesizing activity. Formamidase activity, kynurenin-acetylating activity, and gross tryptophan metabolism in germinated conidia was not influenced by the concentration of nicotinamide in the germination medium. The results obtained indicate that the site of inhibition by nicotinamide is the first step in the pathway, the tryptophan pyrrolase reaction. The data are interpreted as nicotinamide or a product thereof, such as nicotinamide adenine dinucleotide, acting as a repressor of the formation of tryptophan pyrrolase in N. crassa.     

Enhancement of DNA polymerase II activity in E. coli after treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

The G₁(G₀) arrest induced in NRK cells by picolinic acid was preceded by marked changes in iron metabolism. In contrast, picolinic acid did not significantly prevent zinc uptake and changes in intracellular zinc were small and clearly preceded by changes in iron. A kinetic study revealed that iron uptake by NRK cells was rapidly halted by picolinic acid. Experiments with radioiron-labeled cells indicated that picolinic acid, in a dose dependent manner, effectively removed iron from the cells. The dose of picolinic acid that exactly removed iron from the cells was also the concentration that induced the G₁(G₀) arrest. Picolinic acid, therefore, may induce the growth inhibition by selectively withholding iron from the cells. These data strongly suggest that iron availability may be a controlling factor in the initiation of DNA synthesis in NRK cells.     

The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by the nuclear gene cyt-18, functions not only in aminoacylation but also in the splicing of group I introns. Here, we isolated the cognate Podospora anserina mt tyrRS gene, designated yts1, by using the N. crassa cyt-18 gene as a hybridization probe. DNA sequencing of the P. anserina gene revealed an open reading frame (ORF) of 641 amino acids which has significant similarity to other tyrRSs. The yts1 ORF is interrupted by two introns, one near its N terminus at the same position as the single intron in the cyt-18 gene and the other downstream in a region corresponding to the nucleotide-binding fold. The P. anserina yts1+ gene transformed the N. crassa cyt-18-2 mutant at a high frequency and rescued both the splicing and protein synthesis defects. Furthermore, the YTS1 protein synthesized in Escherichia coli was capable of splicing the N. crassa mt large rRNA intron in vitro. Together, these results indicate that YTS1 is a bifunctional protein active in both splicing and protein synthesis. The P. anserina YTS1 and N. crassa CYT-18 proteins share three blocks of amino acids that are not conserved in bacterial or yeast mt tyrRSs which do not function in splicing. One of these blocks corresponds to the idiosyncratic N-terminal domain shown previously to be required for splicing activity of the CYT-18 protein. The other two are located in the putative tRNA-binding domain toward the C terminus of the protein and also appear to be required for splicing. Since the E. coli and yeast mt tyrRSs do not function in splicing, the adaptation of the Neurospora and Podospora spp. mt tyrRSs to function in splicing most likely occurred after the divergence of their common ancestor from yeast.     

3-Hydroxy-3-methylglutaryl CoA reductase and mevalonate kinase of Neurospora crassa

Two enzymes of polyisoprenoid synthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) and mevalonate kinase (ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36), are present in the microsomal and soluble fractions of Neurospora crassa, respectively. HMG CoA reductase specifically uses NADPH as reductant and has a K(m) for dl-HMG CoA of 30 micro M. The activities of HMG CoA reductase and mevalonate kinase are low in conidia and increase threefold during the first 12 hr of stationary growth. Maximum specific activities of both enzymes occur when aerial hyphae and conidia first appear (2 days), but total activities peak later (3-4 days). Addition to the growth media of ergosterol or beta-carotene, alone or in combination, does not affect the specific or total activity of either enzyme. The mevalonate kinase of N. crassa, purified 200-fold to a specific activity of 5 micro moles/min/mg, is free from HMG CoA reductase, phosphomevalonate kinase, ATPase, adenylate kinase, and NADH oxidase activities. Mevalonate kinase specifically requires ATP as cosubstrate and exhibits a marked preference for Mg(2+) over Mn(2+), especially at high ratios of divalent metal ion to ATP. Kinase activity is inhibited by p-hydroxymercuribenzoate, and this inhibition is partially prevented by mevalonate or MgATP. Optimum activity occurs at pH 8.0-8.5 and at about 55 degrees C. The Neurospora kinase, like that of hog liver, has a sequential mechanism for substrate addition. The Michaelis constants obtained were 2.8 mM for dl-mevalonate and 1.8 mM for MgATP(-2). Geranyl pyrophosphate is an inhibitor competitive with MgATP (K(i) = 0.11 mM).     

Inhibition of UDP-N-acetylglucosamine pyrophosphorylase by uridine

UDP-N-acetylglucosamine pyrophosphorylases (UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.23) from baker's yeast and Neurospora crassa IFO 6178 were inhibited by uridine which is the nucleoside moiety of UDP-GlcNAc. The inhibition was shown in both directions of pyrophosphorolysis and of synthesis of UDP-GlcNAc. Kinetic analysis revealed that uridine demonstrated a noncompetitive type of inhibition with UDP-GlcNAc and competitive inhibition with PPi. The Ki values for the baker's yeast enzyme were 1.8 mM for UDP-GlcNAc and 0.16 mM for PPi, and the values for the Neurospora enzyme were 1.1 mM for UDP-GlcNAc and 0.15 mM for PPi, respectively. Uridine did not bind irreversibly to the enzyme, as the activity was restored with dialysis. No other nucleosides caused inhibition of the enzyme activity except uridine. Some uridine derivatives, such as 5-hydroxyuridine, 5,6-dihydrouridine and pseudouridine, also inhibited the enzyme activity. But doexyuridine showed only slight inhibition, and 5'-UMP and orotidine caused no inhibition of the enzyme activity.     

Mutations in Nuclear Gene Cyt-4 of Neurospora Crassa Result in Pleiotropic Defects in Processing and Splicing of Mitochondrial Rnas

The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3' end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5' and 3' end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3' end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.     

Characterization of 5.8S ribosomal ribonucleic acid in Neurospora crassa.

Neurospora crassa ribosomes contain a species of ribonucleic acid (RNA) of molecular weight 54,000, similar to 5.8S ribosomal RNA previously described for other eukaryotic organisms. The 5.8S RNA from N. crassa was found to be released by heat treatment at 60 C from 25S ribosomal RNA but not from 18S ribosomal RNA. The base composition of N. crassa 5.8S RNA was similar to that of 5.8S RNA from Saccharomyces cerevisiae, but differed from animal 5.8S RNA. During the course of this study, it was discovered that N. crassa 25S ribosomal RNA had a number of internal cleavages that may exist in vivo.     

Intracellular protein degradation in Neurospora crassa.

In exponentially growing cultures of Neurospora crassa, the basal rate of protein degradation increases as the constant of the rate of growth decreases, so that in slow growing cells (mu = 0.13) the rate of protein degradation is about 25% of the rate of protein accumulation. During glucose starvation and shift-down transition of growth, the rate of protein degradation is greatly enhanced, and a moderate reduction (about 30%) of the ATP level is observed. Treatment of glucose-starved cells with 2-deoxyglucose reduces the ATP content by 70% and blocks protein degradation. The addition of cycloheximide, given at the onset of glucose starvation, prevents the enhancement of protein degradation; instead cycloheximide is without effect if added when proteolysis has already started. At a supraoptimal temperature (42 degrees C) the basal rate of protein degradation is not stimulated, contrary to the behavior observed in bacteria. Guanosine nucleotides, which appear to have a regulatory role for protein degradation in bacteria, are not found in N. crassa.     

The Goodwin model: simulating the effect of cycloheximide and heat shock on the sporulation rhythm of Neurospora crassa

The Goodwin model is a negative feedback oscillator which describes rather closely the putative molecular mechanism of the circadian clock of Neurospora and Drosophila. An essential feature is that one or two clock proteins are synthesized and degraded in a rhythmic fashion. When protein synthesis in N. crassa (wild-type frq+and long-period mutant frq7) was inhibited by continuous incubation with increasing concentrations of cycloheximide (CHX) the period of the circadian sporulation rhythmicity is only slightly increased. The explanation of this effect may be seen in the inhibition of protein synthesis and protein degradation. In the model, increasing inhibition of both processes led to very similar results with respect to period length. That protein degradation is, in fact, inhibited by CHX is shown by determining protein degradation in N. crassa by means of pulse chase experiments. Phase response curves (PRCs) of the N. crassa sporulation rhythm toward CHX which were reported in the literature and investigated in this paper revealed significant differences between frq+and the long period mutants frq7and csp -1 frq7. These PRCs were also convincingly simulated by the model, if a transient inhibition of protein degradation by CHX is assumed as well as a lower constitutive degradation rate of FRQ-protein in the frq7/ csp -1 frq7mutants. The lower sensitivities of frq7and csp -1 frq7towards CHX may thus be explained by a lower degradation rate of clock protein FRQ7. The phase shifting by moderate temperature pulses (from 25 to 30 degrees C) can also be simulated by the Goodwin model and shows large phase advances at about CT 16-20 as observed in experiments. In case of higher temperature pulses (from 35 to 42 or 45 degrees C=heat shock) the phase position and form of the PRC changes as protein synthesis is increasingly inhibited. It is known from earlier experiments that heat shock not only inhibits the synthesis of many proteins but also inhibits protein degradation. Taking this into account, the Goodwin model also simulates the PRCs of high temperature (heat shock) pulses.     

Isolation and Preparation of Pretyrosine, Accumulated as a Dead-End Metabolite by Neurospora crassa

Pretyrosine is an amino acid intermediate of phenylalanine and/or tyrosine biosyntheses in a variety of organisms. A procedure for the isolation of high-quality pretyrosine as the barium salt is described. Stable solutions of ammonium pretyrosine that are suitable for use as substrate in enzyme assays can be prepared in good yield with relatively few purification steps. A triple mutant of Neurospora crassa, bearing genetic blocks corresponding to each initial enzyme step of the three pathway branchlets leading to the aromatic amino acids, accumulates prephenate and pretyrosine. Although the time courses of prephenate and pretyrosine accumulations were found to be parallel in any given experiment, the ratios of the two metabolites varied as much as 100-fold depending upon such variables as carbon source, temperature of growth, accumulation, and especially the presence of aromatic pathway metabolites. Under appropriate nutritional conditions of accumulation, pretyrosine concentrations in excess of 4 mM in culture supernatant fluids were obtained. Strains individually auxotrophic for phenylalanine or tyrosine accumulate lesser amounts of prephenate and pretyrosine. The metabolic blocks of the mutant result in high intracellular levels of prephenate, which is then partially transaminated to pretyrosine. In N. crassa, pretyrosine is a dead-end metabolite since it is not enzymatically converted to phenylalanine or tyrosine. At a mildly acidic pH, pretyrosine is quantitatively converted to phenylalanine in a nonenzymatic reaction.     

δ-Aminolaevulinate dehydratase, the regulatory enzyme of the haem-biosynthetic pathway in Neurospora crassa

The activity of delta-aminolaevulinate dehydratase is very low in the mould Neurospora crassa compared with the activities detected in bacterial and animal systems. The enzyme is inducible in iron-deficient cultures by addition of iron and is repressed by protoporphyrin. The properties of the purified enzyme indicate its allosteric nature and susceptibility to feedback inhibition by coproporphyrinogen III. Neurospora extracts also contain a protein inhibitor of the enzyme and a small-molecule activator, which appears to be associated with the enzyme. The regulatory function of this enzyme in vivo is correlated with the accumulation of delta-aminolaevulinic acid in normal cultures of N. crassa. The decay curve of the iron-induced enzyme in vivo shows a biphasic pattern, with one of the components showing a half-life of 4-5 min.