Cyclic AMP-induced tyrosinase synthesis in Neurospora crassa

Cyclic AMP induces the synthesis of tyrosinase in $\text{Neurospora crassa}$. Adenine, adenosine, 3′-AMP, 5′-AMP, and 2′,3′-cyclic AMP have no inductive effect while 8-bromocyclic AMP and dibutyryl cyclic AMP are good inducers. Caffeine and theophylline, inhibitors of cyclic AMP phosphodiesterase, also induce tyrosinase. A possible relationship between cyclic AMP induction and previously reported induction by cycloheximide is suggested.     

DNA-mediated genetic changes in Neurospora crassa.

Evidence for genetic transformation in Neurospora crassa is based on the observations that allo-DNA has a specific effect in producing transformants which is abolished by DNAase treatment and that iso-DNA is not effective in transformation. Here, unambiguous evidence for genetic transformation is provided by transfer of a temperature-sensitive inositol requirement from a donor to a recipient strain. Data provided also suggest the role of growth conditions and the involvement of a nuclease gene in the DNA uptake and transformation of N. crassa.     

Pyrimidine synthesis in Neurospora crassa: regulation of enzyme activities

The regulation of several enzymes involved in pyrimidine biosynthesis in Neurospora crassa has been studied. Elevation of ATCase (l-aspartate carbamoyltransferase) activity is found in all pyrimidine-requiring mutants when they are starved for uridine. DHOase (dihydroorotase) is an unstable enzyme, and it is impossible to conclude what type of regulation, if any, controls this enzyme. DHOdehase (dihydroorotate dehydrogenase) activity shows a marked elevation in uridine-starved pyr-2 cultures, a mutant blocked late in the pathway. Several mutants blocked early in the pathway show much smaller increases in DHOdehase activity and possible explanations for this are discussed. Differences in the modes of regulation of the pyrimidine biosynthetic pathways in various organisms are compared.     

The synthesis of alkaline phosphatase in Neurospora crassa

Mutations which affect the regulation of Neurospora repressible alkaline phosphatase do so by altering the rate of de novo alkaline phosphatase synthesis. In regulatory mutants the rate of alkaline phosphatase polypeptide synthesis can vary over a 1000-fold range. Following transfer to phosphate-free medium, the wild-type cell is capable of increasing the rate of synthesis of alkaline phosphatase molecules within 30-45 min.     

ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.     

Ribosomal RNA synthesis in mitochondria of Neurospora crassa

Ribosomal RNA synthesis in Neurospora crassa mitochondria has been investigated by continuous labeling with [5-³H]uracil and pulse-chase experiments. A short-lived 32 S mitochondrial RNA was detected, along with two other short-lived components; one slightly larger than large subunit ribosomal RNA, and the other slightly larger than small subunit ribosomal RNA. The experiments give support to the possibility that 32 S RNA is the precursor of large and small subunit ribosomal RNA's. Both mature ribosomal RNA's compete with 32 S RNA in hybridization to mitochondrial DNA. Quantitative results from such hybridization-competition experiments along with measurements of electrophoretic mobility have been used to construct a molecular size model for synthesis of mitochondrial ribosomal RNA's. The large molecular weight precursor (32 S) of both ribosomal RNA's appears to be 2.4 × 10⁶ daltons in size. Maturation to large subunit RNA (1.28 × 10⁶ daltons) is assumed to involve an intermediate ~1.6 × 10⁶ daltons in size, while cleavage to form small subunit RNA (0.72 × 10⁶ daltons) presumably involves a 0.9 × 10⁶ dalton intermediate. In the maturation process ~22% of the precursor molecule is lost. As is the case for ribosomal RNA's, the mitochondrial precursor RNA has a strikingly low G + C content.     

Glycogen synthesis in the fungus Neurospora crassa.

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [14C]glucose from ADP-[14C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose : glycogen glucosyltransferase as compared with those of the already known UDP glucose : glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-14C]glucose or ADP-[14C]glucose released all the radioactivity as maltose after alpha or beta amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[14C]glucose was the substrate but the stimulation was much greater with ADP-[14C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent &on the presence of a 'primer' of the alpha 1 leads to 4 glucan type.     

The genetics of polyamine synthesis in Neurospora crassa

New mutations of the polyamine pathway of Neurospora crassa fell into three categories. The majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. One mutation, JP100, defining the new spe-2 locus, eliminated S-adenosyl-methionine decarboxylase and led to putrescine accumulation. Revertants of this mutation suggested that the locus encodes the enzyme. Two other mutations, LV105 and JP120, defined a third locus, spe-3. Strains with these mutations also accumulated putrescine and were presumed to lack spermidine synthase activity, which catalyzes the formation of spermidine from putrescine and decarboxylated S-adenosylmethionine. The three spe loci lay within about 20 map units of one another on the right arm of Linkage Group V in the order: centromere-spe-2-spe-1-spe-3. The requirement for spermidine for growth was much less in spe-2 and spe-3 mutants than in spe-1 mutants, which do not accumulate putrescine. This suggested that putrescine fulfills many, but not all, of the functions of spermidine, or that high levels of putrescine render spermidine more effective in its essential roles.     

Defining individual size in the model filamentous fungus Neurospora crassa

It is challenging to apply the tenets of individuality to filamentous fungi: a fungal mycelium can contain millions of genetically diverse but totipotent nuclei, each capable of founding new mycelia. Moreover, a single mycelium can potentially stretch over kilometres, and it is unlikely that its distant parts share resources or have the same fitness. Here, we directly measure how a single mycelium of the model ascomycete Neurospora crassa is patterned into reproductive units (RUs), meaning subpopulations of nuclei that propagate together as spores, and function as reproductive individuals. The density of RUs is sensitive to the geometry of growth; we detected 50-fold smaller RUs when mycelia had expanding frontiers than when they were constrained to grow in one direction only. RUs fragmented further when the mycelial network was perturbed. In mycelia with expanding frontiers, RU composition was strongly influenced by the distribution of genotypes early in development. Our results provide a concept of fungal individuality that is directly connected to reproductive potential, and therefore to theories of how fungal individuals adapt and evolve over time. Our data show that the size of reproductive individuals is a dynamic and environment-dependent property, even within apparently totally connected fungal mycelia.     

Cross-pathway control of ornithine carbamoyltransferase synthesis in Neurospora crassa

The pattern of cross-pathway regulation of the arginine synthetic enzyme ornithine carbamoyltransferase was investigated in Neurospora crassa, using single and double mutant auxotrophic strains starved for their required amino acids. These experiments show that starvation for histidine, tryptophan, isoleucine, valine or arginine can result in derepression of ornithine carbamoyltransferase. Methionine starvation also gave slight derepression, but starvation for lysine or leucine gave little or no effect.     

Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine.

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.