Possible role of a regulatory gene product upon the myo-inositol-1-phosphate synthase production in Neurospora crassa

The regulatory effect of inositol on inositol-1-phosphate synthase in Neurospora crassa strains was studied. Inositol represses enzyme production in the cultures of the wild type and that of the thermosensitive inositol-requiring mutant grown at 22°C. Enzyme activity as well as the quantity of enzyme protein decreased sharply in both strains by increasing concentrations of inositol in the medium. Inositol-requiring strains used in our experiments can be divided into two groups. The first group produces a protein related immunologically to inositol phosphate synthase, but which is enzymatically inactive. The synthesis of this defective enzyme was also repressed by inositol. In the second group, this protein was found to be completely lacking, in both the thermosensitive mutant grown at 37°C, and in a strain requiring inositol due to a reciprocal translocation. The thermostability and the cross immunoelectrophoresis of the enzyme suggest that in the case of the thermosensitive inositol-requiring mutant, the mutation did not occur in the structural gene of the enzyme, but its regulation was probably affected.     

Characterization of a mutation that causes overproduction of inositol in Neurospora crassa

Summary Slow-growing (inl ^+/-) spontaneous mutants have been isolated from an inositol requiring (inl) strain of Neurospora crassa that produces defective myo-inositol-1-phosphate synthase (MIPS), the enzyme responsible for the production of inositol-1-phosphate from glucose-6-phosphate. The defective enzyme has some residual activity. In the inl ^+/- strain the synthesis of the defective enzyme is enhanced, which enables the strain to grow slowly on minimal medium. The mutation (opi1) responsible for the partial inositol independence segregates independently from the inositol locus, and suppresses the inositolless character by overproduction of defective MIPS. opi1 acting upon the wild type (inl ⁺) allele increases MIPS production and causes inositol excretion.     

A RELATIONSHIP BETWEEN CELL WALL STRUCTURE AND COLONIAL GROWTH IN NEUROSPORA CRASSA

de Terra, Noël, and E. L. Tatum. (Rockefeller Inst., New York, N. Y.) A relationship between cell wall structure and colonial growth in Neurospora crassa . Amer.Jour. Bot. 50(7): 669–677. Illus. 1963.—Cell walls were isolated from morphological mutants of Neurospora crassa and from their corresponding wild-type strains. Acid hydrolysates prepared from these cell walls were then studied, qualitatively and quantitatively, with respect to their reducing sugar content. Paper chromatography revealed the presence of glucose and glucosamine in the cell walls of all strains studied. Quantitative analysis has shown that a group of 4 colonial mutants which strongly resemble each other in morphology all have significantly less glucose and more glucosamine per unit weight of cell wall than do their corresponding wild-type strains. These data strongly suggest that a particular type of morphological aberration in Neurospora is associated with similar changes in cell wall composition.     

Formation and regeneration of protoplasts of wild-typeNeurospora crassa

Trichoderma reesei was grown using purified cell walls ofNeurospora crassa as a primary source of carbon. The resulting culture medium contained an undefined mixture ofN. crassa cell-wall digesting enzymes. Protoplasts (cell lacking wall) were formed when youngN. crassa hyphae were treated withTrichoderma mixture. The vast majority of protoplasts resynthesized cell-wall material when washed free of cell-wall digesting enzyme; of these, about 40% regenerated a mycelium.     

Growth and metabolism of inositol-starved Saccharomyces cerevisiae.

Upon starvation for inositol, a phospholipid precursor, an inositol-requiring mutant of Saccharomyces cerevisiae has been shown to die if all other conditions are growth supporting. The growth and metabolism of inositol-starved cells has been investigated in order to determine the physiological state leading to "inositolless death". The synthesis of the major inositol-containing phospholipid ceases within 30 min after the removal of inositol from the growth medium. The cells, however, continue in an apparently normal fashion for one generation (2 h under the growth conditions used in this study). The cessation of cell division is not preceded or accompanied by any detectable change in the rate of macromolecular synthesis. When cell division ceases, the cells remain constant in volume, whereas macromolecular synthesis continues at first at an unchanged rate and eventually at a decreasing rate. Macromolecular synthesis terminates after about 4 h of inositol starvation, at approximately the time when the cells begin to die. Cell death is also accompanied by a decline in cellular potassium and adenosine triphosphate levels. The cells can be protected from inositolless death by several treatments that block cellular metabolism. It is concluded that inositol starvation results in a imbalance between the expansion of cell volume and the accumulation of cytoplasmic constituents. This imbalance is very likely the cause of inositolless death.     

Lipid and cell wall changes in an inositol-requiring mutant of Neurospora crassa.

An inositol deficiency in the inositol-requiring (inl) mutant of Neurospora crassa led to changes in the composition of the inositol-containing lipids and the cell wall. On deficient levels of inositol, phosphatidyl inositol decreased by 23-fold, di(inositolphosphoryl) ceramide decreased by 4-fold, and monoinositolphosphoryl ceramide increased slightly. The inositol deficiency also led to an aberrant hyphal morphology and changes in both the amount of cell wall and the amino sugar content of the cell wall. The glucosamine content of the cell wall decreased by 50%, the galactosamine increased by 50%, but no significant changes were found in the content of the cell wall amino sugar precursors, or in the amino acid, glucose, or total hexose content of the cell wall. Inositol-containing compounds were found associated with purified cell wall material. These compounds were bound tightly to the cell wall but could be removed by treatment with alkali, a treatment which disrupts the cell wall integrity. Possible mechanisms of how changes in lipid composition can affect cell wall biosynthesis are discussed.     

Hyphal wall peptides and colonial morphology in Neurospora crassa

The peptides of the hyphal wall of 23 colonial strains of Neurospora crassa have been compared, both quantitatively and qualitatively, to those of three wild-type strains. We found that all colonial strains examined have a reduced quantity of peptide, ranging from 6.64% to 3.34% of the dry weight of the wall, compared to the wild-type average of 9.35%. The peptides from the walls of all colonial strains except doily eluted from DEAE-cellulose with the same pattern as those from wild-type walls. The aberrant peptides from doily walls did not bind to DEAE-cellulose, suggesting a reduction in the number of acidic residues in these peptides. Although a causal connection between colonial morphology and reduced peptide is not shown, we consider the quantity of peptide in the hyphal wall to be an important determinant in the control of normal morphology and growth.This work was supported by a grant from the National Institutes of Health, GM-16224.     

Cloning and characterization of a gene (arpA) from Aspergillus oryzae encoding an actin-related protein required for normal nuclear distribution and morphology of conidiophores

We have isolated the arpA gene from Aspergillus oryzae as a homologue of the Neurospora crassa ro-4 gene. In N. crassa, mutations in the ro-4 gene, which encodes a major component of the dynactin complex Arp1, causes curling of hyphae and abnormalities in nuclear distribution. The arpA gene contains two introns and encodes a polypeptide of 381 amino acids, with a 78% sequence identity to the N. crassa Arp1. Overexpression of the arpA gene causes a defect in nuclear migration into elongating hyphae of germlings in A. oryzae. We constructed arpA disruptant strains of A. oryzae. The arpA null mutants showed poor growth and hyper-branched mycelia, as well as a nuclear distribution defect. Scanning electron microscopy revealed that the arpA null mutant has an aberrant conidiophore morphology with irregular phialides. Received: 26 January 1999 / Accepted: 22 July 1999     

Liposome-mediated uptake of chloroplasts by plant protoplasts

Summary Liposomes, artificial lipid vesicles, have been used to induce the uptake of isolated spinach chloroplasts into protoplasts of bothNeurospora crassa andDatura innoxia. Chloroplasts showed high oxygen evolution rates preceding uptake and for extended periods of time after uptake. Survival, as measured by pigment retention and oxygen evolution, was much improved over polyethylene glycol induce uptake. Possible future application of liposome-mediated uptake in plant protoplast studies are discussed.     

INTERACTION OF IONIZING AND VISIBLE RADIATION IN MUTATION INDUCTION IN NEUROSPORA CRASSA

Klein , Richard M. (New York Bot. Gdn., New York, N.Y.), and Deana T. Klein. Interaction of ionizing and visible radiation in mutation induction in Neurospora crassa. Amer. Jour. Bot. 49(8): 870–874. 1962.—Conidia of the purple adenineless strain of N. crassa were irradiated with 25 kr of X rays and then exposed to far-red or red radiations or to far-red followed by red radiation. Far-red light, without effect on un-irradiated conidia, augmented the genetic damage caused by X rays as measured by survival (colony count), back mutation to adenine prototrophy, and the induction of mutants affecting colony morphology. Post-X-irradiation with red light ameliorated the severity of X-radiation as measured by survival and back mutation. The potentiation of X-ray-induced genetic damage by far-red light could be completely negated by subsequent exposure to red light.     

A fungal actin-related protein involved in nuclear migration

The ro-4 mutant of the filamentous fungus Neurospora crassa forms distinctive colonies in which hyphae grow into rope-like aggregates. This unusual morphology coincides with a defect in hyphal nuclear migration. The ro-4 gene was cloned from a cosmid library by complementation of the closely linked pab-2 gene. The deduced 380 amino acid protein is most similar to the vertebrate actin-related protein/centractin. The R04 protein is not essential for cell viability, and new strains created by inducing point mutations at the ro-4 locus have a phenotype which is very similar to that of the original mutant. This study provides genetic evidence that an actin-related protein plays a role in nuclear motility. Since nuclear motility is believed to be a microtubule-dependent process, the ro-4 gene product may function as a component of the dynactin complex which activates force generation by cytoplasmic dynein.     

A ras homologue of Neurospora crassa regulates morphology

In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type. The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall synthesis, and conidial formation in N. crassa. Received: 1 October 1996 / Accepted: 9 December 1996     

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

Summary A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10^–9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.     

Growth and Development of Isolated Tips of <Emphasis Type="Italic">Neurospora crassa</Emphasis> Hyphae in a Sorbitol-Containing Medium

It is known that the replacement of easily utilizable glucose with sorbitol in a medium activates a large number of repressed genes and metabolic changes in Neurospora crassa, which manifest themselves at the cellular level in a considerable vacuolization of the cytoplasm. The study of the morphological parameters and growth characteristics of isolated apical fragments of N. crassa hyphae showed that, in sorbitol-containing medium, the isolation from the parent mycelium causes the same disturbances in the coordination of elongation, branching, and septation as those described by us earlier for the glucose-containing medium. The metabolic changes caused by the carbon source replacement do not affect the hyphal-segment size, the distance from the growth point to the first septum, and the morphology and distribution of nuclei.     

Transport properties of N-acyl derivatives of the coprogen and ferrichrysin classes of siderophores inNeurospora crassa

Several derivatives of the coprogen and ferrichrysin classes of siderophores were synthesized as potential affinity labels of the iron uptake system inNeurospora crassa. While only one of these compounds has proved useful as an affinity label, all were recognized and transported byNeurospora crassa. One derivative, chloroacetyl-ferrichrysin, proved to be an unexpectedly potent reversible inhibitor (K ₁=0.4 M) of both ferrichrysin and coprogen uptake, similar to the natural siderophore, ferrirubin. The reported results provide further understanding of the steric and electronic requirements of siderophores for the iron uptake system inNeurospora crassa.Abbreviations amu atomic mass units - DMF dimethylformamide - FAB tast atom bombardment - NMR nuclear magnetic resonance - ppm parts per million - tlc thin layer chromatography     

Demonstration of dihydro-orotate dehydrogenase in Neurospora crassa hyphae with a cytochemical procedure

Synopsis Dihydro-orotate dehydrogenase activity was demonstrated in the hyphae of the fungusNeurospora crassa by a cytochemical technique. The activity was slightly stronger in the hyphal tips thus demonstrating the more intense metabolic activity in these parts of the mycelium. Several control reactions showed that the staining reaction was specific.     

Occlusion of the septal pores of damaged hyphae ofNeurospora crassa by hexagonal crystals

Summary Hyphae ofNeurospora crassa were damaged by flooding colonies with water or cutting them with a razor blade. Loss of cytoplasm from damaged hyphae was reduced by the rapid occlusion of septal pores by hexagonal crystals. Re-growth of these hyphae occurred by the formation of intra-hyphal branches from the plugged septa, the production of side branches just below the septa, or the production of multiple branches from the lateral walls. Small vesicles accumulated behind plugged septa prior to branch production.     

Distribution of ions inNeurospora crassa determined by quantitative electron microprobe analysis

Summary It was investigated whether the low membrane potential at the tip of hyphae ofNeurospora crassa could be described as a diffusion potential. The distribution of Na, K and P inNeurospora was measured by electron microprobe analysis of freeze-dried hyphae. Quantitative determinations were made with freeze-dried gelatin standards and with crystalline standards. The local concentrations of Na and K were in accordance with the theory that at the tip of the hyphe, these ions are distributed passively across the plasma membrane.     

Electrophoretic analysis of the plasma membrane proteins of a mutant of Neurospora crassa which lacks a cell wall

The plasma membrane proteins of a mutant of Neurospora crassa (FGSC No. 326) which lacks a cell wall were analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 180 different proteins were detected in purified plasma membrane preparations. Nonpermeant labeling experiments indicated that approximately 40% of these proteins were exposed on the extracytoplasmic surface of the plasma membranes of these cells. The studies demostrate the complexity of the protein composition of N. crassa 326 plasma membranes to be greater than has been suggested by previous investigations.