PHENOTYPE DISTRIBUTIONS IN ASCI OF NEUROSPORA CRASSA

Fourteen crosses are described from which 3103 asci were dissected and scored with respect to phenotype distribution patterns of 1, 2 or 3 markers. The results illustrate the following points. There may regularly occur preferred ratios, including 8:1, 4:1, 2:1 and 1:2, of first to second division segregation patterns. Variations in this ratio (designated here as S/C, meaning simple to complex phenotype distribution patterns), shown by the same marker in different crosses, may regularly be discontinuous in that the ratio may shift from one preferred value to another. Values of linkage-correction factors applicable to certain crosses suggest that these factors also may constitute a discontinuous series. Control of phenotype distributions in asci by a systematic, but unknown, mechanism is thought to be suggested. Characteristics of the order of isolation of asci, from 2 crosses treated in a special way, are consistent with the possibility that members of adjacently formed, twin asci are frequently, or even regularly, alike with respect to phenotype distribution class.     

CONTROL OF TREHALASE SYNTHESIS IN NEUROSPORA CRASSA

When an aconidial strain (STL6A) of Neurospora crassa is grown on carbon sources such as glucose, maltose, sucrose, etc., trehalase activity per unit weight of mycelium is very low. By contrast, media containing arabinose, glutamic acid, glycine, etc., which support growth only poorly, produce mycelium with very high trehalase activity. Retarding growth limiting the supply of a necessary nutrient, altering the pH and temperature, or adding toxic substances, however, does not derepress trehalase activity. Repression and derepression of trehalase was found to be reversible through the transfer of cultures to appropriate media. It is likely that the increase in trehalase activity results from de novo synthesis because labeled enzyme can be isolated from acrylamide gels after isolation from medium containing C¹⁴-labeled leucine and after purification by other means. These experiments are interpreted in terms of catabolite repression which may be correlated with events during growth and conidiation.     

THIAMINE METABOLISM IN NEUROSPORA CRASSA

Eberhart , Bruce M. (Princeton U., Princeton, New Jersey), and E. L. Tatum . Thiamine metabolism in Neurospora crassa , Amer. Jour. Bot. 48(8): 702–711. Illus. 1961.—Growth adaptation is a regular feature of the vegetative cycle of thi-1 strains of Neurospora crassa but not of thi-2 or thi-3 strains. The adaptive growth of thi-1 strains is paralleled by the production of cocarboxylase and carboxylase levels which are similar to those of wild-type strains. The growth responses of thi-1,2 and thi-1,3 double mutant strains to thiamine indicates that the thi-1 gene is concerned with the utilization (pyrophosphorylation) of thiamine.     

SOMATIC MITOSIS IN NEUROSPORA CRASSA

Ward , E. W. B., and K. W. Ciurysek . (Canada Department of Agriculture, Edmonton, Alberta.) Somatic mitosis in Neurospora crassa. Amer. Jour. Bot. 49(4): 393–399. Illus. 1962.—The process of nuclear division in the vegetative mycelium of Neurospora crassa was studied with the aid of the HCl-Giemsa staining technique. Division followed the conventional mitotic sequence and 7 morphologically distinct chromosomes were demonstrated at metaphase. The findings are discussed in relation to other proposed schemes of nuclear division in the fungi.     

THE RELATIONSHIP OF M-INOSITOL TO MORPHOLOGY IN NEUROSPORA CRASSA

Shatkin , A. J., and E. L. Tatum . (The Rockefeller Institute, New York 21, New York.) The relationship of m-inositol to morphology in Neurospora crassa. Amer. Jour. Bot. 48(9): 760–771. Illus. 1961.—The role of m-inositol and its relationship to morphology in Neurospora crassa have been examined. The growth pattern of the inositolless mutant has been found to be a function of concentration of carbon and nitrogen sources and m-inositol, utilizability of carbon source, ratio of concentrations of sugar to m-inositol, and size of conidial inoculum. Inhibition of mycelial m-inositol uptake has been demonstrated for a number of carbon sources, and the ability of a particular compound to inhibit m-inositol uptake found to be directly related to both its effectiveness as an energy source and its effect on the morphology of the inositol-requirer. Results of cell-fractionation experiments and autoradiography have shown that phospholipid inositol is widely distributed in the hyphae and not bound in a single subcellular organelle. Electron micrographs of cell fractions indicate that inositol is a structural constituent of N. crassa lipoprotein membranes, including plasmalemma, nuclear envelope, mitchondrial membranes, and endoplasmic reticulum. Normal inositol-requiring and wild-type hyphae are similar in ultrastructure. However, sub-optimally cultured, colonial, inositolless hyphae contain large lipid droplets, and the cellular membranes are in various stages of degeneration. Chemical evidence indicates that colonial hyphae do not synthesize more fat than normal hyphae, suggesting that the lipid droplets are products of membrane catabolism. It is proposed that the colonial morphology of the inositol-requiring mutant of N. crassa is due to a metabolic imbalance between membrane synthesis and the synthesis of other cellular constituents.     

SYNCHRONOUS MITOCHONDRIAL DIVISION IN NEUROSPORA CRASSA

Samples of mycelium of Neurospora crassa of known age were harvested from agar plates and examined with the electron microscope. The relative volume of the mitochondria was determined for mycelium of different ages. The volume measurements indicated that the mitochondria were dividing synchronously in fronts 6, 13, and 22½ hr behind the growing hyphal tips. The sequence of mitochondrial division is hypothesized to include mitochondrial cupping followed by division which results in closely associated daughter mitochondria. On the basis of percentages of mitochondrial cupping and association, mitochondrial division was postulated to be occurring at 6, 14, and 26 hr. Close agreement between the mycelial mass doubling time and the calculated mitochondrial mass doubling time indicates that synchronous mitochondrial division is sufficient to maintain growth. The possibility that mitochondrial division is due to intercellular regulation of a mitochondrial genetic system is advanced.     

ENTRAINMENT ELICITS PERIOD AFTEREFFECTS IN NEUROSPORA CRASSA

Circadian clocks continue to oscillate in constant conditions with their own period (τ) and entrain to a cyclic environment by adjusting their intrinsic period to that of the zeitgeber. When circadian clocks are released from entrained to constant conditions, the τ of their initial free-run often depends on the nature of the prior zeitgeber. These postentrainment effects on period (τ-aftereffects) have predominantly been reported for animals but, so far, not fungi. The authors therefore investigated τ aftereffects in the classic circadian model system Neurospora crassa. The standard laboratory strain frq⁺, the short-period mutant frq¹, and the long-period mutant frq⁷ were entrained to 11 different photoperiods in a 24-h day (2–22?h) and to zeitgebers with six different T (16–26?h), and then released to constant darkness. τ-Aftereffects in response to different photoperiods correlated weakly with prior photoperiod in frq⁺ and were unsystematic in both period mutant strains. Strength and direction of the τ-aftereffect in zeitgeber cycles with different T depended on their length and on the strain, showing a negative correlation with zeitgeber length in frq⁺ and positive correlations in frq¹ and frq⁷. It has been proposed that τ-aftereffects are based on interactions of oscillators within a cellular network. The present findings in Neurospora, which grows as a syncytium, suggest that τ-aftereffects also exist in circadian systems based on multioscillatory networks organized at the molecular level. (Author correspondence: roenneberg@lmu.de)     

LOCALIZATION OF STRUCTURAL POLYMERS IN THE CELL WALL OF NEUROSPORA CRASSA

The distribution and localization of structural polymers in the cell wall of Neurospora crassa has been studied by selective removal and light and electron microscope examination. Observations with the light microscope indicated that each polymer by itself can provide structural integrity to the cell wall. Examination by electron microscopy showed that the cell wall consists of an outer layer of thick fibrils, identified chemically as a glucan-peptide-galactosamine complex, and an inner layer made up of β-1,3 glucan and thin fibrils of chitin.     

THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to ¹⁴C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-³H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.     

EVOLUTION AND MITOCHONDRIAL DNA IN NEUROSPORA CRASSA

We studied mitochondrial DNA variability in 19 natural Neurospora crassa isolates and one wild-type isolate to examine evolution of these fungi and their mitochondrial DNA (mtDNA). We combined restriction endonuclease analysis of natural isolate mtDNA with DNA-DNA hybridization to cloned EcoR I fragments of a wild-type genome to discriminate between length mutations and site changes due to nucleotide substitution. Most variability was due to length mutations (insertions and deletions); genome size could vary 25% between pairs of isolates. Length-mutation distribution was not random, nor simply explained by the presence of coding versus noncoding regions. Restriction-site changes were few; the estimated amount of nucleotide substitution per nucleotide between the most divergent pair of isolates was 0.78%. Evolutionary relationships among isolates based on both types of mutations were compatible, and suggest that geographically distinct populations of mitochondrial DNA exist in the biological species, N. crassa. In contrast, no such correlation was shown by the previously determined distribution of nuclear heterokaryon incompatibility genes in the same isolates (Mylyk, 1975, 1976).     

AMINO ACID INTERACTIONS IN NEUROSPORA CRASSA.

Soboren, Josephine (University of California, Los Angeles), and Joseph F. Nyc. Amino acid interactions in Neurospora crassa. J. Bacteriol. 82:20-25. 1961.-A systematic study of the effects of the naturally occurring amino acids on the growth of a wild-type strain of Neurospora crassa focused attention upon l-tryptophan, which exhibits a strong growth inhibitory effect. Further investigation disclosed that other tryptophan metabolites, anthranilic acid, indole, kynurenine, and 3-hydroxykynurenine also inhibit growth. The proposed antimetabolic role of these aromatic compounds explains the poor growth response of certain tryptophan-requiring strains of N. crassa to tryptophan supplements. The growth of normal and mutant strains of N. crassa on media supplemented with tryptophan is influenced by the presence of other amino acids.