Colonial Growth of
            <i>Neurospora</i>

Terra, Noël de; Tatum, E. L.

doi:10.1126/science.134.3485.1066

Cited by 71 publications

(12 citation statements)

References 12 publications

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“…A useful property of Neurospora is that addition of sorbose to the medium results in dramatically increased hyphal branching. Consequently, when cultures are grown on sorbose, they grow as colonies (13). We asked if colonial Neurospora bearing frq-luc-I might also show luminescence.…”

Section: Resultsmentioning

confidence: 99%

“…Sorbose medium containing 1ϫ FGS (0.05% fructose, 0.05% glucose, 2% sorbose), 1ϫ Vogel's medium (46), 50 g/liter biotin, and 1.8% agar was autoclaved for 20 min and was used to induce colonial growth of Neurospora (13). We find that in order to see dependable rhythmicity in colonial Neurospora, rather than autoclaving the FGS components separately (5), we need to autoclave all of the components together, which, incidentally, produces a slight caramelization of the medium.…”

Section: Methodsmentioning

confidence: 99%

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Fully Codon-Optimized luciferase Uncovers Novel Temperature Characteristics of the Neurospora Clock

et al. 2008

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We report the complete reconstruction of the firefly luciferase gene, fully codon optimized for expression in Neurospora crassa. This reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase, shows the highest levels of luciferase activity near the growth front and newly formed conidial bands. Further, we have established a sorbose medium colony assay that will facilitate luciferase-based screens. The signals from sorbose-grown colonies of strains in which the frq promoter drives luciferase exhibit the properties of circadian rhythms and can be tracked for many days to weeks. This reporter now makes it possible to follow the clock in real time, even in strains or under conditions in which the circadian rhythm in conidial banding is not expressed. This property has been used to discover short, ca. 15-h period rhythms at high temperatures, at which banding becomes difficult to observe in race tubes, and to generate a high-resolution temperature phase-response curve.Circadian rhythmicity is an ancient form of biological regulation. The ability of an organism to determine the time of day in order to regulate metabolic events is close to ubiquitous within the eukaryotes and, additionally, is found in more than one cyanobacterial species (17). Although the ability to tell time from a molecularly based oscillator is thematically conserved across many phyla, the outputs that the circadian clock regulates are organismally dependent, and in many systems, reporters are used to monitor these rhythms. There is a rich history of using luminescence for this purpose in circadian biology. Studies using circadianly regulated endogenous luciferase in the marine dinoflagellate Gonyaulax polyedra to follow the clock (34) were precursors for a host of experiments using luminescent reporters in plants (35

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Fully Codon-Optimized luciferase Uncovers Novel Temperature Characteristics of the Neurospora Clock

et al. 2008

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“…The intense peaks observed in the spectrum in The resonance at 251.1 ppm can be assigned to N-acetyl-Dglucosamine either as a free metabolite or as a component of fungal cell walls (22). Among the nitrogens in cellular nucleic acids and free nucleotides, only the N3 resonance of uridine (216.7 ppm) was observed; this resonance probably arises from uridinediphosphoacetylglucosamine and uridinediphosphoglucose, both of which occur in significant amounts in N. crassa (see ref.…”

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confidence: 99%

15N NMR studies of nitrogen metabolism in intact mycelia of Neurospora crassa.

Legerton¹,

Kanamori²,

Weiss³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Suspensions of intact mycelia of Neurospora crassa grown in medium containing '5NH4CI have been found to give well-resolved '5N nuclear magnetic resonance spectra for metabolites that play crucial roles in intermediary nitrogen metabolism. These include the amide nitrogen of glutamine, the aamino nitrogens of glutamate and other amino acids, the guanidino nitrogens of arginine, the ureido nitrogen of citrulline, the side-chain nitrogens of ornithine or lysine, or both, and uridine diphosphates. The turnover time of glutamine in vivo was estimated to be less than 1 hr by using nuclear magnetic resonance spectroscopy in conjunction with tracer methodologies. Applications of these techniques to the study of nitrogen metabolism are discussed. High-resolution EXPERIMENTAL PROCEDURESThe ureaseless strain (LA44) of Neurospora crassa used in these studies was obtained from R. H. Davis. The medium was Vogel's minimal medium supplemented with 1.5% (wt/vol) sucrose (12). Nitrogen-free medium is the same without the addition of NH4NO3. The '5N-substituted ammonium chloride (99% enriched in '5N) was purchased from Kor Isotopes.Cultures were inoculated with an aqueous suspension of washed conidia to a final concentration of approximately 1 X 107 conidia per ml. Conidia were germinated in 1-liter baffled flasks containing 500 ml of culture medium, with aeration provided by shaking at 150 rpm at 16°C for a period of 16-20 hr. The temperature was then raised to 30°C for 3 hr or until growth of the culture was logarithmic as measured by culture turbidity. In some experiments, cultures were exposed to`5NH4Cl for the entire germination period. Alternatively, growing mycelia were transferred to nitrogen-free medium for 3 hr prior to the addition of '5NH4Cl. Where specified, cycloheximide {4-[2-(3,5-dimethyl-2 -oxocyclohexyl)-2 -hydroxyethyl]-2,6 -piperidinedione} was added to the culture medium to a final concentration of 20 ,ug/ml a few minutes before the addition of 15NH4Cl to inhibit the incorporation of '5N-labeled amino acids into proteins (13). The mycelial suspension for the NMR measurements was prepared by collecting the mycelia by filtration and resuspending them in enough medium to make 18 ml of mycelial suspension, which was placed in a 25-mm NMR sample tube. The volume ratio of wet mycelium to medium in the NMR sample tube was approximately 1:1.Amino acid pools were determined by extraction of mycelia with boiling water (14). Pool extracts were evaporated to dryness, redissolved in 0.2 M sodium citrate (pH 2.2), and analyzed on a Beckman 120C amino acid analyzer. Protein determinations were made by the method of Lowry et al. (15) after overnight suspension of mycelia in 0.5 M NaOH at room temperature to solubilize the protein.The '5N NMR spectra were obtained with a Bruker WH 180

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“…L-Sorbose added to the minimal liquid medium containing sucrose induces the wild-type strain to grow in a colonial fashion [39,40]. Hexokinase pro- Variable amounts of rabbit immune sera anti-(yeast hexokinase P) or anti-PI1 were incubated with the isolated hexokinases from N .…”

Section: The Effect Of L-sorbose On the Hexokinase Pattern Of Neurospmentioning

confidence: 99%

The Hexokinases from Wild-Type and Morphological Mutant Strains of Neurospora crassa

Lagos

Ureta

1980

Eur J Biochem

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Four hexose-phosphorylating isozymes (hexokinases a, B, y and 6) were separated by DEAEcellulose column chromatography of high-speed supernatants from Neurospora crassa wild-type strains 77a and RL3-8A. The isozymes displayed broad sugar specificity and Michaelis constants for glucose in the micromolar range. The isozymes were not inhibited by glucose 6-phosphate nor activated by citrate. Molecular weights were estimated as 95 000 by gel filtration. Glucose-specific kinases were not found.Marked quantitative variations in the relative proportion of the hexokinases were observed during growth of the fungus on minimal medium plus sucrose. Hexokinase p was the predominant form (82% of the total activity) during the period of hyphal growth. At maturation time all the isozymes were present at about the same level although a small preponderance of hexokinase fl was still evident.The relative proportions of the hexokinases did not vary when sucrose was replaced by glucose, fructose or casamino acids. When sorbose was added to the minimal medium containing sucrose, a high proportion of hexokinase p was apparent concomitantly with the well known change of growth to a colonial mode. Several morphological mutant strains were examined with respect to the hexokinase profile.Strains COI-16 and crisp showed a marked preponderance of hexokinase p. Extracts from the dapple and dingy strains lacked hexokinases a and 6. Rabbit antisera to commercial preparations of hexokinase PI and PI1 from Saccharomyces cerevisiae were able to inhibit partially the enzyme activity of Neurospora hexokinases y and 6.No effect of the antisera on hexokinases a and B could be observed.Multiple hexokinases were first recognized in the yeast Succhuromyces cerevisiae in 1961 by Trayser and Colowick [l] and by several other research groups afterwards [2,3]. The existence of two hexokinases of broad sugar specificities, designated as PI and PI1 (or A and B), and a rather specific glucokinase, seems now well established. Hexokinases PI and PI1 have been thoroughly studied by a number of investigators with respect to kinetic properties [4] and structural features [5] ; the elucidation of the amino acid sequence of one of the isozymes may be possible soon. Recent detailed genetic studies by Maitra and his group [6,7] and by Fraenkel and coworkers [8] have shown that

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Colonial Growth of Neurospora

Cited by 71 publications

References 12 publications

Fully Codon-Optimized luciferase Uncovers Novel Temperature Characteristics of the Neurospora Clock

Fully Codon-Optimized luciferase Uncovers Novel Temperature Characteristics of the Neurospora Clock

15N NMR studies of nitrogen metabolism in intact mycelia of Neurospora crassa.

The Hexokinases from Wild-Type and Morphological Mutant Strains of Neurospora crassa

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