Up to 36% of Neurospora crassa transformants showing an albino phenotype were recovered by transforming a wild-type strain with different portions of the carotenogenic albino-3 (al-3) and albino-1 (al-1) genes. The presence of the exogenous sequences (which were randomly integrated in ectopic locations) provoked a severe impairment in the expression of the endogenous al-1 or al-3 genes. This phenomenon, which we have termed 'quelling', was found to be spontaneously and progressively reversible, leading to wild-type or intermediate phenotypes. The phenotypic reversion is characterized by a progressive release of the transcriptional inhibition and seems to correlate with a reduction of the number of the ectopic integrated sequences. Moreover, quelling appears to be monodirectional, as, once relieved, it cannot take place again, despite the continuing presence of some of the ectopic sequences in the genome.

Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

/pdf/lessons-from-the-genome-sequence-of-neurospora-crassa-1vsx4nukvq.pdf

Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

Circadian rhythmicity is universally associated with the ability to perceive light, and the oscillators (“clocks”) giving rise to these rhythms, which are feedback loops based on transcription and translation, are reset by light. Although such loops must contain elements of positive and negative regulation, the clock genes analyzed to date—frq in Neurospora andper and tim in Drosophila—are associated only with negative feedback and their biochemical functions are largely inferred. The white collar–1 and white collar–2 genes, both global regulators of photoresponses inNeurospora, encode DNA binding proteins that contain PAS domains and are believed to act as transcriptional activators. Data shown here suggest that wc-1 is a clock-associated gene andwc-2 is a clock component; both play essential roles in the assembly or operation of the Neurospora circadian oscillator. Thus DNA binding and transcriptional activation can now be associated with a clock gene that may provide a positive element in the feedback loop. In addition, similarities between the PAS-domain regions of molecules involved in light perception and circadian rhythmicity in several organisms suggest an evolutionary link between ancient photoreceptor proteins and more modern proteins required for circadian oscillation.

https://science.sciencemag.org/content/sci/276/5313/763.full.pdf

Neurospora wc-1 and wc-2: Transcription, Photoresponses, and the Origins of Circadian Rhythmicity

UV irradiation of mice causes a systemic immune alteration that can be detected either by suppression of the immunologic rejection of UV-induced tumors, or by suppression of contact hypersensitivity (CHS). Suppression of these two immunologic responses has similar photobiologic characteristics and in both cases is associated with the generation of antigen-specific suppressor T cells. To identify whether a specific photoreceptor for this effect exists, the relative wavelength effectiveness (action spectrum) was determined for the UV-induced suppression of CHS. Narrow bands of UV (half bandwidth 3 nm) were used at 10 wavelengths from 250 to 320 nm to obtain dose-response curves. Irradiation with each of these bands of UV caused dose-dependent immunosuppression of CHS, but with differing effectiveness. Immunosuppression was clearly separable from the generation of gross skin damage and inflammation. Further, immunosuppression by the most effective wavelength (270 nm) was associated with the generation of antigen-specific suppressor cells. The action spectrum derived from the dose-response curves has a maximum between 260 and 270 nm, a shoulder at 280-290 nm, and declines steadily to approximately 3% of maximum at 320 nm. The finding of such a clearly defined wavelength dependence implies the presence of a specific photoreceptor for this effect. Removing the stratum corneum by tape stripping before UV irradiation prevented the suppression of CHS using 254-nm radiation, suggesting the photoreceptor is superficially located in the skin. A number of epidermal compounds with absorption spectra similar to the action spectrum are discussed and evaluated with respect to their potential for being the photoreceptor. Based on (a) the close fit of its absorption spectrum to the action spectrum, (b) its superficial location in the stratum corneum, and (c) its photochemical properties, the hypothesis is advanced that the photoreceptor for systemic UV-induced immunosuppression of contact hypersensitivity may be urocanic acid. As such, it may also play a role in UV-induced carcinogenesis via the production of tumor-specific suppressor cells.

/pdf/mechanism-of-immune-suppression-by-ultraviolet-irradiation-37fsp8rym5.pdf

MECHANISM OF IMMUNE SUPPRESSION BY ULTRAVIOLET IRRADIATION IN VIVO I. Evidence for the Existence of a Unique Photoreceptor in Skin and its Role in Photoimmunology

The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.

White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein.

The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-(14)C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [(14)C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [(14)C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [(14)C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [(14)C]geranylgeranyl pyrophosphate from [1-(14)C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino-1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway.

Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa

Positive phototropism of perithecial beaks in the fungus Neurospora crassa has been demonstrated. The effect was shown to be mediated by blue light. When mutants (white collar-1 and white collar-2) which are blocked in the light induction of enzymes in the carotenoid biosynthetic pathway were used as the protoperithecial parent in crosses, the resulting perithecial beaks did not show a phototropic response. However, when wild type, albino-1, albino-2, or albino-3 strains were used as the protoperithecial parent, phototropism occurred.The results show that both photoinduced carotenogenesis and phototropism in N. crassa are controlled by the white collar-1 and white collar-2 loci. Thus, the sensory transduction pathways for the two photoresponses must have some steps in common. The results further support the proposal that the white collar strains are regulatory mutants blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains can carry out light induction but have the albino phenotype because they are each defective for a different enzyme in the carotenoid biosynthetic pathway.

/pdf/genetic-analysis-of-phototropism-of-neurospora-crassa-20o33i3nzy.pdf

Genetic Analysis of Phototropism of Neurospora crassa Perithecial Beaks Using White Collar and Albino Mutants.

An action spectrum for light-induced carotenoid biosynthesis in Neurospora crassa was determined in 4 to 20 nm steps from 260 to 800 nm. Four-day, dark-grown mycelial pads of N. crassa were exposed to varying amounts of monochromatic radiant energy and time. After a 48-hour incubation period at 6 C, carotenoid content was assayed spectrophotometrically in vivo . The action spectrum has maxima at 450 and 481 nm in the visible range and at 280 and 370 nm in the ultraviolet. A pigment synthesized by Neurospora whose absorption spectrum resembles the action spectrum is β-carotene. A model for the regulation of carotenoid biosynthesis in N. crassa is proposed which describes a mechanism by which β-carotene could act as a photoregulator. This carotenoid is suggested to be both photoreceptor for and regulator of carotenoid biosynthesis.

Action Spectrum between 260 and 800 Nanometers for the Photoinduction of Carotenoid Biosynthesis in Neurospora crassa.

The temperature dependence of carotenoid synthesis in Neurospora crassa was investigated. The primary light reaction is independent of temperature, but the amount of carotenoid pigment which subsequently accumulates in the dark is strongly dependent on the temperature during the dark incubation. Carotenoid synthesis shows a sensitivity to both high and low temperatures, and of the temperatures tested, 6 C is optimal. Exposure to temperatures above 6 C for various times immediately following irradiation brings about a temperature-dependent reduction in the amount of carotenoid pigment that is synthesized in a total dark incubation time of 24 hours. This sensitivity to incubations at temperatures above 6 C is reduced by either continuous irradiation during the entire time at the higher temperature or by a short irradiation at the end of this period, and the relative effectiveness of these two types of light treatments is presented. Carotenoid production is also sensitive to amino acid analogues and inhibitors of protein synthesis during a critical period after irradiation. It is proposed that the light reaction leads to the production of a compound which can be degraded in a temperature-dependent competitive reaction. This compound (or a product derived from it) can also induce the de novo synthesis of an enzyme (or enzymes) required for carotenoid production. An alternative hypothesis, that a repressor is directly inactivated in the light reaction, can be ruled out by the results presented.

Roy W. Harding

Papers

Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa

Genetic Analysis of Phototropism of Neurospora crassa Perithecial Beaks Using White Collar and Albino Mutants.

Action Spectrum between 260 and 800 Nanometers for the Photoinduction of Carotenoid Biosynthesis in Neurospora crassa.

The Effect of Temperature on Photo-induced Carotenoid Biosynthesis in Neurospora crassa

Biosynthesis of phytoene from isopentenyl pyrophosphate by a Neurospora enzyme system.