WC-1 and the Proximal GATA Sequence Mediate a Cis-/Trans-Acting Repressive Regulation of Light-Dependent Gene Transcription in the Dark.

Light influences a wide range of physiological processes from prokaryotes to mammals. Neurospora crassa represents an important model system used for studying this signal pathway. At molecular levels, the WHITE COLLAR Complex (WCC), a heterodimer formed by WC-1 (the blue light photo-sensor) and WC-2 (the transcriptional activator), is the critical positive regulator of light-dependent gene expression. GATN (N indicates any other nucleotide) repeats are consensus sequences within the promoters of light-dependent genes recognized by the WCC. The distal GATN is also known as C-box since it is involved in the circadian clock. However, we know very little about the role of the proximal GATN, and the molecular mechanism that controls the transcription of light-induced genes during the dark/light transition it is still unclear. Here we showed a first indication that mutagenesis of the proximal GATA sequence within the target promoter of the albino-3 gene or deletion of the WC-1 zinc finger domain led to a rise in expression of light-dependent genes already in the dark, effectively decoupling light stimuli and transcriptional activation. This is the first observation of cis-/trans-acting repressive machinery, which is not consistent with the light-dependent regulatory mechanism observed in the eukaryotic world so far.

A relationship between carotenoid accumulation and the distribution of species of the fungus Neurospora in Spain.

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.

Signal transduction and amplification in a circadian oscillator: interaction between two colored noises.

The signal transduction and amplification in a Neurospora circadian clock system is studied by using the mechanism of internal signal stochastic resonance (ISSR). Two cases have been investigated: the case of no correlations between multiplicative and additive colored noises and the case of correlations between two noises. The results show that, in both cases, the noise-induced circadian oscillations can be transduced with the phenomenon of internal signal stochastic resonance (ISSR). However, the correlation time and intensity of an additive colored noise play different roles for the ISSR, driven by multiplicative colored noise, while the correlation time and intensity of multiplicative colored noise hardly influence the ISSR driven by additive colored noise. In addition, the ISSR can be amplified or suppressed at an appropriate range of the correlation intensity between two colored noises. The fundamental frequency of noise-induced circadian oscillations is hardly shifted with the increment of the intensity and correlation time of colored noises, which implies that the Neurospora system could be resistant to colored noises, exhibit strong vitality and sustain intrinsic circadian rhythms.

Long and short isoforms of Neurospora clock protein FRQ support temperature-compensated circadian rhythms.

The large (l) and small (s) isoforms of FREQUENCY (FRQ) are elements of interconnected feedback loops of the Neurospora circadian clock. The expression ratio of l-FRQ vs. s-FRQ is regulated by thermosensitive splicing of an intron containing the initiation codon for l-FRQ. We show that this splicing is dependent on light and temperature and displays a circadian rhythm. Strains expressing only l-FRQ or s-FRQ support short and long temperature-compensated circadian rhythms, respectively. The thermosensitive expression ratio of FRQ isoforms influences period length in wt. Our data indicate that differential expression of FRQ isoforms is not required for temperature compensation but rather provides a means to fine-tune period length in response to ambient temperature.

Fungal photoreceptors: sensory molecules for fungal development and behaviour.

Light regulates fungal development and behaviour and activates metabolic pathways. In addition, light is one of the many signals that fungi use to perceive and interact with the environment. In the ascomycete Neurospora crassa blue light is perceived by the white collar (WC) complex, a protein complex formed by WC-1 and WC-2. WC-1 is a protein with a flavin-binding domain and a zinc-finger domain, and interacts with WC-2, another zinc-finger domain protein. The WC complex operates as a photoreceptor and a transcription factor for blue-light responses in Neurospora. Proteins similar to WC-1 and WC-2 have been described in other fungi, suggesting a general role for the WC complex as a fungal receptor for blue light. The ascomycete Aspergillus nidulans uses red light perceived by a fungal phytochrome as a signal to regulate sexual and asexual development. In addition, other photoreceptors, rhodopsins and cryptochromes, have been identified in fungi, but their functional relevance has not been elucidated. The investigation of fungal light responses provides an opportunity to understand how fungi perceive the environment and to identify the mechanisms involved in the regulation by light of cellular development and metabolism.

Posttranslational photomodulation of circadian amplitude.

The transcription-translation feedback loops that form our current view of how the core mechanism of the clock operates is being challenged, as more and more posttranslational events are seen as essential to a full understanding of oscillator function. But in addition to phosphorylation, other processes may be involved. Here, a novel mechanism of posttranslational photomodulation of circadian amplitude is described that uniquely ties together light perception, protein stabilization, and proteolysis. In the process, the waveform of a core clock component is sharpened or "sculpted," resulting in appropriately high amplitude and proper phasing to obtain normal clock function.

PAS domain-mediated WC-1/WC-2 interaction is essential for maintaining the steady-state level of WC-1 and the function of both proteins in circadian clock and light responses of Neurospora.

In the frq-wc-based circadian feedback loops of Neurospora, two PAS domain-containing transcription factors, WHITE COLLAR-1 (WC-1) and WC-2, form heterodimeric complexes that activate the transcription of frequency (frq). FRQ serves two roles in these feedback loops: repressing its own transcription by interacting with the WC complex and positively upregulating the levels of WC-1 and WC-2 proteins. We report here that the steady-state level of WC-1 protein is independently regulated by both FRQ and WC-2 through different posttranscriptional mechanisms. The WC-1 level is extremely low in wc-2 knockout strains, and this low level of expression is independent of wc-1 transcription and FRQ protein expression. In addition, our data show that the PAS domain of WC-2 mediates the interactions of this protein with both WC-1 and FRQ in vivo. Such interactions are essential for maintaining the steady-state level of WC-1 and the proper function of WC-1 and WC-2 in circadian clock and light responses.

Assignment of circadian function for the Neurospora clock gene frequency.

Circadian clocks consist of three elements: entrainment pathways (inputs), the mechanism generating the rhythmicity (oscillator), and the output pathways that control the circadian rhythms. It is difficult to assign molecular clock components to any one of these elements. Experiments show that inputs can be circadianly regulated and outputs can feed back on the oscillator. Mathematical simulations indicate that under- or overexpression of a gene product can result in arrhythmicity, whether the protein is part of the oscillator or substantially part of a rhythmically expressed input pathway. To distinguish between these two possibilities, we used traditional circadian entrainment protocols on a genetic model system, Neurospora crassa.

Blue light photoreception in Neurospora circadian rhythm: evidence for involvement of the flavin triplet state.

The mechanism of the photoreceptor acting on the circadian conidiation rhythm of Neurospora crassa was studied, with the following results: (1) the efficiency of 8-haloflavins as sensitizers increased with their triplet yields. (2) Phase shifts were not abolished by removal of oxygen prior to illumination. (3) Oxygen inhibited phase shifts when introduced into the cultures after light treatment. It is proposed that the blue light photoreceptor for the circadian clock of Neurospora crassa acts (1) from its triplet state, but (2) not via singlet oxygen; (3) signal transduction involves (an) oxygen-sensitive intermediate(s).

Light-induced dephosphorylation of a 33 kDa protein in the wild-type strain of Neurospora crassa: the regulatory mutants wc-1 and wc-2 are abnormal.

Light induces the dephosphorylation of a 33 kdalton protein within 8 min in the wild-type strain of Neurospora crassa. The regulatory mutants, wc-1 and wc-2, have an altered pattern of phosphoproteins in darkness and also after irradiation. Because the wc genes have previously been implicated in photodifferentiation (F. Degli Innocenti and V. E. A. Russo, Genetic analysis of blue light-induced responses in Neurospora crassa, in H. Senger (ed.), Blue Light Effects in Biological Systems, Springer-Verlag, Berlin, Heidelberg, 1984, pp. 213-219), we suggest that protein dephosphorylation may constitute a necessary step in the light-transduction chain of Neurospora crassa.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. V. Irreparable mutants of genotype ad-3A ad-3B, ad-3A ad-3B nic-2, and ad-3B nic-2 result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations.

More extensive complementation tests than those performed initially (Webber and de Serres, 1965) on a series of 832 X-ray-induced specific-locus mutations in the adenine-3 (ad-3) region of a two-component heterokaryon (H-12) of Neurospora crassa (de Serres, 1989a) showed that unexpectedly high frequencies of specific-locus mutations in the ad-3 region have additional, but separate, sites of recessive lethal (RLCL) damage in the immediately adjacent genetic regions. The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than that expected on the basis of target theory and classical models of chromosome structure during interphase (de Serres, 1989a). Genetic fine structure analyses, by means of homology tests with tester strains carrying genetic markers in the ad-3 and immediately adjacent regions, have been performed to map the presumed multiple-locus mutations. In a previous paper (de Serres, 1989c), X-ray-induced irreparable ad-3 mutants of the following genotypes and numbers (ad-3A or ad-3B were analyzed, and the high frequency of multiple-locus mutations was confirmed. In the present paper, X-ray-induced irreparable ad-3 mutants of the following genotypes and numbers (ad-3A ad-3B, ad-3A ad-3B nic-2, and ad-3B nic-2 have also been subjected to the same genetic fine structure analysis. These experiments, in the previous (de Serres, 1989c) and present papers, were designed to determine the extent of the functional inactivation in the ad-3 and immediately adjacent genetic regions in individual mutants classified as presumptive multilocus deletions or multiple-locus mutations.

Fast induction of translatable mRNA by blue light in Neurospora crassa wt: the wc-1 and wc-2 mutants are blind.

After blue-light irradiation of Neurospora crassa (wt) mycelia we observed an increase of about 13 translatable mRNA species within a period of 30 min. The induction of translatable mRNA species followed a specific temporal pattern which permitted the identification of four distinct classes. One of the translatable mRNAs was induced in less than 2 min, while the others showed lag periods of 5, 10 or 20 min from the beginning of illumination. The white collar mutants, wc-1 and wc-2, which do not display any of the blue-light-induced physiological effects tested until now were found to be defective for the photoinduction of translatable mRNAs.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. IV. Irreparable mutants of genotype ad-3A and ad-3B result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations.

The induction of specific-locus mutations in the ad-3 region of Neurospora crassa after X-irradiation was studied in a two-component heterokaryon to determine: (1) the ratio of reparable ad-3 mutants (presumed gene/point mutations, designated ad-3R) to irreparable ad-3 mutants (presumed multilocus deletions, designated ad-3IR), and (2) the induction kinetics of each class (Webber and de Serres, 1965). More extensive genetic tests made subsequently (de Serres, 1989a) on the 832 X-ray-induced specific-locus mutations recovered in those experiments showed that unexpected high frequencies of reparable and irreparable ad-3 mutants are actually multiple-locus mutants that have additional, but separate, sites of recessive lethal (RLCL) damage in the immediately adjacent genetic regions (designated ad-3R + RLCL or ad-3IR + RLCL). The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory (where the frequency of the double mutant is expected to be the product of the frequencies of each single mutant) and classical models of chromosome structure during interphase (de Serres, 1989a). In the present paper, a random sample of 832 X-ray-induced ad-3 mutants of genotype ad-3A or ad-3B that are irreparable have been subjected to more extensive genetic fine-structure analysis. These experiments were designed to determine the extent of the functional inactivation in individual mutants in the ad-3 and immediately adjacent genetic regions in mutants classified as presumptive multilocus deletions or multiple-locus mutations. These experiments have shown that in Neurospora crassa most X-ray-induced irreparable mutants of genotype ad-3A or ad-3B map as a series of overlapping multilocus deletions. Among the 29 irreparable mutants of genotype ad-3A, there are 16 different subgroups of complementation patterns; and among the 63 irreparable mutants of genotype ad-3B, there are also 16 different subgroups. In addition, mutants classified as presumptive multiple-locus mutants result from a variety of separate, but closely linked, sites of genetic damage.(ABSTRACT TRUNCATED AT 400 WORDS)

X-ray-induced specific locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. III. Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions by means of complementation tests.

Genetic fine structure analysis of the ad-3 and immediately adjacent genetic regions was made by means of complementation tests on all possible pairwise combinations of 50 X-ray-induced irreparable adenine-3 mutants (designated ad-3IR). All mutants were induced in either heterokaryon 11 or heterokaryon 12 of Neurospora crassa, 2-component heterokaryons heterozygous for mutants at the 3 closely linked loci ad-3A and ad-3B and nic-2 (nicotinamide-requiring) located about 5.0 map units distal to ad-3B. The complementation tests involved mutants of the following genotypes: 15 ad-3A, 27 ad-3B, 7 ad-3A ad-3B nic-2 and 1 ad-3B nic-2. To facilitate mapping, 5 additional strains (each consisting of a gene/point mutation at the ad-3A or ad-3B locus and a separate site of closely linked recessive lethal damage in the immediately adjacent regions [designated ad-3R + RLCL]) were also included. The data from these complementation tests showed that the majority (46/50) of X-ray-induced irreparable ad-3 mutants mapped as a series of overlapping multilocus deletions that extend both proximally and distally into the immediately adjacent genetic regions, as well as into the 'X' region (a region of unknown, but essential function) between ad-3A and ad-3B. The remaining mutants (4/50) were found to result from a series of closely linked, but separate, mutations (designated multilocus mutations) of the type ad-3IR + RLCL, different from those found in previous studies (de Serres, 1968; de Serres and Brockman, 1968). The data from the present complementation tests have expanded the process of genetic fine structure mapping of the ad-3 and immediately adjacent regions (de Serres, 1968) and defined the presence of the following 11 genetic loci: (a) 4 loci (with either known [i.e. col-1t] or unknown [i.e. unknA]) function proximal to ad-3A: unknA, unknB, col-1t, and col-2t, (b) 4 loci in the 'X' region: unknC, unknD, unknE, and unknF, (c) 2 loci distal to ad-3B: unknG, col-3t, and (d) 1 locus distal to nic-2: unknH.

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. II. More extensive genetic tests reveal an unexpectedly high frequency of multiple-locus mutations.

More extensive genetic tests have been performed on a series of 832 X-ray-induced specific-locus mutations in the ad-3 region of a 2-component heterokaryon (H-12) of Neurospora crassa, reported earlier (Webber and de Serres 1965). Using new tester strains and techniques for performing large-scale genetic tests (heterokaryon, dikaryon and trikaryon) to characterize ad-3 mutants induced in 2-component heterokaryons, new data have been obtained on this sample of X-ray-induced ad-3 mutants. These new data show that unexpectedly high frequencies of both single-locus (gene/point) mutations and multilocus deletions in the ad-3 region have additional, but separate, sites of recessive lethal (RLCL) damage in the immediately adjacent genetic regions. The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory and classical models of chromosome structure during interphase. Current models of interphase chromosome structure in higher eukaryotes as revealed by chromosome "painting" offer a possible explanation of the Neurospora data.

Blue light induces circadian rhythms in the bd mutant of Neurospora: double mutants bd,wc-1 and bd,wc-2 are blind.

This paper describes a new blue light effect for Neurospora crassa, the photoinduction of circadian rhythms in the bd mutant. The wc-1 and wc-2 genes are necessary for this effect.

Repair-resistant mutation in Neurospora.

Chronic UV treatment produces severalfold fewer mutations in Neurospora conidia than does the same total dose of acute UV. Experiments were designed to determine the conditions required for chronic UV mutagenesis. Measurement of the coincidence frequency for two independent mutations revealed the existence of a subset of cells which are mutable by chronic UV. Analysis of forward mutation at the mtr locus showed that the genetic alterations produced by chronic UV were virtually all point mutants, even though the assay system could detect alterations or deletions extending into neighboring genes. A significant fraction of the mutants produced by acute UV were multigenic deletions. The size of the dose-rate effect (acute UV mutation frequency divided by chronic UV mutation frequency) was compared for several different mutation assay systems. Forward mutations (recessive lethals and mtr) gave values ranging from four to nine. For events which were restricted to specific molecular sites (specific reversions and nonsense suppressor mutations), there was a wider range of dose-rate ratios. This suggests that chronic UV mutation may be restricted to certain molecular sequences or configurations.

Gamma-ray-sensitive mutants of Neurospora crassa with characteristics analogous to ataxia telangiectasia cell lines.

Well characterized gamma-ray sensitive mutants of the fungus Neurospora crassa have been screened for characteristics analogous to those of cell lines derived from humans with the genetic disease, ataxia telangiectasia (AT). Two Neurospora mutants, uvs-6 and mus-9, show the AT cell line characteristics of gamma-ray and bleomycin sensitivity, and little or no repression of DNA synthesis following treatment with these agents. Normal human or Neurospora cells show an extensive biphasic DNA synthesis repression (to 50% of control) and when DNA synthesis is analyzed by alkaline sucrose gradient centrifugation, repression of DNA synthesis by low doses of gamma-radiation occurs primarily in low molecular weight (MW) DNA pieces in both organisms. In AT cells and the uvs-6 mutant, no repression in synthesis of low or higher MW DNA is seen at low doses, while the mus-9 mutant shows little repression of high MW DNA, but an intermediate level of low MW DNA synthesis. Both mutants have been shown previously to have an increased level of spontaneous chromosome instability as do AT lines. The uvs-6 and mus-9 mutations are known to be due to two different genes in two different epistatic groups. These results demonstrate that AT-like cellular characteristics can arise from defects in at least two and probably any of several genes, and that lower eukaryotes such as Neurospora can provide an inexpensive and useful model for AT while avoiding the problems inherent in using transformed cell lines.