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f-actin dynamics in neurospora crassa.this study demonstrates the utility of lifeact for the investigation of actin dynamics in neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. lifeact is a 17-amino-acid peptide derived from the nonessential saccharomyces cerevisiae actin-binding protein abp140p. fused to green fluorescent protein (gfp) or red fluorescent protein (tagrfp), lifeact allowed live-cell imaging of actin patches, cable ...201020139238
the role of paaac1 encoding a mitochondrial adp/atp carrier in the biosynthesis of extracellular glycolipids, mannosylerythritol lipids, in the basidiomycetous yeast pseudozyma antarctica.pseudozyma antarctica produces large amounts of the glycolipid biosurfactants known as mannosylerythritol lipids (mel), which show not only excellent surface-active properties but also versatile biochemical actions. a gene homologous with a mitochondrial adp/atp carrier was dominantly expressed in p. antarctica under mel-producing conditions on the basis of previous gene expression analysis. the gene encoding the mitochondrial adp/atp carrier of p. antarctica (paaac1) contained a putative open r ...201020146402
a single mutation in the first transmembrane domain of yeast cox2 enables its allotopic expression.during the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. to further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast cox2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. nuclear-recoded saccharomyces cerevisiae cox2 fused at the amino terminus to various alternati ...201020194738
septum formation is regulated by the rho4-specific exchange factors bud3 and rgf3 and by the landmark protein bud4 in neurospora crassa.rho gtpases have multiple, yet poorly defined functions during cytokinesis. by screening a neurospora crassa knock-out collection for rho guanine nucleotide exchange factor (gef) mutants that phenocopy rho-4 defects (i.e. lack of septa, slow growth, abnormal branching and cytoplasmic leakage), we identified two strains defective in homologues of bud3p and rgf3 of budding and fission yeast respectively. the function of these proteins as rho4-specific gefs was determined by in vitro assays. in viv ...201020199606
nuclear dynamics, mitosis, and the cytoskeleton during the early stages of colony initiation in neurospora crassa.neurospora crassa macroconidia form germ tubes that are involved in colony establishment and conidial anastomosis tubes (cats) that fuse to form interconnected networks of conidial germlings. nuclear and cytoskeletal behaviors were analyzed in macroconidia, germ tubes, and cats in strains that expressed fluorescently labeled proteins. heterokaryons formed by cat fusion provided a rapid method for the imaging of multiple labeled fusion proteins and minimized the potential risk of overexpression a ...201020207852
high-throughput construction of gene deletion cassettes for generation of neurospora crassa knockout strains.the availability of complete genome sequences for a number of biologically important fungi has become an important resource for fungal research communities. however, the functions of many open reading frames (orfs) identified through annotation of whole genome sequences have yet to be determined. the disruption of orfs is a practical method for loss-of-function gene analyses in fungi that are amenable to transformation. unfortunately, the construction of knockout cassettes using traditional dige ...201020238259
visualization of f-actin localization and dynamics with live cell markers in neurospora crassa.filamentous actin (f-actin) plays essential roles in filamentous fungi, as in all other eukaryotes, in a wide variety of cellular processes including cell growth, intracellular motility, and cytokinesis. we visualized f-actin organization and dynamics in living neurospora crassa cells via confocal microscopy of growing hyphae expressing gfp fusions with homologues of the actin-binding proteins fimbrin (fim) and tropomyosin (tpm-1), a subunit of the arp2/3 complex (arp-3) and a recently developed ...201020302965
genetic and molecular characterization of a cryptochrome from the filamentous fungus neurospora crassa.in plants and animals, cryptochromes function as either photoreceptors or circadian clock components. we have examined the cryptochrome from the filamentous fungus neurospora crassa and demonstrate that neurospora cry encodes a dash-type cryptochrome that appears capable of binding flavin adenine dinucleotide (fad) and methenyltetrahydrofolate (mthf). the cry transcript and cry protein levels are strongly induced by blue light in a wc-1-dependent manner, and cry transcript is circadianly regulat ...201020305004
roles of the mdm10, tom7, mdm12, and mmm1 proteins in the assembly of mitochondrial outer membrane proteins in neurospora crassa.the mdm10, mdm12, and mmm1 proteins have been implicated in several mitochondrial functions including mitochondrial distribution and morphology, assembly of beta-barrel proteins such as tom40 and porin, association of mitochondria and endoplasmic reticulum, and maintaining lipid composition of mitochondrial membranes. here we show that loss of any of these three proteins in neurospora crassa results in the formation of large mitochondrial tubules and reduces the assembly of porin and tom40 into ...201020335503
an ap-1-like transcription factor, nap-1, regulates expression of the glutathione s-transferase and nadh:flavin oxidoreductase genes in neurospora crassa.ap-1-like transcription factors play crucial roles in oxidative stress responses in yeast and filamentous fungi. the deletion of an ap-1-like transcription factor gene, nap-1, in neurospora crassa slightly increased its sensitivity to oxidative stressors, including menadione. microarray and quantitative real-time reverse transcriptase-pcr analyses were employed to identify menadione-inducible genes (migs) and the roles of nap-1 in their regulation. n. crassa migs include three putative glutathio ...201020378991
the fungal genetics stock center: a repository for 50 years of fungal genetics research.the fungal genetics stock center (fgsc) was established in 1960 to ensure that important strains used in early genetics research were available to subsequent generations of fungal geneticists. originally, only mutant strains were held. at present, any organism that has had its genome sequenced is a genetic system and so the fgsc has added many new organisms. the fgsc is well integrated in its core community and, as research came to depend on cloned genes, vectors and gene libraries, the fgsc inc ...201020413916
rotenone enhances the antifungal properties of staurosporine.we studied staurosporine-induced cell death in the filamentous fungus neurospora crassa. the generation of reactive oxygen species during the process appears to be an important signaling event, since addition of the antioxidant glutathione prevents the effects of staurosporine on fungal growth. selected mutants with mutations in respiratory chain complex i are extremely sensitive to the drug, stressing the involvement of complex i in programmed cell death. following this finding, we determined t ...201020435699
extensive and varied modifications in histone h2b of wild-type and histone deacetylase 1 mutant neurospora crassa.dna methylation is deficient in a histone deacetylase 1 (hda1) mutant (hda-1) strain of neurospora crassa with inactivated histone deacetylase 1. difference two-dimensional (2d) gels identified the primary histone deacetylase 1 target as histone h2b. acetylation was identified by lc-ms/ms at five different lysines in wild-type h2b and at 11 lysines in hda-1 h2b, suggesting neurospora h2b is a complex combination of different acetylated species. individual 2d gel spots were shifted by single lysi ...201020462202
photobiology in the zygomycota: multiple photoreceptor genes for complex responses to light.light is an environmental signal that modulates many aspects of the biology of zygomycete fungi. light regulation has been investigated in the zygomycetes phycomyces blakesleeanus, mucor circinelloides and pilobolus crystallinus. examples of light regulation include the phototropism of the fruiting bodies, the regulation of the development of reproductive structures, and the activation of the biosynthesis of β-carotene. in fungi blue light is perceived by proteins homologous to wc-1, a neurospor ...201020466063
regulation of fruiting body photomorphogenesis in coprinopsis cinerea.the agaricomycete (homobasidiomycete) coprinopsis cinerea has been used as a model to study the molecular mechanism for photomorphogenesis. molecular genetic analyses of mutants defective in fruiting body (mushroom) photomorphogenesis of c. cinerea identified two genes, dst1 and dst2. dst1 encodes a homolog of wc-1, a fungal blue-light photoreceptor first identified in neurospora crassa, while dst2 encodes a novel protein with a putative flavin adenine dinucleotide (fad)-binding-4 domain. in add ...201020471485
ferrochelatase is a conserved downstream target of the blue light-sensing white collar complex in fungi.light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. previous research has established that conserved proteins, originally called white collar 1 and 2 from the ascomycete neurospora crassa, regulate uv/blue light sensing. homologous proteins function in distant relatives of n. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. here we conduct ...201020488877
expression of a neurospora crassa zinc transporter gene in transgenic nicotiana tabacum enhances plant zinc accumulation without co-transport of cadmium.zinc (zn) is an essential micronutrient required for growth and development of all organisms. deficiency of zn in humans is widespread, affecting 25% of world population and efforts are underway to develop crop plants with high levels of zn in their edible parts. when strategies for enhancing zn in crop plants are designed, it is essential to exclude cadmium (cd), a toxic analogue of zn. in the present work, a high affinity and high specificity zinc transporter gene (tzn1) from neurospora crassa ...201020492552
structure and applications of a rhamnolipid surfactant produced in soybean oil waste.soybean oil soapstock was utilized as an alternative carbon source for the production of rhamnolipids by pseudomonas aeruginosa lbi strain. the chemical composition and properties of the rhamnolipid mixture obtained were determined to define its potential applications. the chemical characterization of the rhamnolipid has revealed the presence of ten different homologues. the monorhamnolipid rhac10c1) and the dirhamnolipid rha2c10c10 were the main components of the mixture that showed predominanc ...201019649781
expression of ribonuclease a and ribonuclease n1 in the filamentous fungus neurospora crassa.in this study, we investigated the ability of the fungus neurospora crassa to produce and secrete two ribonucleases: the heterologous bovine rnase a and the endogenous rnase n(1). a set of expression vectors was constructed, each consisting of an rnase a open reading frame under the control of a specific promoter and each with a specific terminator. n. crassa transformants were analyzed at the transcriptional and protein levels. irrespective of the promoter used, all transformants showed an rnas ...201019662399
purification and characterization of a low molecular weight endo-xylanase from mushroom termitomyces clypeatus.a low molecular weight endo-xylanase (ec 3.2.1.8) was purified from an edible mushroom termitomyces clypeatus grown in submerged medium with oat spelt xylan. xylanase was purified to apparent homogeneity by ammonium sulfate fractionation and gel filtration chromatography. its molecular weight was determined by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 12 kda. the enzyme was found to be most active at 50 degrees c and ph 5.0, being most stab ...201019728166
a comparative study of activity and apparent inhibition of fungal β-glucosidases.β-glucosidases (bgs) from aspergillus fumigates, aspergillus niger, aspergillus oryzae, chaetomium globosum, emericella nidulans, magnaporthe grisea, neurospora crassa, and penicillium brasilianum were purified to homogeneity, and analyzed by isothermal titration calorimetry with respect to their hydrolytic activity and its sensitivity to glucose (product) using cellobiose as substrate. global non-linear regression of several reactions, with or without added glucose, to a product inhibition equa ...201020677177
rna interference pathways in filamentous fungi.rna interference is a conserved homology-dependent post-transcriptional/transcriptional gene silencing mechanism in eukaryotes. the filamentous fungus neurospora crassa is one of the first organisms used for rnai studies. quelling and meiotic silencing by unpaired dna are two rnai-related phenomena discovered in neurospora, and their characterizations have contributed significantly to our understanding of rnai mechanisms in eukaryotes. a type of dna damage-induced small rna, microrna-like small ...201020680389
cazymes analysis toolkit (cat): web service for searching and analyzing carbohydrate-active enzymes in a newly sequenced organism using cazy database.the carbohydrate-active enzyme (cazy) database provides a rich set of manually annotated enzymes that degrade, modify, or create glycosidic bonds. despite rich and invaluable information stored in the database, software tools utilizing this information for annotation of newly sequenced genomes by cazy families are limited. we have employed two annotation approaches to fill the gap between manually curated high-quality protein sequences collected in the cazy database and the growing number of oth ...201020696711
involvement of a putative response regulator brrg-1 in the regulation of sporulation, sensitivity to fungicides, and osmotic stress in botrytis cinerea.the response regulator protein is a core element of two-component signaling pathway. in this study, we investigated functions of brrg-1 of botrytis cinerea, a gene that encodes a putative response regulator protein, which is homologous to rrg-1 in neurospora crassa. the brrg-1 gene deletion mutant δbrrg1-62 was unable to produce conidia. the mutant showed increased sensitivity to osmotic stress mediated by nacl and kcl, and to oxidative stress generated by h(2)o(2). additionally, the mutant was ...201021161211
molecular cloning of nit-2, a regulatory gene required for nitrogen metabolite repression in neurospora crassa.we used an efficient sib-selection procedure to isolate a cosmid clone that complemented a mutated nit-2 gene of neurospora crassa. restriction fragment length polymorphism mapping indicated that the cosmid dna insert was derived from linkage group il, between 5s rdna locus 12 and mt, the region of the n. crassa genome that contains nit-2. we conclude that the cosmid carries the nit-2 gene.20102879771
chorismate synthase: a bifunctional enzyme in neurospora crassa. 20102955201
chitinase from neurospora crassa. 20102976112
the qa repressor gene of neurospora crassa: wild-type and mutant nucleotide sequences.the qa-1s gene, one of two regulatory genes in the qa gene cluster of neurospora crassa, encodes the qa repressor. the qa-1s gene together with the qa-1f gene, which encodes the qa activator protein, control the expression of all seven qa genes, including those encoding the inducible enzymes responsible for the utilization of quinic acid as a carbon source. the nucleotide sequence of the qa-1s gene and its flanking regions has been determined. the deduced coding sequence for the qa-1s protein en ...20103010294
protein chemistry of the neurospora crassa plasma membrane h+-atpase.a highly effective procedure for fragmenting the neurospora crassa plasma membrane h+-atpase and purifying the resulting peptides is described. the enzyme is cleaved with trypsin to form a limit digest containing both hydrophobic and hydrophilic peptides, and the hydrophobic and hydrophilic peptides are then separated by extraction with an aqueous ammonium bicarbonate solution. the hydrophilic peptides are fractionated by sephadex g-25 column chromatography into three pools, and the individual p ...20102903697
purification of vacuolar membranes, mitochondria, and plasma membranes from neurospora crassa and modes of discriminating among the different h+-atpases. 20102906719
circadian rhythms in neurospora crassa: the effects of point mutations on the proteolipid portion of the mitochondrial atp synthetase.five oligomycin-resistant (olir) mutant strains of neurospora crassa were analyzed for their growth rate and for the periodicity of their circadian rhythm. the most resistant strains had periods of 18-19 h while the least resistant strain had a normal period of 21.0 h. there was a rough correlation between the in vivo degree of oligomycin-resistance and the amount of change in the period. several of the olir mutations have been previously described by sebald et al. (1977) in terms of known amino ...20102863735
dna methylation and normal chromosome behavior in neurospora depend on five components of a histone methyltransferase complex, dcdc.methylation of dna and of lysine 9 on histone h3 (h3k9) is associated with gene silencing in many animals, plants, and fungi. in neurospora crassa, methylation of h3k9 by dim-5 directs cytosine methylation by recruiting a complex containing heterochromatin protein-1 (hp1) and the dim-2 dna methyltransferase. we report genetic, proteomic, and biochemical investigations into how dim-5 is controlled. these studies revealed dcdc, a previously unknown protein complex including dim-5, dim-7, dim-9, cu ...201021079689
translocations used to generate chromosome segment duplications in neurospora can disrupt genes and create novel open reading frames.in neurospora crassa, crosses between normal sequence strains and strains bearing some translocations can yield progeny bearing a duplication (dp) of the translocated chromosome segment. here, 30 breakpoint junction sequences of 12 dp-generating translocations were determined. the breakpoints disrupted 13 genes (including predicted genes), and created 10 novel open reading frames. insertion of sequences from lg iii into lg i as translocation t(uk8-18) disrupts the eat-3 gene, which is the orthol ...201021289436
molecular mechanism of the neurospora circadian oscillator.circadian clocks are the internal time-keeping mechanisms for organisms to synchronize their cellular and physiological processes to the daily light/dark cycles. the molecular mechanisms underlying circadian clocks are remarkably similar in eukaryotes. neurospora crassa, a filamentous fungus, is one of the best understood model organisms for circadian research. in recent years, accumulating data have revealed complex regulation in the neurospora circadian clock at transcriptional, posttranscript ...201021203945
role of individual subunits of the neurospora crassa csn complex in regulation of deneddylation and stability of cullin proteins.the cop9 signalosome (csn) is an evolutionarily conserved multifunctional complex that controls ubiquitin-dependent protein degradation in eukaryotes. we found seven csn subunits in neurospora crassa in a previous study, but only one subunit, csn-2, was functionally characterized. in this study, we created knockout mutants for the remaining individual csn subunits in n. crassa. by phenotypic observation, we found that loss of csn-1, csn-2, csn-4, csn-5, csn-6, or csn-7 resulted in severe defects ...201021151958
evolution and diversity of a fungal self/nonself recognition locus.self/nonself discrimination is an essential feature for pathogen recognition and graft rejection and is a ubiquitous phenomenon in many organisms. filamentous fungi, such as neurospora crassa, provide a model for analyses of population genetics/evolution of self/nonself recognition loci due to their haploid nature, small genomes and excellent genetic/genomic resources. in n. crassa, nonself discrimination during vegetative growth is determined by 11 heterokaryon incompatibility (het) loci. cell ...201021124910
the neurospora peptide:n-glycanase ortholog png1 is essential for cell polarity despite its lack of enzymatic activity.secretory proteins are subjected to a stringent endoplasmic reticulum-based quality control system that distinguishes aberrant from correctly folded proteins. the cytoplasmic peptide:n-glycanase cleaves oligosaccharides from misfolded glycoproteins and prepares them for degradation by the 26 s proteasome. in contrast to abundant in vitro data on its enzymatic function, the in vivo relevance of peptide:n-glycanase activity remains unclear. here we show that the png1 ortholog from the filamentous ...201019940117
a complex photoreceptor system mediates the regulation by light of the conidiation genes con-10 and con-6 in neurospora crassa.genes con-10 and con-6 in neurospora crassa are activated during conidiation or after illumination of vegetative mycelia. light activation requires the white-collar complex (wcc), a transcription factor complex composed of the photoreceptor wc-1 and its partner wc-2. we have characterized the photoactivation of con-10 and con-6, and we have identified 300bp required for photoactivation in the con-10 promoter. a complex stimulus-response relationship for con-10 and con-6 photoactivation suggested ...201019932184
protein roles in group i intron rna folding: the tyrosyl-trna synthetase cyt-18 stabilizes the native state relative to a long-lived misfolded structure without compromising folding kinetics.the neurospora crassa cyt-18 protein is a mitochondrial tyrosyl-trna synthetase that also promotes self-splicing of group i intron rnas by stabilizing the functional structure in the conserved core. cyt-18 binds the core along the same surface as a common peripheral element, p5abc, suggesting that cyt-18 can replace p5abc functionally. in addition to stabilizing structure generally, p5abc stabilizes the native conformation of the tetrahymena group i intron relative to a globally similar misfolde ...201019913030
robustness from flexibility in the fungal circadian clock.robustness is a central property of living systems, enabling function to be maintained against environmental perturbations. a key challenge is to identify the structures in biological circuits that confer system-level properties such as robustness. circadian clocks allow organisms to adapt to the predictable changes of the 24-hour day/night cycle by generating endogenous rhythms that can be entrained to the external cycle. in all organisms, the clock circuits typically comprise multiple interloc ...201020576110
atm and atr homologes of neurospora crassa are essential for normal cell growth and maintenance of chromosome integrity.genome integrity is maintained by many cellular mechanisms in eukaryotes. one such mechanism functions during the cell cycle and is known as the dna damage checkpoint. in the filamentous fungus neurospora crassa, mus-9 and mus-21 are homologes of two key factors of the mammalian dna damage checkpoint, atr and atm, respectively. we previously showed that mus-9 and mus-21 mutants are sensitive to dna damage and that each mutant shows a characteristic growth defect: conidia from the mus-9 mutant ha ...201020553930
qip, a protein that converts duplex sirna into single strands, is required for meiotic silencing by unpaired dna.rna interference (rnai) depends on the production of small rna to regulate gene expression in eukaryotes. two rnai systems exist to control repetitive selfish elements in neurospora crassa. quelling targets transgenes during vegetative growth, whereas meiotic silencing by unpaired dna (msud) silences unpaired genes during meiosis. the two mechanisms require common rnai proteins, such as rna-directed rna polymerases, dicers, and argonaute slicers. we have previously demonstrated that, while quell ...201020551436
turgor and net ion flux responses to activation of the osmotic map kinase cascade by fludioxonil in the filamentous fungus neurospora crassa.the internal hydrostatic pressure (turgor) of the filamentous fungus neurospora crassa is regulated at about 400-500 kilopascals, primarily by an osmotic map kinase cascade which activates ion uptake from the extracellular medium and glycerol synthesis. in the absence of hyperosmotic stress, the phenylpyrrole fungicide fludioxonil activates the osmotic map kinase cascade, resulting in cell death. turgor, the electrical potential and net ion fluxes were measured after treatment with fludioxonil. ...201020546911
reinforced postmating reproductive isolation barriers in neurospora, an ascomycete microfungus.maladaptive hybridization promotes reinforcement, selection for stringent reproductive isolation barriers during speciation. reinforcement is suspected when barriers between sympatric populations are stronger than allopatric barriers, and particularly when stronger barriers evolve in the species and sex suffering the greatest costs of hybridization. canonically, reinforcement involves premating barriers. selection for postmating barriers is controversial, but theoretically possible. we examined ...201020546092
deletion of a novel f-box protein, mus-10, in neurospora crassa leads to altered mitochondrial morphology, instability of mtdna and senescence.while mitochondria are renowned for their role in energy production, they also perform several other integral functions within the cell. thus, it is not surprising that mitochondrial dysfunction can negatively impact cell viability. although mitochondria have received an increasing amount of attention in recent years, there is still relatively little information about how proper maintenance of mitochondria and its genomes is achieved. the neurospora crassa mus-10 mutant was first identified thro ...201020516500
characterization of the temperature-sensitive mutations un-7 and png-1 in neurospora crassa.the model filamentous fungus neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. while most of these have been mapped genetically, many remain anonymous. the mutation in the n. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated ncu00651 on linkage group i. other mutations in this gene have been identified that lead to a temperature-sensitiv ...201020502699
catalase evolved to concentrate h2o2 at its active site.catalase is a homo-tetrameric enzyme that has its heme active site deeply buried inside the protein. its only substrate, hydrogen peroxide (h2o2), reaches the heme through a 45 a-long channel. large-subunit catalases, but not small-subunit catalases, have a loop (gate loop) that interrupts the major channel. two accesses lead to a gate that opens the final section of the channel to the heme; gates from the r-related subunits are interconnected. using molecular dynamic simulations of the neurospo ...201020494646
membrane fluidity determines sensitivity of filamentous fungi to chitosan.the antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. we have found that the plasma membrane forms a barrier to chitosan in chitosan-resistant but not chitosan-sensitive fungi. the plasma membranes of chitosan-sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan-resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. a fatty acid desaturase mutant of neurosp ...201020487294
functional characterization of mat1-1-specific mating-type genes in the homothallic ascomycete sordaria macrospora provides new insights into essential and nonessential sexual regulators.mating-type genes in fungi encode regulators of mating and sexual development. heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. to analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted eac ...201020435701
mapping of lysine methylation and acetylation in core histones of neurospora crassa.core histones are susceptible to a variety of post-translational modifications (ptms), among which methylation and acetylation play critical roles in various chromatin-dependent processes. the nature and biological functions of these ptms have been extensively studied in plants, animals, and yeasts. in contrast, the histone modifications in neurospora crassa, a convenient model organism for multicellular eukaryotes, remained largely undefined. in this study, we used several mass spectrometric te ...201020433192
biotransformation of alpha-cedrol and caryophyllene oxide by the fungus neurospora crassa.incubation of alpha-cedrol and caryophyllene oxide with neurospora crassa afforded 12beta-hydroxy cedrol, 10alpha-hydroxy cedrol, and 3beta-hydroxy cedrol, and 12beta-hydroxy caryophyllene oxide as major metabolites, respectively. the antibacterial and radical scavenging activities of the metabolites were evaluated in vitro using broth microdilution and bioauthographic techniques. however, no significant antibacterial and antioxidant activities were observed when compared with those of standard ...201020433062
dna methylation and the formation of heterochromatin in neurospora crassa.studies of the control and function of dna methylation in neurospora crassa have led to a greater understanding of heterochromatin formation. dna methylation in neurospora is dependent on trimethylation of histone h3 lysine 9 (h3k9me3) by the histone methyltransferase, dim-5. the linkage between these two methyl marks is facilitated by heterochromatin protein 1 (hp1), which serves as an adapter protein. hp1 binds to the h3k9me3 and recruits the dna methyltransferase, dim-2. although hp1 links h3 ...201020407471
high efficient gene targeting on the agamous gene in an arabidopsisatlig4 mutant.gene targeting induced by homologous integration of a foreign dna segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. but, integration of introduced dna fragments is predominantly non-homologous in most species. however, we presented high-efficient homologous integration in disruptants of non-homologous end joining (nhej), that is, the ...201020406622
identification of dim-7, a protein required to target the dim-5 h3 methyltransferase to chromatin.functionally distinct chromatin domains are delineated by distinct posttranslational modifications of histones, and in some organisms by differences in dna methylation. proper establishment and maintenance of chromatin domains is critical but not well understood. we previously demonstrated that heterochromatin in the filamentous fungus neurospora crassa is marked by cytosine methylation directed by trimethylated lysine 9 on histone h3 (h3k9me3). h3k9me3 is the product of the dim-5 lysine methylt ...201020404183
de novo assembly of a 40 mb eukaryotic genome from short sequence reads: sordaria macrospora, a model organism for fungal morphogenesis.filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by sanger sequencing. while next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and chip analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size an ...201020386741
effects of prd circadian clock mutations on frq-less rhythms in neurospora.rhythmic conidiation (spore formation) in neurospora crassa provides a model system for investigating the molecular mechanisms of circadian rhythmicity. a feedback loop involving the frq, wc-1, and wc-2 gene products (frq/ wcc) is an important component of the mechanism; however, rhythmic conidiation can still be observed when these gene products are absent. the nature of the oscillator(s) that drives this frq-less rhythmicity (flo) is an important question in neurospora circadian biology. we ha ...201020348458
marker fusion tagging, a new method for production of chromosomally encoded fusion proteins.a new gene-tagging method (marker fusion tagging [mft]) is demonstrated for neurospora crassa and magnaporthe oryzae. translational fusions between the hygromycin b resistance gene and various markers are inserted into genes of interest by homologous recombination to produce chromosomally encoded fusion proteins. this method can produce tags at any position and create deletion alleles that maintain n- and c-terminal sequences. we show the utility of mft by producing enhanced green fluorescent pr ...201020348390
live-cell imaging of microtubule dynamics in hyphae of neurospora crassa.due to the large number of microtubules in the wild-type strain, total internal reflection fluorescence (tirf) microscopy was used to study cortical microtubule dynamics in leading hyphae of neurospora crassa expressing beta-tubulin-gfp. detection of plus-end dynamics of individual microtubule was much improved with this approach compared to the other commonly used methods such as confocal and widefield fluorescence microscopy. in order to address the roles of motor proteins in microtubule dynam ...201020238275
biochemical methods used to study the gene expression and protein complexes in the filamentous fungus neurospora crassa.biochemical approaches are powerful tools for investigating mechanisms of biological processes. here, we describe several biochemical approaches that have been successfully in our laboratory to study the filamentous fungus neurospora crassa. these approaches include protein extraction and western blot analysis, protein purification using epitope-tagged fusion protein, protein immunoprecipitation (ip) and chromatin immunoprecipitation (chip) assays. these methods can also be modified for use in o ...201020238270
gene silencing for functional analysis: assessing rnai as a tool for manipulation of gene expression.the availability of a large number of gene-disrupted mutants (either from natural mutants' collections or from knockout projects) is a great advantage for functional analysis studies. however, disfunction of many fungal genes, involved in key developmental processes, leads to dramatic and pleotropic changes in cell morphology, conferring a major difficulty in studying null mutants. therefore, obtaining variable levels of reduction in gene expression, especially of essential genes or genes whose ...201020238262
the dmm complex prevents spreading of dna methylation from transposons to nearby genes in neurospora crassa.transposable elements are common in genomes and must be controlled. many organisms use dna methylation to silence such selfish dna, but the mechanisms that restrict the methylation to appropriate regions are largely unknown. we identified a jmjc domain protein in neurospora, dna methylation modulator-1 (dmm-1), that prevents aberrant spreading of dna and histone h3k9 methylation from inactivated transposons into nearby genes. mutation of a conserved residue within the jmjc fe(ii)-binding site ab ...201020139222
systematic overrepresentation of dna termini and underrepresentation of subterminal regions among sequencing templates prepared from hydrodynamically sheared linear dna molecules.analysis of fungal genome sequence assemblies reveals that telomeres are poorly represented even though telomeric reads tend to be superabundant. we surmised that the problem might lie in the dna shearing conditions used to create clone libraries for genome sequencing.201020122283
circadian rhythms in neurospora crassa: dynamics of the clock component frequency visualized using a fluorescent reporter.the frequency (frq) gene of neurospora crassa has long been considered essential to the function of this organism's circadian rhythm. increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. to address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins ...201020051268
regulation by blue light of the fluffy gene encoding a major regulator of conidiation in neurospora crassa.the development of asexual spores, that is, the process of conidiation, in the fungus neurospora crassa is increased by light. the fluffy (fl) gene, encoding a major regulator of conidiation, is activated by light. we describe here a detailed characterization of the regulation by blue light of fl in vegetative hyphae. this induction requires the white collar complex (wcc) while the fld protein acts as a dark repressor of fl transcription. we show that the wcc directly regulates fl transcription ...201020026679
transcription of the neurospora crassa 70-kda class heat shock protein genes is modulated in response to extracellular ph changes.heat shock proteins belong to a conserved superfamily of molecular chaperones found in prokaryotes and eukaryotes. these proteins are linked to a myriad of physiological functions. in this study, we show that the n. crassa hsp70-1 (ncu09602.3) and hsp70-2 (ncu08693.3) genes are preferentially expressed in an acidic milieu after 15 h of cell growth in sufficient phosphate at 30 degrees c. no significant accumulation of these transcripts was detected at alkaline ph values. both genes accumulated t ...201019618296
nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis.nitronate monooxygenase (nmo), formerly referred to as 2-nitropropane dioxygenase, is an fmn-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. over the past 5 years, a resurgence of interest on the enzymology of nmo has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. this revi ...201019577534
circadian rhythms in neurospora crassa: downstream effectors.the circadian rhythm in neurospora crassa is exhibited as alternating areas of conidiating and non-conidiating mycelia growth. a significant role in this circadian rhythm is played by the frq (frequency) and wc (white-collar) genes, comprising the "fwc" oscillator. strains lacking the fwc can be restored to rhythmicity, which has been attributed to a second oscillator, called the flo (frq-less oscillator). this study reports additional conditions that allow this rhythmicity to occur. rhythmicity ...201019800017
ubiquitin ligase components cullin4 and ddb1 are essential for dna methylation in neurospora crassa.dna methylation and h3k9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. in the filamentous fungus neurospora crassa, it has been demonstrated that h3k9 trimethylation catalyzed by histone methyltransferase dim-5 is essential for dna methylation. trimethylated h3k9 is recognized by hp1, which then recruits the dna methyltransferase dim-2 to methylate the dna. here, we show that in neurospora, ubiquitin ligase components cullin4 and ddb1 are essent ...201019948733
the de novo cytosine methyltransferase drm2 requires intact uba domains and a catalytically mutated paralog drm3 during rna-directed dna methylation in arabidopsis thaliana.eukaryotic dna cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. this silencing is stable between cell generations as cytosine methylation is maintained epigenetically through dna replication. the arabidopsis thaliana dnmt3 cytosine methyltransferase ortholog domains rearranged methyltransferase2 (drm2) is required for establishment of small interfering rna (sirna) directed dna methylation. in mammals piwi proteins and pir ...201021060858
of switches and hourglasses: regulation of subcellular traffic in circadian clocks by phosphorylation.investigation of the phosphorylation of circadian clock proteins has shown that this modification contributes to circadian timing in all model organisms. phosphorylation alters the stability, transcriptional activity and subcellular localization of clock proteins during the course of a day, such that time-of-day-specific phosphorylation encodes information for measuring time and is crucial for the establishment of an approximately 24-h period. one main feature of molecular timekeeping is the day ...201021052092
degradation of carbohydrate moieties of arabinogalactan-proteins by glycoside hydrolases from neurospora crassa.arabinogalactan-proteins (agps) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. the carbohydrate moieties of agps commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as l-ara and glca are attached. for the present study, an α-l-arabinofuranosidase belonging to glycoside hydrolase family (ghf) 54, ncaraf1, and an endo-β-(1→6)-galactanase of ghf 5, nc6gal, were identified in neurospora crassa. recombinant ncara ...201020932514
dcaf26, an adaptor protein of cul4-based e3, is essential for dna methylation in neurospora crassa.dna methylation is involved in gene silencing and genome stability in organisms from fungi to mammals. genetic studies in neurospora crassa previously showed that the cul4-ddb1 e3 ubiquitin ligase regulates dna methylation via histone h3k9 trimethylation. however, the substrate-specific adaptors of this ligase that are involved in the process were not known. here, we show that, among the 16 ddb1- and cul4-associated factors (dcafs) encoded in the n. crassa genome, three interacted strongly with ...201020885793
sequence requirements for ribosome stalling by the arginine attenuator peptide.the 5' regions of eukaryotic mrnas often contain upstream open reading frames (uorfs). the neurospora crassa arg-2 uorf encodes the 24-residue arginine attenuator peptide (aap). this regulatory uorf-encoded peptide, which is evolutionarily conserved in fungal transcripts specifying an arginine biosynthetic enzyme, functions as a nascent peptide within the ribosomal tunnel and negatively regulates gene expression. the nascent aap causes ribosomes to stall at the uorf stop codon in response to arg ...201020884617
modeling a circadian surface.experiments that systematically varied t, τ, and photoperiod in neurospora crassa revealed that the traditional nonparametric and parametric approaches could not explain entrainment for all of the tested conditions. the authors have developed a new approach to understanding entrainment that incorporates several features of the old paradigms but allows exploration of the underlying mechanisms in synchronized clocks, making extrapolations from constant conditions to entrained state unnecessary. it ...201020876814
a circadian surface of entrainment: varying t, τ, and photoperiod in neurospora crassa.the two major prerequisites for a functional circadian system are the generation of an internal day (circadian cycle) and adjusting its length-and phase-to that of the external day (zeitgeber cycle). the generation of circadian cycles can be observed in constant conditions where organisms show a self-sustained, free-running rhythm. their expression depends on the nature of the constant conditions (e.g., constant darkness, dd, or constant light, ll). the mechanism that synchronizes the circadian ...201020876812
temporal and spatial regulation of gene expression during asexual development of neurospora crassa.in this study we profiled spatial and temporal transcriptional changes during asexual sporulation in the filamentous fungus neurospora crassa. aerial tissue was separated from the mycelium to allow detection of genes specific to each tissue. we identified 2641 genes that were differentially expressed during development, which represents ∼25% of the predicted genes in the genome of this model fungus. on the basis of the distribution of functional annotations of 1102 of these genes, we identified ...201020876563
h2b- and h3-specific histone deacetylases are required for dna methylation in neurospora crassa.neurospora crassa utilizes dna methylation to inhibit transcription of heterochromatin. dna methylation is controlled by the histone methyltransferase dim-5, which trimethylates histone h3 lysine 9, leading to recruitment of the dna methyltransferase dim-2. previous work demonstrated that the histone deacetylase (hdac) inhibitor trichostatin a caused a reduction in dna methylation, suggesting involvement of histone deacetylation in dna methylation. we therefore created mutants of each of the fou ...201020876559
α-1,6-mannosylation of n-linked oligosaccharide present on cell wall proteins is required for their incorporation into the cell wall in the filamentous fungus neurospora crassa.the enzyme α-1,6-mannosyltransferase (och-1) is required for the synthesis of galactomannans attached to the n-linked oligosaccharides of neurospora crassa cell wall proteins. the neurospora crassa och-1 mutant has a tight colonial phenotype and a defective cell wall. a carbohydrate analysis of the och-1 mutant cell wall revealed a 10-fold reduction in the levels of mannose and galactose and a total lack of 1,6-linked mannose residues. analysis of the integral cell wall protein from wild-type an ...201020870880
circadian oscillators in eukaryotes.the biological clock, present in nearly all eukaryotes, has evolved such that organisms can adapt to our planet's rotation in order to anticipate the coming day or night as well as unfavorable seasons. as all modern high-precision chronometers, the biological clock uses oscillation as a timekeeping element. in this review, we describe briefly the discovery, historical development, and general properties of circadian oscillators. the issue of temperature compensation (tc) is discussed, and our pr ...201020836046
vivid interacts with the white collar complex and frequency-interacting rna helicase to alter light and clock responses in neurospora.the photoreceptor and pas/lov protein vivid (vvd) modulates blue-light signaling and influences light and temperature responses of the circadian clock in neurospora crassa. one of the main actions of vvd on the circadian clock is to influence circadian clock phase by regulating levels of the transcripts encoded by the central clock gene frequency (frq). how this regulation is achieved is unknown. here we show that vvd interacts with complexes central for circadian clock and blue-light signaling, ...201020807745
new insights into the circadian clock in chlamydomonas.the unicellular green alga chlamydomonas reinhardtii has long been used in research on circadian rhythm. various circadian rhythms in behavior and physiology including phototaxis, chemotaxis, the cell division cycle, uv sensitivity, stickiness to glass (changes in properties of the cell surface), and starch content are observed in this alga. soon after the isolation of clock mutants in drosophila melanogaster and neurospora crassa, clock mutants were also isolated in chlamydomonas. whereas genes ...201020797685
entrainment elicits period aftereffects in neurospora crassa.circadian clocks continue to oscillate in constant conditions with their own period (tau) 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 tau of their initial free-run often depends on the nature of the prior zeitgeber. these postentrainment effects on period (tau-aftereffects) have predominantly been reported for animals but, so far, not fungi. the authors therefore in ...201020795879
physical interaction between vivid and white collar complex regulates photoadaptation in neurospora.photoadaptation, the ability to attenuate a light response on prolonged light exposure while remaining sensitive to escalating changes in light intensity, is essential for organisms to decipher time information appropriately, yet the underlying molecular mechanisms are poorly understood. in neurospora crassa, vivid (vvd), a small lov domain containing blue-light photoreceptor protein, affects photoadaptation for most if not all light-responsive genes. we report that there is a physical interacti ...201020733070
a proteomic approach towards the identification of the matrix protein content of the two types of microbodies in neurospora crassa.microbodies (peroxisomes) comprise a class of organelles with a similar biogenesis but remarkable biochemical heterogeneity. here, we purified the two distinct microbody family members of filamentous fungi, glyoxysomes and woronin bodies, from neurospora crassa and analyzed their protein content by hplc/esi-ms/ms. in the purified woronin bodies, we unambiguously identified only hexagonal 1 (hex1), suggesting that the matrix is probably exclusively filled with the hex1 hexagonal crystal. the prot ...201020707002
transcription factors in light and circadian clock signaling networks revealed by genomewide mapping of direct targets for neurospora white collar complex.light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. here, we used chromatin immunoprecipitation (chip) sequencing to uncover direct targets of the neurospora crassa circadian regulator white collar complex (wcc). the wcc is a blue-light receptor and the key transcription factor of the circadian oscillator. it controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to lig ...201020675579
cr(vi) removal on fungal biomass of neurospora crassa: the importance of dissolved organic carbons derived from the biomass to cr(vi) reduction.interactions of toxic cr(vi) with renewable biomaterials are considered an important pathway for cr(vi) removal in ecosystems. biomaterials are susceptible to dissolution, and their dissolved derivatives may provide an alternative to surface-involved pathway for scavenging of cr(vi). in this study, dissolved organic carbon (doc) derived from neurospora crassa biomass was investigated. the proportion of cr(vi) reduction by doc to that on biomass was determined to evaluate the importance of doc to ...201020669959
structure and engineering of l-arabinitol 4-dehydrogenase from neurospora crassa.l-arabinitol 4-dehydrogenase (lad) catalyzes the conversion of l-arabinitol into l-xylulose with concomitant nad(+) reduction. it is an essential enzyme in the development of recombinant organisms that convert l-arabinose into fuels and chemicals using the fungal l-arabinose catabolic pathway. here we report the crystal structure of lad from the filamentous fungus neurospora crassa at 2.6 a resolution. in addition, we created a number of site-directed variants of n. crassa lad that are capable o ...201020655316
in vitro activities of the multifunctional rna silencing polymerase qde-1 of neurospora crassa.qde-1 is an rna- and dna-dependent rna polymerase that has functions in the rna silencing and dna repair pathways of the filamentous fungus neurospora crassa. the crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic dna-dependent rna polymerases. however, the specific activities of this multifunctional enzyme are still largely unknown. in this study, we characterized the in vitro activities of the n-terminally trunca ...201020647305
neurospora illuminates fungal photoreception.light not only is indispensable as an energy source for life on earth but also serves as an essential environmental cue conveying the information of daily and seasonal time to organisms across different kingdoms. although the molecular mechanisms underlying light responses are actively explored in various light-sensitive organisms, these studies are either hindered by the complexity of the systems or an incomplete familiarity with the light signaling components involved in the scheme. therefore, ...201020637887
qip, a component of the vegetative rna silencing pathway, is essential for meiosis and suppresses meiotic silencing in neurospora crassa.among the processes that play essential roles in both genome defense and organism survival are those involved in chromosome comparison. they are acutely active in the meiotic cells of neurospora crassa, where they evaluate the mutual identity of homologs by a process we call trans-sensing. when nonsymmetrical regions are found, they are silenced. the known molecular components of this meiotic silencing machinery are related to rna-dependent rna polymerases, argonautes and dicers, suggesting that ...201020592262
real-time fluorescence lifetime imaging system with a 32 x 32 0.13microm cmos low dark-count single-photon avalanche diode array.a compact real-time fluorescence lifetime imaging microscopy (flim) system based on an array of low dark count 0.13microm cmos single-photon avalanche diodes (spads) is demonstrated. fast background-insensitive fluorescence lifetime determination is achieved by use of a recently proposed algorithm called 'integration for extraction method' (iem) [j. opt. soc. am. a 25, 1190 (2008)]. here, iem is modified for a wider resolvability range and implemented on the fpga of the new spad array imager. we ...201020588879
lilbid-mass spectrometry of the mitochondrial preprotein translocase tom.in the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (lilbid-ms) to the outer mitochondrial membrane protein translocon tom to analyze its subunit composition and stoichiometry. with tom core complex, purified at high ph, we demonstrate that a tom core complex of neurospora crassa is composed of at least two tom40 and tom22 molecules, respectively, and more than five small tom subunits between 5.5 and 6.4 kda. we show t ...201021339618
linkage of conidial longevity determinant genes in neurospora crassa.the longevity of conidia of neurospora crassa was previously defined as their ability to grow after aging in a constant environment. the heritable median lifespan of the wild type is 22 days. heritable mutants with lifespans of 5-7 days were previously selected. the pleiotropic colony phenotype of the mutants greatly facilitates genetical analysis of their inheritance. twenty-eight mutants were mapped by recombinational analysis. all but one were located at genes on one arm of the seven chromoso ...20106233462
selection of conidial longevity mutants of neurospora crassa.conidial survival was measured after incubation in white light at 30 degrees c and 85-100% relative humidity. the heritable median lifespan (mls) of the wild-type ( age0 ) was 22 days. spontaneous short-lived mutants (age-) with mls about 7 days occurred among both sexual and asexual progeny of wild-type at a frequency of about 10%. radiation of conidia with near ultraviolet light increased the mutation frequency about 5-9-fold above spontaneous. the apparent spontaneous reversion frequency was ...20096233461
[plant extracts with cytostatic properties growing in cuba. i].the cytostatic activity of aqueous, alcoholic and ketonic extracts of 9 species of superior plants of the families fitolacaceae, compositae, moraceae, zingiberaceae, martiniaceae, mirtaceae, verbenaceae and annonaceae was assessed. the kubas microbiologic method and the fungus ascomiceto neurospora crassa were used in the assessment. the fungus growth was measured in millimeters. inhibition percentages for every case regarding control are reported. the best results were obtained from annona muri ...2009161407
continuous selection for increased linear growth rate in neurospora crassa. 20095111033
the relation between growth, conidiation and trehalase activity in neurospora crassa. 20095373654
reversible phosphorylation subserves robust circadian rhythms by creating a switch in inactivating the positive element.reversible phosphorylation of proteins is ubiquitous in circadian systems, but the role it plays in generating rhythmicity is not completely understood. a common mechanism for most circadian rhythms involves a negative feedback loop between the positive and negative elements. here, we built a minimal model for the neurospora crassa circadian clock based on the core negative feedback loop and the protein frequency (frq)-dependent phosphorylation of the white collar complex (wcc). the model can re ...200919948115
a splice variant of the neurospora crassa hex-1 transcript, which encodes the major protein of the woronin body, is modulated by extracellular phosphate and ph changes.the woronin body, a septal pore-associated organelle specific to filamentous ascomycetes, is crucial for preventing cytoplasmic bleeding after hyphal injury. in this study, we show that t1hex-1 transcript and a variant splicing t2hex-1 transcript are up-regulated at alkaline ph. we also show that both hex-1 transcripts are overexpressed in the preg(c), nuc-1(rip), and pacc(ko) mutant strains of neurospora crassa grown under conditions of phosphate shortage at alkaline ph, suggesting that hex-1 t ...200919071122
a high-density single nucleotide polymorphism map for neurospora crassa.we report the discovery and validation of a set of single nucleotide polymorphisms (snps) between the reference neurospora crassa strain oak ridge and the mauriceville strain (fgsc 2555), of sufficient density to allow fine mapping of most loci. sequencing of mauriceville cdnas and alignment to the completed genomic sequence of the oak ridge strain identified 19,087 putative snps. of these, a subset was validated by cleaved amplified polymorphic sequence (caps), a simple and robust pcr-based ass ...200919015548
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