Shannon entropy as a metric for conditional gene expression in Neurospora crassa.
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ABSTRACT:
SUBMITTER: Ameri AJ
PROVIDER: S-EPMC8049430 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The impact of loci that determine sexual identity upon the asexual, dominant stage of fungal life history has been well studied. To investigate their impact, expression differences between strains of different mating type during asexual development were assayed, with RNA sampled from otherwise largely isogenic mat A and mat a strains of Neurospora crassa at early, middle, and late clonal stages of development. We observed significant differences in overall gene expression between mating types across clonal development, especially at late development stages. The expression levels of mating-type genes and pheromone genes were assayed by reverse transcription and quantitative PCR, revealing expression of pheromone and receptor genes in strains of both mating types in all development stages, and revealing that mating type (mat) genes were increasingly expressed over the course of asexual development. Interestingly, among differentially expressed genes, the mat A genotype more frequently exhibited a higher expression level than mat a, and demonstrated greater transcriptional regulatory dynamism. Significant up-regulation of expression was observed for many late light-responsive genes at late asexual development stages. Further investigation of the impact of light and the roles of light response genes in asexual development of both mating types are warranted.
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Project description:Many fungi form complex three-dimensional fruiting bodies, within which the meiotic machinery for sexual spore production has been considered to be largely conserved over evolutionary time. Indeed, much of what we know about meiosis in plant and animal taxa has been deeply informed by studies of meiosis in Saccharomyces and Neurospora. Nevertheless, the genetic basis of fruiting body development and its regulation in relation to meiosis in fungi is barely known, even within the best studied multicellular fungal model Neurospora crassa. We characterized morphological development and genome-wide transcriptomics in the closely related species Neurospora crassa, Neurospora tetrasperma, and Neurospora discreta, across eight stages of sexual development. Despite diverse life histories within the genus, all three species produce vase-shaped perithecia. Transcriptome sequencing provided gene expression levels of orthologous genes among all three species. Expression of key meiosis genes and sporulation genes corresponded to known phenotypic and developmental differences among these Neurospora species during sexual development. We assembled a list of genes putatively relevant to the recent evolution of fruiting body development by sorting genes whose relative expression across developmental stages increased more in N. crassa relative to the other species. Then, in N. crassa, we characterized the phenotypes of fruiting bodies arising from crosses of homozygous knockout strains of the top genes. Eight N. crassa genes were found to be critical for the successful formation of perithecia. The absence of these genes in these crosses resulted in either no perithecium formation or in arrested development at an early stage. Our results provide insight into the genetic basis of Neurospora sexual reproduction, which is also of great importance with regard to other multicellular ascomycetes, including perithecium-forming pathogens, such as Claviceps purpurea, Ophiostoma ulmi, and Glomerella graminicola.
| S-EPMC4203796 | biostudies-literature
Project description:Precise control of gene expression is a powerful method to elucidate biological function, and protein overexpression is an important tool for industry and biochemistry. Expression of the Neurospora crassa tcu-1 gene (NCU00830), encoding a high-affinity copper transporter, is tightly controlled by copper availability. Excess copper represses, and copper depletion, via the use of a copper chelator, activates expression. The kinetics of induction and repression of tcu-1 are rapid, and the effects are long lived. We constructed a plasmid carrying the bar gene (for glufosinate selection) fused to the tcu-1 promoter. This plasmid permits the generation of DNA fragments that can direct integration of Ptcu-1 into any desired locus. We use this strategy to integrate Ptcu-1 in front of wc-1, a circadian oscillator and photoreceptor gene. The addition of excess copper to the Ptcu-1::wc-1 strain phenocopies a ?wc-1 strain, and the addition of the copper chelator, bathocuproinedisulfonic acid, phenocopies a wc-1 overexpression strain. To test whether copper repression can recapitulate the loss of viability that an essential gene knockout causes, we placed Ptcu-1 upstream of the essential gene, hpt-1. The addition of excess copper drastically reduced the growth rate as expected. Thus, this strategy will be useful to probe the biological function of any N. crassa gene through controlled expression.
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Project description:Nonself recognition is exemplified in the fungal kingdom by the regulation of cell fusion events between genetically different individuals (heterokaryosis). The het-6 locus is one of approximately 10 loci that control heterokaryon incompatibility during vegetative growth of N. crassa. Previously, it was found that het-6-associated incompatibility in Oak Ridge (OR) strains involves two contiguous genes, het-6 and un-24. The OR allele of either gene causes "strong" incompatibility (cell death) when transformed into Panama (PA)-background strains. Several remarkable features of the locus include the nature of these incompatibility genes (het-6 is a member of a repetitive gene family and un-24 also encodes the large subunit of ribonucleotide reductase) and the observation that un-24 and het-6 are in severe linkage disequilibrium. Here, we identify "weak" (slow, aberrant growth) incompatibility activities by un-24PA and het-6PA when transformed separately into OR strains, whereas together they exhibit an additive, strong effect. We synthesized strains with the new allelic combinations un-24PA het-6OR and un-24OR het-6PA, which are not found in nature. These strains grow normally and have distinct nonself recognition capabilities but may have reduced fitness. Comparing the Oak Ridge and Panama het-6 regions revealed a paracentric inversion, the architecture of which provides insights into the evolution of the un-24-het-6 gene complex.
| S-EPMC1569700 | biostudies-literature
Project description:We show that Neurospora crassa has a single histone H1 gene, hH1, which encodes a typical linker histone with highly basic N- and C-terminal tails and a central globular domain. A green fluorescent protein-tagged histone H1 chimeric protein was localized exclusively to nuclei. Mutation of hH1 by repeat-induced point mutation (RIP) did not result in detectable defects in morphology, DNA methylation, mutagen sensitivity, DNA repair, fertility, RIP, chromosome pairing, or chromosome segregation. Nevertheless, hH1 mutants had mycelial elongation rates that were lower than normal on all tested carbon sources. This slow linear growth phenotype, however, was less evident on medium containing ethanol. The pyruvate decarboxylase gene, cfp, was abnormally derepressed in hH1 mutants on ethanol-containing medium. This derepression was also found when an ectopically integrated fusion of the cfp gene promoter to the reporter gene hph was analyzed. Thus, Neurospora histone H1 is required for the proper regulation of cfp, a gene with a key role in the respiratory-fermentative pathway.
| S-EPMC154839 | biostudies-literature
Project description:Filamentous fungi are important model systems for understanding eukaryotic cellular processes, including the study of protein expression. A salient feature of fungi is the ability of the protein-processing machinery to perform all of the extensive posttranslational modifications needed in the complex world of eukaryotic organisms, making them great hosts for production of eukaryotic proteins. In the model organism Neurospora crassa, several regulatable promoters have been used for heterologous gene expression but all suffer from leaky expression absent stimuli or an inability to induce protein expression at levels greater than those seen in vivo. To increase and better control in vivo protein expression in Neurospora, we have harnessed the light-induced vvd promoter. vvd promoter-driven mRNA expression is dependent upon light, shows a graded response, and is rapidly shut off when returned to the dark. The vvd promoter is a highly tunable and regulatable system, which could be a useful instrument for those interested in efficient and controllable gene expression.
| S-EPMC3464113 | biostudies-literature
Project description:An ensemble of genetic networks that describe how the model fungal system, Neurospora crassa, utilizes quinic acid (QA) as a sole carbon source has been identified previously. A genetic network for QA metabolism involves the genes, qa-1F and qa-1S, that encode a transcriptional activator and repressor, respectively and structural genes, qa-2, qa-3, qa-4, qa-x, and qa-y. By a series of 4 separate and independent, model-guided, microarray experiments a total of 50 genes are identified as QA-responsive and hypothesized to be under QA-1F control and/or the control of a second QA-responsive transcription factor (NCU03643) both in the fungal binuclear Zn(II)2Cys6 cluster family. QA-1F regulation is not sufficient to explain the quantitative variation in expression profiles of the 50 QA-responsive genes. QA-responsive genes include genes with products in 8 mutually connected metabolic pathways with 7 of them one step removed from the tricarboxylic (TCA) Cycle and with 7 of them one step removed from glycolysis: (1) starch and sucrose metabolism; (2) glycolysis/glucanogenesis; (3) TCA Cycle; (4) butanoate metabolism; (5) pyruvate metabolism; (6) aromatic amino acid and QA metabolism; (7) valine, leucine, and isoleucine degradation; and (8) transport of sugars and amino acids. Gene products both in aromatic amino acid and QA metabolism and transport show an immediate response to shift to QA, while genes with products in the remaining 7 metabolic modules generally show a delayed response to shift to QA. The additional QA-responsive cutinase transcription factor-1β (NCU03643) is found to have a delayed response to shift to QA. The series of microarray experiments are used to expand the previously identified genetic network describing the qa gene cluster to include all 50 QA-responsive genes including the second transcription factor (NCU03643). These studies illustrate new methodologies from systems biology to guide model-driven discoveries about a core metabolic network involving carbon and amino acid metabolism in N. crassa.
| S-EPMC3114802 | biostudies-literature
Project description:Neurospora crassa has a long history as an excellent model for genetic, cellular, and biochemical research. Although this fungus is known as a saprotroph, it normally appears on burned vegetations or trees after forest fires. However, due to a lack of experimental evidence, the nature of its association with living plants remains enigmatic. Here we report that Scots pine (Pinus sylvestris) is a host plant for N. crassa. The endophytic lifestyle of N. crassa was found in its interaction with Scots pine. Moreover, the fungus can switch to a pathogenic state when its balanced interaction with the host is disrupted. Our data reveal previously unknown lifestyles of N. crassa, which are likely controlled by both environmental and host factors. Switching among the endophytic, pathogenic, and saprotrophic lifestyles confers upon fungi phenotypic plasticity in adapting to changing environments and drives the evolution of fungi and associated plants.
| S-EPMC4038807 | biostudies-literature
Project description:The filamentous fungus Neurospora crassa is a long-studied eukaryotic microbial system amenable to heterologous expression of native and foreign proteins. However, relatively few highly tunable promoters have been developed for this species. In this study, we compare the tcu-1 and nit-6 promoters for controlled expression of a GFP reporter gene in N. crassa. Although the copper-regulated tcu-1 has been previously characterized, this is the first investigation exploring nitrogen-controlled nit-6 for expression of heterologous genes in N. crassa. We determined that fragments corresponding to 1.5-kb fragments upstream of the tcu-1 and nit-6 open reading frames are needed for optimal repression and expression of GFP mRNA and protein. nit-6 was repressed using concentrations of glutamine from 2 to 20 mM and induced in medium containing 0.5-20 mM nitrate as the nitrogen source. Highest levels of expression were achieved within 3 hr of induction for each promoter and GFP mRNA could not be detected within 1 hr after transfer to repressing conditions using the nit-6 promoter. We also performed metabolic profiling experiments using proton NMR to identify changes in metabolite levels under inducing and repressing conditions for each promoter. The results demonstrate that conditions used to regulate tcu-1 do not significantly change the primary metabolome and that the differences between inducing and repressing conditions for nit-6 can be accounted for by growth under nitrate or glutamine as a nitrogen source. Our findings demonstrate that nit-6 is a tunable promoter that joins tcu-1 as a choice for regulation of gene expression in N. crassa.
| S-EPMC4555226 | biostudies-literature
Project description:BackgroundSingle-cell RNA sequencing (scRNA-seq) is the most widely used technique to obtain gene expression profiles from complex tissues. Cell subsets and developmental states are often identified via differential gene expression patterns. Most of the single-cell tools utilized highly variable genes to annotate cell subsets and states. However, we have discovered that a group of genes, which sensitively respond to environmental stimuli with high coefficients of variation (CV), might impose overwhelming influences on the cell type annotation.ResultIn this research, we developed a method, based on the CV-rank and Shannon entropy, to identify these noise genes, and termed them as "sensitive genes". To validate the reliability of our methods, we applied our tools in 11 single-cell data sets from different human tissues. The results showed that most of the sensitive genes were enriched pathways related to cellular stress response. Furthermore, we noticed that the unsupervised result was closer to the ground-truth cell labels, after removing the sensitive genes detected by our tools.ConclusionOur study revealed the prevalence of stochastic gene expression patterns in most types of cells, compared the differences among cell marker genes, housekeeping genes (HK genes), and sensitive genes, demonstrated the similarities of functions of sensitive genes in various scRNA-seq data sets, and improved the results of unsupervised clustering towards the ground-truth labels. We hope our method would provide new insights into the reduction of data noise in scRNA-seq data analysis and contribute to the development of better scRNA-seq unsupervised clustering algorithms in the future.
| S-EPMC8063398 | biostudies-literature