Expression and Visualization of Red Fluorescent Protein (RFP) in Neurospora crassa

We report the expression of Discosoma red fluorescent protein (RFP) and RFP fusion proteins in Neurospora crassa. RFP was expressed under the control of the Neurospora ccg-1 promoter in transformants with single copies integrated at the his-3 locus by gene targeting. Because this RFP gene, tdimer2(12), contains a 677 bp direct tandem repeat of dsRed, RFP constructs underwent RIP at high frequency in rid+ strains. Fusion proteins of RFP to the amino terminus of Neurospora heterochromatin protein 1 (HP1) were localized to heterochromatic foci in Neurospora nuclei, consistent with prior findings with carboxy-terminal HP1-GFP fusion proteins

Expression and visualization of Green Fluorescent Protein (GFP) in Neurospora crassa

We report the first successful imaging of GFP expression in Neurospora crassa. GFP was expressed under the control of the heterologous ToxA promoter from Pyrenophora tritici-repentis in transformants carrying multiple or single copies of the GFP construct. GFP was also detected in ascospores but not during earlier stages of the sexual cycle

Different frequencies of RIP among early vs. late ascospores of Neurospora crassa

We have noticed that the frequency of RIP can be quite variable, even in crosses of the same strains. One possible source of variability is the time at which ascospores are harvested. We reasoned that the earliest ascospores shot from a perithecium might contain DNA that went through relatively few mitotic divisions in pre-meiosis. RIP occurs between fertilization and premeiotic DNA synthesis (Selker et al. 1987 Cell 51:741-752). Thus, early spores might have less exposure to RIP than late spores. Since all ascospores from a perithecium are thought to arise from a single fertilization event, a minimum of 7- 10 divisions are required to account for the number of ascospores normally produced (Perkins and Barry, 1977 Adv. Genet. 211:541-544). It is likely, however, that some ascospore lineages contain fewer divisions than others

Reversal of a Neurospora Translocation by Crossing over Involving Displaced Rdna, and Methylation of the Rdna Segments That Result from Recombination

In translocation OY321 of Neurospora crassa, the nucleolus organizer is divided into two segments, a proximal portion located interstitially in one interchange chromosome, and a distal portion now located terminally on another chromosome, linkage group I. In crosses of Translocation x Translocation, exceptional progeny are recovered nonselectively in which the chromosome sequence has apparently reverted to Normal. Genetic, cytological, and molecular evidence indicates that reversion is the result of meiotic crossing over between homologous displaced rDNA repeats. Marker linkages are wild type in these exceptional progeny. They differ from wild type, however, in retaining an interstitial block of rRNA genes which can be demonstrated cytologically by the presence of a second, small interstitial nucleolus and genetically by linkage of an rDNA restriction site polymorphism to the mating-type locus in linkage group I. The interstitial rDNA is more highly methylated than the terminal rDNA. The mechanism by which methylation enzymes distinguish between interstitial rDNA and terminal rDNA is unknown. Some hypotheses are considered

The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9

<p>Abstract</p> <p>Background</p> <p>Silencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in <it>Neurospora crassa</it>, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized.</p> <p>Results</p> <p>The selectable marker, <it>hph</it>, was inserted at the subtelomere of Linkage Group VR in an <it>nst-1 </it>(n<it>eurospora </it>s<it>ir </it>t<it>wo</it>-1) mutant and was silenced when <it>nst-1 </it>function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, <it>bar</it>, tested at two other subtelomeres, was similarly sensitive to <it>nst-1 </it>function. Mutation of three additional SIR2 homologues, <it>nst-2</it>, <it>nst-3 </it>and <it>nst-5</it>, partially relieved silencing. Two genes showed stronger effects: <it>dim-5</it>, which encodes a histone H3 K9 methyltransferase and <it>hpo</it>, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased.</p> <p>Conclusion</p> <p>We demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in <it>de novo </it>DNA methylation.</p

The common ancestral core of vertebrate and fungal telomerase RNAs

Telomerase is a ribonucleoprotein with an intrinsic telomerase RNA (TER) component. Within yeasts, TER is remarkably large and presents little similarity in secondary structure to vertebrate or ciliate TERs. To better understand the evolution of fungal telomerase, we identified 74 TERs from Pezizomycotina and Taphrinomycotina subphyla, sister clades to budding yeasts. We initially identified TER from Neurospora crassa using a novel deep-sequencing-based approach, and homologous TER sequences from available fungal genome databases by computational searches. Remarkably, TERs from these non-yeast fungi have many attributes in common with vertebrate TERs. Comparative phylogenetic analysis of highly conserved regions within Pezizomycotina TERs revealed two core domains nearly identical in secondary structure to the pseudoknot and CR4/5 within vertebrate TERs. We then analyzed N. crassa and Schizosaccharomyces pombe telomerase reconstituted in vitro, and showed that the two RNA core domains in both systems can reconstitute activity in trans as two separate RNA fragments. Furthermore, the primer-extension pulse-chase analysis affirmed that the reconstituted N. crassa telomerase synthesizes TTAGGG repeats with high processivity, a common attribute of vertebrate telomerase. Overall, this study reveals the common ancestral cores of vertebrate and fungal TERs, and provides insights into the molecular evolution of fungal TER structure and function

A Cytosine Methyltransferase Homologue Is Essential for Sexual Development in Aspergillus nidulans

Background: The genome defense processes RIP (repeat-induced point mutation) in the filamentous fungus Neurospora crassa, and MIP (methylation induced premeiotically) in the fungus Ascobolus immersus depend on proteins with DNA methyltransferase (DMT) domains. Nevertheless, these proteins, RID and Masc1, respectively, have not been demonstrated to have DMT activity. We discovered a close homologue in Aspergillus nidulans, a fungus thought to have no methylation and no genome defense system comparable to RIP or MIP. Principal Findings: We report the cloning and characterization of the DNA methyltransferase homologue A (dmtA) gene from Aspergillus nidulans. We found that the dmtA locus encodes both a sense (dmtA) and an anti-sense transcript (tmdA). Both transcripts are expressed in vegetative, conidial and sexual tissues. We determined that dmtA, but not tmdA, is required for early sexual development and formation of viable ascospores. We also tested if DNA methylation accumulated in any of the dmtA/tmdA mutants we constructed, and found that in both asexual and sexual tissues, these mutants, just like wild-type strains, appear devoid of DNA methylation. Conclusions/Significance: Our results demonstrate that a DMT homologue closely related to proteins implicated in RIP and MIP has an essential developmental function in a fungus that appears to lack both DNA methylation and RIP or MIP. It remains formally possible that DmtA is a bona fide DMT, responsible for trace, undetected DNA methylation that i

Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for a simple, inexpensive platform that allows high-density SNP discovery and genotyping in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, and mapped three traits in two model organisms, using less than half the capacity of one Illumina sequencing run. We demonstrated that different marker densities can be attained by choice of restriction enzyme. Furthermore, we developed a barcoding system for sample multiplexing and fine mapped the genetic basis of lateral plate armor loss in threespine stickleback by identifying recombinant breakpoints in F2 individuals. Barcoding also facilitated mapping of a second trait, a reduction of pelvic structure, by in silico re-sorting of individuals. To further demonstrate the ease of the RAD sequencing approach we identified polymorphic markers and mapped an induced mutation in Neurospora crassa. Sequencing of RAD markers is an integrated platform for SNP discovery and genotyping. This approach should be widely applicable to genetic mapping in a variety of organisms

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, CUL4, and DDB1. Components of DCDC are required for H3K9me3, proper chromosome segregation, and DNA methylation. DCDC-defective strains, but not HP1-defective strains, are hypersensitive to MMS, revealing an HP1-independent function of H3K9 methylation. In addition to DDB1, DIM-7, and the WD40 domain protein DIM-9, other presumptive DCAFs (DDB1/CUL4 associated factors) co-purified with CUL4, suggesting that CUL4/DDB1 forms multiple complexes with distinct functions. This conclusion was supported by results of drug sensitivity tests. CUL4, DDB1, and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains, indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3. DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9. These data support a two-step mechanism for H3K9 methylation in Neurospora