Protocol for the Electroporation of Neurospora Spheroplasts

A protocol for electroporating Neurospora strains is described. This protocol uses spheroplasts for transformation rather than germinated conidia with enzyme-weakened walls. High efficiency transformation is obtained with this system even with batches of Novozyme234 which worked poorly in the PEG based protocol

Use of the Neurospora tyrosinase gene as a reporter gene in transformation experiments

Reporter gene systems have been developed to allow investigators to visually identify transformed cells and to follow the transcriptional activity from promoter regions preceding the reporter genes. These reporter genes encode protein products which can be easily assayed and which can be strained for in the transformed cell

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

Endosomal Wnt signaling proteins control microtubule nucleation in dendrites.

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points

The genome sequence of the filamentous fungus Neurospora crassa

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