Ds-like Restless Deletion Derivatives Occur in Tolypocladium inflatum and Two Foreign Hosts, Neurospora crassa and Penicillium chrysogenum

Windhofer, Frank; Hauck, Katarzyna; Catcheside, David; Kück, Ulrich; Kempken, Frank

doi:10.1006/fgbi.2001.1323

Cited by 20 publications

(15 citation statements)

References 49 publications

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“…Transformation of P. chrysogenum strains was performed using conventional transformation procedures with modifications as described by Windhofer et al (2002). Cultures were inoculated with 2.5-5610 6 spores ml 21 and grown at 27 uC and 120 r.p.m.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

2009

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RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing system that downregulates target gene expression. Here, we provide several lines of evidence for RNA silencing in the industrial β-lactam antibiotic producer Penicillium chrysogenum using the DsRed reporter gene under the control of the constitutive trpC promoter or the inducible xylP promoter. The functional RNAi system was verified by detection of siRNAs that hybridized exclusively with gene-specific 32P-labelled RNA probes. Moreover, when RNAi was used to silence the endogenous PcbrlA morphogene that controls conidiophore development, a dramatic reduction in the formation of conidiospores was observed in 47 % of the corresponding transformants. Evidence that RNAi in P. chrysogenum is dependent on a Dicer peptide was provided with a strain lacking Pcdcl2. In the ΔPcdcl2 background, silencing of the PcbrlA gene was tested. None of the transformants analysed showed a developmental defect. The applicability of the RNAi system in P. chrysogenum was finally demonstrated by silencing the Pcku70 gene to increase homologous recombination frequency. This led to the generation of single and double knockout mutants.

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Section: Methodsmentioning

confidence: 99%

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

2009

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“…As in a, a hybridization experiment with untreated mitochondria (-) is shown as a control. c Untreated ccc DNA (the 11.1-kb plasmid pFW224, containing fungal genomic DNA; Windhofer et al 2002) was introduced into mitochondria by electroporation at 20 kV/cm. As in a, a hybridisation experiment with untreated mitochondria (-) is shown as a control.…”

Section: Transcription and Rna Editing In Isolated Mitochondriamentioning

confidence: 99%

Electroporation of isolated higher-plant mitochondria: transcripts of an introduced cox2 gene, but not an atp6 gene, are edited in organello

Staudinger

Kempken

2003

Mol Gen Genomics

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To facilitate the analysis of RNA processing in plant mitochondria, a method was established for introducing foreign DNA into mitochondria isolated from maize and sorghum. This method permits the uptake of DNA of up to 11 kb into the mitochondrial matrix. In vitro incubation of maize mitochondria in a specific buffer system was found to permit splicing and editing of newly synthesized RNAs for a period of at least 7 h. This was shown both for transcripts of endogenous mitochondrial genes (atp6, cox2) and for transcripts derived from an introduced Arabidopsis thaliana cox2 gene. In contrast, when a Sorghum bicolor atp6 gene was introduced into isolated maize mitochondria, the gene was transcribed, but the RNA was not edited, although all the editing sites in maize and sorghum atp6 RNA are identical. This may indicate the presence of transcript-specific cis -acting regions in the up- or downstream untranslated sequences of the mRNA. The system described here should allow further dissection of the mechanism of RNA editing in plant mitochondria.

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“…Only three reintegration sites were not found in the database, while all others were identified. The very high reintegration frequency of Vader is very promising for development as a mutagenesis tool, as comparable experiments with the Restless transposon did not lead to reintegration after excision (26).…”

Section: Resultsmentioning

confidence: 99%

Suitability of Vader for Transposon-Mediated Mutagenesis in Aspergillus niger

Hihlal¹,

Braumann²,

Berg³

et al. 2011

Appl Environ Microbiol

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The filamentous fungus Aspergillus niger is widely used in biotechnological applications. Strain CBS513.88 is known to harbor 21 copies of the nonautonomous transposon Vader. Upon selection of chlorate-resistant A. niger colonies, one Vader copy was found integrated in the nirA gene. This copy was used for vector construction and development of a transposon-tagging method. Vader showed an excision frequency of about 1 in 2.2 ؋ 10 5 conidiospores. A total of 95 of 97 colonies analyzed exhibited an excision event at the DNA level, and Vader footprints were found. By employing thermal asymmetric interlaced (TAIL)-PCR, the reintegration sites of 21 independent excision events were determined. All reintegration events occurred within or very close to genes. Therefore, this method can be used for transposon mutagenesis in A. niger.

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Ds-like Restless Deletion Derivatives Occur in Tolypocladium inflatum and Two Foreign Hosts, Neurospora crassa and Penicillium chrysogenum

Cited by 20 publications

References 49 publications

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

Electroporation of isolated higher-plant mitochondria: transcripts of an introduced cox2 gene, but not an atp6 gene, are edited in organello

Suitability of Vader for Transposon-Mediated Mutagenesis in Aspergillus niger

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