Methylation of the foreign transposon Restless in vegetative mycelia of Neurospora crassa.
Methylation of foreign and/or repeated sequences in the filamentous fungus Neurospora crassa is believed to be directed against invading transposable elements. To test this hypothesis, the fate of a transposon in N. crassa was investigated. Vectors were constructed which carried the transposon Restless, an active class-II element isolated from the fungus Tolypocladium inflatum. These vectors were introduced into N. crassa strains by protoplast transformation. Two strategies were employed: (1) ectopic multi-copy integration, and (2) site-specific single-copy integration at the his-3 locus. All ectopic transformants exhibited strong methylation as confirmed by Southern hybridization of genomic DNA digested with the methylation-sensitive endonuclease Sau3AI and the methylation-insensitive endonuclease NdeII. Single copies of Restless integrated at the his-3 locus were not methylated. These results are discussed with respect to non-RIP methylation and potential consequences for gene-tagging strategies based on the use of Restless.
Windhofer
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F
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Germany.
Catcheside
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Kempken
F
F
D015139:Blotting, Southern; D019175:DNA Methylation; D004251:DNA Transposable Elements / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D004274:DNA, Recombinant; D015252:Deoxyribonucleases, Type II Site-Specific; C024405:GATC-specific type II deoxyribonucleases