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Related Experiment Videos

Targeted transformation in Coprinus cinereus.

D M Binninger1, L Le Chevanton, C Skrzynia

  • 1Department of Biology and Curriculum in Genetics, University of North Carolina, Chapel Hill 27599-3280.

Molecular & General Genetics : MGG
|June 1, 1991
PubMed
Summary

Investigating DNA in Coprinus cinereus revealed that while gene replacement occurred with various DNA forms, supercoiled DNA was most effective for transformation and targeting. DNA breaks influenced tandem insertion frequencies.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Mycology

Background:

  • Understanding DNA integration is crucial for genetic manipulation in fungi.
  • The basidiomycete Coprinus cinereus is a model organism for fungal genetics.

Purpose of the Study:

  • To investigate how DNA form and size affect the integration and genomic location of transforming DNA in Coprinus cinereus.
  • To compare the efficiency of different DNA substrates for targeted gene replacement and insertion.

Main Methods:

  • Transformation of Coprinus cinereus protoplasts with various forms of the tryptophan synthetase (TRP1) gene.
  • DNA substrates included single-stranded DNA, gapped circular DNA, linear DNA with double-strand breaks, and DNA with flanking sequences.
  • Comparison with supercoiled circular DNA as a control.

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Main Results:

  • Gene replacement events were recovered with all tested DNA types.
  • Supercoiled circular DNA showed comparable or superior transformation and targeting frequencies.
  • Single-stranded DNA reduced tandem insertion frequency, while double-strand breaks increased it.

Conclusions:

  • DNA form and size influence the outcome of transformation in Coprinus cinereus.
  • Supercoiled circular DNA is an efficient substrate for both gene replacement and homologous insertion.
  • These findings aid in optimizing targeted transformation strategies in fungi.