Targeted transformation in Coprinus cinereus.
We examined the influence of DNA form and size on the arrangement and genomic location of transforming DNA sequences in the basidiomycete Coprinus cinereus. Protoplasts with either single or double mutations in the tryptophan synthetase (TRP1) gene were transformed with cloned copies of this gene which contained only a single DNA strand, contained a specific single nick within the C. cinereus sequences (4.8 kb), contained a specific double-strand break, or contained an additional 35 kb of flanking genomic sequences. Gene replacement events were recovered when each DNA type was used. However, none of these substrates offers a substantial improvement in transformation or targeting frequency when compared to supercoiled circular DNA, which has allowed recovery of both gene replacements as well as homologous insertions in 5% of the transformants analyzed. The frequency of transformants carrying tandem insertions with multiple copies of the transforming DNA was reduced when single-stranded DNA was used, and increased when DNA containing double-strand breaks was used. These results have important implications for the efficient design of targeted transformation and co-transformation experiments.
Binninger
D M
DM
Department of Biology and Curriculum in Genetics, University of North Carolina, Chapel Hill 27599-3280.
Le Chevanton
L
L
Skrzynia
C
C
Shubkin
C D
CD
Pukkila
P J
PJ
D015139:Blotting, Southern; D003001:Cloning, Molecular; D003302:Coprinus / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D009690:Nucleic Acid Conformation; D011523:Protoplasts / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic; D014367:Tryptophan Synthase / Q000235:genetics
D004270:DNA, Circular; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D014367:Tryptophan Synthase