Six of the nine analyzed transformants showed the expected 0.7-kb target Omipalisib mw band, indicating the presence of the egfp gene in the transformants (Fig. 3). Southern hybridization analysis of the transformants 5 and 43 was carried out to analyze the mode of integration of the transforming DNA (Fig. 4). The non transformed mycelium does not show any hybridization. The transformants 5 and 43 showed a different pattern of bands. The transformant 43 showed
single bands in each digestion. For the transformant 5, several bands of various sizes were observed. These results demonstrated that the introduced sequence was integrated ectopically into the chromosomal DNA with one or more copy numbers in these transformants. Transcription of egfp in the transformants 5 and 43 was demonstrated by RT-PCR (Fig. 5). Detection of fluorescence was performed in vivo on 2 days grown transformants mycelia on microscopic slides. In Fig. 6, phase-contrast micrographs of transformants (a) and the corresponding images under UV light (b) LBH589 price are shown. Nontransformed mycelium did not show any fluorescence. Scanned
images show a positive fluorescence emission with respect to untransformed control. Fluorescence emission extended to entire hyphae, especially to clamps connection. Similar phenomenon was also observed when poxc promoter-driven reporter plasmid was used for transformation (to be published elsewhere). The P. ostreatus transformants 1, 5, 2, and 43 were analyzed for intracellular fluorescence emission by measuring emission of fluorescence of intracellular protein extracts from 7-day-old mycelium in comparison with the control (nontransformed mycelium; Fig. 7). The entity of fluorescence emission was measured as difference between spectrum area recorded between 500 and 550 nm for the transformant and that of the control sample (nontransformed fungus). The expression of GFP in each of the transformants has proved stable over a 6-month period of repeated subculturing on selective media (data not shown). Difference in intracellular
fluorescence emission was revealed for different transformants that could be ascribed to the different copy numbers and loci of exogen Cell press DNA integration within the fungal genome. Variation in GFP concentration among independent fungal transformants has been observed by other authors (Chalfie et al., 1994; Cubitt et al., 1995). Comparison of intracellular fluorescence emission by transformants growth in the presence and in the absence of copper sulfate showed that metal addition causes an increase in green fluorescence driven by the poxa1b promoter, up to fourfold (20 000 fluorescence unit per 0.05 mg of proteins). It is worth noting that an induction of transcription from a particular promoter sequence was hereby demonstrated by quantitative measurement of fluorescence emission for the first time in basidiomycetes.