PCR products of expected sizes of 905 bp for ble and 1281 bp for vgb were obtained from the transformants while in the wild-type sample, these DNA regions were absent (Fig. 2).
For the ade2 assay, the numbers of Ade+ Ura+ transformants were compared to Ade− Ura+ transformants, while for the leu2 assay Leu+ Ura+ transformants were compared to the number of Leu− Ura+ transformants.
Thereby a 2900 bp fragment indicated successful deletion of phlp1 in the respective transformant while the presence of the wild type fragment (990 bp) revealed ectopic or no integration of the deletion cassette (data not shown).
The highest levels (up to 19·2 mg g−1 d. wt) accumulated in a progeny of a cross between a purple ANT1 and a IFS/ CHI transformant, while the second highest concentration was found in a plant derived from a selfed IFS2-12 transformant.
A predicted extra protein band of 46 kDa was observed in the transformants, while no such band was seen in the negative controls.
Numbers from 1 to 9 and 11 to 19 refer to the arbitrarily chosen transformants, while numbers 10 and 20 refer to the wild strain.
Six hours after transfection, 2 mM tetracycline (Sigma, St Louis, MO, USA) was added to the medium of pcDNA5/FRT/TO transformants while 150 μM isopropyl-b-D-1-thiogalactopyranoside (IPTG, Sigma) was added to the medium of pTUNE transformants for vector induction.
Overexpression of the A. niger ortholog of Pex11, which is known to be involved in peroxisomal proliferation [ 21] only resulted in minor peroxisomal proliferation in the A. niger transformants, while peroxicretion was not enhanced (figure 13).
Control transformations of linearized plasmid DNA alone or PCR amplicons alone did not yield a significant number of transformants (< 20) while the 'complete' transformation contained several hundred transformants (data not shown).
Perhaps most obvious among these aberrant features is petiolule length, (petiolule = stalk connecting leaflet to petiole in a compound leaf), which is distinctly elongated in both diploid and tetraploid E08 transformants while inconspicuous in wild type 'Hawaii 4'.
