Strains carrying null alleles, GFP or cherry-mRFP C-terminally tagged fusions of nuclear transporters, and nuclear and nucleolar markers were generated as described (Yang et al., 2004 (link); Supplemental Figure S2), using Aspergillus fumigatus pyrG, riboB, or pyroA gene as a prototrophic selection marker. To construct an N-terminal GFP-tagged KapJKap123, a null kapJKap123::pyrGAf strain was transformed with a 5′UTR::GFP::KapJORF::3′UTR DNA cassette, and the replacement of the null locus was selected by growing the transformants in regeneration plates containing 2 mg/ml 5-fluoro-orotic acid (5-FOA; Apollo Scientific, Stockport, United Kingdom). After 24 h of incubation at 37°C, a layer of Aspergillus minimal medium containing the required nutrients and supplements plus 2 mg/ml FOA was poured onto the protoplast-regeneration plates. Colonies that grew through this layer of medium were purified to homokaryosis and analyzed by Southern blot to verify the correct integration of the cassette at the null kapJKap123 locus. Strains coexpressing tagged karyopherins and histone H1 (HhoA::mCh) or fibrillarin (An-Fib::mCh) were obtained by step-by-step transformation of MAD1425 with each fusion PCR cassette. Amplification of HhoA and An-Fib tagging cassettes follow standard procedures as described, but the selectable marker in this case was A. fumigatus pyroA gene. Strains MAD2653 (kapLMsn5::gfp; hhoA::mCh) and MAD2654 (kapIPse1::gfp; hhoA::mCh) were obtained by transformation of MAD2446 with the karyopherin-tagging PCR cassette. Strains MAD3772 (kapHKap120::gfp; nls::Dsred) and MAD3771 (kapIPse1::gfp; nls::Dsred) were obtained by transformation of AY02 with the karyopherin-tagging PCR cassette. Strain MAD3817 is a diploid obtained from MAD3773 and MAD3604 haploid strains.
Oligonucleotides used in this study are summarized in Supplementary Table S2. Homologous recombination for each construct was confirmed by both diagnostic PCR and Southern blot analysis.
The expression of tagged proteins was analyzed by Western blotting using standard procedures. Briefly, A. nidulans strains were cultivated for 18 h in fermentation medium (Orejas et al., 1995 (link)), filtered through Miracloth (Calbiochem, La Jolla, CA), squeezed to dry, frozen in dry ice, and lyophilized for 16 h. Protein extraction was carried out as previously described (Araújo-Bazán et al., 2008 (link)), and 50 μg were loaded on an 8% polyacrylamide gel before electrotransfer to nitrocellulose filters. To detect GFP fusions, filters were incubated with anti-GFP mouse monoclonal antibody cocktail (1/5000; Roche, Indianapolis, IN). Actin, used as loading and extract quality control, was detected using mouse anti-actin antibody (1/50,000; ICN Biomedical, Irvine, CA). Peroxidase-conjugated anti-mouse (1/4000; Jackson ImmunoResearch Laboratories, West Grove, PA) was used as secondary antibody.
To characterize respective transporter coding sequence (CDS), cDNA was generated from total RNA extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and following the manufacturer's instructions. Total RNA was isolated from mycelia of a wild-type strain cultured under standard conditions. A single-strand cDNA library was made using the First-Strand cDNA Kit (Roche) and an oligo-dT primer. cDNA for each nuclear transporter was amplified using gene-specific oligonucleotides, and the CDS was confirmed by sequencing.
Oligonucleotides used in this study are summarized in Supplementary Table S2. Homologous recombination for each construct was confirmed by both diagnostic PCR and Southern blot analysis.
The expression of tagged proteins was analyzed by Western blotting using standard procedures. Briefly, A. nidulans strains were cultivated for 18 h in fermentation medium (Orejas et al., 1995 (link)), filtered through Miracloth (Calbiochem, La Jolla, CA), squeezed to dry, frozen in dry ice, and lyophilized for 16 h. Protein extraction was carried out as previously described (Araújo-Bazán et al., 2008 (link)), and 50 μg were loaded on an 8% polyacrylamide gel before electrotransfer to nitrocellulose filters. To detect GFP fusions, filters were incubated with anti-GFP mouse monoclonal antibody cocktail (1/5000; Roche, Indianapolis, IN). Actin, used as loading and extract quality control, was detected using mouse anti-actin antibody (1/50,000; ICN Biomedical, Irvine, CA). Peroxidase-conjugated anti-mouse (1/4000; Jackson ImmunoResearch Laboratories, West Grove, PA) was used as secondary antibody.
To characterize respective transporter coding sequence (CDS), cDNA was generated from total RNA extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) and following the manufacturer's instructions. Total RNA was isolated from mycelia of a wild-type strain cultured under standard conditions. A single-strand cDNA library was made using the First-Strand cDNA Kit (Roche) and an oligo-dT primer. cDNA for each nuclear transporter was amplified using gene-specific oligonucleotides, and the CDS was confirmed by sequencing.
