DNA manipulations, and chkA and chkB deletion strains isolation: DNA manipulations were according to SAMBROOK et al. (2001). Isolation of A. nidulans DNA was performed using standard procedures. DNA fragment probes for Southern analysis were labeled with [-32P]dCTP using the RTS Random Primer DNA labeling System kit (Invitrogen, USA). Using Primer Express Version 1.0 (Applied Biosystems) design software, PCR primers were designed for amplifying every DNA fragment necessary for PCR-mediated deletion technique (KUWAYAMA et al. 2002). For the deletion cassette used in chkACHK1 (AN6073.3) inactivation, the A. fumigatuspyrG gene was used as marker. This fragment was amplified from the pCDA21 plasmid (CHAVEROCHE et al. 2000). The A. fumigatus pyroA (Afu5g08090) gene was PCR-amplified from A. fumigatus CEA17 strain genomic DNA and used as genetic marker in the deletion cassette for the chkBCHK2 (AN4279.3) inactivation. The PCR-mediated construction for the chkA and chkB genes consisted of three initial PCRs that amplified a 5'- and 3'-flanking regions of chkA and chkB genes and a final fusion PCR with the marker gene. For the DNA fragments containing the 5’- and 3’-gene flanking regions, genomic DNA from FGSC A4 strain was used as a template. Table 2 shows the primer sequences and fragment sizes for chkA and chkB 5’ and 3’-flanking regions. The 50 µl amplification mixture included 1 X Platinum Taq DNA Polymerase High Fidelity buffer (Invitrogen), 20 pmol of each primer, 0.4 mM deoxynucleotide triphosphate (dNTP) mix, 1.0 U of Taq Platinum DNA polymerase High Fidelity (Invitrogen), and 500 ng of genomic DNA or 200 ng of plasmid pCDA21. PCR amplifications were carried out in a PTC100 96-well thermal cycler (MJ Research), at 94°C for 2 minutes, and 30 times 94°C for 1 minute, 58 to 60°C (depending on the fragment) for 1 minute, and 68°C for 2 to 3 minutes, followed by an extension step at 68 oC for 2 to 5 minutes. The fusion (overlapping PCR) reaction used as templates equal amounts of the three DNA fragments previously amplified for each gene and the outermost primers numbered as -1 and -4 preceded by the gene name in Table 2 for the cassette amplification. After the reaction, the fusion PCR products were gel-purified with PerfectPrep Gel Cleanup (Eppendorf) according to the manufacturer’s instructions. These fragments were independently transformed in the TNO2A3 [(nkuA) strain (NAYAK et al. 2006)] according to standard protocols (OSMANI et al. 1987) using approximately 5-10 µg of DNA deletion cassette fragments. Transformants were scored for their ability to grow on YAG medium and the gene replacement of the chkA and chkB locus was checked by Southern blot analysis. One candidate of each disruption that showed no additional integration event was chosen and crossed to the wild type strain UI224 in order to eliminate the nkuA mutation. Accordingly, all the deletion strains are wild type for this locus.

Construction of an alcA::gfp::atmA strain: In order to create an N-terminal alcA::gfp fusion construct, a 2 kb fragment of atmA, starting from the ATG was amplified from genomic DNA (FGSC A4 strain) with Taq Platinum DNA polymerase High Fidelity (Invitrogen) using primers ATM AscI and ATM2000 PacI and cloned in the AscI-PacI sites of the pMCB17apx vector (EFIMOV 2003) resulting in the plasmid pMCB17apx-ATM2000. This plasmid was used to transform the wild type recipient strain GR5. Transformants were tested for their ability to grow in YAG and homologous recombination of this plasmid into the atmA locus should result in an N-terminal GFP fusion of the entire atmA gene under the control of the alcA promoter plus a truncated 5’ region under the control of the atmA promoter. Twenty-seven transformants were obtained and three of them presented a very consistent atmA deletion phenotype under repression conditions (YAG glucose 4 %) and wild type phenotype under inducing conditions (MM-G + threonine 100 mM). Homologous integration of the construct was further confirmed by PCR analysis using the primers GFP VE FW and ATM2796 (Table 1).

REFERENCES

Chaveroche, M. K., J. M. Ghigo and C. D’Enfert, 2000 A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res. 28: E97-E104.

Kuwayama, H., S.Obara, T.Morio, M.Katoh, H.Urushiharaet al., 2002

PCR-mediated generation of a gene disruption construct without the use

of DNA ligase and plasmid vectors.Nuclei Acids Res.30:e2.

Nayak, T., E. Szewczyk E, C. E. Oakley, A. Osmani, L. Ukil, et al., 2006 A versatile and efficient gene targeting system for Aspergillus nidulans. Genetics 172: 1557-1566.

Osmani, S. A., G. S.May and N. R.Morris, 1987 Regulation of the mRNA

levels of nimA, a gene required for the G2-M transition in Aspergillus

nidulans.J. Cell Biol.104:1495–1504.

Sambrook, J. and D. W. Russell, 2001 Molecular Cloning: A Laboratory Manual. 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.