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. fumigatus pyrG 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. URUSHIHARA et 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.