Inactivation of the NcPho85 Gene Encoding the Pho85p-Like Protein Kinase of Neurospora crassa

In Saccharomyces cerevisiae, PHO85 encodes the cyclin-dependent protein kinase (Cdk) catalytic subunit, with multiple regulatory roles depending on its association with different cyclin partners (Pcls). Deletion of this gene generates a phenotype of hyper-accumulation of glycogen. To investigate the role of the NcPHO85 protein (337 amino acids) in N. crassa glycogen metabolism we have isolated the gene encoding this protein, including its 5´- and 3´-flanking regions, and the gene inactivation has been performed. An approach used to inactivate the gene was by the RIP (Repeat Induced Point Mutation) process, which introduces point mutations throughout the gene, which may lead to the introduction of a stop codon. For that, a genomic copy of the gene was subcloned into pMSN-1 plasmid and used to transform the FGSC 3957 strain. After conidia transformation, clones containing a double copy of the gene, as shown by Southern blot, were selected. Cells of this clone were crossed with a strain of opposite mating-type, and after two rounds of crossing, one segregant was selected. Cells of this strain exhibited mutations into the gene coding sequence, one of them leading to the introduction of a stop codon at the amino acid position 166, resulting in a truncated protein. This strain showed severe morphologic alterations in solid culture medium, which need to be characterized. Even though we isolated a mutant strain, half of the protein is still being synthesized. An approach to construct a null mutant by gene replacement has also been started. For this procedure, the entire gene coding sequence was replaced by the hygromycin resistance gene by PCR. Thus, a fragment, containing the hygromycin gene plus 1,000 bp of the 5´- and 3´-flanking regions of the Ncpho85 gene, was generated using a fusion PCR approach. The resulting fragment, after DNA sequencing, will be used to transform N. crassa conidia and clones able to grow in a medium containing hygromycin will be selected and analyzed for the inactivation of the gene. Supported by FAPESP, CAPES and CNPq