Three standard approaches to investigate gene function are 1) inactivate the gene (e.g. gene knockouts) and determine the effect on the organism; 2) determine the cellular and subcellular location of the protein; and 3) determine with which other proteins the protein of interest physically interacts. The key to all three approaches is the ability to replace the endogenous gene with altered forms created in vitro (gene replacement). Although gene replacements are the “industry standard”, they have been laborious and time consuming in all eukaryotes except budding yeast (Saccharomyces cerevisiae). Recent technological advances have made gene replacements in several fungi as facile as with yeast. Here we apply this technology to initiate the investigation of six Aspergillus nidulans genes implicated in cell cycle regulation. Five are hypothesized to function with the Anaphase Promoting Complex/ Cyclosome (aka APC/C), which is an ubiquitin ligase that regulates multiple cell cycle events. Two, afrA and afrB, are implicated as cell cycle-stage-specific activators of the APC/C. Three, ubc3, ubc4, and ubc11, are implicated as ubiquitin conjugating enzyme required for APC/C function. The last, sv9, was originally identified as required for nuclear division but has since been implicated in lipid metabolism. We report the successful deletion (knock-out) of all six genes, three of which are essential (afrA, ubc4, and sv9). We also report the isolation of A. nidulans strains that probably contain epitope-tagged versions of the genes for use in future cytological localization and protein-interaction studies.

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