MATERIALS AND METHODS

A. nidulans strains, media, and transformation. The A. nidulans strains used in this study are shown in Table 1. A. nidulans media (ANM) and growth conditions were as described previously (11). Unless stated otherwise, nitrogen sources were used at a final concentration of 10 mM and the carbon source was 1%(wt/vol) glucose. Genetic analysis was carried out by using techniques described previously (8). Strains of A. nidulans were transformed by the method of Andrianopoulos and Hynes (1). Cotransformants were selected by using the selectable marker plasmid pI4 (PyroA) on media lacking pyridoxine (28, 31). Molecular techniques. Standard methods for the manipulation of E. coli cells and DNA were those described by Sambrook et al.(36). The E. coli strain used in this study was NM522. Restriction enzymes (Promega) were used according to manufacturer recommendations. Blunt-ended restriction fragments were created by end filling with the Klenow fragment of DNA polymerase I (Promega). DNA fragments were recovered from agarose gels by using a BresaClean DNA purification kit (Geneworks). DNA fragments were subcloned into plasmid vector pBluescript SK ()(Stratagene) or pGEM-T (Promega). A. nidulans genomic DNA was isolated by the method of Lee and Taylor (20). Total A. nidulans RNA was obtained by using an RNA Red Fast Prep kit (Bio 101). Southern and Northern gels were transferred to Hybond N membranes (Amersham) by using 0.4 and 0.04 M NaOH, respectively.[-32P] dATP (Bresatec) radioactively labeled DNA probes for hybridization were created by using the random hexanucleotide priming method (36). Automated DNA sequencing was performed at the Australian Genome Research Foundation with plasmid DNA prepared by using a High Pure Plasmid kit (Roche).