Rescued

plasmids were sequenced and analysed by restriction digestion to define the site of insertion in the genome. Insertion sites are shown in the cartoon in Fig. 2. In REMI-11, the insertion had occurred at a XhoI site 1820 bp downstream from the start codon of an ABC transporter family gene (AFUA_1G14330) and within the coding region. REMI-56 is an insertion upstream of a major facilitator superfamily (MFS) transporter. The insertional mutation has occurred in the 619 bp intergenic region between two tandemly transcribed genes, 203 bp downstream of a putative sulphate transporter (AFUA_1G05020) BMS-777607 in vitro and 416 bp upstream of a putative MFS transporter (AFUA_1G05010). The REMI insertion is close to the start codon of this ORF and to motifs associated with the core promoter in filamentous fungal genes.

REMI-14D is an insertion in the coding region of triose phosphate isomerase. Sequence analysis of rescued plasmid shows that REMI-14D contains an insertion within the coding region of AFUA_2G11020, the single-copy Angiogenesis inhibitor triose phosphate isomerase gene. The insertion is within the coding region, 452 bp from the start codon. REMI-85 and REMI-103 occur within the coding regions of two hypothetical genes (AFUA_5G07550 and AFUA_4G10880, respectively). These genes have no known function or homology with any gene of known function. AFUA_5G07550 is conserved in all Aspergillus species but poorly conserved in other fungi. The GNAT2 moderately azole-resistant strain REMI-101 has an insertion within the gene coding for the mitochondrial 29.9 KD ubiquinone NADH oxidoreductase subunit of respiratory complex I (AFUA_2G10600). The insertion was shown to occur at a genomic XhoI site, 534 bp downstream from the start codon of the gene. MICs for this strain were 1.0, 0.50 and 4.0 μg mL−1 for ITR, POS and RAV, respectively, versus 0.25, 0.12 and 1.0 μg mL−1 for AF210. REMI-102 was determined to contain an insertion in the promoter

region of the A. fumigatus cyc8 gene orthologue (AFUA_2G11840). The final REMI strain (REMI-116) was shown to have an insertion in the epsin 2 gene (AFUA_6G12570). However, Southern blot analysis suggests that there are at least two insertional events in this strain, and attempts to re-create the phenotype were unsuccessful. Therefore, it seems likely that the insertion in the epsin2 gene and the observed phenotype may be unlinked. As uncharacterised secondary mutations may arise during transformation or REMI, rescued plasmids were used to re-create the azole-resistant phenotype in the parental strain. Because the rescued plasmids contain flanking regions of the original insertion site, this procedure is functionally analogous to commonly used allele replacement methods and recombination between flanking regions and genomic DNA should regenerate the original REMI insertion in a new wild-type or parental strain (Fig. S1). Plasmids were termed p11, p85 etc.