F and Cyp61dw2.R. Red thick arrows along with a number represent the nine exons of the CYP61 gene. Plasmids pBS-Cyp61/Hyg and pBS-Cyp61/Zeo were built by inserting the hygromycin B (HygR, in green) and zeocin (ZeoR, in violet) resistance expression cassettes, respectively, at the EcoRV site of plasmid pBS-gCyp61. To linearize the plasmids for transformation purposes, pBS-Cyp61/Hyg and pBS-Cyp61/Zeo were digested with XbaI. Figure 5 PCR-based analysis of cyp61 – mutants. Each gel shows PCR

reactions performed with different sets of primers and genomic DNA from strains UCD 67–385 (lane 1), 385-CYP61/cyp61 hph (lane 2), 385-cyp61 hph /cyp61 zeo (lane 3), CBS 6938 (lane 4), CBS-cyp61 hph (lane 5), AVHN2 (lane 6), Av2-cyp61 zeo (lane 7), and a negative control without Cell Cycle inhibitor DNA (lane 8). The diagram below each gel represents the amplification target (cyp61 – mutant or wild-type allele) and the size of the expected amplicon. The colors represent the resitance cassettes HygR in green and ZeoR in violet, the CYP61 gene in red and the CYP61

flanking DNA in dark grey. The EcoRV recognition site, where the respective antibiotic resistance marker was inserted to disrupt the CYP61 gene, is also shown. Molecular weight standard were: lambda DNA/Hind III (23.1, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.6 kbp) at the left, and 1 kb DNA ladder (10.0, 8.0, 6.0, 5.0, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.75 and 0.5 kbp) at the right, of each gel. CYP61 gene mutant phenotype evaluation: ergosterol and carotenoid production To analyze and compare the cyp61 – mutant phenotypes,

the seven strains UCD 67–385, 385-CYP61/cyp61 hph , 385-cyp61 hph /cyp61 zeo , CBS 6938, click here CBS-cyp61 hph , AVHN2 and Av2-cyp61 zeo were cultivated in YM complete medium for 5 days at 22°C with constant agitation. Growth was measured by the culture absorbance at 600 nm, and samples were taken after 24, 72 and 120 h of cultivation. The samples were processed to determine the yeast dry weight and to extract sterols, carotenoids and RNA as described in the Materials and Methods section. As in other species, the CYP61 gene is involved in the ergosterol biosynthesis, so we evaluated the sterol production and composition in the cyp61 – mutants by RP-HPLC. Figure  6 shows representative chromatograms obtained from sterols extracted from strains UCD 67–385 BCKDHB and 385-cyp61 hph /cyp61 zeo , representing the parental and the cyp61 – mutant strains, respectively. In wild-type strains, we observed a predominant peak (peak 1) at the 280 nm channel at approximately 18 min with the ergosterol characteristic spectra (Figure  6A), and its identity was confirmed by co-injecting each sample with standard ergosterol (Figure  6B). On the other hand, in the analysis of the sterols from the homozygous and hemizygous cyp61 – mutants, two peaks were observed with retention times close to 15 (peak 2) and 21 min (peak 3) (Figure  6C, Table  3).