Schematic diagram of the reaction catalyzed by Erg3p in yeast.

<p>Erg3p is responsible for introducing a double bond at the C-5 carbon (denoted by the star) of the B-ring of episterol to produce ergosta- 5,7,24(28)- trienol. This step is the second to last step in the biosynthetic pathway to ergosterol.</p

Deletion of the ERG3 gene in yeast.

<p>(A) Schematic diagram of homologous recombination strategy used to delete <i>ERG3</i> in <i>Sacchromyces cerevisiae</i>. PCR primers were designed to amplify the <i>URA3</i> marker gene containing the flanking regions of <i>ERG3</i>. (B) Ura⁺ isolates were screened by PCR to verify proper integration of <i>URA3</i> and deletion of <i>ERG3</i>. Only correctly integrated isolates would show a 459 base pair product using primers internal to <i>URA3</i> and upstream of the flanking homology. Stars denote the positive isolates selected for further analysis. PCR was also used to verify the downstream end of flanking region homology with <i>URA3</i> (data not shown).</p

Amino acid sequence alignment of Sterol C-5 desaturase in different organisms.

<p><i>Arabidopsis thaliana</i> (AtERG3) NCBI Accession Number CAA62079; <i>Zea mays</i> (ZmERG3) ACG38774; <i>Chlamydomonas reinhardtii</i> (CrERG3) XP_001701457; <i>Homo sapiens</i> (HsERG3) BAA33729; <i>Rattus norvegicus</i> (RnERG3) NP_446094; <i>Sacchromyces cerevisiae</i> (ScERG3) NP_013157. Conserved amino acid sequences shared by all organisms are denoted by a star. The highlighted area corresponds to the putative histidine-containing metal binding domain. Dashed lines indicate gaps in the alignment. The bold, italicized font corresponds to the three putative transmembrane spanning regions of the <i>C. reinhardtii ERG3</i> protein.</p

Yeast strains used in this work.

<p>Yeast strains used in this work.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p