Cloning and disruption of the yeast C‐8 sterol isomerase gene

Abstract

The yeastERG2 gene codes for the C-8 sterol isomerase, an enzyme required for the isomerization of the δ8 double bond to the δ7 position in ergosterol biosynthesis. TheERG2 gene was cloned by complementation of a C-8 sterol isomerase mutant strain (erg2). The complementing region of DNA required to restore ergosterol synthesis toerg2 was limited to a 1.0 kbStuI-BglII fragment. In order to determine whether theERG2 gene was essential for yeast viability, aLEU2 gene was inserted into theNdeI site (made blunt) of this 1.0 kb fragment. Transformation of a wild type diploid strain with theERG2 substituted DNA resulted in the generation of viable haploids containing theerg2 null allele (erg2–4∶∶Leu2). These results suggest that the C-8 sterol isomerase activity is not essential for yeast cell viability. This disruption represents the second ergosterol biosynthetic gene in the distal portion of the pathway to be disrupted without adversely affecting cell viability.