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

Abstract

The yeast ERG2 gene codes for the C-8 sterol isomerase, an enzyme required for the isomerization of the delta 8 double bond to the delta 7 position in ergosterol biosynthesis. The ERG2 gene was cloned by complementation of a C-8 sterol isomerase mutant strain (erg2). The complementing region of DNA required to restore ergosterol synthesis to erg2 was limited to a 1.0 kb StuI-BglII fragment. In order to determine whether the ERG2 gene was essential for yeast viability, a LEU2 gene was inserted into the NdeI site (made blunt) of this 1.0 kb fragment. Transformation of a wild type diploid strain with the ERG2 substituted DNA resulted in the generation of viable haploids containing the erg2 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.