Abstract
The target site of the antifungal compound LY214352 [8-chloro-4-(2-chloro-4-fluorophenoxy) quinoline] has been identified through a dual biochemical and molecular-genetics approach. In the molecular-genetics approach, a cosmid library was prepared from an Aspergillus nidulans mutant that was resistant to LY214352 because of a dominant mutation in a single gene. A single cosmid (6A6-6) that could transform an LY214352-sensitive strain of A. nidulans to LY214352-resistance was isolated from the library by sib-selection. Restriction fragments from cosmid 6A6-6 containing the functional resistance gene were identified by transformation, and sequenced. The LY214352-resistance gene coded for a protein of 520 amino acids that had a 34% identity and a 57% similarity in a 333 amino-acid overlap to E. coli dihydroorotate dehydrogenase (DHO-DH). The results of a series of biochemical mechanism-of-action studies initiated simultaneously with molecular-genetic experiments also suggested that DHO-DH was the target of LY214352. Assays measuring the inhibition of DHO-DH activity by LY214352 in a wild-type strain (I50=40 ng/ml) and a highly resistant mutant (I50>100 μg/ml) conclusively demonstrated that DHO-DH is the target site of LY214352 in A. nidulans. Several mutations in the DHO-DH (pyrE) gene that resulted in resistance to LY214352 were identified.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Gustafson, G., Davis, G., Waldron, C. et al. Identification of a new antifungal target site through a dual biochemical and molecular-genetics approach. Curr Genet 30, 159–165 (1996). https://doi.org/10.1007/s002940050115
Issue Date:
DOI: https://doi.org/10.1007/s002940050115