ISSN:
1432-0983
Keywords:
Key words: Dihydroorotate dehydrogenase
;
Fungicide
;
Aspergillus nidulans
;
Resistance gene
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002940050115
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