American Cyanamid Company

Advanced Research & Technology Institute

Martin Bard

Donald R Kirsch

Margaret H Lai

Margaret Hsien-fen Kuh Lai

The invention provides an isolated and purified nucleic acid molecule encoding a,sterol methyltransferase (ERG6). Also provided is a,strain or isolate that has reduced levels of sterol methyltransferase as a result of the disruption of at least one sterol methyltransferase gene. Preferred isolates are more susceptible to a number of sterol synthesis and metabolic inhibitors relative to wild type isolates. Further provided are methods to identify sterol methyltransferase inhibitors and methods to screen for antifungals or metabolic inhibitors which are not normally permeable to the fungal cell.

DNA encoding sterol methyltransferase

A binary assay identifies agents that inhibit sterol .DELTA.14 reductase involved in ergosterol biosynthesis. In the primary screen, sterol .DELTA.14 reductase inhibition by a test sample is assayed by adding the test sample to a culture of Neurospora crassa having an erg-3 mutation and also to a culture of a strain having an erg-1 mutation, comparing the extent of growth inhibition after incubation in the two cultures, and identifying as positives those samples that show growth inhibition in the erg-3 culture exceeding that in the erg-1 culture. In the secondary screen, samples that test positive in the primary screen are reassayed by adding the test sample to a culture of a Saccharomyces cerevisiae strain into which has been introduced multiple copies of a gene encoding sterol .DELTA.14 reductase and also to a strain of S. cerevisiae that does not have the introduced gene; positive samples are identified after incubation by observation that growth inhibition in the culture having no introduced reductase gene exceeds growth inhibition in the culture having the introduced reductase gene. In preferred embodiments, a known inhibitor of sterol .DELTA.14 reductase is employed in solidified media in both the primary and the secondary screens, resulting in an assay that is highly sensitive and specific for the detection of sterol .DELTA.14 reductase inhibitors.

Sterol .DELTA.14 reductase screen

A gene encoding Saccharomyces cerevisiae sterol .DELTA.14 reductase of the ergosterol biosynthetic pathway is isolated and cloned by selecting strains carrying sequences on a 2.mu. based vector for resistance to a morpholine fungicide such as fenpropimorph. Four distinct plasmid inserts which produce morpholine resistance are obtained, and one of these is characterized and sequenced. The purified and isolated DNA sequence encoding sterol .DELTA.14 reductase encodes a polypeptide exhibiting homology to the S. cerevisiae sterol C-24(28) reductase enzyme in the ergosterol biosynthetic pathway.

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Carmel, IN, United States of America

Martin Bard