A 'low-tech, high-throughput' screen of the deletion mutant
collection efficiently identifies karmellae biogenesis genes.
Jennifer Loertscher, Emily Cadera, Dangelei Fox, Clint Matson,
Jeffrey Shaver, Christine Tachibana, Robin Wright
Department of Zoology, University of Washington, Box 351800, Seattle, WA
98195, USA
In Saccharomyces cerevisiae, increased expression of an HMG-CoA
reductase isozyme, Hmg1p, induces stacks of nucleus-associated membranes
called karmellae. The goal of this study was to identify every non-essential yeast gene required for normal growth in the presence of
increased levels of Hmg1p and karmellae. We developed an efficient
protocol for transforming and analyzing the growth of approximately 4500
mutant strains obtained from the Saccharomyces Genome Deletion
Consortium. The strains were transformed with a plasmid that expresses
HMG1 under the control of a galactose-inducible promoter. Growth
of transformants was assessed on glucose and galactose at 16°, 26°, and
37°. The process was completed in just over a month without the use of
robotic automation. In the initial screen, approximately 800 transformed
mutant strains exhibited a galactose-dependent growth phenotype. We
expect that a substantial fraction of these mutants will be eliminated
in secondary screens that distinguish Hmg1p-sensitivity from inability
to metabolize galactose. Most of the mutations identified represent
genes with unknown function. For mutations in genes with known
functions, several major functional categories were observed, including
genes involved in vacuole biogenesis, DNA metabolism, protein synthesis,
membrane transport, and cytoskeleton. This project demonstrates that
global genomic screens can be rapidly and economically conducted without
specialized instrumentation.
Return to YGM 2002 Home at SGD