A Streamlined Process to Phenotypically Profile Heterologous cDNAs in Parallel Using Yeast Cell-Based Assays
Abstract
To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of ∼30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.
[The sequence data described in this paper have been submitted to the data library under accession no. AF359244.]
Footnotes
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Present addresses: 1Chromos Molecular Systems, Burnaby, BC V5A 1W9, Canada; 2Microcide Pharmaceuticals, Mountain View, CA 94043, USA; 3University of California San Francisco Comprehensive Cancer Center, San Francisco, CA 94143, USA.
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↵4 Corresponding author.
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E-MAIL tmelese{at}cc.ucsf.edu; FAX (415) 502-6779.
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Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.191601.
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- Received April 9, 2001.
- Accepted July 6, 2001.
- Cold Spring Harbor Laboratory Press