title: 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 approximately 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.
authors.affiliation: Iconix Pharmaceuticals, Mountain View, California 94043, USA.
authors.forename: S
authors.initials: S
authors.lastname: Tugendreich
authors.forename: E
authors.initials: E
authors.lastname: Perkins
authors.forename: J
authors.initials: J
authors.lastname: Couto
authors.forename: P
authors.initials: P
authors.lastname: Barthmaier
authors.forename: D
authors.initials: D
authors.lastname: Sun
authors.forename: S
authors.initials: S
authors.lastname: Tang
authors.forename: S
authors.initials: S
authors.lastname: Tulac
authors.forename: A
authors.initials: A
authors.lastname: Nguyen
authors.forename: E
authors.initials: E
authors.lastname: Yeh
authors.forename: A
authors.initials: A
authors.lastname: Mays
authors.forename: E
authors.initials: E
authors.lastname: Wallace
authors.forename: T
authors.initials: T
authors.lastname: Lila
authors.forename: D
authors.initials: D
authors.lastname: Shivak
authors.forename: M
authors.initials: M
authors.lastname: Prichard
authors.forename: L
authors.initials: L
authors.lastname: Andrejka
authors.forename: R
authors.initials: R
authors.lastname: Kim
authors.forename: T
authors.initials: T
authors.lastname: Melese
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012680:Sensitivity and Specificity; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases