RESUMO
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.
Assuntos
DNA Complementar/genética , Perfilação da Expressão Gênica/métodos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Clonagem Molecular/métodos , Inibidores Enzimáticos/farmacologia , Vetores Genéticos/genética , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Dados de Sequência Molecular , Mutagênese , Fenótipo , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sensibilidade e Especificidade , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: Real-time polymerase chain reaction (PCR) is a powerful new technique in the evolution of quantitative reverse transcription-PCR assays. With the increased sensitivity and resolution of real-time techniques, the requirements for constitutive expression of endogenous controls have become increasingly stringent. METHODS AND RESULTS: We compare the expression of the mitochondrial gene, adenosine triphosphate synthase 6 (ATPsy6), to the expression of other routinely used endogenous control genes (e.g., beta-actin, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal RNA 18S [18S rRNA], and cyclophilin). In a diverse assortment of tissues and across a wide range of disease stages, ATPsy6 shows a relative steady state of expression compared with other endogenous controls. ATPsy6 gene expression has been used as an endogenous control in a quantitative real-time PCR assay designed to evaluate the expression of potential cancer diagnostic leads across a diverse tissue panel. CONCLUSION: Mitochondrial ATPsy6 serves as a good endogenous control to measure target gene expression independent of the tissue- or disease-specific variation inherent with many housekeeping genes.
