Dominant genetics using a yeast genomic library under the control of a strong inducible promoter

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 23; pp. 11589 - 11593
• Main Authors: Ramer, Sandra W., Elledge, Stephen J., Davis, Ronald W.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.12.1992; National Acad Sciences; National Academy of Sciences
• Subjects: adn; analytical methods; Base Sequence; Biological and medical sciences; Classical genetics, quantitative genetics, hybrids; Deoxyribonucleic acid; DNA; DNA, Fungal - genetics; expresion genica; expression des genes; fenotipos; Fundamental and applied biological sciences. Psychology; Galactose; gene expression; Gene expression regulation; Gene Expression Regulation, Fungal; Genes; Genes, Dominant; Genes, Fungal; Genetic Complementation Test; Genetic screening; Genetic Vectors; Genetics; Genetics of eukaryotes. Biological and molecular evolution; Genomic Library; Genomics; levadura; levure; Libraries; Mating Factor; Molecular Sequence Data; nucleotide; nucleotides; nucleotidos; Oligodeoxyribonucleotides - chemistry; Peptides - genetics; phenotype; Phenotypes; Pheromones; Plasmids; Promoter Regions, Genetic; Pteridophyta, spermatophyta; recombinacion; recombinaison; recombination; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; technique analytique; tecnicas analiticas; Transcription, Genetic; Vegetals; Yeast; Yeasts; Yeast; Induction; Transcription promoter; Gene library; Method; Gene expression; Fungi; Dominance recessiveness; Phenotype; Regulation(control); Isolation; Ascomycetes; Galactose; Saccharomyces cerevisiae; Mating type; Thallophyta
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.23.11589

In Saccharomyces cerevisiae, numerous genes have been identified by selection from high-copy-number libraries based on "multicopy suppression" or other phenotypic consequences of overexpression. Although fruitful, this approach suffers from two major drawbacks. First, high copy number alone may not permit high-level expression of tightly regulated genes. Conversely, other genes expressed in proportion to dosage cannot be identified if their products are toxic at elevated levels. This work reports construction of a genomic DNA expression library for S. cerevisiae that circumvents both limitations by fusing randomly sheared genomic DNA to the strong, inducible yeast GAL1 promoter, which can be regulated by carbon source. The library obtained contains 5 X 10(7) independent recombinants, representing a breakpoint at every base in the yeast genome. This library was used to examine aberrant gene expression in S. cerevisiae. A screen for dominant activators of yeast mating response identified eight genes that activate the pathway in the absence of exogenous mating pheromone, including one previously unidentified gene. One activator was a truncated STE11 gene lacking approximately 1000 base pairs of amino-terminal coding sequence. In two different clones, the same GAL1 promoter-proximal ATG is in-frame with the coding sequence of STE11, suggesting that internal initiation of translation there results in production of a biologically active, truncated STE11 protein. Thus this library allows isolation based on dominant phenotypes of genes that might have been difficult or impossible to isolate from high-copy-number libraries.