Facilitating functional analysis of the Saccharomyces cerevisiae genome using an EGFP-based promoter library and flow cytometry.
A promoter library was generated to facilitate identification of differentially regulated promoters in Saccharomyces cerevisiae. The library was constructed in a vector containing two reporter genes (EGFP and lacZ) divergently arranged about a unique cloning site. Approximately 2x10(5) clones were obtained and a flow cytometer was used to screen the library for copper-induced EGFP expression. A DNA fragment conferring copper-inducible expression of EGFP was rapidly identified. This DNA fragment, which contained several motifs associated with copper and oxidative stress homeostasis, lies upstream of two 'orphan' genes of unknown function. Further studies comparing expression from episomal vs. integrative vectors showed that construction of a similar library using an integrative vector would further enhance rapid identification of genes that are differentially regulated in S. cerevisiae. The ability to identify regulated promoters rapidly should facilitate the functional analysis of the yeast genome by identifying genes induced by specific physiological conditions.
Bell
P J
PJ
Burns Philp Research and Development, 67 Epping Road, North Ryde, New South Wales 2113, Australia. pbell@rna.bio.mq.edu.au
Davies
I W
IW
Attfield
P V
PV
D001483:Base Sequence; D016384:Consensus Sequence; D019327:Copper Sulfate / Q000737:chemistry; D005434:Flow Cytometry; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007763:Lac Operon; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000032:analysis
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D019327:Copper Sulfate