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Facilitating functional analysis of the Saccharomyces cerevisiae genome using an EGFP ‐based promoter library and flow cytometry
Author(s) -
Bell Philip J. L.,
Davies Ian W.,
Attfield Paul V.
Publication year - 1999
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(199912)15:16<1747::aid-yea492>3.0.co;2-2
Subject(s) - biology , promoter , saccharomyces cerevisiae , gene , genome , genomic library , cloning (programming) , genetics , reporter gene , multiple cloning site , computational biology , microbiology and biotechnology , expression vector , gene expression , recombinant dna , peptide sequence , computer science , programming language
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 2×10 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. Copyright © 1999 John Wiley & Sons, Ltd.