Green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae.
The green fluorescent protein (GFP) of Aequorea victoria is now an established marker for gene expression and subcellular localization in budding yeast. Relatively high expression (greater than 2500 copies per cell) of GFP is required for direct microscopic visualization. This report provides a method for studying the expression of less highly expressed genes by the analysis of crude cell extracts--a simple and cheap alternative to the fluorescent activated cell sorter (FACS). The utility of this marker is demonstrated in a study of the expression of the RAD54 gene. It is shown that the induction of the RAD54 promoter leads to the accumulation of Rad54p and of GFP and that the fluorescence induction is correctly regulated. This method should allow the screening of large numbers of novel gene disrupters for their effects on RAD54 expression and so identify trans-acting factors involved in the cellular response to DNA damage.
Walmsley
R M
RM
Department of Biomolecular Sciences, UMIST, Manchester, U.K.
Billinton
N
N
Heyer
W D
WD
D004249:DNA Damage; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods
D005656:Fungal Proteins; D008164:Luminescent Proteins; D009153:Mutagens; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D008741:Methyl Methanesulfonate; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes