Monitoring dynamics of gene expression in yeast during stationary phase.
The commonly used genetic approaches in yeast are designed to identify defects in cell/colony growth. In order to identify genes which control molecular mechanisms during quiescence ('stationary phase'), different tactics are required. We describe the development of a new genetic approach based on the previous observations that gene expression in quiescent Saccharomyces cerevisiae cells is largely repressed. For studying the mechanism controlling the repression of gene expression in stationary phase, we use UBI4-lacZ as a reporter gene. The product of this fusion gene was shown previously to encode an unstable protein in dividing cells. We show here that it is also unstable in stationary cells. We demonstrate that the relatively short half-life of this reporter protein can be utilized to monitor the dynamics of the repression of gene expression during stationary phase in liquid culture, using ACT1 or SSA3 promoters as the model promoters. By adapting a colony color test, we show that the reporter gene can also be used to monitor gene expression in quiescent colonies, thus serving as a tool to screen for defects in the regulation of this process during growth arrest. The utility of the approach was demonstrated by confirming the defects of top1Delta and bcy1Delta cells to appropriately express the ACT1p-UBI4-lacZ in stationary phase. The mutant colonies were easily discernible from wild-type colonies by our color test. Finally, using SSA3p-UBI4-lacZ as the reporter gene, we found that the 5'-untranslated region of SSA3 mRNA is sufficient to repress translation of the reporter mRNA after entry of the cells into stationary phase. The possibility that the short length of the SSA3 5'-untranslated region is a major determinant of the inefficient translation of SSA3p-UBI4-lacZ in stationary phase is discussed.
Paz
I
I
Department of Molecular Microbiology and Biotechnology, Ramat Aviv 69978, Israel.
Meunier
J R
JR
Choder
M
M
D000199:Actins / Q000235:genetics / Q000502:physiology; D029681:Arabidopsis Proteins; D019677:Artificial Gene Fusion; D002453:Cell Cycle / Q000502:physiology*; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D008957:Models, Genetic; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology*; D013997:Time Factors; D014158:Transcription, Genetic
C098619:AAC1 protein, Arabidopsis; D000199:Actins; D029681:Arabidopsis Proteins; D011994:Recombinant Proteins