Global protein expression profiling of budding yeast in response to DNA damage.
Exposure to DNA-damaging agents can activate cell cycle checkpoint and DNA repair processes to ensure genetic integrity. Such exposures also can affect the transcription of many genes required for these processes. In the budding yeast Saccharomyces cerevisiae, changes of global gene expression as a result of a DNA-damaging agent were previously identified by using DNA chip technology. DNA microarray analysis is a powerful tool for identifying genes whose expressions are changed in response to environmental changes. Transcriptional levels, however, do not necessarily reflect cellular protein levels. Green fluorescent protein (GFP) has been widely used as a reporter of gene expression and subcellular protein localization. We have used 4156 yeast strains expressing full-length, chromosome-tagged GFP fusion proteins to monitor changes of protein levels in response to the DNA-damaging agent, methyl methanesulphonate (MMS). Through flow cytometry, we identified 157 proteins whose levels were increased at least three-fold following treatment with MMS. Of 157 responsible genes, transcriptions of 57 were previously not known to be induced by MMS. Immunoblot experiments with tandem affinity-tagged yeast strains under the same experimental conditions confirmed these newly found proteins as inducible. These results suggest, therefore, that the 57 protein expressions are regulated by different mechanisms, such as post-translational modifications, and not by transcriptional regulation.
Lee
Min-Woo
MW
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, 300 Chunchun-dong, Jangan-gu, Suwon, Kyonggi-do 440-746, Republic of Korea.
Kim
Beom-Jun
BJ
Choi
Hyun-Kyung
HK
Ryu
Min-Jung
MJ
Kim
Sang-Bae
SB
Kang
Kyung-Min
KM
Cho
Eun-Jung
EJ
Youn
Hong-Duk
HD
Huh
Won-Ki
WK
Kim
Seong-Tae
ST
D015153:Blotting, Western; D004249:DNA Damage; D004271:DNA, Fungal; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism*
D004271:DNA, Fungal; D005656:Fungal Proteins; D009153:Mutagens; D008741:Methyl Methanesulfonate