Establish a recombinant yeast detection system to study the effect of MIP on transactivation function of hMafF in US2-driven gene transcription.
The human gene MafF (hMafF) is a member of bZip transcription factor Maf family, but it alone cannot activate its target genes. In 2006, a novel hMafF interacting protein (MIP) was identified. Transient transfection assay in Hela cells suggested that co-expression of MIP and hMafF could activate US2-driven transcription. In this work, we constructed a series of plasmids and transformed YM4271 yeast strain to establish a recombinant yeast detection system. In this system, MIP's expression level could be regulated using glucose incubation or galactose-induced incubation. The expression level of reporter gene LacZ in obtained recombinant yeast strains was measured using quantitative liquid assay. By comparing and analyzing the beta-galactosidase activities of different yeast strains or the same yeast strain in different culture media, the effect of MIP on transactivation driven by nUS2-hMafF was finally determined. Only in the presence of both MIP and hMafF could the nUS2-pLacZi reporter in yeast genome be activated. More importantly, this work established a novel recombinant yeast detection system, which may serve as a powerful tool to study the regulatory mechanisms of transcription complex in the future.
Ye
Xiaoxia
X
Department of Histology and Embryology, Guangdong Medical College, Guangdong Zhanjiang 524023, China. yemissing@yahoo.com.cn
Shi
Yan
Y
Huo
Keke
K
Chen
Dong
D
D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D019677:Artificial Gene Fusion; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D051260:MafF Transcription Factor / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D020346:Aquaporins; D005136:Eye Proteins; C484147:MAFF protein, human; D051260:MafF Transcription Factor; D009687:Nuclear Proteins; D014157:Transcription Factors; C030328:aquaporin 0; D001616:beta-Galactosidase