[Cloning and prokaryotic expression of yeast GCN5 and RPD3].
The yeast histone acetyltransferase (HAT) gene gcn5 and histone deacetylase (HDAC) gene rpd3 were cloned from yeast genomic DNA by PCR amplification. The two genes,both with additional 6xHis tag,were subcloned into pBV220 vector to construct expression plasmids pBVgcn5 and pBVrpd3,respectively. Both pBVgcn5 and pBVrpd3 were over-expressed in Escherichia coli upon temperature induction,as revealed by SDS-PAGE. The recombinant GCN5 and RPD3 were purified by using a 6xHis affinity column. The purified GCN5 was tested to possess the HAT activity by using a (14)C-labeling assay. This work has laid down the basis for further in vitro studies into roles of histone acetylation/deacetylation in modulating chromatin conformation and transcription activity.
Chen
Ting
T
Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, China. ct200079@hotmail.com
Lu
Jun
J
Sun
Hui
H
Dong
Mei
M
Han
Song-Yan
SY
Huang
Bai-Qu
BQ