Screening regulatory sequences from bacterial artificial chromosome DNA of alpha- and beta-globin gene clusters.
In the forthcoming postgenomic era, identification of regulatory DNA sequences is becoming increasingly important for characterizing DNA-binding proteins and for elucidating the regulatory mechanisms of gene expression. Presently, there lack efficient methods to broadly screen and identify DNA regulatory elements on a large scale. We established herein an efficient strategy to screen regulatory sequences from bacterial artificial chromosome (BAC) DNAs containing human alpha- and beta-globin gene clusters based on polymerase chain reaction and electrophoretic mobility shift assay (EMSA) techniques without purified transcription factors. Twenty-three subclones derived from alpha-BAC DNA by bulk EMSA selection retained the ability to bind nuclear proteins of K562 cells when retested by EMSA. In 19 clones sequenced, 14 are identical to those registered in GenBank and five have one base difference. All of the 24 randomly picked beta-BAC clones showed specific binding with nuclear proteins of K562 cells. In 11 clones sequenced, eight are identical to those registered in GenBank and three have one base difference. This approach could be particularly powerful if combined with other systematic methods for identifying cis-regulatory DNA elements.
Zhang
Shen
S
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Peking Union Medical College, Beijing, PR China.
Zhang
Hua-Bing
HB
Liu
De-Pei
DP
Li
Xing-Guo
XG
Hao
De-Long
DL
Lv
Xiang
X
Xu
Hai-Ming
HM
Liang
Chih-Chuan
CC
D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D022202:Chromosomes, Artificial, Bacterial; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D005914:Globins / Q000235:genetics*; D006801:Humans; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000378:metabolism
D015534:Trans-Activators; D005914:Globins