Structure and Function
of a Novel Silencer in 5′ Flanking Distal Region of Human β Globin Gene
YANG Yu, YANG
You Yun, LI Hou-Min, ZHU Ding-Er*
( Molecular Biology Reseach Center, Xiangya Medical College, Central South
University, Changsha 410078, China )
Abstract A novel
silencer fragment(310 bp) was recently discovered and identified to locate
between -2 132 bp and -1 822 bp upstream from the cap site of β-globin gene by
gel retardation assay and luciferase reporter gene expression analysis. DNA
footprinting assays were performed to determine the interaction between its DNA
sequence and binding proteins from the nuclear extract of Hela cells. The
results showed that there were two nuclear protein binding sites in
thissilencer, one was the -2 017–2 011 bp sequence “CTTCCGC” and the other was
the -2 006–1 997 bp sequence “CACTTTATTT”. Two sequences were mutated into
“CTTAAGC” and “CACTTAAGTT”,
respectively by two mutagenic primer pairs, in order to construct two mutation
types of the 310 bp fragment. The competitive gel retardation assays showed
that two mutation types of the 310 bp fragment and their four smaller DNA
fragments, which were formed respectively by the digestion of restriction
enzyme BspTI, all lost their competitive ability against the wild type
of 310 bp fragment probe for DNA binding proteins without exception.
Furthermore, the double strand oligonucleotides, which contained both the
sequences of “CTTCCGC” and “CACTTTATTT”, were synthesized, and the competitive
gel retardation assays showed that they competed ability against wild type 310
bp fragment probe for DNA binding proteins. The results suggest that two
binding sites of the nuclear proteins are involved or associated with a potential
DNA-DNA interaction. Moreover, the specific DNA-binding proteins were purified
from the nuclear extract of Hela cells by using “DNA-binding protein
purification kit” for magnetic isolation. In order to identify the purified
DNA-binding proteins, a SDS-PAGE was performed. By using the silver staining,
the PAGE electrophoretogram showed that these two nuclear proteins specifically
bound to these two sites of the silencer, appearing as two definite bands. The
molecular weight of each protein was determined to be 37 kD or 81 kD,
respectively.
Key words β-globin gene； silencer； DNA footprinting； gel retardation assay； DNA-binding protein
*Corresponding author： Tel, 86-731-4805449； e-mail, [email protected]