Screening And Identification Of Regulatory Fragments Among Apo(a)-Plasminogen Cluster With An Improved RERS Method

To meet the needs of self-development and sustain the relative stability of inner-condition in the changeful environment, it is vital for cells to keep a well-organized regulatory network of gene expression, which can capture every subtle changing of the surrounding world or itself, and make timely feedback. Finding out all the nodal points in this stereo network and clarifying the links between them is one of the major tasks in the post-genomic era.Transcriptional regulation is the most important part in the network of gene expression regulation. Present studies indicate that the interactions between trans-acting factors and corresponding DNA cis-acting elements are the basis of chromatin remodeling and gene transcription regulation. One major aim of gene expression regulation research is to isolate and identify genomic regulatory sequences as well as their protein companions and to elucidate the structure basis of their functions.It is difficult to broadly isolate and identify DNA regulatory fragments by using present methods. Some of them can't isolate DNA regulatory fragments in genome-wide efficiently, quickly or in large quantity. Some others may need purified transacting factors or antibodies and only isolate the DNA fragments binding to known proteins. Since transcription regulatory factors are expressed in low level in the cell, it is difficult to obtain enough pure protein for amino acid sequencing or antibody preparation by routine purifying methods. Although a few transcription factors were cloned, their expression pattern and the interaction between DNA and transcription factors were not studied in detail. So the establishment of a highly efficient method for broad isolation and identification of regulatory DNA fragments is still an essential work for the present transcription regulation research.Based on modified whole genome PCR and electrophoretic mobility shift assay (EMSA), our laboratory established a new method to isolate genomic regulatory sequences using crude nuclear extract. The principle is as follows. After sonication and ligation with DNA linker, human genomic DNA is amplified and labeled with isotope by whole genome PCR. Only the PCR product about 200bp is recovered as DNA probe for EMSA with nuclear extract prepared from K562 cells. Genomic DNA in retarded bands in EMSA is...