Analysis Of Binding Sites Between Nuclear Transcription Factor USF1 And Glucose Transporter Gene

The key place of monosaccharide uptake and transport is the small intestine,which mainly embodies in small intestinal villus epithelial cells,epithelial cells containing monosaccharide transport carrier.USF1 is recognized transcription factors that regulating glucose and lipid,and it is finded in the small intestine tissue.In the small intestine of chicken,the monosaccharide transport carrier usually expressed by three kinds of GLUT5,GLUT2 SGLT1 that transfer glucose and fructose.If it is finded the binding site between USF1 and upstream of SGLT1,GLUT2,GLUT5 gene,it will provide a strong basis for the regulation of USF1 sugar transporter gene.In this study,the cells were cultured by primary culture on small intestinal epithelial cells(IEC),and the cells were identified by morphology and immunohistochemistry.The total protein and nuclear protein were extracted and the total protein was extracted and identified by Western-blot.The nucleic acid sequence of the transcription factor binding site predicted by ModuleMasterl.4 and bind TF and so on,and the predicted nucleic acid sequence is markered by Biotin.The extracted nuclear proteins are mixed with prior labeled biotin nucleic acid probes,which is used to conduct the electrophoretic mobility shift assay(EMSA)experiment and analysis the detection results of exposing,that concludes binding sites between transcription factor USF1 and the three sugar transportersThe following results are obtained through a series of experiments:(1)After identification of cell morphology,the cultured cells first appeared irregular polygon "paving stone" structure,after 6?7 days of culture,the polygon explosively growth into another shape like tadpole;by immunohisto chemical identification of cell specific antigens keratin 18 in IEC,and displayed that the cells in the experimental group hole glances into brown.(2)The total protein extracted from the cells was identified by Western-blot,and the specific bands appeared in the 43kD position,and the bands were clear.(3)Detection of biotin labeled nucleic acid sequences,they are signed.(4)After the EMSA experiment between that extracted nuclear proteins and Biotin labeled nucleic acid probe,the result show that the mix between the Num.2 nucleic acid probe in upstream of GLUT2 gene and nuclear proteins has a lagging band and a super shift.And the mix between the Num.S5 nucleic acid probe in upstream of SGLT1 gene and nuclear proteins has a lagging band and a super shift.Other no shift bands.The following conclusions are drawn from the above experiments:(1)The cells were cultured by tissue block method is Chicken small intestine epithelial cells;(2)The total protein extracted from the cell does contain transcription factor USF1,and the protein concentration is high;(3)USF1 binding site in upstream of the GLUT2 gene is 5'-GCAGCAGCACCTGTTGGCATGTGGGGTTCC-3,;USF1 has no binding site in upstream of the GLUT5 gene;USF1 binding site in upstream of the SGLT1 gene is 5'-TACATCTCACCTGCC AGGAT-3'.