Part One Functional Analysis Of Promoter Region In Human CBS Gene

Objective:To define the promoter of CBS gene and determine the minimal promoter region,search for the cis-acting element that is crucial for transcription regulation of CBS gene,identify the core bases of the cis-acting elements which play an important role in the promoter activity.Methods: To construct a series of the luciferase reporter plasmids,various 5’-flanking region DNA fragment of CBS gene had been amplified from HEK293 cells with different primers.The CBS reporter plasmids and Renilla luciferase control reporter plasmid were co-transfected into HEK293 cells,luciferase activity was measured after 24 hours of transfection.The promoter activity region of CBS gene and the cis-acting region were defined by analyzing the results of reporter experiments.To investigate core base of the cis-acting region,different reporter plasmids contained the respective mutantions were constructed by site direct mutagenesis.Results: A series of CBS promoter luciferase reporter plasmids containing different promoter regions were successfully constructed.The longest inserted fragment is 1825 bp,which locates at the region of 4765 to 2941 bp upstream of translation start site(ATG).The promoter activity of reporter plasmid p CBS-4048/-3439 is higher than others.The deletion of the 5’ end reveals that there is a positive functional region-4048 to-4039,the deletion of the 3’ end reveals a positive functional region-3439 to-3520 and a negative functional region-3569 to-3604,respectively.Conclusion: We have identified the functional promoter of CBS gene and demonstrated that two fragments of-4048 to-4039 bp and-3439 to-3520 bp are function as the positive regulation regions,and the region of-3439 to-3520 bp has negative effects on the promoter activity of CBS gene.Objective:To study the effect of ethanol on APP and BACE1 gene expression.Methods:Conventional methods were used to culture 2EB2,SH-SY5 Y and SH105 cells.Plasmids contain APP and BACE1 gene promoter region were co-transfected with control reporter plasmid P3 PRluc into SH-SY5 Y cells respectively.Twelve hours after transfection,cells were treated with different concentrations of alcohol(0,0.4,1.6,3.2 mg / ml)for 24 hours,and then the luciferase activity was measured with a double luciferase assay reagent.The effect of ethanol on the activity of the promoter was determined by comparing the values of Fluc/Rluc with different gradient ethanol intervention.SH-SY5 Y and SH105 cells were used to extract RNA and protein respectively after ethanol intervention for 48 hours.The effects of ethanol on APP and BACE1 expression were confirmed by RT-PCR(m RNA level)and Western blotting(protein level).In addition,2EB2 cells,2 group cells with and without ethanol(3.2 mg / ml)treated for 48 hours,were intervented by 100μg/ml of cyclohexanimide for different time(0,15,30,60 min)before harvested.The effect of ethanol intervention on degradation changes of APP and BACE1 were detected by Western blotting.Results: Compared with the control group,the promoter activity of APP and BACE1 gene in ethanol intervention group was significantly higher than that in control group.The m RNA level of endogenous APP and BACE1 genes in SH-SY5 Y cells was up-regulated under a certain concentration of ethanol.Furthermore,in SH105 cells,higher levels of APP and BACE1 protein expression were detected than in the control group.And,APP and BACE1 protein degradation pathway has not been affected.Conclusion: We have found that ethanol upregulate the expression of APP and BACE1 through activiting the transcription and translation level in cell.