The Effects Of Point Mutation Of Enhancer On GFP Gene Expression And Bioinformatics Analysis

Objective: Enhancer is a cis-acting element that can raise the geneexpression. There are many worthy studies about the mechanism ofupregulating gene expression. The preliminary work of our laboratory foundthat a22bp fragment from SV40PolyA (Namely22R,5’-GTGAAAAAAATGCTTTATTTGT) can activate GFP gene expression.Some variants of22R can activate GFP gene expression, and some of themcan not. For example,4TMI (5＇-GTGAAATAAATGCTTTTTTTGT) canactivate GFP gene expression, and22R4C (5＇-GTGAAAAAAATGCTTTCTTTGT) can not. Why do the similar sequenceshave the different ability to activate gene expression? In order to clarify therole of fragments and a single base in the activation of genes, we mutated22Rsequence. Then we found that AA fragment (5’-GTGAAATAAATGCAAATAAAGT) activated the GFP reporter gene, while7pieA fragment (5’-GTGAAAAAAATGCAAAAAAAGT) can not activateGFP reporter gene. Apparently “T” nucleotides in the7th and17th sites of AAfragment have the role for activating GFP reporter gene. We will knowwhether the other “T” nucleotides in the AA fragment are also involved inactivation of gene process. So we continued to change “T” nucleotides in the2th,11th and22th sites of AA fragment in order to study the impact of “T”mutation on the expression of GFP reporter gene.Methods:1Primers and DNA fragments as templatesPrimers with suitable restriction enzyme sites and DNA fragments weredesigned and synthesized. All of the primers and fragments in this study areshown in table1.2Construction of expression vectorsUsing primers with suitable restriction enzyme sites and the synthesized templates with the sequence of mutations, purpose sequences were amplified.After enzyme cutting, we inserted purpose sequences into pEGFP-C1plasmid,got restructuring plasmid (×1restructuring plasmid). Then×1restructuringplasmids were cutted with Hind Ⅲ/Xba Ⅰ enzymes, larger pieces wereregained with agarose gel electrophoresis (AGE), and HindⅢ/NheⅠenzymecutted them, regaining smaller pieces. Larger pieces and smaller pieces wereligated by T4DNA ligase. We got×2restructuring plasmids. They weredigested with suitable restriction enzymes and were inserted into downstreamof GFP gene in pAlu14(inserting14Alu into downstream of GFP gene inPEGFP-C1plasmid).3Cell culture and cell transfectionThe recombinant plasmids were transiently transfected HeLa cells usingLipofectamineTM2000. GFP protein expression was assessed by fluorescencemicroscopy. On white light and fluorescent light, respectively, pictures of thecells were taken within the same scope.4Flow Cytometry assayFlow Cytometry assay was entrusted to the Fourth Hospital of HebeiMedical University.Results:1Construction of expression vectors and identificationExpression vectors are correct through restriction enzyme andsequencing.2AA sequence eliminated the inhibition of Alu tandem on GFP geneexpressionThe plasmids of C1-AA×2-Alu14, C1-Alu14, pEGFP-C1weretransfected into HeLa cells, and then observed using fluorescence microscopy.The ratios of GFP positive cells was: C1-AA×2-Alu147.39±0.91%，C1-Alu140.69±0.11%，pEGFP-C180.50±1.33%. C1-AA×2-Alu14can eliminate theinhibition of Alu tandem on GFP gene expression.The results of Flow Cytometry assay showed that the ratios of GFPpositive cells of C1-AA×2-Alu14and C1-Alu14were44.96%and21.47%respectively using HeLa as a control (fluorescence intensity>100). If stronger fluorescence cells were counted, the percentages (cell number of fluorescenceintensity>102/total cell number) were5.0%and0.1%respectively. Theseresultes are consistant with those by fluorescence microscopy observation. AAsequence eliminated the inhibition of Alu tandem on GFP gene expression3The effects of point mutations of AA sequence on GFP gene expressionThe recombinant plasmids with point mutation of AA sequence weretransfected into HeLa cells, and then observed using fluorescence microscopy.The ratios of GFP positive cells were:C1-AAMFA×2-Alu143.59±0.27%， C1-AAMFC×2-Alu145.78±1.09%，C1-AAMFG×2-Alu142.95±0.70%，C1-AAMSeA×2-Alu144.02±1.06%，C1-AAMSeC×2-Alu144.28±1.29%，C1-AAMSeG×2-Alu143.70±0.86%，C1-AAMThA×2-Alu147.09±1.60%，C1-AAMThC×2-Alu145.25±1.33%，C1-AAMThG×2-Alu143.02±0.74%， C1-AA×2-Alu147.39±0.91%，C1-Alu140.69±0.11%.The results of Flow Cytometry assay showed that the ratios of GFPpositive cells of the recombinant plasmids were (cell number of fluorescenceintensity>102/total cell number):C1-AAMFA×2-Alu141.87%，C1-AAMFC×2-Alu144.22%，C1-AAMFG×2-Alu140.96%，C1-AAMSeA×2-Alu142.35%，C1-AAMSeC×2-Alu142.67%，C1-AAMSeG×2-Alu142.58%，C1-AAMThA×2-Alu144.78%，C1-AAMThC×2-Alu143.26%，C1-AAMThG×2-Alu142.21%， C1-AA×2-Alu145.00%，C1-Alu140.10%。These resultes are consistant with those by fluorescence microscopyobservation. The recombinant plasmids with point mutation of AA sequencestill can eliminate the inhibition of Alu tandem on GFP gene expression.4Our laboratory found several enhancers, for example,22R（5’-GTGAAAAAAATGCTTTATTTGT），4TMI（5’-GTGAAATAAATGCTTTTTTTGT）， AA（5’-GTGAAATAAATGCAAATAAAGT），5AMI（5’-GTGAATAAAATGCTTTAATTGT）. The common feature of these enhancers is that A, T bases are preponderated and both flanks havesymmetrical trend with TGC center. To study whether the proportion of smallRNA fragments is related with enhancer base, we analyzed RNA smallfragments from expressed gene in five kinds of cells. The results found thatthe contents of UUUU and AAAA are the highest in RNA small fragments ofthess cells. If comparing UUUU with AAAA string, proportion of the UUUUstring is higher than the AAAA.Conclusion:1AA sequence eliminated the inhibition of Alu tandem onGFP gene expression.2The mutations of other three T nucleotides (the2,11,22sites) showthat the three T bases impact the activity of AA enhancer. The enhanceractivity of all mutants was lower than that of AA sequence, and higher thanthat of Alu14.3RNA small fragments in five kinds of cells were analyzed using thestring bioinformatics and found that the proportion of U string was highestfollowed by the A string. U and A strings are preponderated in RNA smallfragments. This phenomenon whether it is related with that we found A, Tbases dominant in enhancer deserves further study.