Hurat1 Gene 5'utr District, +262 / T, +292 C, / G Point Mutation And-424t / C Snp Of The Promoter Activity Of The Impact

Objective:hURAT1 is the mian gene for regulating surem uric acid as well as one of the most important candidate genes of primary gout and hyperuricemia.According to our previous report-5'UTR,+262C/T and +292C/G were only existed in people with hyperuricemia,while not in the normal people.the present vitro function study is designed to find whether 5'UTR,+262C/T and+292C/G affect the activity of promoter of hURAT1,then enhance the uric acid reabsorption of kidney approximate tubule,this mechanism is likely to be the key cause of primary gout and hyperuricemia.Methods:Total genome DNA was prepared from normal human total blood.Then DNA was subjected to PCR amplification the smallest functional unit of hURAT1 gene promoter and 5'UTR. PCR product was cloned into PGL3-basic and then the recombinant vector was transformed into DH5 a Ecoli.Wide type PGL3-basic/hURAT1 was identified by restricting enzyme digestion analysis and DNA sequencing.Mutant type PGL3-basic/hURAT1 was constructed with QuikChange(?)ⅡSite-Directed Mutagenesis Kit.The wild and mutant type PGL3-basic /hURAT1 construction were set as case groups,The eight study groups were transient transfected into OK cell to express, By double luciferase report system to study the influence of hURAT1 5'UTR+262C/T and +292C/G to the activity of promoter.Results:1.The wild type PGL3-basic/hURAT1 construction had been constructed successfully with restriction enzyme digestion and DNA sequencing confirm.2. The mutant type PGL3-basic/hURAT1 construction had been also constructed successfully with restriction enzyme digestion and DNA sequencing confirm.3.The double luciferase report system showed that+262C/T,+292C/G chain with-424T/C respectively may enhance the activity of promoter compared with the wild type.Conclusions:It is certified that the 5'UTR,+262C/T,+292C/G chain with-424T/C respectively may enhance the activity of promoter compared with the wild groups.As a result, the structure and function of hURAT1 may partly change and the surem uric acid reabsorption may increase by this pathway.