CAJ | 학술논문

目的在不同物种间通过保守序列分析和荧光素酶功能测定的方法寻找发现CD2相关蛋白(cD2AP)基因启动子重要的调节成分.方法用BLAST分析软件进行序列比较和同源分析不同种系CD2AP启动子序列,构建人CD2AP启动子不同的缺失变异载体,转染来自不同种类的细胞,测定荧光素酶活性,并用全反式维甲酸处理,观测其对CD2AP启动子活性的影响.结果在人、牛、猪的CD2AP推断的启动子区域进行同源比较发现推断的sp1(specific protein 1)和下游启动子成分高度进化保守,进行性缺失荧光素酶分析表明在人胚肾细胞株HEK-293、非洲绿猴肾细胞株Vero、仓鼠肾细胞株BHK-21中人CD2AP启动子活性有相似的形式,ATG上游500 bp有基本的启动子活性,再向上100 bp启动子活性增加10倍,两个推断的Sp1位点位于该100 bp区域内,全反式维甲酸可下调CD2AP启动子的活性.结论我们初步发现推断的Sp1位点和下游启动子成分在CD2AP启动子调控中起重要作用.
목적 재불동물충간통과보수서렬분석화형광소매공능측정적방법심조발현CD2상관단백(cD2AP)기인계동자중요적조절성분.방법 용BLAST분석연건진행서렬비교화동원분석불동충계CD2AP계동자서렬,구건인CD2AP계동자불동적결실변이재체,전염래자불동충류적세포,측정형광소매활성,병용전반식유갑산처리,관측기대CD2AP계동자활성적영향.결과 재인、우、저적CD2AP추단적계동자구역진행동원비교발현추단적sp1(specific protein 1)화하유계동자성분고도진화보수,진행성결실형광소매분석표명재인배신세포주HEK-293、비주록후신세포주Vero、창서신세포주BHK-21중인CD2AP계동자활성유상사적형식,ATG상유500 bp유기본적계동자활성,재향상100 bp계동자활성증가10배,량개추단적Sp1위점위우해100 bp구역내,전반식유갑산가하조CD2AP계동자적활성.결론 아문초보발현추단적Sp1위점화하유계동자성분재CD2AP계동자조공중기중요작용.
Objective To identify the important regulatory elements in the promoter of human CD2 associated protein(CD2AP) by conserved sequence analysis among different species and luciferase functional detection. Methods The promoter sequences of CD2AP from different species were analyzed by BLAST. Plasmids containing different length of deletion mutations of human CD2AP promoter were constructed. Pro-moter activities were tested in 3 kinds of cells from different species by luciferase analysis and were tested in HEK-293 cells treated with all-trans-retinoic acid. Results Homologous sequence comparison in CD2AP promoter area among human, cattle and pig showed that putative specific protein 1 (Sp1) sites and down-stream promoter element (DPE) were highly evolutional]y conserved. Progressive deletion luciferase analysis of DNA fragments revealed similar promoter activity style among 3 different cell lines from 3 different spe-cies, HEK-293, BHK-21 and Vero cells. One basic promoter activity located within 500 bp upstream of ATG. Fragments of further upstream 100 bp or more had drastically 10 times increased promoter activity. Two putative Sp1 sites were in this 100 bp region. All-trans-retinoic acid decreased the luciferase activity of CD2AP promoter. Conclusion Putative Spl sites and DPE have important functions in the promoter activity of CD2AP.