POMC基因5′上游近端片段的转录调控作用初探

The Role of 5′Proximal Fragment of POMC Gene in the Regulation of Transcription in AtT20 Cells

目的 建立阿片黑皮质素原 (POMC)基因 5′上游近端转录调控体系并初步探讨其作用特点。方法 采用 DNA重组技术构建一系列受大鼠 POMC基因 5′上游近端不同长度片段介导的荧光素酶报告基因质粒 ,以lipofectam ine 包裹质粒转染垂体 ACTH瘤细胞系 (At T2 0 ) ,然后测定荧光素酶活性。结果 成功地构建了含POMC基因 - 34 / +6 3bp、- 16 5 / +6 3bp、- 32 3/ +6 3bp和 - 480 / +6 3bp片段的 4种报告基因质粒 ,其荧光素酶相对表达活性分别为 1、2 .1± 0 .3、3.3± 0 .3和 3.7± 0 .5 ,阳性对照为 4.8± 0 .8。结论 POMC基因的核心启动子比较弱 ,提示 POMC基因可能存在多个转录起始位点 :At T2 0细胞中 POMC基因持续表达的近端组织特异性调控元件主要集中于 - 34 / - 16 5 bp范围内。

Objective Identification of the vital regions in 5′flanking sequence of POMC gene in order to deterimine the characteristics of POMC gene promoter and provide the basis for stadying the effects of multiple hormones and other factors in the POMC gene expression. Methods Construction of four kinds of plasmid recombined by rat POMC gene promoter regions and eukarytic expression reporter gene vector pG12Basic. Each kind of them was identified by restriction maps analysis and DNA sequencing. All recombinant plasmids were transfected into AtT20 cells respectively by using a lipofectaminebased method. Luciferase activity in each celllytate was measured by monolight 2010 luminometer. Results Four recombinant plasmids were constructed, including pGL2POMC480 (rPOMC480/+63),pGL2POMC323 (rPOMC323/+63),pGL2POMC165 (rPOMC165/+63).pGL2POMC34(rPOMC-34/+63) Among them, PGL2POMC34 contains the core promoter which consist of TATA box at -31/-25 bp and transcription start site at +1 bp of POMC gene. It showed that, comparing with the pGL2POMC34, the relative luciferase activity of pGL2POMC165 was 21±03,pGL2POMC323 was 33±03,pGL2POMC480 was 37±05,and the positive control pGL2promoter was 48±08. Conclusions (1)A model system to study the direct effects of various ACTH secretagogues on the dynamics of the POMC gene promoter activity in vitro was established. (2) The studying of POMC promoter basal transcriptional activities identified that the POMC gene has a weak core promoter. The proximal sequence elements were concentrated in the fragment of -34/-165 bp, and the transcriptional activities need POMC 5′ promoter -480 bp or a longer region.