[The role of 5'proximal fragment of POMC gene in the regulation of transcription in AtT20 cells].
OBJECTIVE
Identification of the vital regions in 5'-flanking sequence of POMC gene in order to determine 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 pG12-Basic. 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 lipofectamine-based method. Luciferase activity in each cell-lytate was measured by monolight 2010 luminometer.


RESULTS
Four recombinant plasmids were constructed, including pGL2-POMC480 (rPOMC -480/+63), pGL2-POMC323 (rPOMC -323/+63), pGL2-POMC165 (rPOMC -165/+63). pGL2-POMC34 (rPOMC -34/+63) Among them, PGL2-POMC34 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 pGL2-POMC34, the relative luciferase activity of pGL2-POMC165 was 2.1 +/- 0.3, pGL2-POMC323 was 3.3 +/- 0.3, pGL2-POMC480 was 3.7 +/- 0.5, and the positive control pGL2-promoter was 4.8 +/- 0.8.


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.
Wang
Z
Z
Department of Endocrinology, PUMC Hospital CAMS, PUMC, Beijing 100730, China. biochem@mail.bjmu.edu.cn
Lu
Z
Z
Guo
A
A
D000306:Adrenal Cortex Neoplasms / Q000235:genetics* / Q000473:pathology; D000818:Animals; D017931:DNA Primers; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D010911:Pituitary Neoplasms / Q000235:genetics* / Q000473:pathology; D010957:Plasmids; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D014158:Transcription, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D011333:Pro-Opiomelanocortin