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FUNCTIONAL CHARACTERISATION OF THE HUMAN OVIDUCT-SPECIFIC GLYCOPROTEIN PROMOTER IN HUMAN OVIDUCTAL EPITHELIAL CELL LINE.
Agarwal, Anika1, Yeung, William1, Lee, Kai-Fai1, 1
ABSTRACT- The oviduct-specific glycoprotein (OGP) is a glycoprotein of the chitinase family expressed in the oviduct of many mammalian species. Recent studies have shown that OGP plays an important role in pre-fertilisation reproductive events (sperm capacitation, sperm zona binding, and zona penetration). In order to understand the regulation and expression of the OGP, we cloned the human (HuOGP) and mouse (MOGP) OGP promoters and studied their trans-activities on various cell lines under estrogen stimulation. The 5' flanking sequences of HOGP (AF189710) and MOGP (AF148876) were isolated from genomic DNA using a PCR-based Genome Walker kit and their respective sizes are 2.6kbp and 3.5kbp. DNA sequence analysis for both revealed a number of consensus binding sites for known transcriptional factors. Interestingly, several half-estrogen responsive elements (5'-TGACC-3') were found in the promoter and intron 1 of HuOGP and MOGP, whereas they were present only 5' to the exon 1 in the hamster OGP promoter sequence. All three promoters had an imperfect ERE site (5'-GGTCANNNTGACT-3') located upstream of the transcriptional start sites. The transcriptional start site for HuOGP was found to be 12 bp upstream of the translational start site. The HuOGP promoter deletion constructs were cloned into pBlue-TOPO reporter vector in both orientations and transfected into CHO-K1 and immortalized human oviductal epithelial (OE-E6/E7) cell lines. Our data show that HuOGP fragments can trans-activate reporter constructs only in OE-E6/E7 cells under estrogen stimulation. Interestingly, our results also suggest that potential silencing elements are present in the distal region of the HuOGP promoter. These findings shall facilitate our understanding of the regulation of OGP gene expression and it's role in the process of fertilization. [This project is partially supported by a CRCG grant (HKU) to KFL].
KEY WORDS: human oviduct specific glycoprotein, promoter, oviductal epithelial cells
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