| The rat 20a-hydroxysteroid dehydrogenase (20aHSD) belongs to a family member of aldo-ketoreductases used NADPH as a cofactor and principally converts progesterone to 20a-hydroxyprogesterone as a lower or non-active hormone. Thus 20oHSD acts as a molecular switch and makes the potent progesterone hormone into an inactive potent metabolite. The enzyme of 20aHSD is totally silenced throughout gestation, but the level of both 20aHSD protein and message RNA becomes abruptly and massively high 24 hours before parturition. It is obvious that 20aHSD plays a key role in the initiation of labor. The activity of this enzyme is regulated by an array of hormones, including prolactin (PRL), chorionic gonadotropin (CG) and so on. It was indicated that regulation of 20aHSD activity was not due to post-translational modification by either phosphorylation cycle or glycosylation, but rather at the transcription level of 20aHSD gene expression in previous research papers. Therefore, we have determined the structure of 20aHSD gene and isolated the genomic fragments encoding its promoter region to verify the cis-acting DNA response elements and trans-acting factor, which are involved in hormone regulation on 20aHSD gene expression in our research work. The research work was divided into 3 parts in this thesis:1)2. 5Kb 5' flanking region of the 20oHSD gene was isolated by PCR. The analysis on the 5' flanking region revealed TATA box, putative API and Nur77 response elements, PRL and progesterone response elements motifs related with regulation on 20aHSD gene expression.2)To examine the regulative activity on the rat 20aHSD gene expression, various lengths fragment 1.6Kb, 338bp obtained from 5' flanking promoter region by the digestion of Kpnl, Mlul or Bglll, Hind Illand 2.5Kb fragment were subcloned into pGL3 basic vector, resulting in pGL3~2.5, pGL3-l.6,pGL3-338 constructs. Then the plasmid DNA of pGL3-l. 6, pGL3-338, pGL3-2.5 was respectively co-transfected with pRL-SV40 as an internal control into COS7 or CHO cells. Both Firefly luciferase activity drived by the 20 aHSD promoter from pGL3-l.6, pGL3-338, pGL3-2. 5 and Renilla luciferase activity driven by TK promoter from pRL-TK were measured by the Lumat LB9506 Luminometor and Promega's duel luciferase report assay system. The luciferase activity from each groups was normalized with control Renilla luciferase activity. We found that the expression of luciferase report gene directed by different length fragment from 20aHSD gene 5' flanking promoter region was observed in either transfected CHO cells or COS7 cells as group of pGL3-l. 6>group of pGL3-2. 5>group of pGL3-338.3)Since PRL and CG have been shown to control 20aHSD enzyme activity through its gene expression, both hormone were used to treat COS7 and CHO cells transfected with pGL3-l. 6, pGL3-338, pGL3-2.5 promoter report gene respectively. The inhibition of PRL on luciferase gene expression in the group of transfected CHO cells was observed as pGL3-l.6> pGL3-2.5>pGL3-338. The enhancement of CG on luciferase gene expression in the group of transfected CHO cells was also observed as pGL3-1.6> pGL3-2.5> pGL3-338. It was inferred that the nucleotides responded to PRL or CG regulation on 20ctHSD gene expression were located between -1587-289 at the 20aHSD5' flanking region. By contrary, any obvious reactions were not observed in the group of transfected COS7 cells. We believed that no receptors or signal transduction pathway as closely related with PRL or CG control in COS7 cells.
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