Molecular Cloning Of 5' Flanking Region And Introns Of 20 A Hydroxysteroid Dehydrogenase Gene,Structure Analysis And Measurement On Their Activities Of Regulation Gene Expression

The rat 20a -hydroxysteroid dehydrogenase (20a HSD) 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 20 a HSD acts as a molecular switch and makes the potent progesterone hormone into a inactive potent metabolite. The enzyme of 20a HSD is totally silenced throughout gestation, but the level of both 20a HSD protein and message RNA becomes abruptly and massively high 24hs before parturition. It is obvious that 20a HSD 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 20a HSD activity was not due to post-translational modification by either phosphorylation cycle or glycosylation, but rather at the transcription level of 20a HSD gene expression in previous research papers. Therefore, we have determined the structure of 20a HSD gene and isolated the genomic fragments encoding its promoter region to verify the cis梐cting DNA response elements and trans-acting factor, which are involved in hormone regulation on 20 a HSD gene expression in our research work. The research work was divided into 4 parts in this thesis: 1)2.5Kb 5?flanking region of the 20a HSD gene was isolated by genomic DNA walking. The transcription start site was identificated by primer extension analysis and 5?RACE (rapid amplification of cDNA ends) technique. The nine exons, eight introns and the boundaries between exon and intron of 20a HSD gene were characterized with nested primes PCR designed by the sequence of 20a HSD cDNA. 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 20a HSD gene expression. The fragments from six introns were cloned and sequenced to analysis some putative DNA response elements. We found that the intron2, 3, 4, 6, 7 and 8 were consisted of 768bp, 966bp, lOl4bp, 253bp, 897bp and 2092bp respectively. The 9. 6kb DNA structure from all 20a HSD gene with regulation region was described by DNA sequence and DNA analysis software. 2)To examine the regulative activity on the rat 20a HSD gene expression, various lengths fragment 1. 6Kb 338bp obtained from 5?flanking promoter region by digestion of KpnI, MluI or BglIJ, Hind Hland 2. 5Kp fragment were subcloned into pGL3 basic vector, resulting in pGL3-2. 5, pGL3-1. 6, pGL3-338 constructs. Then the plasmid DNA of pGL3-1. 6, pGL3-338, pGL3-2. 5 was respectively co-transfected with DNA pRL-SV40 as a internal control into COS7 or CHO cells. Both Firefly luciferase activity drived by the 20a HSD promoter from pGL3-1.6, 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 20a HSD gene 5?flanking promoter region was observed in either transfected CHO cells or COS7 cells as group of pGL3-1. 6> group of pGL3-2. 5>group of pGL3-338. 3)Since PRL and CG have been shown to control 20a HSD enzyme activity through its...