Corticotrophin releasing hormone (CRH) and CRH receptor (CRH-R) appear to play a number of important roles in human pregnancy. The purpose of the first part of my project was to clone and sequence CRH-R subtypes from human myometrial biopsies. In order to understand the molecular mechanisms that direct the expression of the CRH-R gene, the objective of the second part of my research project was to clone and characterise the promoter region of the human CRH-R2 gene. Our results demonstrated the presence of multiple CRH-R mRNAs in the human myometrium. Six subtypes of the CRH receptor, 1a, 1b, 1c, 2a, 2b, and 2g, were found in the pregnant human myometrium, whereas only three subtypes, 1a, 1b, and 2b were found in the nonpregnant myometrium. Multiple CRH-R mRNAs have been identified in human myometrium with differential expression pattern during pregnancy, which argues for multiple roles for CRH and/or related peptides in myometrial function and suggests distinct functional roles for each receptor during pregnancy. These findings suggest that CRH and its receptor play an important modulatory role in myometrial function. The genomic organisation of human CRH-R2 alternative exons has been determined in the project. The coding region of the gene spans over 18kb. The genomic orders of the alternative exons are CRH-R2b-2g-2a. The 5'-flanking region of the human CRH-R2 gene was cloned by the genomic walking method. Using 5'RACE, the transcription start sites were mapped. The results suggest that there may be a single promoter regulating the expression of all (2a, 2b and 2g) subtypes. The CRH-R2 5'-flanking sequence has many characteristics of the housekeeping gene promoter. Therefore, we speculated that the strong and housekeeping gene-like activity of the CRH-2R gene promoter may contribute to the ubiquitous expression of the CRH-R2 gene. For functional analysis, 5'-flanking sequences up to-1393 bp were fused to the Chloramphenicol acetyltransferase (CAT) gene and tested using a HEK-293 cell line by transfection CAT assays. The results confirmed that the DNA region indicated demonstrated strong basal promoter activity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:524635 |
| Date | January 2001 |
| Creators | Chen, Jing |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/3084/ |
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