McVie, Alison Jane (1997) Molecular analysis and physical mapping of the human 3beta-hydroxysteroid dehydrogenase sigma 5/sigma 4 isomerase gene family. PhD thesis, University of Glasgow.
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Abstract
3beta-hydroxysteroid dehydrogenase (3beta -HSD) catalyses the conversion of 3beta-hydroxy- Delta5-steroids into the corresponding Delta4-3-ketosteroids and is essential for the biosynthesis of mineralocorticoids, glucocorticoids and sex hormones. Two isoforms encoded by two highly homologous, closely linked 3beta-HSD genes (HSD3B1 and 2) are known to be expressed in humans. However, Southern blot analysis and 3B-HSD type II mutation screening suggested that there was more than two HSD3B genes in humans; therefore, two human genomic gammagem11 libraries were screened with 3beta-HSD type I cDNA and bacteriophage clones containing novel 3beta-HSD sequences were identified. Two of the phage clones were characterised and the segments equivalent to HSD3B1 coding regions were sequenced. The 3beta-HSD coding sequence determined from both of these clones contained frameshift mutations resulting in premature stop codons, and it was concluded that these sequences were unprocessed pseudogene members of the 3beta-HSD gene family. The library screens generated 5 new members of the 3beta-HSD gene family and these were mapped by fluorescent in situ hybridisation (FISH) to chromosome 1p13, the same region of the genome as HSD3B1 and 2. Specific oligonucleotide primer pairs were designed for each gene and using PCR the genes were mapped to a set of 3 overlapping yeast artificial chromosomes (YACs) and 9 overlapping bacterial artificial chromosomes (BACs). The gene order was subsequently confirmed using restriction analysis and 3beta-HSD-specific oligonucleotide probes. The orientation of HSD3B1, 2, Psi1 and Psi4 was determined by extensive restiction analysis of the BACs and the positions of the endpoints in three BAG clones. The estimated length of the entire contig is 500kb with the 3beta-HSD gene cluster over a centrally-based 235kb fragment. cDNA selection techniques were established to detect expressed sequences from the region of the 3beta-HSD gene cluster. Three BAG clones from the contig were biotinylated and hybridised to a placental PGR-amplifiable cDNA library. The hybrids produced were isolated using streptavidin coated magnetic beads. Many 3beta-HSD transcripts were detected from these experiments indicating that the selection process was successful, however no previously identified genes were detected that could be localised to the 3beta-HSD locus. Although, several unknown sequences were discovered which may belong to unidentified genes present within or close to the 3beta-HSD gene cluster.
Item Type: | Thesis (PhD) |
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Qualification Level: | Doctoral |
Additional Information: | Adviser: Roger Sutcliffe. |
Keywords: | Genetics. |
Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology |
Colleges/Schools: | College of Medical Veterinary and Life Sciences |
Date of Award: | 1997 |
Depositing User: | Enlighten Team |
Unique ID: | glathesis:1997-71366 |
Copyright: | Copyright of this thesis is held by the author. |
Date Deposited: | 10 May 2019 10:49 |
Last Modified: | 17 Oct 2022 08:40 |
Thesis DOI: | 10.5525/gla.thesis.71366 |
URI: | https://theses.gla.ac.uk/id/eprint/71366 |
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