Characterisation and Genetic Mapping of Genes With Potential Relevance to Neurodegenerative Disease

McCallion, Andrew Smyth (1997) Characterisation and Genetic Mapping of Genes With Potential Relevance to Neurodegenerative Disease. PhD thesis, University of Glasgow.

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Section 1 I have established a PCR based single strand conformation polymorphism (SSCP) assay for the detection of polymorphisms in PCR amplified genomic fragments. I have used this assay in the analysis of PCR products up to 300 bp in length from putative 3' untranslated regions (3'UTR) of mouse expressed sequence tags (ESTs) and show that an SSCP difference allowing mapping of ESTs on Mus spretus x C57BL/6 crosses is found at >90%. These studies report the mapping of 46 new mouse brain expressed sequence tags (ESTs) and one anonymous sequence tag to localised regions (? 2 cM) of the mouse genetic map. Assignment of these ESTs to chromosomes was carried out by SSCP analysis of sets of typed DNAs from animals in several backcross generations of a breeding programme that generates consomic lines with particular chromosomes deriving from M.spretus (SEG/Pas) on a C57BL/6 background. This allowed specific sets of recombinants to be selected from the European Collaborative Interspecific Backcross (EUCIB) resource for precise mapping. These experiments establish an effective strategy for the genetic mapping of multiple mouse ESTs. The particular EST set which I am mapping is enriched for novel and locally expressed genes of the substantia nigra, and may prove to be a good source of candidate genes for loci involved with neurological disorders. These studies also establish a qualitative RT-PCR screen evaluating the presence or absence of an RNA species in a known tissue at a specific timepoint. This work constitutes a significant step towards the goal of establishing an integrated gene map, combining information on the position of a gene, its sequence and its expression pattern. Section 2 A new mouse oligodendrocyte-specific gene was discovered in a differential screen, and subsequently found to be the mouse homologue of the recently discovered rat gene encoding the myelin-associated oligodendrocytic basic proteins (MOBP); a family of related highly basic myelin protein isoforms. MOBP is a new major component of CNS myelin. I have used SSCP analysis of a radioactively labelled PCR product from the 3'UTR of this gene in an assay to allow rapid genetic mapping, I report that Mobp is localised between D9Mit55 and D9Mit19 at 63.6 +/- IcM distal to the centromere of chromosome nine. Three mouse neurological mutations, spinner, tippy and ducky, map in this region of the mouse genome. The syntenic region of the human genome is 3p21-22. I have cloned the mouse gene Mobp in an endeavour to elucidate its genomic organisation and regulation. These studies demonstrate that the Mobp gene is a complex transcriptional unit. It comprises at eight discrete exons encompassing a genomic region in excess of 14 kb. Mobp also contains alternative internal splice donor/acceptor sites within at least two of its exons. I have utilised an oligo-capping assay in an endeavour to identify the transcription start points (tsp) associated with the Mobp gene. These studies have also revealed the presence of a novel transcriptional unit within the Mobp gene. In excess of 10 kb of sequence has been generated across the genomic region encompassing Mobp. Finally I have generated a gene targeting construct, designed to disrupt the Mobp gene, in an endeavour to begin to elucidate the function of this novel gene. These studies provide the platform for a more detailed analysis of the function of the Mobp gene product and subsequent evaluation of its possible involvement in known neuropathies.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: R Wayne Davies
Keywords: Genetics, Neurosciences
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-75243
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:28
Last Modified: 19 Nov 2019 21:28

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