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Title: Characterisation of the murine gene and the porcine cDNA for lung surfactant protein D and genetic mapping of murine complement components using a novel approach
Author: Lawson, Peter R.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2000
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The work in this thesis describes the isolation and characterisation of the mouse surfactant protein D (SP-D) gene (Sftpd). This gene has been show to span 8 exons across 14 kb of DNA sequence and displays an overall organisation similar to the other collectin genes. The complete 5' untranslated region of the mRNA was also cloned which allowed the identification of the transcription start site for SP-D. Subsequently, the analysis of the promoter region of Sftpd revealed positional conservation of a number of transcription factor binding sites between the murine and human SP-D genes and the bovine conglutinin gene. A single copy SP-D-like gene has been shown to be present in mammals, birds and amphibians. Three polymorphic microsatellites were identified for the SP-D gene. In addition, the murine genes for surfactant protein A, SP-D and mannose binding lectin (MBL-A) were shown to co-segregate within a 5.64 cM area on chromosome 14. PCR cloning identified the presence of a CL-43-like gene in sheep, this represent the second member of the bovidea to contain CL-43. Specific antibodies were raised against mouse SP-A and SP-D by cDNA cloning fragments of these molecules and using them for protein expression. The complete cDNA sequence of the porcine cDNA was cloned. Three unique features were revealed in comparison to SP-D from other species. The collagen region contains an extra cysteine residue, which may have important structural consequences. Two other differences lie within loop regions of the carbohydrate recognition domain, a potential glycosylation site and an insertion of three amino acids, which may have functional implications by influencing carbohydrate binding. The porcine genes for SP-D and SP-A were also shown to co-localise to porcine chromosome 14, which is syntenic with murine chromosome 14. A novel and rapid PCR restriction fragment length polymorphism method was developed to exploit the murine expressed sequence tag (EST) database. This technique circumvents the laborious cDNA or genomic cloning steps of other mapping methods by relying on EST data and the prediction of exon-intron boundaries. This approach revealed that the C3a receptor, C1r and C1s genes form an unexpected complement gene cluster while the second MBL associated serine protease gene does not co-segregate with other complement activating serine protease genes. The gene localisations performed here help to explain the evolution of this group of proteins. This method can easily be applied to the genes of other systems, ranging from the interests of the individual researcher to large scale gene localisation projects.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Genes; Proteins