Ellis, Scott Anthony; (2000) Evolutionary and functional studies of the mouse retroviral restriction gene, Fv1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Fv1 is a gene of mice known to restrict the replication of Murine Leukaemia virus (MLV) by blocking integration by an unknown mechanism. The gene itself is retroviral in origin, and is located on the distal part of chromosome 4. The sequence of markers known to flank Fv1 in the mouse was used to identify sequence from the human homologues of these 2 genes. The construction of primers to these sequences permitted the screening of 2 YAC and a PAC human genomic libraries for clones containing either of these genes. The YAC libraries were negative for both markers. The human region was finally cloned as 2 overlapping PAC clones. This region was sequenced, and the comparison of this sequence and the analogous region in rat to the Fv1 region in mice allowed the determination of what sequence had been lost and gained during the formation of the gene. The Fv1 ORF of mice from across the Mus genus was PCR-amplified, cloned and sequenced. The analysis of this sequence has shown how Fv1 has evolved during the speciation of the Mus genus. By combining this data with what is known of the distribution of endogenous MLV and Fv1 activity among the Mus genus, a scheme of how and why Fv1 has evolved activity has been proposed. The mouse genome was screened for a 'progenitor' sequence(s) that may have given rise to Fv1 during its germline infection of Mus. A mouse 129/SvJ genomic library was screened by hybridisation for the sequences bearing homology to Fv1. Sequences obtained were shown to be the Fv1 gene itself, members of the murine endogenous retroviral-L (MERV-L) family, or had no known sequence homology as determined by BLAST searching. A polymorphism at an EcoR1 site was identified in the Fv1 gene of the 129 mouse that appears to be responsible for the presence of 2 bands that hybridise with an Fv1-specific probe in a 129 genomic southern blot. Fv1null cell lines expressing mutant Fv1 ORFs were established and assayed for Fv1 activity in an assay using pseudotyped MLV. This allowed the contribution of the 3 changes known to be important between the 2 main alleles to be assessed. The first amino acid change in the Fv1 ORF was found to be the major determinant of Fv1 phenotype, with the difference in the C-terminus contributing to a lesser extent. Fv1d allele, a less common allele of Fv1, was shown to be mediated by a single amino add change and not by a change in the level of Fv1 expression.

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