A Model for Mitochondrial Disease Associated With Rearrangements of the Mitochondrial Genome

Glasssmith, Leslie L (1998) A Model for Mitochondrial Disease Associated With Rearrangements of the Mitochondrial Genome. PhD thesis, University of Glasgow.

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Individuals affected by mitochondrial diseases such as Kearns-Sayre syndrome (KSS) frequently possess a heteroplasmic population of mitochondrial DNA molecules consisting of multiple copies of both a wild-type mitochondrial genome and a mitochondrial genome which has undergone a major rearrangement. One hypothesis to account for the pathogenic effects of rearranged mtDNA molecules is that in affected individuals abnormal proteins, encoded by chimeric mitochondrial genes created by the rearrangement of mitochondrial DNA, contribute to mitochondrial dysfunction. In order to investigate the relationship between the expression of a chimeric peptide in mammalian mitochondria and mitochondrial dysfunction, three synthetic genes were constructed. The first, designated PI ("Patient I construct"), was a chimeric version of mouse cytochrome c oxidase I (COXI) based on a rearranged COXI gene found in the mutant mtDNA of a KSS patient (Poulton et al., 1989). The second, PIZ, was an epitope-tagged version of PI, and the third was a full length, universal-code version of mouse COXI. As no transfection system for mammalian mitochondria has been developed, it was necessary to construct genes which could be transcribed in the nucleus and translated in the cytosol, with subsequent import to mitochondria of the encoded protein. The alteration of the coding sequence of COXI from the mammalian mitochondrial genetic code to the universal code required 43 programmed point mutations using site-directed mutagenesis. In order that the proteins encoded by the synthetic genes could be targeted to mitochondria, a sequence encoding the cytochrome c oxidase IV mitochondrial transit peptide was subcloned 5' to the code-corrected genes. The epitope-tagged gene PIZ incorporated an epitope recognised by a mouse monoclonal antibody against a herpes simplex viral protein, to facilitate expression analysis Expression of PI, PIZ and the universal-code version of mouse COXI ail appeared to be highly toxic to E. coli, based both on difficulties experienced in subcloning, and on expression analysis of the constructs. Several hypotheses to account for this are discussed, including the possible interference with assembly/activity of the bacterial cytochrome c oxidase, and potential effects of the amphipathic transit peptide on membrane integrity. Mouse cell-lines which had incorporated both the PI and PIZ constructs were generated. However, the lack of an effective antibody with which to detect PI prohibited characterisation of the PI-transfected cell-lines at the protein level. Northern and Western analysis suggested that PIZ was expressed at both the RNA and protein levels in several PIZ-transfected 3T3 clones. Western analysis also suggested that the PIZ protein may have been correctly processed proteolytically, which would indicate that the protein may be imported into the mitochondria. In order to test whether the chimeric peptide conferred a respiratory phenotype in the 3T3 cell line, assays of growth in media containing one of two alternative carbon sources (glucose or galactose) as the catabolic substrate were performed. Statistical analysis of the cell growth assays suggests that PIZ expression was inhibiting respiration. Staining with mitochondrial- specific dyes also suggested that respiration was inhibited in the PIZ-transfected cell-line so tested (clone PTZ8.3). Clone PIZ8.3 appeared to have incorporated multiple copies of the PIZ transgene. Under growth conditions which increase the requirement for respiration (galactose as the carbon source), clone PIZ8.3 was apparently deleting some copies of the transgene. This would support the conclusion that expression of the transgene is detrimental to respiration and cell survival or growth. The synthetic genes and cell-lines which have been created in this study can contribute to further investigation of the relationship between a chimeric mitochondrial peptide and mitochondrial dysfunction by enabling biochemical analysis of the observed respiration deficiency. They may also facilitate analysis of the pathogenicity in an organism through the creation of transgenic mice. The results obtained in this study support the view that the expression of chimeric genes encoded across rearrangement break-points in mitochondrial DNA has a deleterious effect on mitochondrial function, and hence could play a significant role in mitochondrial disease pathogenesis.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Howry Jacobs
Keywords: Genetics
Date of Award: 1998
Depositing User: Enlighten Team
Unique ID: glathesis:1998-75378
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 20:21
Last Modified: 19 Nov 2019 20:21
URI: https://theses.gla.ac.uk/id/eprint/75378

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