Mutation of the mtATP6 gene, which encodes an essential subunit of the F0F1-ATP synthase (Complex V) in mitochondria, is known to cause a group of related encephalomyopathies. The ATP synthase acts as a hydrogen ion transporter that couples ion dissipation with ATP production. Diseases including NARP (neuropathy ataxia and retinitis pigmentosa) and MILS (maternally inherited Leighs syndrome) are caused by missense mutations in the ATP6 gene. Drosophila melanogaster, the common fruit fly, has a mitochondrial ATP6 missense mutation that models NARP/MILS diseases. Our aim is to develop a transgenic strategy where allotopic expression of a mitochondrial-targeted ATP6 mRNA may serve as a potential gene therapy for these devastating mitochondrial diseases. Mitochondria in metazoans are known to import nuclear encoded 5SrRNAs, which are thought to be essential for mitochondrial protein synthesis. We utilized a cluster of 100 individual 5S rRNA genes found at 56F region of the right arm of chromosome 2 in Drosophila melanogaster. Sequence comparisons revealed 17 groups of genomic variants and 14 processed rRNA counterparts. Identifying which, if any, of the known 5S rRNAs are competent for mitochondrial import was integral to our proposed gene therapy approach. A protocol was developed that utilizes gradient and percoll centrifugation steps to isolate highly purified mitochondria that lack detectable cytosolic contamination. RT-PCR and cloning were used to determine which 5S rRNAs were expressed and localized to the mitochondria. The cytoplasmic and mitochondrial derived clones and gDNA control clones support the assertion that, at least under normal in vivo conditions, ~ 60 % of the identified 5S rRNA genes are not expressed and are likely pseudogenes. One variant, 5S rRNA III, is predominantly expressed and localized to the mitochondria. Also, 8 novel and 3 possible 5S rRNA gene isoforms not currently categorized in sequence databases have been discovered. Clones capable of expressing chimeric rRNA::mRNAs in cells and in vivo were generated. These constructs could later be used to assess the ability of 5S rRNA to direct mitochondrial import of passenger mRNAs.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-09042007-102449 |
| Date | 07 September 2007 |
| Creators | Kotchey, Nicole Marie |
| Contributors | Donald B. DeFranco Ph.D., Gregg Homanics Ph.D., Ferruccio Galbiati Ph.D., Michael Palladino Ph.D. |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-09042007-102449/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0017 seconds