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Mitochondrial Transfer in Saccharomyces cerevisiae

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thesis
posted on 2021-12-07, 14:05 authored by Hummel, Sonja

This thesis investigated mitochondrial transfer in Saccharomyces cerevisiae, between respiratory compromised B18p⁰ recipient and respiratory competent donor cells. The respiratory compromised strain had three red fluorescent proteins tagged to the membrane, nucleus and cytoplasm (triple RFP-B18p⁰) and is referred to as the B18p⁰ strain. B18p⁰ cells did not contain mitochondrial DNA, causing it to be respiratory compromised and required a fermentable carbon source, such as glucose/dextrose, for proliferation. The respiratory competent strain used had a green fluorescent protein tagged to the Tom70 mitochondrial protein (Tom70-GFP) and is referred to as the Tom70 strain. The Tom70 cells contained the nuclear encoded URA3 cassette, allowing for negative selectivity of this strain using 5-FOA.  S. cerevisiae strains were co-cultured together in media containing only non-fermentable carbon sources (YPGE), plated on YPGE plates containing 5-FOA and colonies grown were distinguished post-co-culture based on their distinct phenotypic and genotypic characteristics. Fluorescent analysis of co-culture colonies revealed the presence of 5-FOA resistant Tom70 cells and some red B18p⁰ cells that had acquired the ability to grow on non-fermentable carbon sources. Genotypic analysis revealed that the majority of these red colonies had acquired mtDNA as well as the nuclear encoded, Tom70 specific URA3 cassette. Several permutations of co-cultures were performed, using different ratios of recipient and donor cells and single-gene deletion donor cells.  Purified mitochondria from Tom70 cells were tried to be transferred into B18p⁰ cells using centrifugation forces to induce a higher occurrence frequency of mitochondrial transfer. Metabolic support experiments were conducted to investigate if the Tom70 strain could provide metabolic support to the B18p⁰ strain without mitochondrial transfer.  Results indicate that no permutation induced potential mitochondrial transfer at a higher rate than others. However, results indicate that mitochondrial transfer did occur at low frequencies, potentially through the fusion of respiratory competent and respiratory compromised cells. Forced transfer did not increase the occurrence frequency of B18p⁰ cells to take up mitochondria and Tom70 cells did not provide metabolic support to B18p⁰ cells.

History

Copyright Date

2019-01-01

Date of Award

2019-01-01

Publisher

Te Herenga Waka—Victoria University of Wellington

Rights License

Author Retains Copyright

Degree Discipline

Biomedical Science

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level

Masters

Degree Name

Master of Biomedical Science

Victoria University of Wellington Unit

Centre for Biodiscovery

ANZSRC Type Of Activity code

1 PURE BASIC RESEARCH

Victoria University of Wellington Item Type

Awarded Research Masters Thesis

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

McConnell, Melanie; Munkacsi, Andrew