Impact of Yih1 and Gcn2 on filamentous growth in yeast : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Manawatu, New Zealand. EMBARGOED until 12th January 2027.
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Date
2024
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Massey University
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Abstract
Filamentous growth occurs in yeast in response to nutrient limitation. It is characterized by elongated cell shape, cells that remain connected after cytokinesis and invasive growth. Invasive growth describes the ability of the filaments to invade surfaces, such as solid agar mediums. This study presents evidence that General control non-depressible 2 (Gcn2) can promote invasive growth in yeast. Gcn2 is an important component of the General Amino Acid Control (GAAC) pathway in yeast. Gcn2 is activated by amino acid starvation and promotes cell survival. Active Gcn2 changes the gene expression profile of the cell and upregulates genes required to overcome the stressor and down regulates those that are not. Yeast Impact Homolog (Yih1) is a competitive inhibitor of Gcn2 as it binds the co-effector protein, Gcn1, to prevent the Gcn1-Gcn2 interaction that is required for Gcn2 activation. This study aimed to optimize a technique to evaluate filamentous growth in various Saccharomyces cerevisiae strains. It used Ʃ1278b strains to optimize the plate washing assay, microscopy and the flocculation test as methods for visualizing filamentous growth. This study outlines the strengths and weaknesses of each method in evaluating filamentous growth and the individual characteristics of filamentous growth. This study also aimed to find a potential role for Yih1 or Gcn2 during invasive growth. Expression studies were performed, involving Yih1 and Gcn2 and the plate washing assay was used to determine the change in invasiveness. We found a significant decrease in invasive growth when a Yih1 fragment that binds actin and Gcn1 was overexpressed. We also saw a significant increase in invasive growth when Gcn2 was overexpressed under starvation conditions and when a constitutively active form of Gcn2 was overexpressed. Finally, this study also aimed to evaluate filamentous growth in non-filamentous, S288C background strains. We introduced a functional FLO8 gene on a plasmid into the H1511/BY4741 diploid strain and restored filamentous growth. We present evidence that S288C background strains have accumulated genomic mutations, in addition to the FLO8 mutation, which make them non-filamentous. Understanding invasive growth is important as it is characteristic of many pathogenic fungal species and has been shown to be required for pathogenesis in many cases. Also, further characterizing the various roles of Gcn2 and Yih1 is paramount to further understand how abnormal Gcn2 function promotes various diseases and disorders.
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Embargoed until 12th January 2027