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Title: Adapting yeast to the study of higher eukaryotes : 'humanisation' of histone H4
Author: Rushton, Sally
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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Seven of the eight differences between the yeast and human histone H4 amino acid sequences fall within the globular histone fold domain. The other is a conservative isoleucine to value substitution in the more flexible N-terminal tail (I21V). A yeast mutant expressing a form of histone H4 with the seven globular mutations is unable to grow on non-fermentable carbon sources. Through Southern blotting and DAPI staining, this phenotype is shown to be due to loss of mitochondrial DNA (mtDNA). This leads to non-functional electron transport, and defines the strain as a ‘petite’ mutant. This strain also shows a genome-wide reduction of histone H4 acetylation levels to 30% of WT. Further incorporation of the I21V substitution generates a strain expressing human histone H4. This highly conservative mutation at an apparently distal site confers a partial rescue to its parent strain. MtDNA is not lost, and growth on non-fermentable media is restored. Histone H4 acetylation is at 60% of WT. The final histone H4 mutant of the set contains I21V in isolation. This strain exhibits heightened growth on non-fermentable media, and normal histone H4 acetylation levels. Microarray experiments were designed to allow cross referencing between two complementary sets of genome wide expression data. This enabled changes in gene expression down-stream of mtDNA loss to be ‘filtered out’. This analysis identifies genes which may have a novel, but indirect role in mtDNA maintenance. Additionally they reveal that the altered levels of histone H4 acetylation are not due to changes in transcript levels of enzymes conferring or removing these modifications. The effect is a more direct consequence of the mutations. I hypothesise that the interaction between globular domain and N-terminal tail mutations may be at the level of gene silencing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available