Hill, James (1991) Genetic manipulation and biochemical studies of Saccharomyces Cerevisiae. PhD thesis, University of Warwick.

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Abstract

The brewing properties of an industrial strain of Saccharomyces cerevisiae were investigated by laboratory scale brewing trials in the presence or absence of an uncoupler of oxidative phosphorylation (SX-1). When SX-1 was added the change in specific gravity of brewing wort with respect to time was less, yeast produced less biomass and more ethanol per unit drop in specific gravity than the control. Similar fermentation properties were observed for C2, a haploid laboratory yeast strain with the ability to ferment maltose. Recombinant DNA technology was used to generate a C2 pet mutant, specifically in the ATP12 gene, which encodes a protein essential for mitochondrial ATP synthesis. In brewing trials a comparison of C2 and C2:AATP 12 shows similar results to C2 fermentations in the presence or absence of SX-1 although the effects of SX-1 are more dramatic than with C2:AA TP 12. Together, the results of chemical studies and gene disruption mutagenesis suggest that mitochondrial ATP synthesis affects nuclear functions, although the possibility that results obtained are a consequence of changes to the yeast mitochondrial DNA cannot be eliminated.

Problems experienced with yeast DNA transformation protocols lead to the development of a new transformation method that is quicker and more efficient than the standard protocol. Initial studies revealed that DMSO could enhance yeast transformation efficiency, and that the optimal concentration of DMSO used is strain specific. The point at which DMSO was added was found to be important, with maximal transformation efficiency achieved when DMSO was added just before heat shocking. The optimised protocol for S. cerevisiae JRY188 routinely enhanced transformation IS- to 25-fold compared with a control transformation protocol. The osmotic condition was found to be important for DNA uptake as transformation was inhibited if yeast were washed in 1M sorbitol and selected on plates containing the same. Significantly, DMSO enhanced transformation even in the absence of captions, therefore this method may prove useful for yeasts which transform poorly by existing cationic-based yeast transformation methods.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QD Chemistry Q Science > QK Botany
Library of Congress Subject Headings (LCSH):	Saccharomyces cerevisiae
Official Date:	September 1991
Dates:	Date Event September 1991 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Griffiths, D. E.
Sponsors:	Science and Engineering Research Council (Great Britain) ; Whitbread and Company
Format of File:	pdf
Extent:	xi, 146 leaves : illustrations
Language:	eng

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