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Title: Quantitative measurement of the Ca²⁺-signature in living hyphae of Neurospora crassa, and a genomic analysis of Ca²⁺-signalling machinery in filamentous fungi
Author: Zelter, Alexander
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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The aims of this research were to develop and an aequorin-based approach for measuring cytosolic Ca2+ ([Ca2+]c) in living hyphae of Neurospora crassa and to use this method to investigate the contribution of individual proteins to the generation of the specific Ca2+-signatures associated with [Ca2+]c transients. Molecular and genomic methods were also used to identify Ca2+-signalling proteins in Neurospora crass, Aspergillus fumigatus and Magnaporthe grisea. Results confirmed that a reliable method for the quantitative measurement of [Ca2+]c in living N. crassa hyphae had been developed with the aequorin reporter system. This method was used to characterise Ca2+-signatures in N. crassa in response to (a) mechanical perturbation, (b) hypo-osmotic shock and (c) high external Ca2+ under different environmental conditions. Ca2+-signatures in response to these stimuli were shown to have a unique set of characteristics in response to each stimulus. These characteristics were apparent under all the conditions tested. Ca2+-signatures in response to the three stimuli were measured in wild-type N. crassa treated with Ca2+ antagonists and agonists and in untreated mutant strains of N. crassa compromised in Ca2+-signalling. In each case, differences in Ca2+-signatures could be quantitatively measured. Cloning of the cot-4 gene in the cot-4 morphological mutant of N. crassa showed it to encode the catalytic subunit of calcineurin, a Ca2+/calmodulin dependent protein phosphatase. An analysis of the genomes of N. crassa, A. fumigatus and M. grisea identified for the first time, many of the key Ca2+-signalling proteins present in filamentous fungi. An inventory of Ca2+-signalling proteins in filamentous fungi is an important starting point for reverse genetic and physiological approaches aiming at elucidating the biological significance of these proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available