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Title: The development of transgenic aequorin as an indicator for cytosolic free calcium in Neurospora crassa
Author: Collis, Amanda J.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1996
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The aim of this project was to develop techniques for the measurement of cytosolic free calcium ([Ca2+]c) in Neurospora crassa, using the Ca2+-sensitive photoprotein aequorin. An in vitro assay of aequorin luminescence was developed for the screen of apoaequorin yield in primary transformants. The assay confirmed that the apoprotein produced by N. crassa was fully functional. The yield of apoaequorin from both transformant series was low, at best 13 fg/μg total soluble protein. Isolation of homokaryotic derivatives from primary transformants improved this level by up to 11-fold, though this was still 32-times lower than that obtained in apoaequorin transformed Nicotiana plumbaginifolia. Possible reasons for poor transgene expression were explored. Southern analysis showed that virtually all transformants contained at least one intact apoaequorin expression cassette. Northern analysis revealed an apoaequorin mRNA which corresponded to the expected transcript size. Apoaequorin mRNA abundance in N. crassa primary transformants was at best approximately 2-fold less than in N. plumbaginifolia, this was not reflected in the protein yield which was almost 400-fold lower. Northern analysis confirmed that the block acting on apoaequorin yield must be at translation or beyond. The codon requirements of apoaequorin and codon usage in a number of apoaequorin transformed species was examined. In general, species which showed a much more compatible codon usage produced more apoaequorin. Apoaequorin was shown to be somewhat unstable in N. crassa with a half life of approximately 45 minutes, which may be a contributory factor to the low yield. Methods were developed whereby the transformed strains were able to report changes in their [Ca2+]c. Both apoaequorin and coelenterazine were shown to be non-toxic, and aequorin was regenerated in cells simply by incubation with coelenterazine. Background sources of luminescence were identified and methods for lysing N. crassa cells were developed. Lysis is a prerequisite for the calibration of Ca2+ concentration from aequorin light emission.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available