Type of Document Dissertation Author Brumfield, Kristy Marie Author's Email Address firstname.lastname@example.org URN etd-10022009-140919 Title Investigations of Chlamydomonas reinhardtii Ergosterol Biosynthesis Degree Doctor of Philosophy (Ph.D.) Department Biological Sciences Advisory Committee
Advisor Name Title Moroney, James V. Committee Chair Bartlett, Sue Committee Member Donze, David Committee Member Longstreth, David J. Committee Member Moore, Thomas S. Committee Member Cooper, Richard Dean's Representative Keywords
- Chlamydomonas reinhardtii
Date of Defense 2009-08-14 Availability unrestricted AbstractErgosterol is the major sterol found in the membranes of Chlamydomonas reinhardtii. While past studies have identified some ergosterol mutants in C. reinhardtii, very little is
known about sterol biosynthesis pathways in this species. With the elucidation of the Chlamydomonas genome, bioinformatics analysis has allowed us to determine potential genes involved in ergosterol biosynthesis. With this knowledge, a working model of the pathway was designed for future analysis. Several of the ergosterol biosynthetic genes were analyzed in respect to their role and involvement in flagellar regeneration. These genes were upregulated during the regrowth of the flagella. Also Chlamydomonas strains lacking flagella were analyzed by Q-RT PCR to determine what role ergosterol biosynthetic genes played in the absence of their flagella. Finally, one of the genes with
homology to the yeast sterol C-5 desaturase, ERG3, was chosen for further analysis. To test whether ERG3 of C. reinhardtii had a similar function, yeast Saccharomyces cerevisiae ERG3 knockout strains were created to assess whether a plasmid expressing the Chlamydomonas ERG3 could complement the deletion. These erg3 null mutants were transformed with a vector expressing ERG3 cDNA from C. reinhardtii driven by the yeast ADH1 promoter, and this expression was able to restore ergosterol biosynthesis and reverse phenotypes associated with lack of ERG3 function. Complementation of these erg3 null phenotypes strongly suggests that ERG3 in C. reinhardtii functions as a sterol C-5 desaturase. Results from this dissertation provides the groundwork for future experimentation in the field of sterol lipid research in Chlamydomonas reinhardtii.
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