Creating deletion strains to study the role of the argonaute and dicer genes in RNA interference in Cryptococcus neoformans
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Alan Dale Curtis II (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Indrani Bose
Abstract: Cryptococcus neoformans is a spherical, encapsulated, basidiomycetous yeast and
the causative agent of cryptococcosis, a form of meningitis that affects the central
nervous system of immunocompromised individuals (immunocompromised means
patients with compromised immune systems). Since the 1980’s and the emergence of the
AIDS epidemic, much study has been concentrated on this fungus because cryptococcosis
is 100% fatal in untreated patients. Even with treatment, the condition does not always
decrease in severity, and no major advancements in antifungal drugs have been made in a
decade. Recently, Cryptococcus has been shown to possess the necessary machinery for
RNA interference (RNAi). RNAi is a method of post-transcriptional gene silencing that
may increase cryptococcal survival within mammalian hosts by controlling gene
expression at various stages of the life cycle through heterochromatin and euchromatin
rearrangement. RNAi was first described in Caenorhabditis elegans in 1998 by Andrew
Fire and his colleagues, and is best studied in Drosophila melanogaster. The pathway
includes argonaute and dicer proteins that aid in highly specific degradation of mRNA in vivo. To date, no organism has been shown to utilize RNAi as a factor for virulence,
nor should it be thought of as such; however, this pathway may indeed play a role in the
formation of certain virulence factors for Cryptococcus, including the transcription of
heat stress response genes required for thermotolerance, oxidative stress response genes
such as those required for melanin and mannitol biosynthesis, as well as the formation of
the polysaccharide capsule. Two separate, yet equally effective methods were used in
order to create deletion constructs of the AGO1 and AGO2 genes. Overlap PCR and
traditional cloning methods were employed. While overlap PCR was optimized,
molecular cloning techniques were also used to create the complementation constructs for
the above genes.
Through overlap PCR, this study resulted in the creation of deletion constructs for
the AGO1 and AGO2 genes, which are thought to play a role in RNAi. This study also
resulted in the creation of a complementation construct for the ago1 deletion. These
constructs can be biolistically inserted into C. neoformans to produce the corresponding
deletion strains. Once these strains are produced, future experiments will include many
phenotypic studies between them and other known strains, as well as an eventual murine
virulence study.
Creating deletion strains to study the role of the argonaute and dicer genes in RNA interference in Cryptococcus neoformans
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Created on 7/1/2009
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2009
- Keywords
- Argonaute, Cryptococcus neoformans, Dicer, RNA Interference, Virulence
- Subjects
- Cryptococcus neoformans
- Cryptococcus
- Genetic regulation