Analysis of Sense and Antisense Transcripts during the Giardia lamblia Lifecycle

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DESCRIPTION (provided by applicant): This proposal is centered on understanding the transcriptional and post-transcriptional mechanisms that regulate gene control in Giardia lamblia. We are particularly interested in understanding the connection between the abundant antisense transcripts and gene expression in this intestinal protozoan pathogen. Developing a strong understanding ofthe parasite's molecular biology and gene regulation will enable researchers to develop new chemotherapeutic treatments to disrupt the critical life stages of the Giardia parasite. Our hypothesis is that the parasite uses a combination of mechanisms to regulate global gene expression during developmental periods. The research focuses on three areas of gene regulation; (1) mRNA stability, (2) chromatin-level control of gene regulation, and (3) examination of post-transcriptional modifications that affect gene expression. The specific aims are to examine the stability of sense/antisense transcripts in trophozoites and cysts, to investigate the effect of a drug that affects chromatin remodeling oh sense/antisense transcript production, and to understand the modifications and fates of mRNA sense and antisense transcripts. To measure mRNA stability, we will treat cells with the transcription inhibitor Actinomycin D and examine the rates of degradation of sense and antisense transcripts for a specified list of genes using qRT-PCR. To examine the effect of chromatin level control, specifically methylation, on transcription, we will conduct drug studies using 5-Azacytidine and quantify sense and antisense transcripts levels as above. During excystation, we will investigate the 5' modification of transcripts using RNA ligase- mediated RT-PCR. FISH will be used to image transcript localization within the cell. Giardiasis, like many other waterborne parasitic diseases is prevalent in areas that lack basic sanitation and adequate plumbing. Critical to the parasite's survival is its ability to enter and exit the cyst stage. Understanding ofthe parasite's molecular biology is a priority if we are to develop novel chemotherapeutic treatments for giardiasis.