Thesis

The characterization of obi-1 transcripts in response to changes in cGMP levels in the nematode Pristionchus pacificus

Interactions between organisms involve highly specific modes of communication. The nematode Pristionchus pacificus displays a nematode-invertebrate interaction with its host beetle Exomala Orientalis known as necromeny. In order to determine molecular factors mediating oriental beetle pheromone odor sensing, a forward genetic screen was previously performed for a mutants that were unable to sense the beetle’s sex pheromone Z-7-tetradecen-2-one (ZTDO). One of the isolated Oriental Beetle pheromone Insensitive mutants Ppa-obi-1, displayed the stronger loss of attraction phenotype. Cloning of the full length sequence revealed that the longest Ppa-obi-1 transcript is 1,607 bp long and at least 5 splice variants were identified by sequencing. Ppa-obi-1 as well as Ppa-egl-4 transcriptional abundance was measured by Real Time PCR assay. Chemosensory defects found in Ppa-obi-1, as well as decreased levels of Ppa-obi-1 transcript found in Ppa-obi-1(tu404) mutants following cGMP treatment strongly implicate that Ppa-OBI-1 may be a regulator of cGMP levels to modulate host pheromone recognition. Ppa-obi-1p::cDNA::3’UTR construct was made for transgenic rescue of Ppa-obi-1(tu404) mutant phenotype and translational reporter gene Ppa-obi-1p::cDNA::GFP::3’UTR fusion was created for a protein expression patterns characterization. We also searched for orthologous P. pacificus’ glial cells and neuronal sequences known to be highly enriched in Caenorhabditis elegans. Using a candidate gene approach, the primers were generated to potential P. pacificus orthologs of known C. elegans genes that are highly expressed in the sheath cells and neurons. This study describes 10 of the best-hit reciprocal orthologs of glia enriched transcripts and 3 potential neuronal markers in P. pacificus based on partial cDNA and 5’ UTR sequences. Promoter GFP and mCherry reporter transgenes were made to monitor expression of these putative glial markers in P. pacificus.

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