Abstract
Caenorhabditis elegans is a multicellular androdioecious eukaryotic organism that serves as a model for developmental and genetic studies due to its completely sequenced genome. The application of reverse genetics methodologies such as RNAi and use of fluorescent tags to investigate gene expression makes this nematode an ideal model to explore metal toxicity.A new family W08E(12.2-12.5) of cysteine rich (13%) protein coding genes containing four isoforms (W08E12.2, W08E12.3, W08E12.4 and W08E12.5 consecutively aligned on chromosome IV were investigated. W08E(12.2-12.5) are ≥ 90% similar in their coding region and the promoters of W08E12.3 and W08E12.4 display an identity of 100%. Occurrence of such highly similar genes consecutively in the compact worm genome highlights the importance of their study. Cloning and sequencing of the genomic regions of the isoforms confirmed the presence and order of all the four genes within the worm genome. Bioinformatic screening of the isoforms predicted the presence of putative metal binding sites similar to metallothioneins within the respective promoter regions. From the bioinformatics screening it was demonstrated that the existence of highly similar W08E(12.2-12.5) isoforms is a rare (0.6%) event and only highly essential genes required for the nematode survival occur at that frequency within the worm.
Transgenic-GFP tagged worms showed constitutive expression of PW08E12.3/4::GFP in the pharyngeal region which is regulated by metals (Cd, Zn, Cu). The transgenic worm metal exposure along with the qPCR assays of the metal exposed nematodes revealed strong metal responsiveness of the gene family. Knockdown by RNAi (W08E12.3/4/5) was used as a tool to characterize the phenotype of the gene family. The phenotypic studies determined that the growth of the nematode is affected (drastically in the presence of metals) upon knockdown of these genes. The RNAi metal exposed worms were further screened by X-ray laser imaging at Stanford Linear Accelerator Centre (SLAC), which revealed vast Zn accumulation in the pharyngeal area of the W08E(12.3-12.5) knockdown worms demonstrating the role of the gene family in metal (Zn) sensing/ homeostasis/detoxification.
In order to characterize the function at the protein level, the genes were sub cloned into an intein tag fused vector but the approach failed due to internal cleavage of the protein from the tag. The S-tag based PET vector was used as an alternative to express the W08E12.3 protein. Purification of the tagged protein enabled the determination of the metal binding properties of the protein by utilizing pH titration and metal displacement assays. The assays performed with the W08E12.3 protein depicted its strong Zn binding capability which correlates with the metal accumulation data. Taken together, it can be concluded that W08E(12.2-12.5) gene family is regulated transcriptionally by metals and is also metal binding in nature. Future studies of the W08E(12.2-12.5) family might add to ongoing efforts to develop enhanced biomarkers for metal toxicosis.
| Date of Award | 1 Apr 2016 |
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| Original language | English |
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| Supervisor | Stephen Sturzenbaum (Supervisor) & Wolfgang Maret (Supervisor) |