Nerve degeneration is the basis for many devastating diseases such as Alzheimer’s and Parkinson’s. In order to understand this process, it is first necessary to understand gene expression in nerve cells. One such gene that may be involved in neurodegeneration in the model organism C. elegans encodes the transcription factor DAF-19. Previous experiments compared gene expression from daf-19 mutant and wild-type worms to identify 176 potential downstream target genes of daf-19. In addition, several search algorithms identified a relatively conserved DNA sequence motif, dubbed the Sandbox, in 69 of these target genes. We hypothesize that DAF-19 could bind to this sequence, controlling expression of the genes. I am testing this hypothesis with four candidate genes; these genes thus may have a role in maintaining synaptic function.
The aim of this project is to successfully clone putative control regions of W03G1.7, M01F1.4, Y51H7C.1, and F58E2.3 into reporter vectors upstream of the coding region of green fluorescent protein to characterize the target genes expression pattern. The presence of the putative control regions of the genes Y51H7C.1 and F58E2.3 are currently being confirmed. Chimeric reporter genes containing the putative upstream control regions of the genes W03G1.1 or M01F1.4 and the coding region of green fluorescent protein were successfully constructed and microinjected into the gonad of daf-19 mutant worms. Confocal microscopy was used to determine whether these genes are expressed in neurons and/or hypodermal tissue, as is daf-19. Unexpectedly, expression of GFP driven by the W03G1.7 control region was not observed in worms lacking the DAF-19 transcription factor. If comparisons of this expression pattern to that in wild type worms differs in the neurons or hypodermis, it may be concluded that this gene is DAF-19 dependent and regulated by the Sandbox.
GFP expression patterns in M01F1.4 transgenic worms are observed to be localized in the intestinal cells; however, it was later found that the captured putative control region of this gene does not actually contain the Sandbox motif. Therefore, results determining if the Sandbox plays a role in the regulation of this gene are inconclusive.
Level of Honors
Elizabeth De Stasio
Zuleger, Brenda, "A Molecular Approach to the Analysis of Neuronal Protein Regulation in C. elegans" (2012). Lawrence University Honors Projects. 24.