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Molecular identification of let-56 and characterization of its regulatory elements in Caenorhabditis elegans

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(Thesis) M.Sc.
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let-56 was generated by EMS mutagenesis in a screen for essential genes in the unc-22(IV) region of C. elegans. The let-56 locus is the largest developmentally required mutagenic target in the area. By sequencing seven of twelve alleles, I have determined the molecular identity of let-56 to be ZK829.4, encoding glutamate dehydrogenase. GDH catalyzes the reversible formation of α-ketoglutarate and ammonia from glutamate, and mutations in GDH are associated with hyperinsulinism/hyperammonemia syndrome in humans. To gain better understanding of GDH activity, I have investigated mechanisms of its regulation. By analyzing let-56’s 5’ promoter region, I have identified a 31bp window containing a transcription factor binding site. Using chimeric GFP reporter constructs, I have also demonstrated the importance of let-56’s 3’ UTR in downregulating gene expression. In addition, by mimicking a mutation found in individuals with HI/HA, I have shown that C. elegans GDH also plays a role in insulin regulation.
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