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Resources » Paper

van Gilst MR et al. (2000) West Coast Worm Meeting "Molecular Mechanisms of Daf-12"

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    Status:
    Publication type:
    Meeting_abstract
    WormBase ID:
    WBPaper00017009

    van Gilst MR, Yamamoto KR, Antebi A, & Shostak Y (2000). Molecular Mechanisms of Daf-12 presented in West Coast Worm Meeting. Unpublished information; cite only with author permission.

    Dauer larva formation in Caenorhabditis elegans is regulated by the intracellular receptor daf-12, which functions at the convergence of TGF-b, cGMP, and insulin-like signaling pathways. daf-12 has also been implicated in the regulation of C. elegans lifespan and developmental age. Alleles of daf-12 with distinct protein sequence alteration spartially uncouple its phenotypic effects. As a step toward understanding the molecular biology of Daf-12 function, we identified specific DNA sites in the C. elegans genome bound by the protein in vitro. We developed an in vitro selection and amplification method that, using immobilized recombinant daf-12 DNA binding domain, yielded a series of specific C. elegans genomic DNA fragments. We inserted some of these fragments into yeast reporter plasmids and showed that Daf-12 selectively activated transcription of the reporter genes in Saccharomyces cerevisiae. Hence, C. elegans DNA fragments bound specifically by Daf-12 in vitro display Daf-12 response element activity in vivo. Intracellular receptors are multidomain proteins with characteristic DNA binding, putative ligand binding, and potentially ligand-controlled transcriptional regulatory domains. We used the heterologous yeast expression system to investigate contributions of different Daf-12 regions to transcriptional regulation. We found thatDaf-12 derivatives with truncated ligand binding domains are potent transcriptional activators, and mapped an activation domain to the "hinge region" just downstream of the DNA binding domain. In future work, we shall determine whether the identified response elements are linked to Daf-12regulated target genes in C. elegans. Similarly, we shall examine the roles of the activation domain in Daf-12 biology and molecular function in the animal.


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