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Characterization of downstream elements in a Raf-1 pathway
G J Liaw1, E Steingrimsson, F Pignoni
1Department of Biology, University of California, Los Angeles 90024.
Abstract:
At the poles of the Drosophila embryo, cell fate is established by a pathway that begins with the activation of a membrane-associated tyrosine kinase (the torso gene product); this then leads to activation of a serine/threonine kinase (Drosophila Raf-1). Activated Raf-1 then leads, by an undefined mechanism, to the transcriptional activation of the tailless (tll) gene; the tll gene product, itself a transcription factor, subsequently regulates the expression of an array of target genes. To further define this pathway, we have utilized sequence comparison between Drosophila melanogaster and Drosophila virilis to identify conserved elements in the tll promoter region. As assessed by DNase I footprinting and promoter dissection experiments, two of these elements are potential regulatory targets of Raf-1-activated transcription factors. Sequence comparison also reveals that the unique residues in the DNA-binding domain of the tll protein, the next component in the pathway, are conserved. One of these residues, the alanine after the last cysteine in the first zinc finger, may be responsible for part of the difference between the tll protein DNA binding site and the closely related half-site of the retinoid/estrogen receptors. Consistent with the rapid turnover of the tll protein, it contains a PEST sequence (rich in proline, glutamate and aspartate, serine, and threonine) that is also conserved.
Insights
Investigating the Drosophila embryo
Area of Science:
- Developmental biology
- Genetics
- Molecular biology
Background:
- Cell fate determination in Drosophila embryos involves a signaling pathway initiated by torso tyrosine kinase.
- This pathway activates Drosophila Raf-1, a serine/threonine kinase, which then upregulates the tailless (tll) gene transcription.
Purpose of the Study:
- To elucidate the regulatory mechanisms of the tailless (tll) gene.
- To identify conserved regulatory elements in the tll promoter region and understand their role in the signaling pathway.
Main Methods:
- Comparative sequence analysis of tll promoter regions between Drosophila melanogaster and Drosophila virilis.
- DNase I footprinting and promoter dissection experiments to identify regulatory elements.
- Sequence analysis of the tll protein's DNA-binding domain.
Main Results:
- Identified conserved elements in the tll promoter, two of which are potential targets for Raf-1-activated transcription factors.
- Revealed conserved unique residues in the tll protein's DNA-binding domain, potentially explaining differences from retinoid/estrogen receptors.
- Confirmed the conservation of a PEST sequence in the tll protein, correlating with its rapid turnover.
Conclusions:
- The study defines key regulatory elements in the tll promoter targeted by the torso-Raf signaling pathway.
- Identified conserved structural features in the tll protein's DNA-binding domain and its PEST sequence, contributing to its function and regulation.