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A screen for mutations that prevent lethality caused by expression of activated sevenless and Ras1 in the Drosophila
A Maixner1, T P Hecker, Q N Phan
1Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
Ras1 plays a critical role in receptor tyrosine kinase (RTK) signal transduction pathways that function during Drosophila development. We demonstrate that mis-expression of constitutively active forms of Ras1 (Ras1V12) and the Sevenless (Sev) RTK (SevS11) during embryogenesis causes lethality due to inappropriate activation of RTK/Ras1 signaling pathways. Genetic and molecular data indicate that the rate of SevS11/sev-Ras1V12 lethality is sensitive to the expression level of both transgenes. To identify genes that encode components of RTK/Ras1 signaling pathways or modulators of RNA polymerase II transcription, we took advantage of the dose-sensitivity of the system and screened for second site mutations that would dominantly suppress the lethality. The collection of identified suppressors includes the PR55 subunit of Protein Phosphatase 2A indicating that downstream of Sev and Ras1 this subunit acts as a negative regulator of phosphatase activity. The isolation of mutations in the histone deacetylase RPD3 suggests that it functions as positive regulator of sev enhancer-driven transcription. Finally, the isolation of mutations in the Trithorax group gene devenir and the characterized allelism with the Breathless RTK encoding gene provides evidence for Ras1-mediated regulation of homeotic genes.
Insights
Mis-expressing Ras1 and Sevenless receptor tyrosine kinase (RTK) in Drosophila causes lethality. Suppressor mutations identified new regulators of RTK/Ras1 signaling and transcription, including Protein Phosphatase 2A and RPD3.
Area of Science:
- Developmental Biology
- Molecular Genetics
- Signal Transduction
Background:
- Ras1 is crucial for receptor tyrosine kinase (RTK) signaling in Drosophila development.
- Overactivation of RTK/Ras1 pathways during embryogenesis leads to lethality.
Purpose of the Study:
- Identify components of RTK/Ras1 signaling pathways.
- Discover modulators of RNA polymerase II transcription.
- Utilize a dose-sensitive lethality system to screen for suppressors.
Main Methods:
- Generated a dose-sensitive lethal Drosophila model by mis-expressing active Ras1 (Ras1V12) and Sevenless RTK (SevS11).
- Conducted a genetic screen for second-site mutations that dominantly suppress the observed lethality.
- Analyzed suppressor mutations to identify novel pathway components and transcriptional regulators.
Main Results:
- Identified the PR55 subunit of Protein Phosphatase 2A as a negative regulator downstream of Sev/Ras1.
- Isolated mutations in the histone deacetylase RPD3, suggesting a positive role in sev enhancer-driven transcription.
- Found mutations in the Trithorax group gene devenir, with allelism to Breathless RTK, indicating Ras1-mediated regulation of homeotic genes.
Conclusions:
- The study reveals novel negative and positive regulators of RTK/Ras1 signaling and transcription in Drosophila.
- PR55 and RPD3 are implicated in modulating RTK/Ras1 pathway output and transcriptional control.
- Ras1 signaling influences homeotic gene regulation through pathways involving Trithorax group genes and RTKs.