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Inhibitory Smads (I-Smads) regulate TGF-β/BMP signaling through multiple distinct mechanisms, but whether different tissues preferentially employ specific mechanisms remains unknown. To address this question, we performed structure–function analyses of the Drosophila I-Smad, Dad, and its vertebrate orthologs Smad6 and Smad7 in neural and wing tissues, measuring outputs of BMP signaling in vivo. We identified a 24–amino acid putative DNA-binding domain within the MH1 domain of Dad that is essential for inhibitory function in wing tissue but unessential in neural tissue. Structural analyses revealed that ΔDNA-binding domain disrupts a β-hairpin structure homologous to R-Smad DNA-binding regions. We also found that Dad requires an intact MH1 domain to disrupt wing development, whereas either MH1 or MH2 can independently disrupt BMP signaling in motor neurons. These findings support a model where Dad functions through MH1-mediated transcriptional regulation in wing primordium, but through multiple mechanisms in neurons. Comparative analysis revealed that vertebrate I-Smad orthologs also show tissue-specific activity patterns, with structural predictions suggesting that Smad6 retains ancestral DNA-binding capacity, whereas Smad7 has evolved enhanced MH2-mediated functions. These results reveal context-dependent mechanisms of I-Smads that further the understanding of TGF-β/BMP pathway regulation. © 2026, Life Science Alliance, LLC. All rights reserved.
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One exciting class of future genetic devices could be those deployed in microbes that join complex microbial environments in the wild. We sought to determine whether genetic parts designed for monoculture are predictable when used in co-culture by testing constitutive Anderson promoters driving the expression of chromoproteins from a plasmid. In Escherichia coli monoculture, a high copy number origin of replication causes stochastic expression regardless of promoter strength, and high constitutive Anderson promoter strength leads to selection for inactivating mutations, resulting in inconsistent chromoprotein expression. Medium- and low-strength constitutive Anderson promoters function more predictably in E. coli monoculture but experience an increase in inactivating mutations when grown in co-culture over many generations with Pseudomonas aeruginosa. Expression from regulated promoters instead of constitutive Anderson promoters can lead to stable expression in a complex wastewater culture. Overall, we show intraspecies selection for inactivating mutations due to a competitive growth advantage for E. coli that do not express the genetic device compared to their peers that retain the functional device. We show additional interspecies selection against the functional device when E. coli is co-cultured with another organism. Together, these two selection pressures create a significant barrier to genetic device function in microbial communities that we overcome by utilizing a regulated E. coli promoter. Future strategies for genetic device design in microorganisms that need to function in a complex microbial environment should focus on regulated promoters and/or strategies that give the microorganism carrying the device a selective or growth advantage. IMPORTANCE: First-generation biotechnology focused on genetic devices designed for use in monoculture conditions. One class of next-generation biotechnology devices could be designed to function in complex ecosystems with other organisms, so we sought to create conditions where the genetic device retained function when the organism carrying it is in co-culture with other organisms. We discovered that when the genetic device is a significant resource burden on the organism carrying the device, mutations will be selected for due to intraspecies and interspecies selection pressures, and the device will be rendered non-functional. Therefore, genetic device design for complex ecosystems in next-generation biotechnology needs to balance functionality of the genetic device with the need to reduce resource burden on the organism carrying it.