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Abstract Timber rattlesnakes (Crotalus horridus) face escalating threats in the Northeastern Appalachians, including habitat fragmentation, human encroachment, and the fungal pathogenOphidiomyces ophiodiicola. Using untargeted sequencing of DNA extracted from scale clips, we generated both host whole-genome and metagenomic data for 97 snakes from eight populations. Analysis of the snake genomes shows the populations surveyed exhibit relatively low levels of inbreeding and are genetically distinct, but that the degree of separation correlates only weakly with geographic distance. A genome-wide association analysis identified a locus associated with black-to-yellow color variation that contains an aldehyde dehydrogenase gene (ALDH4A1) related to genes involved in hair color differences in humans. Metagenomic analysis showed thatO. ophiodiicolaread counts were generally higher in snakes exhibiting clinical signs of Snake Fungal Disease, but some visually asymptomatic snakes had high pathogen loads. Together, these findings highlight the dual utility of untargeted sequencing for population genetics and pathogen surveillance, providing a foundation for future studies of adaptation, disease dynamics, and conservation in this declining species. 

Abstract Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both messenger RNA (mRNA) and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using hybridization chain reaction Flow-FISH (HCR Flow-FISH) for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to define the transcription start and splice sites of common synthetic promoters and independently vary the defined promoter and 5′ UTR sequences in HCR Flow-FISH. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for building more robust transgenic systems.