Project description:Understanding and predicting the relationships between genotype and phenotype is often challenging, largely due to the complex nature of eukaryotic gene regulation. A step towards this goal is to map how phenotypic variation evolves through genomic changes that modify gene regulatory interactions. Using the Prairie Rattlesnake (Crotalus viridis) and related species, we integrate mRNA-seq, proteomic, ATAC-seq and whole genome resequencing data to understand how specific evolutionary modifications to gene regulatory network components produce variation in venom gene expression. Through comparisons within and between species, we find a remarkably high degree of gene expression and regulatory network variation across even a shallow level of evolutionary divergence. We use these data to test hypotheses about the roles of specific trans-factors and cis-regulatory elements, how these roles may vary across venom genes and gene families, and how variation in regulatory systems drive variation in venom phenotypes. Our results illustrate that variation in chromatin and genotype at regulatory elements plays major roles in modulating expression. However, we also find that enhancer deletions, variation in transcription-factor expression, and variation in activity of the insulator protein CTCF also impact downstream venom phenotypes. Our findings provide insight into the diversity and gene-specificity of gene regulatory features and highlight the value of comparative studies to link gene regulatory network variation to phenotypic variation.
Project description:We generated ATAC-seq data for pre- and post-extraction venom gland samples and H3K4me3, H3K27ac, and CTCF ChIP-seq from post-extraction venom gland samples from the Prairie Rattlesnake to investigate patterns of chromatin accessibility, transcription factor binding, and insulation during venom production, and to identify open promoters and active enhancer regions.
Project description:The venom color variation of C. d. terrificus (Cdt) is attributed to the presence of the toxin LAAO. However, the driving mechanisms of such variability have not been studied and identified so far. During the venom milking routine at Butantan Institute, we have noticed that most of the venoms of captive Cdt specimens show a yellowish color, while most of the venoms of wild specimens are white. Here we describe a comparative analysis of long-term captive (LTC) and recently wild-caught (RWC) Cdt, focusing on the enzyme LAAO. For the identification of LAAO in individual venoms, four different approaches were employed: evaluation of the enzymatic activity, SDS-PAGE, Western blotting and ELISA. In addition, mass spectrometry analysis was performed using pooled samples. Although some variation among these methodologies was observed, it was clear the significative higher percentage of individual venom samples presenting LAAO in LTC venoms. LAAO was identified in 60-80% LTC specimens and in only 10-12% of RWC specimens. Furthermore, this enzyme accounts for 5.6% of total venom proteins of LTC Cdt pooled venom, while it corresponds to only 0.7% of RWC Cdt pooled venom. These findings strongly suggest that the captive maintenance increases the expression of LAAO in Cdt venom.
Project description:Both single cell and bulk RNA sequencing was performed on expanding or differentiating snake venom gland organoids (from Aspidelaps Lubricus Cowlesi and Naja Nivea), or tissue (Aspidelaps Lubricus Cowlesi). Bulk RNA sequencing from the snake venom gland, liver and pancreas was performed to construct a de novo transcriptome using Trinity.
Project description:In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium bicarinatum and to unveil the potential of its products, high-throughput expressed sequence tags were generated using Illumina paired-end sequencing technology. A total of 212,371,758 pairs of quality-filtered, 100-base-pair Illumina reads were obtained. The de novo assemblies yielded 36,042 contigs for which 27,873 have at least one predicted ORF among which 59.77% produce significant hits in the available databases. The investigation of the reads mapping toxin class revealed a high diversification with the major part consistent with the classical hymenopteran venom protein signature represented by venom allergen (33.3%) followed by a diverse toxin-expression profile including several distinct isoforms of phospholipase A1 and A2, venom serine protease, hyaluronidase, protease inhibitor and secapin. Moreover, our results revealed for the first time the presence of toxin-like peptides that have been previously identified from unrelated venomous animals such as waprin-like (snakes) and agatoxins (spiders and conus). 300 ant specimens from the species Tetramorium bicarinatum were dissected in order to extract the RNA from their venom gland, The whole ant body was used as a reference,
Project description:While cellular transcripts encode rich information that provide key features to understand the molecular basis of snake venom variation, their presence/ abundance does not necessary imply/correlate in the translation of a functional (protein) product. In this study we carried out an analysis of the venom gland proteome of Bothrops jararaca taking into account two distinct phases of its ontogenetic development (i.e. newborn and adult specimens) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed wider dynamic range for toxins when comparing to non-toxins and a dynamic proteome rearrangement in cellular proteins upon B. jararaca development. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins related to transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results showed that the variation in the expression levels of cellular proteins gives rise to an even higher variation in the dynamic range of the expressed toxins. Upon ageing, the molecular constraints related to protein synthesis together with ecological traits would likely have an impact on the toxin repertoire, which, in the case of B. jararaca species, would enable the species to deal with different prey types during its lifespan.
Project description:Diachasmimorpha longicaudata parasitoid wasps carry a symbiotic poxvirus, known as DlEPV, within the female wasp venom gland. We sequenced RNA from venom gland tissue to identify DlEPV orthologs for 3 conserved poxvirus core genes. The DlEPV ORFs identified from this transcriptome were used to design primers for downstream RT-qPCR analysis and RNAi knockdown experiments.