Biology subjects

John F Mulley

Publications and source records attributed to John F Mulley.

5 recordsLinked to original sources

bioRxiv2015

Snake venom gland cDNA sequencing using the Oxford Nanopore MinION portable DNA sequencer

Portable DNA sequencers such as the Oxford Nanopore MinION device have the potential to be truly disruptive technologies, facilitating new approaches and analyses and, in some cases, taking sequencing out of the lab and into the field. However, the capabilities of these technologies are still being revealed. Here we show that single-molecule cDNA sequencing using the MinION accurately characterises venom toxin-encoding genes in the painted saw-scaled viper, Echis coloratus. We find the raw sequencing error rate to be around 12%, improved to 0-2% with hybrid error correction and 3% with de novo error correction. Our corrected data provides full coding sequences and 5' and 3' UTRs for 29 of 33 candidate venom toxins detected, far superior to Illumina data (13/40 complete) and Sanger-based ESTs (15/29). We suggest that, should the current pace of improvement continue, the MinION will become the default approach for cDNA sequencing in a variety of species.

Genetics↗

bioRxiv2014

Genomic and transcriptomic insights into the regulation of snake venom production

The gene regulatory mechanisms underlying the rapid replenishment of snake venom following expenditure are currently unknown. Using a comparative transcriptomic approach we find that venomous and non-venomous species produce similar numbers of secreted products in their venom or salivary glands and that only one transcription factor (Tbx3) is expressed in venom glands but not salivary glands. We also find evidence for temporal variation in venom production. We have generated a draft genome sequence for the painted saw-scaled viper, Echis coloratus, and identified conserved transcription factor binding sites in the upstream regions of venom genes. We find binding sites to be conserved across members of the same gene family, but not between gene families, indicating that multiple gene regulatory networks are involved in venom production. Finally, we suggest that negative regulation may be important for rapid activation of the venom replenishment cycle.

Genetics↗

bioRxiv2014

Restriction and recruitment - gene duplication and the origin and evolution of snake venom toxins

Snake venom has been hypothesised to have originated and diversified via a process that involves duplication of genes encoding body proteins with subsequent recruitment of the copy to the venom gland, where natural selection acts to develop or increase toxicity. However, gene duplication is known to be a rare event in vertebrate genomes and the recruitment of duplicated genes to a novel expression domain (neofunctionalisation) is an even rarer process that requires the evolution of novel combinations of transcription factor binding sites in upstream regulatory regions. Therefore, whilst this hypothesis concerning the evolution of snake venom is therefore very unlikely and should be regarded with caution, it is nonetheless often assumed to be established fact, hindering research into the true origins of snake venom toxins. To critically evaluate this hypothesis we have generated transcriptomic data for body tissues and salivary and venom glands from five species of venomous and non-venomous reptiles. Our comparative transcriptomic analysis of these data reveals that snake venom does not evolve via the hypothesised process of duplication and recruitment of genes encoding body proteins. Indeed, our results show that many proposed venom toxins are in fact expressed in a wide variety of body tissues, including the salivary gland of non-venomous reptiles and that these genes have therefore been restricted to the venom gland following duplication, not recruited. Thus snake venom evolves via the duplication and subfunctionalisation of genes encoding existing salivary proteins. These results highlight the danger of the elegant and intuitive ?just-so story? in evolutionary biology.

Evolutionary Biology↗

bioRxiv2014

Testing the Toxicofera: comparative reptile transcriptomics casts doubt on the single, early evolution of the reptile venom system

Background The identification of apparently conserved gene complements in the venom and salivary glands of a diverse set of reptiles led to the development of the Toxicofera hypothesis the idea that there was a single, early evolution of the venom system in reptiles. However, this hypothesis is based largely on relatively small scale EST-based studies of only venom or salivary glands and toxic effects have been assigned to only some of these putative Toxcoferan toxins in some species. We set out to investigate the distribution of these putative venom toxin transcripts in order to investigate to what extent conservation of gene complements may reflect a bias in previous sampling efforts. Results We have carried out the first large-scale test of the Toxicofera hypothesis and found it lacking in a number of regards. Our quantitative transcriptomic analyses of venom and salivary glands and other body tissues in five species of reptile, together with the use of available RNA-Seq datasets for additional species shows that the majority of genes used to support the establishment and expansion of the Toxicofera are in fact expressed in multiple body tissues and most likely represent general maintenance or housekeeping genes. The apparent conservation of gene complements across the Toxicofera therefore reflects an artefact of incomplete tissue sampling. In other cases, the identification of a non-toxic paralog of a gene encoding a true venom toxin has led to confusion about the phylogenetic distribution of that venom component. Conclusions Venom has evolved multiple times in reptiles. In addition, the misunderstanding regarding what constitutes a toxic venom component, together with the misidentification of genes and the classification of identical or near-identical sequences as distinct genes has led to an overestimation of the complexity of reptile venoms in general, and snake venom in particular, with implications for our understanding of (and development of treatments to counter) the molecules responsible for the physiological consequences of snakebite.

Evolutionary Biology↗

bioRxiv2014

Transcriptomic analysis of the lesser spotted catshark (Scyliorhinus canicula) pancreas, liver and brain reveals molecular level conservation of vertebrate pancreas function

BackgroundUnderstanding the evolution of the vertebrate pancreas is key to understanding its functions. The chondrichthyes (cartilaginous fish such as sharks and rays) have been suggested to possess the most ancient example of a distinct pancreas with both hormonal (endocrine) and digestive (exocrine) roles, although the lack of genetic, genomic and transcriptomic data for cartilaginous fish has hindered a more thorough understanding of the molecular-level functions of the chondrichthyan pancreas, particularly with respect to their \"unusual\" energy metabolism (where ketone bodies and amino acids are the main oxidative fuel source) and their paradoxical ability to both maintain stable blood glucose levels and tolerate extensive periods of hypoglycemia. In order to shed light on some of these processes we have carried out the first large-scale comparative transcriptomic survey of multiple cartilaginous fish tissues: the pancreas, brain and liver of the lesser spotted catshark, Scyliorhinus canicula.\n\nResultsWe generated a mutli-tissue assembly comprising 86,006 contigs, of which 44,794 were assigned to a particular tissue or combination of tissue based on mapping of sequencing reads. We have characterised transcripts encoding genes involved in insulin regulation, glucose sensing, transcriptional regulation, signaling and digestion, as well as many peptide hormone precursors and their receptors for the first time. Comparisons to published mammalian pancreas transcriptomes reveals that mechanisms of glucose sensing and insulin regulation used to establish and maintain a stable internal environment are conserved across jawed vertebrates and likely pre-date the vertebrate radiation. Conservation of pancreatic hormones and genes encoding digestive proteins support the single, early evolution of a distinct pancreatic gland with endocrine and exocrine functions in vertebrates, although the peptide diversity of the early vertebrate pancreas has been overestimated as a result of the use of cross-reacting antisera in earlier studies. A three hormone islet organ is therefore the basal vertebrate condition, later elaborated upon only in the tetrapod lineage.\n\nConclusionsThe cartilaginous fish are a great untapped resource for the reconstruction of patterns and processes of vertebrate evolution and new approaches such as those described in this paper will greatly facilitate their incorporation into the rank of \"model organism\".

Genetics↗