Books by Stephen Mackessy
REPORTS www.BioTechniques.com REPORTS Reports Vol. 65 | 2018 No. 6 339 DNA barcoding provides a r... more REPORTS www.BioTechniques.com REPORTS Reports Vol. 65 | 2018 No. 6 339 DNA barcoding provides a rapid and effi cient means to determine taxonomic status of specimens using one or a few common genetic markers. This technique involves amplifying and sequencing relatively short regions of DNA (e.g., cytochrome b in animals) from a large number of organisms that display a high level of variation between species, but not within species. These reference genetic markers are used to identify species, to characterize new species, and ultimately to develop a large-scale system of classifi cation that is broadly applicable across a wide variety of taxa [1, 2]. Although DNA barcoding lacks the depth of information acquired from sequencing many genetic regions, it compensates for this with broad applicability and high throughput potential across highly divergent species. Ultimately, the success of DNA barcoding relies on the depth and breadth of a genetic library of reference sequences. Mitochondrial regions cytochrome C oxidase I (COI) and cytochrome b (cyt b) DNA barcoding is a simple technique used to develop a large-scale system of classifi cation that is broadly applicable across a wide variety of taxa. DNA-based analysis of snake venoms can provide a system of clas-sifi cation independent of currently accepted taxonomic relationships by generating DNA barcodes spe-cifi c to each venom sample. DNA purifi cation from dried snake ven-oms has previously required large amounts of starting material, has resulted in low yields and inconsistent amplification, and was possible with front-fanged snakes only. Here, we present a modi-fi ed DNA extraction protocol applied to venoms of both front-and rear-fanged snakes that requires signifi cantly less starting material (1 mg) and yields suffi cient amounts of DNA for successful PCR amplifi cation of regions commonly used for DNA barcoding. Crotalus simus tzabcan METHOD SUMMARY Modifications to a commercial DNA extraction kit (cultured cells protocol) allow purification of DNA from snake venoms for DNA-barcoding efforts. The outlined protocol uses ~100x less venom than previously possible and is applicable to both front-and rear-fanged venomous snakes. .
Toxins, 2018
The use of-omics technologies allows for the characterization of snake venom composition at a fas... more The use of-omics technologies allows for the characterization of snake venom composition at a fast rate and at high levels of detail. In the present study, we investigated the protein content of Red-headed Krait (Bungarus flaviceps) venom. This analysis revealed a high diversity of snake venom protein families, as evidenced by high-throughput mass spectrometric analysis. We found all six venom protein families previously reported in a transcriptome study of the venom gland of B. flaviceps, including phospholipases A 2 (PLA 2 s), Kunitz-type serine proteinase inhibitors (KSPIs), three-finger toxins (3FTxs), cysteine-rich secretory proteins (CRISPs), snaclecs, and natriuretic peptides. A combined approach of automated database searches and de novo sequencing of tandem mass spectra, followed by sequence similarity searches, revealed the presence of 12 additional toxin families. De novo sequencing alone was able to identify 58 additional peptides, and this approach contributed significantly to the comprehensive description of the venom. Abundant protein families comprise 3FTxs (22.3%), KSPIs (19%), acetylcholinesterases (12.6%), PLA 2 s (11.9%), venom endothelial growth factors (VEGFs, 8.4%), nucleotidases (4.3%), and C-type lectin-like proteins (snaclecs, 3.3%); an additional 11 toxin families are present at significantly lower concentrations, including complement depleting factors, a family not previously detected in Bungarus venoms. The utility of a multifaceted approach toward unraveling the proteome of snake venoms, employed here, allowed detection of even minor venom components. This more in-depth knowledge of the composition of B. flaviceps venom facilitates a better understanding of snake venom molecular evolution, in turn contributing to more effective treatment of krait bites. Key Contribution: We describe the deep proteomic mining of Bungarus flaviceps venom that allowed us to identify the presence of 18 protein families, including several low abundance toxins. Utilization of this approach facilitates the understanding of the evolution of venom complexity and the differential expression of dominant venom toxins by allowing detection/identification of even trace venom components.
Papers by Stephen Mackessy
Reptile Venoms and Toxins
CRC Press eBooks, May 1, 2021
Journal of Heredity, Jan 27, 2021
Male-biased mutation rates occur in a diverse array of organisms. The ratio of male-to-female mut... more Male-biased mutation rates occur in a diverse array of organisms. The ratio of male-to-female mutation rate may have major ramifications for evolution across the genome, and for sex-linked genes in particular. In ZW species, the Z chromosome is carried by males two-thirds of the time, leading to the prediction that male-biased mutation rates will have a disproportionate effect on the evolution of Z-linked genes relative to autosomes and the W chromosome. Colubroid snakes (including colubrids, elapids, and viperids) have ZW sex determination, yet male-biased mutation rates have not been well studied in this group. Here we analyze a population genomic dataset from rattlesnakes to quantify genetic variation within and genetic divergence between species. We use a new method for unbiased estimation of population genetic summary statistics to compare variation between the Z chromosome and autosomes and to calculate net nucleotide differentiation between species. We find evidence for a 2.03-fold greater mutation rate in male rattlesnakes relative to females, corresponding to an average μ Z /μ A ratio of 1.1. Our results from snakes are quantitatively similar to birds, suggesting that male-biased mutation rates may be a common feature across vertebrate lineages with ZW sex determination.
Toxicon, Oct 1, 1996
Phospholipase A, (PLA,), an enzyme found in most snake venoms, catalyzes the hydrolysis of phosph... more Phospholipase A, (PLA,), an enzyme found in most snake venoms, catalyzes the hydrolysis of phospholipids in biological membranes, and some have presynaptic neurotoxic activity. A synthetic substrate, 4-nitro-3-(octanoyloxy)benzoic acid, was synthesized and purified on a silica gel column using a published method. This substrate was used to develop an endpoint assay which is rapid and requires a minimum of equipment. This aqueous assay system allowed enzyme activity to be examined without the use of radioactive substrates or organic solvents, minimizing waste disposal concerns. Whole venoms, partially purified enzyme isolated from Crotalus mitchelli pyrrhus venom, tissue extracts and commercial preparations were employed as sources of PLA?. Results show that this method is a convenient and specific assay for PLA, from several sources and is particularly suited for assaying large numbers of fractions generated during purification procedures.
Venom composition of adult Western Diamondback Rattlesnakes (Crotalus atrox) maintained under controlled diet and environmental conditions shows only minor changes
Toxicon, Jun 1, 2019
Many species of snakes produce venom as a chemical means of procuring potentially fractious prey.... more Many species of snakes produce venom as a chemical means of procuring potentially fractious prey. Studies have increasingly focused on venom compositional variation between and within individual snakes of the same species/subspecies, with significant differences often being observed. This variation in composition has been attributed to differences in age, season, diet, and environment, suggesting that these factors could help explain the inter- and intra-specific variation found in some snake venoms, perhaps via some type of feedback mechanism(s). To address several of these possible sources of variation, this study utilized wild-caught Western Diamondback Rattlesnakes (Crotalus atrox) from Cochise Co., AZ. Sixteen adult C. atrox were maintained in the lab on a diet of NSA mice for eight months to determine whether venom composition changed in captivity under a static diet in a stable environment. Reducing 1-D SDS-PAGE, fibrinogen degradation assays, reversed-phase HPLC, and MALDI-TOF mass spectrometry revealed only minor differences over time within individuals. Venom L-amino acid oxidase (LAAO) and phosphodiesterase activities significantly increased over the course of captivity, with no changes occurring in azocasein metalloproteinase, kallikrein-like serine proteinase (KLSP), or thrombin-like serine proteinase (TLSP) activities. Snake total length was positively correlated with TLSP activity and negatively correlated with LAAO and KLSP activity. There was typically a much higher degree of variation between individuals than within individuals for all analyses performed and measurements collected. Because the overall "fingerprint" of each snake's venom remained more/less constant, it is concluded that biologically significant changes in venom composition did not occur within individual C. atrox as a function of captivity/diet. However, this study does indicate that differences in activity levels do occur in minor venom enzyme components, but the differences observed are likely to be of minimal significance to the production of antivenom or to subsequent treatment of human envenomations.
The unique Duvernoy's secretion of the brown tree snake (Boiga irregularis)
Toxicon, 1991
Recently, bites by the colubrid Boiga irregularis (brown tree snake) in infants and young childre... more Recently, bites by the colubrid Boiga irregularis (brown tree snake) in infants and young children on Guam have produced severe systemic reactions which bear some resemblance to classical manifestations of neurotoxic venom poisoning. This study demonstrates that the Duvernoy's secretion which elicits these reactions is a remarkably simple venom secretion with comparatively low toxicity and generally weak enzymatic activity. The intravenous LD50 for Swiss-Webster mice was approximately 80 mg/kg; significant neurotoxic manifestations were not observed in mouse trials. Deaths of lethally challenged mice occurred within minutes of injection, and appeared to result from cardiopulmonary crises. Duvernoy's secretion yields, protein content, enzyme activities, electrophoretic data and toxicity characteristics of the secretion are presented.
Toxicon, Dec 1, 2000
R.E. Hill and S.P. Mackessy. Characterization of venom (Duvernoy's secretion) from twelve species... more R.E. Hill and S.P. Mackessy. Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon XX, xx±yy, 2000. Ð Venomous colubrids, which include more than 700 snake species worldwide, represent a vast potential source of novel biological compounds. The present study characterized venom (Duvernoy's gland secretion) collected from twelve species of opisthoglyphous (rear-fanged) colubrid snakes, an extremely diverse assemblage of nonvenomous to highly venomous snakes. Most venoms displayed proteolytic activity (casein), though activity levels varied considerably. Low phosphodiesterase activity was detected in several venoms (Amphiesma stolata, Diadophis punctatus, Heterodon nasicus kennerlyi, H. n. nasicus and Thamnophis elegans vagrans ), and acetylcholinesterase was found in Boiga irregularis saliva and venom, but no venoms displayed hyaluronidase, thrombin-like or kallikrein-like activities. High phospholipase A 2 (PLA 2 ) activity was found in Trimorphodon biscutatus lambda venom, and moderate levels were detected in Boiga dendrophila and D. p. regalis venoms as well as B. dendrophila and H. n. nasicus salivas. Non-reducing SDS± PAGE revealed 7±20 protein bands (3.5 to over 200 kD, depending on species) for all venoms analyzed, and electrophoretic pro®les of venoms were typically quite distinct from saliva pro®les. Components from A. stolata, Hydrodynastes gigas, Tantilla nigriceps and T. e. vagrans venoms showed protease activity when run on gelatin zymogram gels. N-terminal
CRC Press eBooks, Jul 14, 2009
Handbook of Venoms and Toxins of Reptiles
CRC Press eBooks, Apr 19, 2016
... 155 Bhadrapura L. Dhananjaya, Bannikuppe S. Vishwanath, and Cletus JM D'Souz... more ... 155 Bhadrapura L. Dhananjaya, Bannikuppe S. Vishwanath, and Cletus JM D'Souza 8 Chapter Snake Venom Phospholipase A2 Enzymes ... 259 Ana Gisele C. Neves-Ferreira, Richard H. Valente, Jonas Perales, and Gilberto B. Domont II SectIon I Reptile Venom toxins 1 Chapter ...
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Books by Stephen Mackessy
Papers by Stephen Mackessy