Identification of sodium channel toxins from marine cone snails of the subgenera Textilia and Afonsoconus.

Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1-NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure-activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.

Rapid expansion of the protein disulfide isomerase gene family facilitates the folding of venom peptides.

Formation of correct disulfide bonds in the endoplasmic reticulum is a crucial step for folding proteins destined for secretion. Protein disulfide isomerases (PDIs) play a central role in this process. We report a previously unidentified, hypervariable family of PDIs that represents the most diverse gene family of oxidoreductases described in a single genus to date. These enzymes are highly expressed specifically in the venom glands of predatory cone snails, animals that synthesize a remarkably diverse set of cysteine-rich peptide toxins (conotoxins). Enzymes in this PDI family, termed conotoxin-specific PDIs, significantly and differentially accelerate the kinetics of disulfide-bond formation of several conotoxins. Our results are consistent with a unique biological scenario associated with protein folding: The diversification of a family of foldases can be correlated with the rapid evolution of an unprecedented diversity of disulfide-rich structural domains expressed by venomous marine snails in the superfamily Conoidea.

Specialized insulin is used for chemical warfare by fish-hunting cone snails.

More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.

Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins.

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 (Conus Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Conus Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.

Hormone-like peptides in the venoms of marine cone snails.

The venoms of cone snails (genus Conus) are remarkably complex, consisting of hundreds of typically short, disulfide-rich peptides termed conotoxins. These peptides have diverse pharmacological targets, with injection of venom eliciting a range of physiological responses, including sedation, paralysis and sensory overload. Most conotoxins target the prey's nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules in the venoms of cone snails we systematically mined the venom gland transcriptomes of several cone snail species and examined secreted venom peptides in dissected and injected venom of the Australian cone snail Conus victoriae. Using this approach we identified several novel hormone/neuropeptide-like toxins, including peptides similar to the bee brain hormone prohormone-4, the mollusc ganglia neuropeptide elevenin, and thyrostimulin, a member of the glycoprotein hormone family, and confirmed the presence of insulin. We confirmed that at least two of these peptides are not only expressed in the venom gland but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers an exciting new avenue for the discovery of novel pharmacological tools and drug candidates, complementary to conotoxins.

The Venom Repertoire of Conus gloriamaris (Chemnitz, 1777), the Glory of the Sea.

The marine cone snail Conus gloriamaris is an iconic species. For over two centuries, its shell was one of the most prized and valuable natural history objects in the world. Today, cone snails have attracted attention for their remarkable venom components. Many conotoxins are proving valuable as research tools, drug leads, and drugs. In this article, we present the venom gland transcriptome of C. gloriamaris, revealing this species' conotoxin repertoire. More than 100 conotoxin sequences were identified, representing a valuable resource for future drug discovery efforts.

A disulfide tether stabilizes the block of sodium channels by the conotoxin µO§-GVIIJ.

A cone snail venom peptide, µO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. µO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVß2 or rNaVß4, but not that of rNaVß1 or rNaVß3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVß- and NaVα-subunits are present in native neurons.

Combined proteomic and transcriptomic interrogation of the venom gland of Conus geographus uncovers novel components and functional compartmentalization.

Cone snails are highly successful marine predators that use complex venoms to capture prey. At any given time, hundreds of toxins (conotoxins) are synthesized in the secretory epithelial cells of the venom gland, a long and convoluted organ that can measure 4 times the length of the snail's body. In recent years a number of studies have begun to unveil the transcriptomic, proteomic and peptidomic complexity of the venom and venom glands of a number of cone snail species. By using a combination of DIGE, bottom-up proteomics and next-generation transcriptome sequencing the present study identifies proteins involved in envenomation and conotoxin maturation, significantly extending the repertoire of known (poly)peptides expressed in the venom gland of these remarkable animals. We interrogate the molecular and proteomic composition of different sections of the venom glands of 3 specimens of the fish hunter Conus geographus and demonstrate regional variations in gene expression and protein abundance. DIGE analysis identified 1204 gel spots of which 157 showed significant regional differences in abundance as determined by biological variation analysis. Proteomic interrogation identified 342 unique proteins including those that exhibited greatest fold change. The majority of these proteins also exhibited significant changes in their mRNA expression levels validating the reliability of the experimental approach. Transcriptome sequencing further revealed a yet unknown genetic diversity of several venom gland components. Interestingly, abundant proteins that potentially form part of the injected venom mixture, such as echotoxins, phospholipase A2 and con-ikots-ikots, classified into distinct expression clusters with expression peaking in different parts of the gland. Our findings significantly enhance the known repertoire of venom gland polypeptides and provide molecular and biochemical evidence for the compartmentalization of this organ into distinct functional entities.

Elucidation of the molecular envenomation strategy of the cone snail Conus geographus through transcriptome sequencing of its venom duct.

BACKGROUND: The fish-hunting cone snail, Conus geographus, is the deadliest snail on earth. In the absence of medical intervention, 70% of human stinging cases are fatal. Although, its venom is known to consist of a cocktail of small peptides targeting different ion-channels and receptors, the bulk of its venom constituents, their sites of manufacture, relative abundances and how they function collectively in envenomation has remained unknown. RESULTS: We have used transcriptome sequencing to systematically elucidate the contents the C. geographus venom duct, dividing it into four segments in order to investigate each segment's mRNA contents. Three different types of calcium channel (each targeted by unrelated, entirely distinct venom peptides) and at least two different nicotinic receptors appear to be targeted by the venom. Moreover, the most highly expressed venom component is not paralytic, but causes sensory disorientation and is expressed in a different segment of the venom duct from venoms believed to cause sensory disruption. We have also identified several new toxins of interest for pharmaceutical and neuroscience research. CONCLUSIONS: Conus geographus is believed to prey on fish hiding in reef crevices at night. Our data suggest that disorientation of prey is central to its envenomation strategy. Furthermore, venom expression profiles also suggest a sophisticated layering of venom-expression patterns within the venom duct, with disorientating and paralytic venoms expressed in different regions. Thus, our transcriptome analysis provides a new physiological framework for understanding the molecular envenomation strategy of this deadly snail.

A very short, functionally constrained sequence diagnoses cone snails in several Conasprella clades.

The traditional taxonomy of ca. 700 cone snails assigns all species to a single genus, Conus Linnaeus 1758. However, an increasing body of evidence suggests that some belong to a phylogenetically distinct clade that is sometimes referred to as Conasprella. Previous work (Kraus et al., 2011) showed that a short (259 bp) conserved intronic sequence (CIS) of the γ-glutamyl carboxylase gene (intron 9) can be used to delineate deep phylogenetic relationships among some groups of Conus. The work described here uses intron 9 (338 bp) to resolve problematic relationships among the conasprellans and to distinguish them from Conus proper. Synapomorphic mutations at just 39 sites can resolve several groups within Conasprella because the informative region of intron 9 is so well conserved that the phylogenetic signal is not obscured by homoplasies at conflicting sites. Intron 9 also unambiguously distinguishes Conasprella as a whole from Conus because the conserved regions that are so well conserved within each group are not alignable and clearly not homologous between them. This pattern suggests that expression of the γ-glutamyl carboxylase gene may have undergone a functionally significant change in Conus or Conasprella shortly after they diverged.

Characterization of the Conus bullatus genome and its venom-duct transcriptome.

BACKGROUND: The venomous marine gastropods, cone snails (genus Conus), inject prey with a lethal cocktail of conopeptides, small cysteine-rich peptides, each with a high affinity for its molecular target, generally an ion channel, receptor or transporter. Over the last decade, conopeptides have proven indispensable reagents for the study of vertebrate neurotransmission. Conus bullatus belongs to a clade of Conus species called Textilia, whose pharmacology is still poorly characterized. Thus the genomics analyses presented here provide the first step toward a better understanding the enigmatic Textilia clade. RESULTS: We have carried out a sequencing survey of the Conus bullatus genome and venom-duct transcriptome. We find that conopeptides are highly expressed within the venom-duct, and describe an in silico pipeline for their discovery and characterization using RNA-seq data. We have also carried out low-coverage shotgun sequencing of the genome, and have used these data to determine its size, genome-wide base composition, simple repeat, and mobile element densities. CONCLUSIONS: Our results provide the first global view of venom-duct transcription in any cone snail. A notable feature of Conus bullatus venoms is the breadth of A-superfamily peptides expressed in the venom duct, which are unprecedented in their structural diversity. We also find SNP rates within conopeptides are higher compared to the remainder of C. bullatus transcriptome, consistent with the hypothesis that conopeptides are under diversifying selection.

Against expectation: a short sequence with high signal elucidates cone snail phylogeny.

A short (259 nucleotide) conserved intronic sequence (CIS) is surprisingly informative for delineating deep phylogenetic relationships in cone snails. Conus species previously have been assigned to clades based on the evidence from mitochondrial 12S and 16S rRNA gene sequences (1129 bp). Despite their length, these genes lack the phylogenetic information necessary to resolve the relationships among the clades. Here we show that the relationships can be inferred from just 46 sites in the very short CIS sequence (a portion of "intron 9" of the γ-glutamyl carboxylase gene). This is counterintuitive because in short sequences sampling error (noise) often drowns out phylogenetic signal. The intron 9 CIS is rich in synapomorphies that define the divergence patterns among eight clades of worm- and fish-hunting Conus, and it contains almost no homoplasy. Parsimony, maximum likelihood and Bayesian analyses of the combined sequences (mt rRNA+CIS) confirm most of the relationships among 23 Conus sequences. This phylogeny implies that fish-hunting behavior evolved at least twice during the history of Conus-once among New World species and independently in the Indo-Pacific clades.

A rapidly diverging superfamily of peptide toxins in venomous Gemmula species.

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.

Divergence of the Venom Exogene Repertoire in Two Sister Species of Turriconus.

The genus Conus comprises approximately 700 species of venomous marine cone snails that are highly efficient predators of worms, snails, and fish. In evolutionary terms, cone snails are relatively young with the earliest fossil records occurring in the Lower Eocene, 55 Ma. The rapid radiation of cone snail species has been accompanied by remarkably high rates of toxin diversification. To shed light on the molecular mechanisms that accompany speciation, we investigated the toxin repertoire of two sister species, Conus andremenezi and Conus praecellens, that were until recently considered a single variable species. A total of 196 and 250 toxin sequences were identified in the venom gland transcriptomes of C. andremenezi and C. praecellens belonging to 25 and 29 putative toxin gene superfamilies, respectively. Comparative analysis with closely (Conus tribblei and Conus lenavati) and more distantly related species (Conus geographus) suggests that speciation is associated with significant diversification of individual toxin genes (exogenes) whereas the expression pattern of toxin gene superfamilies within lineages remains largely conserved. Thus, changes within individual toxin sequences can serve as a sensitive indicator for recent speciation whereas changes in the expression pattern of gene superfamilies are likely to reflect more dramatic differences in a species' interaction with its prey, predators, and competitors.

Complete mitochondrial DNA sequence of a Conoidean gastropod, Lophiotoma (Xenuroturris) cerithiformis: gene order and gastropod phylogeny.

We have determined the first complete nucleotide sequence of the mitochondrial genome of a venomous mollusc, the Conoidean gastropod, Lophiotoma (Xenuroturris) cerithiformis. It is 15,380 nucleotide pairs (ntp) and encodes 13 proteins, two ribosomal RNAs and 22 tRNAs of the mitochondrion's own protein synthesizing system. The protein mRNAs, ribosomal RNAs and 13 of the tRNAs are transcribed from the same strand, the remaining tRNAs from the other strand. The longest segment of unassigned sequence is 139 ntp and includes a 82 ntp segment that is a perfect inverted repeat sequence of 37 ntp separated by 8 nt. The gene arrangement of L. cerithiformis mtDNA shows remarkable similarity to the gene arrangements of mtDNAs of the vetigastropod Haliotis rubra, the polyplacophoran Katharina tunicata and the cephalopod Octopus vulgaris, but differs dramatically from the gene arrangements found in the mtDNAs of pulmonate and opisthobranch gastropods, as well as mtDNAs of bivalves and scaphopods. A single sixteen gene inversion that distinguishes L. cerithiformis mtDNA from mtDNAs of H. rubra, K. tunicata and O. vulgaris is shared by mtDNA of a littorinomorph gastropod Littorina saxitalis, suggesting a close relationship of conoidean and littorinomorph gastropods.

αS-conotoxin GVIIIB potently and selectively blocks α9α10 nicotinic acetylcholine receptors.

Although acetylcholine is widely utilized in vertebrate nervous systems, nicotinic acetylcholine receptors (nAChRs), including the α9α10 subtype, also are expressed in a wide variety of non-neuronal cells. These cell types include cochlear hair cells, adrenal chromaffin cells and immune cells. α9α10 nAChRs present in these cells may respectively play roles in protection from noise-induced hearing loss, response to stress and neuroprotection. Despite these critical functions, there are few available selective ligands to confirm mechanistic hypothesis regarding the role of α9α10 nAChRs. Conus, has been a rich source of ligands for receptors and ion channels. Here, we identified Conus geographus venom as a lead source for a novel α9α10 antagonist. The active component was isolated and the encoding gene cloned. The peptide signal sequence and cysteine arrangement had the signature of the σ-conotoxin superfamily. Previously isolated σ-conotoxin GVIIIA, also from Conus geographus, targets the 5-HT3 receptor. In contrast, αS-GVIIIB blocked the α9α10 nAChR with an IC50 of 9.8 nM, yet was inactive at the 5-HT3 receptor. Pharmacological characterization of αS-GVIIIB shows that it is over 100-fold selective for the α9α10 nAChR compared to other nAChR subtypes. Thus, the S-superfamily represents a novel conotoxin scaffold for flexibly targeting a variety of receptor subtypes. Functional competition studies utilized distinct off-rate kinetics of conotoxins to identify the α10/α9 nAChR interface as the site of αS-GVIIIB binding; this adds to the importance of the (+) face of the α10 rather than the (+) face of the α9 nAChR subunit as critical to binding of α9α10-targeted conotoxins.

Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus.

Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells.

Novel venom peptides from the cone snail Conus pulicarius discovered through next-generation sequencing of its venom duct transcriptome.

The venom peptides (i.e., conotoxins or conopeptides) that species in the genus Conus collectively produce are remarkably diverse, estimated to be around 50,000 to 140,000, but the pace of discovery and characterization of these peptides have been rather slow. To date, only a minor fraction have been identified and studied. However, the advent of next-generation DNA sequencing technologies has opened up opportunities for expediting the exploration of this diversity. The whole transcriptome of a venom duct from the vermivorous marine snail C. pulicarius was sequenced using the 454 sequencing platform. Analysis of the data set resulted in the identification of over eighty unique putative conopeptide sequences, the highest number discovered so far from a Conus venom duct transcriptome. More importantly, majority of the sequences were potentially novel, many with unexpected structural features, hinting at the vastness of the diversity of Conus venom peptides that remains to be explored. The sequences represented at least 14 major superfamilies/types (disulfide- and non-disulfide-rich), indicating the structural and functional diversity of conotoxins in the venom of C. pulicarius. In addition, the contryphans were surprisingly more diverse than what is currently known. Comparative analysis of the O-superfamily sequences also revealed insights into the complexity of the processes that drive the evolution and diversification of conotoxins.

Vitamin K-dependent gamma-glutamylcarboxylation: an ancient posttranslational modification.

The vitamin K-dependent carboxylase carries out the posttranslational modification of specific glutamate residues in proteins to gamma-carboxy glutamic acid (Gla) in the presence of reduced vitamin K, molecular oxygen, and carbon dioxide. In the process, reduced vitamin K is converted to vitamin K epoxide, which is subsequently reduced to vitamin K, by vitamin K epoxide reductase (VKOR) for use in the carboxylation reaction. The modification has a wide range of physiological implications, including hemostasis, bone calcification, and signal transduction. The enzyme interacts with a high affinity gamma-carboxylation recognition sequence (gamma-CRS) of the substrate and carries out multiple modifications of the substrate before the product is released. This mechanism ensures complete carboxylation of the Gla domain of the coagulation factors, which is essential for their biological activity. gamma-Carboxylation, originally discovered in mammals, is widely distributed in the animal kingdom. It has been characterized in sea squirt (Ciona intestinalis), in flies (Drosophila melanogaster), and in marine snails (Conus textile), none of which have a blood coagulation system similar to mammals. The cone snails express a large array of gamma-carboxylated peptides that modulate the activity of ion channels. These findings have led to the suggestion that gamma-carboxylation is an extracellular posttranslational modification that antedates the divergence of molluscs, arthropods, and chordates. I will first summarize recent understanding of gamma-carboxylase and gamma-carboxylation gleaned from experiments using the mammalian enzyme, and then I will briefly describe the available information on gamma-carboxylation in D. melanogaster and C. textile.

The mitochondrial genome of Conus textile, coxI-coxII intergenic sequences and Conoidean evolution.

The cone snails belong to the superfamily Conoidea, comprising approximately 10,000 venomous marine gastropods. We determined the complete mitochondrial DNA sequence of Conus textile. The gene order is identical in Conus textile, Lophiotoma cerithiformis (another Conoidean gastropod), and the neogastropod Ilyanassa obsoleta, (not in the superfamily Conoidea). However, the intergenic interval between the coxI and coxII genes was much longer in C. textile (165bp) than in any other previously analyzed gastropod. We used the intergenic region to evaluate evolutionary patterns. In most neogastropods and three conidean families the intergenic interval is small (<30 nucleotides). Within Conus, the variation is from 130 to 170bp, and each different clade within Conus has a narrower size distribution. In Conasprella, a subgenus traditionally assigned to Conus, the intergenic regions vary between 200 and 500bp, suggesting that the species in Conasprella are not congeneric with Conus. The intergenic region was used for phylogenetic analysis of a group of fish-hunting Conus, despite the short length resolution was better than using standard markers. Thus, the coxI-coxII intergenic region can be used both to define evolutionary relationships between species in a clade, and to understand broad evolutionary patterns across the large superfamily Conoidea.