Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

Trocarin D (TroD), a venom prothrombin activator from Tropidechis carinatus, shares similar structure and function with blood coagulation factor Xa [Tropidechis carinatus FX (TrFX) a]. Their distinct physiologic roles are due to their distinct expression patterns. The genes of TroD and TrFX are highly similar, except for promoter and intron 1, indicating that TroD has probably evolved by duplication of FX, the plasma counterpart. The promoter insertion in TroD accounts for the elevated but not venom gland-specific expression. Here we examined the roles of 3 insertions and 2 deletions in intron 1 of TroD in the regulation of expression using luciferase as a reporter. By systematic deletions, we showed that a 209 bp region within the second insertion silences expression in mammalian and unmilked venom gland cells. Through bioinformatics analysis, we identified 5 AG-rich motifs in this region. All except the 5th motif are important for silencing function. YY1, Sp3 and HMGB2 were identified to bind these AG-rich motifs and silence gene expression in mammalian cells. Similar AG-rich motif clusters are also found in other toxin genes but not in their physiologic counterparts. Thus, AG-rich motifs contribute to regulation of expression of TroD, and probably other toxin genes.-Han, S. X., Kwong, S., Ge, R., Kolatkar, P. R., Woods, A. E., Blanchet, G., Kini, R. M. Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

Is the COI barcoding gene involved in speciation through intergenomic conflict?

We here test the proposition that changes in the barcoding region of COI are commonly involved in speciation through intergenomic conflict. We demonstrate that this is unlikely given that even with incomplete taxon sampling, 78-90% of closely-related animal species have identical COI amino acid sequences. In addition, in those cases where amino acid substitutions between closely related species are observed, the inter- and intra-specific substitution patterns are very similar and/or lack consistent differences in the number, position and type of amino acid change. Overall, we conclude that there is little evidence for a widespread involvement of the barcoding gene in speciation.

An update on DNA barcoding: low species coverage and numerous unidentified sequences.

DNA barcoding was proposed in 2003, the Consortium for the Barcode of Life was established in 2004, and the movement has since attracted more than $80 million funding. Here we investigate how many species of multicellular animals have been barcoded. We compare the numbers in a public database (GenBank as of January 2012) with those in the Barcode of Life Database (BOLD) and find that GenBank contains COI (cytochrome c oxidase subunit 1) sequences for ca. 60 000 species while BOLD reports barcodes for ca. 150 000 species. The discrepancy is likely due to a large amount of unpublished data in BOLD. Overall, the species coverage remains sparse, growth rates are low, and the barcode accumulation curve for Metazoa is linear with only 4788 species having been added in 2011. In addition, the vast majority of species in the public database (73%) were barcoded by projects that are unlikely to be related to the DNA barcoding movement. Particularly surprising was the large number of DNA barcodes in GenBank that were not identified to species (Jan 2012: 74%), with insect barcodes often being identified only to order. Of these several hundred thousand have since been suppressed by NCBI because they did not satisfy the iBOL/GenBank early release agreement. Species coverage is considerably better for target taxa of DNA barcoding campaigns (e.g. birds, fishes, Lepidoptera), although it also falls short of published campaign targets. © The Willi Hennig Society 2012.

The recruitment of blood coagulation factor X into snake venom gland as a toxin: the role of promoter cis-elements in its expression.

Trocarin D is a prothrombin activator from the Tropidechis carinatus venom. It is a functional and structural homologue to mammalian blood coagulation factor Xa. Trocarin D is hypothesised to have evolved from its factor X counterpart (TrFX) through gene duplication and recruitment. The genes of trocarin D and TrFX have significant sequence identities, except for insertions/deletions in their intron 1 and promoter regions. In trocarin D intron 1 region, there are three insertions and two deletions. In trocarin D promoter region, there is a novel 264 bp insertion which has potential cis-elements. This insertion is termed as Venom Recruitment/Switch Element (VERSE) and is hypothesised to account for switching the low-level constitutive expression of factor X in the liver to the high-level inducible expression of trocarin D in the venom gland. To understand the role of VERSE in the trocarin D expression, its cis-elements were characterised by luciferase assays in mammalian cell lines as well as snake venom gland cells. The ability of VERSE to drive luciferase expression is comparable to that of the trocarin D promoter. The predicted cis-elements are important in promoting expression as their mutagenesis resulted in lower luciferase expression. VERSE minimal core promoter and three novel cis-elements (two up-regulatory and one suppressor elements) were identified using deletion/site-directed mutagenesis studies. VERSE is primarily responsible for the increase of trocarin D expression. The insertions/deletions within trocarin D intron 1 need to be characterised for their role in tissue-specific and inducible expression of trocarin D.