Resolving the evolutionary relationships of molluscs with phylogenomic tools.

Molluscs (snails, octopuses, clams and their relatives) have a great disparity of body plans and, among the animals, only arthropods surpass them in species number. This diversity has made Mollusca one of the best-studied groups of animals, yet their evolutionary relationships remain poorly resolved. Open questions have important implications for the origin of Mollusca and for morphological evolution within the group. These questions include whether the shell-less, vermiform aplacophoran molluscs diverged before the origin of the shelled molluscs (Conchifera) or lost their shells secondarily. Monoplacophorans were not included in molecular studies until recently, when it was proposed that they constitute a clade named Serialia together with Polyplacophora (chitons), reflecting the serial repetition of body organs in both groups. Attempts to understand the early evolution of molluscs become even more complex when considering the large diversity of Cambrian fossils. These can have multiple dorsal shell plates and sclerites or can be shell-less but with a typical molluscan radula and serially repeated gills. To better resolve the relationships among molluscs, we generated transcriptome data for 15 species that, in combination with existing data, represent for the first time all major molluscan groups. We analysed multiple data sets containing up to 216,402 sites and 1,185 gene regions using multiple models and methods. Our results support the clade Aculifera, containing the three molluscan groups with spicules but without true shells, and they support the monophyly of Conchifera. Monoplacophora is not the sister group to other Conchifera but to Cephalopoda. Strong support is found for a clade that comprises Scaphopoda (tusk shells), Gastropoda and Bivalvia, with most analyses placing Scaphopoda and Gastropoda as sister groups. This well-resolved tree will constitute a framework for further studies of mollusc evolution, development and anatomy.

E. coli RNase III(E38A) generates discrete-sized products from long dsRNA.

Ribonuclease III (RNase III) represents a highly conserved family of double-strand-specific endoribonucleases that are important for RNA processing and post-transcriptional gene regulation in both prokaryotes and eukaryotes. We constructed a single amino acid substitution (E38A) of RNase III that shows a unique and useful enzymatic activity. It produces a dsRNA product of a discrete size migrating as 23 base pairs (bp) when given a long dsRNA as a substrate in an easy-to-control reaction. We demonstrate that the RNase III(E38A) mutant produces the 23-bp dsRNA product by making a double-strand cleavage of the long dsRNA substrate with the product being protected from further digestion. Using the hairpin RNA R1.1 as a substrate, RNase III(E38A) cleaves at the primary site and remains bound to the RNA, thereby preventing cleavage at the secondary site. The 23-bp dsRNA product is demonstrated to be a pool of dsRNAs representative of the long dsRNA substrate and has RNA interference activity in mammalian tissue culture transfection experiments. The RNA interference activity suggests that the 23-bp dsRNA product has typical 2-nucleotide 3' overhangs and behaves as siRNA thereby making it a useful tool in RNA interference experiments.