Assembly and annotation of full mitochondrial genomes for the corn rootworm species, Diabrotica virgifera virgifera and Diabroticabarberi (Insecta: Coleoptera: Chrysomelidae), using Next Generation Sequence data

Complete mitochondrial genomes for two corn rootworm species, Diabrotica virgifera virgifera (16,747 bp) and Diabrotica barberi (16,632; Insecta: Coleoptera: Chrysomelidae), were assembled from Illumina HiSeq2000 read data. Annotation indicated that the order and orientation of 13 protein coding genes (PCGs), and 22 tRNA and 2 rRNA sequences were in typical of insect mitochondrial genomes. Non-standard nad4 and cox3 stop codons were composed of single T nucleotides and likely completed by adenylation, and atypical TTT start codons was predicted for both D. v. virgifera and D. barberinad1 genes. The D. v. virgifera and D. barberi haplotypes showed 819 variable nucleotide positions within PCG regions (7.36% divergence), which suggest that speciation may have occurred ~ 3.68 million years ago assuming a linear rate of short-term substitution. Phylogenetic analyses of Coleopteran MtD genome show clustering based on family level, and may have the capacity to resolve the evolutionary history within this Order of insects.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae)

Potassium inward rectifier K_IR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the K_IR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of K_IR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. K_IR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian K_IR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of K_IR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed K_IR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle K_IR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 K_IR2.1 sequences showed that all disease causing K_IR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake K_IR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC₅₀ ∼6 μM) inward rectifier currents.