Evidence of cryptic and pseudocryptic speciation in the Paracalanus parvus species complex (Crustacea, Copepoda, Calanoida), supplementary material

Introduction Many marine planktonic crustaceans such as copepods have been considered as widespread organisms. However, the growing evidence for cryptic and pseudo-cryptic speciation has emphasized the need of re-evaluating the status of copepod species complexes in molecular and morphological studies to get a clearer picture about pelagic marine species as evolutionary units and their distributions. This study analyses the molecular diversity of the ecologically important Paracalanus parvus species complex. Its seven currently recognized species are abundant and also often dominant in marine coastal regions worldwide from temperate to tropical oceans. Results COI and Cytochrome b sequences of 160 specimens of the Paracalanus parvus complex from all oceans were obtained. Furthermore, 42 COI sequences from GenBank were added for the genetic analyses. Thirteen distinct molecular operational taxonomic units (MOTU) and two single sequences were revealed with cladistic analyses (Maximum Likelihood, Bayesian Inference), of which seven were identical with results from species delimitation methods (barcode gaps, ABDG, GMYC, Rosenberg's P(AB)). In total, 10 to 12 putative species were detected and could be placed in three categories: (1) temperate geographically isolated, (2) warm-temperate to tropical wider spread and (3) circumglobal warm-water species. Conclusions The present study provides evidence of cryptic or pseudocryptic speciation in the Paracalanus parvus complex. One major insight is that the species Paracalanus parvus s.s. is not panmictic, but may be restricted in its distribution to the northeastern Atlantic.

SMART, a simple modular architecture research tool: Identification of signaling domains

Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.

Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism

We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.

Addition of diazomethane to armchair single-walled carbon nanotubes and their reaction sequences : A computational study

The sidewall additions of diazomethane to (n, n), n = 3–10 armchair single-walled carbon nanotubes (SWCNTs) on two different orientations of C–C bonds have been studied using the ONIOM(B3LYP/6-31G(d):PM3) approach. The binding energies of SWCNTs complexes with CH2N2, CH2 and their transition-state structures were computed at the B3LYP/6-31G(d) level. The effects of diameters of armchair SWCNTs on their binding energies were studied. Relative reactivities of all the SWCNTs and their complexes based on their frontier orbital energies gaps are reported.

Complete genome sequences of Helicoverpa armigera Single Nucleopolyhedrovirus Strains AC53 and H25EA1 from Australia

We report here the genome sequences of two alphabaculoviruses of Helicoverpa spp. from Australia: AC53, used in the biopesticides ViVUS and ViVUS Max, and H25EA1, used in in vitro production studies.

Bi-criteria single machine scheduling problem with a learning effect: Aneja-Nair method to obtain the set of optimal sequences

In this paper, we consider the bi-criteria single machine scheduling problem of n jobs with a learning effect. The two objectives considered are the total completion time (TC) and total absolute differences in completion times (TADC). The objective is to find a sequence that performs well with respect to both the objectives: the total completion time and the total absolute differences in completion times. In an earlier study, a method of solving bi-criteria transportation problem is presented. In this paper, we use the methodology of solvin bi-criteria transportation problem, to our bi-criteria single machine scheduling problem with a learning effect, and obtain the set of optimal sequences,. Numerical examples are presented for illustrating the applicability and ease of understanding.

Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein

The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease P1. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.

Polypurine-polypyrimidine sequences adopt unwound structure in pBR322 form V DNA as probed by single-hit analysis of HpaII sites.

Structure at the polypurine-polypyrimidine sequences flanking the HpaII sites (CCGG) in pBR322 form V DNA was probed employing single-hit analysis using HpaII restriction endonuclease. Reduced cleavage efficiency of HpaII sites flanked by polypurine-polypyrimidine sequences suggested that under high torsional stress these sequences adopt unwound structures rendering these sites insensitive to restriction enzyme cleavage. In addition to polypurine-polypyrimidine sequences. HpaII sites flanked by alternating purine-pyrimidine sequence, a potential motif of left handed Z-DNA, were also found to be resistant to HpaII cleavage. Results obtained from various studies implicating structure sensitivity of restriction endonucleases and methylases were compiled and a direct correlation was observed between the occurrence of altered sites in a domain and its G/C content in pBR322 form V DNA.

Sequence context analysis in the mouse genome: Single nucleotide polymorphisms and CpG island sequences

A genome-wide view of sequence mutability in mice is still limited, although biologists usually assume the same scenario for mice as for humans. In this study, we examined the sequence context in the local environment of 482,528 mouse single nucleotide po

Mining expressed sequences for single nucleotide polymorphisms in Pacific abalone Haliotis discus hannai

Although single nucleotide polymorphisms (SNPs) are important resources for population genetics, pedigree analysis and genomic mapping, such loci have not been reported in Pacific abalone so far. In this study, a bioinformatics strategy was adopted to discover SNPs within the expressed sequences (ESTs) of Pacific abalone, Haliotis discus hannai, and furthermore, polymerase chain reaction direct sequencing (PCR-DS) and allele-specific PCR (AS-PCR) were used for SNPs detection and genotype scoring respectively. A total of 5893 ESTs were assembled and 302 putative SNPs were identified. The average density of SNPs in ESTs was 1%. Fifty-two sets of sequencing primers were designed from SNPs flanking ESTs to amplify the genomic DNA, and 13 could generate products of expected size. Polymerase chain reaction direct sequencing of the amplification products from pooled DNA samples revealed 40 polymorphic SNP loci. Using a modified tetra-primer AS-PCR, seven mitochondrial and six nuclear SNPs were typed and characterized among 37 wild abalones. In conclusion, it is feasible to discover SNPs from number limited ESTs and the AS-PCR as a simple, robust and reliable assay could be a primary method for small- and medium-scale SNPs detection in abalones as well as other non-model organisms.

rbcL sequences indicate a single evolutionary origin of multinucleate cells in the red algal tribe Callithamnieae.

In the Ceramiaceae, one of the largest families of the red algae, there are from 1 to 4000 nuclei in each vegetative cell, but each tribe is homogeneous with respect to the uninucleate/multinucleate character state, except for the Callithamnieae. The goals of this study were to analyze rbcL gene sequences to clarify the evolution of taxa within the tribe Callithamnieae and to evaluate the potential evolutionary significance of the development of multinucleate cells in certain taxa. The genus Aglaothamnion, segregated from Callithamnion because it is uninucleate, was paraphyletic in all analyses. Callithamnion (including Aristothamnion) was monophyletic although not robustly so, apparently due to variations between taxa in rate of sequence evolution. Morphological synapomorphies were identified at different depths in the tree, supporting the molecular phylogenetic analysis. The uninucleate character state is ancestral in this tribe. The evolution of multinucleate cells has occurred once in the Callithamnieae. Multiple nuclei in each cell may combine the benefits of small C values (rapid cell cycle) with large cells (permitting morphological elaboration) while maintaining a constant ratio of nuclear volume: cytoplasmic volume.

Analysis of 16S rDNA sequences from pathogenic Leptospira serovars and use of single nucleotide polymorphisms for rapid speciation by D-HPLC

Leptospira have a worldwide distribution and include important zoonotic pathogens yet diagnosis and differentiation still tend to rely on traditional bacteriological and serological approaches. In this study a 1.3 kb fragment of the rrs gene (16S rDNA) was sequenced from a panel of 22 control strains, representing serovars within the pathogenic species Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri, to identify single nucleotide polymorphisms (SNPs). These were identified in the 5' variable region of the 16S sequence and a 181 bp PCR fragment encompassing this region was used for speciation by Denaturing High Performance Liquid Chromatography (D-HPLC). This method was applied to eleven additional species, representing pathogenic, non-pathogenic and intermediate species and was demonstrated to rapidly differentiate all but 2 of the non-pathogenic Leptospira species. The method was applied successfully to infected tissues from field samples proving its value for diagnosing leptospiral infections found in animals in the UK. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

Regulatory sequences of the porcine THBD gene facilitate endothelial-specific expression of bioactive human thrombomodulin in single- and multitransgenic pigs.

BACKGROUND Among other mismatches between human and pig, incompatibilities in the blood coagulation systems hamper the xenotransplantation of vascularized organs. The provision of the porcine endothelium with human thrombomodulin (hTM) is hypothesized to overcome the impaired activation of protein C by a heterodimer consisting of human thrombin and porcine TM. METHODS We evaluated regulatory regions of the THBD gene, optimized vectors for transgene expression, and generated hTM expressing pigs by somatic cell nuclear transfer. Genetically modified pigs were characterized at the molecular, cellular, histological, and physiological levels. RESULTS A 7.6-kb fragment containing the entire upstream region of the porcine THBD gene was found to drive a high expression in a porcine endothelial cell line and was therefore used to control hTM expression in transgenic pigs. The abundance of hTM was restricted to the endothelium, according to the predicted pattern, and the transgene expression of hTM was stably inherited to the offspring. When endothelial cells from pigs carrying the hTM transgene--either alone or in combination with an aGalTKO and a transgene encoding the human CD46-were tested in a coagulation assay with human whole blood, the clotting time was increased three- to four-fold (P<0.001) compared to wild-type and aGalTKO/CD46 transgenic endothelial cells. This, for the first time, demonstrated the anticoagulant properties of hTM on porcine endothelial cells in a human whole blood assay. CONCLUSIONS The biological efficacy of hTM suggests that the (multi-)transgenic donor pigs described here have the potential to overcome coagulation incompatibilities in pig-to-primate xenotransplantation.