ANNODA: tool for integrating molecular-biological annotation data

Collecting, analyzing, and making Molecularbiological annotation data accessible in different public data sources is still an ongoing project. Integration of such data from these data sources might lead to valuable biological knowledge. There are numerous annotation data and only some of those are structured. The number and contents of related sources are continuously increasing. In addition, the existing data sources have their own storage structure and implementation. As a result, these could lead to a limitation in the combining of annotation. Here, we proposed a tool, called ANNODA, for integrating Molecular-biological annotation data. Unlike the past work on database interoperation in the bioinformatics community, this database design uses web-links which are very useful for interactive navigation and meanwhile it also supports automated large-scale analysis tasks.

Widespread convergence in toxin resistance by predictable molecular evolution

The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na(+)/K(+)-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of 2 of the 12 amino acids comprising the Na(+)/K(+)-ATPase H1-H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na(+)/K(+)-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.

Functional analysis of yeast snoRNA and snRNA 3' end formation mediated by uncoupling of cleavage and polyadenylation

Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3′-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3′ portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

Dimerization of retroviral RNA genomes : an inseparable pair

Many viruses carry more than one segment of nucleic acid into the virion particle, but retroviruses are the only known group of viruses that contain two identical (or nearly identical) copies of the RNA genome within the virion. These RNA genomes are non-covalently joined together through a process known as genomic RNA dimerization. Uniquely, the RNA dimerization of the retroviral genome is of crucial importance for efficient retroviral replication. In this article, our current understanding of the relationship between retroviral genome conformation, dimerization and replication is reviewed.

Mitochondrial DNA analysis of field populations of Helicoverpa armigera (Lepidoptera : Noctuidae) and of its relationship to H. zea

Background
Helicoverpa armigera and H. zea are amongst the most significant polyphagous pest lepidopteran species in the Old and New Worlds respectively. Separation of H. armigera and H. zea is difficult and is usually only achieved through morphological differences in the genitalia. They are capable of interbreeding to produce fertile offspring. The single species status of H. armigera has been doubted, due to its wide distribution and plant host range across the Old World. This study explores the global genetic diversity of H. armigera and its evolutionary relationship to H zea.

Results
We obtained partial (511 bp) mitochondrial DNA (mtDNA) Cytochrome Oxidase-I (COI) sequences for 249 individuals of H. armigera sampled from Australia, Burkina Faso, Uganda, China, India and Pakistan which were associated with various host plants. Single nucleotide polymorphisms (SNPs) within the partial COI gene differentiated H. armigera populations into 33 mtDNA haplotypes. Shared haplotypes between continents, low F-statistic values and low nucleotide diversity between countries (0.0017 – 0.0038) suggests high mobility in this pest. Phylogenetic analysis of four major Helicoverpa pest species indicates that H. punctigera is basal to H. assulta, which is in turn basal to H. armigera and H. zea. Samples from North and South America suggest that H. zea is also a single species across its distribution. Our data reveal short genetic distances between H. armigera and H. zea which seem to have been established via a founder event from H. armigera stock at around 1.5 million years ago.

Conclusion
Our mitochondrial DNA sequence data supports the single species status of H. armigera across Africa, Asia and Australia. The evidence for inter-continental gene flow observed in this study is consistent with published evidence of the capacity of this species to migrate over long distances. The finding of high genetic similarity between Old World H. armigera and New World H. zea emphasises the need to consider work on both pests when building pest management strategies for either.

Expression and purification of soluble recombinant full length HIV-1 Pr55(Gag) protein in Escherichia coli

The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.

Swine influenza virus PA and neuraminidase gene reassortment into human H1N1iInfluenza Virus is associated with an altered pathogenic phenotype linked to increased MIP-2 expression

Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression. IMPORTANCE: Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

Lipids in Amyloid-β processing, aggregation, and toxicity

Aggregation of amyloid-beta (Aβ) peptide is the major event underlying neuronal damage in Alzheimer's disease (AD). Specific lipids and their homeostasis play important roles in this and other neurodegenerative disorders. The complex interplay between the lipids and the generation, clearance or deposition of Aβ has been intensively investigated and is reviewed in this chapter. Membrane lipids can have an important influence on the biogenesis of Aβ from its precursor protein. In particular, increased cholesterol in the plasma membrane augments Aβ generation and shows a strong positive correlation with AD progression. Furthermore, apolipoprotein E, which transports cholesterol in the cerebrospinal fluid and is known to interact with Aβ or compete with it for the lipoprotein receptor binding, significantly influences Aβ clearance in an isoform-specific manner and is the major genetic risk factor for AD. Aβ is an amphiphilic peptide that interacts with various lipids, proteins and their assemblies, which can lead to variation in Aβ aggregation in vitro and in vivo. Upon interaction with the lipid raft components, such as cholesterol, gangliosides and phospholipids, Aβ can aggregate on the cell membrane and thereby disrupt it, perhaps by forming channel-like pores. This leads to perturbed cellular calcium homeostasis, suggesting that Aβ-lipid interactions at the cell membrane probably trigger the neurotoxic cascade in AD. Here, we overview the roles of specific lipids, lipid assemblies and apolipoprotein E in Aβ processing, clearance and aggregation, and discuss the contribution of these factors to the neurotoxicity in AD.

EST-PAC HPC - a web portal for high-throughput EST annotation and protein sequence prediction

Expressed Sequence Tags (ESTs) are short DNA sequences generated by sequencing the transcribed cDNAs coming from a gene expression. They can provide significant functional, structural and evolutionary information and thus are a primary resource for gene discovery. EST annotation basically refers to the analysis of unknown ESTs that can be performed by database similarity search for possible identities and database search for functional prediction of translation products. Such kind of annotation typically consists of a series of repetitive tasks which should be automated, and be customizable and amenable to using distributed computing resources. Furthermore, processing of EST data should be done efficiently using a high performance computing platform. In this paper, we describe an EST annotator, EST-PACHPC, which has been developed for harnessing HPC resources potentially from Grid and Cloud systems for high throughput EST annotations. The performance analysis of EST-PACHPC has shown that it provides substantial performance gain in EST annotation.

Molecular recognition of DNA by rigid [n]-polynorbornane-derived bifunctional intercalators: synthesis and evaluation of their binding properties

We have exploited the concept of multivalency in the context of DNA recognition, using novel chemistry to synthesize a new type of bis-intercalator with unusual sequence-selectivity. Bis-intercalation has been observed previously, but design principles for de novo construction of such molecules are not known. Our compounds feature two aromatic moieties projecting from a rigid, polynorbornane-based scaffold. The length and character of the backbone as well as the identity of the intercalators were varied, resulting in mono- or divalent recognition of the double helix with varying affinity. Our lead compound proved to be a moderately sequence-selective bis-intercalator with an unwinding angle of 27 and a binding constant of about 8 M. 9-Aminoacridine rings were preferred over acridine carboxamides or naphthalimides, and a rigid [3]-polynorbornane scaffold was superior to a [5]-polynorbornane. The flexibility of the linker connecting the rings to the scaffold, although less influential, could affect the strength and character of the DNA binding.

Information integration in molecular bioscience

Integrating information in the molecular biosciences involves more than the cross-referencing of sequences or structures. Experimental protocols, results of computational analyses, annotations and links to relevant literature form integral parts of this information, and impart meaning to sequence or structure. In this review, we examine some existing approaches to integrating information in the molecular biosciences. We consider not only technical issues concerning the integration of heterogeneous data sources and the corresponding semantic implications, but also the integration of analytical results. Within the broad range of strategies for integration of data and information, we distinguish between platforms and developments. We discuss two current platforms and six current developments, and identify what we believe to be their strengths and limitations. We identify key unsolved problems in integrating information in the molecular biosciences, and discuss possible strategies for addressing them including semantic integration using ontologies, XML as a data model, and graphical user interfaces as integrative environments.

Molecular Genetic Evidence for a New Species of Bream of the Genus Acanthopagrus Peters (Perciformes: Sparidae)

Fishes of the genus Acanthopagrus are found throughout the coastal waters of Asia and Australia with several species being of commercial significance. In this study, genetic comparisons are made between widely disjunct populations of Acanthopagrus australis (Günther) from Australian and Taiwanese waters and among samples of A. butcheri (Munro), A. berda, (Forskal), A schlegeli (Day) and A. latus (Houttuyn) using mitochondrial DNA sequences obtained from the control region. The mean interspecific pairwise sequence divergence for all species is 17%, while the divergence between A. australis from Australia and that of Taiwan is slightly larger at 18%. These values are considerably higher than those found for intraspecific control region comparisons in some fish species. Phylogenetic analyses indicate that A. australis from Australia is more closely related to the Australian species A. butcheri than to A. australis from Taiwan. These findings suggest that the northern and southern hemisphere forms of A. australis are not monophyletic, with the former possibly representing a new undescribed species of Acanthopagrus.

Molecular cloning of natriuretic peptides from the heart of reptiles : loss of ANP in diapsid reptiles and birds

Atrial natriuretic peptide (ANP) and B-type NP (BNP) are hormones involved in homeostatic control of body fluid and cardiovascular regulation. Both ANP and BNP have been cloned from the heart of mammals, amphibians, and teleost fishes, while an additional cardiac peptide, ventricular NP, has been found in selected species of teleost fish. However, in chicken, BNP is the primary cardiac peptide identified thus far. In contrast, the types of NP/s present in the reptilian heart are unknown, representing a considerable gap in our understanding of NP evolution. In the present study, we cloned and sequenced a BNP cDNA from the atria of representative species of reptile, including crocodile, lizard, snake, and tortoise. In addition, we cloned BNP from the pigeon atria. The reptilian and pigeon BNP cDNAs had ATTTA repeats in the 3′ untranslated region, as observed in all vertebrate BNP mRNAs. A high sequence homology was evident when comparing reptile and pigeon preproBNP with the previously identified chicken preproBNP. In particular, the predicted mature BNP-29 was identical between crocodile, tortoise, and chicken, with pigeon having a single amino acid substitution; lizard and snake BNP had seven and nine substitutions, respectively. Furthermore, an ANP cDNA could only be cloned from the tortoise atria. Since ANP was not isolated from the heart of any non-chelonian reptile and appears to be absent in birds, we propose that the ANP gene has been lost after branching of the turtles in the amniote line. This data provides new avenues for research on NP function in reptiles.

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using 14C-labeled S-adenosyl-L-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-14C]AdoMet or [carboxyl-14C]AdoMet yielded the sequence H2N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu10654 and Pro10655 as the major sites of derivatization. [carboxyl-14C]AdoMet in addition labeled Tyr10650. Chymotryptic digestion generated the radiolabeled peptide H2N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu2128 and Pro2129, which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.

Molecular identification of Staphylococcus xylosus MAK2, a new α-l-rhamnosidase producer

A bacterial strain, MAK-2, was isolated as a producer of α-l-rhamnosidase from a soil sample of Dehradoon, India. The strain was identified based on morphology, physiological tests and 16S rDNA analysis. The phylogenetic analysis based on the 16S rDNA sequence, identified the isolate as Staphylococcus xylosus, a nonpathogenic member of CNS (coagulase-negative staphylococci) family. The strain was capable of producing α-l-rhamnosidase by hydrolysing flavonoids thus confirming potential application in the citrus-processing industry.