Sequence analysis of cyclodextrin glycosyltransferase from the alkaliphilic Bacillus agaradhaerens

The gene encoding an alkaline active cyclodextrin glycosyltransferase (CGTase) from the alkaliphilic B. agaradhaerens LS-3C was cloned and sequenced. It encodes a mature polypeptide of 679 amino acids with a molecular mass of 76488 Da. The deduced amino acid sequence of the mature CGTase revealed 99 and 95% identity to the CGTase sequences from the other B. agaradhaerens strains, DSM 8721^T and 9948, respectively. The next closest identity was of 59% with B. clarkii enzyme. CGTases from B. agaradhaerens, B. clarkii, and B. firmus/lentus formed a phylogenetically separated cluster from the other CGTases of Bacillus spp. origin. A number of usually conserved residues in the CGTases were found to be replaced in the sequence of B. agaradhaerens enzyme. The sequence analysis indicated the enzyme to be close to the so-called `intermediary enzymes' in the -amylase family.     

Detection of sequence polymorphisms in red junglefowl and White Leghorn ESTs

Over 16,000 high quality expressed sequence tags (ESTs) from red junglefowl (RJ) and White Leghorn (WL) brain and testis cDNA libraries were generated. Here, we have used this resource for detection of single nucleotide polymorphisms (SNPs), and also completed full-length sequencing of 46 pairs of clones, representing the same gene from both the RJ and WL libraries. From the main set of ESTs, which were assembled using Phrap, 746 putative SNPs were identified, of which 76% were transitions and 24% were transversions. A subset of SNPs was evaluated by sequence analysis of five RJ and five WL birds. Nine of 12 SNPs were verified in this limited sample, suggesting that a majority of the putative polymorphisms documented in this study represent real SNPs. During full-length sequencing of the 46 RJ/WL clones 100 SNPs were identified, which translated to a frequency of 1.90 SNPs/1000 bp. The number of transitions and transversions were 77% and 23%, respectively, and the proportion of non-synonymous vs. synonymous SNPs was 20% and 80%, respectively. Four large insertions/deletions were identified between the RJ and WL full-length sequences, and they appear to represent different splice variants.     

An immunostimulatory DNA sequence from a probiotic strain of Bifidobacterium longum inhibits IgE production in vitro

The immunostimulatory oligodeoxynucleotide (ODN) BL07 (5'-GCGTCGGTTTCGGTGCTCAC-3') was identified from the genomic DNA of the probiotic strain Bifidobacterium longum BB536. ODN BL07 stimulated B-lymphocyte proliferation and induced interleukin-12 (IL-12) production in macrophage-like J774.1 cells. ODNs BL07 and BL07S (modified with phosphorothioate backbone) significantly inhibited immunoglobulin E (IgE) production and stimulated interferon-gamma (IFN-gamma) and IL-12 production, but did not affect IL-4 secretion in murine splenic cells of ovalbumin-primed BALB/c mice. These ODNs also significantly inhibited production of IgE in purified murine B cells in the presence of IL-4 and anti-CD40. The results suggest the potential of ODNs BL07 and BL07S in preventing IgE-related immune responses and the possible involvement of ODN BL07 in the antiallergic efficacy of B. longum BB536.     

Homology between the seed cytolysin enterolobin and bacterial aerolysins

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian treeEnterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins fromAeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences ofA. hydrophila andA. sobria aerolysins showed several similar regions with many residue identites. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.     

Structural signatures of enzyme binding pockets from order-independent surface alignment: a study of metalloendopeptidase and NAD binding proteins

Detecting similarities between local binding surfaces can facilitate identification of enzyme binding sites and prediction of enzyme functions, and aid in our understanding of enzyme mechanisms. Constructing a template of local surface characteristics for a specific enzyme function or binding activity is a challenging task, as the size and shape of the binding surfaces of a biochemical function often vary. Here we introduce the concept of signature binding pockets, which captures information on preserved and varied atomic positions at multiresolution levels. For proteins with complex enzyme binding and activity, multiple signatures arise naturally in our model, forming a signature basis set that characterizes this class of proteins. Both signatures and signature basis sets can be automatically constructed by a method called SOLAR (Signature Of Local Active Regions). This method is based on a sequence-order-independent alignment of computed binding surface pockets. SOLAR also provides a structure-based multiple sequence fragment alignment to facilitate the interpretation of computed signatures. By studying a family of evolutionarily related proteins, we show that for metzincin metalloendopeptidase, which has a broad spectrum of substrate binding, signature and basis set pockets can be used to discriminate metzincins from other enzymes, to predict the subclass of metzincins functions, and to identify specific binding surfaces. Studying unrelated proteins that have evolved to bind to the same NAD cofactor, we constructed signatures of NAD binding pockets and used them to predict NAD binding proteins and to locate NAD binding pockets. By measuring preservation ratio and location variation, our method can identify residues and atoms that are important for binding affinity and specificity. In both cases, we show that signatures and signature basis set reveal significant biological insight.     

A Cysteine Endopeptidase (“Dionain”) Is Involved in the Digestive Fluid of Dionaea muscipula (Venus’s Fly-trap)

The carnivorous plant Dionaea muscipula (Venus’s flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS–PAGE band protein. The name “dionain” is proposed for the enzyme.     

Amylase gene expression patterns in Helicoverpa armigera upon feeding on a range of host plants

Venoms contain highly complex mixtures that typically include hundreds of different components and have evolved independently in a diverse range of animals including platypuses, shrews, snakes, lizards, fishes, echinoderms, spiders, wasps, centipedes, sea snails, cephalopods, jellyfish and sea anemones. Many venom genes evolved through gene duplication. Gene duplication occurs in all domains of life and provides the raw substrate from which novel function arise. In this review, we focus on the role that gene duplication has played in the origin and diversification of venom genes. We outline the selective advantages of venom gene duplicates and the role that selection has played in the retention of these duplicates. We use toxin gene intermediates to help trace the evolution of toxin innovation. We also focus on other genomic processes, such as exon and domain duplications, in venom evolution. Finally, we conclude by focusing on the use of high throughput sequencing technology in understanding venom evolution.