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PubMed2005

Comparing alignment methods for inferring the history of the new world lizard genus Mabuya (Squamata: Scincidae).

The rapid increase in the ability to generate molecular data, and the focus on model-based methods for tree reconstruction have greatly advanced the use of phylogenetics in many fields. The recent flurry of new analytical techniques has focused almost solely on tree reconstruction, whereas alignment issues have received far less attention. In this paper, we use a diverse sampling of gene regions from lizards of the genus Mabuya to compare the impact, on phylogeny estimation, of new maximum likelihood alignment algorithms with more widely used methods. Sequences aligned under different optimality criteria are analyzed using partitioned Bayesian analysis with independent models and parameter settings for each gene region, and the most strongly supported phylogenetic hypothesis is then used to test the hypothesis of two colonizations of the New World by African scincid lizards. Our results show that the consistent use of model-based methods in both alignment and tree reconstruction leads to trees with more optimal likelihood scores than the use of independent criteria in alignment and tree reconstruction. We corroborate and extend earlier evidence for two independent colonizations of South America by scincid lizards. Relationships within South American Mabuya are found to be in need of taxonomic revision, specifically complexes under the names M. heathi, M. agilis, and M. bistriata (sensu, M.T. Rodrigues, Papeis Avulsos de Zoologia 41 (2000) 313).

Animals↗

PubMed1994

Biochemical characterization of Bacillus thuringiensis cytolytic delta-endotoxins.

The entomocidal delta-endotoxins CytA and CytB produced by Bacillus thuringiensis (Bt) subspecies israelensis and kyushuensis respectively showed a similar level of toxicity to mosquito larvae but were not toxic to the larvae of the lepidopteran Manduca sexta. CytA and CytB are also similar in sequence, predicted secondary structure and alpha-helical content, the only obvious difference being a C-terminal fifteen residue 'tail' on CytB. Investigations of the function, if any, of the CytB C-terminal 'tail' showed that this delta-endotoxin is highly expressed and forms inclusions in an acrylstalliferous Bt mutant without the aid of the 20 kDa 'helper' protein from Bt subspecies israelensis which is essential for CytA inclusion formation. After proteinase K treatment, CytA and CytB were processed to virtually the same points in a sequence alignment and were equally haemolytic in vitro. However, the results suggested that unprocessed CytB differs from unprocessed CytA in that the former is not haemolytic.

Amino Acid Sequence↗

PubMed2003

Phylogenetic systematics and evolution of primate-derived Pneumocystis based on mitochondrial or nuclear DNA sequence comparison.

Previous studies have demonstrated that the agent of Pneumocystis pneumonia (PcP), Pneumocystis carinii, is actually a complex of eukaryotic organisms, and cophylogeny could explain the distribution of the hosts and parasites. In the present work, we tested the hypothesis of cophylogeny between the primate-derived Pneumocystis group and their hosts. Specific strains isolated from 20 primate species, including humans, were used to produce a phylogeny of the parasites. Aligned sequences corresponding to DNA sequences of three genes (DHPS, mtSSU-rRNA, and mtLSU-rRNA) were separately analyzed and then combined in a single data set. The resulting parasite phylogeny was compared with different controversial phylogenies for the hosts. This comparison demonstrated that, depending upon which topology is accepted for the hosts, at least 61% and perhaps 77% of the homologous nodes of the respective cladograms of the hosts and parasites may be interpreted as resulting from codivergence events. This finding and the high specificity of these parasites suggests that cophylogeny may be considered the dominant pattern of evolution for Pneumocystis organisms, representing a new example of parallel evolution between primates and their specific parasites. Because the phylogeny of Pneumocystis followed very closely the differentiation of their hosts at the species level, the study of the parasites could provide valuable information on the phylogeny of their hosts. We used this information to explore controversial hypotheses of the phylogeny of the Platyrrhini by comparison with the phylogeny of their specific Pneumocystis parasites. If these organisms were closely associated as lung parasites with primates through the ages, the hypothesis of the Pneumocystis spp. being new pathogenic agents could be refuted. However, these organisms are opportunistic symbionts, becoming pathogenic whenever the immunological defences of their hosts decline. This study also provides support for the hypothesis that the different Pneumocystis species are genetically independent organisms, helping to clarify their taxonomic status.

Animals↗

PubMed1993

Taxonomic relationships between distinct potato virus Y isolates based on detailed comparisons of the viral coat proteins and 3'-nontranslated regions.

Detailed comparisons were made of the sequences of the coat protein (CP) cistrons and 3'-nontranslated regions (3'-NTR) of 21 (geographically) distinct isolates of potato virus Y (PVY) and a virus isolate initially described as pepper mottle virus (PepMoV). Multiple sequence alignments and phylogenetic relationships based on these alignments resulted into a subgrouping of virus isolates which largely corresponded with the historical strain differentiation based on biological criteria as host range, symptomatology and serology. Virus isolates belonging to the same subgroup shared a number of characteristic CP amino acid and 3'-NTR nucleotide residues indicating that, by using sequences from the 3'-terminal region of the potyvirus genome, a distinction could be made between different isolates of one virus species as well as between different virus species. RNA secondary structure analysis of the 3'-NTR of twelve PVY isolates revealed four major stem-loop structures of which, surprisingly, the loop sequences gave a similar clustering of isolates as resulting from the overall comparisons of CP and 3'-NTR sequences. This implies a biological significance of these structural elements.

Amino Acid Sequence↗

PubMed2003

Search-based optimization.

The problem of determining the minimum cost hypothetical ancestral sequences for a given cladogram is known to be NP-complete (Wang and Jiang, 1994). Traditionally, point estimations of hypothetical ancestral sequences have been used to gain heuristic, upper bounds on cladogram cost. These include procedures with such diverse approaches as non-additive optimization of multiple sequence alignment, direct optimization (Wheeler, 1996), and fixed-state character optimization (Wheeler, 1999). A method is proposed here which, by extending fixed-state character optimization, replaces the estimation process with a search. This form of optimization examines a diversity of potential state solutions for cost-efficient hypothetical ancestral sequences and can result in greatly more parsimonious cladograms. Additionally, such an approach can be applied to other NP-complete phylogenetic optimization problems such as genomic break-point analysis.

Animals↗

PubMed1996

A novel clade of protistan parasites near the animal-fungal divergence.

Sequences of nuclear-encoded small-subunit rRNA genes have been determined for representatives of the enigmatic genera Dermocystidium, Ichthyophonus, and Psorospermium, protistan parasites of fish and crustaceans. The small-subunit rRNA genes from these parasites and from the "rosette agent" (also a parasite of fish) together form a novel, statistically supported clade. Phylogenetic analyses demonstrate this clade to diverge near the animal-fungal dichotomy, although more precise resolution is problematic. In the most parsimonious and maximally likely phylogenetic frameworks inferred from the most stably aligned sequence regions, the clade constitutes the most basal branch of the metazoa; but within a limited range of model parameters, and in some analyses that incorporate less well-aligned sequence regions, an alternative topology in which it diverges immediately before the animal-fungal dichotomy was recovered. Mitochondrial cristae of Dermocystidium spp. are flat, whereas those of Ichthyophonus hoferi appear tubulovesiculate. These results extend our understanding of the types of organisms from which metazoa and fungi may have evolved.

Animals↗

PubMed2001

Purification, molecular cloning, and properties of a beta-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae.

Two beta-glycosidases (M(r) 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (betaGly1 and betaGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E379 (nucleophile) and E169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant beta-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellodextrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified beta-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose beta-1,4-glucosidic bond is 21.2 s(-1) x M(-1). The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.

Amino Acid Sequence↗

PubMed2006

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein.

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

Adrenal Hyperplasia, Congenital↗

PubMed1997

Three-dimensional comparative modeling of RNA.

Comparative sequence analysis and ERNA-3D software were used to model the three-dimensional structure of the small domain of signal recognition particle RNA. RNA secondary structures were established by allowing only phylogenetically-supported base pairs. The folding of the RNA molecules was constrained further to include a well-supported pseudoknot. Helical sections were oriented coaxially where a continuous helical stack was formed in the RNA of another species. Finally, RNA helices were placed at distances that preserved the connectivity of the molecule with the smallest number of single-stranded nucleotide residues as identified from the aligned sequences. We show that the comparative three-dimensional structure modeling approach is an extremely powerful tool as it requires only a critical number of carefully aligned sequences.

Bacillus subtilis↗

PubMed2005

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli.

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.

Amino Acid Sequence↗

PubMed2006

Panta rhei (QAlign2): an open graphical environment for sequence analysis.

MOTIVATION: The first version of the graphical multiple sequence alignment environment QAlign was published in 2003. Heavy response from the molecular-biological user community clearly demonstrated the need for such a platform. RESULTS: Panta rhei extends QAlign by several features. Major redesigns on the user interface, for instance, allow users to flexibily compose views for multiple projects. The new sequence viewer handles datasets with arbitrarily many and arbitrarily large sequences that may still be edited by guided block moving. More distance-based algorithms are available to interactively reconstruct phylogenetic trees which can now also be zoomed and navigated graphicaly. AVAILABILITY: Executables and the JAVA source code are available under the Apache license at http://gi.cebitec.uni-bielefeld.de/qalign CONTACT: qalign@cebitec.uni-bielefeld.de.

Algorithms↗

PubMed2000

Purification, characterisation and cDNA sequencing of pyruvate decarboxylase from Zygosaccharomyces bisporus.

Cells of the wild-type yeast strain Zygosaccharomyces bisporus CBS 702 form alpha-hydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the non-oxidative decarboxylation of pyruvate and other 2-oxo-acids, was purified and characterised. The active enzyme is homotetrameric (alpha4) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent Km value 1.73 mM; Hill coefficient 2.10). A cDNA library was screened using a PCR-based procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies (> 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.

DNA, Complementary↗

PubMed1989

Hydrophobic cluster analysis of the primary sequences of alpha-amylases.

The amino acid sequences of 18 alpha-amylases have been compared by hydrophobic cluster analysis. The method was first calibrated with two alpha-amylases (Aspergillus oryzae and pig pancreas) whose three-dimensional structures are known. It was then applied to the other alpha-amylases resulting in straightforward sequence alignments which could be used for structure prediction. It was found that all alpha-amylases which were investigated display the same basic super-secondary structure with a (beta alpha)8 barrel. Most of the secondary structure elements of the protein cores could be assigned to segments of the amino acid sequences. In addition, six sub-families could be identified, based upon specific similarities occurring in the variable regions of alpha-amylases.

Amino Acid Sequence↗

PubMed2005

Amino acid recognition by Venus flytrap domains is encoded in an 8-residue motif.

A motif foramino acid recognition by proteins or domains of the periplasmic binding protein-like I superfamily has been identified. An initial pattern of 5 residues was based on a multiple sequence alignment of selected proteins of that fold family and on common structural features observed in the crystal structure of some members of the family [leucine isoleucine valine binding protein (LIVBP), leucine binding protein (LBP), and metabotropic glutamate receptor type 1 (mGlu1R) amino terminal domain)]. This pattern was used against the PIR-NREF sequence database and further refined to retrieve all sequences of proteins that belong to the family and eliminate those that do not belong to it. A motif of 8 residues was finally selected to build up the general signature. A total of 232 sequences were retrieved. They were found to belong to only three families of proteins: bacterial periplasmic binding proteins (PBP, 71 sequences), family 3 (or C) of G-protein coupled receptor (GPCR) (146 sequences), and plant putative ionotropic glutamate receptors (iGluR, 15 sequences). PBPs are known to adopt a bilobate structure also named Venus flytrap domain, or LIVBP domain in the present case. Family 3/C GPCRs are also known to hold such a domain. However, for plant iGluRs, it was previously detected by classical similarity searches but not specifically described. Thus plant iGluRs carry two Venus flytrap domains, one that binds glutamate and an additional one that would be a modulatory LIVBP domain. In some cases, the modulator binding to that domain would be an amino acid.

Amino Acid Motifs↗

PubMed1994

MIF proteins are not glutathione transferase homologs.

Although macrophage migration inhibitory factor (MIF) proteins conjugate glutathione, sequence analysis does not support their homology to other glutathione transferases. Glutathione transferases are not detected with MIF proteins in searches of protein sequence databases, and MIF proteins do not share significant sequence similarity with glutathione transferases. Homology cannot be demonstrated by multiple sequence alignment or evolutionary tree construction; such methods assume that the proteins being analyzed are homologous.

Animals↗

PubMed2006

Genetic relatedness of six North-Indian butterfly species (Lepidoptera :Pieridae) based on 16S rRNA sequence analysis.

The present work involves the assessment of level of genetic relatedness or divergence amongst the six North-Indian species of Lepidoptera belonging to family Pieridae and sub family Pierinae on the basis of sequence variation of 16S ribosomal RNA. The PCR amplified products of these species were directly sequenced using ABI Prism BigDye Terminator Sequencing Kits (Applied Biosystems). The multiple nucleotide sequence alignment analysis has revealed several differences across these species. Significantly high percentage of A + T base composition content ranging between 73.13% (Ixias pyrene ) and 79.20 % (Pieris brassica) was observed in studied species. The percentage divergence in the investigated species of Pieridae family varied from 5.5% to 21.7%. The two species of Catopsilia revealed minimum sequence divergence of only 5.5%, whereas the other two groups of Ixias and Pieris revealed 15.5% and 8.6% sequence divergence, respectively. Pieris canidia and Ixias pyrene are genetically most divergent (21.7%) amongst the studied lepidopteran species. Phylogenetic analysis based on 16S rRNA nucleotide sequence revealed grouping of six species of Lepidoptera in the form of two different clusters, each cluster being represented by two species from the same genera. The separate taxonomic grouping of these Indian species has been observed when compared with several species of Piernae and Coliadinae subfamilies from other country isolates.

Animals↗

PubMed1998

Comparative analysis of human and macaque monkey CD4: differences in formaldehyde lability and conformation.

Here we characterized macaque monkey CD4 by flow cytometry. The results showed that relatively lower fluorescence intensity was observed depending on the monoclonal antibodies (mAbs) used for staining; Leu-3a exhibited four-fold lower intensity than Nu-Th/i, and that formaldehyde fixation dramatically reduced fluorescence intensity of macaque CD4+ cells stained with Leu-3a but not of human cells. Nu-Th/i is therefore preferable for the analysis of macaque CD4. Pretreatment of either mAb inhibited the other mAb binding to human CD4. On the contrary, Nu-Th/i inhibited Leu-3a binding but Leu-3a poorly blocked Nu-Th/i binding to the macaque CD4. These results indicate that Leu-3a and Nu-Th/i epitopes are conserved in macaque CD4 but Leu-3a epitope is conformationally cryptic and/or fragile, resulting in the lower affinity. Amino acid sequence alignment of CD4 domain 1 shows that the substitutions outside the linear Leu-3a epitope may determine these characteristics of macaque CD4.

Amino Acid Sequence↗

PubMed2002

A novel index which precisely derives protein coding regions from cross-species genome alignments.

We introduce here a novel index which precisely derives protein coding regions from cross-species genome alignments. The index is deeply related to frame recovery observed in coding sequence alignments, that is, if insertions or deletions of nucleotides causes frame shifts in coding regions, other in-dels which recover the reading frames will be often observed in the vicinity. In contrast, such frame recoveries are not observed in other conserved regions. We prepared two gene models: a model which finds gene by using sequence similarity and intrinsic gene measures (basic model), and the other model which finds gene by using frame recovery index in addition to sequence similarity and intrinsic gene measures (frame recovery model). We evaluated the prediction accuracies of the two models, and our benchmark test revealed that frame recovery model significantly improved the prediction accuracy in comparison with basic model.

Abstracting and Indexing↗