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PubMed2006

A jumping profile Hidden Markov Model and applications to recombination sites in HIV and HCV genomes.

BACKGROUND: Jumping alignments have recently been proposed as a strategy to search a given multiple sequence alignment A against a database. Instead of comparing a database sequence S to the multiple alignment or profile as a whole, S is compared and aligned to individual sequences from A. Within this alignment, S can jump between different sequences from A, so different parts of S can be aligned to different sequences from the input multiple alignment. This approach is particularly useful for dealing with recombination events. RESULTS: We developed a jumping profile Hidden Markov Model (jpHMM), a probabilistic generalization of the jumping-alignment approach. Given a partition of the aligned input sequence family into known sequence subtypes, our model can jump between states corresponding to these different subtypes, depending on which subtype is locally most similar to a database sequence. Jumps between different subtypes are indicative of intersubtype recombinations. We applied our method to a large set of genome sequences from human immunodeficiency virus (HIV) and hepatitis C virus (HCV) as well as to simulated recombined genome sequences. CONCLUSION: Our results demonstrate that jumps in our jumping profile HMM often correspond to recombination breakpoints; our approach can therefore be used to detect recombinations in genomic sequences. The recombination breakpoints identified by jpHMM were found to be significantly more accurate than breakpoints defined by traditional methods based on comparing single representative sequences.

Algorithms↗

PubMed2003

MAVG: locating non-overlapping maximum average segments in a given sequence.

SUMMARY: MAVG is a software tool for finding k non-overlapping maximum-average segments that are sufficiently long in a given sequence of real numbers, for any k > 0. It has applications in several areas of biomolecular sequence analysis including locating GC-rich regions and CpG islands in a genomic sequence, and annotating multiple sequence alignments. AVAILABILITY: http://iubio.bio.indiana.edu/soft/molbio/pattern/cpg_islands/.

Algorithms↗

PubMed2004

Relation between protein stability, evolution and structure, as probed by carboxylic acid mutations.

Native proteins are marginally stable. Low thermodynamic stability may actually be advantageous, although the accumulation of neutral, destabilizing mutations may have also contributed to it. In any case, once marginal stability has been reached, it appears plausible that mutations at non-constrained positions become fixed in the course of evolution (due to random drift) with frequencies that roughly reflect the mutation effects on stability ("pseudo-equilibrium hypothesis"). We have found that all glutamate-->aspartate mutations in wild-type Escherichia coli thioredoxin are destabilizing, as well as most of the aspartate-->glutamate mutations. Furthermore, the effect of these mutations on thioredoxin thermodynamic stability shows a robust correlation with the frequencies of occurrence of the involved residues in several-hundred sequence alignments derived from a BLAST search. These results provide direct and quantitative experimental evidence for the pseudo-equilibrium hypothesis and should have general consequences for the interpretation of mutation effects on protein stability, as they suggest that residue environments in proteins may be optimized for stabilizing interactions to a remarkable degree of specificity. We also provide evidence that such stabilizing interactions may be detected in sequence alignments, and briefly discuss the implications of this possibility for the derivation of structural information (on native and denatured states) from comparative sequence analyses.

Aspartic Acid↗

PubMed2000

Altered subunit communication in subfamilies of trimeric dUTPases.

The enzyme dUTPase is essential in preventing uracil incorporation into DNA. Design of antagonists against this novel chemotherapeutic target requires identification of species-specific differences in the structure and mechanism of the enzyme. This task is now approached via comparisons of available crystallographic structures of dUTPases from Homo sapiens, Escherichia coli, and retroviruses. The eukaryotic protein uniquely displays polar and charged amino acid residues participating in threefold intersubunit interactions. In bacterial and retroviral dUTPases, threefold interactions are mainly hydrophobic. The residues responsible for this contrast are mapped in multiple sequence alignment to positions differently and characteristically conserved in distinct evolutionary branches. The general feature of this contrast is further strengthened by a second eukaryotic model structure constructed using comparative modeling. The dUTPase cDNA from Drosophila melanogaster was identified, sequenced, and the model structure of the encoded polypeptide displayed a polar hydrogen-bonding network of threefold interactions, identically to the human structure. Results allow clear distinction between two subfamilies of trimeric dUTPases where altered subunit communication may account for a functional difference in the catalytic cycle.

Amino Acid Sequence↗

PubMed1998

Molecular modelling and the biosynthesis of apolipoprotein B containing lipoproteins.

APOBEC-1 is the cytidine deaminase. We show by sequence alignment, molecular modelling and mutagenesis, that it is related in crystal structure to the cytidine deaminase of Escherichia coli (ECCDA). The two enzymes are both homodimers with composite active sites formed with loops from each monomer. In the sequence of APOBEC-1, three gaps compared to ECCDA match the size and contour of the minimal RNA substrate. We propose a model in which the asymmetric binding of one active site to the substrate cytidine which is positioned by the downstream binding of the product uridine and that this helps to target the other active site for deamination.

APOBEC-1 Deaminase↗

PubMed1997

Glutathione synthetase: similarities of the proteins from Schizosaccharomyces pombe and Arabidopsis thaliana.

Glutathione synthetase predicted from the reported gene sequence from Schizosaccharomyces pombe is substantially smaller than the equivalent protein predicted from the cDNAs sequenced from Arabidopsis thaliana, Saccharomyces cerevisiae and other eukaryotes. Sequence alignments of the proteins encoded by the cDNA clones for glutathione synthetase from Arabidopsis and S. pombe show that the Arabidopsis protein contains 200 extra amino acids at the N-terminus. In order to test if this sequence is essential in the function of the protein, the full-length Arabidopsis protein and as two N-terminal deletions (Delta67-71 and Delta67-200) were expressed in S. pombe mutant MN101, which lacks endogenous glutathione synthetase activity. Although the wild-type plant cDNA could complement the yeast mutation, neither deletion mutant was able to restore glutathione-dependent cadmium resistance. When the three proteins were expressed as fusion proteins in Escherichia coli, they accumulated to the same level, but only the plasmid containing the full-length cDNA, pFLAG222, produced detectable enzyme activity in vitro. These results suggested that the N-terminus of the Arabidopsis glutathione synthetase is essential for its function and opened up the possibility that there was a sequencing error in the reported S. pombe sequence. Therefore the gsh2 sequence from wild-type S. pombe and the mutant strain MN101 were determined. The wild-type S. pombe gsh2 encodes a protein that is about the same length as that found in Arabidopsis, and the MN101 mutation involves a frameshift mutation early in the glutathione synthetase reading frame.

Amino Acid Sequence↗

PubMed1998

Equus caballus gelsolin--cDNA sequence and protein structural implications.

We have generated and characterized the cDNA from equine smooth muscle that encodes gelsolin, an actin-modulating protein. Overlapping cDNA clones synthesized by the reverse transcriptase/polymerase chain reaction and clones isolated from a horse genomic library provided the complete primary structure for the intracellular isoform of gelsolin, while cDNA complemented with protein sequence data produced the full-length primary transcript of the gelsolin isoform found circulating in equine plasma. The deduced amino acid sequences of the intracellular and secreted versions of equine gelsolin infer polypeptides of 731 and 755 residues with apparent molecular masses of 80.7 kDa and 83.2 kDa, respectively. Multiple sequence alignment analysis of equine, human, porcine, and murine orthologs of gelsolin demonstrates prominent similarities among all of these proteins, with the horse and human molecules exhibiting the largest degree of likeness with respect to polypeptide length and overall sequence composition. Both horse and human plasma gelsolins are comprised of 755 amino acids with 94% of the residues identical, while the degree of sequence identity in the shorter (731 residues) cytoplasmic gelsolins is 95%. Analysis of the sequences and structures of the six related domains that comprise gelsolin emphasizes the strong correlation that exists between primary structural conservation among mammalian gelsolins and maintenance of the three-dimensional domain fold characteristic of members of this protein family.

Amino Acid Sequence↗

PubMed1998

A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase.

The leader (L) proteinases of aphthoviruses (foot-and-mouth disease viruses) and equine rhinovirus serotypes 1 and 2 cleave themselves from the growing polyprotein. This cleavage occurs intramolecularly between the C terminus of the L proteinases and the N terminus of the subsequent protein VP4. The foot-and-mouth disease virus enzyme has been shown, in addition, to cleave at least one cellular protein, the eukaryotic initiation factor 4G. Mechanistically, inhibitor studies and sequence analysis have been used to classify the L proteinases as papain-like cysteine proteinases. However, sequence identity within the L proteinases themselves is low (between 18% and 32%) and only 14% between the L proteinases and papain. Secondary structure predictions, sequence alignments that take into account the positions of the essential catalytic residues, and structural considerations have been used in this study to investigate more closely the relationships between the L proteinases and papain. In spite of the low sequence identities, the analyses strongly suggest that the L proteinases of foot-and-mouth disease virus and of equine rhinovirus 1 have a similar overall fold to that of papain. Regions in the L proteinases corresponding to all five alpha-helices and seven beta-sheets of papain could be identified. Further comparisons with the proteinase bleomycin hydrolase, which also displays a papain topology in spite of important differences in size and amino acid sequence, support these conclusions and suggest how a C-terminal extension, present in all three L proteinases, and predicted to be an alpha-helix, might enable C-terminal self-processing to occur.

Amino Acid Sequence↗

PubMed2004

Noncoding regulatory sequences of Ciona exhibit strong correspondence between evolutionary constraint and functional importance.

We show that sequence comparisons at different levels of resolution can efficiently guide functional analyses of regulatory regions in the ascidians Ciona savignyi and Ciona intestinalis. Sequence alignments of several tissue-specific genes guided discovery of minimal regulatory regions that are active in whole-embryo reporter assays. Using the Troponin I (TnI) locus as a case study, we show that more refined local sequence analyses can then be used to reveal functional substructure within a regulatory region. A high-resolution saturation mutagenesis in conjunction with comparative sequence analyses defined essential sequence elements within the TnI regulatory region. Finally, we found a significant, quantitative relationship between function and sequence divergence of noncoding functional elements. This work demonstrates the power of comparative sequence analysis between the two Ciona species for guiding gene regulatory experiments.

Animals↗

PubMed1998

Alternative macrophage activation-associated CC-chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2-associated expression pattern.

We have cloned a novel human CC-chemokine, alternative macrophage activation-associated CC-chemokine (AMAC)-1. The isolated cDNA clone (803 bp) shows a single open reading frame of 267-bp coding for 89 amino acid residues; mature AMAC-1 protein is predicted to consist of 69 amino acids with a m.w. of 7855. Sequence alignment and 3D-modeling show the typical structural characteristics of CC-chemokines with special features in the receptor-activating domain. AMAC-1 is most closely related to MIP-1 alpha with a cDNA and protein sequence homology of 55% and 59%, respectively. However, the expression pattern of AMAC-1 is directly opposite to that of MIP-1 alpha. While MIP-1 alpha is induced by classical macrophage mediators such as LPS and is inhibited by IL-4 and glucocorticoids, AMAC-1 is specifically induced in macrophages by alternative macrophage mediators such as IL-4, IL-13, and IL-10. Expression of AMAC-1 is inhibited by IFN-gamma while glucocorticoids exert a slightly positive synergistic effect in combination with IL-4. Peripheral blood monocytes do not express AMAC-1; time course experiments show that monocyte-to-macrophage differentiation is a prerequisite for AMAC-1 expression. Expression of AMAC-1 by granulocyte-macrophage CSF/IL-4-induced, monocyte-derived dendritic cells is complex; in mature adherent dendritic cells, however, only minor AMAC-1 mRNA expression was found. In vivo, AMAC-1 is expressed by alveolar macrophages from healthy persons, smokers, and asthmatic patients. In conclusion, AMAC-1 is a novel CC-chemokine whose expression is induced in alternatively activated macrophages by Th2-associated cytokines; thus, AMAC-1 may be involved in the APC-dependent T cell development in inflammatory and immune reactions.

Amino Acid Sequence↗

PubMed2006

PANDIT: an evolution-centric database of protein and associated nucleotide domains with inferred trees.

PANDIT is a database of homologous sequence alignments accompanied by estimates of their corresponding phylogenetic trees. It provides a valuable resource to those studying phylogenetic methodology and the evolution of coding-DNA and protein sequences. Currently in version 17.0, PANDIT comprises 7738 families of homologous protein domains; for each family, DNA and corresponding amino acid sequence multiple alignments are available together with high quality phylogenetic tree estimates. Recent improvements include expanded methods for phylogenetic tree inference, assessment of alignment quality and a redesigned web interface, available at the URL http://www.ebi.ac.uk/goldman-srv/pandit.

Databases, Nucleic Acid↗

PubMed2000

Structural similarities and evolutionary relationships in chloride-dependent alpha-amylases.

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion. This search provides 38 alpha-amylases potentially binding a chloride ion. All belong to animals, including mammals, birds, insects, acari, nematodes, molluscs, crustaceans and are also found in three extremophilic Gram-negative bacteria. An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species. On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca(2+) and Cl(-) binding sites, a protease-like catalytic triad and disulfide bonds. The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dependent alpha-amylases.

Amino Acid Sequence↗

PubMed1998

Characterization of cDNAs encoding a new family of tetraspanins from schistosomes--the Sj25 family.

The tetraspanins, also known as members of the transmembrane 4 superfamily (TM4SF), comprise an assemblage of surface antigens reported from mammalian and other vertebrate cells, from schistosomes, from fruit flies and from Caenorhabditis elegans. Tetraspanins are characterized by the presence of four hydrophobic domains, which are presumed to be membrane-spanning, and specific conserved motifs. A novel cDNA encoding a new tetraspanin, termed TE736 (tetraspanin 736), was isolated and characterized from the human blood fluke Schistosoma japonicum. Nucleotide and deduced amino acid sequences of the cDNA revealed that TE736 was similar to the previously characterized Sm23/Sj23/Sh23 species homologues and to Sj25/TM4 from schistosomes, and to other tetraspanins. Comparison of hydropathicity profiles of TE736, Sj25/TM4 and two novel tetraspanin-like sequences from S. mansoni showed that they contain four potential transmembrane domains like other tetraspanins. Sequence alignments showed there are four highly conserved, cysteine residues on the second predicted extracellular loop of TE736. A phylogenetic comparison of the relationship of approximately 30 tetraspanins from mammals and other groups revealed that TE736, Sj25/TM4 and the two sequences from S. mansoni formed an independent family of tetraspanins. We have termed these tetraspanins from schistosomes the Sj25 family. TE736 appeared to be encoded by a single gene and to be expressed in at least two life cycle stages of S. japonicum.

Amino Acid Sequence↗

PubMed2003

Protein-protein interactions: structurally conserved residues distinguish between binding sites and exposed protein surfaces.

Polar residue hot spots have been observed at protein-protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface. Consequently, hot spots can be used to define binding epitopes. We further show a correspondence between energy hot spots and structurally conserved residues. The number of structurally conserved residues, particularly of high ranking energy hot spots, increases with the binding site contact size. This finding may suggest that effectively dispersing hot spots within a large contact area, rather than compactly clustering them, may be a strategy to sustain essential key interactions while still allowing certain protein flexibility at the interface. Thus, most conserved polar residues at the binding interfaces confer rigidity to minimize the entropic cost on binding, whereas surrounding residues form a flexible cushion. Furthermore, our finding that similar residue hot spots occur across different protein families suggests that affinity and specificity are not necessarily coupled: higher affinity does not directly imply greater specificity. Conservation of Trp on the protein surface indicates a highly likely binding site. To a lesser extent, conservation of Phe and Met also imply a binding site. For all three residues, there is a significant conservation in binding sites, whereas there is no conservation on the exposed surface. A hybrid strategy, mapping sequence alignment onto a single structure illustrates the possibility of binding site identification around these three residues.

Amino Acid Sequence↗

PubMed1989

Sequence, organization, transcription and evolution of RNA polymerase subunit genes from the archaebacterial extreme halophiles Halobacterium halobium and Halococcus morrhuae.

The genes for the four largest subunits, A, B', B" and C, of the DNA-dependent RNA polymerase were cloned from the extreme halophile Halobacterium halobium and sequenced and their transcription was analyzed. The downstream half of this gene cluster from another extreme halophile Halococcus morrhuae was also cloned, sequenced and its transcription products characterized. The H. halobium genes were transcribed into a common transcript from an upstream promoter in the order B", B', A and C. They are flanked by, and co-transcribed with, two smaller genes coding for 75 and 139 amino acid residues, respectively. Immediately downstream from these genes were two open reading frames that are homologous to ribosomal proteins S12 and S7 from Escherichia coli. In both extreme halophiles these genes were transcribed from their own promoter, but in Hc. morrhuae there was also considerable read-through from the RNA polymerase genes. Sequence alignment studies showed that the combined B" + B' subunits are equivalent to the B subunits of the eukaryotic polymerases I and II and to the eubacterial beta subunit, while the combined A + C subunits correspond to the A subunits of eukaryotic RNA polymerases I, II and III and to the eubacterial beta' subunit. The sequence similarity to the eukaryotic subunits was always much higher than to the eubacterial subunits. Conserved sequence regions within the individual subunits were located which are likely to constitute functionally important domains; they include sites associated with rifampicin and alpha-amanitin binding and two possible zinc binding fingers. Phylogenetic analyses based on sequence alignments confirmed that the extreme halophiles belong to the archaebacterial kingdom.

Archaea↗

PubMed2004

Local graph alignment and motif search in biological networks.

Interaction networks are of central importance in postgenomic molecular biology, with increasing amounts of data becoming available by high-throughput methods. Examples are gene regulatory networks or protein interaction maps. The main challenge in the analysis of these data is to read off biological functions from the topology of the network. Topological motifs, i.e., patterns occurring repeatedly at different positions in the network, have recently been identified as basic modules of molecular information processing. In this article, we discuss motifs derived from families of mutually similar but not necessarily identical patterns. We establish a statistical model for the occurrence of such motifs, from which we derive a scoring function for their statistical significance. Based on this scoring function, we develop a search algorithm for topological motifs called graph alignment, a procedure with some analogies to sequence alignment. The algorithm is applied to the gene regulation network of Escherichia coli.

Algorithms↗

PubMed2002

Performance evaluation of a new algorithm for the detection of remote homologs with sequence comparison.

A detailed analysis of the performance of hybrid, a new sequence alignment algorithm developed by Yu and coworkers that combines Smith Waterman local dynamic programming with a local version of the maximum-likelihood approach, was made to access the applicability of this algorithm to the detection of distant homologs by sequence comparison. We analyzed the statistics of hybrid with a set of nonhomologous protein sequences from the SCOP database and found that the statistics of the scores from hybrid algorithm follows an Extreme Value Distribution with lambda approximately 1, as previously shown by Yu et al. for the case of artificially generated sequences. Local dynamic programming was compared to the hybrid algorithm by using two different test data sets of distant homologs from the PFAM and COGs protein sequence databases. The studies were made with several score functions in current use including OPTIMA, a new score function originally developed to detect remote homologs with the Smith Waterman algorithm. We found OPTIMA to be the best score function for both both dynamic programming and the hybrid algorithms. The ability of dynamic programming to discriminate between homologs and nonhomologs in the two sets of distantly related sequences is slightly better than that of hybrid algorithm. The advantage of producing accurate score statistics with only a few simulations may overcome the small differences in performance and make this new algorithm suitable for detection of homologs in conjunction with a wide range of score functions and gap penalties.

Algorithms↗

PubMed2006

Plant fatty acid (ethanol) amide hydrolases.

Fatty acid amide hydrolase (FAAH) plays a central role in modulating endogenous N-acylethanolamine (NAE) levels in vertebrates, and, in part, constitutes an "endocannabinoid" signaling pathway that regulates diverse physiological and behavioral processes in animals. Recently, an Arabidopsis FAAH homologue was identified which catalyzed the hydrolysis of NAEs in vitro suggesting a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, we provide evidence to support this concept by identifying candidate FAAH genes in monocots (Oryza sativa) and legumes (Medicago truncatula), which have similar, but not identical, exon-intron organizations. Corresponding M. truncatula and rice cDNAs were isolated and cloned into prokaryotic expression vectors and expressed as recombinant proteins in Escherichia coli. NAE amidohydrolase assays confirmed that these proteins indeed catalyzed the hydrolysis of 14C-labeled NAEs in vitro. Kinetic parameters and inhibition properties of the rice FAAH were similar to those of Arabidopsis and rat FAAH, but not identical. Sequence alignments and motif analysis of plant FAAH enzymes revealed a conserved domain organization for these members of the amidase superfamily. Five amino-acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the FAAH sequences of six plant species. Homology modeling of the plant FAAH proteins using the rat FAAH crystal structure as a template revealed a conserved protein core that formed the active site of each enzyme. Collectively, these results indicate that plant and mammalian FAAH proteins have similar structure/activity relationships despite limited overall sequence identity. Defining the molecular properties of NAE amidohydrolase enzymes in plants will help to better understand the metabolic regulation of NAE lipid mediators.

Amidohydrolases↗