Multiple Sequence Comparisons Research Articles

An Efficient Cis-Element Discovery Method Using Multiple Sequence Comparisons Based on Evolutionary Relationships

The discovery of cis-element control motifs in noncoding DNA poses a difficult problem in genome analysis. Functional analysis by means of reporter constructs expressed in transgenic organisms is the most reliable method, but is by itself time-consuming and expensive. Searching noncoding DNA for known control motifs by sequence analysis is problematic, since protein binding motifs are short, in the range of 8–10 bp, and occur frequently by chance. Heretofore, the most reliable sequence analysis method has been the comparison of homologous sequence domains in related but moderately evolutionarily divergent species such as, for example, mouse and human. In such pairwise combinations, control regions are conserved because they serve a vital function and can be identified by their similar sequences. Single pairwise comparisons, however, allow the discovery of conserved sequence strings only at low resolution and without specific identity. We have investigated the possibility of using multiple sequence comparisons to correct these shortcomings. We applied this method to the Hoxc8 early enhancer region that has been previously analyzed in depth by functional methods and through its application successfully identified known protein binding cis-element motifs. Candidate protein binding sites could also be identified. This method, based on evolutionarily related sequence comparisons, should be quite useful as a prescreening step prior to functional analysis with corresponding savings in time and resources.

Pancreatic reg and a conserved bioactive fragment are mitogenic through the MAPK P38 pathway

Pancreatic reg proteins are exocrine proteins which are mitogenic to pancreatic ductal and ∃-cells (1,2). Using multiple alignment sequence comparison of the reg proteins with known mitogenic activity, we identified an amino acid sequence (RegP) highly conserved between rat, mouse and human reg’s (3), and postulated that it is the bioactive fragment. This sequence contained an ‘WIGL’ amino acid motif, considered important in calcium-dependant lectins. We also postulated that the action of reg would be through activation of mitogen-activated protein kinases (MAPK).

The characterization of a measure of information discrepancy

A Function of Degree of Disagreement (FDOD) function has been introduced in the study of information discrepancies among multiple information sources [W. Fang, Mathematical Social Sciences 28 (2) (1994) 85–111; J. Global Optim. 11 (1997) 387–408]. It possesses some peculiar properties and can be applied in some areas, in which the traditional measures of information discrepancy are not valid, for example, in the study of multiple DNA sequence comparison. In this paper, based on the ideas concerned with Shannon entropy and measures of difference, we investigate its characterization. The final results and even intermediate results show a close connection between the FDOD function and Shannon entropy.

Reidentification of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus pseudofirmus OF4.

With a view toward verifying the original classification of alkaliphilic Bacillus firmus OF4, physiological and biochemical characteristics were more extensively catalogued than in original studies, and this catalog was supplemented with 16S rDNA sequence homology and more extensive DNA-DNA hybridization analyses. Phylogenetic analysis of this alkaliphile based on the comparison of multiple 16S rDNA sequences from Bacillus species indicated that this strain is most closely related to Bacillus pseudofirmus. Consistently, in the DNA-DNA hybridization analysis of the alkaliphile and Bacillus reference strains, the highest level of DNA-DNA relatedness (96%) was found between the alkaliphile and the B. pseudofirmus type strain (DSM 8715(T)). The findings support the conclusion that this alkaliphile strain is more closely related to B. pseudofirmus than to B. firmus, and we propose the future use of the designation B. pseudofirmus OF4.

Fast assignment of protein structures to sequences using the intermediate sequence library PDB-ISL.

For large-scale structural assignment to sequences, as in computational structural genomics, a fast yet sensitive sequence search procedure is essential. A new approach using intermediate sequences was tested as a shortcut to iterative multiple sequence search methods such as PSI-BLAST. A library containing potential intermediate sequences for proteins of known structure (PDB-ISL) was constructed. The sequences in the library were collected from a large sequence database using the sequences of the domains of proteins of known structure as the query sequences and the program PSI-BLAST. Sequences of proteins of unknown structure can be matched to distantly related proteins of known structure by using pairwise sequence comparison methods to find homologues in PDB-ISL. Searches of PDB-ISL were calibrated, and the number of correct matches found at a given error rate was the same as that found by PSI-BLAST. The advantage of this library is that it uses pairwise sequence comparison methods, such as FASTA or BLAST2, and can, therefore, be searched easily and, in many cases, much more quickly than an iterative multiple sequence comparison method. The procedure is roughly 20 times faster than PSI-BLAST for small genomes and several hundred times for large genomes. Sequences can be submitted to the PDB-ISL servers at http://stash.mrc-lmb.cam.ac.uk/PDB_ISL/ or http://cyrah.ebi.ac.uk:1111/Serv/PDB_ISL/ and can be downloaded from ftp://ftp.ebi.ac.uk/pub/contrib/jong/PDB_+ ++ISL/ CONTACT: sat@mrc-lmb.cam.ac.uk and jong@ebi.ac.uk

Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif

The unique catalytic characteristics of adenosine kinase (Adk) and its stage-specific differential activity pattern have made this enzyme a prospective target for chemotherapeutic manipulation in the purine-auxotrophic parasitic protozoan Leishmania donovani. However, nothing is known about the structure of the parasite Adk. We report here the cloning of its gene and the characterization of the gene product. The encoded protein, consisting of 345 amino acid residues with a calculated molecular mass of 37173 Da, shares limited but significant similarity with sugar kinases and inosine-guanosine kinase of microbial origin, supporting the notion that these enzymes might have the same ancestral origin. The identity of the parasite enzyme with the corresponding enzyme from two other sources so far described was only 40%. Furthermore, 5' RNA mapping studies indicated that the Adk gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon (spliced leader) occurring at nt -160 from the predicted translation initiation site. The biochemical properties of the recombinant enzyme were similar to those of the enzyme isolated from leishmanial cells. The intrinsic tryptophan fluorescence of the enzyme was substrate-sensitive. On the basis of a multiple protein-alignment sequence comparison and ATP-induced fluorescence quenching in the presence or the absence of KI and acrylamide, the docking site for ATP has been provisionally identified and shown to have marked divergence from the consensus P-loop motif reported for ATP- or GTP-binding proteins from other sources.

Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif

The unique catalytic characteristics of adenosine kinase (Adk) and its stage-specific differential activity pattern have made this enzyme a prospective target for chemotherapeutic manipulation in the purine-auxotrophic parasitic protozoan Leishmania donovani. However, nothing is known about the structure of the parasite Adk. We report here the cloning of its gene and the characterization of the gene product. The encoded protein, consisting of 345 amino acid residues with a calculated molecular mass of 37173 Da, shares limited but significant similarity with sugar kinases and inosine-guanosine kinase of microbial origin, supporting the notion that these enzymes might have the same ancestral origin. The identity of the parasite enzyme with the corresponding enzyme from two other sources so far described was only 40%. Furthermore, 5ʹ RNA mapping studies indicated that the Adk gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon (spliced leader) occurring at nt -160 from the predicted translation initiation site. The biochemical properties of the recombinant enzyme were similar to those of the enzyme isolated from leishmanial cells. The intrinsic tryptophan fluorescence of the enzyme was substrate-sensitive. On the basis of a multiple protein-alignment sequence comparison and ATP-induced fluorescence quenching in the presence or the absence of KI and acrylamide, the docking site for ATP has been provisionally identified and shown to have marked divergence from the consensus P-loop motif reported for ATP- or GTP-binding proteins from other sources.

Heat shock protein 70 family: multiple sequence comparisons, function, and evolution.

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus "matches" 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins.

Multiple sequence comparison — a peptide matching approach

We present in this paper a peptide matching approach to the multiple comparison of a set of protein sequences. This approach consists in looking for all the words that are common to q of these sequences, where q is a parameter. The comparison between words is done by using as reference an object called a model. In the case of proteins, a model is a product of subsets of the alphabet Σ of the amino acids. These subsets belong to a cover of Σ, that is, their union covers all of Σ. A word is said to be an instance of a model if it belongs to the model. A further flexibility is introduced in the comparison by allowing for up to e errors in the comparison between a word and a model. These errors may concern gaps or substitutions not allowed by the cover. A word is said to be this time an occurrence of a model if the Levenshtein distance between it and an instance of the model is inferior or equal to e. This corresponds to what we call a Set-Levenshtein distance between the occurrences and the model itself. Two words are said to be similar if there is at least one model of which both are occurrences. In the special case where e = 0, the occurrences of a model are simply its instances. If a model M has occurrences in at least q of the sequences of the set, M is said to occur in the set. The algorithm presented here is an efficient and exact way of looking for all the models, of a fixed length k or of the greatest possible length k max , that occur in a set of sequences. It is linear in the total length n of the sequences and proportional to ( e + 2)(2 e+ 1) k e+1 p e+1 g k where k ⪡ n is a small value in all practical situations, p is the number of sets in the cover and g is related to the latter's degree of nontransitivity. Models are closely related to what is called a consensus in the biocomputing area, and covers are a good way of representing complex relationships between the amino acids.

Relatedness and phylogeny within the family of periplasmic chaperones involved in the assembly of pili or capsule-like structures of gram-negative bacteria.

The structure of a Salmonella enterica serovar typhi gene located within the fim gene cluster and encoding a putative periplasmic chaperone-like protein involved in the assembly of type 1 pili was determined. This gene, named fimC, has the ability to encode a 26-kDa polypeptide which is similar, at the sequence level, to the PapD periplasmic chaperonin mediating the assembly of P pili of Escherichia coli, as well as to other periplasmic chaperone-like proteins involved in the biogenesis of pili or capsule-like structures of various Gram-negative bacteria. A comprehensive search through the literature and sequence databases identified 31 (putative) bacterial proteins that can be included in this protein family on the basis of sequence similarity. Results of a multiple sequence comparison analysis showed that several residues, including most of those known to be critical in maintaining the three-dimensional structure of PapD, are either conserved or conservatively substituted in all these proteins, suggesting an overall similar folding for all of them. It was also evident that members of this family are clustered into different subfamilies according to structural and phyletic data.

Detection of human genomic mutations by chemical single-reaction DNA sequencing

▼The ideal DNA sequencing method should yield complete and unambiguous information in a single electrophoretic pattern and should be simple, rapid and economical. A single-lane sequencing procedure is available, based upon the dideoxy Sanger methodology. This method cannot a priori be compressed further. Chemical DNA sequencing offers the potential of absolute compression: if one could obtain unambiguous and complete sequence information in a single pattern, then several different DNAs, each labeled with a different fluorochrome, could be analyzed in the same electrophoretic lane. This procedure could be quite relevant for mass genetic screenings. We have developed a chemical method (Ref. 1, 2) that yields a one-reaction one-lane sequence determination, thus allowing fast, multiple, simultaneous sequencing and direct sequence comparisons in the same electrophoretic lane. The method is based on the base-selective degradation (G>A>C>T) of DNA by N-methyl formamide (see Fig. 1). The chemical rationale of this method is as follows. Amides (as determined for formamide, N-methyl formamide and Ndimethyl formamide) react with DNA according to a multistep mechanism. (a) Degradation of the purine and pyrimidine bases: for purines, degradation occurs by nucleophilic attack in C-8, leading to degradative C-8-ring opening of the imidazole ring; for pyrimidines by nucleophilic attack in C-6, leading to degradative C-6-ring opening of the pyrimidine ring. (b) β-Elimination of the sugar-protons with consequent cleavage of the phosphate bonds. N-Methyl formamide has proved to be the best compound to carry out the sequencing reaction because of its efficiencies in base

Multiple Sequence Comparison and Consistency on Multipartite Graphs

Calculation of dot-matrices is a widespread tool in biological sequence comparison. As a visual aid they are used in pairwise sequence comparison but so far have been of little help in the simultaneous comparison of several sequences. Viewing dot-matrices as projections of unknown n-dimensional points we consider the multiple alignment problem (for n sequences) as an n-dimensional image reconstruction problem with noise. We model this situation using a multipartite graph and introduce a notion of "consistency" on such a graph. From this perspective we introduce and develop the filtering method due to Vingron and Argos ( J. Mol. Biol. 218 (1991), 33-43). We discuss a conjecture of theirs regarding the number of iterations their algorithm requires and demonstrate that this number may be large. An improved version of the original algorithm is introduced that avoids costly dot-matrix multiplications and runs in O( n 3 · L 3) time ( L is the length of the longest sequence and n is the number of sequences). This is equivalent to only one iteration of the original algorithm. We further consider the relationship between consistency and transitivity and introduce a hierarchy of notions linking consistency and transitivity. Finally applications to biological sequence comparison will be presented.

A multiple sequence comparison method

This article presents a new method for the comparison of multiple macromolecular sequences. It is based on a hierarchical sequence synthesis procedure that does not require any a priori knowledge of the molecular structure of the sequences or the phylogenetic relations among the sequences. It differs from the existing methods as it has the capability of: (i) generating a statistical-structural model of the sequences through a synthesis process that detects homologous groups of the sequences, and (ii) aligning the sequences while the taxonomic tree of the sequences is being constructed in one single phase. It produces superior results when compared with some existing methods.

A multiple sequence comparison method

A multiple sequence comparison method

Alignment and comparison of multiple related DNA sequences can be improved by consideration of known patterns of mutation

The rate at which bases are replaced in mammalian DNA is influenced by the sequence involved. I have modified a multiple sequence alignment and comparison program to take account of the observed patterns of base replacement in mammals, and used the program to analyse 100 human Alu sequences. The results show that using a sequence matching matrix based on the actual rate of base replacement, as opposed to arbitrary base matching matrix, gives significant differences in the way that sequences are aligned and compared with each other. Use of the observed matching matrix gives significantly better discrimination between sub-families of Alu sequences than conventional methods, and shows that comparison systems which take account of the mechanism of mutation give more biologically realistic results.

A survey of multiple sequence comparison methods

A survey of multiple sequence comparison methods

Evolutionary conservation of intron position in a subfamily of genes encoding carbohydrate-recognition domains

The structure of the gene encoding a chicken liver receptor, the chicken hepatic lectin, which mediates endocytosis of glycoproteins has been established. The coding sequence is divided into six exons separated by five introns. The first three exons correspond to separate functional domains of the receptor polypeptide (cytoplasmic tail, transmembrane sequence, and extracellular neck region), while the final three exons encode the Ca(2+)-dependent carbohydrate-recognition domain. These results, as well as computer-assisted multiple sequence comparisons, establish this receptor as the evolutionary homolog of the mammalian asialoglycoprotein receptors. It is interesting that the chicken receptor falls into a subfamily of proteins along with the mammalian asialoglycoprotein receptors, since the saccharide-binding specificity of the chicken receptor resembles more closely that of a different set of calcium-dependent animal lectins, which includes the mannose-binding proteins. The portions of the genes encoding the carbohydrate-recognition domains of these proteins lack introns. The results suggest that divergence of intron-containing and intron-lacking carbohydrate-recognition domains preceded shuffling events in which other functional domains were associated with the carbohydrate-recognition domains. This was followed by further divergence, generating a variety of saccharide-binding specificities.

Parallel computation of multiple biological sequence comparisons

A parallel implementation of an efficient method for comparison of multiple DNA sequences is presented. The method is described in terms of a conceptual tree data structure for the sequences to be compared. The parallel algorithm shows efficient utilization of processors on an Encore Multimax computer in a sample comparison of 11 sequences totaling over 4000 bases. Timing data show the strong influence of computer system details on this parallel program. Also presented is a graphics program for displaying multiple sequence comparison output data. The display is capable of representing large volumes of multiple sequence comparison data in a single plot. The program has several additional features that allow closer examination of subsets of sequences. A display of matches from the sample comparison reflects the known structure of these sequences.

Sequence divergence and open regions of RNA secondary structures in the envelope regions of the 17 human immunodeficiency virus isolates.

Genetic variation during the course of infection of an individual is a remarkable feature of the acquired immune deficiency syndrome (AIDS) disease. This variation has been studied for the envelope protein encoding regions of seventeen different sequences from various isolates of human immunodeficiency virus (HIV) using multiple sequence comparison and calculation of variability. The open regions with little intramolecular base pairing in these envelope sequences are predicted by a recently developed statistical method. The minimum length L for a run of hypervariable sites, conserved sites, or open regions that gives significance at the 1% (or 0.1%) level is then determined by a scan statistical method. The results show that significant clusters of open regions predicted at the RNA levels correlate with significant clusters of hypervariable sites in the HIV envelope gene. Those significant genomic variations in HIVs seem to be manifested mainly in the extracellular portion of the envelope protein. Twelve potential antigenic determinants are predicted using an antigenic index method. Interestingly, the majority of the significant hypervariable regions in the exterior envelope protein (gp120) were predicted potential epitopes.