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PubMed1998

Hidden Markov models for detecting remote protein homologies.

MOTIVATION: A new hidden Markov model method (SAM-T98) for finding remote homologs of protein sequences is described and evaluated. The method begins with a single target sequence and iteratively builds a hidden Markov model (HMM) from the sequence and homologs found using the HMM for database search. SAM-T98 is also used to construct model libraries automatically from sequences in structural databases. METHODS: We evaluate the SAM-T98 method with four datasets. Three of the test sets are fold-recognition tests, where the correct answers are determined by structural similarity. The fourth uses a curated database. The method is compared against WU-BLASTP and against DOUBLE-BLAST, a two-step method similar to ISS, but using BLAST instead of FASTA. RESULTS: SAM-T98 had the fewest errors in all tests-dramatically so for the fold-recognition tests. At the minimum-error point on the SCOP (Structural Classification of Proteins)-domains test, SAM-T98 got 880 true positives and 68 false positives, DOUBLE-BLAST got 533 true positives with 71 false positives, and WU-BLASTP got 353 true positives with 24 false positives. The method is optimized to recognize superfamilies, and would require parameter adjustment to be used to find family or fold relationships. One key to the performance of the HMM method is a new score-normalization technique that compares the score to the score with a reversed model rather than to a uniform null model. AVAILABILITY: A World Wide Web server, as well as information on obtaining the Sequence Alignment and Modeling (SAM) software suite, can be found at http://www.cse.ucsc.edu/research/compbi o/ CONTACT: karplus@cse.ucsc.edu; http://www.cse.ucsc.edu/karplus

Algorithms↗

PubMed2005

Phylogenetic analysis of six species of pacific abalone (Haliotidae) based on DNA sequences of 16s rRNA and cytochrome c oxidase subunit I mitochondrial genes.

Six species of abalones (Haliotidae) are found on the Korean coasts. Identification and characterization of these abalones are usually based on morphologic characters. In this research we compared the partial sequences of the mitochondrial 16S ribosomal RNA and cytochrome c oxidase subunit I genes to identify species using molecular data and to determine their phylogenetic relationships. Sequence alignments and phylogenetic analysis revealed that the 6 species fell into 2 distinct groups which were genetically distant from each other and exhibited little internal phylogenetic resolution. One group included Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea, while the other group contained H. diversicolor supertexta and H. diversicolor diversicolor. The 16S rRNA sequences were relatively more conserved than to the COI sequences, but both gene sequences provided sufficient phylogenetic information to distinguish among the 6 species of Pacific abalone, and thus could be valuable molecular characters for species identification.

Animals↗

PubMed1989

Structural analysis of homologous repeated domains in alpha-actinin and spectrin.

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.

Actinin↗

PubMed2002

Cloning and characterization of the chick Oct binding factor OBF-1.

We have cloned the chicken homolog of OBF-1, chOBF-1, which comprises 256 amino acids, and exhibits only 65% overall identity to the human and mouse OBF-1 proteins. Amino acid sequence alignment revealed the putative Oct-binding sequence, RPYQGVRVKEPVKELL(K/R)RKRG, which is conserved among chicken, mouse and human. chOBF-1 protein was demonstrated to bind chicken Oct-1 protein by the in vitro immunoprecipitation experiment, and chOBF-1 was shown to functionally activate the chicken immunoglobulin (Ig) light chain promoter in the NIH 3T3 cell. Taken together, these data indicate that the Ig gene transcription machinery, including Oct-1 and OBF-1, has been highly conserved in vertebrate evolution.

3T3 Cells↗

PubMed2003

The identification of a conserved domain in both spartin and spastin, mutated in hereditary spastic paraplegia.

Multiple sequence alignment has revealed the presence of a sequence domain of approximately 80 amino acids in two molecules, spartin and spastin, mutated in hereditary spastic paraplegia. The domain, which corresponds to a slightly extended version of the recently described ESP domain of unknown function, was also identified in VPS4, SKD1, RPK118, and SNX15, all of which have a well established and consistent role in endosomal trafficking. Recent functional information indicates that spastin is likely to be involved in microtubule interaction. With this new information relating to its likely function, we propose the more descriptive name 'MIT' (contained within microtubule-interacting and trafficking molecules) for the domain and predict endosomal trafficking as the principal functionality of all molecules in which it is present.

Adenosine Triphosphatases↗

PubMed1999

Genetical and immunological analysis of recent Asian type A and O foot-and-mouth disease virus isolates.

This report extends the knowledge on the epizootical situation of foot-and-mouth disease in Asia. RNA from six samples of type A and five of type O virus, isolated between 1987 and 1997 in Bangladesh, Iran, Malaysia and Turkey, was subjected to reverse transcription-dependent polymerase chain reactions that amplify large parts of the capsid protein VP1 encoding genome region. The amplification products were sequenced, and the sequences aligned to each other and to published sequences. This showed the type O isolates of 1987-1997 from Bangladesh to be of same genotype and closely related to isolates of 1988 and later from Saudi Arabia, 1990 from India, 1996 from Greece and Bulgaria, and 1997 from Iran. Among the analyzed type A isolates, those of 1992 and 1996 from Turkey were of same genotype and related to previously described isolates of 1987 from Iran and of 1992 from Saudi Arabia. The isolate of 1997 from Malaysia was found to be related to isolates from Thailand of 1993 and 1996. The isolates of 1987 from Bangladesh and 1997 from Iran, however, represent different so far not described genotypes. Monoclonal antibodies, raised against the vaccine production strains A22 Iraq, Asial Shamir, O1 Kaufbeuren and O1 Manisa, and the recent type A field isolates Saudi Arabia/92 and Albania/96, were used in an ELISA to compare the reaction patterns of many of the field isolates. The monoclonal antibodies were further characterized for virus-neutralizing activity and binding to trypsinized homologous virus. The failure of neutralizing antibodies in binding to trypsinized homologous as well as to heterologous virus suggested the epitopes to reside at the major antigenic component of the virus, which is the capsid protein VP1. Two non-neutralizing antibodies that bind to trypsin-sensitive epitopes cross-reacted, however, with heterologous virus. This indicates the existence of a trypsin-sensitive antigenic site outside of VP1. In summary, the results obtained by ELISA confirm the observed sequence differences, but indicate further sequence differences at minor antigenic sites that do not reside on VP1.

Amino Acid Sequence↗

PubMed2001

Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export.

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.

Active Transport, Cell Nucleus↗

PubMed1994

PHD--an automatic mail server for protein secondary structure prediction.

By the middle of 1993, > 30,000 protein sequences has been listed. For 1000 of these, the three-dimensional (tertiary) structure has been experimentally solved. Another 7000 can be modelled by homology. For the remaining 21,000 sequences, secondary structure prediction provides a rough estimate of structural features. Predictions in three states range between 35% (random) and 88% (homology modelling) overall accuracy. Using information about evolutionary conservation as contained in multiple sequence alignments, the secondary structure of 4700 protein sequences was predicted by the automatic e-mail server PHD. For proteins with at least one known homologue, the method has an expected overall three-state accuracy of 71.4% for proteins with at least one known homologue (evaluated on 126 unique protein chains).

Algorithms↗

PubMed2004

Nucleotide diversity on the ovine Y chromosome.

To investigate the impact of male-mediated introgression during the evolution of sheep breeds, a sequencing approach was used to identify single nucleotide polymorphisms (SNPs) from the male-specific region of the ovine Y chromosome (MSY). A total of 4380 bp, which comprised nine fragments from five MSY genes was sequenced within a panel of 14 males from seven breeds. Sequence alignment identified a single segregating site, an A/G SNP located approximately 1685 bp upstream of the ovine SRY gene. The resulting estimation of nucleotide diversity (piY = 0.90 +/- 0.50 x 10(-4)) falls towards the lower end of estimates from other species. This was compared with the nucleotide diversity estimated from the autosomal component of the genome. Sequence analysis of 2933 bp amplified from eight autosomal genes revealed a nucleotide diversity (piA = 2.15 +/- 0.27 x 10(-3)) higher than previously reported for sheep. Following adjustment for the contrasting influence of effective population size and a male biased mutation rate, comparison revealed that approximately 10% of the expected nucleotide diversity is present on the ovine Y chromosome.

Animals↗

PubMed2004

Structural and functional analysis of ataxin-2 and ataxin-3.

Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3) are autosomal-dominantly inherited, neurodegenerative diseases caused by CAG repeat expansions in the coding regions of the genes encoding ataxin-2 and ataxin-3, respectively. To provide a rationale for further functional experiments, we explored the protein architectures of ataxin-2 and ataxin-3. Using structure-based multiple sequence alignments of homologous proteins, we investigated domains, sequence motifs, and interaction partners. Our analyses focused on presumably functional amino acids and the construction of tertiary structure models of the RNA-binding Lsm domain of ataxin-2 and the deubiquitinating Josephin domain of ataxin-3. We also speculate about distant evolutionary relationships of ubiquitin-binding UIM, GAT, UBA and CUE domains and helical ANTH and UBX domain extensions.

Amino Acid Sequence↗

PubMed2000

A region of the transmembrane regulatory protein ToxR that tethers the transcriptional activation domain to the cytoplasmic membrane displays wide divergence among Vibrio species.

The virulence regulatory protein ToxR of Vibrio cholerae is unique in that it contains a cytoplasmic DNA-binding-transcriptional activation domain, a transmembrane domain, and a periplasmic domain. Although ToxR and other transmembrane transcriptional activators have been discovered in other bacteria, little is known about their mechanism of activation. Utilizing degenerate oligonucleotides and PCR, we have amplified internal toxR gene sequences from seven Vibrio and Photobacterium species and subspecies, demonstrating that toxR is an ancestral gene of the family Vibrionaceae. Sequence alignment of all available ToxR amino acid sequences revealed a region between the transcriptional activation and transmembrane domains that displays wide divergence among Vibrio species. We hypothesize that this region merely tethers the transcriptional activation domain to the cytoplasmic membrane and thus can tolerate wide divergence and multiple insertions and deletions. The divergence in the tether region at the nucleotide level may provide a useful tool for the distinction of Vibrio and Photobacterium species.

Amino Acid Sequence↗

PubMed1999

Immunohistochemical detection of somatostatin sst2a receptors in the lymphatic, smooth muscular, and peripheral nervous systems of the human gastrointestinal tract: facts and artifacts.

The cellular distribution of the somatostatin sst2A receptor protein was investigated in the lymphatic, smooth muscular, and nervous components of the human gastrointestinal tract using subtype-specific antibody R2-88 for immunohistochemical staining of cryostat and formalin-fixed, paraffin-embedded tissue sections. Germinal centers of intestinal lymphatic follicles were immunostained, exhibiting a predominantly plasma membrane localization of the receptor. Similarly, nerve fibers and cells in the submucosal and myenteric plexus were stained for sst2A. Antibody preabsorption with 100 nmol/L antigen peptide abolished staining in all of these tissues, and immunohistochemical staining correlated with the labeling observed after receptor autoradiography using the sst2-preferring radioligand 125I-[Tyr3]octreotide. Cytoplasmic immunostaining was detected in gastrointestinal smooth muscle cells and was inhibited by antibody pre-absorption with antigen peptide. However, 125I-[Tyr3]octreotide autoradiography was negative, and Western blots showed no band at the usual 70-90 kDa location for sst2A. Instead, a band was observed at 205 kDa. This band comigrated with the rabbit myosin standard, which was also stained with R2-88, although antibody sensitivity for myosin was less than 0.002% of that for the sst2A receptor. Rigorous computer-based sequence analysis demonstrated the peptide sequence chosen for antibody production was unique. Moreover, standard sequence alignment protocols were unable to identify the sequences in myosin responsible for the observed reactivity with the R2-88 antiserum. The observed cross-reactivity emphasizes the need for extensive controls to prove the specificity of immunostaining for such low abundance proteins as receptors even when the peptide sequence chosen for antibody production is unique. This study demonstrates for the first time the presence of specific sst2A receptor protein by immunohistochemistry in the human gastrointestinal lymphatic and nervous components, but not in gastrointestinal circular and longitudinal smooth muscle.

Animals↗

PubMed1996

Phylogenetic analysis of the env gene of HIV-1 isolates taking into account individual nucleotide substitution rates.

OBJECTIVE: To estimate the relative substitution rate of the individual positions in an alignment of HIV-1 env sequences coding for areas V3, V4, V5, and the beginning of gp41, and to study phylogenetic relationships between HIV-1 strains taking into account these substitution rate estimates. DESIGN: Phylogenetic comparison of 145 HIV-1 strains classified in HIV-1 group M, subtypes A-H and isolated from patients of 24 different geographical origins. METHODS: A new method recently developed for measuring the substitution rates of the individual nucleotides in a sequence alignment was applied to an alignment of env gene sequences. From the resulting substitution rate distribution, an equation was derived that describes the relationship between dissimilarity and evolutionary distance better than equations previously available. Phylogenetic trees were then constructed from matrices of distances computed using this new equation. RESULTS: 'Substitution rate calibration' offers detailed information on the relative substitution rate or variability of the nucleotides in the env gene. A large phylogenetic tree of 145 env gene sequences constructed by neighbour-joining and taking into account the substitution rate spectrum for this gene, clearly shows the existence of the eight subtypes A-H, all supported at a bootstrap level of 90% or higher. Intersubtype distances were between 0.25 and 0.38, which is considerably higher than those found in trees not considering differences in substitution rates among different alignment positions. CONCLUSIONS: Evolutionary distances are seriously underestimated when individual substitution rates are not considered in the estimation evolutionary distances. Furthermore, due to the more accurate estimation of evolutionary distances, naturally occurring HIV-1 intersubtype recombinants could be recognized more easily.

Base Sequence↗

PubMed2005

Evidence for essential catalytic determinants for human erythrocyte pyrimidine 5'-nucleotidase.

Human erythrocyte pyrimidine 5'-nucleotidase, PN-I, catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates. The enzyme also possesses phosphotransferase activity, transferring phosphate groups between pyrimidine nucleoside monophosphates and various pyrimidine nucleosides. Deficiency of the enzyme activity is associated with a hemolytic anemia. PN-I cDNA has been expressed in Escherichia coli, yielding a fully active recombinant enzyme, which was purified to homogeneity and extensively characterized. Multiple sequence alignment of PN-I and homologues proteins revealed the existence of conserved regions, whose importance in catalysis was examined by performing experiments designed to intercept covalent intermediates as strongly suggested by our previous kinetic studies. Furthermore, a functional analysis of the enzyme was carried out through site-directed mutagenesis designed on the basis of the sequence of the identified conserved regions as well as mutations observed in PN-I-deficient patients.

5'-Nucleotidase↗

PubMed1999

Crystal structure of the amino-terminal domain of N-ethylmaleimide-sensitive fusion protein.

The cytosolic ATPase N-ethylmaleimide-sensitive fusion protein (NSF) disassembles complexes of membrane-bound proteins known as SNAREs, an activity essential for vesicular trafficking. The amino-terminal domain of NSF (NSF-N) is required for the interaction of NSF with the SNARE complex through the adaptor protein alpha-SNAP. The crystal structure of NSF-N reveals two subdomains linked by a single stretch of polypeptide. A polar interface between the two subdomains indicates that they can move with respect to one another during the catalytic cycle of NSF. Structure-based sequence alignments indicate that in addition to NSF orthologues, the p97 family of ATPases contain an amino-terminal domain of similar structure.

Adenosine Triphosphatases↗

PubMed1998

Detailed comparison of two molecular models of the human CD40 ligand with an x-ray structure and critical assessment of model-based mutagenesis and residue mapping studies.

The interactions between the B cell receptor CD40 and its ligand on T cells are critical for the integrity of immune responses. The human CD40 ligand gp39, a tumor necrosis factor-like protein, has been the subject of intense efforts to identify the receptor-binding site and to analyze naturally occurring mutations that compromise gp39 function in vivo. These investigations relied heavily on molecular models of gp39, built in the presence of only approximately 25% sequence identity to tumor necrosis factor. The x-ray structure of gp39 has made it possible to assess modeling accuracy and to evaluate the results of model-based mutagenesis analyses. Although the models display local errors, their accuracy was sufficient to predict the CD40-binding site, to map natural mutations, and to rationalize their effects. One of five gp39 residues critical for CD40 binding was displaced in the models, and 1 of 21 point mutants was incorrectly classified. Factors most important for the reliability of the molecular models and their successful applications were valid sequence alignments and the focus of experimental studies on regions of high prediction confidence. Analysis of mutagenesis experiments correlated with anti-gp39 monoclonal antibody binding studies to assess the conformational integrity of mutant proteins.

Amino Acid Sequence↗

PubMed2003

Pyranose oxidase identified as a member of the GMC oxidoreductase family.

Fungal pyranose oxidase is a flavoenzyme whose preferred substrate among several monosaccharides is D-glucose. After a comprehensive analysis of conserved features in a structure-based multiple sequence alignment of homologous proteins, we could classify this enzyme into the GMC oxidoreductase family. The identified homology also suggests a three-dimensional protein structure similar to the functionally related glucose oxidase.

Amino Acid Sequence↗

PubMed2003

Conserved residue clustering and protein structure prediction.

Protein residues that are critical for structure and function are expected to be conserved throughout evolution. Here, we investigate the extent to which these conserved residues are clustered in three-dimensional protein structures. In 92% of the proteins in a data set of 79 proteins, the most conserved positions in multiple sequence alignments are significantly more clustered than randomly selected sets of positions. The comparison to random subsets is not necessarily appropriate, however, because the signal could be the result of differences in the amino acid composition of sets of conserved residues compared to random subsets (hydrophobic residues tend to be close together in the protein core), or differences in sequence separation of the residues in the different sets. In order to overcome these limits, we compare the degree of clustering of the conserved positions on the native structure and on alternative conformations generated by the de novo structure prediction method Rosetta. For 65% of the 79 proteins, the conserved residues are significantly more clustered in the native structure than in the alternative conformations, indicating that the clustering of conserved residues in protein structures goes beyond that expected purely from sequence locality and composition effects. The differences in the spatial distribution of conserved residues can be utilized in de novo protein structure prediction: We find that for 79% of the proteins, selection of the Rosetta generated conformations with the greatest clustering of the conserved residues significantly enriches the fraction of close-to-native structures.

Acyl Coenzyme A↗