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PubMed2001

Phylogenetic analysis of chloroplast small-subunit rRNA genes of the genus Euglena Ehrenberg.

Almost complete sequences of plastid SSU rDNA (16S rDNA) from 17 species belonging to the order Euglenales (sensu Nemeth, 1997; Shi et al., 1999) were determined and used to infer phylogenetic relationships between 10 species of Euglena, three of Phacus, and one of each of Colacium, Lepocinclis, Strombomonas, Trachelomonas and Eutreptia. The maximum-parsimony (MP), maximum-likelihood (ML) and distance analyses of the unambiguously aligned sequence fragments imply that the genus Euglena is not monophyletic. Parsimony and distance methods divide Euglenaceae into two sister groups. One comprises of representatives from the subgenera Phacus, Lepocinclis and Discoglena (sensu Zakryś, 1986), whereas the other includes members of Euglena and Calliglena subgenera (sensu Zakryś, 1986), intermixed with representatives of Colacium, Strombomonas and Trachelomonas. In all analyses subgenera Euglena--together with Euglena polymorpha (representative of the subgenus Calliglena)--and Discoglena--together with Phacus and Lepocinclis--form two well-defined clades. The data clearly indicate that a substantial revision of euglenoid systematics is very much required, nevertheless it must await while more information can be gathered, allowing resolution of outstanding relationships.

Animals↗

PubMed1985

Primary structure of the A chain of human complement-classical-pathway enzyme C1r. N-terminal sequences and alignment of autolytic fragments and CNBr-cleavage peptides.

Activated human complement-classical-pathway enzyme C1r has previously been shown to undergo autolytic cleavages occurring in the A chain [Arlaud, Villiers, Chesne & Colomb (1980) Biochim. Biophys. Acta 616, 116-129]. Chemical analysis of the autolytic products confirms that the A chain undergoes two major cleavages, generating three fragments, which have now been isolated and characterized. The N-terminal alpha fragment (approx. 210 residues long) has a blocked N-terminus, as does the whole A chain, whereas N-terminal sequences of fragments beta and gamma (approx. 66 and 176 residues long respectively) do not, and their N-terminal sequences were determined. Fragments alpha, beta and gamma, which are not interconnected by disulphide bridges, are located in this order within C1r A chain. Fragment gamma is disulphide-linked to the B chain of C1r, which is C-terminal in the single polypeptide chain of precursor C1r. CNBr cleavage of C1r A chain yields seven major peptides, CN1b, CN4a, CN2a, CN1a, CN3, CN4b and CN2b, which were positioned in that order, on the basis of N-terminal sequences of the methionine-containing peptides generated from tryptic cleavage of the succinylated (3-carboxypropionylated) C1r A chain. About 60% of the sequence of C1r A chain (440-460 residues long) was determined, including the complete sequence of the C-terminal 95 residues. This region shows homology with the corresponding parts of plasminogen and chymotrypsinogen and, more surprisingly, with the alpha 1 chain of human haptoglobin 1-1, a serine proteinase homologue.

Amino Acids↗

PubMed2005

Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain.

PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of alpha(1''-->2') or alpha(1'''-->2'') O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG 'signature sequence' [vDFA-X3-GGg-X6-8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610-795 in bovine PARG.

Amino Acid Sequence↗

PubMed2004

VisRD--visual recombination detection.

SUMMARY: VisRD, a program for visual recombination detection in a sequence alignment is presented. VisRD is written in Java and is designed to complement the multi-purpose phylogenetic software package SplitsTree4. AVAILABILITY: The software is freely available from http://www.lcb.uu.se/~vmoulton/software/visrd/

Algorithms↗

PubMed2006

Hairpins in a Haystack: recognizing microRNA precursors in comparative genomics data.

UNLABELLED: Recently, genome-wide surveys for non-coding RNAs have provided evidence for tens of thousands of previously undescribed evolutionary conserved RNAs with distinctive secondary structures. The annotation of these putative ncRNAs, however, remains a difficult problem. Here we describe an SVM-based approach that, in conjunction with a non-stringent filter for consensus secondary structures, is capable of efficiently recognizing microRNA precursors in multiple sequence alignments. The software was applied to recent genome-wide RNAz surveys of mammals, urochordates, and nematodes. AVAILABILITY: The program RNAmicro is available as source code and can be downloaded from http://www.bioinf.uni-leipzig/Software/RNAmicro.

Algorithms↗

PubMed2004

Are protein-protein interfaces more conserved in sequence than the rest of the protein surface?

Protein interfaces are thought to be distinguishable from the rest of the protein surface by their greater degree of residue conservation. We test the validity of this approach on an expanded set of 64 protein-protein interfaces using conservation scores derived from two multiple sequence alignment types, one of close homologs/orthologs and one of diverse homologs/paralogs. Overall, we find that the interface is slightly more conserved than the rest of the protein surface when using either alignment type, with alignments of diverse homologs showing marginally better discrimination. However, using a novel surface-patch definition, we find that the interface is rarely significantly more conserved than other surface patches when using either alignment type. When an interface is among the most conserved surface patches, it tends to be part of an enzyme active site. The most conserved surface patch overlaps with 39% (+/- 28%) and 36% (+/- 28%) of the actual interface for diverse and close homologs, respectively. Contrary to results obtained from smaller data sets, this work indicates that residue conservation is rarely sufficient for complete and accurate prediction of protein interfaces. Finally, we find that obligate interfaces differ from transient interfaces in that the former have significantly fewer alignment gaps at the interface than the rest of the protein surface, as well as having buried interface residues that are more conserved than partially buried interface residues.

Amino Acid Sequence↗

PubMed2001

The Bacillus thuringiensis cyt genes for hemolytic endotoxins constitute a gene family.

In the same way that cry genes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, the cyt genes for hemolytic toxins seem to compose another set of highly related genes in Bacillus thuringiensis. Although the occurrence of Cyt hemolytic factors in B. thuringiensis has been typically associated with mosquitocidal strains, we have recently shown that cyt genes are also present in strains with different pathotypes; this is the case for the morrisoni subspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such as B. thuringiensis subsp. israelensis and subsp. morrisoni PG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50 B. thuringiensis strains revealed that cyt2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of many B. thuringiensis strains.

Amino Acid Sequence↗

PubMed2005

Antigenic domain 1 is required for oligomerization of human cytomegalovirus glycoprotein B.

Human cytomegalovirus (HCMV) glycoprotein B (gB) is an abundant virion envelope protein that has been shown to be essential for the infectivity of HCMV. HCMV gB is also one of the most immunogenic virus-encoded proteins, and a significant fraction of virus neutralizing antibodies are directed at gB. A linear domain of gB designated AD-1 (antigenic domain 1) represents a dominant antibody binding site on this protein. AD-1 from clinical isolates of HCMV exhibits little sequence variation, suggesting that AD-1 plays an essential role in gB structure or function. We investigated this possibility by examining the role of AD-1 in early steps of gB synthesis. Our results from studies using eukaryotic cells indicated that amino acid (aa) 635 of the gB sequence represented the carboxyl-terminal limit of this domain and that deletion of aa 560 to 640 of the gB sequence resulted in loss of AD-1 expression. AD-1 was shown to be required for oligomerization of gB. Mutation of cysteine at either position 573 or 610 in AD-1 resulted in loss of its reactivity with AD-1-specific monoclonal antibodies and gB oligomerization. Infectious virus could not be recovered from HCMV bacterial artificial chromosomes following introduction of these mutations into the HCMV genome, suggesting that AD-1 was an essential structural domain required for gB function in the replicative cycle of HCMV. Sequence alignment of AD-1 with homologous regions of gBs from other herpesviruses demonstrated significant relatedness, raising the possibility that this domain may contribute to multimerization of gBs in other herpesviruses.

Amino Acid Sequence↗

PubMed1994

A complete cDNA sequence for core I protein subunit of human ubiquinol-cytochrome c reductase.

Core I protein is the largest subunit of ubiquinol-cytochrome c reductase. We have isolated a complete cDNA clone of 1575 bp encoding the precursor to this protein by screening a human fibroblast cDNA library. Nucleotide sequence comparison showed that the human core I protein cDNA is about 85% homologous with the reported bovine counter part but with a large difference in the length of coding region which consists of 480 amino acids in human and 362 amino acids in bovine. Human core I protein is presumed to contain a presequence of 34 amino acids. Amino acid sequence alignment study showed that the predicted human core I protein has a significant homology with other members of matrix processing peptidase (MPP) and processing enhancing protein (PEP) family.

Amino Acid Sequence↗

PubMed2005

Microevolution in prehistoric Andean populations: chronologic mtDNA variation in the desert valleys of northern Chile.

Archeological evidence suggests that the iconographic and technological developments that took place in the highlands around Lake Titicaca in the Central Andean region had an influence on the cultural elaborations of the human groups in the valleys and the Pacific coast of northern Chile. In a previous communication, we were able to show, by means of a distance analysis, that a craniofacial differentiation accompanied the process of cultural evolution in the valleys (Rothhammer and Santoro [2001] Lat. Am. Antiq. 12:59-66). Recently, numerous South Amerindian mtDNA studies were published, and more accurate molecular techniques to study ancient mtDNA are available. In view of these recent developments, we decided 1) to study chronological changes of ancient mtDNA haplogroup frequencies in the nearby Lluta, Azapa, and Camarones Valleys, 2) to identify microevolutionary forces responsible for such changes, and 3) to compare ancient mtDNA haplogroup frequencies with previous data in order to validate craniometrical results and to reconstruct the biological history of the prehistoric valley groups in the context of their interaction with culturally more developed highland populations. From a total of 97 samples from 83 individuals, 68 samples (61 individuals) yielded amplifications for the fragments that harbor classical mtDNA markers. The haplogroup distribution among the total sample was as follows: 26.2%, haplogroup A; 34.4%, haplogroup B; 14.8%, haplogroup C; 3.3%, haplogroup D; and 21.3%, other haplogroups. Haplogroup B tended to increase, and haplogroup A to decrease during a 3,900-year time interval. The sequence data are congruent with the haplogroup analysis. In fact, the sequencing of hypervariable region I of 30 prehistoric individuals revealed 43 polymorphic sites. Sequence alignment and subsequent phylogenetic tree construction showed two major clusters associated with the most common restriction haplogroups. Individuals belonging to haplogroups C and D tended to cluster together with nonclassical lineages.

Base Sequence↗

PubMed2000

Identification of Asp197 as the catalytic nucleophile in the family 38 alpha-mannosidase from bovine kidney lysosomes.

Bovine kidney lysosomal alpha-mannosidase is a family 38 alpha-mannosidase involved in the degradation of glycoproteins. The mechanism-based reagent, 5-fluoro-beta-L-gulosyl fluoride, was used to trap a glycosyl-enzyme intermediate, thereby labelling the catalytic nucleophile of this enzyme. After proteolytic digestion and high performance liquid chromatography/tandem mass spectrometry (MS) analysis, a labelled peptide was localised, and the sequence: HIDPFGHSRE determined by fragmentation tandem MS analysis. Taking into consideration sequence alignments of this region with those of other alpha-mannosidases of the same family, this result strongly suggests that the catalytic nucleophile in this enzyme is Asp197.

Amino Acid Sequence↗

PubMed1999

Comparative sequence analysis of classical swine fever virus isolates from the epizootic in The Netherlands in 1997-1998.

Sixteen classical swine fever virus (CSFV) field isolates from outbreaks of classical swine fever from the period between February 1997 and March 1998 in the Netherlands were sequence analysed. Parts of the 5' noncoding region (5'NCR) and the E1/E2 gene were sequenced after RT-PCR. The obtained sequences were compared with isolates of recent outbreaks in Europe and those of former outbreaks in the Netherlands. Sequence alignment of the 5'NCR region (321 bp) revealed that the isolates of the Dutch outbreak of 1997-1998 were closely linked to an isolate of the CSF outbreak that started in Paderborn, Germany in 1996. A relatively large fragment of the E1/E2 gene of 850 bp, including the antigenic region of E2, which is one of the most variable regions of the CSFV genome, was sequenced to determine whether this region can be used for epidemiology within an epizootic. Epidemiological tracing of transmission of virus was followed, starting from the first isolate and a line of five generations of viruses was analysed. Besides this, new isolates which could not be epidemiologically linked to preceding ones were also characterised. Differences between the isolates of the Dutch outbreak were minor both for the linked as well as for the non-linked isolates, indicating that all isolates have a common origin. Furthermore, our data show for the first time the genetic stability of CSFV even in the highly variable antigenic region of the E2 gene during a major epidemic lasting more than 1 year.

Animals↗

PubMed1999

Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis.

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine.

Amide Synthases↗

PubMed2004

Numerous novel annotations of the human genome sequence supported by a 5'-end-enriched cDNA collection.

A collection of 90,000 human cDNA clones generated to increase the fraction of "full-length" cDNAs available was analyzed by sequence alignment on the human genome assembly. Five hundred fifty-two gene models not found in LocusLink, with coding regions of at least 300 bp, were defined by using this collection. Exon composition proposed for novel genes showed an average of 4.7 exons per gene. In 20% of the cases, at least half of the exons predicted for new genes coincided with evolutionary conserved regions defined by sequence comparisons with the pufferfish Tetraodon nigroviridis. Among this subset, CpG islands were observed at the 5' end of 75%. In-frame stop codons upstream of the initiator ATG were present in 49% of the new genes, and 16% contained a coding region comprising at least 50% of the cDNA sequence. This cDNA resource also provided candidate small protein-coding genes, usually not included in genome annotations. In addition, analysis of a sample from this cDNA collection indicates that approximately 380 gene models described in LocusLink could be extended at their 5' end by at least one new exon. Finally, this cDNA resource provided an experimental support for annotations based exclusively on predictions, thus representing a resource substantially improving the human genome annotation.

5' Untranslated Regions↗

PubMed2003

Bioinformatics methods to predict protein structure and function. A practical approach.

Protein structure prediction by using bioinformatics can involve sequence similarity searches, multiple sequence alignments, identification and characterization of domains, secondary structure prediction, solvent accessibility prediction, automatic protein fold recognition, constructing three-dimensional models to atomic detail, and model validation. Not all protein structure prediction projects involve the use of all these techniques. A central part of a typical protein structure prediction is the identification of a suitable structural target from which to extrapolate three-dimensional information for a query sequence. The way in which this is done defines three types of projects. The first involves the use of standard and well-understood techniques. If a structural template remains elusive, a second approach using nontrivial methods is required. If a target fold cannot be reliably identified because inconsistent results have been obtained from nontrivial data analyses, the project falls into the third type of project and will be virtually impossible to complete with any degree of reliability. In this article, a set of protocols to predict protein structure from sequence is presented and distinctions among the three types of project are given. These methods, if used appropriately, can provide valuable indicators of protein structure and function.

Algorithms↗

PubMed1994

Essential histidine at the active site of sorghum leaf NADP-dependent malate dehydrogenase.

Chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) is a key enzyme in the photosynthetic CO2 fixation pathway of C4-plants. The presence of a histidine at its active site has been proposed, based on sequence alignment with nonchloroplastic NAD-dependent malate dehydrogenases. In order to investigate this hypothesis, the effect of diethylpyrocarbonate on the sorghum leaf enzyme has been tested. Diethylpyrocarbonate strongly inhibited NADP-MDH activity, its effect being dramatically decreased in the presence of substrates and reversed by hydroxylamine. When diethylpyrocarbonate-inactivated NADP-MDH was cleaved with trypsin, one peptide with increased absorbance at 240 nm was detected. Sequencing of this peptide and analysis by mass spectrometry demonstrated that histidine 229 was modified by diethylpyrocarbonate. This amino acid was changed to an alanine by site-directed mutagenesis, and the modified protein was produced in Escherichia coli. It was similar to the plant enzyme except that it was totally inactive. Taken together, these results indicate that His229 is an essential residue in the active site of sorghum NADP-MDH.

Amino Acid Sequence↗

PubMed1994

Design of interchain disulfide bonds in the framework region of the Fv fragment of the monoclonal antibody B3.

The Fv fragments are the smallest units of antibodies that retain the specific antigen binding characteristics of the whole molecule and are being used for the diagnosis and therapy of human diseases. These are noncovalently associated heterodimers of the heavy (VH) and the light (VL) chain variable domains, which, without modification, tend to dissociate, unfold, and/or nonspecifically aggregate. The fragment is usually stabilized by producing it as a single chain recombinant molecule in which the two chains are linked by means of a short polypeptide linker. An alternative strategy is to connect the two chains by means of an interchain disulfide bond. We used molecular graphics and other modeling tools to identify two possible interchain disulfide bond sites in the framework region of the Fv fragment of the monoclonal mouse antibody (mAb) B3. The mAb B3 binds to many human cancer cells and is being used in the development of a new anticancer agent. The two sites identified are VH44-VL105 and VH111-VL48. (VH44-VL100 and VH105-VL43 in the numbering scheme of Kabat et al., "Sequence of Proteins of Immunological Interest," U.S. DHHS, NIH publication No. 91-3242, 1991). This design was recently tested using the chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of mAb B3 with the engineered disulfide bond at VH44-VL105 (Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A. 90:7538, 1993). The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable. Because these disulfide bond sites are in the framework region, they can be located from sequence alignment alone. We expect that the disulfide bond at these sites will stabilize the Fv fragment of most antibodies and the antigen-specific portion of the T-cell receptors, which are homologous.

ADP Ribose Transferases↗

PubMed2005

Molecular characterization of new members of the Hevea brasiliensis hevein multigene family and analysis of their promoter region in rice.

The cloning of hevein genes from Hevea brasiliensis was undertaken with the objective to isolate useful promoters to drive transgene expression in genetically engineered rubber tree. Four different full length genes were cloned by library screening and a fifth, a partial gene, by adaptor-anchored PCR. Sequence alignment revealed that hevein genes, although highly conserved in their transcribed region, diverged in two groups, with major differences in their promoter region, suggesting a more rapid evolution of the upstream regulatory functions of the genes than the downstream functions of their protein products. The promoter regions from two hevein genes representative of each group were isolated and analyzed in rice. Although both were functional, only the longest promoter sequence (PHev2.1) conferred a high level of expression to the transgene in various tissues of this heterologous host. It was in addition up-regulated by mechanical wounding and fungal infection in leaves. A number of potential cis-regulatory elements were identified in silico and are discussed in view of the expression profiles observed in rice.

Base Sequence↗