PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Sequence Alignment”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,081 records · Page 60Linked to original sources
PubMed2001

Cloning and expression of a ferritin subunit for Galleria mellonella.

Ferritin was purified from iron-fed Galleria mellonella hemolymph by ultra centrifugation and FPLC (Superose 6). SDS-PAGE revealed three bands of 26, 30, and 32 kDa. The ferritin 26 kDa subunit cDNA was obtained from RT-PCR using primer designed from N-terminal sequence analysis. 5'-RACE was used to obtain the complete protein coding sequence. The sequence encodes a 211 amino acid polypeptide including a 20 amino acid leader peptide. An IRE (iron-responsive element) sequence with a predicted stem-loop structure was present in the 5'-UTR of ferritin mRNA. Sequence alignment has a sequence identity with Calpodes ethlius (S)(74%), Drosophila melanogaster (50%), and Aedes aegypti (39%). Northern blot analysis indicated that there were 1.5- and 1.75-fold increases in the expression of ferritin mRNA after iron-fed fat body and midgut, respectively. Also, we confirmed that the ferritin mRNA is not expressed in adult ovary and testis. Arch.

Amino Acid Sequence↗
PubMed1997

Evaluation of algorithms used for cross-species proteome characterisation.

The ability to effectively search databases for the identification of protein spots from two-dimensional electrophoresis gels has become an essential step in the study of microbial proteomes. A variety of analytical techniques are currently being employed during protein characterisation. A number of algorithms used to search databases, accessible via the World Wide Web, depend upon information concerning N- and C-terminal microsequence, amino acid composition, and peptide-mass fingerprinting. The effectiveness of nine such algorithms, as well as COMBINED (software developed in this laboratory for identifying proteins across species boundaries) was examined. Fifty-four ribosomal proteins from the Mycoplasma genitalium genome, and 72 amino acyl tRNA synthetases from the Haemophilus influenzae, M. genitalium and Methanococcus jannaschii genomes were chosen for study. These proteins were selected because they represent a wide range of sequence identities across species boundaries (22.7-100% identity), as detected by standard sequence alignment tools. Such sequence variation allowed for a statistical comparison of algorithm success measured against published sequence identity. The ability of analytical techniques used in protein characterisation and associated database query programs to detect identity at the functional group level was examined for proteins with low levels of homology at the gene/protein sequence level. The significance of these theoretical data manipulations provided the means to predict the utility of data acquired experimentally for non-sequence-dependent software in proteome analysis. The data obtained also predicted that 'sequence tagging' of peptide fingerprints would need to be accompanied by at least 11-20 residues of amino acid sequence for it to be widely used for protein characterisation across species boundaries.

Algorithms↗
PubMed1996

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I.

The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.

Amino Acid Sequence↗
PubMed2000

Trypanosoma brucei: four tandemly linked genes for fatty acyl-CoA synthetases.

We have cloned four acyl CoA synthetase (ACS) genes from Trypanosoma brucei strain 927. Each of these genes encodes a polypeptide about 78 kDa in size and all four contain the "ACS signature motif." Sequence alignments indicate that these proteins are 46%-95% identical in amino acid sequence. Interestingly, three of them share almost identical C-termini (about 215 amino acid residues). Southern blots suggest that these genes are present in a single copy, and Northern blots reveal that all four are expressed in both bloodstream and procyclic trypanosomes.

Amino Acid Sequence↗
PubMed1999

Non-functional conserved residues in globins and their possible role as a folding nucleus.

Structure-based sequence alignment of 728 sequences of different globin subfamilies shows that in each subfamily there are two clusters of consensually conserved residues. The first is the well-known "functional" cluster which includes six heme-binding conserved residues (Phe CD1, His F8; aliphatic E11, FG5; hydrophobic F4, G5) and seven other conserved residues (Pro C2; aliphatic H19; hydrophobic B10, B13, B14, CD4, E4) that do not bind the heme but belong to its immediate neighborhood. The second cluster revealed here (aliphatic A8, G16, G12; aromatic A12; hydrophobic H8 and possibly H12) is distant from the heme. It is entirely non-polar and includes one turn (i, i+4 positions) from each of helices A, G, and H. It is known that A, G, and H helices formed at the earliest stage of apomyoglobin folding remain relatively stable in the equilibrium molten globule state, and are likely to be tightly packed with each other in this state. We have shown the existence of two similar conserved clusters in c -type cytochromes, heme-binding and distal from the heme. The second cluster in c -cytochromes includes one turn from each of the N and C-terminal alpha-helices. These N and C-terminal helices in cytochrome c are formed at the earliest stage of protein folding, remain relatively stable in the molten globule state, and are tightly packed with each other in this state, similar to the observed behavior of the globins. At least these two large protein families (c -type cytochromes and globins) have a close similarity in the existence and mutual positions of non-functional conserved residues. We assume that non-functional conserved residues are requisite for the fast and correct folding of both of these protein families into their stable 3D structures.

Amino Acid Sequence↗
PubMed2005

Cloning and characterization of OsORC2, a new member of rice origin recognition complex.

In eukaryotic cells, the origin recognition complex (ORC) governs the initiation site of DNA replication and formation of the prereplication complex. The isolation, characterization and tissue-specific expression of a putative ORC subunit 2 (OsORC2) in Oryza sativa is described here. A novel cDNA fragment encoding rice ORC2 was isolated by screening the subtractive library, which had a higher expression level in inflorescence meristem than in shoot apical meristem. The full-length cDNA of rice ORC2 was obtained by the method of rapid amplification of cDNA ends, which contained an 1140 bp open reading frame encoding a 379 amino acid polypeptide. Sequence alignment shows that there is a high homology between the deduced amino sequence of OsORC2 and maize ORC2 (85%). The tissue-specific expression pattern of OsORC2 reveals that it is abundant in roots, seedling and inflorescence meristem, while its expression level is much lower in mature leaves and shoot.

Amino Acid Sequence↗
PubMed1984

Characterization of translational inhibitors from Phytolacca americana. Amino-terminal sequence determination and antibody-inhibitor conjugates.

Two translational inhibitors (pokeweed antiviral protein and pokeweed antiviral protein II) isolated from the leaves of the pokeweed plant, Phytolacca americana, were characterized as to their behavior during reverse-phase HPLC and their amino-terminal sequences. Alignment of the sequences demonstrated that a substantial degree of homology was present (10 of 29 identical residues). Pokeweed antiviral protein was shown by reverse-phase chromatography to be composed of at least two components, pokeweed antiviral proteina and pokeweed antiviral proteinb, which comigrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, shared identical N-terminal amino-acid sequences through residue 31, and had similar specific activities in a cell-free translation inhibition assay. Pokeweed antiviral protein II was covalently coupled to a monoclonal antibody that recognizes the transferrin receptor (anti-transferrin receptor). The disulfide-linked conjugate inhibited protein synthesis in the human breast tumor cell line MCF-7, whereas anti-transferrin receptor, pokeweed antiviral protein II, or an immunotoxin composed of an irrelevant antiserum and pokeweed antiviral protein II, were nontoxic. The inhibitory dose 50% of anti-transferrin receptor-pokeweed antiviral protein II for MCF-7 cells was 0.7 nM, whereas the corresponding ricin A chain conjugate (anti-transferrin receptor-ricin A chain) was more potent with a inhibitory dose 50% of 0.1 nM. Pokeweed antiviral protein II can be added to the growing list of translation inhibitors that are effective as components of immunotoxins in vitro. Additional studies will be needed to determine whether pokeweed antiviral protein II immunotoxins provide advantageous properties for in vivo applications.

Amino Acid Sequence↗
PubMed1993

Nucleotide and deduced amino acid sequence of the envelope gene of the Vasilchenko strain of TBE virus; comparison with other flaviviruses.

A strain of tick-borne encephalitis virus known as Vasilchenko (Vs) exhibits relatively low virulence characteristics in monkeys, Syrian hamsters and humans. The gene encoding the envelope glycoprotein of this virus was cloned and sequenced. Alignment of the sequence with those of other known tick-borne flaviviruses and identification of the recognised amino acid genetic marker EHLPTA confirmed its identity as a member of the TBE complex. However, Vs virus was distinguishable from eastern and western tick-borne serotypes by the presence of the sequence AQQ at amino acid positions 232-234 and also by the presence of other specific amino acid substitutions which may be genetic markers for these viruses and could determine their pathogenetic characteristics. When compared with other tick-borne flaviviruses, Vs virus had 12 unique amino acid substitutions including an additional potential glycosylation site at position (315-317). The Vs virus strain shared closest nucleotide and amino acid homology (84.5% and 95.5% respectively) with western and far eastern strains of tick-borne encephalitis virus. Comparison with the far eastern serotype of tick-borne encephalitis virus, by cross-immunoelectrophoresis of Vs virions and PAGE analysis of the extracted virion proteins, revealed differences in surface charge and virus stability that may account for the different virulence characteristics of Vs virus. These results support and enlarge upon previous data obtained from molecular and serological analysis.

Amino Acid Sequence↗
PubMed1994

Crystallography of halophilic ribosome: the isolation of an internal ribonucleoprotein complex.

Crystals of 50S ribosomal subunits from Haloarcula marismortui diffracting to 2.9 A resolution were grown. Because of their large unit cells and the extremely weak diffracting power, almost all X-ray crystallographic analysis of these crystals must be performed with intense synchrotron radiation. At ambient temperature, all ribosomal crystals decay upon the first instance of X-irradiation. To overcome this severe sensitivity, procedures for data collection at cryo temperature were developed. Under these conditions the crystals can be irradiated for periods sufficient for the collection of more than one data set from an individual crystal (days or weeks) with no observable damage. They also can be stored for months, to resume interrupted measurements. To assist the interpretation of the anticipated electron density map, a specific internal nucleoprotein complex of protein HmaL1 and a stretch of H23S rRNA was isolated from the halophilic ribosome. The fragments of the 23S rRNA protected by the protein from nuclease digestion were sequenced. Alignment of the sequences of some archaebacterial L1-specific RNA fragments to the corresponding parts of eubacterial and eukaryotic rDNAs, localized the sequence identities to two distinct regions. Chimeric complexes were reconstituted with the corresponding E. coli ribosomal components, indicating a rather high homology, despite the evolution distance. A feasible secondary structure of the rRNA stretch participating in this complex was found to be compatible with the one proposed for the corresponding part in the E. coli ribosomal RNA.

Base Sequence↗
PubMed2006

Molecular cloning and expression analysis of a RanBP2 zinc finger protein gene in upland cotton (Gossypium hirsutum L.).

Gossypol is an important resistant substance of Gossypium, and its storage organ is pigment gland. Although, the relationship between gossypol and pigment gland has been studied for a long time, the development mechanism of pigment gland has not been revealed up to now in molecular perspective. On the basis of differentially expressed cDNAs fragments at the stage of the cotton gland development using suppression subtractive hybridization (SSH), the complete cDNA sequence of a novel RanBP2 zinc finger protein (ZFP) gene was cloned by rapid amplification of cDNA ends (RACE) from upland cotton (Gossypium hirsutum L.), Xiangmian 18. The cotton RanBP2 ZFP cDNA (GenBank accession number: DQ173926) is 717 base pair (bp) long with an open reading frame encoding 139 amino acids, which encodes a 15.6 kDa protein. The cotton RanBP2 ZFP had three Ran-binding protein (RanBP) two zinc finger motifs and belonged to RanBP2 ZFP family. There is a 292-base non-coding sequence at 3' cDNA end, which includes polyA sequence. Sequence alignment analysis revealed that the cDNA nucleotide and its deduced amino acid sequence are moderately identical to the putative ZFP from other species. The mRNA expressing profiles of the novel ZFP gene was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The result showed that it expressed at different development stages of gland, including the undeveloped stage, developing stage, developed stage and cotyledon stage. However, with the development of pigment gland, the mRNA levels in the gland-developed seed and cotyledon were increased to about 1.5 and 2 folds of that in gland-undeveloped seed, respectively, which suggested that the novel ZFP played a role in the development of the cotton gland.

Amino Acid Sequence↗
PubMed2005

A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus.

Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) of swine HEV. Different concentrations of swine HEV (120-1.2PID50) spiked into a surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan RT-PCR assay for the detection of HEV in clinical and environmental samples.

Animals↗
PubMed2003

Annealing function of GroEL: structural and bioinformatic analysis.

The Escherichia coli chaperonin system, GroEL-GroES, facilitates folding of substrate proteins (SPs) that are otherwise destined to aggregate. The iterative annealing mechanism suggests that the allostery-driven GroEL transitions leading to changes in the microenvironment of the SP constitutes the annealing action of chaperonins. To describe the molecular basis for the changes in the nature of SP-GroEL interactions we use the crystal structures of GroEL (T state), GroEL-ATP (R state) and the GroEL-GroES-(ADP)(7) (R" state) complex to determine the residue-specific changes in the accessible surface area and the number of tertiary contacts as a result of the T-->R-->R" transitions. We find large changes in the accessible area in many residues in the apical domain, but relatively smaller changes are associated with residues in the equatorial domain. In the course of the T-->R transition the microenvironment of the SP changes which suggests that GroEL is an annealing machine even without GroES. This is reflected in the exposure of Glu386 which loses six contacts in the T-->R transition. We also evaluate the conservation of residues that participate in the various chaperonin functions. Multiple sequence alignments and chemical sequence entropy calculations reveal that, to a large extent, only the chemical identities and not the residues themselves important for the nominal functions (peptide binding, nucleotide binding, GroES and substrate protein release) are strongly conserved. Using chemical sequence entropy, which is computed by classifying aminoacids into four types (hydrophobic, polar, positively charged and negatively charged) we make several new predictions that are relevant for peptide binding and annealing function of GroEL. We identify a number of conserved peptide binding sites in the apical domain which coincide with those found in the 1.7 A crystal structure of 'mini-chaperone' complexed with the N-terminal tag. Correlated mutations in the HSP60 family, that might control allostery in GroEL, are also strongly conserved. Most importantly, we find that charged solvent-exposed residues in the T state (Lys 226, Glu 252 and Asp 253) are strongly conserved. This leads to the prediction that mutating these residues, that control the annealing function of the SP, can decrease the efficacy of the chaperonin function.

Algorithms↗
PubMed1997

Study of the 16S-23S ribosomal DNA internal spacer of Coxiella burnetii.

The complete 16S-23S ribosomal DNA (rDNA) internal transcribed spacer (ITS) of 22 isolates of the obligate intracellular bacterium Coxiella burnetii, the agent of Q fever, were amplified by the polymerase chain reaction (PCR) and sequenced using an automated laser fluorescent DNA sequencer. The ITS measured 497 base pairs (bp) and encoded isoleucine-tRNA and alanine-tRNA. The comparison of the sequence alignments of the 22 C. burnetii strains revealed very high levels of sequence similitary (> 99%) although they had different geographic origins and phenotypic characteristics. Sequencing of the 16S-23S rDNA ITS of C. burnetii could be utilized for identification of the bacterium but is not applicable to studies of epidemiology, virulence and taxonomy.

Animals↗
PubMed2005

An olive pollen protein with allergenic activity, Ole e 10, defines a novel family of carbohydrate-binding modules and is potentially implicated in pollen germination.

CBMs (carbohydrate-binding modules) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. They have been frequently identified by amino acid sequence alignments, but only a few have been experimentally established to have a carbohydrate-binding activity. A small olive pollen protein, Ole e 10 (10 kDa), has been described as a major inducer of type I allergy in humans. In the present study, the ability of Ole e 10 to bind several polysaccharides has been analysed by affinity gel electrophoresis, which demonstrated that the protein bound 1,3-beta-glucans preferentially. Analytical ultracentrifugation studies confirmed binding to laminarin, at a protein/ligand ratio of 1:1. The interaction of Ole e 10 with laminarin induced a conformational change in the protein, as detected by CD and fluorescence analyses, and an increase of 3.6 degrees C in the thermal denaturation temperature of Ole e 10 in the presence of the glycan. These results, and the absence of alignment of the sequence of Ole e 10 with that of any classified CBM, indicate that this pollen protein defines a novel family of CBMs, which we propose to name CBM43. Immunolocalization of Ole e 10 in mature and germinating pollen by transmission electron microscopy and confocal laser scanning microscopy demonstrated the co-localization of Ole e 10 and callose (1,3-beta-glucan) in the growing pollen tube, suggesting a role for this protein in the metabolism of carbohydrates and in pollen tube wall re-formation during germination.

Allergens↗
PubMed1998

Active sites residues of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase (CPT-II).

The carnitine acyltransferases which catalyse the reversible transfer of fatty acyl groups between carnitine and coenzyme A have been proposed to contain a catalytic histidine. Here, the chemical reactivity of active site groups has been used to demonstrate differences between the active sites of beef liver carnitine octanoyltransferase (COT) and carnitine palmitoyltransferase-II (CPT-II). Treatment of CPT-II with the histidine-selective reagent, diethyl pyrocarbonate (DEPC), resulted in simple linear pseudo-first-order kinetics. The reversal of the inhibition by hydroxylamine and the pKa (7.1) of the modified residue indicated that the residue was a histidine. The order of the inactivation kinetics showed that 1mol of histidine was modified per mol of CPT-II. When COT was treated with DEPC the kinetics of inhibition were biphasic with an initial rapid loss of activity followed by a slower loss of activity. The residue reacting in the faster phase of inhibition was not a histidine but possibly a serine. The modification of this residue did not lead to complete loss of activity suggesting that a direct role in catalysis is unlikely. It was deduced that the residue modified by DEPC in the slower phase was a lysine and indeed fluorodinitrobenzene (FDNB) inactivated COT with linear pseudo-first-order kinetics. The COT peptide containing the FDNB-labelled lysine was isolated and sequenced. Alignment of this sequence placed it 10 amino acids downstream of the putative active-site histidine.

Amino Acid Sequence↗
PubMed2000

Genetic characterization and phylogenetic analysis of HIV-1 subtype C from Uganda.

To better understand the emergence of subtype C and its potential impact on vaccine efforts in Uganda, we have characterized subtype C sequences from Uganda (n = 13), Zimbabwe (n = 11), Mozambique (n = 5), South Africa (n = 4), and India (n = 3). Phylogenetic analysis of subtype C sequences in the env gp41 gene region revealed multiple subclusters within subtype C. Further, while most Ugandan specimen subclustered together, other subclusters did not reflect a clear geographic location. The nucleotide divergence within the Ugandan subset was 8.2% (6.1-9.8%) compared with 9.5% (2.5-15%) for the other subtype C gp41 sequences. The protein sequence alignment revealed marked sequence conservation of major immunodominant epitopes within the gp41 region.

HIV Envelope Protein gp41↗