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PubMed2005

Characterization of canarypox-like viruses infecting endemic birds in the Galápagos Islands.

The presence of avian pox in endemic birds in the Galápagos Islands has led to concern that the health of these birds may be threatened by avipoxvirus introduction by domestic birds. We describe here a simple polymerase chain reaction-based method for identification and discrimination of avipoxvirus strains similar to the fowlpox or canarypox viruses. This method, in conjunction with DNA sequencing of two polymerase chain reaction-amplified loci totaling about 800 bp, was used to identify two avipoxvirus strains, Gal1 and Gal2, in pox lesions from yellow warblers (Dendroica petechia), finches (Geospiza spp.), and Galápagos mockingbirds (Nesomimus parvulus) from the inhabited islands of Santa Cruz and Isabela. Both strains were found in all three passerine taxa, and sequences from both strains were less than 5% different from each other and from canarypox virus. In contrast, chickens in Galápagos were infected with a virus that appears to be identical in sequence to the characterized fowlpox virus and about 30% different from the canarypox/Galápagos group viruses in the regions sequenced. These results indicate the presence of canarypox-like viruses in endemic passerine birds that are distinct from the fowlpox virus infecting chickens on Galápagos. Alignment of the sequence of a 5.9-kb region of the genome revealed that sequence identities among Gal1, Gal2, and canarypox viruses were clustered in discrete regions. This indicates that recombination between poxvirus strains in combination with mutation led to the canarypox-like viruses that are now prevalent in the Galápagos.

Amino Acid Sequence↗

PubMed2004

A new approach for rapidly reshaping single-chain antibody in vitro by combining DNA shuffling with ribosome display.

Antibody reshaping is an effective way to reduce the immunogenicity while maintaining or improving the affinity of murine antibodies. This paper describe a new in vitro approach for rapidly reshaping murine antibodies by combining DNA shuffling with ribosome display. With the new method, a reshaping anti-4-1BB single-chain antibody (scFv), Re-4B4-1 scFv, which bound to its antigen (4-1BB) specifically and strongly, was selected from a reshaping library. These results proved definitely the feasibility of the new designed approach for antibody reshaping.

Amino Acid Sequence↗

PubMed2004

Combining evolutionary and structural information for local protein structure prediction.

We study the effects of various factors in representing and combining evolutionary and structural information for local protein structural prediction based on fragment selection. We prepare databases of fragments from a set of non-redundant protein domains. For each fragment, evolutionary information is derived from homologous sequences and represented as estimated effective counts and frequencies of amino acids (evolutionary frequencies) at each position. Position-specific amino acid preferences called structural frequencies are derived from statistical analysis of discrete local structural environments in database structures. Our method for local structure prediction is based on ranking and selecting database fragments that are most similar to a target fragment. Using secondary structure type as a local structural property, we test our method in a number of settings. The major findings are: (1) the COMPASS-type scoring function for fragment similarity comparison gives better prediction accuracy than three other tested scoring functions for profile-profile comparison. We show that the COMPASS-type scoring function can be derived both in the probabilistic framework and in the framework of statistical potentials. (2) Using the evolutionary frequencies of database fragments gives better prediction accuracy than using structural frequencies. (3) Finer definition of local environments, such as including more side-chain solvent accessibility classes and considering the backbone conformations of neighboring residues, gives increasingly better prediction accuracy using structural frequencies. (4) Combining evolutionary and structural frequencies of database fragments, either in a linear fashion or using a pseudocount mixture formula, results in improvement of prediction accuracy. Combination at the log-odds score level is not as effective as combination at the frequency level. This suggests that there might be better ways of combining sequence and structural information than the commonly used linear combination of log-odds scores. Our method of fragment selection and frequency combination gives reasonable results of secondary structure prediction tested on 56 CASP5 targets (average SOV score 0.77), suggesting that it is a valid method for local protein structure prediction. Mixture of predicted structural frequencies and evolutionary frequencies improve the quality of local profile-to-profile alignment by COMPASS.

Algorithms↗

PubMed2002

Phylogenetic relationships in rhinonyssid mites (Acari: Rhinonyssidae) based on ribosomal DNA sequences: insights for the discrimination of closely related species.

The complete internal transcribed spacer 1 (ITS1), 5.8S rDNA and ITS2 region of the ribosomal DNA from 11 species of rhinonyssid mites ( Tinaminyssus columbae, T. minisetosum, T. sartbaevi, T. bubulci, T. melloi, T. streptopelioides, Sternostoma fulicae, S. boydi, S. strandtmanni, S. turdi, Rhinonyssus tringae) were sequenced to assess the utility of this genomic region in resolving taxonomic questions in this group and to estimate phylogenetic relationships between species. Two different geographic locations of T. melloi and T. streptopelioides were analyzed to detect intraspecies variation. Our study shows that ribosomal sequences can help to discriminate between T. melloi and T. sartbaevi, which are morphologically very close and difficult to separate by classic methods. The resulting phylogenetic tree shows some differences from the current taxonomy of the family Rhinonyssidae. This study appeals for the revision of the taxonomic status of S. boydi and closely related species which parasitize aquatic birds and suggests the synonymy of S. boydi and S. strandtmanni, despite the different hosts of the two mites.

Acaridae↗

PubMed1996

Selection of peptides that bind to the HLA-A2.1 molecule by molecular modelling.

Cytotoxic T lymphocytes recognize antigenic peptides in association with major histocompatibility complex class I proteins. Although a large set of class I binding peptides has been described, it is not yet easy to search for potentially antigenic peptides without synthesis of a panel of peptides, and subsequent binding assays. In order to predict HLA-A2.1-restricted antigenic epitopes, a computer model of the HLA-A2.1 molecule was established using X-ray crystallography data. In this model nonameric peptide sequences were aligned. In a molecular dynamics (MD) simulation with two sets of peptides known to be presented by HLA-A2.1, it was important to know the anchor amino acid residue preference and the distance between the anchor residues. We show here that the peptides bound to the HLA-A2.1 model structure possess a side chain of C-terminal anchor residue oriented into the binding groove with different distances between the two anchor residues from 15 to 21A. We also synthesized a set of nonamer peptides containing amino acid sequences of Hepatitis B virus protein that were selected on the basis of previously described HLA-A2.1 specific motifs. When results obtained from the MD simulation were compared with functional binding assays using the TAP-deficient cell line T2, it was evident that the MD simulation method improves prediction of the HLA-A2.1 binding epitope sequence. These results suggest that this approach can provide a way to predict peptide epitopes and search for antigenic regions in sequences in a variety of antigens without screening a large number of synthetic peptides.

Amino Acid Sequence↗

PubMed2004

Protein fiber linear dichroism for structure determination and kinetics in a low-volume, low-wavelength couette flow cell.

High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 microL), low-wavelength (down to 180 nm), low-pathlength (100 microm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-beta-sheet amyloid fibers of the Alzheimer's derived protein Abeta and the long-chain assemblies of alpha1-antitrypsin polymers.

Actins↗

PubMed2003

Identification of a new HLA allele, A*1114, in a Chinese family.

A novel HLA-A allele, A*1114, was initially detected in two generations of a Chinese family by unusual polymerase chain reaction based sequence-specific primers ( PCR-SSP) reaction patterns and ambiguous sequence-based typing (SBT). Molecular cloning and sequencing analysis indicated that this new allele differs from HLA-A*1102 by three nucleotide substitutions in exon 3, 524 A-->G, 526 G-->C, and 527 C-->G, thus changing codon 175 from His to Arg (CAT-->CGT) and codon 176 from Ala to Arg (GCG-->CGG). Segregation analysis showed that the proband inherited his mother's HLA haplotype A*1114, B*5801, DRB1*1405. The serologic equivalent of A*1114 is a split antigen HLA-A11.2. A PCR-SSP method was developed to distinguish A*1114 from other A*11 alleles. No further individuals with A*1114 were found in 5000 Chinese bone marrow donors.

Alleles↗

PubMed2000

PatSearch: a pattern matcher software that finds functional elements in nucleotide and protein sequences and assesses their statistical significance.

MOTIVATION: The identification of sequence patterns involved in gene regulation and expression is a major challenge in molecular biology. In this paper we describe a novel algorithm and the software for searching nucleotide and protein sequences for complex nucleotide patterns including potential secondary structure elements, also allowing for mismatches/mispairings below a user-fixed threshold, and assessing the statistical significance of their occurrence through a Markov chain simulation. RESULTS: The application of the proposed algorithm allowed the identification of some functional elements, such as the Iron Responsive Element, the Histone stem-loop structure and the Selenocysteine Insertion Sequence, located in the mRNA untranslated regions of post-transcriptionally regulated genes with the assessment of sensitivity and selectivity of the searching method. AVAILABILITY: A Web interface is available at: http://bigarea.area.ba.cnr.it:8000/EmbIT/Pats earch.html.

3' Untranslated Regions↗

PubMed2004

Analysis and prediction of leucine-rich nuclear export signals.

We present a thorough analysis of nuclear export signals and a prediction server, which we have made publicly available. The machine learning prediction method is a significant improvement over the generally used consensus patterns. Nuclear export signals (NESs) are extremely important regulators of the subcellular location of proteins. This regulation has an impact on transcription and other nuclear processes, which are fundamental to the viability of the cell. NESs are studied in relation to cancer, the cell cycle, cell differentiation and other important aspects of molecular biology. Our conclusion from this analysis is that the most important properties of NESs are accessibility and flexibility allowing relevant proteins to interact with the signal. Furthermore, we show that not only the known hydrophobic residues are important in defining a nuclear export signals. We employ both neural networks and hidden Markov models in the prediction algorithm and verify the method on the most recently discovered NESs. The NES predictor (NetNES) is made available for general use at http://www.cbs.dtu.dk/.

Active Transport, Cell Nucleus↗

PubMed1995

Identification of the Chlamydia trachomatis RecA-encoding gene.

DNA sequencing of the major outer membrane protein (MOMP) gene (omp1) from Chlamydia trachomatis shows that some strains have a mosaic structure suggestive of homologous recombination between two distinct omp1 genes. On the basis of this conjecture, we attempted to clone by complementation and sequence the chlamydial recA homolog from C. trachomatis serovar L2. Chlamydial genomic DNA was partially restricted with XbaI, and fragments of 2 to 4 kb were ligated into pUC19. The recombinant plasmid was electroporated into Escherichia coli HB101 (RecA-), and colonies were selected in the presence of methyl methanesulfonate (MMS). A 2.1-kb fragment of C. trachomatis DNA in pUC19 conferred relative MMS resistance to E. coli HB101. When this recombinant plasmid (pX203) was electroporated into E. coli JC14604 (RecA- lacZ), lac+ recombinants were isolated. Rabbit polyclonal antibodies produced to purified E. coli RecA were immunoreactive in an immunoblot assay with a 35-kDa antigen in RecA- strains of E. coli transformed with pX203. The 2.1-kb insert was cycle sequenced by the dideoxy chain termination method. An open reading frame of 1,056 bp encoding 352 amino acids that had 44% sequence identity with E. coli RecA was identified. The finding of a recA homolog in C. trachomatis suggests that homologous recombination may occur in this organism. The cloned C. trachomatis RecA-encoding gene will be useful for the construction of a recA mutant once a gene transfer system is developed for chlamydiae.

Amino Acid Sequence↗

PubMed2002

Establishment of a mini-gene expression database for bladder tumor.

BACKGROUND AND PURPOSE: Transitional cell carcinoma has been diagnosed mostly in urinary bladder in southern Taiwan and has an exceptionally high mortality rate. To identify the genes associated with bladder cancer, we investigated differential gene expression. Six bladder tumor cDNA libraries were constructed and their sequences were compared and analyzed. METHODS: mRNA from bladder tumor cell lines or tissue samples were used to construct two regular cDNA libraries and four subtractive cDNA libraries after subtractive hybridization with cDNA derived from normal bladder epithelial cells. Subsequently, more than 100 cDNA inserts from each library were randomly isolated and sequenced, followed by sequence comparisons with nucleotide and protein sequence databases. RESULTS: After searching the Basic Local Alignment Search Tool (Blast) databases, the cDNA nucleotide sequences were grouped into novel, known, and common gene categories. Since tumor nucleotide sequences are informative and valuable for research, they were organized as a mini-gene expression database (http://bladder.nhri.org.tw/). Interestingly, in one subtractive cDNA library, the ATPase 6 gene was found to be highly expressed in normal bladder epithelial cells and elevated levels of ATPase 6 mRNA were later confirmed by reverse transcription-polymerase chain reaction. However, the role of ATPase 6 in bladder tumorigenesis remains to be investigated. CONCLUSIONS: The establishment of this database is an important step to enable systematic screening for bladder tumor-associated genes and may also be useful in developing diagnostic and/or therapeutic applications.

Adenosine Triphosphatases↗

PubMed2003

cDNA cloning of growth hormone from giant panda (Ailuropoda melanoleuca) and its expression in Escherichia coli.

A cDNA encoding Ailuropoda melanoleuca growth hormone (AmGH) was isolated from pituitary total RNA using RT-PCR and expressed in Escherichia coli. This is the first report of a GH nucleotide and amino acid (aa) sequence from giant panda. The open reading frame of AmGH (651 bp) encodes a precursor of 216 aa comprising a 26 aa signal peptide and a 190 aa mature protein with four cysteine residues similar to the typical primary structure of mammalian GH precursor. AmGH shares a high degree of identity (54-98.9%) with that of mammals, birds and amphibians, but a very low identity with bony fish GH (only 20-30%). The mature AmGH exhibits striking similarity to that of putative ancestral GH with a difference of only two residues, indicating a very slow basal rate of molecular evolution. The DNA fragment encoding mature AmGH was then subcloned into the pGEX-4T-1 expression vector and highly expressed in E. coli host BL21 with IPTG induction. The expressed proteins fused to GST were found to be sequestered into inclusion bodies and therefore the NaOH method was employed to solubilize the inclusion bodies; the proteins were further purified by Glutathione Sepharose 4B affinity chromatography. The production and purification of GST-AmGH reported here provide a basis for further studies on the biological activity of AmGH.

Amino Acid Sequence↗

PubMed2005

How old are the extant lineages of Toxoplasma gondii?

Most known isolates of Toxoplasma gondii belong to one of only three lineages, which are presumed to be clonal. Three models have been proposed for the evolutionary relationship of these lineages to the other extant lineages: Model (a) proposing that all lineages are derived from a most recent common ancestor (MRCA) in the distant past, Model (b) that all lineages are derived from a MRCA in the very recent past, and Model (c) that the clonal lineages share a recent MRCA but are related to the other lineages only in the distant past. Here, I test these models using DNA intron and coding-sequence data for loci at 14 genes, using three different methods to calculate the time of the MRCA. All of the calculations agree that the MRCA of the clonal lineages was > 70% of the age of the MRCA of all lineages, thus favouring Model (a). The MRCA may have existed approximately 150,000 years ago, with the clonal lineages expanding in prevalence approximately 10,000 years ago.

Animals↗

PubMed1988

Complete amino acid sequence of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

The complete amino acid sequence of 6-phospho-fructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was determined by direct analysis of the S-carboxamidomethyl protein. A complete set of nonoverlapping peptides was produced by cleavage with a combination of cyanogen bromide and specific proteolytic enzymes. The active enzyme is a dimer of two identical polypeptide chains composed of 470 amino acids each. The NH2-terminal amino acid residue of the polypeptide chain was shown to be N-acetylserine by fast atom bombardment mass spectrometry of the purified N-terminal tetradecapeptide isolated after cleavage of the intact S-carboxamidomethylated protein with lysyl endoproteinase (Achromobacter protease I). Alignment of the set of unique peptides was accomplished by the analysis of selected overlapping peptides generated by proteolytic cleavage of the intact protein and the larger purified cyanogen bromide peptides with trypsin, Staphylococcus aureus V8 protease, and lysyl endoproteinase. Four nonoverlapping peptides were aligned by comparison with the amino acid sequence predicted from a partial cDNA clone encoding amino acid positions 166-470 of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Colosia, A.D., Lively, M., El-Maghrabi, M. R., and Pilkis, S. J. (1987) Biochem. Biophys. Res. Commun. 143, 1092-1098). The nucleotide sequence of the cDNA corroborated the peptide sequence determined by direct methods. A search of the Protein Identification Resource protein sequence database revealed that the overall amino acid sequence appears to be unique since no obviously homologous sequences were identified. However, a 100-residue segment of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (residues 250-349), including the active site histidine residue of the bisphosphatase domain, was found to be homologous to the active site regions of yeast phosphoglycerate mutase and human bisphosphoglycerate mutase.

Amino Acid Sequence↗

PubMed1997

Dynamite: a flexible code generating language for dynamic programming methods used in sequence comparison.

We have developed a code generating language, called Dynamite, specialised for the production and subsequent manipulation of complex dynamic programming methods for biological sequence comparison. From a relatively simple text definition file Dynamite will produce a variety of implementations of a dynamic programming method, including database searches and linear space alignments. The speed of the generated code is comparable to hand written code, and the additional flexibility has proved invaluable in designing and testing new algorithms. An innovation is a flexible labelling system, which can be used to annotate the original sequences with biological information. We illustrate the Dynamite syntax and flexibility by showing definitions for dynamic programming routines (i) to align two protein sequences under the assumption that they are both poly-topic transmembrane proteins, with the simultaneous assignment of transmembrane helices and (ii) to align protein information to genomic DNA, allowing for introns and sequencing error.

Algorithms↗

PubMed2004

Dynamic structure of vesicle-bound melittin in a variety of lipid chain lengths by solid-state NMR.

Solid-state 31P- and 13C-NMR spectra were recorded in melittin-lecithin vesicles composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Highly ordered magnetic alignments were achieved with the membrane surface parallel to the magnetic field above the gel-to-liquid crystalline phase transition temperature (Tc). Using these magnetically oriented vesicle systems, dynamic structures of melittin bound to the vesicles were investigated by analyzing the 13C anisotropic and isotropic chemical shifts of selectively 13C-labeled carbonyl carbons of melittin under the static and magic-angle spinning conditions. These results indicate that melittin molecules adopt an alpha-helical structure and laterally diffuse to rotate rapidly around the membrane normal with tilt angles of the N-terminal helix being -33 degrees and -36 degrees and those of the C-terminal helix being 21 degrees and 25 degrees for DLPC and DPPC vesicles, respectively. The rotational-echo double-resonance method was used to measure the interatomic distance between [1-13C]Val8 and [15N]Leu13 to further identify the bending alpha-helical structure of melittin to possess the interhelical angles of 126 degrees and 119 degrees in DLPC and DPPC membranes, respectively. These analyses further lead to the conclusion that the alpha-helices of melittin molecules penetrate the hydrophobic cores of the bilayers incompletely as a pseudo-trans-membrane structure and induce fusion and disruption of vesicles.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

PASS2: a semi-automated database of protein alignments organised as structural superfamilies.

PASS2 is a nearly automated version of CAMPASS and contains sequence alignments of proteins grouped at the level of superfamilies. This database has been created to fall in correspondence with SCOP database (1.53 release) and currently consists of 110 multi-member superfamilies and 613 superfamilies corresponding to single members. In multi-member superfamilies, protein chains with no more than 25% sequence identity have been considered for the alignment and hence the database aims to address sequence alignments which represent 26 219 protein domains under the SCOP 1.53 release. Structure-based sequence alignments have been obtained by COMPARER and the initial equivalences are provided automatically from a MALIGN alignment and subsequently augmented using STAMP4.0. The final sequence alignments have been annotated for the structural features using JOY4.0. Several interesting links are provided to other related databases and genome sequence relatives. Availability of reliable sequence alignments of distantly related proteins, despite poor sequence identity and single-member superfamilies, permit better sampling of structures in libraries for fold recognition of new sequences and for the understanding of protein structure-function relationships of individual superfamilies. The database can be queried by keywords and also by sequence search, interfaced by PSI-BLAST methods. Structure-annotated sequence alignments and several structural accessory files can be retrieved for all the superfamilies including the user-input sequence. The database can be accessed from http://www.ncbs.res.in/%7Efaculty/mini/campass/pass.html.

Amino Acid Sequence↗

PubMed1994

Determination of membrane-bound fragments of cytochrome P-450 2B4.

Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segments 273-314 and 427-491. Epitope mapping of the domain next to the N-terminus (residues 21-119) of the isolated hemoprotein was performed with the help of a peptide-scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane-penetrating part of this domain was found except for the fragment 1-21. A model of structure of P-450 2B4 was computed by comparison with the structure of cytochrome P-450cam on the basis of an alignment of 47 cytochromes P-450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody-reactive epitopes of the region 21-119, are located at the membrane-facing side in this model.

Amino Acid Sequence↗