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PubMed2006

Comparative sequence analysis of envelope protein genes of Indian buffalopox virus isolates.

Buffalopox virus (BPXV) is considered to be a close variant of vaccinia virus (VACV), the prototype member of the genus Orthopoxvirus. In the present study, we have analyzed the sequences of H3L, A27L, and D8L gene-homologues of VACV in BPXV to elucidate its genetic relationship to VACV and other orthopoxviruses (OPVs). Products of these genes have been shown to be important in attachment of VACV to host cell surface receptors during viral entry. Additionally, the A27L gene is also responsible for cell fusion during infection, while the H3L gene is required for synthesis of the highly immunogenic major envelope protein p35. Full-length nucleotide sequences of H3L, A27L, and D8L genes of three BPXV isolates were determined by PCR amplification, cloning, and sequencing. The nucleotide (nt) sequence and the deduced amino acid (aa) sequences were compared with published sequences from other members of the genus Orthopoxvirus. Comparative sequence analysis of all the three genes revealed high sequence identity of BPXV isolates with VACV (close to 99% sequence identity) at both the nt and aa level. Phylogenetic analysis based on the deduced aa sequences of the H3L, A27L, and D8L genes also showed that BPXVs are more closely related to VACV than to any of the other OPVs.

Amino Acid Sequence↗

PubMed2003

[Gene sequence analysis of SARS-associated coronavirus by nested RT-PCR].

OBJECTIVE: To explore an effective means for the detection of Severe Acute Respiratory Syndrome (SARS)- associated coronavirus. METHODS: The RNAs of the virus contained in the sputum samples from established SARS patients were extracted and reversely transcripted, followed by nested PCR using the reversely transcripted cDNA as the template. The PCR products were cloned then into the pMD18-T vectors, followed by sequence analysis. RESULTS: Specific fragments were amplified from the sputum samples of SARS patients, which were confirmed by DNA cloning and sequencing to belong to SARS-associated coronavirus. The Result of Blast shows only the difference in one nucleic acid from the TOR2 strain of SARS-associated coronavirus. CONCLUSION: Sequence analysis has confirmed the existence of SARS-associated coronavirus in the sputum samples of SARS patients, and nested RT-PCR is a quick, easy, and convenient way for the detection of the virus.

Base Sequence↗

PubMed2001

An analysis of the interactions between the Sem-5 SH3 domain and its ligands using molecular dynamics, free energy calculations, and sequence analysis.

The Src-homology-3 (SH3) domain of the Caenorhabditis elegans protein Sem-5 binds proline-rich sequences. It is reported that the SH3 domains broadly accept amide N-substituted residues instead of only recognizing prolines on the basis of side chain shape or rigidity. We have studied the interactions between Sem-5 and its ligands using molecular dynamics (MD), free energy calculations, and sequence analysis. Relative binding free energies, estimated by a method called MM/PBSA, between different substitutions at sites -1, 0, and +2 of the peptide are consistent with the experimental data. A new method to calculate atomic partial charges, AM1-BCC method, is also used in the binding free energy calculations for different N-substitutions at site -1. The results are very similar to those obtained from widely used RESP charges in the AMBER force field. AM1-BCC charges can be calculated more rapidly for any organic molecule than can the RESP charges. Therefore, their use can enable a broader and more efficient application of the MM/PBSA method in drug design. Examination of each component of the free energy leads to the construction of van der Waals interaction energy profiles for each ligand as well as for wild-type and mutant Sem-5 proteins. The profiles and free energy calculations indicate that the van der Waals interactions between the ligands and the receptor determine whether an N- or a Calpha-substituted residue is favored at each site. A VC value (defined as a product of the conservation percentage of each residue and its van der Waals interaction energy with the ligand) is used to identify several residues on the receptor that are critical for specificity and binding affinity. This VC value may have a potential use in identifying crucial residues for any ligand-protein or protein-protein system. Mutations at two of those crucial residues, N190 and N206, are examined. One mutation, N190I, is predicted to reduce the selectivity of the N-substituted residue at site -1 of the ligand and is shown to bind similarly with N- and Calpha-substituted residues at that site.

Animals↗

PubMed1997

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor.

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.

Amino Acid Sequence↗

PubMed1995

Genetic stability of cold-adapted A/Leningrad/134/47/57 (H2N2) influenza virus: sequence analysis of live cold-adapted reassortant vaccine strains before and after replication in children.

We previously reported that the A/Leningrad/134/47/57 (H2N2) cold-adapted virus (A/Len/47) used in preparing reassortant live attenuated vaccines for children acquired 14 (11 coding) mutations in genes coding for proteins other than haemagglutinin and neuraminidase during cold-adaptation. Preservation of these mutations in genomes of viruses isolated from children on the second, fifth, or eighth day after vaccination was examined by sequence analysis. The sequence data demonstrated that all nine coding mutations selected for examination were conserved in the genomes of all 11 strains investigated, indicating that the mutations accompanying cold-adaptation and attenuation of the A/Len/47 master vaccine are highly stable.

Acclimatization↗

PubMed1993

Cloning and sequence analysis of the meso-diaminopimelate decarboxylase gene from Bacillus methanolicus MGA3 and comparison to other decarboxylase genes.

The lysA gene of Bacillus methanolicus MGA3 was cloned by complementation of an auxotrophic Escherichia coli lysA22 mutant with a genomic library of B. methanolicus MGA3 chromosomal DNA. Subcloning localized the B. methanolicus MGA3 lysA gene into a 2.3-kb SmaI-SstI fragment. Sequence analysis of the 2.3-kb fragment indicated an open reading frame encoding a protein of 48,223 Da, which was similar to the meso-diaminopimelate (DAP) decarboxylase amino acid sequences of Bacillus subtilis (62%) and Corynebacterium glutamicum (40%). Amino acid sequence analysis indicated several regions of conservation among bacterial DAP decarboxylases, eukaryotic ornithine decarboxylases, and arginine decarboxylases, suggesting a common structural arrangement for positioning of substrate and the cofactor pyridoxal 5'-phosphate. The B. methanolicus MGA3 DAP decarboxylase was shown to be a dimer (M(r) 86,000) with a subunit molecular mass of approximately 50,000 Da. This decarboxylase is inhibited by lysine (Ki = 0.93 mM) with a Km of 0.8 mM for DAP. The inhibition pattern suggests that the activity of this enzyme in lysine-overproducing strains of B. methanolicus MGA3 may limit lysine synthesis.

Amino Acid Sequence↗

PubMed1998

Sequences annotated by structure: a tool to facilitate the use of structural information in sequence analysis.

With the aim of bridging the gap between protein sequence and structural analyses, we have developed a tool to aid the identification of new protein sequences by recognizing distant homologues using structural information. The tool generates sequence annotated by structure (SAS) files, applying structural information derived from structural analyses to a given protein sequence. A World Wide Web interface allows a given sequence to be submitted either for structural annotation or, where its structure is unknown, for search and alignment against sequences of known structure. In both cases, SAS will colour residues in the sequence of known structure according to a selection of properties, including secondary structure, interatomic contacts and active site information. SAS can also be used to inspect properties of a single structure.

Amino Acid Sequence↗

PubMed1995

Use of maximum length sequence analysis in newborn hearing testing.

The use of maximum length sequence analysis (MLSA) with rapid click rates may be of clinical value in newborn hearing screening because a greater number of individual responses can be signal averaged without adding to test time. To examine the potential clinical value of MLSA in newborn screening, auditory brainstem responses (ABRs) from 50 premature newborns were studied. ABRs were acquired with conventional signal averaging at four stimulus intensity levels (50 dB, 40 dB, 30 dB, and 20 dB nHL) using a click rate of 33.3/sec. These responses were directly compared with the ABRs acquired with MLSA using a rate of 227.3/sec. MLSA and conventional signal averaging yielded similar results with no statistically significant differences in the number of responses detected. Across babies, the overall quality of the tracings slightly favored MLSA, particularly when recording conditions were poor. Though the results of this investigation do not support the use of MLSA as the primary technique in newborn screening in the neonatal intensive care unit (NICU), they do support consideration of the use of MLSA as an alternative technique when the responses obtained with conventional signal averaging are poorly defined.

Acoustic Stimulation↗

PubMed2004

Vector NTI, a balanced all-in-one sequence analysis suite.

Vector NTI is a well-balanced desktop application integrated for molecular sequence analysis and biological data management. It has a centralised database and five application modules: Vector NTI, AlignX, BioAnnotator, ContigExpress and GenomBench. In this review, the features and functions available in this software are examined. These include database management, primer design, virtual cloning, alignments, sequence assembly, 3D molecular viewer and internet tools. Some problems encountered when using this software are also discussed. It is hoped that this review will introduce this software to more molecular biologists so they can make better-informed decisions when choosing computational tools to facilitate their everyday laboratory work. This tool can save time and enhance analysis but it requires some learning on the user's part and there are some issues that need to be addressed by the developer.

Algorithms↗

PubMed1987

Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.

Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.

Amino Acid Sequence↗

PubMed2004

Genetic diversity among Pasteurella multocida strains of avian, bovine, ovine and porcine origin from England and Wales by comparative sequence analysis of the 16S rRNA gene.

Genetic diversity among 86 Pasteurella multocida isolates was investigated by comparative sequence analysis of a 1468 bp fragment of the 16S rRNA gene. The strains included 79 field isolates recovered from birds (poultry) (22), cattle (21), pigs (26) and sheep (10) within England and Wales, four Asian isolates associated with bovine haemorrhagic septicaemia, and the type strains of the three subspecies of P. multocida. Dulcitol and sorbitol fermentation patterns were also determined to establish correlations between subspecies status and phylogenetic relatedness. Nineteen 16S rRNA types were identified, but these were clustered into two distinct phylogenetic lineages, A and B. Sequences within lineages A and B had a mean number of nucleotide differences of 21.12+/-3.90. Isolates within lineage A were associated with birds, cattle, pigs and sheep, whereas those belonging to lineage B were recovered from birds and a cat. Eighty-seven per cent of the isolates were classified as P. multocida subsp. multocida by dulcitol and sorbitol fermentation patterns, but these have diverse 16S rRNA gene sequences that were represented in both lineages A and B. Avian P. multocida subsp. septica isolates were associated exclusively with lineage B, but bovine P. multocida subsp. septica isolates were present in lineage A. P. multocida subsp. gallicida isolates of avian, bovine and porcine origin represent a homogeneous group within lineage A, but they have the same 16S rRNA type as certain P. multocida subsp. multocida isolates. These findings provide strong support for the view that dulcitol and sorbitol fermentation patterns are inaccurate indicators of genetic relatedness among P. multocida strains. Avian capsular type B isolates and capsular type B and E isolates associated with haemorrhagic septicaemia of cattle and water buffaloes are closely related and form a distinct cluster within lineage A. The current subspecies nomenclature of P. multocida neither accurately reflects the 16S rRNA-based phylogenetic relationships among isolates nor does it adequately encompass the full range of diversity within the species. The study provides a 16S rRNA-based evolutionary framework that will form the basis of further studies into the genetic diversity of P. multocida and will also help in the reclassification of the species.

Animals↗

PubMed1994

Evolutionary affiliation of the marine nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067, derived by 16S ribosomal RNA sequence analysis.

The 16S rRNA sequence of Trichodesmium sp. strain NIBB 1067 was determined and used for the construction of a distance tree and bootstrap analysis. The tree shows that, among the available cyanobacterial 16S rRNA sequences, Trichodesmium NIBB 1067 has Oscillatoria PCC 7515 as its closest relative, presenting 94.9% of sequence similarity with the latter strain. This is in contrast to a difference of 9 mol% G+C in mean genomic DNA base composition between the two organisms. Nevertheless, the genotypic heterogeneity presented by a number of strains assigned to the genus Oscillatoria hinders a taxonomic decision on the separate existence of the genera Trichodesmium and Oscillatoria. The sequence of the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes was also determined, as a possible marker to study inter- and intraspecific variability. The ITS contains the genes coding for tRNA(Ile) and tRNA(Ala) and its total length is 547 nucleotides. In six out of eight sequenced clones, there is a duplication of 29 nucleotides, surrounding the 5' end of the tRNA(Ile).

Base Sequence↗

PubMed1990

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5'end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191, 337-446], was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, Mr 46,265) and 469 amino acids (E3, Mr 49,193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-oxo-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.

Acetyltransferases↗

PubMedDate not supplied

DNA sequence analysis of bacterial toxic heavy metal resistances.

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be "linked" together on multiple resistance plasmids. For Cd2+, AsO2-, AsO4(3)-, Hg2+, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. The cadA ATPase of S. aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd2+ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K+ ATPases of Escherichia coli and Streptococcus faecalis, the H+ ATPases of yeast and Neurospora, the Na+/K+ ATPases of vertebrate animals, and the Ca2+ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in the mer system include a DNA-binding regulatory protein from the merR gene and a Hg2+ transport system consisting of a periplasmic Hg2(+)-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.

Amino Acid Sequence↗

PubMed2004

[Cloning and sequence analysis of cDNA encoding PPAR from goose].

The peroxisome proliferator activated receptor (PPAR) belongs to a large family of nuclear receptors. This study was designed to clone and sequence analysis of cDNA encoding PPAR from goose. The RT-PCR method was developed to clone the cDNA, and the lengths of cDNA encoding PPARalpha and PPARgamma were 1407bp and 1428bp respectively. The cDNAs of the two genes were cloned and sequenced for the first time. The identities of CDS of PPARalpha and PPARgamma gene were 87.43% and 92.00% by homologous comparison among goose and other five species, and that were 93.38% and 96.95% in amino acid sequences. The further analysis among seventeen species including goose showed that the identities of PPAR genes were low(66.18%) among different sub-type (alpha,gamma,beta) of PPAR genes and that was high for the same sub-type of PPAR genes: PPARalpha, PPARgamma and PPARbeta (or PPARdelta) were 84.80%, 86.23% and 87.36% respectively. The results showed that these two genes are conservative in the process of evolution and has important physiological function for the growth and development of birds and mammals. The results of the present study will benefit the further study of relationship between PPAR genes and the growth and development, especially in fat metabolism of goose.

Amino Acid Sequence↗

PubMed1993

BK virus antigenic variants: sequence analysis within the capsid VP1 epitope.

DNA sequences for the VP1 gene which codes for the major capsid protein of BK virus (BKV) and may be responsible for antigenic variability were determined for seven BKV isolates. The observed sequence differences and those previously reported correlate with the typing of isolates into four groups by haemagglutination inhibition. Amino acid coding alterations were found to be clustered within residues 61 to 83. Each antigenic group was found to have a characteristic amino acid sequence between residues 61 and 83. Several clones originating from a single isolate, although differing slightly in restriction enzyme digestion patterns, were found to be identical in this region. The VP1 sequences of three of the four groups were analysed by hydropathy plots and two hydrophilic areas of high antigenicity were identified. One of these corresponds to residues 61 to 83 and it is postulated that this region is the epitope responsible for serotypic differences between BK isolates.

Amino Acid Sequence↗

PubMed1999

Sequence analysis of heparan sulphate and heparin oligosaccharides.

The biological activity of heparan sulphate (HS) and heparin largely depends on internal oligosaccharide sequences that provide specific binding sites for an extensive range of proteins. Identification of such structures is crucial for the complete understanding of glycosaminoglycan (GAG)-protein interactions. We describe here a simple method of sequence analysis relying on the specific tagging of the sugar reducing end by 3H radiolabelling, the combination of chemical scission and specific enzymic digestion to generate intermediate fragments, and the analysis of the generated products by strong-anion-exchange HPLC. We present full sequence data on microgram quantities of four unknown oligosaccharides (three HS-derived hexasaccharides and one heparin-derived octasaccharide) which illustrate the utility and relative simplicity of the technique. The results clearly show that it is also possible to read sequences of inhomogeneous preparations. Application of this technique to biologically active oligosaccharides should accelerate progress in the understanding of HS and heparin structure-function relationships and provide new insights into the primary structure of these polysaccharides.

Animals↗

PubMed1995

Isolation and sequence analysis of the chicken GABAA receptor alpha 1-subunit gene promoter.

A genomic clone containing the 5'-flanking sequence of the chicken GABAA receptor alpha 1-subunit-encoding gene (GabR alpha 1) was isolated and characterized. An intron was found to interrupt the 5'-untranslated region. The transcription start point (tsp) was determined by primer extension, RNase protection and the amplification of chick brain first-strand cDNA. DNA sequence analysis revealed a number of putative transcriptional regulatory motifs, including a TATA box 30 nucleotides upstream from the tsp, and that this region is a CpG island. While there is conservation between the chicken and human GabR alpha 1 sequences, the chicken GabR alpha 1 promoter has a different structure to those reported for the GABAA receptor beta 3- and delta-subunit-encoding genes.

Animals↗