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PubMed2001

Typing of Candida albicans isolates by sequence analysis of the cytochrome b gene and differentiation from Candida stellatoidea.

Including type strains, mitochondrial cytochrome b genes of 32 strains of Candida albicans and 6 strains of Candida stellatoidea, presently treated as a synonym for C. albicans, were partially sequenced. Analysis of 396-bp nucleotide sequences of the strains under investigation divided C. albicans isolates into three types: type I, type II, and type III; however, strains of C. stellatoidea represented distinct type IV isolates. Deduced amino acid sequences of type I, type II, and type III were identical and differed from that of type IV by one amino acid. Genotypes (rDNA type) of the test strains were also checked. Cytochrome b typing did not correlate with genotyping, and different genotypes occurred for one cytochrome b type. This study shows that cytochrome b gene sequences are useful for analyzing the genetic relatedness of C. albicans isolates and effective for differentiating C. stellatoidea from C. albicans.

Amino Acid Sequence↗

PubMed1999

[Genotype and sequence analysis of merozoite surface protein 1 of Plasmodium falciparum isolates in Yunnan province].

AIM: To identify the genotype of merozoite surface protein 1 of Plasmodium falciparum in Yunnan Province and explore the polymorphism of MSP1 genes in geographical characteristics and genetics. METHODS: Nested polymerase chain reaction was applied to genotyping of P. falciparum isolated in Yunnan Province. Some of parasite alleles were sequenced by dye primer cycle sequencing. RESULTS: In 30 P. falciparum infections, 38 different alleles were found. Of them, the dominant allele was a variant of MAD20, while was K1 less and the RO33 was few. In addition, incidences of mixed allele infections were observed. Sequence analysis showed that DNA sequences of MAD20, K1 and RO33 alleles from Yunnan were highly homologous with those of international standard strains, respectively. CONCLUSION: Composition and sequence characteristics of P. falciparum arasite population in the endemic area can be detected by genotyping with MSP1 as genetic marker, which would be useful for the prevention and treatment of malaria.

Alleles↗

PubMed1990

Characterization of the African HIV-1 isolate CBL-4 (RUT) by partial sequence analysis and virus neutralization with peptide antibody and antisense phosphate-methylated DNA.

The HIV-1 isolate CBL-4 (RUT), originating from Tanzania, was characterized using a comprehensive virus-typing system. This system included sequence analysis of the region coding for the neutralization domain in the third variable region (V3) of the external envelope and of the tat responsive (TAR) region after polymerase chain reaction (PCR) amplification of these sequences from cellular DNA in the CBL-4 (RUT) producer line. Based on independent cluster analysis of TAR and V3 sequences the CBL-4 (RUT) virus was positioned closest to the Z6 (and ELI) African virus family. The V3 amino acid sequence on the surface of the virus particle was confirmed by the inhibition of neutralization of CBL-4 (RUT) by a synthetic peptide derived from the nucleic acid sequence. Using antisense phosphate-methylated DNA covering the TAR loop region of LAV-1/HTLV-IIIB, inhibition of HTLV-IIIB and HTLV-IIIRF infection was seen, whereas no inhibition was observed for CBL-4 (RUT), indicating two or more mismatches in the TAR loop region, a characteristic shared with Z6 virus, but not with ELI. We propose a virus-typing system based on sequence analysis confirmed by virus neutralization with a peptide binding antibody and inhibition by antisense phosphate-methylated DNA to group viruses for laboratory use and vaccine design.

Acquired Immunodeficiency Syndrome↗

PubMed2004

Combining text mining and sequence analysis to discover protein functional regions.

Recently presented protein sequence classification models can identify relevant regions of the sequence. This observation has many potential applications to detecting functional regions of proteins. However, identifying such sequence regions automatically is difficult in practice, as relatively few types of information have enough annotated sequences to perform this analysis. Our approach addresses this data scarcity problem by combining text and sequence analysis. First, we train a text classifier over the explicit textual annotations available for some of the sequences in the dataset, and use the trained classifier to predict the class for the rest of the unlabeled sequences. We then train a joint sequence text classifier over the text contained in the functional annotations of the sequences, and the actual sequences in this larger, automatically extended dataset. Finally, we project the classifier onto the original sequences to determine the relevant regions of the sequences. We demonstrate the effectiveness of our approach by predicting protein sub-cellular localization and determining localization specific functional regions of these proteins.

Algorithms↗

PubMed1993

Electroblotting proteolytic products from native gel for direct N-terminal sequence analysis: an approach for studying protein-protein interaction.

Proteins which are electroblotted from native gels onto polyvinylidene difluoride (PVDF) membranes are suitable for detailed structural analysis. This method, in conjunction with limited proteolysis and N-terminal sequencing, has been used to study the molecular interactions between native protein molecules. The interaction between recombinant interleukin-2 (rIL-2) and its receptor (rIL-2R alpha) was examined as a model system. The working strategy consists of (i) proteolysis of rIL-2R alpha and rIL-2R alpha/rIL-2 complex, (ii) separation of the major proteolytic products by native polyacrylamide gel electrophoresis followed by electroblotting onto PVDF membrane, and (iii) sequence analysis of the blotted protein bands for the identification of peptide regions sensitive to proteolysis. Results have indicated that the exon 3 encoded region in rIL-2R alpha is sensitive to proteolysis regardless whether it is complexed with rIL-2 or not. This suggests that no major conformational changes occur in rIL-2R alpha during interaction with rIL-2. This electroblotting approach is, therefore, useful for studying protein-protein interaction in solution.

Animals↗

PubMed2005

Cloning and sequence analysis of llama (lama glama) Th2 (IL-4, IL-10 and IL-13) cytokines.

This paper describes the cloning and sequence analysis of the cDNAs encoding the T helper (Th) 2 cytokines of llama including interleukin-4 (IL-4), IL-10 and IL-13. The cDNAs encoding for IL-4, IL-10 and IL-13 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-4, IL-10 and IL-13 were found to be 402, 537 and 411 bp in length, with open reading frames encoding 133, 178 or 136 amino acids, respectively. Homology analyses of nucleotide and deduced amino acid sequences of llama IL-4, IL-10 and IL-13 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla (pig, cattle) and Perissodactyla (horse).

Amino Acid Sequence↗

PubMed2003

Sequence analysis of kgp in Porphyromonas gingivalis isolates from periodontitis patients.

The aim of the present study was to determine sequence variation in the Lys-x-specific protease (Kgp) encoding gene kgp of Porphyromonas gingivalis and to analyze its association with periodontal disease severity. Pooled subgingival plaque samples were obtained from the six most severely affected sites of 102 patients with periodontitis. Sequence analysis of the kgp gene in 23 clinical P. gingivalis isolates resulted in the identification of two distinct kgp types (kgp-I and kgp-II) according to sequence differences in the region encoding the catalytic domain. Restriction analysis revealed that 59 of the 102 patients were colonized by kgp-I and 43 by kgp-II. Patients harboring kgp-I or kgp-II showed no significant difference in the severity of periodontal disease as assessed by pocket probing depth and bleeding on probing following adjustment for smoking habit and age. Moreover, no differences in proteolytic activity of Kgp-I and Kgp-II were detected. The results indicated that two kgp types are maintained in natural populations of P. gingivalis.

Adhesins, Bacterial↗

PubMedDate not supplied

[Development and role of comparative sequence analysis in medical virology].

INTRODUCTION: Development of the polymerase chain reaction and deoxyribonucleic acid sequencing techniques has enabled precise identification, classification and taxonomy of viruses. COMPARATIVE SEQUEENCE ANALYSIS: (Comparative sequence analysis methods can be used in medical virology for many practical purposes. They may be classified into three broad categories: I - reconstruction of genealogical relationships between individual viral isolatesfor detection and monitoring of sources, reservoirs and modes of viral transmission; II - virus genotyping, that is determination of relationships between genetic types of viruses and their phenotypic properties, which has important implications for immunoprophylaxis, therapy and prognosis of viral diseases, and III investigation of functional properties of defined viral sequences, of special importance for explanation of viral pathogenesis and design of antiviral drugs. FUTURE PROSPECTS: The combination of DNA sequencing with polymerase chain reaction following reverse transcription with the use of random primers offers a universal means for diagnosis of viral infections.

DNA, Viral↗

PubMed2004

Cyst morphology and sequence analysis of the small subunit rDNA and ef1 alpha identifies a novel Giardia genotype in a quenda (Isoodon obesulus) from Western Australia.

Sequence analysis of the small subunit ribosomal DNA (SSU-rDNA) and elongation factor 1 alpha (ef1 alpha) was performed on Giardia cysts isolated from faeces collected from a quenda (Isoodon obesulus) in the southwest of Western Australia. The SSU-rDNA and ef1 alpha were sequenced in their entirety and correspondingly aligned with the published sequence information of other known species and genotypes of Giardia. Phylogenetic analysis of the SSU-rDNA and ef1 alpha sequences identified the quenda isolate as a novel genotype of Giardia not previously reported. We believe that this quenda Giardia isolate constitutes a distinct species, which may be endemic within the Australian native fauna.

Animals↗

PubMed1996

Sequence analysis of a 10 kb DNA fragment from yeast chromosome VII reveals a novel member of the DnaJ family.

We report the sequence analysis of a 10 kb DNA fragment of Saccharomyces cerevisiae chromosome VII. This sequence contains four complete open reading frames (ORFs) of greater than 100 amino acids. There are also two incomplete ORFs flanking the extremes: one of these, G2868, is the 5' part of the SCS3 gene (Hosaka et al., 1994). ORFs G2853 and G2856 correspond to the genes CEG1, coding for the alfa subunit of the mRNA guanylyl transferase and a 3' gene of unknown function previously sequenced (Shibagaki et al., 1992). G2864 is identical to SOH1 also reported (Fan and Klein, 1994).

Amino Acid Sequence↗

PubMed1994

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.

Actinomycetales↗

PubMed2003

[A new method for amplification and sequence analysis of the full-length of HBV genome].

OBJECTIVES: To develop a new method for amplifying and sequencing the full-length of HBV genome. METHODS: A pair of primers located at the nick region of HBV molecule and a thermostable polymerase with high fidelity and sensitivity were used. After cloning the PCR products into a plasmid, the sequences of HBV genome were analyzed. RESULTS: The full-length of HBV genome were acquired using this method. The sensitivity and fidelity of the new method were also analyzed. The least quantity of initial templates was 10(2) and the artificial mutation rate was 1.2 bp/kb. CONCLUSION: This method can be used in amplification and sequence analysis of the full-length of HBV genome on a large scale.

DNA Primers↗

PubMedDate not supplied

Identification of the Goodpasture antigen, alpha 3(IV) NC1, and four other NC1 domains of type IV collagen, by amino-terminal sequence analysis of human glomerular basement membrane separated by two-dimensional electrophoresis.

Two-dimensional (2-D) electrophoresis of collagenase-digested human glomerular basement membrane (GBM), containing the Goodpasture antigen, revealed a range of monomeric (24-30 kD) and dimeric (43-56 kD) subunits present across a pI range of 3-10. Five distinct alpha(IV)-chains were identified by amino-terminal sequence analysis of 18 of these components transferred to polyvinylidene difluoride membrane. The positions of the well-characterised 26-kD alpha 1(IV)-chain and 24-kD alpha 2(IV)-chain were confirmed. A highly cationic 28-kD monomer was identified as the alpha 3(IV)-chain, while more neutral 28-kD monomers were found to contain the alpha 4(IV)-chain. Sequences from neutral 26-kD monomers corresponded to the known cDNA sequence of the alpha 5(IV)-chain. The presence of charge isoforms of the alpha 1(IV)- and alpha 4(IV)-chains was confirmed by identification of several monomers with different pI but the same sequence. Sequence analysis of dimeric components demonstrated homodimers of alpha 1(IV), alpha 2(IV) and alpha 4(IV), and suggested the presence of heterodimers of alpha 3/alpha 5 and alpha 1/alpha 5. 2-D Western blots of human GBM, with anti-GBM autoantibodies, a monoclonal antibody (P1) to the Goodpasture antigen and a monoclonal antibody to the alpha 3(IV)-chain, demonstrated that the major autoantigenic epitope was localised to the alpha 3(IV)-chain, but that there was also reactivity with the alpha 4(IV)-chain.

Amino Acid Sequence↗

PubMed2003

Sequence analysis of cyclodextrin glycosyltransferase from the alkaliphilic Bacillus agaradhaerens.

The gene encoding an alkaline active cyclodextrin glycosyltransferase (CGTase) from the alkaliphilic B. agaradhaerens LS-3C was cloned and sequenced. It encodes a mature polypeptide of 679 amino acids with a molecular mass of 76,488 Da. The deduced amino acid sequence of the mature CGTase revealed 99 and 95% identity to the CGTase sequences from the other B. agaradhaerens strains, DSM 8721T and 9948, respectively. The next closest identity was of 59% with B. clarkii enzyme. CGTases from B. agaradhaerens, B. clarkii, and B. firmus/lentus formed a phylogenetically separated cluster from the other CGTases of Bacillus spp. origin. A number of usually conserved residues in the CGTases were found to be replaced in the sequence of B. agaradhaerens enzyme. The sequence analysis indicated the enzyme to be close to the so-called 'intermediary enzymes' in the alpha-amylase family.

Amino Acid Sequence↗

PubMed1994

Cloning and sequence analysis of the human SNAP25 cDNA.

During the screening of human brain-specific cDNAs by a modified differential hybridization analysis, we found a clone which was highly and specifically expressed in the (adult) brain. This clone was subjected to nucleotide sequence analysis and proven to be a nearly full-length cDNA of a human homologue of the previously reported mouse synaptosomal-associated protein 25 (mSNAP25). The human SNAP25 (hSNAP25) and mSNAP25 showed perfect amino-acid sequence conservation.

Base Sequence↗

PubMedDate not supplied

The TR-DNA region carrying the auxin synthesis genes of the Agrobacterium rhizogenes agropine-type plasmid pRiA4: nucleotide sequence analysis and introduction into tobacco plants.

We have determined the nucleotide sequence of a 6-kilobase fragment of the Agrobacterium rhizogenes plasmid pRiA4 TR-region that carries genes (aux1 and aux2) responsible for auxin biosynthesis in transformed plant cells. Sequence analysis revealed two open reading frames corresponding to proteins of 749 amino acids for the aux1 gene and 466 amino acids for the aux2 gene. We observed significant similarity between the amino acid sequences deduced from the pRiA4 aux genes and those of the auxin biosynthesis genes of A. tumefaciens octopine-type Ti plasmids, the iaaM and iaaH genes of Pseudomonas savastanoi, and different genes of the pRiA4 TL-region; however, the 5'-flanking regions of the pRi and pTi auxin biosynthesis genes were found to be completely different. Transgenic tobacco plants containing this entire 6-kilobase fragment of the pRiA4 TR-region have been obtained. Regenerated plants are phenotypically normal. The aux1 gene is not or is very weakly expressed in these plants, but expression of the aux2 gene leads to a modified root phenotype when plants are grown on medium containing an auxin precursor (naphthalene acetamide).

Agrobacterium tumefaciens↗

PubMed2001

Chemical C-terminal protein sequence analysis: improved sensitivity, length of degradation, proline passage, and combination with edman degradation.

Use of a C-terminal sequencer with modified solvents, reagent concentrations, chromatographic parameters, temperatures, and reaction cartridge geometry yields four sets of improvements in chemical degradations. They are increased sensitivity, longer runs, passage of Pro residues, and practical combination with N-terminal degradation. Over 200 proteins and protein fragments with sizes between 20 and 600 residues were analyzed. C-terminal sequences could be interpreted for more than 10 residues at high picomole sample levels, while the 10-pmol level gave 4-5 residues. The average initial yield was 15% but up to 30% could be achieved. The improved performance allowed combination of C- and N-terminal degradations from the same sample application. After initial Edman degradation, the sample is moved to the C-terminal instrument for continued sequencing. Proteins available in limited amount are thereby efficiently analyzed. Lys, modified from the N-terminal degradation, may be detected as the alkylated thiohydantoin-phenylthiocarbamyl-Lys derivative in the C-terminal degradation. Notably, C-terminal sequence analysis could be proceeded through Pro residues which unexpectedly were no absolute hindrance. The improved technique provides characterization of truncation patterns and microheterogeneities in proteins down to the 10-pmol level and is a useful approach for analysis of N-terminally blocked polypeptides.

Mass Spectrometry↗

PubMed1999

MHC class I genes of the channel catfish: sequence analysis and expression.

Four cDNAs encoding the major histocompatibility complex (MHC) class I alpha chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I alpha chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I alpha and beta2-microglobulin (beta2m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 Mr glycoprotein (presumably class I alpha) was associated with beta2m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of beta2m with the MHC class I alpha chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts.

Amino Acid Sequence↗