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PubMed1997

Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis.

Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xylenol and harbours the plasmid RP4, was mated with a plasmid-free derivative of Pseudomonas putida NCIB 9869, strain RA713, which cannot grow on 2,5-xylenol. Some RA713 transconjugants, initially selected on 2,5-xylenol, were found to carry RP4 plasmids that had acquired additional fragments (designated Xin) which ranged in size from 2 kb to approximately 26 kb instability of DNA inserts in RP4::Xin hybrid plasmids was observed. The smallest insert present in a stable RP4::Xin6 hybrid plasmid, termed Xin6, yielded multiple bands when it was used as a probe with digested P25X chromosomal DNA. Sequence analysis of Xin6 led to the discovery of an open reading frame with homology to the maturases of group II introns. The Xin6 insert also exhibited several features characteristic of a group II intron. These included the presence of the consensus sequence GUGYG at the 5' and and RAY at the 3' end of the intron. RNA secondary structure modelling of Xin6 also revealed the presence of perfectly conserved domains V and VI. Differences were detected in the Xin6 hybridization profiles of several P25X catabolic mutants that have lost the ability to grow on 2,5-xylenol. In these mutants the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the Xin6 group II intron. This is the first reported group II intron isolated from Pseudomonas spp. and the first time that the mobility of a bacterial group II intron has been demonstrated.

Amino Acid Sequence↗

PubMed2004

Biochemical and functional characterization of protein kinase CK2 in ascidian Ciona intestinalis oocytes at fertilization. Cloning and sequence analysis of cDNA for alpha and beta subunits.

The ubiquitous and pleiotropic dual specificity protein kinase CK2 has been studied and characterized in many organisms, from yeast to mammals. Generally, the enzyme is composed of two catalytic (alpha and/or alpha') and two regulatory (beta) subunits, forming a differently assembled tetramer. Although prone to controversial interpretation, the function of CK2 has been associated with fundamental biological processes such as signal transduction, cell cycle progression, cell growth, apoptosis, and transcription. Less known is the role of CK2 during meiosis and the early phase of embryogenesis. In this work, we studied CK2 activity during oocyte activation, a process occurring at the end of oocyte maturation and triggered by fertilization. In ascidian Ciona intestinalis, an organism whose complete genome has been published recently, CK2 was constitutively active in unfertilized and fertilized oocytes. The enzymatic activity oscillated through meiosis showing three major peaks: soon after fertilization (metaphase I exit), before metaphase II, and at the exit from metaphase II. Biochemical analysis of CK2 subunit composition in activated oocytes indicated that CK2-alpha was catalytically active as a monomer, independently from its regulatory subunit beta; however, CK2-beta was only detectable in unfertilized oocytes where it was associated with a bona fide identified ascidian mitogen-activated protein kinase. After fertilization, CK2-beta was undetectable, suggesting its rapid degradation. Protein sequence analysis of CK2-alpha and -beta cDNA indicated a high identity compared with vertebrate homologs. In addition, the absence of putative phosphorylation sites for Cdc2 kinase on both alpha and beta subunits suggested an important role for CK2 in regulating meiotic cell cycle in C. intestinalis oocytes.

Amino Acid Sequence↗

PubMed1994

Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.

A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens. The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations. Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons. Calli induced from scutella were very good starting materials. A strain of A. tumefaciens that carried a so-called 'super-binary' vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture.

Agrobacterium tumefaciens↗

PubMed1995

Sequence analysis of the Streptococcus mutans Ingbritt dexA gene encoding extracellular dextranase.

The complete nucleotide sequence (3,747 bp) of the dextranase gene (dexA) and flanking regions of the chromosome of Streptococcus mutans Ingbritt (serotype c) were determined. The open reading frame for dexA was 2,550 bp, ending with a stop codon TGA. A putative ribosome-binding site, promoter preceding the start codon, and potential stem-loop structure were identified. The presumed dextranase protein (DexA) consisting of 850 amino acids was estimated to have a molecular size of 94,536 Da and a pI of 4.79. The nucleotide sequence and the deduced amino acid sequences of S. mutans dexA exhibited homologies of 57.8% and 47.0%, respectively, to those of Streptococcus sobrinus dex. The homologous region of dex of S. sobrinus was in the N-terminal half. The C terminus of DexA consisted of a hexapeptide LPQTGD, followed by 7 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged hexapeptide tail, which have been reported as a common structure of C termini of not only the surface-associated proteins of Gram-positive cocci but also the extracellular enzymes such as beta-fructosidase of S. mutans and dextranase of S. sobrinus. The DexA protein had no significant homology with the glucosyltransferases, the glucan-binding protein, or the dextranase inhibitor of mutans streptococci.

Amino Acid Sequence↗

PubMed2003

The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene.

A taxonomic study was carried out on eight strains of Saccharomyces boulardii. Morphological and physiological characteristics were consistent with those of Saccharomyces cerevisiae. Sequences of the D1/D2 domain of the 26S rDNA were identical for all strains examined and had a similarity value of 100% compared to sequences of the type strain of S. cerevisiae (CBS 1171T) and strain S288c. For all S. boulardii isolates was found the exact same ITS1-5.8S rDNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171(T) (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii isolates and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171(T) (96.6%). The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from strains of this species. The results of the present study strongly indicate a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species.

DNA, Fungal↗

PubMed2001

16S ribosomal DNA sequence analysis distinguishes biotypes of Streptococcus bovis: Streptococcus bovis Biotype II/2 is a separate genospecies and the predominant clinical isolate in adult males.

We characterized 22 human clinical strains of Streptococcus bovis by genotypic (16S rRNA gene sequence analysis [MicroSeq]; Applied Biosystems, Foster City, Calif.) and phenotypic (API 20 Strep and Rapid ID32 Strep systems (bioMerieux Vitek, Hazelton, Mo.) methods. The strains, isolated from blood, cerebrospinal fluid (CSF), and urine, formed two distinct 16S ribosomal DNA sequence clusters. Three strains which were associated with endocarditis urinary tract infection (UTI), and sepsis clustered with the S. bovis type strain ATCC 33317 (cluster 1); other closely related type strains were S. equinus and S. infantarius. Nineteen strains clustered at a distance of about 2.5% dissimilarity to the S. bovis type strain (cluster 2) and were associated with central nervous system (CNS) disease in addition to endocarditis, UTI, and sepsis. All strains were distinct from S. gallolyticus. Within cluster 2, a single strain grouped with ATCC strain 43143 (cluster 2a) and may be phenotypically distinct. All the other strains formed a second subgroup (cluster 2b) that was biochemically similar to S. bovis biotype II/2 (mannitol negative and beta galactosidase, alpha galactosidase, beta glucuronidase, and trehalose positive). The API 20 Strep system identified isolates of cluster 2b as S. bovis biotype II/2, those of cluster 1 as S. bovis biotype II/1, and that of cluster 2a as S. bovis biotype I. There was an excellent correlation of biotype and genotype: S. bovis biotype II/2 isolates form a separate genospecies distinct from the S. bovis, S. gallolyticus, and S. infantarius type strains and are the most common isolates in adult males.

Adult↗

PubMed2005

Comparison between sequence analysis and a line probe assay for testing genotypic resistance of human immunodeficiency virus type 1 to antiretroviral drugs.

The purpose of this study was to compare a line probe assay (LiPA) with sequence analysis for the detection of mutations conferring resistance to nucleoside and non-nucleoside inhibitors in human immunodeficiency reverse transcriptase and protease inhibitors. The limitations for interpreting LiPA make it unacceptable for routine clinical practice.

Anti-HIV Agents↗

PubMed2001

Genetic heterogeneity of Borrelia burgdorferi sensu lato in the southern United States based on restriction fragment length polymorphism and sequence analysis.

Fifty-six strains of Borrelia burgdorferi sensu lato, isolated from ticks and vertebrate animals in Missouri, South Carolina, Georgia, Florida, and Texas, were identified and characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons. A total of 241 to 258 bp of intergenic spacers between tandemly duplicated rrf (5S) and rrl (23S) was amplified by PCR. MseI and DraI restriction fragment polymorphisms were used to analyze these strains. PCR-RFLP analysis results indicated that the strains represented at least three genospecies and 10 different restriction patterns. Most of the strains isolated from the tick Ixodes dentatus in Missouri and Georgia belonged to the genospecies Borrelia andersonii. Excluding the I. dentatus strains, most southern strains, isolated from the ticks Ixodes scapularis and Ixodes affinis, the cotton rat (Sigmodon hispidus), and cotton mouse (Peromyscus gossypinus) in Georgia and Florida, belonged to Borrelia burgdorferi sensu stricto. Seven strains, isolated from Ixodes minor, the wood rat (Neotoma floridana), the cotton rat, and the cotton mouse in South Carolina and Florida, belonged to Borrelia bissettii. Two strains, MI-8 from Florida and TXW-1 from Texas, exhibited MseI and DraI restriction patterns different from those of previously reported genospecies. Eight Missouri tick strains (MOK-3a group) had MseI patterns similar to that of B. andersonii reference strain 21038 but had a DraI restriction site in the spacer. Strain SCGT-8a had DraI restriction patterns identical to that of strain 25015 (B. bissettii) but differed from strain 25015 in its MseI restriction pattern. Strain AI-1 had the same DraI pattern as other southern strains in the B. bissettii genospecies but had a distinct MseI profile. The taxonomic status of these atypical strains needs to be further evaluated. To clarify the taxonomic positions of these atypical Borrelia strains, the complete sequences of rrf-rrl intergenic spacers from 20 southeastern and Missouri strains were determined. The evolutionary and phylogenetic relationships of these strains were compared with those of the described genospecies in the B. burgdorferi sensu lato species complex. The 20 strains clustered into five separate lineages on the basis of sequence analysis. MI-8 and TXW-1 appeared to belong to two different undescribed genospecies, although TXW-1 was closely related to Borrelia garinii. The MOK-3a group separated into a distinct deep branch in the B. andersonii lineage. PCR-RFLP analysis results and the results of sequence analyses of the rrf-rrl intergenic spacer confirm that greater genetic heterogeneity exists among B. burgdorferi sensu lato strains isolated from the southern United States than among strains isolated from the northern United States. The B. andersonii genospecies and its MOK-3a subgroup are associated with the I. dentatus-cottontail rabbit enzootic cycle, but I. scapularis was also found to harbor a strain of this genospecies. Strains that appear to be B. bissettii in our study were isolated from I. minor and the cotton mouse, cotton rat, and wood rat. The B. burgdorferi sensu stricto strains from the south are genetically and phenotypically similar to the B31 reference strain.

Animals↗

PubMed2001

Sequence analysis of fibroblast growth factor receptor 2 ( FGFR2 ) in Japanese patients with craniosynostosis.

Recently, mutations of the fibroblast growth factor receptor ( FGFR ) genes have been detected in syndromic craniosynostosis. We examined nucleotide sequences of FGFR2 in Japanese craniosynostosis patients (Crouzon syndrome: 9 cases; Apert syndrome: 6 cases; scaphocephaly: 3 cases as non-syndromic patients) by polymerase chain reaction (PCR) followed by direct sequencing methods. The results demonstrated FGFR2 heterozygous mutations at codons 252, 290 of exon 7, and at codon 342, 354 of exon 9 in Crouzon syndromes. In Apert syndrome patients, Ser252Trp and Pro253Arg were detected in five and one patients, respectively. No mutation was detected in one case of Crouzon, all cases of scaphocephaly and healthy individuals. Thus far sequence analysis of FGFR2 in syndromic craniosynostosis has been reported in many white patients, whereas in Japanese only several cases have been studied. The current study with 18 patients confirmed that a similar series of mutations occur in Japanese patients as in white patients regardless of ethnicity and environment. The frequency of the mutation was 82% (9/11 cases) in Japanese Crouzon patients. The ratio of S252W:P253R was 5 : 1 in Japanese Apert patients. Moreover, in Japanese Apert patients, complication rate of cleft palate was 60% for mutation of Ser252Trp and 0 of 2 patients for Pro253Arg, with their syndactyly score being 4.90 and 5.50, respectively.

Acrocephalosyndactylia↗

PubMed2005

De novo sequence analysis of N-terminal sulfonated peptides after in-gel guanidination.

Here we report a novel approach in which gel-separated proteins are guanidinated in-gel prior to enzymatic cleavage. In contrast to previously described techniques, this procedure allows the extracted tryptic peptides to be N-terminal sulfonated without any further sample purification. The derivatized peptides were subsequently fragmented using a matrix-assisted laser desorption/ionization time of flight/time of flight instrument. The approach facilitates the de novo sequence analysis and allows obtaining longer stretches of amino acid sequence information. We demonstrate that the obtained information can be used to identify proteins using a sequence similarity search algorithm. The technique was compared to the standard peptide mass fingerprint approach, applied either in-gel or in solution, using a number of sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated model proteins. Finally, the new protocol was applied on a proteomic study of two-dimensional PAGE separated proteins from Shewanella oneidensis. More than 50 proteins from this organism were identified using sub-picomol quantities of protein, and peptide sequences of up to 20 amino acid residues in length have been determined.

Algorithms↗

PubMed2003

Multilocus sequence analysis of Porphyromonas gingivalis indicates frequent recombination.

In this study, the genetic relationship of 19 Porphyromonas gingivalis isolates from patients with periodontitis was investigated by multilocus sequence analysis. Internal 400-600 bp DNA fragments of the 10 chromosomal genes ef-tu, ftsQ, hagB, gpdxJ, pepO, mcmA, dnaK, recA, pga and nah were amplified by PCR and sequenced. No two isolates were identical at all 10 loci. Phylogenetic analyses indicated a panmictic population structure of P. gingivalis. Split decomposition analysis, calculation of homoplasy ratios and analyses of clustered polymorphisms all indicate that recombination plays a major role in creating the genetic heterogeneity of P. gingivalis. A standardized index of association of 0.0898 indicates that the P. gingivalis genes analysed are close to linkage equilibrium.

Chromosomes, Bacterial↗

PubMed2003

Characterization of chicken CENP-A and comparative sequence analysis of vertebrate centromere-specific histone H3-like proteins.

Centromere protein A (CENP-A) is a centromere-specific histone H3 variant conserved amongst all eukaryotes. We have isolated the chicken gene for CENP-A (GgCENP-A). It encodes a 131-amino-acid polypeptide that possesses an average identity of 54% with human CENP-A, reaching 69% in the histone-fold domain. The gene spans 1.7 kb of genomic DNA and contains four exons that range in size from 78 to 186 bp. The exon/intron organisation of the chicken gene is conserved with its mammalian counterparts in the carboxy-terminal histone-fold domain (exons 2 to 4), consistent with the strong conservation of this domain at the amino acid level. Sequence analysis of the chicken CENP-A locus revealed that the gene is located within the class III genes of the major histocompatibility complex (MHC), and extended the previously defined limit of the compact chicken MHC complex. We compared the sequences of CENP-A from mammals, chicken and fishes and thereby identified conserved motifs in the otherwise variable amino-terminal tail that may be important for functional reasons. We also identified evolutionarily variable regions within the conserved histone-fold domain. We found that loop 1 between the first and second alpha-helix is the region that diverged most widely. This finding is in agreement with evolutionary studies in Drosophila species, and suggests that this domain could play a role in species-specific centromere targeting of CENP-A. In addition, protein sequence comparison of several vertebrate species revealed that the RT-PCR strategy we have developed for isolating the chicken centromeric histone H3 variant gene should be applicable to the isolation of CENP-A from a wide range of vertebrates.

Amino Acid Sequence↗

PubMed2006

Biomarker discovery by CE-MS enables sequence analysis via MS/MS with platform-independent separation.

CE-MS is a successful proteomic platform for the definition of biomarkers in different body fluids. Besides the biomarker defining experimental parameters, CE migration time and molecular weight, especially biomarker's sequence identity is an indispensable cornerstone for deeper insights into the pathophysiological pathways of diseases or for made-to-measure therapeutic drug design. Therefore, this report presents a detailed discussion of different peptide sequencing platforms consisting of high performance separation method either coupled on-line or off-line to different MS/MS devices, such as MALDI-TOF-TOF, ESI-IT, ESI-QTOF and Fourier transform ion cyclotron resonance, for sequencing indicative peptides. This comparison demonstrates the unique feature of CE-MS technology to serve as a reliable basis for the assignment of peptide sequence data obtained using different separation MS/MS methods to the biomarker defining parameters, CE migration time and molecular weight. Discovery of potential biomarkers by CE-MS enables sequence analysis via MS/MS with platform-independent sample separation. This is due to the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly correlates with their CE-MS migration time/molecular weight coordinates. This uniqueness facilitates the independent entry of different sequencing platforms for peptide sequencing of CE-MS-defined biomarkers from highly complex mixtures.

Amino Acid Sequence↗

PubMed1969

Polynucleotide sequence analysis by sequential base elimination: 3'-terminus of phage Q-beta RNA.

The nucleotide sequence of a 3'-terminal fragment obtained by ribonuclease T(1) hydrolysis of the ribonucleic acid from bacteriophage Qbeta has been determined by an improved method of sequence analysis which involves sequential removal of bases by periodate oxidation and beta-elimination. The results obtained from ten such oxidation-elimination cycles and from the alkaline hydrolysis of the remaining oligonucleotide indicate that the first 16 nucleotides at the 3'-terminus of this ribonucleic acid have the sequence: -G-C-C-C-U-C-U-C-U-C-C-U-C-C-C-A.

Bacteriophages↗

PubMed2004

Sequence analysis of the MHC class II DRB alleles in Alpine chamois (Rupicapra r. rupicapra).

This study characterises the expressed MHC class II DRB gene and its genetic variation in exon 2 among 59 Alpine chamois (Rupicapra r. rupicapra) from several locations in the Eastern Alps plus one population in the Western Alps. The expressed DRB gene contains all the expected characteristics including all of the conserved residues found in other mammalian species. The isolated expressed DRB gene from chamois can be considered orthologous to MHC class II DRB genes in other mammalian species. Cattle primers yielded exon 2 sequences of 284 bp length. Sequence analysis revealed 19 DRB exon 2 alleles among 59 chamois. Alleles exhibited a high degree of nucleotide and amino acid polymorphism, with most amino acid variations occurring at positions forming the peptide-binding sites. A significantly higher rate of non-synonymous (dN = 0.053+/-0.0132) than synonymous (dS = 0.007+/-0.003) substitutions indicated positive selection for diversity in the DRB locus.

Amino Acid Sequence↗

PubMed2001

Hemochromatosis (HFE) gene sequence analysis of formalin-fixed, paraffin-embedded liver biopsy specimens.

BACKGROUND: Hereditary hemochromatosis (HH) is a common disease predominantly characterized by mutations of the HFE gene. METHODS AND RESULTS: We investigated the utility of HFE gene sequence analysis in the diagnosis of HH in 61 prospectively accrued formalin-fixed, paraffin-embedded liver biopsy specimens with clinical or histologic features suggestive of HH. Mutations in codons 63 or 282 of the HFE gene were identified by direct sequencing; in 21 of these samples, quantitative hepatic iron testing was also performed. Changes characteristic of HH were present in 16 (26%) of the cases, and 54% of the cases showed HFE gene mutations. The most common alteration was homozygous mutation of codon 282 (11 cases, 18%), followed by the combined 63 + 282 heterozygous mutation (3 cases, 5%). Two cases (3%) showed biallelic mutation of codon 63. The other 28 cases (46%) showed no sequence abnormalities. Weak iron staining did not exclude HH; intense staining did not reliably predict HH. CONCLUSION: When HH is clinically and/or histologically suspected, HFE gene sequencing of formalin-fixed, paraffin-embedded liver biopsy specimens is a rapid and cost-effective approach to genotypic diagnosis of HH.

Age Factors↗

PubMed2001

Comparative sequence analysis of the plasmid-encoded regulator of enteropathogenic Escherichia coli Strains.

Enteropathogenic Escherichia coli (EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator (per). Considerable variation in gene content of EAF plasmids from different strains was seen. However, bfpA, the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and per genes were found in 96.8% of strains. Sequence analysis of the per operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5' two-thirds of the perA gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in perA leading to premature truncation and consequent inactivation of the gene were identified. Cloned perA, -B, and -C genes from these strains, unlike those from strains with a functional operon, failed to activate the LEE1 operon and bfpA transcriptional fusions or to complement a per mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive per operons were deficient in virulence protein expression. The context in which the perABC operon occurs on the EAF plasmid varies. The sequence upstream of the per promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS1294-homologous sequence.

Amino Acid Sequence↗

PubMed1998

Macaque MDC family of proteins: sequence analysis, tissue distribution and processing in the male reproductive tract.

A large number of sequence-related, cysteine-rich membrane proteins containing metalloproteinase-like and disintegrin-like domains (the MDC protein family) have been identified in mammalian tissues from a variety of species. Previous studies in the macaque (Macaca fascicularis) have led to the cDNA cloning and sequence analysis of a number of MDC proteins which are abundantly expressed in the male reproductive tract. We now describe the distribution of seven of these macaque MDC transcripts in a range of different tissues. This description includes a novel macaque testis-derived MDC, tMDC III, whose full-length sequence is reported for the first time. In addition, polyclonal antisera have been used to localize a number of these MDC proteins to spermatogenic cells in testis sections, and to demonstrate their processing on the sperm surface during epididymal transit.

ADAM Proteins↗