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PubMed1994

Nucleic acid sequence analysis software packages.

Software packages have made sequence analysis easier and more accessible to research scientists in many fields. The majority of these packages are routinely upgraded to include improved and innovative programs for primer design, sequence assembly and improvements in database searching programs. The interfaces of many packages have also been improved, greatly facilitating their use. In the future, researchers can expect to be able to access a growing number of network applications (e.g. database searching and exon prediction programs) from within software packages, making them accessible in familiar formats. Increased network access from within software packages will also make more databases, and more frequently updated databases, available to a larger research community.

Base Sequence↗
PubMed1984

GENAS: a database system for nucleic acid sequence analysis.

A database system, named GENAS (GENe Analyzing System), for computer analysis of sequence was constructed using Adbis which is a relational database management system (1). GENAS enables us to retrieve any sequence data from EMBL nucleotide sequence data library (2) and readily to analyze them (if necessary, together with private data) by various application programs in a interactive manner. Analysis of structure of replication origin of replicons was demonstrated using this system.

Base Sequence↗
PubMed2005

Comparison of sequence analysis and INNO-LiPA HBV DR line probe assay in patients with chronic hepatitis B.

The aim of this study was to compare direct sequence analysis of partial HBV pol gene and Inno-LiPA HBV DR in serum samples of 120 chronic hepatitis B patients sent to the Clinical Microbiology Laboratory of Ege University Hospital because of lamivudine resistance. Sequence analysis was performed on ABI Prism 310 Genetic Analyzer. Comparison of Inno-LiPA and sequence results obtained by double-blind evaluation showed full agreement (both at rt180 and rt204) in 58.8% of samples. Visually rechecking of the electropherograms increased this rate to 68.3% Codon based rates are 81.7% and 75.8% at rt180 and rt204 respectively. LiPA detected variants in additional 12 (10%) samples, but missed one variant sample (both rt180 and rt204) and one sample was indeterminate due to poor probe binding. LiPA allows determination of mixed variants and seems to be more sensitive and simple for routine testing even though sequence analysis is still the gold standard for detecting new variants.

DNA Probes↗
PubMed1995

Rapid detection and characterization of foot-and-mouth disease virus by restriction enzyme and nucleotide sequence analysis of PCR products.

Reverse transcription coupled with PCR was used for the detection of foot-and-mouth disease virus serotypes A, C, and O in organ extracts from experimentally infected cattle. Primers were selected from conserved sequences flanking the genome region coding for the major antigenic site of the capsid located in the C-terminal part of viral protein 1 (VP1). Because this region of the capsid is highly variable its coding sequence is considered to be the most appropriate for the characterization of virus isolates and, therefore, for the determination of the epidemiological relationships between viruses of the same serotype. For differentiation between serotypes and for detailed characterization of individual virus isolates restriction enzyme cleavage and nucleotide sequence analysis of the respective PCR products were carried out. In order to minimize the time required for sample preparation from clinical material, viral RNA was released from particles by heating the sample for 5 min at 90 degrees C. Finally, an air thermocycler was used, which allows performance of a PCR of 30 cycles in approximately 20 min. The results show that reverse transcription PCR followed by restriction enzyme analysis and/or nucleotide sequence analysis of the PCR products is useful for the rapid detection and differentiation of foot-and-mouth disease virus.

Animals↗
PubMed1997

New horizons in sequence analysis.

An ever increasing number of protein sequences are being compared, partly because of the availability of full sets of protein sequences from several completed genome-sequencing projects. The resulting problem of scale has shifted the emphasis of sequence analysis method development from sensitivity and flexibility, which relies on manual intervention and interpretation, to the automatic generation of results of known reliability.

Amino Acid Sequence↗
PubMed1993

Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies.

An insertion sequence (IS) element of Brucella ovis, named IS6501, was isolated and its complete nucleotide sequence determined. IS6501 is 836 bp in length and occurs 20-35 times in the B. ovis genome and 5-15 times in other Brucella species. Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch. IS6501 presents significant similarity (53.4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence. A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A. tumefaciens and with the transposase of Mycobacterium tuberculosis. IS6501 is present in all Brucella strains we have tested. Restriction fragment length polymorphism of reference and field strains of two species (B. melitensis and B. ovis) was studied using either pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe. The genome of B. melitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns. In contrast, the number of IS copies in the B. ovis genome is around 30 and the different field strains can be differentiated by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Mutation screening of EXT1 and EXT2 by direct sequence analysis and MLPA in patients with multiple osteochondromas: splice site mutations and exonic deletions account for more than half of the mutations.

Multiple osteochondromas (MO) is an autosomal dominant condition, caused by mutations in either the EXT1 or the EXT2 gene. The DNA of a cohort of 35 patients, clinically suspected to be affected with MO, was screened for mutations by a combination of direct sequence analysis and multiplex ligation-dependent probe amplification (MLPA). In this cohort, 26 pathogenic gene alterations were found (74%). With sequence analysis mutations were detected in 22 patients (63%). In total, 10 mutations were detected in the EXT1 and 12 in the EXT2 gene. The number of the splice site mutations detected was larger than expected from the literature. In addition, with the MLPA four deletions of one or more exons were found in this cohort. Two patients, of whom one had a negative family history, showed deletions of exon 1 of the EXT1 gene, which is possibly a deletion hot spot. In patients suspected to be affected by MO, we recommend a quantitative analysis such as MLPA, followed by direct sequence analysis for the screening of the EXT1 and EXT2 genes.

Cohort Studies↗
PubMed2005

[Isolation and sequence analysis of a omega-gliadin homologous gene from wheat].

The DNA sequence of a full-length Triticum astivum CV. Jinan 177omega-gliadin homologous gene (omega1236) containing partial 5' and 3' flanking sequences with no intron was cloned by genomic PCR-based technology. The omega1236 sequence possibly encode a putative 47.2 kDa protein except for eight stop codons at amino acid residue positions 87, 117, 125, 157, 198, 313, 357 and 365 respectively. All the eight stop codons were caused by base transition. Sequence analysis revealed that omega1236 had 98% homology to a omega-gliadin gene of wheat (AB059812). Like all other gliadin gene families characterized in cereals, this gene possessed all the features in other plant reported previously. Phylogenetic analysis of the completely sequenced gene as well as those omega-genes in wheat, omega-secalin and C-horden genes in rye and barley, and alpha-, beta- and gamma-gliadin genes in wheat indicated that the omega1236 was more closely related to omega-gliadin gene family, much less homology to alpha- , beta- and gamma-gliadin gene families. Short peptide was produced in the culture of transformed E. coli induced by IPTG in early 2 h. It indicated that stop codon would be in omega1236. The result is consistent with that of the sequenced gene. The present paper could accumulate data useful for both omega-gliadin gene cloning by PCR and the study on structures and functions of these genes.

Amino Acid Sequence↗
PubMed1997

The arginine deiminase pathway in Rhizobium etli: DNA sequence analysis and functional study of the arcABC genes.

Sequence analysis upstream of the Rhizobium etli fixLJ homologous genes revealed the presence of three open reading frames homologous to the arcABC genes of Pseudomonas aeruginosa. The P. aeruginosa arcABC genes code for the enzymes of the arginine deiminase pathway: arginine deiminase, catabolic ornithine carbamoyltransferase (cOTCase), and carbamate kinase. OTCase activities were measured in free-living R. etli cells and in bacteroids isolated from bean nodules. OTCase activity in free-living cells was observed at a different pH optimum than OTCase activity in bacteroids, suggesting the presence of two enzymes with different characteristics and different expression patterns of the corresponding genes. The characteristics of the OTCase isolated from the bacteroids were studied in further detail and were shown to be similar to the properties of the cOTCase of P. aeruginosa. The enzyme has a pH optimum of 6.8 and a molecular mass of approximately 450 kDa, is characterized by a sigmoidal carbamoyl phosphate saturation curve, and exhibits a cooperativity for carbamoyl phosphate. R. etli arcA mutants, with polar effects on arcB and arcC, were constructed by insertion mutagenesis. Bean nodules induced by arcA mutants were still able to fix nitrogen but showed a significantly lower acetylene reduction activity than nodules induced by the wild type. No significant differences in nodule dry weight, plant dry weight, and number of nodules were found between the wild type and the mutants. Determination of the OTCase activity in extracts from bacteroids revealed a strong decrease in activity of this enzyme in the arcA mutant compared to the wild-type strain. Finally, we observed that expression of an R. etli arcA-gusA fusion was strongly induced under anaerobic conditions.

Acetylene↗
PubMed2001

Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions.

Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.

Bacterial Typing Techniques↗
PubMed1999

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter.

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na+ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na+ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na+/H+ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs.

ATP-Binding Cassette Transporters↗
PubMed2000

Identification of non-tuberculous mycobacteria: 16S rRNA gene sequence analysis vs. conventional methods.

In a retrospective study, 45 clinical isolates of non-tuberculous mycobacteria were identified to the species level by biochemical profile, gas liquid chromatography and partial sequence analysis of 16S rRNA, and were found to represent 13 different species. The results of sequence analysis showed 100% identity with conventional tests for 34 isolates (76%) and could identify species such as M. bohemicum which are difficult to characterise with conventional methods. Most of the discrepant results for the remaining 11 isolates resulted in species of the same group of mycobacteria. Based on these findings. we concluded that direct sequence analysis of amplified 16S rRNA gene is a promising rapid and accurate method for species determination of non-tuberculous mycobacteria.

Bacterial Typing Techniques↗
PubMed1990

Molecular cloning and sequence analysis of the Sta58 major antigen gene of Rickettsia tsutsugamushi: sequence homology and antigenic comparison of Sta58 to the 60-kilodalton family of stress proteins.

The scrub typhus 58-kilodalton (kDa) antigen (Sta58) of Rickettsia tsutsugamushi is a major protein antigen often recognized by humans infected with scrub typhus rickettsiae. A 2.9-kilobase HindIII fragment containing a complete sta58 gene was cloned in Escherichia coli and found to express the entire Sta58 antigen and a smaller protein with an apparent molecular mass of 11 kDa (Stp11). DNA sequence analysis of the 2.9-kilobase HindIII fragment revealed two adjacent open reading frames encoding proteins of 11 (Stp11) and 60 (Sta58) kDa. Comparisons of deduced amino acid sequences disclosed a high degree of homology between the R. tsutsugamushi proteins Stp11 and Sta58 and the E. coli proteins GroES and GroEL, respectively, and the family of primordial heat shock proteins designated Hsp10 Hsp60. Although the sequence homology between the Sta58 antigen and the Hsp60 protein family is striking, the Sta58 protein appeared to be antigenically distinct among a sample of other bacterial Hsp60 homologs, including the typhus group of rickettsiae. The antigenic uniqueness of the Sta58 antigen indicates that this protein may be a potentially protective antigen and a useful diagnostic reagent for scrub typhus fever.

Amino Acid Sequence↗
PubMed1979

Molecular cloning and sequence analysis of adult chicken betal globin cDNA.

The molecular cloning and nucleotide sequence analysis of adult chicken beta globin mRNA is reported. DNA sequences derived from in vitro transcrption of globin mRNA were purified and amplified as recombinant DNA using the plasmid pBR322. Sequence analysis of several clones coding for beta globin strongly suggests that transcription errors may be generated near the 5' end of transcripts in vitro by reverse transcription. The complete sequence of the longest beta globin insert containing 51 bases of the 5' untranslated region as well as the complete coding and 3' untranslated regions has been determined.

Amino Acid Sequence↗
PubMed1994

Comparative amino acid sequence analysis of Thermotoga maritima beta-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between beta-glucosidases of the BGA family and other families of beta-1,4-glycosyl hydrolases.

The primary structure of the bglA gene region encoding a beta-glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51,545 Da. The T. maritima beta-glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15-20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity beta-glucosidase and as a member of the beta-glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family beta-glucosidases, a beta-xylosidase, beta-1,4-glycanases of the enzyme family F (mostly xylanases), and other families of beta-1,4-glycosyl hydrolases. This result indicates that BGA beta-glucosidases may comprise one enzyme family within a large 'enzyme order' of retaining beta-glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.

Amino Acid Sequence↗
PubMed2025

Genetic predisposition to systemic inflammatory proteins is causally associated with inflammatory bowel disease: Insights from multi-omics association study and single-cell RNA-sequencing analysis.

Systemic inflammatory proteins have been reported to be related to inflammatory bowel disease (IBD) in previous observational research. However, their causal links remain obscure. Herein, we performed a Mendelian randomization (MR) analysis to analyze the causality between systemic inflammatory proteins and IBD. Genetic variants related to systemic inflammatory proteins were extracted from a meta-analysis of genome-wide association study (GWAS) data of 8293 European participants. Summary statistics of IBD diverse subtypes were obtained from the international IBD genetic consortium (IIBDGC). We conducted multi-omics method and MR study to detect the causal links through integrating GWAS and protein quantity trait loci (pQTL) data. Inverse variance weighted (IVW) approach was utilized as the dominated analysis method. Moreover, complementary approaches such as MR-Egger intercept test, Cochran Q test and leave-one-out analysis were utilized to validate pleiotropy and heterogeneity. Finally, single-cell RNA-sequencing analysis was performed to detect the expression of significant genes. For IBD, IVW estimates suggested that genetically predicted IL-10 and IL-13 were suggestively associated with an elevated risk of IBD (IL-10: OR: 1.12, 95% CI: 1.00-1.24, P = .04; IL-13: OR: 1.09, 95% CI: 1.01-1.18, P = .023), while CXCL10 was suggestively linked to a lower risk of IBD (CXCL10: OR: 0.90, 95% CI: 0.82-0.99, P = .037). For Crohn disease (CD), the IVW approach provided evidence to sustain that genetically determined IL-13 and CCL3 had a suggestive association with a higher risk of CD (IL-13: OR: 1.13, 95% CI: 1.02-1.26, P = .023; CCL3: OR: 1.22, 95% CI: 1.03-1.45, P = .018). Sensitivity analysis did not explore any heterogeneity and pleiotropy. Our findings supported the causal relationships between 4 specific inflammatory proteins (IL-10, IL-13, CXCL10, and CCL3) and the risk of IBD and CD, thereby providing promising biomarkers of various subtypes stratification and new insights for the prevention and therapeutic target of IBD.

Humans↗
PubMed1992

Sequence analysis of polymerase chain reaction amplified t(14;18) chromosomal breakpoints in formalin fixed, paraffin wax embedded follicular lymphoma.

AIMS: To determine whether junctional sequences of rearranged chromosomes can be amplified by use of the polymerase chain reaction (PCR) and whether direct sequence analysis of the PCR products is possible, using DNA from formalin fixed, paraffin wax embedded biopsy specimens. METHODS: DNA was extracted from paraffin wax embedded, formalin fixed lymphoma specimens, and junctional sequences of rearranged chromosomes were amplified by the PCR. The products were used as templates for asymmetrical PCR. Subsequently, direct sequence analysis was performed using the chain termination method. RESULTS: Formalin fixed, paraffin wax embedded biopsy specimens and PCR amplification could be used to determine the nucleotide sequences of junctional regions of rearranged chromosomes t(14;18) from patients with follicular lymphoma. CONCLUSION: The identification of junctional sequences of the translocation in follicular lymphoma provides a molecular "fingerprint" of t(14;18) of the lymphoma of an individual patient and can be used for the detection of clone specific DNA in any biopsy tissue obtained from the patient. The strategy used for rapid sequence analysis of PCR amplified DNA sequences will be useful in many areas of molecular pathology.

Base Sequence↗