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PubMed2006

Cloning and sequence analysis of maize FAD2 gene.

Delta-12 desaturases are involved in the conversion of oleic acid to linoleic acid in plant. Based on the conserved oligo amino acid residues of the published delta-12 desaturase genes from other higher plant species, a cDNA fragment was amplified by RT-PCR (reverse transcriptase-polymerase chain reaction) from the total RNA of immature maize embryos. According to bioinformation analysis of the cDNA sequence, a specific fragment of FAD2 gene was isolated by RT-PCR from immature maize embryos, and DNA of the same length was amplified from maize genome. Results of sequence analysis indicate that they are all 1 164 bp long, and have just an open reading frame (ORF) coding for 387 amino acids, and there is no intron in the FAD2 ORF (GenBank accession DQ496227). The deduced amino acid sequence of the cloned FAD2 showed high identity to those of other plant delta-12 fatty acid desaturases. It contains three histidine motifs and two long stretches of hydrophobic residues, indicative of an integral membrane protein spanning membrane four times. Analysis by semi-quantitive RT-PCR showed that FAD2 was strongly expressed in maize immature embryos than in leaves, stems and roots.

Amino Acid Sequence↗

PubMed2002

[Cloning of Ig Fab genes of patients with chronic B lymphocytic leukemia and sequences analysis of their variable regions].

OBJECTIVE: Sequences analysis of Ig variable regions from the peripheral blood mononuclear cells (PBMC) of patients with chronic B lymphocytic leukemia. METHODS: Total RNA was isolated from PBMC of patients with chronic B lymphocytic leukemia, oligo-dT-primed cDNA was synthesized from RNA. The cDNA was amplified by Taq DNA polymerase with a set of specific 5' primers corresponding to Ig FR1 and 3' primers corresponding to CH1 (C micro /C) or CL (Ckappa/Clambda), the PCR products of variable regions of Ig heavy (IgH) and light (IgL) chains were sequenced by ABI PRISM Dye terminator cycle sequencing ready reaction kit and ABI PRISM 310 Genetic Analyzer. The gene homology of variable regions of IgH and IgL chains was compared by using DNA tools 5.1 system and "the international immunogenetics database". RESULTS: Four light chains and 3 heavy chains were amplified from 4 and 3 patients respectively. Homology analysis of the sequences of 4 light chains and 3 heavy chains were performed by DNA tools system. The sequences of light chains are high homologous. And the sequences of heavy chains are quite different. The homologous analysis of the sequences of variable region by using "the international immunogenetics database" showed that the sequences were higher homologous to idiotype gene of some B lymphocytic leukemia. Four VL genes belong to human Ig Vkappa subgroup I, 2 of 3 VH genes belong to VH3 family and 1 belongs to VH5 family. CONCLUSION: Ig genes have idiotype and same disease may have same idiotype.

Base Sequence↗

PubMed1998

Line probe assay for detection of human immunodeficiency virus type 1 mutations conferring resistance to nucleoside inhibitors of reverse transcriptase: comparison with sequence analysis.

We compared the line probe assay (LiPA) to sequence analysis for the detection of mutations conferring resistance to nucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Plasma samples from 40 patients who had received zidovudine, dideoxyinosine, and dideoxycytosine, alone or in combination, and who were enrolled in the ALTIS 2 clinical trial (lamivudine [3TC] plus stavudine) were tested at enrollment and at week 24. RT PCR products from plasma were used for LiPA, and DNA was used for sequence analysis. LiPA gave uninterpretable results for 8.5% of the analyzed codons corresponding to 63 samples, mainly for codons 41, 69, and 70. Several minor discrepancies between the two methods occurred, mainly due to the ability of LiPA to detect mixed populations while sequence analyses detect a single homogeneous population. LiPA is suitable for detecting mixed populations and easy to implement in clinical laboratories and might be useful for epidemiological surveys of primary HIV-1 resistance.

Anti-HIV Agents↗

PubMed2003

Regulatory sequence analysis tools.

The web resource Regulatory Sequence Analysis Tools (RSAT) (http://rsat.ulb.ac.be/rsat) offers a collection of software tools dedicated to the prediction of regulatory sites in non-coding DNA sequences. These tools include sequence retrieval, pattern discovery, pattern matching, genome-scale pattern matching, feature-map drawing, random sequence generation and other utilities. Alternative formats are supported for the representation of regulatory motifs (strings or position-specific scoring matrices) and several algorithms are proposed for pattern discovery. RSAT currently holds >100 fully sequenced genomes and these data are regularly updated from GenBank.

5' Flanking Region↗

PubMed1991

Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence-based DNA sequencing.

A new method of signal analysis for automated fluorescence-based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400-450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed served in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.

Base Sequence↗

PubMed2006

Leishmania major: genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer.

Protozoan parasites of Leishmania major are the causative agents of cutaneous leishmaniasis in different parts of Iran. We applied PCR-based methods to analyze L. major parasites isolated from patients with active lesions from different geographic areas in Iran in order to understand DNA polymorphisms within L. major species. Twenty-four isolates were identified as L. major by RFLP analysis of the ribosomal internal transcribed spacer 1 (ITS1) amplicons. These isolates were further studied by single-strand conformation polymorphism (SSCP) analysis and sequencing of ITS1 and ITS2. Data obtained from SSCP analysis of the ITS1 and ITS2 loci revealed three and four different patterns among all studied samples, respectively. Sequencing of ITS1 and ITS2 confirmed the results of SSCP analysis and showed the potential of the PCR-SSCP method for assessing genetic heterogeneity within L. major. Different patterns in ITS1 were due to substitution of one nucleotide, whereas in ITS2 the changes were defined by variation in the number of repeats in two polymorphic microsatellites. In total five genotypic groups LmA, LmB, LmC, LmD and LmE were identified among L. major isolates. The most frequent genotype, LmA, was detected in isolates collected from different endemic areas of cutaneous leishmaniasis in Iran. Genotypes LmC, LmD and LmE were found only in the new focus of CL in Damghan (Semnan province) and LmB was identified exclusively among isolates of Kashan focus (Isfahan province). The distribution of genetic polymorphisms suggests the existence of distinct endemic regions of L. major in Iran.

Animals↗

PubMed1999

Identity and geometry of a base triple in 16S rRNA determined by comparative sequence analysis and molecular modeling.

Comparative sequence analysis complements experimental methods for the determination of RNA three-dimensional structure. This approach is based on the concept that different sequences within the same gene family form similar higher-order structures. The large number of rRNA sequences with sufficient variation, along with improved covariation algorithms, are providing us with the opportunity to identify new base triples in 16S rRNA. The three-dimensional conformations for one of our strongest candidates involving U121 (C124:G237) and/or U121 (U125:A236) (Escherichia coli sequence and numbering) are analyzed here with different molecular modeling tools. Molecular modeling shows that U121 interacts with C124 in the U121 (C124:G237) base triple. This arrangement maintains isomorphic structures for the three most frequent sequence motifs (approximately 93% of known bacterial and archaeal sequences), is consistent with chemical reactivity of U121 in E. coli ribosomes, and is geometrically favorable. Further, the restricted set of observed canonical (GU, AU, GC) base-pair types at positions 124:237 and 125:236 is consistent with the fact that the canonical base-pair sets (for both base pairs) that are not observed in nature prevent the formation of the 121 (124:237) base triple. The analysis described here serves as a general scheme for the prediction of specific secondary and tertiary structure base pairing where there is a network of correlated base changes.

Base Pairing↗

PubMed1998

alpha-Amylase gene of thermophilic Streptomyces sp. TO1: nucleotide sequence, transcriptional and amino acid sequence analysis.

The nucleotide sequence of a 1860-bp region encoding a thermostable alpha-amylase of Streptomyces sp. TO1 was determined. Frame analysis revealed the presence of a 1359-bp long open reading frame (amy TO1) encoding a 453 amino acid protein with a deduced M(r) of 49 kDa. Northern blot analysis revealed that amy TO1 gene was expressed as approximately 1.5-kbp monocistronic transcript in both SL1326/pLM1 and Streptomyces sp. TO1 strains. Primer extension experiments indicated that the transcriptional start site lies 30 bp upstream of the ATG start codon, and allowed the identification of -35 (TTGCTG) and -10 (TACGCG) eubacterial-like promoter sequences. Amy TO1 exhibits strong amino acid identities with those from other Streptomyces species with a maximum of 78% with S. thermoviolaceus alpha-amylase. Nevertheless, subtle amino acid changes such as the substitution of four conserved residues found at similar positions in other Streptomyces alpha-amylases by proline residues, and the substitution of three conserved hydrophilic amino acids by hydrophobic ones in Amy TO1 might account for the thermostable properties of Amy TO1.

Amino Acid Sequence↗

PubMed1990

Sequence analysis of bovine lens aldose reductase.

The covalent structure of bovine lens aldose reductase (alditol-NADP+ oxidoreductase, EC 1.1.1.21) was determined by sequence analysis of peptides generated by specific and chemical cleavage of the homogeneous apoenzyme. Peptides, purified by reverse-phase high performance liquid chromatography were subjected to compositional analysis and sequencing by gas-phase automated Edman degradation. Aldose reductase was found to contain 315 amino acid residues. The enzyme is blocked at the amino terminus, and mass spectrometry was employed to identify the blocking acetyl group and to sequence the amino-terminal tryptic peptide. The aldose reductase was shown to contain no carbohydrate despite the fact that the enzyme contains the consensus sequence -Asn-Lys-Thr- for N-linked glycosylation. Comparative sequence analysis and application of algorithms for prediction of secondary structure and nucleotide binding domains are consistent with the view that aldose reductase is a double-domain protein with a beta-alpha-beta secondary structural organization. The NADPH binding site appears to be associated with the amino-terminal half of the enzyme. Modeling studies based on the tertiary structures of dihydrofolate and glutathione reductases indicate that the NADPH binding site begins at Lys-11 and continues with a beta-alpha-beta fold characteristic of nucleotide binding proteins.

Aldehyde Reductase↗

PubMed1996

Compression and genetic sequence analysis.

A novel approach to genetic sequence analysis is presented. This approach, based on compression of algorithms, has been launched simultaneously by Grumbach and Tahi, Milosavljevic and Rivals. To reduce the description of an object, a compression algorithm replaces some regularities in the description by special codes. Thus a compression algorithm can be applied to a sequence in order to study the presence of those regularities all over the sequence. This paper explains this ability, gives examples of compression algorithms already developed and mentions their applications. Finally, the theoretical foundations of the approach are presented in an overview of the algorithmic theory of information.

Algorithms↗

PubMed1997

Third complementarity-determining-region sequence analysis of lymphocytic interstitial pneumonia: most cases demonstrate a minor monoclonal population hidden among normal lymphocyte clones.

We analyzed the third complementarity-determining region (CDR3) of the immunoglobulin heavy chain (IgH) gene in five patients with lymphoid interstitial pneumonia (LIP) through a two-step polymerase chain reaction (PCR) and sequencing analysis. By sequencing analysis of the PCR products, morphologic LIP could be divided into two groups: a polyclonal type and a minor monoclonal type. Because of their high frequencies, minor monoclonal clones seemed to be neoplastic clones hidden in normally reactive lymphocyte clones. Consequently, only the polyclonal type might have represented true LIP. Sequencing of the PCR products from open-chest biopsy and transbronchial lung biopsy (TBLB) specimens obtained 8 yr later in one patient with minor monoclonal type LIP confirmed this possibility. In true LIP, six of 20 lymphocyte clones showed 67 to 86% homology with lymphocyte clones derived from fetal tissue. In three of these six clones, the D-region (N-D-N) lengths were very short, whereas four clones showed a high homology with autoreactive lymphocytes (rheumatoid factor, anti-DNA antibody, and G6-positive lymphocytes). Since rheumatoid factors, anti-DNA antibodies, and G6 are autoreactive antibodies, immature B cells stimulated by autoantigens might play some role in the pathogenesis of true LIP.

Adult↗

PubMed1998

Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: application of mtDNA sequence analysis to a forensic case.

The sequence of the two hypervariable segments of the mitochondrial DNA (mtDNA) control region was generated for 101 random Austrian Caucasians. A total of 86 different mtDNA sequences was observed, where 11 sequences were shared by more than 1 individual, 7 sequences were shared by 2 individuals and 4 sequences were shared by 3 individuals. One of the four most common mtDNA sequences in Austrians is also the most common sequence in both U.S. and British Caucasians, found in approximately 3.0% of Austrians, 4.0% of British, and 3.9% of U.S. Caucasians. Of the remaining three common Austrian sequences, one was not observed in either U.S. or British Caucasians. However, three British Caucasians exhibited a similar sequence type. Therefore, this particular cluster of sequence polymorphisms may represent a common "European" mtDNA sequence type. In general, Austrian Caucasians show little deviation from other Caucasian databases of European descent. Finally, mtDNA sequence analysis was applied to a forensic case, where hairs found at a crime scene matched the control hairs from the suspect.

Austria↗

PubMed1995

Isolation and sequence analysis of human cadherin-6 complementary DNA for the full coding sequence and its expression in human carcinoma cells.

The expression pattern of E- and P-cadherin in human carcinomas has been reported by many laboratories. However, little is known about the involvement of other cadherin types in human carcinomas. cDNA clones for a cadherin molecule were isolated from a cDNA library of human hepatocellular carcinoma cells which lacked E- and P-cadherin expression but exhibited cell aggregation activity mediated by an unknown cadherin, and they were subjected to sequence analysis. The overlapped clones covered 4315 nucleotides and were found to encode a typical cadherin molecule consisting of 790 amino acids. Since the deduced amino acid sequence was identical to a partially available human cadherin-6 sequence except for two amino acid residues, the clones were considered to be human cadherin-6 cDNAs encoding the entire open reading frame. The deduced amino acid sequence also showed extremely high homology with recently reported rat K-cadherin, 97% for the putative mature protein, suggesting that cadherin-6 is the human counterpart of rat K-cadherin. Expression of cadherin-6 in various human normal tissues and carcinoma cells was examined by Northern blot analysis using a specific probe corresponding to the signal and precursor sequence. Among normal tissues examined, brain, cerebellum, and kidney showed strong expression of cadherin-6, whereas lung, pancreas, and gastric mucosa showed weak expression. Transcripts of cadherin-6 were not detected in normal liver, whereas four of six hepatocellular carcinoma cell lines examined expressed cadherin-6 abundantly. As reported for rat K-cadherin, three renal carcinoma cell lines also expressed cadherin-6 strongly. The most interesting finding was obtained for small cell lung carcinoma lines. Among 15 of such cell lines examined, all of 11 cadherin-6-positive lines were classified into the classic type, whereas the negative cell lines were all of the variant type. The present results suggest that besides E- and P-cadherin, other cadherin molecules are expressed in human cancers and are responsible for additional biological properties of the carcinoma cells.

Base Sequence↗

PubMed2005

Isolation of culturable yeasts from market wines and evaluation of the 5.8S-ITS rDNA sequence analysis for identification purposes.

AIMS: To test the possibility that wines available in the marketplace may contain culturable yeasts and to evaluate the 5.8S-ITS rDNA sequence analysis as adequate means for the identification of isolates. METHODS AND RESULTS: As a case study, typical Greek wines were surveyed. Sequence analysis of the 5.8S-ITS rDNA was tested for its robustness in species or strain identification. Sixteen isolates could be assigned into the species Brettanomyces bruxellensis, Saccharomyces cerevisiae and Rhodotorula pinicola, whereas four isolates could not be safely identified. B. bruxellensis was the dominant species present in house wines, while non-Saccharomyces sp. were viable in aged wines of high alcohol content. CONCLUSIONS: Yeast population depends on postfermentation procedures or storage conditions. Although 5.8S-ITS rDNA sequence analysis is generally a rapid method to identify wine yeast isolates at the species level, or even below that, it may not be sufficient for some genera. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to show that commercial wines may possess diverse and potentially harmful yeast populations. The knowledge of yeasts able to reside in this niche environment is essential towards integrated quality assurance programmes. For selected species, the 5.8S-ITS rDNA sequence analysis is a rapid and accurate means.

DNA, Ribosomal Spacer↗

PubMed2004

[Molecular taxonomy and identification of pathogenic fungi based on DNA sequence analysis].

Although approximately 80,000 fungi are known, less than 1% are associated with human infection. However, their taxonomy has long been insufficient. During the last decade, DNA sequence analysis was introduced to the taxonomy of pathogenic fungi. Taxonomic advances in the field of medical mycology are helping to identify the causative agents of infectious diseases accurately, facilitating diagnosis and treatment. For example, Malassezia furfur was long considered the major microflora in atopic dermatitis, yet recent studies have indicated that this is not the case, as M. furfur is taxonomically heterogeneous and consists of five species. DNA sequence analysis resolved its taxonomic heterogeneity. Similar examples can be seen in "Candida albicans and C. dubliniensis" and "Trichosporon cutaneum and T. asahii". DNA sequence analysis also enables accurate identification of fungi. At present, almost all pathogenic fungi can be identified by determining the D1/D2 26S rDNA and ITS region of rRNA gene. This paper describes the practical taxonomy and identification of pathogenic fungi based on DNA sequence analysis.

Fungi↗

PubMed1996

Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum: nucleotide sequence analysis of the aceB, aceC, aceD and aceE genes.

Sequence analysis of a 5.323 kb chromosomal DNA fragment from Acetobacter xylinum involved in the biosynthesis of the exopolysaccharide acetan, revealed the presence of four ace genes designated aceB, aceC, aceD and aceE. Comparison of translated gene sequences to the databanks was used to assign putative gene functions. AceB displayed strong homology to a glucose-diphosphoprenyl beta, D-glucose transferase from Xanthomonas campestris, while AceC was homologous to a cellobiosyl-diphosphoprenyl alpha, D-mannose transferase from the same organism. Thus these genes encode enzymes catalyzing the second and third steps of the acetan biosynthetic pathway. AceD and AceE were homologous to ExoP and ExoT respectively from Rhizobium meliloti and are likely to be involved in acetan polymerization and export.

Amino Acid Sequence↗

PubMed1998

W2H: WWW interface to the GCG sequence analysis package.

MOTIVATION: The user-friendly, graphical X-windows interface (WPI) to the GCG sequence analysis package can often not be used due to the lack of an X-server on PC or Macintosh computers. Because Web browsers like Netscape are much more common on those platforms, we decided to develop W2H, a WWW interface to the GCG Sequence Analysis Software Package with nearly the same functionality as the X-windows interface WPI. RESULTS: The new WWW interface (W2H) to the GCG Sequence Analysis Software Package (Wisconsin Package) supports modern Web technologies, like client-pull method, or embedded scripting language, and provides a reasonable platform independence. The interface is quite comprehensive with advanced features like sequence selector, search set builder, enzyme chooser, access to sequence databases, uploading client files to the GCG server or displaying and manipulating graphical outputs in addition to GCG analysis programs. W2H also manages secure access to both GCG server and user data. For special environments, like workshops, conferences and company intranets, there is a special mode (Intranet mode) with less security constraints. The behaviour of W2H is mostly controlled by meta-data files describing the applications and giving a base for dynamic creation of HTML documents. This paper presents mainly the development approaches used, and architectural design aspects of W2H. AVAILABILITY: W2H is available by ftp://ftp.ebi.ac. uk/pub/software/unix/w2h or ftp://genome.dkfz-heidelberg.de/pub/w2h CONTACT: m.senger@ebi.ac.uk

Computational Biology↗

PubMed1991

VOSTORG: a package of microcomputer programs for sequence analysis and construction of phylogenetic trees.

VOSTORG is a new, versatile package of programs for the inference and presentation of phylogenetic trees, as well as an efficient tool for nucleotide (nt) and amino acid (aa) sequence analysis (sequence input, verification, alignment, construction of consensus, etc.). On appropriately equipped systems, these data can be displayed on a video monitor or printed as required. They are implemented on IBM PC/XT/AT/PS-2 or compatible computers and hardware graphic support is recommended. The package is designed to be easily handled by occasional computer users and yet it is powerful enough for experienced professionals.

Amino Acid Sequence↗