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Uma abordagem para detecção e remoção de artefatos em sequencias ESTs; An approach to detect and remove artifacts in EST sequences

Christian Baudet
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 01/12/2006 Português
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O sequenciamento de ESTs (Expressed Sequence Tag) [2] e uma tecnica que trabalha com bibliotecas de cDNAs tendo como objetivo a obtençao de uma boa aproximaçao para o ?ndice genico, que e a listagem de genes existentes no genoma do organismo estudado. Antes da serem analisadas, as sequencias obtidas do sequenciamento dos ESTs devem ser processadas para eliminaçao de artefatos. Artefatos sao trechos que nao pertencem ao organismo ou que possuem baixa qualidade ou baixa complexidade. Trechos de vetores, adaptadores e caudas poli-A podem ser citados como exemplos de artefatos. A eliminaçao dos artefatos deve ser feita para que a an´alise das sequencias produzidas no projeto nao seja prejudicada por estes ?ru?dos?. Por exemplo, artefatos presentes em sequencias freq¨uentemente produzem erros em processos de clusterizaçao, pois eles podem determinar se sequencias serao unidas em um mesmo cluster ou separadas em clusters diferentes. Observando a importancia da realizaçao de um bom processo de limpeza das sequencias, o trabalho desenvolvido nesta dissertaçao teve como principal objetivo a obtençao de um conjunto eficiente de procedimentos de detecçao e remoçao de artefatos. Este conjunto foi produzido a partir de uma nova estrategia de deteçao de artefatos. Normalmente...

Expressed sequence tag analysis of khat (Catha edulis) provides a putative molecular biochemical basis for the biosynthesis of phenylpropylamino alkaloids

Hagel,Jillian M.; Krizevski,Raz; Kilpatrick,Korey; Sitrit,Yaron; Marsolais,Frédéric; Lewinsohn,Efraim; Facchini,Peter J.
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2011 Português
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Khat (Catha edulis Forsk.) is a flowering perennial shrub cultivated for its neurostimulant properties resulting mainly from the occurrence of (S)-cathinone in young leaves. The biosynthesis of (S)-cathinone and the related phenylpropylamino alkaloids (1S,2S)-cathine and (1R,2S)-norephedrine is not well characterized in plants. We prepared a cDNA library from young khat leaves and sequenced 4,896 random clones, generating an expressed sequence tag (EST) library of 3,293 unigenes. Putative functions were assigned to > 98% of the ESTs, providing a key resource for gene discovery. Candidates potentially involved at various stages of phenylpropylamino alkaloid biosynthesis from L-phenylalanine to (1S,2S)-cathine were identified.

Discovery of three genes specifically expressed in human prostate by expressed sequence tag database analysis

Vasmatzis, George; Essand, Magnus; Brinkmann, Ulrich; Lee, Byungkook; Pastan, Ira
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 06/01/1998 Português
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A procedure is described to discover genes that are specifically expressed in human prostate. The procedure involves searching the expressed sequence tag (EST) database for genes that have many related EST sequences from human prostate cDNA libraries but none or few from nonprostate human libraries. The selected candidate EST clones were tested by RNA dot blots to examine tissue specificity and by Northern blots to examine the transcript size of the corresponding mRNA. The computer analysis identified 15 promising genes that were previously unidentified. When seven of these were examined in an RNA hybridization experiment, three were found to be prostate specific. The genes identified could be useful in the targeted therapy of prostate cancer. The procedure can easily be applied to discover genes specifically expressed in other organs or tumors.

Linking yeast genetics to mammalian genomes: identification and mapping of the human homolog of CDC27 via the expressed sequence tag (EST) data base.

Tugendreich, S; Boguski, M S; Seldin, M S; Hieter, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/11/1993 Português
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We describe a strategy for quickly identifying and positionally mapping human homologs of yeast genes to cross-reference the biological and genetic information known about yeast genes to mammalian chromosomal maps. Optimized computer search methods have been developed to scan the rapidly expanding expressed sequence tag (EST) data base to find human open reading frames related to yeast protein sequence queries. These methods take advantage of the newly developed BLOSUM scoring matrices and the query masking function SEG. The corresponding human cDNA is then used to obtain a high-resolution map position on human and mouse chromosomes, providing the links between yeast genetic analysis and mapped mammalian loci. By using these methods, a human homolog of Saccharomyces cerevisiae CDC27 has been identified and mapped to human chromosome 17 and mouse chromosome 11 between the Pkca and Erbb-2 genes. Human CDC27 encodes an 823-aa protein with global similarity to its fungal homologs CDC27, nuc2+, and BimA. Comprehensive cross-referencing of genes and mutant phenotypes described in humans, mice, and yeast should accelerate the study of normal eukaryotic biology and human disease states.

A Comprehensive Rice Transcript Map Containing 6591 Expressed Sequence Tag Sites

Wu, Jianzhong; Maehara, Tomoko; Shimokawa, Takanori; Yamamoto, Shinichi; Harada, Chizuko; Takazaki, Yuka; Ono, Nozomi; Mukai, Yoshiyuki; Koike, Kazuhiro; Yazaki, Jyunshi; Fujii, Fumiko; Shomura, Ayahiko; Ando, Tsuyu; Kono, Izumi; Waki, Kazunori; Yamamoto,
Fonte: American Society of Plant Biologists Publicador: American Society of Plant Biologists
Tipo: Artigo de Revista Científica
Publicado em /03/2002 Português
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To determine the chromosomal positions of expressed rice genes, we have performed an expressed sequence tag (EST) mapping project by polymerase chain reaction–based yeast artificial chromosome (YAC) screening. Specific primers designed from 6713 unique EST sequences derived from 19 cDNA libraries were screened on 4387 YAC clones and used for map construction in combination with genetic analysis. Here, we describe the establishment of a comprehensive YAC-based rice transcript map that contains 6591 EST sites and covers 80.8% of the rice genome. Chromosomes 1, 2, and 3 have relatively high EST densities, approximately twice those of chromosomes 11 and 12, and contain 41% of the total EST sites on the map. Most of the EST-dense regions are distributed on the distal regions of each chromosome arm. Genomic regions flanking the centromeres for most of the chromosomes have lower EST density. Recombination frequency in these regions is suppressed significantly. Our EST mapping also shows that 40% of the assigned ESTs occupy only ∼21% of the entire genome. The rice transcript map has been a valuable resource for genetic study, gene isolation, and genome sequencing at the Rice Genome Research Program and should become an important tool for comparative analysis of chromosome structure and evolution among the cereals.

Uneven distribution of expressed sequence tag loci on maize pachytene chromosomes

Anderson, Lorinda K.; Lai, Ann; Stack, Stephen M.; Rizzon, Carene; Gaut, Brandon S.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /01/2006 Português
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Examining the relationships among DNA sequence, meiotic recombination, and chromosome structure at a genome-wide scale has been difficult because only a few markers connect genetic linkage maps with physical maps. Here, we have positioned 1195 genetically mapped expressed sequence tag (EST) markers onto the 10 pachytene chromosomes of maize by using a newly developed resource, the RN-cM map. The RN-cM map charts the distribution of crossing over in the form of recombination nodules (RNs) along synaptonemal complexes (SCs, pachytene chromosomes) and allows genetic cM distances to be converted into physical micrometer distances on chromosomes. When this conversion is made, most of the EST markers used in the study are located distally on the chromosomes in euchromatin. ESTs are significantly clustered on chromosomes, even when only euchromatic chromosomal segments are considered. Gene density and recombination rate (as measured by EST and RN frequencies, respectively) are strongly correlated. However, crossover frequencies for telomeric intervals are much higher than was expected from their EST frequencies. For pachytene chromosomes, EST density is about fourfold higher in euchromatin compared with heterochromatin, while DNA density is 1.4 times higher in heterochromatin than in euchromatin. Based on DNA density values and the fraction of pachytene chromosome length that is euchromatic...

A Chromosome Bin Map of 16,000 Expressed Sequence Tag Loci and Distribution of Genes Among the Three Genomes of Polyploid Wheat

Qi, L. L.; Echalier, B.; Chao, S.; Lazo, G. R.; Butler, G. E.; Anderson, O. D.; Akhunov, E. D.; Dvořák, J.; Linkiewicz, A. M.; Ratnasiri, A.; Dubcovsky, J.; Bermudez-Kandianis, C. E.; Greene, R. A.; Kantety, R.; La Rota, C. M.; Munkvold, J. D.; Sorrells
Fonte: Genetics Society of America Publicador: Genetics Society of America
Tipo: Artigo de Revista Científica
Publicado em /10/2004 Português
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Because of the huge size of the common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) genome of 17,300 Mb, sequencing and mapping of the expressed portion is a logical first step for gene discovery. Here we report mapping of 7104 expressed sequence tag (EST) unigenes by Southern hybridization into a chromosome bin map using a set of wheat aneuploids and deletion stocks. Each EST detected a mean of 4.8 restriction fragments and 2.8 loci. More loci were mapped in the B genome (5774) than in the A (5173) or D (5146) genomes. The EST density was significantly higher for the D genome than for the A or B. In general, EST density increased relative to the physical distance from the centromere. The majority of EST-dense regions are in the distal parts of chromosomes. Most of the agronomically important genes are located in EST-dense regions. The chromosome bin map of ESTs is a unique resource for SNP analysis, comparative mapping, structural and functional analysis, and polyploid evolution, as well as providing a framework for constructing a sequence-ready, BAC-contig map of the wheat genome.

ESTpass: a web-based server for processing and annotating expressed sequence tag (EST) sequences

Lee, Byungwook; Hong, Taehui; Byun, Sang Jin; Woo, Taeha; Choi, Yoon Jeong
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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We present a web-based server, called ESTpass, for processing and annotating sequence data from expressed sequence tag (EST) projects. ESTpass accepts a FASTA-formatted EST file and its quality file as inputs, and it then executes a back-end EST analysis pipeline consisting of three consecutive steps. The first is cleansing the input EST sequences. The second is clustering and assembling the cleansed EST sequences using d2_cluster and CAP3 programs and producing putative transcripts. From the CAP3 output, ESTpass detects chimeric EST sequences which are confirmed through comparison with the nr database. The last step is annotating the putative transcript sequences using RefSeq, InterPro, GO and KEGG gene databases according to user-specified options. The major advantages of ESTpass are the integration of cleansing and annotating processes, rigorous chimeric EST detection, exhaustive annotation, and email reporting to inform the user about the progress and to send the analysis results. The ESTpass results include three reports (summary, cleansing and annotation) and download function, as well as graphic statistics. They can be retrieved and downloaded using a standard web browser. The server is available at http://estpass.kobic.re.kr/.

ESTExplorer: an expressed sequence tag (EST) assembly and annotation platform

Nagaraj, Shivashankar H.; Deshpande, Nandan; Gasser, Robin B.; Ranganathan, Shoba
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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The analysis of expressed sequence tag (EST) datasets offers a rapid and cost-effective approach to elucidate the transcriptome of an organism, but requiring several computational methods for assembly and annotation. ESTExplorer is a comprehensive workflow system for EST data management and analysis. The pipeline uses a ‘distributed control approach’ in which the most appropriate bioinformatics tools are implemented over different dedicated processors. Species-specific repeat masking and conceptual translation are in-built. ESTExplorer accepts a set of ESTs in FASTA format which can be analysed using programs selected by the user. After pre-processing and assembly, the dataset is annotated at the nucleotide and protein levels, following conceptual translation. Users may optionally provide ESTExplorer with assembled contigs for annotation purposes. Functionally annotated contigs/ESTs can be analysed individually. The overall outputs are gene ontologies, protein functional identifications in terms of mapping to protein domains and metabolic pathways. ESTExplorer has been applied successfully to annotate large EST datasets from parasitic nematodes and to identify novel genes as potential targets for parasite intervention. ESTExplorer runs on a Linux cluster and is freely available for the academic community at http://estexplorer.biolinfo.org.

Chasing Migration Genes: A Brain Expressed Sequence Tag Resource for Summer and Migratory Monarch Butterflies (Danaus plexippus)

Zhu, Haisun; Casselman, Amy; Reppert, Steven M.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 09/01/2008 Português
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North American monarch butterflies (Danaus plexippus) undergo a spectacular fall migration. In contrast to summer butterflies, migrants are juvenile hormone (JH) deficient, which leads to reproductive diapause and increased longevity. Migrants also utilize time-compensated sun compass orientation to help them navigate to their overwintering grounds. Here, we describe a brain expressed sequence tag (EST) resource to identify genes involved in migratory behaviors. A brain EST library was constructed from summer and migrating butterflies. Of 9,484 unique sequences, 6068 had positive hits with the non-redundant protein database; the EST database likely represents ∼52% of the gene-encoding potential of the monarch genome. The brain transcriptome was cataloged using Gene Ontology and compared to Drosophila. Monarch genes were well represented, including those implicated in behavior. Three genes involved in increased JH activity (allatotropin, juvenile hormone acid methyltransfersase, and takeout) were upregulated in summer butterflies, compared to migrants. The locomotion-relevant turtle gene was marginally upregulated in migrants, while the foraging and single-minded genes were not differentially regulated. Many of the genes important for the monarch circadian clock mechanism (involved in sun compass orientation) were in the EST resource...

Comparative Analysis of Expressed Sequence Tag (EST) Libraries in the Seagrass Zostera marina Subjected to Temperature Stress

Reusch, Thorsten B. H.; Veron, Amelie S.; Preuss, Christoph; Weiner, January; Wissler, Lothar; Beck, Alfred; Klages, Sven; Kube, Michael; Reinhardt, Richard; Bornberg-Bauer, Erich
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Português
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Global warming is associated with increasing stress and mortality on temperate seagrass beds, in particular during periods of high sea surface temperatures during summer months, adding to existing anthropogenic impacts, such as eutrophication and habitat destruction. We compare several expressed sequence tag (EST) in the ecologically important seagrass Zostera marina (eelgrass) to elucidate the molecular genetic basis of adaptation to environmental extremes. We compared the tentative unigene (TUG) frequencies of libraries derived from leaf and meristematic tissue from a control situation with two experimentally imposed temperature stress conditions and found that TUG composition is markedly different among these conditions (all P < 0.0001). Under heat stress, we find that 63 TUGs are differentially expressed (d.e.) at 25°C compared with lower, no-stress condition temperatures (4°C and 17°C). Approximately one-third of d.e. eelgrass genes were characteristic for the stress response of the terrestrial plant model Arabidopsis thaliana. The changes in gene expression suggest complex photosynthetic adjustments among light-harvesting complexes, reaction center subunits of photosystem I and II, and components of the dark reaction. Heat shock encoding proteins and reactive oxygen scavengers also were identified...

Quantitative Gene Expression Profiles in Real Time From Expressed Sequence Tag Databases

FUNARI, VINCENT A.; VOEVODSKI, KONSTANTIN; LEYFER, DIMITRY; YERKES, LAURA; CRAMER, DONALD; TOLAN, DEAN R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
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An accumulation of expressed sequence tag (EST) data in the public domain and the availability of bioinformatic programs have made EST gene expression profiling a common practice. However, the utility and validity of using EST databases (e.g., dbEST) has been criticized, particularly for quantitative assessment of gene expression. Problems with EST sequencing errors, library construction, EST annotation, and multiple paralogs make generation of specific and sensitive qualitative and quantitative expression profiles a concern. In addition, most EST-derived expression data exists in previously assembled databases. The Virtual Northern Blot (VNB) (http://tlab.bu.edu/vnb.html) allows generation, evaluation, and optimization of expression profiles in real time, which is especially important for alternatively spliced, novel, or poorly characterized genes. Representative gene families with variable nucleotide sequence identity, tissue specificity, and levels of expression (bcl-xl, aldoA, and cyp2d9) are used to assess the quality of VNB’s output. The profiles generated by VNB are more sensitive and specific than those constructed with ESTs listed in preindexed databases at UCSI and NCBI. Moreover, quantitative expression profiles produced by VNB are comparable to quantization obtained from Northern blots and qPCR. The VNB pipeline generates real-time gene expression profiles for single-gene queries that are both qualitatively and quantitatively reliable.

Immune gene discovery by expressed sequence tag (EST) analysis of hemocytes in the ridgetail white prawn Exopalaemon carinicauda

Duan, Yafei; Liu, Ping; Li, Jitao; Li, Jian; Chen, Ping
Fonte: Academic Press Publicador: Academic Press
Tipo: Artigo de Revista Científica
Publicado em /01/2013 Português
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The ridgetail white prawn Exopalaemon carinicauda is one of the most important commercial species in eastern China. However, little information of immune genes in E. carinicauda has been reported. To identify distinctive genes associated with immunity, an expressed sequence tag (EST) library was constructed from hemocytes of E. carinicauda. A total of 3411 clones were sequenced, yielding 2853 ESTs and the average sequence length is 436 bp. The cluster and assembly analysis yielded 1053 unique sequences including 329 contigs and 724 singletons. Blast analysis identified 593 (56.3%) of the unique sequences as orthologs of genes from other organisms (E-value < 1e-5). Based on the COG and Gene Ontology (GO), 593 unique sequences were classified. Through comparison with previous studies, 153 genes assembled from 367 ESTs have been identified as possibly involved in defense or immune functions. These genes are categorized into seven categories according to their putative functions in shrimp immune system: antimicrobial peptides, prophenoloxidase activating system, antioxidant defense systems, chaperone proteins, clottable proteins, pattern recognition receptors and other immune-related genes. According to EST abundance, the major immune-related genes were thioredoxin (141...

Development and Validation of Single Nucleotide Polymorphism (SNP) Markers from an Expressed Sequence Tag (EST) Database in Olive Flounder (Paralichthys olivaceus)

Kim, Jung Eun; Lee, Young Mee; Lee, Jeong-Ho; Noh, Jae Koo; Kim, Hyun Chul; Park, Choul-Ji; Park, Jong-Won; Kim, Kyung-Kil
Fonte: Korean Society of Developmental Biology Publicador: Korean Society of Developmental Biology
Tipo: Artigo de Revista Científica
Publicado em /12/2014 Português
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To successful molecular breeding, identification and functional characterization of breeding related genes and development of molecular breeding techniques using DNA markers are essential. Although the development of a useful marker is difficult in the aspect of time, cost and effort, many markers are being developed to be used in molecular breeding and developed markers have been used in many fields. Single nucleotide polymorphisms (SNPs) markers were widely used for genomic research and breeding, but has hardly been validated for screening functional genes in olive flounder. We identified single nucleotide polymorphisms (SNPs) from expressed sequence tag (EST) database in olive flounder; out of a total 4,327 ESTs, 693 contigs and 514 SNPs were detected in total EST, and these substitutions include 297 transitions and 217 transversions. As a result, 144 SNP markers were developed on the basis of 514 SNP to selection of useful gene region, and then applied to each of eight wild and culture olive flounder (total 16 samples). In our experimental result, only 32 markers had detected polymorphism in sample, also identified 21 transitions and 11 transversions, whereas indel was not detected in polymorphic SNPs. Heterozygosity of wild and cultured olive flounder using the 32 SNP markers is 0.34 and 0.29...

Gene discovery and transcript analyses in the corn smut pathogen Ustilago maydis: expressed sequence tag and genome sequence comparison

Ho, Eric C. H.; Cahill, Matt J.; Saville, Barry J.
Fonte: Universidade de Cambridge Publicador: Universidade de Cambridge
Tipo: Article; Published Version
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Abstract Background Ustilago maydis is the basidiomycete fungus responsible for common smut of corn and is a model organism for the study of fungal phytopathogenesis. To aid in the annotation of the genome sequence of this organism, several expressed sequence tag (EST) libraries were generated from a variety of U. maydis cell types. In addition to utility in the context of gene identification and structure annotation, the ESTs were analyzed to identify differentially abundant transcripts and to detect evidence of alternative splicing and anti-sense transcription. Results Four cDNA libraries were constructed using RNA isolated from U. maydis diploid teliospores (U. maydis strains 518 ? 521) and haploid cells of strain 521 grown under nutrient rich, carbon starved, and nitrogen starved conditions. Using the genome sequence as a scaffold, the 15,901 ESTs were assembled into 6,101 contiguous expressed sequences (contigs); among these, 5,482 corresponded to predicted genes in the MUMDB (MIPS Ustilago maydis database), while 619 aligned to regions of the genome not yet designated as genes in MUMDB. A comparison of EST abundance identified numerous genes that may be regulated in a cell type or starvation-specific manner. The transcriptional response to nitrogen starvation was assessed using RT-qPCR. The results of this suggest that there may be cross-talk between the nitrogen and carbon signalling pathways in U. maydis. Bioinformatic analysis identified numerous examples of alternative splicing and anti-sense transcription. While intron retention was the predominant form of alternative splicing in U. maydis...

Polymorphism of human Alpha class glutathione transferases

Tetlow, Natasha; Liu, Dan; Board, Philip
Fonte: Lippincott Williams & Wilkins Publicador: Lippincott Williams & Wilkins
Tipo: Artigo de Revista Científica
Português
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The recognition of the importance and utility of single nucleotide polymorphisms has generated an interest in the development of new strategies for their identification. Analysis of the Expressed Sequence Tag (EST) database can provide a rapid and efficient means of identifying polymorphisms. Screening of the Alpha class glutathione transferases (GSTs) in the EST database identified 10 putative polymorphisms in the coding region of the GSTA1 and GSTA2 genes, six of which were subsequently verified by sequence analysis. Polymerase chain reaction/restriction fragment length polymorphism analysis revealed the existence of three variants, a silent base substitution, K125K (G365A) in GSTA1, and T112S and E210A in GSTA2, in European Australian, African and Chinese populations. The variant isoforms of GSTA2 were expressed in Escherichia coli, purified, and enzymatically characterized. Modelling of the two GSTA2 polymorphisms into a three-dimensional structure of GSTA2, and characterization of their enzymatic properties, has shown that the structure and function of the wild-type GSTA2-2 isoenzyme is not significantly altered by these polymorphisms. This report demonstrates that analysis of the EST database provides a rapid and efficient means of identifying variant proteins.

Establishment of a root proteome reference map for the model legume medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting

Mathesius, Ulrike; Keijzers, G; Natera, Siria Helen Anna; Weinman, Jeremy; Djordjevic, Michael; Rolfe, Barry
Fonte: Wiley-VCH Verlag GMBH Publicador: Wiley-VCH Verlag GMBH
Tipo: Artigo de Revista Científica
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We have established a proteome reference map for Medicago truncatula root proteins using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation-related genes and proteins. Over 2500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4-7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105 000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.

Discovery of a functional polymorphism in human glutathione transferase Zeta by expressed sequence Tag database analysis

Blackburn, Anneke; Tzeng, Huey-Fen; Anders, Michael; Board, Philip
Fonte: Lippincott Williams & Wilkins Publicador: Lippincott Williams & Wilkins
Tipo: Artigo de Revista Científica
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Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest. The human zeta class glutathione transferase GSTZ1 has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic. This report identifies three GSTZ1 alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region, GSTZ1*A - A94A124; GSTZ1*B - A94G124; GSTZ1*C - G94G124. Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 for Z1*A, Z1*B and Z1*C, respectively. These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32 to Glu and Arg42 to Gly substitutions, respectively. The variant proteins were expressed in Escherichia coli as 6X His-tagged proteins and purified by Ni-agarose column chromatography. Examination of the activities of recombinant proteins revealed that GSTZ1a-1a displayed differences in activity towards several substrates compared with GSTZ1b-1b and GSTZ1c-1c, including 3.6-fold higher activity towards dichloroacetate. This report demonstrates the discovery of a functional polymorphism by analysis of the EST database. (C) 2000 Lippincott Williams and Wilkins.

Database Analysis and Gene Discovery in Parmacogenetics

Board, Philip; Tetlow, Natasha; Blackburn, Anneke; Chelvanayagam, Gareth
Fonte: Walter de Gruyter Publicador: Walter de Gruyter
Tipo: Artigo de Revista Científica
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The global genome research effort has resulted in the creation of extensive DNA and protein sequence data-bases that are a valuable resource for the identification of new genes and polymorphic variants of enzymes of pharmacogenetic interest. Previously undescribed members of gene families with novel functions and substrate specificities can be identified by database searching and sequence alignment strategies. Since the expressed sequence tag (EST) database contains sequences from many individuals, it can be searched for evidence of polymorphisms that can significantly influence enzyme function. The different approaches to these forms of analysis are reviewed and illustrated with examples from the glutathione transferase gene family.

IDENTIFICATION OF NOVEL GLUTATHIONE TRANSFERASES AND POLYMORPHIC VARIANTS BY EXPRESSED SEQUENCE TAG DATABASE ANALYSIS

Board, Philip; Chelvanayagam, Gareth; Jermiin, Lars; Tetlow, Natasha; Tzeng, Huey-Fen; Anders, Michael; Blackburn, Anneke
Fonte: American Society for Pharmacology and Experimental Therapeutics Publicador: American Society for Pharmacology and Experimental Therapeutics
Tipo: Artigo de Revista Científica
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The human expressed sequence tag (EST) database can be searched by different sequence alignment strategies to identify new members of gene families and allelic variants. To illustrate the value of database analysis for gene discovery, we have focused on the glutathione S-transferase (GST) super family, an approach that has led to the identification of the Zeta class. The Zeta class GSTs catalyze the glutathione-dependent biotransformation of α-haloacids and the isomerization of maleylacetoacetic acid to fumarylacetoacetic acid, an essential step in the catabolism of tyrosine. Allelic variants of the GST Z1 and GST A2 genes have also been identified by EST database analysis. One GST Z1 variant (GST Z1A) has significantly higher activity with dichloroacetic acid as a substrate than other GST Z1 isoforms. This variant may be important in the clinical treatment of lactic acidosis where dichloroacetic acid is prescribed. Our experience with the application of EST database searching methods suggests that it may be productively applied to other gene families of pharmacogenetic interest.