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A survey of canine expressed sequence tags and a display of their annotations through a flexible web-based interface.

We have initially sequenced approximately 8,000 canine expressed sequence tags (ESTs) from several complementary DNA (cDNA) libraries: testes, whole brain, and Madin-Darby canine kidney (MDCK) cells. Analysis of these sequences shows that they provide partial sequence information for about 5%-10% of the canine genes. An analysis pipeline has been created to cluster the ESTs and to map individual ESTs as well as clustered ESTs to both the human genome and the human proteome. Gene ontology (GO) terms have been assigned to the ESTs and clusters based on their top matches to the International Protein Index (IPI) set of human proteins. The data generated is stored in a MySQL relational database for analysis and display. A Web-based Perl script has been written to display the analyzed data to the scientific community.

Animals↗

PubMed2001

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis.

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

Animals↗

PubMed2006

Common plantain. A collection of expressed sequence tags from vascular tissue and a simple and efficient transformation method.

The vascular tissue of higher plants consists of specialized cells that differ from all other cells with respect to their shape and size, their organellar composition, their extracellular matrix, the type of their plasmodesmata, and their physiological functions. Intact and pure vascular tissue can be isolated easily and rapidly from leaf blades of common plantain (Plantago major), a plant that has been used repeatedly for molecular studies of phloem transport. Here, we present a transcriptome analysis based on 5,900 expressed sequence tags (ESTs) and 3,247 independent mRNAs from the Plantago vasculature. The vascular specificity of these ESTs was confirmed by the identification of well-known phloem or xylem marker genes. Moreover, reverse transcription-polymerase chain reaction, macroarray, and northern analyses revealed genes and metabolic pathways that had previously not been described to be vascular specific. Moreover, common plantain transformation was established and used to confirm the vascular specificity of a Plantago promoter-beta-glucuronidase construct in transgenic Plantago plants. Eventually, the applicability and usefulness of the obtained data were also demonstrated for other plant species. Reporter gene constructs generated with promoters from Arabidopsis (Arabidopsis thaliana) homologs of newly identified Plantago vascular ESTs revealed vascular specificity of these genes in Arabidopsis as well. The presented vascular ESTs and the newly developed transformation system represent an important tool for future studies of functional genomics in the common plantain vasculature.

Arabidopsis↗

PubMed1996

A novel profile of expressed sequence tags for zinc finger encoding genes from the poorly differentiated exocrine pancreatic cell line AR4IP.

Genes encoding for C2H2 zinc finger proteins are known to regulate normal cell proliferation and differentiation and have often been found to be mutated in different forms of cancer. We are interested in understanding the role of these genes as regulators of cell proliferation and differentiation in the exocrine pancreas. Therefore, we have generated expressed sequence tags (ESTs) encoding pancreas-enriched zinc finger peptides using the polymerase chain reaction and hybridization techniques [Adams, M.D. et al. (1991) Science, 252, 1651-1656]. Here we report the primary structure and expression pattern of 18 different zinc finger-encoding cDNAs (DZF-1-18) from the azaserine-derived tumoral cell line AR4IP which displays a poorly differentiated phenotype. Sequence analysis shows that all of these clones encode peptides which share the consensus DNA-binding motif with the Drosophila zinc finger transcription factor krüppel. High stringency Northern blot analysis shows that eight different zinc finger transcripts are expressed at high levels in normal adult rat pancreas and therefore constitute good candidates to play a role as transcription factors in exocrine pancreatic cells.

Animals↗

PubMed2004

Anchoring 9,371 maize expressed sequence tagged unigenes to the bacterial artificial chromosome contig map by two-dimensional overgo hybridization.

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 x 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize.

Chromosomes, Artificial, Bacterial↗

PubMed2004

A comprehensive nonredundant expressed sequence tag collection for the developing Rattus norvegicus heart.

Congenital heart defects affect approximately 1,000,000 people in the United States, with 40,000 new births contributing to that number every year. A large percentage of these defects can be attributed to septal defects. We assembled a nonredundant collection of over 12,000 expressed sequence tags (ESTs) from a total of 30,000 ESTs, with the ultimate goal of identifying spatially and/or temporally regulated genes during heart septation. These ESTs were compiled from nonnormalized, normalized, and serially subtracted cDNA libraries derived from two sets of tissue samples. The first includes microdissected rat hearts from embryonic (E) days E13, E15, and E16.5-E18.5 and adult heart. The second includes hearts from embryonic days E17, E19, and E21 and postnatal (P) days P1, P12, P74, and P200. Over 6,000 novel ESTs were identified in the libraries derived from these two sets of tissues, all of which have been contributed to the NCBI rat UniGene collection. It is anticipated that such EST and cDNA clone resources will prove invaluable to gene expression studies aimed at the understanding of the molecular mechanisms underlying heart septation defects.

Animals↗

PubMed2003

Expressed sequence tags from the Closterium peracerosum-strigosum-littorale complex, a unicellular charophycean alga, in the sexual reproduction process.

We obtained genetic information on sexual reproduction of the Closterium peracerosum-strigosum-littorale complex, a unicellular charophycean alga. Normalized cDNA libraries were constructed from cells in the sexual reproduction process, and a total of 1190 5'-end expressed sequence tags were established. Since 604 of these ESTs were classified into 174 non-redundant sequences, these 1190 ESTs include 760 unique sequences. Similarity search against a public non-redundant protein database indicated that 390 unique sequences had significant similarity to registered sequences. Among these 390 sequences, 3 were identical to and 4 were homologous to previously identified sex-pheromone genes. According to our study, 370 of 760 unique sequences are likely to be novel transcripts. These cDNA clones and EST sequence information may would be helpful for future functional analyses using DNA array technologies.

Amino Acid Sequence↗

PubMed2000

Developing expressed sequence tags (ESTs) from polymorphic transcript-derived fragments (TDFs) in cassava (Manihot esculenta Crantz).

We applied the cDNA-AFLP (amplified fragment length polymorphism) technique to mRNA from the parents of a cassava (Manihot esculenta) genetic mapping population, and obtained more than 500 transcript-derived fragments (TDFs) that were unique in either parent. A subset of 50 TDFs were cloned and sequenced. Sequence alignment of the expressed sequence tags (ESTs) revealed mostly genes of unknown function. Six of the TDFs were mapped on to the cassava genetic map. We also demonstrated by genetic mapping of the TDFs, as RFLP (restriction fragment length polymorphism) markers, that TDFs are more polymorphic than random cDNAs. Generation of ESTs as differentially expressed sequences, in time or between different varieties, is proposed as a way of developing ESTs around specific traits for the candidate locus approach to mapping complex traits.

Chromosome Mapping↗

PubMed2003

Identification of a novel sugar transporter homologue strongly expressed in maturing stem vascular tissues of sugarcane by expressed sequence tag and microarray analysis.

The ability of sugarcane to accumulate sucrose provides an experimental system for the study of gene expression determining carbohydrate partitioning and metabolism. A sequence survey of 7242 ESTs derived from the sucrose-accumulating, maturing stem revealed that transcripts for carbohydrate metabolism gene sequences (CMGs) are relatively rare in this tissue. However, within the CMG group, putative sugar transporter ESTs form one of the most abundant classes observed. A combination of EST analysis and microarray and northern hybridization revealed that one of the putative sugar transporter types, designated PST type 2a, was the most abundant and most strongly differentially expressed CMG in maturing stem tissue. PST type 2a is homologous to members of the major facilitator super-family of transporters, possessing 12 predicted transmembrane domains and a sugar transport conserved domain, interrupted by a large cytoplasmic loop. Its transcript was localized to phloem companion cells and associated parenchyma in maturing stem, suggesting a role in sugar translocation rather than storage. In addition, other categories of CMGs show evidence of coordinated expression, such as enzymes involved in sucrose synthesis and cleavage, and a majority of enzymes involved in glycolysis and the pentose phosphate pathway. This study demonstrates the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques for studies of the developmental regulation of metabolic enzymes and potential transporters in sugarcane.

Amino Acid Sequence↗

PubMed2001

Gene identification in the obligate fungal pathogen Blumeria graminis by expressed sequence tag analysis.

Powdery mildew of barley is caused by the obligate fungal pathogen Blumeria graminis f. sp. hordei. Haploid conidia of B. graminis, landing on the barley leaf, germinate to form first a primary germ tube and then an appressorial germ tube. The appressorial germ tube differentiates into a mature appressorium from which direct penetration of host epidermis occurs. Here we present data on 4908 expressed sequence tags obtained from B. graminis conidia. The combined sequences represent 2676 clones describing 1669 individual genes. Comparison with sequences from other pathogenic and nonpathogenic fungi defines hypotheses on the genes required for pathogenicity and growth on the host. The putative roles of some of the identified genes are discussed.

Ascomycota↗

PubMed1999

Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.

Abscisic Acid↗

PubMed2000

Analysis of expressed sequence tags of Porphyra yezoensis.

Single direct partial sequencing of anonymous cDNA clones was performed to obtain genetic information on red algae Porphyra yezoensis of which genetic information is not available. This expressed sequence tags (EST) analysis revealed 81 clones (42%) had significant homologies to known genes in GenBank. Of these clones, eight are related to known algal genes, whereas above 90% of the EST clones were newly identified in algae. Putative functional categories of these clones showed that the most abundant genes were involved in stress and defense mechanisms and that the next abundant genes were associated with protein synthetic pathways.

DNA, Complementary↗

PubMed2006

Analysis of expressed sequence tags from a naked foraminiferan Reticulomyxa filosa.

Foraminifers are a major component of modern marine ecosystems and one of the most important oceanic producers of calcium carbonate. They are a key phylogenetic group among amoeboid protists, but our knowledge of their genome is still mostly limited to a few conserved genes. Here, we report the first study of expressed genes by means of expressed sequence tag (EST) from the freshwater naked foraminiferan Reticulomyxa filosa. Cluster analysis of 1630 valid ESTs enabled the identification of 178 groups of related sequences and 871 singlets. Approximately 50% of the putative unique 1059 ESTs could be annotated using Blast searches against the protein database SwissProt + TrEMBL. The EST database described here is the first step towards gene discovery in Foraminifera and should provide the basis for new insights into the genomic and transcriptomic characteristics of these interesting but poorly understood protists.

Animals↗

PubMed2005

Identification of genes with a biocontrol function in Trichoderma harzianum mycelium using the expressed sequence tag approach.

Species of Trichoderma are commercially applied as biological control agents against plant fungal pathogens based on different mechanisms, such as the production of antifungal metabolites, competition for space and nutrients and mycoparasitism. But the integrated biocontrol mechanism of Trichoderma harzianum is not well explored at the genetic level. This study aimed to initiate the preliminary development of an expressed sequence tag (EST) database for T. harzianum and thereby gain potentially useful information on Trichoderma gene sequences in order to elucidate the integrated biocontrol mechanism. Partial sequencing of anonymous cDNA clones is a widely used technique for gene identification. A directional cDNA library has been constructed from mycelium of T. harzianum and 3298 clones have been randomly selected, subjected to single-pass sequencing from the 5' end of the vector, and identified by sequence similarity searches against gene sequences in international databases. Of the 3298 mycelium clones, 2174 exhibit similarity to known genes and 451 to known ESTs, while 673 represent novel gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes encoding proteins such as enzymes, structural proteins, and regulatory factors. A significant proportion of genes identified in the mycelium were involved in processes related to mycoparasitism and fungicidal metabolites, as would be expected in biocontrol fungus. These results present the successful application of EST analysis in T. harzianum and provide a preliminary indication of gene expression in mycelium.

Base Sequence↗

PubMed2003

Mapping of 1400 expressed sequence tags in the bovine genome using a somatic cell hybrid panel.

A bovine/hamster hybrid cell panel consisting of 30 independent hybrids was developed to locate genes. Polymerase chain reaction analysis of 279 microsatellites on the cattle linkage map in this panel revealed the presence of all chromosomes in either entire or fragmented form. Among primer pairs prepared from bovine 3'-expressed sequence tags (ESTs), 1400 ESTs were assigned to specific chromosomes, of which 1303 were newly assigned in this study, and mapped 854 (61%) to 1 of 192 chromosomal segments using this panel. The regional mapping of new genes to cattle chromosomes can be rapidly achieved using this panel.

Animals↗

PubMed2004

Mapping of 99 new microsatellite-derived loci in rye (Secale cereale L.) including 39 expressed sequence tags.

The genetic map of rye contains predominantly restriction fragment length polymorphism (RFLP) markers but also a limited number of microsatellite markers, which are known to be more reliable and easier to apply. We report here the saturation of the genomic map of rye with additional microsatellite-derived markers that we obtained from the rye expressed sequence tag (EST) databases and the Gatersleben collection of wheat microsatellite markers (WMS). A total of 99 loci (39 EST and 60 WMS) were mapped into the RFLP frameworks of four rye mapping populations consisting of 139, 64, 58 and 60 RFLPs, respectively. For another ten WMS loci, which amplified PCR products not polymorphic in any of the mapping populations, chromosome and chromosome arm locations were determined using wheat-rye addition lines. Chromosomes 1R, 2R, 3R, 4R, 5R, 6R and 7R were enriched with 9, 19, 9, 13, 27, 16 and 16 microsatellite loci, respectively. The microsatellite loci mapped were evenly distributed along the chromosomes, which is important for the further application of these markers for gene mapping or diversity studies in rye. Forty-four of the WMS loci mapped in rye were found to be homologous to those mapped in bread wheat ( Triticum aestivum L.).

Chromosome Mapping↗

PubMed2006

Differentially displayed expressed sequence tags in Melipona scutellaris (Hymenoptera, Apidae, Meliponini) development.

We have compared gene expression, using the Differential Display Reverse Transcriptase-Polymerase Chain Reaction (DDRT-PCR) technique, by means of mRNA profile in Melipona scutellaris during ontogenetic postembryonic development, in adult worker, and in both Natural and Juvenile Hormone III-induced adult queen. Six, out of the nine ESTs described here, presented differentially expressed in the phases L1 or L2, or even in both of them, suggesting that key mechanisms to the development of Melipona scutellaris are regulated in these stages. The combination HT11G-AP05 revealed in L1 and L2 a product which matches to thioredoxin reductase protein domain in the Clostridium sporogenes, an important protein during cellular oxidoreduction processes. This study represents the first molecular evidence of differential gene expression profiles toward a description of the genetic developmental traits in the genus Melipona.

Animals↗

PubMed2005

Characterization and comparison of microsatellites derived from repeat-enriched libraries and expressed sequence tags.

The construction of high-density linkage maps for use in identifying loci underlying important traits requires the development of large numbers of polymorphic genetic markers spanning the entire genome at regularly spaced intervals. As part of our efforts to develop markers for rainbow trout (Oncorhynchus mykiss), we performed a comparison of allelic variation between microsatellite markers developed from expressed sequence tag (EST) data and anonymous markers identified from repeat-enriched libraries constructed from genomic DNA. A subset of 70 markers (37 from EST databases and 33 from repeat enriched libraries) was characterized with respect to polymorphism information content (PIC), number of alleles, repeat number, locus duplication within the genome and ability to amplify in other salmonid species. Higher PIC was detected in dinucleotide microsatellites derived from ESTs than anonymous markers (72.7% vs. 54.0%). In contrast, dinucleotide repeat numbers were higher for anonymous microsatellites than for EST derived microsatellites (27.4 vs.18.1). A higher rate of cross-species amplification was observed for EST microsatellites. Approximately half of each marker type was duplicated within the genome. Unlike single-copy markers, amplification of duplicated microsatellites in other salmonids was not correlated to phylogenetic distance. Genomic microsatellites proved more useful than EST derived microsatellites in discriminating among the salmonids. In total, 428 microsatellite markers were developed in this study for mapping and population genetic studies in rainbow trout.

Alleles↗