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PubMed2005

Expressed sequence tags from the midgut and an epithelial cell line of Chironomus tentans: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels.

Expressed sequence tags (ESTs) were generated from two Chironomus tentans cDNA libraries, constructed from an embryo epithelial cell line and from larva midgut tissue. 8584 5'-end ESTs were generated and assembled into 3110 tentative unique transcripts, providing the largest contribution of C. tentans sequences to public databases to date. Annotation using Blast gave 1975 (63.5%) transcripts with a significant match in the major gene/protein databases, 1170 with a best match to Anopheles gambiae and 480 to Drosophila melanogaster. 1091 transcripts (35.1%) had no match to any database. Studies of open reading frames suggest that at least 323 of these contain a coding sequence, indicating that a large proportion of the genes in C. tentans belong to previously unknown gene families.

Animals↗

PubMed2001

Generation and mapping of expressed sequence tags from virus-infected swine macrophages.

In an effort to identify genes that have a major effect on macrophage function during viral infection, we employed differential display reverse transcription (DDRT)-PCR to capture expressed sequence tags (ESTs) of swine alveolar macrophages infected by the porcine reproductive and respiratory syndrome virus (PRRSV). Sequence analyses showed that approximately 60% of these ESTs had significant similarity (> or =93%) to known pig ESTs or genes or matched sequences from other species with homology > or =80%. To determine chromosomal localization, PCR-based mapping was performed across either swine somatic cell hybrid or radiation hybrid panels. A total of 48 porcine viral response ESTs were mapped via the swine somatic cell panel or the INRA-Minnesota porcine Radiation Hybrid (IMpRH) panel (LOD > 6.0). Northern blot analyses confirmed PRRSV-induced altered transcript expression for several ESTs, including a 2'-5' oligoadenylate synthetase and a putative dual-specificity phosphatase. These virus-response ESTs represent good candidate genes for understanding PRRSV pathogenesis and for dissecting host genes which may have major effect on disease resistance.

2',5'-Oligoadenylate Synthetase↗

PubMed2002

Analysis of expressed sequence tags (EST) obtained from common carp, Cyprinus carpio L., head kidney cells after stimulation by two mitogens, lipopolysaccharide and concanavalin-A.

A representative cDNA library from mRNA obtained from lipopolysaccharide and concanavalin-A-induced head kidney cells of carp, Cyprinus carpio, was constructed. Two hundred single pass and partially sequenced clones (AU183343 to AU183542) were generated from expressed sequence tags (ESTs) and these were searched for homology in the DDBJ/GENBANK with blastN and blastX programs. Clones matching known genes were classified according to their function and distribution. One hundred and twenty-nine genes showed homology with known genes in databases, whereas 71 (35.5%) clones did not show any significant homology to sequences in the public database. Known genes also showed homology to fish genes deposited in the database. Twenty-two clones (11%), encoding 16 different sequences, were identified as putative biodefense and oncogenes, associated with an immune response. High expression of lysozyme (3%) was detected. Putatively identified biodefense-related sequences such as Lectin type 2, MHC class II invariant chain, mcl-1a and lysozyme were aligned with known homologues from the database and the percentage identity determined. A time course evaluation of gene expression due to mitogen stimulation by RT-PCR revealed the above mentioned gene homologues were switched on early during the cell proliferation.

Amino Acid Sequence↗

PubMed2005

Expressed sequence tags from the Yukon ecotype of Thellungiella reveal that gene expression in response to cold, drought and salinity shows little overlap.

Thellungiella salsuginea (also known as T. halophila) is a close relative of Arabidopsis that is very tolerant of drought, freezing, and salinity and may be an appropriate model to identify the molecular mechanisms underlying abiotic stress tolerance in plants. We produced 6578 ESTs, which represented 3628 unique genes (unigenes), from cDNA libraries of cold-, drought-, and salinity-stressed plants from the Yukon ecotype of Thellungiella. Among the unigenes, 94.1% encoded products that were most similar in amino acid sequence to Arabidopsis and 1.5% had no match with a member of the family Brassicaceae. Unigenes from the cold library were more similar to Arabidopsis sequences than either drought- or salinity-induced sequences, indicating that latter responses may be more divergent between Thellungiella and Arabidopsis. Analysis of gene ontology using the best matched Arabidopsis locus showed that the Thellungiella unigenes represented all biological processes and all cellular components, with the highest number of sequences attributed to the chloroplast and mitochondria. Only 140 of the unigenes were found in all three abiotic stress cDNA libraries. Of these common unigenes, 70% have no known function, which demonstrates that Thellungiella can be a rich resource of genetic information about environmental responses. Some of the ESTs in this collection have low sequence similarity with those in Genbank suggesting that they may encode functions that may contribute to Thellungiella's high degree of stress tolerance when compared with Arabidopsis. Moreover, Thellungiella is a closer relative of agriculturally important Brassica spp. than Arabidopsis, which may prove valuable in transferring information to crop improvement programs.

Algorithms↗

PubMed2001

Analysis of expressed sequence tags from a cDNA library of somatic nuclear transfer-derived cloned bovine whole foetus.

The expression profile of genes in specific tissues is studied through analysing expressed sequence tags (ESTs) and provides useful information for characterizing gene function and tissue physiology. Analysis of ESTs is achieved by partial sequencing and characterization of clones isolated randomly from cDNA libraries. In the present study, we analysed the genes expressed in the somatic nuclear transfer-derived cloned bovine foetus in the early period of foetal development. To this aim, we constructed a directionally cloned cDNA library from somatic nuclear transfer-derived cloned 60 day-old whole foetus of cattle and sequenced 3' end of 510 randomly isolated clones. By BLASTN analysis, we identified 403 unique clones: 186 showed homology to previously identified genes, 123 matched uncharacterized ESTs and 94 showed no significant matches to sequences already present in DNA databases. Analysis of these cDNA clones revealed that this library contained a variety of functional genes, while foetuin, insulin-like growth factor 2, collagen type I alpha I and maternal G10 transcript genes were the most abundant transcripts. Our study allowed the establishment of a first list of genes expressed in bovine whole foetus. In future, the list of genes might help facilitate the understanding of physiology of foetal development in somatic nuclear transfer-derived cloned bovine foetus.

Animals↗

PubMed2000

Identification of differentially expressed genes in cardiac hypertrophy by analysis of expressed sequence tags.

Cardiac hypertrophy is an adaptive response to chronic hemodynamic overload. We employed a whole-genome approach using expressed sequence tags (ESTs) to characterize gene transcription and identify new genes overexpressed in cardiac hypertrophy. Analysis of general transcription patterns revealed a proportional increase in transcripts related to cell/organism defense and a decrease in transcripts related to cell structure and motility in hypertrophic hearts compared to normal hearts. Detailed comparison of individual gene expression identified 64 genes potentially overexpressed in hypertrophy, of 232 candidate genes derived from a set of 77,692 cardiac ESTs, including 47,856 ESTs generated in our laboratory. Of these, 29 were good candidates (P < 0.0002) and 35 were weaker candidates (P < 0.005). RT-PCR of a number of these candidate genes demonstrated correspondence of EST-based predictions of gene expression with in vitro levels. Consistent with an organ under various stresses, up to one-half of the good candidates predicted to exhibit differential expression were genes potentially involved in stress response. Analyses of general transcription patterns and of single-gene expression levels were also suggestive of increased protein synthesis in the hypertrophic myocardium. Overall, these results depict a scenario compatible with current understanding of cardiac hypertrophy. However, the identification of several genes not previously known to exhibit increased expression in cardiac hypertrophy (e.g., prostaglandin D synthases; CD59 antigen) also suggests a number of new avenues for further investigation. These data demonstrate the utility of genome-based resources for investigating questions of cardiovascular biology and medicine.

Adult↗

PubMed1997

Chromosome assignment of 115 expressed sequence tags (ESTs) from human skeletal muscle.

The chromosome assignment of 115 expressed sequence tags (ESTs) from human skeletal muscle, 101 of which identify unknown human genes, is reported. The ESTs were selected among over 4,000 obtained from systematic sequencing of a skeletal muscle cDNA library containing 3' portions of the mRNAs. Chromosome assignments were obtained by PCR amplification of two panels of human x rodent somatic cell hybrids. Analysis of these preliminary data suggests a nonrandom distribution of muscle ESTs in the human chromosome complement. The unexpected occurrence of multiple chromosome localizations for some ESTs is discussed.

Adult↗

PubMed1997

Evaluation of cDNA libraries from different developmental stages of Schistosoma mansoni for production of expressed sequence tags (ESTs).

A comparative study of the gene expression profile in different developmental stages of Schistosoma mansoni has been initiated based on the expressed sequence tag (EST) approach. A total of 1401 ESTs were generated from seven different cDNA libraries constructed from four distinct stages of the parasite life cycle. The libraries were first evaluated for their quality for a large-scale cDNA sequencing program. Most of them were shown to have less than 20% useless clones and more than 50% new genes. The redundancy of each library was also analyzed, showing that one adult worm cDNA library was composed of a small number of highly frequent genes. When comparing ESTs from distinct libraries, we could detect that most genes were present only in a single library, but others were expressed in more than one developmental stage and may represent housekeeping genes in the parasite. When considering only once the genes present in more than one library, a total of 466 unique genes were obtained, corresponding to 427 new S. mansoni genes. From the total of unique genes, 20.2% were identified based on homology with genes from other organisms, 8.3% matched S. mansoni characterized genes and 71.5% represent unknown genes.

Animals↗

PubMed1997

Molecular characterisation of an expressed sequence tag locus of Toxoplasma gondii encoding the micronemal protein MIC2.

The expressed sequence tag (EST) dataset of Toxoplasma gondii provides a wealth of information towards gene discovery. The complete cDNA and genomic sequence of EST tgc050 locus shows that it contains five copies of the conserved thrombospondin (TSP)-like motif present in a number of molecules with adhesive properties. A conserved region implicated with the adhesive characteristic of another group of proteins including several integrins, is also present in this molecule. The protein encoded by this sequence (rc50) is strongly recognised by monoclonal antibodies to MIC2. Affinity purified anti-rc50 antisera specifically reacted with a single protein of identical molecular mass as MIC2 and exclusively labeled the micronemes of T. gondii by cryo-immunoelectron microscopy. These results demonstrate that c50 encodes for MIC2, a previously characterised microneme protein of T. gondii. The extensive sequence similarity across multiple protein domains provides evidence that the protein encoded by this locus is the homologue to the Etp100 microneme protein of Eimeria tenella.

Amino Acid Sequence↗

PubMed2003

Analysis of the genes expressed in Clonorchis sinensis adults using the expressed sequence tag approach.

Clonorchis sinensis is a biliary tract parasite, which infects over 30 million people in China, Korea and Southeast Asia through the ingestion of undercooked freshwater fish that harbour the infective metacercariae. The genes expressed in C. sinensis adults were identified in order to develop novel drugs, better diagnostics and vaccines for the parasite. The C. sinensis cDNA library was constructed and DNA sequencing was performed with 450 randomly selected clones. Four hundred and fifteen clones contained the amino-acid-encoding sequences. The functions of these genes could be assigned by DNA sequence homology. Basic Local Alignment Search Tool X analysis showed that 277 of the 415 clones were strongly matched ( P<10(-9)) to previously identified proteins, while the remaining 138 fell into the "no database match" category. Among the clones matching previously identified proteins, 220 putatively identified genes were sorted into seven functional categories. These included the genes associated with energy metabolism (38), gene expression/RNA metabolism (21), regulatory/signalling components (14), protein metabolism/sorting (98), the structure/cytoskeleton (29), membrane transporters (ten) and antigenic proteins (ten). The remaining 57 clones were not included in these categories. The dataset included the genes encoding the proteases, a lipid binding protein, the antigen proteins and the other genes of interest from a diagnostics, drug or vaccine development viewpoint. The present expressed sequence tag analysis proved to be an effective tool for examining gene expression and identified several important genes which increase and complement our knowledge of the biology of C. sinensis.

Animals↗

PubMed2000

Improving quality of expressed sequence tag (EST) databases: recovery of reversed, antisense cDNA sequences.

Expressed sequence tag (EST) databases contain a significant number (5-20%) of reversed, antisense, cDNA sequences that can be recognized by the label "reversed clone: similarity on wrong strand" in the annotations to the sequence. Despite this high number of altered sequences, no attempt has been made to explain the alteration in molecular terms, or to evaluate their effect on the quality of the information curated in EST databases. In this paper we try to explain the way these altered sequences are originated, and propose a plausible mechanism: a "double priming" of the first strand oligo-dT primer at both ends of nascent cDNAs. In this way, a symmetrical cDNA intermediate is generated, an intermediate that can be cloned after partial digestion with the restriction enzyme used for the directional cloning. Furthermore, when "secondary" priming takes place inside the cDNA, the chain synthesized is prone to be truncated prematurely, with the subsequent loss of upstream information. One of the most subtle effects of this cloning alteration is the generation of virtual open reading frames (ORFs) in sequences with no homologues available for comparison. Nevertheless, and according to our model and our data, the "double priming mechanism" does not shift the ORF effected, so antisense sequences should be considered as normal ones after a simple transformation in their inverse-complementary forms.

Artifacts↗

PubMed2006

Immune gene discovery by expressed sequence tag analysis of spleen in the duck (Anas platyrhynchos).

To search for immune relevant genes of the duck we have conducted an expressed sequence tag (EST) project. Duck immune genes are relevant to disease resistance in agriculture and in intervention in avian influenza outbreaks through vaccination of ducks. We sequenced 3168 clones from a spleen cDNA library of a White Pekin duck, Anas platyrhynchos. We constructed an EST analysis pipeline enabling (1) quality checking and clustering, (2) identification based on BLAST results, (3) annotation using Gene Ontology, and (4) submission to the dbEST database. In total, 208 genes were relevant to the duck immune system, which we have divided into categories: (1) leukocyte receptors, (2) lectin-like immunoreceptors, (3) cytokines and chemokines, (4) transcription factors (5) mediators of antigen processing or apoptosis, and (6) innate effectors. We compared 425 homologous sequences between the duck and the closest genetic model organism, chicken. Homologous genes had regions sharing 80 to 99 percent nucleotide identity, however immune-relevant genes were less conserved than other genes.

Animals↗

PubMed1994

Isolation of differentially expressed sequence tags from human breast cancer.

Identification of quantitative changes in gene expression that occur in the malignant mammary gland, if sufficiently characterized, may yield novel molecular markers which may be useful in the diagnosis and treatment of human breast cancer. Using modifications of a previously documented technique, the differential display polymerase chain reaction, we describe the isolation of differentially expressed sequence tags, short complementary DNA fragments corresponding to mRNAs that are differentially expressed in breast cancer biopsies, as compared to normal breast tissue controls. Direct sequencing and expression analysis of two sequence tags demonstrate that they represent sequences which are overexpressed in a number of breast carcinoma cell lines. A paradigm for generating a catalogue of these sequence tags is discussed.

Base Sequence↗

PubMed2005

Analysis of organ-specific, expressed genes in Oncidium orchid by subtractive expressed sequence tags library.

The pseudobulb of Oncidium orchid plays a key role in water, carbohydrate, and other nutrition support during floral development, yet a large scale of gene expression analysis involved in the metabolisms have not been evaluated. By subtracting RsaI-digested cDNAs of leaf from those of psuedobulb, an efficient subtractive cDNA library was developed. In total, 1080 subtractive expressed sequence tags (ESTs) were obtained. Analysis revealed approximately 636 unique gene parts, 120 clusters and 516 singles. Of these sequences, 74.8% were annotated on the database of NCBI GenBank. Peroxidase, sodium/dicarboxylate cotransporter, and mannose-binding lectin were highly expressed. Some gene profiles were identified as related to carbohydrate metabolism involved in mannan, pectin, starch and sucrose biosynthesis. A large fraction of the ESTs (35%) were classified into transportation, stress-related, cell cycle, or regulatory functions. Most genes that were differentially expressed are important in early flowering development, carbohydrate metabolism and stress-response physiology. This efficient organ-specific EST library represented an explicit transcriptome profile of Oncidium pseudobulb.

Base Sequence↗

PubMed2005

Analyzing the potato abiotic stress transcriptome using expressed sequence tags.

To further increase our understanding of responses in potato to abiotic stress and the potato transcriptome in general, we generated 20 756 expressed sequence tags (ESTs) from a cDNA library constructed by pooling mRNA from heat-, cold-, salt-, and drought-stressed potato leaves and roots. These ESTs were clustered and assembled into a collection of 5240 unique sequences with 3344 contigs and 1896 singleton ESTs. Assignment of gene ontology terms (GOSlim/Plant) to the sequences revealed that 8101 assignments could be made with a total of 3863 molecular function assignments. Alignment to a set of 78 825 ESTs from other potato cDNA libraries derived from root, leaf, stolon, tuber, germinating eye, and callus tissues revealed 1476 sequences unique to abiotic stressed potato leaf and root tissue. Sequences present within the 5240 sequence set had similarity to genes known to be involved in abiotic stress responses in other plant species such as transcription factors, stress response genes, and signal transduction processes. In addition, we identified a number of genes unique to the abiotic stress library with unknown function, providing new candidate genes for investigation of abiotic stress responses in potato.

Arabidopsis↗

PubMed2000

Generation of 7137 non-redundant expressed sequence tags from a legume, Lotus japonicus.

For comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 22,983 5' end expressed sequence tags (ESTs) were accumulated from normalized and size-selected cDNA libraries constructed from young (2 weeks old) plants. The EST sequences were clustered into 7137 non-redundant groups. Similarity search against public non-redundant protein database indicated that 3302 groups showed similarity to genes of known function, 1143 groups to hypothetical genes, and 2692 were novel sequences. Homologues of 5 nodule-specific genes which have been reported in other legume species were contained in the collected ESTs, suggesting that the EST source generated in this study will become a useful tool for identification of genes related to legume-specific biological processes. The sequence data of individual ESTs are available at the web site: http://www.kazusa.or.jp/en/plant/lotus/EST/.

Codon↗

PubMed2002

Generation of expressed sequence tags from a normalized porcine skeletal muscle cDNA library.

Recent developments in microarray technologies permit scientists to analyze expression of thousands of genes simultaneously in diverse biological systems. In an effort to provide integrated resources for application of microarray technologies to studies of skeletal muscle growth and development in swine, we have constructed a normalized cDNA library from porcine skeletal muscle. The effectiveness of normalization was evaluated by DNA sequencing of clones randomly picked from the library before and after normalization, and also by Southern blot hybridization using probes representing abundant transcripts. Our data suggests that the normalization procedure successfully reduced the highly abundant cDNA species in the normalized library. To date, a total of 782 EST (expressed sequence tag) sequences have been generated from this normalized library (687 ESTs) and the original library (95 ESTs). The sequence information of these ESTs plus their BLAST results has been made available through a web accessible database (http://nbfgc.msu.edu). Cluster analysis of the data indicates that a total of 742 unique sequences are present in this collection. BLASTN search of the 742 EST sequences against the public database (dbEST) revealed that 139 had no significant matches (E-value > 10(-15)) to porcine ESTs already entered in the database, suggesting the possibility of their specific expression in porcine skeletal muscle. Generation of non-redundant ESTs from this library will allow us to construct cDNA microarrays for identification of gene expression changes that regulate muscle growth and affect meat quality in swine.

Animals↗

PubMed1994

Analysis of expressed sequence tags from Plasmodium falciparum.

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Animals↗