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Comparative analysis of the Arabidopsis and rice expressed sequence tag (EST) sets.

Large numbers of expressed sequence tags (ESTs) have now been generated from a variety of model organisms. In plants, substantial collections of ESTs are available for Arabidopsis and rice, in each case representing significant proportions of the estimated total numbers of genes. Large-scale comparisons of Arabidopsis and rice sequences are especially interesting due to the fact that these two species are representatives of the two subclasses of the flowering plants (Dicotyledonae and Monocotyledonae, respectively). Here we present the results of systematic analysis of the Arabidopsis and rice EST sets. Non-redundant sets of sequences from Arabidopsis and rice were first separately derived and then combined so that gene families in common between the two species could be identified. Our results show that 58% of non-singleton ESTs are derived from genes in gene families common to the two species. These gene families constitute the basis of a core set of higher plant genes.

Arabidopsis↗

PubMed2004

Study of simple sequence repeat (SSR) markers from wheat expressed sequence tags (ESTs).

The increasing availability of expressed sequence tags (ESTs) in wheat ( Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. We examined 170,746 wheat ESTs from the public (International Triticeae EST Cooperative) and Génoplante databases, previously clustered in contigs, for the presence of di- to hexanucleotide simple sequence repeats (SSRs). Analysis of 46,510 contigs identified 3,530 SSRs, which represented 7.5% of the total number of contigs. Only 74% of the sequences allowed primer pairs to be designed, 70% led to an amplification product, mainly of a high quality (68%), and 53% exhibited polymorphism for at least one cultivar among the eight tested. Even though dinucleotide SSRs were less represented than trinucleotide SSRs (15.5% versus 66.5%, respectively), the former showed a much higher polymorphism level (83% versus 46%). The effect of the number and type of repeats is also discussed. The development of new EST-SSRs markers will have important implications for the genetic analysis and exploitation of the genetic resources of wheat and related species and will provide a more direct estimate of functional diversity.

Breeding↗

PubMed2003

Characterization of soybean genomic features by analysis of its expressed sequence tags.

We analyzed 314,254 soybean expressed sequence tags (ESTs), including 29,540 from our laboratory and 284,714 from GenBank. These ESTs were assembled into 56,147 unigenes. About 76.92% of the unigenes were homologous to genes from Arabidopsis thaliana ( Arabidopsis). The putative products of these unigenes were annotated according to their homology with the categorized proteins of Arabidopsis. Genes corresponding to cell growth and/or maintenance, enzymes and cell communication belonged to the slow-evolving class, whereas genes related to transcription regulation, cell, binding and death appeared to be fast-evolving. Soybean unigenes with no match to genes within the Arabidopsis genome were identified as soybean-specific genes. These genes were mainly involved in nodule development and the synthesis of seed storage proteins. In addition, we also identified 61 genes regulated by salicylic acid, 1,322 transcription factor genes and 326 disease resistance-like genes from soybean unigenes. SSR analysis showed that the soybean genome was more complex than the Arabidopsis and the Medicago truncatula genomes. GC content in soybean unigene sequences is similar to that in Arabidopsis and M. truncatula. Furthermore, the combined analysis of the EST database and the BAC-contig sequences revealed that the total gene number in the soybean genome is about 63,501.

Chromosomes, Artificial, Bacterial↗

PubMed1998

Regional assignment and expression analysis of 29 expressed sequence tags mapped to chromosome 3.

Of 311 expressed sequenced tags (ESTs) mapped to single human chromosomes by analysis of a monochromosome somatic cell hybrid panel, 29 were localized to chromosome 3. Analysis of somatic cell hybrid lines containing different regions of chromosome 3 has enabled the regional assignment of these 29 ESTs to 13 of 23 intervals covering chromosome 3. Northern analysis of 25 of the EST clones has provided information on the pattern of expression of potential genes represented by these transcripts in 16 human tissue types. Nine of the clones hybridized solely to a transcript(s) in the testis, 12 hybridized to transcripts in testis and other tissues, and 4 hybridized with transcripts in testis and other tissues but in addition have testis-specific transcript sizes. These ESTs will provide useful markers throughout chromosome 3 for the development of physical and transcription maps. In addition, they provide candidate genes for disease loci mapping to the intervals defined by the chromosome 3 deletion panel.

Chromosome Mapping↗

PubMed2002

Generation and analysis of 113 adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags.

OBJECTIVE: To rapidly and economically obtain knowledge about adult stage Schistosoma japonicum (Chinese strain) expressed genes using expressed sequence tag (EST). METHODS: A directional cDNA library constructed from Schistosoma japonicum (Chinese strain) adult stage RNA was used to generate expressed sequence tags (ESTs). These were compared against an EMBL-parasites database and GENBANK database by BLASTn and BLASTx. RESULTS: A total of 314 phage clones were randomly selected for generating expressed sequence tags (ESTs). From these clones, 132 EST-quality sequence were obtained. Among these EST-quality sequences, 113 ESTs were successfully submitted to the dbEST at GenBanK. A total of 7.6% of these EST-quality sequences were previously identified sequence of Schistosoma japonicum, while 4.5% were putatively identified sequences of Schistosoma japonicum. A total of 23.5% of these EST-quality sequences were putatively identified sequence of Schistosoma mansoni or other organisms. 57.6% had no matches in the database and were classified as unknown sequences. Most ESTs with the putative protein identified belonged to housekeeping proteins. Information about several interesting genes was found. CONCLUSION: Partial cDNA sequencing to generate expressed sequence tags (ESTs) has the potential to rapidly and economically increase our knowledge about adult stage Schistosoma japonicum (Chinese strain) expressed genes.

Animals↗

PubMed2003

Profiling genes expressed in human fetal cartilage using 13,155 expressed sequence tags.

OBJECTIVE: To analyze the gene expression profile of human fetal cartilage by expressed sequence tags (ESTs). METHODS: A human fetal cartilage (8-12 weeks) cDNA library was constructed using the lambda ZAP Express vector. ESTs were obtained by partial sequencing of cDNA clones. The basic local alignment search tool algorithm was used to compare all generated ESTs to known sequences. RESULTS: A total of 13,155 ESTs were analyzed, of which 8696 ESTs (66.1%) matched known genes, 53 ESTs (0.4%) were putatively novel (with no match) and the rest matched other ESTs, genomic DNA and repetitive sequences. Importantly, we identified 2448 unique known genes through non-redundancy analysis of the known gene matches, which were then functionally categorized. The tissue specificity of this library was reflected by its EST profile of the extracellular matrix (ECM) proteins. Collagens were the major transcripts, representing 68.5% of the ECM proteins. Proteoglycans were the second most abundant, constituting 9.5%. Collagen type II was the most abundant gene of all. Glypican 3, decorin and aggrecan were the major transcripts of proteoglycans. Many genes involved in cartilage development were identified, such as insulin-like growth factor-II, its receptor and binding proteins, connective tissue growth factor and fibroblast growth factors. Proteases and their regulatory factors were also identified, including matrix metalloprotease 2 and tissue inhibitor of metalloproteinase 1. CONCLUSIONS: The EST approach is an effective way of characterizing the genes expressed in cartilage. These data represent the most extensive molecular information on human fetal cartilage to date. The availability of this information will serve as a basis for further research to identify genes that are essential in cartilage development.

Cartilage, Articular↗

PubMed2000

Analysis of Medicago truncatula nodule expressed sequence tags.

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.

Blotting, Northern↗

PubMed2004

Expressed sequence tag (EST) analysis of Gregarine gametocyst development.

Gregarines are protozoan parasites of invertebrates in the phylum Apicomplexa. We employed an expressed sequence tag strategy in order to dissect the molecular processes of sexual or gametocyst development of gregarines. Expressed sequence tags provide a rapid way to identify genes, particularly in organisms for which we have very little molecular information. Analysis of approximately 1800 expressed sequence tags from the gametocyst stage revealed highly expressed genes related to cell division and differentiation. Evidence was found for the role of degradation and recycling in gametocyst development. Numerous additional genes uncovered by expressed sequence tag sequencing should provide valuable tools to investigate gametocyst development as well as for molecular phylogenetics, and comparative genomics in this important group of parasites.

Animals↗

PubMed2005

Analysis of expressed sequence tags of the water flea Daphnia magna.

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.

Animals↗

PubMed2005

Characterisation of the two most abundant genes in the Haemonchus contortus expressed sequence tag dataset.

Analysis of the Haemonchus contortus Expressed sequence tag (EST) dataset revealed that almost 10% of all ESTs (1719 ESTs) belong to a family of related genes. Close analysis of the ESTs suggests that these represent two genes (called here Hc-nim-1 and Hc-nim-2) with multiple alleles of each. These genes show significant similarity to two genes from Caenorhabditis elegans, F54D5.3 (Wormbase accession WBGene00010049, corresponding protein WP:CE28033) and F54D5.4 (WBGene00010050, WP:CE03409) of unknown function. Reverse transcriptase coupled-PCR showed that both genes are transcribed from the L4 stage onwards and are transcribed in both male and female adult worms. A partial bacterial recombinant of the Hc-NIM-1 protein was made and used to raise antiserum in rabbits which recognised a 19 kDa antigen in the water soluble protein fraction of adult worms. By immunohistochemistry, the Hc-NIM-1 protein was localised in the hypodermis of the pharyngeal region of adult worms but not posterior in the hypodermis surrounding the reproductive tract. To investigate the function of this novel protein family we conducted a RNA interference experiment for the homologuous proteins in C. elegans. No visible phenotype was detected after simultaneous RNAi treatment for both Ce-F54D5.3 and Ce-F54D5.4.

Amino Acid Sequence↗

PubMed2003

Expressed sequence tag profiling identifies developmental and anatomic partitioning of gene expression in the mouse prostate.

BACKGROUND: The prostate gland is an organ with highly specialized functional attributes that serves to enhance the fertility of mammalian species. Much of the information pertaining to normal and pathological conditions affecting the prostate has been obtained through extensive developmental, biochemical and genetic analyses of rodent species. Although important insights can be obtained through detailed anatomical and histological assessments of mouse and rat models, further mechanistic explanations are greatly aided through studies of gene and protein expression. RESULTS: In this article we characterize the repertoire of genes expressed in the normal developing mouse prostate through the analysis of 50,562 expressed sequence tags derived from 14 mouse prostate cDNA libraries. Sequence assemblies and annotations identified 15,009 unique transcriptional units of which more than 600 represent high quality assemblies without corresponding annotations in public gene expression databases. Quantitative analyses demonstrate distinct anatomical and developmental partitioning of prostate gene expression. This finding may assist in the interpretation of comparative studies between human and mouse and guide the development of new transgenic murine disease models. The identification of several novel genes is reported, including a new member of the beta-defensin gene family with prostate-restricted expression. CONCLUSIONS: These findings suggest a potential role for the prostate as a defensive barrier for entry of pathogens into the genitourinary tract and, further, serve to emphasize the utility of the continued evaluation of transcriptomes from a diverse repertoire of tissues and cell types.

Amino Acid Sequence↗

PubMed2001

An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families.

Amino Acid Motifs↗

PubMed1994

A catalogue of genes in the cardiovascular system as identified by expressed sequence tags.

The heart, which is composed of all the cellular components of the circulatory system, is a representative organ for obtaining genes expressed in the cardiovascular system in normal and disease states. We used partial sequences of cDNA clones, or expressed sequence tags, to identify and tag genes expressed in this organ. More than 3500 partial sequences representing > 3000 cDNA clones have been obtained from either the 5' or 3' end of inserts derived from human heart cDNA libraries. Of 3132 cDNA clones analyzed by sequence similarity searching against the GenBank/EMBL data bases, 1485 (47.4%) were found to represent additional, previously undiscovered genes, whereas 267 clones were matched to human brain expressed sequence tags. Clones matching to known genes were catalogued according to their putative structural and cellular functions. cDNA probes from reverse-transcribed mRNAs of fetal and adult hearts were used to study differential expression of selected clones in cardiac development. Cataloguing genes expressed in the heart may provide insight into the genes involved in health and cardiovascular disease.

Adult↗

PubMed2004

Large-scale analysis of the barley transcriptome based on expressed sequence tags.

To provide resources for barley genomics, 110,981 expressed sequence tags (ESTs) were generated from 22 cDNA libraries representing tissues at various developmental stages. This EST collection corresponds to approximately one-third of the 380,000 publicly available barley ESTs. Clustering and assembly resulted in 14,151 tentative consensi (TCs) and 11 073 singletons, altogether representing 25 224 putatively unique sequences. Of these, 17.5% showed no significant similarity to other barley ESTs present in dbEST. More than 41% of all barley genes are supposed to belong to multigene families and approximately 4% of the barley genes undergo alternative splicing. Based on the functional annotation of the set of unique sequences, the functional category 'Energy' was further analysed to reveal tissue- and stage-specific differences in gene expression. Hierarchical clustering of 362 differentially expressed TCs resulted in the identification of seven major clusters. The clusters reflect biochemical pathways predominantly activated in specific tissues and at various developmental stages. During seed germination glycolysis could be identified as the most predominant biochemical pathway. Germination-specific glycolysis is characterized by the coordinated expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase, whose antagonistic actions possibly regulate the flux of amino acids into protein biosynthesis and gluconeogenesis respectively. The expression of defence-related and antioxidant genes during germination might be controlled by the ethylene-signalling pathway as concluded from the coordinated expression of those genes and the transcription factors (TF) EIN3 and EREBPG. Moreover, because of their predominant expression in germinating seeds, TF of the AP2 and MYB type are presumably major regulators of germination.

Expressed Sequence Tags↗

PubMed2005

Analysis of expressed sequence tags from the venom ducts of Conus striatus: focusing on the expression profile of conotoxins.

Cone snails (genus Conus) are predatory marine gastropods that use venom peptides for interacting with prey, predators and competitors. A majority of these peptides, generally known as conotoxins demonstrate striking selectivity in targeting specific subtypes of ion channels and neurotransmitter receptors. So they are not only useful tools in neuroscience to characterize receptors and receptor subtypes, but offer great potential in new drug research and development as well. Here, a cDNA library from the venom ducts of a fish-hunting cone snail species, Conus striatus is described for the generation of expressed sequence tags (ESTs). A total of 429 ESTs were grouped into 137 clusters or singletons. Among these sequences, 221 were toxin sequences, accounting for 52.1% (corresponding to 19 clusters) of all transcripts. A-superfamily (132 ESTs) and O-superfamily conotoxins (80 ESTs) constitute the predominant toxin components. Some non-disulfide-rich Conus peptides were also found. The expression profile of conotoxins also explained to some extent the pharmacological and physiological reactions elicited by this typical piscivorous species. For the first time, a nonstop transcript of conotoxin was identified, which is suggestive that alternative polyadenylation may be a means of post-transcriptional regulation of conotoxin production. A comparison analysis of these conotoxins reveals the different variation and divergence patterns in these two superfamilies. Our investigations indicate that focal hyper-mutation, block substitution and exon shuffling are three main mechanisms leading to the conotoxin diversity in a species. The comprehensive set of Conus gene sequences allowed the identification of the representative classes of conotoxins and related components, which may lay the foundation for further research and development of conotoxins.

Amino Acid Sequence↗

PubMed2004

Gene expression profile of the rat eye iridocorneal angle: NEIBank expressed sequence tag analysis.

PURPOSE: To characterize gene expression pattern in the combined tissues of the rat iridocorneal angle by expressed sequence tag (EST) analysis, as part of the NEIBank project. METHODS: RNA was extracted from dissected tissues of the rat iridocorneal angle (iris, ciliary body, trabecular meshwork, and Schlemm's canal) and used to construct unamplified, non-normalized cDNA libraries in the pSPORT1 vector. Approximately 5000 clones were sequenced from the 5'-end. Clones were clustered and identified using the GRIST software, a procedure based on BLAST comparisons. Complete sequences of several novel cDNAs showing eye-preferred expression patterns were obtained. The expression patterns of several genes have been investigated by Northern blot and in situ hybridization, as well as by RT-PCR. RESULTS: After analysis and removal of non-mRNA sequences, 2195 independent clusters, potentially representing individual eye angle-expressed clones were obtained. The expression profile of the combined rat eye angle tissues was more similar to that of the human iris than to human trabecular meshwork. Several cDNAs encoding transcription factors essential for normal eye development and function including Pax-6, Six3, c-Maf, Maf1, Sox-4, Foxc1, Rx, and Ldb2 were present among sequenced clones. A number of tested cDNAs showed eye-preferred expression patterns. Myocilin, which is abundant in human eye angle tissues, was not observed in the rat collection; however, transcripts for three other olfactomedin-domain proteins were seen. Latrotoxin receptor (CL1AA) and optimedin were shown to be expressed in the iris and ciliary body, as well as in the ganglion and inner nuclear cell layers of the retina, whereas the rat orthologue of the human HNOEL-iso gene was expressed in the iris and sclera and less actively in the trabecular meshwork, retina, and optic nerve. CONCLUSIONS: The iridocorneal libraries are a good source of novel uncharacterized genes and molecular markers for the tissues of the eye angle. Although myocilin is not abundantly expressed in rat eye angle, other olfactomedin-containing genes are expressed there and may play important roles in normal eye function and disease.

Amino Acid Sequence↗

PubMed1992

Expressed sequence tags and chromosomal localization of cDNA clones from a subtracted retinal pigment epithelium library.

Expressed sequence tags (ESTs) provide useful molecular landmarks for physical mapping and identify the position of an expressed region in the genome. The use of subtracted cDNA libraries enriched for tissue-specific genes as a source of ESTs should reduce the repetitive isolation of constitutively expressed sequences. We report here the sequence tags from the 3'-end region of 58 new directionally cloned cDNAs from a subtracted human retinal pigment epithelium (RPE) cell line library. Eight of the cDNAs have been assigned to human chromosomes using PCR-based EST assays. Chromosomal mapping of subtracted RPE cDNA clones may also help in identifying candidate genes for inherited eye diseases.

Base Sequence↗

PubMed2004

[Isolation and expression of novel expressed sequence tags (ESTs) from ovarian follicles of Shaoxing ducks].

Three expressed sequence tags ( ESTs), SXDF0201 (271 bp), SXDF0202 (200 bp) and SXDF0203 (173 bp), were isolated from ovarian follicles of Shaoxing ducks by using silver staining mRNA differential display. GenBank/BLAST analysis revealed that SXDF0201 was not homologous to any of the published sequences from all species, indicating that it was a novel EST and was then registered in GenBank (GenBank Accession No.: CB072629), while SXDF0202 and SXDF0203 were found to be highly homologous to seven known chicken ESTs and chicken mRNA for gizzard smooth muscle myosin heavy chain. 5'-RACE was employed to extend the SXDF0201 to 544 bp which was confirmed as novel in BLAST search. The temporal and spatial expression of SXDF0201 and SXDF0202 were also investigated with semi-quantitative RT-PCR. The result showed that: both SXDF0201 and SXDF0202 were found to be expressed in hypothalamus, pituitary, muscle, liver, and fat tissues of Shaoxing ducks; SXDF0201 was expressed significantly higher in ovaries of 30-day-old Shaoxing ducks compared with that of 60-day-old (P < 0.05) and 90-day-old (P = 0.015), but the expression of SXDF0202 showed no difference throughout the ovarian development; granulose layers expressed higher SXDF0201 than theca layers in almost all hierarchical follicles, the expression of SXDF0202 in granulose layers increased along with follicular maturation (P < 0.01) from Fw to F3 follicles, but decreased dramatically to the lowest in F1 follicles (P < 0.01). In theca layers, the highest expression of SXDF0202 was found in Fw follicles (P < 0.01).

Animals↗