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PubMed2003

Comparative analyses of potato expressed sequence tag libraries.

The cultivated potato (Solanum tuberosum) shares similar biology with other members of the Solanaceae, yet has features unique within the family, such as modified stems (stolons) that develop into edible tubers. To better understand potato biology, we have undertaken a survey of the potato transcriptome using expressed sequence tags (ESTs) from diverse tissues. A total of 61,940 ESTs were generated from aerial tissues, below-ground tissues, and tissues challenged with the late-blight pathogen (Phytophthora infestans). Clustering and assembly of these ESTs resulted in a total of 19,892 unique sequences with 8,741 tentative consensus sequences and 11,151 singleton ESTs. We were able to identify a putative function for 43.7% of these sequences. A number of sequences (48) were expressed throughout the libraries sampled, representing constitutively expressed sequences. Other sequences (13,068, 21%) were uniquely expressed and were detected only in a single library. Using hierarchal and k means clustering of the EST sequences, we were able to correlate changes in gene expression with major physiological events in potato biology. Using pair-wise comparisons of tuber-related tissues, we were able to associate genes with tuber initiation, dormancy, and sprouting. We also were able to identify a number of characterized as well as novel sequences that were unique to the incompatible interaction of late-blight pathogen, thereby providing a foundation for further understanding the mechanism of resistance.

Cluster Analysis↗

PubMed2000

Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii.

To characterize genes whose expression is induced in carbon-stress conditions, 12,969 and 13,450 5'-end expressed sequence tags (ESTs) were generated from cells grown in low-CO2 and high-CO2 conditions of the unicellular green alga, Chlamydomonas reinhardtii. These ESTs were clustered into 4436 and 3566 non-redundant EST groups, respectively. Comparison of their sequences with those of 3433 non-redundant ESTs previously generated from the cells under the standard growth condition indicated that 2665 and 1879 EST groups occurred only in the low-CO2 and high-CO2 populations, respectively. It was also noted that 96.2% and 96.0% of the cDNA species respectively obtained from the low-CO2 and high-CO2 conditions had no similar EST sequence deposited in the public databases. The EST species identified only in the low-CO2 treated cells included genes previously reported to be expressed specifically in low-CO2 acclimatized cells, suggesting that the ESTs generated in this study will be a useful source for analysis of genes related to carbon-stress acclimatization. The sequence information and search results of each clone will appear at the web site: http://www.kazusa.or.jp/en/plant/chlamy/EST/.

Animals↗

PubMed2005

Expressed sequence tags from the laboratory-grown miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom and mining for single nucleotide polymorphisms and insertions/deletions in tomato cultivars.

Laboratory-grown miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom has attracted attention as a host for functional genomics research. In this study, we generated 35,824 expressed sequence tags (ESTs) from leaves and fruits of Micro-Tom. The ESTs comprised 10,287 unigenes (5007 contigs and 5280 singletons), including 1858 novel tomato unigenes. Of the 18 unigenes that shared strong homology with tobacco chloroplast genome sequences, one unigene was likely derived from polyadenylated transcripts of the atpH gene. Interestingly, ESTs for vacuolar invertase, pectate lyase and alcohol acyl transferase were underrepresented in the Micro-Tom data set. From all of the ESTs, we mined 2039 candidate single nucleotide polymorphisms (SNPs) and 121 candidate insertions and deletions (indels) based on homology with four tomato inbred lines, E6203, R11-13, Rio Grande PtoR and R11-12, and a wild relative, L. pennellii TA56, for which sequence data was publicly available with more than 5000 entries. Direct genome sequencing of several SNP or indel sites in Micro-Tom and L. esculentum E6203 suggested that more than 69% of the candidate sites were truly polymorphic, making them useful for the preparation of DNA markers.

DNA, Complementary↗

PubMedDate not supplied

The Brugia malayi genome project: expressed sequence tags and gene discovery.

To advance and facilitate molecular studies of Brugia malayi, one of the causative agents of human lymphatic filariasis, an expressed sequence tag (EST)-based gene discovery programme has been carried out. Over 22,000 ESTs have been produced and deposited in the public databases by a consortium of laboratories from endemic and non-endemic countries. The ESTs have been analysed using custom informatic tools to reveal patterns of individual gene expression that may point to potential targets for future research on anti-filarial drugs and vaccines. Many genes first discovered as ESTs are now being analysed by researchers for immunodiagnostic, vaccine and drug target potential. Building on the success of the B. malayi EST programme, significant EST datasets are being generated for a number of other major parasites of humans and domesticated animals, and model parasitic species.

Animals↗

PubMed2004

Generation and analysis of expressed sequence tags from the salt-tolerant mangrove species Avicennia marina (Forsk) Vierh.

Salinization poses an increasingly serious problem in coastal and agricultural areas with negative effects on plant productivity and yield. Avicennia marina is a pantropical mangrove species that can survive in highly saline conditions. As a first step towards the characterization of genes that contribute to combating salinity stress, the construction of a cDNA library of A. marina genes is reported here. Random expressed sequence tag (EST) sequencing of 1,841 clones produced 1,602 quality reads. These clones were classified into functional categories, and BLAST: comparisons revealed that 113 clones were homologous to genes earlier implicated in stress responses, of which the dehydrins are the most predominant in this category. Of the ESTs analyzed, 30% showed homology to previously uncharacterized genes in the public plant databases. Of these 30%, 52 clones were selected for reverse Northern analysis: 26 were shown to be up-regulated and five shown to be down-regulated. The results obtained by reverse Northern analysis were confirmed by Northern analysis for three clones.

Amino Acid Sequence↗

PubMed2005

[Molecular screening for moyamoya disease by use of expressed sequence tag on chromosome 3p].

Moyamoya disease is a well-known cerebrovascular disorder of unknown pathogenesis affecting terminal portion of internal carotid arteries and causing ischemic attacks. Its familial occurrence suggests genetic background. We hypothesized that paternally imprinted gene might be associated with this disorder. To identify the expressed sequence tags (ESTs) with monoallelic expressions on chromosome 3, we used mouse A9 hybrid cells having human chromosome 3. Two ESTs showed only maternal expression in mouse A9 hybrid cells, and four showed non-expression in the lymphocytes derived from moyamoya patients. Although these ESTs are clustered on the same 150 kb region, we finally failed to identify cDNA in this region.

Adult↗

PubMed2005

Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides.

Fusarium verticillioides (teleomorph Gibberella moniliformis) is a pathogen of maize worldwide and produces fumonisins, a family of mycotoxins that have been associated with several animal diseases as well as cancer in humans. In this study, we sought to identify fungal genes that affect fumonisin production and/or the plant-fungal interaction. We generated over 87,000 expressed sequence tags from nine different cDNA libraries that correspond to 11,119 unique sequences and are estimated to represent 80% of the genomic complement of genes. A comparative analysis of the libraries showed that all 15 genes in the fumonisin gene cluster were differentially expressed. In addition, nine candidate fumonisin regulatory genes and a number of genes that may play a role in plant-fungal interaction were identified. Analysis of over 700 FUM gene transcripts from five different libraries provided evidence for transcripts with unspliced introns and spliced introns with alternative 3' splice sites. The abundance of the alternative splice forms and the frequency with which they were found for genes involved in the biosynthesis of a single family of metabolites as well as their differential expression suggest they may have a biological function. Finally, analysis of an EST that aligns to genomic sequence between FUM12 and FUM13 provided evidence for a previously unidentified gene (FUM20) in the FUM gene cluster.

Amino Acid Sequence↗

PubMed2005

Analysis of 686 expressed sequence tags (ESTs) from the intertidal harpacticoid copepod Tigriopus japonicus (Crustacea, Copepoda).

The intertidal harpacticoid copepod Tigriopus japonicus is an important species in the study of marine pollution. To facilitate molecular biomonitoring using T. japonicus, we constructed a T. japonicus unidirectional cDNA library using lambdaZAP expression vector, excised to pBluescript vector with the aid of helper phage, and analyzed 686 randomly picked expressed sequence tags (ESTs) from this species. From the 686 ESTs sequenced, we found several functional genes such as vitellin, kinases and potential detoxification-related genes. We are now preparing a T. japonicus cDNA chip for molecular ecotoxicological studies. In this paper, we discuss the potential use of T. japonicus ESTs and their importance in ecotoxicology.

Animals↗

PubMed2004

Physical and linkage mapping of mammary-derived expressed sequence tags in cattle.

This study describes the physical and linkage mapping of 42 gene-associated markers developed from mammary gland-derived expressed sequence tags to the cattle genome. Of the markers, 25 were placed on the USDA reference linkage map and 37 were positioned on the Roslin 3000-rad radiation hybrid (RH) map, with 20 assignments shared between the maps. Although no novel regions of conserved synteny between the cattle and the human genomes were identified, the coverage was extended for bovine chromosomes 3, 7, 15, and 29 compared with previously published comparative maps between human and bovine genomes. Overall, these data improve the resolution of the human-bovine comparative maps and will assist future efforts to integrate bovine RH and linkage map data.

Animals↗

PubMed2006

Gene expression profiling of human HBV- and/or HCV-associated hepatocellular carcinoma cells using expressed sequence tags.

Liver cancer is one of the leading causes of cancer death worldwide. To identify novel target genes that are related to liver carcinogenesis, we examined new genes that are differentially expressed in human hepatocellular carcinoma (HCC) cell lines and tissues based on the expressed sequence tag (EST) frequency. Eleven libraries were constructed from seven HCC cell lines and three normal liver tissue samples obtained from Korean patients. An analysis of gene expression profiles for HCC was performed using the frequency of ESTs obtained from these cDNA libraries. Genes were identified (n=120) as being either up- or down-regulated in human liver cancer cells. Among these, 14 genes (FTL, K-ALPHA1, LDHA, RPL4, ENO1, ANXA2, RPL9, RPL10, RPL13A, GNB2L1, AMBP, GC, A1BG, and SERPINC1), in addition to previously well-known liver cancer related genes, were confirmed to be differentially expressed in seven liver cancer cell lines and 17 HCC tissues by semi-quantitative RT-PCR. In addition, 73 genes, in which there was a significant difference (P>0.99) between HBV- and HCV-associated HCC cells, were selected. Of these, expression patterns of 14 (RPLP0, AKR1C, KRT8, GPX4, RPS15, ID1, RPS21, VIM, EEF1G, EIF4A1, HLA-C, FN1, CD44, and RPS10) were confirmed by semi-quantitative RT-PCR in four of HBV- and three of HCV-associated HCC cell lines. Among those genes, an immunohistochemical analysis for ANXA2 showed that it is expressed at high levels in HCC. Using an analysis of EST frequency, the newly identified genes, especially ANXA2, represent potential biomarkers for HCC and useful targets for elucidating the molecular mechanisms associated with HCC involving virological etiology.

Biomarkers, Tumor↗

PubMedDate not supplied

Expressed sequence tags for the chicken genome from a normalized 10-day-old White Leghorn whole embryo cDNA library: 1. DNA sequence characterization and linkage analysis.

Expressed sequence tags (ESTs) provide a rapid and reliable method for gene discovery as well as a resource for the large-scale analysis of gene expression of known and unknown genes. Here we describe a normalized cDNA library developed from a 10-day-old White Leghorn chicken whole embryo. The utility of the library was evaluated by partial sequencing of 99 randomly selected insert-containing clones and the analysis of EST-targeted genomic regions for single nucleotide polymorphisms (SNPs) in the East Lansing chicken reference DNA mapping panel. Using stringent match criteria of percent identity of 80 or higher across a length of 50 or more bases, 46 ESTs matched database sequences including previously reported Gallus gallus genes. Thirty-seven of the 50 primer pairs developed from 50 unique ESTs amplified a single fragment. The size of the 37 amplicons ranged from 276 to 693 bp for a total of 17,508 and an average of 473. About 70% of the SNPs detected were either G-->A or C-->T transition. The number of SNPs detected within the amplicons from EST-targeted genomic regions ranged from 0 to 4 for a total of 65 and a frequency of about 1 every 470 bases. About 35% of the amplicons contained only 1 SNP, while 19% had 4 SNPs. Using the SNPs that were informative in the East Lansing reference panel, 17 ESTs were mapped on the East Lansing chicken genetic map. The ESTs described, as well as the nucleotide variants identified within the EST-targeted genomic regions, represent significant resources for genome analysis in the chicken.

Animals↗

PubMed2002

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PURPOSE: To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. METHODS: A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. RESULTS: The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. CONCLUSIONS: The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

Adult↗

PubMed2002

Expressed sequence tag analysis of blood cells in the vanadium-rich ascidian, Ascidia sydneiensis samea--a survey of genes for metal accumulation.

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10(7) times that found in seawater. The vanadium ions are stored in vacuoles located within vanadium-containing blood cells, vanadocytes. To investigate the phenomenon, an expressed sequence tag analysis (EST) of a cDNA library of Ascidia sydneiensis samea blood cells was carried out. Three hundred clones were obtained and sequenced by EST analysis. A similarity search revealed that 158 of the clones (52.7%) were known genes, and 142 of the clones (47.3%) did not have any similarity to genes registered in the SwissProt database. According to the functions of their genes the identified EST clones were categorized into eight types of clones; these consisted of genes; metal-related proteins (29 clones), signal transduction (22 clones), protein synthesis (17 clones), nuclear proteins (17 clones), cytoskeleton and motility (14 clones), energy conversion (3 clones), hypothetical proteins (11 clones), and others (45 clones). The ferritin homologue has a high degree of similarity to that of mammals; the iron-binding sites of ferritin are well conserved including His-118 which is important for capturing Fe(2+), also works as a ligand for VO(2+).

Amino Acid Sequence↗

PubMed2005

Insights into a dinoflagellate genome through expressed sequence tag analysis.

BACKGROUND: Dinoflagellates are important marine primary producers and grazers and cause toxic "red tides". These taxa are characterized by many unique features such as immense genomes, the absence of nucleosomes, and photosynthetic organelles (plastids) that have been gained and lost multiple times. We generated EST sequences from non-normalized and normalized cDNA libraries from a culture of the toxic species Alexandrium tamarense to elucidate dinoflagellate evolution. Previous analyses of these data have clarified plastid origin and here we study the gene content, annotate the ESTs, and analyze the genes that are putatively involved in DNA packaging. RESULTS: Approximately 20% of the 6,723 unique (11,171 total 3'-reads) ESTs data could be annotated using Blast searches against GenBank. Several putative dinoflagellate-specific mRNAs were identified, including one novel plastid protein. Dinoflagellate genes, similar to other eukaryotes, have a high GC-content that is reflected in the amino acid codon usage. Highly represented transcripts include histone-like (HLP) and luciferin binding proteins and several genes occur in families that encode nearly identical proteins. We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X. We speculate this histone may be retained for its role in DNA double-strand break repair. CONCLUSION: This is the most extensive collection to date of ESTs from a toxic dinoflagellate. These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production.

Amino Acid Sequence↗

PubMed2005

An in silico mining for simple sequence repeats from expressed sequence tags of zebrafish, medaka, Fundulus, and Xiphophorus.

Teleost fish genome projects involving model species are resulting in a rapid accumulation of genomic and expressed DNA sequences in public databases. The expressed sequence tags (ESTs) collected in the databases can be mined for the analysis of both structural and functional genomics. In this study, we in silico analyzed 49,430 unigenes representing a total of 692,654 ESTs from four model fish for their potential use in developing simple sequence repeats (SSRs), or microsatellites. After bioinformatical mining, a total of 3,018 EST derived SSRs (EST-SSRs) were identified for 2,335 SSR containing ESTs (SSR-ESTs). The frequency of identified SSR-ESTs ranged from 1.5% for Xiphophorus to 7.3% for zebrafish. The dinucleotide repeat motif is the most abundant SSR, accounting for 47%, 52%, 64%, and 78% for medaka, Fundulus, zebrafish, and Xiphophorus, respectively. Simulation analysis suggests that a majority of these EST-SSRs have sufficient flanking sequences for polymerase chain reaction (PCR) primer design. Comparative DNA sequence analyses of SSR-ESTs identified several cross-species SSRs and sequences that may be used as cross-reference genes in comparative studies. For example, the flanking sequences of one SSR (CTG)n within the pituitary tumor-transforming gene (PTTG) 1 interacting protein (PTTGIP), showed conservation spanning the medaka, Fundulus, human, and mouse genomes. This study provides a large body of information on EST-SSRs that can be useful for the development of polymorphic markers, gene mapping, and comparative genome analysis. Functional analysis of these SSR-ESTs may reveal their role in metabolism and gene evolution of these model species.

Amino Acid Sequence↗

PubMed1995

Regional assignment of 30 expressed sequence tags on human chromosome 7 using a somatic cell hybrid panel.

The regional assignments of 30 expressed sequence tags (ESTs) on human chromosome 7 were determined by studying the segregation of their PCR-amplified products in a panel of mouse somatic cell hybrids. ESTs are important molecular landmarks for physical mapping and can be considered as tags to candidate genes for genetically linked human inherited diseases. These results contribute further potential gene sequences to the transcriptional map of chromosome 7.

Animals↗

PubMed2002

Bioinformatics and reanalysis of subtracted expressed sequence tags from the human ciliary body: Identification of novel biological functions.

PURPOSE: The ciliary body is largely known for its major roles in the regulation of aqueous humor secretion, intraocular pressure, and accommodation of the lens. In this review article we applied bioinformatics to re-examine hundreds of expressed sequence tags (ESTs) previously isolated by subtractive hybridization from a human ciliary body library [1]. The DNA sequences of these clones have been recently added to the web site of NEIBank. METHODS: DNA sequence comparisons of subtracted ESTs were performed against all entries in the last available release of the non-redundant database containing GenBank, EMBL, DDBJ and PDB sequences using the BlastN program accessed through NCBI's BLAST services on the internet (NCBI). Sequences were also compared and mapped using the Blast search program provided through the Internet by the Human Genome Project (UCSC). RESULTS: A total number of 284 independent ESTs were classified in 17 functional groups. Analysis of their relationships allowed to define the expression of five major groups of known genes: (i) protein synthesis, folding, secretion and degradation (20%); (ii) energy supply and biosynthesis (12%); (iii) contractility and cytoskeleton structure (6%); (iv) cellular signaling and cell cycle regulation (7%); and (v) nerve cell related tasks (2%), including neuropeptide processing and putative non-visual phototransduction and circadian rhythm control. The largest group contain unidentified sequences, a total of 105 sequences, accounting for 37% of ESTs. The unidentified sequences show similarity to genomic non-coding regions, or genes of unknown function. CONCLUSIONS: The most highly represented EST, correspond to myocilin, a gene involved in glaucoma. The data also confirms the secretory functions of the ciliary epithelium, and its high metabolism; the presence of a neuroendocrine peptidergic system presumably involved in the regulation of the intraocular pressure and/or aqueous humor secretion. Additional genes may be related to a non-visual phototransduction cascade and/or to circadian rhythms. Overall this initial group of subtracted ESTs can lead to uncover novel physiological functions of the ciliary body in normal and in disease, as well as novel candidate genes for ocular diseases.

Ciliary Body↗

PubMed1999

Identification of abundantly expressed novel and conserved genes from the infective larval stage of Toxocara canis by an expressed sequence tag strategy.

Larvae of Toxocara canis, a nematode parasite of dogs, infect humans, causing visceral and ocular larva migrans. In noncanid hosts, larvae neither grow nor differentiate but endure in a state of arrested development. Reasoning that parasite protein production is orientated to immune evasion, we undertook a random sequencing project from a larval cDNA library to characterize the most highly expressed transcripts. In all, 266 clones were sequenced, most from both 3' and 5' ends, and similarity searches against GenBank protein and dbEST nucleotide databases were conducted. Cluster analyses showed that 128 distinct gene products had been found, all but 3 of which represented newly identified genes. Ninety-five genes were represented by a single clone, but seven transcripts were present at high frequencies, each composing >2% of all clones sequenced. These high-abundance transcripts include a mucin and a C-type lectin, which are both major excretory-secretory antigens released by parasites. Four highly expressed novel gene transcripts, termed ant (abundant novel transcript) genes, were found. Together, these four genes comprised 18% of all cDNA clones isolated, but no similar sequences occur in the Caenorhabditis elegans genome. While the coding regions of the four genes are dissimilar, their 3' untranslated tracts have significant homology in nucleotide sequence. The discovery of these abundant, parasite-specific genes of newly identified lectins and mucins, as well as a range of conserved and novel proteins, provides defined candidates for future analysis of the molecular basis of immune evasion by T. canis.

3' Untranslated Regions↗