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PubMed2001

An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library.

The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3' and 14,408 5') from over 16,000 of these clones. Clustering of 10,654 high-quality 3' ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.

Animals↗

PubMed2004

Evidence of systematic expressed sequence tag IMAGE clone cross-hybridization on cDNA microarrays.

We present evidence of a potentially serious source of error intrinsic to all spotted cDNA microarrays that use IMAGE clones of expressed sequence tags (ESTs). We found that a high proportion of these EST sequences contain 5'-end poly(dT) sequences that are remnants from the oligo(dT)-primed reverse transcription of polyadenylated mRNA templates used to generate EST cDNA for sequence clone libraries. Analysis of expression data from two single-dye cDNA microarray experiments showed that ESTs whose sequences contain repeats of consecutive 5'-end dT residues appeared to be strongly coexpressed, while expression data of all other sequences exhibited no such pattern. Our analysis suggests that expression data from sequences containing 5' poly(dT) tracts are more likely to be due to systematic cross-hybridization of these poly(dT) tracts than to true mRNA coexpression. This indicates that existing data generated by cDNA microarrays containing IMAGE clone ESTs should be filtered to remove expression data containing significant 5' poly(dT) tracts.

Adipocytes↗

PubMed2003

Correlated clustering and virtual display of gene expression patterns in the wheat life cycle by large-scale statistical analyses of expressed sequence tags.

Compared to rice, wheat exhibits characteristic growth habits and contains complex genome constituents. To assess global changes in gene expression patterns in the wheat life cycle, we conducted large-scale analysis of expressed sequence tags (ESTs) in common wheat. Ten wheat tissues were used to construct cDNA libraries: crown and root from 14-day-old seedlings; spikelet from early and late flowering stages; spike at the booting stage, heading date and flowering date; pistil at the heading date; and seeds at 10 and 30 days post-anthesis. Several thousand colonies were randomly selected from each of these 10 cDNA libraries and sequenced from both 5' and 3' ends. Consequently, a total of 116 232 sequences were accumulated and classified into 25 971 contigs based on sequence homology. By computing abundantly expressed ESTs, correlated expression patterns of genes across the tissues were identified. Furthermore, relationships of gene expression profiles among the 10 wheat tissues were inferred from global gene expression patterns. Genes with similar functions were grouped with one another by clustering gene expression profiles. This technique might enable estimation of the functions of anonymous genes. Multidimensional analysis of EST data that is analogous to the microarray experiments may offer new approaches to functional genomics of plants.

Contig Mapping↗

PubMed1997

Identification of the gene encoding the human mitochondrial RNA polymerase (h-mtRPOL) by cyberscreening of the Expressed Sequence Tags database.

A gene cloning strategy based on the screening of the Expressed Sequence Tags database (dbEST) using sequences of mitochondrial housekeeping proteins of yeast was employed to identify the cDNA encoding the precursor of the human mitochondrial RNA polymerase (h-mtRPOL). The 3831 bp h-mtRPOL cDNA is located on chromosome 19p13.3 and encodes a protein of 1230 amino acid residues. The protein sequence shows significant homologies with sequences corresponding to mitochondrial RNA polymerases from lower eukaryotes, and to RNA polymerases from several bacteriophages. The mitochondrial RNA polymerase carries out the central activity of mitochondrial gene expression and, by providing the RNA primers for replication-initiation, is also implicated in the maintenance and propagation of the mitochondrial genome. Genes involved in the control of mtDNA replication and gene expression are attractive candidates for human disorders due to abnormalities of nucleo-mitochondrial intergenomic signalling. The availability of the h-mtRPOL cDNA will allow us to test its role in mitochondrial pathology. In addition, we propose the 'cyberscreening' of dbEST, based on yeast/human cross-species comparison, as a powerful, simple, rapid and inexpensive method, that may accelerate several-fold the molecular dissection of the human mitochondrial proteome.

Amino Acid Sequence↗

PubMed2004

Monitoring early differentiation events in human embryonic stem cells by massively parallel signature sequencing and expressed sequence tag scan.

To identify genes that may be involved in the process of human embryonic stem cell (hESC) differentiation, we profiled gene expression by expressed sequenced tag (EST) enumeration and massively parallel signature sequencing (MPSS) using RNA samples from feeder-free cultures of undifferentiated (passages 40-50) and differentiated (day 14) H1, H7, and H9 lines. MPSS and EST scan analysis showed good concordance and identified a large number of genes that changed rapidly as cultures transition from a pluripotent to a differentiated state. These included known and unknown ES cell-specific genes as well as a large number of known genes that were altered as cells differentiate. A subset of genes that were either up- or down-regulated were selected and their differential expression confirmed by a variety of independent methods, including comparison of expression after further differentiation, publicly available databases, and direct assessments by reverse transcriptase (RT)-PCR and immunocytochemistry. The analysis identified markers unique to the hESC and embryoid bodies (hEBs) stage as well as signaling pathways that likely regulate differentiation. The data generated can be used to monitor the state of hESC isolated by different laboratories using independent methods and maintained under differing culture conditions.

Cell Culture Techniques↗

PubMed1999

Expressed sequence tags from immature female sexual organ of a liverwort, Marchantia polymorpha.

A total of 970 expressed sequence tag (EST) clones were generated from immature female sexual organ of a liverwort, Marchantia polymorpha. The 376 ESTs resulted in 123 redundant groups, thus the total number of unique sequences in the EST set was 717. Database search by BLAST algorithm showed that 302 of the unique sequences shared significant similarities to known nucleotide or amino acid sequences. Six unique sequences showed significant similarities to genes that are involved in flower development and sexual reproduction, such as cynarase, fimbriata-associated protein and S-receptor kinase genes. The remaining unique 415 sequences have no significant similarity with any database-registered genes or proteins. The redundant 123 ESTs implied the presence of gene families and abundant transcripts of unknown identity. Analyses of the coding sequences of 61 unique sequences, which contained no ambiguous bases in the predicted coding regions, highly homologous to known sequences at the amino acid level with a similarity score greater than 400, and with stop codons at similar positions as their possible orthologues, indicated the presence of biased codon usage and higher GC content within the coding sequences (50.4%) than that within 3' flanking sequences (41.9%).

Amino Acid Sequence↗

PubMed2006

Generation and analysis of expressed sequence tags from NaCl-treated Glycine soja.

BACKGROUND: Salinization causes negative effects on plant productivity and poses an increasingly serious threat to the sustainability of agriculture. Wild soybean (Glycine soja) can survive in highly saline conditions, therefore provides an ideal candidate plant system for salt tolerance gene mining. RESULTS: As a first step towards the characterization of genes that contribute to combating salinity stress, we constructed a full-length cDNA library of Glycine soja (50109) leaf treated with 150 mM NaCl, using the SMART technology. Random expressed sequence tag (EST) sequencing of 2,219 clones produced 2,003 cleaned ESTs for gene expression analysis. The average read length of cleaned ESTs was 454 bp, with an average GC content of 40%. These ESTs were assembled using the PHRAP program to generate 375 contigs and 696 singlets. The resulting unigenes were categorized according to the Gene Ontology (GO) hierarchy. The potential roles of gene products associated with stress related ESTs were discussed. We compared the EST sequences of Glycine soja to that of Glycine max by using the blastn algorithm. Most expressed sequences from wild soybean exhibited similarity with soybean. All our EST data are available on the Internet (GenBank_Accn: DT082443-DT084445). CONCLUSION: The Glycine soja ESTs will be used to mine salt tolerance gene, whose full-length cDNAs will be obtained easily from the full-length cDNA library. Comparison of Glycine soja ESTs with those of Glycine max revealed the potential to investigate the wild soybean's expression profile using the soybean's gene chip. This will provide opportunities to understand the genetic mechanisms underlying stress response of plants.

Arabidopsis↗

PubMed2000

Characterisation of complementary DNAs from the expressed sequence tag analysis of life cycle stages of Laminaria digitata (Phaeophyceae).

Laminariales (Phaeophyceae, Heterokonta) are characterised by a heteromorphic digenetic life cycle with a filamentous, microscopic gametophyte and a highly evolved, macroscopic sporophyte. With the ultimate goal of comparing gene expression in each life cycle stage, complementary DNA libraries were constructed from sporophytes and gametophytes of Laminaria digitata. A set of ca. 500 expressed sequence tags (EST) was generated from each life history phase, by single-run partial sequencing of randomly picked cDNA clones. Comparison of the EST deduced amino acid sequences with database protein sequences assigned a putative identity for 39% of the 412 gametophyte clones and 48% of the 493 sporophyte clones sequenced thus far. These data represent more than 152 different proteins now probably identified in L. digitata. Several of those newly identified proteins are of interest to our understanding of the molecular physiology of kelps, for example their carbon-concentrating mechanisms, cell wall biosynthesis and halogen metabolism. EST analysis also confirmed that genes with long 3'-UTRs are widespread in Laminariales and the study of 5'-UTRs allowed the identification of a Kozak consensus sequence, c(A/C)A(A/C)CAUGGc(G/T). Several potential developmentally regulated differences in gene expression are discussed.

Algal Proteins↗

PubMed2006

Tissue expression map of a large number of expressed sequence tags and its application to in silico screening of stress response genes in common wheat.

In order to assess global changes in gene expression patterns in stress-induced tissues, we conducted large-scale analysis of expressed sequence tags (ESTs) in common wheat. Twenty-one cDNA libraries derived from stress-induced tissues, such as callus, as well as liquid cultures and abiotic stress conditions (temperature treatment, desiccation, photoperiod, moisture and ABA) were constructed. Several thousand colonies were randomly selected from each of these 21 cDNA libraries and sequenced from both the 5' and 3' ends. By computing abundantly expressed ESTs, correlated expression patterns of genes across the tissues were monitored. Furthermore, the relationships between gene expression profiles among the stress-induced tissues were inferred from the gene expression patterns. Multi-dimensional analysis of EST data is analogous to microarray experiments. As an example, genes specifically induced and/or suppressed by cold acclimation and heat-shock treatments were selected in silico. Four hundred and ninety genes showing fivefold induction or 218 genes for suppression in comparison to the control expression level were selected. These selected genes were annotated with the BLAST search. Furthermore, gene ontology was conducted for these genes with the InterPro search. Because genes regulated in response to temperature treatment were successfully selected, this method can be applied to other stress-treated tissues. Then, the method was applied to screen genes in response to abiotic stresses such as drought and ABA treatments. In silico selection of screened genes from virtual display should provide a powerful tool for functional plant genomics.

Abscisic Acid↗

PubMed2000

Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes.

Functional genomics approaches, which use combined computational and expression-based analyses of large amounts of sequence information, are emerging as powerful tools to accelerate the comprehensive understanding of cellular metabolism in specialized tissues and whole organisms. As part of an ongoing effort to identify genes of essential oil (monoterpene) biosynthesis, we have obtained sequence information from 1,316 randomly selected cDNA clones, or expressed sequence tags (ESTs), from a peppermint (Mentha x piperita) oil gland secretory cell cDNA library. After bioinformatic selection, candidate genes putatively involved in essential oil biosynthesis and secretion have been subcloned into suitable expression vectors for functional evaluation in Escherichia coli. On the basis of published and preliminary data on the functional properties of these clones, it is estimated that the ESTs involved in essential oil metabolism represent about 25% of the described sequences. An additional 7% of the recognized genes code for proteins involved in transport processes, and a subset of these is likely involved in the secretion of essential oil terpenes from the site of synthesis to the storage cavity of the oil glands. The integrated approaches reported here represent an essential step toward the development of a metabolic map of oil glands and provide a valuable resource for defining molecular targets for the genetic engineering of essential oil formation.

DNA, Complementary↗

PubMed2000

Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.

Several methods have been developed for the comprehensive analysis of gene expression in complex biological systems. Generally these procedures assess either a portion of the cellular transcriptome or a portion of the cellular proteome. Each approach has distinct conceptual and methodological advantages and disadvantages. We have investigated the application of both methods to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cells. This pathway is of critical importance for the development and progression of prostate cancer. Of clinical importance, modulation of androgens remains the mainstay of treatment for patients with advanced disease. To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate-derived expressed sequence tags (ESTs). A total of 55000 prostate ESTs were assembled into a set of 15953 clusters putatively representing 15953 distinct transcripts. These clusters were used to construct cDNA microarrays suitable for examining the androgen-response pathway at the level of transcription. The expression of 20 genes was found to be induced by androgens. This cohort included known androgen-regulated genes such as prostate-specific antigen (PSA) and several novel complementary DNAs (cDNAs). Protein expression profiles of androgen-stimulated prostate cancer cells were generated by two-dimensional electrophoresis (2-DE). Mass spectrometric analysis of androgen-regulated proteins in these cells identified the metastasis-suppressor gene NDKA/nm23, a finding that may explain a marked reduction in metastatic potential when these cells express a functional androgen receptor pathway.

DNA, Complementary↗

PubMed1995

Chromosomal localization and expressed sequence tag generation of clones from a normalized human adult thymus cDNA library.

Expressed sequence tags (ESTs) from 298 clones have been generated from a randomly primed, normalized human adult thymus cDNA library. We describe the chromosomal localization of 136 of these ESTs by PCR-based mapping to a human monochromosomal somatic cell hybrid panel. Data base similarities to known genes are also described. A subset (n = 18) of these randomly primed ESTs extended the sequence of ESTs from other tissues currently in dbEST. Of the nonrepetitive human adult thymus ESTs generated in this study, 237 (79.5%) have no similarity to current data base entries. This would suggest that our collection contains approximately 100 new coding regions from thymus tissue, a large proportion of which likely will represent the middle regions of genes. The mapped ESTs should prove useful as new gene-based markers for mapping and candidate gene hunting, particularly when anchored to a well-developed physical map of the human genome.

Chromosome Mapping↗

PubMed2006

Expressed sequence tags from loblolly pine embryos reveal similarities with angiosperm embryogenesis.

The process of embryogenesis in gymnosperms differs in significant ways from the more widely studied process in angiosperms. To further our understanding of embryogenesis in gymnosperms, we have generated Expressed Sequence Tags (ESTs) from four cDNA libraries constructed from un-normalized, normalized, and subtracted RNA populations of zygotic and somatic embryos of loblolly pine (Pinus taeda L.). A total of 68,721 ESTs were generated from 68,131 cDNA clones. Following clustering and assembly, these sequences collapsed into 5,274 contigs and 6,880 singleton sequences for a total of 12,154 non-redundant sequences. Searches of a non-identical amino acid database revealed a putative homolog for 9,189 sequences, leaving 2,965 sequences with no known function. More extensive searches of additional plant sequence data sets revealed a putative homolog for all but 1,388 (11.4%) of the sequences. Using gene ontologies, a known function could be assigned for 5,495 of the 12,154 total non-redundant sequences with 13,633 associations in total assigned. When compared to approximately 72,000 sequences in a collated P. taeda transcript assembly derived from >245,000 ESTs derived from root, xylem, stem, needles, pollen cone, and shoot ESTs, 3,458 (28.5%) of the non-redundant embryo sequences were unique and thereby provide a valuable addition to development of a complete loblolly pine transcriptome. To assess similarities between angiosperm and gymnosperm embryo development, we examined our EST collection for putative homologs of angiosperm genes implicated in embryogenesis. Out of 108 angiosperm embryogenesis-related genes, homologs were present for 83 of these genes suggesting that pine contains similar genes for embryogenesis and that our RNA sampling methods were successful. We also identified sequences from the pine embryo transcriptome that have no known function and may contribute to the programming of gene expression and embryo development.

Amino Acid Sequence↗

PubMed2006

Confirming single nucleotide polymorphisms from expressed sequence tag datasets derived from three cattle cDNA libraries.

Using the Phred/Phrap/Polyphred/Consed pipeline established in the National Livestock Research Institute of Korea, we predicted candidate coding single nucleotide polymorphisms (cSNPs) from 7,600 expressed sequence tags (ESTs) derived from three cDNA libraries (liver, M. longissimus dorsi, and intermuscular fat) of Hanwoo (Korean native cattle) steers. From the 7,600 ESTs, 829 contigs comprising more than two EST reads were assembled using the Phrap assembler. Based on the contig analysis, 201 candidate cSNPs were identified in 129 contigs, in which transitions (69%) outnumbered transversions (31%). To verify whether the predicted cSNPs are real, 17 SNPs involved in lipid and energy metabolism were selected from the ESTs. Twelve of these were confirmed to be real while five were identified as artifacts, possibly due to expressed sequence tag sequence error. Further analysis of the 12 verified cSNPs was performed using the program BLASTX. Five were identified as nonsynonymous cSNPs, five were synonymous cSNPs, and two SNPs were located in 3'-UTRs. Our data indicated that a relatively high SNP prediction rate (71%) from a large EST database could produce abundant cSNPs rapidly, which can be used as valuable genetic markers in cattle.

Animals↗

PubMed2004

Evaluation of cleaved amplified polymorphic sequence markers for Chamaecyparis obtusa based on expressed sequence tag information from Cryptomeria japonica.

We have developed and evaluated sequence-tagged site (STS) primers based on expressed sequence-tag information derived from sugi (Cryptomeria japonica) for use in hinoki (Chamaecyparis obtusa), a species that belongs to a different family (although it appears to be fairly closely related to sugi). Of the 417 C. japonica STS primer pairs we screened, 120 (approximately 30%) were transferable and provided specific PCR amplification products from 16 C. obtusa plus trees. We used haploid megagametophytes to investigate the homology of 80 STS fragments between C. obtusa and C. japonica and to identify orthologous loci. Nearly 90% of the fragments showed high (>70%) degrees of similarity between the species, and 35 STSs indicated homology to entries with the same putative function in a public DNA database. Of the 120 STS fragments amplified, 72 showed restriction fragment length polymorphisms; in addition, the CC2430 primers detected amplicon length polymorphism. We assessed the inheritance pattern of 27 cleaved amplified polymorphic sequence markers, using 20 individuals from the segregation population. All the markers analyzed were consistent with the marker inheritance patterns obtained from the screening panel, and no markers (except CC2716) showed significant (P<0.01) deviation from the expected segregation ratio. In total, 136 polymorphic markers were developed using C. japonica-based STS primers without any sequence modification. In addition, the applicability of STS-based markers developed in one species to other species was found to closely reflect the evolutionary distance between the species, which is roughly concordant with the difference between their rbcL sequences. We plan to use these markers for genetic studies in C. obtusa. Most of the markers should also provide reliable anchor loci for comparative mapping studies of the C. obtusa and C. japonica genomes.

Base Sequence↗

PubMed1997

Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation.

Amino Acid Sequence↗

PubMed2003

Expressed sequence tag analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence and pathogenicity genes.

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5' and 3' ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.

Base Sequence↗

PubMed2000

Analysis of expressed sequence tags of flower buds in Lotus japonicus.

In order to study gene expression in a reproductive organ, we constructed a cDNA library of mature flower buds in Lotus japonicus, and characterized expressed sequence tags (ESTs) of 842 clones randomly selected. The EST sequences were clustered into 718 non-redundant groups. From BLAST and FASTA search analyses of both protein and DNA databases, 58.5% of the EST groups showed significant sequence similarities to known genes. Several genes encoding these EST clones were identified as pollen-specific genes, such as pectin methylesterase, ascorbate oxidase, and polygalacturonase, and as homologous genes involved in pollen-pistil interaction. Comparison of these EST sequences with those derived from the whole plant of L. japonicus, revealed that 64.8% of EST sequences from the flower buds were not found in EST sequences of the whole plant. Taken together, the EST data from flower buds generated in this study is useful in dissecting gene expression in floral organ of L. japonicus.

Ascorbate Oxidase↗