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Results for “Expressed Sequence Tags”

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PubMed2000

Preliminary profile of the Cryptosporidium parvum genome: an expressed sequence tag and genome survey sequence analysis.

Cryptosporidium parvum is a protozoan enteropathogen that infects humans and animals and causes a pronounced diarrheal disease that can be life-threatening in immunocompromised hosts. No specific chemo- or immunotherapies exist to treat cryptosporidiosis and little molecular information is available to guide development of such therapies. To accelerate gene discovery and identify genes encoding potential drug and vaccine targets we constructed sporozoite cDNA and genomic DNA sequencing libraries from the Iowa isolate of C. parvum and determined approximately 2000 sequence tags by single-pass sequencing of random clones. Together, the 567 expressed sequence tags (ESTs) and 1507 genome survey sequences (GSSs) totaled one megabase (1 mb) of unique genomic sequence indicating that approximately 10% of the 10.4 mb C. parvum genome has been sequence tagged in this gene discovery expedition. The tags were used to search the public nucleic acid and protein databases via BLAST analyses, and 180 ESTs (32%) and 277 GSSs (18%) exhibited similarity with database sequences at smallest sum probabilities P(N)< or =10(-8). Some tags encoded proteins with clear therapeutic potential including S-adenosylhomocysteine hydrolase, histone deacetylase, polyketide/fatty-acid synthases, various cyclophilins, thrombospondin-related cysteine-rich protein and ATP-binding-cassette transporters. Several anonymous ESTs encoded proteins predicted to contain signal peptides or multiple transmembrane spanning segments suggesting they were destined for membrane-bound compartments, the cell surface or extracellular secretion. One-hundred four simple sequence repeats were identified within the nonredundant sequence tag collection with (TAA)(> or =6)/(TTA)(> or =6) and (TA)(> or = 10)/(AT)(> or =10 ) being the most prevalent, occurring 40 and 15 times, respectively. Various cellular RNAs and their genes were also identified including the small and large ribosomal RNAs, five tRNAs, the U2 small nuclear RNA, and the small and large virus-like, double-stranded RNAs. This investigation has demonstrated that survey sequencing is an efficient procedure for gene discovery and genome characterization and has identified and sequence tagged many C. parvum genes encoding potential therapeutic targets.

Amino Acid Sequence↗

PubMed2006

Analysis of expressed sequence tags from the harmful alga, Prymnesium parvum (Prymnesiophyceae, Haptophyta).

A cDNA library was constructed from late log-phase cultures of the ichthyotoxin-producing haptophyte, Prymnesium parvum. Approximately 6,300 single-pass 5' nucleotide sequences (out of nearly 8,000 randomly isolated clones) achieved length and quality criteria for incorporation into an expressed sequence tag (EST) database. Collectively, 3,415 unique genes (tentative unigenes [TUGs]) were assembled. Database searches revealed approximately 1,100 TUG (34%) that encode proteins of known or putative function (expect values E < or = 1.0 x 10(-10)), representing most functional classes of proteins and many metabolic pathways. In addition to proteins involved in photosynthesis and protein synthesis/degradation, one of the most frequently encountered transcripts putatively encodes an ABC-type phosphate transport system component. Active (and efficient?) phosphate transport may correlate with rapid growth, perhaps explaining how this alga out-competes other species when it blooms and becomes toxic under low-phosphate conditions. The majority of TUGs, including 12 of the 50 most commonly encountered transcripts, encode potentially novel proteins, some of which are probably involved in synthesis and secretion of the unique prymnesin toxins. This work suggests that future studies on the expression of many of these genes during rapid growth should provide insight into the genetic basis of bloom formation and toxicity.

DNA, Algal↗

PubMed2004

Identification and analysis of safener-inducible expressed sequence tags in Populus using a cDNA microarray.

Safeners are the chemicals used to protect plants from detrimental effects of herbicides, but their mode of action at the molecular level is not well understood. As an initial step towards understanding the molecular mechanism of safener action in trees, homologous genes in hybrid poplar (Populus nigra x Populus maximowiczii) that were induced by a safener were identified. We here describe the identification of differentially expressed genes in Populus that are induced by Concep-III, a herbicide safener. Expressed sequence tags (ESTs) enriched for transcriptionally induced genes were isolated by suppressive subtractive hybridization (SSH). The SSH library cDNA inserts were used to construct a cDNA microarray for high-throughput validation of the up-regulated expression of safener-induced genes. Single-pass and partial sequences of 1,344 safener-induced ESTs were assembled into 418 singletons and 328 clusters, but the putative functions of almost 53% of the ESTs are not known. Genes encoding proteins involved in all three different phases of safener action, viz., oxidation, conjugation, and sequestration, were found in the SSH library. Almost 75% of genes that showed greater than 2-fold expression upon safener treatment were redundant in the SSH library. The expression pattern for selected genes was validated by reverse transcription-polymerase chain reaction. A few safener-induced genes that were not previously reported to be induced by safeners, but which may have a role in herbicide metabolism, were identified. The newly identified genes could have potential for application in genetic engineering of plants for herbicide detoxification and tolerance.

Acetophenones↗

PubMed1996

Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the expressed sequence tags database.

As an approach to characterizing all human ATP-binding cassette (ABC) superfamily genes, a search of the human expressed sequence tag (EST) database was performed using sequences from known ABC genes. A total of 105 clones, containing sequences of potential ABC genes, were identified, representing 21 distinct genes. This brings the total number of characterized human ABC genes from 12 to 33. The new ABC genes were mapped by PCR on somatic cell and radiation hybrid panels and yeast artificial chromosomes (YACs). The genes are located on human chromosomes 1, 2, 3, 4, 6, 7, 10, 12, 13, 14, 16, 17 and X; at locations distinct from previously mapped members of the superfamily. The characterized genes display extensive diversity in sequence and expression pattern and this information was utilized to determine potential structural, functional and evolutionary relationships to previously characterized members of the ABC superfamily.

ATP-Binding Cassette Transporters↗

PubMed2001

Identification of novel transcribed sequences on human chromosome 22 by expressed sequence tag mapping.

To identify sequences on the human genome that are actually transcribed, we mapped expressed sequence tags (ESTs) of long cDNAs ranging from 4 kb to 7 kb along a 33.4-Mb sequence of human chromosome 22, the first human chromosome entirely sequenced. By the EST mapping of 30,683 long cDNAs in silico, 603 cDNA sequences were found to locate on chromosome 22 and classified into 169 clusters. Comparison of the genomic loci of these cDNA sequences with 679 genes already annotated on chromosome 22q revealed that 46 clusters represented newly identified transcribed sequences. To further characterize these sequences, we sequenced 12 cDNAs in their entirety out of 46 clusters. Of these 12 cDNAs, 6 were predicted to include a protein-coding region while the remaining 6 were unlikely to encode proteins. Interestingly, 3 out of the 12 cDNAs had the nucleotide sequences of the opposite strands of the genes previously annotated, which suggested that these genomic regions were transcribed bi-directionally. In addition to these newly identified 12 cDNAs, another 12 cDNAs were entirely sequenced since these cDNAs were likely to contain new information about the predicted protein-coding sequences previously annotated. In the cases of KIAA1670 and KIAA1672, these single cDNA sequences covered two separately annotated transcribed regions. For example, the sequence of a clone for KIAA1670 indicated that the CHKL and CPT1B genes were co-transcribed as a contiguous transcript without making both the protein-coding regions fused. In conclusion, the mapping of ESTs derived from long cDNAs followed by sequencing of the entire cDNAs provided indispensable information for the precise annotation of genes on the genome together with ESTs derived from short cDNAs.

Brain Chemistry↗

PubMedDate not supplied

Expressed sequence tag analysis of the dinoflagellate Lingulodinium polyedrum during dark phase.

To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light-dark cycle. A total of 4324 5'-end sequence tags were isolated. The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database. Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae. We also isolated three bioluminescence-related (luciferase and two luciferin-binding proteins [LBP]) and 37 photosynthesis-related genes. Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene. These cDNA clones and EST sequence data should provide a powerful resource for future genome-wide functional analyses for uncharacterized genes.

Animals↗

PubMed2006

Water stress-responsive genes in loblolly pine (Pinus taeda) roots identified by analyses of expressed sequence tag libraries.

Drought stress is the principal cause of seedling mortality in pine forests of the southeastern United States and in many other forested regions around the globe. As part of a larger effort to discover loblolly pine genes, this study subjected rooted cuttings of three unrelated pine genotypes to three watering regimens. Expressed sequence tags (ESTs) were obtained from both the 3' and 5' ends of 12,918 randomly selected cDNAs generated from root tissues. These ESTs were clustered to identify 6,765 unique transcripts (UniScripts) derived from 6,202 putative unique genes (UniGenes-S). Tentative annotations were assigned on the basis of BLASTX comparisons to the Protein Information Resource Nonredundant Reference (PIR-NREF) database. Expression levels of 42 UniScripts varied with high statistical significance with respect to treatment. Many of them resembled gene products shown to be important for drought tolerance in other species, including dehydrins, endochitinases, cytochrome P450 enzymes, pathogenesis-related proteins and various late-embryogenesis abundant (LEA) gene products. Similarly, expression levels of 110 UniScripts varied with high statistical significance among genotypes, indicating that gene expression patterns in this species are much more dependent on genotype than on treatment. Most of the water stress-induced pine UniScripts that appeared to encode products resembling drought tolerance factors in other species were most highly induced in a single genotype, suggesting that particularly useful adaptive alleles for drought tolerance might exist within the collection of cDNAs characterized from this genotype. Mining and visualizing the complete data set, as well as downloading of both EST and UniScript contig sequences, are possible using MAGIC Gene Discovery at http://fungen.org/genediscovery/.

Acclimatization↗

PubMed2005

Differential display-mediated identification of three drought-responsive expressed sequence tags in tea [Camellia sinensis (L.) O. Kuntze].

There is no information on drought-modulated gene(s) in tea [Camellia sinensis (L.) O. Kuntze], a woody and perennial plant of commercial importance. Using differential display of mRNA, three drought-modulated expressed sequence tags (ESTs) were identified. Northern and BLAST analysis revealed that clone dr1 (drought-responsive), induced only by drought but not by ABA, showed significant scores with PR-5 (pathogenesis related) family of PR-protein gene. Another clone dr2, repressed by drought but not by ABA, had nucleotide repeats for polyasparate that are also present in chicken calsequestrin-like mRNA. Clone dr3, responded similarly to clone dr2 but did not show significant homology with the reported genes, hence appears to be novel. Identification of these ESTs is an initial step to clone the full length genes and their promoters.

Adaptation, Physiological↗

PubMed2006

The heavy metal hyperaccumulator Thlaspi caerulescens expresses many species-specific genes, as identified by comparative expressed sequence tag analysis.

Thlaspi caerulescens is a natural zinc (Zn), cadmium (Cd) and nickel (Ni) hyperaccumulator and an emerging plant model species to study heavy metal hyperaccumulation and tolerance. This paper describes the analysis of the first expressed sequence tag (EST) collection from T. caerulescens. This collection is a new resource to unravel the molecular basis of plant metal homeostasis, tolerance and hyperaccumulation. In total, 4289 ESTs were generated originating from Zn-exposed root and shoot tissues, leading to 3709 T. caerulescens assembled partial cDNA sequences (unigenes). In comparison to Arabidopsis or other publicly available plant sequences, a fraction of c. 8% of the T. caerulescens unigenes (TcUGs) had no significant similarity with any known DNA sequence and, so far, these sequences are T. caerulescens specific. Three per cent of the TcUGs correspond to Arabidopsis thaliana orthologues that, as yet, have not been found to be expressed. The T. caerulescens transcriptome generally relates very well to the A. thaliana transcriptome, although, compared with other closely related species, a relatively large number of T. caerulescens-specific transcripts were found. T. caerulescens also expresses a relatively large number of genes which are expressed at a very low level in A. thaliana.

Adaptation, Physiological↗

PubMed2005

Expressed sequence tag analysis of the soybean rust pathogen Phakopsora pachyrhizi.

Soybean rust is caused by the obligate fungal pathogen Phakopsora pachyrhizi Sydow. A unidirectional cDNA library was constructed using mRNA isolated from germinating P. pachyrhizi urediniospores to identify genes expressed at this physiological stage. Single pass sequence analysis of 908 clones revealed 488 unique expressed sequence tags (ESTs, unigenes) of which 107 appeared as multiple copies. BLASTX analysis identified 189 unigenes with significant similarities (Evalue<10(-5)) to sequences deposited in the NCBI non-redundant protein database. A search against the NCBI dbEST using the BLASTN algorithm revealed 32 ESTs with high or moderate similarities to plant and fungal sequences. Using the Expressed Gene Anatomy Classification, 31.7% of these ESTs were involved in primary metabolism, 14.3% in gene/protein expression, 7.4% in cell structure and growth, 6.9% in cell division, 4.8% in cell signaling/cell communication, and 4.8% in cell/organism defense. Approximately 29.6% of the identities were to hypothetical proteins and proteins with unknown function.

Basidiomycota↗

PubMed2002

Isolation of porcine expressed sequence tags for the construction of a first genomic transcript map of the skeletal muscle in pig.

To identify genes with effects on meat quality and production traits we developed an adult porcine skeletal muscle cDNA library. After pre-screening this library with seven genes highly expressed in skeletal muscle, 385 non-hybridizing clones were sequenced from both ends to yield 510 expressed sequence tags (ESTs). Together with those ESTs previously generated from this library, we have produced 701 porcine skeletal muscle ESTs. These ESTs were grouped into 306 different cDNA species and compared with the human skeletal muscle transcriptional profiles obtained from different databases. Furthermore we mapped 107 of these cDNAs using a somatic cell hybrid panel with genes mapping over all the autosomes (except on chromosome 11) and on chromosome X. The mapping of these cDNAs contributed to the construction of a first genomic transcript map of the skeletal muscle tissue in pig.

Animals↗

PubMed1993

Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library.

A human infant brain cDNA library, made specifically for production of expressed sequence tags (ESTs) was evaluated by partial sequencing of over 1,600 clones. Advantages of this library, constructed for EST sequencing, include the use of directional cloning, size selection, very low numbers of mitochondrial and ribosomal transcripts, short polyA tails, few non-recombinants and a broad representation of transcripts. 37% of the clones were identified, based on matches to over 320 different genes in the public databases. Of these, two proteins similar to the Alzheimer's disease amyloid precursor protein were identified.

Amino Acid Sequence↗

PubMed1993

3,400 new expressed sequence tags identify diversity of transcripts in human brain.

We present the results of the partial sequencing of over 3,400 expressed sequence tags (ESTs) from human brain cDNA clones, which increases the number of distinct genes expressed in the brain, that are represented by ESTs, to about 6,000. By choosing clones in an unbiased manner, it is possible to construct a profile of the transcriptional activity of the brain at different stages. Proteins that comprise the cytoskeleton are the most abundant; however, a large variety of regulatory proteins are also seen. About half of the ESTs predicted to contain a protein-coding region have no matches in the public peptide databases and may represent new gene families.

Amino Acid Sequence↗

PubMed2002

Stage-specific expressed sequence tags obtained during preimplantation bovine development by differential display RT-PCR and suppression subtractive hybridization.

Differential display RT-PCR (DDRT-PCR) and suppression subtractive hybridization (SSH) were applied in order to detect preimplantation stage-specific expressed sequence tags (ESTs) of bovine embryos. Seventeen ESTs were detected from the differential display RT-PCR approach. All clones but two showed homology to genes or ESTs known in human, cattle or other species. One of the clones similar to H. sapiens mRNA for KIAA1764 protein was used exemplarily to quantify the transcripts by real-time PCR. The result of quantitative differential screening was found to be in agreement with DDRT-PCR banding patterns. In the second approach, a blastocyst-stage enriched cDNA library was constructed using SSH of blastocyst versus morula transcripts. The 71 clones that were analysed represent 33 distinct loci including candidate genes for the regulative processes during differentiation of inner cell mass (ICM) and trophoblast cells and the initial phase of embryo implantation, such as galectin-3 and fibronectin. As revealed by real-time PCR, the mRNA level of galectin-3 was three times higher in the blastocyst stage than in the morula stage. DDRT-PCR and SSH are both powerful tools for the identification of stage-specific expressed gene in preimplantation bovine embryos. Real-time PCR allows to test and confirm the outcome and to add quantitative data of selected transcripts of interest.

Animals↗

PubMed2003

Exploiting plant somatic radiation hybrids for physical mapping of expressed sequence tags.

Methods are described for the optimisation of the generation of radiation hybrids suitable for physical mapping of a plant (barley) genome. A combination of PCR-based technologies, involving the use of whole genome, mixed primer and hemi-nested primer amplifications, can greatly extend their utility for the physical mapping of expressed sequence tags (ESTs). Using panels of hybrids and ESTs, donor DNA retention and individual marker retention frequencies for the expressed portion of the barley genome in the hybrids were estimated.

Expressed Sequence Tags↗

PubMed2004

Interaction of Sclerotinia sclerotiorum with a resistant Brassica napus cultivar: expressed sequence tag analysis identifies genes associated with fungal pathogenesis.

Sclerotinia sclerotiorum is a ubiquitous necrotrophic fungal pathogen capable of infecting a wide range of plants. To identify genes involved in fungal development and pathogenesis we generated 2232 expressed sequence tags (ESTs) from two cDNA libraries constructed using either mycelia grown in pectin medium or tissues from infected Brassica napus stems. A total of 774 individual fungal genes were identified of which 39 were represented only among the infected plant EST collection. Annotation of 534 unigenes was possible following the categories applied to Saccharomyces cerevisiae and the Universal Gene Ontology scheme. cDNAs were identified that encoded potential pathogenicity factors including four endopolygalacturonases, two exopolygalacturonases, and several metabolite transporters. The potential role of these genes, as well as those encoding signal transduction factors, in the infection process is discussed.

Ascomycota↗

PubMed1997

Expression of CYP71B7, a cytochrome P450 expressed sequence Tag from Arabidopsis thaliana.

The systematic sequencing of anonymous cDNA clones (expressed sequence tags or ESTs) from the plant Arabidopsis thaliana has identified a number of cDNAs with similarity to known cytochrome P450 sequences. The partial sequence of one of these cDNAs, 5G6, indicated that it was likely to encode a full-length cytochrome P450 monooxygenase (cyt P450) sequence. In this paper we describe the complete sequence of this clone, which has been designated CYP71B7 in accordance with the nomenclature for the cyt P450 gene superfamily. The cDNA was used to determine the pattern of expression of the corresponding gene in A. thaliana. Northern hybridization analysis indicated that maximal expression of CYP71B7 occurred in rosette leaves. Weaker hybridizing bands were also detected by Northern analysis of RNA from roots, leaves, flowers, and siliques. No expression could be detected in stem tissue. Southern analysis indicated that the CYP71B7 gene was likely to exist as a single copy in the genome of A. thaliana. CYP71B7 was expressed episomally in yeast, and microsomes prepared from transgenic yeast exhibited a carbon monoxide difference spectrum characteristic of cyt P450. Microsomes from yeast expressing CYP71B7 were assayed for enzymatic activity with synthetic model cyt P450 substrates. Microsomes from yeast cells expressing CYP71B7 or those from control cells exhibited no detectable NADPH-supported 7-ethoxycoumarin or 7-ethoxyresorufin deethylase activities. However, in the presence of cumene hydroperoxide, activity was observed with microsomes from cells expressing CYP71B7 with 7-ethoxycoumarin as substrate. Organic hydroperoxides are well known to support cyt P450 catalysis in the absence of electrons from NADPH. The yeast microsomes contained high levels of endogenous NADPH-ferricytochrome P450 reductase (CPR) activity. The data suggest that this A. thaliana cyt P450, although expressed in an active form, is incapable of accepting electrons from the endogenous yeast CPR protein.

7-Alkoxycoumarin O-Dealkylase↗

PubMed2000

A new set of Arabidopsis expressed sequence tags from developing seeds. The metabolic pathway from carbohydrates to seed oil.

Large-scale single-pass sequencing of cDNAs from different plants has provided an extensive reservoir for the cloning of genes, the evaluation of tissue-specific gene expression, markers for map-based cloning, and the annotation of genomic sequences. Although as of January 2000 GenBank contained over 220,000 entries of expressed sequence tags (ESTs) from plants, most publicly available plant ESTs are derived from vegetative tissues and relatively few ESTs are specifically derived from developing seeds. However, important morphogenetic processes are exclusively associated with seed and embryo development and the metabolism of seeds is tailored toward the accumulation of economically valuable storage compounds such as oil. Here we describe a new set of ESTs from Arabidopsis, which has been derived from 5- to 13-d-old immature seeds. Close to 28,000 cDNAs have been screened by DNA/DNA hybridization and approximately 10,500 new Arabidopsis ESTs have been generated and analyzed using different bioinformatics tools. Approximately 40% of the ESTs currently have no match in dbEST, suggesting many represent mRNAs derived from genes that are specifically expressed in seeds. Although these data can be mined with many different biological questions in mind, this study emphasizes the import of photosynthate into developing embryos, its conversion into seed oil, and the regulation of this pathway.

Arabidopsis↗