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Mapping of 14 expressed sequence tags (ESTs) from porcine skeletal muscle by somatic cell hybrid analysis.

Chromosomal assignments are reported for fourteen porcine expressed sequence tags (ESTs)--CALM1, CRYAB, MYH7, MYL1, PDK4, PGAM2, PYGM, REV3L, RFC1, SLN, SPTBN1, SRM160, TPM1 and YWHAG. The ESTs were derived from our porcine skeletal muscle cDNA library. The ESTs sequences selected for mapping included the presence of the 3'-untranslated region. The assignments were performed using two independent somatic cell hybrid panels providing the possibility of confirmation of the results obtained. The observed localizations are compared with the locations predicted from heterologous (human-pig, pig-human) chromosome painting data and knowledge of the map locations of the human homologues. These results add new information to the porcine genome transcript map.

Animals↗

RED: the analysis, management and dissemination of expressed sequence tags.

The Rancourt EST Database (RED) is a web-based system for the analysis, management, and dissemination of expressed sequence tags (ESTs). RED represents a flexible template DNA sequence database that can be easily manipulated to suit the needs of other laboratories undertaking mid-size sequencing projects.

Animals↗

Electronic identification and chromosomal assignment by radiation hybrid mapping of human expressed sequence tags corresponding to new potassium channel genes.

Human homologues of 36 Caenorhabditis elegans potassium channels were identified by expressed sequence tag (EST) database searching. This approach was combined with radiation hybrid mapping to localize new potassium channel genes in the human genome. In addition, several ESTs whose location was already known were also identified as cDNAs encoding additional potassium channels. The identification and mapping of all these genes will make them useful tools for mutation detection in neurological as well as other human diseases.

Animals↗

Chromosome bin map of expressed sequence tags in homoeologous group 1 of hexaploid wheat and homoeology with rice and Arabidopsis.

A total of 944 expressed sequence tags (ESTs) generated 2212 EST loci mapped to homoeologous group 1 chromosomes in hexaploid wheat (Triticum aestivum L.). EST deletion maps and the consensus map of group 1 chromosomes were constructed to show EST distribution. EST loci were unevenly distributed among chromosomes 1A, 1B, and 1D with 660, 826, and 726, respectively. The number of EST loci was greater on the long arms than on the short arms for all three chromosomes. The distribution of ESTs along chromosome arms was nonrandom with EST clusters occurring in the distal regions of short arms and middle regions of long arms. Duplications of group 1 ESTs in other homoeologous groups occurred at a rate of 35.5%. Seventy-five percent of wheat chromosome 1 ESTs had significant matches with rice sequences (E < or = e(-10)), where large regions of conservation occurred between wheat consensus chromosome 1 and rice chromosome 5 and between the proximal portion of the long arm of wheat consensus chromosome 1 and rice chromosome 10. Only 9.5% of group 1 ESTs showed significant matches to Arabidopsis genome sequences. The results presented are useful for gene mapping and evolutionary and comparative genomics of grasses.

Arabidopsis↗

Analysis of expressed sequence tag loci on wheat chromosome group 4.

A total of 1918 loci, detected by the hybridization of 938 expressed sequence tag unigenes (ESTs) from 26 Triticeae cDNA libraries, were mapped to wheat (Triticum aestivum L.) homoeologous group 4 chromosomes using a set of deletion, ditelosomic, and nulli-tetrasomic lines. The 1918 EST loci were not distributed uniformly among the three group 4 chromosomes; 41, 28, and 31% mapped to chromosomes 4A, 4B, and 4D, respectively. This pattern is in contrast to the cumulative results of EST mapping in all homoeologous groups, as reported elsewhere, that found the highest proportion of loci mapped to the B genome. Sixty-five percent of these 1918 loci mapped to the long arms of homoeologous group 4 chromosomes, while 35% mapped to the short arms. The distal regions of chromosome arms showed higher numbers of loci than the proximal regions, with the exception of 4DL. This study confirmed the complex structure of chromosome 4A that contains two reciprocal translocations and two inversions, previously identified. An additional inversion in the centromeric region of 4A was revealed. A consensus map for homoeologous group 4 was developed from 119 ESTs unique to group 4. Forty-nine percent of these ESTs were found to be homoeologous to sequences on rice chromosome 3, 12% had matches with sequences on other rice chromosomes, and 39% had no matches with rice sequences at all. Limited homology (only 26 of the 119 consensus ESTs) was found between wheat ESTs on homoeologous group 4 and the Arabidopsis genome. Forty-two percent of the homoeologous group 4 ESTs could be classified into functional categories on the basis of blastX searches against all protein databases.

Chromosome Mapping↗

Analysis of expressed sequence tags of retinal pigment epithelium: cystatin C is an abundant transcript.

In order to identify genes that are expressed in the retinal pigment epithelium (RPE), randomly chosen clones of a cDNA library of cultured human foetal RPE cells were analyzed by sequencing. Of 164 informative expressed sequence tags (ESTs), 88 matched the sequences of 74 genes for proteins of known or presumed function. Approximately a third of these represented genes with involvement in gene/protein expression, with a major subcategory concerned with protein turnover. In particular, the gene coding for precursor cystatin C was represented by 3 independent ESTs, and plaque hybridization estimated the frequency of cystatin C clones in the library to be 1.3%. Cystatin C mRNA in cultured RPE cells was confirmed by Northern blotting and by reverse transcription polymerase chain reaction (RT-PCR) with identification of the cystatin C sequence as the product of the reaction. The survey also revealed 25 novel human sequences representing genes that are active in RPE. One of these was localized near a recently identified, new autosomal recessive retinitis pigmentosa locus. In conclusion, the findings specifically demonstrate the unexpected presence of cystatin C mRNA at fairly high abundance in cultured human RPE cells, and, more generally, serve as a model study establishing the usefulness of the EST approach for further characterizing the molecular basis of the activities of the RPE.

Cells, Cultured↗

Generation and analysis of 280,000 human expressed sequence tags.

We report the generation of 319,311 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' and 3' ends of 194,031 human cDNA clones. Our goal has been to obtain tag sequences from many different genes and to deposit these in the publicly accessible Data Base for Expressed Sequence Tags. Highly efficient automatic screening of the data allows deposition of the annotated sequences without delay. Sequences have been generated from 26 oligo(dT) primed directionally cloned libraries, of which 18 were normalized. The libraries were constructed using mRNA isolated from 17 different tissues representing three developmental states. Comparisons of a subset of our data with nonredundant human mRNA and protein data bases show that the ESTs represent many known sequences and contain many that are novel. Analysis of protein families using Hidden Markov Models confirms this observation and supports the contention that although normalization reduces significantly the relative abundance of redundant cDNA clones, it does not result in the complete removal of members of gene families.

Adult↗

Generation of expressed sequence tags as physical landmarks in the genome of Trypanosoma brucei.

Previous molecular genetic studies on the African trypanosome have focused on only a few genes and gene products, the majority of which are concerned with surface antigenic variation; consequently, an insignificant number of the genes of this organism have been characterized to date. In order to: (1) identify new genes and analyze their expression profile, (2) generate expressed sequence tags (ESTs) for derivation of a physical map of the trypanosome genome, and (3) make available the partial sequence information and the corresponding clones for general biomedical research on the parasite, we have performed single-pass sequencing of random, directionally cloned cDNAs from a bloodstream form Trypanosoma brucei rhodesiense library. Analysis of 2128 such ESTs sequenced so far in this study showed significant similarities [BLASTX P(n)-value < 10(-4), and a match > 10 amino acid residues] with proteins whose genes have been described in diverse organisms including man, rodents, kinetoplastids, yeasts and plants. A number of the ESTs encode homologues of proteins involved in various functions including signal reception and transduction, cell division, gene regulation, DNA repair and replication, general metabolism, and structural integrity. Although some of these genes may have been expected to be present in the African trypanosomes, the majority of them had not previously been described in these organisms. A large proportion, 768 individual ESTs (36%, representing 385 different transcripts), had a significant homology with genes described in organisms other than the African trypanosomes; however, 15% of the ESTs were from genes already described in trypanosomes. Among the ESTs analysed were 462 distinct known genes, only 77 of which have been described in T. brucei. Approximately 52% of the ESTs did not show any significant homology with the sequences in any of the public domain databases.

Animals↗

A survey of gene expression and diversity in the venom glands of the pitviper snake Bothrops insularis through the generation of expressed sequence tags (ESTs).

In order to produce a global panorama of the transcriptional activity of snake venom glands and to correlate with its venom composition, we constructed a DNA complementary to RNA library from the venom glands of the Viperidae snake Bothrops insularis for the generation of expressed sequence tags (ESTs). Sequences from 610 independent clones were grouped in 297 clusters, revealing the putative identification of 210 distinct gene products. Toxin sequences correspond to 56% of all transcripts (85 clusters), being the metalloproteinases (23%) and the bradykinin-potentiating peptides (11%) the major components. This approach revealed a new highly expressed toxin similar to vascular endothelial growth factor, which was recently reported (J. Biol. Chem. 276 (2001) 39836). Among the 125 clusters matching cellular proteins, the major part represents molecules involved in gene and protein expression, notably in disulfide bond assembly, reflecting a high specialization of this tissue for toxin synthesis. An unusual representation of retrotransposon-like sequences was also found and could be related to the occurrence and diversity of many paralogous forms of toxins in the venom gland. Our B. insularis dbEST allowed the identification of the most common classes of toxins present in Viperidae venoms, which parallels the complex hemorrhagic effects evoked by the venom on the prey. In addition, it provides the first comprehensive set of reptilian gene sequences described so far.

Amino Acid Sequence↗

Identification of expressed sequence tags in an alkali grass (Puccinellia tenuiflora) cDNA library.

Alkali grass (Puccinellia tenuiflora), a monocotyledonous halophyte, can serve as a model of salt tolerance in monocotyledon crops. To elucidate the molecular events associated with salt tolerance in alkali grass, we generated a directional cDNA library from leaves treated with the alkali salt, NaHCO3. Large-scale sequencing of the cDNA library identified 2942 ESTs representing 2366 non-redundant transcripts. These have been deposited in the dbEST division of GenBank. BLASTX evaluation indicated that 1274 of the ESTs were homologous to various known genes/proteins in a broad range of organisms, especially gramineae species. The other 1092 ESTs, which showed little if any homology to known sequences, were considered novel. Based on the encoded proteins, the 1274 identified ESTs fell into 12 functional categories, of which four were abundant, namely metabolism (18.84%), transcription (12.48%), unclassified (11.22%) and cell rescue/defense (9.66%). The 162 unique transcripts corresponding to possible salt-related genes were also identified. This study provides an overview of gene expression in NaHCO3-stressed alkali grass, as well as useful information for further investigation of salt resistance in plants.

Cloning, Molecular↗

Generation of 10,154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis.

A total of 10,154 5'-end expressed sequence tags (EST) were established from the normalized and size-selected cDNA libraries of a marine red alga, Porphyra yezoensis. Among the ESTs, 2140 were unique species, and the remaining 8014 were grouped into 1127 species. Database search of the 3267 non-redundant ESTs by BLAST algorithm showed that the sequences of 1080 species (33.1%) have similarity to those of registered genes from various organisms including higher plants, mammals, yeasts, and cyanobacteria, while 2187 (66.9%) are novel. Codon usage analysis in the coding regions of 101 non-redundant EST groups showing significant similarity to known genes indicated the higher GC contents at the third position of codons (79.4%) than the first (62.2%) and the second position (45.0%), suggesting that the genome has been exposed to high GC pressure during evolution. The sequence data of individual ESTs are available at the web site http://www.kazusa.or.jp/en/plant/porphyra/EST/.

Algorithms↗

prot4EST: translating expressed sequence tags from neglected genomes.

BACKGROUND: The genomes of an increasing number of species are being investigated through generation of expressed sequence tags (ESTs). However, ESTs are prone to sequencing errors and typically define incomplete transcripts, making downstream annotation difficult. Annotation would be greatly improved with robust polypeptide translations. Many current solutions for EST translation require a large number of full-length gene sequences for training purposes, a resource that is not available for the majority of EST projects. RESULTS: As part of our ongoing EST programs investigating these "neglected" genomes, we have developed a polypeptide prediction pipeline, prot4EST. It incorporates freely available software to produce final translations that are more accurate than those derived from any single method. We show that this integrated approach goes a long way to overcoming the deficit in training data. CONCLUSIONS: prot4EST provides a portable EST translation solution and can be usefully applied to >95% of EST projects to improve downstream annotation. It is freely available from http://www.nematodes.org/PartiGene.

Animals↗

Mining of assembled expressed sequence tag (EST) data for protein families: application to the G protein-coupled receptor superfamily.

The availability of large expressed sequence tag (EST) databases has led to a revolution in the way new genes are identified. Mining of these databases using known protein sequences as queries is a powerful technique for discovering orthologous and paralogous genes. The scientist is often confronted, however, by an enormous amount of search output owing to the inherent redundancy of EST data. In addition, high search sensitivity often cannot be achieved using only a single member of a protein superfamily as a query. In this paper a technique for addressing both of these issues is described. Assembled EST databases are queried with every member of a protein superfamily, the results are integrated and false positives are pruned from the set. The result is a set of assemblies enriched in members of the protein superfamily under consideration. The technique is applied to the G protein-coupled receptor (GPCR) superfamily in the construction of a GPCR Resource. A novel full-length human GPCR identified from the GPCR Resource is presented, illustrating the utility of the method.

Amino Acid Sequence↗

Comparative mapping of expressed sequence tags containing microsatellites in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Comparative genomics, through the integration of genetic maps from species of interest with whole genome sequences of other species, will facilitate the identification of genes affecting phenotypes of interest. The development of microsatellite markers from expressed sequence tags will serve to increase marker densities on current salmonid genetic maps and initiate in silico comparative maps with species whose genomes have been fully sequenced. RESULTS: Eighty-nine polymorphic microsatellite markers were generated for rainbow trout of which at least 74 amplify in other salmonids. Fifty-five have been associated with functional annotation and 30 were mapped on existing genetic maps. Homologous sequences were identified for 20 of the EST containing microsatellites to identify comparative assignments within the tetraodon, mouse, and/or human genomes. CONCLUSION: The addition of microsatellite markers constructed from expressed sequence tag data will facilitate the development of high-density genetic maps for rainbow trout and comparative maps with other salmonids and better studied species.

Alleles↗

[Development of EST (Expressed Sequence Tags) Marker in Chinese Cabbage and its Transferability to Repeseed.].

28 pairs of primers were designed according to the expressed sequence tags in Chinese cabbage. After testing on the annealing temperature and the concentration of primer, dNTP and MgCl2, a suitable PCR system was established. Under the condition of reaction system developed, primers designed specific to ESTs were screened against genomic DNA of inbreed line A from which the cDNA library was constructed. Among them, 18 pairs of primers showed the amplification. Then all the primers available in line A were subjected to PCR for DNAs from 17 cabbage varieties. Polymorphism was detected by electrophoresis with agarose gel, and 10 of 18 primer sets could reveal polymorphisms among cabbage varieties, which accounted for 55.6% of primers selected. To examine the transferability of EST markers developed in cabbage, all primers were further used for PCR-mediated amplification of genomic DNA from different varieties of rapeseeds. Of 28 pairs of primers, 24 were able to produce amplified product(s) and 18 showed polymorphisms, accounting for 85.7% and 64.3% of total primers respectively. All of 18 primer sets that amplified in cabbage also showed amplified products in rapeseed and 13 of them were polymorphic. Even amongst the 10 primer sets that were unable to amplify in cabbage, 6 pairs produced amplification and 5 could reveal the polymorphism in rapeseeds. Results obtained in the present paper proved that developing polymorphic markers based on EST could be feasible and this kind of marker would be transferable to closed related species.

Brassica↗

Redundancy based detection of sequence polymorphisms in expressed sequence tag data using autoSNP.

UNLABELLED: AutoSNP is a program to detect single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (indels) in expressed sequence tag (EST) data. The program uses d2cluster and cap3 to cluster and align EST sequences, and uses redundancy to differentiate between candidate SNPs and sequence errors. Candidate polymorphisms are identified as occurring in multiple reads within an alignment. For each candidate SNP, two measures of confidence are calculated, the redundancy of the polymorphism at a SNP locus and the co segregation of the candidate SNP with other SNPs in the alignment. AVAILABILITY: The program was written in PERL and is freely available to non-commercial users by request from the authors.

Cluster Analysis↗

Generation and chromosome mapping of expressed sequence tags (ESTs) from a human infant thymus.

In an effort to identify novel genes that are expressed differentially in an infant thymus, we constructed an oligo-d(T) primed cDNA library from a human infant thymus followed by single-run partial sequencing to generate expressed sequence tags (ESTs). Characterization of more than 1400 sequences enabled us to convert human thymus transcripts into 1223 useful ESTs. These ESTs consisted of 613 (50.1%) showing homology to known human genes, 51 (4.2%) matching to genes from other species, 289 (23.6%) matching ESTs of unknown functions, and 182 (14.9%) being novel transcripts. The expression profile of an infant thymus features a high number of genes related to cell division-DNA synthesis and gene-protein expression, indicating the active growth stage of an infant thymus. To identify the chromosomal localization of 43 thymus ESTs, PCR-based mapping was performed using a human-rodent somatic cell hybrid or radiation hybrid mapping panel. The results indicated that several novel genes were determined to be located in the vicinity of previously mapped disease loci; histidinemia loci, plasminogen Tochigi disease loci, Ehlers-Danlos syndrome, hypertriglyceridemia, thyroid resistance locus, ocular albinism, galactosemia, porphyria variegata, Charcot-Marie-tooth disease, FEOM (fibrosis of extraocular muscles), Prader-Willi syndrome.

Animals↗

[Finding of new FCC1/HN antigenic expressed sequence tag(ESTs) of Plasmodium falciparum].

AIM: To sequence the strong positive clones obtained by immuno-screening of Plasmodium falciparum FCC1/HN lambda gt11 cDNA expression library, and to elucidate the antigenic expressed sequence tags through sequencing the cDNA insert of these positive clones, and new antigenic ESTs could serve as a resource to pursue their corresponding antigen genes. METHODS: cDNA inserts of positive lambda gt11 phage clones were amplified by PCR. The PCR products, after purification, were cloned into the M13 mp18 sequencing vector. Single-stranded M13 DNA was prepared and sequenced. Then the acquired sequences were compared in homologies with EMBL/GenBank database on the PC/GENE software system and searched in NCBI (National Center for Biotechnology Information) GenBank using BLAST (Basic Local Alignment Search Tool) commond. RESULTS: Sequence C03 was part of the known P. falciparum antigenic heat shock protein 70 (Pfhsp70) gene, while the other 5 sequences were new P. falciparum antigenic expressed sequence tags (ESTs). CONCLUSION: The 5 new antigenic ESTs generated could serve as the breaking through points in our efforts to find out new P. falciparum antigen genes.

Animals↗