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PubMed1998

Genetic variability in white leghorns revealed by chicken liver expressed sequence tags.

A total of 92 expressed sequence tags from chicken liver (CLEST) were searched for homology with known genes. Among the CLEST, 29% had no sequence similarities with known genes, 34% showed sequence similarity to rRNA, 9% to mitochondrial genes, 23% to known nuclear genes, and 5% to human expressed sequence tags. Among the nuclear CLEST (excluding rRNA), clones with sequence similarity to aldolase B were represented four times, whereas all the other clones represented unique genes. The presence of MspI and TaqI restriction fragment length polymorphisms (RFLP) associated with CLEST were analyzed by bulk Southern blotting in 16 strains of White Leghorn chickens derived from five different genetic bases. No RFLP were observed with rRNA CLEST and a single MspI RFLP was observed with mitochondrial CLEST. The nuclear CLEST with sequence similarity to known nuclear genes were grouped into two classes on the basis of their involvement in intermediary metabolism. Among the nine genes coding for metabolic enzymes, all but one were polymorphic at MspI and/or TaqI sites in at least one of the strains, whereas among the other genes six of nine were polymorphic. The average frequency of clones revealing RFLP per cDNA clone and restriction enzyme for the two classes were 0.7 and 0.3, respectively. The analysis indicated that in White Leghorns, RFLP markers in the vicinity of nuclear CLEST are relatively frequent. Further, RFLP in the vicinity of genes coding for metabolic enzymes were significantly more frequent than near genes coding for other proteins.

Animals↗
PubMed2004

Profiling of gender-specific gene expression for Trichostrongylus vitrinus (Nematoda: Strongylida) by microarray analysis of expressed sequence tag libraries constructed by suppressive-subtractive hybridisation.

Gender-specific gene expression in Trichostrongylus vitrinus (order Strongylida) was investigated by constructing male- and female-specific gene archives using a suppressive-subtractive hybridisation approach, sequencing of expressed sequence tags from these archives, comparison with genes of Caenorhabditis elegans and other organisms, and expression profiling of a representative subset of 716 expressed sequence tags by microarray and macroarray analysis. Of these T. vitrinus expressed sequence tags, 391 had sequence homology to C. elegans genes. Of the remaining expressed sequence tags, 62 had homology to genes of other species of parasitic nematodes, and 263 expressed sequence tags had no significant homology. Expression profiling showed gender-specific expression for 561 of the 716 T. vitrinus expressed sequence tags. Male-specific protein kinases and protein phosphatases, major sperm proteins and enzymes involved in carbohydrate metabolism were abundant in the cDNA archive. Female-specific vitellogenins, heat-shock proteins and chaperonins were also highly represented. Genes involved in a number of cellular processes, such as ubiquitination and proteasome function, gene transcription, cell signalling, protein-protein interactions and chromatin assembly and function were also expressed in a gender-specific manner. The potential roles of these genes in gametogenesis, embryogenesis and reproduction in the parasitic nematode are discussed in relation to the known roles of their homologues in C. elegans.

Animals↗
PubMed1994

Expressed sequence tags (EST) identify genes preferentially expressed in catfish chemosensory tissues.

Expressed sequence tags (ESTs) for the catfish (Ictalurus punctatus) were identified and characterized by shotgun sequencing coupled to Northern analysis. We have identified and characterized a number of cDNA clones from a catfish olfactory mucosal library that show differential tissue expression including several that are enriched in chemosensory tissue. Among the novel cDNA clones studied were an olfactory specific beta-tubulin and a novel member of the S-100 family of calcium-binding proteins that is highly expressed in barbel, olfactory mucosa and gill, but not in brain. Several clones of low abundance mRNAs were also identified, including one manifesting a basic-helix-loop-helix (b-HLH) motif that is typical of many transcription factors. Additional cDNA clones whose mRNAs are differentially expressed, but are of unknown function, were also obtained. These results demonstrate the case with which novel gene products enriched in chemosensory tissues can be identified.

Amino Acid Sequence↗
PubMed2001

Comparative expressed-sequence-tag analysis of differential gene expression profiles in BmNPV-infected BmN cells.

To compare the gene expression profiles of uninfected and Bombyx mori nucleopolyhedrovirus (BmNPV)-infected BmN cells, we constructed four cDNA libraries for mock-infected cells, and cells at 2, 6, and 12 h postinfection (h.p.i.). A total of 2645 partial sequences obtained for expressed-sequence-tags (ESTs) from the libraries were categorized using BLAST searches of the public database and the BmNPV genome sequence. The following proportions of BmNPV-derived ESTs were observed: 0.4, 4.5, and 57% at 2, 6, and 12 h.p.i, respectively. Moreover, 31 BmNPV open reading frames (ORFs) were newly identified for transcripts and the baculovirus-repeated ORFs (bro) showed the highest levels of expression in the 12 h.p.i. library. Most of the host genes decreased in number as the infection progressed. However, several, including cytochrome c oxidase 1, increased in the late stages of infection. Two apoptosis-related host genes were also identified.

Amino Acid Sequence↗
PubMed2000

Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis.

Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest. The human zeta class glutathione transferase GSTZ1 has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic. This report identifies three GSTZ1 alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region, GSTZ1*A-A94A124; GSTZ1*B-A94G124; GSTZ1*C-G94G124. Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 for Z1*A, Z1*B and Z1*C, respectively. These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32 to Glu and Arg42 to Gly substitutions, respectively. The variant proteins were expressed in Escherichia coli as 6X His-tagged proteins and purified by Ni-agarose column chromatography. Examination of the activities of recombinant proteins revealed that GSTZ1a-1a displayed differences in activity towards several substrates compared with GSTZ1b-1b and GSTZ1c-1c, including 3.6-fold higher activity towards dichloroacetate. This report demonstrates the discovery of a functional polymorphism by analysis of the EST database.

Acetates↗
PubMed2004

Expressed sequence tags: medium-throughput protocols.

Generating expressed sequence tags is a simple, cheap, and efficient way to sample the genome of a target organism. An expressed sequence tag (EST) is a single-pass sequence derived from a single complementary DNA (cDNA) clone, and the sequence serves to identify the gene from which it derives. We present a set of tested laboratory protocols for setting up and performing an EST analysis of any chosen species. These medium-throughput protocols do not require dedicated genomics equipment, such as robots, and focus on the use of microtiter plates and multichannels. Using these protocols, a single competent research worker should be able to generate 2000 ESTs in 1 mo. In a nonnormalized library, these 2000 ESTs should identify between 1000 and 1500 different genes, and thus possibly between 10 and 20% of the genes of any target parasite.

Base Sequence↗
PubMed2003

Comparative mapping in the pig: localization of 214 expressed sequence tags.

In total, 214 ESTs (Expressed Sequence Tags) were assigned to the porcine gene map by using somatic cell hybrid mapping, radiation hybrid mapping, and FISH. The ESTs were isolated from a porcine small intestine cDNA library on the basis of significant sequence identity with human annotated genes. In total, 390 primer pairs were designed primarily in the 3' UTR of the sequences. Overall, 58.6% of the ESTs were successfully mapped by this approach. In total, 191 of the localizations are in agreement with the human comparative map, strongly indicating that these represent true orthologous genes. The remaining 23 ESTs provide new comparative mapping data, which should be considered as preliminary until confirmed by other studies. Our mapping efforts provide a significant contribution to the porcine map as well as to the comparative map for human and pig.

Animals↗
PubMed2001

Identification of novel glutathione transferases and polymorphic variants by expressed sequence tag database analysis.

The human expressed sequence tag (EST) database can be searched by different sequence alignment strategies to identify new members of gene families and allelic variants. To illustrate the value of database analysis for gene discovery, we have focused on the glutathione S-transferase (GST) super family, an approach that has led to the identification of the Zeta class. The Zeta class GSTs catalyze the glutathione-dependent biotransformation of alpha-haloacids and the isomerization of maleylacetoacetic acid to fumarylacetoacetic acid, an essential step in the catabolism of tyrosine. Allelic variants of the GST Z1 and GST A2 genes have also been identified by EST database analysis. One GST Z1 variant (GST Z1A) has significantly higher activity with dichloroacetic acid as a substrate than other GST Z1 isoforms. This variant may be important in the clinical treatment of lactic acidosis where dichloroacetic acid is prescribed. Our experience with the application of EST database searching methods suggests that it may be productively applied to other gene families of pharmacogenetic interest.

Amino Acid Sequence↗
PubMed2006

Use of a large-scale Triticeae expressed sequence tag resource to reveal gene expression profiles in hexaploid wheat (Triticum aestivum L.).

The US Wheat Genome Project, funded by the National Science Foundation, developed the first large public Triticeae expressed sequence tag (EST) resource. Altogether, 116,272 ESTs were produced, comprising 100,674 5' ESTs and 15 598 3' ESTs. These ESTs were derived from 42 cDNA libraries, which were created from hexaploid bread wheat (Triticum aestivum L.) and its close relatives, including diploid wheat (T. monococcum L. and Aegilops speltoides L.), tetraploid wheat (T. turgidum L.), and rye (Secale cereale L.), using tissues collected from various stages of plant growth and development and under diverse regimes of abiotic and biotic stress treatments. ESTs were assembled into 18,876 contigs and 23,034 singletons, or 41,910 wheat unigenes. Over 90% of the contigs contained fewer than 10 EST members, implying that the ESTs represented a diverse selection of genes and that genes expressed at low and moderate to high levels were well sampled. Statistical methods were used to study the correlation of gene expression patterns, based on the ESTs clustered in the 1536 contigs that contained at least 10 5' EST members and thus representing the most abundant genes expressed in wheat. Analysis further identified genes in wheat that were significantly upregulated (p < 0.05) in tissues under various abiotic stresses when compared with control tissues. Though the function annotation cannot be assigned for many of these genes, it is likely that they play a role associated with the stress response. This study predicted the possible functionality for 4% of total wheat unigenes, which leaves the remaining 96% with their functional roles and expression patterns largely unknown. Nonetheless, the EST data generated in this project provide a diverse and rich source for gene discovery in wheat.

Cluster Analysis↗
PubMed1999

Expressed-sequence-tag approach to identify differentially expressed genes following peripheral nerve axotomy.

Gene expression profiles in the rat hypoglossal nucleus after axotomy were demonstrated using expressed-sequence-tag (EST) approach. To demonstrate the gene-expression profiles after axotomy, nerve-transected hypoglossal nuclei were dissected and collected from about 1000 rats, with which a cDNA library was constructed. More than 750 clones were sub-cloned and sequenced from the library. The clones which hit frequently are likely to be associated with mitochondrial respiratory chain, cytoskeletal protein and protein synthesis. One hundred three clones from among the sequenced clones were further processed for histological screening using unilateral-hypoglossal nerve-transected brain sections by in situ hybridization histochemistry. In situ hybridization study revealed that 26% of clones examined showed upregulated expression of mRNA in response to axotomy. They included genes encoding proteins associated with glucose, lipid and protein metabolism, cytoskeleton, neurotransmission and immune reaction. The present EST analysis may have an advantage in targeting genes which are associated with nerve injury with a good efficacy, as compared with other methods such as differential display and subtraction.

Animals↗
PubMed2006

Comparative analysis of expressed sequence tags (ESTs) of ginseng leaf.

The expressed sequence tags (ESTs) referenced in this report are the first transcriptomes in a leaf from a half-shade ginseng plant. A cDNA library was constructed from samples of the leaves of 4-year-old Panax ginseng plants, which were cultured in a field. The 2,896 P. ginseng cDNA clones represent 1,576 unique sequences, consisting of 1,167 singletons and 409 contig sequences. BLAST comparisons of the cDNAs in GenBank's non-redundant databases revealed that 2,579 of the 2,896 cDNAs (89.1%) exhibited a high degree of sequence homology to genes from other organisms. The majority of the identified transcripts were found to be genes related with energy, metabolism, subcellular localization, and protein synthesis and transport. The chlorophyll a/b-binding protein ESTs in the ginseng leaf samples manifested a substantially higher level of expression than was observed in other plant leaves. The ESTs involved in ginsenoside biosynthesis were also identified and discussed.

Expressed Sequence Tags↗
PubMed2003

Gene identification and expression analysis of 86,136 Expressed Sequence Tags (EST) from the rice genome.

Expressed Sequence Tag (EST) analysis has pioneered genome-wide gene discovery and expression profiling. In order to establish a gene expression index in the rice cultivar indica, we sequenced and analyzed 86,136 ESTs from nine rice cDNA libraries from the super hybrid cultivar LYP9 and its parental cultivars. We assembled these ESTs into 13,232 contigs and leave 8,976 singletons. Overall, 7,497 sequences were found similar to existing sequences in GenBank and 14,711 are novel. These sequences are classified by molecular function, biological process and pathways according to the Gene Ontology. We compared our sequenced ESTs with the publicly available 95,000 ESTs from japonica, and found little sequence variation, despite the large difference between genome sequences. We then assembled the combined 173,000 rice ESTs for further analysis. Using the pooled ESTs, we compared gene expression in metabolism pathway between rice and Arabidopsis according to KEGG. We further profiled gene expression patterns in different tissues, developmental stages, and in a conditional sterile mutant, after checking the libraries are comparable by means of sequence coverage. We also identified some possible library specific genes and a number of enzymes and transcription factors that contribute to rice development.

Arabidopsis↗
PubMed2004

A pilot-scale expressed sequence tag analysis of Beauveria bassiana gene expression reveals a tripeptidyl peptidase that is differentially expressed in vivo.

The entomopathogen Beauveria bassiana is a dimorphic fungus that displays an in vivo-specific, yeast-like parasitic phase. In order to study the transcriptome of B. bassiana during this unique developmental phase, we developed a method to harvest in vivo B. bassiana cells from infected Manduca sexta larvae. The infected hemolymph was collected just prior to insect death and subjected to gradient centrifugation, which allowed for separation of the B. bassiana in vivo-produced cells from remaining insect hemocytes. Total RNA was extracted from the harvested fungal cells and used to construct a cDNA library that is representative of B. bassiana gene expression in vivo. Expressed Sequence Tags (ESTs) were generated and led to the cloning of two protease genes. One of these proteases was identified as a tripeptidyl peptidase (Bb TPP). The Bb TPP protease was shown to be up-regulated during infection, and identification of a signal peptide suggested that the enzyme is secreted in the host hemolymph. Although its activity and role have yet to be characterized, the Bb TPP protease appears as a likely candidate for being involved in B. bassiana pathogenesis. The identification of this novel, up-regulated protease also suggests that random sequencing from our in vivo cDNA library may be a valuable step towards identifying biologically active metabolites produced in vivo by B. bassiana.

Aminopeptidases↗
PubMed1999

Human cochlear expressed sequence tags provide insight into cochlear gene expression and identify candidate genes for deafness.

To identify candidate genes for human hearing disorders and to understand better human hearing at the molecular level, we constructed a human cochlear cDNA library. An aliquot of the unsubtracted cochlear library was contributed to the IMAGE Consortium at Lawrence Livermore National Laboratory for the generation of expressed sequence tags (ESTs) by the Merck/WashU EST project. Over 4000 ESTs were developed from the cochlear cDNA library and deposited in the GenBank EST database. Sequence clustering shows that the majority of clones are in low copy numbers, demonstrating the high complexity of the library. The sequences of 1388 cochlear ESTs (33%) match 517 known human genes. Among these are genes previously shown to cause both syndromic and non-syndromic hearing loss. A number of the cochlear ESTs show high homology to non-human genes, suggesting new gene family members or human homologs of animal genes. We also report the chromosomal map positions of 437 cochlear ESTs. These provide positional candidate genes for 18 different non-syndromic hearing disorders. A Human Cochlear EST Database web site (http://www.bwh.partners. org/pathology ) has been created to provide access to the cochlear clone data for gene discovery investigations.

Animals↗
PubMed2003

Expressed sequence tag survey of gene expression in the scab mite Psoroptes ovis--allergens, proteases and free-radical scavengers.

Psoroptes ovis, the causative agent of sheep scab, is an important ectoparasitic mite infecting sheep, goats and cattle. Infection is characterized by an extensive dermatitis, scab formation and intense itching. Initial focal lesions spread outwards, coalesce and may extend over the whole body. The host response to infestation has all the characteristics of an immediate-type hypersensitivity reaction but the mite antigens and allergens which initiate this response are almost completely undefined. Here, 507 randomly selected cDNAs derived from a mixed population of P. ovis were sequenced and the resultant nucleotide sequences subjected to Cluster analysis and Blast searches. This analysis yielded 280 clusters of which 49 had > 1 sequence with 24 showing significant Blast X homology to another protein in the databases. There were 231 sequences which appeared on one occasion and 109 of these showed significant Blast X homology to other sequences in the databases. This analysis identified homologues of 9 different types of allergens which have been characterized in other allergic conditions such as responses to house dust mites. It also identified a number of cysteine proteases which may contribute to lesion development as well as several free-radical scavenging enzymes which may protect the mite from host immune effector responses.

Allergens↗
PubMed2004

Estimating and comparing the rates of gene discovery and expressed sequence tag (EST) frequencies in EST surveys.

MOTIVATION: Expressed sequence tag (EST) surveys are an efficient way to characterize large numbers of genes from an organism. The rate of gene discovery in an EST survey depends on the degree of redundancy of the cDNA libraries from which sequences are obtained. However, few statistical methods have been developed to assess and compare redundancies of various libraries from preliminary EST surveys. RESULTS: We consider statistics for the comparison of EST libraries based upon the frequencies with which genes occur in subsamples of reads. These measures are useful in determining which one of several libraries is more likely to yield new genes in future reads and what proportion of additional reads one might want to take from the libraries in order to be likely to obtain new genes. One approach is to compare single sample measures that have been successfully used in species estimation problems, such as coverage of a library, defined as the proportion of the library that is represented in the given sample of reads. Another single library measure is an estimate of the expected number of additional genes that will be found in a new sample of reads. We also propose statistics that jointly use data from all the libraries. Analogous formulas for coverage and the expected numbers of new genes are presented. These measures consider coverage in a single library based upon reads from all libraries and similarly, the expected numbers of new genes that will be discovered by taking reads from all libraries with fixed proportions. Together, the statistics presented provide useful comparative measures for the libraries that can be used to guide sampling from each of the libraries to maximize the rate of gene discovery. Finally, we present tests for whether genes are equally represented or expressed in a set of libraries. Binomial and chi2 tests are presented for gene-by-gene comparisons of expression. Overall tests of the equality of proportional representation are presented and multiple comparisons issues are addressed. These methods can be used to evaluate changes in gene expression reflected in the composition of EST libraries prepared from different tissue types or cells exposed to different environmental conditions. AVAILABILITY: Software will be made available at http://www.mathstat.dal.ca/~tsusko

Algorithms↗