Expressed sequence tag analysis of Eimeria-stimulated intestinal intraepithelial lymphocytes in chickens

Intraepithelial lymphocytes (IELs) play a critical role in protective immune response to intestinal pathogens such as Eimeria, the etiologic agent of avian coccidiosis. A list of genes expressed by intestinal IELs of Eimeria-infected chickens was compiled using the expressed sequence tag (EST) strategy. The 14,409 ESTs consisted of 1851 clusters and 7595 singletons, which revealed 9446 unique genes in the data set. Comparison of the sequence data with chicken DNA sequences in GenBank identified 125 novel clones. This EST library will provide a valuable resource for profiling global gene expression in normal and pathogen-infected chickens and identifying additional unique immune-related genes.     

A survey of genes in Eimeria tenella merozoites by EST sequencing

A study of about 500 expressed sequence tags (ESTs), derived from a merozoite cDNA library, was initiated as an approach to generate a larger pool of gene information on Eimeria tenella. Of the ESTs, 47.7% had matches with entries in the databases, including ribosomal proteins, metabolic enzymes and proteins with other functions, of which 14.3% represented previously known E. tenella genes. Thus over 50% of the ESTs had no significant database matches. The E. tenella EST dataset contained a range of highly abundant genes comparable with that found in the EST dataset of T. gondii and may thus reflect the importance of such molecules in the biology of the apicomplexan organisms. However, comparison of the two datasets revealed very few homologies between sequences of apical organelle molecules, and provides evidence for sequence divergence between these closely-related parasites. The data presented underpin the potential value of the EST strategy for the discovery of novel genes and may allow for a more rapid increase in the knowledge and understanding of gene expression in the merozoite life cycle stage of Eimeria spp.     

Conservation and change in structural and 5′ flanking sequences of esterase 6 in siblingDrosophila species

Esterase 6 (Est-6/EST6) is the major β-carboxylesterase inD. melanogaster and its siblingsD. simulans andD. mauritiana. It is expressed in several tissues but its major site of expression is the sperm ejaculatory duct of the adult male. Although EST6 activity affects reproductive fitness, there are high levels of electrophoretic and activity polymorphism, at least withinD. melanogaster andD. simulans. Here we present the nucleotide sequences of anEst-6 allele and its flanking regions from each ofD. simulans andD. mauritiana and compare them with the publishedD. melanogaster sequences. As might be expected, replacement sites are significantly less divergent than exon silent sites in all comparisons, suggesting that selection is acting to maintain EST6 structure and function among the three species. Nevertheless, the ratio of the levels of replacement to silent site divergence is still much higher forEst-6 than for seven of ten other genes (including both isozyme-coding loci) for which comparable data have been published for these species. This is consistent with the high levels of EST6 electrophoretic polymorphism withinD. melanogaster andD. simulans and implies that selective constraints against amino acid change are relatively weak for EST6. By contrast, comparisons involving promotor sequences show that the level of divergence in the first 350bp 5′ of the gene is significantly lower than those for four of the six other loci for which comparable data have been published for these species. In particular, there are two perfectly conserved stretches (−1 to −158bp and −219 to −334bp) each over 100bp long included in this 350bp region. Thus the data suggest a relatively low level of selective constraint on the amino acid sequence of EST6 but a relatively high level of constraint on sequences affecting aspects of its expression.     

A comparative genomic analysis of ESTs from <Emphasis Type="Italic">Ustilago maydis</Emphasis>

A large-scale comparative genomic analysis of unisequence sets obtained from an Ustilago maydis EST collection was performed against publicly available EST and genomic sequence datasets from 21 species. We annotated 70% of the collection based on similarity to known sequences and recognized protein signatures. Distinct grouping of the ESTs, defined by the presence or absence of similar sequences in the species examined, allowed the identification of U. maydis sequences present only (1) in fungal species, (2) in plants but not animals, (3) in animals but not plants, or (4) in all three eukaryotic lineages assessed. We also identified 215 U. maydis genes that are found in the ascomycete but not in the basidiomycete genome sequences searched. Candidate genes were identified for further functional characterization. These include 167 basidiomycete-specific sequences, 58 fungal pathogen-specific sequences (including 37 basidiomycete pathogen-specific sequences), and 18 plant pathogen-specific sequences, as well as two sequences present only in other plant pathogen and plant species.Supplemental Excel Table 1 used for analysis and the derivation of Fig. 3 as well as supplemental Tables 2 and 3 are available at All ESTs used in this analysis have been submitted to GenBank. The accession numbers are CF638289–CF645747, CF663122–CF663127, and CD487847–CD490309 (Supplemental Table 3)     

Identification and characterization of new plant microRNAs using EST analysis

Seventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST databases by comparing all previously known Arabidopsis miRNAs to GenBank‘s plant EST databases with BLAST algorithms. After removing the EST sequences with high numbers (more than 2) of mismatched nucleotides, a total of 812 EST contigs were identified. After predicting and scoring the RNA secondary structure of the 812 EST sequences using mFold software, 338 new potential miRNAs were identified in 60 plant species, miRNAs are widespread. Some microRNAsmay highly conserve in the plant kingdom, and they may have the same ancestor in very early evolution. There is no nucleotide substitution in most miRNAs among many plant species. Some of the new identified potential miRNAs may be induced and regulated by environmental biotic and abiotic stresses. Some may be preferentially expressed in specific tissues, and are regulated by developmental switching. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes. A large number of miRNAs exist in different plant species and play important roles in plant developmental switching and plant responses to environmental abiotic and biotic stresses as well as signal transduction. Environmental stresses and developmental switching may be the signals for synthesis and regulation of miRNAs in plants. A model for miRNA induction and expression, and gene regulation by miRNA is hypothesized.     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

The neuropeptidomics of Ixodes scapularis synganglion

Ticks (Ixodoidea) likely transmit the greatest variety of human and animal pathogens of any arthropod vector. Despite their medical significance little data is available about the messenger molecules in the central nervous system that coordinate all physiological processes in these animals, including behaviour. In our study, we performed the first comprehensive neuropeptidomic analysis of a tick species by using MALDI-TOF mass spectrometry. Specifically we analyzed the neuropeptides in the synganglion of Ixodes scapularis. The forthcoming sequence of the genome of this species will represent the first genomic analysis of a member of the large subphylum Chelicerata. For our approach we used information from predicted neuropeptide precursor sequences found in EST databases [Christie, AE. Neuropeptide discovery in Ixodoidea: an in silico investigation using publicly accessible expressed sequence tags. Gen Comp Endocrinol 2008;157:174–185] as well as data obtained by complete de novo sequencing. The direct tissue profiling yielded 20 neuropeptides from 12 neuropeptide precursors. The sequences of these neuropeptides are not as unique as predicted; a comparison with the peptidome of other invertebrates shows a close relationship with insect neuropeptides. This work will provide a resource for studying tick neurobiology and will hopefully also help to identify novel targets for tick and tick-borne disease control.     

Lengths and nucleotide sequences of the internal spacers of nuclear ribosomal DNA in gymnosperms and pteridophytes

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA were analyzed in species belonging to gymnosperms and pteridophytes. The lengths of the ITSs of sixteen species of gymnosperms and seven species of pteridophytes were estimated. The gymnosperms have ITS1 regions larger than those observed in the pteridophytes and angiosperms (ca. 610–3100 bp versus 159–360 bp). On the other hand, the ITS2 regions appear to be of a conserved length (182–370 bp). We have determined the complete nucleotide sequences of ITS regions from four gymnosperm species and five pteridophyte species by cloning the PCR products. Sequence analysis showed the presence of three short tandem arranged subrepeats of about 70 bp in the 1112 bp ITS1 ofEphedra fragilis. Pyrimidine rich (about 90%) DNA segments of 40–50 bp were observed in the ITS1 ofGinkgo biloba. A highly conserved 16 bp long sequence known to be present in the ITS1 of the angiosperm species has been also found in the ITS1 ofCycas revoluta, Taxus baccata andEphedra fragilis. Dedicated to Prof.Emilio Battaglia.     

Integrative approaches to determining Csl function

While there is an ever-increasing amount of information regarding cellulose synthase catalytic subunits (CesA) and their role in the formation of the cell wall, the remainder of the enzymes that synthesize structural cell wall polysaccharides are unknown. The completion of the Arabidopsis genome and the wealth of the sequence information from other plant genome projects provide a rich resource for determining the identity of these enzymes. Arabidopsis contains six families of genes related to cellulose synthase, the cellulose synthase-like (Csl) genes. Our laboratory is taking a multidisciplinary approach to determine the function of the Csl genes, incorporating genomic, genetic and biochemical data. Information from expressed sequence tag (EST) projects has revealed the presence of Csl genes in all plant species with a significant number of ESTs. Certain Csl families appear to be missing from some species. For example, no examples of CslG ESTs have been found in rice or maize. Microarray data and reporter constructs are being used to determine the expression pattern of the CesA and Csl genes in Arabidopsis. Mutations and insertion events have been identified in a majority of the genes in the Arabidopsis CesA superfamily and are being characterized by phenotypic and biochemical analysis. While we cannot yet link the function of any of the Csl genes to their respective products, the expression and localization of these genes is consistent with the expected expression pattern of polysaccharide synthases that contribute to the primary cell wall.     

Snipping polymorphisms from large EST collections in barley (<Emphasis Type="Italic">Hordeum vulgare </Emphasis>L.)

The public EST (expressed sequence tag) databases represent an enormous but heterogeneous repository of sequences, including many from a broad selection of plant species and a wide range of distinct varieties. The significant redundancy within large EST collections makes them an attractive resource for rapid pre-selection of candidate sequence polymorphisms. Here we present a strategy that allows rapid identification of candidate SNPs in barley (Hordeum vulgare L.) using publicly available EST databases. Analysis of 271,630 EST sequences from different cDNA libraries, representing 23 different barley varieties, resulted in the generation of 56,302 tentative consensus sequences. In all, 8171 of these unigene sequences are members of clusters with six or more ESTs. By applying a novel SNP detection algorithm (SNiPpER) to these sequences, we identified 3069 candidate inter-varietal SNPs. In order to verify these candidate SNPs, we selected a small subset of 63 present in 36 ESTs. Of the 63 SNPs selected, we were able to validate 54 (86%) using a direct sequencing approach. For further verification, 28 ESTs were mapped to distinct loci within the barley genome. The polymorphism information content (PIC) and nucleotide diversity () values of the SNPs identified by the SNiPpER algorithm are significantly higher than those that were obtained by random sequencing. This demonstrates the efficiency of our strategy for SNP identification and the cost-efficient development of EST-based SNP-markers.The first two authors contributed equally to this work     

An expressed sequence tag (EST) library from developing fruits of an Hawaiian endemic mint (Stenogyne rugosa, Lamiaceae): characterization and microsatellite markers

Background  

The endemic Hawaiian mints represent a major island radiation that likely originated from hybridization between two North American polyploid lineages. In contrast with the extensive morphological and ecological diversity among taxa, ribosomal DNA sequence variation has been found to be remarkably low. In the past few years, expressed sequence tag (EST) projects on plant species have generated a vast amount of publicly available sequence data that can be mined for simple sequence repeats (SSRs). However, these EST projects have largely focused on crop or otherwise economically important plants, and so far only few studies have been published on the use of intragenic SSRs in natural plant populations. We constructed an EST library from developing fleshy nutlets of Stenogyne rugosa principally to identify genetic markers for the Hawaiian endemic mints.     

Analysis and comparison of a set of expressed sequence tags of the parthenogenetic water flea <Emphasis Type="Italic">Daphnia carinata</Emphasis>

The water flea Daphnia carinata (D. carinata) reproduces both sexually and parthenogenetically, yet little is known about the genes involved in these processes. To further clarify the reproductive biology of Daphnia and elucidate their unique mechanism of reproductive transformation, we have generated and characterized an expressed sequence tag (EST) data set from D. carinata. A set of 1,495 clusters were generated from sequencing 3,072 randomly chosen clones from a parthenogenetic, juvenile water flea cDNA library. The nucleic acid and deduced amino acid sequences were compared with known GenBank sequences. Functional annotation found that 959 clusters showed significant homology with known genes involved in a broad range of activities, including metabolism, translation, development and reproduction, as well as genes involved in sensing environmental factors. We speculate that genes involved in development and reproduction, along with genes that allow the organism to sense changes in the environment, play important roles in the process of parthenogenetic reproduction and could be markers of the early steps of sexual differentiation. Additionally, 86% of the D. Carinata unique sequences could be stringently mapped to the D. pulex genome, of which 125 mapped to intergenic and intronic regions on the current assembly. Our results provide practical insight into crustacean reproductive biology, in addition to establishing a new animal model for reproductive and developmental biology. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Xiaoqian Xu and Shuhui Song contributed equally to this work. Nucleotide sequence data reported are available in the GenBank databases under the accession numbers: GD269049−GD272045.     

Identification and cross-species amplification of EST derived SSR markers in different bamboo species

The availability of expressed sequence data derived from gene discovery programs became an alternative source of mining simple sequence repeat (SSR) and developing inexpensive genetic markers for the crop improvements. In present study, 10 express sequence tags (EST)-SSR markers were identified from Bambusa oldhamii public sequence data base. Transferability to 25 species of Bambusoideae ranged from 30% to 100%. The number of alleles detected per locus ranged from 2 to 10. All the newly identified SSR markers were found to be moderately to highly polymorphic with an average Polymorphic Information Content (PIC) value of 0.54. As these loci represents transcribed region and recorded high level of cross transferability and reliable amplification across the species, demonstrating the utility of these markers for functional and genetic analyses of bamboo species.     

Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.     

Screening of ESTs from Mytilus for the detection of SSR markers in Mytilus californianus

A set of expressed sequence tag‐simple sequence repeat (EST‐SSR) markers for the genus Mytilus was developed through bioinformatic mining of the GenBank public database. A total of 33 782 EST sequences from GenBank were downloaded and screened for di‐, tri‐ and tetranucleotide, with 1274 EST containing SSR markers. Nine microsatellite markers were characterized in Mytilus californianus with a number of alleles per locus ranging from two to six, and total observed and expected heterozygosities ranging from 0.490 to 0.730 and from 0.510 to 0.860 respectively. Cross‐species amplification was achieved in several other species, confirming the usefulness of these markers in Mytilus genetics.