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PubMed2002

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays.

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.

Acetates↗
PubMedDate not supplied

Expressed sequence tags from poplar wood tissues--a comparative analysis from multiple libraries.

Xylogenesis involves successive developmental processes--cambial division, cell expansion and differentiation, cell death--each occurring along a gradient from the cambium to the pith of the stem. Taking advantage of the high level of organisation of wood tissues, we isolated cambial zone (CZ), differentiating xylem (DX) and mature xylem (MX) from both tension wood (TW) and opposite wood (OW) of bent poplars. Four different cDNA libraries were then constructed and used to generate 10,062 EST, reflecting the genes expressed in the different wood tissues. For the most abundant clusters, the EST distributions were compared between libraries in order to identify genes specific or over-represented at some specific developmental stages. They clearly showed a developmental shift between CZ and DX, whereas there is a continuity of development between DX and MX. CZ was mainly characterized by clusters of genes involved in cell cycle, protein synthesis and fate. Interestingly, two clusters with no assigned function were found specific to the cambial zone. In DX and MX, clusters were mostly involved in methylation of lignin precursors and microtubule cytoskeleton. In addition, in DX, EST from TW and OW were compared: five clusters of arabinogalactan proteins, one for sucrose synthase and one for fructokinase were specific or over-represented in TW. Moreover, a putative transcription factor and a cluster of unknown function were also identified in DX-TW. The informative comparison of multiple libraries prepared from wood tissues led to the identification of genes--some with still unknown functions--putatively involved in xylogenesis and tension wood formation.

Cell Cycle↗
PubMed1999

Cloning of novel temperature-related expressed sequence tags in rat testis during spermatogenesis.

Spermatogenesis needs the relatively cool environment of the scrotum in most mammals, it would be arrested when the testis was exposed to abdominal temperature. In this study, we have used a differential display PCR technique (DD-PCR) to screen temperature-related ESTs during spermatogenesis (TRS) in scrotal testes through a unilateral cryptorchid rat model after in situ analysis of testis cell DNA fragmentation. We reported here the cloning and sequencing of three such ESTs: TRS1, TRS3, and TRS4. Northern blot analysis confirmed that they were expressed specifically in scrotal testes. In situ hybridization showed that TRS1 was mainly expressed in the spermatocytes and the round spermatids in scrotal testis. Homology searches revealed that TRS1 and TRS3 were unknown cDNA sequences, and TRS4 was identical to a known EST whose function had not been reported. TRS1, TRS2, and TRS3 were first found to be temperature-related during spermatogenesis.

Animals↗
PubMed2001

Genomic characterization of human SEC14L1 splice variants within a 17q25 candidate tumor suppressor gene region and identification of an unrelated embedded expressed sequence tag.

Human SEC14L1 shows partial sequence homology to the budding yeast SEC14 protein and the Japanese flying squid retinal-binding protein and was previously generally localized to 17q25. We more precisely mapped SEC14L1 within a discrete region of 17q25 that likely harbors at least one putative breast and ovarian tumor suppressor gene. We determined that this gene consists of 18 exons ranging in size from 70 bp (exon 11) to 3088 bp (exon 17) and spanning at least 58 kb of DNA. Exon 17 contained a highly polymorphic variable number of tandem repeats (VNTR) and was present only in the larger ubiquitously expressed 5.5-kb transcript. The 3.0-kb ubiquitously expressed transcript included sequences at the beginning of exon 17 (designated exon 17a) and the end of exon 17 (designated exon 18), but lacked the internal 2439 bp of exon 17, including the VNTR. This alternative splicing resulted in a predicted protein of 719 residues from the smaller transcript with four more terminal amino acids than the 715 residue protein predicted from the larger transcript. EST H49244 spanned exon 11 of SEC14L1 and was specifically expressed in human peripheral blood leukocytes. One intragenic single nucleotide polymorphism (SNP) was confirmed. SEC14L1 contained the CRAL/TRIO domain also found in alpha-tocopherol transfer protein (TTPA) and cellular retinaldehyde-binding protein (CRALBP). As retinoids have been shown to inhibit the growth of breast cancer cells, loss of the proposed SEC14L1 retinal-binding function may contribute to breast tumorigenesis. As TTPA and CRALBP have been implicated in retinitis pigmentosa (RP), altered SEC14L1 expression may contribute to RP in previously unlinked families. Coding exon-specific PCR primers were designed to aid in future expression and mutational analyses.

Adult↗
PubMed2005

Expressed sequence tags from Diabrotica virgifera virgifera midgut identify a coleopteran cadherin and a diversity of cathepsins.

The Western corn rootworm is the major pest of corn in the USA and has recently become the target for insect-resistant transgenic crops. Transgenic crops have switched the focus for identifying insecticide targets from the insect nervous system to the midgut. Here we describe a collection of 691 sequences from the Western corn rootworm midgut, 27% of which predict proteins with no matches in current databases. Of the remaining sequences, most predict proteins with either catalytic (62%) or binding (19%) functions, as expected for proteins expressed in the insect midgut. The utility of this approach for the identification of targets for novel toxins is demonstrated by analysis of the first coleopteran cadherin gene, a putative Bt receptor, and a large class of cysteine-proteases, the cathepsins.

Amino Acid Sequence↗
PubMed2006

Expressed sequence tags from feline uterine library.

Partial nucleotide sequences of 634 cDNAs randomly isolated from a feline uterine cDNA library (Stratagene) were determined by single pass sequencing. Homology search of the sequences to the non-redundant nucleotide databases revealed that 83% of the cDNAs matched registered feline or non-feline genes. Based on the gene identifications, these genes were predicted to be related with immunological, biochemical and regulatory functions in cats. Interestingly, the rest 17% of the cDNAs did not show homology to gene or EST sequence present in the nucleotide and protein databases, suggesting that these cDNAs include novel genes expressed only in the Felidae. This large scale sequencing of uterine cDNA will provide a useful molecular source for research not only towards health and disease conditions in cats but also in different fields of science where genetic information from cats will be of interest.

Animals↗
PubMed1998

Assembly of a complete zebrafish mitochondrial 16S rRNA gene from overlapping expressed sequence tags.

A complete zebrafish mitochondrial 16S ribosomal RNA gene was assembled from our existing zebrafish EST clones by aligning them with the carp mitochondria 16S rRNA sequence. The overall homology between our assembled zebrafish mt 16S rRNA and the carp mt 16S rRNA is 83.5%. As the number of zebrafish ESTs grows, the assembly of more full-length cDNA sequences from overlapping EST data could be expected.

Animals↗
PubMed2005

Comparison of quantitative trait loci for adaptive traits between oak and chestnut based on an expressed sequence tag consensus map.

A comparative genetic and QTL mapping was performed between Quercus robur L. and Castanea sativa Mill., two major forest tree species belonging to the Fagaceae family. Oak EST-derived markers (STSs) were used to align the 12 linkage groups of the two species. Fifty-one and 45 STSs were mapped in oak and chestnut, respectively. These STSs, added to SSR markers previously mapped in both species, provided a total number of 55 orthologous molecular markers for comparative mapping within the Fagaceae family. Homeologous genomic regions identified between oak and chestnut allowed us to compare QTL positions for three important adaptive traits. Colocation of the QTL controlling the timing of bud burst was significant between the two species. However, conservation of QTL for height growth was not supported by statistical tests. No QTL for carbon isotope discrimination was conserved between the two species. Putative candidate genes for bud burst can be identified on the basis of colocations between EST-derived markers and QTL.

Adaptation, Physiological↗
PubMed2005

Expressed sequence tags analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, kidney and spleen tissues following vaccination against Vibrio anguillarum and Aeromonas salmonicida.

To investigate the response of Atlantic halibut to vaccination and pathogen exposure, a cDNA library was constructed from liver, kidney and spleen mRNA collected following vaccination against Vibrio anguillarum and Aeromonas salmonicida. After sequencing 1114 clones 1072 (96.23%) readable sequences were obtained of which 106 sequences are the first reported from the fish. Of these, 182 clones (16.98%) contained cell/organism defence genes including immunoglobulin light chain, MHC class I and II, interferon consensus sequence binding protein, B-cell receptor-associated protein, early B-cell factor, 10 complement components, heat shock protein 70 and 90, antimicrobial peptides hepcidin type 1 and 2, and CC chemokine (macrophage inflammatory protein-1 beta-like chemokine, MIP-1beta). Expression of MIP-1beta-like was elevated in the kidney and spleen at 1, 2, 7 and 14 days post vaccination. Functional genes involved in cellular processes of hematopoietic tissues were also identified. These results indicate that this cDNA library contains many important genes involved in the immune response, making it an important resource for studying the response of Atlantic halibut to vaccination or pathogen exposure.

Aeromonas salmonicida↗
PubMed2004

Evolutionary expressed sequence tag analysis of Drosophila female reproductive tracts identifies genes subjected to positive selection.

Genes whose products are involved in reproduction include some of the fastest-evolving genes found within the genomes of several organisms. Drosophila has long been used to study the function and evolutionary dynamics of genes thought to be involved in sperm competition and sexual conflict, two processes that have been hypothesized to drive the adaptive evolution of reproductive molecules. Several seminal fluid proteins (Acps) made in the Drosophila male reproductive tract show evidence of rapid adaptive evolution. To identify candidate genes in the female reproductive tract that may be involved in female-male interactions and that may thus have been subjected to adaptive evolution, we used an evolutionary bioinformatics approach to analyze sequences from a cDNA library that we have generated from Drosophila female reproductive tracts. We further demonstrate that several of these genes have been subjected to positive selection. Their expression in female reproductive tracts, presence of signal sequences/transmembrane domains, and rapid adaptive evolution indicate that they are prime candidates to encode female reproductive molecules that interact with rapidly evolving male Acps.

Animals↗
PubMed2005

Genetic linkage maps of the red flour beetle, Tribolium castaneum, based on bacterial artificial chromosomes and expressed sequence tags.

A genetic linkage map was constructed in a backcross family of the red flour beetle, Tribolium castaneum, based largely on sequences from bacterial artificial chromosome (BAC) ends and untranslated regions from random cDNA's. In most cases, dimorphisms were detected using heteroduplex or single-strand conformational polymorphism analysis after specific PCR amplification. The map incorporates a total of 424 markers, including 190 BACs and 165 cDNA's, as well as 69 genes, transposon insertion sites, sequence-tagged sites, microsatellites, and amplified fragment-length polymorphisms. Mapped loci are distributed along 571 cM, spanning all 10 linkage groups at an average marker separation of 1.3 cM. This genetic map provides a framework for positional cloning and a scaffold for integration of the emerging physical map and genome sequence assembly. The map and corresponding sequences can be accessed through BeetleBase (http://www.bioinformatics.ksu.edu/BeetleBase/).

Animals↗
PubMed1999

A large scale structural analysis of cDNAs in a unicellular green alga, Chlamydomonas reinhardtii. I. Generation of 3433 non-redundant expressed sequence tags.

To understand genetic information carried in a unicellular green alga, Chlamydomonas reinhardtii, normalized and size-selected cDNA libraries were constructed from cells at photoautotrophic growth, and a total of 11,571 5'-end sequence tags were established. These sequences were grouped into 3433 independent EST species. Similarity search against the public non-redundant protein database indicated that 817 groups showed significant similarity to registered sequences, of which 140 were of previously identified C. reinhardtii genes, but the remaining 2616 species were novel sequences. The coverage of full-length protein coding regions was estimated to be over 60%. These cDNA clones and EST sequence information will provide a powerful source for the future genome-wide functional analysis of uncharacterized genes.

Animals↗
PubMed2000

An expressed sequence tag database of T-cell-enriched activated chicken splenocytes: sequence analysis of 5251 clones.

The cDNA and gene sequences of many mammalian cytokines and their receptors are known. However, corresponding information on avian cytokines is limited due to the lack of cross-species activity at the functional level or strong homology at the molecular level. To improve the efficiency of identifying cytokines and novel chicken genes, a directionally cloned cDNA library from T-cell-enriched activated chicken splenocytes was constructed, and the partial sequence of 5251 clones was obtained. Sequence clustering indicates that 2357 (42%) of the clones are present as a single copy, and 2961 are distinct clones, demonstrating the high level of complexity of this library. Comparisons of the sequence data with known DNA sequences in GenBank indicate that approximately 25% of the clones match known chicken genes, 39% have similarity to known genes in other species, and 11% had no match to any sequence in the database. Several previously uncharacterized chicken cytokines and their receptors were present in our library. This collection provides a useful database for cataloging genes expressed in T cells and a valuable resource for future investigations of gene expression in avian immunology. A chicken EST Web site (http://udgenome. ags.udel. edu/chickest/chick.htm) has been created to provide access to the data, and a set of unique sequences has been deposited with GenBank (Accession Nos. AI979741-AI982511). Our new Web site (http://www. chickest.udel.edu) will be active as of March 3, 2000, and will also provide keyword-searching capabilities for BLASTX and BLASTN hits of all our clones.

Animals↗
PubMed2004

Characterization of the protein processing and secretion pathways in a comprehensive set of expressed sequence tags from Trichoderma reesei.

Trichoderma reesei is a filamentous fungus widely used as an efficient protein producer and known to secrete large quantities of biomass degrading enzymes. Much work has been done aimed at improving the secretion efficiency of this fungus. It is generally accepted that the major bottlenecks in secretion are protein folding and ornamentation steps in this pathway. In an attempt to identify genes involved in these steps, the 5' ends of 21888 cDNA clones were sequenced from which a unique set of over 5000 were also 3' sequenced. Using annotation tools Gene Ontology terms were assigned to 2732 of the sequences. Homologs to the majority of Aspergillus niger's Srg genes as well as a number of homologs to genes involved in protein folding and ornamentation pathways were identified.

Computational Biology↗