Intellectual property rights in genes and gene fragments: a registration solution for expressed sequence tags.
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Biotechnology↗
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Developing microsatellite markers from cDNA: a tool for adding expressed sequence tags to the genetic linkage map of the chicken.
A chicken embryonic cDNA library was screened with a (TG)13 probe in order to develop polymorphic microsatellite markers. The redundancy of the embryonic cDNA library with a chicken brain cDNA library, which was used for microsatellite development in a previous study, was extremely high. Of the 300 (TG)13 positive clones, only 80 were unique for the embryonic cDNA library. Still, nine expressed sequences derived from the embryonic cDNA library were mapped in the Wageningen (WAU) resource population. In addition seven microsatellite markers from the chicken brain cDNA library, which were monomorphic or unlinked in the two international reference families in the previous study, were also mapped in the WAU population. Three of the 16 mapped chicken expressed sequence tags (ESTs) showed relatively high percentages of sequence similarity to sequences found in other species. As two of these genes, RAB6 and ZFX/ZFY, have been mapped in humans, they contribute to the comparative map of the chicken.
Animals↗
Expressed sequence tags of Chinese cabbage flower bud cDNA.
We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.
Cloning, Molecular↗
Expressed Sequence Tags from Developing Castor Seeds.
To expand the availability of genes encoding enzymes and structural proteins associated with storage lipid synthesis and deposition, partial nucleotide sequences, or expressed sequence tags (ESTs), were obtained for 743 cDNA clones derived from developing seeds of castor (Ricinus communis L.). Enrichment for seed-specific cDNA clones was obtained by selecting clones that did not detectably hybridize to first-strand cDNA from leaf mRNA. Similarly, clones that hybridized to storage proteins or other highly abundant mRNA species from developing seeds were selected against. To enrich for endomembrane-associated proteins, some clones were selected for sequencing by immunological screening with antibodies prepared against partially purified endoplasmic reticulum membranes. Comparison of the deduced amino acid sequences of the ESTs with the public data bases resulted in the assignment of putative identities of 49% of the clones selected by differential hybridization and 71% of the clones selected by immunological screening. Open reading frames in 100 of the ESTs exhibited higher homology to 78 different nonplant gene products than to any previously known plant gene product.
Journal Article↗
Use of expressed sequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillus nidulans.
The use of microarrays in the analysis of gene expression is becoming widespread for many organisms, including yeast. However, although the genomes of a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here, we describe the construction and validation of microarrays for the fungus Aspergillus nidulans using PCR products from a 4092 EST conidial germination library. An experiment was designed to validate these arrays by monitoring the expression profiles of known genes following the addition of 1% (w/v) glucose to wild-type A. nidulans cultures grown to mid-exponential phase in Vogel's minimal medium with ethanol as the sole carbon source. The profiles of genes showing statistically significant differential expression following the glucose up-shift are presented and an assessment of the quality and reproducibility of the A. nidulans arrays discussed.
Aspergillus nidulans↗
Chromosomal assignment of 38 human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels.
We have localized 38 human brain cDNA sequences to individual human chromosomes. PCR primers were designed from expressed sequence tags and tested for specific amplification from human genomic DNA. The sizes of amplification products from DNA of somatic cell hybrid mapping panels were determined electrophoretically using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis.
Animals↗
Expressed sequence tags identify a human isolog of the suil translation initiation factor.
The complete cDNA sequence of a human isolog of the yeast suil translation initiation factor gene was obtained by assembling over 40 expressed sequence tags (ESTs) for this gene obtained from a variety of tissue-specific cDNA libraries. The human suilisol gene product is a 113 amino-acid polypeptide similar to proteins known from yeast, rice, mosquito, and Methanococcus. The identification of suilisol illustrates the utility of assemblies of independent ESTs for deriving full-length cDNA sequences for new human genes.
Amino Acid Sequence↗
Expressed sequence tag analysis of the bradyzoite stage of Toxoplasma gondii: identification of developmentally regulated genes.
Toxoplasma gondii is a protozoan parasite responsible for widespread infections in humans and animals. Two major asexual forms are produced during the life cycle of this parasite: the rapidly dividing tachyzoite and the more slowly dividing, encysted bradyzoite. To further study the differentiation between these two forms, we have generated a large number of expressed sequence tags (ESTs) from both asexual stages. Previously, we obtained data on approximately 7,400 ESTs from tachyzoites (J. Ajioka et al., Genome Res. 8:18-28, 1998). Here, we report the results from analysis of approximately 2,500 ESTs from bradyzoites purified from the cysts of infected mice. We also report the results from analysis of 760 ESTs from parasites induced to differentiate from tachyzoites to bradyzoites in vitro. Comparison of the data sets from bradyzoites and tachyzoites reveals many previously uncharacterized sequence clusters which are largely or completely specific to one or other developmental stage. This class includes a bradyzoite-specific form of enolase. Combined with the previously identified bradyzoite-specific form of lactate dehydrogenase, this finding suggests significant differences in flux through the lower end of the glycolytic pathway in this stage. Thus, the generation of this data set provides valuable insights into the metabolism and growth of the parasite in the encysted form and represents a substantial body of information for further study of development in Toxoplasma.
Amino Acid Sequence↗
[Expressed sequence tags (ESTs) analysis of the oral gland of Lampetra japonica].
A cDNA library (2.1 x 10(6) pfu/mL) was constructed from the oral gland of Lampetra japonica. After random selection of clones for sequencing, 1 323 clones with inserts longer than 100 bp and with good chromato-grams were obtained. Using BlastX and BlastN programs, We found 653 ESTs (49.36%) that shared significant homology with known sequences in protein or nucleotide databases of NCBI, including 328 ESTs homologous to known sequences of Petromazonidae animals. These ESTs were classified into 11 different functional categories,the highest proportion of which was related to protein synthesis. Out of the 1323 ESTs, 162 non-redundant contigs were assembled from 547 sequences after alignment using software at the threshold of more than 90% homology over a minimum of 20 base pairs. There were eight full-length cDNAs with complete open reading frame. Further studies on the ESTs in the library may be helpful to elucidate the components of Lampetra japonica oral gland and to understand the function of those proteins at the molecular level.
Animals↗
Expressed sequence tags from cultured cells of rice (Oryza sativa L.) under stressed conditions: analysis of transcripts of genes engaged in ATP-generating pathways.
Large-scale sequencing of randomly selected cDNA clones was used to isolate numerous genes in rice (Oryza sativa L.). Total RNA used for cDNA synthesis was prepared from suspension-cultured cells of rice grown under stressed conditions, such as in saline or nitrogen-starvation conditions. A total of 780 cDNA clones were partially sequenced and about 15% could be identified as putative genes. In the library constructed under saline conditions, we identified several genes associated with signal transduction, such as protein kinase and small GTP-binding protein genes. Many stress-related genes were isolated from both the saline and nitrogen-starvation libraries. These results indicate that stress treatment of suspension-cultured cells makes it possible to efficiently isolate various types of plant genes. To examine the usefulness of such tagged cDNAs for the study of gene expression in a specific metabolic pathway, we analyzed mRNA levels of genes engaged in the ATP-generating pathways in cultured cells of rice under different stresses, such as 20% sucrose, salt stress, cold stress and nitrogen-starvation stress. The results suggest that the coordinated induction of several genes in key steps under stressed conditions may be essential for activation of the entire energy-producing pathway to maintain homeostasis in rice cells. Expressed sequence tags identified by random cDNA sequencing provide the opportunity to generate a transcript map of rice genes.
Adaptation, Biological↗
Identification of novel 'expressed sequence tags' within the FHIT gene locus in human chromosome region 3p14.2.
Losses of genetic material within human chromosome regions (HCR) 3p12-p14 and 3p21-p22 are observed in various neoplasias, suggesting tumor suppressor gene (TSG) loci within these regions. HCR 3p14 is particularly interesting as it contains the t(3;8) translocation breakpoint of a hereditary renal cell carcinoma, the FRA3B fragile site, and DNA markers deleted in several types of human cancer. We here report on the identification of five novel 'expressed sequence tags' (ESTs) within 3p14.2 which map proximal to exon 9 of the candidate TSG, FHIT. These ESTs may be valuable for elucidation of the supposed TSG content in 3p14.2.
Acid Anhydride Hydrolases↗
Identification of genes up-regulated during conidiation of Fusarium oxysporum through expressed sequence tag analysis.
Fusarium oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. F. oxysporum produces microconidia and macroconidia in carboxymethyl cellulose-added liquid medium (CMCLM) and exhibits vegetative growth without conidiation in complete liquid medium (CLM). The cDNA libraries were constructed using mRNAs from CLM and CMCLM cultures. A total of 1288 and 1353 clones from CLM (vegetative growth) and CMCLM (conidiation) libraries, respectively, were sequenced, and 641 and 626 unique genes were identified. Of these unique genes, only 130 ( approximately 20%) were common in the two libraries, indicating different patterns of gene expression during vegetative growth and conidiation. The expression levels of 496 CMCLM-specific genes were compared during vegetative growth and conidiation by cDNA dot-blot differential hybridization and real-time quantitative PCR analyses, and 42 genes were identified to display >5-fold increases in mRNA abundance during conidiation. These genes provide ideal candidates for further studies directed at understanding fungal conidiogenesis and its molecular regulation.
DNA, Fungal↗
Potential indicators of stress response identified by expressed sequence tag analysis of hemocytes and embryos from the American oyster, Crassostrea virginica.
A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.
Journal Article↗
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.
Bone Marrow Cells↗
Caenorhabditis elegans expressed sequence tags identify gene families and potential disease gene homologues.
A database containing mapped partial cDNA sequences from Caenorhabditis elegans will provide a ready starting point for identifying nematode homologues of important human genes and determining their functions in C. elegans. A total of 720 expressed sequence tags (ESTs) have been generated from 585 clones randomly selected from a mixed-stage C. elegans cDNA library. Comparison of these ESTs with sequence databases identified 422 new C. elegans genes, of which 317 are not similar to any sequences in the database. Twenty-six new genes have been mapped by YAC clone hybridization. Members of several gene families, including cuticle collagens, GTP-binding proteins, and RNA helicases were discovered. Many of the new genes are similar to known or potential human disease genes, including CFTR and the LDL receptor.
Amino Acid Sequence↗
[Application of expressed sequence tag (EST) in parasite genome research].
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Animals↗
Trypanosoma rangeli Transcriptome Project: Generation and analysis of expressed sequence tags.
Trypanosoma rangeli is an important hemoflagellate parasite of several mammalian species in Central and South America, sharing geographical areas, vectors and reservoirs with T. cruzi, the causative agent of Chagas disease. Thus, the occurrence of single and/or mixed infections, including in humans, must be expected and are of great importance for specific diagnosis and epidemiology. In comparison to several Trypanosomatidae species, the T. rangeli biology and genome are little known, reinforcing the needs of a gene discovery initiative. The T. rangeli transcriptome initiative aims to promote gene discovery through the generation of expressed sequence tags (ESTs) and Orestes (ORF ESTs) from both epimastigote and trypomastigote forms of the parasite, allowing further studies of the parasite biology, taxonomy and phylogeny.
Journal Article↗
Chromosome assignment of human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels.
Sixty-three human brain cDNA sequences were newly assigned to individual human chromosomes. Ten of these were subregionally localized, and one was also mapped in the mouse genome. Four previously reported assignments were refined. PCR primers were designed from expressed sequence tags (ESTs) and tested for specific amplification from human genomic DNA. DNA was then amplified, often in multiplexed PCR reactions, using DNA from somatic cell hybrid mapping panels as templates. The amplification products were identified using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis. Thirteen newly localized cDNAs exhibited homology to previously reported sequences. EST01471 was shown to correspond to human microtubule-associated protein 1B (MAP1B), confirming the previous mapping of this gene to human chromosome 5. Other genes tentatively assigned to chromosomes based on these results were a component of the signal peptide receptor of the endoplasmic reticulum (EST00745) and a cyclic AMP-regulated phosphoprotein (EST01041) on chromosome 1, a protein phosphatase 2A 55-kDa regulatory subunit (EST01650) on chromosome 4, an NAD(P) transhydrogenase (EST01744) on chromosome 5, ribosomal proteins L1a or L1b (EST01627) and L18a (EST01583), a brain transcription factor (BF-1, EST00795) on chromosome 14, a milk fat globule membrane-related protein (EST01678) on chromosome 15, a putative peptide initiation factor (EST00675) on chromosome 17, thiosulfate sulfurtransferase (TST) on chromosome 22, and moesin (MSN) (EST00896) and a human equivalent of rat spot 14 (S14) (EST00887) on Xp11-->cen and Xpter-->p21.3, respectively.
Animals↗