Porcine gene discovery by normalized cDNA-library sequencing and EST cluster assembly
Scott C. FahrenkrugTimothy P. L. SmithB. A. FrekingJennifer ChoJoseph WhiteJeff ValletT. WiseG. A. RohrerGeo PerteaRăzvan SultanaJohn QuackenbushJ. W. Keele
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Aim To rapidly and economically obtain the new genes of adult stage Schistosoma japonicum (Chinese strain) by expressed sequence tag (EST) strategy.Methods A directional cDNA library constructed from Schistosoma japonicum (Chinese strain ) adult stage RNA was used to generate expressed sequence tags(ESTs) and the ESTs obtained were compared against EMBL-parasites database and GenBank datebase by BLASTn and BLASTx.Results A total 200 phage clones were randomly selected for generating expressed sequence tags(ESTs).From these clones,we obtained 76 EST-quality sequence.A total of 7.9% of these EST-quality sequences were identified suquence of Schistosoma japonicum,while 5.3% were putatively identified sequence of Schistosoma japonicum.A total of 22.4% of these EST-quality sequences were putatively identified sequence of Schistosoma mansoni or other organisms,and 59.2% which had no matches in the database were classified as unknow sequence.Among these EST-quality sequences,66 ESTs were successfully submitted to the dbEST at GenBank.In addition,some interesting genes were found.Conclusion Partial cDNA sequencing to get expressed sequence tags(ESTs) was a rapid and economical method to discover new genes of adult stage Schistosoma japonicum(Chinese strain).
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Aim To isolate and identify expressed Schistosoma japonicum(Chinese strain) genes from a directional cDNA library by expressed sequence Tags (EST) strategy for searching Schistosoma vaccine candidates and Schistosoma Genome Project Methods the cDNA clones were selected randomly,and the ESTs were obtained by PCR directed sequencing from 5’ end of the sequence,then the ESTs were compared with all sequences in GenBank data bases The novel ESTs were submitted to NCBI dbEST and GenBank for obtaining accession numbers Results 100 recombinant clones were picked up from the cDNA,and the inserts of cDNA were identified by PCR 25 ESTs were generated by PCR directed sequencing Among them,2 ESTs belonged to“identified genes of Schistosoma mansoni”for they showed matched with Schistosome sequences in the databases 19 ESTs were defined as novel genes EST sequences for they showed only partial homology with non Schistosome genes or other organism in the database These novel genes ESTs were submitted into NCBI dbEST and were given the accession numbers of sequence in GenBank Conclusion This study shows that EST strategy,i e picking up recombinant cDNA clones and obtaining partial sequences of cDNAS can been used to discover Schistosoma japonicum expressed genes
Suppression subtractive hybridization
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Sanger sequencing
clone (Java method)
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Powdery mildew is a serious disease of wheat in China. As part of ITEC (International Triteace EST cooperation), EST (expressed sequence tags) technique was used to explore the gene expression in leaf induced by Erysiphe graminis DC. A conventional cDNA library was constructed, and a total of 1 500 clones picked randomly from the library were sequenced, three hundred and eighty_seven ESTs of them were unique, which got the Accession Number in GenBank. About 49.4% ESTs showed significant similarity to functions of known sequences in GenBank. There are 196 ESTs' with functions not able to be determined, and eighty_four ESTs were demonstrated to be novel sequences. High_density dot membranes from unique clones were produced, and several disease resistance related genes were screened by differential hybridization.
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EST, as an expressed sequence tag, represents a cDNA sequence of a gene expressed in a specific tissue and a specific stage. Plant EST project is an important part of the genomics project, in which a huge amount of the species' ESTs are to be obtained,which will then be used to reveal biological mechanisms of the species' life activities at the transcript level. This paper reviews research progress in EST projects of Triticeae crops, mainly including the ESTs number in GenBank database deposited, and their applications to developing molecular markers, enriching genetic or physical maps, comparing genomics, and identifying new genes and expressed genes. In addition, the unsolved problems and application prospects of ESTs projects of Triticeae crops are analyzed.
Triticeae
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Functional Genomics
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The cDNA library of Fungi (E-20) were constructed using the SMARTIM cDNA library construction lit. After having constructed the cDNA library, the titer and kthe recombination rate of the unamplified library were detected and then amplified. Then, ten clones were selected randomly and sequenced. the titer and the recomination rate of the unamplified library were about 1.0×10~(6). pfu/ml and 98.9 %, and the titer and the capacity of the amplified library were about 3×10~(8) pfu/ml and 4×10~(10). Tne expressed sequence taqs (ESTs) were gained from ten clones being sequenced. Blasting in the GenBank, one sample shows high homology with the data of NADH-ubiquinone oxidoreductase (NuoC) gene. Accomplishment of this paper is an initial key work for building the resource information databank of the fungus (E-20).
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To acquire and analyze adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags and new genes from an adult S. japonicum cDNA library, and to search new vaccine candidates and drug targets.A cDNA library was constructed from adult stage S. japonicum. Clones were selected randomly from the cDNA library and were sequenced. ESTs and new genes were acquired after analysis in GenBank databases by BLAST and other programs. All ESTs and new genes were submitted to GenBank and received accession numbers.149 ESTs were acquired from a total 382 clones that were randomly selected from the adult S. japonicum cDNA library. All ESTs were successfully submitted to the dbEST at Genbank. Some of them were homologous with sequences of male, female, egg, schistosomula, cercaria and miracidia of S. japonicum. 18 new genes of adult S. japonicum were acquired. Some genes were housekeeping genes and some genes might be interesting as vaccine candidates or drugs targets.The EST strategy is a rapid, efficient and economical method to acquire ESTs and to discover new genes of adult stage S. japonicum from cDNA libraries.
Housekeeping gene
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The pineal gland is the circadian oscillator in the chicken, regulating diverse functions ranging from egg laying to feeding. Here, we describe the isolation and characterization of expressed sequence tags (ESTs) isolated from a chicken pineal gland cDNA library. A total of 192 unique sequences were analysed and submitted to GenBank; 6p of the ESTs matched neither GenBank cDNA sequences nor the newly assembled chicken genomic DNA sequence, three ESTs aligned with sequences designated to be on the Zlrandom, while one matched a W chromosome sequence and could be useful in cataloguing functionally important genes on this sex chromosome. Additionally, single nucleotide polymorphisms (SNPs) were identified and validated in 10 ESTs that showed 98p or higher sequence similarity to known chicken genes. Here, we have described resources that may be useful in comparative and functional genomic analysis of genes expressed in an important organ, the pineal gland, in a model and agriculturally important organism. Copyright © 2005 John Wiley & Sons, Ltd.
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genomic DNA
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In order to monitor quality of the chicken hypothalamus cDNA library constructed and whether it could be used to construct the expression profile of hypothalamus,we sequenced and primarily analyzed 12 ESTs from randomly selected clones.By BLASTn and FASTA analysis in GenBank,1 EST could find homology in chicken,4 had matching sequences in turkey or other species,1 had homologous EST sequence in EST database,6 were function unknown gene.All 12 ESTs sequence were loaded into GenBank.
Homology
Sequence homology
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