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Graeme Wistow - One of the best experts on this subject based on the ideXlab platform.
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Expressed Sequence Tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers
BMC Neuroscience, 2009Co-Authors: Steven L Bernstein, Yan Guo, Katherine Peterson, Graeme WistowAbstract:Background The optic nerve is a pure white matter central nervous system (CNS) tract with an isolated blood supply, and is widely used in physiological studies of white matter response to various insults. We examined the gene expression profile of human optic nerve (ON) and, through the NEIBANK online resource, to provide a resource of Sequenced verified cDNA clones. An un-normalized cDNA library was constructed from pooled human ON tissues and was used in Expressed Sequence Tag (EST) analysis. Location of an abundant oligodendrocyte marker was examined by immunofluorescence. Quantitative real time polymerase chain reaction (qRT-PCR) and Western analysis were used to compare levels of expression for key calcium channel protein genes and protein product in primate and rodent ON. Results Our analyses revealed a profile similar in many respects to other white matter related tissues, but significantly different from previously available ON cDNA libraries. The previous libraries were found to include specific markers for other eye tissues, suggesting contamination. Immune/inflammatory markers were abundant in the new ON library. The oligodendrocyte marker QKI was abundant at the EST level. Immunofluorescence revealed that this protein is a useful oligodendrocyte cell-type marker in rodent and primate ONs. L-type calcium channel EST abundance was found to be particularly low. A qRT-PCR-based comparative mammalian species analysis reveals that L-type calcium channel expression levels are significantly lower in primate than in rodent ON, which may help account for the class-specific difference in responsiveness to calcium channel blocking agents. Several known eye disease genes are abundantly Expressed in ON. Many genes associated with normal axonal function, mRNAs associated with axonal transport, inflammation and neuroprotection are observed. Conclusion We conclude that the new cDNA library is a faithful representation of human ON and EST data provide an initial overview of gene expression patterns in this tissue. The data provide clues for tissue-specific and species-specific properties of human ON that will help in design of therapeutic models.
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Expressed Sequence Tag analysis of guinea pig cavia porcellus eye tissues for neibank
Molecular Vision, 2008Co-Authors: M F Simpanya, Graeme Wistow, James Gao, Larry L David, Frank J Giblin, Kenneth P MittonAbstract:PURPOSE To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank Expressed Sequence Tags. METHODS RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were Sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS). RESULTS Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA Sequences and had significant homologies to other mammalian Sequences in GenBank. Complete cDNA Sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome Sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea. CONCLUSIONS Genomic analysis of guinea pig eye tissues provides Sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.
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Expressed Sequence Tag analysis of zebrafish eye tissues for neibank
Molecular Vision, 2005Co-Authors: Thomas S Vihtelic, James Gao, James M Fadool, Kimberley A Thornton, David R Hyde, Graeme WistowAbstract:PURPOSE To characterize gene expression patterns in various tissues of the zebrafish (Danio rerio) eye and identify zebrafish orthologs of human genes by Expressed Sequence Tag (EST) analysis for NEIBank. METHODS mRNA was extracted from adult zebrafish eye tissues, including lenses, anterior segments (minus lens), retinas, posterior segments lacking retinas, and whole eyes. Five different cDNA libraries were constructed in the pCMVSport6 vector. Approximately 4,000 clones from each library were Sequenced and analyzed using various bioinformatics programs. RESULTS The analysis yielded approximately 2,500 different gene clusters for each library. Combining data from the five libraries produced 10,392 unique gene clusters. GenBank accession numbers were identified for 37.6% (3,906) of the total gene clusters in the combined libraries and approximately 50% were linked to Unigene clusters in the current database. Several new crystallin genes, including two gammaN-crystallins, and a second major intrinsic protein (MIP) were identified in the lens library. In addition, a zebrafish homolog of cochlin (COCH), a gene that may play a role in the pathogenesis of human glaucoma, was identified in the anterior segment library. Surprisingly, no clear ortholog of the major retinal transcription factor Nrl was identified. CONCLUSIONS The zebrafish eye tissue cDNA libraries are a useful resource for comparative gene expression analysis. These libraries will complement the cDNA libraries made for the Zebrafish Gene Collection (ZGC) and provide an additional source for gene identification and characterization in the vertebrate eye.
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Expressed Sequence Tag analysis of human rpe choroid for the neibank project over 6000 non redundant transcripts novel genes and splice variants
Molecular Vision, 2002Co-Authors: Graeme Wistow, Steven L Bernstein, M K Wyatt, Robert N Fariss, A Behal, J W Touchman, G Bouffard, D Smith, Katherine PetersonAbstract:PURPOSE The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe Expressed Sequence Tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. METHODS A cDNA library (cs) was made from human RPE/choroid and Sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. RESULTS Ten thousand individual Sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. CONCLUSIONS The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.
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Expressed Sequence Tag analysis of adult human lens for the neibank project over 2000 non redundant transcripts novel genes and splice variants
Molecular Vision, 2002Co-Authors: Graeme Wistow, Steven L Bernstein, M K Wyatt, A Behal, J W Touchman, G Bouffard, D Smith, Katherine PetersonAbstract:PURPOSE To explore the expression profile of the human lens and to provide a resource for microarray studies, Expressed Sequence Tag (EST) analysis has been performed on cDNA libraries from adult lenses. METHODS A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were Sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 Sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. RESULTS The lens library (by) contains a low percenTage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes Expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. CONCLUSIONS The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.
Lining Zhao - One of the best experts on this subject based on the ideXlab platform.
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Diversity Analysis in Cannabis sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers.
PloS one, 2014Co-Authors: Chunsheng Gao, Pengfei Xin, Changbiao Wang, Gonggu Zang, Chaohua Cheng, Qing Tang, Ping Chen, Lining ZhaoAbstract:Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple Sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of Expressed Sequence Tag simple Sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.
Shoba Ranganathan - One of the best experts on this subject based on the ideXlab platform.
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ESTExplorer: An Expressed Sequence Tag (EST) assembly and annotation platform
Nucleic Acids Research, 2007Co-Authors: Shivashankar H Nagaraj, Nandan Deshpande, Robin B Gasser, Shoba RanganathanAbstract:The analysis of Expressed Sequence Tag (EST) datasets offers a rapid and cost-effective approach to elucidate the transcriptome of an organism, but requiring several computational methods for assembly and annotation. ESTExplorer is a comprehensive workflow system for EST data management and analysis. The pipeline uses a 'distributed control approach' in which the most appropriate bioinformatics tools are implemented over different dedicated processors. Species-specific repeat masking and conceptual translation are in-built. ESTExplorer accepts a set of ESTs in FASTA format which can be analysed using programs selected by the user. After pre-processing and assembly, the dataset is annotated at the nucleotide and protein levels, following conceptual translation. Users may optionally provide ESTExplorer with assembled contigs for annotation purposes. Functionally annotated contigs/ESTs can be analysed individually. The overall outputs are gene ontologies, protein functional identifications in terms of mapping to protein domains and metabolic pathways. ESTExplorer has been applied successfully to annotate large EST datasets from parasitic nematodes and to identify novel genes as potential targets for parasite intervention. ESTExplorer runs on a Linux cluster and is freely available for the academic community at http://estexplorer.biolinfo.org.
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a hitchhiker s guide to Expressed Sequence Tag est analysis
Briefings in Bioinformatics, 2006Co-Authors: Shivashankar H Nagaraj, Robin B Gasser, Shoba RanganathanAbstract:Expressed Sequence Tag (EST) sequencing projects are underway for numerous organisms, generating millions of short, single-pass nucleotide Sequence reads, accumulating in EST databases. Extensive computational strategies have been developed to organize and analyse both small- and large-scale EST data for gene discovery, transcript and single nucleotide polymorphism analysis as well as functional annotation of putative gene products. We provide an overview of the significance of ESTs in the genomic era, their properties and the applications of ESTs. Methods adopted for each step of EST analysis by various research groups have been compared. Challenges that lie ahead in organizing and analysing the ever increasing EST data have also been identified. The most appropriate software tools for EST pre-processing, clustering and assembly, database matching and functional annotation have been compiled (available online from http://biolinfo.org/EST). We propose a road map for EST analysis to accelerate the effective analyses of EST data sets. An investigation of EST analysis platforms reveals that they all terminate prior to downstream functional annotation including gene ontologies, motif/pattern analysis and pathway mapping.
Chunsheng Gao - One of the best experts on this subject based on the ideXlab platform.
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Diversity Analysis in Cannabis sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers.
PloS one, 2014Co-Authors: Chunsheng Gao, Pengfei Xin, Changbiao Wang, Gonggu Zang, Chaohua Cheng, Qing Tang, Ping Chen, Lining ZhaoAbstract:Cannabis sativa L. is an important economic plant for the production of food, fiber, oils, and intoxicants. However, lack of sufficient simple Sequence repeat (SSR) markers has limited the development of cannabis genetic research. Here, large-scale development of Expressed Sequence Tag simple Sequence repeat (EST-SSR) markers was performed to obtain more informative genetic markers, and to assess genetic diversity in cannabis (Cannabis sativa L.). Based on the cannabis transcriptome, 4,577 SSRs were identified from 3,624 ESTs. From there, a total of 3,442 complementary primer pairs were designed as SSR markers. Among these markers, trinucleotide repeat motifs (50.99%) were the most abundant, followed by hexanucleotide (25.13%), dinucleotide (16.34%), tetranucloetide (3.8%), and pentanucleotide (3.74%) repeat motifs, respectively. The AAG/CTT trinucleotide repeat (17.96%) was the most abundant motif detected in the SSRs. One hundred and seventeen EST-SSR markers were randomly selected to evaluate primer quality in 24 cannabis varieties. Among these 117 markers, 108 (92.31%) were successfully amplified and 87 (74.36%) were polymorphic. Forty-five polymorphic primer pairs were selected to evaluate genetic diversity and relatedness among the 115 cannabis genotypes. The results showed that 115 varieties could be divided into 4 groups primarily based on geography: Northern China, Europe, Central China, and Southern China. Moreover, the coefficient of similarity when comparing cannabis from Northern China with the European group cannabis was higher than that when comparing with cannabis from the other two groups, owing to a similar climate. This study outlines the first large-scale development of SSR markers for cannabis. These data may serve as a foundation for the development of genetic linkage, quantitative trait loci mapping, and marker-assisted breeding of cannabis.
Delin Duan - One of the best experts on this subject based on the ideXlab platform.
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development of Expressed Sequence Tag derived microsatellite markers for saccharina laminaria japonica
Journal of Applied Phycology, 2010Co-Authors: Fuli Liu, Xiuliang Wang, Jianting Yao, Delin DuanAbstract:Expressed Sequence Tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy-Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parenTage, and mapping studies of L. japonica.
