Sample records for catfish expressed sequence

  1. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan


    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

  2. Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

    Indian Academy of Sciences (India)

    Vikas Anathy; Thayanithy Venugopal; Ramanathan Koteeswaran; Thavamani J Pandian; Sinnakaruppan Mathavan


    A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

  3. Assembly of 500,000 inter-specific catfish expressed sequence tags and large scale gene-associated marker development for whole genome association studies

    Energy Technology Data Exchange (ETDEWEB)

    Catfish Genome Consortium; Wang, Shaolin; Peatman, Eric; Abernathy, Jason; Waldbieser, Geoff; Lindquist, Erika; Richardson, Paul; Lucas, Susan; Wang, Mei; Li, Ping; Thimmapuram, Jyothi; Liu, Lei; Vullaganti, Deepika; Kucuktas, Huseyin; Murdock, Christopher; Small, Brian C; Wilson, Melanie; Liu, Hong; Jiang, Yanliang; Lee, Yoona; Chen, Fei; Lu, Jianguo; Wang, Wenqi; Xu, Peng; Somridhivej, Benjaporn; Baoprasertkul, Puttharat; Quilang, Jonas; Sha, Zhenxia; Bao, Baolong; Wang, Yaping; Wang, Qun; Takano, Tomokazu; Nandi, Samiran; Liu, Shikai; Wong, Lilian; Kaltenboeck, Ludmilla; Quiniou, Sylvie; Bengten, Eva; Miller, Norman; Trant, John; Rokhsar, Daniel; Liu, Zhanjiang


    Background-Through the Community Sequencing Program, a catfish EST sequencing project was carried out through a collaboration between the catfish research community and the Department of Energy's Joint Genome Institute. Prior to this project, only a limited EST resource from catfish was available for the purpose of SNP identification. Results-A total of 438,321 quality ESTs were generated from 8 channel catfish (Ictalurus punctatus) and 4 blue catfish (Ictalurus furcatus) libraries, bringing the number of catfish ESTs to nearly 500,000. Assembly of all catfish ESTs resulted in 45,306 contigs and 66,272 singletons. Over 35percent of the unique sequences had significant similarities to known genes, allowing the identification of 14,776 unique genes in catfish. Over 300,000 putative SNPs have been identified, of which approximately 48,000 are high-quality SNPs identified from contigs with at least four sequences and the minor allele presence of at least two sequences in the contig. The EST resource should be valuable for identification of microsatellites, genome annotation, large-scale expression analysis, and comparative genome analysis. Conclusions-This project generated a large EST resource for catfish that captured the majority of the catfish transcriptome. The parallel analysis of ESTs from two closely related Ictalurid catfishes should also provide powerful means for the evaluation of ancient and recent gene duplications, and for the development of high-density microarrays in catfish. The inter- and intra-specific SNPs identified from all catfish EST dataset assembly will greatly benefit the catfish introgression breeding program and whole genome association studies.

  4. Molecular cloning, sequencing and tissue expression of vasotocin and isotocin precursor genes from Ostariophysian catfishes: Phylogeny and evolutionary considerations in teleosts

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    Putul eBanerjee


    Full Text Available Basic and neutral neurohypophyseal (NH nonapeptides have evolved from vasotocin (VT by a gene duplication at the base of the gnathostome lineage. In teleosts, VT and IT are the basic and neutral peptides, respectively. In the present study, VT and IT precursor genes of Heteropneustes fossilis and Clarias batrachus (Siluriformes, Ostariophysi were cloned and sequenced. The channel catfish Icatalurus punctatus NH precursor sequences were obtained from EST database. The catfish NH sequences were used along with the available Acanthopterygii and other vertebrate NH precursor sequences to draw phylogenetic inference on the evolutionary history of the teleost NH peptides. Synteny analysis of the NH gene loci in various teleost species was done to complement the phylogenetic analysis. In H. fossilis, the NH transcripts were also sequenced from the ovary. The cloned genes and the deduced precursor proteins showed conserved characteristics of the NH nonapeptide precursors. The genes are expressed in brain and ovary (follicular envelope of H. fossilis with higher transcript abundance in the brain. The addition of the catfish sequences in the phylogenetic analysis revealed that the VT and IT precursors of the species-rich superorders of teleosts have a distinct phylogenetic history with the Acanthopterygii VT and IT precursors sharing a less evolutionary distance and the Ostariophysi VT and IT having a greater evolutionary distance. The genomic location of VT and IT precursors, and synteny analysis of the NH loci lend support to the phylogenetic inference and suggest a footprint of fish- specific whole genome duplication (3R and subsequent diploidization in the NH loci. The VT and IT precursor genes are most likely lineage-specific paralogs resulting from differential losses of the 3R NH paralogs in the two superorders. The independent yet consistent retention of VT and IT in the two superorders might be directed by a stringent ligand-receptor selectivity.

  5. Isolation of yellow catfish β-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein. (United States)

    Ge, Jiachun; Dong, Zhangji; Li, Jingyun; Xu, Zhiqiang; Song, Wei; Bao, Jie; Liang, Dong; Li, Junbo; Li, Kui; Jia, Wenshuang; Zhao, Muzi; Cai, Yongxiang; Yang, Jiaxin; Pan, Jianlin; Zhao, Qingshun


    Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of β-actin gene in yellow catfish. The sequence is 1,017 bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own β-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.

  6. Molecular Cloning and Sequencing of Channel Catfish, Ictalurus punctatus, Cathepsin H and L cDNA (United States)

    Cathepsin H and L, a lysosomal cysteine endopeptidase of the papain family, are ubiquitously expressed and involve in antigen processing. In this communication, the channel catfish cathepsin H and L transcripts were sequenced and analyzed. Total RNA from tissues was extracted and cDNA libraries we...

  7. cDNA sequence and protein bioinformatics analyses of MSTN in African catfish (Clarias gariepinus). (United States)

    Kanjanaworakul, Poonmanee; Sawatdichaikul, Orathai; Poompuang, Supawadee


    Myostatin, also known as growth differentiation factor 8, has been identified as a potent negative regulator of skeletal muscle growth. The purpose of this study was to characterize and predict function of the myostatin gene of the African catfish (Cg-MSTN). Expression of Cg-MSTN was determined at three growth stages to establish the relationship between the levels of MSTN transcript and skeletal muscle growth. The partial cDNA sequence of Cg-MSTN was cloned by using published information from its congener walking catfish (Cm-MSTN). The Cg-MSTN was 1194 bp in length encoding a protein of 397 amino acids. The deduced MSTN sequence exhibited key functional sites similar to those of other members of the TGF-β superfamily, especially, the proteolytic processing site (RXXR motif) and nine conserved cysteines at the C-terminal. Expression of MSTN appeared to be correlated with muscle development and growth of African catfish. Protein bioinformatics revealed that the primary sequence of Cg-MSTN shared 98 % sequence identity with that of walking catfish Cm-MSTN with only two different residues, [Formula: see text]. and [Formula: see text]. The proposed model of Cg-MSTN revealed the key point mutation [Formula: see text] causing a 7.35 Å shorter distance between the N- and C-lobes and an approximately 11° narrow angle than those of Cm-MSTN. The substitution of a proline residue near the proteolytic processing site which altered the structure of myostatin may play a critical role in reducing proteolytic activity of this protein in African catfish.

  8. Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque (United States)

    : Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

  9. Comparative analysis of catfish BAC end sequences with the zebrafish genome

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    Abernathy Jason


    Full Text Available Abstract Background Comparative mapping is a powerful tool to transfer genomic information from sequenced genomes to closely related species for which whole genome sequence data are not yet available. However, such an approach is still very limited in catfish, the most important aquaculture species in the United States. This project was initiated to generate additional BAC end sequences and demonstrate their applications in comparative mapping in catfish. Results We reported the generation of 43,000 BAC end sequences and their applications for comparative genome analysis in catfish. Using these and the additional 20,000 existing BAC end sequences as a resource along with linkage mapping and existing physical map, conserved syntenic regions were identified between the catfish and zebrafish genomes. A total of 10,943 catfish BAC end sequences (17.3% had significant BLAST hits to the zebrafish genome (cutoff value ≤ e-5, of which 3,221 were unique gene hits, providing a platform for comparative mapping based on locations of these genes in catfish and zebrafish. Genetic linkage mapping of microsatellites associated with contigs allowed identification of large conserved genomic segments and construction of super scaffolds. Conclusion BAC end sequences and their associated polymorphic markers are great resources for comparative genome analysis in catfish. Highly conserved chromosomal regions were identified to exist between catfish and zebrafish. However, it appears that the level of conservation at local genomic regions are high while a high level of chromosomal shuffling and rearrangements exist between catfish and zebrafish genomes. Orthologous regions established through comparative analysis should facilitate both structural and functional genome analysis in catfish.

  10. Molecular characterization, phylogenetic analysis and expression patterns of five protein arginine methyltransferase genes of channel catfish, Ictalurus punctatus (Rafinesque). (United States)

    Yeh, Hung-Yueh; Klesius, Phillip H


    Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMT), has recently emerged as an important modification in the regulation of gene expression. In this communication, we identified and characterized the channel catfish orthologs to human PRMT 1, 3, 4 and 5, and PRMT4 like. Each PRMT nucleic acid sequence has an open reading frame (ORF) and 3'-untranslated regions. Each ORF appears to encode 361, 587 and 458 amino acid residues for PRMT1, PRMT4 and variant, respectively. The partial ORF of PRMT3 and PRMT5 encode 292 and 563 amino acids, respectively. By comparison with the human counterparts, each channel catfish PRMT also has conserved domains. For expression profile, the channel catfish PRMT1 transcript was detected by RT-PCR in spleens, anterior kidneys, livers, intestines, skin and gills of fish examined. Except in liver, the PRMT3 transcript was detected in all catfish tissues examined. However, the PRMT4 cDNA was detected in livers from all three catfish and gills from two fish, but not other tissues. This information will enable us to further elucidate PRMT functions in channel catfish.

  11. The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts (United States)

    Liu, Zhanjiang; Liu, Shikai; Yao, Jun; Bao, Lisui; Zhang, Jiaren; Li, Yun; Jiang, Chen; Sun, Luyang; Wang, Ruijia; Zhang, Yu; Zhou, Tao; Zeng, Qifan; Fu, Qiang; Gao, Sen; Li, Ning; Koren, Sergey; Jiang, Yanliang; Zimin, Aleksey; Xu, Peng; Phillippy, Adam M.; Geng, Xin; Song, Lin; Sun, Fanyue; Li, Chao; Wang, Xiaozhu; Chen, Ailu; Jin, Yulin; Yuan, Zihao; Yang, Yujia; Tan, Suxu; Peatman, Eric; Lu, Jianguo; Qin, Zhenkui; Dunham, Rex; Li, Zhaoxia; Sonstegard, Tad; Feng, Jianbin; Danzmann, Roy G.; Schroeder, Steven; Scheffler, Brian; Duke, Mary V.; Ballard, Linda; Kucuktas, Huseyin; Kaltenboeck, Ludmilla; Liu, Haixia; Armbruster, Jonathan; Xie, Yangjie; Kirby, Mona L.; Tian, Yi; Flanagan, Mary Elizabeth; Mu, Weijie; Waldbieser, Geoffrey C.


    Catfish represent 12% of teleost or 6.3% of all vertebrate species, and are of enormous economic value. Here we report a high-quality reference genome sequence of channel catfish (Ictalurus punctatus), the major aquaculture species in the US. The reference genome sequence was validated by genetic mapping of 54,000 SNPs, and annotated with 26,661 predicted protein-coding genes. Through comparative analysis of genomes and transcriptomes of scaled and scaleless fish and scale regeneration experiments, we address the genomic basis for the most striking physical characteristic of catfish, the evolutionary loss of scales and provide evidence that lack of secretory calcium-binding phosphoproteins accounts for the evolutionary loss of scales in catfish. The channel catfish reference genome sequence, along with two additional genome sequences and transcriptomes of scaled catfishes, provide crucial resources for evolutionary and biological studies. This work also demonstrates the power of comparative subtraction of candidate genes for traits of structural significance. PMID:27249958

  12. Molecular Cloning, Expression and Genome Organization of Channel Catfish (Ictalurus punctatus) Matrix Metalloproteinase-9 (United States)

    In the course of studying pathogenesis of enteric septicemia of catfish, we noted that channel catfish matrix metalloproteinase-9 (MMP-9) gene was up-regulated after Edwardsiella ictaluri infection. In this study, we cloned, sequenced using the RACE (rapid amplification of cDNA ends) method and cha...

  13. Growth hormone from striped catfish (Pangasianodon hypophthalmus): genomic organization, recombinant expression and biological activity. (United States)

    Poen, Sinothai; Pornbanlualap, Somchai


    Growth hormone is an essential polypeptide required for normal growth and development of vertebrates. In this report, striped catfish (Pangasianodon hypophthalmus) growth hormone gene and cDNA were isolated by reverse transcriptase-polymerase chain reaction. The striped catfish growth hormone (scGH) encoding gene contains 5 exons and 4 introns. The cDNA sequence of the scGH gene contains a 603bp open reading frame and encodes for a 200-aa protein consisting of a putative 22-aa signal peptide and the mature 178-aa protein. The recombinant histidine-tagged scGH protein which expressed in Escherichia coli as inclusion bodies was unfolded, refolded and purified to near-homogeneity by Ni(2+)-NTA chromatography. Analysis of the secondary structure content by CD spectroscopy showed that the α-helical content of the refolded scGH is 55%. Elucidation of the folding pathway of scGH by fluorescence spectroscopy showed that denaturation transition of scGH is coincident and cooperative, consistent with the two-state denaturation mechanism. The purified scGH was biologically active and exhibited growth-promoting activity in striped catfish, but not tilapia.

  14. Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish. (United States)

    Kobayashi, Y; Peterson, B C; Waldbieser, G C


    This study tested the hypothesis that increased growth in channel catfish is associated with expression of the genes that code for uncoupling proteins (UCP) 2 and 3, members of the mitochondrial channel proteins involved in nutrient sensing and metabolism. The specific objective was to contrast the levels of UCP2 messenger RNA (mRNA) in fast vs slow growing catfish as well as in fed vs fasted catfish. Two distinct UCP2 transcripts were identified and named UCP2a and UCP2b, respectively. Nucleotide and amino acid sequence of catfish UCP2s were highly similar to UCP2 and other UCPs from other fish and mammals (>75%). Expression of UCP2a mRNA was detectable at very low levels in various metabolically active tissues, whereas the expression of UCP2b mRNA was readily detectable in the muscle and heart. In a 21-wk feeding study, fish that grew faster had a greater percent body fat at the end of the study (P muscle was increased (P growth and associated fat accumulation appears to be independent of muscle UCP2b mRNA expression and UCP2b-mediated mechanisms.

  15. Mitochondrial cytochrome b sequence variations and phylogeny of the East Asian bagrid catfishes

    Institute of Scientific and Technical Information of China (English)


    The mitochondrial DNA cytochrome b gene was sequenced from 8 bagrid catfishes in China. Aligned with cytochrome b sequences from 9 bagrid catfishes in Japan, Korea and Russia retrieved from GenBank, and selected Silurus meridionalis, Liobagrus anguillicauda, Liobagrus reini and Phenacogrammus interruptus as outgroups, we constructed a matrix of 21 DNA sequences. The Kimura's two-parameter distances were calculated and molecular phylogenetic trees were constructed by using the maximum parsimony (MP) and neighbor-joining (NJ) methods. The results show that (i) there exist 3-bp deletions of mitochondrial cytochrome b gene compared with cypriniforms and characiforms; (ii) the molecular phylogenetic tree suggests that bagrid catfishes form a monophyletic group, and the genus Mystus is the earliest divergent in the East Asian bagrid catfishes, as well as the genus Pseudobagrus is a monophyletic group but the genus Pelteobagrus and Leiocassis are complicated; and (iii) the evolution rate of the East Asian bagrids mitochondrial cytochrome b gene is about 0.18%~0.30% sequence divergence per million years.

  16. Expression of immune genes in skin of channel catfish immunized with live theronts of Ichthyophthirius multifiliis (United States)

    There is limited information on innate and adaptive immune gene expression in the skin of channel catfish, Ictalurus punctatus immunized with Ichthyophthirius multifiliis (Ich). The objective of this study was to evaluate differential expression of innate and adaptive immune genes, including immunog...

  17. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish. (United States)

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang


    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish.

  18. Modulation of lipocalin-type prostaglandin D2 synthase expression in catfish seminal vesicles by thyroid disrupting agents and hormones. (United States)

    Sreenivasulu, Gunti; Pavani, Ayinampudi; Sudhakumari, Cheni-Chery; Dutta-Gupta, Aparna; Senthilkumaran, Balasubramanian


    Thyroid hormones play crucial role in several biological processes including reproduction. Disruption of normal thyroid status by environmental contaminants can cause severe impairment in reproductive functions. In our previous study, we reported down-regulation of a protein in seminal vesicular fluid of air-breathing catfish, Clarias gariepinus during experimentally induced hyperthyroidism. N-terminal amino acid sequence analysis followed by search in sequence database denoted it to be lipocalin-type prostaglandin D2 synthase (ptgds-b). In the present study, we cloned full-length cDNA of ptgds-b based on the N-terminal amino acid sequence. Surprisingly, Northern blot as well as RT-PCR analysis demonstrated the presence of ptgds-b transcript predominantly in seminal vesicles and developing testis. Further, ptgds-b mRNA significantly decreased in seminal vesicles following L-thyroxine overdose while there was an increased expression of ptgds-b after depletion of thyroid hormone by thiourea and withdrawal of the treatments reverted this effect. Treatment of catfish with human chorionic gonadotropin and estradiol significantly reduced ptgds-b expression. Taken together, we report ptgds-b as a thyroid hormone regulated protein in the seminal vesicles in addition to gonadotropin and estradiol. Further studies might explain the exclusive presence of ptgds-b in seminal vesicles and developing testis yet present data evaluated it as a putative biomarker for thyroid hormone disruption.

  19. G-protein coupled receptor 18 (GPR18) in channel catfish: Expression analysis and efficacy as immunostimulant against Aeromonas hydrophila infection (United States)

    The objectives of this study were: 1) to determine the transcriptional profiles of G-protein coupled receptor 18 (GPR18) in channel catfish after infection with A. hydrophila compared to that in healthy catfish; 2) to determine whether over-expression of GPR18 in catfish gill cells will offer protec...

  20. Draft genome sequence of Pseudomonas mosselii Gil3, isolated from catfish and antagonistic against hypervirulent Aeromonas hydrophila (United States)

    Pseudomonas mosselii Gil3 was isolated from a catfish that survived from lethal challenge with hypervirulent Aeromonas hydrophila (vAh). When assayed in vitro, the bacterium showed antagonism against vAh. Sequence analysis revealed that the genome of P. mosselii Gil3 encodes numerous aromatic metabo...

  1. Peroxisome proliferator-activated receptor gamma (PPARγ) in yellow catfish Pelteobagrus fulvidraco: molecular characterization, mRNA expression and transcriptional regulation by insulin in vivo and in vitro. (United States)

    Zheng, Jia-Lang; Zhuo, Mei-Qin; Luo, Zhi; Pan, Ya-Xiong; Song, Yu-Feng; Huang, Chao; Zhu, Qing-Ling; Hu, Wei; Chen, Qi-Liang


    Peroxisome proliferator-activated receptor gamma (PPARγ) is ligand-inducible transcription factor and has important roles in lipid metabolism, cell proliferation and inflammation. In the present study, yellow catfish Pelteobagrus fulvidraco PPARγ cDNA was isolated from liver by RT-PCR and RACE, and its molecular characterization and transcriptional regulation by insulin in vivo and in vitro were determined. The generation of PPARγ1 and PPARγ2 was due to alternative promoter of PPARγ gene. PPARγ1 and PPARγ2 mRNA covered 2426 bp and 2537 bp, respectively, with an open reading frame (ORF) of 1584 bp encoding 527 amino acid residues. Yellow catfish PPARγ gene was organized in a manner similar to that of their mammalian homologs, implying a modular organization of the protein's domains. A comparison between the yellow catfish PPARγ amino acid sequence and the correspondent sequences of several other species revealed the identity of 55-76.2%. Two PPARγ transcripts (PPARγ1 and PPARγ2) mRNAs were expressed in a wide range of tissues, but the abundance of each PPARγ mRNA showed the tissue- and developmental stage-dependent expression patterns. Intraperitoneal injection of insulin in vivo significantly stimulated the mRNA expression of total PPARγ and PPARγ1, but not PPARγ2 in the liver of yellow catfish. In contrast, incubation of hepatocytes with insulin in vitro increased the mRNA levels of PPARγ1, PPARγ2 and total PPARγ. To our knowledge, for the first time, the present study provides evidence that PPARγ1 and PPARγ2 are differentially expressed with and among tissues during different developmental stages and also regulated by insulin both in vivo and in vitro, which serves to increase our understanding on PPARγ physiological function in fish.


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    Huria Marnis


    Full Text Available Faster growing African catfish can be produced by transgenesis. This study was conducted to investigate the distribution and expression of growth hormone gene (PhGH in various organs of the transgenic African catfish (Clarias gariepinus founder (F0. Transgene was detected using the PCR method in various organs, namely pituitary, brain, liver, heart, spleen, kidney, intestine, stomach, muscle, caudal fin, gill and eye. Transgene expression levels were analyzed using the method of reverse transcriptase-polymerase chain reaction (RT-PCR, -actin gene used as internal controls. The results showed that the PhGH was detected and expressed in all organs of the transgenic African catfish founder. The high level of PhGH expression was found in the liver, pituitary, intestine and brain; smaller amounts were detectable in muscle, spleen, kidneys, heart, and stomach, caudal fin, gill and eyes, range from 0.02-0.75 PhGH/-actin mRNA. The expression levels of PhGH had positive correlation with tissue and body size (P<0.05.


    Institute of Scientific and Technical Information of China (English)

    张娟; 张其中; 张占会; 崔淼


    热休克蛋白70(HSPTO)与生物体的抗胁迫能力密切相关.本文采用RACE(Rapid amplification of cDNA ends)技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白(HSC70)基因及其cDNA.该cDNA全长2245bp,包括5'非编码区82bp,3'非编码区225bp,开放阅读框(ORF)1938bp,编码645个氨基酸组成的蛋白质.黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似.其中,最长内含子(873bp)位于5'端非编码区,其余内含子(长度在80-251bp之间不等)均在编码区以内.黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%.RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p0.05).%Heat shock protein 70 (HSP70s) act as a role of chaperone and play a key function in cytoprotection and cytore-pair, including protein assembly, correct folding, and membrane translocation, it also enhance the organisms' immunity and enduration to stressors. Yellow catfish (Pelteobagrus fulvidraco) is an important cultured species in China. In order to illuminate molecular mechanism of the HSP70 family members in the catfish against stressors and diseases, it is necessary to clone the gene and cDNA sequence of HSP70 family members in the first instance. Therefore, the gene and its cDNA of a HSP70 family member were cloned in yellow catfish, and mRNA expression of the gene was studied in various tissues and organs of the catfish under heat-treated or unstressed condition.A full length cDNA of 2245 bp was cloned in the gill of yellow catfish with RACE (rapid amplification of cDNA ends) technique. The cDNA contained an open reading frame (ORF) of 1938 bp, 5

  4. Draft genome sequences of four virulent aeromonas hydrophila strains from catfish aquaculture (United States)

    Since 2009, a clonal group of virulent Aeromonas hydrophila (VAh) strains has been causing severe disease in the catfish aquaculture industry in the Southeastern United States. Here, we report draft genomes of four A. hydrophila isolates from catfish aquaculture that represent this clonal group....

  5. Mitochondrial 16S rRNA sequence variations and phylogeny of the Chinese sisorid catfishes

    Institute of Scientific and Technical Information of China (English)

    GUO Xianguang; ZHANG Yaoguang; HE Shunping; CHEN Yiyu


    Partial sequences of mitochondrial 16S rRNA gene were obtained by PCR amplification for comparisons among nine species of glyptosternoid fishes and six species of non-glyptosternoids representing 10 sisorid genera. There are compositional biases in the A-rich unpaired regions and G-rich paired regions. A-G transitions are primarily responsible for the Ts/Tv bias in unpaired regions. The overall substitution rate in unpaired regions is almost two times higher than that in the paired regions. Saturation plots at comparable levels of sequence divergence demonstrate no saturation effects. Phylogenetic analyses using both maximum likelihood and Bayesian methods support the monophyly of Sisoridae. Chinese sisorid catfishes are composed of two major lineages, one represented by (Gagata (Bagarius, Glyptothorax)) and the other by "glyptosternoids + Pseudecheneis".The glyptosternoids may not be a monophyletic group. A previous hypothesis referring to Pseudecheneis as the sister group of monophyletic glyptosternoids, based on morphological evidence, is not supported by the molecular data.Pseudecheneis is shown to be a sister taxon of Glaridoglanis.Pareuchiloglanis might be paraphyletic with Pseudexostoma and Euchiloglanis. Our results also support the hypothesis that Pareuchiloglanis anteanalis might be considered as the synonyms of Pareuchiloglanis sinensis, and genus Euchiloglanis might have only one valid species, Euchiloglanis davidi.

  6. L-Rhamnose-binding lectins (RBLs) in channel catfish, Ictalurus punctatus: Characterization and expression profiling in mucosal tissues. (United States)

    Thongda, Wilawan; Li, Chao; Luo, Yupeng; Beck, Benjamin H; Peatman, Eric


    Rhamnose-binding lectins (RBLs) have recently emerged as important molecules in the context of innate immunity in teleost fishes. Previously, using RNA-seq technology, we observed marked up-regulation of a RBL in channel catfish (Ictalurus punctatus) gill following a challenge with the bacterial pathogen Flavobacterium columnare. Furthermore, the magnitude of RBL up-regulation positively correlated with disease susceptibility. Moving forward from these findings, we wished to more broadly understand RBL function, diversity, and expression kinetics in channel catfish. Therefore, in the present study we characterized the RBL gene family present in select channel catfish tissues and profiled family member expression after challenge with two different Gram-negative bacterial pathogens. Here, six RBLs were identified from channel catfish and were designated IpRBL1a, IpRBL1b, IpRBL1c, IpRBL3a, IpRBL3b, and IpRBL5a. These RBLs contained carbohydrate recognition domains (CRD) ranging from one to three domains and each CRD contained the conserved motifs of -YGR- and -DPC-. Despite a level of structural conservation, the catfish RBLs showed low full-length identity with RBLs from outside the order Siluriformes. IpRBL expression after bacterial infection varied depending on both pathogen and tissue type, suggesting that IpRBLs may exert disparate functions or exhibit distinct tissue-selective roles in the host immune response to bacterial pathogens.

  7. Draft genome sequences of three Aeromonas hybrophila isolates from catfish and tilapia (United States)

    Aeromonas hydrophila is a Gram-negative bacteria that is particularly adapted to freshwater environments and can cause severe infections in fish and humans. Here we report the draft genomes of three A. hydrophila catfish and tilapia isolates....

  8. Gene expression analysis in gonads and brain of catfish Clarias batrachus after the exposure of malathion. (United States)

    Prathibha, Y; Murugananthkumar, R; Rajakumar, A; Laldinsangi, C; Sudhakumari, C C; Mamta, S K; Dutta-Gupta, A; Senthilkumaran, B


    Pesticides like malathion have the potential to disrupt development and reproduction of aquatic organisms including fishes. To investigate the likely consequences of malathion exposure at low doses in juvenile catfish, Clarias batrachus, we studied the expression pattern of genes encoding certain transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes which are known to be involved in gonadal development along with histological changes. To compare further, we also analyzed certain brain specific genes related to gonadal axis. Fifty days post hatch catfish fingerlings were exposed continuously to 1 and 10 µg/L of malathion for 21 days. Results from these experiments indicated that transcript levels of various genes were altered by the treatments, which may further affect the gonadal development either directly or indirectly through brain. Histological analysis revealed slow progression of spermatogenesis in testis, while in ovary, the oil droplet oocytes were found to be higher after treatment (10 µg/L). Our findings revealed that the exposure of malathion, even at low doses, hinder or modulate early gonadal development differentially by targeting gene expression pattern of transcription factors, activin A, sex steroid or orphan nuclear receptors and steroidogenic enzymes with an evidence on histological changes. Further, some of the genes showed differential expression at the level of brain in male and female sex after the exposure of malathion.

  9. Molecular identification and expression analysis of a goose-type lysozyme (LysG) gene in yellow catfish Pelteobagrus fulvidraco. (United States)

    Liu, Qiu-Ning; Xin, Zhao-Zhe; Zhang, Dai-Zhen; Jiang, Sen-Hao; Chai, Xin-Yue; Li, Chao-Feng; Zhou, Chun-Lin; Tang, Bo-Ping


    Lysozymes, innate immunity molecules, play a vital role in immune response to pathogens. The yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae) is an economically important fish in China. The aim of this study was to quantify expression of the P. fulvidraco LysG gene (a g-type lysozyme) in response to pathogen-associated molecular patterns (PAMP) challenge. First, the P. fulvidraco LysG gene (PfLysG) was cloned and characterized. The full-length cDNA of PfLysG is 1323 bp, including a 5'-untranslated region (UTR) of 131 bp, a 3'-UTR of 634 bp, and an open reading frame of 558 bp encoding a polypeptide of 185 amino acids, which contains a transglycosylase SLT domain (Pfam01464). The predicted molecular weight of the protein is 20.52 kDa with a pI of 9.08. Two catalytic residues and seven N-acetyl-D-glucosamine binding sites are conserved in the sequence and there is no predicted signal peptide. The deduced PfLysG protein sequence has 84%, 76% and 69% percent identity with the LysGs from Ictalurus furcatus, Danio rerio, and Salmo salar, respectively. The predicted tertiary structure of PfLysG is very similar to that from other animals. Phylogenetic analysis showed that PfLysG is closely related to those from Teleostei. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that PfLysG was expressed in all examined tissues and most highly expressed in head kidney, spleen, and intestine. After simulated pathogen challenge with lipopolysaccharide and polyriboinosinic polyribocytidylic acid, respectively, the mRNA expression of PfLysG was upregulated significantly at different time points. The results suggest that the identified g-type lysozyme of P. fulvidraco is involved in innate immune responses.

  10. Draft Genome Sequences of Three Aeromonas hydrophila Isolates from Catfish and Tilapia (United States)

    Tekedar, Hasan C.; Kumru, Salih; Kalindamar, Safak; Karsi, Attila; Waldbieser, Geoffrey C.; Sonstegard, Tad; Schroeder, Steven G.; Liles, Mark R.; Griffin, Matt J.


    ABSTRACT Aeromonas hydrophila is a Gram-negative bacterium that is particularly adapted to freshwater environments and can cause severe infections in fish and humans. Here, we report the draft genomes of three A. hydrophila catfish and tilapia isolates. PMID:28104665

  11. The channel catfish genome sequence provides insights into the evolution of scale formation in teleosts (United States)

    Channel catfish (Ictalurus punctatus), characteristic of its scaleless skin and prominent barbels, is an ideal species to study osteogenesis, development of appendages, olfactory sensing, and immunological adaptation. It is the leading aquaculture species in the United States. A high quality refere...

  12. Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens (United States)

    Iwanowicz, Luke R.; Stafford, James L.; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W.; Blazer, Vicki


    Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

  13. Molecular cloning and expression analysis of fushi tarazu factor 1 in the brain of air-breathing catfish, Clarias gariepinus.

    Directory of Open Access Journals (Sweden)

    Parikipandla Sridevi

    Full Text Available BACKGROUND: Fushi tarazu factor 1 (FTZ-F1 encodes an orphan nuclear receptor belonging to the nuclear receptor family 5A (NR5A which includes adrenal 4-binding protein or steroidogenic factor-1 (Ad4BP/SF-1 and liver receptor homologue 1 (LRH-1 and plays a pivotal role in the regulation of aromatases. METHODOLOGY/PRINCIPAL FINDINGS: Present study was aimed to understand the importance of FTZ-F1 in relation to brain aromatase (cyp19a1b during development, recrudescence and after human chorionic gonadotropin (hCG induction. Initially, we cloned FTZ-F1 from the brain of air-breathing catfish, Clarias gariepinus through degenerate primer RT-PCR and RACE. Its sequence analysis revealed high homology with other NR5A1 group members Ad4BP/SF-1 and LRH-1, and also analogous to the spatial expression pattern of the latter. In order to draw functional correlation of cyp19a1b and FTZ-F1, we analyzed the expression pattern of the latter in brain during gonadal ontogeny, which revealed early expression during gonadal differentiation. The tissue distribution both at transcript and protein levels revealed its prominent expression in brain along with liver, kidney and testis. The expression pattern of brain FTZ-F1 during reproductive cycle and after hCG induction, in vivo was analogous to that of cyp19a1b shown in our earlier study indicating its involvement in recrudescence. CONCLUSIONS/SIGNIFICANCE: Based on our previous results on cyp19a1b and the present data, it is plausible to implicate potential roles for brain FTZ-F1 in ovarian differentiation and recrudescence process probably through regulation of cyp19a1b in teleosts. Nevertheless, these interactions would require primary coordinated response from ovarian aromatase and its related transcription factors.

  14. Chronic hyperosmotic stress inhibits renal Toll-Like Receptors expression in striped catfish (Pangasianodon hypophthalmus, Sauvage) exposed or not to bacterial infection. (United States)

    Schmitz, Mélodie; Baekelandt, Sébastien; Bequet, Sandrine; Kestemont, Patrick


    Toll-like Receptors (TLRs) are the first innate receptors in recognizing pathogen-associated molecular patterns. In fish, upregulation of toll-like receptors during infection has been largely demonstrated while the effects of abiotic stressors on their expression remain poorly investigated. In this study, striped catfish were submitted during 20 days to three salinity profiles (freshwater, low saline water, saline water), followed by injection of a bacterial strain of Edwardsiella ictaluri. The expression of TLRs 1, 3, 4, 5, 7, 9, 19, 21, and 22 was measured in kidney at different time points in non infected and infected striped catfish. Infection induced overexpression of TLRs 1, 3, 4, 5, 7, 21 and 22. With elevated salinity, the expression of all TLRs, except TLR5, was severely decreased, particularly after bacterial infection. The TLRs responsiveness of striped catfish facing bacterial disease and salinity stress and possible consequences on striped catfish immune response's efficiency are discussed.

  15. The Innate Immune-Related Genes in Catfish

    Directory of Open Access Journals (Sweden)

    Weidong Liu


    Full Text Available Catfish is one of the most important aquaculture species in America (as well as in Asia and Africa. In recent years, the production of catfish has suffered massive financial losses due to pathogen spread and breakouts. Innate immunity plays a crucial role in increasing resistance to pathogenic organisms and has generated increasing interest in the past few years. This review summarizes the current understanding of innate immune-related genes in catfish, including pattern recognition receptors, antimicrobial peptides, complements, lectins, cytokines, transferrin and gene expression profiling using microarrays and next generation sequencing technologies. This review will benefit the understanding of innate immune system in catfish and further efforts in studying the innate immune-related genes in fish.

  16. Complete genome sequence of the highly virulent Aeromonas hydrophila AL09-71 isolated from disease channel catfish from West Alabama (United States)

    The highly virulent Aeromonas hydrophila isolate AL09-71 was cultured from infected channel catfish during the 2009 disease outbreak in West Alabama. The complete genome of this highly virulent Aeromonas hydrophila isolate AL09-71 is 5,023,861 bp in size. The genome has 4489 coding sequences, 11 cop...

  17. Characterization of gamma-crystallin from a catfish: structural characterization of one major isoform with high methionine by cDNA sequencing. (United States)

    Pan, F M; Chang, W C; Lin, C H; Hsu, A L; Chiou, S H


    gamma-Crystallin is the major and most abundant lens protein present in the eye lens of most teleostean fishes. To facilitate structural characterization of gamma-crystallins isolated from the lens of the catfishes (Clarias fuscus), a cDNA mixture was synthesized from the poly(A)+mRNA isolated from fresh eye lenses, and amplification by polymerase chain reaction (PCR) was adopted to obtain cDNAs encoding various gamma-crystallins. Plasmids of transformed E. coli strain JM109 containing amplified gamma-crystallin cDNAs were purified and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing more than five clones containing DNA inserts of 0.52 kb revealed the presence of one major isoform with a complete reading frame of 534 base pairs, covering a gamma-crystallin (gamma M1) with a deduced protein sequence of 177 amino acids excluding the initiating methionine. It was of interest to find that this crystallin of pI 9.1 contains a high-methionine content of 15.3% in contrast to those gamma-crystallins of low-methionine content from most mammalian lenses. Sequence comparisons of catfish gamma M1-crystallin with those published sequences of gamma-crystallins from carp, bovine and mouse lenses indicate that there is approx. an 82% sequence homology between the catfish and the carp species of piscine class whereas only 51-58% homology is found between mammals and the catfish. Moreover the differences in the hydropathy profiles for these two groups of gamma-crystallins, i.e. one with a high-methionine content from teleostean fishes and the other with a low-methionine content from mammalian species, reflect a distinct variance in the polarity distributions of surface amino acids in these crystallins.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Chicken-type lysozyme in channel catfish: Expression analysis, lysozyme activity and efficacy as immunostimulant against Aeromonas hydrophila infection (United States)

    To understand whether chicken-type lysozyme (Lys-c) in channel catfish was induced by infection of Aeromonas hydrophila, the transcriptional levels of Lys-c in skin, gut, liver, spleen, posterior kidney, and blood cells in healthy channel catfish was compared to that in channel catfish infected with...

  19. Expression of leptin-like mRNA in channel catfish (Ictalurus punctatus) is induced by exposure to Edwardsiella ictaluri but is independent of energy status (United States)

    Leptin is a key pleiotropic cytokine involved in regulation of food intake and energy homeostasis in mammals. In channel catfish, the presence of a messenger RNA that encodes a leptin-like peptide (LLP) has been described. The objectives of our studies were to characterize tissue expression of LLP...

  20. Identification and characterization of TCRgamma and TCRdelta chains in channel catfish, Ictalurus punctatus (United States)

    Channel catfish, Ictalurus punctatus, T cell receptors (TCR) gamma and delta were identified by mining of expressed sequence tag databases and full length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), (diversity; D), joining ...

  1. Complete genome sequence of channel catfish epidemic isolate Aeromonas hydrophila ML09-119 (United States)

    Aeromonas hydrophila is a Gram-negative, rod-shaped, mesophilic bacteria that infects both aquatic poikilothermic animals and mammals, including humans. Here, we present the complete genome sequence of Aeromonas hydrophila ML-09-119, which represents a clonal group of A. hydrophila isolates causing ...

  2. Expression Patterns of Cytochrome P450 Aromatase Genes During Ovary Development and Their Responses to Temperature Stress in Female Yellow Catfish (Pelteobagrus fulvidraco)

    Institute of Scientific and Technical Information of China (English)

    LIU Miao; QI Baoxia; WEN Haishen; HE Feng; LI Jifang; SHI Dan; HU Jian; ZHANG Yuanqing; MA Ruiqin; MU Weijie


    Cytochrome P450 aromatase (P450arom) plays a pivotal role in ovary development.In this study,we used semi-quantitative reverse transcription PCR (RT-PCR) to analyze spatiotemporal expressions of two P450arom genes (CYP1gA and CYP19B)and their responses to temperature stress in female yellow catfish (Pelteobagrusfulvidraco).Tissue distribution pattern of CYP19showed that CYP19B was abundantly expressed in fish brain and ovary (brain>ovary),but weakly in intestines,whereas CYP19Awas exclusively expressed in ovary.Semi-quantitative RT-PCR analyses showed high transcript abundance of both CYP19A and CYP19B in the ovarian reproductive cycle,corresponding with serum estradiol-17β (E2) levels.Increases in aromatases,serum E2 and testosterone (T) levels in fish exposed to higher temperature indicated stimulation of ovarian maturation and recrudescence by heat stress in stages Ⅱ and V during the ovarian cycle,whereas associated decreases in stage III suggested vitellogenesis inhibition by heat stress.Gene expression of CYP19 was closely related to levels of serum E2.Results demonstrated CYP19 played a crucial role in the reproductive cycle of female yellow catfish.Different temperature stress affected CYP19 gene expression in the fish ovarian reproductive cycle.Associated P450arom genes could be useful for studying physiological aspects of yellow catfish.

  3. Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17β on mRNA expression. (United States)

    Chaube, R; Rawat, A; Inbaraj, R M; Bobe, J; Guiguen, Y; Fostier, A; Joy, K P


    Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the

  4. RNA-Seq reveals expression signatures of genes involved in oxygen transport, protein synthesis, folding, and degradation in response to heat stress in catfish. (United States)

    Liu, Shikai; Wang, Xiuli; Sun, Fanyue; Zhang, Jiaren; Feng, Jianbin; Liu, Hong; Rajendran, K V; Sun, Luyang; Zhang, Yu; Jiang, Yanliang; Peatman, Eric; Kaltenboeck, Ludmilla; Kucuktas, Huseyin; Liu, Zhanjiang


    Temperature is one of the most prominent abiotic factors affecting ectotherms. Most fish species, as ectotherms, have extraordinary ability to deal with a wide range of temperature changes. While the molecular mechanism underlying temperature adaptation has long been of interest, it is still largely unexplored with fish. Understanding of the fundamental mechanisms conferring tolerance to temperature fluctuations is a topic of increasing interest as temperature may continue to rise as a result of global climate change. Catfish have a wide natural habitat and possess great plasticity in dealing with environmental variations in temperature. However, no studies have been conducted at the transcriptomic level to determine heat stress-induced gene expression. In the present study, we conducted an RNA-Seq analysis to identify heat stress-induced genes in catfish at the transcriptome level. Expression analysis identified a total of 2,260 differentially expressed genes with a cutoff of twofold change. qRT-PCR validation suggested the high reliability of the RNA-Seq results. Gene ontology, enrichment, and pathway analyses were conducted to gain insight into physiological and gene pathways. Specifically, genes involved in oxygen transport, protein folding and degradation, and metabolic process were highly induced, while general protein synthesis was dramatically repressed in response to the lethal temperature stress. This is the first RNA-Seq-based expression study in catfish in response to heat stress. The candidate genes identified should be valuable for further targeted studies on heat tolerance, thereby assisting the development of heat-tolerant catfish lines for aquaculture.

  5. Expressing stochastic filters via number sequences


    Capponi, A.; Farina, A; Pilotto, C.


    We generalize the results presented in [1] regarding the relation between the Kalman filter and the Fibonacci sequence. We consider more general filtering models and relate the finite dimensional Kalman and Benes filters to the Fibonacci sequence and to the Golden Section. We also prove that Fibonacci numbers may be expressed as the convolution of the Fibonacci and Padovan sequence, thus extending the connection between stochastic filtering and Fibonacci sequence to the Padovan sequence.

  6. Expression, Purification and Antibacterial Activity of NK-Lysin Mature Peptides from the Channel Catfish (Ictalurus punctatus

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    Shurui Cai


    Full Text Available Antimicrobial peptides (AMPs are small peptides and play important roles in host innate immune response against microbial invasion. Aquatic animals secrete different kinds of antimicrobial peptides which have antimicrobial activity towards microorganisms. NK-lysins, mature peptides produced by cytotoxic T lymphocytes and natural killer cells, are comprised of 74–78 amino acid residues, demonstrating broad-spectrum antimicrobial activity against bacteria, fungi, protozoa, and parasites. In this study, three distinct NK-lysin mature peptide (mNKLs, transcripts (76 amino acid residues cloned from the channel catfish (Ictalurus punctatus head kidney were ligated into plasmid vector pET-32a(+ to express the mNKLs fusion protein. The fusion protein was successfully expressed in E. coli Rosetta (DE3 under optimized conditions. After purification by affinity column chromatography, the fusion protein was successfully cleaved by enterokinase and released the peptide mNKLs. Tricine-SDS-PAGE results showed that mNKLs (approximately 8.6 kDa were successfully expressed. The purified peptide mNKLs exhibited antibacterial activity against Staphylococcus aureus and E. coli.

  7. 18S rRNA gene sequencing identifies a novel species of Henneguya parasitizing the gills of the channel catfish (Ictaluridae). (United States)

    Rosser, Thomas G; Griffin, Matt J; Quiniou, Sylvie M A; Khoo, Lester H; Pote, Linda M


    In the southeastern USA, the channel catfish Ictalurus punctatus is a host to at least eight different species of myxozoan parasites belonging to the genus Henneguya, four of which have been characterized molecularly using sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. However, only two of these have confirmed life cycles that involve the oligochaete Dero digitata as the definitive host. During a health screening of farm-raised channel catfish, several fish presented with deformed primary lamellae. Lamellae harbored large, nodular, white pseudocysts 1.25 mm in diameter, and upon rupturing, these pseudocysts released Henneguya myxospores, with a typical lanceolate-shaped spore body, measuring 17.1 ± 1.0 μm (mean ± SD; range = 15.0-19.3 μm) in length and 4.8 ± 0.4 μm (3.7-5.6 μm) in width. Pyriform-shaped polar capsules were 5.8 ± 0.3 μm in length (5.1-6.4 μm) and 1.7 ± 0.1 μm (1.4-1.9 μm) in width. The two caudal processes were 40.0 ± 5.1 μm in length (29.5-50.0 μm) with a spore length of 57.2 ± 4.7 (46.8-66.8 μm). The contiguous SSU rRNA gene sequence obtained from myxospores of five excised cysts did not match any Henneguya sp. in GenBank. The greatest sequence homology (91% over 1,900 bp) was with Henneguya pellis, associated with blister-like lesions on the skin of blue catfish Ictalurus furcatus. Based on the unique combination of pseudocyst and myxospore morphology, tissue location, host, and SSU rRNA gene sequence data, we report this isolate to be a previously unreported species, Henneguya bulbosus sp. nov.

  8. cis sequence effects on gene expression

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    Jacobs Kevin


    Full Text Available Abstract Background Sequence and transcriptional variability within and between individuals are typically studied independently. The joint analysis of sequence and gene expression variation (genetical genomics provides insight into the role of linked sequence variation in the regulation of gene expression. We investigated the role of sequence variation in cis on gene expression (cis sequence effects in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. We estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature. Results We generated gene expression profiling data at N = 697 candidate genes from N = 30 lymphoblastoid cell lines for this study and used available candidate gene resequencing data at N = 552 candidate genes to identify N = 30 candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. We used two additive models and the haplotype phylogeny scanning approach of Templeton (Tree Scanning to evaluate association between individual SNPs, all SNPs at a gene, and diplotypes, with log-transformed gene expression. SNPs and diplotypes at eight candidate genes exhibited statistically significant (p cis sequence effects in our study, respectively. Conclusion Based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. Despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies.

  9. Characterization of the isoforms of type IIb sodium-dependent phosphate cotransporter (Slc34a2) in yellow catfish, Pelteobagrus fulvidraco, and their vitamin D3-regulated expression under low-phosphate conditions. (United States)

    Chen, Pei; Huang, Yanqing; Bayir, Abdulkadir; Wang, Chunfang


    In this study, two isoforms slc34a2 genes (type IIb sodium-dependent phosphate cotransporter), slc34a2a2 and slc34a2b, were cloned from intestine and kidney of yellow catfish (Pelteobagrus fulvidraco), with rapid amplification of cDNA ends. The structure differences and the regulation effects of dietary VD3 under low phosphorus were compared among three isoforms of slc34a2 in yellow catfish. The predicted Slc34a2a2 and Slc34a2b proteins match 65 % and 53.8 % sequence identity, with Slc34a2a1, respectively. The membrane-spanning domains were different among these three isoforms. Intestinal Slc34a2a1 and Slc34a2a2 proteins had eight and eleven transmembrane domains, while renal Slc34a2b protein had nine. The tissue distribution study showed that same as slc34a2a1, slc34a2a2 mRNA was mainly distributed in intestine and slc34a2b mRNA in kidney. The effect of vitamin D3 (VD3) level on slc34a2 subfamily expression under low-phosphate conditions, induced by the addition of 0 (VD0), 324 (VD1), 1243 (VD2), 3621 (VD3), 8040 (VD4), or 22700 (VD5) IU VD3/kg feed, was assessed by qPCR. The dose-responsive expression of intestinal slc34a2a2 and high expression of intestinal slc34a2a2 in VD5 together with peak expression of kidney slc34a2b in VD3 coincided with the accumulation of body phosphate content. These data suggested that appropriate level of dietary VD3 up-regulated slc34a2a1, slc34a2a2, and slc34a2b mRNA levels, which increased phosphate retention. In conclusion, the current study provided another possible approach to improve dietary phosphate utilization by adding appropriate level of VD3 to a low-phosphate diet to regulate intestinal and renal slc34a2 gene expression and thus minimize the excretion of phosphorus in yellow catfish.

  10. Early exposure of 17α-ethynylestradiol and diethylstilbestrol induces morphological changes and alters ovarian steroidogenic pathway enzyme gene expression in catfish, Clarias gariepinus. (United States)

    Sridevi, P; Chaitanya, R K; Prathibha, Y; Balakrishna, S L; Dutta-Gupta, A; Senthilkumaran, B


    Environmental estrogens are major cause of endocrine disruption in vertebrates, including aquatic organisms. Teleosts are valuable and popular models for studying the effects of endocrine disrupting chemicals (EDCs) in the environment. In the present study, we investigated the changes caused by exposure to the synthetic estrogens 17α-ethynylestradiol (EE2 ) and diethylstilbesterol (DES) during early stages of growth and sex differentiation of air-breathing catfish, Clarias gariepinus, at the morphological, histological, and molecular levels. Catfish hatchlings, 0 day post hatch (dph) were exposed continuously to sublethal doses of EE2 (50 ng/L) and DES (10 ng/L) until 50 dph and subsequently monitored for ovarian structural changes and alteration in the gene expression of steroidogenic enzymes till adulthood. Treated fish exhibited morphological deformities such as spinal curvature, stunted growth, and yolk-sac fluid retention. In addition to ovarian atrophy, DES-treated fish showed either rudimentary or malformed ovaries. Detailed histological studies revealed precocious oocyte development as well as follicular atresia. Further, transcript levels of various steroidogenic enzyme and transcription factor genes were altered in response to EE2 and DES. Activity of the rate-limiting enzyme of estrogen biosynthesis, aromatase, in the ovary as well as the brain of treated fish was in accordance with transcript level changes. These developmental and molecular effects imparted by EE2 and DES during early life stages of catfish could demonstrate the deleterious effects of estrogen exposure and provide reliable markers for estrogenic EDCs exposure in the environment.

  11. Molecular characterization and expression analysis of a novel cystatin-like gene in a hypoxia-tolerant Indian catfish, Clarias batrachus [Linnaeus, 1758]. (United States)

    Mohindra, Vindhya; Tripathi, Ratnesh K; Singh, Akanksha; Singh, Balvinder


    A novel member of Cystatin superfamily was identified from Indian catfish, Clarias batrachus, in response to oxidation stress induced by environmental hypoxia. Integrated genomic approaches, expression profiling and computational techniques showed that CbCystatin had putative cystatin/monelin like domain and might be a transmembrane and/or intermediate protein in signaling pathways. CbCystatin was found to be clustered into family 2 Cystatins. At transcriptional level, its expression was significantly up-regulated in response to short as well as long periods (more than 20 fold) of hypoxia, suggesting its positive association with oxygen concentrations lower than physiological concentrations.

  12. Effect of ammonia-N and pathogen challenge on complement component 8α and 8β expression in the darkbarbel catfish Pelteobagrus vachellii. (United States)

    Qin, Chuanjie; Shao, Ting; Zhao, Daxian; Duan, Huiguo; Wen, Zhengyong; Yuan, Dengyue; Li, Huatao; Qi, Zemin


    The complement components C8α and C8β mediate the formation of the membrane attack complex (MAC) to resist pathogenic bacteria and play important roles in innate immunity. Full-length complement C8α (Pv-C8α) and C8β (Pv-C8β) cDNA were identified in the darkbarbel catfish Pelteobagrus vachellii, and their mRNA expression levels were analyzed after ammonia-N and pathogen treatment. The Pv-C8α gene contained 1983 bp, including a 1794-bp open reading frame (ORF) encoding 598 amino acids. The Pv-C8β gene contained 1952 bp, including a 1761-bp ORF encoding 587 amino acids. Pv-C8α and Pv-C8β had the highest amino acid identity with rainbow trout Oncorhynchus mykiss C8α (62%) and Japanese flounder Paralichthys olivaceus C8β (83%), respectively. Sequence analysis indicated that both Pv-C8α and Pv-C8β contained a thrombospondin type-1 (TSP1) domain, a low-density lipoprotein receptor class A (LDLR-A) domain, a membrane attack complex/perforin (MACPF) domain and an epidermal growth factor-like (EGF-like) domain. In addition, Pv-C8α and Pv-C8β were mainly distributed in the liver, head kidney, spleen, and eggs. Under ammonia-N stress, the Pv-C8α and Pv-C8β mRNA levels significantly decreased (P spleen. After Aeromonas hydrophila challenge, the Pv-C8α and Pv-C8β mRNA levels significantly increased (P spleen. The present study indicated that Pv-C8α and Pv-C8β exhibited important immune responses to infection and that ammonia-N in water decreased the immune responses of Pv-C8α and Pv-C8β.

  13. Genomic organisation of the channel catfish Mx1 gene and characterisation of multiple channel catfish Mx gene promoters. (United States)

    Plant, Karen P; Thune, Ronald L


    In order to further characterise channel catfish (Ictalurus punctatus) Mx1, studies were initiated to amplify and clone the Mx1 promoter into a reporter vector, pGL3basic. Initially the Mx1 gene was amplified from genomic DNA and was found to have 12 exons and 11 introns, spanning a region over 6 kilobases (kb) in length. The Mx1 promoter was amplified using genome walking and during this process four additional Mx promoters were identified, suggesting the presence of five Mx genes in the channel catfish. All five promoters possess an interferon stimulated response element (ISRE) and the Mx1 promoter possessed two potential NF-kappabeta transcription sites. Following cloning each construct was transiently transfected into COS-7 and EPC cells for 24h and treated with 5 microg/ml poly I:C for 24h. An increase in expression of the reporter gene in response to poly I:C was noted in both cell lines in the pGL3Mx1 construct only. However, the reporter gene was also constitutively expressed in these cells. Constitutive expression was also observed in channel catfish ovary cells transiently transfected with pGL3Mx1 only. Treatment with 5 microg/ml poly I:C did not increase this expression, which may be due to high levels of cell death in this difficult to transfect cell line. The constitutive expression observed implies that a repressor element is missing in the 390 base pair sequence of the Mx1 promoter used in this study. These results suggest that only channel catfish Mx1 is involved in the type I interferon pathway and that the presence of an ISRE in a regulatory region is not necessarily indicative of a role in the type I interferon response.

  14. Towards the Ictalurid Catfish Transcriptome: Generation and Analysis of 31,215 Catfish ESTs.


    Dunham Rex; Muir William; Liu Lei; Turan Cemal; Simmons Micah; Serapion Jerry; Somridhivej Benjaporn; Nandi Samiran; Kucuktas Huseyin; Xu Peng; Baoprasertkul Puttharat; He Chongbo; Feng Jinian; Wang Shaolin; Peatman Eric


    Abstract Background EST sequencing is one of the most efficient means for gene discovery and molecular marker development, and can be additionally utilized in both comparative genome analysis and evaluation of gene duplications. While much progress has been made in catfish genomics, large-scale EST resources have been lacking. The objectives of this project were to construct primary cDNA libraries, to conduct initial EST sequencing to generate catfish EST resources, and to obtain baseline inf...

  15. In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus). (United States)

    Hang, Bui Thi Bich; Milla, Sylvain; Gillardin, Virginie; Phuong, Nguyen Thanh; Kestemont, Patrick


    Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedly stimulates fish immune system. However, mechanisms driving this immunomodulatory effect are yet unknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression of striped catfish (Pangasianodon hypophthalmus), juvenile fish (20-25 g) were injected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol and glucose were rather low and did not differ (p<0.05) among treatments. All LPS treatments differed regarding blood cell count and immune variables such as plasma and spleen lysozyme, complement activity and antibody titer, 3mg LPS/kg yielding best results; red blood cell count was not affected by LPS treatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% for treatment 3, 15, 45 mg LPS/kg fish and control respectively. Proteomic analysis of peripheral blood mononuclear cells (PBMC) confirmed that LPS induced differentially over-expressed immune proteins such as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such as Wap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catfish injected with E.coli LPS enhanced innate immune responses.

  16. Expression characteristics of potential biomarker genes in Tra catfish, Pangasianodon hypophthalmus, exposed to trichlorfon. (United States)

    Sinha, Amit Kumar; Vanparys, Caroline; De Boeck, Gudrun; Kestemont, Patrick; Wang, Neil; Nguyen, Phuong Thanh; Scippo, Marie-Louise; De Coen, Wim; Robbens, Johan


    Trichlorfon (TRC) is the most common organophosphorous insecticide used in aquaculture practices in Southeast Asian countries. Indiscriminate use of TRC can either damage or alter the enzymatic and hormonal activities in the living organisms. In this present study, therefore, toxicogenomic analyses using real time PCR was used to characterize expression levels of various genes in Pangasianodon hypophthalmus after exposure to three concentrations, the 96 h 1/100LC(50) (0.01 mg/L), the 96 h 110LC(50) (0.1 mg/L) and the 96 h 12LC(50) (0.5 mg/L) of TRC for 6 h, 24 h, 96 h, 7 days, 14 days, 28 days and 56 days respectively. The expression kinetics of stress and other cellular toxicity representative genes such as heat shock protein70 (HSP70), growth hormone, acetylcholinesterase (AChE), trypsinogen, cytochrome P4501B (CYP1B) and cytochrome oxidase subunit 1 (COI) were investigated in liver and gills. TRC at a level of 0.1 mg/L and 0.5 mg/L induced a time and dose-dependent increase in the expression of the HSP70, COI and CYPIB while the transcript level of AChE, growth hormone and trypsinogen were significantly down-regulated. These results could permit to develop a "molecular biomarker system" which can be applied as a first-tier method of identifying contaminant exposure before effects at population level occur.

  17. JAK and STAT members of yellow catfish Pelteobagrus fulvidraco and their roles in leptin affecting lipid metabolism. (United States)

    Wu, Kun; Tan, Xiao-Ying; Xu, Yi-Huan; Chen, Qi-Liang; Pan, Ya-Xiong


    The present study clones and characterizes the full-length cDNA sequences of members in JAK-STAT pathway, explores their mRNA tissue expression and the biological role in leptin influencing lipid metabolism in yellow catfish Pelteobagrus fulvidraco. Full-length cDNA sequences of five JAKs and seven STAT members, including some splicing variants, were obtained from yellow catfish. Compared to mammals, more members of the JAKs and STATs family were found in yellow catfish, which provided evidence that the JAK and STAT family members had arisen by the whole genome duplications during vertebrate evolution. All of these members were widely expressed across the eleven tissues (liver, white muscle, spleen, brain, gill, mesenteric fat, anterior intestine, heart, mid-kidney, testis and ovary) but at the variable levels. Intraperitoneal injection in vivo and incubation in vitro of recombinant human leptin changed triglyceride content and mRNA expression of several JAKs and STATs members, and genes involved in lipid metabolism. AG490, a specific inhibitor of JAK2-STAT pathway, partially reversed leptin-induced effects, indicating that the JAK2a/b-STAT3 pathway exerts main regulating actions of leptin on lipid metabolism at transcriptional level. Meanwhile, the different splicing variants were differentially regulated by leptin incubation. Thus, our data suggest that leptin activated the JAK/STAT pathway and increases the expression of target genes, which partially accounts for the leptin-induced changes in lipid metabolism in yellow catfish.

  18. Dynamic expression of 11β-hydroxylase during testicular development, recrudescence and after hCG induction, in vivo and in vitro in catfish, Clarias batrachus. (United States)

    Rajakumar, Anbazhagan; Senthilkumaran, Balasubramanian


    Cytochrome P450 11β-hydroxylase (11β-h), is involved in the production of 11-hydroxytestosterone, an immediate precursor for 11-ketotestosterone (11-KT), a potent androgen in teleosts. To understand the role of 11β-h in gonadal development, maturation, function and recrudescence in an annually reproducing teleost, the present study was conducted using Clarias batrachus. Four forms of 11β-h cDNA, regular type (2.253 kb), variant 1 (1.290 kb), variant 2 (1.223 kb) and variant 3 (1.978 kb) were identified from the testis of catfish which expressed ubiquitously with high levels in testis. 11β-h transcripts were detected as early as 0 days post hatch further, stage- and sex-dependent increase in the 11β-h transcripts were seen during gonadal differentiation/development. In addition, high expression of 11β-h (regular type) in pre-spawning phase was detected. Corroboratively, levels of 11-KT in serum and testicular tissue was high during pre-spawning and spawning phases, which might facilitate initiation and normal progression of spermatogenesis. The expression of 11β-h was high after human chorionic gonadotropin induction in vivo (all forms), and in vitro (regular type). Immunohistochemical and immunofluorescence localization showed the presence of 11β-h in Sertoli and interstitial/Leydig cells of the testis. These results suggest that 11β-h is involved in late stages of testicular development, together with the regulation of seasonal reproductive cycle in catfish.

  19. Gonadal transcriptomic analysis of yellow catfish (Pelteobagrus fulvidraco): identification of sex-related genes and genetic markers. (United States)

    Lu, Jianguo; Luan, Peixian; Zhang, Xiaofeng; Xue, Shuqun; Peng, Lina; Mahbooband, Shahid; Sun, Xiaowen


    Yellow catfish (Pelteobagrus fulvidraco) has been recognized as a vital freshwater aquaculture species in East and Southeast Asia. In addition to its commercial interest, it is also attracted much attention because of its value in studying sex-determination mechanisms. A comprehensive gonadal transcriptome analysis is believed to provide a resource for genome annotation, candidate gene identification, and molecular marker development. Herein, we performed a de novo assembly of yellow catfish gonad transcriptome by high-throughput Illumina sequencing. A total of 82,123 contigs were obtained, ranging from 351 to 21,268 bp, and N50 of 2,329 bp. Unigenes of 21,869 in total were identified. Of these, 229 and 1,188 genes were found to be specifically expressed in XY gonad tissue for 1 yr and 2 yr old yellow catfish, respectively; correspondingly, 51 and 40 genes were identified in XX gonad tissue at those two stages. Gene ontology and KEGG analysis were conducted and classified all contigs into different categories. A large number of unigenes involved in sex determination were identified, as well as microsatellites and SNP variants. The expression patterns of sex-related genes were then validated by quantitative real-time PCR (qRT-PCR) suggesting the high reliability of RNA-Seq results. In this study, the transcriptome of yellow catfish gonad was first sequenced, assembled, and characterized; it provides a valuable genomic resource for better understanding of yellow catfish sex determination as well as development of molecular markers, thereby assisting in the production of monosex yellow catfish for aquaculture.

  20. Influence of environmental ammonia on the production of nitric oxide and expression of inducible nitric oxide synthase in the freshwater air-breathing catfish (Heteropneustes fossilis)

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Mahua G. [Biochemical Adaptation Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022 (India); Saha, Nirmalendu, E-mail: [Biochemical Adaptation Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022 (India)


    Highlights: Black-Right-Pointing-Pointer High environmental ammonia caused more production and accumulation of NO in air-breathing catfish (Heteropneustes fossilis). Black-Right-Pointing-Pointer Hyper-ammonia stress caused induction and zonal specific expression of iNOS enzyme protein, mRNA expression in different tissues. Black-Right-Pointing-Pointer Activation of NF{kappa}B that resulted under hyper-ammonia stress was believed to be the cause of induction of iNOS gene. - Abstract: Nitric oxide (NO) is a highly versatile and unique ubiquitous signaling molecule, and is known to play diverse physiological functions in mammals including those of adaptation to various stresses. The present study reports on the influence of exposure to high external ammonia (HEA) on the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), that produces NO from L-arginine in the freshwater air-breathing catfish (Heteropneustes fossilis), which is reported to tolerate a very HEA. Some levels of NO were found to be present in all the tissues and also in plasma of control fish, which further enhanced significantly in fishes treated with high concentrations of environmental ammonia (25 and 50 mM ammonium chloride) for 7 days, accompanied by more efflux of NO from the perfused liver. This was accomplished by the induction of iNOS activity in different tissues of fish exposed to HEA, which otherwise was not detectable in control fish. Exposure to 25 mM ammonium chloride also led to a significant expression of iNOS protein in different tissues, followed by further increase at 50 mM ammonium chloride. Further, there was an increase in the expression of iNOS mRNA in ammonia-treated fish, thus suggesting that the expression of iNOS gene under hyper-ammonia stress was probably regulated at the transcriptional level. Immunocytochemical analysis indicated that the expression of iNOS in different tissues was zonal specific and not expressed uniformly

  1. Sixteen EST-linked microsatellite markers in Günther's walking catfish, Clarias macrocephalus. (United States)

    Sukkorntong, Chantapim; Panprommin, Dutrudi; Poompuang, Supawadee


    Twenty-seven new microsatellite sequences were identified by screening 2029 expressed sequence tags from Günther's walking catfish, Clarias macrocephalus. Sixteen loci were polymorphic with the number of alleles ranging from two to 16 per locus and the observed and expected heterozygosities ranging from 0.4667 to 0.9333 and from 0.427 to 0.8819 per locus, respectively. Cross-species amplifications of all 16 primer pairs were tested in four other species of catfish including Clarias gariepinus, Pangasius hypophthalmus, Pangasius larnaudii and Pangasianodon gigas. Eleven loci were found to amplify in other species, with the number of polymorphic loci ranging from one in P. larnaudii to nine in C. gariepinus.

  2. Complete genome sequence of the highly virulent Aeromonas hydrophila AL09-71 isolated from diseased channel catfish in West Alabama (United States)

    Aeromonas hydrophila AL09-71 was isolated from diseased channel catfish in West Alabama during a 2009 disease outbreak. The full genome of A. hydrophila AL09-71 is 5,023,861 bp. The availability of this genome will allow comparative genomics to identify genes involved in pathogenesis or immunogens f...

  3. Analysis of expressed sequence tags from Plasmodium falciparum. (United States)

    Chakrabarti, D; Reddy, G R; Dame, J B; Almira, E C; Laipis, P J; Ferl, R J; Yang, T P; Rowe, T C; Schuster, S M


    An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

  4. Male-biased genes in catfish as revealed by RNA-Seq analysis of the testis transcriptome.

    Directory of Open Access Journals (Sweden)

    Fanyue Sun

    Full Text Available BACKGROUND: Catfish has a male-heterogametic (XY sex determination system, but genes involved in gonadogenesis, spermatogenesis, testicular determination, and sex determination are poorly understood. As a first step of understanding the transcriptome of the testis, here, we conducted RNA-Seq analysis using high throughput Illumina sequencing. METHODOLOGY/PRINCIPAL FINDINGS: A total of 269.6 million high quality reads were assembled into 193,462 contigs with a N50 length of 806 bp. Of these contigs, 67,923 contigs had hits to a set of 25,307 unigenes, including 167 unique genes that had not been previously identified in catfish. A meta-analysis of expressed genes in the testis and in the gynogen (double haploid female allowed the identification of 5,450 genes that are preferentially expressed in the testis, providing a pool of putative male-biased genes. Gene ontology and annotation analysis suggested that many of these male-biased genes were involved in gonadogenesis, spermatogenesis, testicular determination, gametogenesis, gonad differentiation, and possibly sex determination. CONCLUSION/SIGNIFICANCE: We provide the first transcriptome-level analysis of the catfish testis. Our analysis would lay the basis for sequential follow-up studies of genes involved in sex determination and differentiation in catfish.

  5. Male-Biased Genes in Catfish as Revealed by RNA-Seq Analysis of the Testis Transcriptome (United States)

    Sun, Fanyue; Liu, Shikai; Gao, Xiaoyu; Jiang, Yanliang; Perera, Dayan; Wang, Xiuli; Li, Chao; Sun, Luyang; Zhang, Jiaren; Kaltenboeck, Ludmilla; Dunham, Rex; Liu, Zhanjiang


    Background Catfish has a male-heterogametic (XY) sex determination system, but genes involved in gonadogenesis, spermatogenesis, testicular determination, and sex determination are poorly understood. As a first step of understanding the transcriptome of the testis, here, we conducted RNA-Seq analysis using high throughput Illumina sequencing. Methodology/Principal Findings A total of 269.6 million high quality reads were assembled into 193,462 contigs with a N50 length of 806 bp. Of these contigs, 67,923 contigs had hits to a set of 25,307 unigenes, including 167 unique genes that had not been previously identified in catfish. A meta-analysis of expressed genes in the testis and in the gynogen (double haploid female) allowed the identification of 5,450 genes that are preferentially expressed in the testis, providing a pool of putative male-biased genes. Gene ontology and annotation analysis suggested that many of these male-biased genes were involved in gonadogenesis, spermatogenesis, testicular determination, gametogenesis, gonad differentiation, and possibly sex determination. Conclusion/Significance We provide the first transcriptome-level analysis of the catfish testis. Our analysis would lay the basis for sequential follow-up studies of genes involved in sex determination and differentiation in catfish. PMID:23874634

  6. Identification of Bacillus strains for biological control of catfish pathogens. (United States)

    Ran, Chao; Carrias, Abel; Williams, Malachi A; Capps, Nancy; Dan, Bui C T; Newton, Joseph C; Kloepper, Joseph W; Ooi, Ei L; Browdy, Craig L; Terhune, Jeffery S; Liles, Mark R


    Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7) CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (Pbiological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.

  7. Efficient assembly and annotation of the transcriptome of catfish by RNA-Seq analysis of a doubled haploid homozygote

    Directory of Open Access Journals (Sweden)

    Liu Shikai


    Full Text Available Abstract Background Upon the completion of whole genome sequencing, thorough genome annotation that associates genome sequences with biological meanings is essential. Genome annotation depends on the availability of transcript information as well as orthology information. In teleost fish, genome annotation is seriously hindered by genome duplication. Because of gene duplications, one cannot establish orthologies simply by homology comparisons. Rather intense phylogenetic analysis or structural analysis of orthologies is required for the identification of genes. To conduct phylogenetic analysis and orthology analysis, full-length transcripts are essential. Generation of large numbers of full-length transcripts using traditional transcript sequencing is very difficult and extremely costly. Results In this work, we took advantage of a doubled haploid catfish, which has two sets of identical chromosomes and in theory there should be no allelic variations. As such, transcript sequences generated from next-generation sequencing can be favorably assembled into full-length transcripts. Deep sequencing of the doubled haploid channel catfish transcriptome was performed using Illumina HiSeq 2000 platform, yielding over 300 million high-quality trimmed reads totaling 27 Gbp. Assembly of these reads generated 370,798 non-redundant transcript-derived contigs. Functional annotation of the assembly allowed identification of 25,144 unique protein-encoding genes. A total of 2,659 unique genes were identified as putative duplicated genes in the catfish genome because the assembly of the corresponding transcripts harbored PSVs or MSVs (in the form of pseudo-SNPs in the assembly. Of the 25,144 contigs with unique protein hits, around 20,000 contigs matched 50% length of reference proteins, and over 14,000 transcripts were identified as full-length with complete open reading frames. The characterization of consensus sequences surrounding start codon and the stop

  8. Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags

    DEFF Research Database (Denmark)

    de Souza, S J; Camargo, A A; Briones, M R;


    by EST or full length cDNA sequences available in GenBank but not utilized in the initial annotation of the first human chromosome sequence. Thus despite representing less than 15% of all expressed human sequences in the public databases at the time of the present analysis, ORESTES sequences defined 48......Transcribed sequences in the human genome can be identified with confidence only by alignment with sequences derived from cDNAs synthesized from naturally occurring mRNAs. We constructed a set of 250,000 cDNAs that represent partial expressed gene sequences and that are biased toward the central...... coding regions of the resulting transcripts. They are termed ORF expressed sequence tags (ORESTES). The 250,000 ORESTES were assembled into 81,429 contigs. Of these, 1, 181 (1.45%) were found to match sequences in chromosome 22 with at least one ORESTES contig for 162 (65.6%) of the 247 known genes...

  9. Catfish culture in Southeast Asia


    Adan, R. I. Y.


    Catfish rank fifth in the world in terms of fresh and brackishwater fish culture. In Asia and the Pacific, the Clariidae family dominates production, representing nearly 80% of the total catfish production. Among the most cultured species are Clarias batrachus, C. macrocephalus, C. gariepinus. The domestic market generally absorbs catfish produce in Asia, although high-producing countries like Thailand and Vietnam engage in export. There are two basic markets for catfish: live fish and proces...

  10. Effect of nutrient restriction and re-feeding on calpain family genes in skeletal muscle of channel catfish (Ictalurus punctatus.

    Directory of Open Access Journals (Sweden)

    Elena Preziosa

    Full Text Available BACKGROUND: Calpains, a superfamily of intracellular calcium-dependent cysteine proteases, are involved in the cytoskeletal remodeling and wasting of skeletal muscle. Calpains are generated as inactive proenzymes which are activated by N-terminal autolysis induced by calcium-ions. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we characterized the full-length cDNA sequences of three calpain genes, clpn1, clpn2, and clpn3 in channel catfish, and assessed the effect of nutrient restriction and subsequent re-feeding on the expression of these genes in skeletal muscle. The clpn1 cDNA sequence encodes a protein of 704 amino acids, Clpn2 of 696 amino acids, and Clpn3 of 741 amino acids. Phylogenetic analysis of deduced amino acid sequences indicate that catfish Clpn1 and Clpn2 share a sequence similarity of 61%; catfish Clpn1 and Clpn3 of 48%, and Clpn2 and Clpn3 of only 45%. The domain structure architectures of all three calpain genes in channel catfish are similar to those of other vertebrates, further supported by strong bootstrap values during phylogenetic analyses. Starvation of channel catfish (average weight, 15-20 g for 35 days influenced the expression of clpn1 (2.3-fold decrease, P<0.05, clpn2 (1.3-fold increase, P<0.05, and clpn3 (13.0-fold decrease, P<0.05, whereas the subsequent refeeding did not change the expression of these genes as measured by quantitative real-time PCR analysis. Calpain catalytic activity in channel catfish skeletal muscle showed significant differences only during the starvation period, with a 1.2- and 1.4- fold increase (P<0.01 after 17 and 35 days of starvation, respectively. CONCLUSION/SIGNIFICANCE: We have assessed that fasting and refeeding may provide a suitable experimental model to provide us insight into the role of calpains during fish muscle atrophy and how they respond to changes in nutrient supply.

  11. Effects of the fish spawning inducer ovaprim on vasotocin receptor gene expression in brain and ovary of the catfish Heteropneustes fossilis with a note on differential transcript expression in ovarian follicles. (United States)

    Rawat, A; Chaube, R; Joy, K P


    Ovaprim (OVP), a commercial formulation of a salmon GnRH analogue and the dopamine receptor-2 blocker domperidone, is a successful spawning inducer for fish breeding. It induces a preovulatory surge in LH, which stimulates the synthesis of a maturation-inducing steroid (MIS, 17,20β-dihydroxy-4-pregnen-3-one) that initiates germinal vesicle breakdown (GVBD) and ovulation. Coincidently, the OVP treatment also stimulates vasotocin (VT) secretion in the brain and ovary of the catfish Heteropneustes fossilis that also stimulates the synthesis of the MIS. VT mediates its effect through V1- and V2-type receptors. In the present study in the catfish, we report that OVP stimulates the expression of VT receptor genes v1a1, v1a2 and v2a in the brain and ovary. A single intraperitoneal administration of OVP (0.5μL/g body weight) or incubation of post-vitellogenic ovarian follicles with 5μL/mL OVP, for 0, 4, 8, 12, 16, and 24h stimulated ovulation and GVBD, respectively, in a time-dependent manner. The OVP treatment in vivo stimulated brain VT receptor transcript levels 4h onwards. The peak expression was noticed at 12h (v1a1), 8 and 12h (v1a2), and 8, 12 and 16h (v2a), coinciding with FOM and ovulation. The VT receptor genes are expressed in the ovarian follicles compartmentally; both v1a1 and v1a2 are expressed in the isolated follicular layer (theca and granulosa) but absent in denuded oocytes. V2a is expressed in the denuded oocytes and not in the follicular layer. The OVP injection stimulated the v1a1 and v1a2 expression from 4h onwards in both intact follicle and isolated follicular layer, the peak expression was observed at 16h. The v2a expression was up-regulated in both intact follicles and denuded oocytes at 4h (denuded oocytes) or 8h (intact follicle) onwards with the peak expression at 12h and 16h (denuded oocytes) or at 16h (intact follicles). Under in vitro conditions, the OVP incubations elicited similar pattern of changes with the peak stimulation at 16h for

  12. Expression analysis and localization of wt1, ad4bp/sf-1 and gata4 in the testis of catfish, Clarias batrachus: Impact of wt1-esiRNA silencing. (United States)

    Murugananthkumar, Raju; Senthilkumaran, Balasubramanian


    In teleosts, a comprehensive role or interaction of wt1, ad4bp/sf-1 and gata4 genes in relation to gonadal development and/or recrudescence was never attempted. Present study aimed to identify the involvement of these genes during testicular development of catfish, Clarias batrachus. Dominant expression of wt1 and gata4 was observed in developing and adult testis, while ad4bp/sf-1 showed steady expression. Localization of these genes in adult testis revealed their presence in spermatogonia, spermatocytes and interstitial/Leydig cells. Significant high expression during pre-spawning and spawning phases, and upregulated levels of these genes after hCG induction authenticated gonadotropic regulation. Transient silencing of wt1-esiRNA displayed decrease in wt1 expression, which further downregulated the expression of ad4bp/sf-1 and gata4, and certain steroidogenic enzyme genes related to androgen production. These results suggest that wt1 might target ad4bp/sf-1 and gata4 expression, and also have regulatory influence either indirectly or directly on the steroidogenic enzyme genes of catfish.

  13. BOB.1 of the channel catfish, Ictalurus punctatus: not a transcriptional coactivator? (United States)

    Richard, Mara L Lennard; Hikima, Jun-ichi; Wilson, Melanie R; Miller, Norman W; Cunningham, Charles; Warr, Gregory W


    Expression of the immunoglobulin heavy chain (IGH) locus of the channel catfish (Ictalurus punctatus) is driven by the Emu3' enhancer, whose core region contains two octamer motifs and a muE5 site. Orthologues of the Oct1 and Oct2 transcription factors have been cloned in the channel catfish and shown to bind to the octamer motifs within the core enhancer. While catfish Oct2 is an activator of transcription, catfish Oct1 failed to drive transcription and may act as a negative regulator of IGH transcription. In mammals, the Oct co-activator BOB.1 (B cell Oct-binding protein1, also known as OCA-B and OBF-1) greatly enhances the transcriptional activity of Oct factors and plays an important role in the development of the immune system. An orthologue of BOB.1 has been cloned in the catfish, and its function characterized. The POU binding domain of the catfish BOB.1 was found to be 95% identical at the amino acid level with the binding domain of human BOB.1, and all the residues directly involved in binding to the Oct-DNA complex were conserved. Despite this conservation, catfish BOB.1 failed to enhance transcriptional activation mediated by endogenous or co-transfected catfish Oct2, and failed to rescue the activity of the inactive catfish Oct1. Electrophoretic mobility shift assays showed that catfish BOB.1 was capable of binding both catfish Oct1 and Oct2 when they formed a complex with the Oct motif. Analysis of recombinant chimeric catfish and human BOB.1 proteins demonstrated that the failure to drive transcription was due to the lack of a functional activation domain within the catfish BOB.1.

  14. Characterization and Development of EST-SSR Markers Derived from Transcriptome of Yellow Catfish

    Directory of Open Access Journals (Sweden)

    Jin Zhang


    Full Text Available Yellow catfish (Pelteobagrus fulvidraco is one of the most important freshwater fish due to its delicious flesh and high nutritional value. However, lack of sufficient simple sequence repeat (SSR markers has hampered the progress of genetic selection breeding and molecular research for yellow catfish. To this end, we aimed to develop and characterize polymorphic expressed sequence tag (EST–SSRs from the 454 pyrosequencing transcriptome of yellow catfish. Totally, 82,794 potential EST-SSR markers were identified and distributed in the coding and non-coding regions. Di-nucleotide (53,933 is the most abundant motif type, and AC/GT, AAT/ATT, AAAT/ATTT are respective the most frequent di-, tri-, tetra-nucleotide repeats. We designed primer pairs for all of the identified EST-SSRs and randomly selected 300 of these pairs for further validation. Finally, 263 primer pairs were successfully amplified and 57 primer pairs were found to be consistently polymorphic when four populations of 48 individuals were tested. The number of alleles for the 57 loci ranged from 2 to 17, with an average of 8.23. The observed heterozygosity (HO, expected heterozygosity (HE, polymorphism information content (PIC and fixation index (fis values ranged from 0.04 to 1.00, 0.12 to 0.92, 0.12 to 0.91 and −0.83 to 0.93, respectively. These EST-SSR markers generated in this study could greatly facilitate future studies of genetic diversity and molecular breeding in yellow catfish.

  15. Response of CYP1A Gene expression in fish liver of catfish (Ariopsis felis) from Gulf of Mexico and their relationship with the genetic variability. (United States)

    Zapata-Perez, Omar; Sanchez-Teyer, Lorenzo F; Perez-Nunez, Maria T; Arroyo-Herrera, Ana L; Moreno, Adriana Quiroz; Albores-Medina, Arnulfo


    We determined the hepatic Cytochrome P4501A (CYP1A) mRNA and Ethoxyresorufin-O-deethylase (EROD) activities in the fish, Ariopsis felis, from highly polluted to relatively pristine regions in the southwest Gulf of Mexico and their relationship with the genetic polymorphisms of the same fish. We hypothesized that a high genetic variation reflects interindividual variability in levels of CYP1A mRNA underlying the pathway culminating in EROD induction caused by the environmental contaminants. Catfish from Laguna de Mecoacan exhibited marked induction of CYP1A mRNA and high levels of hepatic EROD activities, whereas fish from Laguna de Celestun showed no induction of CYP1A mRNA and moderately low levels of EROD activities. In contrast, the similarity index considering all samples varied from 0.4 to 0.87, showing a wide range of variation. A dendrogram showed a clear grouping of fish collected from the Laguna de Terminos, Rio Coatzacoalcos and Laguna de Celestun, with discrete subgroups according to region. In contrast, fish from Laguna de Mecoacan were grouped together completely separate from the rest of the fish. Despite the low number of fish from Mecoacan (a high bootstrap support was observed in this group), the results indicated a significant genetic variability in comparison with the other ecosystems included. The differential level of expression of CYP1A and the EROD activity observed among the ecosystems analyzed could be due to the high range of genetic variation, with special emphasis on fish collected in Mecoacan where it is possible to find a subspecies of Ariopsis felis.

  16. Identification of Bacillus strains for biological control of catfish pathogens.

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    Chao Ran

    Full Text Available Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC and motile aeromonad septicaemia (MAS, respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7 CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05. A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.

  17. Sex-biased miRNAs in gonad and their potential roles for testis development in yellow catfish. (United States)

    Jing, Jing; Wu, Junjie; Liu, Wei; Xiong, Shuting; Ma, Wenge; Zhang, Jin; Wang, Weimin; Gui, Jian-Fang; Mei, Jie


    Recently, YY super-male yellow catfish had been created by hormonal-induced sex reversal and sex-linked markers, which provides a promising research model for fish sex differentiation and gonad development, especially for testis development. MicroRNAs (miRNAs) have been revealed to play crucial roles in the gene regulation and gonad development in vertebrates. In this study, three small RNA libraries constructed from gonad tissues of XX female, XY male and YY super-male yellow catfish were sequenced. The sequencing data generated a total of 384 conserved miRNAs and 113 potential novel miRNAs, among which 23, 30 and 14 miRNAs were specifically detected in XX ovary, XY testis, and YY testis, respectively. We observed relative lower expression of several miR-200 family members, including miR-141 and miR-429 in YY testis compared with XY testis. Histological analysis indicated a higher degree of testis maturity in YY super-males compared with XY males, as shown by larger spermatogenic cyst, more spermatids and fewer spermatocytes in the spermatogenic cyst. Moreover, five miR-200 family members were significantly up-regulated in testis when treated by 17α-ethinylestradiol (EE2), high dose of which will impair testis development and cell proliferation. The down-regulation of miR-141 and 429 coincides with the progression of testis development in both yellow catfish and human. At last, the expression pattern of nine arbitrarily selected miRNAs detected by quantitative RT-PCR was consistent with the Solexa sequencing results. Our study provides a comprehensive miRNA transcriptome analysis for gonad of yellow catfish with different sex genotypes, and identifies a number of sex-biased miRNAs, some of that are potentially involved in testis development and spermatogenesis.

  18. Comparative transcriptional analysis reveals distinct expression patterns of channel catfish genes after the first infection and re-infection with Aeromonas hydrophila (United States)

    To determine whether transcriptional levels of channel catfish (Ictalurus punctatus) genes are differentially regulated between a first infection with Aeromonas hydrophila and a re-infection, suppression subtractive hybridization (SSH) was performed in this study using anterior kidney cDNA after the...

  19. Identification of two uncoupling protein 2 mRNA in channel catfish: their tissue distribution and changes in their expression after prolonged fasting and refeeding (United States)

    Muscle growth in fish is regulated by complex interactions of neural and endocrine factors that regulate food intake and factors that regulate muscle nutrient metabolism. Our previous studies showed that channel catfish selected for increased growth consumed more food than those with slower growth....

  20. Predicting tissue-specific expressions based on sequence characteristics

    KAUST Repository

    Paik, Hyojung


    In multicellular organisms, including humans, understanding expression specificity at the tissue level is essential for interpreting protein function, such as tissue differentiation. We developed a prediction approach via generated sequence features from overrepresented patterns in housekeeping (HK) and tissue-specific (TS) genes to classify TS expression in humans. Using TS domains and transcriptional factor binding sites (TFBSs), sequence characteristics were used as indices of expressed tissues in a Random Forest algorithm by scoring exclusive patterns considering the biological intuition; TFBSs regulate gene expression, and the domains reflect the functional specificity of a TS gene. Our proposed approach displayed better performance than previous attempts and was validated using computational and experimental methods.

  1. Development of expressed sequence tag and expressed sequence tag–simple sequence repeat marker resources for Musa acuminata (United States)

    Passos, Marco A. N.; de Oliveira Cruz, Viviane; Emediato, Flavia L.; de Camargo Teixeira, Cristiane; Souza, Manoel T.; Matsumoto, Takashi; Rennó Azevedo, Vânia C.; Ferreira, Claudia F.; Amorim, Edson P.; de Alencar Figueiredo, Lucio Flavio; Martins, Natalia F.; de Jesus Barbosa Cavalcante, Maria; Baurens, Franc-Christophe; da Silva, Orzenil Bonfim; Pappas, Georgios J.; Pignolet, Luc; Abadie, Catherine; Ciampi, Ana Y.; Piffanelli, Pietro; Miller, Robert N. G.


    Background and aims Banana (Musa acuminata) is a crop contributing to global food security. Many varieties lack resistance to biotic stresses, due to sterility and narrow genetic background. The objective of this study was to develop an expressed sequence tag (EST) database of transcripts expressed during compatible and incompatible banana–Mycosphaerella fijiensis (Mf) interactions. Black leaf streak disease (BLSD), caused by Mf, is a destructive disease of banana. Microsatellite markers were developed as a resource for crop improvement. Methodology cDNA libraries were constructed from in vitro-infected leaves from BLSD-resistant M. acuminata ssp. burmaniccoides Calcutta 4 (MAC4) and susceptible M. acuminata cv. Cavendish Grande Naine (MACV). Clones were 5′-end Sanger sequenced, ESTs assembled with TGICL and unigenes annotated using BLAST, Blast2GO and InterProScan. Mreps was used to screen for simple sequence repeats (SSRs), with markers evaluated for polymorphism using 20 diploid (AA) M. acuminata accessions contrasting in resistance to Mycosphaerella leaf spot diseases. Principal results A total of 9333 high-quality ESTs were obtained for MAC4 and 3964 for MACV, which assembled into 3995 unigenes. Of these, 2592 displayed homology to genes encoding proteins with known or putative function, and 266 to genes encoding proteins with unknown function. Gene ontology (GO) classification identified 543 GO terms, 2300 unigenes were assigned to EuKaryotic orthologous group categories and 312 mapped to Kyoto Encyclopedia of Genes and Genomes pathways. A total of 624 SSR loci were identified, with trinucleotide repeat motifs the most abundant in MAC4 (54.1 %) and MACV (57.6 %). Polymorphism across M. acuminata accessions was observed with 75 markers. Alleles per polymorphic locus ranged from 2 to 8, totalling 289. The polymorphism information content ranged from 0.08 to 0.81. Conclusions This EST collection offers a resource for studying functional genes, including

  2. Recombinant goose-type lysozyme in channel catfish: lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection. (United States)

    Pridgeon, Julia W; Klesius, Phillip H; Dominowski, Paul J; Yancey, Robert J; Kievit, Michele S


    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection.

  3. A blackberry (Rubus L.) expressed sequence tag library for the development of simple sequence repeat markers (United States)

    A blackberry (Rubus L.) expressed sequence tag (EST) library was produced for developing simple sequence repeat (SSR) markers from the tetraploid blackberry cultivar, Merton Thornless, the source of the thornless trait in commercial cultivars. RNA was extracted from young expanding leaves and used f...

  4. Assessment of microcystin concentration in carp and catfish: a case study from Lakshmikund pond, Varanasi, India. (United States)

    Singh, Shweta; Asthana, Ravi Kumar


    The present study was conducted to analyse microcystin concentrations in Lakshmikund pond, Varanasi, India, as well as in carp and catfish of the pond. The concentrations of microcystin were found well above the WHO guidelines (1 µg/L) both for the dissolved and particulate fractions of bloom samples. The microcystin concentrations in different organs of carp and catfish were in the following sequence; liver > gut > kidney > gall bladder > gills > muscles and gut > liver > kidney > gall bladder > gills > muscles, respectively. The bioaccumulation of microcystin in carp and catfish was negatively correlated with body weight, and showed species specificity. The higher bioaccumulation of microcystin in muscles of catfish (>tenfold) over carp indicates a possible threat to human beings on consumption of catfish. Therefore, to avoid animal and human intoxication, routine analyses of microcystin in pond water as well as fishes are strongly recommended.

  5. Expression profiling and comparative sequence derived insights into lipid metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Callow, Matthew J.; Rubin, Edward M.


    Expression profiling and genomic DNA sequence comparisons are increasingly being applied to the identification and analysis of the genes involved in lipid metabolism. Not only has genome-wide expression profiling aided in the identification of novel genes involved in important processes in lipid metabolism such as sterol efflux, but the utilization of information from these studies has added to our understanding of the regulation of pathways participating in the process. Coupled with these gene expression studies, cross species comparison, searching for sequences conserved through evolution, has proven to be a powerful tool to identify important non-coding regulatory sequences as well as the discovery of novel genes relevant to lipid biology. An example of the value of this approach was the recent chance discovery of a new apolipoprotein gene (apo AV) that has dramatic effects upon triglyceride metabolism in mice and humans.

  6. Analysis of Seed-expressed Sequence Tags in Triticum aestivum

    Institute of Scientific and Technical Information of China (English)

    LIJia-Rui; WANGFang; ZHAOXiang-Yu; DONGYu-Xiu; ZHANGLi-Yuan; ZHANGXian-Sheng


    To isolate seed-expressed sequences, a cDNA library was constructed using wheat ( Triticumaest/vum L) seed tissues at 12 d after pollination. Plasmid DNAs of 10 000 clones randomly picked out fromthe library were prepared. The preparation of high density filters were made with the Biomek 2000 HDRTsystem, and then hybridized separately with three probes prepared by reverse transcription of RNA ofunpollinated ovary, embryo and endosperm. Based on the hybridization results, 800 clones expressed inembryo and/or endosperm were chosen for further analysis of expressed sequence tags (ESTs). Finally,216 different genes were identified preliminarily. Of them, 24 (11.5%) were considered identical to knownwheat genes, 122 (56%) were identified as putative new plant genes which may be involved in seed storageproteins, biochemical metabolisms, development, and other biological processes of seeds, while 70 (32.5%)sequence identities could not be determined.

  7. Catfish production using intensive aeration (United States)

    For the last 3 years, researchers at UAPB and NWAC have been monitoring and verifying production yields in intensively aerated catfish ponds with aeration rates greater than 6 hp/acre. We now have three years of data on commercial catfish production in intensively aerated ponds. With stocking densi...

  8. Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus). (United States)

    Murata, Jun; Bienzle, Dorothee; Brandle, Jim E; Sensen, Christoph W; De Luca, Vincenzo


    The Madagascar periwinkle (Catharanthus roseus) is well known to produce the chemotherapeutic anticancer agents, vinblastine and vincristine. In spite of its importance, no expressed sequence tag (EST) analysis of this plant has been reported. Two cDNA libraries were generated from RNA isolated from the base part of young leaves and from root tips to select 9,824 random clones for unidirectional sequencing, to yield 3,327 related sequences and 1,696 singletons by cluster analysis. Putative functions of 3,663 clones were assigned, from 5,023 non-redundant ESTs to establish a resource for transcriptome analysis and gene discovery in this medicinal plant.

  9. On the expressiveness of single-pass instruction sequences

    CERN Document Server

    Bergstra, J A


    We perceive programs as single-pass instruction sequences. A single-pass instruction sequence under execution is considered to produce a behaviour to be controlled by some execution environment. Threads as considered in basic thread algebra model such behaviours. We show that all regular threads, i.e. threads that can only be in a finite number of states, can be produced by single-pass instruction sequences without jump instructions if use can be made of Boolean registers. We also show that, in the case where goto instructions are used instead of jump instructions, a bound to the number of labels restricts the expressiveness.

  10. Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco. (United States)

    Ke, F; Zhang, H B; Wang, Y; Hou, L F; Dong, H J; Wang, Z F; Pan, G W; Cao, X Y


    This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR).

  11. Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

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    Nour Hammoudeh


    Full Text Available Leishmania Homologue of receptors for Activated C Kinase (LACK antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica.The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR technique.The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed.Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.

  12. Analysis of Expressed Sequence Tags from Liver Tissue in Swine

    Institute of Scientific and Technical Information of China (English)

    LI Ning; ZHAO Zhi-hui; LIU Zhao-liang; ZHAO Xing-bo; LIAN Zhen-xing; WU Chang-xin


    In order to study the expression of function gene and its effect on metabolic control and other physiological function in liver, 438 expressed sequence tags (ESTs) were determined, which were from a cDNA library of porcine liver tissue. The results showed that the nucleotide sequences of 186 ESTs have already presented in GenBank database, and 37 ESTs could be found the homology with human and other species,while the others were not identified. 45 full length insertion of the clones randomly isolated from cDNA library were also completely sequenced with different size, and the results showed that 19 of them were functionknown genes, 11 had no open reading frame ( ORF )at all and 15 had ORF but the function were not elucidated yet.

  13. Sequence and expression analysis of gaps in human chromosome 20

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Seemann, Stefan; Mang, Yuan;


    The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 (chr 20) currently has three unfinished gaps remaining on its q-arm. All three gaps are within gene-dense regions and....../or overlap disease-associated loci, including the DLGAP4 locus. In this study, we sequenced ~99% of all three unfinished gaps on human chr 20, determined their complete genomic sizes and assessed epigenetic profiles using a combination of Sanger sequencing, mate pair paired-end high-throughput sequencing...... and chromatin, methylation and expression analyses. We found histone 3 trimethylated at Lysine 27 to be distributed across all three gaps in immortalized B-lymphocytes. In one gap, five novel CpG islands were predominantly hypermethylated in genomic DNA from peripheral blood lymphocytes and human cerebellum...

  14. Transcriptional profiles of multiple genes in the anterior kidney of channel catfish vaccinated with an attenuated Aeromonas hydrophila. (United States)

    Mu, Xingjiang; Pridgeon, Julia W; Klesius, Phillip H


    A total of 22 uniquely expressed sequence tags (ESTs) were identified from channel catfish anterior kidney subtractive cDNA library at 12 h post vaccination with an attenuated Aeromonas hydrophila (AL09-71 N+R). Of the 22 ESTs, six were confirmed to be significantly (P < 0.05) induced by the vaccination. Of 88 channel catfish genes selected from literature, 14 were found to be significantly (P < 0.05) upregulated by the vaccination. The transcriptional levels of the total 20 genes induced by the vaccination were then compared to that induced by the virulent parent A. hydrophila (AL09-71) at different time points. At 3 h post vaccination (hpv) or infection (hpi), Na(+)/K(+) ATPase α subunit was upregulated the most. At 6 and 12 hpv or hpi, hepcidin and interleukin-1β were induced the highest. At 24 hpv or hpi, hepcidin was upregulated the most, followed by lysozyme c. At 48 hpi, lysozyme c and hepcidin were significantly induced. When vaccinated fish were challenged by AL09-71, relative percent of survival of vaccinated fish were 100% at 14 days post vaccination (dpv). Transcriptional levels of toll-like receptor 5 and hepcidin were significantly upregulated in vaccinated fish at 14 dpv. Taken together, our results suggest that vaccination with attenuated A. hydrophila mimics infection by live bacteria, inducing multiple immune genes in channel catfish.

  15. Novel circovirus in European catfish (Silurus glanis). (United States)

    Lőrincz, Márta; Dán, Adám; Láng, Mária; Csaba, György; Tóth, Adám György; Székely, Csaba; Cságola, Attila; Tuboly, Tamás


    Circular single-stranded DNA viral genomes had been identified worldwide in different species and in environmental samples. Among them, viruses belonging to the genus Circovirus of the family Circoviridae are present in birds and pigs, and recently, they were detected in barbels. The present study reports the identification of a new circovirus in fish. PCR amplification and sequencing were used to identify the novel circular DNA virus in European catfish (Silurus glanis). Full genome characterization and phylogenetic analysis showed that the virus belonged to the family Circoviridae and that it was distantly related to the previously described barbel circovirus.

  16. Expressed sequence tags (ESTs and simple sequence repeat (SSR markers from octoploid strawberry (Fragaria × ananassa

    Directory of Open Access Journals (Sweden)

    Bies Dawn H


    Full Text Available Abstract Background Cultivated strawberry (Fragaria × ananassa represents one of the most valued fruit crops in the United States. Despite its economic importance, the octoploid genome presents a formidable barrier to efficient study of genome structure and molecular mechanisms that underlie agriculturally-relevant traits. Many potentially fruitful research avenues, especially large-scale gene expression surveys and development of molecular genetic markers have been limited by a lack of sequence information in public databases. As a first step to remedy this discrepancy a cDNA library has been developed from salicylate-treated, whole-plant tissues and over 1800 expressed sequence tags (EST's have been sequenced and analyzed. Results A putative unigene set of 1304 sequences – 133 contigs and 1171 singlets – has been developed, and the transcripts have been functionally annotated. Homology searches indicate that 89.5% of sequences share significant similarity to known/putative proteins or Rosaceae ESTs. The ESTs have been functionally characterized and genes relevant to specific physiological processes of economic importance have been identified. A set of tools useful for SSR development and mapping is presented. Conclusion Sequences derived from this effort may be used to speed gene discovery efforts in Fragaria and the Rosaceae in general and also open avenues of comparative mapping. This report represents a first step in expanding molecular-genetic analyses in strawberry and demonstrates how computational tools can be used to optimally mine a large body of useful information from a relatively small data set.

  17. Apolipoprotein A1 in channel catfish: Transcriptional analysis, antimicrobial activity, and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection (United States)

    The objectives of this study were to: 1) determine transcriptional profiles of apolipoprotein A1 (ApoA1) in collected channel catfish tissues after infection with A. hydrophila by bath immersion; 2) investigate whether recombinant channel catfish apolipoprotein A1 produced in E. coli expression syst...

  18. Recombinant goose-type lysozyme in channel catfish: Lysozyme activity and efficacy as plasmid DNA immunostimulant against Aeromonas hydrophila infection (United States)

    The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme g (CC-Lys-g) produced in E. coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme g plasmid DNA could be used as an immunostimulant to protect chann...

  19. Quantitative modeling of a gene's expression from its intergenic sequence.

    Directory of Open Access Journals (Sweden)

    Md Abul Hassan Samee


    Full Text Available Modeling a gene's expression from its intergenic locus and trans-regulatory context is a fundamental goal in computational biology. Owing to the distributed nature of cis-regulatory information and the poorly understood mechanisms that integrate such information, gene locus modeling is a more challenging task than modeling individual enhancers. Here we report the first quantitative model of a gene's expression pattern as a function of its locus. We model the expression readout of a locus in two tiers: 1 combinatorial regulation by transcription factors bound to each enhancer is predicted by a thermodynamics-based model and 2 independent contributions from multiple enhancers are linearly combined to fit the gene expression pattern. The model does not require any prior knowledge about enhancers contributing toward a gene's expression. We demonstrate that the model captures the complex multi-domain expression patterns of anterior-posterior patterning genes in the early Drosophila embryo. Altogether, we model the expression patterns of 27 genes; these include several gap genes, pair-rule genes, and anterior, posterior, trunk, and terminal genes. We find that the model-selected enhancers for each gene overlap strongly with its experimentally characterized enhancers. Our findings also suggest the presence of sequence-segments in the locus that would contribute ectopic expression patterns and hence were "shut down" by the model. We applied our model to identify the transcription factors responsible for forming the stripe boundaries of the studied genes. The resulting network of regulatory interactions exhibits a high level of agreement with known regulatory influences on the target genes. Finally, we analyzed whether and why our assumption of enhancer independence was necessary for the genes we studied. We found a deterioration of expression when binding sites in one enhancer were allowed to influence the readout of another enhancer. Thus, interference

  20. Survey of transposable elements in sugarcane expressed sequence tags (ESTs

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    Rossi Magdalena


    Full Text Available The sugarcane expressed sequence tag (SUCEST project has produced a large number of cDNA sequences from several plant tissues submitted or not to different conditions of stress. In this paper we report the result of a search for transposable elements (TEs revealing a surprising amount of expressed TEs homologues. Of the 260,781 sequences grouped in 81,223 fragment assembly program (Phrap clusters, a total of 276 clones showed homology to previously reported TEs using a stringent cut-off value of e-50 or better. Homologous clones to Copia/Ty1 and Gypsy/Ty3 groups of long terminal repeat (LTR retrotransposons were found but no non-LTR retroelements were identified. All major transposon families were represented in sugarcane including Activator (Ac, Mutator (MuDR, Suppressor-mutator (En/Spm and Mariner. In order to compare the TE diversity in grasses genomes, we carried out a search for TEs described in sugarcane related species O.sativa, Z. mays and S. bicolor. We also present preliminary results showing the potential use of TEs insertion pattern polymorphism as molecular markers for cultivar identification.

  1. Characterization of simple sequence repeats (SSRs from Phlebotomus papatasi (Diptera: Psychodidae expressed sequence tags (ESTs

    Directory of Open Access Journals (Sweden)

    Hamarsheh Omar


    Full Text Available Abstract Background Phlebotomus papatasi is a natural vector of Leishmania major, which causes cutaneous leishmaniasis in many countries. Simple sequence repeats (SSRs, or microsatellites, are common in eukaryotic genomes and are short, repeated nucleotide sequence elements arrayed in tandem and flanked by non-repetitive regions. The enrichment methods used previously for finding new microsatellite loci in sand flies remain laborious and time consuming; in silico mining, which includes retrieval and screening of microsatellites from large amounts of sequence data from sequence data bases using microsatellite search tools can yield many new candidate markers. Results Simple sequence repeats (SSRs were characterized in P. papatasi expressed sequence tags (ESTs derived from a public database, National Center for Biotechnology Information (NCBI. A total of 42,784 sequences were mined, and 1,499 SSRs were identified with a frequency of 3.5% and an average density of 15.55 kb per SSR. Dinucleotide motifs were the most common SSRs, accounting for 67% followed by tri-, tetra-, and penta-nucleotide repeats, accounting for 31.1%, 1.5%, and 0.1%, respectively. The length of microsatellites varied from 5 to 16 repeats. Dinucleotide types; AG and CT have the highest frequency. Dinucleotide SSR-ESTs are relatively biased toward an excess of (AXn repeats and a low GC base content. Forty primer pairs were designed based on motif lengths for further experimental validation. Conclusion The first large-scale survey of SSRs derived from P. papatasi is presented; dinucleotide SSRs identified are more frequent than other types. EST data mining is an effective strategy to identify functional microsatellites in P. papatasi.

  2. De novo Assembly and Analysis of the Chilean Pencil Catfish Trichomycterus areolatus Transcriptome (United States)

    Schulze, Thomas T.; Ali, Jonathan M.; Bartlett, Maggie L.; McFarland, Madalyn M.; Clement, Emalie J.; Won, Harim I.; Sanford, Austin G.; Monzingo, Elyssa B.; Martens, Matthew C.; Hemsley, Ryan M.; Kumar, Sidharta; Gouin, Nicolas; Kolok, Alan S.; Davis, Paul H.


    Trichomycterus areolatus is an endemic species of pencil catfish that inhabits the riffles and rapids of many freshwater ecosystems of Chile. Despite its unique adaptation to Chile's high gradient watersheds and therefore potential application in the investigation of ecosystem integrity and environmental contamination, relatively little is known regarding the molecular biology of this environmental sentinel. Here, we detail the assembly of the Trichomycterus areolatus transcriptome, a molecular resource for the study of this organism and its molecular response to the environment. RNA-Seq reads were obtained by next-generation sequencing with an Illumina® platform and processed using PRINSEQ. The transcriptome assembly was performed using TRINITY assembler. Transcriptome validation was performed by functional characterization with KOG, KEGG, and GO analyses. Additionally, differential expression analysis highlights sex-specific expression patterns, and a list of endocrine and oxidative stress related transcripts are included. PMID:27672404

  3. Development of a 690 K SNP array in catfish and its application for genetic mapping and validation of the reference genome sequence (United States)

    Zeng, Qifan; Fu, Qiang; Li, Yun; Waldbieser, Geoff; Bosworth, Brian; Liu, Shikai; Yang, Yujia; Bao, Lisui; Yuan, Zihao; Li, Ning; Liu, Zhanjiang


    Single nucleotide polymorphisms (SNPs) are capable of providing the highest level of genome coverage for genomic and genetic analysis because of their abundance and relatively even distribution in the genome. Such a capacity, however, cannot be achieved without an efficient genotyping platform such as SNP arrays. In this work, we developed a high-density SNP array with 690,662 unique SNPs (herein 690 K array) that were relatively evenly distributed across the entire genome, and covered 98.6% of the reference genome sequence. Here we also report linkage mapping using the 690 K array, which allowed mapping of over 250,000 SNPs on the linkage map, the highest marker density among all the constructed linkage maps. These markers were mapped to 29 linkage groups (LGs) with 30,591 unique marker positions. This linkage map anchored 1,602 scaffolds of the reference genome sequence to LGs, accounting for over 97% of the total genome assembly. A total of 1,007 previously unmapped scaffolds were placed to LGs, allowing validation and in few instances correction of the reference genome sequence assembly. This linkage map should serve as a valuable resource for various genetic and genomic analyses, especially for GWAS and QTL mapping for genes associated with economically important traits. PMID:28079141

  4. Analysis of expressed sequence tags from the Ulva prolifera (Chlorophyta)

    Institute of Scientific and Technical Information of China (English)

    牛建峰; 胡海岩; 胡松年; 王广策; 彭光; 孙松


    In 2008,a green tide broke out before the sailing competition of the 29th Olympic Games in Qingdao.The causative species was determined to be Enteromorpha prolifera (Ulva prolifera O.F.Müller),a familiar green macroalga along the coastline of China.Rapid accumulation of a large biomass of floating U.prolifera prompted research on different aspects of this species.In this study,we constructed a nonnormalized cDNA library from the thalli of U.prolifera and acquired 10 072 high-quality expressed sequence tags ...

  5. Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi (United States)

    Kong, Hyun-Hee; Hwang, Mee-Yeul; Kim, Hyo-Kyung


    Randomly selected 435 clones from Acanthamoeba healyi cDNA library were sequenced and a total of 387 expressed sequence tags (ESTs) had been generated. Based on the results of BLAST search, 130 clones (34.4%) were identified as the genes enconding surface proteins, enzymes for DNA, energy production or other metabolism, kinases and phosphatases, protease, proteins for signal transduction, structural and cytoskeletal proteins, cell cycle related proteins, transcription factors, transcription and translational machineries, and transporter proteins. Most of the genes (88.5%) are newly identified in the genus Acanthamoeba. Although 15 clones matched the genes of Acanthamoeba located in the public databases, twelve clones were actin gene which was the most frequently expressed gene in this study. These ESTs of Acanthamoeba would give valuable information to study the organism as a model system for biological investigations such as cytoskeleton or cell movement, signal transduction, transcriptional and translational regulations. These results would also provide clues to elucidate factors for pathogenesis in human granulomatous amoebic encephalitis or keratitis by Acanthamoeba. PMID:11441502

  6. A blackberry (Rubus L. expressed sequence tag library for the development of simple sequence repeat markers

    Directory of Open Access Journals (Sweden)

    Main Dorrie S


    Full Text Available Abstract Background The recent development of novel repeat-fruiting types of blackberry (Rubus L. cultivars, combined with a long history of morphological marker-assisted selection for thornlessness by blackberry breeders, has given rise to increased interest in using molecular markers to facilitate blackberry breeding. Yet no genetic maps, molecular markers, or even sequences exist specifically for cultivated blackberry. The purpose of this study is to begin development of these tools by generating and annotating the first blackberry expressed sequence tag (EST library, designing primers from the ESTs to amplify regions containing simple sequence repeats (SSR, and testing the usefulness of a subset of the EST-SSRs with two blackberry cultivars. Results A cDNA library of 18,432 clones was generated from expanding leaf tissue of the cultivar Merton Thornless, a progenitor of many thornless commercial cultivars. Among the most abundantly expressed of the 3,000 genes annotated were those involved with energy, cell structure, and defense. From individual sequences containing SSRs, 673 primer pairs were designed. Of a randomly chosen set of 33 primer pairs tested with two blackberry cultivars, 10 detected an average of 1.9 polymorphic PCR products. Conclusion This rate predicts that this library may yield as many as 940 SSR primer pairs detecting 1,786 polymorphisms. This may be sufficient to generate a genetic map that can be used to associate molecular markers with phenotypic traits, making possible molecular marker-assisted breeding to compliment existing morphological marker-assisted breeding in blackberry.

  7. Complete mitochondrial genome of the Southern catfish (Silurus meridionalis Chen) and Chinese catfish (S. asotus Linnaeus): Structure, phylogeny, and intraspecific variation. (United States)

    Wang, Q R; Xu, C; Xu, C R; Wang, R J


    The complete mitochondrial genome of the Southern catfish (Silurus meridionalis) and the Chinese catfish (S. asotus), was determined using the long and accurate polymerase chain reaction (LA-PCR) method. The mitochondrial DNA nucleotide sequences of S. meridionalis and S. asotus were compared with those of 47 other catfish species in the same order. The total length of mitochondrial DNA for S. meridionalis and S. asotus was 16,526 and 16,525 bp, respectively, and included 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a non-coding control region. This mitochondrial gene arrangement is identical to that observed in other Siluriformes. To determine the relative phylogenetic positions of S. meridionalis and S. asotus, and to discover phylogenetic relationships among 24 families of Siluriformes, analyses were conducted, based on mitochondrial DNA, 12S ribosomal RNA, 16S ribosomal RNA, and 13 protein-coding gene sequence data sets. Phylogenetic analyses were congruent with a basal split of the order into Clupeiformes, Characiformes, Cypriniformes, and Siluriformes, and supported a closer relationship of the Southern catfish (family Siluridae) and the Chinese catfish (family Siluridae) to Pimelodidae than to Bagridae. We concluded that these two species are part of a molecular clade that is different from that proposed in recent studies, in which Amblycipitidae appears as a sister group. Our results showed Amblycipitidae appearing as the most basal extant, and Bagridae appearing as a sister group of Cranoglanididae and Pangasiidae. The Siluriformes showed close phylogenetic relationship to the Characiformes.

  8. Analysis of simple sequence repeats markers derived from Phytophthora sojae expressed sequence tags

    Institute of Scientific and Technical Information of China (English)

    ZHU Zhendong; HUO Yunlong; WANG Xiaoming; HUANG Junbin; WU Xiaofei


    Five thousand and eight hundred publicly available expressed sequence tags (ESTs) of Phytophthora sojae were electronically searched and 415 simple sequence repeats (SSRs) were identified in 369 ESTs. The average density of SSRs was one SSR per 8.9 kb of EST sequence screened. The most frequent repeats were trinucleotide repeats (50.1%) and the least frequent were tetranucleotide repeats (8.2%). Forty primer pairs were designed and tested on 5 strains of P. sojae. Thirty-three primer pairs had successful PCR amplifications. Of the 33 functional primer pairs, 28 primer pairs produced characteristic SSR bands of the expected size, and 15 primer pairs (45.5%) detected polymorphism among 5 tested strains of P. sojae. Based on the polymorphisms detected with 20 EST-SSR markers, the 5 tested strains of P. sojae were clustered into 3 groups. In this study, the SSR markers of P. sojae were developed for the first time. These markers could be useful for identification, genetic variation study, and molecular mapping of P. sojae and its relative species.

  9. 76 FR 10433 - Mandatory Inspection of Catfish and Catfish Products (United States)


    ... the final rule. For purposes of convenience, this proposed rule uses the term ``catfish'' to refer to... (head on), mainly to restaurants and institutions. Waste materials and byproducts from heading, gutting... 1991 NJ Restaurant Salmonella 10 6 0 1999 FL Private Home Unknown 2 0 0 1999 FL...

  10. Identification of Expressed Resistance Gene Analogs from Peanut (Arachis hypogaea L.) Expressed Sequence Tags

    Institute of Scientific and Technical Information of China (English)

    Zhanji Liu; Suping Feng; Manish K.Pandey; Xiaoping Chen; Albert K.Culbreath; Rajeev K.Varshney; Baozhu Guo


    Low genetic diversity makes peanut (Arachis hypogaea L.) very vulnerable to plant pathogens,causing severe yield loss and reduced seed quality.Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat (NBS-LRR) resistance genes (R) have been identified,but a small portion with expressed transcripts has been found.We aimed to identify resistance gene analogs (RGAs) from peanut expressed sequence tags (ESTs) and to develop polymorphic markers.The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases.A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered,and assembled 156 contigs and 229 singletons as peanut-expressed RGAs.There were 69 that encoded for NBS-LRR proteins,191 that encoded for protein kinases,82 that encoded for LRR-PK/transmembrane proteins,28 that encoded for Toxin reductases,11 that encoded for LRR-domain containing proteins and four that encoded for TM-domain containing proteins.Twenty-eight simple sequence repeats (SSRs)were identified from 25 peanut expressed RGAs.One SSR polymorphic marker (RGA121) was identified.Two polymerase chain reaction-based markers (Ahsw-1 and Ahsw-2) developed from RGA013 were homologous to the Tomato Spotted Wilt Virus (TSWV) resistance gene.All three markers were mapped on the same linkage group AhlV.These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.

  11. Transcriptome analysis of Anopheles stephensi embryo using expressed sequence tags

    Indian Academy of Sciences (India)

    Kaustubh Gokhale; Deepak P Patil; Dhiraj P Dhotre; Rajnikant Dixit; Murlidhar J Mendki; Milind S Patole; Yogesh S Shouche


    Germ band retraction (GBR) stage is one of the important stages during insect development. It is associated with an extensive epithelial morphogenesis and may also be pivotal in generation of morphological diversity in insects. Despite its importance, only a handful of studies report the transcriptome repertoire of this stage in insects. Here, we report generation, annotation and analysis of ESTs from the embryonic stage (16–22 h post fertilization) of laboratory-reared Anopheles stephensi mosquitoes. A total of 1002 contigs were obtained upon clustering of 1140 high-quality ESTs, which demonstrates an astonishingly low transcript redundancy (12.1%). Putative functions were assigned only to 213 contigs (21%), comprising mainly of transcripts encoding protein synthesis machinery. Approximately 78% of the transcripts remain uncharacterized, illustrating a lack of sequence information about the genes expressed in the embryonic stages of mosquitoes. This study highlights several novel transcripts, which apart from insect development, may significantly contribute to the essential biological complexity underlying insect viability in adverse environments. Nonetheless, the generated sequence information from this work provides a comprehensive resource for genome annotation, microarray development, phylogenetic analysis and other molecular biology applications in entomology.

  12. Anti-DNA antibodies: Sequencing, cloning, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Barry, M.M.


    To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

  13. LOX: Inferring level of expression from diverse methods of census sequencing

    KAUST Repository

    Zhang, Zhang


    Summary: We present LOX (Level Of eXpression) that estimates the Level Of gene eXpression from high-throughput-expressed sequence datasets with multiple treatments or samples. Unlike most analyses, LOX incorporates a gene bias model that facilitates integration of diverse transcriptomic sequencing data that arises when transcriptomic data have been produced using diverse experimental methodologies. LOX integrates overall sequence count tallies normalized by total expressed sequence count to provide expression levels for each gene relative to all treatments as well as Bayesian credible intervals. © The Author 2010. Published by Oxford University Press. All rights reserved.

  14. Catfish Preservation using Porphyra Yezoensis Composites Preservatives

    Directory of Open Access Journals (Sweden)

    Zhi-Gang Qian


    Full Text Available This study aims to preserve fresh catfish meat by using Porphyra Yezoensis extract, chitosan and lactic acid Nisin. The composite preservative obtained by sensory evaluation can effectively maintain the color, odor and texture of fresh catfish meat, as well as inhibit bacterial growth. Results show that treatment using a preservative solution (Porphyra Yezoensis extract 10%, Nisin 0.2% and chitosan 15% extended the shelf life of the fresh catfish meat from 12 h to 24 h when stored at room temperature and from 6 d to 9 d when stored at 4°C. These results provide a practical method of preserving fresh catfish meat.

  15. A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery. (United States)

    Thanh, Nguyen Minh; Jung, Hyungtaek; Lyons, Russell E; Chand, Vincent; Tuan, Nguyen Viet; Thu, Vo Thi Minh; Mather, Peter


    The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9ppt which showed best growth performance. Total sequence data generated was 467.8Mbp, consisting of 4,116,424 reads with an average length of 112bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

  16. Potency of blue catfish, Ictalurus furcatus (individual vs pooled) sperm to fertilize stripped channel catfish, I. punctatus eggs on the production and performance of progeny (United States)

    Channel x blue hybrid catfish is the desired genotype for US farm-raised catfish industry. Induced spawning of gravid channel catfish, followed by fertilization of stripped eggs with blue catfish sperm is the only reliable means to produce hybrid catfish embryos in hatcheries. Hybrid catfish fry p...

  17. Isolation and characterization of gene sequences expressed in cotton fiber

    Directory of Open Access Journals (Sweden)

    Taciana de Carvalho Coutinho


    Full Text Available ABSTRACT Cotton fiber are tubular cells which develop from the differentiation of ovule epidermis. In addition to being one of the most important natural fiber of the textile group, cotton fiber afford an excellent experimental system for studying the cell wall. The aim of this work was to isolate and characterise the genes expressed in cotton fiber (Gossypium hirsutum L. to be used in future work in cotton breeding. Fiber of the cotton cultivar CNPA ITA 90 II were used to extract RNA for the subsequent generation of a cDNA library. Seventeen sequences were obtained, of which 14 were already described in the NCBI database (National Centre for Biotechnology Information, such as those encoding the lipid transfer proteins (LTPs and arabinogalactans (AGP. However, other cDNAs such as the B05 clone, which displays homology with the glycosyltransferases, have still not been described for this crop. Nevertheless, results showed that several clones obtained in this study are associated with cell wall proteins, wall-modifying enzymes and lipid transfer proteins directly involved in fiber development.

  18. ESTExplorer: an expressed sequence tag (EST) assembly and annotation platform. (United States)

    Nagaraj, Shivashankar H; Deshpande, Nandan; Gasser, Robin B; Ranganathan, Shoba


    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

  19. Water quality in hybrid catfish ponds after partial fish harvest (United States)

    Intensification of United States catfish aquaculture involves hybrid catfish ('channel catfish Ictalurus punctatus x ' blue catfish I. furcatus) grown in ponds with abundant aeration and high feeding rates. High feeding rates cause water quality deterioration because most of the nitrogen, phosphorus...

  20. Finding noncoding RNA transcripts from low abundance expressed sequence tags

    Institute of Scientific and Technical Information of China (English)

    Chenghai Xue; Fei Li; Fei Li


    It has been proved that noncoding RNA (ncRNA) genes are much more numerous than expected.However,it remains a difficult task to identify ncRNAs with either computational algorithms or biological experiments.Recent reports have suggested that ncRNAs may also appear in the expressed sequence tags (EST's) database.Nevertheless,intergenic ESTs have received little attention and are poorly annotated owing to their low abundance.Here,we have developed a computational strategy for discovering ncRNA genes from human ESTs.We first collected ESTs that are located in the intergenic regions and do not have detailed annotations.The intergenic regions were divided into non-overlapping 50-nt windows and PhastCons scores obtained from the UCSC database were assigned to these windows.We kept conserved windows that had PhastCons scores of over 0.8 and that had at least three supporting ESTs to act as seeds.Each cluster of ESTs corresponding to the seeds was assembled into a long contig.We used two criteria to screen for ncRNA transcripts from these contigs:the first was that the longest predicted open reading frame was less than 300 nt and the second was that the likely Pol-Ⅱ promoters exist within 2 000 nt upstream or downstream of the contigs.As a result,118 novel ncRNA genes were identified from human low abundance ESTs.Of seven randomly selected candidates,six were transcribed in human 2BS cells as shown by RT-PCR.Our work proves that the EST is a 'hidden treasure' for detecting novel ncRNA genes.

  1. Gene Identification and Expression Analysis of 86,136 Expressed Sequence Tags (EST) from the Rice Genome

    Institute of Scientific and Technical Information of China (English)

    Yan Zhou; Lin Ye; Li Lin; Jun Li; Xuegang Wang; Hao Xu; Yibin Pan; Wei Lin; Wei Tian; Jing Liu; Liping Wei; Jiabin Tang; Siqi Liu; Huanming Yang; Jun Yu; Jian Wang; Michael G. Walker; Xiuqing Zhang; Jun Wang; Songnian Hu; Huayong Xu; Yajun Deng; Jianhai Dong


    Expressed Sequence Tag (EST) analysis has pioneered genome-wide gene discovery and expression profiling. In order to establish a gene expression index in the rice cultivar indica, we sequenced and analyzed 86,136 ESTs from nine rice cDNA libraries from the super hybrid cultivar LYP9 and its parental cultivars. We assembled these ESTs into 13,232 contigs and leave 8,976 singletons. Overall, 7,497 sequences were found similar to the existing sequences in GenBank and 14,711 are novel. These sequences are classified by molecular function, biological process and pathways according to the Gene Ontology. We compared our sequenced ESTs with the publicly available 95,000 ESTs from japonica, and found little sequence variation, despite the large difference between genome sequences. We then assembled the combined 173,000 rice ESTs for further analysis. Using the pooled ESTs, we compared gene expression in metabolism pathway between rice and Avabidopsis according to KEGG. We further profiled gene expression patterns in different tis sues, developmental stages, and in a conditional sterile mutant, after checking the libraries are comparable by means of sequence coverage. We also identified some possible library specific genes and a number of enzymes and transcription factors that contribute to rice development.

  2. Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

    Institute of Scientific and Technical Information of China (English)

    Kai XIAO; Gui-Hua BAI; Brett F CARVER


    To enrich differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) nearisogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to Al stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg2+ transportation, and other functions. Under Al stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways.The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.

  3. Alternative Splicing and Expression Profile Analysis of Expressed Sequence Tags in Domestic Pig

    Institute of Scientific and Technical Information of China (English)

    Liang Zhang; Lin Tao; Lin Ye; Ling He; Yuan-Zhong Zhu; Yue-Dong Zhu; Yan Zhou


    Domestic pig (Sus scrofa domestica) is one of the most important mammals to humans. Alternative splicing is a cellular mechanism in eukaryotes that greatly increases the diversity of gene products. Expression sequence tags (ESTs) have been widely used for gene discovery, expression profile analysis, and alternative splicing detection. In this study, a total of 712,905 ESTs extracted from 101 different nonnormalized EST libraries of the domestic pig were analyzed. These EST libraries cover the nervous system, digestive system, immune system, and meat production related tissues from embryo, newborn, and adult pigs, making contributions to the analysis of alternative splicing variants as well as expression profiles in various stages of tissues. A modified approach was designed to cluster and assemble large EST datasets, aiming to detect alternative splicing together with EST abundance of each splicing variant. Much efforts were made to classify alternative splicing into different types and apply different filters to each type to get more reliable results. Finally, a total of 1,223 genes with average 2.8 splicing variants were detected among 16,540 unique genes. The overview of expression profiles would change when we take alternative splicing into account.

  4. The World Demand for Catfish Pangasius

    DEFF Research Database (Denmark)

    Nguyen, Thong Tien; Roth, Eva; Nielsen, Max


    . Direct elasticity including own- and cross- price elasticity and income elasticity are calculated to show how consumers from different markets of the world prefer for the Pangasius catfish. We found that catfish products have big room of market demand, indicated by absolute values of own price...

  5. Sequencing, analysis, and annotation of expressed sequence tags for Camelus dromedarius.

    Directory of Open Access Journals (Sweden)

    Abdulaziz M Al-Swailem

    Full Text Available Despite its economical, cultural, and biological importance, there has not been a large scale sequencing project to date for Camelus dromedarius. With the goal of sequencing complete DNA of the organism, we first established and sequenced camel EST libraries, generating 70,272 reads. Following trimming, chimera check, repeat masking, cluster and assembly, we obtained 23,602 putative gene sequences, out of which over 4,500 potentially novel or fast evolving gene sequences do not carry any homology to other available genomes. Functional annotation of sequences with similarities in nucleotide and protein databases has been obtained using Gene Ontology classification. Comparison to available full length cDNA sequences and Open Reading Frame (ORF analysis of camel sequences that exhibit homology to known genes show more than 80% of the contigs with an ORF>300 bp and approximately 40% hits extending to the start codons of full length cDNAs suggesting successful characterization of camel genes. Similarity analyses are done separately for different organisms including human, mouse, bovine, and rat. Accompanying web portal, CAGBASE (, hosts a relational database containing annotated EST sequences and analysis tools with possibility to add sequences from public domain. We anticipate our results to provide a home base for genomic studies of camel and other comparative studies enabling a starting point for whole genome sequencing of the organism.

  6. A first generation BAC-based physical map of the channel catfish genome

    Directory of Open Access Journals (Sweden)

    Waldbieser Geoffrey C


    Full Text Available Abstract Background Channel catfish, Ictalurus punctatus, is the leading species in North American aquaculture. Genetic improvement of catfish is performed through selective breeding, and genomic tools will help improve selection efficiency. A physical map is needed to integrate the genetic map with the karyotype and to support fine mapping of phenotypic trait alleles such as Quantitative Trait Loci (QTL and the effective positional cloning of genes. Results A genome-wide physical map of the channel catfish was constructed by High-Information-Content Fingerprinting (HICF of 46,548 Bacterial Artificial Chromosomes (BAC clones using the SNaPshot technique. The clones were assembled into contigs with FPC software. The resulting assembly contained 1,782 contigs and covered an estimated physical length of 0.93 Gb. The validity of the assembly was demonstrated by 1 anchoring 19 of the largest contigs to the microsatellite linkage map 2 comparing the assembly of a multi-gene family to Restriction Fragment Length Polymorphism (RFLP patterns seen in Southern blots, and 3 contig sequencing. Conclusion This is the first physical map for channel catfish. The HICF technique allowed the project to be finished with a limited amount of human resource in a high throughput manner. This physical map will greatly facilitate the detailed study of many different genomic regions in channel catfish, and the positional cloning of genes controlling economically important production traits.

  7. Putative roles for a rhamnose binding lectin in Flavobacterium columnare pathogenesis in channel catfish Ictalurus punctatus. (United States)

    Beck, Benjamin H; Farmer, Bradley D; Straus, David L; Li, Chao; Peatman, Eric


    Columnaris disease, caused by the bacterial pathogen Flavobacterium columnare, continues to be a major problem worldwide and commonly leads to tremendous losses of both wild and cultured freshwater fish, particularly in intensively farmed aquaculture species such as channel catfish. Despite its ecologic and economic impacts, the fundamental molecular mechanisms of the host immune response to this pathogen remain unclear. While F. columnare can induce marked pathologic changes in numerous ectopic tissues, the adhesion of F. columnare to the gill in particular is strongly associated with pathogen virulence and host susceptibility. Recently, in this regard, using RNA-seq expression profiling we found that a rhamnose-binding lectin (RBL) was dramatically upregulated in the gill of fish infected with F. columnare (as compared to naïve fish). Thus, in the present study we sought to further characterize and understand the RBL response in channel catfish (Ictalurus punctatus). We first identified two distinct catfish families with differential susceptibilities to columnaris disease; one family was found to be completely resistant while the other was susceptible (0% mortality versus 18.3% respectively, P catfish RBL that persisted for at least 24 h (P catfish to different doses of the putative RBL ligands l-rhamnose and d-galactose, and found that these sugars, protected channel catfish against columnaris disease, likely through competition with F. columnare binding of host RBL. Finally, we examined the role of nutritional status on RBL regulation and found that RBL expression was upregulated (>120-fold; P < 0.05) in fish fasted for 7 d (as compared to fish fed to satiation daily), yet expression levels returned to those of satiated fish within 4 h after re-feeding. Collectively, these findings highlight putative roles for RBL in the context of columnaris disease and reveal new aspects linking RBL regulation to feed availability.

  8. Developing expressed sequence tag libraries and the discovery of simple sequence repeat markers for two species of raspberry (Rubus L.) (United States)

    Background: Due to a relatively high level of codominant inheritance and transferability within and among taxonomic groups, simple sequence repeat (SSR) markers are important elements in comparative mapping and delineation of genomic regions associated with traits of economic importance. Expressed S...


    Energy Technology Data Exchange (ETDEWEB)



    A recent, popular method of finding promoter sequences is to look for conserved motifs up-stream of genes clustered on the basis of expression data. This method presupposes that the clustering is correct. Theoretically, one should be better able to find promoter sequences and create more relevant gene clusters by taking a unified approach to these two problems. We present a likelihood function for a sequence-expression model giving a joint likelihood for a promoter sequence and its corresponding expression levels. An algorithm to estimate sequence-expression model parameters using Gibbs sampling and Expectation/Maximization is described. A program, called kimono, that implements this algorithm has been developed and the source code is freely available over the internet.

  10. Comparative genomic analysis of bacteriophages specific to the channel catfish pathogen Edwardsiella ictaluri

    Directory of Open Access Journals (Sweden)

    Mead David A


    Full Text Available Abstract Background The bacterial pathogen Edwardsiella ictaluri is a primary cause of mortality in channel catfish raised commercially in aquaculture farms. Additional treatment and diagnostic regimes are needed for this enteric pathogen, motivating the discovery and characterization of bacteriophages specific to E. ictaluri. Results The genomes of three Edwardsiella ictaluri-specific bacteriophages isolated from geographically distant aquaculture ponds, at different times, were sequenced and analyzed. The genomes for phages eiAU, eiDWF, and eiMSLS are 42.80 kbp, 42.12 kbp, and 42.69 kbp, respectively, and are greater than 95% identical to each other at the nucleotide level. Nucleotide differences were mostly observed in non-coding regions and in structural proteins, with significant variability in the sequences of putative tail fiber proteins. The genome organization of these phages exhibit a pattern shared by other Siphoviridae. Conclusions These E. ictaluri-specific phage genomes reveal considerable conservation of genomic architecture and sequence identity, even with considerable temporal and spatial divergence in their isolation. Their genomic homogeneity is similarly observed among E. ictaluri bacterial isolates. The genomic analysis of these phages supports the conclusion that these are virulent phages, lacking the capacity for lysogeny or expression of virulence genes. This study contributes to our knowledge of phage genomic diversity and facilitates studies on the diagnostic and therapeutic applications of these phages.

  11. Widespread Differential Expression of Coding Region and 3' UTR Sequences in Neurons and Other Tissues. (United States)

    Kocabas, Arif; Duarte, Terence; Kumar, Saranya; Hynes, Mary A


    Mature messenger RNAs (mRNAs) consist of coding sequence (CDS) and 5' and 3' UTRs, typically expected to show similar abundance within a given neuron. Examining mRNA from defined neurons, we unexpectedly show extremely common unbalanced expression of cognate 3' UTR and CDS sequences; many genes show high 3' UTR relative to CDS, others show high CDS to 3' UTR. In situ hybridization (19 of 19 genes) shows a broad range of 3' UTR-to-CDS expression ratios across neurons and tissues. Ratios may be spatially graded or change with developmental age but are consistent across animals. Further, for two genes examined, a 3' UTR-to-CDS ratio above a particular threshold in any given neuron correlated with reduced or undetectable protein expression. Our findings raise questions about the role of isolated 3' UTR sequences in regulation of protein expression and highlight the importance of separately examining 3' UTR and CDS sequences in gene expression analyses.

  12. Cloning, sequencing and expression analysis of the NAR promoter activated during hyphal stage of Magnaporthe grisea

    Institute of Scientific and Technical Information of China (English)


    The promoter of N4R gene in Magnaporthe grisea was isolated and sequenced. The promoter sequences contained the "TATA" box, the "CAAT" box, and binding sites for fungal regulatory proteins. Programs that predict promoter sequences indicated that promoter sequence lies between locations 430 and 857 of the NAR promoter fragment. GFP expression under the NAR promoter and NAR transcript analysis revealed that this promoter is activated primarily at the mycelial stage in the rice blast fungus and could be used to express native or extrinsic genes in the mycelia of the rice blast fungus.

  13. Sequence and gene expression evolution of paralogous genes in willows. (United States)

    Harikrishnan, Srilakshmy L; Pucholt, Pascal; Berlin, Sofia


    Whole genome duplications (WGD) have had strong impacts on species diversification by triggering evolutionary novelties, however, relatively little is known about the balance between gene loss and forces involved in the retention of duplicated genes originating from a WGD. We analyzed putative Salicoid duplicates in willows, originating from the Salicoid WGD, which took place more than 45 Mya. Contigs were constructed by de novo assembly of RNA-seq data derived from leaves and roots from two genotypes. Among the 48,508 contigs, 3,778 pairs were, based on fourfold synonymous third-codon transversion rates and syntenic positions, predicted to be Salicoid duplicates. Both copies were in most cases expressed in both tissues and 74% were significantly differentially expressed. Mean Ka/Ks was 0.23, suggesting that the Salicoid duplicates are evolving by purifying selection. Gene Ontology enrichment analyses showed that functions related to DNA- and nucleic acid binding were over-represented among the non-differentially expressed Salicoid duplicates, while functions related to biosynthesis and metabolism were over-represented among the differentially expressed Salicoid duplicates. We propose that the differentially expressed Salicoid duplicates are regulatory neo- and/or subfunctionalized, while the non-differentially expressed are dose sensitive, hence, functionally conserved. Multiple evolutionary processes, thus drive the retention of Salicoid duplicates in willows.

  14. Transcriptome generation and analysis from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758) through normalized cDNA library. (United States)

    Singh, Akanksha; Barman, A S; Sood, Neeraj; Mohindra, Vindhya


    Catfishes are commercially important fish for both the fisheries and aquaculture industry. Clarias batrachus, an Indian catfish species is economically important owing to its high demand. A normalized cDNA library was constructed from spleen of the Indian catfish to identify genes associated with immune function. One thousand nine hundred thirty seven ESTs were submitted to the GenBank with an average read length of approximately 700 bp. Clustering analysis of ESTs yielded 1,698 unique sequences, including 184 contigs and 1,514 singletons. Significant homology to known genes was found by homology searches against data in GenBank in 576 (34 %) ESTs, including similarity to functionally annotated unigenes for 158 ESTs. Additionally, 433 ESTs revealed similarity to unigenes and ESTs in the dbEST but the remaining 658 EST sequences (39 %) did not match any sequence in GenBank. Of a total of 1,698 ESTs generated, 65 ESTs were found to be associated with immune functions. Gene Ontology and KEGG pathway analyses of C. batrachus ESTs collectively revealed a preponderance of immune relevant pathways apart from the presence of pathways involved in protein processing, localization, folding and protein degradation. This study constitutes first EST analysis of lymphoid organ in aquaculturally important Indian catfish species and could pave the way for further research of immune-related genes and functional genomics in this catfish.

  15. Pre and postprandial changes in orexigenic and anorexigenic factors in channel catfish Ictalurus punctatus (United States)

    We examined pre- and postprandial changes in the expression of plasma ghrelin (GHRL) and mRNAs encoding GRLN, cocaine and amphetamine regulated transcript (CART), neuropeptide Y (NPY), and cholecystokinin (CCK) in channel catfish. Fish were either offered feed (Fed) or fasted (Unfed). Feeding incr...

  16. Characterization of Expressed Sequence Tags From a Gallus gallus Pineal Gland cDNA Library



    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; 6% 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...

  17. Development of 'Serunding' from African catfish

    DEFF Research Database (Denmark)

    Juhari, Nurul Hanisah Binti; MK, Zainol; MM, Masduki

    This research was conducted to develop serunding from African catfish (Clarias gariepinus). Proximate analysis, physical analysis, ascorbic acid analysis, mineral analysis and sensory evaluation were carried out to determine the nutrient compositions and consumer acceptance towards the products. ...

  18. Analyses of an expressed sequence tag library from Taenia solium, Cysticerca.

    Directory of Open Access Journals (Sweden)

    Jonas Lundström

    Full Text Available BACKGROUND: Neurocysticercosis is a disease caused by the oral ingestion of eggs from the human parasitic worm Taenia solium. Although drugs are available they are controversial because of the side effects and poor efficiency. An expressed sequence tag (EST library is a method used to describe the gene expression profile and sequence of mRNA from a specific organism and stage. Such information can be used in order to find new targets for the development of drugs and to get a better understanding of the parasite biology. METHODS AND FINDINGS: Here an EST library consisting of 5760 sequences from the pig cysticerca stage has been constructed. In the library 1650 unique sequences were found and of these, 845 sequences (52% were novel to T. solium and not identified within other EST libraries. Furthermore, 918 sequences (55% were of unknown function. Amongst the 25 most frequently expressed sequences 6 had no relevant similarity to other sequences found in the Genbank NR DNA database. A prediction of putative signal peptides was also performed and 4 among the 25 were found to be predicted with a signal peptide. Proposed vaccine and diagnostic targets T24, Tsol18/HP6 and Tso31d could also be identified among the 25 most frequently expressed. CONCLUSIONS: An EST library has been produced from pig cysticerca and analyzed. More than half of the different ESTs sequenced contained a sequence with no suggested function and 845 novel EST sequences have been identified. The library increases the knowledge about what genes are expressed and to what level. It can also be used to study different areas of research such as drug and diagnostic development together with parasite fitness via e.g. immune modulation.

  19. Catfish stings: A report of two cases

    Directory of Open Access Journals (Sweden)

    Gholamali Dorooshi


    Full Text Available Venomous catfish stings are a common environment hazard worldwide. Although these stings are often innocuous, significant morbidity may result from stings, including severe pain, retained foreign bodies, infection, respiratory compromise, arterial hypotension, and cardiac dysrhythmias. Treatment included hot water immersion, analgesia, wound exploration, and prophylactic antibiotics. In this article, two cases of stings by catfish referred to the poison center of Noor Hospital, Isfahan University of Medical Sciences and their treatments have been reported.

  20. Mining of haplotype-based expressed sequence tag single nucleotide polymorphisms in citrus


    Chen, Chunxian; Gmitter Jr, Fred G


    Background Single nucleotide polymorphisms (SNPs), the most abundant variations in a genome, have been widely used in various studies. Detection and characterization of citrus haplotype-based expressed sequence tag (EST) SNPs will greatly facilitate further utilization of these gene-based resources. Results In this paper, haplotype-based SNPs were mined out of publicly available citrus expressed sequence tags (ESTs) from different citrus cultivars (genotypes) individually and collectively for...

  1. Sequencing and bacterial expression of a novel murine alpha interferon gene

    Institute of Scientific and Technical Information of China (English)

    王焱; 王征宇; 周鸣南; 蔡菊娥; 孙兰英; 刘新垣; B.L.Daugherty; S.Pestka


    A murine new alpha interferon gene (mIFN-αB) was found by primer-based sequencing method in a murine genomic DNA library. The gene was cloned and its sequence was determined. It was expressed in Escherichia coli under the control of the PL promoter which resulted in antiviral activity on mouse L-cells. The sequence of mlFN-αB has been accepted by GENEBANK.

  2. Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

    DEFF Research Database (Denmark)

    Studer, Bruno; Asp, Torben; Frei, Ursula;


    An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification...

  3. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    DEFF Research Database (Denmark)

    Fromont-Racine, M; Bucchini, D; Madsen, O;


    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific tra...... of the transgene was observed in cell types other than beta-islet cells.......Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...

  4. Evaluation of potential candidate genes involved in salinity tolerance in striped catfish (Pangasianodon hypophthalmus) using an RNA-Seq approach. (United States)

    Nguyen, Tuan Viet; Jung, Hyungtaek; Nguyen, Thanh Minh; Hurwood, David; Mather, Peter


    Increasing salinity levels in freshwater and coastal environments caused by sea level rise linked to climate change is now recognized to be a major factor that can impact fish growth negatively, especially for freshwater teleost species. Striped catfish (Pangasianodon hypophthalmus) is an important freshwater teleost that is now widely farmed across the Mekong River Delta in Vietnam. Understanding the basis for tolerance and adaptation to raised environmental salinity conditions can assist the regional culture industry to mitigate predicted impacts of climate change across this region. Attempt of next generation sequencing using the ion proton platform results in more than 174 million raw reads from three tissue libraries (gill, kidney and intestine). Reads were filtered and de novo assembled using a variety of assemblers and then clustered together to generate a combined reference transcriptome. Downstream analysis resulted in a final reference transcriptome that contained 60,585 transcripts with an N50 of 683 bp. This resource was further annotated using a variety of bioinformatics databases, followed by differential gene expression analysis that resulted in 3062 transcripts that were differentially expressed in catfish samples raised under two experimental conditions (0 and 15 ppt). A number of transcripts with a potential role in salinity tolerance were then classified into six different functional gene categories based on their gene ontology assignments. These included; energy metabolism, ion transportation, detoxification, signal transduction, structural organization and detoxification. Finally, we combined the data on functional salinity tolerance genes into a hypothetical schematic model that attempted to describe potential relationships and interactions among target genes to explain the molecular pathways that control adaptive salinity responses in P. hypophthalmus. Our results indicate that P. hypophthalmus exhibit predictable plastic regulatory responses

  5. Analysis of expressed sequence tags from Prunus mume flower and fruit and development of simple sequence repeat markers

    Directory of Open Access Journals (Sweden)

    Gao Zhihong


    Full Text Available Abstract Background Expressed Sequence Tag (EST has been a cost-effective tool in molecular biology and represents an abundant valuable resource for genome annotation, gene expression, and comparative genomics in plants. Results In this study, we constructed a cDNA library of Prunus mume flower and fruit, sequenced 10,123 clones of the library, and obtained 8,656 expressed sequence tag (EST sequences with high quality. The ESTs were assembled into 4,473 unigenes composed of 1,492 contigs and 2,981 singletons and that have been deposited in NCBI (accession IDs: GW868575 - GW873047, among which 1,294 unique ESTs were with known or putative functions. Furthermore, we found 1,233 putative simple sequence repeats (SSRs in the P. mume unigene dataset. We randomly tested 42 pairs of PCR primers flanking potential SSRs, and 14 pairs were identified as true-to-type SSR loci and could amplify polymorphic bands from 20 individual plants of P. mume. We further used the 14 EST-SSR primer pairs to test the transferability on peach and plum. The result showed that nearly 89% of the primer pairs produced target PCR bands in the two species. A high level of marker polymorphism was observed in the plum species (65% and low in the peach (46%, and the clustering analysis of the three species indicated that these SSR markers were useful in the evaluation of genetic relationships and diversity between and within the Prunus species. Conclusions We have constructed the first cDNA library of P. mume flower and fruit, and our data provide sets of molecular biology resources for P. mume and other Prunus species. These resources will be useful for further study such as genome annotation, new gene discovery, gene functional analysis, molecular breeding, evolution and comparative genomics between Prunus species.

  6. Delineation of Cis-Acting Sequences Required for Expression of Drosophila Mojavensis Adh-1 (United States)

    Bayer, C. A.; Curtiss, S. W.; Weaver, J. A.; Sullivan, D. T.


    The control of expression of the Adh-1 gene of Drosophila mojavensis has been analyzed by transforming ADH null Drosophila melanogaster hosts with P element constructs which contain D. mojavensis Adh-1 having deletions of different extent in the 5' and 3' ends. Adh-1 expression in the D. melanogaster hosts is qualitatively similar to expression in D. mojavensis, although expression is quantitatively lower in transformants. Deletions of the 5' end indicate that information required for normal temporal and tissue expression in larvae is contained within 70 bp of the transcription start site. However, deletion constructs to -70 are deficient in ovarian nurse cell expression, whereas the additional upstream sequences present in constructs containing deletions to -257 do support expression in the ovary. Comparison of the nucleotide sequence in the -257 to -70 region of Adh-1 of four species: D. mojavensis and Drosophila arizona, which express Adh-1 in the ovary, and Drosophila mulleri and Drosophila navojoa, which do not, has led to the identification of regions of sequence similarity that correlate with ovary expression. One of these bears a striking similarity to a conserved sequence located upstream of the three heat shock genes that have constitutive ovarian expression and may be an ovarian control element. We have identified an aberrant aspect of Adh-1 expression. In transformants which carry an Adh-1 gene without a functional upstream Adh-2 gene Adh-1 expression continues into the adult stage instead of ceasing at the onset of metamorphosis. In transformants with a functional Adh-2 gene, Adh-1 expression ceases in the third larval instar stage and aberrant expression in the adult stage does not occur. PMID:1317314

  7. Analysis and Functional Annotation of an Expressed Sequence Tag Collection for Tropical Crop Sugarcane (United States)

    Vettore, André L.; da Silva, Felipe R.; Kemper, Edson L.; Souza, Glaucia M.; da Silva, Aline M.; Ferro, Maria Inês T.; Henrique-Silva, Flavio; Giglioti, Éder A.; Lemos, Manoel V.F.; Coutinho, Luiz L.; Nobrega, Marina P.; Carrer, Helaine; França, Suzelei C.; Bacci, Maurício; Goldman, Maria Helena S.; Gomes, Suely L.; Nunes, Luiz R.; Camargo, Luis E.A.; Siqueira, Walter J.; Van Sluys, Marie-Anne; Thiemann, Otavio H.; Kuramae, Eiko E.; Santelli, Roberto V.; Marino, Celso L.; Targon, Maria L.P.N.; Ferro, Jesus A.; Silveira, Henrique C.S.; Marini, Danyelle C.; Lemos, Eliana G.M.; Monteiro-Vitorello, Claudia B.; Tambor, José H.M.; Carraro, Dirce M.; Roberto, Patrícia G.; Martins, Vanderlei G.; Goldman, Gustavo H.; de Oliveira, Regina C.; Truffi, Daniela; Colombo, Carlos A.; Rossi, Magdalena; de Araujo, Paula G.; Sculaccio, Susana A.; Angella, Aline; Lima, Marleide M.A.; de Rosa, Vicente E.; Siviero, Fábio; Coscrato, Virginia E.; Machado, Marcos A.; Grivet, Laurent; Di Mauro, Sonia M.Z.; Nobrega, Francisco G.; Menck, Carlos F.M.; Braga, Marilia D.V.; Telles, Guilherme P.; Cara, Frank A.A.; Pedrosa, Guilherme; Meidanis, João; Arruda, Paulo


    To contribute to our understanding of the genome complexity of sugarcane, we undertook a large-scale expressed sequence tag (EST) program. More than 260,000 cDNA clones were partially sequenced from 26 standard cDNA libraries generated from different sugarcane tissues. After the processing of the sequences, 237,954 high-quality ESTs were identified. These ESTs were assembled into 43,141 putative transcripts. Of the assembled sequences, 35.6% presented no matches with existing sequences in public databases. A global analysis of the whole SUCEST data set indicated that 14,409 assembled sequences (33% of the total) contained at least one cDNA clone with a full-length insert. Annotation of the 43,141 assembled sequences associated almost 50% of the putative identified sugarcane genes with protein metabolism, cellular communication/signal transduction, bioenergetics, and stress responses. Inspection of the translated assembled sequences for conserved protein domains revealed 40,821 amino acid sequences with 1415 Pfam domains. Reassembling the consensus sequences of the 43,141 transcripts revealed a 22% redundancy in the first assembling. This indicated that possibly 33,620 unique genes had been identified and indicated that >90% of the sugarcane expressed genes were tagged. PMID:14613979

  8. Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo). (United States)

    Sunde, Roger A; Sunde, Gavin R; Sunde, Colin M; Sunde, Milton L; Evenson, Jacqueline K


    The minimum Se requirement for male turkey poults is 0.3 μg Se/g--three times higher than requirements found in rodents--based on liver and gizzard glutathione peroxidase-4 (GPX4) and GPX1 activities. In addition, turkey liver GPX4 activity is 10-fold higher and GPX1 activity is 10-fold lower than in rats, and both GPX1 and GPX4 mRNA levels are dramatically down-regulated by Se deficiency. Currently, the sequences of all annotated turkey selenoprotein transcripts and proteins in the NCBI database are only "predicted." Thus we initiated cloning and sequencing of the full turkey selenoprotein transcriptome to demonstrate expression of selenoprotein transcripts in the turkey, and to develop tools to investigate Se regulation of the full selenoproteome. Total RNA was isolated from six tissues of Se-adequate adult tom turkeys, and used to prepare reverse-transcription cDNA libraries. PCR primers were designed, based initially on chicken, rodent, porcine, bovine and human sequences and later on turkey shotgun cloning sequences. We report here the cloning of full transcript sequences for 9 selenoproteins, and 3'UTR portions for 15 additional selenoproteins, which include SECIS elements in 22 3'UTRs, and in-frame Sec (UGA) codons within coding regions of 19 selenoproteins, including 12 Sec codons in SEPP1. In addition, we sequenced the gap between two contigs from the shotgun cloning of the turkey genome, and found the missing sequence for the turkey Sec-tRNA. RTPCR was used to determine the relative transcript expression in 6 tissues. GPX3 expression was high in all tissues except kidney, GPX1 expression was high in kidney, SEPW1 expression was high in heart, gizzard and muscle, and SELU expression was high in liver. SEPP2, a selenoprotein not found in mammals, was highly expressed in liver but not in other tissues. In summary, transcripts for 24 selenoproteins are expressed in the turkey, not just predicted.

  9. Expression of the human glucokinase gene: important roles of the 5' flanking and intron 1 sequences.

    Directory of Open Access Journals (Sweden)

    Yi Wang

    Full Text Available BACKGROUND: Glucokinase plays important tissue-specific roles in human physiology, where it acts as a sensor of blood glucose levels in the pancreas, and a few other cells of the gut and brain, and as the rate-limiting step in glucose metabolism in the liver. Liver-specific expression is driven by one of the two tissue-specific promoters, and has an absolute requirement for insulin. The sequences that mediate regulation by insulin are incompletely understood. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the liver-specific expression of the human glucokinase gene we compared the structures of this gene from diverse mammals. Much of the sequence located between the 5' pancreatic beta-cell-specific and downstream liver-specific promoters of the glucokinase genes is composed of repetitive DNA elements that were inserted in parallel on different mammalian lineages. The transcriptional activity of the liver-specific promoter 5' flanking sequences were tested with and without downstream intronic sequences in two human liver cells lines, HepG2 and L-02. While glucokinase liver-specific 5' flanking sequences support expression in liver cell lines, a sequence located about 2000 bases 3' to the liver-specific mRNA start site represses gene expression. Enhanced reporter gene expression was observed in both cell lines when cells were treated with fetal calf serum, but only in the L-02 cells was expression enhanced by insulin. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the normal liver L-02 cell line may be a better model to understand the regulation of the liver-specific expression of the human glucokinase gene. Our results also suggest that sequences downstream of the liver-specific mRNA start site have important roles in the regulation of liver-specific glucokinase gene expression.

  10. Novel expressed sequence tags of an alpine-cold plant species,Gymnadenia conopsea (Orchidaceae)

    Institute of Scientific and Technical Information of China (English)


    Gymnadenia conopsea,an alpine Orchidaceae plant,was one of the widely used Tibetan traditional medicines.In this study,we sequenced total 105 expressed sequence tags (ESTs) from a full-length cDNA expression library constructed by the Oligo-capping technique.The further bioinformatic analyses suggested that the 65 represented unique sequences showed high homology to previously identified genes in other plants:30 sequences matched to other uncharacterized expressed sequence tags (ESTs),and 10 sequences showed no good matches to available sequences in DNA databases.Gene ontology annotation by InterProScan indicated that many of these cDNAs (7 percent) have no known molecular functions and may be unique to G.conopsea.Fifty-five ESTs with matched proteins were involved in a series of diverse functions,in which molecular function such as "binding" (42.9 percent) and "catalytic activity" (25.0 percent) were the most frequent functions of the cDNAs.This cDNA library provided a critical basis for further investigation of functional genes expression under cold stress in this alpine species.In addition,13 ESTs-based polymerase chain reaction (PCR) primers were designed and can also be used for genotypic identification and for the genetic diversity analysis of G.conopsea and its closely related species.

  11. The complete mitochondrial genome of the armored catfish, Hypostomus plecostomus (Siluriformes: Loricariidae). (United States)

    Liu, Shikai; Zhang, Jiaren; Yao, Jun; Liu, Zhanjiang


    The complete mitochondrial genome of the armored catfish, Hypostomus plecostomus, was determined by next generation sequencing of genomic DNA without prior sample processing or primer design. Bioinformatics analysis resulted in the entire mitochondrial genome sequence with length of 16,523 bp. The H. plecostomus mitochondrial genome is consisted of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region, showing typical circular molecule structure of mitochondrial genome as in other vertebrates. The whole genome base composition was estimated to be 31.8% A, 27.0% T, 14.6% G, and 26.6% C, with A/T bias of 58.8%. This work provided the H. plecostomus mitochondrial genome sequence which should be valuable for species identification, phylogenetic analysis and conservation genetics studies in catfishes.

  12. Expressed sequence tags from heat-shocked seagrass Zostera noltii (Hornemann) from its southern distribution range

    NARCIS (Netherlands)

    Massa, Sonia I.; Pearson, Gareth A.; Aires, Tania; Kube, Michael; Olsen, Jeanine L.; Reinhardt, Richard; Serrao, Ester A.; Arnaud-Haond, Sophie


    Predicted global climate change threatens the distributional ranges of species worldwide. We identified genes expressed in the intertidal seagrass Zostera midi during recovery from a simulated low tide heat-shock exposure. Five Expressed Sequence Tag (EST) libraries were compared, corresponding to f

  13. DNA barcoding of commercially important catfishes in the Philippines. (United States)

    Quilang, Jonas P; Yu, Shiny Cathlynne S


    Many species of catfish are important resources for human consumption, for sport fishing and for use in aquarium industry. In the Philippines, some species are cultivated and some are caught in the wild for food and a few introduced species have become invasive. In this study, DNA barcoding using the mitochondrial cytochrome c oxidase I (COI) gene was done on commercially and economically important Philippine catfishes. A total of 75 specimens belonging to 11 species and 5 families were DNA barcoded. The genetic distances were computed and Neighbor-Joining (NJ) trees were constructed based on the Kimura 2-Parameter (K2P) method. The average K2P distances within species, genus, family and order were 0.2, 8.2, 12.7 and 21.9%, respectively. COI sequences clustered according to their species designation for 7 of the 11 catfishes. DNA barcoding was not able to discriminate between Arius dispar and A. manillensis and between Pterygoplichthys disjunctivus and P. pardalis. The morphological characters that are used to distinguish between these species do not complement molecular identification through DNA barcoding. DNA barcoding also showed that Clarias batrachus from the Philippines is different from the species found in India and Thailand, which supports earlier suggestions based on morphology that those found in India should be designated as C. magur and those in mainland Southeast Asia as C. aff. batrachus "Indochina". This study has shown that DNA barcoding can be used for species delineation and for tagging some species for further taxonomic investigation, which has implications on proper management and conservation strategies.

  14. Insights into a dinoflagellate genome through expressed sequence tag analysis

    Directory of Open Access Journals (Sweden)

    Bonaldo Maria F


    Full Text Available Abstract Background Dinoflagellates are important marine primary producers and grazers and cause toxic "red tides". These taxa are characterized by many unique features such as immense genomes, the absence of nucleosomes, and photosynthetic organelles (plastids that have been gained and lost multiple times. We generated EST sequences from non-normalized and normalized cDNA libraries from a culture of the toxic species Alexandrium tamarense to elucidate dinoflagellate evolution. Previous analyses of these data have clarified plastid origin and here we study the gene content, annotate the ESTs, and analyze the genes that are putatively involved in DNA packaging. Results Approximately 20% of the 6,723 unique (11,171 total 3'-reads ESTs data could be annotated using Blast searches against GenBank. Several putative dinoflagellate-specific mRNAs were identified, including one novel plastid protein. Dinoflagellate genes, similar to other eukaryotes, have a high GC-content that is reflected in the amino acid codon usage. Highly represented transcripts include histone-like (HLP and luciferin binding proteins and several genes occur in families that encode nearly identical proteins. We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X. We speculate this histone may be retained for its role in DNA double-strand break repair. Conclusion This is the most extensive collection to date of ESTs from a toxic dinoflagellate. These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production.

  15. Leveraging human genomic information to identify nonhuman primate sequences for expression array development

    Directory of Open Access Journals (Sweden)

    Boyle Nicholas F


    Full Text Available Abstract Background Nonhuman primates (NHPs are essential for biomedical research due to their similarities to humans. The utility of NHPs will be greatly increased by the application of genomics-based approaches such as gene expression profiling. Sequence information from the 3' end of genes is the key resource needed to create oligonucleotide expression arrays. Results We have developed the algorithms and procedures necessary to quickly acquire sequence information from the 3' end of nonhuman primate orthologs of human genes. To accomplish this, we identified terminal exons of over 15,000 human genes by aligning mRNA sequences with genomic sequence. We found the mean length of complete last exons to be approximately 1,400 bp, significantly longer than previous estimates. We designed primers to amplify genomic DNA, which included at least 300 bp of the terminal exon. We cloned and sequenced the PCR products representing over 5,500 Macaca mulatta (rhesus monkey orthologs of human genes. This sequence information has been used to select probes for rhesus gene expression profiling. We have also tested 10 sets of primers with genomic DNA from Macaca fascicularis (Cynomolgus monkey, Papio hamadryas (Baboon, and Chlorocebus aethiops (African green monkey, vervet. The results indicate that the primers developed for this study will be useful for acquiring sequence from the 3' end of genes for other nonhuman primate species. Conclusion This study demonstrates that human genomic DNA sequence can be leveraged to obtain sequence from the 3' end of NHP orthologs and that this sequence can then be used to generate NHP oligonucleotide microarrays. Affymetrix and Agilent used sequences obtained with this approach in the design of their rhesus macaque oligonucleotide microarrays.

  16. Differential expression in normal-adenoma-carcinoma sequence suggests complex molecular carcinogenesis in colon. (United States)

    Lee, Seungkoo; Bang, Seunghyun; Song, Kyuyoung; Lee, Inchul


    The majority of colon cancers develop from pre-existing adenomas. We analyzed the expression profiles in the sequence of normal colon crypts, adenomas and early-stage carcinomas using microdissected cells from tubular adenomas with foci of malignant transformation. Differentially expressed genes were detected between normal-adenoma and adenoma-carcinoma, and were grouped according to the patterns of expression changes in the sequence. Down-regulated genes in the sequence included PLA2G2A, TSPAN1, PDCD4, FCGBP, AATK, EPLIN, FABP1, AGR2, MTUS1, TSC1, galectin 4 and MT1F. PLA2G2A has been shown to suppress colon tumorigenesis in mice, but the pathobiological role in humans has been controversial. Our data showed continuous down-regulation of PLA2G2A in the sequence supporting an implication in human colon cancer. Tumor suppressor and/ or proapoptotic activities have also been reported in other genes. Up-regulated genes included ribosomal proteins, IER3 and TPR. TGF-beta2 and matrix metalloproteinase 23B were up-regulated in carcinoma but not in adenoma, supporting the pathobiological roles in malignant transformation. Differentially expressed genes partly coincided with those in the adenoma-carcinoma sequence of the stomach, which was published previously, suggesting a partial overlap between the adenoma-carcinoma sequences of the colon and stomach.

  17. Reducing size variation in hybrid catfish culture through graded partial harvest (United States)

    Hybrid catfish (' channel catfish Ictalurus punctatus x ' blue catfish I. furcatus) are becoming a major contributor to catfish aquaculture in the United States. Because of unique production characteristics of hybrid catfish, rapid farmer adoption of hybrids has outpaced appropriate management resea...

  18. Generation and analysis of expressed sequence tags from the ciliate protozoan parasite Ichthyophthirius multifiliis

    Directory of Open Access Journals (Sweden)

    Arias Covadonga


    Full Text Available Abstract Background The ciliate protozoan Ichthyophthirius multifiliis (Ich is an important parasite of freshwater fish that causes 'white spot disease' leading to significant losses. A genomic resource for large-scale studies of this parasite has been lacking. To study gene expression involved in Ich pathogenesis and virulence, our goal was to generate expressed sequence tags (ESTs for the development of a powerful microarray platform for the analysis of global gene expression in this species. Here, we initiated a project to sequence and analyze over 10,000 ESTs. Results We sequenced 10,368 EST clones using a normalized cDNA library made from pooled samples of the trophont, tomont, and theront life-cycle stages, and generated 9,769 sequences (94.2% success rate. Post-sequencing processing led to 8,432 high quality sequences. Clustering analysis of these ESTs allowed identification of 4,706 unique sequences containing 976 contigs and 3,730 singletons. These unique sequences represent over two million base pairs (~10% of Plasmodium falciparum genome, a phylogenetically related protozoan. BLASTX searches produced 2,518 significant (E-value -5 hits and further Gene Ontology (GO analysis annotated 1,008 of these genes. The ESTs were analyzed comparatively against the genomes of the related protozoa Tetrahymena thermophila and P. falciparum, allowing putative identification of additional genes. All the EST sequences were deposited by dbEST in GenBank (GenBank: EG957858–EG966289. Gene discovery and annotations are presented and discussed. Conclusion This set of ESTs represents a significant proportion of the Ich transcriptome, and provides a material basis for the development of microarrays useful for gene expression studies concerning Ich development, pathogenesis, and virulence.

  19. Contig sequences and their annotation (amino acid sequence and results of homology search), and expression profile - Dicty_cDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available Dicty_cDB Contig sequences and their annotation (amino acid sequence and results of homology search), and expre...s of homology search), and expression profile Description of data contents Contig...TogoDB edition only. Annotation Translated Amino Acid sequence Representative ), which is the program to predict the localization of proteins in the cell. Expression prof...o acid sequence and results of homology search), and expression profile - Dicty_cDB | LSDB Archive ...

  20. Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing

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    Robles José A


    Full Text Available Abstract Background RNA sequencing (RNA-Seq has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods. Results Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices. Conclusions This work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library (technical replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates.

  1. SpiroESTdb: a transcriptome database and online tool for sparganum expressed sequences tags

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    Kim Dae-Won


    Full Text Available Abstract Background Sparganum (plerocercoid of Spirometra erinacei is a parasite that possesses the remarkable ability to survive by successfully modifying its physiology and morphology to suit various hosts and can be found in various tissues, even the nervous system. However, surprisingly little is known about the molecular function of genes that are expressed during the course of the parasite life cycle. To begin to decipher the molecular processes underlying gene function, we constructed a database of expressed sequence tags (ESTs generated from sparganum. Findings SpiroESTdb is a web-based information resource that is built upon the annotation and curation of 5,655 ESTs data. SpiroESTdb provides an integrated platform for expressed sequence data, expression dynamics, functional genes, genetic markers including single nucleotide polymorphisms and tandem repeats, gene ontology and KEGG pathway information. Moreover, SpiroESTdb supports easy access to gene pages, such as (i curation and query forms, (ii in silico expression profiling and (iii BLAST search tools. Comprehensive descriptions of the sparganum content of all sequenced data are available, including summary reports. The contents of SpiroESTdb can be viewed and downloaded from the web ( Conclusions This integrative web-based database of sequence data, functional annotations and expression profiling data will serve as a useful tool to help understand and expand the characterization of parasitic infections. It can also be used to identify potential industrial drug targets and vaccine candidate genes.

  2. Citrus plastid-related gene profiling based on expressed sequence tag analyses

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    Tercilio Calsa Jr.


    Full Text Available Plastid-related sequences, derived from putative nuclear or plastome genes, were searched in a large collection of expressed sequence tags (ESTs and genomic sequences from the Citrus Biotechnology initiative in Brazil. The identified putative Citrus chloroplast gene sequences were compared to those from Arabidopsis, Eucalyptus and Pinus. Differential expression profiling for plastid-directed nuclear-encoded proteins and photosynthesis-related gene expression variation between Citrus sinensis and Citrus reticulata, when inoculated or not with Xylella fastidiosa, were also analyzed. Presumed Citrus plastome regions were more similar to Eucalyptus. Some putative genes appeared to be preferentially expressed in vegetative tissues (leaves and bark or in reproductive organs (flowers and fruits. Genes preferentially expressed in fruit and flower may be associated with hypothetical physiological functions. Expression pattern clustering analysis suggested that photosynthesis- and carbon fixation-related genes appeared to be up- or down-regulated in a resistant or susceptible Citrus species after Xylella inoculation in comparison to non-infected controls, generating novel information which may be helpful to develop novel genetic manipulation strategies to control Citrus variegated chlorosis (CVC.

  3. Sequencing and expression analysis of hepcidin mRNA in donkey (Equus asinus liver

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    José P. Oliveira-Filho


    Full Text Available The hypoferremia that is observed during systemic inflammatory processes is mediated by hepcidin, which is a peptide that is mainly synthesized in the livers of several mammalian species. Hepcidin plays a key role in iron metabolism and in the innate immune system. It's up-regulation is particularly useful during acute inflammation, and it restricts the iron availability that is necessary for the growth of pathogenic microorganisms. In this study, the hepcidin mRNA of Equus asinus has been characterized, and the expression of donkey hepcidin in the liver has been determined. The donkey hepcidin sequence has an open reading frame (ORF of 261 nucleotides, and the deduced corresponding protein sequence has 86 amino acids. The amino acid sequence of donkey hepcidin was most homologous to Equus caballus (98%. The mature donkey hepcidin sequence (25 amino acids was 100% homologous to the equine mature hepcidin and has eight conserved cysteine residues that are found in all of the investigated hepcidin sequences. The expression profile of donkey hepcidin in the liver was high and was similar to the reference gene expression. The donkey hepcidin sequence was deposited in GenBankTM (HQ902884 and may be useful for additional studies on iron metabolism and the inflammatory process in this species.

  4. Complement regulatory protein genes in channel catfish and their involvement in disease defense response. (United States)

    Jiang, Chen; Zhang, Jiaren; Yao, Jun; Liu, Shikai; Li, Yun; Song, Lin; Li, Chao; Wang, Xiaozhu; Liu, Zhanjiang


    Complement system is one of the most important defense systems of innate immunity, which plays a crucial role in disease defense responses in channel catfish. However, inappropriate and excessive complement activation could lead to potential damage to the host cells. Therefore the complement system is controlled by a set of complement regulatory proteins to allow normal defensive functions, but prevent hazardous complement activation to host tissues. In this study, we identified nine complement regulatory protein genes from the channel catfish genome. Phylogenetic and syntenic analyses were conducted to determine their orthology relationships, supporting their correct annotation and potential functional inferences. The expression profiles of the complement regulatory protein genes were determined in channel catfish healthy tissues and after infection with the two main bacterial pathogens, Edwardsiella ictaluri and Flavobacterium columnare. The vast majority of complement regulatory protein genes were significantly regulated after bacterial infections, but interestingly were generally up-regulated after E. ictaluri infection while mostly down-regulated after F. columnare infection, suggesting a pathogen-specific pattern of regulation. Collectively, these findings suggested that complement regulatory protein genes may play complex roles in the host immune responses to bacterial pathogens in channel catfish.

  5. Influences on gene expression in vivo by a Shine-Dalgarno sequence

    DEFF Research Database (Denmark)

    Jin, Haining; Zhao, Qing; Gonzalez de Valdivia, Ernesto I;


    The Shine-Dalgarno (SD+: 5'-AAGGAGG-3') sequence anchors the mRNA by base pairing to the 16S rRNA in the small ribosomal subunit during translation initiation. We have here compared how an SD+ sequence influences gene expression, if located upstream or downstream of an initiation codon....... The positive effect of an upstream SD+ is confirmed. A downstream SD+ gives decreased gene expression. This effect is also valid for appropriately modified natural Escherichia coli genes. If an SD+ is placed between two potential initiation codons, initiation takes place predominantly at the second start site...

  6. Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

    Institute of Scientific and Technical Information of China (English)

    李琪; 束婧; 赵翠; 刘士凯; 孔令锋; 郑小东


    Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai).Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences,after redundancy elimination.Seventeen polymorphic EST-SSRs were developed.The number of alleles per locus varied from 2-17,with an average of 6.8 alleles per locus.The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922,respective...

  7. Molecular cloning, sequence characterization and expression pattern of Rab18 gene from watermelon (Citrullus lanatus). (United States)

    Xinli, Xiao; Lei, Peng


    The complete mRNA sequence of watermelon Rab18 gene was amplified through the rapid amplification of cDNA ends (RACE) method. The full-length mRNA was 1010 bp containing a 645 bp open reading frame, which encodes a protein of 214 amino acids. Sequence analysis revealed that watermelon Rab18 protein shares high homology with the Rab18 of cucumber (99%), muskmelon (98%), Morus notabilis (90%), tomato (89%), wine grape (89%) and potato (88%). Phylogenetic analysis revealed that watermelon Rab18 gene has a closer genetic relationship with Rab18 gene of cucumber and muskmelon. Tissue expression profile analysis indicated that watermelon Rab18 gene was highly expressed in root, stem and leaf, moderately expressed in flower and weakly expressed in fruit.

  8. Expressed Sequence Tag-Simple Sequence Repeat (EST-SSR Marker Resources for Diversity Analysis of Mango (Mangifera indica L.

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    Natalie L. Dillon


    Full Text Available In this study, a collection of 24,840 expressed sequence tags (ESTs generated from five mango (Mangifera indica L. cDNA libraries was mined for EST-based simple sequence repeat (SSR markers. Over 1,000 ESTs with SSR motifs were detected from more than 24,000 EST sequences with di- and tri-nucleotide repeat motifs the most abundant. Of these, 25 EST-SSRs in genes involved in plant development, stress response, and fruit color and flavor development pathways were selected, developed into PCR markers and characterized in a population of 32 mango selections including M. indica varieties, and related Mangifera species. Twenty-four of the 25 EST-SSR markers exhibited polymorphisms, identifying a total of 86 alleles with an average of 5.38 alleles per locus, and distinguished between all Mangifera selections. Private alleles were identified for Mangifera species. These newly developed EST-SSR markers enhance the current 11 SSR mango genetic identity panel utilized by the Australian Mango Breeding Program. The current panel has been used to identify progeny and parents for selection and the application of this extended panel will further improve and help to design mango hybridization strategies for increased breeding efficiency.

  9. Evaluation of cleaved amplified polymorphic sequence markers for Chamaecyparis obtusa based on expressed sequence tag information from Cryptomeria japonica. (United States)

    Matsumoto, A; Tsumura, Y


    We have developed and evaluated sequence-tagged site (STS) primers based on expressed sequence-tag information derived from sugi (Cryptomeria japonica) for use in hinoki (Chamaecyparis obtusa), a species that belongs to a different family (although it appears to be fairly closely related to sugi). Of the 417 C. japonica STS primer pairs we screened, 120 (approximately 30%) were transferable and provided specific PCR amplification products from 16 C. obtusa plus trees. We used haploid megagametophytes to investigate the homology of 80 STS fragments between C. obtusa and C. japonica and to identify orthologous loci. Nearly 90% of the fragments showed high (>70%) degrees of similarity between the species, and 35 STSs indicated homology to entries with the same putative function in a public DNA database. Of the 120 STS fragments amplified, 72 showed restriction fragment length polymorphisms; in addition, the CC2430 primers detected amplicon length polymorphism. We assessed the inheritance pattern of 27 cleaved amplified polymorphic sequence markers, using 20 individuals from the segregation population. All the markers analyzed were consistent with the marker inheritance patterns obtained from the screening panel, and no markers (except CC2716) showed significant (Pobtusa. Most of the markers should also provide reliable anchor loci for comparative mapping studies of the C. obtusa and C. japonica genomes.

  10. Gene discovery and transcript analyses in the corn smut pathogen Ustilago maydis: expressed sequence tag and genome sequence comparison

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    Saville Barry J


    Full Text Available Abstract Background Ustilago maydis is the basidiomycete fungus responsible for common smut of corn and is a model organism for the study of fungal phytopathogenesis. To aid in the annotation of the genome sequence of this organism, several expressed sequence tag (EST libraries were generated from a variety of U. maydis cell types. In addition to utility in the context of gene identification and structure annotation, the ESTs were analyzed to identify differentially abundant transcripts and to detect evidence of alternative splicing and anti-sense transcription. Results Four cDNA libraries were constructed using RNA isolated from U. maydis diploid teliospores (U. maydis strains 518 × 521 and haploid cells of strain 521 grown under nutrient rich, carbon starved, and nitrogen starved conditions. Using the genome sequence as a scaffold, the 15,901 ESTs were assembled into 6,101 contiguous expressed sequences (contigs; among these, 5,482 corresponded to predicted genes in the MUMDB (MIPS Ustilago maydis database, while 619 aligned to regions of the genome not yet designated as genes in MUMDB. A comparison of EST abundance identified numerous genes that may be regulated in a cell type or starvation-specific manner. The transcriptional response to nitrogen starvation was assessed using RT-qPCR. The results of this suggest that there may be cross-talk between the nitrogen and carbon signalling pathways in U. maydis. Bioinformatic analysis identified numerous examples of alternative splicing and anti-sense transcription. While intron retention was the predominant form of alternative splicing in U. maydis, other varieties were also evident (e.g. exon skipping. Selected instances of both alternative splicing and anti-sense transcription were independently confirmed using RT-PCR. Conclusion Through this work: 1 substantial sequence information has been provided for U. maydis genome annotation; 2 new genes were identified through the discovery of 619

  11. The cytochrome P450 genes of channel catfish: their involvement in disease defense responses as revealed by meta-analysis of RNA-Seq datasets (United States)

    Zhang, Jiaren; Yao, Jun; Wang, Ruijia; Zhang, Yu; Liu, Shikai; Sun, Luyang; Jiang, Yanliang; Feng, Jianbin; Liu, Nannan; Nelson, David; Waldbieser, Geoff; Liu, Zhanjiang


    Background Cytochrome P450s (CYPs) encode one of the most diverse enzyme superfamily in nature. They catalyze oxidative reactions of endogenous molecules and exogenous chemicals. Methods We identified CYPs genes through in silico analysis using EST, RNA-Seq and genome databases of channel catfish. Phylogenetic analyses and conserved syntenic analyses were conducted to determine their identities and orthologies. Meta-analysis of RNA-Seq databases was conducted to analyze expression profile of CYP genes following bacterial infection. Results A full set of 61 CYP genes were identified and characterized in channel catfish. Phylogenetic tree and conserved synteny provided strong evidence of their identities and orthorlogy. Lineage-specific gene duplication was evident in a number of clans in channel catfish. CYP46A1 is missing in the catfish genome as observed with syntenic analysis and RT-PCR analysis. Thirty CYPs were found up- or down-regulated in liver, while seven and eight CYPs were observed regulated in intestine and gill following bacterial infection. Conclusion We systematically identified and characterized a full set of 61 CYP genes in channel catfish and studied their expression profiles after bacterial infection. Strikingly large numbers of CYP genes appear to be involved in the bacterial defense processes. General significance This work provides an example to systematically study CYP genes in non-model species. Moreover, it provides a basis for further toxicological and physiological studies in channel catfish. PMID:24780645

  12. Probing the effect of promoters on noise in gene expression using thousands of designed sequences. (United States)

    Sharon, Eilon; van Dijk, David; Kalma, Yael; Keren, Leeat; Manor, Ohad; Yakhini, Zohar; Segal, Eran


    Genetically identical cells exhibit large variability (noise) in gene expression, with important consequences for cellular function. Although the amount of noise decreases with and is thus partly determined by the mean expression level, the extent to which different promoter sequences can deviate away from this trend is not fully known. Here, we present a high-throughput method for measuring promoter-driven noise for thousands of designed synthetic promoters in parallel. We use it to investigate how promoters encode different noise levels and find that the noise levels of promoters with similar mean expression levels can vary more than one order of magnitude, with nucleosome-disfavoring sequences resulting in lower noise and more transcription factor binding sites resulting in higher noise. We propose a kinetic model of gene expression that takes into account the nonspecific DNA binding and one-dimensional sliding along the DNA, which occurs when transcription factors search for their target sites. We show that this assumption can improve the prediction of the mean-independent component of expression noise for our designed promoter sequences, suggesting that a transcription factor target search may affect gene expression noise. Consistent with our findings in designed promoters, we find that binding-site multiplicity in native promoters is associated with higher expression noise. Overall, our results demonstrate that small changes in promoter DNA sequence can tune noise levels in a manner that is predictable and partly decoupled from effects on the mean expression levels. These insights may assist in designing promoters with desired noise levels.

  13. Whole Blood Transcriptome Sequencing Reveals Gene Expression Differences between Dapulian and Landrace Piglets


    Hu, Jiaqing; Yang, Dandan; Chen, Wei; Li, Chuanhao; Wang, Yandong; Zeng, Yongqing; Wang, Hui


    There is little genomic information regarding gene expression differences at the whole blood transcriptome level of different pig breeds at the neonatal stage. To solve this, we characterized differentially expressed genes (DEGs) in the whole blood of Dapulian (DPL) and Landrace piglets using RNA-seq (RNA-sequencing) technology. In this study, 83 DEGs were identified between the two breeds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified immun...

  14. Nucleic and amino acid sequences relating to a novel transketolase, and methods for the expression thereof

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Lange, Bernd Markus (Pullman, WA); McCaskill, David G. (Pullman, WA)


    cDNAs encoding 1-deoxyxylulose-5-phosphate synthase from peppermint (Mentha piperita) have been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences (SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7) are provided which code for the expression of 1-deoxyxylulose-5-phosphate synthase from plants. In another aspect the present invention provides for isolated, recombinant DXPS proteins, such as the proteins having the sequences set forth in SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. In other aspects, replicable recombinant cloning vehicles are provided which code for plant 1-deoxyxylulose-5-phosphate synthases, or for a base sequence sufficiently complementary to at least a portion of 1-deoxyxylulose-5-phosphate synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding a plant 1-deoxyxylulose-5-phosphate synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant 1-deoxyxylulose-5-phosphate synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant 1-deoxyxylulose-5-phosphate synthase may be used to obtain expression or enhanced expression of 1-deoxyxylulose-5-phosphate synthase in plants in order to enhance the production of 1-deoxyxylulose-5-phosphate, or its derivatives such as isopentenyl diphosphate (BP), or may be otherwise employed for the regulation or expression of 1-deoxyxylulose-5-phosphate synthase, or the production of its products.

  15. A score system for quality evaluation of RNA sequence tags: an improvement for gene expression profiling

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    Pinheiro Daniel G


    Full Text Available Abstract Background High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE, Massively Parallel Signature Sequencing (MPSS or Sequencing-by-Synthesis (SBS represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. Results This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. Conclusion These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at S3T source code and datasets can also be downloaded from the aforementioned website.

  16. Two lamprey Hedgehog genes share non-coding regulatory sequences and expression patterns with gnathostome Hedgehogs.

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    Shungo Kano

    Full Text Available Hedgehog (Hh genes play major roles in animal development and studies of their evolution, expression and function point to major differences among chordates. Here we focused on Hh genes in lampreys in order to characterize the evolution of Hh signalling at the emergence of vertebrates. Screening of a cosmid library of the river lamprey Lampetra fluviatilis and searching the preliminary genome assembly of the sea lamprey Petromyzon marinus indicate that lampreys have two Hh genes, named Hha and Hhb. Phylogenetic analyses suggest that Hha and Hhb are lamprey-specific paralogs closely related to Sonic/Indian Hh genes. Expression analysis indicates that Hha and Hhb are expressed in a Sonic Hh-like pattern. The two transcripts are expressed in largely overlapping but not identical domains in the lamprey embryonic brain, including a newly-described expression domain in the nasohypophyseal placode. Global alignments of genomic sequences and local alignment with known gnathostome regulatory motifs show that lamprey Hhs share conserved non-coding elements (CNE with gnathostome Hhs albeit with sequences that have significantly diverged and dispersed. Functional assays using zebrafish embryos demonstrate gnathostome-like midline enhancer activity for CNEs contained in intron2. We conclude that lamprey Hh genes are gnathostome Shh-like in terms of expression and regulation. In addition, they show some lamprey-specific features, including duplication and structural (but not functional changes in the intronic/regulatory sequences.

  17. Cloning, Sequencing, and Expression of Selenoprotein Transcripts in the Turkey (Meleagris gallopavo.

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    Roger A Sunde

    Full Text Available The minimum Se requirement for male turkey poults is 0.3 μg Se/g--three times higher than requirements found in rodents--based on liver and gizzard glutathione peroxidase-4 (GPX4 and GPX1 activities. In addition, turkey liver GPX4 activity is 10-fold higher and GPX1 activity is 10-fold lower than in rats, and both GPX1 and GPX4 mRNA levels are dramatically down-regulated by Se deficiency. Currently, the sequences of all annotated turkey selenoprotein transcripts and proteins in the NCBI database are only "predicted." Thus we initiated cloning and sequencing of the full turkey selenoprotein transcriptome to demonstrate expression of selenoprotein transcripts in the turkey, and to develop tools to investigate Se regulation of the full selenoproteome. Total RNA was isolated from six tissues of Se-adequate adult tom turkeys, and used to prepare reverse-transcription cDNA libraries. PCR primers were designed, based initially on chicken, rodent, porcine, bovine and human sequences and later on turkey shotgun cloning sequences. We report here the cloning of full transcript sequences for 9 selenoproteins, and 3'UTR portions for 15 additional selenoproteins, which include SECIS elements in 22 3'UTRs, and in-frame Sec (UGA codons within coding regions of 19 selenoproteins, including 12 Sec codons in SEPP1. In addition, we sequenced the gap between two contigs from the shotgun cloning of the turkey genome, and found the missing sequence for the turkey Sec-tRNA. RTPCR was used to determine the relative transcript expression in 6 tissues. GPX3 expression was high in all tissues except kidney, GPX1 expression was high in kidney, SEPW1 expression was high in heart, gizzard and muscle, and SELU expression was high in liver. SEPP2, a selenoprotein not found in mammals, was highly expressed in liver but not in other tissues. In summary, transcripts for 24 selenoproteins are expressed in the turkey, not just predicted.

  18. Exploring PTX3 expression in Sus scrofa cardiac tissue using RNA sequencing. (United States)

    Cabiati, Manuela; Caselli, Chiara; Savelli, Sara; Prescimone, Tommaso; Lionetti, Vincenzo; Giannessi, Daniela; Del Ry, Silvia


    The prototypic long pentraxin PTX3 is a novel vascular inflammatory marker sharing similarities with the classic short pentraxin (C-reactive protein). PTX3 is rapidly produced and released by several cell types in response to local inflammation of the cardiovascular system. Plasma PTX3 levels are very low in normal conditions and increase in heart failure (HF) patients with advancing NYHA functional class, but its exact role during HF pathogenetic mechanisms is not yet established. No data about PTX3 cardiac expression in normal and pathological conditions are currently available, either in human or in large-size animals. Of the latter, the pig has a central role in "in vivo" clinical settings but its genome has not been completely sequenced and the PTX3 gene sequence is still lacking. The aim of this study was to sequence the PTX3 in Sus scrofa, whose sequence is not yet present in GenBank. Utilizing our knowledge of this sequence, PTX3 mRNA expression was evaluated in cardiac tissue of normal (n=6) and HF pigs (n=5), obtained from the four chambers. To sequence PTX3 gene in S. scrofa, the high homology between Homo sapiens and S. scrofa was exploited. Pig PTX3 mRNA was sequenced using polymerase chain reaction primers designed from human consensus sequences. The DNA, obtained from different RT-PCR reactions, was sequenced using the Sanger method. S. scrofa PTX3 mRNA, 1-336 bp, was submitted to GenBank (ID: GQ412351). The sequence obtained from pig cardiac tissue shared an 84% sequence identity with human homolog. The presence of PTX3 mRNA expression was detected in all the cardiac chambers sharing an increase after 3 weeks of pacing compared to controls (p=0.036 HF right atrium vs. N; p=0.022, HF left ventricle vs. N). Knowledge of the PTX3 sequence could be a useful starting point for future studies devoted to better understanding the specific role of this molecule in the pathogenesis of cardiovascular diseases.

  19. Expressed sequence tags as a tool for phylogenetic analysis of placental mammal evolution.

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    Morgan Kullberg

    Full Text Available BACKGROUND: We investigate the usefulness of expressed sequence tags, ESTs, for establishing divergences within the tree of placental mammals. This is done on the example of the established relationships among primates (human, lagomorphs (rabbit, rodents (rat and mouse, artiodactyls (cow, carnivorans (dog and proboscideans (elephant. METHODOLOGY/PRINCIPAL FINDINGS: We have produced 2000 ESTs (1.2 mega bases from a marsupial mouse and characterized the data for their use in phylogenetic analysis. The sequences were used to identify putative orthologous sequences from whole genome projects. Although most ESTs stem from single sequence reads, the frequency of potential sequencing errors was found to be lower than allelic variation. Most of the sequences represented slowly evolving housekeeping-type genes, with an average amino acid distance of 6.6% between human and mouse. Positive Darwinian selection was identified at only a few single sites. Phylogenetic analyses of the EST data yielded trees that were consistent with those established from whole genome projects. CONCLUSIONS: The general quality of EST sequences and the general absence of positive selection in these sequences make ESTs an attractive tool for phylogenetic analysis. The EST approach allows, at reasonable costs, a fast extension of data sampling from species outside the genome projects.

  20. Depletion of Shine-Dalgarno Sequences Within Bacterial Coding Regions Is Expression Dependent

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    Chuyue Yang


    Full Text Available Efficient and accurate protein synthesis is crucial for organismal survival in competitive environments. Translation efficiency (the number of proteins translated from a single mRNA in a given time period is the combined result of differential translation initiation, elongation, and termination rates. Previous research identified the Shine-Dalgarno (SD sequence as a modulator of translation initiation in bacterial genes, while codon usage biases are frequently implicated as a primary determinant of elongation rate variation. Recent studies have suggested that SD sequences within coding sequences may negatively affect translation elongation speed, but this claim remains controversial. Here, we present a metric to quantify the prevalence of SD sequences in coding regions. We analyze hundreds of bacterial genomes and find that the coding sequences of highly expressed genes systematically contain fewer SD sequences than expected, yielding a robust correlation between the normalized occurrence of SD sites and protein abundances across a range of bacterial taxa. We further show that depletion of SD sequences within ribosomal protein genes is correlated with organismal growth rates, supporting the hypothesis of strong selection against the presence of these sequences in coding regions and suggesting their association with translation efficiency in bacteria.

  1. Edwardsiella tarda and Aeromonas hydrophila isolated from diseased Southern flounder (Paralichthys lethostigma) are virulent to channel catfish and Nile tilapia

    Institute of Scientific and Technical Information of China (English)

    Julia W Pridgeon; Phillip H Klesius; Gregory A Lewbart; Harry V Daniels; Megan Jacob


    Objective:To identify bacteria isolated from diseased Southern flounder and determine whether they are virulent to channel catfish and Nile tilapia. Methods:Gram-negative bacteria isolates were recovered from five tissues of diseased Southern flounder (Paralichthys lethostigma). The isolates were subjected to biochemical and molecular identification followed by virulence study in fish. Results: Based on biochemical analysis, the 25 isolates were found to share homologies with either Edwardsiella tarda (E. tarda) or Aeromonas hydrophila (A. hydrophila). Based on sequencing results of partial 16S rRNA gene, 15 isolates shared 100%identities with the 16S rRNA sequence of previously identified E. tarda strain TX1, whereas the other 10 isolates shared 100%identities with the 16S rRNA sequence of previously identified A. hydrophila strain An4. When healthy fish were exposed to flounder isolate by intracoelomic injection, the LD50 values of flounder isolate E. tarda to channel catfish or Nile tilapia [(10±2) g] were 6.1í104 and 1.1í107 CFU/fish, respectively, whereas that of flounder isolate A. hydrophila to channel catfish and Nile tilapia were 1.4í107 and 5.6í107 CFU/fish, respectively. Conclusions: This is the first report that E. tarda and A. hydrophila isolated from diseased Southern flounder are virulent to catfish and tilapia.

  2. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, Saket; Ely, Abdullah; Bloom, Kristie; Weinberg, Marc S. [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa); Arbuthnot, Patrick, E-mail: [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)


    RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.

  3. Molecular characterization, sequence analysis and tissue expression of a porcine gene – MOSPD2

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    Yang Jie


    Full Text Available The full-length cDNA sequence of a porcine gene, MOSPD2, was amplified using the rapid amplification of cDNA ends method based on a pig expressed sequence tag sequence which was highly homologous to the coding sequence of the human MOSPD2 gene. Sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 491 amino acids that has high homology with the motile sperm domain-containing protein 2 (MOSPD2 of five species: horse (89%, human (90%, chimpanzee (89%, rhesus monkey (89% and mouse (85%; thus, it could be defined as a porcine MOSPD2 gene. This novel porcine gene was assigned GeneID: 100153601. This gene is structured in 15 exons and 14 introns as revealed by computer-assisted analysis. The phylogenetic analysis revealed that the porcine MOSPD2 gene has a closer genetic relationship with the MOSPD2 gene of horse. Tissue expression analysis indicated that the porcine MOSPD2 gene is generally and differentially expressed in the spleen, muscle, skin, kidney, lung, liver, fat and heart. Our experiment is the first to establish the primary foundation for further research on the porcine MOSPD2 gene.

  4. Quantitative miRNA expression analysis: comparing microarrays with next-generation sequencing

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Salomon, Jesper; Søkilde, Rolf


    Recently, next-generation sequencing has been introduced as a promising, new platform for assessing the copy number of transcripts, while the existing microarray technology is considered less reliable for absolute, quantitative expression measurements. Nonetheless, so far, results from the two te...

  5. Transcriptome analysis of Loxosceles laeta (Araneae, Sicariidae spider venomous gland using expressed sequence tags

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    Almeida Diego D


    Full Text Available Abstract Background The bite of spiders belonging to the genus Loxosceles can induce a variety of clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis, and persistent inflammation. In order to examine the transcripts expressed in venom gland of Loxosceles laeta spider and to unveil the potential of its products on cellular structure and functional aspects, we generated 3,008 expressed sequence tags (ESTs from a cDNA library. Results All ESTs were clustered into 1,357 clusters, of which 16.4% of the total ESTs belong to recognized toxin-coding sequences, being the Sphingomyelinases D the most abundant transcript; 14.5% include "possible toxins", whose transcripts correspond to metalloproteinases, serinoproteinases, hyaluronidases, lipases, C-lectins, cystein peptidases and inhibitors. Thirty three percent of the ESTs are similar to cellular transcripts, being the major part represented by molecules involved in gene and protein expression, reflecting the specialization of this tissue for protein synthesis. In addition, a considerable number of sequences, 25%, has no significant similarity to any known sequence. Conclusion This study provides a first global view of the gene expression scenario of the venom gland of L. laeta described so far, indicating the molecular bases of its venom composition.

  6. Non-recursive expressions for even-index Bernoulli numbers: A remarkable sequence of determinants


    Van Malderen, Renaat


    Bernoulli numbers are usually expressed in terms of their lower index numbers (recursive). This paper gives explicit formulas for Bernoulli numbers of even index. The formulas contain a remarkable sequence of determinants. The value of these determinants for variable dimension is investigated.

  7. 3'-end sequencing for expression quantification (3SEQ from archival tumor samples.

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    Andrew H Beck

    Full Text Available Gene expression microarrays are the most widely used technique for genome-wide expression profiling. However, microarrays do not perform well on formalin fixed paraffin embedded tissue (FFPET. Consequently, microarrays cannot be effectively utilized to perform gene expression profiling on the vast majority of archival tumor samples. To address this limitation of gene expression microarrays, we designed a novel procedure (3'-end sequencing for expression quantification (3SEQ for gene expression profiling from FFPET using next-generation sequencing. We performed gene expression profiling by 3SEQ and microarray on both frozen tissue and FFPET from two soft tissue tumors (desmoid type fibromatosis (DTF and solitary fibrous tumor (SFT (total n = 23 samples, which were each profiled by at least one of the four platform-tissue preparation combinations. Analysis of 3SEQ data revealed many genes differentially expressed between the tumor types (FDR<0.01 on both the frozen tissue (approximately 9.6K genes and FFPET (approximately 8.1K genes. Analysis of microarray data from frozen tissue revealed fewer differentially expressed genes (approximately 4.64K, and analysis of microarray data on FFPET revealed very few (69 differentially expressed genes. Functional gene set analysis of 3SEQ data from both frozen tissue and FFPET identified biological pathways known to be important in DTF and SFT pathogenesis and suggested several additional candidate oncogenic pathways in these tumors. These findings demonstrate that 3SEQ is an effective technique for gene expression profiling from archival tumor samples and may facilitate significant advances in translational cancer research.

  8. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans (United States)

    Abbott, Lynn; Alshiekh-Nasany, Ruham; Mitschow, Charles


    In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart. PMID:27703814

  9. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

    Directory of Open Access Journals (Sweden)

    Dipak K. Dube


    Full Text Available In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4 each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

  10. Tumor transcriptome sequencing reveals allelic expression imbalances associated with copy number alterations. (United States)

    Tuch, Brian B; Laborde, Rebecca R; Xu, Xing; Gu, Jian; Chung, Christina B; Monighetti, Cinna K; Stanley, Sarah J; Olsen, Kerry D; Kasperbauer, Jan L; Moore, Eric J; Broomer, Adam J; Tan, Ruoying; Brzoska, Pius M; Muller, Matthew W; Siddiqui, Asim S; Asmann, Yan W; Sun, Yongming; Kuersten, Scott; Barker, Melissa A; De La Vega, Francisco M; Smith, David I


    Due to growing throughput and shrinking cost, massively parallel sequencing is rapidly becoming an attractive alternative to microarrays for the genome-wide study of gene expression and copy number alterations in primary tumors. The sequencing of transcripts (RNA-Seq) should offer several advantages over microarray-based methods, including the ability to detect somatic mutations and accurately measure allele-specific expression. To investigate these advantages we have applied a novel, strand-specific RNA-Seq method to tumors and matched normal tissue from three patients with oral squamous cell carcinomas. Additionally, to better understand the genomic determinants of the gene expression changes observed, we have sequenced the tumor and normal genomes of one of these patients. We demonstrate here that our RNA-Seq method accurately measures allelic imbalance and that measurement on the genome-wide scale yields novel insights into cancer etiology. As expected, the set of genes differentially expressed in the tumors is enriched for cell adhesion and differentiation functions, but, unexpectedly, the set of allelically imbalanced genes is also enriched for these same cancer-related functions. By comparing the transcriptomic perturbations observed in one patient to his underlying normal and tumor genomes, we find that allelic imbalance in the tumor is associated with copy number mutations and that copy number mutations are, in turn, strongly associated with changes in transcript abundance. These results support a model in which allele-specific deletions and duplications drive allele-specific changes in gene expression in the developing tumor.

  11. Sperm competition shapes gene expression and sequence evolution in the ocellated wrasse. (United States)

    Dean, Rebecca; Wright, Alison E; Marsh-Rollo, Susan E; Nugent, Bridget M; Alonzo, Suzanne H; Mank, Judith E


    Gene expression differences between males and females often underlie sexually dimorphic phenotypes, and the expression levels of genes that are differentially expressed between the sexes are thought to respond to sexual selection. Most studies on the transcriptomic response to sexual selection treat sexual selection as a single force, but postmating sexual selection in particular is expected to specifically target gonadal tissue. The three male morphs of the ocellated wrasse (Symphodus ocellatus) make it possible to test the role of postmating sexual selection in shaping the gonadal transcriptome. Nesting males hold territories and have the highest reproductive success, yet we detected feminization of their gonadal gene expression compared to satellite males. Satellite males are less brightly coloured and experience more intense sperm competition than nesting males. In line with postmating sexual selection affecting gonadal gene expression, we detected a more masculinized expression profile in satellites. Sneakers are the lowest quality males and showed both de-masculinization and de-feminization of gene expression. We also detected higher rates of gene sequence evolution of male-biased genes compared to unbiased genes, which could at least in part be explained by positive selection. Together, these results reveal the potential for postmating sexual selection to drive higher rates of gene sequence evolution and shape the gonadal transcriptome profile.

  12. Gene expression analysis of volatile-rich male flowers of dioecious Pandanus fascicularis using expressed sequence tags. (United States)

    Vinod, M S; Sankararamasubramanian, H M; Priyanka, R; Ganesan, G; Parida, Ajay


    Pandanus fascicularis is dioecious with the female plant producing a non-scented fruit while the male produces a flower rich in volatiles. The essential oil extracted from the flowers is economically exploited as a natural flavouring agent as well as for its therapeutic properties. Molecular dissection of this distinct flower for identifying the genes responsible for its aroma by way of expressed sequence tags (ESTs) has not been initiated in spite of its economic viability. A male flower-specific cDNA library was constructed and 977 ESTs were generated. CAP3 analysis performed on the dataset revealed 83 contigs (549 ESTs) and 428 singlets, thereby yielding a total of 511 unigenes. Functional annotation using the BLAST2GO software resulted in 1952 Gene ontology (GO) functional classification terms for 621 sequences. Unknown proteins were further analysed with InterProScan to determine their functional motifs. RNA gel blot analysis of 26 functionally distinct transcripts potentially involved in flowering and volatile generation, using vegetative and reproductive tissues of both the sexes, revealed differential expression profiles. In addition to an overview of genes expressed, candidate genes with expression that are modulated predominantly in the male inflorescence were also identified. This is the first report on generation of ESTs to determine the subset of genes that can be used as potential candidates for future attempts aimed towards its genetic and genome analysis including metabolic engineering of floral volatiles in this economically important plant.

  13. DEET toxicity to channel catfish sac fry (United States)

    The combination of open facilities, moisture, and warm weather during channel catfish Ictalurus punctatus spawning season causes mosquito-infestation problems. A common solution to mosquito problems in hatcheries is to use mosquito repellents applied to exposed skin and clothing. DEET (N,N-diethyl-...

  14. Virulent Aeromonas hydrophila in channel catfish (United States)

    In this study, we investigated factors that predisposed catfish to motile aeromonas septicemia (MAS) caused by virulent Aeromonas hydrophila (vAh). Our results revealed that wounding on fish body surface was a prerequisite for vAh infection and disease development. A reproducible waterborne challeng...

  15. Large-scale Identification of Expressed Sequence Tags (ESTs from Nicotianatabacum by Normalized cDNA Library Sequencing

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    Alvarez S Perez


    Full Text Available An expressed sequence tags (EST resource for tobacco plants (Nicotianatabacum was established using high-throughput sequencing of randomly selected clones from one cDNA library representing a range of plant organs (leaf, stem, root and root base. Over 5000 ESTs were generated from the 3’ ends of 8000 clones, analyzed by BLAST searches and categorized functionally. All annotated ESTs were classified into 18 functional categories, unique transcripts involved in energy were the largest group accounting for 831 (32.32% of the annotated ESTs. After excluding 2450 non-significant tentative unique transcripts (TUTs, 100 unique sequences (1.67% of total TUTs were identified from the N. tabacum database. In the array result two genes strongly related to the tobacco mosaic virus (TMV were obtained, one basic form of pathogenesis-related protein 1 precursor (TBT012G08 and ubiquitin (TBT087G01. Both of them were found in the variety Hongda, some other important genes were classified into two groups, one of these implicated in plant development like those genes related to a photosynthetic process (chlorophyll a-b binding protein, photosystem I, ferredoxin I and III, ATP synthase and a further group including genes related to plant stress response (ubiquitin, ubiquitin-like protein SMT3, glycine-rich RNA binding protein, histones and methallothionein. The interesting finding in this study is that two of these genes have never been reported before in N. tabacum (ubiquitin-like protein SMT3 and methallothionein. The array results were confirmed using quantitative PCR.

  16. Diversity Analysis in Cannabis sativa Based on Large-Scale Development of Expressed Sequence Tag-Derived Simple Sequence Repeat Markers


    Chunsheng Gao; Pengfei Xin; Chaohua Cheng; Qing Tang; Ping Chen; Changbiao Wang; Gonggu Zang; Lining Zhao


    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 SS...

  17. Generation and analysis of expressed sequence tags (ESTs for marker development in yam (Dioscorea alata L.

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    Robert Asiedu


    Full Text Available Abstract Background Anthracnose (Colletotrichum gloeosporioides is a major limiting factor in the production of yam (Dioscorea spp. worldwide. Availability of high quality sequence information is necessary for designing molecular markers associated with resistance. However, very limited sequence information pertaining to yam is available at public genome databases. Therefore, this collaborative project was developed for genetic improvement and germplasm characterization of yams using molecular markers. The current investigation is focused on studying gene expression, by large scale generation of ESTs, from one susceptible (TDa 95-0310 and two resistant yam genotypes (TDa 87-01091, TDa 95-0328 challenged with the fungus. Total RNA was isolated from young leaves of resistant and susceptible genotypes and cDNA libraries were sequenced using Roche 454 technology. Results A total of 44,757 EST sequences were generated from the cDNA libraries of the resistant and susceptible genotypes. Greater than 56% of ESTs were annotated using MapMan Mercator tool and Blast2GO search tools. Gene annotations were used to characterize the transcriptome in yam and also perform a differential gene expression analysis between the resistant and susceptible EST datasets. Mining for SSRs in the ESTs revealed 1702 unique sequences containing SSRs and 1705 SSR markers were designed using those sequences. Conclusion We have developed a comprehensive annotated transcriptome data set in yam to enrich the EST information in public databases. cDNA libraries were constructed from anthracnose fungus challenged leaf tissues for transcriptome characterization, and differential gene expression analysis. Thus, it helped in identifying unique transcripts in each library for disease resistance. These EST resources provide the basis for future microarray development, marker validation, genetic linkage mapping and QTL analysis in Dioscorea species.

  18. Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression. (United States)

    Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J


    The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

  19. An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data.

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    Daniel Ramsköld


    Full Text Available The parts of the genome transcribed by a cell or tissue reflect the biological processes and functions it carries out. We characterized the features of mammalian tissue transcriptomes at the gene level through analysis of RNA deep sequencing (RNA-Seq data across human and mouse tissues and cell lines. We observed that roughly 8,000 protein-coding genes were ubiquitously expressed, contributing to around 75% of all mRNAs by message copy number in most tissues. These mRNAs encoded proteins that were often intracellular, and tended to be involved in metabolism, transcription, RNA processing or translation. In contrast, genes for secreted or plasma membrane proteins were generally expressed in only a subset of tissues. The distribution of expression levels was broad but fairly continuous: no support was found for the concept of distinct expression classes of genes. Expression estimates that included reads mapping to coding exons only correlated better with qRT-PCR data than estimates which also included 3' untranslated regions (UTRs. Muscle and liver had the least complex transcriptomes, in that they expressed predominantly ubiquitous genes and a large fraction of the transcripts came from a few highly expressed genes, whereas brain, kidney and testis expressed more complex transcriptomes with the vast majority of genes expressed and relatively small contributions from the most expressed genes. mRNAs expressed in brain had unusually long 3'UTRs, and mean 3'UTR length was higher for genes involved in development, morphogenesis and signal transduction, suggesting added complexity of UTR-based regulation for these genes. Our results support a model in which variable exterior components feed into a large, densely connected core composed of ubiquitously expressed intracellular proteins.

  20. Functional analysis of cis-acting sequences regulating root-specific expression in transgenic tobacco

    Institute of Scientific and Technical Information of China (English)


    Two different length fragments, RSF1 and RSF2 which contained the cis-acting sequences of root-spe- cific gene TobRB7, were isolated from tobacco genome. The abilities of these fragments to direct root-specific expression were studied by fusing them to the β-glucuronidase (GUS) report gene with different directions. After the recombined vectors were transformed into tobacco, the expression pattern was performed by histochemical staining and the quantitative analysis of GUS activity. The data suggested that the cis-acting element of TobRB7 gene direct GUS expression not only as root-specific but also as bidirectional. In our studies, the short fragment, RSF2, performed stronger activity than RSF1 with any direction. The stronger activity of GUS expression was determined by reverse inserting of RSF1 or RSF2 than positive inserting.

  1. Hematological- and Neurological-Expressed Sequence 1 Gene Products in Progenitor Cells during Newt Retinal Development

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    Tatsushi Goto


    Full Text Available Urodele amphibians such as Japanese common newts have a remarkable ability to regenerate their injured neural retina, even as adults. We found that hematological- and neurological-expressed sequence 1 (Hn1 gene was induced in depigmented retinal pigment epithelial (RPE cells, and its expression was maintained at later stages of newt retinal regeneration. In this study, we investigated the distribution of the HN1 protein, the product of the Hn1 gene, in the developing retinas. Our immunohistochemical analyses suggested that the HN1 protein was highly expressed in an immature retina, and the subcellular localization changed during this retinogenesis as observed in newt retinal regeneration. We also found that the expression of Hn1 gene was not induced in mouse after retinal removal. Our results showed that Hn1 gene can be useful for detection of undifferentiated and dedifferentiated cells during both newt retinal development and regeneration.

  2. In-depth cDNA Library Sequencing Provides Quantitative Gene Expression Profiling in Cancer Biomarker Discovery

    Institute of Scientific and Technical Information of China (English)

    Wanling Yang; Dingge Ying; Yu-Lung Lau


    procedures may allow detection of many expres-sion features for less abundant gene variants. With the reduction of sequencing cost and the emerging of new generation sequencing technology, in-depth sequencing of cDNA pools or libraries may represent a better and powerful tool in gene expression profiling and cancer biomarker detection. We also propose using sequence-specific subtraction to remove hundreds of the most abundant housekeeping genes to in-crease sequencing depth without affecting relative expression ratio of other genes, as transcripts from as few as 300 most abundantly expressed genes constitute about 20% of the total transcriptome. In-depth sequencing also represents a unique ad-vantage of detecting unknown forms of transcripts, such as alternative splicing variants, fusion genes, and regulatory RNAs, as well as detecting mutations and polymorphisms that may play important roles in disease pathogenesis.

  3. Analysis of expressed sequence tags from the venom ducts of Conus striatus: focusing on the expression profile of conotoxins. (United States)

    Pi, Canhui; Liu, Yun; Peng, Can; Jiang, Xiuhua; Liu, Junliang; Xu, Bin; Yu, Xuesong; Yu, Yanghong; Jiang, Xiaoyu; Wang, Lei; Dong, Meiling; Chen, Shangwu; Xu, An-Long


    Cone snails (genus Conus) are predatory marine gastropods that use venom peptides for interacting with prey, predators and competitors. A majority of these peptides, generally known as conotoxins demonstrate striking selectivity in targeting specific subtypes of ion channels and neurotransmitter receptors. So they are not only useful tools in neuroscience to characterize receptors and receptor subtypes, but offer great potential in new drug research and development as well. Here, a cDNA library from the venom ducts of a fish-hunting cone snail species, Conus striatus is described for the generation of expressed sequence tags (ESTs). A total of 429 ESTs were grouped into 137 clusters or singletons. Among these sequences, 221 were toxin sequences, accounting for 52.1% (corresponding to 19 clusters) of all transcripts. A-superfamily (132 ESTs) and O-superfamily conotoxins (80 ESTs) constitute the predominant toxin components. Some non-disulfide-rich Conus peptides were also found. The expression profile of conotoxins also explained to some extent the pharmacological and physiological reactions elicited by this typical piscivorous species. For the first time, a nonstop transcript of conotoxin was identified, which is suggestive that alternative polyadenylation may be a means of post-transcriptional regulation of conotoxin production. A comparison analysis of these conotoxins reveals the different variation and divergence patterns in these two superfamilies. Our investigations indicate that focal hyper-mutation, block substitution and exon shuffling are three main mechanisms leading to the conotoxin diversity in a species. The comprehensive set of Conus gene sequences allowed the identification of the representative classes of conotoxins and related components, which may lay the foundation for further research and development of conotoxins.

  4. Replication error deficient and proficient colorectal cancer gene expression differences caused by 3'UTR polyT sequence deletions

    DEFF Research Database (Denmark)

    Wilding, Jennifer L; McGowan, Simon; Liu, Ying


    , and have distinct pathologies. Regulatory sequences controlling all aspects of mRNA processing, especially including message stability, are found in the 3'UTR sequence of most genes. The relevant sequences are typically A/U-rich elements or U repeats. Microarray analysis of 14 RER+ (deficient) and 16 RER......- (proficient) colorectal cancer cell lines confirms a striking difference in expression profiles. Analysis of the incidence of mononucleotide repeat sequences in the 3'UTRs, 5'UTRs, and coding sequences of those genes most differentially expressed in RER+ versus RER- cell lines has shown that much...... of this differential expression can be explained by the occurrence of a massive enrichment of genes with 3'UTR T repeats longer than 11 base pairs in the most differentially expressed genes. This enrichment was confirmed by analysis of two published consensus sets of RER differentially expressed probesets for a large...

  5. Isolation, sequence identification and tissue expression profiles of 3 novel porcine genes: ASPA, NAGA, and HEXA. (United States)

    Shu, Xianghua; Liu, Yonggang; Yang, Liangyu; Song, Chunlian; Hou, Jiafa


    The complete coding sequences of 3 porcine genes - ASPA, NAGA, and HEXA - were amplified by the reverse transcriptase polymerase chain reaction (RT-PCR) based on the conserved sequence information of the mouse or other mammals and referenced pig ESTs. These 3 novel porcine genes were then deposited in the NCBI database and assigned GeneIDs: 100142661, 100142664 and 100142667. The phylogenetic tree analysis revealed that the porcine ASPA, NAGA, and HEXA all have closer genetic relationships with the ASPA, NAGA, and HEXA of cattle. Tissue expression profile analysis was also carried out and results revealed that swine ASPA, NAGA, and HEXA genes were differentially expressed in various organs, including skeletal muscle, the heart, liver, fat, kidney, lung, and small and large intestines. Our experiment is the first one to establish the foundation for further research on these 3 swine genes.

  6. Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing. (United States)

    Khuansuwan, Sataree; Gamse, Joshua T


    We describe a method for isolating RNA suitable for high-throughput RNA sequencing (RNA-seq) from small numbers of fluorescently labeled cells isolated from live zebrafish (Danio rerio) embryos without using costly, commercially available columns. This method ensures high cell viability after dissociation and suspension of cells and gives a very high yield of intact RNA. We demonstrate the utility of our new protocol by isolating RNA from fluorescence activated cell sorted (FAC sorted) pineal complex neurons in wild-type and tbx2b knockdown embryos at 24 hours post-fertilization. Tbx2b is a transcription factor required for pineal complex formation. We describe a bioinformatics pipeline used to analyze differential expression following high-throughput sequencing and demonstrate the validity of our results using in situ hybridization of differentially expressed transcripts. This protocol brings modern transcriptome analysis to the study of small cell populations in zebrafish.

  7. Nucleotide sequence of maize dwarf mosaic virus capsid protein gene and its expression in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    赛吉庆; 康良仪; 黄忠; 史春霖; 田波; 谢友菊


    The 3’-terminal 1 279 nucleotide sequence of maize dwarf mosaic virus (MDMV) genome has been determined. This sequence contains an open reading frame of 1023 nudeotides and a 3’ -non-coding region of 256 nucleotides. The open reading frame includes all of the coding regions for the viral capsid protein (CP) and part of the viral nuclear inclusion protein (Nib). The predicted viral CP consists of 313 amino acid residues with a calculated molecular weight of 35400. The amino acid sequence of the viral CP derived from MDMV cDNA shows about 47%-54% homology to that of 4 other potyviruses. The viral CP gene was constructed in frame with the lacZ gene in pUC19 plasmid and expressed in E. coli cells. The fusion polypeptide positively reacted in Western blot with an antiserum prepared against the native viral CP.

  8. Expressed Sequence Tags from the oomycete Plasmopara halstedii, an obligate parasite of the sunflower

    Directory of Open Access Journals (Sweden)

    Mouzeyar Said


    Full Text Available Abstract Background Sunflower downy mildew is a major disease caused by the obligatory biotrophic oomycete Plasmopara halstedii. Little is known about the molecular mechanisms underlying its pathogenicity. In this study we used a genomics approach to gain a first insight into the transcriptome of P. halstedii. Results To identify genes from the obligatory biotrophic oomycete Plasmopara halstedii that are expressed during infection in sunflower (Helianthus annuus L. we employed the suppression subtraction hybridization (SSH method from sunflower seedlings infected by P. halstedii. Using this method and random sequencing of clones, a total of 602 expressed sequence tags (ESTs corresponding to 230 unique sequence sets were identified. To determine the origin of the unisequences, PCR primers were designed to amplify these gene fragments from genomic DNA isolated either from P. halstedii sporangia or from Helianthus annuus. Only 145 nonredundant ESTs which correspond to a total of 373 ESTs (67.7% proved to be derived from P. halstedii genes and that are expressed during infection in sunflower. A set of 87 nonredundant sequences were identified as showing matches to sequences deposited in public databases. Nevertheless, about 7% of the ESTs seem to be unique to P. halstedii without any homolog in any public database. Conclusion A summary of the assignment of nonredundant ESTs to functional categories as well as their relative abundance is listed and discussed. Annotation of the ESTs revealed a number of genes that could function in virulence. We provide a first glimpse into the gene content of P. halstedii. These resources should accelerate research on this important pathogen.

  9. p21WAF1/CIP1 gene DNA sequencing and its expression in human osteosarcoma

    Institute of Scientific and Technical Information of China (English)

    廖威明; 张春林; 李佛保; 曾炳芳; 曾益新


    Background Mutation and expression change of p21WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21WAF1/CIP1 gene in human osteosarcoma, p21WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis.Methods p21WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistry, respectively. Results The occurrence of P21 protein in osteosarcoma was 17.78% (8/45), and p21WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma, there were 17 cases (47.22%) with C→T at position 609; 10 normal blood samples' DNA sequence analysis yielded 8 cases (80.00%) with C→T at the same position. Conclusions Along with the increase of malignancy, the expression of p21WAF1/CIP1mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21WAF1/CIP1 gene mutation should be investigated. Our research leads to the location of p21WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients, which can provide a basis for further research.

  10. Molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer myostatin gene

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    Smith-Keune Carolyn


    Full Text Available Abstract Background Myostatin (MSTN is a member of the transforming growth factor-β superfamily that negatively regulates growth of skeletal muscle tissue. The gene encoding for the MSTN peptide is a consolidate candidate for the enhancement of productivity in terrestrial livestock. This gene potentially represents an important target for growth improvement of cultured finfish. Results Here we report molecular characterization, tissue expression and sequence variability of the barramundi (Lates calcarifer MSTN-1 gene. The barramundi MSTN-1 was encoded by three exons 379, 371 and 381 bp in length and translated into a 376-amino acid peptide. Intron 1 and 2 were 412 and 819 bp in length and presented typical GT...AG splicing sites. The upstream region contained cis-regulatory elements such as TATA-box and E-boxes. A first assessment of sequence variability suggested that higher mutation rates are found in the 5' flanking region with several SNP's present in this species. A putative micro RNA target site has also been observed in the 3'UTR (untranslated region and is highly conserved across teleost fish. The deduced amino acid sequence was conserved across vertebrates and exhibited characteristic conserved putative functional residues including a cleavage motif of proteolysis (RXXR, nine cysteines and two glycosilation sites. A qualitative analysis of the barramundi MSTN-1 expression pattern revealed that, in adult fish, transcripts are differentially expressed in various tissues other than skeletal muscles including gill, heart, kidney, intestine, liver, spleen, eye, gonad and brain. Conclusion Our findings provide valuable insights such as sequence variation and genomic information which will aid the further investigation of the barramundi MSTN-1 gene in association with growth. The finding for the first time in finfish MSTN of a miRNA target site in the 3'UTR provides an opportunity for the identification of regulatory mutations on the

  11. Annotated expressed sequence tags and xenobiotic detoxification in the aphid Myzus persicae (Sulzer)

    Institute of Scientific and Technical Information of China (English)



    Aphids (Hemiptera: Aphididae) are phytophagous insects that are importantagricultural pests. The enormous negative economic impacts caused by aphids worldwide arewell known, and are mostly due to their high multiplication rate and the transmission ofphytopathogenic viruses. Aphid management strategies mainly involve chemical treatmentswhich are pollutants and are increasingly inefficient, since aphids have developed multipleinsecticide-resistant mechanisms. Among the most economically important species is thegreen peach aphid Myzuspersicae Sulzer (Aphididae: Macrosiphini), which is able to colonizea wide range of host plants belonging to many different families, and transmits numerous plantviruses. Because of its large prevalence, M. persicae has been the target of massive insecticidetreatments; consequently, it has evolved several insecticide-resistant mechanisms. In thiswork, a collection of expressed genes from M. persicae is presented in order to identify putativegenes involved in xenobiotic detoxification. After cDNA cloning and sequencing, 959expressed sequence tags (EST) were annotated. Most sequences matched known genescorresponded to metabolism proteins (26%), ribosomal proteins (23%) and structural proteins(8%). Among them, several sequences corresponded to proteins putatively involved in sensing,degradation or detoxification of plant xenobiotic products.

  12. Construction and characterization of an expressed sequenced tag library for the mosquito vector Armigeres subalbatus

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    Tsai Shih-Feng


    Full Text Available Abstract Background The mosquito, Armigeres subalbatus, mounts a distinctively robust innate immune response when infected with the nematode Brugia malayi, a causative agent of lymphatic filariasis. In order to mine the transcriptome for new insight into the cascade of events that takes place in response to infection in this mosquito, 6 cDNA libraries were generated from tissues of adult female mosquitoes subjected to immune-response activation treatments that lead to well-characterized responses, and from aging, naïve mosquitoes. Expressed sequence tags (ESTs from each library were produced, annotated, and subjected to comparative analyses. Results Six libraries were constructed and used to generate 44,940 expressed sequence tags, of which 38,079 passed quality filters to be included in the annotation project and subsequent analyses. All of these sequences were collapsed into clusters resulting in 8,020 unique sequence clusters or singletons. EST clusters were annotated and curated manually within ASAP (A Systematic Annotation Package for Community Analysis of Genomes web portal according to BLAST results from comparisons to Genbank, and the Anopheles gambiae and Drosophila melanogaster genome projects. Conclusion The resulting dataset is the first of its kind for this mosquito vector and provides a basis for future studies of mosquito vectors regarding the cascade of events that occurs in response to infection, and thereby providing insight into vector competence and innate immunity.

  13. Comparative susceptibility of Channel Catfish, Blue Catfish, and their hybrid cross to experimental challenge with Bolbophorus damnificus (Digenea: Bolbophoridae) cercariae. (United States)

    Griffin, Matt J; Reichley, Stephen R; Khoo, Lester H; Ware, Cynthia; Greenway, Terrence E; Mischke, Charles C; Wise, David J


    The digenetic trematode Bolbophorus damnificus has been implicated in significant losses in catfish aquaculture since the late 1990s. The complex life cycle sequentially involves the American white pelican Pelecanus erythrorhynchos, the marsh rams horn snail Planorbella trivolvis, and Channel Catfish Ictalurus punctatus. Research supports anecdotal reports from the industry, suggesting that the hybrid of Channel Catfish×Blue Catfish I. furcatus is less susceptible to disease agents that have been historically prohibitive to Channel Catfish production, namely the gram-negative bacteria Edwardsiella ictaluri and Flavobacterium columnare, as well as the myxozoan parasite Henneguya ictaluri. This current research compared the susceptibility of Channel Catfish, Blue Catfish, and their hybrid cross to an experimental challenge by B. damnificus. Fish were exposed to 0, 100, 200, and 400 B. damnificus cercariae per fish, and the numbers of metacercariae per fish were determined 14 d postchallenge. Metacercariae were recovered from all challenged fish. There were no significant differences among fish groups challenged with the same dose, suggesting Channel and Blue Catfish and their hybrid are comparably susceptible to B. damnificus infection. As such, it is recommended that producers raising hybrid catfish remain diligent in controlling populations of the snail intermediate host to prevent production losses attributed to B. damnificus, especially when loafing pelicans have been observed at the aquaculture operation.

  14. Generation and analysis of the expressed sequence tags from the mycelium of Ganoderma lucidum.

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    Yen-Hua Huang

    Full Text Available Ganoderma lucidum (G. lucidum is a medicinal mushroom renowned in East Asia for its potential biological effects. To enable a systematic exploration of the genes associated with the various phenotypes of the fungus, the genome consortium of G. lucidum has carried out an expressed sequence tag (EST sequencing project. Using a Sanger sequencing based approach, 47,285 ESTs were obtained from in vitro cultures of G. lucidum mycelium of various durations. These ESTs were further clustered and merged into 7,774 non-redundant expressed loci. The features of these expressed contigs were explored in terms of over-representation, alternative splicing, and natural antisense transcripts. Our results provide an invaluable information resource for exploring the G. lucidum transcriptome and its regulation. Many cases of the genes over-represented in fast-growing dikaryotic mycelium are closely related to growth, such as cell wall and bioactive compound synthesis. In addition, the EST-genome alignments containing putative cassette exons and retained introns were manually curated and then used to make inferences about the predominating splice-site recognition mechanism of G. lucidum. Moreover, a number of putative antisense transcripts have been pinpointed, from which we noticed that two cases are likely to reveal hitherto undiscovered biological pathways. To allow users to access the data and the initial analysis of the results of this project, a dedicated web site has been created at

  15. Bioinformatic identification of microRNAs and their target genes from Solanum tuberosum expressed sequence tags

    Institute of Scientific and Technical Information of China (English)


    MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene post-transcriptional expression in plants and animals. Low levels of some miRNAs and time- and tissue-specific expression patterns lead to the difficulty for experimental identification of miRNAs. Here we present a bioinformatic approach for expressed sequence tags (ESTs) prediction of novel miRNAs as well as their targets in Solanum tuberosum. We blasted the databases of S. Tuberosum ESTs to search for potential miRNAs, using previously known miRNA sequences from Arabidopsis, rice and other plant species. By analyzing parameters of plant precursors, including secondary structure, stem length and conservation of miRNAs, and following a variety of filtering criteria, a total of 22 potential miRNAs were detected. Using the newly identified miRNA sequences, we were able to further blast the S. Tuberosum mRNA database and detected 75 potential targets of miRNAs in S. Tuberosum. According to the mRNA annotations provided by the National Center for Biotechnology Information (NCBI) (, most of the miRNA target genes were predicted to encode transcription factors that regulate cell growth and development, signaling, and metabolism.

  16. Sequence evolution and expression regulation of stress-responsive genes in natural populations of wild tomato. (United States)

    Fischer, Iris; Steige, Kim A; Stephan, Wolfgang; Mboup, Mamadou


    The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced) gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives.

  17. Sequence evolution and expression regulation of stress-responsive genes in natural populations of wild tomato.

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    Iris Fischer

    Full Text Available The wild tomato species Solanum chilense and S. peruvianum are a valuable non-model system for studying plant adaptation since they grow in diverse environments facing many abiotic constraints. Here we investigate the sequence evolution of regulatory regions of drought and cold responsive genes and their expression regulation. The coding regions of these genes were previously shown to exhibit signatures of positive selection. Expression profiles and sequence evolution of regulatory regions of members of the Asr (ABA/water stress/ripening induced gene family and the dehydrin gene pLC30-15 were analyzed in wild tomato populations from contrasting environments. For S. chilense, we found that Asr4 and pLC30-15 appear to respond much faster to drought conditions in accessions from very dry environments than accessions from more mesic locations. Sequence analysis suggests that the promoter of Asr2 and the downstream region of pLC30-15 are under positive selection in some local populations of S. chilense. By investigating gene expression differences at the population level we provide further support of our previous conclusions that Asr2, Asr4, and pLC30-15 are promising candidates for functional studies of adaptation. Our analysis also demonstrates the power of the candidate gene approach in evolutionary biology research and highlights the importance of wild Solanum species as a genetic resource for their cultivated relatives.

  18. Gene expression profiling of TIR-domain-containing adaptor molecule (TICAM)in channel catfish Ictalurus punctatus challenged with different pathogens including bacteria and virus%斑点叉尾(鱼回)TICAM在细菌和病毒感染后的基因表达特征

    Institute of Scientific and Technical Information of China (English)

    王启龙; 李敏; 路飏; 黄爱平; 曾令兵; 王文琪; 陈松林; 沙珍霞


    In mammals, Toll-IL-1 receptor (TIR) domain-containing adaptor molecule 1(TICAM-1) is a signaling adaptor for TLR3 and TLR4 that activates the transcription factors IRF-3, NF-kB, and AP-1, leading to the induction of type I interferon and cytokines. TICAM is also identified in some fish species, however, the gene expression profiling of TICAM is largely unknown in teleosts. Because bacteria such as Aeromonas hydrophila , Streptococcus spp. And Edwardsiella tarda and viruses such as channel catfish virus cause a multisystemic disease responsible for severe losses in channel catfish aquaculture in China. In this study, gene expression profiling of TICAM in different immune tissues(iver, headkidney, spleen,and intestine) after infection with these pathogens assayed by quantitative RT-PCR was described. After infection with A. Hydrophila, TICAM was up-regulated approximately 2. 3-fold at 24 h in liver and 1. 9-fold at 12 h in spleen, while expression of this gene was down-regulated in headkidney and intestine, with the lowest expression as 0. 15-fold at 48 h in headkidney, 0. 53-fold at 24 h in intestine, respectively. TICAM was up-regulated drastically in liver, spleen, headkidney and intestine after infection with Streptococcus spp. It reached the highest level with 23-fold in liver at 7 d post infection, and it increased about 10 times in headkidney and spleen after infection. The expression of TICAM increased in all tested tissues after infection with E. Tarda, especially it was up-regulated to the highest (23. 1-fold) at 7d in spleen. After infection with channel catfish virus, the gene TICAM expression was up-regulated in liver, headkidney and intestine moderately, with the highest expression of 3. 7-fold in liver at 72 h, 2. 8-fold in headkidney at 7 d, 1. 5-fold at 24 h in intestine. However, it was down-regulated in spleen,and its lowest expression was 0. 13-fold at 24 h. In conclusion, the results of this study suggest that the TICAM gene may play crucial

  19. Tumor transcriptome sequencing reveals allelic expression imbalances associated with copy number alterations.

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    Brian B Tuch

    Full Text Available Due to growing throughput and shrinking cost, massively parallel sequencing is rapidly becoming an attractive alternative to microarrays for the genome-wide study of gene expression and copy number alterations in primary tumors. The sequencing of transcripts (RNA-Seq should offer several advantages over microarray-based methods, including the ability to detect somatic mutations and accurately measure allele-specific expression. To investigate these advantages we have applied a novel, strand-specific RNA-Seq method to tumors and matched normal tissue from three patients with oral squamous cell carcinomas. Additionally, to better understand the genomic determinants of the gene expression changes observed, we have sequenced the tumor and normal genomes of one of these patients. We demonstrate here that our RNA-Seq method accurately measures allelic imbalance and that measurement on the genome-wide scale yields novel insights into cancer etiology. As expected, the set of genes differentially expressed in the tumors is enriched for cell adhesion and differentiation functions, but, unexpectedly, the set of allelically imbalanced genes is also enriched for these same cancer-related functions. By comparing the transcriptomic perturbations observed in one patient to his underlying normal and tumor genomes, we find that allelic imbalance in the tumor is associated with copy number mutations and that copy number mutations are, in turn, strongly associated with changes in transcript abundance. These results support a model in which allele-specific deletions and duplications drive allele-specific changes in gene expression in the developing tumor.

  20. Identification and Validation of Single Nucleotide Polymorphisms in Poplar Using Publicly Expressed Sequence Tags

    Institute of Scientific and Technical Information of China (English)

    Bo ZHANG; Yan ZHOU; Liang ZHANG; Qiang ZHUGE; Ming-Xiu WANG; Min-Ren HUANG


    By using assembled expressed sequence tags (ESTs) from 14 different cDNA libraries that contain 84 132 sequences reads, 556 Populus candidate single nucleotide polymorphisms (SNPs) were identified. Because traces were not available from dbEST (,stringent filters were used to identify reliable candidate SNPs. Sequences analysis indicated that the main types of substitutions among candidate SNPs were A/G and T/C transitions, which accounted for 22.0% and 30.8%, respectively. One hundred and ten candidate SNPs were tested. As a result, 38 candidate SNPs were confirmed by directed sequencing of PCR products amplified from six different individuals. Thirteen new SNPs in intron regions were found and multiple SNPs were found to be located in both intron and exon regions of four contigs. Heterozygosis was found in all 47 candidate sites and five SNP sites were heterozygous in all six samples. This is the first report of SNP identification in a tree species which reveals that assembled ESTs from multiple libraries of the public database may provide a rich source of comparative sequences for an SNP search in the poplar genome.

  1. Rice bicoid-related cDNA sequence and its expression during early embryogenesis

    Institute of Scientific and Technical Information of China (English)


    Bicoid is one of the important Drosophila maternal genes involved in the control of embryo polarity and larvae segmentation.To clone and characterize the rice bicoid-related genes,one cDNA clone,Rb24 (EMBL accession number: AJ2771380),was isolated by screening of rice unmature seed cDNA library.Sequence analysis indicates that Rb24 contains a putative amino acid sequence,which is homologous to unique 8 amino acids sequence within Drosophila bicoid homeodomain (50% identity,75% similarity) and involves a lys-9 in putative helix 3.Northern blot analysis of rice RNA has shown that this sequence is expressed in a tissue-specific manner.The transcript was detected strongly in young panicles,but less in young leaves and roots.This results are further confirmed with paraffin section in situ hybridization.The signal is intensive in rice globular embryo and located at the apical tip of the embryo,then,along with the development of embryo,the signal is getting reduced and transfers into both sides of embryo.The existence of bicoid-related sequence in rice embryo and the similarity of polar distribution of bicoid and Rb24 mRNA in early embryo development may implicates a conserved maternal regulation mechanism of body axis presents in Drosophila and in rice.

  2. Classification, expression pattern and comparative analysis of sugarcane expressed sequences tags (ESTs encoding glycine-rich proteins (GRPs

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    Fusaro Adriana


    Full Text Available Since the isolation of the first glycine-rich proteins (GRPs in plants a wealth of new GRPs have been identified. The highly specific but diverse expression pattern of grp genes, taken together with the distinct sub-cellular localization of some GRP groups, clearly indicate that these proteins are involved in several independent physiological processes. Notwithstanding the absence of a clear definition of the role of GRPs in plant cells, studies conducted with these proteins have provided new and interesting insights into the molecular biology and cell biology of plants. Complexly regulated promoters and distinct mechanisms for the regulation of gene expression have been demonstrated and new protein targeting pathways, as well as the exportation of GRPs from different cell types have been discovered. These data show that GRPs can be useful as markers and/or models to understand distinct aspects of plant biology. In this paper, the structural and functional features of these proteins in sugarcane (Saccharum officinarum L. are summarized. Since this is the first description of GRPs in sugarcane, special emphasis has been given to the expression pattern of these GRP genes by studying their abundance and prevalence in the different cDNA-libraries of the Sugarcane Expressed Sequence Tag (SUCEST project . The comparison of sugarcane GRPs with GRPs from other species is also discussed.

  3. A sequence-based approach to identify reference genes for gene expression analysis

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    Chari Raj


    Full Text Available Abstract Background An important consideration when analyzing both microarray and quantitative PCR expression data is the selection of appropriate genes as endogenous controls or reference genes. This step is especially critical when identifying genes differentially expressed between datasets. Moreover, reference genes suitable in one context (e.g. lung cancer may not be suitable in another (e.g. breast cancer. Currently, the main approach to identify reference genes involves the mining of expression microarray data for highly expressed and relatively constant transcripts across a sample set. A caveat here is the requirement for transcript normalization prior to analysis, and measurements obtained are relative, not absolute. Alternatively, as sequencing-based technologies provide digital quantitative output, absolute quantification ensues, and reference gene identification becomes more accurate. Methods Serial analysis of gene expression (SAGE profiles of non-malignant and malignant lung samples were compared using a permutation test to identify the most stably expressed genes across all samples. Subsequently, the specificity of the reference genes was evaluated across multiple tissue types, their constancy of expression was assessed using quantitative RT-PCR (qPCR, and their impact on differential expression analysis of microarray data was evaluated. Results We show that (i conventional references genes such as ACTB and GAPDH are highly variable between cancerous and non-cancerous samples, (ii reference genes identified for lung cancer do not perform well for other cancer types (breast and brain, (iii reference genes identified through SAGE show low variability using qPCR in a different cohort of samples, and (iv normalization of a lung cancer gene expression microarray dataset with or without our reference genes, yields different results for differential gene expression and subsequent analyses. Specifically, key established pathways in lung

  4. Conservation and divergence of plant LHP1 protein sequences and expression patterns in angiosperms and gymnosperms. (United States)

    Guan, Hexin; Zheng, Zhengui; Grey, Paris H; Li, Yuhua; Oppenheimer, David G


    Floral transition is a critical and strictly regulated developmental process in plants. Mutations in Arabidopsis LIKE HETEROCHROMATIN PROTEIN 1 (AtLHP1)/TERMINAL FLOWER 2 (TFL2) result in early and terminal flowers. Little is known about the gene expression, function and evolution of plant LHP1 homologs, except for Arabidopsis LHP1. In this study, the conservation and divergence of plant LHP1 protein sequences was analyzed by sequence alignments and phylogeny. LHP1 expression patterns were compared among taxa that occupy pivotal phylogenetic positions. Several relatively conserved new motifs/regions were identified among LHP1 homologs. Phylogeny of plant LHP1 proteins agreed with established angiosperm relationships. In situ hybridization unveiled conserved expression of plant LHP1 in the axillary bud/tiller, vascular bundles, developing stamens, and carpels. Unlike AtLHP1, cucumber CsLHP1-2, sugarcane SoLHP1 and maize ZmLHP1, rice OsLHP1 is not expressed in the shoot apical meristem (SAM) and the OsLHP1 transcript level is consistently low in shoots. "Unequal crossover" might have contributed to the divergence in the N-terminal and hinge region lengths of LHP1 homologs. We propose an "insertion-deletion" model for soybean (Glycine max L.) GmLHP1s evolution. Plant LHP1 homologs are more conserved than previously expected, and may favor vegetative meristem identity and primordia formation. OsLHP1 may not function in rice SAM during floral induction.

  5. Synthesize emotion expression sequences%多模情感序列的生成

    Institute of Scientific and Technical Information of China (English)

    周伟; 张明敏


    A novel method is proposed to generate multimodal expressions by given emotion states.In order to generate more complex animation than Niewiadomski' s algorithm, this method constructs a practical datasets of body and hands movements for every emotional dimension.Based on the datasets and special restrains of these clips,the system searches over for several clips related to current emotion state and then blends them into multimodal expression sequences.Compared with the previous method,the expression sequences are under a high-level control thus the virtual human can act much more realistic, lifelike and fluidly emotion expressions.%提出一种多模情感序列合成的方法.针对Niewiadomski的方法中生成动作较单一的问题,该方法细化了情感表达通道,为更多的人体部位构建所属的动作片段库,并根据运动段之间的时序与空间限制合成能够表达相关情感的动作序列.实验表明,该方法能够构建真实丰富的情感输出.

  6. Expressed sequence tag analysis of functional genes associated with adventitious rooting in Liriodendron hybrids. (United States)

    Zhong, Y D; Sun, X Y; Liu, E Y; Li, Y Q; Gao, Z; Yu, F X


    Liriodendron hybrids (Liriodendron chinense x L. tulipifera) are important landscaping and afforestation hardwood trees. To date, little genomic research on adventitious rooting has been reported in these hybrids, as well as in the genus Liriodendron. In the present study, we used adventitious roots to construct the first cDNA library for Liriodendron hybrids. A total of 5176 expressed sequence tags (ESTs) were generated and clustered into 2921 unigenes. Among these unigenes, 2547 had significant homology to the non-redundant protein database representing a wide variety of putative functions. Homologs of these genes regulated many aspects of adventitious rooting, including those for auxin signal transduction and root hair development. Results of quantitative real-time polymerase chain reaction showed that AUX1, IRE, and FB1 were highly expressed in adventitious roots and the expression of AUX1, ARF1, NAC1, RHD1, and IRE increased during the development of adventitious roots. Additionally, 181 simple sequence repeats were identified from 166 ESTs and more than 91.16% of these were dinucleotide and trinucleotide repeats. To the best of our knowledge, the present study reports the identification of the genes associated with adventitious rooting in the genus Liriodendron for the first time and provides a valuable resource for future genomic studies. Expression analysis of selected genes could allow us to identify regulatory genes that may be essential for adventitious rooting.

  7. Sequence analysis and prokaryotic expression of Giardia lamblia α-18 giardin gene. (United States)

    Wu, Sheng; Yu, Xingang; Abdullahi, Auwalu Yusuf; Hu, Wei; Pan, Weida; Shi, Xianli; Tan, Liping; Song, Meiran; Li, Guoqing


    To study the genetic variation and prokaryotic expression of α18 giardin gene of Giardia lamblia zoonotic assemblage A and host-specific assemblage F, the α18 genes were amplified from G. lamblia assemblages A and F by PCR and sequenced. The PCR product was cloned into the prokaryotic expression vector pET-28a(+) and the positive recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) strain for the expression. The expressed α18 giardin fusion protein was validated by SDS-PAGE and Western blot analysis, and purified by Ni-Agarose resin. The putative sequence of α18 giardin amino acid was analyzed by bioinformatics software. Results showed that the α18 giardin gene was 861 bp in length, encoding 286 amino acids; it was 100% homologous between human-derived and dog-derived G. lamblia assemblage A, but it was 86.8% homologous with G. lamblia assemblage F (cat-derived). Giardin α18 was about 36 kDa in molecular weight, with good reactivity. Prediction based on in silico analyses: it had hydrophobicity, without signal peptide and transmembrane domain, and contained 11 alpha regions, 13 beta sheets, 1 beta turn and 7 random coils in secondary structure. The above information would lay the foundation for research about the subcellular localization and biological function of α18 giardin in G. lamblia.

  8. Optimizing de novo transcriptome assembly and extending genomic resources for striped catfish (Pangasianodon hypophthalmus). (United States)

    Thanh, Nguyen Minh; Jung, Hyungtaek; Lyons, Russell E; Njaci, Isaac; Yoon, Byoung-Ha; Chand, Vincent; Tuan, Nguyen Viet; Thu, Vo Thi Minh; Mather, Peter


    Striped catfish (Pangasianodon hypophthalmus) is a commercially important freshwater fish used in inland aquaculture in the Mekong Delta, Vietnam. The culture industry is facing a significant challenge however from saltwater intrusion into many low topographical coastal provinces across the Mekong Delta as a result of predicted climate change impacts. Developing genomic resources for this species can facilitate the production of improved culture lines that can withstand raised salinity conditions, and so we have applied high-throughput Ion Torrent sequencing of transcriptome libraries from six target osmoregulatory organs from striped catfish as a genomic resource for use in future selection strategies. We obtained 12,177,770 reads after trimming and processing with an average length of 97bp. De novo assemblies were generated using CLC Genomic Workbench, Trinity and Velvet/Oases with the best overall contig performance resulting from the CLC assembly. De novo assembly using CLC yielded 66,451 contigs with an average length of 478bp and N50 length of 506bp. A total of 37,969 contigs (57%) possessed significant similarity with proteins in the non-redundant database. Comparative analyses revealed that a significant number of contigs matched sequences reported in other teleost fishes, ranging in similarity from 45.2% with Atlantic cod to 52% with zebrafish. In addition, 28,879 simple sequence repeats (SSRs) and 55,721 single nucleotide polymorphisms (SNPs) were detected in the striped catfish transcriptome. The sequence collection generated in the current study represents the most comprehensive genomic resource for P. hypophthalmus available to date. Our results illustrate the utility of next-generation sequencing as an efficient tool for constructing a large genomic database for marker development in non-model species.

  9. MicroRNA expression signatures of bladder cancer revealed by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Yonghua Han

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are a class of small noncoding RNAs that regulate gene expression. They are aberrantly expressed in many types of cancers. In this study, we determined the genome-wide miRNA profiles in bladder urothelial carcinoma by deep sequencing. METHODOLOGY/PRINCIPAL FINDINGS: We detected 656 differentially expressed known human miRNAs and miRNA antisense sequences (miRNA*s in nine bladder urothelial carcinoma patients by deep sequencing. Many miRNAs and miRNA*s were significantly upregulated or downregulated in bladder urothelial carcinoma compared to matched histologically normal urothelium. hsa-miR-96 was the most significantly upregulated miRNA and hsa-miR-490-5p was the most significantly downregulated one. Upregulated miRNAs were more common than downregulated ones. The hsa-miR-183, hsa-miR-200b ∼ 429, hsa-miR-200c ∼ 141 and hsa-miR-17 ∼ 92 clusters were significantly upregulated. The hsa-miR-143 ∼ 145 cluster was significantly downregulated. hsa-miR-182, hsa-miR-183, hsa-miR-200a, hsa-miR-143 and hsa-miR-195 were evaluated by Real-Time qPCR in a total of fifty-one bladder urothelial carcinoma patients. They were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium (p < 0.001 for each miRNA. CONCLUSIONS/SIGNIFICANCE: To date, this is the first study to determine genome-wide miRNA expression patterns in human bladder urothelial carcinoma by deep sequencing. We found that a collection of miRNAs were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium, suggesting that they might play roles as oncogenes or tumor suppressors in the development and/or progression of this cancer. Our data provide novel insights into cancer biology.

  10. Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach.

    Directory of Open Access Journals (Sweden)

    Ândrea Ribeiro-dos-Santos

    Full Text Available BACKGROUND: While microRNAs (miRNAs play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia. METHODOLOGY/PRINCIPAL FINDINGS: A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05. Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451 and could be considered part of the expression pattern of the healthy gastric tissue. CONCLUSIONS/SIGNIFICANCE: This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.

  11. Effect of Lactobacillus plantarum C014 on Innate Immune Response and Disease Resistance against Aeromonas hydrophila in Hybrid Catfish

    Directory of Open Access Journals (Sweden)

    Sureerat Butprom


    Full Text Available A bacterial strain isolated from intestines of hybrid catfish (Clarias gariepinus Male × Clarias macrocephalus Female exhibited an in vitro inhibitory effect on a fish pathogen, Aeromonas hydrophila TISTR 1321. By using the 16S rDNA sequence analysis, it was identified as Lactobacillus plantarum C014. To examine whether L. plantarum C014 had potential for use as an immunostimulant and biocontrol agent in hybrid catfish, the fish diet supplemented with L. plantarum C014 (107 CFU/g diet was prepared and used for the in vivo investigation of its effect on innate immune response and disease resistance of hybrid catfish. Two innate immune response parameters, phagocytic activity of blood leukocytes and plasma lysozyme activity, were significantly enhanced in the treated fish after 45 days of feeding. Feeding the fish with the L. plantarum C014 supplemented diet for 45 days before challenging them with A. hydrophila at the dose of LD50 could reduce the mortality rate of the fish from 50% (in control group to 0% (in treated group. Based on its origin and beneficial effect on innate immune response and disease resistance, L. plantarum C014 may be a potential candidate for use as a natural and safe immunostimulant and biocontrol agent in hybrid catfish.

  12. Production Practices of Commercial Catfish Producers in Northeast Louisiana. (United States)

    Letlow, Richard M.; Verma, Satish

    A study determined the yields obtained and the production practices followed by Louisiana commercial catfish farmers in Catahoula, Concordia, and Franklin Parishes. This information was used by the Louisiana Cooperative Extension Service (LCES) faculty to develop a catfish education program. The study: (1) determined use of selected recommended…

  13. Cannibalism in single-batch hybrid catfish production ponds (United States)

    Hybrid catfish are more efficiently harvested by seining than are Channel Catfish. Due to that, and their faster growth, hybrids are typically produced in “single-batch” production systems, either in intensively-aerated commercial ponds or in split-pond systems. In either production system, hybrids...

  14. Serpins in rice: protein sequence analysis, phylogeny and gene expression during development

    Directory of Open Access Journals (Sweden)

    Francis Sheila E


    Full Text Available Abstract Background Most members of the serpin family of proteins are potent, irreversible inhibitors of specific serine or cysteine proteinases. Inhibitory serpins are distinguished from members of other families of proteinase inhibitors by their metastable structure and unique suicide-substrate mechanism. Animal serpins exert control over a remarkable diversity of physiological processes including blood coagulation, fibrinolysis, innate immunity and aspects of development. Relatively little is known about the complement of serpin genes in plant genomes and the biological functions of plant serpins. Results A structurally refined amino-acid sequence alignment of the 14 full-length serpins encoded in the genome of the japonica rice Oryza sativa cv. Nipponbare (a monocot showed a diversity of reactive-centre sequences (which largely determine inhibitory specificity and a low degree of identity with those of serpins in Arabidopsis (a eudicot. A new convenient and functionally informative nomenclature for plant serpins in which the reactive-centre sequence is incorporated into the serpin name was developed and applied to the rice serpins. A phylogenetic analysis of the rice serpins provided evidence for two main clades and a number of relatively recent gene duplications. Transcriptional analysis showed vastly different levels of basal expression among eight selected rice serpin genes in callus tissue, during seedling development, among vegetative tissues of mature plants and throughout seed development. The gene OsSRP-LRS (Os03g41419, encoding a putative orthologue of Arabidopsis AtSerpin1 (At1g47710, was expressed ubiquitously and at high levels. The second most highly expressed serpin gene was OsSRP-PLP (Os11g11500, encoding a non-inhibitory serpin with a surprisingly well-conserved reactive-centre loop (RCL sequence among putative orthologues in other grass species. Conclusions The diversity of reactive-centre sequences among the putatively

  15. Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brahmachari Samir K


    Full Text Available Abstract Background Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes. These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes. Results A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed. The sequence was inserted within a nucleosome positioned upstream of the β-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10Upstream Activating Sequences (UASg. Upon derepression with galactose, β-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences. This reduction in expression is correlated with reduced transcription. Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells. Conclusion We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription. Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells.

  16. A new genus and species of proteocephalidean (Cestoda) from Clarias catfishes (Siluriformes: Clariidae) in Africa. (United States)

    de Chambrier, Alain; Scholz, Tomás; Beletew, Moges; Mariaux, Jean


    A new proteocephalidean cestode is described from 2 catfishes, Clarias gariepinus (type host) and C. cf. anguillaris (Siluriformes: Clariidae), from Ethiopia (type locality), Sudan, Tanzania, and Zimbabwe, and a new genus, Barsonella, is proposed to accommodate it. The genus belongs to the Proteocephalinae because its genital organs (testes, ovary, vitellarium, and uterus) are situated in the medulla. Barsonella lafoni, the type and only species of the new genus, is characterized mainly by the possession of an additional opening of each sucker; circular musculature on the anterior margin of suckers, serving as a sphincter; a small thin-walled glandular apical organ; absence of well-developed osmoregulatory canals in mature, pregravid, and gravid proglottids; and a large strobila, up to 173 mm long and 3.2 mm wide. Species of Marsypocephalus Wedl, 1861 (Marsypocephalinae), other large-sized proteocephalidean tapeworms occurring sympatrically in African catfishes (Clarias and Heterobranchus) and also possessing a sphincter-like, circular musculature on the anterior part of suckers, differ from B. lafoni in the absence of an additional sucker opening and glandular apical organ, the cortical position of the testes, well-developed osmoregulatory canals throughout the strobila, and a large cirrus sac. Proteocephalus glanduligerus (Janicki, 1928), another cestode parasitic in Clarias spp. in Africa, is much smaller than B. lafoni (maximum length 15 mm), has suckers without additional opening and circular musculature on the suckers, a large-sized glandular organ, much larger than suckers, and well-developed osmoregulatory canals. Comparison of partial sequences of the 28S rRNA gene for 7 samples of B. lafoni from 2 different hosts and 4 localities in Ethiopia, Sudan, and Tanzania has shown a very low genetic variability. In a limited phylogenetic analysis, B. lafoni formed a clade with Corallobothrium solidum Fritsch, 1886 (Proteocephalidae: Corallobothriinae), an African

  17. Characterization of soybean genomic features by analysis of its expressed sequence tags

    DEFF Research Database (Denmark)

    Tian, Ai-Guo; Wang, Jun; Cui, Peng


    We analyzed 314,254 soybean expressed sequence tags (ESTs), including 29,540 from our laboratory and 284,714 from GenBank. These ESTs were assembled into 56,147 unigenes. About 76.92% of the unigenes were homologous to genes from Arabidopsis thaliana ( Arabidopsis). The putative products of these......We analyzed 314,254 soybean expressed sequence tags (ESTs), including 29,540 from our laboratory and 284,714 from GenBank. These ESTs were assembled into 56,147 unigenes. About 76.92% of the unigenes were homologous to genes from Arabidopsis thaliana ( Arabidopsis). The putative products...... to be fast-evolving. Soybean unigenes with no match to genes within the Arabidopsis genome were identified as soybean-specific genes. These genes were mainly involved in nodule development and the synthesis of seed storage proteins. In addition, we also identified 61 genes regulated by salicylic acid, 1......,322 transcription factor genes and 326 disease resistance-like genes from soybean unigenes. SSR analysis showed that the soybean genome was more complex than the Arabidopsis and the Medicago truncatula genomes. GC content in soybean unigene sequences is similar to that in Arabidopsis and M. truncatula. Furthermore...

  18. Large scale in silico identification of MYB family genes from wheat expressed sequence tags. (United States)

    Cai, Hongsheng; Tian, Shan; Dong, Hansong


    The MYB proteins constitute one of the largest transcription factor families in plants. Much research has been performed to determine their structures, functions, and evolution, especially in the model plants, Arabidopsis, and rice. However, this transcription factor family has been much less studied in wheat (Triticum aestivum), for which no genome sequence is yet available. Despite this, expressed sequence tags are an important resource that permits opportunities for large scale gene identification. In this study, a total of 218 sequences from wheat were identified and confirmed to be putative MYB proteins, including 1RMYB, R2R3-type MYB, 3RMYB, and 4RMYB types. A total of 36 R2R3-type MYB genes with complete open reading frames were obtained. The putative orthologs were assigned in rice and Arabidopsis based on the phylogenetic tree. Tissue-specific expression pattern analyses confirmed the predicted orthologs, and this meant that gene information could be inferred from the Arabidopsis genes. Moreover, the motifs flanking the MYB domain were analyzed using the MEME web server. The distribution of motifs among wheat MYB proteins was investigated and this facilitated subfamily classification.

  19. Computational identification of citrus microRNAs and target analysis in citrus expressed sequence tags. (United States)

    Song, C; Jia, Q; Fang, J; Li, F; Wang, C; Zhang, Z


    MicroRNAs (miRNAs) are a new family of small RNA molecules found in plants and animals. We developed a comprehensive strategy for identifying new miRNA homologues by mining the repository of available citrus expressed sequence tags (ESTs). By adopting a range of filtering criteria, we identified a total of 38 potential miRNAs--nine, five, nine and 15 miRNAs in Citrus trifoliata (ctr-miRNAs), C. clementina (ccl-miRNAs), C. reticulata (crt-miRNAs) and C. sinensis (csi-miRNAs), respectively--from more than 430,000 EST sequences in citrus. Using the potential miRNA sequences, we conducted a further BLAST search of the mRNA database and found six potential target genes in these citrus species. Eight miRNAs were selected in order to verify their existence in citrus using Northern blotting, and the functions of several miRNAs in miRNA-mediated gene regulation are experimentally suggested. It appears that all these miRNAs regulate expression of their target genes by cleavage, which is the most common situation in gene regulation mediated by plant miRNAs. Our achievement in identifying new miRNAs in citrus provides a powerful incentive for further studies on the important roles of these miRNAs.

  20. Expressed sequence tags from the oomycete fish pathogen Saprolegnia parasitica reveal putative virulence factors

    Directory of Open Access Journals (Sweden)

    van West Pieter


    Full Text Available Abstract Background The oomycete Saprolegnia parasitica is one of the most economically important fish pathogens. There is a dramatic recrudescence of Saprolegnia infections in aquaculture since the use of the toxic organic dye malachite green was banned in 2002. Little is known about the molecular mechanisms underlying pathogenicity in S. parasitica and other animal pathogenic oomycetes. In this study we used a genomics approach to gain a first insight into the transcriptome of S. parasitica. Results We generated 1510 expressed sequence tags (ESTs from a mycelial cDNA library of S. parasitica. A total of 1279 consensus sequences corresponding to 525944 base pairs were assembled. About half of the unigenes showed similarities to known protein sequences or motifs. The S. parasitica sequences tended to be relatively divergent from Phytophthora sequences. Based on the sequence alignments of 18 conserved proteins, the average amino acid identity between S. parasitica and three Phytophthora species was 77% compared to 93% within Phytophthora. Several S. parasitica cDNAs, such as those with similarity to fungal type I cellulose binding domain proteins, PAN/Apple module proteins, glycosyl hydrolases, proteases, as well as serine and cysteine protease inhibitors, were predicted to encode secreted proteins that could function in virulence. Some of these cDNAs were more similar to fungal proteins than to other eukaryotic proteins confirming that oomycetes and fungi share some virulence components despite their evolutionary distance Conclusion We provide a first glimpse into the gene content of S. parasitica, a reemerging oomycete fish pathogen. These resources will greatly accelerate research on this important pathogen. The data is available online through the Oomycete Genomics Database 1.

  1. Transcriptome sequencing and expression analysis of terpenoid biosynthesis genes in Litsea cubeba.

    Directory of Open Access Journals (Sweden)

    Xiao-Jiao Han

    Full Text Available BACKGROUND: Aromatic essential oils extracted from fresh fruits of Litsea cubeba (Lour. Pers., have diverse medical and economic values. The dominant components in these essential oils are monoterpenes and sesquiterpenes. Understanding the molecular mechanisms of terpenoid biosynthesis is essential for improving the yield and quality of terpenes. However, the 40 available L. cubeba nucleotide sequences in the public databases are insufficient for studying the molecular mechanisms. Thus, high-throughput transcriptome sequencing of L. cubeba is necessary to generate large quantities of transcript sequences for the purpose of gene discovery, especially terpenoid biosynthesis related genes. RESULTS: Using Illumina paired-end sequencing, approximately 23.5 million high-quality reads were generated. De novo assembly yielded 68,648 unigenes with an average length of 834 bp. A total of 38,439 (56% unigenes were annotated for their functions, and 35,732 and 25,806 unigenes could be aligned to the GO and COG database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG, 16,130 unigenes were assigned to 297 KEGG pathways, and 61 unigenes, which contained the mevalonate and 2-C-methyl-D-erythritol 4-phosphate pathways, could be related to terpenoid backbone biosynthesis. Of the 12,963 unigenes, 285 were annotated to the terpenoid pathways using the PlantCyc database. Additionally, 14 terpene synthase genes were identified from the transcriptome. The expression patterns of the 16 genes related to terpenoid biosynthesis were analyzed by RT-qPCR to explore their putative functions. CONCLUSION: RNA sequencing was effective in identifying a large quantity of sequence information. To our knowledge, this study is the first exploration of the L. cubeba transcriptome, and the substantial amount of transcripts obtained will accelerate the understanding of the molecular mechanisms of essential oils biosynthesis. The

  2. Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice. (United States)

    Püschel, A W; Gruss, P; Westerfield, M


    Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice. We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog. pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain including the eye, the olfactory bulb and the pituitary gland express pax-6. Cells at the midbrain-hindbrain junction express eng genes and are separated from the neighboring pax-6 regions by several cells that express neither gene, indicating a complex subdivision of this region. pax-6 expression appears during processes when cell-to-cell signalling is thought to be important, for example during induction of the eye and regionalization of the spinal cord and brain, suggesting that it may be one component mediating the response to inductive interactions.

  3. Development and Characterisation of Irap Markers From Expressed Retrotransposon-like sequences in Pinus sylvestris L.

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    Voronova Angelika


    Full Text Available Conifer genomes are large and stably diploid, in contrast to angiosperms, which are more variable both in genome size and ploidy. Conifer genomes are characterised by multiple gene families and pseudogenes, contain large inter-gene regions and a considerable proportion of repetitive sequences. All members of plant retrotransposon orders have been identified in gymnosperm genomes, however active elements have not been described. Investigation of transposable elements in Scots pine (Pinus sylvestris L. could offer insights into transposon-mediated reorganisation under stress conditions in complex and ancient plant genomes. Nine Pinus sylvestris specific markers were developed to hypothetical long terminal repeats (LTRs from differentially expressed retrotransposon-like fragments after heat stress and insect damage. Genetic diversity of 150 trees from a naturally regenerated pine stand was investigated using the IRAP method. The developed markers revealed high levels of genetic diversity and were able to distinguish subpopulations growing in long-term differential environmental conditions. Somaclonal variation was also investigated using these markers and polymorphic fragments were identified between ramets of Scots pine clones growing in two different plantations, possibly indicating evidence of recent transposition events. Sequencing of the polymorphic fragments identified two groups of sequences containing LTR sequences of an unknown retrotransposon with homology to the LTRs of the Copia-17-PAb-I element.

  4. The Effect of Addition Vitamin E on Catfish Oil Stability

    Directory of Open Access Journals (Sweden)

    Clara Maria Kusharto


    Full Text Available Unsaturated fatty acids contained oils which sensitive to oxidation caused by heat, light andoxygen. The oxidized oil known harmful to the body. One of the effort to prevent the oxidationprocess is by adding antioxidants stability of oil catfish and shelf life of the oil. The experimentalstudy was applied by adding vitamin E to the oil as much as 0.67 mg / g PUFA compared with thecontrol oil. Oil was stored with the Schaal Oven Test method, which are stored at a temperatureof 600C. The study showed that, based on the parameters of oxidation (free fatty acids, peroxidevalue, anisidin numbers, and the number of total oxidation, catfish oil were added vitamin Emore stabilized than the catfish oil without added vitamin E. The catfish fish oil in the form of softgell capsules added vitamin E could maintain stability in appropriate with IFOS up to 23 months.Keywords: Catfish oil, vitamin E, stability, Schaal Oven Test

  5. Identification of Hyaloperonospora arabidopsidis transcript sequences expressed during infection reveals isolate-specific effectors.

    Directory of Open Access Journals (Sweden)

    Adriana Cabral

    Full Text Available Biotrophic plant pathogens secrete effector proteins that are important for infection of the host. The aim of this study was to identify effectors of the downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa that are expressed during infection of its natural host Arabidopsis thaliana. Infection-related transcripts were identified from Expressed Sequence Tags (ESTs derived from leaves of the susceptible Arabidopsis Ws eds1-1 mutant inoculated with the highly virulent Hpa isolate Waco9. Assembly of 6364 ESTs yielded 3729 unigenes, of which 2164 were Hpa-derived. From the translated Hpa unigenes, 198 predicted secreted proteins were identified. Of these, 75 were found to be Hpa-specific and six isolate Waco9-specific. Among 42 putative effectors identified there were three Elicitin-like proteins, 16 Cysteine-rich proteins and 18 host-translocated RXLR effectors. Sequencing of alleles in different Hpa isolates revealed that five RXLR genes show signatures of diversifying selection. Thus, EST analysis of Hpa-infected Arabidopsis is proving to be a powerful method for identifying pathogen effector candidates expressed during infection. Delivery of the Waco9-specific protein RXLR29 in planta revealed that this effector can suppress PAMP-triggered immunity and enhance disease susceptibility. We propose that differences in host colonization can be conditioned by isolate-specific effectors.

  6. PPARG: Gene Expression Regulation and Next-Generation Sequencing for Unsolved Issues

    Directory of Open Access Journals (Sweden)

    Valerio Costa


    Full Text Available Peroxisome proliferator-activated receptor gamma (PPARγ is one of the most extensively studied ligand-inducible transcription factors (TFs, able to modulate its transcriptional activity through conformational changes. It is of particular interest because of its pleiotropic functions: it plays a crucial role in the expression of key genes involved in adipogenesis, lipid and glucid metabolism, atherosclerosis, inflammation, and cancer. Its protein isoforms, the wide number of PPARγ target genes, ligands, and coregulators contribute to determine the complexity of its function. In addition, the presence of genetic variants is likely to affect expression levels of target genes although the impact of PPARG gene variations on the expression of target genes is not fully understood. The introduction of massively parallel sequencing platforms—in the Next Generation Sequencing (NGS era—has revolutionized the way of investigating the genetic causes of inherited diseases. In this context, DNA-Seq for identifying—within both coding and regulatory regions of PPARG gene—novel nucleotide variations and haplotypes associated to human diseases, ChIP-Seq for defining a PPARγ binding map, and RNA-Seq for unraveling the wide and intricate gene pathways regulated by PPARG, represent incredible steps toward the understanding of PPARγ in health and disease.

  7. Analysis of expressed sequence tags of a marine red alga,Gracilaria lemaneiformis

    Institute of Scientific and Technical Information of China (English)


    The expressed sequence tags (EST) has been proved to be a useful tool for discovering and identifying functional genes, especially in some species whose genetic information is unavailable. A total of 180 ESTs have been generated from a cDNA library of gametophytic Gracilaria lemaneiformis in this study. These clones are clustered into 151 groups, among which 8 groups are highly homologous to chloroplast genes and are abundant in the library. After searching for matches in the EST database of red alga, 22 groups are found to match with the registered ESTs of Rhodophyta and 6 with Gracilaria. Searching in the protein database reveal that 73 non-redundant clones have significant similarity to some known sequences, the majority of which are involved in photosynthesis, DNA transcription or translation, and 6, 4 and 3 clones are associated with growth or development, signal transduction and stress or defense response, respectively.

  8. Patterns of homoeologous gene expression shown by RNA sequencing in hexaploid bread wheat.

    KAUST Repository

    Leach, Lindsey J


    BACKGROUND: Bread wheat (Triticum aestivum) has a large, complex and hexaploid genome consisting of A, B and D homoeologous chromosome sets. Therefore each wheat gene potentially exists as a trio of A, B and D homoeoloci, each of which may contribute differentially to wheat phenotypes. We describe a novel approach combining wheat cytogenetic resources (chromosome substitution \\'nullisomic-tetrasomic\\' lines) with next generation deep sequencing of gene transcripts (RNA-Seq), to directly and accurately identify homoeologue-specific single nucleotide variants and quantify the relative contribution of individual homoeoloci to gene expression. RESULTS: We discover, based on a sample comprising ~5-10% of the total wheat gene content, that at least 45% of wheat genes are expressed from all three distinct homoeoloci. Most of these genes show strikingly biased expression patterns in which expression is dominated by a single homoeolocus. The remaining ~55% of wheat genes are expressed from either one or two homoeoloci only, through a combination of extensive transcriptional silencing and homoeolocus loss. CONCLUSIONS: We conclude that wheat is tending towards functional diploidy, through a variety of mechanisms causing single homoeoloci to become the predominant source of gene transcripts. This discovery has profound consequences for wheat breeding and our understanding of wheat evolution.

  9. Single-Cell RNA Sequencing Identifies Extracellular Matrix Gene Expression by Pancreatic Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    David T. Ting


    Full Text Available Circulating tumor cells (CTCs are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.


    Institute of Scientific and Technical Information of China (English)

    王克勤; 吕新跃; 吴钢; 薛红; 陈保生


    A rabbit anti-serum to tree shrew apolipoprotein C-I (apo C-l) was used to screen an expression cDNA li-braDy constructed by us from tree shrew (TS) liver tissue. Two apo C-I cDNA clones were obtained. The longerone consists of 380 nucleotides, including 21 bp and 95 bp at the 5' and 3' end of the non-translated region srespectively, and a 2 64-bp fragment in an open reading frame encoding 88 amino acids prepropeptide which con-ta-ins 26 amino acids of signal peptide and a mature protein (62 amino acids). Comparing the amino-acid se-quence deduced from this cDNA with those of the published mammalian apo C-Is reveals that it shared some struc-tural similarity with zat, mouse and dog apo C-l, but it had 5 more amino acids than that of human and baboon.The expression of apo C-I mRNA in 8 different tissues were also assayed with Northern blot. The results demonstrat-ed that liver had the highest expression, intestine had much less expression and no expression in other tissues,which is much different from human and other species. This study has laid down a good foundation for further study-ing on the function and the stucture of tree shrew apo C-I gene.``

  11. First Season Catfish Farming. A Workbook for Beginning Pond and Cage Culture of Channel Catfish. Teacher Edition and Student Edition. (United States)

    Oklahoma State Board of Vocational and Technical Education, Stillwater. Curriculum and Instructional Materials Center.

    This workbook, comprised of both the teacher and student editions, presents guidelines useful for first-year catfish farmers in Oklahoma using pond or cage cultures to raise channel catfish. The teacher edition is a set of unit guidelines only. Contents include a list of suggested readings, important addresses with types of information available…

  12. Oral vaccination of channel catfish against enteric septicemia of catfish (ESC) using a live attenuated Edwardsiella ictaluri isolate (United States)

    Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are t...

  13. Oral vaccination of channel catfish against enteric septicemia of catfish using a live attenuated Edwardsiella ictaluri isolate (United States)

    Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is the most problematic bacterial disease affecting catfish aquaculture in the southeastern United States. Efforts to develop an effective ESC vaccine have had limited industrial success. In commercial settings, ESC vaccines are...

  14. Analysis of expressed sequence tags of the cyclically parthenogenetic rotifer Brachionus plicatilis.

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    Koushirou Suga

    Full Text Available BACKGROUND: Rotifers are among the most common non-arthropod animals and are the most experimentally tractable members of the basal assemblage of metazoan phyla known as Gnathifera. The monogonont rotifer Brachionus plicatilis is a developing model system for ecotoxicology, aquatic ecology, cryptic speciation, and the evolution of sex, and is an important food source for finfish aquaculture. However, basic knowledge of the genome and transcriptome of any rotifer species has been lacking. METHODOLOGY/PRINCIPAL FINDINGS: We generated and partially sequenced a cDNA library from B. plicatilis and constructed a database of over 2300 expressed sequence tags corresponding to more than 450 transcripts. About 20% of the transcripts had no significant similarity to database sequences by BLAST; most of these contained open reading frames of significant length but few had recognized Pfam motifs. Sixteen transcripts accounted for 25% of the ESTs; four of these had no significant similarity to BLAST or Pfam databases. Putative up- and downstream untranslated regions are relatively short and AT rich. In contrast to bdelloid rotifers, there was no evidence of a conserved trans-spliced leader sequence among the transcripts and most genes were single-copy. CONCLUSIONS/SIGNIFICANCE: Despite the small size of this EST project it revealed several important features of the rotifer transcriptome and of individual monogonont genes. Because there is little genomic data for Gnathifera, the transcripts we found with no known function may represent genes that are species-, class-, phylum- or even superphylum-specific; the fact that some are among the most highly expressed indicates their importance. The absence of trans-spliced leader exons in this monogonont species contrasts with their abundance in bdelloid rotifers and indicates that the presence of this phenomenon can vary at the subphylum level. Our EST database provides a relatively large quantity of transcript

  15. Sequencing and identification of expressed Schistosoma mansoni genes by random selection of cDNA clones from a directional library

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    Glória R. Franco


    Full Text Available We have initiated a gene discovery program in Schistosoma mansoni based on the technique of Expressed Sequence Tags (ESTs, i.e. partial sequences of cDNAs obtained from single passes in automatic DNA sequencers. ESTs can be used to identify genese onf the basis of their homology whith sequences from other species deposited in DNA or protein databases. Trasncripts with sequences without matches in teh databases may represent novel parasite-specific genes. This approach has shown to be very efficient and in less than two years a broad range of novel genes has already been ascertained, more than doubling the number of known S. mansoni genes.

  16. Molecular cloning, sequencing and expression in Escherichia coli cells Thermus thermophilus leucyl-tRNA synthetase

    Directory of Open Access Journals (Sweden)

    Kovalenko O. P.


    Full Text Available Aim. Cloning and sequencing of the T. thermophilus leucyl-tRNA synthetase (LeuRSTT followed by the creation of genetically engineered construct for protein expression in E.coli cells and its purification. Methods. Searching for the LeuRSTT gene was performed by Southern blot hybridization with chromosomal DNA, where digoxigenin-labeled PCR fragments of DNA were used as probes. Results. The gene of T. thermophilus HB27 leucyl-tRNA synthetase was cloned and sequenced. The open reading frame encodes a polypeptide chain of 878 amino acid residues in length (molecular mass 101 kDa. Comparison of the amino acid sequence of T. thermophilus LeuRS with that of the enzymes from other organisms showed that LeuRSTT was a part of the group of similar enzymes of prokaryotes, formed by the proteins of protobacteriae, rickettsia and mitochondria of eukaryotes. The resulting phylogenetic tree of LeuRSs reveals dichotomous branching into two lines: prokaryotic/eukaryotic mitochondrial and arhaeal/eukaryotic cytosolic proteins. Differences between prokaryotic and arhaeal branches of the LeuRSs phylogenetic tree are primarily due to the structure of two domains of the enzyme – the editing and the C-terminal. T. thermophilus LeuRS was expressed in E. coli cells by cloning the corresponding gene into pET29b vector. Conclusions. The cloned T. thermophilus leuS gene and expressed recombinant protein will be used for structural and functional studies on LeuRSTT, including X-ray analysis of the enzyme and its mutant forms in complex with different substrates

  17. Generation and analysis of expressed sequence tags from the medicinal plant Salvia miltiorrhiza

    Institute of Scientific and Technical Information of China (English)

    SPENCER; David; F


    Salvia miltiorrhiza a well-known traditional Chinese herb.Its roots have been formulated and used clinically for the treatment of various diseases.However,little genetic information has so far been available and this fact has become a major obstacle for molecular studies.To address this lack of genetic information,an Expressed Sequence Tag (EST) library from whole plantlets of S.miltiorrhiza was generated.From the 12959 cDNA clones that were randomly selected and subjected to single-pass sequencing from their 5′ ends,10288 ESTs (with sizes≥100 bp) were selected and assembled into 1288 contigs,leaving 2937 singletons,for a total of 4225 unigenes.These were analyzed using BLASTX (against protein databases),RPS-BLAST (against a conserved domain database) as well as the web-based KEGG Automatic Annotation Server for metabolic enzyme assignment.Based on the metabolic enzyme assignment,expression patterns of 14 secondary metabolic enzyme genes in different organs and under different treatments were verified using real-time PCR analysis.Additionally,a total of 122 microsatellites were identified from the ESTs,with 89 having sufficient flanking sequences for primer design.This set of ESTs represents a significant proportion of the S.miltiorrhiza transcriptome,and gives preliminary insights into the gene complement of S.miltiorrhiza.They will prove useful for uncovering secondary metabolic pathways,analyzing cDNA-array based gene expression,genetic manipulation to improve yield of desirable secondary products,and molecular marker identification.

  18. G-stack modulated probe intensities on expression arrays - sequence corrections and signal calibration

    Directory of Open Access Journals (Sweden)

    Fasold Mario


    Full Text Available Abstract Background The brightness of the probe spots on expression microarrays intends to measure the abundance of specific mRNA targets. Probes with runs of at least three guanines (G in their sequence show abnormal high intensities which reflect rather probe effects than target concentrations. This G-bias requires correction prior to downstream expression analysis. Results Longer runs of three or more consecutive G along the probe sequence and in particular triple degenerated G at its solution end ((GGG1-effect are associated with exceptionally large probe intensities on GeneChip expression arrays. This intensity bias is related to non-specific hybridization and affects both perfect match and mismatch probes. The (GGG1-effect tends to increase gradually for microarrays of later GeneChip generations. It was found for DNA/RNA as well as for DNA/DNA probe/target-hybridization chemistries. Amplification of sample RNA using T7-primers is associated with strong positive amplitudes of the G-bias whereas alternative amplification protocols using random primers give rise to much smaller and partly even negative amplitudes. We applied positional dependent sensitivity models to analyze the specifics of probe intensities in the context of all possible short sequence motifs of one to four adjacent nucleotides along the 25meric probe sequence. Most of the longer motifs are adequately described using a nearest-neighbor (NN model. In contrast, runs of degenerated guanines require explicit consideration of next nearest neighbors (GGG terms. Preprocessing methods such as vsn, RMA, dChip, MAS5 and gcRMA only insufficiently remove the G-bias from data. Conclusions Positional and motif dependent sensitivity models accounts for sequence effects of oligonucleotide probe intensities. We propose a positional dependent NN+GGG hybrid model to correct the intensity bias associated with probes containing poly-G motifs. It is implemented as a single-chip based calibration

  19. Using Genome-Referenced Expressed Sequence Tag Assembly to Analyze the Origin and Expression Patterns of Gossypium hirsutum Transcripts

    Institute of Scientific and Technical Information of China (English)

    Xiang Jin; Qin Li; Guanghui Xiao; Yu-Xian Zhu


    We assembled a total of 297,239 Gossypium hirsutum (Gh,a tetraploid cotton,AADD) expressed sequence tag (EST) sequences that were available in the National Center for Biotechnology Information database,with reference to the recently published G.raimondii (Gr,a diploid cotton,DD) genome,and obtained 49,125 UniGenes.The average lengths of the UniGenes were increased from 804 and 791 bp in two previous EST assemblies to 1,019 bp in the current analysis.The number of putative cotton UniGenes with lengths of 3 kb or more increased from 25 or 34 to 1,223.As a result,thousands of originally independent G.hirsutum ESTs were aligned to produce large contigs encoding transcripts with very long open reading frames,indicating that the G.raimondii genome sequence provided remarkable advantages to assemble the tetraploid cotton transcriptome.Significant different distribution patterns within several GO terms,including transcription factor activity,were observed between D-and A-derived assemblies.Transcriptome analysis showed that,in a tetraploid cotton cell,29,547 UniGenes were possibly derived from the D subgenome while another 19,578 may come from the A subgenome.Finally,some of the in silico data were confirmed by reverse transcription polymerase chain reaction experiments to show the changes in transcript levels for several gene families known to play key role in cotton fiber development.We believe that our work provides a useful platform for functional and evolutionary genomic studies in cotton.

  20. No evidence for intercohort cannibalism in mixed-size cultures of food-size and fingerling hybrid catfish (channel catfish x blue catfish) grown in ponds in winter or summer. (United States)

    Hybrid catfish (' Channel Catfish Ictalurus punctatus X ' Blue Catfish I. furcatus) are normally harvested by seining single-batch pond production systems in fall or winter. Ponds are typically restocked without draining. There is concern that without completely draining the pond after harvest, food...

  1. Biochemical, textural, microbiological and sensory attributes of gutted and ungutted sutchi catfish (Pangasianodon hypophthalmus) stored in ice. (United States)

    Viji, P; Tanuja, S; Ninan, George; Lalitha, K V; Zynudheen, A A; Binsi, P K; Srinivasagopal, T K


    Pangasianodon hypophthalmus (sutchi catfish) is a fresh water catfish extensively being cultured in the South East Asian countries in the recent years. The present study provides the first report on the effects of gutting on the quality characteristics of aquacultured sutchi catfish stored in ice. pH of whole ungutted and gutted catfish didn't show significant difference (p > 0.05) during ice storage period. Total Volatile Base Nitrogen (TVB-N), Alpha Amino Nitrogen (AAN), Free Fatty Acids (FFA) and Thio Barbituric Acid Reactive Substance (TBARS) were lower in gutted fish compared to whole ungutted fish at any particular day during ice storage. However, gutted fish expressed higher rate of primary lipid oxidation than ungutted fish. Textural degradation of the fish muscle as indicated by hardness, cohesiveness, springiness and chewiness was lower in gutted fish. Results of sensory evaluation revealed that gutting has significantly improved the sensory quality of the fish. However, microbiological analysis revealed higher Total Plate Count (TPC) and Enterobactereaceae count in gutted fish. The shelf life of gutted and whole ungutted sutchi cat fish as determined by microbiological analysis was 16-18 days and 18-20 days respectively while storage in ice.

  2. Expression and sequence analyses of serum amyloid A in the Syrian hamster. (United States)

    Webb, C F; Tucker, P W; Dowton, S B


    Reactive amyloidosis occurs during chronic inflammation and involves deposition of amyloid A (AA) fibrils in many organs. Amyloid A is derived by proteolysis from serum amyloid A component (SAA), a major acute-phase reactant in many species. Since spontaneous amyloidosis occurs commonly in Syrian hamsters, we have studied the structure and expression of SAA genes during inflammation in these animals. Two cDNA clones and one genomic clone were sequenced, suggesting that Syrian hamster SAA is encoded by at least two genes. Hepatic mRNA analyses showed that SAA was inducible in many hamster organs during acute inflammation. These studies also demonstrated that SAA mRNA for one isotype is maximally expressed at a site of local tissue damage.

  3. Plant Omics: Isolation, Identification, and Expression Analysis of Cytochrome P450 Gene Sequences from Coleus forskohlii. (United States)

    Awasthi, Praveen; Mahajan, Vidushi; Rather, Irshad Ahmad; Gupta, Ajai Prakash; Rasool, Shafaq; Bedi, Yashbir S; Vishwakarma, Ram A; Gandhi, Sumit G


    The omics analyses of plants and the agrigenomics field offer the opportunity to better characterize our ecosystems. In this context, characterization of cytochrome P450 genes (CYP450s), which constitute one of the largest gene families in plants, is important. They play vital roles in biosynthesis of secondary metabolites, phytohormones as well as in detoxification of harmful chemicals. Tuberous roots of Coleus forskohlii accumulate forskolin, a potent and reversible activator of adenylate cyclase, as well as other related diterpenoids. Coleus forskohlii is also known to produce rosmarinic acid, genkwanin (7-O-methylapigenin), and guaiacol glycerin. We report here the isolation of CYP450s from C. forskohlii, expression profiling of CYP450s in different tissues, and how different elicitors/stresses regulate the expression of different CYP450 sequences. Degenerate primers, designed from the conserved regions of CYP450s, were used to amplify fragments from cDNA of C. forskohlii and a library was prepared. Sequences homologous to CYP450s were assembled into seven distinct gene fragments (CfP450C1-C7), belonging to seven CYP450 families. Expression profiling of CYP450s showed that the transcripts of CfP450C1, CfP450C4, CfP450C5, CfP450C6, and CfP450C7 were prominent in aerial tissues (flower, young leaf, and mature leaf), whereas expression of CfP450C3 was dominant in root and root tip. CfP450C2 showed higher expression in flowers and roots as compared to other tissues. Expression profiles of CYP450s, in response to different stresses (abscisic acid, methyl jasmonate, salicylic acid, 2, 4-dichloro-phenoxyacetic acid, UVA, and wounding) were also studied. This study has isolated CYP450s from C. forskohlii, and will help to understand their regulation as well as their functions. This is the first report on the isolation and expression analysis of CYP450s from this herb.

  4. Whole Blood Transcriptome Sequencing Reveals Gene Expression Differences between Dapulian and Landrace Piglets. (United States)

    Hu, Jiaqing; Yang, Dandan; Chen, Wei; Li, Chuanhao; Wang, Yandong; Zeng, Yongqing; Wang, Hui


    There is little genomic information regarding gene expression differences at the whole blood transcriptome level of different pig breeds at the neonatal stage. To solve this, we characterized differentially expressed genes (DEGs) in the whole blood of Dapulian (DPL) and Landrace piglets using RNA-seq (RNA-sequencing) technology. In this study, 83 DEGs were identified between the two breeds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified immune response and metabolism as the most commonly enriched terms and pathways in the DEGs. Genes related to immunity and lipid metabolism were more highly expressed in the DPL piglets, while genes related to body growth were more highly expressed in the Landrace piglets. Additionally, the DPL piglets had twofold more single nucleotide polymorphisms (SNPs) and alternative splicing (AS) than the Landrace piglets. These results expand our knowledge of the genes transcribed in the piglet whole blood of two breeds and provide a basis for future research of the molecular mechanisms underlying the piglet differences.

  5. RNA Sequencing Reveals that Kaposi Sarcoma-Associated Herpesvirus Infection Mimics Hypoxia Gene Expression Signature (United States)

    Viollet, Coralie; Davis, David A.; Tekeste, Shewit S.; Reczko, Martin; Pezzella, Francesco; Ragoussis, Jiannis


    Kaposi sarcoma-associated herpesvirus (KSHV) causes several tumors and hyperproliferative disorders. Hypoxia and hypoxia-inducible factors (HIFs) activate latent and lytic KSHV genes, and several KSHV proteins increase the cellular levels of HIF. Here, we used RNA sequencing, qRT-PCR, Taqman assays, and pathway analysis to explore the miRNA and mRNA response of uninfected and KSHV-infected cells to hypoxia, to compare this with the genetic changes seen in chronic latent KSHV infection, and to explore the degree to which hypoxia and KSHV infection interact in modulating mRNA and miRNA expression. We found that the gene expression signatures for KSHV infection and hypoxia have a 34% overlap. Moreover, there were considerable similarities between the genes up-regulated by hypoxia in uninfected (SLK) and in KSHV-infected (SLKK) cells. hsa-miR-210, a HIF-target known to have pro-angiogenic and anti-apoptotic properties, was significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Interestingly, expression of KSHV-encoded miRNAs was not affected by hypoxia. These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Therefore, targeting hypoxic pathways may be a useful way to develop therapeutic strategies for KSHV-related diseases. PMID:28046107

  6. Whole Blood Transcriptome Sequencing Reveals Gene Expression Differences between Dapulian and Landrace Piglets

    Directory of Open Access Journals (Sweden)

    Jiaqing Hu


    Full Text Available There is little genomic information regarding gene expression differences at the whole blood transcriptome level of different pig breeds at the neonatal stage. To solve this, we characterized differentially expressed genes (DEGs in the whole blood of Dapulian (DPL and Landrace piglets using RNA-seq (RNA-sequencing technology. In this study, 83 DEGs were identified between the two breeds. Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG pathway analyses identified immune response and metabolism as the most commonly enriched terms and pathways in the DEGs. Genes related to immunity and lipid metabolism were more highly expressed in the DPL piglets, while genes related to body growth were more highly expressed in the Landrace piglets. Additionally, the DPL piglets had twofold more single nucleotide polymorphisms (SNPs and alternative splicing (AS than the Landrace piglets. These results expand our knowledge of the genes transcribed in the piglet whole blood of two breeds and provide a basis for future research of the molecular mechanisms underlying the piglet differences.

  7. Parasin Ⅰ : Prokaryotic Expression, Purification and Antimicrobial Activity Analysis of a Recombination Catfish Antimicrobial Peptide%重组鲢鱼抗菌肽parasin Ⅰ原核表达、纯化与抗菌活性

    Institute of Scientific and Technical Information of China (English)

    赵华; 张艳艳; 汤加勇; 李珂; 周继昌; 王康宁


    This study was conducted to construct parasin I expression vector, produce the recombinant peptide in the E. coli expression system, and analyze the antibacterial activity of the recombinant parasin I . According to the amino acid sequence of mature peptide of parasin I and codon bias of E. coli, a 57 bp cDNA fragment was synthesized. The recombinant vector p£7B2-Para I in E. coli was constructed by PCR, and a sequence encoding endonuclease site of Xa factor was cloned into the 5'-upstream of the parasin I gene. The recombinant vector was delivered into E. coli Rosetta ( DE3) cells and isopropyl (3-D-thiogalactoside (IPTG) was used to induce recombinant fusion protein under different temperatures (37 and 20 t). The results showed that the recombinant fusion protein reached about 45% to 50% of the total protein in E. coli, which had a molecular mass of 21 ku. The fusion protein induced under low temperature (20 t) mainly existed in dissolvable form. After purified by affinity chromatography, the recombinant parasin I showed potent anti-Staphylococ-cus aureus activity after digested by Xa fator. An efficient expression system in E. coli for the production of a recombinant parasin I with high yield are successfully constructed in this study, and the recombinant protein shows potent anti-bacterial activity after digested by Xa fator. [ Chinese Journal of Animal Nutrition, 2012, 24(9) : 1731-1736]%本文旨在构建抗菌肽parasin Ⅰ大肠杆菌重组表达载体,表达人工重组parasin Ⅰ,并检测其抑菌活性.根据parasin Ⅰ的成熟肽序列和大肠杆菌密码子偏好性,人工合成1段57 bp的基因编码cDNA,通过PCR技术构建大肠杆菌重组表达质粒pET32-Para Ⅰ,并在parasin Ⅰ基因5′-端引入Xa因子酶切位点.重组载体转化大肠杆菌Rosetta(DE3)菌株后,在不同温度(37和20℃)条件下对阳性转化子进行异丙基硫代半乳糖苷(IPTG)诱导表达.结果表明:IPTG成功诱导了1个21 ku的融合蛋白表达,

  8. Mining of expressed sequence tag libraries of cacao for microsatellite markers using five computational tools

    Indian Academy of Sciences (India)

    Aikkal Riju; M. K. Rajesh; P. T. P. Fasila Sherin; A. Chandrasekar; S. Elain Apshara; Vadivel Arunachalam


    Expressed sequence tags (ESTs) provide researchers with a quick and inexpensive route for discovering new genes, data on gene expression and regulation, and also provide genic markers that help in constructing genome maps. Cacao is an important perennial crop of humid tropics. Cacao EST sequences, as available in the public domain, were downloaded and made into contigs. Microsatellites were located in these ESTs and contigs using five softwares (MISA, TRA, TROLL, SSRIT and SSR primer). MISA gave maximum coverage of SSRs in cacao ESTs and contigs, although TRA was able to detect higher order (>5-mer) repeats. The frequency of SSRs was one per 26.9 kb in the known set of ESTs. One-third of the repeats in EST-contigs were found to be trimeric. A few rare repeats like 21-mer repeat were also located. A/T repeats were most abundant among the mononucleotide repeats and the AG/GA/TC/CT type was the most frequent among dimerics. Flanking primers were designed using Primer3 program and verified experimentally for PCR amplification. The results of the study are made available freely online database ( Seven primer pairs amplified genomic DNA isolated from leaves were used to screen a representative set of 12 accessions of cacao.

  9. Cloning, sequencing, and expression of interferon-γ from elk in North America (United States)

    Sweeney, Steven J.; Emerson, Carlene; Eriks, Inge S.


    Eradication of Mycobacterium bovis relies on accurate detection of infected animals, including potential domestic and wildlife reservoirs. Available diagnostic tests lack the sensitivity and specificity necessary for accurate detection, particularly in infected wildlife populations. Recently, an in vitro diagnostic test for cattle which measures plasma interferon-gamma (IFN-γ) levels in blood following in vitro incubation with M. bovis purified protein derivative has been enveloped. This test appears to have increased sensitivity over traditional testing. Unfortunately, it does not detect IFN-γ from Cervidae. To begin to address this problem, the IFN-γ gene from elk (Cervus elaphus) was cloned, sequenced, expressed, and characterized. cDNA was cloned from mitogen stimulated peripheral blood mononuclear cells. The predicted amino acid (aa) sequence was compared to known sequences from cattle, sheep, goats, red deer (Cervus elaphus), humans, and mice. Biological activity of the recombinant elk IFN-γ (rElkIFN-γ) was confirmed in a vesicular stomatitis virus cytopathic effect reduction assay. Production of monoclonal antibodies to IFN-γ epitopes conserved between ruminant species could provide an important tool for the development of reliable, practical diagnostic assays for detection of a delayed type hypersensitivity response to a variety of persistent infectious agents in ruminants, including M. bovis and Brucella abortus. Moreover, development of these reagents will aid investigators in studies to explore immunological responses of elk that are associated with resistance to infectious diseases.

  10. Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

    Institute of Scientific and Technical Information of China (English)


    Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide.Reverse-transcription Polymerase Chain Reaction (RT-PCR) was performed using cDNAs as templates from melonHuangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named mp-19was cloned and sequenced. The Open Reading Frame (ORF) of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of mp-19. The obvious expression differences detected by semi-quantitative RTPCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

  11. Expressed sequences tags of the anther smut fungus, Microbotryum violaceum, identify mating and pathogenicity genes

    Directory of Open Access Journals (Sweden)

    Devier Benjamin


    Full Text Available Abstract Background The basidiomycete fungus Microbotryum violaceum is responsible for the anther-smut disease in many plants of the Caryophyllaceae family and is a model in genetics and evolutionary biology. Infection is initiated by dikaryotic hyphae produced after the conjugation of two haploid sporidia of opposite mating type. This study describes M. violaceum ESTs corresponding to nuclear genes expressed during conjugation and early hyphal production. Results A normalized cDNA library generated 24,128 sequences, which were assembled into 7,765 unique genes; 25.2% of them displayed significant similarity to annotated proteins from other organisms, 74.3% a weak similarity to the same set of known proteins, and 0.5% were orphans. We identified putative pheromone receptors and genes that in other fungi are involved in the mating process. We also identified many sequences similar to genes known to be involved in pathogenicity in other fungi. The M. violaceum EST database, MICROBASE, is available on the Web and provides access to the sequences, assembled contigs, annotations and programs to compare similarities against MICROBASE. Conclusion This study provides a basis for cloning the mating type locus, for further investigation of pathogenicity genes in the anther smut fungi, and for comparative genomics.


    Zhu, Hui; Wang, Wen-Xiu; Wang, Bao-Qin; Zhu, Xiao-Fu; Wu, Xu-Jin; Ma, Qing-Yi; Chen, De-Kun


    The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. Interferon-gamma (IFN-γ) is the only member of type □ IFN and is vital for the regulation of host adapted immunity and inflammatory response. Little is known aboutthe FN-γ gene and its roles in giant panda.In this study, IFN-γ gene of Qinling giant panda was amplified from total blood RNA by RT-CPR, cloned, sequenced and analysed. The open reading frame (ORF) of Qinling giant panda IFN-γ encodes 152 amino acidsand is highly similar to Sichuan giant panda with an identity of 99.3% in cDNA sequence. The IFN-γ cDNA sequence was ligated to the pET32a vector and transformed into E. coli BL21 competent cells. Expression of recombinant IFN-γ protein of Qinling giant panda in E. coli was confirmed by SDS-PAGE and Western blot analysis. Biological activity assay indicated that the recombinant IFN-γ protein at the concentration of 4-10 µg/ml activated the giant panda peripheral blood lymphocytes,while at 12 µg/mlinhibited. the activation of the lymphocytes.These findings provide insights into the evolution of giant panda IFN-γ and information regarding amino acid residues essential for their biological activity.

  13. Expressed sequence tags in venomous tissue of Scorpaena plumieri (Scorpaeniformes: Scorpaenidae

    Directory of Open Access Journals (Sweden)

    Fábio L. S. Costa

    Full Text Available Species of the family Scorpaenidae are responsible for accidents and sporadic casualties by the shore they inhabit. The species Scorpaena plumierifrom this family populate the Northeastern and Eastern coast of Brazil causing human envenomation characterized by local and systemic symptoms. In experimental animals the venom induces cardiotoxic, hypotensive, and airway respiratory effects. As first step to identify the venom components we isolated gland mRNA to produce a cDNA library from the fish gland. This report describes the partial sequencing of 356 gland transcripts from S. plumieri. BLAST analysis of transcripts showed that 30% were unknown sequences, 17% hypothetical proteins, 17% related to metabolic enzymes, 14% belonged to signal transducing functions and the remaining groups (7-8% composed by gene related with expressing proteins, regulatory proteins and structural proteins. A considerable number of these EST were not found in available databases suggesting the existence of new proteins and/or functions yet to be discovered. By screening the library with antibodies against a lectin fraction from S. plumierivenom we identified several clones whose DNA sequence showed similarities with lectins found in fish. In silicoanalysis of these clones confirm the identity of these molecules in the venom gland of S. plumieri.

  14. Rabbit serum amyloid protein A: expression and primary structure deduced from cDNA sequences. (United States)

    Rygg, M; Marhaug, G; Husby, G; Dowton, S B


    Serum amyloid A protein (SAA), the precursor of amyloid protein A (AA) in deposits of secondary amyloidosis, is an acute phase plasma apolipoprotein produced by hepatocytes. The primary structure of SAA demonstrates high interspecies homology. Several isoforms exist in individual species, probably with different amyloidogenic potential. The nucleotide sequences of two different rabbit serum amyloid A cDNA clones have been analysed, one (corresponding to SAA1) 569 base pairs (bp) long and the other (corresponding to SAA2) 513 bp long. Their deduced amino acid sequences differ at five amino acid positions, four of which are located in the NH2-terminal region of the protein. The deduced amino acid sequence of SAA2 corresponds to rabbit protein AA previously described except for one amino acid in position 22. Eighteen hours after turpentine stimulation, rabbit SAA mRNA is abundant in liver, while lower levels are present in spleen. None of the other extrahepatic organs studied showed any SAA mRNA expression. A third mRNA species (1.9 kb) hybridizing with a single-stranded RNA probe transcribed from the rabbit SAA cDNA, was identified. SAA1 and SAA2 mRNA were found in approximately equal amounts in turpentine-stimulated rabbit liver, but seem to be coordinately decreased after repeated inflammatory stimulation.

  15. Gene expression profiling of liver cancer stem cells by RNA-sequencing.

    Directory of Open Access Journals (Sweden)

    David W Y Ho

    Full Text Available BACKGROUND: Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90(+ liver cancer stem cells (CSCs in hepatocellular carcinoma (HCC. Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq to compare the gene expression profiling of CD90(+ cells sorted from tumor (CD90(+CSCs with parallel non-tumorous liver tissues (CD90(+NTSCs and elucidate the roles of putative target genes in hepatocarcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CD90(+ cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90(+ cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90(+CSCs and CD90(+NTSCs, and validated by quantitative real-time PCR (qRT-PCR on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes between CD90(+CSCs and CD90(+NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90(+CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3, a member of glypican family, was markedly elevated in CD90(+CSCs compared to CD90(+NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90(+CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90(+CSCs in liver tumor tissues. CONCLUSIONS

  16. Efficacy of Florfenicol for Control of Mortality Associated with Edwardsiella ictaluri in Three Species of Catfish. (United States)

    Gaunt, Patricia S; Chatakondi, Nagaraj; Gao, Dana; Endris, Richard


    The efficacy of florfenicol for control of mortality associated with Edwardsiella icatluri was studied in fingerlings of Channel Catfish Ictalurus puntatus (Delta strain), Blue Catfish I. furcatus (D&B strain), and a hybrid catfish (Delta strain Channel Catfish × D&B strain Blue Catfish). On day 0, fish were immersion challenged in 65-L aquaria. For each of the three species of catfish, 10 aquaria were randomly assigned to two treatment groups, either treated with florfenicol at 0 mg/kg of body weight (unmedicated feed) or at 10 mg/kg (medicated feed). Fish were treated for 10 consecutive days, monitored for mortality during this treatment period, and observed for 14 d afterwards. Post observation, all survivors were humanely euthanized in tricaine methanesulfonate, cultured for E. ictaluri, and examined for gross pathology. The mean cumulative percent mortality from enteric septicemia of catfish (ESC) challenge among the three genotypes of catfish did not differ between Blue Catfish, hybrid, and Channel Catfish in treated or control groups. However, the florfenicol-treated fish had a significantly lower mean cumulative mortality (6%) than the controls (78%). All genotypes of catfish tested were responsive to treatment with florfenicol-medicated feed for control of mortality associated with ESC. There were no significant differences in mortality associated with hybrid catfish, blue catfish, and Channel Catfish (Delta strain).

  17. 淮河野生鲇鱼线粒体Cyt b基因的序列变异与遗传结构分析%Genetic Structure and Variation of Wild Chinese Catfish from Huaihe River Based on Cytochrome b Gene Sequences

    Institute of Scientific and Technical Information of China (English)

    肖明松; 崔峰; 康健; 张晓红


    本研究利用线粒体DNA细胞色素b(Cyt b)基因序列分析淮河信阳段、淮滨段、蚌埠段、洪泽湖及其支流颍河、淠河和池河的野生鲇鱼(Silurus asotus)种群遗传结构及种群历史.结果表明,在841 bp的同源序列中,7个种群共检测到变异位点40个,占全部序列的4.76%,121个个体共检测到32种单倍型;7个种群的平均单倍型多样性(h)、核甘酸多样性(Pi)分别为0.884 8、0.003 8,表明淮河野生鲇鱼种群的遗传多样性水平较高.7个种群间的遗传分化指数Fst为0.115 0,仅12.92%的变异来自种群间(AMOVA分析),基因交流值为3.85,种群间K2-P遗传距离为0.002 ~0.009,显示鲇鱼种群间没有发生明显的地理分化.NJ树揭示7个种群的个体组成2个谱系,但这2个谱系与地理分布并不相关.中性检验、错配分析和Network网络亲缘关系分析皆表明鲇鱼种群有过种群扩张,扩张时间约在0.17~0.29百万年前的中更新世中期和末期.%The Chinese Catfish Silurus asotus is a commercially valuable aquaculture fish in some regions of China and Japan, and it is widely distributed throughout the freshwater reservoirs, lakes and rivers of China. Due to overfishing, environmental pollution, water conservancy, human production activities and other causes, wild Chinese Catfish has declined rapidly. Its fishery resource has been gradually exhausted in the Huaihe River Basin in the past decades. In order to analyze the genetic structure and demographic history of S. asotus, the mitochondrial cytochrome b ( Cyt b) gene sequences were used in the study. The results showed that Cyt b gene sequence contained 841 bp nucleotides and the T, C, A and G contents were 28. 59% , 29. 55% , 27. 44% and 14. 42% , respectively. A total of 40 nucleotide sites and 32 haplotypes were obtained from 121 individuals collected in 7 different regions, including Xinyang, Huaibin, Bengbu, Hongze Lake, Yinghe River, Pihe River and Chihe River. The

  18. Generation, analysis and functional annotation of expressed sequence tags from the ectoparasitic mite Psoroptes ovis

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    Kenyon Fiona


    Full Text Available Abstract Background Sheep scab is caused by Psoroptes ovis and is arguably the most important ectoparasitic disease affecting sheep in the UK. The disease is highly contagious and causes and considerable pruritis and irritation and is therefore a major welfare concern. Current methods of treatment are unsustainable and in order to elucidate novel methods of disease control a more comprehensive understanding of the parasite is required. To date, no full genomic DNA sequence or large scale transcript datasets are available and prior to this study only 484 P. ovis expressed sequence tags (ESTs were accessible in public databases. Results In order to further expand upon the transcriptomic coverage of P. ovis thus facilitating novel insights into the mite biology we undertook a larger scale EST approach, incorporating newly generated and previously described P. ovis transcript data and representing the largest collection of P. ovis ESTs to date. We sequenced 1,574 ESTs and assembled these along with 484 previously generated P. ovis ESTs, which resulted in the identification of 1,545 unique P. ovis sequences. BLASTX searches identified 961 ESTs with significant hits (E-value P. ovis ESTs. Gene Ontology (GO analysis allowed the functional annotation of 880 ESTs and included predictions of signal peptide and transmembrane domains; allowing the identification of potential P. ovis excreted/secreted factors, and mapping of metabolic pathways. Conclusions This dataset currently represents the largest collection of P. ovis ESTs, all of which are publicly available in the GenBank EST database (dbEST (accession numbers FR748230 - FR749648. Functional analysis of this dataset identified important homologues, including house dust mite allergens and tick salivary factors. These findings offer new insights into the underlying biology of P. ovis, facilitating further investigations into mite biology and the identification of novel methods of intervention.

  19. Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues (United States)

    García-Chequer, A.J.; Méndez-Tenorio, A.; Olguín-López, G.; Sánchez-Vallejo, C.; Isa, P.; Arias, C.F.; Torres, J.; Hernández-Angeles, A.; Ramírez-Ortiz, M.A.; Lara, C.; Cabrera-Muñoz,; Sadowinski-Pine, S.; Bravo-Ortiz, J.C.; Ramón-García, G.; Diegopérez-Ramírez, J.; Ramírez-Reyes, G.; Casarrubias-Islas, R.; Ramírez, J.; Orjuela, M.; Ponce-Castañeda, M.V.


    Retinoblastoma (Rb) is a pediatric intraocular malignancy and probably the most robust clinical model on which genetic predisposition to develop cancer has been demonstrated. Since deletions in chromosome 13 have been described in this tumor, we performed next generation sequencing to test whether recurrent losses could be detected in low coverage data. We used Illumina platform for 13 tumor tissue samples: two pools of 4 retinoblastoma cases each and one pool of 5 medulloblastoma cases (raw data can be found at We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5). From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at number GSE11488). The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press) [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma. PMID:26937470

  20. Robust global microRNA expression profiling using next-generation sequencing technologies. (United States)

    Tam, Shirley; de Borja, Richard; Tsao, Ming-Sound; McPherson, John D


    miRNAs are a class of regulatory molecules involved in a wide range of cellular functions, including growth, development and apoptosis. Given their widespread roles in biological processes, understanding their patterns of expression in normal and diseased states will provide insights into the consequences of aberrant expression. As such, global miRNA expression profiling of human malignancies is gaining popularity in both basic and clinically driven research. However, to date, the majority of such analyses have used microarrays and quantitative real-time PCR. With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal many more options. To make effective use of these different platforms, the strengths and pitfalls of several miRNA profiling technologies were assessed, including a microarray platform, NGS technologies and the NanoString nCounter System. Overall, NGS had the greatest detection sensitivity, largest dynamic range of detection and highest accuracy in differential expression analysis when compared with gold-standard quantitative real-time PCR. Its technical reproducibility was high, with intrasample correlations of at least 0.95 in all cases. Furthermore, miRNA analysis of formalin-fixed, paraffin-embedded (FFPE) tissue was also evaluated. Expression profiles between paired frozen and FFPE samples were similar, with Spearman's ρ>0.93. These results show the superior sensitivity, accuracy and robustness of NGS for the comprehensive profiling of miRNAs in both frozen and FFPE tissues.

  1. RNA expression profile of calcified bicuspid, tricuspid, and normal human aortic valves by RNA sequencing. (United States)

    Guauque-Olarte, Sandra; Droit, Arnaud; Tremblay-Marchand, Joël; Gaudreault, Nathalie; Kalavrouziotis, Dimitri; Dagenais, Francois; Seidman, Jonathan G; Body, Simon C; Pibarot, Philippe; Mathieu, Patrick; Bossé, Yohan


    The molecular mechanisms leading to premature development of aortic valve stenosis (AS) in individuals with a bicuspid aortic valve are unknown. The objective of this study was to identify genes differentially expressed between calcified bicuspid aortic valves (BAVc) and tricuspid valves with (TAVc) and without (TAVn) AS using RNA sequencing (RNA-Seq). We collected 10 human BAVc and nine TAVc from men who underwent primary aortic valve replacement. Eight TAVn were obtained from men who underwent heart transplantation. mRNA levels were measured by RNA-Seq and compared between valve groups. Two genes were upregulated, and none were downregulated in BAVc compared with TAVc, suggesting a similar gene expression response to AS in individuals with bicuspid and tricuspid valves. There were 462 genes upregulated and 282 downregulated in BAVc compared with TAVn. In TAVc compared with TAVn, 329 genes were up- and 170 were downregulated. A total of 273 upregulated and 147 downregulated genes were concordantly altered between BAVc vs. TAVn and TAVc vs. TAVn, which represent 56 and 84% of significant genes in the first and second comparisons, respectively. This indicates that extra genes and pathways were altered in BAVc. Shared pathways between calcified (BAVc and TAVc) and normal (TAVn) aortic valves were also more extensively altered in BAVc. The top pathway enriched for genes differentially expressed in calcified compared with normal valves was fibrosis, which support the remodeling process as a therapeutic target. These findings are relevant to understand the molecular basis of AS in patients with bicuspid and tricuspid valves.

  2. Comparative analysis of differentially expressed sequence tags of sweet orange and mandarin infected with Xylella fastidiosa

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    Alessandra A. de Souza


    Full Text Available The Citrus ESTs Sequencing Project (CitEST conducted at Centro APTA Citros Sylvio Moreira/IAC has identified and catalogued ESTs representing a set of citrus genes expressed under relevant stress responses, including diseases such as citrus variegated chlorosis (CVC, caused by Xylella fastidiosa. All sweet orange (Citrus sinensis L. Osb. varieties are susceptible to X. fastidiosa. On the other hand, mandarins (C. reticulata Blanco are considered tolerant or resistant to the disease, although the bacterium can be sporadically detected within the trees, but no disease symptoms or economic losses are observed. To study their genetic responses to the presence of X. fastidiosa, we have compared EST libraries of leaf tissue of sweet orange Pêra IAC (highly susceptible cultivar to X. fastidiosa and mandarin ‘Ponkan’ (tolerant artificially infected with the bacterium. Using an in silico differential display, 172 genes were found to be significantly differentially expressed in such conditions. Sweet orange presented an increase in expression of photosynthesis related genes that could reveal a strategy to counterbalance a possible lower photosynthetic activity resulting from early effects of the bacterial colonization in affected plants. On the other hand, mandarin showed an active multi-component defense response against the bacterium similar to the non-host resistance pattern.

  3. Illumina next generation sequencing data and expression microarrays data from retinoblastoma and medulloblastoma tissues

    Directory of Open Access Journals (Sweden)

    A.J. García-Chequer


    We first created an in silico reference profile generated from a human sequenced genome (GRCh37p5. From this data we calculated an integrity score to get an overview of gains and losses in all chromosomes; we next analyzed each chromosome in windows of 40 kb length, calculating for each window the log2 ratio between reads from tumor pool and in silico reference. Finally we generated panoramic maps with all the windows whether lost or gained along each chromosome associated to its cytogenetic bands to facilitate interpretation. Expression microarrays was done for the same samples and a list of over and under expressed genes is presented here. For this detection a significance analysis was done and a log2 fold change was chosen as significant (raw data can be found at number GSE11488. The complete research article can be found at Cancer Genetics journal (Garcia-Chequer et al., in press [1]. In summary here we provide an overview with visual graphics of gains and losses chromosome by chromosome in retinoblastoma and medulloblastoma, also the integrity score analysis and a list of genes with relevant expression associated. This material can be useful to researchers that may want to explore gains and losses in other malignant tumors with this approach or compare their data with retinoblastoma.

  4. Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi. (United States)

    Carballar-Lejarazú, Rebeca; Jasinskiene, Nijole; James, Anthony A


    Malaria parasites are transmitted to humans by mosquitoes of the genus Anopheles, and these insects are the targets of innovative vector control programs. Proposed approaches include the use of genetic strategies based on transgenic mosquitoes to suppress or modify vector populations. Although substantial advances have been made in engineering resistant mosquito strains, limited efforts have been made in refining mosquito transgene expression, in particular attenuating the effects of insertions sites, which can result in variations in phenotypes and impacts on fitness due to the random integration of transposon constructs. A promising strategy to mitigate position effects is the identification of insulator or boundary DNA elements that could be used to isolate transgenes from the effects of their genomic environment. We applied quantitative approaches that show that exogenous insulator-like DNA derived from the Drosophila melanogaster gypsy retrotransposon can increase and stabilize transgene expression in transposon-mediated random insertions and recombinase-catalyzed, site-specific integrations in the malaria vector mosquito, Anopheles stephensi. These sequences can contribute to precise expression of transgenes in mosquitoes engineered for both basic and applied goals.

  5. Sequencing, Expression and Diagnostic Application of the Nucleoprotein Gene of Xinjiang Hemorrhagic Fever Virus

    Institute of Scientific and Technical Information of China (English)

    马本江; 杭长寿; 解燕乡; 王世文


    In order to analyze the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV), viral RNA was amplified by RT-PCR by using the proof-reading DNA polymerase to produce the complete NP gene. The PCR product was sequenced, analyzed for phylogenesis and cloned into the expression vector pE132a and the recombinant plasmid expressed in E. coil BL-21 with high yield. The primarily purified fused protein.was used to coat ELISA plates for the detect antibodies. It was found the similarities between NP gene of BA88166 and other XHFVs in nucleotide level and amino acid contents were very significant, and the NP gene of BA88166 encoded a nucleoprotein with 482 amino acid and a deduced molecular weight (MW) of 54 kDa. Western blot assay showed that the fusion protein expressed in bacteria possessed good antigenicity. The results with ELISA for the detection of the human and animal sera collected in endemic areas were found to be in good accordance to the clinical diagnosis. It concluded that the relations of NP genes of XHFV BA88166 and other XHFVs appeared to be evolutionally close. The methodologies established in this study were accurate, specific, rapid and reproducible for the clinical examinations and epidemiological survey.

  6. Transcriptome sequencing of gene expression in the brain of the HIV-1 transgenic rat.

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    Ming D Li

    Full Text Available The noninfectious HIV-1 transgenic (HIV-1Tg rat was developed as a model of AIDs-related pathology and immune dysfunction by manipulation of a noninfectious HIV-1(gag-pol virus with a deleted 3-kb SphI-MscI fragment containing the 3' -region of gag and the 5' region of pol into F344 rats. Our previous studies revealed significant behavioral differences between HIV-1Tg and F344 control rats in their performance in the Morris water maze and responses to psychostimulants. However, the molecular mechanisms underlying these behavioral differences remain largely unknown. The primary goal of this study was to identify differentially expressed genes and enriched pathways affected by the gag-pol-deleted HIV-1 genome. Using RNA deep sequencing, we sequenced RNA transcripts in the prefrontal cortex, hippocampus, and striatum of HIV-1Tg and F344 rats. A total of 72 RNA samples were analyzed (i.e., 12 animals per group × 2 strains × 3 brain regions. Following deep-sequencing analysis of 50-bp paired-end reads of RNA-Seq, we used Bowtie/Tophat/Cufflinks suites to align these reads into transcripts based on the Rn4 rat reference genome and to measure the relative abundance of each transcript. Statistical analyses on each brain region in the two strains revealed that immune response- and neurotransmission-related pathways were altered in the HIV-1Tg rats, with brain region differences. Other neuronal survival-related pathways, including those encoding myelin proteins, growth factors, and translation regulators, were altered in the HIV-1Tg rats in a brain region-dependent manner. This study is the first deep-sequencing analysis of RNA transcripts associated the HIV-1Tg rat. Considering the functions of the pathways and brain regions examined in this study, our findings of abnormal gene expression patterns in HIV-1Tg rats suggest mechanisms underlying the deficits in learning and memory and vulnerability to drug addiction and other psychiatric disorders

  7. Sequencing, physical organization and kinetic expression of the patulin biosynthetic gene cluster from Penicillium expansum. (United States)

    Tannous, Joanna; El Khoury, Rhoda; Snini, Selma P; Lippi, Yannick; El Khoury, André; Atoui, Ali; Lteif, Roger; Oswald, Isabelle P; Puel, Olivier


    Patulin is a polyketide-derived mycotoxin produced by numerous filamentous fungi. Among them, Penicillium expansum is by far the most problematic species. This fungus is a destructive phytopathogen capable of growing on fruit, provoking the blue mold decay of apples and producing significant amounts of patulin. The biosynthetic pathway of this mycotoxin is chemically well-characterized, but its genetic bases remain largely unknown with only few characterized genes in less economic relevant species. The present study consisted of the identification and positional organization of the patulin gene cluster in P. expansum strain NRRL 35695. Several amplification reactions were performed with degenerative primers that were designed based on sequences from the orthologous genes available in other species. An improved genome Walking approach was used in order to sequence the remaining adjacent genes of the cluster. RACE-PCR was also carried out from mRNAs to determine the start and stop codons of the coding sequences. The patulin gene cluster in P. expansum consists of 15 genes in the following order: patH, patG, patF, patE, patD, patC, patB, patA, patM, patN, patO, patL, patI, patJ, and patK. These genes share 60-70% of identity with orthologous genes grouped differently, within a putative patulin cluster described in a non-producing strain of Aspergillus clavatus. The kinetics of patulin cluster genes expression was studied under patulin-permissive conditions (natural apple-based medium) and patulin-restrictive conditions (Eagle's minimal essential medium), and demonstrated a significant association between gene expression and patulin production. In conclusion, the sequence of the patulin cluster in P. expansum constitutes a key step for a better understanding of the mechanisms leading to patulin production in this fungus. It will allow the role of each gene to be elucidated, and help to define strategies to reduce patulin production in apple-based products.

  8. Temperature Influences on the Expression of GFP Promoted by the Upstream Sequence of cpcB from Arthrospira platensis

    Institute of Scientific and Technical Information of China (English)


    In order to investigate the regulation mechanism of the phycocyanin gene, a series of functional analyses of the upstream sequence of cpcB gene from Arthrospira platensis were conducted in E. coli with green fluorescent protein encoding gene (gfp) as the reporter. Results showed that the gfp gene could express at a high level under the promotion of the upstream sequence, suggesting the existence of some strong promoter elements in it. The expression of GFP was influenced by temperature. Higher temperature led to higher expression level. The bioinformatics analyses followed by mutation analyses on the secondary structure of translation initiation region (TIR) revealed that RNA thermosensor might account for the temperature regulation.

  9. Temperature influences on the expression of GFP promoted by the upstream sequence of cpcB from Arthrospira platensis (United States)

    Lu, Yongzhong; Zhang, Xuecheng


    In order to investigate the regulation mechanism of the phycocyanin gene, a series of functional analyses of the upstream sequence of cpcB gene from Arthrospira platensis were conducted in E. coli with green fluorescent protein encoding gene (gfp) as the reporter. Results showed that the gfp gene could express at a high level under the promotion of the upstream sequence, suggesting the existence of some strong promoter elements in it. The expression of GFP was influenced by temperature. Higher temperature led to higher expression level. The bioinformatics analyses followed by mutation analyses on the secondary structure of translation initiation region (TIR) revealed that RNA thermosensor might account for the temperature regulation.

  10. MicroRNA Expression Profile in Penile Cancer Revealed by Next-Generation Small RNA Sequencing.

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    Li Zhang

    Full Text Available Penile cancer (PeCa is a relatively rare tumor entity but possesses higher morbidity and mortality rates especially in developing countries. To date, the concrete pathogenic signaling pathways and core machineries involved in tumorigenesis and progression of PeCa remain to be elucidated. Several studies suggested miRNAs, which modulate gene expression at posttranscriptional level, were frequently mis-regulated and aberrantly expressed in human cancers. However, the miRNA profile in human PeCa has not been reported before. In this present study, the miRNA profile was obtained from 10 fresh penile cancerous tissues and matched adjacent non-cancerous tissues via next-generation sequencing. As a result, a total of 751 and 806 annotated miRNAs were identified in normal and cancerous penile tissues, respectively. Among which, 56 miRNAs with significantly different expression levels between paired tissues were identified. Subsequently, several annotated miRNAs were selected randomly and validated using quantitative real-time PCR. Compared with the previous publications regarding to the altered miRNAs expression in various cancers and especially genitourinary (prostate, bladder, kidney, testis cancers, the most majority of deregulated miRNAs showed the similar expression pattern in penile cancer. Moreover, the bioinformatics analyses suggested that the putative target genes of differentially expressed miRNAs between cancerous and matched normal penile tissues were tightly associated with cell junction, proliferation, growth as well as genomic instability and so on, by modulating Wnt, MAPK, p53, PI3K-Akt, Notch and TGF-β signaling pathways, which were all well-established to participate in cancer initiation and progression. Our work presents a global view of the differentially expressed miRNAs and potentially regulatory networks of their target genes for clarifying the pathogenic transformation of normal penis to PeCa, which research resource also

  11. Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee. (United States)

    Whitfield, Charles W; Band, Mark R; Bonaldo, Maria F; Kumar, Charu G; Liu, Lei; Pardinas, Jose R; Robertson, Hugh M; Soares, M Bento; Robinson, Gene E


    To accelerate the molecular analysis of behavior in the honey bee (Apis mellifera), we created expressed sequence tag (EST) and cDNA microarray resources for the bee brain. Over 20,000 cDNA clones were partially sequenced from a normalized (and subsequently subtracted) library generated from adult A. mellifera brains. These sequences were processed to identify 15,311 high-quality ESTs representing 8912 putative transcripts. Putative transcripts were functionally annotated (using the Gene Ontology classification system) based on matching gene sequences in Drosophila melanogaster. The brain ESTs represent a broad range of molecular functions and biological processes, with neurobiological classifications particularly well represented. Roughly half of Drosophila genes currently implicated in synaptic transmission and/or behavior are represented in the Apis EST set. Of Apis sequences with open reading frames of at least 450 bp, 24% are highly diverged with no matches to known protein sequences. Additionally, over 100 Apis transcript sequences conserved with other organisms appear to have been lost from the Drosophila genome. DNA microarrays were fabricated with over 7000 EST cDNA clones putatively representing different transcripts. Using probe derived from single bee brain mRNA, microarrays detected gene expression for 90% of Apis cDNAs two standard deviations greater than exogenous control cDNAs. [The sequence data described in this paper have been submitted to Genbank data library under accession nos. BI502708-BI517278. The sequences are also available at

  12. Analysis of cDNA sequence, protein structure and expression of parotid secretory protein in pig

    Institute of Scientific and Technical Information of China (English)

    YIN Haifang; FAN Baoliang; ZHAO Zhihui; LIU Zhaoliang; FEI Jing; LI Ning


    Parotid secretory protein (PSP) secreted abundantly in saliva, whose function is related with the anti-bacterial effect. The PSP cDNA has been isolated from pig parotid glands by 3′ and 5′ rapid amplification of cDNA end (RACE),based on the conserved signal peptide region among the known mammalian PSP. Theresult of homologous comparison shows that pig PSP and human PSP shares the high identity at the level of the primary, secondary and tertiary protein structure. A search for functionally significant protein motifs revealed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu- X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is important to its function. RT-PCR, Dot blot and Northern blot analysis demonstrated that PSP was strongly expressed in parotid glands, but not in other tissues.

  13. Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

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    Ravi Chandra Yandamuri


    Full Text Available eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.

  14. Gene Expression Profiling of Development and Anthocyanin Accumulation in Kiwifruit (Actinidia chinensis Based on Transcriptome Sequencing.

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    Wenbin Li

    Full Text Available Red-fleshed kiwifruit (Actinidia chinensis Planch. 'Hongyang' is a promising commercial cultivar due to its nutritious value and unique flesh color, derived from vitamin C and anthocyanins. In this study, we obtained transcriptome data of 'Hongyang' from seven developmental stages using Illumina sequencing. We mapped 39-54 million reads to the recently sequenced kiwifruit genome and other databases to define gene structure, to analyze alternative splicing, and to quantify gene transcript abundance at different developmental stages. The transcript profiles throughout red kiwifruit development were constructed and analyzed, with a focus on the biosynthesis and metabolism of compounds such as phytohormones, sugars, starch and L-ascorbic acid, which are indispensable for the development and formation of quality fruit. Candidate genes for these pathways were identified through MapMan and phylogenetic analysis. The transcript levels of genes involved in sucrose and starch metabolism were consistent with the change in soluble sugar and starch content throughout kiwifruit development. The metabolism of L-ascorbic acid was very active, primarily through the L-galactose pathway. The genes responsible for the accumulation of anthocyanin in red kiwifruit were identified, and their expression levels were investigated during kiwifruit development. This survey of gene expression during kiwifruit development paves the way for further investigation of the development of this uniquely colored and nutritious fruit and reveals which factors are needed for high quality fruit formation. This transcriptome data and its analysis will be useful for improving kiwifruit genome annotation, for basic fruit molecular biology research, and for kiwifruit breeding and improvement.

  15. Sequence Conservation and Differential Expression of Marek's Disease Virus MicroRNAs▿ (United States)

    Morgan, Robin; Anderson, Amy; Bernberg, Erin; Kamboj, Sachin; Huang, Emily; Lagasse, Grace; Isaacs, Grace; Parcells, Mark; Meyers, Blake C.; Green, Pamela J.; Burnside, Joan


    Marek's disease virus (MDV), a herpesvirus that causes a lymphoproliferative disorder in chickens, encodes a number of microRNAs derived primarily from two locations in the MDV genome. One cluster of microRNA genes flanks the meq oncogene, and a second cluster is found within the latency-associated transcript (LAT) region. The sequences of MDV microRNAs from a collection of field and reference strains with various levels of virulence were compared and found to be highly conserved. However, microRNAs from the meq cluster were detected at higher levels in lymphomas caused by a form of the virus designated very virulent plus (vv+; strain 615K, also known as T. King) than in those caused by a less virulent (very virulent [vv]) form (RB1B). For example, levels of mdv1-miR-M4, which shares a seed sequence with miR-155, a microRNA implicated in B-cell lymphoma, were threefold higher and levels of mdv1-miR-M2*/3p were more than sixfold higher in vv+ MDV-induced tumors than in vv MDV-induced tumors. In contrast, levels of the microRNAs from the LAT cluster were equivalent in tumors produced by vv and vv+ strains. Additionally, mdv1-miR-M4 is the MDV microRNA most highly expressed in tumors, where it accounts for 72% of all MDV microRNAs, as determined by deep sequencing. These data suggest that the meq cluster microRNAs play an important role in the pathogenicity of MDV. PMID:18842708

  16. Expressed sequence tag analysis of Antarctic hairgrass Deschampsia antarctica from King George Island, Antarctica. (United States)

    Lee, Hyoungseok; Cho, Hyun Hee; Kim, Il-Chan; Yim, Joung Han; Lee, Hong Kum; Lee, Yoo Kyung


    Deschampsia antarctica is the only monocot that thrives in the tough conditions of the Antarctic region. It is an invaluable resource for the identification of genes associated with tolerance to various environmental pressures. In order to identify genes that are differentially regulated between greenhouse-grown and Antarctic field-grown plants, we initiated a detailed gene expression analysis. Antarctic plants were collected and greenhouse plants served as controls. Two different cDNA libraries were constructed with these plants. A total of 2,112 cDNA clones was sequenced and grouped into 1,199 unigene clusters consisting of 243 consensus and 956 singleton sequences. Using similarity searches against several public databases, we constructed a functional classification of the ESTs into categories such as genes related to responses to stimuli, as well as photosynthesis and metabolism. Real-time PCR analysis of various stress responsive genes revealed different patterns of regulation in the different environments, suggesting that these genes are involved in responses to specific environmental factors.

  17. Revised sequence and expression of cyclin B cDNA from the starfish Asterina pectinifera. (United States)

    Miyake, Y; Deshimaru, S; Toraya, T


    Cyclin B cDNA was cloned from the ovary of the starfish Asterina pectinifera and analyzed by RT-PCR and 3'- and 5'-RACE techniques. The cDNA consists of a 0.13-kb upstream untranslated region, a 1.22-kb coding region, and a 0.86-kb downstream untranslated region. The open reading frame encoded a polypeptide of 404 amino acid residues with a calculated molecular weight of 45,692. All the characteristic sequences, such as destruction and cyclin boxes, cyclin B motif, and cytoplasmic retention and nuclear export signals, were found in the newly cloned cyclin B cDNA. The deduced amino acid sequence of the cyclin B cDNA was highly homologous in the middle and carboxy terminal regions to that from mature eggs of the same organism, but quite different in the amino terminal region. Evidence was obtained which suggested that this cyclin B is expressed in immature and maturing oocytes and is the same as that cloned from mature eggs.

  18. Primary analysis of the expressed sequence tags in a pentastomid nymph cDNA library.

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    Full Text Available BACKGROUND: Pentastomiasis is a rare zoonotic disease caused by pentastomids. Despite their worm-like appearance, they are commonly placed into a separate sub-class of the subphylum Crustacea, phylum Arthropoda. However, until now, the systematic classification of the pentastomids and the diagnosis of pentastomiasis are immature, and genetic information about pentastomid nylum is almost nonexistent. The objective of this study was to obtain information on pentastomid nymph genes and identify the gene homologues related to host-parasite interactions or stage-specific antigens. METHODOLOGY/PRINCIPAL FINDINGS: Total pentastomid nymph RNA was used to construct a cDNA library and 500 colonies were sequenced. Analysis shows one hundred and ninety-seven unigenes were identified. In which, 147 genes were annotated, and 75 unigenes (53.19% were mapped to 82 KEGG pathways, including 29 metabolism pathways, 29 genetic information processing pathways, 4 environmental information processing pathways, 7 cell motility pathways and 5 organismal systems pathways. Additionally, two host-parasite interaction-related gene homologues, a putative Kunitz inhibitor and a putative cysteine protease. CONCLUSION/SIGNIFICANCE: We first successfully constructed a cDNA library and gained a number of expressed sequence tags (EST from pentastomid nymphs, which will lay the foundation for the further study on pentastomids and pentastomiasis.

  19. Expressed sequence tags analysis of a liver tissue cDNA library from a highly inbred minipig line

    Institute of Scientific and Technical Information of China (English)

    CHEN You-nan; TAN Wei-dong; LU Yan-rong; QIN Sheng-fang; LI Sheng-fu; ZENG Yang-zhi; BU Hong; LI You-ping; CHENG Jing-qiu


    Background Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore,we investigated the liver expression profile of a highly inbred minipig line.Methods A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme.Results Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences.Conclusion These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

  20. Water hardness influences Flavobacterium columnare pathogenesis in channel catfish (United States)

    Studies were conducted to determine aspects of water chemistry responsible for large differences in pathogenesis and mortality rates in challenges of channel catfish Ictalurus punctatus with Flavobacterium columnare; challenges were conducted in water supplying the Stuttgart National Aquaculture Res...

  1. The Effect of Addition Vitamin E on Catfish Oil Stability

    Directory of Open Access Journals (Sweden)

    Clara M. Kusharto


    Full Text Available Unsaturated fatty acids contained oils which sensitive to oxidation caused by heat, light and oxygen. The oxidized oil known harmful to the body. One of the effort to prevent the oxidation process is by adding antioxidants stability of oil catfish and shelf life of the oil. The experimental study was applied by adding vitamin E to the oil as much as 0.67 mg / g PUFA compared with the control oil. Oil was stored with the Schaal Oven Test method, which are stored at a temperature of 600C. The study showed that, based on the parameters of oxidation (free fatty acids, peroxide value, anisidin numbers, and the number of total oxidation, catfish oil were added vitamin E more stabilized than the catfish oil without added vitamin E. The catfish fish oil in the form of soft gell capsules added vitamin E could maintain stability in appropriate with IFOS up to 23 months.

  2. Comparisons between Arabidopsis thaliana and Drosophila melanogaster in relation to Coding and Noncoding Sequence Length and Gene Expression

    Directory of Open Access Journals (Sweden)

    Rachel Caldwell


    Full Text Available There is a continuing interest in the analysis of gene architecture and gene expression to determine the relationship that may exist. Advances in high-quality sequencing technologies and large-scale resource datasets have increased the understanding of relationships and cross-referencing of expression data to the large genome data. Although a negative correlation between expression level and gene (especially transcript length has been generally accepted, there have been some conflicting results arising from the literature concerning the impacts of different regions of genes, and the underlying reason is not well understood. The research aims to apply quantile regression techniques for statistical analysis of coding and noncoding sequence length and gene expression data in the plant, Arabidopsis thaliana, and fruit fly, Drosophila melanogaster, to determine if a relationship exists and if there is any variation or similarities between these species. The quantile regression analysis found that the coding sequence length and gene expression correlations varied, and similarities emerged for the noncoding sequence length (5′ and 3′ UTRs between animal and plant species. In conclusion, the information described in this study provides the basis for further exploration into gene regulation with regard to coding and noncoding sequence length.

  3. Trophic relations of introduced flathead catfish in an atlantic river (United States)

    Baumann, Jessica R.; Kwak, Thomas J.


    The flathead catfish Pylodictis olivaris is a large piscivore that is native to the Mississippi and Rio Grande river drainages but that has been widely introduced across the United States. River ecologists and fisheries managers are concerned about introduced flathead catfish populations because of the negative impacts on native fish communities or imperiled species associated with direct predation and indirect competition from this apex predator. We studied the trophic relations of introduced flathead catfish in an Atlantic river to further understand the effects on native fish communities. Crayfish (Astacidea) occurred most frequently in the flathead catfish diet, while sunfish Lepomis spp. comprised the greatest percentage by weight. Neither of two sympatric imperiled fish species (the federally endangered Cape Fear shiner Notropis mekistocholas and the Carolina redhorse Moxostoma sp., a federal species of concern) was found in any diet sample. An ontogenetic shift in diet was evident when flathead catfish reached about 300 mm, and length significantly explained the variation in the percent composition by weight of sunfish and darters Etheostoma and Percina spp. Flathead catfish showed positive prey selectivity for taxa that occupied similar benthic microhabitat, highlighting the importance of opportunistic feeding and prey encounter rates. Flathead catfish displayed a highly variable diel feeding chronology during July, when they had a mean stomach fullness of 0.32%, but then showed a single midday feeding peak during August (mean fullness = 0.52%). The gastric evacuation rate increased between July (0.40/h) and August (0.59/h), as did daily ration, which more than doubled between the 2 months (3.06% versus 7.37%). Our findings increase the understanding of introduced flathead catfish trophic relations and the degree of vulnerability among prey taxa, which resource managers may consider in fisheries management and conservation of native fish populations and

  4. Discovery of differentially expressed genes in cashmere goat (Capra hircus) hair follicles by RNA sequencing. (United States)

    Qiao, X; Wu, J H; Wu, R B; Su, R; Li, C; Zhang, Y J; Wang, R J; Zhao, Y H; Fan, Y X; Zhang, W G; Li, J Q


    The mammalian hair follicle (HF) is a unique, highly regenerative organ with a distinct developmental cycle. Cashmere goat (Capra hircus) HFs can be divided into two categories based on structure and development time: primary and secondary follicles. To identify differentially expressed genes (DEGs) in the primary and secondary HFs of cashmere goats, the RNA sequencing of six individuals from Arbas, Inner Mongolia, was performed. A total of 617 DEGs were identified; 297 were upregulated while 320 were downregulated. Gene ontology analysis revealed that the main functions of the upregulated genes were electron transport, respiratory electron transport, mitochondrial electron transport, and gene expression. The downregulated genes were mainly involved in cell autophagy, protein complexes, neutrophil aggregation, and bacterial fungal defense reactions. According to the Kyoto Encyclopedia of Genes and Genomes database, these genes are mainly involved in the metabolism of cysteine and methionine, RNA polymerization, and the MAPK signaling pathway, and were enriched in primary follicles. A microRNA-target network revealed that secondary follicles are involved in several important biological processes, such as the synthesis of keratin-associated proteins and enzymes involved in amino acid biosynthesis. In summary, these findings will increase our understanding of the complex molecular mechanisms of HF development and cycling, and provide a basis for the further study of the genes and functions of HF development.

  5. Cloning and Sequencing of the Pokeweed Antiviral Protein Gene and Its Expression in E. coli

    Institute of Scientific and Technical Information of China (English)

    CHEN Ding-hu; WANG Xi-feng; LI Li; ZHOU Guang-he


    The total RNA was isolated from pokeweed (Phytolacca americana ) leaves using the method of guanidine isothiocyanite and used as a template to amplify the deleted mutant pokeweed antiviral protein (PAP) gene by RT-PCR and then the gene was cloned into the pGEMR-T vector. The sequencing results showed that the PAP gene consisted of 711nt, which was 99.6% identical to the PAP gene reported by Lin et al (1991). The IPTG-inducible expression vector containing the PAP gene was constructed and transferred into the E. coli strain BL21 (DE3)-plysS. A specific protein was produced after induction with 0.4m mol/L IPTG and its molecular weight was 26ku. The results of the double diffusion on the agar plate and the western blotting test showed that the protein produced in E. coli was highly identical with the PAP extracted by a Frenchman from French pokeweed leaves. These revealed that PAP gene was actually achieved and exactly expressed in E . coli.

  6. Gene Expression Versus Sequence for Predicting Function:Glia Maturation Factor Gamma Is Not A Glia Maturation Factor

    Institute of Scientific and Technical Information of China (English)



    It is standard practice,whenever a researcher finds a new gene,to search databases for genes that have a similar sequence.It is not standard practice,whenever a researcher finds a new gene,to search for genes that have similar expression(coexpression).Failure to perform co-expression searches has lead to incorrect conclusions about the likely function of new genes,and has lead to wasted laboratory attempts to confirm functions incorrectly predicted.We present here the example of Glia Maturation Factor gamma(GMF-gamma).Despite its name,it has not been shown to participate in glia maturation.It is a gene of unknown function that is similar in sequence to GMF-beta.The sequence homology and chromosomal location led to an unsuccessful searchfor GMF-gamma mutations in glioma.We examined GMF-gamma expression in 1432 human cDNA libraries.Highest expression occurs in phagocytic,antigen-presenting and other hematopoietic cells.We found GMF-gamma mRNA in almost every tissue examined,with expression in nervous tissue no higher than in any other tissue.Our evidence indicates that GMF-gamma participates in phagocytosis in antigen presenting cells.Searches for genes with similar sequences should be supplemented with searches for genes with similar expression to avoid incorrect predictions.

  7. Gene Expression Versus Sequence for Predicting Function: Glia Maturation Factor Gamma Is Not A Glia Maturation Factor

    Institute of Scientific and Technical Information of China (English)

    Michael G. Walker


    It is standard practice, whenever a researcher finds a new gene, to search databases for genes that have a similar sequence. It is not standard practice, whenever a researcher finds a new gene, to search for genes that have similar expression (coexpression). Failure to perform co-expression searches has lead to incorrect conclusions about the likely function of new genes, and has lead to wasted laboratory attempts to confirm functions incorrectly predicted. We present here the example of Glia Maturation Factor gamma (GMF-gamma). Despite its name, it has not been shown to participate in glia maturation. It is a gene of unknown function that is similar in sequence to GMF-beta. The sequence homology and chromosomal location led to an unsuccessful search for GMF-gamma mutations in glioma.We examined GMF-gamma expression in 1432 human cDNA libraries. Highest expression occurs in phagocytic, antigen-presenting and other hematopoietic cells.We found GMF-gamma mRNA in almost every tissue examined, with expression in nervous tissue no higher than in any other tissue. Our evidence indicates that GMF-gamma participates in phagocytosis in antigen presenting cells. Searches for genes with similar sequences should be supplemented with searches for genes with similar expression to avoid incorrect predictions.

  8. Development of Expressed Sequence Tag (EST)-based Cleaved Amplified Polymorphic Sequence (CAPS) markers of tea plant and their application to cultivar identification. (United States)

    Ujihara, Tomomi; Taniguchi, Fumiya; Tanaka, Jun-Ichi; Hayashi, Nobuyuki


    To develop cleaved amplified polymorphic sequence (CAPS) markers for cultivar identification of the tea leaf, 5 primer pairs designed on the basis of genes that encode proteins related to nitrogen assimilation and 26 primer pairs based on expressed sequence tag (EST) sequences of the root of tea plant were screened. From combinations of primer pair and restriction enzyme that showed polymorphism among tea plants, 16 markers were selected and applied to DNA fingerprinting of Japanese tea cultivars. Sixty-three cultivars, except for a bud sport (Kiraka) and its original cultivar (Yabukita) and a pair that was the progeny of the same crossing parent (Harumoegi and Sakimidori), were distinguished from one another. By combining the 16 markers with previously developed CAPS markers and observing the physical appearance, 67 cultivars were distinguishable. The cultivars involve approximately 95% of total tea cultivating area in Japan; therefore, about 95% of tea leaves produced in Japan can be authenticated by labeling their cultivars.

  9. Long CAG repeat sequence and protein expression of androgen receptor considered as prognostic indicators in male breast carcinoma.

    Directory of Open Access Journals (Sweden)

    Yan-Ni Song

    Full Text Available BACKGROUND: The androgen receptor (AR expression and the CAG repeat length within the AR gene appear to be involved in the carcinogenesis of male breast carcinoma (MBC. Although phenotypic differences have been observed between MBC and normal control group in AR gene, there is lack of correlation analysis between AR expression and CAG repeat length in MBC. The purpose of the study was to investigate the prognostic value of CAG repeat lengths and AR protein expression. METHODS: 81 tumor tissues were used for immunostaining for AR expression and CAG repeat length determination and 80 normal controls were analyzed with CAG repeat length in AR gene. The CAG repeat length and AR expression were analyzed in relation to clinicopathological factors and prognostic indicators. RESULTS: AR gene in many MBCs has long CAG repeat sequence compared with that in control group (P = 0.001 and controls are more likely to exhibit short CAG repeat sequence than MBCs. There was statistically significant difference in long CAG repeat sequence between AR status for MBC patients (P = 0.004. The presence of long CAG repeat sequence and AR-positive expression were associated with shorter survival of MBC patients (CAG repeat: P = 0.050 for 5y-OS; P = 0.035 for 5y-DFS AR status: P = 0.048 for 5y-OS; P = 0.029 for 5y-DFS, respectively. CONCLUSION: The CAG repeat length within the AR gene might be one useful molecular biomarker to identify males at increased risk of breast cancer development. The presence of long CAG repeat sequence and AR protein expression were in relation to survival of MBC patients. The CAG repeat length and AR expression were two independent prognostic indicators in MBC patients.

  10. Population characteristics of channel catfish near the northern edge of their distribution: implications for management (United States)

    Carter-Lynn, K. P.; Quist, Michael


    Channel catfish, Ictalurus punctatus (Rafinesque), populations in six lakes in northern Idaho, USA, were sampled to describe their population characteristics. During the summers of 2011 and 2012, 4864 channel catfish were sampled. Channel catfish populations had low to moderate catch rates, and length structure was dominated by fish catfish were in good body condition. All populations were maintained by stocking age-1 or age-2 fish. Growth of fish reared in thermally enriched environments prior to stocking was fast compared to other North American channel catfish populations. After stocking, growth of channel catfish declined rapidly. Once stocked, cold water temperatures, prey resources and (or) genetic capabilities limited growth. Total annual mortality of age 2 and older channel catfish was generally catfish population dynamics and highlights important considerations associated with their ecology and management.

  11. Verification of otolith identity used by fisheries scientists for aging channel catfish (United States)

    Long, James M.; Stewart, David R.


    Previously published studies of the age estimation of channel catfish Ictalurus punctatus based on otoliths have reported using the sagittae, whereas it is likely they were actually using the lapilli. This confusion may have resulted because in catfishes (ostariophyseans) the lapilli are the largest of the three otoliths, whereas in nonostariophysean fish the sagittae are the largest. Based on (1) scanning electron microscope microphotographs of channel catfish otoliths, (2) X-ray computed tomography scans of a channel catfish head, (3) descriptions of techniques used to removed otoliths from channel catfish reported in the literature, and (4) a sample of channel catfish otoliths received from fisheries biologists from around the country, it is clear that lapilli are most often used for channel catfish aging studies, not sagittae, as has been previously reported. Fisheries scientists who obtain otoliths from channel catfish can use the information in this paper to correctly identify otolith age.

  12. Co-transcriptomic Analysis by RNA Sequencing to Simultaneously Measure Regulated Gene Expression in Host and Bacterial Pathogen

    KAUST Repository

    Ravasi, Timothy


    Intramacrophage pathogens subvert antimicrobial defence pathways using various mechanisms, including the targeting of host TLR-mediated transcriptional responses. Conversely, TLR-inducible host defence mechanisms subject intramacrophage pathogens to stress, thus altering pathogen gene expression programs. Important biological insights can thus be gained through the analysis of gene expression changes in both the host and the pathogen during an infection. Traditionally, research methods have involved the use of qPCR, microarrays and/or RNA sequencing to identify transcriptional changes in either the host or the pathogen. Here we describe the application of RNA sequencing using samples obtained from in vitro infection assays to simultaneously quantify both host and bacterial pathogen gene expression changes, as well as general approaches that can be undertaken to interpret the RNA sequencing data that is generated. These methods can be used to provide insights into host TLR-regulated transcriptional responses to microbial challenge, as well as pathogen subversion mechanisms against such responses.

  13. Identifying sugarcane expressed sequences associated with nutrient transporters and peptide metal chelators

    Directory of Open Access Journals (Sweden)

    Antonio Figueira


    Full Text Available Plant nutrient uptake is an active process, requiring energy to accumulate essential elements at higher levels in plant tissues than in the soil solution, while the presence of toxic metals or excess of nutrients requires mechanisms to modulate the accumulation of ions. Genes encoding ion transporters isolated from plants and yeast were used to identify sugarcane putative homologues in the sugarcane expressed sequence tag (SUCEST database. Five cluster consensi with sequence homology to plant high-affinity phosphate transporter genes were identified. One cluster consensus allowed the prediction of a full-length protein containing 541 amino acids, with 81% amino acid identity to the Nicotiana tabacum NtPT1 gene, consisting of 12 membrane-spanning domains divided by a large hydrophilic charged region. Putative homologues to Arabidopsis thaliana micronutrient transporter genes were also detected in some of the SUCEST libraries. Iron uptake in grasses involves the release of the phytosiderophore mugeneic acid (MA which chelate Fe3+ which is then absorbed by a specific transporter. Sugarcane expressed sequence tag (EST homologous to genes coding for three enzymes of the mugeneic acid biosynthetic pathway [nicotianamine synthase; nicotianamine transferase; and putative mugeneic acid synthetase (ids3] and a putative Fe3+-phytosiderophore transporter were detected. Seven sugarcane sequence clusters were identified with strong homology to members of the ZIP gene family (ZIP1, ZIP3, ZIP4, IRT1 and ZNT1, while four clusters homologous to ZIP2 and three to ZAT were found. Homologues to members of another gene family, Nramp, which code for broad-specificity transition metal transporters were also detected with constitutive expression. Partial transcripts homologous to genes encoding gamma-glutamylcysteine synthetase, glutathione synthetase, and phytochelatin synthase (responsible for biosynthesis of the metal chelator phytochelatin and all four types of the

  14. Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum. (United States)

    Wankhede, Dhammaprakash Pandhari; Biswas, Dipul Kumar; Rajkumar, Subramani; Sinha, Alok Krishna


    Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

  15. Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression. (United States)

    Li, Yue; Syed, Junetha; Suzuki, Yuki; Asamitsu, Sefan; Shioda, Norifumi; Wada, Takahito; Sugiyama, Hiroshi


    ATR-X (α-thalassemia/mental retardation X-linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α-globin gene cluster. The VNTR sequence, which contains the potential G-quadruplex-forming sequence CGC(GGGGCGGGG)n , is involved in the downregulation of α-globin expression. We investigated G-quadruplex and i-motif formation in single-stranded DNA and long double-stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G-quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G-quadruplex formation by the VNTR sequence downregulates the expression of α-globin genes and that ATRX might bind to and resolve the G-quadruplex.

  16. Price Volatility Spillover in Agricultural Markets: An Examination of U.S. Catfish Markets


    Buguk, Cumhur; Hudson, Darren; Hanson, Terrill R.


    Price volatility spillovers in the U.S. catfish supply chain are analyzed based on monthly price data from 1980 through 2000 for catfish feed, its ingredients, and farm- and wholesale-level catfish. The exponential generalized autoregressive conditional heteroskedasticity (EGARCH) model was used to test univariate volatility spillovers for prices in the supply chain. Strong price volatility spillover from feeding material (corn, soybeans, menhaden) to catfish feed and farm- and wholesale-leve...

  17. Transgene transmission in South American catfish (Rhamdia quelen) larvae by sperm-mediated gene transfer

    Indian Academy of Sciences (India)

    Tiago Collares; Vinicius Farias Campos; Fabiana Kömmling Seixas; Paulo V Cavalcanti; Odir A Dellagostin; Heden Luiz M Moreira; João Carlos Deschamps


    The silver catfish (Rhamdia quelen) is an endemic American fish species. The sperm of each species has its own peculiarities and biological characteristics, which influence the success of mass DNA transfer methods. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfish. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/rehydrated (DR), (2) dehydrated/rehydrated/electroporated (DRE), (3) electroporated (E), (4) incubated with seminal plasma (INC); and (5) incubated in the absence of seminal plasma (INCSP). Sperm motility, time of activity duration (TAD), fertilization rate (FR), hatching rate (HR) and sperm morphology were also evaluated. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%; DR 40%; E 25%; INC 5% and INCSP 25%. The rates of embryo EGFP expression were: DRE 63%; DR 44%; E 34%; INC 8% and INCSP 38%. The fertilization rate in the control and DRE treatments groups were higher than in the DR group, but the E, INC and INCSP treatment groups had the lowest rate. The hatching rates of the DRE, DR and control groups were higher than in the INCSP, INC and E treatment groups ( > 0.05). There were no differences among the DRE and DR, E and DR, E and INCSP groups in expression and PCR positivity rates of enhanced green fluorescent protein (EGFP) in embryos. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups. To the best of our knowledge, this is the first report on transgene transmission of exogenous DNA into silver catfish larvae through SMGT technology

  18. Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences. (United States)

    Marhaug, G; Husby, G; Dowton, S B


    The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.

  19. The oligodeoxynucleotide sequences corresponding to never-expressed peptide motifs are mainly located in the non-coding strand

    Directory of Open Access Journals (Sweden)

    Bickis Mik


    Full Text Available Abstract Background We study the usage of specific peptide platforms in protein composition. Using the pentapeptide as a unit of length, we find that in the universal proteome many pentapeptides are heavily repeated (even thousands of times, whereas some are quite rare, and a small number do not appear at all. To understand the physico-chemical-biological basis underlying peptide usage at the proteomic level, in this study we analyse the energetic costs for the synthesis of rare and never-expressed versus frequent pentapeptides. In addition, we explore residue bulkiness, hydrophobicity, and codon number as factors able to modulate specific peptide frequencies. Then, the possible influence of amino acid composition is investigated in zero- and high-frequency pentapeptide sets by analysing the frequencies of the corresponding inverse-sequence pentapeptides. As a final step, we analyse the pentadecamer oligodeoxynucleotide sequences corresponding to the never-expressed pentapeptides. Results We find that only DNA context-dependent constraints (such as oligodeoxynucleotide sequence location in the minus strand, introns, pseudogenes, frameshifts, etc. provide a coherent mechanistic platform to explain the occurrence of never-expressed versus frequent pentapeptides in the protein world. Conclusions This study is of importance in cell biology. Indeed, the rarity (or lack of expression of specific 5-mer peptide modules implies the rarity (or lack of expression of the corresponding n-mer peptide sequences (with n

  20. Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase

    Directory of Open Access Journals (Sweden)

    Garg Neha


    Full Text Available Abstract Background Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase. Results In this study, complete cDNA encoding laccase (Lac from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac. Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac. Conclusion Laccase of C. bulleri was

  1. 40 CFR 408.10 - Applicability; description of the farm-raised catfish processing subcategory. (United States)


    ...-raised catfish processing subcategory. 408.10 Section 408.10 Protection of Environment ENVIRONMENTAL... SOURCE CATEGORY Farm-Raised Catfish Processing Subcategory § 408.10 Applicability; description of the farm-raised catfish processing subcategory. The provisions of this subpart are applicable to...

  2. Detection and quantification of virulent Aeromonas hydrophila in channel catfish tissues following waterborne challenge (United States)

    The aim of this study was to understand the pathogenesis of motile aeromonas septicemia caused by virulent A. hydrophila (vAh) in channel catfish, Ictalurus punctatus. Adipose fin clipped catfish were challenged with vAh using waterborne challenge method and the distribution of vAh in catfish tissue...

  3. Pyrethroid toxicity in silver catfish, Rhamdia quelen

    Directory of Open Access Journals (Sweden)

    Francisco P. Montanha


    Full Text Available This study aimed to determine both the lethal and sublethal concentrations of Cypermethrin in young Silver Catfish (Brazilian "Jundiá", Rhamdia quelen on aquatic environment during 96 hours, as well as to determine the Cypermethrin and Deltamethrin sublethal concentrations during the initial embryonic development period of Rhamdia quelen, and to verify their respective rates of fertilization, hatching and survival. Pyrethroid nowadays is a widely used insecticide, which presents a high toxicity to fish. In order to determine lethal and sublethal concentrations, 120 silver catfish were used; each one had an average weight of 59.58±4.50g and an average size of 20.33±2.34cm. Concentrations used were 0, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0, 10.0, 15.0 and 20.0mg of Cypermethrin per liter of water (mg/L. Fish were exposed to the product in 30-liter fish tanks. In each fish tank there were four fishes and the product was applied three times, i.e., a total of twelve fish were exposed to the product at each application, and a total of 120 fish during the entire experiment (n=120. In order to determine the Cypermethrin and Deltamethrin sublethal concentrations during the initial embryonic development, ovulation induction was performed on female fishes using hormones, and then and egg collection was performed. The eggs were then hydrated and fertilized in Cypermethrin and Deltamethrin in different concentrations: 0.001, 0.01, 0.1, 1.0 and 10.0mg/L of Cypermethrin and 0.001, 0.01, 0.1, 0.5 and 1.0mg/L of Deltamethrin, in addition to the control group (0mg/L. After fertilization, the eggs were kept in containers with the respective pesticides of Cypermethrin and Deltamethrin until hatching, when hatching rate was verified. Then the alevins, from the hatching, were kept on their respective concentrations of Cypermethrin and Deltamethrin so that the survival rate could be analyzed regarding the tested insecticides, during both 12-hour and 24-hour periods

  4. Acute toxicity of copper to sea catfish

    Energy Technology Data Exchange (ETDEWEB)

    Steele, C.W.


    Sea catfish (Arius felis) were exposed to aqueous solutions of reagent grade cupric chloride in artificial seawater (30.0 +/- 2.0 0/00, 21-23/sup 0/C) in four static bioassays. The 24, 48, 72 and 96 h LC/sub 50/ were calculated and found to be 5.43, 4.17, 3.57 and 2.40 mg 1./sup -1/ copper, respectively. Experimental concentrations of copper producing subtle behavioral changes in this species correspond to less than 0.3% of the 72 h LC/sub 50/. Based on this comparison with literature values, a new, maximum 'safe' concentration for copper in marine waters of 0.01 mg 1./sup -1/ is proposed.

  5. Channel catfish hemoglobin genes: identification, phylogenetic and syntenic analysis, and specific induction in response to heat stress. (United States)

    Feng, Jianbin; Liu, Shikai; Wang, Xiuli; Wang, Ruijia; Zhang, Jiaren; Jiang, Yanliang; Li, Chao; Kaltenboeck, Ludmilla; Li, Jiale; Liu, Zhanjiang


    Hemoglobins transport oxygen from gill to inner organs in fish, and this process is affected by temperature, one of the major environmental factors for fish. The hemoglobin gene clusters have been well studied in humans and several model fish species, but remain largely unknown in catfish. Here, eight α- and six β-hemoglobin genes were identified and characterized in channel catfish. Genomic synteny analysis showed that these hemoglobin genes were separated into two unlinked clusters, the MN cluster containing six α- and six β-hemoglobin genes, and the LA cluster consisting of two α-hemoglobin genes. Channel catfish hemoglobin genes were ubiquitously expressed in all the 10 tested tissues from healthy fish, but exhibited higher expression level in spleen, head kidney, and trunk kidney. In response to heat stress, hemoglobin genes, especially MN Hbα4, MN Hbα5, MN Hbα6, MN Hbβ4, MN Hbβ5, MN Hbβ6, LA Hbα1, and LA Hbα2, presumably the embryonic hemoglobin genes, were drastically up-regulated in the gill and head kidney of heat-tolerant fishes, but not in these tissues of the heat-intolerant fish, suggesting the importance of the embryonic hemoglobin genes in coping with the low oxygen conditions under heat stress.

  6. Human chorionic somatomammotropin and growth hormone gene expression in rat pituitary tumor cells is dependent on proximal promoter sequences

    Energy Technology Data Exchange (ETDEWEB)

    Nachtigal, M.W.; Nickel, B.E.; Klassen, M.E.; Cattini, P.A. (Univ. of Manitoba, Winnipeg (Canada)); Zhang, Wengang; Eberhardt, N.L. (Univ. of California, San Francisco (USA))


    Human placental chorionic somatomammotropin (hCS-A or hCS-B) and pituitary growth hormone (hGH-N) are related by structure and function. The hCS-A gene is expressed in rat pituitary tumor (GC) cells after gene transfer. Deletion of hCS-A 5{prime}-flanking DNA reveals repressor activity upstream of nucleotide {minus}132, and a region essential for expression in GC cells between nucleotides {minus}94 and {minus}61. The sequences in this region differ from the equivalent hGH-N gene DNA by one nucleotide, and include the binding site for a pituitary-specific factor (GHF-1), required for hGH-N expression in GC cells. Exchange of hGH-N with hCS-A gene DNA in this region maintains expression in GC cells. By contrast, modification of these sequences blocks expression. These data indicate that proximal promoter sequences, equivalent to those bound by GHF-1 on the hGH-N gene, are required for hCS-A expression in GC cells.

  7. Manipulating the Prion Protein Gene Sequence and Expression Levels with CRISPR/Cas9.

    Directory of Open Access Journals (Sweden)

    Lech Kaczmarczyk

    Full Text Available The mammalian prion protein (PrP, encoded by Prnp is most infamous for its central role in prion diseases, invariably fatal neurodegenerative diseases affecting humans, food animals, and animals in the wild. However, PrP is also hypothesized to be an important receptor for toxic protein conformers in Alzheimer's disease, and is associated with other clinically relevant processes such as cancer and stroke. Thus, key insights into important clinical areas, as well as into understanding PrP functions in normal physiology, can be obtained from studying transgenic mouse models and cell culture systems. However, the Prnp locus is difficult to manipulate by homologous recombination, making modifications of the endogenous locus rarely attempted. Fortunately in recent years genome engineering technologies, like TALENs or CRISPR/Cas9 (CC9, have brought exceptional new possibilities for manipulating Prnp. Herein, we present our observations made during systematic experiments with the CC9 system targeting the endogenous mouse Prnp locus, to either modify sequences or to boost PrP expression using CC9-based synergistic activation mediators (SAMs. It is our hope that this information will aid and encourage researchers to implement gene-targeting techniques into their research program.

  8. Bioinformatic analysis of expressed sequence tags from sporophyte of Porphyra yezoensis (Bagiaceae, Rhodophyta)

    Institute of Scientific and Technical Information of China (English)

    XU Minjun; MAO Yunxiang; ZHANG Xuecheng; ZHOU Xiaojun; SUI Zhenghong; ZHOU Hailin; LI Jinhong


    A total of 719 expressed sequence tags (EST) clustered into 329 non-redundant EST groups are obtained from the sporophyte cDNA library of red algae, Porphyra yezoensis. Gene Ontology (GO) analysis is employed in characterizing 60 strictest annotated unique genes out of the 329 EST groups and some domains such as COX1, Sod_ Fe-C, GST-N, SHMT, and RNase_ PH related to the enz ymes and proteins functioning in cells have been identified by HMMPFAM search. As its leafy gametophyte, the similar codon usage with strong bias is found in P. yezoensis filamentous sporophyte, regardless of some differences found in given amino acids. The average GC content of the 329 unique genes is 53.0 %. In contrast, the third nucleotide of codon exhibits a higher GC content (72 % ) than that of the first (58 % ) and the second (42 % ) nucleotides. Similarity search of the present study shows a novel EST ratio of 60.2 %,which is against the Porphyra ESTs database, suggesting further investigations towards elucidating the characteristics of Porphyra functional genome.

  9. Analysis of expressed sequence tags from Citrus sinensis L. Osbeck infected with Xylella fastidiosa

    Directory of Open Access Journals (Sweden)

    Alessandra A. de Souza


    Full Text Available In order to understand the genetic responses resulting from physiological changes that occur in plants displaying citrus variegated chlorosis (CVC symptoms, we adopted a strategy of comparing two EST libraries from sweet orange [Citrus sinensis (L. Osbeck]. One of them was prepared with plants showing typical CVC symptoms caused by Xylella fastidiosa and the other with non-inoculated plants. We obtained 15,944 ESTs by sequencing the two cDNA libraries. Using an in silico hybridization strategy, 37 genes were found to have significant variation at the transcriptional level. Within this subset, 21 were up-regulated and 16 were down-regulated in plants with CVC. The main functional categories of the down-regulated transcripts in plants with CVC were associated with metabolism, protein modification, energy and transport facilitation. The majority of the up-regulated transcripts were associated with metabolism and defense response. Some transcripts associated with adaptation to stress conditions were up-regulated in plants with CVC and could explain why plants remain alive even under severe water and nutritional stress. Others of the up-regulated transcripts are related to defense response suggesting that sweet orange plants activate their defense machinery. The genes associated with stress response might be expressed as part of a secondary response related to physiological alterations caused by the infection.

  10. Sequence motifs and prokaryotic expression of the reptilian paramyxovirus fusion protein (United States)

    Franke, J.; Batts, W.N.; Ahne, W.; Kurath, G.; Winton, J.R.


    Fourteen reptilian paramyxovirus isolates were chosen to represent the known extent of genetic diversity among this novel group of viruses. Selected regions of the fusion (F) gene were sequenced, analyzed and compared. The F gene of all isolates contained conserved motifs homologous to those described for other members of the family Paramyxoviridae including: signal peptide, transmembrane domain, furin cleavage site, fusion peptide, N-linked glycosylation sites, and two heptad repeats, the second of which (HRB-LZ) had the characteristics of a leucine zipper. Selected regions of the fusion gene of isolate Gono-GER85 were inserted into a prokaryotic expression system to generate three recombinant protein fragments of various sizes. The longest recombinant protein was cleaved by furin into two fragments of predicted length. Western blot analysis with virus-neutralizing rabbit-antiserum against this isolate demonstrated that only the longest construct reacted with the antiserum. This construct was unique in containing 30 additional C-terminal amino acids that included most of the HRB-LZ. These results indicate that the F genes of reptilian paramyxoviruses contain highly conserved motifs typical of other members of the family and suggest that the HRB-LZ domain of the reptilian paramyxovirus F protein contains a linear antigenic epitope. ?? Springer-Verlag 2005.

  11. Characterization of Shigella type 1 fimbriae: expression, FimA sequence, and phase variation. (United States)

    Snellings, N J; Tall, B D; Venkatesan, M M


    This study documents the presence of type 1 fimbriae on Shigella and confirms these mannose-sensitive adherence structures to be bona fide components of the Shigella surface. While laboratory-passaged Shigella strains and lyophilized clinical isolates failed to express type 1 fimbriae, 6 of 20 recent clinical isolates, including 4 Shigella flexneri strains, 1 Shigella boydii strain, and 1 Shigella dysenteriae strain, produced type 1 fimbriae as detected by mannose-sensitive hemagglutination (MSHA) and electron microscopy. Optimal production of a predominantly Fim+ population required serial passage every 48 to 72 h in unshaken brain heart infusion broth at 37 degrees C. Fim+ Shigella cultures were capable of reversibly switching to a non-MSHA, afimbriated phase during serial aerobic cultivation on tryptic soy agar plates. The amino acid sequence of S. flexneri type 1 FimA contained 18 substitutions compared to that of Escherichia coli fimbrillin. Indirect immunoelectron microscopy suggested the presence of both shared and unique epitopes on E. coli and S. flexneri type 1 fimbriae. Random phase variation between fimbriated and afimbriated states in Shigella was accompanied by the genomic rearrangement associated with phase variation in E. coli.

  12. Sequence and expression characteristics of a nuclear-encoded chloroplast sigma factor from mustard (Sinapis alba). (United States)

    Kestermann, M; Neukirchen, S; Kloppstech, K; Link, G


    Plant chloroplasts contain transcription factors that functionally resemble bacterial sigma factors. We have cloned the full-length cDNA from mustard (Sinapis alba) for a 53 kDa derived polypeptide that contains similarity to regions 1.2-4.2 of sigma70-type factors. The amino acid sequence at the N-terminus has characteristics of a chloroplast transit peptide. An in vitro synthesized polypeptide containing this region was shown to be imported into the chloroplast and processed. The recombinant factor lacking the N-terminal extension was expressed in Escherichia coli and purified. It confers the ability on E.coli core RNA polymerase to bind specifically to a DNA fragment that contains the chloroplast psbA promoter. Transcription of the psbA template by E.coli core enzyme in the presence of recombinant SIG1 results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site. Together, these data suggest that the mature protein acts as one of the chloroplast transcription factors in mustard. RNA gel blot hybridization reveals a transcript at approximately 1.8 kb, which is more abundant in light-grown than in dark-grown mustard seedlings.


    Institute of Scientific and Technical Information of China (English)

    肖明松; 崔峰; 康健; 马玉涵


    The Chinese longsnout catfish is a semi-migratory fish which is commercially valuable in China. Due to overfishing, environmental pollution, and other human disturbances, the populations of this species have declined rapidly and disappeared in many river systems in the past decades. Currently, the Chinese longsnout catfish mainly inhabits the main streams of the Yangtze River and rarely found in lakes. At present, the Chinese longsnout catfish achieved appropriate scale farming in Sichuan, Guangdong and other places. However, seldom study was reported about analysis of population genetic structure using molecular markers. To protect and exploit this rare species effectively, investigations on population structures, resources and artificial reproduction have been conducted. In this study, the mitochon-drial DNA control region were used to analyze genetic diversity and structure of 7 cultivated and natural populations of Chinese longsnout catfish collected from Meishan, Shishou, Huainan, Chongqing, Wuhan, Anqing and Nanjing named Meishan population, Shishou population, Huainan population, Chongqing population, Wuhan population, Anqing population and Nanjing population separately. The results showed the length of this region (D-loop) contained 790 bp nu-cleotides and the T, C, A and G contents were 31.5%, 25.3%, 29.1% and 14.1% respectively. Twenty-seven nucleotide sites and 18 haplotypes were found in 3 cultivated populations of Chinese longsnout catfish. Thirty-five nucleotide sites and 36 haplotypes were found in 4 natural populations of Chinese longsnout catfish. The average haplotype diversity and nucleotide diversity of cultivated populations of Chinese longsnout catfish were relatively low (Hd=0.8867±0.0013, Pi =0.0056±0.0013). The level of genetic differentiation was relatively low (0.0014-0.1125). Molecular phylogenetic tree and statistical parsimony network constructed by NJ method and statistical parsimony principles showed individuals from the same stock

  14. CASCAD : a database of annotated candidate single nucleotide polymorphisms associated with expressed sequences

    NARCIS (Netherlands)

    Guryev, Victor; Berezikov, Eugene; Cuppen, Edwin


    BACKGROUND: With the recent progress made in large-scale genome sequencing projects a vast amount of novel data is becoming available. A comparative sequence analysis, exploiting sequence information from various resources, can be used to uncover hidden information, such as genetic variation. Althou

  15. Expressed sequence tag-derived polymorphic SSR markers for Fucus serratus and amplification in other species of Fucus

    NARCIS (Netherlands)

    Coyer, J. A.; Hoarau, G.; Beszteri, B.; Pearson, G.; Olsen, J. L.


    The seaweed genus Fucus is a dominant component of intertidal shores throughout the North Atlantic and North Pacific and has been the focus of considerable developmental, ecological, and evolutionary research for the past century. Here, we present details of 21 expressed sequence tag-derived simple

  16. Detecting cognizable trends of gene expression in a time series RNA-sequencing experiment: a bootstrap approach

    Indian Academy of Sciences (India)



    Study of temporal trajectory of gene expression is important. RNA sequencing is popular in genome-scale studies of transcription. Because of high expenses involved, many time-course RNA sequencing studies are challenged by inadequacy of sample sizes. This poses difficulties in conducting formal statistical tests of significance of null hypotheses. We propose a bootstrap algorithm to identify ‘cognizable’ ‘time-trends’ of gene expression. Properties of the proposed algorithm are derived using a simulation study. The proposed algorithm captured known ‘time-trends’ in the simulated data with a high probability of success, even when sample sizes were small (n<10). The proposed statistical method is efficient and robust to capture ‘cognizable’ ‘time-trends’ in RNA sequencing data.

  17. Autochthonous Gut Bacteria in Two Indian Air-breathing Fish, Climbing Perch (Anabas testudineus) and Walking Catfish (Clarias batrachus): Mode of Association, Identification and Enzyme Producing Ability. (United States)

    Banerjee, Goutam; Dan, Suhas K; Nandi, Ankita; Ghosh, Pinki; Ray, Arun K


    Scanning electron microscopy (SEM) was used to define the location of epithelium-associated bacteria in the gastrointestinal (GI) tract of two Indian air-breathing fish, the climbing perch (Anabas testudineus) and walking catfish (Clarias batrachus). The SEM examination revealed substantial numbers of rod shaped bacterial cells associated with the microvillus brush borders of enterocytes in proximal (PI) and distal regions (DI) of the GI tract of both the fish species. Ten (two each from the PI and DI of climbing perch and three each from the PI and DI of walking catfish) isolated bacterial strains were evaluated for extracellular protease, amylase and cellulase production quantitatively. All the bacterial strains exhibited high cellulolytic activity compared to amylolytic and proteolytic activites. Only two strains, CBH6 and CBH7, isolated from the DI of walking catfish exhibited high proteolytic activity. Maximum cellulase activity was exhibited by the strain, CBF2, isolated from the PI of climbing perch. Six most promising enzyme-producing adherent bacterial strains were identified by 16S rDNA gene sequence analysis. The strain ATH1 (isolated from climbing perch) showed high similarity fo Bacillus amyloliquefaciens whereas, the remaining five strains (isolated from walking catfish) were most closely related to Bacillus licheniformis.

  18. Effect of type and concentration of water hardness on the fertilization and hatching success of channel X blue F1 hybrid catfish eggs (United States)

    Consistent and improved performance of channel x blue F1 hybrid catfish fingerlings in production ponds in the US farm raised catfish industry has prompted an increase in demand of hybrid catfish fingerlings even at higher prices compared to commonly raised channel catfish. Hybrid catfish fry are e...

  19. Suspending mammalian LHRHa-injected channel catfish, Ictalurus punctatus in individual nylon-mesh bag reduces stress and improves reproductive performance (United States)

    Hormone induced spawning of channel catfish held communally in tanks is a reliable method to produce channel catfish, Ictalurus punctatus ' x blue catfish, I. furcatus ' F1 hybrid catfish fry. However, mature catfish are crowded, and repeatedly handled during the process of induced ovulation. Repe...

  20. Expressed Sequence Tags Analysis and Design of Simple Sequence Repeats Markers from a Full-Length cDNA Library in Perilla frutescens (L.

    Directory of Open Access Journals (Sweden)

    Eun Soo Seong


    Full Text Available Perilla frutescens is valuable as a medicinal plant as well as a natural medicine and functional food. However, comparative genomics analyses of P. frutescens are limited due to a lack of gene annotations and characterization. A full-length cDNA library from P. frutescens leaves was constructed to identify functional gene clusters and probable EST-SSR markers via analysis of 1,056 expressed sequence tags. Unigene assembly was performed using basic local alignment search tool (BLAST homology searches and annotated Gene Ontology (GO. A total of 18 simple sequence repeats (SSRs were designed as primer pairs. This study is the first to report comparative genomics and EST-SSR markers from P. frutescens will help gene discovery and provide an important source for functional genomics and molecular genetic research in this interesting medicinal plant.



    Carel LIGEON; Jolly, Curtis M.; Jackson, John D.


    The effects of increased exports from NAFTA member countries on the U.S. domestic catfish industry were evaluated. Results showed that the quantity of catfish imported will fall if the domestic price of catfish falls relative to the import price. Past imports have no effect on present imports. The income elasticity was negative indicating that imported catfish may be an inferior good. Doubling present levels of imports from NAFTA member countries is not a threat to the U.S. catfish industry.

  2. Generation and Analysis of Expressed Sequence Tags (ESTs) from Muscle Full-Length cDNA Library of Wujin Pig

    Institute of Scientific and Technical Information of China (English)

    ZHAO Su-mei; LIU Yong-gang; PAN Hong-bing; ZHANG Xi; GE Chang-rong; JIA Jun-jing; GAO Shi-zheng


    Porcine skeletal muscle genes play a major role in determining muscle growth and meat quality. Construction of a full-length cDNA library is an effective way to understand the expression of functional genes in muscle tissues. In addition, novel genes for further research could be identiifed in the library. In this study, we constructed a full-length cDNA library from porcine muscle tissue. The estimated average size of the cDNA inserts was 1076 bp, and the cDNA fullness ratio was 86.2%. A total of 1058 unique sequences with 342 contigs (32.3%) and 716 singleton (67.7%) expressed sequence tags (EST) were obtained by clustering and assembling. Meanwhile, 826 (78.1%) ESTs were categorized as known genes, and 232 (21.9%) ESTs were categorized as unknown genes. 65 novel porcine genes that exhibit no identity in the TIGR gene index ofSus scrofa and 124 full-length sequences with unknown functions were deposited in the dbEST division of GenBank (accession numbers: EU650784-EU650788, GE843306, GH228978-GH229100). The abundantly expressed genes in porcine muscle tissue were related to muscle ifber development, energy metabolism and protein synthesis. Gene ontology analysis showed that sequences expressed in porcine muscle tissue contained a high percentage of binding activity, catalytic activity, structural molecule activity and motor activity, which involved mainly in metabolic, cellular and developmental process, distributed mainly in intracellular region. The sequence data generated in this study would provide valuable information for identifying porcine genes expressed in muscle tissue and help to advance the study on the structure and function of genes in pigs.

  3. High-density interspecific genetic linkage mapping provides insights into genomic incompatibility between channel catfish and blue catfish. (United States)

    Liu, S; Li, Y; Qin, Z; Geng, X; Bao, L; Kaltenboeck, L; Kucuktas, H; Dunham, R; Liu, Z


    Catfish is the leading aquaculture species in the United States. The interspecific hybrid catfish produced by mating female channel catfish with male blue catfish outperform both of their parent species in a number of traits. However, mass production of the hybrids has been difficult because of reproductive isolation. Investigations of genome structure and organization of the hybrids provide insights into the genetic basis for maintenance of species divergence in the face of gene flow, thereby helping develop strategies for introgression and efficient production of the hybrids for aquaculture. In this study, we constructed a high-density genetic linkage map using the hybrid catfish system with the catfish 250K SNP array. A total of 26,238 SNPs were mapped to 29 linkage groups, with 12,776 unique marker positions. The linkage map spans approximately 3240 cM with an average intermarker distance of 0.25 cM. A fraction of markers (986 of 12,776) exhibited significant deviation from the expected Mendelian ratio of segregation, and they were clustered in major genomic blocks across 15 LGs, most notably LG9 and LG15. The distorted markers exhibited significant bias for maternal alleles among the backcross progenies, suggesting strong selection against the blue catfish alleles. The clustering of distorted markers within genomic blocks should lend insights into speciation as marked by incompatibilities between the two species. Such findings should also have profound implications for understanding the genomic evolution of closely related species as well as the introgression of hybrid production programs in aquaculture.

  4. A survey of canine expressed sequence tags and a display of their annotations through a flexible web-based interface. (United States)

    Palmer, L E; O'Shaughnessy, A L; Preston, R R; Santos, L; Balija, V S; Nascimento, L U; Zutavern, T L; Henthorn, P S; Hannon, G J; McCombie, W R


    We have initially sequenced approximately 8,000 canine expressed sequence tags (ESTs) from several complementary DNA (cDNA) libraries: testes, whole brain, and Madin-Darby canine kidney (MDCK) cells. Analysis of these sequences shows that they provide partial sequence information for about 5%-10% of the canine genes. An analysis pipeline has been created to cluster the ESTs and to map individual ESTs as well as clustered ESTs to both the human genome and the human proteome. Gene ontology (GO) terms have been assigned to the ESTs and clusters based on their top matches to the International Protein Index (IPI) set of human proteins. The data generated is stored in a MySQL relational database for analysis and display. A Web-based Perl script has been written to display the analyzed data to the scientific community.

  5. Generation and analysis of expressed sequence tags from a cDNA library of the fruiting body of Ganoderma lucidum

    Directory of Open Access Journals (Sweden)

    Li Xiwen


    Full Text Available Abstract Background Little genomic or trancriptomic information on Ganoderma lucidum (Lingzhi is known. This study aims to discover the transcripts involved in secondary metabolite biosynthesis and developmental regulation of G. lucidum using an expressed sequence tag (EST library. Methods A cDNA library was constructed from the G. lucidum fruiting body. Its high-quality ESTs were assembled into unique sequences with contigs and singletons. The unique sequences were annotated according to sequence similarities to genes or proteins available in public databases. The detection of simple sequence repeats (SSRs was preformed by online analysis. Results A total of 1,023 clones were randomly selected from the G. lucidum library and sequenced, yielding 879 high-quality ESTs. These ESTs showed similarities to a diverse range of genes. The sequences encoding squalene epoxidase (SE and farnesyl-diphosphate synthase (FPS were identified in this EST collection. Several candidate genes, such as hydrophobin, MOB2, profilin and PHO84 were detected for the first time in G. lucidum. Thirteen (13 potential SSR-motif microsatellite loci were also identified. Conclusion The present study demonstrates a successful application of EST analysis in the discovery of transcripts involved in the secondary metabolite biosynthesis and the developmental regulation of G. lucidum.

  6. Isolation, Sequence Analysis and Expression Profile of a Novel Swine Gene Differentially Expressed in the Longissimus Dorsi Muscle Tissues from Landrace×Large White Cross-combination

    Institute of Scientific and Technical Information of China (English)

    Yong-Gang LIU; Yuan-Zhu XIONG; Chang-Yan DENG


    The mRNA differential display technique was performed to investigate the differences in gene expression in the Longissimus dorsi muscle tissues from Landrace×Large White cross-combination. One novel gene that was differentially expressed was identified using semi-quantitative reverse transcriptasepolymerase chain reaction (RT-PCR) and its complete cDNA sequence was obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 260 amino acids that contains the putative conserved domain of the carbonic anhydrase,and this protein has high homology with the carbonic anhydrase Ⅲ (CA-Ⅲ) of four species-mouse (91%),horse (91%), rat (89%) and human (86%)-so that it can be defined as swine carbonic anhydrase Ⅲ. The phylogenetic tree analysis revealed that the swine CA-Ⅲ has a closer genetic relationship with the horse CA-Ⅲ than with those of mouse, rat and human. The tissue expression analysis indicated that the swine CA-Ⅲ gene is generally expressed in most tissues. Our experiment is the first to establish the primary foundation for further research on the swine CA-Ⅲ gene.

  7. Genome-wide analysis reveals diverged patterns of codon bias, gene expression, and rates of sequence evolution in picea gene families. (United States)

    De La Torre, Amanda R; Lin, Yao-Cheng; Van de Peer, Yves; Ingvarsson, Pär K


    The recent sequencing of several gymnosperm genomes has greatly facilitated studying the evolution of their genes and gene families. In this study, we examine the evidence for expression-mediated selection in the first two fully sequenced representatives of the gymnosperm plant clade (Picea abies and Picea glauca). We use genome-wide estimates of gene expression (>50,000 expressed genes) to study the relationship between gene expression, codon bias, rates of sequence divergence, protein length, and gene duplication. We found that gene expression is correlated with rates of sequence divergence and codon bias, suggesting that natural selection is acting on Picea protein-coding genes for translational efficiency. Gene expression, rates of sequence divergence, and codon bias are correlated with the size of gene families, with large multicopy gene families having, on average, a lower expression level and breadth, lower codon bias, and higher rates of sequence divergence than single-copy gene families. Tissue-specific patterns of gene expression were more common in large gene families with large gene expression divergence than in single-copy families. Recent family expansions combined with large gene expression variation in paralogs and increased rates of sequence evolution suggest that some Picea gene families are rapidly evolving to cope with biotic and abiotic stress. Our study highlights the importance of gene expression and natural selection in shaping the evolution of protein-coding genes in Picea species, and sets the ground for further studies investigating the evolution of individual gene families in gymnosperms.

  8. Water quality and plankton communities in hybrid catfish (female channel catfish, Ictalurus punctatus x male blue catfish, I. furcatus) ponds after partial fish harvest (United States)

    Twelve, 0.4-ha ponds were stocked with 10,000 hybrid catfish fingerlings in March 2015. Six ponds were partially harvested in August to remove fish larger than ~ 0.57 kg. All remaining fish were removed in October and November. Partial harvest of faster-growing fish removed ~26% of fish initially st...

  9. Sugarcane expressed sequences tags (ESTs encoding enzymes involved in lignin biosynthesis pathways

    Directory of Open Access Journals (Sweden)

    Ramos Rose Lucia Braz


    Full Text Available Lignins are phenolic polymers found in the secondary wall of plant conductive systems where they play an important role by reducing the permeability of the cell wall to water. Lignins are also responsible for the rigidity of the cell wall and are involved in mechanisms of resistance to pathogens. The metabolic routes and enzymes involved in synthesis of lignins have been largely characterized and representative genes that encode enzymes involved in these processes have been cloned from several plant species. The synthesis of lignins is liked to the general metabolism of the phenylpropanoids in plants, having enzymes (e.g. phenylalanine ammonia-lyase (PAL, cinnamate 4-hydroxylase (C4H and caffeic acid O-methyltransferase (COMT common to other processes as well as specific enzymes such as cinnamoyl-CoA reductase (CCR and cinnamyl alcohol dehydrogenase (CAD. Some maize and sorghum mutants, shown to have defective in CAD and/or COMT activity, are easier to digest because they have a reduced lignin content, something which has motivated different research groups to alter the lignin content and composition of model plants by genetic engineering try to improve, for example, the efficiency of paper pulping and digestibility. In the work reported in this paper, we have made an inventory of the sugarcane expressed sequence tag (EST coding for enzymes involved in lignin metabolism which are present in the sugarcane EST genome project (SUCEST database. Our analysis focused on the key enzymes ferulate-5-hydroxylase (F5H, caffeic acid O-methyltransferase (COMT, caffeoyl CoA O-methyltransferase (CCoAOMT, hydroxycinnamate CoA ligase (4CL, cinnamoyl-CoA reductase (CCR and cinnamyl alcohol dehydrogenase (CAD. The comparative analysis of these genes with those described in other species could be used as molecular markers for breeding as well as for the manipulation of lignin metabolism in sugarcane.

  10. A hybrid distance measure for clustering expressed sequence tags originating from the same gene family.

    Directory of Open Access Journals (Sweden)

    Keng-Hoong Ng

    Full Text Available BACKGROUND: Clustering is a key step in the processing of Expressed Sequence Tags (ESTs. The primary goal of clustering is to put ESTs from the same transcript of a single gene into a unique cluster. Recent EST clustering algorithms mostly adopt the alignment-free distance measures, where they tend to yield acceptable clustering accuracies with reasonable computational time. Despite the fact that these clustering methods work satisfactorily on a majority of the EST datasets, they have a common weakness. They are prone to deliver unsatisfactory clustering results when dealing with ESTs from the genes derived from the same family. The root cause is the distance measures applied on them are not sensitive enough to separate these closely related genes. METHODOLOGY/PRINCIPAL FINDINGS: We propose a hybrid distance measure that combines the global and local features extracted from ESTs, with the aim to address the clustering problem faced by ESTs derived from the same gene family. The clustering process is implemented using the DBSCAN algorithm. We test the hybrid distance measure on the ten EST datasets, and the clustering results are compared with the two alignment-free EST clustering tools, i.e. wcd and PEACE. The clustering results indicate that the proposed hybrid distance measure performs relatively better (in terms of clustering accuracy than both EST clustering tools. CONCLUSIONS/SIGNIFICANCE: The clustering results provide support for the effectiveness of the proposed hybrid distance measure in solving the clustering problem for ESTs that originate from the same gene family. The improvement of clustering accuracies on the experimental datasets has supported the claim that the sensitivity of the hybrid distance measure is sufficient to solve the clustering problem.

  11. Book review: Conservation, ecology, and management of catfish: The second international symposium (United States)

    Long, James M.


    This is the second publication in a series published by the American Fisheries Society devoted to catfish research, biology, and management, which was organized as a symposium held in St. Louis Missouri, in 2010 and builds upon the first symposium that was held in Davenport, Iowa, in 1998 (Irwin et al. 1999).  The organizations of the second symposium stated that this was to "serve as an addendum to Catfish 2000" and "to cover the four corners of catfish science: catfish biology, ecology, management, and conservation" (p. xi).  The book meets this standard easily.  With 64 pages organized into nine sections (plenary, catfishes as sport fish, non-game catfishes, nonnative catfishes, movement and habitat use, sampling and population assessment, age and growth, behavior, and future directions), this publication covers these four corners and more.

  12. Edwardsiella ictaluri infection in Pangasius catfish imported from West Bengal into the Southern Caribbean. (United States)

    Phillips, A C N; Reichley, S R; Ware, C; Griffin, M J


    In response to a mortality event, seven Pangasius catfish (Pangasianodon hypophthalmus) were submitted to the University of the West Indies, School of Veterinary Medicine, Trinidad and Tobago, for diagnostic evaluation. These fish were part of a consignment that arrived from Kolkata two weeks earlier. Fish presented with perianal haemorrhage and blister-like swellings on the skin which ruptured to leave ulcers. Edwardsiella ictaluri was consistently recovered from the brain and skin. Repetitive sequence-mediated PCR analysis revealed genetic fingerprints consistent with E. ictaluri isolates from farm-raised channel catfish in Mississippi, USA. Plasmid analysis of the case isolates identified two unique plasmids that differ slightly in conformation and content from the pEI1 and pEI2 plasmids described for E. ictaluri from other fish hosts. The case isolates were also PCR negative for several E. ictaluri virulence factors. The biological implications of these genetic differences are unclear and warrant further study. This is the first report and documentation of E. ictaluri infection in Trinidad and Tobago, suggesting the pathogen may have been introduced concurrently with the importation of fish. This report emphasizes the importance of adequate health screenings of imported lots to minimize the threat of introducing E. ictaluri to non-endemic areas.

  13. A second rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein expression in a cell-free system. (United States)

    Frassanito, Anna Maria; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valtere; Gualtieri, Paolo


    Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619 bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175 bp consisting of 849 bp coding DNA sequence and 4 introns of 326 bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 α-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy.

  14. Isolation of expressed sequences encoded by the human Xq terminal portion using microclone probes generated by laser microdissection

    Energy Technology Data Exchange (ETDEWEB)

    Yokoi, H.; Kang, X. (Univ. of Ottawa Faculty of Medicine, Ontario (Canada)); Hadano, Shinji; Kogi, Mieko; Wakasa, Kyo (Tokai Univ. School of Medicine, Kanagawa (Japan)); Ikeda, J.E. (Univ. of Ottawa Faculty of Medicine, Ontario (Canada) Tokai Univ. School of Medicine, Kanagawa (Japan) National Institute of Agrobiological Resources, Ibaraki (Japan))


    The genes that cause a variety of neurologic and neuromuscular disorders have been mapped to the distal region of Xq. In an effort to isolate genes from this area, a regional genomic library of the distal 30% of Xq was constructed from a single metaphase spread by means of laser microdissection and single unique primer-polymerase chain reaction. Using pooled probes of 1000 clones from the genomic library, human brain cDNA libraries were screened for expressed sequences encoded by this region. From the 250,000 cDNA clones screened so far, 10 nonoverlapping sequences that mapped back to the target portion were isolated. The complete nucleotide sequences of these cDNA clones have been determined. Analysis of the sequences indicates that none has significant similarity to previously characterized primate genes. One sequence mapping to Xq27.3-qter contained an open reading frame of 281 amino acids and was expressed in every tissue tested. This gene, as well as others isolated in this manner, may prove to be a candidate gene for heritable disorders mapping to this region. 36 refs., 6 figs., 2 tabs.

  15. Expression of cassini, a murine gamma-satellite sequence conserved in evolution, is regulated in normal and malignant hematopoietic cells

    Directory of Open Access Journals (Sweden)

    Arutyunyan Anna


    Full Text Available Abstract Background Acute lymphoblastic leukemia (ALL cells treated with drugs can become drug-tolerant if co-cultured with protective stromal mouse embryonic fibroblasts (MEFs. Results We performed transcriptional profiling on these stromal fibroblasts to investigate if they were affected by the presence of drug-treated ALL cells. These mitotically inactivated MEFs showed few changes in gene expression, but a family of sequences of which transcription is significantly increased was identified. A sequence related to this family, which we named cassini, was selected for further characterization. We found that cassini was highly upregulated in drug-treated ALL cells. Analysis of RNAs from different normal mouse tissues showed that cassini expression is highest in spleen and thymus, and can be further enhanced in these organs by exposure of mice to bacterial endotoxin. Heat shock, but not other types of stress, significantly induced the transcription of this locus in ALL cells. Transient overexpression of cassini in human 293 embryonic kidney cells did not increase the cytotoxic or cytostatic effects of chemotherapeutic drugs but provided some protection. Database searches revealed that sequences highly homologous to cassini are present in rodents, apicomplexans, flatworms and primates, indicating that they are conserved in evolution. Moreover, CASSINI RNA was induced in human ALL cells treated with vincristine. Surprisingly, cassini belongs to the previously reported murine family of γ-satellite/major satellite DNA sequences, which were not known to be present in other species. Conclusions Our results show that the transcription of at least one member of these sequences is regulated, suggesting that this has a function in normal and transformed immune cells. Expression of these sequences may protect cells when they are exposed to specific stress stimuli.

  16. Hypoxia influences expression profile of Pleckstrin homology-like domain, family A, member 2 in Indian catfish, Clarias batrachus (Linnaeus, 1758): A new candidate gene for hypoxia tolerance in fish

    Indian Academy of Sciences (India)

    Vindhya Mohindra; Ratnesh K Tripathi; Prabhaker Yadav; Rajeev K Singh; Kuldeep K Lal


    Several physiologically important genes were found to be regulated by hypoxia at the transcriptional level. The Pleckstrin homology-like domain, family A, member 2 (PHLDA2) gene was previously identified as an imprinted gene. The present study was aimed to determine the structure of complete cDNA and the deduced protein of PHLDA2 along with analysing the changes in its mRNA expression in Clarias batrachus tissues under hypoxic conditions. The complete cDNA of CbPHLDA2 gene consisted of 1009 nucleotides with an open reading frame of 417 nucleotides. The deduced CbPHLDA2 protein of 139 amino acids shared high homology with PHLD2A of other fishes as well as that of vertebrates. Importantly, a single amino acid (asparagine/lysine) insertion was identified in the PH domain of CbPHLDA2 and other fishes, which was absent in other vertebrates studied. Furthermore, under normoxic conditions, CbPHLDA2 was constitutively expressed with varying levels in analysed tissues. Short- and long-term hypoxia exposure resulted in significant changes in the expression of CbPHLDA2 in liver, spleen, head kidney, brain and muscle in a time-dependent manner. The results suggested that CbPHLDA2 might play an important role for adaptive significance under hypoxia.

  17. Nucleotide sequence and spatial expression pattern of a drought- and abscisic Acid-induced gene of tomato. (United States)

    Plant, A L; Cohen, A; Moses, M S; Bray, E A


    The nucleotide sequence of le16, a tomato (Lycopersicon esculentum Mill.) gene induced by drought stress and regulated by abscisic acid specifically in aerial vegetative tissue, is presented. The single open reading frame contained within the gene has the capacity to encode a polypeptide of 12.7 kilodaltons and is interrupted by a small intron. The predicted polypeptide is rich in leucine, glycine, and alanine and has an isoelectric point of 8.7. The amino terminus is hydrophobic and characteristic of signal sequences that target polypeptides for export from the cytoplasm. There is homology (47.2% identity) between the amino terminus of the LE 16 polypeptide and the corresponding amino terminal domain of the maize phospholipid transfer protein. le16 was expressed in drought-stressed leaf, petiole, and stem tissue and to a much lower extent in the pericarp of mature green tomato fruit and developing seeds. No expression was detected in the pericarp of red fruit or in drought-stressed roots. Expression of le16 was also induced in leaf tissue by a variety of other abiotic stresses including polyethylene glycol-mediated water deficit, salinity, cold stress, and heat stress. None of these stresses or direct applications of abscisic acid induced the expression of le16 in the roots of the same plants. The unique expression characteristics of this gene indicates that novel regulatory mechanisms, in addition to endogenous abscisic acid, are involved in controlling gene expression.

  18. Sequence and mRNA expression of nonclassical SLA class I genes SLA-7 and SLA-8. (United States)

    Crew, Mark D; Phanavanh, Bounleut; Garcia-Borges, Carmen N


    Given the prominent position of pig endothelial cells in pig-to-human xenotransplantation and the role of classical and nonclassical MHC class I proteins in T and NK cell recognition, the expression of pig MHC (SLA) class I genes in a pig aortic endothelial cell line (AOC cells) was examined. Using a primer corresponding to a highly conserved region of exon 4, RT-PCR analysis of SLA class I expression in AOC cells revealed not only expression of the classical SLA class I ( SLA-1, -2, and -3) genes, but also SLA class I transcripts corresponding to SLA nonclassical class I (class Ib) genes SLA-6 and SLA-8. Further analysis of SLA class Ib expression in porcine aortic endothelial cells using SLA class I gene-specific primers confirmed SLA-6 and SLA-8 expression and also demonstrated expression of SLA-7. While SLA-6 has been relatively well characterized, no data regarding bona fide SLA-7 and SLA-8 transcripts have been reported. Therefore, cDNAs containing the complete open reading frames of SLA-6, -7, and -8 were obtained. Compared to an SLA-1 protein sequence, the predicted SLA-7 and -8 protein sequences exhibited most sequence divergence in alpha1, alpha2, and cytoplasmic domains. Expression of SLA-6, -7, and -8 was examined by RT-PCR using RNA prepared from a variety of tissues. SLA-6 transcripts were detected in every tissue examined. Except for brain, SLA-8 transcripts were similarly widespread. SLA-7 exhibited more limited tissue distribution.

  19. Efficient gusA Transient Expression in Porphyra yezoensis Protoplasts Mediated by Endogenous Beta-tubulin Flanking Sequences

    Institute of Scientific and Technical Information of China (English)

    GONG Qianhong; YU Wengong; DAI Jixun; LIU Hongquan; XU Rifu; GUAN Huashi; PAN Kehou


    Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are tmknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'- and 3'-flanking regions (Tub5'and Tub3') up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively,into pA, a derivative of pCAT(R)3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3 '. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

  20. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells. (United States)

    Xu, Zhen; Chen, Feng; Zhang, Lingling; Lu, Jing; Xu, Peng; Liu, Guang; Xie, Xuemin; Mu, Wenli; Wang, Yajun; Liu, Depei


    Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

  1. An analysis of expressed sequence tags of developing castor endosperm using a full-length cDNA library

    Directory of Open Access Journals (Sweden)

    Wallis James G


    Full Text Available Abstract Background Castor seeds are a major source for ricinoleate, an important industrial raw material. Genomics studies of castor plant will provide critical information for understanding seed metabolism, for effectively engineering ricinoleate production in transgenic oilseeds, or for genetically improving castor plants by eliminating toxic and allergic proteins in seeds. Results Full-length cDNAs are useful resources in annotating genes and in providing functional analysis of genes and their products. We constructed a full-length cDNA library from developing castor endosperm, and obtained 4,720 ESTs from 5'-ends of the cDNA clones representing 1,908 unique sequences. The most abundant transcripts are genes encoding storage proteins, ricin, agglutinin and oleosins. Several other sequences are also very numerous, including two acidic triacylglycerol lipases, and the oleate hydroxylase (FAH12 gene that is responsible for ricinoleate biosynthesis. The role(s of the lipases in developing castor seeds are not clear, and co-expressing of a lipase and the FAH12 did not result in significant changes in hydroxy fatty acid accumulation in transgenic Arabidopsis seeds. Only one oleate desaturase (FAD2 gene was identified in our cDNA sequences. Sequence and functional analyses of the castor FAD2 were carried out since it had not been characterized previously. Overexpression of castor FAD2 in a FAH12-expressing Arabidopsis line resulted in decreased accumulation of hydroxy fatty acids in transgenic seeds. Conclusion Our results suggest that transcriptional regulation of FAD2 and FAH12 genes maybe one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm. The full-length cDNA library will be used to search for additional genes that affect ricinoleate accumulation in seed oils. Our EST sequences will also be useful to annotate the castor genome, which whole sequence is being generated by shotgun sequencing at

  2. De novo transcriptome sequencing and comparative analysis of differentially expressed genes in Gossypium aridum under salt stress. (United States)

    Xu, Peng; Liu, Zhangwei; Fan, Xinqi; Gao, Jin; Zhang, Xia; Zhang, Xianggui; Shen, Xinlian


    Salinity stress is one of the most serious factors that impede the growth and development of various crops. Wild Gossypium species, which are remarkably tolerant to salt water immersion, are valuable resources for understanding salt tolerance mechanisms of Gossypium and improving salinity resistance in upland cotton. To generate a broad survey of genes with altered expression during various stages of salt stress, a mixed RNA sample was prepared from the roots and leaves of Gossypium aridum plants subjected to salt stress. The transcripts were sequenced using the Illumina sequencing platform. After cleaning and quality checks, approximately 41.5 million clean reads were obtained. Finally, these reads were eventually assembled into 98,989 unigenes with a mean size of 452 bp. All unigenes were compared to known cluster of orthologous groups (COG) sequences to predict and classify the possible functions of these genes, which were classified into at least 25 molecular families. Variations in gene expression were then examined after exposing the plants to 200 mM NaCl for 3, 12, 72 or 144 h. Sequencing depths of approximately six million raw tags were achieved for each of the five stages of salt stress. There were 2634 (1513 up-regulated/1121 down-regulated), 2449 (1586 up-regulated/863 down-regulated), 2271 (946 up-regulated/1325 down-regulated) and 3352 (933 up-regulated/2419 down-regulated) genes that were differentially expressed after exposure to NaCl for 3, 12, 72 and 144 h, respectively. Digital gene expression analysis indicated that pathways involved in "transport", "response to hormone stimulus" and "signaling" play important roles during salt stress, while genes involved in "protein kinase activity" and "transporter activity" undergo major changes in expression during early and later stages of salt stress, respectively.

  3. Analysis of Gene Expression Profile Induced by Water Stress in Upland Rice (Oryza sativa L. var. IRAT109)Seedlings using Subtractive Expressed Sequence Tags Library

    Institute of Scientific and Technical Information of China (English)


    To identify the water stress induced genes of upland rice cultivar IRAT109, which is resistant to drought, a subtractive cDNA library was developed from polyethylene glycol- (PEG) treated and non-treated seedlings by suppression subtractive hybridization, from which 2112 recombinant colonies were obtained. Eight hundred clones were selected randomly for sequencing analysis, and 384 unique expressed sequence tags (ESTs) were obtained. They were found to be involved in diverse biological processes, such as metabolism, transcription, signal transduction, protein synthesis and others. Notably a number of known functional genes in drought tolerance, including genes related to biosynthesis of osmoprotectants,defense against active oxygen, removal of toxic compounds, recovery of proteins and reinforcement of cell wall were also found in the study. Several genes related to deleterious responses were upregulated by PEG stress. The differential expression patterns of 11 SSH-derived ESTs were confirmed by real-time polymerase chain reaction.

  4. Global Transcriptome Analysis of the Tentacle of the Jellyfish Cyanea capillata Using Deep Sequencing and Expressed Sequence Tags: Insight into the Toxin- and Degenerative Disease-Related Transcripts.

    Directory of Open Access Journals (Sweden)

    Guoyan Liu

    Full Text Available Jellyfish contain diverse toxins and other bioactive components. However, large-scale identification of novel toxins and bioactive components from jellyfish has been hampered by the low efficiency of traditional isolation and purification methods.We performed de novo transcriptome sequencing of the tentacle tissue of the jellyfish Cyanea capillata. A total of 51,304,108 reads were obtained and assembled into 50,536 unigenes. Of these, 21,357 unigenes had homologues in public databases, but the remaining unigenes had no significant matches due to the limited sequence information available and species-specific novel sequences. Functional annotation of the unigenes also revealed general gene expression profile characteristics in the tentacle of C. capillata. A primary goal of this study was to identify putative toxin transcripts. As expected, we screened many transcripts encoding proteins similar to several well-known toxin families including phospholipases, metalloproteases, serine proteases and serine protease inhibitors. In addition, some transcripts also resembled molecules with potential toxic activities, including cnidarian CfTX-like toxins with hemolytic activity, plancitoxin-1, venom toxin-like peptide-6, histamine-releasing factor, neprilysin, dipeptidyl peptidase 4, vascular endothelial growth factor A, angiotensin-converting enzyme-like and endothelin-converting enzyme 1-like proteins. Most of these molecules have not been previously reported in jellyfish. Interestingly, we also characterized a number of transcripts with similarities to proteins relevant to several degenerative diseases, including Huntington's, Alzheimer's and Parkinson's diseases. This is the first description of degenerative disease-associated genes in jellyfish.We obtained a well-categorized and annotated transcriptome of C. capillata tentacle that will be an important and valuable resource for further understanding of jellyfish at the molecular level and information

  5. Cloning, sequencing and expression of a xylanase gene from the maize pathogen Helminthosporium turcicum

    DEFF Research Database (Denmark)

    Degefu, Y.; Paulin, L.; Lübeck, Peter Stephensen


    A gene encoding an endoxylanase from the phytopathogenic fungus Helminthosporium turcicum Pass. was cloned and sequenced. The entire nucleotide sequence of a 1991 bp genomic fragment containing an endoxylanase gene was determined. The xylanase gene of 795 bp, interrupted by two introns of 52 and ...

  6. Whole genome sequencing of sugarbeet and identification of differentially expressed genes regulating beet curly top resistance (United States)

    The genome of KDH13 doubled haploid line has been sequenced using Illumina HiSeq2000 NGS platform. This line (PI663862) was released by USDA-ARS as a genetic stock resistant to beet curly top. Sequencing of a standard paired end and a 2kb-insert mate-pair genomic libraries, constructed from a leaf ...

  7. Stress in African catfish (Clarias gariepinus) following overland transportation. (United States)

    Manuel, Remy; Boerrigter, Jeroen; Roques, Jonathan; van der Heul, Jan; van den Bos, Ruud; Flik, Gert; van de Vis, Hans


    Of the many stressors in aquaculture, transportation of fish has remained poorly studied. The objective of this study was therefore to assess the effects of a (simulated) commercial transportation on stress physiology of market-size African catfish (Clarias gariepinus). Catfish weighing approximately 1.25 kg were returned to the farm after 3 h of truck-transportation, and stress-related parameters were measured for up to 72 h following return. Recovery from transportation was assessed through blood samples measuring plasma cortisol, glucose and non-esterified fatty acids (NEFA) and gill histology. Also, the number of skin lesions was compared before and after transport. Pre-transport handling and sorting elevated plasma cortisol levels compared to unhandled animals (before fasting). Plasma cortisol levels were further increased due to transportation. In control fish, plasma cortisol levels returned to baseline values within 6 h, whereas it took 48 h to reach baseline values in transported catfish. Plasma glucose and NEFA levels remained stable and were similar across all groups. Transported catfish did not, on average, have more skin lesions than the handling group, but the number of skin lesions had increased compared to unhandled animals. The macroscopic condition of the gills was similar in control, transported and unhandled catfish; however, light microscopy and immunohistochemistry revealed atypical morphology and chloride cell migration normally associated with adverse water conditions. From our data, we conclude that transportation may be considered a strong stressor to catfish that may add to other stressors and thus inflict upon the welfare of the fish.

  8. A stochastic analysis of tractor overturn costs on catfish farms. (United States)

    Ibendahl, G A; Stephens, W B; Myers, M L


    An area of health and safety risk in agriculture that can be especially dangerous is catfish farming. One of the potential sources of injuries on catfish farms is tractor overturns that often result in crushing injuries. There is likely a higher probability of tractor overturns on a catfish farm than on a traditional crop farm due to the conditions that prevail on catfish farms. A catfish farm requires tractor movement near pond levees and water, and these levees have steep banks. Many of the activities on a catfish farm, such as mowing, feeding, and pond maintenance, require operating a tractor near a pond levee. Rollover protection structures (ROPS) on tractors can help to minimize the injuries caused by tractor overturns. ROPS do not lessen the probability of overturns, but ROPS mitigate the expected injury severity and lower the associated costs of an overturn. Despite the benefits of ROPS, not every tractor is so equipped. Some earlier work indicated that the cost to retrofit older tractors might outweigh the expected benefits. This article uses stochastic (i.e., randomly determined) analysis to determine if risk-averse farmers are more likely than risk-neutral farmers to retrofit tractors with ROPS. For this analysis, a distribution function of injury costs should an overturn occur was developed for both ROPS and non-ROPS tractors, and a Monte Carlo simulation was conducted. Results indicate that many risk-averse producers would be willing to retrofit older tractors with ROPS. However producers who are risk-neutral probably will not retrofit. These results might explain why not all tractors have been retrofitted despite the long-term availability of retrofit kits.

  9. Structural organization, sequence, and expression of the mouse HEXA gene encoding the alpha subunit of hexosaminidase A. (United States)

    Wakamatsu, N; Benoit, G; Lamhonwah, A M; Zhang, Z X; Trasler, J M; Triggs-Raine, B L; Gravel, R A


    Genomic clones of the mouse HEXA gene encoding the alpha subunit of lysosomal beta-hexosaminidase A have been isolated, analyzed, and sequenced. The HEXA gene spans approximately 26 kb and consists of 14 exons and 13 introns. The 5' flanking region of the gene has three candidate GC boxes and a number of potential promoter and regulatory elements. Promoter analysis using deletion constructs of 5' flanking sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) gene showed that 150 bp of 5' sequence was sufficient for expression in transfected monkey kidney COS cells. Determination of the sequence of the 5' end of the Hex alpha mRNA by an "anchor-ligation PCR" procedure showed that transcription is initiated from a cluster of sites centered -42, -32, and -21 bp from the first in-frame ATG. Northern blot analysis from 11 different tissues showed over five times the steady-state level of Hex alpha mRNA in testis as compared to that found in three different brain regions; the lowest level (about 1/3 of brain) was found in liver. Comparison of the 5' flanking sequence with that of the human HEXA gene revealed 78% identity within the first 100 bp. These data suggest that the mouse HEXA gene is controlled mainly by sequences located within 150 bp of the 5' flanking region, and we speculate that it may have a role, not only in brain and other tissues, but also in reproductive function in the adult male mouse.

  10. Molecular diversification based on analysis of expressed sequence tags from the venom glands of the Chinese bird spider Ornithoctonus huwena. (United States)

    Jiang, Liping; Peng, Li; Chen, Jinjun; Zhang, Yongqun; Xiong, Xia; Liang, Songping


    The bird spider Ornithoctonus huwena is one of the most venomous spiders in China. Its venom has been investigated but usually only the most abundant components have been analyzed. To characterize the primary structure of O. huwena toxins, a list of transcripts within the venom gland were made using the expressed sequence tag (EST) strategy. We generated 468 ESTs from a directional cDNA library of O. huwena venom glands. All ESTs were grouped into 24 clusters and 65 singletons, of which 68.00% of total ESTs belong to toxin-like sequences, 13.00% are similar to body peptide transcripts and 19.00% have no significant similarity to any known sequences. Precursors of all toxin-like sequences can be classified into eight different superfamilies (HWTX-I superfamily, HWTX-II superfamily, HWTX-X superfamily, HWTX-XIV superfamily, HWTX-XV superfamily, HWTX-XVI superfamily, HWTX-XVII superfamily, HWTX-XVIII superfamily) except HWTX-XI and HWTX-XIII, according to the identity of their precursor sequences. The results have predictive value for the discovery of various groups of pharmacologically distinct toxins in complex venoms, and for understanding the relationship of spider toxin evolution based on the diversification of cDNA sequences, primary structure of precursor peptides, three-dimensional structure motifs and biological functions.

  11. Mapping the transcription start points of the Staphylococcus aureus eap, emp, and vwb promoters reveals a conserved octanucleotide sequence that is essential for expression of these genes. (United States)

    Harraghy, Niamh; Homerova, Dagmar; Herrmann, Mathias; Kormanec, Jan


    Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp.

  12. Facial Expression Recognition from Video Sequences Based on Spatial-Temporal Motion Local Binary Pattern and Gabor Multiorientation Fusion Histogram

    Directory of Open Access Journals (Sweden)

    Lei Zhao


    Full Text Available This paper proposes novel framework for facial expressions analysis using dynamic and static information in video sequences. First, based on incremental formulation, discriminative deformable face alignment method is adapted to locate facial points to correct in-plane head rotation and break up facial region from background. Then, spatial-temporal motion local binary pattern (LBP feature is extracted and integrated with Gabor multiorientation fusion histogram to give descriptors, which reflect static and dynamic texture information of facial expressions. Finally, a one-versus-one strategy based multiclass support vector machine (SVM classifier is applied to classify facial expressions. Experiments on Cohn-Kanade (CK + facial expression dataset illustrate that integrated framework outperforms methods using single descriptors. Compared with other state-of-the-art methods on CK+, MMI, and Oulu-CASIA VIS datasets, our proposed framework performs better.

  13. Helicobacter pylori cagA Promoter Region Sequences Influence CagA Expression and Interleukin 8 Secretion. (United States)

    Ferreira, Rui M; Pinto-Ribeiro, Ines; Wen, Xiaogang; Marcos-Pinto, Ricardo; Dinis-Ribeiro, Mário; Carneiro, Fátima; Figueiredo, Ceu


    Heterogeneity at the Helicobacter pylori cagA gene promoter region has been linked to variation in CagA expression and gastric histopathology. Here, we characterized the cagA promoter and expression in 46 H. pylori strains from Portugal. Our results confirm the relationship between cagA promoter region variation and protein expression originally observed in strains from Colombia. We observed that individuals with intestinal metaplasia were all infected with H. pylori strains containing a specific cagA motif. Additionally, we provided novel functional evidence that strain-specific sequences in the cagA promoter region and CagA expression levels influence interleukin 8 secretion by the host gastric epithelial cells.

  14. Comparative analysis of expressed sequence tags from three castes and two life stages of the termite Reticulitermes flavipes

    Directory of Open Access Journals (Sweden)

    Steller Matthew M


    Full Text Available Abstract Background Termites (Isoptera are eusocial insects whose colonies consist of morphologically and behaviorally specialized castes of sterile workers and soldiers, and reproductive alates. Previous studies on eusocial insects have indicated that caste differentiation and behavior are underlain by differential gene expression. Although much is known about gene expression in the honey bee, Apis mellifera, termites remain relatively understudied in this regard. Therefore, our objective was to assemble an expressed sequence tag (EST data base for the eastern subterranean termite, Reticulitermes flavipes, for future gene expression studies. Results Soldier, worker, and alate caste and two larval cDNA libraries were constructed, and approximately 15,000 randomly chosen clones were sequenced to compile an EST data base. Putative gene functions were assigned based on a BLASTX Swissprot search. Categorical in silico expression patterns for each library were compared using the R-statistic. A significant proportion of the ESTs of each caste and life stages had no significant similarity to those in existing data bases. All cDNA libraries, including those of non-reproductive worker and soldier castes, contained sequences with putative reproductive functions. Genes that showed a potential expression bias among castes included a putative antibacterial humoral response and translation elongation protein in soldiers and a chemosensory protein in alates. Conclusions We have expanded upon the available sequences for R. flavipes and utilized an in silico method to compare gene expression in different castes of an eusocial insect. The in silico analysis allowed us to identify several genes which may be differentially expressed and involved in caste differences. These include a gene overrepresented in the alate cDNA library with a predicted function of neurotransmitter secretion or cholesterol absorption and a gene predicted to be involved in protein

  15. Inhibition of expression of virulence genes of Yersinia pestis in Escherichia coli by external guide sequences and RNase P. (United States)

    Ko, Jae-hyeong; Izadjoo, Mina; Altman, Sidney


    External guide sequences (EGSs) targeting virulence genes from Yersinia pestis were designed and tested in vitro and in vivo in Escherichia coli. Linear EGSs and M1 RNA-linked EGSs were designed for the yscN and yscS genes that are involved in type III secretion in Y. pestis. RNase P from E. coli cleaves the messages of yscN and yscS in vitro with the cognate EGSs, and the expression of the EGSs resulted in the reduction of the levels of these messages of the virulence genes when those genes were expressed in E. coli.

  16. Conserved sequence motifs upstream from the co-ordinately expressed vitellogenin and apoVLDLII genes of chicken. (United States)

    van het Schip, F; Strijker, R; Samallo, J; Gruber, M; Geert, A B


    The vitellogenin and apoVLDLII yolk protein genes of chicken are transcribed in the liver upon estrogenization. To get information on putative regulatory elements, we compared more than 2 kb of their 5' flanking DNA sequences. Common sequence motifs were found in regions exhibiting estrogen-induced changes in chromatin structure. Stretches of alternating pyrimidines and purines of about 30-nucleotides long are present at roughly similar positions. A distinct box of sequence homology in the chicken genes also appears to be present at a similar position in front of the vitellogenin genes of Xenopus laevis, but is absent from the estrogen-responsive egg-white protein genes expressed in the oviduct. In front of the vitellogenin (position -595) and the VLDLII gene (position -548), a DNA element of about 300 base-pairs was found, which possesses structural characteristics of a mobile genetic element and bears homology to the transposon-like Vi element of Xenopus laevis.

  17. Development, characterization and cross species amplification of polymorphic microsatellite markers from expressed sequence tags of turmeric (Curcuma longa L.). (United States)

    Siju, S; Dhanya, K; Syamkumar, S; Sasikumar, B; Sheeja, T E; Bhat, A I; Parthasarathy, V A


    Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST-SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.

  18. Preparing and Analyzing Expressed Sequence Tags (ESTs) Library for the Mammary Tissue of Local Turkish Kivircik Sheep (United States)

    Omeroglu Ulu, Zehra; Ulu, Salih; Un, Cemal; Ozdem Oztabak, Kemal; Altunatmaz, Kemal


    Kivircik sheep is an important local Turkish sheep according to its meat quality and milk productivity. The aim of this study was to analyze gene expression profiles of both prenatal and postnatal stages for the Kivircik sheep. Therefore, two different cDNA libraries, which were taken from the same Kivircik sheep mammary gland tissue at prenatal and postnatal stages, were constructed. Total 3072 colonies which were randomly selected from the two libraries were sequenced for developing a sheep ESTs collection. We used Phred/Phrap computer programs for analysis of the raw EST and readable EST sequences were assembled with the CAP3 software. Putative functions of all unique sequences and statistical analysis were determined by Geneious software. Total 422 ESTs have over 80% similarity to known sequences of other organisms in NCBI classified by Panther database for the Gene Ontology (GO) category. By comparing gene expression profiles, we observed some putative genes that may be relative to reproductive performance or play important roles in milk synthesis and secretion. A total of 2414 ESTs have been deposited to the NCBI GenBank database (GW996847–GW999260). EST data in this study have provided a new source of information to functional genome studies of sheep.

  19. Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expression. (United States)

    Kapoor, Divya; Chandrayan, Sanjeev Kumar; Ahmed, Shubbir; Guptasarma, Purnananda


    The formation of secondary structure in oligonucleotide DNA is known to lead to "compression" artifacts in electropherograms produced through DNA sequencing. Separately, the formation of secondary structure in mRNA is known to suppress translation; in particular, when such structures form in a region covered by the ribosome either during, or shortly after, initiation of translation. Here, we demonstrate how a DNA sequencing compression artifact provides important clues to the location(s) of translation-suppressing secondary structural elements in mRNA. Our study involves an engineered version of a gene sourced from Rhodothermus marinus encoding an enzyme called Cel12A. We introduced this gene into Escherichia coli with the intention of overexpressing it, but found that it expressed extremely poorly. Intriguingly, the gene displayed a remarkable compression artifact during DNA sequencing electrophoresis. Selected "designer" silent mutations destroyed the artifact. They also simultaneously greatly enhanced the expression of the cel12A gene, presumably by destroying stable mRNA structures that otherwise suppress translation. We propose that this method of finding problem mRNA sequences is superior to software-based analyses, especially if combined with low-temperature CE.

  20. Global Identification of Significantly Expressed Genes in Developing Endosperm of Rice by Expression Sequence Tags and cDNA Array Approaches

    Institute of Scientific and Technical Information of China (English)

    Qichao Tu; Haitao Dong; Haigen Yao; Yongqi Fang; Cheng'en Dai; Hongmei Luo; Jian Yao; Dong Zhao; Debao Li


    Rice endosperm plays a very important role in seedling germination and determines the qualities of fice grain.Although studies on specific gene categories in endosperm have been carried out,global view of gene expression at a transcription level in rice endosperm is still limited.To gain a better understanding of the global and tissue-specific gene expression profiles in rice endosperm,a cDNA library from rice endosperm of immature seeds was sequenced.A cDNA array was constructed based on the tentative unique transcripts derived from expression sequence tag (EST) assembling results and then hybridized with cONAs from five different tissues or organs including endosperm,embryo,leaf,stem and root of rice.Significant redundancy was found for genes encoding prolamin,glutelin,allergen,and starch synthesis proteins,accounting for~34% of the total ESTs obtained.The cDNA array revealed 87 significantly expressed genes in endosperm compared with the other four organs or tissues.These genes included 13 prolamin family proteins,17 glutelin family proteins,12 binding proteins,nine catalytic proteins and four ribosomal proteins,indicating a complicated biological processing in rice endosperm.In addition,Northern verification of 1,4-alpha-glucan branching enzyme detected two isoforms in rice endosperm,the larger one of which only existed in endosperm.

  1. Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

    Energy Technology Data Exchange (ETDEWEB)

    Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.


    Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

  2. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M;


    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed......, was determined to be on distal mouse Chromosome (Chr) 9 by analysis of two sets of multilocus crosses....

  3. Recoding method that removes inhibitory sequences and improves HIV gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Rabadan, Raul; Krasnitz, Michael; Robins, Harlan; Witten, Daniela; Levine, Arnold


    The invention relates to inhibitory nucleotide signal sequences or "INS" sequences in the genomes of lentiviruses. In particular the invention relates to the AGG motif present in all viral genomes. The AGG motif may have an inhibitory effect on a virus, for example by reducing the levels of, or maintaining low steady-state levels of, viral RNAs in host cells, and inducing and/or maintaining in viral latency. In one aspect, the invention provides vaccines that contain, or are produced from, viral nucleic acids in which the AGG sequences have been mutated. In another aspect, the invention provides methods and compositions for affecting the function of the AGG motif, and methods for identifying other INS sequences in viral genomes.

  4. ConiferEST: an integrated bioinformatics system for data reprocessing and mining of conifer expressed sequence tags (ESTs

    Directory of Open Access Journals (Sweden)

    Carter Kikia


    Full Text Available Abstract Background With the advent of low-cost, high-throughput sequencing, the amount of public domain Expressed Sequence Tag (EST sequence data available for both model and non-model organism is growing exponentially. While these data are widely used for characterizing various genomes, they also present a serious challenge for data quality control and validation due to their inherent deficiencies, particularly for species without genome sequences. Description ConiferEST is an integrated system for data reprocessing, visualization and mining of conifer ESTs. In its current release, Build 1.0, it houses 172,229 loblolly pine EST sequence reads, which were obtained from reprocessing raw DNA sequencer traces using our software – WebTraceMiner. The trace files were downloaded from NCBI Trace Archive. ConiferEST provides biologists unique, easy-to-use data visualization and mining tools for a variety of putative sequence features including cloning vector segments, adapter sequences, restriction endonuclease recognition sites, polyA and polyT runs, and their corresponding Phred quality values. Based on these putative features, verified sequence features such as 3' and/or 5' termini of cDNA inserts in either sense or non-sense strand have been identified in-silico. Interestingly, only 30.03% of the designated 3' ESTs were found to have an authenticated 5' terminus in the non-sense strand (i.e., polyT tails, while fewer than 5.34% of the designated 5' ESTs had a verified 5' terminus in the sense strand. Such previously ignored features provide valuable insight for data quality control and validation of error-prone ESTs, as well as the ability to identify novel functional motifs embedded in large EST datasets. We found that "double-termini adapters" were effective indicators of potential EST chimeras. For all sequences with in-silico verified termini/terminus, we used InterProScan to assign protein domain signatures, results of which are available

  5. Use of vaccination against enteric septicemia of catfish and columnaris disease by the U.S. catfish industry. (United States)

    Bebak, Julie; Wagner, Bruce


    Vaccination is an effective strategy used for the protection of food animals against infectious diseases. A 2010 U.S. Department of Agriculture questionnaire examined U.S. catfish industry use (in 2009) of two commercial vaccines that provide protection against enteric septicemia of catfish (ESC) and columnaris disease, catfish producers' opinions regarding the percentage of vaccinated fish they expect to be protected, and producers' general expectations regarding survival of vaccinated fish compared with unvaccinated fish. During 2009, 9.7% of the total fingerling operations used one or both vaccines; 12.3% of the total industry fry production was vaccinated against ESC, and 17.0% was vaccinated against columnaris disease. Of the producers who grew food-sized catfish to harvest, 6.7% used vaccinated catfish. The farms that did not use vaccinated fish for grow out had a mean size of 63.4 water surface hectares (156.6 water surface acres). The operations that used vaccinated fish were larger (mean size = 206.6 water surface hectares, or 510.6 water surface acres). The producers that stocked ESC-vaccinated fish for grow out represented 19.0% of the total water surface area of food fish production; producers that stocked columnaris-vaccinated fish represented 16.6% of the total area. Of the producers that stocked ESC-vaccinated catfish, 41.9% thought that survival was better in vaccinated fish than in unvaccinated fish; of the producers that stocked columnaris-vaccinated catfish, 46.2% thought that vaccinated fish displayed better survival. However, 37.5% of producers that used the ESC vaccine and 39.7% of producers that used the columnaris vaccine did not know whether vaccination improved survival rates. When all producers were asked about their expectations regarding the percentage of vaccinated fish that would be protected from disease, 52.4% responded that they expected 100% of their fish to be protected. More producer information about reasonable expectations

  6. De novo sequence analysis of cytochrome P450 1-3 genes expressed in ostrich liver with highest expression of CYP2G19. (United States)

    Kawai, Yusuke K; Watanabe, Kensuke P; Ishii, Akihiro; Ohnuma, Aiko; Sawa, Hirofumi; Ikenaka, Yoshinori; Ishizuka, Mayumi


    The cytochrome P450 (CYP) 1-3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1-3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2-10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism.

  7. In silico differential display of defense-related expressed sequence tags from sugarcane tissues infected with diazotrophic endophytes

    Directory of Open Access Journals (Sweden)

    Lambais Marcio R.


    Full Text Available The expression patterns of 277 sugarcane expressed sequence tags (EST-contigs encoding putative defense-related (DR proteins were evaluated using the Sugarcane EST database. The DR proteins evaluated included chitinases, beta-1,3-glucanases, phenylalanine ammonia-lyases, chalcone synthases, chalcone isomerases, isoflavone reductases, hydroxyproline-rich glycoproteins, proline-rich glycoproteins, peroxidases, catalases, superoxide dismutases, WRKY-like transcription factors and proteins involved in cell death control. Putative sugarcane WRKY proteins were compared and their phylogenetic relationships determined. A hierarchical clustering approach was used to identify DR ESTs with similar expression profiles in representative cDNA libraries. To identify DR ESTs differentially expressed in sugarcane tissues infected with Gluconacetobacter diazotrophicus or Herbaspirillum rubrisubalbicans, 179 putative DR EST-contigs expressed in non-infected tissues (leaves and roots and/or infected tissues were selected and arrayed by similarity of their expression profiles. Changes in the expression levels of 124 putative DR EST-contigs, expressed in non-infected tissues, were evaluated in infected tissues. Approximately 42% of these EST-contigs showed no expression in infected tissues, whereas 15% and 3% showed more than 2-fold suppression in tissues infected with G. diazotrophicus or H. rubrisubalbicans, respectively. Approximately 14 and 8% of the DR EST-contigs evaluated showed more than 2-fold induction in tissues infected with G. diazotrophicus or H. rubrisubalbicans, respectively. The differential expression of clusters of DR genes may be important in the establishment of a compatible interaction between sugarcane and diazotrophic endophytes. It is suggested that the hierarchical clustering approach can be used on a genome-wide scale to identify genes likely involved in controlling plant-microorganism interactions.

  8. Identification of anhydrobiosis-related genes from an expressed sequence tag database in the cryptobiotic midge Polypedilum vanderplanki (Diptera; Chironomidae). (United States)

    Cornette, Richard; Kanamori, Yasushi; Watanabe, Masahiko; Nakahara, Yuichi; Gusev, Oleg; Mitsumasu, Kanako; Kadono-Okuda, Keiko; Shimomura, Michihiko; Mita, Kazuei; Kikawada, Takahiro; Okuda, Takashi


    Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.

  9. Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers. (United States)

    Gao, Chunsheng; Xin, Pengfei; Cheng, Chaohua; Tang, Qing; Chen, Ping; Wang, Changbiao; Zang, Gonggu; Zhao, Lining


    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.

  10. Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers.

    Directory of Open Access Journals (Sweden)

    Chunsheng Gao

    Full Text Available 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.

  11. Cloning, sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene. (United States)

    De Zoysa, P A; Connerton, I F; Watson, D C; Johnston, J R


    The cloned NADP-specific glutamate dehydrogenase (GDH) genes of Aspergillus nidulans (gdhA) and Neurospora crassa (am) have been shown to hybridize under reduced stringency conditions to genomic sequences of the yeast Schwanniomyces occidentalis. Using 5' and 3' gene-specific probes, a unique 5.1 kb BclI restriction fragment that encompasses the entire Schwanniomyces sequence has been identified. A recombinant clone bearing the unique BclI fragment has been isolated from a pool of enriched clones in the yeast/E. coli shuttle vector pWH5 by colony hybridization. The identity of the plasmid clone was confirmed by functional complementation of the Saccharomyces cerevisiae gdh-1 mutation. The nucleotide sequence of the Schw. occidentalis GDH gene, which consists of 1380 nucleotides in a continuous reading frame of 459 amino acids, has been determined. The predicted amino acid sequence shows considerable homology with GDH proteins from other fungi and significant homology with all other available GDH sequences.

  12. Cloning, sequencing and expression of the gene encoding the extracellular metalloprotease of Aeromonas caviae. (United States)

    Kawakami, K; Toma, C; Honma, Y


    A gene (apk) encoding the extracellular protease of Aeromonas caviae Ae6 has been cloned and sequenced. For cloning the gene, the DNA genomic library was screened using skim milk LB agar. One clone harboring plasmid pKK3 was selected for sequencing. Nucleotide sequencing of the 3.5 kb region of pKK3 revealed a single open reading frame (ORF) of 1,785 bp encoding 595 amino acids. The deduced polypeptide contained a putative 16-amino acid signal peptide followed by a large propeptide. The N-terminal amino acid sequence of purified recombinant protein (APK) was consistent with the DNA sequence. This result suggested a mature protein of 412 amino acids with a molecular mass of 44 kDa. However, the molecular mass of purified recombinant APK revealed 34 kDa by SDS-PAGE, suggesting that further processing at the C-terminal region took place. The 2 motifs of zinc binding sites deduced are highly conserved in the APK as well as in other zinc metalloproteases including Vibrio proteolyticus neutral protease, Emp V from Vibrio vulnificus, HA/P from Vibrio cholerae, and Pseudomonas aeruginosa elastase. Proteolytic activity was inhibited by EDTA, Zincov, 1,10-phenanthroline and tetraethylenepentamine while unaffected by the other inhibitors tested. The protease showed maximum activity at pH 7.0 and was inactivated by heating at 80 C for 15 min. These results together suggest that APK belongs to the thermolysin family of metalloendopeptidases.

  13. Invasion of South American suckermouth armoured catfishes Pterygoplichthys spp. (Loricariidae in Kerala, India - a case study

    Directory of Open Access Journals (Sweden)

    A. Bijukumar


    Full Text Available This paper documents the occurrence of the exotic South American suckermouth armoured catfishes (Loricariidae of the genus Pterygoplichthys spp. in the drainages of Thiruvananthapuram City, Kerala.  The morphological taxonomy revealed that the specimens are closely related to Pterygoplichthys disjunctivus (Weber, 1991 and P. pardalis (Castelnau, 1855, in addition to intermediary forms of unknown identity.  DNA barcoding using the mitochondrial DNA cytochrome c oxidase 1 (CO1 also failed to establish the identity of the invaded species.  There may be possible hybridisation in aquariums or in fish farms or in the wild, but this needs to be found out aided by detailed studies incorporating different molecular markers and with sequences of topotypes.  The possible threats due to Pterygoplichthys spp. invasion and management options are discussed in the paper. 

  14. On Sciadeichthys (Selenaspis) Walrechti, a new South American catfish

    NARCIS (Netherlands)

    Boeseman, M.


    Two large specimens of a hitherto unknown species of neotropical catfish have recently been found dead and washed ashore beneath the dike along the Westerscheldt near Biezelinge, Zeeland, at a mutual distance of approximately 300 meters. Both were in excellent condition and have been presented to th

  15. Controlling fungus on channel catfish eggs with peracetic acid (United States)

    There is much interest in the use of peracetic acid (PAA) to treat pathogens in aquaculture. It is a relatively new compound and is approved for use in Europe, but not in the United States. This study determined the effectiveness of PAA for fungus control on channel catfish Ictalurus punctatus egg...

  16. The Neotropical whale catfishes (Siluriformes: Cetopsidae: Cetopsinae), a revisionary study


    Richard P. Vari; Carl J. Ferraris Jr.; PINNA, Mário C. C. de


    The catfishes of the subfamily Cetopsinae of the Neotropical family Cetopsidae are revised. Four genera, Cetopsidium new genus, Cetopsis, Denticetopsis, and Paracetopsis Bleeker are recognized as valid. Bathycetopsis, Hemicetopsis, and Pseudocetopsis are considered synonyms of Cetopsis and Paracetopsis Eigenmann & Bean and Cetopsogiton synonyms of Paracetopsis. Thirty-seven species are recognized in the Cetopsinae. Cetopsidium includes six species: C. ferreirai, new species, rio Trombetas...

  17. Semen collection and preservation in African catfish, Clarias gariepinus

    NARCIS (Netherlands)

    Viveiros, A.T.M.


    Stock improvement using quantitative and molecular genetics is an essential part of nowadays production of farm animals and fish. To achieve this in aquaculture, germplasm of both parental sexes should be obtained in a life-saving manner. In captivity, male African catfish, Clariasgariepin

  18. Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection

    DEFF Research Database (Denmark)

    Sousa-Nunes, Rita; Rana, Amer Ahmed; Kettleborough, Ross;


    This article investigates the expression patterns of 160 genes that are expressed during early mouse development. The cDNAs were isolated from 7.5 d postcoitum (dpc) endoderm, a region that comprises visceral endoderm (VE), definitive endoderm, and the node-tissues that are required for the initi...

  19. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans. (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S


    Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

  20. The expression sequence tag is an effective method for screening DNA segments that predict urinary bladder transitional cell carcinoma prognosis

    Directory of Open Access Journals (Sweden)

    Yang PS


    Full Text Available Pei-Shan Yang,1,* Yu-Chao Hsu,1,2,* Yu-Hsiang Lin,1,2 Cheng-Pang Hou,1,2 Chien-Lun Chen,1,2 Phei-Lang Chang,1–3 Horng-Heng Juang,3,4 Ke-Hung Tsui1–3 1Department of Urology, Chang Gung Memorial Hospital at Linkou, Taiwan, Republic of China; 2School of Medicine, Chang Gung University, Taiwan, Republic of China; 3Bioinformation Center, Chang Gung Memorial Hospital, Taiwan, Republic of China; 4Department of Anatomy, School of Medicine, Chang Gung University, Taiwan, Republic of China; Kwei-Shan, Tao-Yuan, Taiwan, Republic of China *These authors contributed equally to this workPurpose: We validated the use of expression sequence tags (ESTs as an effective method of screening for DNA segments that could predict urothelial cell carcinoma and for identifying ESTs with such predictive value.Patients and methods: From 2004 to 2009, eleven patients were enrolled in this study: six with high-grade bladder carcinoma and five with low-grade bladder carcinoma. ESTs were used to screen for differential gene expression in a high-grade cell line (MGH-U1 and in a premalignant cell line (MGH-U4. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction were used to validate the degree of EST expression and the prognostic value of ESTs.Results: Apoferritin H subunit (FTH1 protein exhibited increased expression in high-grade bladder carcinoma compared with that seen in low-grade carcinoma. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction both supported the higher expression of FTH1 in high-grade urothelial carcinoma.Conclusion: ESTs are useful for detecting the FTH1 protein, which predicts the prognosis of patients with bladder carcinoma.Keywords: expression sequence tag, transitional cell carcinoma, FTH1

  1. A comprehensive transcriptome provides candidate genes for sex determination/differentiation and SSR/SNP markers in yellow catfish. (United States)

    Chen, Xin; Mei, Jie; Wu, Junjie; Jing, Jing; Ma, Wenge; Zhang, Jin; Dan, Cheng; Wang, Weimin; Gui, Jian-Fang


    Sex dimorphic growth pattern has significant theory and application implications in fish. Recently, a Y- and X-specific allele marker-assisted sex control technique has been developed for mass production of all-male population in yellow catfish (Pelteobagrus fulvidraco), but the genetic information for sex determination and sex control breeding has remained unclear. Here, we attempted to provide the first insight into a comprehensive transcriptome covering multiple tissues from XX females, XY males, and YY super-males of yellow catfish by using 454 GS-FLX platform, for a better assembly and gene coverage. A total of 1,202,933 high quality reads (about 540 Mbp) were obtained and assembled into 28,297 contigs and 141,951 singletons. BLASTX searches against the NCBI non-redundant protein database (nr) led a total of 52,564 unique sequences including 18,748 contigs and 33,816 singletons to match 25,669 known or predicted unique proteins. All of them with annotated function were categorized by gene ontology (GO) analysis, and 712 were assigned to reproduction and reproductive process. Some potential genes relevant to reproductive system including steroid hormone biosynthesis and GnRH (gonadotropin-releasing hormone) signaling pathway were further identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis; and at least 21 sex determination and differentiation-related genes, such as Dmrt1, Sox9a/b, Cyp19b, WT1, and AMH were identified and characterized. Additionally, a total of 82,794 simple sequence repeats (SSRs), 26,450 single nucleotide polymorphisms (SNPs), and 4,145 insertions and deletions (INDELs) were revealed from the transcriptome data. Therefore, the current transcriptome resources highlight further studies on sex-control breeding in yellow catfish and will benefit future studies on reproduction and sex determination in teleost fish.

  2. Rat serum amyloid P component. Analysis of cDNA sequence and gene expression. (United States)

    Dowton, S B; McGrew, S D


    cDNA clones for rat serum amyloid P component (SAP) were isolated, and the derived amino acid sequence for pre-SAP was determined from the complete nucleotide sequence. Rat SAP is encoded by approximately 1 kb of mRNA, and the mature SAP protein is predicted to be 208 amino acids long. An increase in hepatic mRNA levels for rat SAP was found after administration of lipopolysaccharide, and SAP mRNA levels in livers of unstimulated male rats were lower than in hepatic RNA from female rats.

  3. Effect of the Flanking Sequence Architecture of Translation Initiation AUG Codon on Gene Expression Level in Rice

    Institute of Scientific and Technical Information of China (English)

    LIU Qing-po; TAN Jun; XUE Qing-zhong


    The relationship between the codon usage bias, gene expression level and the AUG context (from -20 to +6 positions relative to the initiator AUG codon) was examined in 541unigene sequences of rice. A significant correlation for CAI values (codon adaptation index) was observed at five nucleotide positions (-19, -18, -9, -4, +5), eight (-19, -18,-14, -9, -6, -4, -1, +5) for CPP (codon preference parameter), and seven (-18, -16, -15,-9, -7, -1, + 6) for mRNA abundance in the flanking sequence of the initiator AUG codon respectively, but a significantly positive correlation for both CAI and CPP at two positions (-4 and +5), indicating that both those positions are evolutionally under the natural selection constraint at the translational level. By site-directed mutagenesis at seven specific positions (-18, -16, -15, -9, -7, -1 and + 6) for allergenic protein that had the highest mRNA abundance in this study, its expression level decreased dramatically 63.3 and 72.5% respectively, indicating the importance of those 7 positions for gene expression. A highly positive correlation (r= 0.625, P< 0.01) between AUGCAI and GC content in the flanking sequence of the initiator AUG codon showed a more effective higher GC content on translation initiation efficiency. The strong preference for G or C at those 8 positions (-6, -5, -3, -2, -1, +4, +5 and +6) in the AUG context suggested that an important factor in modulation of the translation efficiency, as well as synonymous codon usage bias, particularly in highly expressed genes.

  4. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat (United States)

    Goswami, Suneha; Kumar, Ranjeet R.; Dubey, Kavita; Singh, Jyoti P.; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C.; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C.; Kala, Yugal K.; Singh, Gyanendra P.; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D.


    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat—a novel step toward the development of

  5. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat. (United States)

    Goswami, Suneha; Kumar, Ranjeet R; Dubey, Kavita; Singh, Jyoti P; Tiwari, Sachidanand; Kumar, Ashok; Smita, Shuchi; Mishra, Dwijesh C; Kumar, Sanjeev; Grover, Monendra; Padaria, Jasdeep C; Kala, Yugal K; Singh, Gyanendra P; Pathak, Himanshu; Chinnusamy, Viswanathan; Rai, Anil; Praveen, Shelly; Rai, Raj D


    Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h) wheat cv. HD2985 by suppression subtractive hybridization (SSH). We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger's sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs). Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD, and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs). We observed eight different types of post-translational modifications (PTMs) in the DEPs corresponds to the cloned ESTs-147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH) in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant), as compared to HD2329 (thermosusceptible) during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat-a novel step toward the development of "climate-smart" wheat.

  6. SSH analysis of endosperm transcripts and characterization of heat stress regulated expressed sequence tags in bread wheat

    Directory of Open Access Journals (Sweden)

    Suneha Goswami


    Full Text Available Heat stress is one of the major problems in agriculturally important cereal crops, especially wheat. Here, we have constructed a subtracted cDNA library from the endosperm of HS-treated (42°C for 2 h wheat cv. HD2985 by suppression subtractive hybridization (SSH. We identified ~550 recombinant clones ranging from 200 to 500 bp with an average size of 300 bp. Sanger’s sequencing was performed with 205 positive clones to generate the differentially expressed sequence tags (ESTs. Most of the ESTs were observed to be localized on the long arm of chromosome 2A and associated with heat stress tolerance and metabolic pathways. Identified ESTs were BLAST search using Ensemble, TriFLD and TIGR databases and the predicted CDS were translated and aligned with the protein sequences available in pfam and InterProScan 5 databases to predict the differentially expressed proteins (DEPs. We observed eight different types of post-translational modifications (PTMs in the DEPs corresponds to the cloned ESTs—147 sites with phosphorylation, 21 sites with sumoylation, 237 with palmitoylation, 96 sites with S-nitrosylation, 3066 calpain cleavage sites, and 103 tyrosine nitration sites, predicted to sense the heat stress and regulate the expression of stress genes. Twelve DEPs were observed to have transmembrane helixes (TMH in their structure, predicted to play the role of sensors of HS. Quantitative Real-Time PCR of randomly selected ESTs showed very high relative expression of HSP17 under HS; up-regulation was observed more in wheat cv. HD2985 (thermotolerant, as compared to HD2329 (thermosusceptible during grain-filling. The abundance of transcripts was further validated through northern blot analysis. The ESTs and their corresponding DEPs can be used as molecular marker for screening or targeted precision breeding program. PTMs identified in the DEPs can be used to elucidate the thermotolerance mechanism of wheat – a novel step towards the development of

  7. Effects of Endosulfan on Predator-Prey Interactions Between Catfish and Schistosoma Host Snails. (United States)

    Monde, Concillia; Syampungani, Stephen; Van den Brink, Paul J


    The effect of the pesticide endosulfan on predator-prey interactions between catfish and Schistosoma host snails was assessed in static tank experiments. Hybrid catfish (Clarias gariepinus × C. ngamensis) and Bulinus globosus were subjected to various endosulfan concentrations including an untreated control. The 48- and 96-h LC50 values for catfish were 1.0 and snails were 1137 and 810 µg/L. To assess sublethal effects on the feeding of the catfish on B. globosus, endosulfan concentrations between 0.03 and 1.0 µg/L were used. Predation was significantly greater (p snails using fish may be affected in endosulfan-polluted aquatic systems of Southern Africa because it has been found present at concentrations that are indicated to cause lethal effects on the evaluated hybrid catfish and to inhibit the predation of snails by this hybrid catfish.

  8. Analysis of RNA-Dependent RNA Polymerase Sequence of Infectious Flacherie Virus Isolated in China and Its Expression in BmN Cells

    Institute of Scientific and Technical Information of China (English)

    LI Ming-qian; LU Qi-neng; WU Xiao-Feng; MAN Nan-a; CHEN Xiao-xue; JIN Wei; LU Xing-meng


    Full gene sequence of RNA-dependent RNA polymerase (RdRp) from Bombyx mori infectious flacherie virus isolated in Zhejiang Province, China (Zhejiang01/CHN/2002) was cloned. The sequence was 1 920 nucleotides in length coding 639 amino acid residues. Sequences comparison of RdRp showed Zhejiang01/CHN/2002 was 99.7% nucleotide sequence and 99.1% amino acids sequence homology with Japanese strain. The RdRp sequence was aligned with 8 representative picorna(-like) viruses and 8 highly conserved regions were detected. The result indicated their relevance function. Phylogenetic tree of 14 picorna(-like) viruses which RdRp presumed protein sequences revealed that the viruses from Iflavirus genus formed an independent clade. The RdRp was successfully expressed in Bran cells using Bac-to-Bac expression system.

  9. Gene expression profiling by high throughput sequencing to determine signatures for the bovine receptive uterus at early gestation

    Directory of Open Access Journals (Sweden)

    Veerle Van Hoeck


    Full Text Available The uterus plays a central role among the reproductive tissues in the context of early embryo-maternal communication and a successful pregnancy depends on a complex series of endometrial molecular and cellular events. The factors responsible for the initial interaction between maternal and embryonic tissues, leading to the establishment of pregnancy, remain poorly understood. In this context, Illumina's next-generation sequencing technology has been used to discover the uterine transcriptome signature that is favourable for ongoing pregnancy. More specifically, the present report documents on a retrospective in vivo study in which data on pregnancy outcome were linked to uterine gene expression signatures on day 6 (bovine model. Using the RNA-Seq method, 14.654 reference genes were effectively analysed for differential expression between pregnant and non-pregnant uterine tissue. Transcriptome data revealed that 216 genes were differently expressed when comparing uterine tissue from pregnant and non-pregnant cows. All read sequences were deposited in the Sequence Read Archive (SRA of the NCBI ( An overview of the gene expression data has been deposited in NCBI's Gene Expression Omnibus (GEO and is accessible through GEO Series accession number GSE65117. This allows the research community to enhance reproducibility and allows for new discoveries by comparing datasets of signatures linked to receptivity and/or pregnancy success. The resulting information can serve as tool to identify valuable and urgently needed biomarkers for scoring maternal receptivity and even for accurate detection of early pregnancy, which is a matter of cross-species interest. Beyond gene expression analysis as a marker tool, the RNA-Seq information on pregnant uterine tissue can be used to gain novel mechanistic insights, such as by identifying alternative splicing events, allele-specific expression, and rare and novel transcripts that might

  10. The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach (United States)


    Background Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences. Results About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein. An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins. Conclusions The use of the combined approaches has allowed to discriminate between cellular

  11. Transcriptome sequencing and differential gene expression analysis in Viola yedoensis Makino (Fam. Violaceae) responsive to cadmium (Cd) pollution

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Jian [Key Laboratory of Biology and Genetic Improvement of Maize in Southwest Region, Ministry of Agriculture, Maize Research Institute of Sichuan Agricultural University, Wenjiang, Sichuan (China); Luo, Mao [Drug Discovery Research Center of Luzhou Medical College, Luzhou, Sichuan (China); Zhu, Ye; He, Ying; Wang, Qin [Department of Pharmacy of Luzhou Medical College, Luzhou, Sichuan (China); Zhang, Chun, E-mail: [Department of Pharmacy of Luzhou Medical College, Luzhou, Sichuan (China)


    Viola yedoensis Makino is an important Chinese traditional medicine plant adapted to cadmium (Cd) pollution regions. Illumina sequencing technology was used to sequence the transcriptome of V. yedoensis Makino. We sequenced Cd-treated (VIYCd) and untreated (VIYCK) samples of V. yedoensis, and obtained 100,410,834 and 83,587,676 high quality reads, respectively. After de novo assembly and quantitative assessment, 109,800 unigenes were finally generated with an average length of 661 bp. We then obtained functional annotations by aligning unigenes with public protein databases including NR, NT, SwissProt, KEGG and COG. In addition, 892 differentially expressed genes (DEGs) were investigated between the two libraries of untreated (VIYCK) and Cd-treated (VIYCd) plants. Moreover, 15 randomly selected DEGs were further validated with qRT-PCR and the results were highly accordant with the Solexa analysis. This study firstly generated a successful global analysis of the V. yedoensis transcriptome and it will provide for further studies on gene expression, genomics, and functional genomics in Violaceae. - Highlights: • A de novo assembly generated 109,800 unigenes and 5,4479 of them were annotated. • 31,285 could be classified into 26 COG categories. • 263 biosynthesis pathways were predicted and classified into five categories. • 892 DEGs were detected and 15 of them were validated by qRT-PCR.

  12. Bioinformatic prediction, deep sequencing of microRNAs and expression analysis during phenotypic plasticity in the pea aphid, Acyrthosiphon pisum

    Directory of Open Access Journals (Sweden)

    Leterme Nathalie


    Full Text Available Abstract Background Post-transcriptional regulation in eukaryotes can be operated through microRNA (miRNAs mediated gene silencing. MiRNAs are small (18-25 nucleotides non-coding RNAs that play crucial role in regulation of gene expression in eukaryotes. In insects, miRNAs have been shown to be involved in multiple mechanisms such as embryonic development, tissue differentiation, metamorphosis or circadian rhythm. Insect miRNAs have been identified in different species belonging to five orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera and Orthoptera. Results We developed high throughput Solexa sequencing and bioinformatic analyses of the genome of the pea aphid Acyrthosiphon pisum in order to identify the first miRNAs from a hemipteran insect. By combining these methods we identified 149 miRNAs including 55 conserved and 94 new miRNAs. Moreover, we investigated the regulation of these miRNAs in different alternative morphs of the pea aphid by analysing the expression of miRNAs across the switch of reproduction mode. Pea aphid microRNA sequences have been posted to miRBase: Conclusions Our study has identified candidates as putative regulators involved in reproductive polyphenism in aphids and opens new avenues for further functional analyses.

  13. Cloning, sequence identification and expression profile analysis of α-L-fucosidase gene from the Mediterranean fruit fly Ceratitis capitata. (United States)

    Intra, Jari; Perotti, Maria-Elisa; Pasini, Maria Enrica


    The Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae) is one of the most destructive agricultural pests, a polyphagus insect of relevant economic importance and is widespread in many regions around the world. It is the best-studied fruit fly pest at genetic and molecular level and much has been learned on its ecology and behaviour. An α-L-fucosidase has been recently hypothesized to be involved in sperm-egg interactions in Drosophila melanogaster and in other Drosophila species. Here, a complete cDNA encoding a putative α-L-fucosidase of the medfly was amplified using the reverse polymerase chain reaction (RT-PCR) with degenerate based on the conserved coding sequence information of several insect α-L-fucosidases, cloned and sequenced (GenBank accession no. FJ177429). The coding region consisted of 1482 bp which encoded a 485-residues protein (named CcFUCA) with a predicted molecular mass of 56.1 kDa. The deduced protein sequence showed 75% amino acid identity to D. melanogaster α-L-fucosidase, and in fact the phylogenetic tree analysis revealed that CcFUCA had closer relationships with the α-L-fucosidases of drosophilid species. The tissue expression analysis indicated that CcFuca was expressed in a single transcript in all tissues, suggesting a ubiquitous localization pattern of the encoded protein. Our findings provide novel insights on a gene encoding a protein potentially involved in primary gamete interactions in C. capitata.

  14. Cloning, sequencing and functional expression of cytosolic malate dehydrogenase from Taenia solium: Purification and characterization of the recombinant enzyme. (United States)

    Nava, Gabriela; Laclette, Juan P; Bobes, Raúl; Carrero, Julio C; Reyes-Vivas, Horacio; Enriquez-Flores, Sergio; Mendoza-Hernández, Guillermo; Plancarte, Agustín


    We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. K(cat) values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962s(-1), respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56Kb genomic contig assembly is also reported.

  15. Sequence, evolution and tissue expression patterns of an epidermal type I keratin from the shark Scyliorhinus stellaris. (United States)

    Schaffeld, Michael; Höffling, Simon; Jürgen, Markl


    From the shark Scyliorhinus stellaris we cloned and sequenced a cDNA encoding a novel type I keratin, termed SstK10. By MALDI-MS peptide mass fingerprinting of cytoskeletal proteins separated on polyacrylamide gels, we assigned SstK10 to a 46-kDa protein which is the major epidermal type I ("IE") keratin in this fish and is specifically expressed in stratified epithelia. In a phylogenetic tree based on type I keratin sequences and with lamprey keratins applied as outgroup, SstK10 branches off in a rather basal position. This tree strongly supports the concept that teleost keratins and tetrapod keratins resulted from two independent gene radiation processes. The only exception is human K18 because its orthologs have been found in all jawed vertebrates (Gnathostomata) studied; in the tree, they form a common, most early branch, with the shark version, SstK18, in the most basal position. Thus, the sequences of SstK10 and SstK18 also favor the classical view of vertebrate evolution that considers the cartilaginous fishes as the most ancient living Gnathostomata. To determine the overall expression patterns of epidermal ("E") and simple epithelial ("S") keratins in this shark, we furthermore tested a panel of monoclonal anti-keratin antibodies by immunofluorescence microscopy of frozen tissue sections, and in immunoblots of cytoskeletal preparations, demonstrating that immunodetection of specific keratins is a convenient method to characterize epithelial tissues in shark.

  16. Comparative analysis of gene expression at early seedling stage between a rice hybrid and its parents using a cDNA microarray of 9198 uni-sequences

    Institute of Scientific and Technical Information of China (English)

    HUANG; Yi; LI; Lihua; CHEN; Ying; LI; Xianghua; XU; Caiguo; WANG; Shiping; ZHANG; Qifa


    Using a cDNA microarray consisting of 9198 expressed sequence tags, we surveyed the gene expression profiles in shoots and roots of a rice hybrid, Liangyoupei 9 and its parents Peiai 64s and 93-11 at 72 h after germination. A total of 8587 sequences had detectable signals in both shoots and roots of the three genotypes. A total of 1571 sequences exhibited significant (P<0.01) expression differences in shoots or roots among the three genotypes, of which 121 showed expression polymorphisms in both shoots and roots, and 870 revealed significant expression differences between the hybrid and one of the parents. The expression polymorphism of the sequences was associated with the functional categories of the sequences. They occurred more frequently in categories of carbohydrate, energy and lipid metabolisms and stress response than expected, while less frequently in categories of amino acid metabolism, transcription and translation regulation, and signal transduction. A total of 214 sequences exhibited significant (P<0.05) mid-parent heterosis in expression, of which 117 had homology to genes with known functions, assigned in the categories of basic metabolism, genetic information processing, cell growth and death, signal transduction, transportation and stress response. The results may provide useful information for exploring the relationship between gene expression polymorphism and phenotypic variation, and for characterizing the molecular mechanism of seedling development and heterosis in rice.

  17. The S-layer gene of Lactobacillus helveticus CNRZ 892 : cloning, sequence and heterologous expression

    NARCIS (Netherlands)

    Callegari, M.L.; Riboli, B.; Sanders, J.W; Cocconcelli, P.S.; Kok, J.; Venema, G; Morelli, L.


    Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (slpH) has been cloned in Escherichia coli and sequenced. slpH consists of 440 codons and is preceded by a ribosome-binding site (RB

  18. Update of the Gene Discovery Program in Schistosoma mansoni with the Expressed Sequence Tag Approach

    Directory of Open Access Journals (Sweden)

    Élida ML Rabelo


    Full Text Available Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of these genes (81% had not previously been described in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor

  19. Molecular Cloning and Expression of Sequence Variants of Manganese Superoxide Dismutase Genes from Wheat (United States)

    Reactive oxygen species (ROS) are very harmful to living organisms due to the potential oxidation of membrane lipids, DNA, proteins, and carbohydrates. Transformed E.coli strain QC 871, superoxide dismutase (SOD) double-mutant, with three sequence variant MnSOD1, MnSOD2, and MnSOD3 manganese supero...

  20. A tandem-repeat galectin-9 involved in immune response of yellow catfish, Pelteobagrus fulvidraco, against Aeromonas hydrophila. (United States)

    Wang, Yun; Ke, Fei; Ma, Jingjing; Zhou, Shuaibang


    Galectins exclusively recognize and bind β-galactoside on cell surface by carbohydrate recognition domain (CRD). In spite of extensive study of mammalian galectin importance in immune system, little is known about that of fish. To study the immune response of yellow catfish to pathogens, a tandem-repeat galectin-9 from yellow catfish was identified and named PfGAL9. Its full-length cDNA was 1314 bp, including a 117 bp of 5' untranslated region (UTR), a 951 bp of open reading frame (ORF), and a 246 bp of 3' UTR. The ORF encoded 316 amino acids (35.12 KDa), shared the highest 78% identity with the predicted galectin-9 of Ictalurus punctatus. This protein possessed two distinct CRDs with two highly conserved sugar binding motifs. Quantitative PCR showed that PfGAL9 was lowly expressed in skin, gill, fin, muscle, heart, and intestine, highly expressed in tested immune tissues (head kidney, trunk kidney, liver, spleen, and blood) in normal body. After inactivated Aeromonas hydrophila challenge, PfGAL9 was remarkably increased in head kidney and liver in a time-dependent manner. The recombinant protein was expressed in Escherichia coli, which not only agglutinated but also bond all examined bacteria. The binding activities are consistent with the size of aggregates formed by agglutinated bacteria. The agglutination must depend on its direct interaction with bacteria. These results suggested that PfGAL9 was involved in the innate immune response against bacterial infection and clearance of pathogens in yellow catfish.

  1. Iron status of channel catfish Ictalurus punctatus affected by channel catfish anemia and response to parenteral iron (United States)

    Originally reported in 1983, channel catfish anemia (CCA), also ‘white lip’ or ‘no blood,’ is a major idiopathic disease affecting commercial production in the Mississippi Delta region of the USA. Affected individuals are characterized by lethargy, anorexia, extreme pallor, and packed cell volumes o...

  2. 76 FR 26655 - Public Meetings on the Proposed Rule for Mandatory Inspection of Catfish and Catfish Products (United States)


    ... Control Point (HACCP) regulations for seafood (9 CFR part 123), including catfish, and to other...(b)), known as the 2008 Farm Bill, amended the Federal Meat Inspection Act (FMIA) to provide that... transportation, facilities and sanitation, requirements for Sanitation Standard Operation Procedures and...

  3. cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the indianmeal moth, Plodia interpunctella. (United States)

    Zhu, Y C; Oppert, B; Kramer, K J; McGaughey, W H; Dowdy, A K


    Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two

  4. Mitochondrial genome sequence and expression profiling for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae.

    Directory of Open Access Journals (Sweden)

    Venu M Margam

    Full Text Available We report the assembly of the 14,054 bp near complete sequencing of the mitochondrial genome of the legume pod borer (LPB, Maruca vitrata (Lepidoptera: Crambidae, which we subsequently used to estimate divergence and relationships within the lepidopteran lineage. The arrangement and orientation of the 13 protein-coding, 2 rRNA, and 19 tRNA genes sequenced was typical of insect mitochondrial DNA sequences described to date. The sequence contained a high A+T content of 80.1% and a bias for the use of codons with A or T nucleotides in the 3rd position. Transcript mapping with midgut and salivary gland ESTs for mitochondrial genome annotation showed that translation from protein-coding genes initiates and terminates at standard mitochondrial codons, except for the coxI gene, which may start from an arginine CGA codon. The genomic copy of coxII terminates at a T nucleotide, and a proposed polyadenylation mechanism for completion of the TAA stop codon was confirmed by comparisons to EST data. EST contig data further showed that mature M. vitrata mitochondrial transcripts are monocistronic, except for bicistronic transcripts for overlapping genes nd4/nd4L and nd6/cytb, and a tricistronic transcript for atp8/atp6/coxIII. This processing of polycistronic mitochondrial transcripts adheres to the tRNA punctuated cleavage mechanism, whereby mature transcripts are cleaved only at intervening tRNA gene sequences. In contrast, the tricistronic atp8/atp6/coxIII in Drosophila is present as separate atp8/atp6 and coxIII transcripts despite the lack of an intervening tRNA. Our results indicate that mitochondrial processing mechanisms vary between arthropod species, and that it is crucial to use transcriptional information to obtain full annotation of mitochondrial genomes.

  5. Mitochondrial genome sequence and expression profiling for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae). (United States)

    Margam, Venu M; Coates, Brad S; Hellmich, Richard L; Agunbiade, Tolulope; Seufferheld, Manfredo J; Sun, Weilin; Ba, Malick N; Sanon, Antoine; Binso-Dabire, Clementine L; Baoua, Ibrahim; Ishiyaku, Mohammad F; Covas, Fernando G; Srinivasan, Ramasamy; Armstrong, Joel; Murdock, Larry L; Pittendrigh, Barry R


    We report the assembly of the 14,054 bp near complete sequencing of the mitochondrial genome of the legume pod borer (LPB), Maruca vitrata (Lepidoptera: Crambidae), which we subsequently used to estimate divergence and relationships within the lepidopteran lineage. The arrangement and orientation of the 13 protein-coding, 2 rRNA, and 19 tRNA genes sequenced was typical of insect mitochondrial DNA sequences described to date. The sequence contained a high A+T content of 80.1% and a bias for the use of codons with A or T nucleotides in the 3rd position. Transcript mapping with midgut and salivary gland ESTs for mitochondrial genome annotation showed that translation from protein-coding genes initiates and terminates at standard mitochondrial codons, except for the coxI gene, which may start from an arginine CGA codon. The genomic copy of coxII terminates at a T nucleotide, and a proposed polyadenylation mechanism for completion of the TAA stop codon was confirmed by comparisons to EST data. EST contig data further showed that mature M. vitrata mitochondrial transcripts are monocistronic, except for bicistronic transcripts for overlapping genes nd4/nd4L and nd6/cytb, and a tricistronic transcript for atp8/atp6/coxIII. This processing of polycistronic mitochondrial transcripts adheres to the tRNA punctuated cleavage mechanism, whereby mature transcripts are cleaved only at intervening tRNA gene sequences. In contrast, the tricistronic atp8/atp6/coxIII in Drosophila is present as separate atp8/atp6 and coxIII transcripts despite the lack of an intervening tRNA. Our results indicate that mitochondrial processing mechanisms vary between arthropod species, and that it is crucial to use transcriptional information to obtain full annotation of mitochondrial genomes.

  6. Isolated yeast promoter sequence and a method of regulated heterologous expression (United States)

    Gao, Johnway; Skeen, Rodney S.; Hooker, Brian S.; Anderson, Daniel B.


    The present invention provides the promoter clone discovery of a glucoamylase gene of a starch utilizing yeast strain Schwanniomyces castellii. The isolated glucoamylase promoter is an inducible promoter, which can regulate strong gene expression in starch culture medium.

  7. Comprehensive profiling of EBV gene expression in nasopharyngeal carcinoma through paired-end transcriptome sequencing. (United States)

    Hu, Lijuan; Lin, Zhirui; Wu, Yanheng; Dong, Juqin; Zhao, Bo; Cheng, Yanbing; Huang, Peiyu; Xu, Lihua; Xia, Tianliang; Xiong, Dan; Wang, Hongbo; Li, Manzhi; Guo, Ling; Kieff, Elliott; Zeng, Yixin; Zhong, Qian; Zeng, Musheng


    The latent expression pattern of Epstein-Barr Virus (EBV) genes in nasopharyngeal carcinoma (NPC) has been extensively investigated, and the expression of several lytic genes in NPC has been reported. However, comprehensive information through EBV transcriptome analysis in NPC is limited. We performed paired-end RNA-seq to systematically and comprehensively characterize the expression of EBV genes in NPC tissue and C666-1 NPC cell line, which consistently carries EBV. In addition to the transcripts restricted to type II latency infection, the type III latency EBNA3s genes and a substantial number of lytic genes, such as BZLF1, BRLF1, and BMRF1, were detected through RNA-seq and were further verified in C666-1 cells and NPC tissue through realtime PCR.We also performed clustering analysis to classify NPC patient groups in terms of EBV gene expression, which presented two subtypes of NPC samples. Results revealed interesting patterns of EBV gene expression in NPC patients. This clustering was correlated with many signaling pathways, such as those related to heterotrimeric G-protein signaling, inflammation mediated by chemokine and cytokine signaling, ribosomes, protein metabolism, influenza infection, and ECM-receptor interaction. Our combined findings suggested that the expression of EBV genes in NPC is restricted not only to type II latency genes but also to type III latency and lytic genes. This study provided further insights into the potential role of EBV in the development of NPC.

  8. Effective inhibition of human cytomegalovirus gene expression by DNA-based external guide sequences

    Institute of Scientific and Technical Information of China (English)

    Zhifeng Zeng; Hongjian Li; Yueqing Li; Yanwei Cui; Qi Zhou; Yi Zou; Guang Yang; Tianhong Zhou


    To investigate whether a 12 nucleotide DNA-based miniEGSs can silence the expression of human cytomegalovirus(HCMV)UL49 gene efficiently,A HeLa cell line stably expressing UL49 gene was constructed and the putative miniEGSs(UL49-miniEGSs)were assayed in the stable cell line.Quantitative RT-PCR and western blot resuits showed a reduction of 67%in UL49expression level in HeLa cells that were transfected with UL49-miniEGSs.It was significantly different from that of mock and control miniEGSs(TK-miniEGSs)which were 1 and 7%,respectively.To further confirm the gene silence directed by UL49-miniEGSs with human RNase P,a mutant of UL49-miniEGSs was constructedand a modified 5'RACE was carried out.Data showed that the inhibition of UL49 gene expression directed by UL49-miniEGSs was RNase P-dependent and the clea vage of UL49 mRNA by RNase P was site specific.As a result,the length of DNA-based miniEGSs that could silence gene expression efficiently was only 12 nt.That is significantly less than any other Oligonucleotide-based method of gene inactivation known SO far.MiniEGSs may represent novel gene-targeting agents for the inhibition of viral genes and other human disease reiated gene expression.

  9. Flavonoid Biosynthesis Genes Putatively Identified in the Aromatic Plant Polygonum minus via Expressed Sequences Tag (EST Analysis

    Directory of Open Access Journals (Sweden)

    Zamri Zainal


    Full Text Available P. minus is an aromatic plant, the leaf of which is widely used as a food additive and in the perfume industry. The leaf also accumulates secondary metabolites that act as active ingredients such as flavonoid. Due to limited genomic and transcriptomic data, the biosynthetic pathway of flavonoids is currently unclear. Identification of candidate genes involved in the flavonoid biosynthetic pathway will significantly contribute to understanding the biosynthesis of active compounds. We have constructed a standard cDNA library from P. minus leaves, and two normalized full-length enriched cDNA libraries were constructed from stem and root organs in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis. Thus, large‑scale sequencing of P. minus cDNA libraries identified 4196 expressed sequences tags (ESTs which were deposited in dbEST in the National Center of Biotechnology Information (NCBI. From the three constructed cDNA libraries, 11 ESTs encoding seven genes were mapped to the flavonoid biosynthetic pathway. Finally, three flavonoid biosynthetic pathway-related ESTs chalcone synthase, CHS (JG745304, flavonol synthase, FLS (JG705819 and leucoanthocyanidin dioxygenase, LDOX (JG745247 were selected for further examination by quantitative RT-PCR (qRT-PCR in different P. minus organs. Expression was detected in leaf, stem and root. Gene expression studies have been initiated in order to better understand the underlying physiological processes.

  10. Nucleotide sequence and expression of relaxin-like gonad-stimulating peptide gene in starfish Asterina pectinifera. (United States)

    Haraguchi, Shogo; Ikeda, Narumi; Abe, Michiko; Tsutsui, Kazuyoshi; Mita, Masatoshi


    Starfish gonad-stimulating substance (GSS) is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Because GSS belongs to the relaxin-like peptide family, we propose renaming for starfish gonadotropic hormone as relaxin-like gonad-stimulating peptide (RGP). This study examined the primary structure and expression regulation of the RGP gene in starfish Asterina pectinifera. RGP consisted of 3896 base pairs (bp) divided over two exons, exon 1 of 208 bp and exon 2 of 2277 bp, and one intron of 1411 bp. Promoter sequences, CAAT and TATA boxes, were present in the 5'-upstream region of the coding DNA sequence of RGP. The transcript was 2485 bases (b) in length. The AAUAAA polyadenylation signal was found in 3'-untranslated region over 2kb away from the stop codon. This showed that only 14% of the RGP mRNA was translated into the peptide, because a size of the open-reading frame was 351 b. Furthermore, an analysis by using real-time quantitative PCR with specific primers for RGP showed that mRNA of RGP was expressed at high levels in the radial nerves. Expression was also observed in the cardiac stomachs, although the level was low, and trace levels were detected in the gonads, pyloric caeca and tube feet. This result suggests that the RGP gene is transcribed mainly in the radial nerves of A. pectinifera.

  11. Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster (United States)

    Lau, Yun-Fai; Kan, Yuet Wai


    We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

  12. RNA Sequence Analysis of Human Huntington Disease Brain Reveals an Extensive Increase in Inflammatory and Developmental Gene Expression.

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    Adam Labadorf

    Full Text Available Huntington's Disease (HD is a devastating neurodegenerative disorder that is caused by an expanded CAG trinucleotide repeat in the Huntingtin (HTT gene. Transcriptional dysregulation in the human HD brain has been documented but is incompletely understood. Here we present a genome-wide analysis of mRNA expression in human prefrontal cortex from 20 HD and 49 neuropathologically normal controls using next generation high-throughput sequencing. Surprisingly, 19% (5,480 of the 28,087 confidently detected genes are differentially expressed (FDR<0.05 and are predominantly up-regulated. A novel hypothesis-free geneset enrichment method that dissects large gene lists into functionally and transcriptionally related groups discovers that the differentially expressed genes are enriched for immune response, neuroinflammation, and developmental genes. Markers for all major brain cell types are observed, suggesting that HD invokes a systemic response in the brain area studied. Unexpectedly, the most strongly differentially expressed genes are a homeotic gene set (represented by Hox and other homeobox genes, that are almost exclusively expressed in HD, a profile not widely implicated in HD pathogenesis. The significance of transcriptional changes of developmental processes in the HD brain is poorly understood and warrants further investigation. The role of inflammation and the significance of non-neuronal involvement in HD pathogenesis suggest anti-inflammatory therapeutics may offer important opportunities in treating HD.

  13. Differential microRNA expression between shoots and rhizomes in Oryza longistaminata using high-throughput RNA sequencing

    Institute of Scientific and Technical Information of China (English)

    Ying; Zong; Liyu; Huang; Ting; Zhang; Qiao; Qin; Wensheng; Wang; Xiuqin; Zhao; Fengyi; Hu; Binying; Fu; Zhikang; Li


    Plant microRNAs(miRNAs)play important roles in biological processes such as development and stress responses.Although the diverse functions of miRNAs in model organisms have been well studied,their function in wild rice is poorly understood.In this study,high-throughput small RNA sequencing was performed to characterize tissue-specific transcriptomes in Oryza longistaminata.A total of 603 miRNAs,380 known rice miRNAs,72 conserved plant miRNAs,and151 predicted novel miRNAs were identified as being expressed in aerial shoots and rhizomes.Additionally,99 and 79 miRNAs were expressed exclusively or differentially,respectively,in the two tissues,and 144 potential targets were predicted for the differentially expressed miRNAs in the rhizomes.Functional annotation of these targets suggested that transcription factors,including squamosa promoter binding proteins and auxin response factors,function in rhizome growth and development.The expression levels of several miRNAs and target genes in the rhizomes were quantified by RT-PCR,and the results indicated the existence of complex regulatory mechanisms between the miRNAs and their targets.Eight target cleavage sites were verified by RNA ligase-mediated rapid 5′end amplification.These results provide valuable information on the composition,expression and function of miRNAs in O.longistaminata,and will aid in understanding the molecular mechanisms of rhizome development.

  14. Transient and Stable GFP Expression in Germ Cells by the vasa Regulatory Sequences from the Red Seabream (Pagrus major

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    Fan Lin, Qinghua Liu, Mingyou Li, Zhendong Li, Ni Hong, Jun Li, Yunhan Hong


    Full Text Available Primordial germ cells (PGCs are the precursors of gametes responsible for genetic transmission to the next generation. They provide an ideal system for cryopreservation and restoration of biodiversity. Recently, considerable attention has been raised to visualize, isolate and transplant PGCs within and between species. In fish, stable PGC visualization in live embryo and individual has been limited to laboratory fish models such as medaka and zebrafish. One exception is the rainbow trout, which represents the only species with aquaculture importance and has GFP-labeled germ cells throughout development. PGCs can be transiently labeled by embryonic injection of mRNA containing green fluorescence protein gene (GFP and 3'-untranslated region (3'-UTR of a maternal germ gene such as vasa, nos1, etc. Stable PGC labeling can be achieved through production of transgenic animals by some transcriptional regulatory sequences from germ genes, such as the vasa promoter and 3'-UTR. In this study, we reported the functional analyses of the red seabream vasa (Pmvas regulatory sequences, using medaka as a model system. It was showed that injection of GFP-Pmvas3'UTR mRNA was able to label medaka PGCs during embryogenesis. Besides, we have constructed pPmvasGFP transgenic vector, and established a stable transgenic medaka line exhibiting GFP expression in germ cells including PGCs, mitotic and meiotic germ cells of both sexes, under control of the Pmvas transcriptional regulatory sequences. It is concluded that the Pmvas regulatory sequences examined in this study are sufficient for germ cell expression and labeling.

  15. Palatability of diets for channel catfish that contain amprolium or salinomycin using feed conversion ratio as criterion (United States)

    Two feeding studies were conducted to evaluate the palatability of diets for juvenile Channel Catfish Ictalurus punctatus that contained the poultry anticoccidial drugs Amprolium or Salinomycin, which are candidates to control the Channel Catfish myxozoan parasite, Henneguya ictaluri. These chemothe...

  16. ESTPiper – a web-based analysis pipeline for expressed sequence tags

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    Tang Zuojian


    Full Text Available Abstract Background EST sequencing projects are increasing in scale and scope as the genome sequencing technologies migrate from core sequencing centers to individual research laboratories. Effectively, generating EST data is no longer a bottleneck for investigators. However, processing large amounts of EST data remains a non-trivial challenge for many. Web-based EST analysis tools are proving to be the most convenient option for biologists when performing their analysis, so these tools must continuously improve on their utility to keep in step with the growing needs of research communities. We have developed a web-based EST analysis pipeline called ESTPiper, which streamlines typical large-scale EST analysis components. Results The intuitive web interface guides users through each step of base calling, data cleaning, assembly, genome alignment, annotation, analysis of gene ontology (GO, and microarray oligonucleotide probe design. Each step is modularized. Therefore, a user can execute them separately or together in batch mode. In addition, the user has control over the parameters used by the underlying programs. Extensive documentation of ESTPiper's functionality is embedded throughout the web site to facilitate understanding of the required input and interpretation of the computational results. The user can also download intermediate results and port files to separate programs for further analysis. In addition, our server provides a time-stamped description of the run history for reproducibility. The pipeline can also be installed locally, allowing researchers to modify ESTPiper to suit their own needs. Conclusion ESTPiper streamlines the typical process of EST analysis. The pipeline was initially designed in part to support the Daphnia pulex cDNA sequencing project. A web server hosting ESTPiper is provided at to now support projects of all size. The software is also freely available from the authors for

  17. Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability. (United States)

    Fehrmann, Steffen; Bottin-Duplus, Hélène; Leonidou, Andri; Mollereau, Esther; Barthelaix, Audrey; Wei, Wu; Steinmetz, Lars M; Yvert, Gaël


    Living systems may have evolved probabilistic bet hedging strategies that generate cell-to-cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent P(met17)-GFP reporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell-to-cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced P(met17)-GFP expression variability. At a second locus, cis-regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transporter genes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.

  18. Candidate genes revealed by a genome scan for mosquito resistance to a bacterial insecticide: sequence and gene expression variations

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    David Jean-Philippe


    Full Text Available Abstract Background Genome scans are becoming an increasingly popular approach to study the genetic basis of adaptation and speciation, but on their own, they are often helpless at identifying the specific gene(s or mutation(s targeted by selection. This shortcoming is hopefully bound to disappear in the near future, thanks to the wealth of new genomic resources that are currently being developed for many species. In this article, we provide a foretaste of this exciting new era by conducting a genome scan in the mosquito Aedes aegypti with the aim to look for candidate genes involved in resistance to Bacillus thuringiensis subsp. israelensis (Bti insecticidal toxins. Results The genome of a Bti-resistant and a Bti-susceptible strains was surveyed using about 500 MITE-based molecular markers, and the loci showing the highest inter-strain genetic differentiation were sequenced and mapped on the Aedes aegypti genome sequence. Several good candidate genes for Bti-resistance were identified in the vicinity of these highly differentiated markers. Two of them, coding for a cadherin and a leucine aminopeptidase, were further examined at the sequence and gene expression levels. In the resistant strain, the cadherin gene displayed patterns of nucleotide polymorphisms consistent with the action of positive selection (e.g. an excess of high compared to intermediate frequency mutations, as well as a significant under-expression compared to the susceptible strain. Conclusion Both sequence and gene expression analyses agree to suggest a role for positive selection in the evolution of this cadherin gene in the resistant strain. However, it is unlikely that resistance to Bti is conferred by this gene alone, and further investigation will be needed to characterize other genes significantly associated with Bti resistance in Ae. aegypti. Beyond these results, this article illustrates how genome scans can build on the body of new genomic information (here, full

  19. Development and characterization of 1,827 expressed sequence tag-derived simple sequence repeat markers for ramie (Boehmeria nivea L. Gaud.

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    Touming Liu

    Full Text Available Ramie (Boehmeria nivea L. Gaud is one of the most important natural fiber crops, and improvement of fiber yield and quality is the main goal in efforts to breed superior cultivars. However, efforts aimed at enhancing the understanding of ramie genetics and developing more effective breeding strategies have been hampered by the shortage of simple sequence repeat (SSR markers. In our previous study, we had assembled de novo 43,990 expressed sequence tags (ESTs. In the present study, we searched these previously assembled ESTs for SSRs and identified 1,685 ESTs (3.83% containing 1,878 SSRs. Next, we designed 1,827 primer pairs complementary to regions flanking these SSRs, and these regions were designated as SSR markers. Among these markers, dinucleotide and trinucleotide repeat motifs were the most abundant types (36.4% and 36.3%, respectively, whereas tetranucleotide, pentanucleotide, and hexanucleotide motifs represented <10% of the markers. The motif AG/CT was the most abundant, accounting for 28.74% of the markers. One hundred EST-SSR markers (97 SSRs located in genes encoding transcription factors and 3 SSRs in genes encoding cellulose synthases were amplified using polymerase chain reaction for detecting 24 ramie varieties. Of these 100 markers, 98 markers were successfully amplified and 81 markers were polymorphic, with 2-6 alleles among the 24 varieties. Analysis of the genetic diversity of all 24 varieties revealed similarity coefficients that ranged from 0.51 to 0.80. The EST-SSRs developed in this study represent the first large-scale development of SSR markers for ramie. These SSR markers could be used for development of genetic and physical maps, quantitative trait loci mapping, genetic diversity studies, association mapping, and cultivar fingerprinting.

  20. Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing

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    Dorothee Pflueger


    Full Text Available TFE3 translocation renal cell carcinoma (tRCC is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study,we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNAread-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2 point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs. The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P<.001 and 22 papillaryRCCs (P<.05-.07. Immunohistochemical analysis of selected genes from the tRCC gene signature showed significantly higher eukaryotic translation elongation factor 1 alpha 2 (EEF1A2 and Contactin 3 (CNTN3 expression in 16 TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in over 200 ccRCCs (P < .0001, both.

  1. Characterization of high-level expression and sequencing of the Escherichia coli K-12 cynS gene encoding cyanase.


    Sung, Y C; Anderson, P. M.; Fuchs, J A


    Restriction fragments containing the gene encoding cyanase, cynS, without its transcriptional regulatory sequences were placed downstream of lac and tac promoters in various pUC derivatives to maximize production of cyanase. Plasmid pSJ105, which contains the cynS gene and an upstream open reading frame, gave the highest expression of cyanase. Approximately 50% of the total soluble protein in stationary-phase cultures of a lac-deleted strain containing plasmid pSJ105 was cyanase. The inserted...

  2. Language trees support the express-train sequence of Austronesian expansion. (United States)

    Gray, R D; Jordan, F M


    Languages, like molecules, document evolutionary history. Darwin observed that evolutionary change in languages greatly resembled the processes of biological evolution: inheritance from a common ancestor and convergent evolution operate in both. Despite many suggestions, few attempts have been made to apply the phylogenetic methods used in biology to linguistic data. Here we report a parsimony analysis of a large language data set. We use this analysis to test competing hypotheses--the "express-train" and the "entangled-bank" models--for the colonization of the Pacific by Austronesian-speaking peoples. The parsimony analysis of a matrix of 77 Austronesian languages with 5,185 lexical items produced a single most-parsimonious tree. The express-train model was converted into an ordered geographical character and mapped onto the language tree. We found that the topology of the language tree was highly compatible with the express-train model.

  3. RNA-sequence analysis of gene expression from honeybees (Apis mellifera) infected with Nosema ceranae (United States)

    Fougeroux, André; Petit, Fabien; Anselmo, Anna; Gorni, Chiara; Cucurachi, Marco; Cersini, Antonella; Granato, Anna; Cardeti, Giusy; Formato, Giovanni; Mutinelli, Franco; Giuffra, Elisabetta; Williams, John L.; Botti, Sara


    Honeybees (Apis mellifera) are constantly subjected to many biotic stressors including parasites. This study examined honeybees infected with Nosema ceranae (N. ceranae). N. ceranae infection increases the bees energy requirements and may contribute to their decreased survival. RNA-seq was used to investigate gene expression at days 5, 10 and 15 Post Infection (P.I) with N. ceranae. The expression levels of genes, isoforms, alternative transcription start sites (TSS) and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation suggesting that bees use a range of tactics to cope with the stress of N. ceranae infection. N. ceranae infection may cause reduced immune function in the bees by: (i)disturbing the host amino acids metabolism (ii) down-regulating expression of antimicrobial peptides (iii) down-regulation of cuticle coatings and (iv) down-regulation of odorant binding proteins. PMID:28350872

  4. Geometric Feature-Based Facial Expression Recognition in Image Sequences Using Multi-Class AdaBoost and Support Vector Machines

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    Joonwhoan Lee


    Full Text Available Facial expressions are widely used in the behavioral interpretation of emotions, cognitive science, and social interactions. In this paper, we present a novel method for fully automatic facial expression recognition in facial image sequences. As the facial expression evolves over time facial landmarks are automatically tracked in consecutive video frames, using displacements based on elastic bunch graph matching displacement estimation. Feature vectors from individual landmarks, as well as pairs of landmarks tracking results are extracted, and normalized, with respect to the first frame in the sequence. The prototypical expression sequence for each class of facial expression is formed, by taking the median of the landmark tracking results from the training facial expression sequences. Multi-class AdaBoost with dynamic time warping similarity distance between the feature vector of input facial expression and prototypical facial expression, is used as a weak classifier to select the subset of discriminative feature vectors. Finally, two methods for facial expression recognition are presented, either by using multi-class AdaBoost with dynamic time warping, or by using support vector machine on the boosted feature vectors. The results on the Cohn-Kanade (CK+ facial expression database show a recognition accuracy of 95.17% and 97.35% using multi-class AdaBoost and support vector machines, respectively.

  5. Sequencing and transcriptional analysis of the Streptococcus thermophilus histamine biosynthesis gene cluster: factors that affect differential hdcA expression

    DEFF Research Database (Denmark)

    Calles-Enríquez, Marina; Hjort, Benjamin Benn; Andersen, Pia Skov;


    Histamine, a toxic compound that is formed by the decarboxylation of histidine through the action of microbial decarboxylases, can accumulate in fermented food products. From a total of 69 Streptococcus thermophilus strains screened, two strains, CHCC1524 and CHCC6483, showed the capacity...... to produce histamine. The hdc clusters of S. thermophilus CHCC1524 and CHCC6483 were sequenced, and the factors that affect histamine biosynthesis and histidine-decarboxylating gene (hdcA) expression were studied. The hdc cluster began with the hdcA gene, was followed by a transporter (hdcP), and ended...... acquisition through a horizontal transfer mechanism. Transcriptional analysis of the hdc cluster revealed the existence of a polycistronic mRNA covering the three genes. The histidine-decarboxylating gene (hdcA) of S. thermophilus demonstrated maximum expression during the stationary growth phase, with high...

  6. Laccase Gene Family in Cerrena sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis

    Directory of Open Access Journals (Sweden)

    Jie Yang


    Full Text Available Laccases are a class of multi-copper oxidases with industrial potential. In this study, eight laccases (Lac1–8 from Cerrena sp. strain HYB07, a white-rot fungus with high laccase yields, were analyzed. The laccases showed moderate identities to each other as well as with other fungal laccases and were predicted to have high redox potentials except for Lac6. Selected laccase isozymes were heterologously expressed in the yeast Pichia pastoris, and different enzymatic properties were observed. Transcription of the eight laccase genes was differentially regulated during submerged and solid state fermentation, as shown by quantitative real-time polymerase chain reaction and validated reference genes. During 6-day submerged fermentation, Lac7 and 2 were successively the predominantly expressed laccase gene, accounting for over 95% of all laccase transcripts. Interestingly, accompanying Lac7 downregulation, Lac2 transcription was drastically upregulated on days 3 and 5 to 9958-fold of the level on day 1. Consistent with high mRNA abundance, Lac2 and 7, but not other laccases, were identified in the fermentation broth by LC-MS/MS. In solid state fermentation, less dramatic differences in transcript abundance were observed, and Lac3, 7 and 8 were more highly expressed than other laccase genes. Elucidating the properties and expression profiles of the laccase gene family will facilitate understanding, production and commercialization of the fungal strain and its laccases.

  7. Analyzing Plasmodium falciparum erythrocyte membrane protein 1 gene expression by a next generation sequencing based method

    DEFF Research Database (Denmark)

    Jespersen, Jakob S.; Petersen, Bent; Seguin-Orlando, Andaine;


    Plasmodium falciparum is responsible for most cases of severe malaria and causes >1 million deaths every year. The particular virulence of this Plasmodium species is highly associated with the expression of certain members of the Plasmodium falciparum erythrocyte membrane protein 1(PfEMP1) family...

  8. Heme regulates the expression in Saccharomyces cerevisiae of chimaeric genes containing 5'-flanking soybean leghemoglobin sequences

    DEFF Research Database (Denmark)

    Jensen, E O; Marcker, K A; Villadsen, IS


    The TM1 yeast mutant was transformed with a 2 micron-derived plasmid (YEp24) which carries a chimaeric gene containing the Escherichia coli chloramphenicol acetyl transferase (CAT) gene fused to the 5'- and 3'-flanking regions of the soybean leghemoglobin (Lb) c3 gene. Expression of the chimaeric...

  9. NHE-1 sequence and expression in toad, snake and fish red blood cells

    DEFF Research Database (Denmark)

    Thomsen, Steffen Nyegaard; Wang, Tobias; Kristensen, Torsten

    Red blood cells (RBC) from reptiles appear not to express regulatory volume increase (RVI) upon shrinkage (Kristensen et al., 2008). In other vertebrates, the RVI response is primarily mediated by activation of the Na+/H+ exchanger (NHE-1) and we, therefore decided to investigate whether red cell...

  10. Identiifcation of novel and differentially expressed microRNAs in ovine ovary and testis tissues using solexa sequencing and bioinformatics

    Institute of Scientific and Technical Information of China (English)

    CHANG Wei-hua; ZHANG Yong; CHENG Zhang-rui; ZHAO Xing-xu; WANG Juan-hong; MA You-ji; HU Jun-jie; ZHANG Quan-wei


    MicroRNAs (miRNAs) are smal , single stranded, non-coding RNA molecules, about 19–25 nucleotides in length, which regulate the development and functions of reproductive system of mammal. To discover novel miRNAs and identify the differential expression of them in ovine ovary and testis tissues, the study constructed two libraries by using next-generation sequencing technologies (Solexa high-throughput sequencing technique). As a result, 9 321 775 and 9 511 538 clean reads were obtained from the ovary and testis separately, which included 130 562 (90 genes of ovary) and 56 272 (85 genes of testis) of known miRNAs and 486 potential novel miRNAs reads. In this study, a total of 65 conserved miRNAs were sig-niifcantly differential y expressed (P<0.01) between the two samples. Among them, 28 miRNAs were up-regulated and 3 miRNAs were down-regulated on ovary compared with testis. In addition, the known miRNAs with the highest expression level (5 miRNAs) and 30 novel miRNAs with the functions related to reproduction were validated using the real-time quan-titative RT-PCR. Moreover, the gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that differential y expressed miRNAs were involved in ovary and testis physiology, including signal transduction, gonad development, sex differentiation, gematogenesis, fertilization and embryo development. The results wil be helpful to facilitate studies on the regulation of miRNAs during ruminant reproduction.

  11. Structure, sequence, expression, and chromosomal localization of the human V{sub 1a} vasopressin receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Thibonnier, M.; Graves, M.K.; Wagner, M.S. [Case Western Reserve Univ. School of Medicine, Cleveland, OH (United States)] [and others


    We recently reported the structure and functional expression of a human V{sub 1a} vasopressin receptor (V{sub 1a}R) cDNA isolated from human liver cDNA libraries. To understand further the expression and regulation of the V{sub 1a}R, we now describe the genomic characteristics, tissue expression, chromosomal localization, and regional mapping of the human V{sub 1a}R gene, AVPR1A. Tissue distribution of the human V{sub 1a}R mRNA explored by Northern blot analysis of various human tissues or organs revealed the presence of a 5.5-kb mRNA transcript expressed in the liver and to a lesser degree in the heart, the kidney, and skeletal muscle. Screening of human genomic libraries revealed that the human AVPR1A gene is included entirely within a 6.4-kb rated by a 2.2-kb intron located before the corresponding seventh transmembrane domain of the receptor sequence. The first exon also contains 2 kb of 5{prime}-untranslated region, and the second exon includes 1 kb of 3{prime}-untranslated region. 5{prime}-RACE analysis of human liver mRNA by PCR localized the V{sub 1a}R mRNA transcription start site 1973 bp upstream of the translation the intron sequence were used as primers in polymerase chain reaction (PCR) analysis of human/rodent somatic cell hybrids. AVPR1A was localized by PCR analysis of a somatic cell hybrid panel to chromosome 12. Fluorescence in situ hybridization using a yeast artificial chromosome physically mapped AVPR1A to region 12q14-q15. 34 refs., 4 figs.

  12. Mapping of heterologous expressed sequence tags as an alternative to microarrays for study of defense responses in plants

    Directory of Open Access Journals (Sweden)

    Postnikova Olga A


    Full Text Available Abstract Background Microarray technology helped to accumulate an immense pool of data on gene expression changes in response to different environmental factors. Yet, computer- generated gene profiling using expressed sequence tags (EST represents a valuable alternative to microarrays, which allows efficient discovery of homologous sequences in evolutionarily different species and comparison of gene sets on the whole genome scale. In this study, we used publicly available EST database derived from different plant species infected with a variety of pathogens, to generate an expression profile of homologous genes involved in defense response of a model organism, Arabidopsis thaliana. Results EST-driven prediction identified 4,935 genes (16% of the total Arabidopsis genome which, according to the origin of EST sets, were associated with defense responses in the reference genome. Profiles of defense-related genes, obtained by mapping of heterologous EST, represent putative Arabidopsis homologs of the corresponding species. Comparison of these profiles in pairs and locating common genes allowed estimating similarity between defense-related gene sets of different plant species. To experimentally support computer data, we arbitrarily selected a number of transcription factor genes (TF detected by EST mapping. Their expression levels were examined by real-time polymerase chain reaction during infection with yellow strain of Cucumber mosaic virus, a compatible virus systemically infecting Arabidopsis. We observed that 65% of the designated TF were upregulated in accordance with the EST-generated profile. Conclusion We demonstrated that heterologous EST mapping may be efficiently used to reveal genes involved in host defense responses to pathogens. Upregulated genes identified in this study substantially overlap with those previously obtained by microarrays.

  13. Gene expression analysis of starch metabolism using mRNAseq and the potato genome sequence

    DEFF Research Database (Denmark)

    Sønderkær, Mads; Kloosterman, Bjorn; Bachem, Christian


    starch yield than presently possible, detailed knowledge about starch metabolism is crucial. Accumulation of carbohydrates in the form of starch in potato tubers is the result of both anabolic and catabolic processes. These processes are highly redundant in terms of gene isoforms and multiple metabolic......, starch synthesis takes place not only in tubers but also in leaves in the form of transient starch during the day, which is consumed in the absence of photosynthesis during the night. This poster will present the results of a transcriptome analysis based on the draft potato genome sequence v3. Samples...

  14. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang


    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  15. Effects of no feeding, maintenance feeding, and refeeding on production and processing characteristics of market-size hybrid catfish (United States)

    A pond study was initiated to evaluate effects of no feeding, maintenance feeding, and refeeding on production and processing characteristics of market-size hybrid catfish (female Channel Catfish Ictalurus punctatus × male Blue Catfish I. furcatus). Fish with an average weight of 644 g were stocked ...

  16. Changes of serum myeloperoxidase and nitric oxide in the early stage of Edwardsiella ictaluri infection in channel catfish, Ictalurus punctatus (United States)

    Enteric septicemia of catfish (ESC), caused by Edwardsiella ictaluri, is an important farm-raised channel catfish, Ictalurus punctatus (Rafinesque), disease. The development of a monitoring system for assessing the catfish health status in hatcheries and ponds is in great demanding. Because of the...

  17. Seasonal Differences in Steroids and Maturation-related Genes in Channel Catfish Under Normal and Accelerated Thermoperiods (United States)

    Selective breeding of channel catfish, Ictalurus punctatus, is hampered by a long generation time. Female channel catfish typically spawn when they are 3-years-old; however, a low percentage of spawning may be observed at two years of age. Mature female channel catfish can spawn once annually. Their...

  18. Survey of glycine-rich proteins (GRPs in the Eucalyptus expressed sequence tag database (ForEST

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    Silvia Nora Bocca


    Full Text Available The occurrence of quasi-repetitive glycine-rich peptides has been reported in different organisms. Glycine-rich regions are proposed to be involved in protein-protein interactions in some mammalian protein families. In plants, a set of glycine-rich proteins (GRPs was characterized several years ago, and since then a wealth of new GRPs have been identified. GRPs may have very diverse sub-cellular localization and functions. The only common feature among all different GRPs is the presence of glycine-rich repeat domains. The expression of genes encoding GRPs is developmentally regulated, and also induced, in several plant genera, by physical, chemical and biological factors. In addition to the highly modulated expression, several GRPs also show tissue-specific localization. GRPs specifically expressed in xylem, phloem, epidermis, anther tapetum and roots have been described. In this paper, the structural and functional features of these proteins in Eucalyptus are summarized. Since this is the first description of GRPs in this species, particular emphasis has been given to the expression pattern of these genes by analyzing their abundance and prevalence in the different cDNA-libraries of the Eucalyptus Genome Sequencing Project Consortium (ForEST. The comparison of GRPs from Eucalyptus and other species is also discussed.

  19. Identification of novel and differentially expressed MicroRNAs of dairy goat mammary gland tissues using solexa sequencing and bioinformatics.

    Directory of Open Access Journals (Sweden)

    Zhibin Ji

    Full Text Available MicroRNAs are small, noncoding RNA molecules that regulate gene expression at the post-transcriptional level and play an important role in various biological processes. Although most microRNAs expression profiles studies have been performed in humans or rodents, relatively limited knowledge also exists in other mammalian species. The identification of the full repertoire of microRNAs expressed in the lactating mammary gland of Capra hircus would significantly increase our understanding of the physiology of lactating mammary glands. In this study, two libraries were constructed using the lactating mammary gland tissues of Laoshan dairy goats (Capra hircus during peak and late lactation. Solexa high-throughput sequencing technique and bioinformatics were used to determine the abundance and differential expression of the microRNAs between peak and late lactation. As a result, 19,044,002 and 7,385,833 clean reads were obtained, respectively, and 1,113 conserved known microRNAs and 31 potential novel microRNA candidates were identified. A total of 697 conserved microRNAs were significantly differentially expressed with a P-value<0.01, 272 microRNAs were up-regulated and 425 microRNAs were down-regulated during peak lactation. The results were validated using real-time quantitative RT-PCR. 762,557 annotated mRNA transcripts were predicted as putative target gene candidates. The GO annotation and KEGG pathway analysis suggested that differentially expressed microRNAs were involved in mammary gland physiology, including signal transduction, and cell-cell and cell-extracellular communications. This study provided the first global of the microRNA in Capra hircus and expanded the repertoire of microRNAs. Our results have great significance and value for the elucidation of complex regulatory networks between microRNAs and mRNAs and for the study of mammary gland physiology and lactation.

  20. Spider web glue: two proteins expressed from opposite strands of the same DNA sequence. (United States)

    Choresh, Omer; Bayarmagnai, Battuya; Lewis, Randolph V


    The various silks that make up the web of the orb web spiders have been studied extensively. However, success in prey capture depends as much on the web glue as on the fibers. Spider silk glue, which is considered one of the strongest and most effective biological glues, is an aqueous solution secreted from the orb weaving spider's aggregate glands and coats the spiral prey capturing threads of their webs. Studies identified the major component of the glue as microscopic nodules made of a glycoprotein. This study describes two newly discovered proteins that form the glue-glycoprotein of the golden orb weaving spider Nephila clavipes . Our results demonstrate that both proteins contain unique 110 amino acid repetitive domains that are encoded by opposite strands of the same DNA sequence. Thus, the genome of the spider encodes two distinct yet functionally related genes by using both strands of an identical DNA sequence. Moreover, the closest match for the nonrepetitive region of one of the proteins is chitin binding proteins. The web glue appears to have evolved a substantial level of sophistication matching that of the spider silk fibers.

  1. Relative effectiveness of carp pituitary extract, luteinizing hormone releasing hormone analog LHRHa injections and LHRHa implants for producing hybrid catfish fry (United States)

    Adoption of the hybrid catfish (channel catfish, Ictalruus punctatus, female x blue catfish, I. furcatus, male) is increasing in the catfish industry. The most effective way to produce fry is hormone induced spawning of females coupled with hand stripping and in vitro fertilization. The success of...

  2. Comparison of RNAi Sequences in Insect-Resistant Plants to Expressed Sequences of a Beneficial Lady Beetle: A Closer Look at Off-Target Considerations. (United States)

    Allen, Margaret L


    Sequences obtained from transcriptomes of the lady beetle Coleomegilla maculata were compared to those designed for incorporation into crops. Searches of the transcriptomes identified sequences as the most likely to be closely similar to the sequences described in RNAi plant incorporated products. Some proposed prime RNAi pest management targets were also used to identify predicted orthologs from C. maculata. The lady beetle sequences were aligned with sequences from corn rootworms and Colorado potato beetles and, as appropriate in the case of targets, regions of similarity were compared with the genetic model organism for beetles, Tribolium castaneum. Some high levels of nucleotide identity were identified, particularly with an actin-derived sequence from Colorado potato beetle. This actin-derived sequence shared identical sequences with the lady beetle and a parasitic wasp.

  3. Comparison of RNAi Sequences in Insect-Resistant Plants to Expressed Sequences of a Beneficial Lady Beetle: A Closer Look at Off-Target Considerations

    Directory of Open Access Journals (Sweden)

    Margaret L. Allen


    Full Text Available Sequences obtained from transcriptomes of the lady beetle Coleomegilla maculata were compared to those designed for incorporation into crops. Searches of the transcriptomes identified sequences as the most likely to be closely similar to the sequences described in RNAi plant incorporated products. Some proposed prime RNAi pest management targets were also used to identify predicted orthologs from C. maculata. The lady beetle sequences were aligned with sequences from corn rootworms and Colorado potato beetles and, as appropriate in the case of targets, regions of similarity were compared with the genetic model organism for beetles, Tribolium castaneum. Some high levels of nucleotide identity were identified, particularly with an actin-derived sequence from Colorado potato beetle. This actin-derived sequence shared identical sequences with the lady beetle and a parasitic wasp.

  4. An evaluation of agreement between pectoral spines and otoliths for estimating ages of catfishes (United States)

    Olive, J.A.; Schramm, Harold; Gerard, Patrick D.; Irwin, E.


    Otoliths have been shown to provide more accurate ages than pectoral spine sections for several catfish populations; but sampling otoliths requires euthanizing the specimen, whereas spines can be sampled non-lethally. To evaluate whether, and under what conditions, spines provide the same or similar age estimates as otoliths, we examined data sets of individual fish aged from pectoral spines and otoliths for six blue catfish Ictalurus furcatus populations (n=420), 14 channel catfish Ictalurus punctatus populations (n=997), and 10 flathead catfish Pylodictus olivaris populations (n=947) from lotic and lentic waters throughout the central and eastern U.S. Logistic regression determined that agreement between ages estimated from otoliths and spines was consistently related to age, but inconsistently related to growth rate. When modeled at mean growth rate, we found at least 80% probability of no difference in spine- and otolith-assigned ages up to ages 4 and 5 for blue and channel catfish, respectively. For flathead catfish, an 80% probability of agreement between spine- and otolith-assigned ages did not occur at any age due to high incidence of differences in assigned ages even for age-1 fish. Logistic regression models predicted at least 80% probability that spine and otolith ages differed by ≤1 year up to ages 13, 16, and 9 for blue, channel, and flathead catfish, respectively. Age-bias assessment found mean spine-assigned age differed by less than 1 year from otolith-assigned age up to ages 19, 9, and 17 for blue catfish, channel catfish, and flathead catfish, respectively. These results can be used to help guide decisions about which structure is most appropriate for estimating catfish ages for particular populations and management objectives.

  5. Characterization of edible film fabricated with channel catfish (Ictalurus punctatus) gelatin extract using selected pretreatment methods. (United States)

    Zhang, S; Wang, Y; Herring, J L; Oh, J-H


    Farm-raised catfish are important to the economy of the southeastern states in the United States, and catfish processing produces about 55% of by-products for inexpensive sale. Therefore, the utilization of catfish by-products is of great interest to the catfish industry. The objectives of this research were to determine the optimum pretreatment method to extract catfish gelatin for edible film application, and to characterize physical, mechanical, and barrier properties of edible films fabricated with catfish skin gelatin. Catfish skins obtained from a local plant were treated with 6 selected pretreatment methods. The main extraction was performed with deionized water at 50 degrees C after pretreatment. The gelatin yield was calculated and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to characterize molecular weight (MW) profile. Color, tensile strength (TS), elongation, and water barrier property were determined to characterize the fabricated catfish gelatin films. From the results of gelatin yield, color, SDS-PAGE, as well as mechanical and barrier properties of the film, the pretreatment method with 0.25 M NaOH and 0.09 M acetic acid, followed by extraction at 50 degrees C for 3 h, was determined as the optimum extraction method. The catfish gelatin exhibited higher MW fractions than commercial mammalian gelatin. The catfish gelatin extracts possessed film-forming properties determined by TS, elongation, and water vapor permeability (WVP) comparable to those of commercial mammalian gelatin. The selected formula for catfish gelatin film was determined as 1% gelatin and 20% glycerol, resulting in greatest TS and lowest WVP.

  6. Molecular cloning, sequence identification and expression profile of domestic guinea pig (Cavia porcellus UGT1A1 gene

    Directory of Open Access Journals (Sweden)

    Yang Deming


    Full Text Available Domestic guinea pig is a model animal for human disease research. Uridine diphosphate glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1 is an important human disease-related gene. In this study, the complete coding sequence of domestic guinea pig gene UGT1A1 was amplified by reverse transcription-polymerase chain reaction. The open reading frame of the domestic guinea pig UGT1A1 gene is 1602 bp in length and was found to encode a protein of 533 amino acids. Sequence analysis revealed that the UGT1A1 protein of domestic guinea pig shared high homology with the UGT1A1 proteins of degu (84%, damara mole-rat (84%, human (80%, northern white-cheeked gibbon (80%, Colobus angolensis palliatus (80% and golden snub-nosed monkey (79%. This gene contains five exons and four introns, as revealed by the computer-assisted analysis. The results also showed that the domestic guinea pig UGT1A1 gene had a close genetic relationship with the UGT1A1 gene of degu. The prediction of transmembrane helices showed that domestic guinea pig UGT1A1 might be a transmembrane protein. Expression profile analysis indicated that the domestic guinea pig UGT1A1 gene was differentially expressed in detected domestic guinea pig tissues. Our experiment laid a primary foundation for using the domestic guinea pig as a model animal to study the UGT1A1-related human diseases.

  7. [Genomic DNA sequences and functional expression, purification of BmalphaTX14 neurotoxin from scorpion Buthus martensii Karsch]. (United States)

    Lü, Meng; Wang, Kun; Cao, Zhi-Jian; Jiang, Da-He; Mao, Xin; Li, Wen-Xin


    Based on the full-length cDNA of BmalphaTX14 from Chinese scorpion Buthus martensii Karsch (BmK), gene of the mature peptide of BmalphaTX14 was cloned into the yeast expression vector pPIC9K. After transforming, screening and inducing, tricine-SDS-PAGE and Western blot proved that rBmalphaTX14 protein was expressed in the medium for up to 84 hours, getting nearly 120 mg/L. Recombinant BmalphaTX14 was purified rapidly and efficiently through Ni-NTA-agarose, polyethylene glycol precipitation and gel filtration chromatography. The purified rBmalphaTX14 proved to have the anti-insect activity by toxicity assay. Meanwhile, genomic gene of BmalphaTX14 was cloned and sequenced by PCR method, sequence analysis of this gene showed that BmalphaTX14 had an intron of 408 base pairs located at the signal peptide encoding region, which was similar with the characteristic of other alpha-type sodium ion-channel toxin. Considering both the genomic organization and the peptide function, BmaTX14 proved to be a membership belonging to alpha-type sodium ion-channel toxin.

  8. Sequence and gene expression of chloroquine resistance transporter (pfcrt in the association of in vitro drugs resistance of Plasmodium falciparum

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    Bray Patrick G


    Full Text Available Abstract Background Plasmodium falciparum chloroquine resistance (CQR transporter protein (PfCRT is known to be the important key of CQR. Recent studies have definitively demonstrated a link between mutations in the gene pfcrt and resistance to chloroquine in P. falciparum. Although these mutations are predictive of chloroquine resistance, they are not quantitatively predictive of the degree of resistance. Methods In this study, a total of 95 recently adapted P. falciparum isolates from Thailand were included in the analysis. Parasites were characterized for their drug susceptibility phenotypes and genotypes with respect to pfcrt. From the original 95 isolates, 20 were selected for complete pfcrt sequence analysis. Results Almost all of the parasites characterized carried the previously reported mutations K76T, A220S, Q271E, N326S, I356T and R371I. On complete sequencing, isolates were identified with novel mutations at K76A and E198K. There was a suggestion that parasites carrying E198K were less resistant than those that did not. In addition, pfcrt and pfmdr1 gene expression were investigated by real-time PCR. No relationship between the expression level of either of these genes and response to drug was observed. Conclusion Data from the present study suggest that other genes must contribute to the degree of resistance once the resistance phenotype is established through mutations in pfcrt.

  9. Species-specific markers provide molecular genetic evidence for natural introgression of bullhead catfishes in Hungary (United States)

    Béres, Beatrix; Kánainé Sipos, Dóra; Müller, Tamás; Staszny, Ádám; Farkas, Milán; Bakos, Katalin; Urbányi, Béla


    Since three bullhead catfish species were introduced to Europe in the late 19th century, they have spread to most European countries. In Hungary, the brown bullhead (Ameiurus nebulosus) was more widespread in the 1970s–1980s, but the black bullhead (Ameiurus melas) has gradually supplanted since their second introduction in 1980. The introgressive hybridization of the two species has been presumed based on morphological examinations, but it has not previously been supported by genetic evidence. In this study, 11 different Hungarian habitats were screened with a new species-specific nuclear genetic, duplex PCR based, marker system to distinguish the introduced catfish species, Ameiurus nebulosus, Ameiurus melas, and Ameiurus natalis, as well as the hybrids of the first two. More than 460 specimens were analyzed using the above markers and additional mitochondrial sequence analyses were also conducted on >25% of the individuals from each habitat sampled. The results showed that only 7.9% of the specimens from two habitats belonged to Ameiurus nebulosus, and 92.1% were classified as Ameiurus melas of all habitats, whereas the presence of Ameiurus natalis was not detected. Two specimens (>0.4%) showed the presence of both nuclear genomes and they were identified as hybrids of Ameiurus melas and Ameiurus nebulosus. An additional two individuals showed contradicting results from the nuclear and mitochondrial assays as a sign of a possible footprint of introgressive hybridization that might have happened two or more generations before. Surprisingly, the level of hybridization was much smaller than expected based on the analyses of the North American continent’s indigenous stock from the hybrid zones. This phenomenon has been observed in several invasive fish species and it is regarded as an added level of complexity in the management of their rapid adaptation. PMID:28265489

  10. Regulation of gene expression in Mycoplasmas: contribution from Mycoplasma hyopneumoniae and Mycoplasma synoviae genome sequences

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    Humberto Maciel França Madeira


    Full Text Available This report describes the transcription apparatus of Mycoplasma hyopneumoniae (strains J and 7448 and Mycoplasma synoviae, using a comparative genomics approach to summarize the main features related to transcription and control of gene expression in mycoplasmas. Most of the transcription-related genes present in the three strains are well conserved among mycoplasmas. Some unique aspects of transcription in mycoplasmas and the scarcity of regulatory proteins in mycoplasma genomes are discussed.

  11. Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

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    Bendahmane Abdelhafid


    Full Text Available Abstract Background Melon (Cucumis melo, an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs and 3,073 single nucleotide polymorphisms (SNPs in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but

  12. Natural variation in CBF gene sequence, gene expression and freezing tolerance in the Versailles core collection of Arabidopsis thaliana

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    Brunel Dominique


    Full Text Available Abstract Background Plants from temperate regions are able to withstand freezing temperatures due to a process known as cold acclimation, which is a prior exposure to low, but non-freezing temperatures. During acclimation, a large number of genes are induced, bringing about biochemical changes in the plant, thought to be responsible for the subsequent increase in freezing tolerance. Key regulatory proteins in this process are the CBF1, 2 and 3 transcription factors which control the expression of a set of target genes referred to as the "CBF regulon". Results To assess the role of the CBF genes in cold acclimation and freezing tolerance of Arabidopsis thaliana, the CBF genes and their promoters were sequenced in the Versailles core collection, a set of 48 accessions that maximizes the naturally-occurring genetic diversity, as well as in the commonly used accessions Col-0 and WS. Extensive polymorphism was found in all three genes. Freezing tolerance was measured in all accessions to assess the variability in acclimated freezing tolerance. The effect of sequence polymorphism was investigated by evaluating the kinetics of CBF gene expression, as well as that of a subset of the target COR genes, in a set of eight accessions with contrasting freezing tolerance. Our data indicate that CBF genes as well as the selected COR genes are cold induced in all accessions, irrespective of their freezing tolerance. Although we observed different levels of expression in different accessions, CBF or COR gene expression was not closely correlated with freezing tolerance. Conclusion Our results indicate that the Versailles core collection contains significant natural variation with respect to freezing tolerance, polymorphism in the CBF genes and CBF and COR gene expression. Although there tends to be more CBF and COR gene expression in tolerant accessions, there are exceptions, reinforcing the idea that a complex network of genes is involved in freezing tolerance

  13. Expressed sequence tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers

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    Peterson Katherine


    Full Text Available Abstract 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

  14. Dissection of two soybean QTL conferring partial resistance to Phytophthora sojae through sequence and gene expression analysis

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    Wang Hehe


    Full Text Available Abstract Background Phytophthora sojae is the primary pathogen of soybeans that are grown on poorly drained soils. Race-specific resistance to P. sojae in soybean is gene-for-gene, although in many areas of the US and worldwide there are populations that have adapted to the most commonly deployed resistance to P. sojae ( Rps genes. Hence, this system has received increased attention towards identifying mechanisms and molecular markers associated with partial resistance to this pathogen. Several quantitative trait loci (QTL have been identified in the soybean cultivar ‘Conrad’ that contributes to the expression of partial resistance to multiple P. sojae isolates. Results In this study, two of the Conrad QTL on chromosome 19 were dissected through sequence and expression analysis of genes in both resistant (Conrad and susceptible (‘Sloan’ genotypes. There were 1025 single nucleotide polymorphisms (SNPs in 87 of 153 genes sequenced from Conrad and Sloan. There were 304 SNPs in 54 genes sequenced from Conrad compared to those from both Sloan and Williams 82, of which 11 genes had SNPs unique to Conrad. Eleven of 19 genes in these regions analyzed with qRT-PCR had significant differences in fold change of transcript abundance in response to infection with P. sojae in lines with QTL haplotype from the resistant parent compared to those with the susceptible parent haplotype. From these, 8 of the 11 genes had SNPs in the upstream, untranslated region, exon, intron, and/or downstream region. These 11 candidate genes encode proteins potentially involved in signal transduction, hormone-mediated pathways, plant cell structural modification, ubiquitination, and basal resistance. Conclusions These findings may indicate a complex defense network with multiple mechanisms underlying these two soyb