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Sample records for twenty-three expressed sequence

  1. Expressing stochastic filters via number sequences

    OpenAIRE

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

    2010-01-01

    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.

  2. cis sequence effects on gene expression

    Directory of Open Access Journals (Sweden)

    Jacobs Kevin

    2007-08-01

    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.

  3. In Vitro Conservation of Twenty-Three Overexploited Medicinal Plants Belonging to the Indian Sub Continent

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    Priyanka Verma

    2012-01-01

    Full Text Available Twenty-three pharmaceutically important plants, namely, Elaeocarpus spharicus, Rheum emodi, Indigofera tinctoria, Picrorrhiza kurroa, Bergenia ciliata, Lavandula officinalis, Valeriana wallichii, Coleus forskohlii, Gentiana kurroo, Saussurea lappa, Stevia rebaudiana, Acorus calamus, Pyrethrum cinerariaefolium, Aloe vera, Bacopa monnieri, Salvia sclarea, Glycyrrhiza glabra, Swertia cordata, Psoralea corylifolia, Jurinea mollis, Ocimum sanctum, Paris polyphylla, and Papaver somniferum, which are at the verge of being endangered due to their overexploitation and collection from the wild, were successfully established in vitro. Collections were made from the different biodiversity zones of India including Western Himalaya, Northeast Himalaya, Gangetic plain, Western Ghats, Semiarid Zone, and Central Highlands. Aseptic cultures were raised at the morphogenic level of callus, suspension, axillary shoot, multiple shoot, and rooted plants. Synseeds were also produced from highly proliferating shoot cultures of Bacopa monnieri, Glycyrrhiza glabra, Stevia rebaudiana, Valeriana wallichii, Gentiana kurroo, Lavandula officinalis, and Papaver somniferum. In vitro flowering was observed in Papaver somniferum, Psoralea corylifolia, and Ocimum sanctum shoots cultures. Out of 23 plants, 18 plants were successfully hardened under glasshouse conditions.

  4. In vitro conservation of twenty-three overexploited medicinal plants belonging to the Indian sub continent.

    Science.gov (United States)

    Verma, Priyanka; Mathur, Ajay Kumar; Jain, Sheetal Prasad; Mathur, Archana

    2012-01-01

    Twenty-three pharmaceutically important plants, namely, Elaeocarpus spharicus, Rheum emodi, Indigofera tinctoria, Picrorrhiza kurroa, Bergenia ciliata, Lavandula officinalis, Valeriana wallichii, Coleus forskohlii, Gentiana kurroo, Saussurea lappa, Stevia rebaudiana, Acorus calamus, Pyrethrum cinerariaefolium, Aloe vera, Bacopa monnieri, Salvia sclarea, Glycyrrhiza glabra, Swertia cordata, Psoralea corylifolia, Jurinea mollis, Ocimum sanctum, Paris polyphylla, and Papaver somniferum, which are at the verge of being endangered due to their overexploitation and collection from the wild, were successfully established in vitro. Collections were made from the different biodiversity zones of India including Western Himalaya, Northeast Himalaya, Gangetic plain, Western Ghats, Semiarid Zone, and Central Highlands. Aseptic cultures were raised at the morphogenic level of callus, suspension, axillary shoot, multiple shoot, and rooted plants. Synseeds were also produced from highly proliferating shoot cultures of Bacopa monnieri, Glycyrrhiza glabra, Stevia rebaudiana, Valeriana wallichii, Gentiana kurroo, Lavandula officinalis, and Papaver somniferum. In vitro flowering was observed in Papaver somniferum, Psoralea corylifolia, and Ocimum sanctum shoots cultures. Out of 23 plants, 18 plants were successfully hardened under glasshouse conditions.

  5. Predicting gene expression from sequence: a reexamination.

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    Yuan Yuan

    2007-11-01

    Full Text Available Although much of the information regarding genes' expressions is encoded in the genome, deciphering such information has been very challenging. We reexamined Beer and Tavazoie's (BT approach to predict mRNA expression patterns of 2,587 genes in Saccharomyces cerevisiae from the information in their respective promoter sequences. Instead of fitting complex Bayesian network models, we trained naïve Bayes classifiers using only the sequence-motif matching scores provided by BT. Our simple models correctly predict expression patterns for 79% of the genes, based on the same criterion and the same cross-validation (CV procedure as BT, which compares favorably to the 73% accuracy of BT. The fact that our approach did not use position and orientation information of the predicted binding sites but achieved a higher prediction accuracy, motivated us to investigate a few biological predictions made by BT. We found that some of their predictions, especially those related to motif orientations and positions, are at best circumstantial. For example, the combinatorial rules suggested by BT for the PAC and RRPE motifs are not unique to the cluster of genes from which the predictive model was inferred, and there are simpler rules that are statistically more significant than BT's ones. We also show that CV procedure used by BT to estimate their method's prediction accuracy is inappropriate and may have overestimated the prediction accuracy by about 10%.

  6. Analysis of expressed sequence tags from Plasmodium falciparum.

    Science.gov (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

    1994-07-01

    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.

  7. 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;

    2000-01-01

    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...

  8. TWENTY-THREE-YEAR LONG-TERM HEALTH OUTCOME AFTER THE WAR IN VUKOVAR.

    Science.gov (United States)

    Habek, Dubravko; Dujaković, Tatjana; Habek, Jasna Cerkez; Jurković, Ivana

    2016-03-01

    Results of the first research of this kind on the 23-year long-term outcome in children born during the war in Vukovar are presented. This retrospective clinical study surveyed the potential 23-year long-term consequences and morbidity of children born between May 1, 1991 and November 19, 1991, during the siege and occupation of Vukovar. Data were obtained from women having delivered their babies in that period and from delivery protocols of the Department of Gynecology and Obstetrics, Vukovar County Hospital. According to the survey and the data collected, there were 9 (3.98%) preterm deliveries, 60 (81%) of the total of 77 subjects were breastfed, 14 (19%) were not breastfed, while three babies died in the postpartum period. However, the breastfeeding period was evidently shorter, as only 10 women breastfed for a period longer than 6 months, while the mean length of the breastfeeding period was 9.9 weeks, i.e. 2.5 months. Allergy-related illnesses and proneness to infections in childhood and preschool age were found in 27.3% and 16.9% of children, respectively, while two children developed diabetes type 1. One child had atopic diathesis, two started speaking after the age of two, one child started walking late (after 20 months) and started speaking after the age of two, one child had loud sound phobia, and one used to overreact and express anger in inconvenient situations, all of them being of female gender and born prematurely. Cognitive and attention disorders and stress reactions were found in 6.5% of the children. Regular elementary school education was completed by 74 (96%) children, while three (3.8%) children experienced failure at school due bad behavior, i.e. delinquency. At the age of 23, 34 (46%) children had developed bad habits, i.e. 33 of them smoked, 2 were addicted to alcohol, whereas one was addicted to both smoking and alcohol. Forty (54%) subjects did not use any harmful substances. Psychiatric disorders related to anxiety, depression or other

  9. Comparative analyses of potato expressed sequence tag libraries.

    Science.gov (United States)

    Ronning, Catherine M; Stegalkina, Svetlana S; Ascenzi, Robert A; Bougri, Oleg; Hart, Amy L; Utterbach, Teresa R; Vanaken, Susan E; Riedmuller, Steve B; White, Joseph A; Cho, Jennifer; Pertea, Geo M; Lee, Yuandan; Karamycheva, Svetlana; Sultana, Razvan; Tsai, Jennifer; Quackenbush, John; Griffiths, Helen M; Restrepo, Silvia; Smart, Christine D; Fry, William E; Van Der Hoeven, Rutger; Tanksley, Steve; Zhang, Peifen; Jin, Hailing; Yamamoto, Miki L; Baker, Barbara J; Buell, C Robin

    2003-02-01

    The cultivated potato (Solanum tuberosum) shares similar biology with other members of the Solanaceae, yet has features unique within the family, such as modified stems (stolons) that develop into edible tubers. To better understand potato biology, we have undertaken a survey of the potato transcriptome using expressed sequence tags (ESTs) from diverse tissues. A total of 61,940 ESTs were generated from aerial tissues, below-ground tissues, and tissues challenged with the late-blight pathogen (Phytophthora infestans). Clustering and assembly of these ESTs resulted in a total of 19,892 unique sequences with 8,741 tentative consensus sequences and 11,151 singleton ESTs. We were able to identify a putative function for 43.7% of these sequences. A number of sequences (48) were expressed throughout the libraries sampled, representing constitutively expressed sequences. Other sequences (13,068, 21%) were uniquely expressed and were detected only in a single library. Using hierarchal and k means clustering of the EST sequences, we were able to correlate changes in gene expression with major physiological events in potato biology. Using pair-wise comparisons of tuber-related tissues, we were able to associate genes with tuber initiation, dormancy, and sprouting. We also were able to identify a number of characterized as well as novel sequences that were unique to the incompatible interaction of late-blight pathogen, thereby providing a foundation for further understanding the mechanism of resistance.

  10. Comparative Analyses of Potato Expressed Sequence Tag Libraries1

    Science.gov (United States)

    Ronning, Catherine M.; Stegalkina, Svetlana S.; Ascenzi, Robert A.; Bougri, Oleg; Hart, Amy L.; Utterbach, Teresa R.; Vanaken, Susan E.; Riedmuller, Steve B.; White, Joseph A.; Cho, Jennifer; Pertea, Geo M.; Lee, Yuandan; Karamycheva, Svetlana; Sultana, Razvan; Tsai, Jennifer; Quackenbush, John; Griffiths, Helen M.; Restrepo, Silvia; Smart, Christine D.; Fry, William E.; van der Hoeven, Rutger; Tanksley, Steve; Zhang, Peifen; Jin, Hailing; Yamamoto, Miki L.; Baker, Barbara J.; Buell, C. Robin

    2003-01-01

    The cultivated potato (Solanum tuberosum) shares similar biology with other members of the Solanaceae, yet has features unique within the family, such as modified stems (stolons) that develop into edible tubers. To better understand potato biology, we have undertaken a survey of the potato transcriptome using expressed sequence tags (ESTs) from diverse tissues. A total of 61,940 ESTs were generated from aerial tissues, below-ground tissues, and tissues challenged with the late-blight pathogen (Phytophthora infestans). Clustering and assembly of these ESTs resulted in a total of 19,892 unique sequences with 8,741 tentative consensus sequences and 11,151 singleton ESTs. We were able to identify a putative function for 43.7% of these sequences. A number of sequences (48) were expressed throughout the libraries sampled, representing constitutively expressed sequences. Other sequences (13,068, 21%) were uniquely expressed and were detected only in a single library. Using hierarchal and k means clustering of the EST sequences, we were able to correlate changes in gene expression with major physiological events in potato biology. Using pair-wise comparisons of tuber-related tissues, we were able to associate genes with tuber initiation, dormancy, and sprouting. We also were able to identify a number of characterized as well as novel sequences that were unique to the incompatible interaction of late-blight pathogen, thereby providing a foundation for further understanding the mechanism of resistance. PMID:12586867

  11. Chapter Twenty Three

    African Journals Online (AJOL)

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    Only women are qualified to converse with these ancestors as men are banned ..... Ibitokun, B.M. Dance as Ritual and Entertainment in the Gelede of the Ketu- Yoruba Sub- ... 'Art as Historical Product' in The Modern Tradition: Backgrounds of.

  12. Development of expressed sequence tag and expressed sequence tag–simple sequence repeat marker resources for Musa acuminata

    Science.gov (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.

    2012-01-01

    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

  13. Predicting tissue-specific expressions based on sequence characteristics

    KAUST Repository

    Paik, Hyojung

    2011-04-30

    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.

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

    Science.gov (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...

  15. 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

    2004-01-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Callow, Matthew J.; Rubin, Edward M.

    2001-12-19

    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.

  17. Yeast DNA sequences initiating gene expression in Escherichia coli.

    Science.gov (United States)

    Lewin, Astrid; Tran, Thi Tuyen; Jacob, Daniela; Mayer, Martin; Freytag, Barbara; Appel, Bernd

    2004-01-01

    DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology. We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S. cerevisiae and measuring the luminescence of transformed E. coli. Fifty-four out of 100 randomly analysed S. cerevisiae DNA sequences caused considerable gene expression in E. coli. Determination of transcription start sites within six selected yeast sequences in E. coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites. Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

  18. On the expressiveness of single-pass instruction sequences

    CERN Document Server

    Bergstra, J A

    2008-01-01

    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.

  19. Expressed sequence tags from Madagascar periwinkle (Catharanthus roseus).

    Science.gov (United States)

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

    2006-08-07

    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.

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

    Directory of Open Access Journals (Sweden)

    Nour Hammoudeh

    2014-12-01

    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.

  1. 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

    2002-01-01

    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.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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...

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

    Directory of Open Access Journals (Sweden)

    Bies Dawn H

    2005-06-01

    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.

  4. Sequencing and cardiac expression of Apelin in Sus Scrofa.

    Science.gov (United States)

    Del Ry, Silvia; Cabiati, Manuela; Raucci, Serena; Simioniuc, Anca; Caselli, Chiara; Prescimone, Tommaso; Giannessi, Daniela

    2009-10-01

    In humans, the Apelin gene is located on chromosome Xq25-26.1 and it encodes a 77-aminoacid prepropeptide. Considerable sequence homology exists across different mammalian species. Apelin is emerging as an important regulator of cardiovascular homeostasis but, at present, few data from humans are available and further studies are necessary to better define its role in cardiovascular pathophysiology. The role and function of Apelin in cardiovascular system could be reliably investigated in experimental models devoid of confounding effects reflecting only the natural history of the disease. The pig constitutes a model largely used in experimental pathology where it has a central role in "in vivo" clinical settings. Sus Scrofa genoma is not completely sequenced and Apelin gene is still lacking. Aim of this study was to sequence the Apelin in Sus Scrofa for future applications to molecular biology studies. Using the guanidinium thyocyanate-phenol-chloroform method, we extracted total RNA from samples obtained from heart of mouse and from atrium and ventricle of normal pigs. Pig Apelin mRNA was sequenced using polymerase chain reaction primers designed from mouse consensus sequences. A partial sequence of Sus Scrofa Apelin mRNA, 1-201 pb, was submitted to GenBank (accession number FJ362603). The bands obtained from pig cardiac tissue shared a 99% sequence identity with Mus musculus and 90% with Rattus norvegicus. The knowledge of Apelin sequence can be an useful starting point for future studies devoted to better understand the possible alterations of Apelin mRNA expression in different cardiac diseases.

  5. RESULTS OF THE INTERNATIONAL INTERLABORATORY COMPARISON OF MGMT PROMOTER METHYLATION ANALYSIS INVOLVING TWENTY-THREE ACADEMIC CENTERS IN GERMANY, AUSTRIA AND THE NETHERLANDS

    Science.gov (United States)

    Reifenberger, Guido; Malzkorn, B.; Acker, T.; Bettstetter, M.; Buslei, R.; von Deimling, A.; Dietmaier, W.; Dubbink, H.J.; Eigenbrod, S.; Garvalov, B.K.; Gerstenmaier, U.; Giese, A.; Haase, D.; Hasselblatt, M.; Kirches, E.; Koch, A.; Marienfeld, R.; Mittelbronn, M.; Montesinos-Rongen, M.; Pagenstecher, A.; Riemenschneider, M.J.; Prinz, M.; Romeike, B.; Roos, A.; Spiegl-Kreinecker, S.; Schittenhelm, J.; Schlegel, J.; Thal, D.R.; Tops, B.B.J.; Weis, J.; Westphal, G.; Worm, K.; Felsberg, J.

    2014-01-01

    BACKGROUND: Molecular testing for MGMT promoter methylation has become of clinical importance in the diagnostic assessment of malignant gliomas since test results may guide therapeutic decision making, in particular in elderly patients with glioblastoma. However, the patterns and extent of MGMT promoter methylation may vary from tumor to tumor, and standardized approaches for its routine diagnostic assessment are lacking. Thus, external quality assessment (EQA) measures are required to ensure accuracy and reproducibility of results across different laboratories. METHODS: We performed an interlaboratory comparison of MGMT promoter methylation analysis involving twenty-three academic institutions in Germany, Austria and the Netherlands. Two different test rounds were carried out, the first one using high molecular weight DNA extracted from frozen tissue samples of 20 tumors and the second one using formalin-fixed paraffin-embedded tissue sections of 16 tumors. All samples were centrally retrieved from the CNS tumor tissue bank at Heinrich Heine University Düsseldorf. Each participating center evaluated the same set of samples using the locally established methods. Results were centrally collected, together with information on the individual assays and the number of tests carried out per year. RESULTS: Methylation specific-PCR was the most commonly used method at the participating centers. Other less common techniques included pyrosequencing of bisulfite-modified DNA, MethyQESD (methylation-quantification of endonuclease-resistant DNA), MLPA (Multiplex Ligation-dependent Probe Amplification), and PCR-based fragment analysis. MGMT testing results showed a good overall concordance across the participating laboratories for those tumors that either had strongly methylated or clearly unmethylated MGMT promoter sequences. However, poor concordance was obtained for cases with only weak or partial MGMT promoter methylation. CONCLUSIONS: Our study provides an overview of the

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

    Directory of Open Access Journals (Sweden)

    Md Abul Hassan Samee

    2014-03-01

    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

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

    Directory of Open Access Journals (Sweden)

    Rossi Magdalena

    2001-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Hamarsheh Omar

    2011-09-01

    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.

  9. Microsatellite markers derived from Calophyllum inophyllum (Clusiaceae) expressed sequence tags.

    Science.gov (United States)

    Setsuko, Suzuki; Uchiyama, Kentaro; Sugai, Kyoko; Hanaoka, So; Yoshimaru, Hiroshi

    2012-01-01

    Robust markers are required (inter alia) for assessing origins of Calophyllum inophyllum populations on the Bonin Islands, Japan. Therefore, informative expressed sequence tag (EST)-based microsatellite or simple sequence repeat (SSRs) markers in the species were sought. Using 135378 ESTs derived from de novo pyrosequencing, primers for 475 EST-SSRs were developed, 48 of which were tested for PCR amplification. Thirty-six of the 48 primers showed clear amplification, with 23 displaying polymorphism in sampled populations. Expected heterozygosity in the samples from the Bonin Islands and Ryukyu Islands populations ranged from 0.041 to 0.697 and from 0.041 to 0.773, respectively. As EST-SSRs are potentially tightly linked with functional genes, and reportedly more transferable to related species than anonymous genomic SSRs, the developed primers have utility for future studies of the origins, genetic structure, and conservation of C. inophyllum and related species.

  10. 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

    2010-01-01

    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 ...

  11. 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

    2008-06-01

    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.

  12. Expressed sequence tags (ESTs) analysis of Acanthamoeba healyi

    Science.gov (United States)

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

    2001-01-01

    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

  13. Molecular chaperone genes in the sugarcane expressed sequence database (SUCEST

    Directory of Open Access Journals (Sweden)

    Júlio C. Borges

    2001-12-01

    Full Text Available Some newly synthesized proteins require the assistance of molecular chaperones for their correct folding. Chaperones are also involved in the dissolution of protein aggregates making their study significant for both biotechnology and medicine and the identification of chaperones and stress-related protein sequences in different organisms is an important task. We used bioinformatic tools to investigate the information generated by the Sugarcane Expressed Sequence Tag (SUCEST genome project in order to identify and annotate molecular chaperones. We considered that the SUCEST sequences belonged to this category of proteins when their E-values were lower than 1.0e-05. Our annotation shows that 4,164 of the 5’ expressed sequence tag (EST sequences were homologous to molecular chaperones, nearly 1.8% of all the 5’ ESTs sequenced during the SUCEST project. About 43% of the chaperones which we found were Hsp70 chaperones and its co-chaperones, 10% were Hsp90 chaperones and 13% were peptidyl-prolyl cis, trans isomerase. Based on the annotation results we predicted 156 different chaperone gene subclasses in the sugarcane genome. Taken together, our results indicate that genes which encode chaperones were diverse and abundantly expressed in sugarcane cells, which emphasizes their biological importance.Algumas proteínas ao serem sintetizadas necessitam do auxílio de chaperones moleculares para seu correto enovelamento. Chaperones também estão envolvidas na dissolução de agregados protéicos, fazendo com que seu estudo seja de relevância biotecnológica e médica. Portanto, a identificação de seqüências de chaperones moleculares é uma tarefa importante. Nós usamos ferramentas de bioinformática para procurar informações geradas pelo sugarcane EST Genome Project (SUCEST a fim de identificar e anotar chaperones e proteínas relacionas ao estresse. As seqüências do SUCEST eram anotadas como pertencentes a uma categoria de proteínas se o E

  14. 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

    2004-01-01

    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.

  15. 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

    2013-01-01

    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.

  16. 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

    2013-06-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Barry, M.M.

    1992-01-01

    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.

  18. Mining expressed sequence tags identifies cancer markers of clinical interest

    Directory of Open Access Journals (Sweden)

    Skrabanek Lucy

    2006-11-01

    Full Text Available Abstract Background Gene expression data are a rich source of information about the transcriptional dis-regulation of genes in cancer. Genes that display differential regulation in cancer are a subtype of cancer biomarkers. Results We present an approach to mine expressed sequence tags to discover cancer biomarkers. A false discovery rate analysis suggests that the approach generates less than 22% false discoveries when applied to combined human and mouse whole genome screens. With this approach, we identify the 200 genes most consistently differentially expressed in cancer (called HM200 and proceed to characterize these genes. When used for prediction in a variety of cancer classification tasks (in 24 independent cancer microarray datasets, 59 classifications total, we show that HM200 and the shorter gene list HM100 are very competitive cancer biomarker sets. Indeed, when compared to 13 published cancer marker gene lists, HM200 achieves the best or second best classification performance in 79% of the classifications considered. Conclusion These results indicate the existence of at least one general cancer marker set whose predictive value spans several tumor types and classification types. Our comparison with other marker gene lists shows that HM200 markers are mostly novel cancer markers. We also identify the previously published Pomeroy-400 list as another general cancer marker set. Strikingly, Pomeroy-400 has 27 genes in common with HM200. Our data suggest that a core set of genes are responsive to the deregulation of pathways involved in tumorigenesis in a variety of tumor types and that these genes could serve as transcriptional cancer markers in applications of clinical interest. Finally, our study suggests new strategies to select and evaluate cancer biomarkers in microarray studies.

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

    KAUST Repository

    Zhang, Zhang

    2010-06-10

    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.

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

    Directory of Open Access Journals (Sweden)

    Taciana de Carvalho Coutinho

    2016-06-01

    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.

  1. ESTExplorer: an expressed sequence tag (EST) assembly and annotation platform.

    Science.gov (United States)

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

    2007-07-01

    The analysis of expressed sequence tag (EST) datasets offers a rapid and cost-effective approach to elucidate the transcriptome of an organism, but requiring several computational methods for assembly and annotation. ESTExplorer is a comprehensive workflow system for EST data management and analysis. The pipeline uses a 'distributed control approach' in which the most appropriate bioinformatics tools are implemented over different dedicated processors. Species-specific repeat masking and conceptual translation are in-built. ESTExplorer accepts a set of ESTs in FASTA format which can be analysed using programs selected by the user. After pre-processing and assembly, the dataset is annotated at the nucleotide and protein levels, following conceptual translation. Users may optionally provide ESTExplorer with assembled contigs for annotation purposes. Functionally annotated contigs/ESTs can be analysed individually. The overall outputs are gene ontologies, protein functional identifications in terms of mapping to protein domains and metabolic pathways. ESTExplorer has been applied successfully to annotate large EST datasets from parasitic nematodes and to identify novel genes as potential targets for parasite intervention. ESTExplorer runs on a Linux cluster and is freely available for the academic community at http://estexplorer.biolinfo.org.

  2. 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

    2008-01-01

    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.

  3. 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

    2003-01-01

    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.

  4. 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

    2007-01-01

    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.

  5. 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

    2005-01-01

    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.

  6. 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 express...s of homology search), and expression profile Description of data contents Contig...o acid sequence and results of homology search), and expression profile - Dicty_cDB | LSDB Archive ...

  7. Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

    Science.gov (United States)

    Martin, Dorrelyn P.; Miya, Jharna; Reeser, Julie W.; Roychowdhury, Sameek

    2017-01-01

    RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 – 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations. PMID:27585245

  8. Studies of a Biochemical Factory: Tomato Trichome Deep Expressed Sequence Tag Sequencing and Proteomics1[W][OA

    Science.gov (United States)

    Schilmiller, Anthony L.; Miner, Dennis P.; Larson, Matthew; McDowell, Eric; Gang, David R.; Wilkerson, Curtis; Last, Robert L.

    2010-01-01

    Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces β-caryophyllene and α-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells. PMID:20431087

  9. Molecular cloning, expression analysis and sequence prediction of ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... comparison of the amino acid sequences from C/EBPβ cloned in this study and those from different ... subcutaneous fat was the highest among all the analyzed tissues, and the relative quantity ..... Carbohydrate Metabolism.

  10. Strategies for undertaking expressed sequence tag (EST) projects.

    Science.gov (United States)

    Clifton, Sandra W; Mitreva, Makedonka

    2009-01-01

    Complementary DNA (cDNA) sequencing can be used to sample an organism's transcriptome, and the generated EST sequences can be used for a variety of purposes. They are especially important for enhancing the utility of a genome sequence or for providing a gene catalog for a genome that has not or will not be sequenced. In planning and executing a cDNA project, several criteria must be considered. One should clearly define the project purpose, including organism tissue(s) choice, whether those tissues should be pooled, ability to acquire adequate amounts of clean and well-preserved tissue, choice of type(s) of library, and construction of a library (or libraries) that is compatible with project goals. In addition, one must possess the skills to construct the library (or libraries), keeping in mind the number of clones that will be necessary to meet the project requirements. If one is inexperienced in cDNA library construction, it might be wise to outsource the library production and/or sequence and analysis to a sequencing center or to a company that specializes in those activities. One should also be aware that new sequencing platforms are being marketed that may offer simpler protocols that can produce cDNA data in a more rapid and economical manner. Of course, the bioinformatics tools will have to be in place to de-convolute and aid in data analysis for these newer technologies. Possible funding sources for these projects include well-justified grant proposals, private funding, and/or collaborators with available funds.

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

    Science.gov (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...

  12. FINDING REGULATORY ELEMENTS USING JOINT LIKELIHOODS FOR SEQUENCE AND EXPRESSION PROFILE DATA.

    Energy Technology Data Exchange (ETDEWEB)

    IAN HOLMES, UC BERKELEY, CA, WILLIAM J. BRUNO, LANL

    2000-08-20

    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.

  13. Widespread Differential Expression of Coding Region and 3' UTR Sequences in Neurons and Other Tissues.

    Science.gov (United States)

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

    2015-12-16

    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.

  14. 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)

    2007-01-01

    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.

  15. Sequence and gene expression evolution of paralogous genes in willows.

    Science.gov (United States)

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

    2015-12-22

    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.

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

    OpenAIRE

    2005-01-01

    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. PASSIOMA: Exploring Expressed Sequence Tags during Flower Development in Passiflora spp.

    Directory of Open Access Journals (Sweden)

    Lucas Cutri

    2012-01-01

    Full Text Available The genus Passiflora provides a remarkable example of floral complexity and diversity. The extreme variation of Passiflora flower morphologies allowed a wide range of interactions with pollinators to evolve. We used the analysis of expressed sequence tags (ESTs as an approach for the characterization of genes expressed during Passiflora reproductive development. Analyzing the Passiflora floral EST database (named PASSIOMA, we found sequences showing significant sequence similarity to genes known to be involved in reproductive development such as MADS-box genes. Some of these sequences were studied using RT-PCR and in situ hybridization confirming their expression during Passiflora flower development. The detection of these novel sequences can contribute to the development of EST-based markers for important agronomic traits as well as to the establishment of genomic tools to study the naturally occurring floral diversity among Passiflora species.

  18. Analysis of expressed sequence tags of the water flea Daphnia magna.

    Science.gov (United States)

    Watanabe, Hajime; Tatarazako, Norihisa; Oda, Shigeto; Nishide, Hiroyo; Uchiyama, Ikuo; Morita, Masatoshi; Iguchi, Taisen

    2005-08-01

    To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.

  19. 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.

  20. 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

    1997-01-01

    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.

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

    OpenAIRE

    Chen, Chunxian; Gmitter Jr, Fred G

    2013-01-01

    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...

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

    NARCIS (Netherlands)

    Studer, B.; Asp, T.; Frei, U.; Hentrup, S.; Meally, H.; Guillard, A.; Barth, S.; Muylle, H.; Roldan-Ruiz, I.; Barre, P.; Boucoiran, C.F.S.; Stunnenberg, G.; Dolstra, O.; Skot, L.; Skot, K.P.; Turner, B.; Humphreys, M.; Kolliker, R.; Roulund, N.; Nielsen, K.K.; Lubberstedt, T.

    2008-01-01

    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 was teste

  3. 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

    2010-07-01

    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.

  4. 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;

    1990-01-01

    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...

  5. Delineation of Cis-Acting Sequences Required for Expression of Drosophila Mojavensis Adh-1

    Science.gov (United States)

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

    1992-01-01

    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

  6. Analysis and Functional Annotation of an Expressed Sequence Tag Collection for Tropical Crop Sugarcane

    Science.gov (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

    2003-01-01

    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

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

    Science.gov (United States)

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

    2015-01-01

    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.

  8. 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.

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    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.

  10. 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

    2011-01-01

    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

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

    Directory of Open Access Journals (Sweden)

    Bonaldo Maria F

    2005-05-01

    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.

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

    DEFF Research Database (Denmark)

    Willenbrock, Hanni; Salomon, Jesper; Søkilde, Rolf

    2009-01-01

    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...... technologies have only been compared based on biological data, leading to the conclusion that, although they are somewhat correlated, expression values differ significantly. Here, we use synthetic RNA samples, resembling human microRNA samples, to find that microarray expression measures actually correlate...... better with sample RNA content than expression measures obtained from sequencing data. In addition, microarrays appear highly sensitive and perform equivalently to next-generation sequencing in terms of reproducibility and relative ratio quantification....

  13. 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

    1990-01-01

    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...... of the transgene was observed in cell types other than beta-islet cells....

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

    Directory of Open Access Journals (Sweden)

    Boyle Nicholas F

    2005-11-01

    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.

  15. Differential expression in normal-adenoma-carcinoma sequence suggests complex molecular carcinogenesis in colon.

    Science.gov (United States)

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

    2006-10-01

    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.

  16. The heavy metal hyperaccumulator Thlaspi caerulescens expresses many species-specific genes, as identified by comparative expressed sequence tag analysis

    NARCIS (Netherlands)

    Rigola, D.; Fiers, M.W.E.J.; Vurro, E.; Aarts, M.G.M.

    2006-01-01

    ¿ Thlaspi caerulescens is a natural zinc (Zn), cadmium (Cd) and nickel (Ni) hyperaccumulator and an emerging plant model species to study heavy metal hyperaccumulation and tolerance. This paper describes the analysis of the first expressed sequence tag (EST) collection from T. caerulescens. This

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

    Directory of Open Access Journals (Sweden)

    Arias Covadonga

    2007-06-01

    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.

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

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    Robles José A

    2012-09-01

    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.

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

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    Kim Dae-Won

    2012-03-01

    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 (http://pathod.cdc.go.kr/spiroestdb. 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.

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

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    Tercilio Calsa Jr.

    2007-01-01

    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.

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

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    José P. Oliveira-Filho

    2012-10-01

    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.

  2. 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

    2010-01-01

    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...

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    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...

  4. 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

    2014-01-01

    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.

  5. Evaluation of cleaved amplified polymorphic sequence markers for Chamaecyparis obtusa based on expressed sequence tag information from Cryptomeria japonica.

    Science.gov (United States)

    Matsumoto, A; Tsumura, Y

    2004-12-01

    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.

  6. 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

    2007-09-01

    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

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

    Science.gov (United States)

    Xinli, Xiao; Lei, Peng

    2015-03-04

    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. Probing the effect of promoters on noise in gene expression using thousands of designed sequences.

    Science.gov (United States)

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

    2014-10-01

    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.

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

    OpenAIRE

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

    2016-01-01

    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...

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

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    Pinheiro Daniel G

    2009-06-01

    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 http://gdm.fmrp.usp.br/s3t/. S3T source code and datasets can also be downloaded from the aforementioned website.

  11. 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)

    2001-01-01

    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.

  12. 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.

  13. 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.

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

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    Chuyue Yang

    2016-11-01

    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.

  15. 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.

  16. Exploring PTX3 expression in Sus scrofa cardiac tissue using RNA sequencing.

    Science.gov (United States)

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

    2012-02-10

    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.

  17. De novo transcriptome sequencing of axolotl blastema for identification of differentially expressed genes during limb regeneration

    Science.gov (United States)

    2013-01-01

    Background Salamanders are unique among vertebrates in their ability to completely regenerate amputated limbs through the mediation of blastema cells located at the stump ends. This regeneration is nerve-dependent because blastema formation and regeneration does not occur after limb denervation. To obtain the genomic information of blastema tissues, de novo transcriptomes from both blastema tissues and denervated stump ends of Ambystoma mexicanum (axolotls) 14 days post-amputation were sequenced and compared using Solexa DNA sequencing. Results The sequencing done for this study produced 40,688,892 reads that were assembled into 307,345 transcribed sequences. The N50 of transcribed sequence length was 562 bases. A similarity search with known proteins identified 39,200 different genes to be expressed during limb regeneration with a cut-off E-value exceeding 10-5. We annotated assembled sequences by using gene descriptions, gene ontology, and clusters of orthologous group terms. Targeted searches using these annotations showed that the majority of the genes were in the categories of essential metabolic pathways, transcription factors and conserved signaling pathways, and novel candidate genes for regenerative processes. We discovered and confirmed numerous sequences of the candidate genes by using quantitative polymerase chain reaction and in situ hybridization. Conclusion The results of this study demonstrate that de novo transcriptome sequencing allows gene expression analysis in a species lacking genome information and provides the most comprehensive mRNA sequence resources for axolotls. The characterization of the axolotl transcriptome can help elucidate the molecular mechanisms underlying blastema formation during limb regeneration. PMID:23815514

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

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    Yang Jie

    2017-01-01

    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.

  19. 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: Patrick.Arbuthnot@wits.ac.za [Antiviral Gene Therapy Research Unit, University of the Witwatersrand (South Africa)

    2009-11-20

    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.

  20. Generation and Analysis of Expressed Sequence Tags from Olea europaea L.

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    Nehir Ozdemir Ozgenturk

    2010-01-01

    Full Text Available Olive (Olea europaea L. is an important source of edible oil which was originated in Near-East region. In this study, two cDNA libraries were constructed from young olive leaves and immature olive fruits for generation of ESTs to discover the novel genes and search the function of unknown genes of olive. The randomly selected 3840 colonies were sequenced for EST collection from both libraries. Readable 2228 sequences for olive leaf and 1506 sequences for olive fruit were assembled into 205 and 69 contigs, respectively, whereas 2478 were singletons. Putative functions of all 2752 differentially expressed unique sequences were designated by gene homology based on BLAST and annotated using BLAST2GO. While 1339 ESTs show no homology to the database, 2024 ESTs have homology (under 80% with hypothetical proteins, putative proteins, expressed proteins, and unknown proteins in NCBI-GenBank. 635 EST's unique genes sequence have been identified by over 80% homology to known function in other species which were not previously described in Olea family. Only 3.1% of total EST's was shown similarity with olive database existing in NCBI. This generated EST's data and consensus sequences were submitted to NCBI as valuable source for functional genome studies of olive.

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

    OpenAIRE

    Van Malderen, Renaat

    2005-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Almeida Diego D

    2008-06-01

    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.

  3. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    Tetranectin is a plasminogen-binding tetrameric protein originally isolated from plasma. Expression of tetranectin appears ubiquitous, although particularly high expression is noted in the stroma of malignant tumors and during mineralization. To dissect the molecular basis of tetranectin gene......% identity and 87% similarity at the amino acid level. Sequence comparisons between mouse and human tetranectin and some C-type lectins confirmed a complete conservation in the position of six cysteines as well as numerous other amino acid residues, indicating an essential structure for potential function......(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...

  4. 3'-end sequencing for expression quantification (3SEQ from archival tumor samples.

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Dipak K. Dube

    2016-01-01

    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.

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

    Science.gov (United States)

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

    2016-01-01

    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

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

    Science.gov (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

    2010-02-19

    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.

  8. Sperm competition shapes gene expression and sequence evolution in the ocellated wrasse.

    Science.gov (United States)

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

    2017-01-01

    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.

  9. Gene expression analysis of volatile-rich male flowers of dioecious Pandanus fascicularis using expressed sequence tags.

    Science.gov (United States)

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

    2010-07-15

    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.

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

    Directory of Open Access Journals (Sweden)

    Alvarez S Perez

    2014-12-01

    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.

  11. 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

    2001-09-01

    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.

  12. 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

    2013-03-01

    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.

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

    OpenAIRE

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

    2014-01-01

    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...

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

    Science.gov (United States)

    Narina, Satya S; Buyyarapu, Ramesh; Kottapalli, Kameswara Rao; Sartie, Alieu M; Ali, Mohamed I; Robert, Asiedu; Hodeba, Mignouna J D; Sayre, Brian L; Scheffler, Brian E

    2011-02-09

    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. 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. 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.

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

    Directory of Open Access Journals (Sweden)

    Robert Asiedu

    2011-02-01

    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.

  16. Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

    Science.gov (United States)

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

    2000-12-01

    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.

  17. Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Rolland

    2016-01-01

    Full Text Available The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson's disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications.

  18. Microarray analysis of expressed sequence tags from haustoria of the rust fungus Uromyces fabae.

    Science.gov (United States)

    Jakupović, Mirza; Heintz, Manuel; Reichmann, Peter; Mendgen, Kurt; Hahn, Matthias

    2006-01-01

    Rust fungi are plant parasites which colonise host tissue with an intercellular mycelium that forms haustoria within living plant cells. To identify genes expressed during biotrophic growth, EST sequencing was performed with a haustorium-specific cDNA library from Uromyces fabae. One thousand seventeen ESTs were generated, which assembled into 530 contigs. Several of the most frequently represented sequences in the EST database were identical to the in planta induced genes (PIGs) identified previously (Hahn, M., Mendgen, K., 1997. Characterisation of in planta-induced rust genes isolated from a haustorium-specific cDNA library, Mol. Plant-Microbe Interact. 10, 427-437). Virus-encoded sequences were identified, providing evidence for two novel RNA mycoviruses in U. fabae. Microarray hybridisation revealed many cDNAs that were significantly activated in rust-infected leaves compared to germinated uredospores. Very strong in planta expression was found for two PIGs encoding putative metallothioneins. Furthermore, several genes involved in ribosome biogenesis and translation, glycolysis, amino acid metabolism, stress response, and detoxification showed an increased expression in the parasitic mycelium. These data indicate a strong shift in gene expression in rust fungi between germination and the biotrophic stage of development.

  19. A leader sequence capable of enhancing RNA expression and protein synthesis in mammalian cells.

    Science.gov (United States)

    Wellensiek, Brian P; Larsen, Andrew C; Flores, Julia; Jacobs, Bertram L; Chaput, John C

    2013-10-01

    Many applications in biotechnology require human proteins generated from human cells. Stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. Transient expression can circumvent this problem, but protein yields are generally too low for most applications. Here we report a novel 37-nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. This sequence was identified by in vitro selection and functions in a transient vaccinia-based cytoplasmic expression system. Vectors containing this sequence produce microgram levels of protein in just 6 h from a small-scale expression in 10(6) cells. This level of protein synthesis is ideal for high throughput production of human proteins, and could be scaled to generate milligram quantities of protein. The technology is compatible with a broad range of cell lines, accepts plasmid and linear DNA, and functions with viruses that are approved for use under BSL1 conditions. We suggest that these advantages provide a powerful method for generating human protein in mammalian cells. © 2013 The Protein Society.

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

    Directory of Open Access Journals (Sweden)

    Daniel Ramsköld

    2009-12-01

    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.

  1. A novel approach to sequence validating protein expression clones with automated decision making

    Directory of Open Access Journals (Sweden)

    Mohr Stephanie E

    2007-06-01

    Full Text Available Abstract Background Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation. Results We have developed an Automated Clone Evaluation (ACE system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set. Conclusion ACE was designed to facilitate high throughput clone sequence

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

    Directory of Open Access Journals (Sweden)

    Tatsushi Goto

    2012-01-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    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.

  4. 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

    2009-01-01

    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.

  5. Analysis of expressed sequence tags from the venom ducts of Conus striatus: focusing on the expression profile of conotoxins.

    Science.gov (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

    2006-02-01

    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.

  6. 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

    1995-01-01

    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.

  7. Identification of differentially expressed genes during development of the zebrafish pineal complex using RNA sequencing.

    Science.gov (United States)

    Khuansuwan, Sataree; Gamse, Joshua T

    2014-11-01

    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.

  8. Isolation, sequence identification and tissue expression profiles of 3 novel porcine genes: ASPA, NAGA, and HEXA.

    Science.gov (United States)

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

    2008-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Mouzeyar Said

    2007-12-01

    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.

  10. A global definition of expression context is conserved between orthologs, but does not correlate with sequence conservation

    Directory of Open Access Journals (Sweden)

    Snel Berend

    2006-01-01

    Full Text Available Abstract Background The massive scale of microarray derived gene expression data allows for a global view of cellular function. Thus far, comparative studies of gene expression between species have been based on the level of expression of the gene across corresponding tissues, or on the co-expression of the gene with another gene. Results To compare gene expression between distant species on a global scale, we introduce the "expression context". The expression context of a gene is based on the co-expression with all other genes that have unambiguous counterparts in both genomes. Employing this new measure, we show 1 that the expression context is largely conserved between orthologs, and 2 that sequence identity shows little correlation with expression context conservation after gene duplication and speciation. Conclusion This means that the degree of sequence identity has a limited predictive quality for differential expression context conservation between orthologs, and thus presumably also for other facets of gene function.

  11. 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

    2010-01-01

    , 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...

  12. The First Molecular Identification of an Olive Collection Applying Standard Simple Sequence Repeats and Novel Expressed Sequence Tag Markers

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    Soraya Mousavi

    2017-07-01

    Full Text Available Germplasm collections of tree crop species represent fundamental tools for conservation of diversity and key steps for its characterization and evaluation. For the olive tree, several collections were created all over the world, but only few of them have been fully characterized and molecularly identified. The olive collection of Perugia University (UNIPG, established in the years’ 60, represents one of the first attempts to gather and safeguard olive diversity, keeping together cultivars from different countries. In the present study, a set of 370 olive trees previously uncharacterized was screened with 10 standard simple sequence repeats (SSRs and nine new EST-SSR markers, to correctly and thoroughly identify all genotypes, verify their representativeness of the entire cultivated olive variation, and validate the effectiveness of new markers in comparison to standard genotyping tools. The SSR analysis revealed the presence of 59 genotypes, corresponding to 72 well known cultivars, 13 of them resulting exclusively present in this collection. The new EST-SSRs have shown values of diversity parameters quite similar to those of best standard SSRs. When compared to hundreds of Mediterranean cultivars, the UNIPG olive accessions were splitted into the three main populations (East, Center and West Mediterranean, confirming that the collection has a good representativeness of the entire olive variability. Furthermore, Bayesian analysis, performed on the 59 genotypes of the collection by the use of both sets of markers, have demonstrated their splitting into four clusters, with a well balanced membership obtained by EST respect to standard SSRs. The new OLEST (Olea expressed sequence tags SSR markers resulted as effective as the best standard markers. The information obtained from this study represents a high valuable tool for ex situ conservation and management of olive genetic resources, useful to build a common database from worldwide olive

  13. 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

    2004-01-01

    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.

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

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    Smith-Keune Carolyn

    2008-02-01

    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

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

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    Tsai Shih-Feng

    2007-12-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    C.C. FIGUEROA; N. PRUNIER-LETERME; C. RISPE; F. SEPULVEDA; E. FUENTES-CONTRERAS; B. SABATER-MUNOZ; J.-C. SIMON; D. TAGU

    2007-01-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    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) (http://www.ncbi.nlm.nih.gov/), most of the miRNA target genes were predicted to encode transcription factors that regulate cell growth and development, signaling, and metabolism.

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

    Directory of Open Access Journals (Sweden)

    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 http://csb2.ym.edu.tw/est/.

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

    Science.gov (United States)

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

    2013-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Comparison of hybridization-based and sequencing-based gene expression technologies on biological replicates

    Directory of Open Access Journals (Sweden)

    Cepko Connie L

    2007-06-01

    Full Text Available Abstract Background High-throughput systems for gene expression profiling have been developed and have matured rapidly through the past decade. Broadly, these can be divided into two categories: hybridization-based and sequencing-based approaches. With data from different technologies being accumulated, concerns and challenges are raised about the level of agreement across technologies. As part of an ongoing large-scale cross-platform data comparison framework, we report here a comparison based on identical samples between one-dye DNA microarray platforms and MPSS (Massively Parallel Signature Sequencing. Results The DNA microarray platforms generally provided highly correlated data, while moderate correlations between microarrays and MPSS were obtained. Disagreements between the two types of technologies can be attributed to limitations inherent to both technologies. The variation found between pooled biological replicates underlines the importance of exercising caution in identification of differential expression, especially for the purposes of biomarker discovery. Conclusion Based on different principles, hybridization-based and sequencing-based technologies should be considered complementary to each other, rather than competitive alternatives for measuring gene expression, and currently, both are important tools for transcriptome profiling.

  2. Identification of expressed resistance gene-like sequences by data mining in 454-derived transcriptomic sequences of common bean (Phaseolus vulgaris L.).

    Science.gov (United States)

    Liu, Zhanji; Crampton, Mollee; Todd, Antonette; Kalavacharla, Venu

    2012-03-23

    Common bean (Phaseolus vulgaris L.) is one of the most important legumes in the world. Several diseases severely reduce bean production and quality; therefore, it is very important to better understand disease resistance in common bean in order to prevent these losses. More than 70 resistance (R) genes which confer resistance against various pathogens have been cloned from diverse plant species. Most R genes share highly conserved domains which facilitates the identification of new candidate R genes from the same species or other species. The goals of this study were to isolate expressed R gene-like sequences (RGLs) from 454-derived transcriptomic sequences and expressed sequence tags (ESTs) of common bean, and to develop RGL-tagged molecular markers. A data-mining approach was used to identify tentative P. vulgaris R gene-like sequences from approximately 1.69 million 454-derived sequences and 116,716 ESTs deposited in GenBank. A total of 365 non-redundant sequences were identified and named as common bean (P. vulgaris = Pv) resistance gene-like sequences (PvRGLs). Among the identified PvRGLs, about 60% (218 PvRGLs) were from 454-derived sequences. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed that PvRGLs were actually expressed in the leaves of common bean. Upon comparison to P. vulgaris genomic sequences, 105 (28.77%) of the 365 tentative PvRGLs could be integrated into the existing common bean physical map. Based on the syntenic blocks between common bean and soybean, 237 (64.93%) PvRGLs were anchored on the P. vulgaris genetic map and will need to be mapped to determine order. In addition, 11 sequence-tagged-site (STS) and 19 cleaved amplified polymorphic sequence (CAPS) molecular markers were developed for 25 unique PvRGLs. In total, 365 PvRGLs were successfully identified from 454-derived transcriptomic sequences and ESTs available in GenBank and about 65% of PvRGLs were integrated into the common bean genetic map. A total of 30

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Comparative Analysis and Functional Annotation of a Large Expressed Sequence Tag Collection of Apple

    Directory of Open Access Journals (Sweden)

    Ksenija Gasic

    2009-03-01

    Full Text Available A total of 34 apple ( × Borkh. cDNA libraries were constructed from root, leaf, bud, shoot, flower, and fruit tissues, at various developmental stages and/or under biotic or abiotic stress conditions, and of several genotypes. From these libraries, 190,425 clones were partially sequenced from the 5′ end and 42,619 clones were sequenced from the 3′ end, and a total of 182,241 high-quality expressed sequence tags (ESTs were obtained. These coalesced into 23,442 tentative contigs and 9843 singletons, for a total of 33,825 apple unigenes. Functional annotation of this unigene set revealed an even distribution of apple sequences among the three main gene ontology categories. Of ∼33,000 apple unigenes, 8437 (25% had no detectable homologs ( >0.1 in the genome. When the entire apple unigene set was compared with the entire citrus [ (L. Osbeck] unigene set and the poplar ( Torr. & Gray predicted proteome, both members of the core eudicot and rosids clade, 13,521 of apple unigenes matched one or more sequences in citrus, while 25,817 had counterparts in the poplar protein database. Apple––citrus–poplar comparisons revealed closer evolutionary relationships between apple and poplar than with the other two species. Genes involved in basic metabolic pathways appear to be largely conserved among apple, citrus, poplar, and .

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

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    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.

  6. 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

    2005-01-01

    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 (http://www.ncbi.nlm.nih.gov/dbEST/index.html),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.

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

    Directory of Open Access Journals (Sweden)

    Fusaro Adriana

    2001-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Chari Raj

    2010-08-01

    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

  9. Sequence diversity and differential expression of major phenylpropanoid-flavonoid biosynthetic genes among three mango varieties.

    Science.gov (United States)

    Hoang, Van L T; Innes, David J; Shaw, P Nicholas; Monteith, Gregory R; Gidley, Michael J; Dietzgen, Ralf G

    2015-07-30

    Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3'-hydroxylase (F3'H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and

  10. Expressed sequence tag analysis of functional genes associated with adventitious rooting in Liriodendron hybrids.

    Science.gov (United States)

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

    2016-06-24

    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.

  11. Sequence analysis and prokaryotic expression of Giardia lamblia α-18 giardin gene.

    Science.gov (United States)

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

    2016-03-01

    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.

  12. Conservation and divergence of plant LHP1 protein sequences and expression patterns in angiosperms and gymnosperms.

    Science.gov (United States)

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

    2011-05-01

    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.

  13. 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

    2011-01-01

    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.

  14. Impacts of Neanderthal-Introgressed Sequences on the Landscape of Human Gene Expression.

    Science.gov (United States)

    McCoy, Rajiv C; Wakefield, Jon; Akey, Joshua M

    2017-02-23

    Regulatory variation influencing gene expression is a key contributor to phenotypic diversity, both within and between species. Unfortunately, RNA degrades too rapidly to be recovered from fossil remains, limiting functional genomic insights about our extinct hominin relatives. Many Neanderthal sequences survive in modern humans due to ancient hybridization, providing an opportunity to assess their contributions to transcriptional variation and to test hypotheses about regulatory evolution. We developed a flexible Bayesian statistical approach to quantify allele-specific expression (ASE) in complex RNA-seq datasets. We identified widespread expression differences between Neanderthal and modern human alleles, indicating pervasive cis-regulatory impacts of introgression. Brain regions and testes exhibited significant downregulation of Neanderthal alleles relative to other tissues, consistent with natural selection influencing the tissue-specific regulatory landscape. Our study demonstrates that Neanderthal-inherited sequences are not silent remnants of ancient interbreeding but have measurable impacts on gene expression that contribute to variation in modern human phenotypes. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. An expressed sequence tag survey of gene expression in the pond snail Lymnaea stagnalis, an intermediate vector of trematodes [corrected].

    Science.gov (United States)

    Davison, A; Blaxter, M L

    2005-05-01

    The pond snail Lymnaea stagnalis is an intermediate vector for the liver fluke Fasciola hepatica, a common parasite of ruminants and humans. Yet, despite being a disease of medical and economic importance, as well as a potentially useful comparative tool, the genetics of the relationship between Lymnaea and Fasciola has barely been investigated. As a complement to forthcoming F. hepatica expressed sequence tags (ESTs), we generated 1320 ESTs from L. stagnalis central nervous system (CNS) libraries. We estimate that these sequences derive from 771 different genes, of which 374 showed significant similarity to proteins in public databases, and 169 were similar to ESTs from the snail vector Biomphalaria glabrata. These L. stagnalis ESTs will provide insight into the function of the snail CNS, as well as the molecular components of behaviour and response to parasitism. In the future, the comparative analysis of Lymnaea/Fasciola with Biomphalaria/Schistosoma will help to understand both conserved and divergent aspects of the host-parasite relationship. The L. stagnalis ESTs will also assist gene prediction in the forthcoming B. glabrata genome sequence. The dataset is available for searching on the world-wide web at http://zeldia.cap.ed.ac.uk/mollusca.html.

  16. Characterization of transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles.

    Science.gov (United States)

    Guo, Shaogui; Liu, Jingan; Zheng, Yi; Huang, Mingyun; Zhang, Haiying; Gong, Guoyi; He, Hongju; Ren, Yi; Zhong, Silin; Fei, Zhangjun; Xu, Yong

    2011-09-21

    Cultivated watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] is an important agriculture crop world-wide. The fruit of watermelon undergoes distinct stages of development with dramatic changes in its size, color, sweetness, texture and aroma. In order to better understand the genetic and molecular basis of these changes and significantly expand the watermelon transcript catalog, we have selected four critical stages of watermelon fruit development and used Roche/454 next-generation sequencing technology to generate a large expressed sequence tag (EST) dataset and a comprehensive transcriptome profile for watermelon fruit flesh tissues. We performed half Roche/454 GS-FLX run for each of the four watermelon fruit developmental stages (immature white, white-pink flesh, red flesh and over-ripe) and obtained 577,023 high quality ESTs with an average length of 302.8 bp. De novo assembly of these ESTs together with 11,786 watermelon ESTs collected from GenBank produced 75,068 unigenes with a total length of approximately 31.8 Mb. Overall 54.9% of the unigenes showed significant similarities to known sequences in GenBank non-redundant (nr) protein database and around two-thirds of them matched proteins of cucumber, the most closely-related species with a sequenced genome. The unigenes were further assigned with gene ontology (GO) terms and mapped to biochemical pathways. More than 5,000 SSRs were identified from the EST collection. Furthermore we carried out digital gene expression analysis of these ESTs and identified 3,023 genes that were differentially expressed during watermelon fruit development and ripening, which provided novel insights into watermelon fruit biology and a comprehensive resource of candidate genes for future functional analysis. We then generated profiles of several interesting metabolites that are important to fruit quality including pigmentation and sweetness. Integrative analysis of metabolite and digital gene expression

  17. 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.

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

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    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.

  19. Expression of a new chimeric protein with a highly repeated sequence in tobacco cells.

    Science.gov (United States)

    Saumonneau, Amélie; Rottier, Karine; Conrad, Udo; Popineau, Yves; Guéguen, Jacques; Francin-Allami, Mathilde

    2011-07-01

    In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS(W)) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS(M)-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.

  20. Nucleotides upstream of the Kozak sequence strongly influence gene expression in the yeast S. cerevisiae.

    Science.gov (United States)

    Li, Jing; Liang, Qiang; Song, Wenjiang; Marchisio, Mario Andrea

    2017-01-01

    In the yeast Saccharomyces cerevisiae, as in every eukaryotic organism, the mRNA 5(')-untranslated region (UTR) is important for translation initiation. However, the patterns and mechanisms that determine the efficiency with which ribozomes bind mRNA, the elongation of ribosomes through the 5(')-UTR, and the formation of a stable translation initiation complex are not clear. Genes that are highly expressed in S. cerevisiae seem to prefer a 5(')-UTR rich in adenine and poor in guanine, particularly in the Kozak sequence, which occupies roughly the first six nucleotides upstream of the START codon. We measured the fluorescence produced by 58 synthetic versions of the S. cerevisiae minimal CYC1 promoter (pCYC1min), each containing a different 5(')-UTR. First, we replaced with adenine the last 15 nucleotides of the original pCYC1min 5(')-UTR-a theoretically optimal configuration for high gene expression. Next, we carried out single and multiple point mutations on it. Protein synthesis was highly affected by both single and multiple point mutations upstream of the Kozak sequence. RNAfold simulations revealed that significant changes in the mRNA secondary structures occur by mutating more than three adenines into guanines between positions -15 and -9. Furthermore, the effect of point mutations turned out to be strongly context-dependent, indicating that adenines placed just upstream of the START codon do not per se guarantee an increase in gene expression, as previously suggested. New synthetic eukaryotic promoters, which differ for their translation initiation rate, can be built by acting on the nucleotides upstream of the Kozak sequence. Translation efficiency could, potentially, be influenced by another portion of the 5(')-UTR further upstream of the START codon. A deeper understanding of the role of the 5(')-UTR in gene expression would improve criteria for choosing and using promoters inside yeast synthetic gene circuits.

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

    Directory of Open Access Journals (Sweden)

    Francis Sheila E

    2012-09-01

    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

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

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    Brahmachari Samir K

    2001-10-01

    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.

  3. Large scale in silico identification of MYB family genes from wheat expressed sequence tags.

    Science.gov (United States)

    Cai, Hongsheng; Tian, Shan; Dong, Hansong

    2012-10-01

    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.

  4. Molecular cloning, sequencing, and expression in Escherichia coli of the potato virus Y cytoplasmic inclusion gene.

    Science.gov (United States)

    Ohshima, K; Inoue, A K; Shikata, E

    1993-01-01

    Complete nucleotide sequences of cytoplasmic inclusion (CI) genes of two strains of potato virus Y (PVY) were determined from six polymerase chain reaction (PCR)-amplified cDNA clones. The size of the CI genes of both ordinary (PVY-O) and necrotic strains (PVY-T13) was 1902 nucleotides, with a sequence homology of 83.4%. Comparison of the predicted amino acid sequences showed more than 90% homology. When these were compared with those of other potyviruses, the homology ranged from 53 to 61%. cDNAs of all or a part of the PVY-O CI gene containing an additional initiation codon (ATG) at the 5' end and a stop codon at the 3' end were constructed by PCR amplification and cloned into an Escherichia coli expression vector, pKK 223-3. Complete and truncated PVY-O CI proteins were successfully produced in E. coli as judged by reactivities with PVY-O CI protein-specific antiserum. To our knowledge, this is the first report on expression of PVY CI proteins in E. coli.

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

    DEFF Research Database (Denmark)

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

    2004-01-01

    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...

  6. Computational identification of citrus microRNAs and target analysis in citrus expressed sequence tags.

    Science.gov (United States)

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

    2010-11-01

    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.

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

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    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

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

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    van West Pieter

    2005-08-01

    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.

  9. angaGEDUCI: Anopheles gambiae gene expression database with integrated comparative algorithms for identifying conserved DNA motifs in promoter sequences

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    Ribeiro Jose Marcos C

    2006-05-01

    Full Text Available Abstract Background The completed sequence of the Anopheles gambiae genome has enabled genome-wide analyses of gene expression and regulation in this principal vector of human malaria. These investigations have created a demand for efficient methods of cataloguing and analyzing the large quantities of data that have been produced. The organization of genome-wide data into one unified database makes possible the efficient identification of spatial and temporal patterns of gene expression, and by pairing these findings with comparative algorithms, may offer a tool to gain insight into the molecular mechanisms that regulate these expression patterns. Description We provide a publicly-accessible database and integrated data-mining tool, angaGEDUCI, that unifies 1 stage- and tissue-specific microarray analyses of gene expression in An. gambiae at different developmental stages and temporal separations following a bloodmeal, 2 functional gene annotation, 3 genomic sequence data, and 4 promoter sequence comparison algorithms. The database can be used to study genes expressed in particular stages, tissues, and patterns of interest, and to identify conserved promoter sequence motifs that may play a role in the regulation of such expression. The database is accessible from the address http://www.angaged.bio.uci.edu. Conclusion By combining gene expression, function, and sequence data with integrated sequence comparison algorithms, angaGEDUCI streamlines spatial and temporal pattern-finding and produces a straightforward means of developing predictions and designing experiments to assess how gene expression may be controlled at the molecular level.

  10. Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice.

    Science.gov (United States)

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

    1992-03-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Voronova Angelika

    2014-07-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Valerio Costa

    2010-01-01

    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.

  13. 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.

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

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    David T. Ting

    2014-09-01

    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.

  16. Transcriptome expression profiling of fur color formation in domestic rabbits using Solexa sequencing.

    Science.gov (United States)

    Qin, L Z; Wang, W Z; Shi, L J; Wan, X Y; Yan, X R; Weng, Q Q; Wu, X S

    2016-04-25

    Fur color is an important, genetically determined characteristic of domestic rabbits, and rabbit furs are of great economic value. To investigate the molecular genetics associated with fur color determination in domestic rabbits, we used Solexa-sequencing technology to probe gene expression in dorsal skin tissues sampled from full-sibling Rex rabbits of different colors. The number of expressed genes in each sample was approximately 14,700. Among the top 30 genes and transcription factors with the highest reads per kilobase per million values, the elongation factor-alpha 1 gene was highly expressed in all samples, as were genes of the ribosomal protein and keratin gene families. Compared with the chinchilla (C) Rex rabbit control sample, the numbers of genes in the black (B) and white (W) rabbit samples were 1809 and 460, respectively, and the number of common differentially expressed genes was 257. Clustering analysis of these 257 genes revealed that 32 were up-regulated in sample B and down-regulated in sample W. Of these 32 genes, we identified some that are related to fur formation, including Tyrosinase-related protein 1 (TYRP1) and Tyrosinase (TYR), as well as genes with unknown functions. Quantitative real-time polymerase chain reaction was used to verify the expression patterns of those genes. The findings are expected to provide reference for the further study of fur color formation in rabbits.

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

    KAUST Repository

    Leach, Lindsey J

    2014-04-11

    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.

  18. THE TREE SHREW APOLIPOPROTEIN C-I cDNA: SEQUENCE AND ITS EXPRESSION

    Institute of Scientific and Technical Information of China (English)

    王克勤; 吕新跃; 吴钢; 薛红; 陈保生

    2001-01-01

    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.``

  19. Expressed sequence tags related to nitrogen metabolism in maize inoculated with Azospirillum brasilense.

    Science.gov (United States)

    Pereira-Defilippi, L; Pereira, E M; Silva, F M; Moro, G V

    2017-05-31

    The relative quantitative real-time expression of two expressed sequence tags (ESTs) codifying for key enzymes in nitrogen metabolism in maize, nitrate reductase (ZmNR), and glutamine synthetase (ZmGln1-3) was performed for genotypes inoculated with Azospirillum brasilense. Two commercial single-cross hybrids (AG7098 and 2B707) and two experimental synthetic varieties (V2 and V4) were raised under controlled greenhouse conditions, in six treatment groups corresponding to different forms of inoculation and different levels of nitrogen application by top-dressing. The genotypes presented distinct responses to inoculation with A. brasilense. Increases in the expression of ZmNR were observed for the hybrids, while V4 only displayed a greater level of expression when the plants received nitrogenous fertilization by top-dressing and there was no inoculation. The expression of the ZmGln1-3EST was induced by A. brasilense in the hybrids and the variety V4. In contrast, the variety V2 did not respond to inoculation.

  20. 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

  1. 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

    2010-01-01

    Salvia miltiorrhiza Bge.is 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.

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

    Directory of Open Access Journals (Sweden)

    Kovalenko O. P.

    2011-12-01

    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

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

    Directory of Open Access Journals (Sweden)

    Glória R. Franco

    1995-04-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Fasold Mario

    2010-04-01

    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

  5. 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

    2013-01-01

    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.

  6. Expression and sequence analyses of serum amyloid A in the Syrian hamster.

    Science.gov (United States)

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

    1989-05-30

    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.

  7. Plant Omics: Isolation, Identification, and Expression Analysis of Cytochrome P450 Gene Sequences from Coleus forskohlii.

    Science.gov (United States)

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

    2015-12-01

    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.

  8. RNA Sequencing Reveals that Kaposi Sarcoma-Associated Herpesvirus Infection Mimics Hypoxia Gene Expression Signature

    Science.gov (United States)

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

    2017-01-01

    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

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

    Science.gov (United States)

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

    2016-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Jiaqing Hu

    2016-01-01

    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.

  11. Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy.

    Science.gov (United States)

    Ames, E G; Lawson, M J; Mackey, A J; Holmes, J W

    2013-09-01

    Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are re-expressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (Phypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Devier Benjamin

    2007-08-01

    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.

  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. MOLECULAR CLONING, SEQUENCING, EXPRESSION AND BIOLOGICAL ACTIVITY OF GIANT PANDA (AILUROPODA MELANOLEUCA) INTERFERON-GAMMA.

    Science.gov (United States)

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

    2012-06-29

    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.

  15. Cloning, sequencing, and expression of interferon-γ from elk in North America

    Science.gov (United States)

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

    2001-01-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    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.

  17. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5)

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, H.D.; Tucker, W.Q.J.; Hort, Y.; Martinson, M.E.; Mayo, G.; Clutterbuck, E.J.; Sanderson, C.J.; Young, I.G.

    1987-10-01

    The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in lambda phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (approx. 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia.

  18. Rabbit serum amyloid protein A: expression and primary structure deduced from cDNA sequences.

    Science.gov (United States)

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

    1991-12-01

    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.

  19. In silico identification of coffee genome expressed sequences potentially associated with resistance to diseases

    Science.gov (United States)

    2010-01-01

    Sequences potentially associated with coffee resistance to diseases were identified by in silico analyses using the database of the Brazilian Coffee Genome Project (BCGP). Keywords corresponding to plant resistance mechanisms to pathogens identified in the literature were used as baits for data mining. Expressed sequence tags (ESTs) related to each of these keywords were identified with tools available in the BCGP bioinformatics platform. A total of 11,300 ESTs were mined. These ESTs were clustered and formed 979 EST-contigs with similarities to chitinases, kinases, cytochrome P450 and nucleotide binding site-leucine rich repeat (NBS-LRR) proteins, as well as with proteins related to disease resistance, pathogenesis, hypersensitivity response (HR) and plant defense responses to diseases. The 140 EST-contigs identified through the keyword NBS-LRR were classified according to function. This classification allowed association of the predicted products of EST-contigs with biological processes, including host defense and apoptosis, and with molecular functions such as nucleotide binding and signal transducer activity. Fisher's exact test was used to examine the significance of differences in contig expression between libraries representing the responses to biotic stress challenges and other libraries from the BCGP. This analysis revealed seven contigs highly similar to catalase, chitinase, protein with a BURP domain and unknown proteins. The involvement of these coffee proteins in plant responses to disease is discussed. PMID:21637594

  20. 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

    2009-08-01

    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 (http://riju.byethost31.com/cocoa/). Seven primer pairs amplified genomic DNA isolated from leaves were used to screen a representative set of 12 accessions of cacao.

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

    Science.gov (United States)

    Chen, Chunxian; Gmitter, Fred G

    2013-11-01

    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. 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 comparison. There were a total of 567,297 ESTs belonging to 27 cultivars in varying numbers and consequentially yielding different numbers of haplotype-based quality SNPs. Sweet orange (SO) had the most (213,830) ESTs, generating 11,182 quality SNPs in 3,327 out of 4,228 usable contigs. Summed from all the individually mining results, a total of 25,417 quality SNPs were discovered - 15,010 (59.1%) were transitions (AG and CT), 9,114 (35.9%) were transversions (AC, GT, CG, and AT), and 1,293 (5.0%) were insertion/deletions (indels). A vast majority of SNP-containing contigs consisted of only 2 haplotypes, as expected, but the percentages of 2 haplotype contigs varied widely in these citrus cultivars. BLAST of the 25,417 25-mer SNP oligos to the Clementine reference genome scaffolds revealed 2,947 SNPs had "no hits found", 19,943 had 1 unique hit / alignment, 1,571 had one hit and 2+ alignments per hit, and 956 had 2+ hits and 1+ alignment per hit. Of the total 24,293 scaffold hits, 23,955 (98.6%) were on the main scaffolds 1 to 9, and only 338 were on 87 minor scaffolds. Most alignments had 100% (25/25) or 96% (24/25) nucleotide identities, accounting for 93% of all the alignments. Considering almost all the nucleotide discrepancies in the 24/25 alignments were at the SNP sites, it served well as in silico validation of these SNPs, in addition to and consistent with the rate (81%) validated by sequencing and SNaPshot assay. High-quality EST-SNPs from different citrus genotypes were detected, and

  2. 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

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

    Directory of Open Access Journals (Sweden)

    Kenyon Fiona

    2011-07-01

    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.

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

    Science.gov (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, Ma.de.L.; 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.

    2016-01-01

    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 http://www.ebi.ac.uk/ena/data/view/PRJEB6630). 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 http://www.ncbi.nlm.nih.gov/geo/accession 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

  5. Robust global microRNA expression profiling using next-generation sequencing technologies.

    Science.gov (United States)

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

    2014-03-01

    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.

  6. An Ambystoma mexicanum EST sequencing project: analysis of 17,352 expressed sequence tags from embryonic and regenerating blastema cDNA libraries

    Science.gov (United States)

    Habermann, Bianca; Bebin, Anne-Gaelle; Herklotz, Stephan; Volkmer, Michael; Eckelt, Kay; Pehlke, Kerstin; Epperlein, Hans Henning; Schackert, Hans Konrad; Wiebe, Glenis; Tanaka, Elly M

    2004-01-01

    Background The ambystomatid salamander, Ambystoma mexicanum (axolotl), is an important model organism in evolutionary and regeneration research but relatively little sequence information has so far been available. This is a major limitation for molecular studies on caudate development, regeneration and evolution. To address this lack of sequence information we have generated an expressed sequence tag (EST) database for A. mexicanum. Results Two cDNA libraries, one made from stage 18-22 embryos and the other from day-6 regenerating tail blastemas, generated 17,352 sequences. From the sequenced ESTs, 6,377 contigs were assembled that probably represent 25% of the expressed genes in this organism. Sequence comparison revealed significant homology to entries in the NCBI non-redundant database. Further examination of this gene set revealed the presence of genes involved in important cell and developmental processes, including cell proliferation, cell differentiation and cell-cell communication. On the basis of these data, we have performed phylogenetic analysis of key cell-cycle regulators. Interestingly, while cell-cycle proteins such as the cyclin B family display expected evolutionary relationships, the cyclin-dependent kinase inhibitor 1 gene family shows an unusual evolutionary behavior among the amphibians. Conclusions Our analysis reveals the importance of a comprehensive sequence set from a representative of the Caudata and illustrates that the EST sequence database is a rich source of molecular, developmental and regeneration studies. To aid in data mining, the ESTs have been organized into an easily searchable database that is freely available online. PMID:15345051

  7. Genomic convergence analysis of schizophrenia: mRNA sequencing reveals altered synaptic vesicular transport in post-mortem cerebellum.

    Directory of Open Access Journals (Sweden)

    Joann Mudge

    Full Text Available Schizophrenia (SCZ is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six, matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ.

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

    Directory of Open Access Journals (Sweden)

    A.J. García-Chequer

    2016-03-01

    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 http://www.ncbi.nlm.nih.gov/geo/accession 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.

  9. Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Natalie A Twine

    Full Text Available Recent studies strongly indicate that aberrations in the control of gene expression might contribute to the initiation and progression of Alzheimer's disease (AD. In particular, alternative splicing has been suggested to play a role in spontaneous cases of AD. Previous transcriptome profiling of AD models and patient samples using microarrays delivered conflicting results. This study provides, for the first time, transcriptomic analysis for distinct regions of the AD brain using RNA-Seq next-generation sequencing technology. Illumina RNA-Seq analysis was used to survey transcriptome profiles from total brain, frontal and temporal lobe of healthy and AD post-mortem tissue. We quantified gene expression levels, splicing isoforms and alternative transcript start sites. Gene Ontology term enrichment analysis revealed an overrepresentation of genes associated with a neuron's cytological structure and synapse function in AD brain samples. Analysis of the temporal lobe with the Cufflinks tool revealed that transcriptional isoforms of the apolipoprotein E gene, APOE-001, -002 and -005, are under the control of different promoters in normal and AD brain tissue. We also observed differing expression levels of APOE-001 and -002 splice variants in the AD temporal lobe. Our results indicate that alternative splicing and promoter usage of the APOE gene in AD brain tissue might reflect the progression of neurodegeneration.

  10. Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi.

    Science.gov (United States)

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

    2013-04-30

    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.

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

    Directory of Open Access Journals (Sweden)

    Alessandra A. de Souza

    2007-01-01

    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.

  12. RNA expression profile of calcified bicuspid, tricuspid, and normal human aortic valves by RNA sequencing.

    Science.gov (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

    2016-10-01

    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.

  13. 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

    2004-01-01

    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.

  14. 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

  15. Sequencing, physical organization and kinetic expression of the patulin biosynthetic gene cluster from Penicillium expansum.

    Science.gov (United States)

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

    2014-10-17

    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.

  16. 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)

    2007-01-01

    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.

  17. Temperature influences on the expression of GFP promoted by the upstream sequence of cpcB from Arthrospira platensis

    Science.gov (United States)

    Lu, Yongzhong; Zhang, Xuecheng

    2007-07-01

    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.

  18. Combined Analysis of ChIP Sequencing and Gene Expression Dataset in Breast Cancer.

    Science.gov (United States)

    Liu, Pengfei; Jiang, Wenhua; Zhou, Shiyong; Gao, Jun; Zhang, Huilai

    2017-04-01

    Breast cancer is a common malignancy in women and contribute largely to the cancer related death. The purpose of this study is to confirm the roles of GATA3 and identify potential biomarkers of breast cancer. Chromatin Immunoprecipitation combined with high-throughput sequencing (ChIP-Seq) (GSM1642515) and gene expression profiles (GSE24249) were downloaded from the Gene Expression Omnibus (GEO) database. Bowtie2 and MACS2 were used for the mapping and peak calling of the ChIP-Seq data respectively. ChIPseeker, a R bioconductor package was adopted for the annotation of the enriched peaks. For the gene expression profiles, we used affy and limma package to do normalization and differential expression analysis. The genes with fold change >2 and adjusted P-Value ChIP-Seq and gene expression profiles. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for the functional enrichment analysis of overlapping genes between the target genes and differential expression genes (DEGs). What's more, the protein-protein interaction (PPI) network of the overlapping genes was obtained through the Human Protein Reference Database (HPRD). A total of 46,487 peaks were identified for GATA3 and out of which, 3256 ones were found to located at -3000 ~ 0 bp from the transcription start sites (TSS) of their nearby gene. A total of 236 down- and 343 up-regulated genes were screened out in GATA3 overexpression breast cancer samples compared with those in control. The combined analysis of ChIP-Seq and gene expression dataset showed GATA3 act as a repressor in breast cancer. Besides, 68 overlaps were obtained between the DEGs and genes included in peaks located at -3000 ~ 0 bp from TSS. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to cancer progression and gene regulation were found to be enriched in those overlaps. In the PPI network, NDRG1, JUP and etc. were found to directly interact with large number of

  19. 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

  20. Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee.

    Science.gov (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

    2002-04-01

    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 http://titan.biotec.uiuc.edu/bee/honeybee_project.htm.

  1. 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

    2003-01-01

    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.

  2. Sequence Conservation and Differential Expression of Marek's Disease Virus MicroRNAs▿

    Science.gov (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

    2008-01-01

    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

  3. 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.

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

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    Ravi Chandra Yandamuri

    2014-10-01

    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.

  5. Revised sequence and expression of cyclin B cDNA from the starfish Asterina pectinifera.

    Science.gov (United States)

    Miyake, Y; Deshimaru, S; Toraya, T

    2001-05-01

    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.

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

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    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.

  7. Primary Analysis of the Expressed Sequence Tags in a Pentastomid Nymph cDNA Library

    Science.gov (United States)

    Yuan, Zhongying; Yin, Jianhai; Zang, Wei; Xu, Yuxin; Lu, Weiyuan; Wang, Yanjuan; Wang, Ying; Cao, Jianping

    2013-01-01

    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. PMID:23437150

  8. Expressed sequence tag analysis of Antarctic hairgrass Deschampsia antarctica from King George Island, Antarctica.

    Science.gov (United States)

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

    2008-04-30

    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.

  9. 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

    2007-01-01

    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.

  10. 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

    2015-01-01

    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.

  11. dictyExpress: a web-based platform for sequence data management and analytics in Dictyostelium and beyond.

    Science.gov (United States)

    Stajdohar, Miha; Rosengarten, Rafael D; Kokosar, Janez; Jeran, Luka; Blenkus, Domen; Shaulsky, Gad; Zupan, Blaz

    2017-06-02

    Dictyostelium discoideum, a soil-dwelling social amoeba, is a model for the study of numerous biological processes. Research in the field has benefited mightily from the adoption of next-generation sequencing for genomics and transcriptomics. Dictyostelium biologists now face the widespread challenges of analyzing and exploring high dimensional data sets to generate hypotheses and discovering novel insights. We present dictyExpress (2.0), a web application designed for exploratory analysis of gene expression data, as well as data from related experiments such as Chromatin Immunoprecipitation sequencing (ChIP-Seq). The application features visualization modules that include time course expression profiles, clustering, gene ontology enrichment analysis, differential expression analysis and comparison of experiments. All visualizations are interactive and interconnected, such that the selection of genes in one module propagates instantly to visualizations in other modules. dictyExpress currently stores the data from over 800 Dictyostelium experiments and is embedded within a general-purpose software framework for management of next-generation sequencing data. dictyExpress allows users to explore their data in a broader context by reciprocal linking with dictyBase-a repository of Dictyostelium genomic data. In addition, we introduce a companion application called GenBoard, an intuitive graphic user interface for data management and bioinformatics analysis. dictyExpress and GenBoard enable broad adoption of next generation sequencing based inquiries by the Dictyostelium research community. Labs without the means to undertake deep sequencing projects can mine the data available to the public. The entire information flow, from raw sequence data to hypothesis testing, can be accomplished in an efficient workspace. The software framework is generalizable and represents a useful approach for any research community. To encourage more wide usage, the backend is open

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

    Science.gov (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

    2016-09-02

    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.

  13. 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

    2002-01-01

    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.

  14. In vitro analysis of expression vectors for DNA vaccination of horses: the effect of a Kozak sequence

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    Torsteinsdóttir Sigurbjörg

    2008-11-01

    Full Text Available Abstract One of the prerequisite for developing DNA vaccines for horses are vectors that are efficiently expressed in horse cells. We have analysed the ectopic expression of the human serum albumin gene in primary horse cells from different tissues. The vectors used are of pcDNA and pUC origin and include the cytomegalovirus (CMV promoter. The pUC vectors contain CMV intron A whereas the pcDNA vectors do not. Insertion of intron A diminished the expression from the pcDNA vectors whereas insertion of a Kozak sequence upstream of the gene in two types of pUC vectors increased significantly the in vitro expression in primary horse cells derived from skin, lung, duodenum and kidney. We report for the first time the significance of full consensus Kozak sequences for protein expression in horse cells in vitro.

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

    Science.gov (United States)

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

    2011-03-09

    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.

  16. 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)

    MichaelG.Walker

    2003-01-01

    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.

  17. 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

    2003-01-01

    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.

  18. 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.

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

    KAUST Repository

    Ravasi, Timothy

    2016-01-24

    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.

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

    Directory of Open Access Journals (Sweden)

    Antonio Figueira

    2001-12-01

    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

  1. GenEST, a powerful bidirectional link between cDNA sequence data and gene expression profiles generated by cDNA-AFLP

    NARCIS (Netherlands)

    Qin Ling,; Prins, P.; Jones, J.T.; Popeijus, H.; Smant, G.; Bakker, J.; Helder, J.

    2001-01-01

    The release of vast quantities of DNA sequence data by large-scale genome and expressed sequence tag (EST) projects underlines the necessity for the development of efficient and inexpensive ways to link sequence databases with temporal and spatial expression profiles. Here we demonstrate the power

  2. Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum.

    Science.gov (United States)

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

    2013-12-01

    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.

  3. Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression.

    Science.gov (United States)

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

    2016-05-17

    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.

  4. Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences.

    Science.gov (United States)

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

    1990-06-15

    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.

  5. 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

    2010-07-01

    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

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

    Directory of Open Access Journals (Sweden)

    Garg Neha

    2012-10-01

    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

  7. 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))

    1989-06-12

    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.

  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

    2006-01-01

    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

    2007-01-01

    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

    Science.gov (United States)

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

    2006-01-01

    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. 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.

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

    Science.gov (United States)

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

    1998-06-01

    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.

  13. Characterization of Shigella type 1 fimbriae: expression, FimA sequence, and phase variation.

    Science.gov (United States)

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

    1997-06-01

    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.

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

    NARCIS (Netherlands)

    Guryev, Victor; Berezikov, Eugene; Cuppen, Edwin

    2005-01-01

    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.

    2009-01-01

    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. 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

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

    Indian Academy of Sciences (India)

    SHATAKSHEE CHATTERJEE; PARTHA P. MAJUMDER; PRIYANKA PANDEY

    2016-09-01

    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.

  18. 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

    2015-01-01

    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.

  19. A survey of canine expressed sequence tags and a display of their annotations through a flexible web-based interface.

    Science.gov (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

    2003-01-01

    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.

  20. AllelicImbalance: An R/ bioconductor package for detecting, managing, and visualizing allele expression imbalance data from RNA sequencing

    DEFF Research Database (Denmark)

    Gådin, Jesper R.; van't Hooft, Ferdinand M.; Eriksson, Per

    2015-01-01

    Background: One aspect in which RNA sequencing is more valuable than microarray-based methods is the ability to examine the allelic imbalance of the expression of a gene. This process is often a complex task that entails quality control, alignment, and the counting of reads over heterozygous single......-nucleotide polymorphisms. Allelic imbalance analysis is subject to technical biases, due to differences in the sequences of the measured alleles. Flexible bioinformatics tools are needed to ease the workflow while retaining as much RNA sequencing information as possible throughout the analysis to detect and address...... the possible biases. Results: We present AllelicImblance, a software program that is designed to detect, manage, and visualize allelic imbalances comprehensively. The purpose of this software is to allow users to pose genetic questions in any RNA sequencing experiment quickly, enhancing the general utility...

  1. 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

    2014-01-01

    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.

  2. 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

    2010-03-01

    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.

  3. Effects of Age and Estrogen on Skeletal Gene Expression in Humans as Assessed by RNA Sequencing

    Science.gov (United States)

    Fujita, Koji; Nicks, Kristy M.; Cunningham, Julie M.; Atkinson, Elizabeth J.; Therneau, Terry M.; McCready, Louise K.; Peterson, James M.; Drake, Matthew T.; Monroe, David G.; Khosla, Sundeep

    2015-01-01

    Precise delineation of the specific genes and pathways altered with aging and estrogen (E) therapy may lead to new skeletal biomarkers and the development of novel bone therapeutics. Previous human bone studies, however, have been limited by only examining pre-specified genes and pathways. High-throughput RNA sequencing (RNAseq), on the other hand, offers an unbiased approach to examine the entire transcriptome. Here we present an RNAseq analysis of human bone samples, obtained from iliac crest needle biopsies, to yield the first in vivo interrogation of all genes and pathways that may be altered in bone with aging and E therapy in humans. 58 healthy women were studied, including 19 young women (mean age ± SD, 30.3 ± 5.4 years), 19 old women (73.1 ± 6.6 years), and 20 old women treated with 3 weeks of E therapy (70.5 ± 5.2 years). Using generally accepted criteria (false discovery rate [q] < 0.10), aging altered a total of 678 genes and 12 pathways, including a subset known to regulate bone metabolism (e.g., Notch). Interestingly, the LEF1 transcription factor, which is a classical downstream target of the Wnt/β-catenin signaling pathway, was significantly downregulated in the bones from the old versus young women; consistent with this, LEF1 binding sites were significantly enriched in the promoter regions of the differentially expressed genes in the old versus young women, suggesting that aging was associated with alterations in Wnt signaling in bone. Further, of the 21 unique genes altered in bone by E therapy, the expression of INHBB (encoding for the inhibin, beta B polypeptide), which decreased with aging (by 0.6-fold), was restored to young adult levels in response to E therapy. In conclusion, our data demonstrate that aging alters a substantial portion of the skeletal transcriptome, whereas E therapy appears to have significant, albeit less wide-ranging effects. These data provide a valuable resource for the potential identification of novel biomarkers

  4. 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

    2005-01-01

    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.

  5. A hybrid distance measure for clustering expressed sequence tags originating from the same gene family.

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    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.

  6. Sugarcane expressed sequences tags (ESTs encoding enzymes involved in lignin biosynthesis pathways

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    Ramos Rose Lucia Braz

    2001-01-01

    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.

  7. Genome-wide analysis reveals diverged patterns of codon bias, gene expression, and rates of sequence evolution in picea gene families.

    Science.gov (United States)

    De La Torre, Amanda R; Lin, Yao-Cheng; Van de Peer, Yves; Ingvarsson, Pär K

    2015-03-05

    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. A second rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein expression in a cell-free system.

    Science.gov (United States)

    Frassanito, Anna Maria; Barsanti, Laura; Passarelli, Vincenzo; Evangelista, Valtere; Gualtieri, Paolo

    2013-08-05

    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.

  9. 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))

    1994-04-01

    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.

  10. Expression of cassini, a murine gamma-satellite sequence conserved in evolution, is regulated in normal and malignant hematopoietic cells

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    Arutyunyan Anna

    2012-08-01

    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.

  11. Expression of cassini, a murine gamma-satellite sequence conserved in evolution, is regulated in normal and malignant hematopoietic cells.

    Science.gov (United States)

    Arutyunyan, Anna; Stoddart, Sonia; Yi, Sun-ju; Fei, Fei; Lim, Min; Groffen, Paula; Feldhahn, Niklas; Groffen, John; Heisterkamp, Nora

    2012-08-23

    Acute lymphoblastic leukemia (ALL) cells treated with drugs can become drug-tolerant if co-cultured with protective stromal mouse embryonic fibroblasts (MEFs). 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. 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.

  12. Nucleotide sequence and spatial expression pattern of a drought- and abscisic Acid-induced gene of tomato.

    Science.gov (United States)

    Plant, A L; Cohen, A; Moses, M S; Bray, E A

    1991-11-01

    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.

  13. Sequence and mRNA expression of nonclassical SLA class I genes SLA-7 and SLA-8.

    Science.gov (United States)

    Crew, Mark D; Phanavanh, Bounleut; Garcia-Borges, Carmen N

    2004-05-01

    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.

  14. 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

    2007-01-01

    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.

  15. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells.

    Science.gov (United States)

    Xu, Zhen; Chen, Feng; Zhang, Lingling; Lu, Jing; Xu, Peng; Liu, Guang; Xie, Xuemin; Mu, Wenli; Wang, Yajun; Liu, Depei

    2016-10-01

    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.

  16. 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

    2007-07-01

    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

  17. De novo transcriptome sequencing and comparative analysis of differentially expressed genes in Gossypium aridum under salt stress.

    Science.gov (United States)

    Xu, Peng; Liu, Zhangwei; Fan, Xinqi; Gao, Jin; Zhang, Xia; Zhang, Xianggui; Shen, Xinlian

    2013-08-01

    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.

  18. 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)

    2007-01-01

    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.

  19. 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

    2001-01-01

    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 ...

  20. Whole genome sequencing of sugarbeet and identification of differentially expressed genes regulating beet curly top resistance

    Science.gov (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 ...

  1. 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

  2. Structural organization, sequence, and expression of the mouse HEXA gene encoding the alpha subunit of hexosaminidase A.

    Science.gov (United States)

    Wakamatsu, N; Benoit, G; Lamhonwah, A M; Zhang, Z X; Trasler, J M; Triggs-Raine, B L; Gravel, R A

    1994-11-01

    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.

  3. Molecular diversification based on analysis of expressed sequence tags from the venom glands of the Chinese bird spider Ornithoctonus huwena.

    Science.gov (United States)

    Jiang, Liping; Peng, Li; Chen, Jinjun; Zhang, Yongqun; Xiong, Xia; Liang, Songping

    2008-06-15

    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.

  4. 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.

    Science.gov (United States)

    Harraghy, Niamh; Homerova, Dagmar; Herrmann, Mathias; Kormanec, Jan

    2008-01-01

    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.

  5. Tissue factor expression in the metaplasia-adenoma-carcinoma sequence of gastric cancer in a European population.

    Science.gov (United States)

    Lo, L; Valentine, H; Harrison, J; Hayes, S; Welch, I; Pritchard, S; West, C; Ang, Y

    2012-09-25

    Tissue factor (TF), which has a role in normal tissue haemostasis, was reported to be aberrantly expressed, associated with higher microvascular density and a poor prognosis in intestinal-type gastric adenocarcinoma in the Japanese population. This is the first study to look at the relationship of TF and the metaplasia-adenoma-carcinoma sequence (MACS) of gastric cancer in a European population. The expression of TF was examined immunohistochemically in 191 gastric tissue samples: (13: normal; 18: intestinal metaplasia; 160: gastric adenocarcinoma) from the European population. TF was not expressed in normal gastric mucosal cells. A strong intensity of staining was found in intestinal metaplasia cells but in 2 of 18 samples. TF expression increased with advancing stage of gastric cancer (Pgastric cancers preferentially expressed TF (34%, P=0.04). In comparison with the Japanese study, TF was not expressed at a higher level in intestinal vs diffuse-type gastric cancers and expression had 'no prognostic' significance. TF may be involved in tumour progression along the MACS of gastric cancer in the European population and is shown to start in precancerous lesions. However, clinical features may differ due to differences in biological features in the two populations, as reflected by differences in TF expression profile.

  6. Helicobacter pylori cagA Promoter Region Sequences Influence CagA Expression and Interleukin 8 Secretion.

    Science.gov (United States)

    Ferreira, Rui M; Pinto-Ribeiro, Ines; Wen, Xiaogang; Marcos-Pinto, Ricardo; Dinis-Ribeiro, Mário; Carneiro, Fátima; Figueiredo, Ceu

    2016-02-15

    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.

  7. 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

    2017-01-01

    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.

  8. RNA sequencing identifies gene expression profile changes associated with β-estradiol treatment in U2OS osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Chen B

    2017-07-01

    Full Text Available Bin Chen, Zude Liu, Jidong Zhang, Hantao Wang, Bo Yu Department of Orthopedic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China Abstract: This study was conducted to identify gene expression profile changes associated with β-estradiol (E2 treatment in U2OS osteosarcoma cells by high-throughput RNA sequencing (RNA-seq. Two U2OS cell samples treated with E2 (15 µmol/L and two untreated control U2OS cell samples were subjected to RNA-seq. Differentially expressed genes (DEGs between the groups were identified, and main biological process enrichment was performed using gene ontology (GO analysis. A protein–protein interaction (PPI network was constructed using Cytoscape based on the Human Protein Reference Database. Finally, NFKB1 expression was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR. The map ratios of the four sequenced samples were >65%. In total, 128 upregulated and 92 downregulated DEGs were identified in E2 samples. After GO enrichment, the downregulated DEGs, such as AKT1, were found to be mainly enriched in cell cycle processes, whereas the upregulated DEGs, such as NFKB1, were involved in the regulation of gene expression. Moreover, AKT1 (degree =117 and NFKB1 (degree =72 were key nodes with the highest degrees in the PPI network. Similarly, the results of qRT-PCR confirmed that E2 upregulated NFKB1 expression. The results suggest that E2 upregulates the expression of NFKB1, ATF7IP, and HDAC5, all of which are involved in the regulation of gene expression and transcription, but downregulates that of TCF7L2, ALCAM, and AKT, which are involved in Wnt receptor signaling through β-catenin and morphogenesis in U2OS osteosarcoma cells. Keywords: differentially expressed genes, Wnt receptor signaling, β-catenin, protein-protein interaction network

  9. 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

    2010-08-01

    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

  10. Inhibition of expression of virulence genes of Yersinia pestis in Escherichia coli by external guide sequences and RNase P.

    Science.gov (United States)

    Ko, Jae-hyeong; Izadjoo, Mina; Altman, Sidney

    2008-08-01

    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.

  11. Conserved sequence motifs upstream from the co-ordinately expressed vitellogenin and apoVLDLII genes of chicken.

    Science.gov (United States)

    van het Schip, F; Strijker, R; Samallo, J; Gruber, M; Geert, A B

    1986-11-11

    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.

  12. Development, characterization and cross species amplification of polymorphic microsatellite markers from expressed sequence tags of turmeric (Curcuma longa L.).

    Science.gov (United States)

    Siju, S; Dhanya, K; Syamkumar, S; Sasikumar, B; Sheeja, T E; Bhat, A I; Parthasarathy, V A

    2010-02-01

    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.

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

    DEFF Research Database (Denmark)

    Studer, Bruno; Asp, Torben; Frei, Ursula

    2008-01-01

    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...... was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight...... genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage...

  14. Cloning, sequencing, expression and structural investigation of mnemiopsin from Mnemiopsis leidyi: an attempt toward understanding Ca2+-regulated photoproteins.

    Science.gov (United States)

    Aghamaali, Mahmoud Reza; Jafarian, Vahab; Sariri, Reyhaneh; Molakarimi, Maryam; Rasti, Behnam; Taghdir, Majid; Sajedi, Reza Hasan; Hosseinkhani, Saman

    2011-12-01

    A comparison of the two most famous groups of calcium-regulated photoproteins, cnidarians and ctenophores, showed unexpectedly high degree of structural similarity regardless of their low sequence identity. It was suggested these photoproteins can play an important role in understanding the structural basis of bioluminescence activity. Based on this postulate, in this study the cDNA of mnemiopsin from luminous ctenophore Mnemiopsis leidyi was cloned, expressed, purified and sequenced. The purified cDNA, with 621 base pairs, coded a 206 residues protein. Sequence of mnemiopsin showed 93.5 and 51% similarity to other ctenophore proteins and cnidarians, respectively. The cDNA encoding apo-mnemiopsin of M. leidyi was expressed in Escherichia coli. The purified apo-protein showed a single band on SDS-PAGE (molecular weight ~27 kDa). A semi-synthetic mnemiopsin was prepared using coelenterazine and EDTA and its luminescence activity was measured in the presence of CaCl(2). The results showed an optimum pH of 9.0 and lower calcium sensitivity compared to aequorin. Comparison of amino acid residues in substrate binding site indicated that binding pocket of ctenophores contains less aromatic residues than cnidarians. This can lead to a decline in the number of stacking interactions between substrate and protein which can affect the stability of coelenterazine in binding cavity. Structural comparison of photoproteins with low sequence identity and high 3D structural similarity, can present a new insight into the mechanism of light emission in photoproteins.

  15. Generation and analysis of expressed sequence tags from Trypanosoma cruzi trypomastigote and amastigote cDNA libraries.

    Science.gov (United States)

    Agüero, Fernán; Abdellah, Karim Ben; Tekiel, Valeria; Sánchez, Daniel O; González, Antonio

    2004-08-01

    We have generated 2771 expressed sequence tags (ESTs) from two cDNA libraries of Trypanosoma cruzi CL-Brener. The libraries were constructed from trypomastigote and amastigotes, using a spliced leader primer to synthesize the cDNA second strand, thus selecting for full-length cDNAs. Since the libraries were not normalized nor pre-screened, we compared the representation of transcripts between the two using a statistical test and identify a subset of transcripts that show apparent differential representation. A non-redundant set of 1619 reconstructed transcripts was generated by sequence clustering. This dataset was used to perform similarity searches against protein and nucleotide databases. Based on these searches, 339 sequences could be assigned a putative identity. One thousand one-hundred and sixteen sequences in the non-redundant clustered dataset (68.8%) are new expression tags, not represented in the T. cruzi epimastigote ESTs that are in the public databases. Additional information is provided online at http://genoma.unsam.edu.ar/projects/tram. To the best of our knowledge these are the first ESTs reported for the life cycle stages of T. cruzi that occur in the vertebrate host.

  16. Preparing and Analyzing Expressed Sequence Tags (ESTs) Library for the Mammary Tissue of Local Turkish Kivircik Sheep

    Science.gov (United States)

    Omeroglu Ulu, Zehra; Ulu, Salih; Un, Cemal; Ozdem Oztabak, Kemal; Altunatmaz, Kemal

    2017-01-01

    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.

  17. Using DNA sequencing electrophoresis compression artifacts as reporters of stable mRNA structures affecting gene expression.

    Science.gov (United States)

    Kapoor, Divya; Chandrayan, Sanjeev Kumar; Ahmed, Shubbir; Guptasarma, Purnananda

    2007-11-01

    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.

  18. 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

    2016-08-23

    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.

  19. Recoding method that removes inhibitory sequences and improves HIV gene expression

    Science.gov (United States)

    Rabadan, Raul; Krasnitz, Michael; Robins, Harlan; Witten, Daniela; Levine, Arnold

    2016-08-23

    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.

  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

    2008-01-01

    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. ConiferEST: an integrated bioinformatics system for data reprocessing and mining of conifer expressed sequence tags (ESTs

    Directory of Open Access Journals (Sweden)

    Carter Kikia

    2007-05-01

    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

  2. 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.

    1987-12-01

    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.

  3. The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags

    Science.gov (United States)

    Brentani, Helena; Caballero, Otávia L.; Camargo, Anamaria A.; da Silva, Aline M.; da Silva, Wilson Araújo; Neto, Emmanuel Dias; Grivet, Marco; Gruber, Arthur; Guimaraes, Pedro Edson Moreira; Hide, Winston; Iseli, Christian; Jongeneel, C. Victor; Kelso, Janet; Nagai, Maria Aparecida; Ojopi, Elida Paula Benquique; Osorio, Elisson C.; Reis, Eduardo M. R.; Riggins, Gregory J.; Simpson, Andrew John George; de Souza, Sandro; Stevenson, Brian J.; Strausberg, Robert L.; Tajara, Eloiza H.; Verjovski-Almeida, Sergio

    2003-01-01

    Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define ≈23,500 genes, of which only ≈1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers. PMID:14593198

  4. Transcriptome sequencing study implicates immune-related genes differentially expressed in schizophrenia: new data and a meta-analysis

    Science.gov (United States)

    Sanders, A R; Drigalenko, E I; Duan, J; Moy, W; Freda, J; Göring, H H H; Gejman, P V

    2017-01-01

    We undertook an RNA sequencing (RNAseq)-based transcriptomic profiling study on lymphoblastoid cell lines of a European ancestry sample of 529 schizophrenia cases and 660 controls, and found 1058 genes to be differentially expressed by affection status. These differentially expressed genes were enriched for involvement in immunity, especially the 697 genes with higher expression in cases. Comparing the current RNAseq transcriptomic profiling to our previous findings in an array-based study of 268 schizophrenia cases and 446 controls showed a highly significant positive correlation over all genes. Fifteen (18%) of the 84 genes with significant (false discovery rateRNAseq and array data sets (797 cases and 1106 controls) showed 169 additional genes (besides those found in the primary RNAseq-based analysis) to be differentially expressed, and provided further evidence of immune gene enrichment. In addition to strengthening our previous array-based gene expression differences in schizophrenia cases versus controls and providing transcriptomic support for some genes implicated by other approaches for schizophrenia, our study detected new genes differentially expressed in schizophrenia. We highlight RNAseq-based differential expression of various genes involved in neurodevelopment and/or neuronal function, and discuss caveats of the approach. PMID:28418402

  5. Two-step amplification of the human PPT sequence provides specific gene expression in an immunocompetent murine prostate cancer model.

    Science.gov (United States)

    Dzojic, H; Cheng, W-S; Essand, M

    2007-03-01

    The recombinant prostate-specific PPT sequence comprises a prostate-specific antigen enhancer, a PSMA enhancer and a TARP promoter. It is transcriptionally active in human prostate cancer cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer, it has no detectable transcriptional activity. Herein, we describe that the PPT sequence in combination with a two-step transcriptional amplification (TSTA) system becomes active also in murine prostate cancer cells. An adenovirus with TSTA-amplified PPT-controlled expression of the luciferase reporter gene, Ad[PPT/TSTA-Luc], has up to 100-fold higher prostate-specific transcriptional activity than a non-amplified PPT-based adenovirus, Ad[PPT-Luc], in human cells. In addition, Ad[PPT/TSTA-Luc] confers prostate-specific transgene expression in murine cells, with an activity that is approximately 23% of Ad[CMV-Luc] in the transgenic adenocarcinoma of the mouse prostate (TRAMP)-C2 cells. Moreover, to visualize luciferase expression in living mice a charge-coupled device camera was used. Ad[PPT/TSTA-Luc] yielded approximately 30-fold higher transgene expression than Ad[PPT-Luc] in LNCaP tumor xenografts. Importantly, Ad[PPT/TSTA-Luc] also showed activity in murine TRAMP-C2 tumors, whereas Ad[PPT-Luc] activity was undetectable. These results highlight that the recombinant PPT sequence is active in murine prostate cancer cells when augmented by a TSTA system. This finding opens up for preclinical studies with prostate-specific therapeutic gene expression in immunocompetent mice.

  6. Novel promoter sequence required for manganese regulation of manganese peroxidase isozyme 1 gene expression in Phanerochaete chrysosporium.

    Science.gov (United States)

    Ma, Biao; Mayfield, Mary B; Godfrey, Bruce J; Gold, Michael H

    2004-06-01

    Manganese peroxidase (MnP) is a major, extracellular component of the lignin-degrading system produced by the wood-rotting basidiomycetous fungus Phanerochaete chrysosporium. The transcription of MnP-encoding genes (mnps) in P. chrysosporium occurs as a secondary metabolic event, triggered by nutrient-nitrogen limitation. In addition, mnp expression occurs only under Mn2+ supplementation. Using a reporter system based on the enhanced green fluorescent protein gene (egfp), we have characterized the P. chrysosporium mnp1 promoter by examining the effects of deletion, replacement, and translocation mutations on mnp1 promoter-directed egfp expression. The 1,528-bp mnp1 promoter fragment drives egfp expression only under Mn2+-sufficient, nitrogen-limiting conditions, as required for endogenous MnP production. However, deletion of a 48-bp fragment, residing 521 bp upstream of the translation start codon in the mnp1 promoter, or replacement of this fragment with an unrelated sequence resulted in egfp expression under nitrogen limitation, both in the absence and presence of exogenous Mn2+. Translocation of the 48-bp fragment to a site 120 bp downstream of its original location resulted in Mn2+-dependent egfp expression under conditions similar to those observed with the wild-type mnp1 promoter. These results suggest that the 48-bp fragment contains at least one Mn2+-responsive cis element. Additional promoter-deletion experiments suggested that the Mn2+ element(s) is located within the 33-bp sequence at the 3' end of the 48-bp fragment. This is the first promoter sequence containing a Mn2+-responsive element(s) to be characterized in any eukaryotic organism. Copyright 2004 American Society for Microbiology

  7. Analysis and comparison of a set of expressed sequence tags of the parthenogenetic water flea Daphnia carinata.

    Science.gov (United States)

    Xu, Xiaoqian; Song, Shuhui; Wang, Qun; Qin, Fen; Liu, Kan; Zhang, Xiaowei; Hu, Songnian; Zhao, Yunlong

    2009-08-01

    The water flea Daphnia carinata (D. carinata) reproduces both sexually and parthenogenetically, yet little is known about the genes involved in these processes. To further clarify the reproductive biology of Daphnia and elucidate their unique mechanism of reproductive transformation, we have generated and characterized an expressed sequence tag (EST) data set from D. carinata. A set of 1,495 clusters were generated from sequencing 3,072 randomly chosen clones from a parthenogenetic, juvenile water flea cDNA library. The nucleic acid and deduced amino acid sequences were compared with known GenBank sequences. Functional annotation found that 959 clusters showed significant homology with known genes involved in a broad range of activities, including metabolism, translation, development and reproduction, as well as genes involved in sensing environmental factors. We speculate that genes involved in development and reproduction, along with genes that allow the organism to sense changes in the environment, play important roles in the process of parthenogenetic reproduction and could be markers of the early steps of sexual differentiation. Additionally, 86% of the D. Carinata unique sequences could be stringently mapped to the D. pulex genome, of which 125 mapped to intergenic and intronic regions on the current assembly. Our results provide practical insight into crustacean reproductive biology, in addition to establishing a new animal model for reproductive and developmental biology.

  8. Accurate Profiling of Gene Expression and Alternative Polyadenylation with Whole Transcriptome Termini Site Sequencing (WTTS-Seq)

    Science.gov (United States)

    Zhou, Xiang; Li, Rui; Michal, Jennifer J.; Wu, Xiao-Lin; Liu, Zhongzhen; Zhao, Hui; Xia, Yin; Du, Weiwei; Wildung, Mark R.; Pouchnik, Derek J.; Harland, Richard M.; Jiang, Zhihua

    2016-01-01

    Construction of next-generation sequencing (NGS) libraries involves RNA manipulation, which often creates noisy, biased, and artifactual data that contribute to errors in transcriptome analysis. In this study, a total of 19 whole transcriptome termini site sequencing (WTTS-seq) and seven RNA sequencing (RNA-seq) libraries were prepared from Xenopus tropicalis adult and embryo samples to determine the most effective library preparation method to maximize transcriptomics investigation. We strongly suggest that appropriate primers/adaptors are designed to inhibit amplification detours and that PCR overamplification is minimized to maximize transcriptome coverage. Furthermore, genome annotation must be improved so that missing data can be recovered. In addition, a complete understanding of sequencing platforms is critical to limit the formation of false-positive results. Technically, the WTTS-seq method enriches both poly(A)+ RNA and complementary DNA, adds 5′- and 3′-adaptors in one step, pursues strand sequencing and mapping, and profiles both gene expression and alternative polyadenylation (APA). Although RNA-seq is cost prohibitive, tends to produce false-positive results, and fails to detect APA diversity and dynamics, its combination with WTTS-seq is necessary to validate transcriptome-wide APA. PMID:27098915

  9. De novo sequence analysis of cytochrome P450 1-3 genes expressed in ostrich liver with highest expression of CYP2G19.

    Science.gov (United States)

    Kawai, Yusuke K; Watanabe, Kensuke P; Ishii, Akihiro; Ohnuma, Aiko; Sawa, Hirofumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2013-09-01

    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.

  10. MicroRNA deep sequencing reveals chamber-specific miR-208 family expression patterns in the human heart.

    Science.gov (United States)

    Kakimoto, Yu; Tanaka, Masayuki; Kamiguchi, Hiroshi; Hayashi, Hideki; Ochiai, Eriko; Osawa, Motoki

    2016-05-15

    Heart chamber-specific mRNA expression patterns have been extensively studied, and dynamic changes have been reported in many cardiovascular diseases. MicroRNAs (miRNAs) are also important regulators of normal cardiac development and functions that generally suppress gene expression at the posttranscriptional level. Recent focus has been placed on circulating miRNAs as potential biomarkers for cardiac disorders. However, miRNA expression levels in human normal hearts have not been thoroughly studied, and chamber-specific miRNA expression signatures in particular remain unclear. We performed miRNA deep sequencing on human paired left atria (LA) and ventricles (LV) under normal physiologic conditions. Among 438 miRNAs, miR-1 was the most abundant in both chambers, representing 21% of the miRNAs in LA and 26% in LV. A total of 25 miRNAs were differentially expressed between LA and LV; 14 were upregulated in LA, and 11 were highly expressed in LV. Notably, the miR-208 family in particular showed prominent chamber specificity; miR-208a-3p and miR-208a-5p were abundant in LA, whereas miR-208b-3p and miR-208b-5p were preferentially expressed in LV. Subsequent real-time polymerase chain reaction analysis validated the predominant expression of miR-208a in LA and miR-208b in LV. Human atrial and ventricular tissues display characteristic miRNA expression signatures under physiological conditions. Notably, miR-208a and miR-208b show significant chamber-specificity as do their host genes, α-MHC and β-MHC, which are mainly expressed in the atria and ventricles, respectively. These findings might also serve to enhance our understanding of cardiac miRNAs and various heart diseases. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. 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.

    2001-01-01

    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.

  12. Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers.

    Science.gov (United States)

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

    2014-01-01

    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.

  13. 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.

  14. Identification of anhydrobiosis-related genes from an expressed sequence tag database in the cryptobiotic midge Polypedilum vanderplanki (Diptera; Chironomidae).

    Science.gov (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

    2010-11-12

    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.

  15. Cloning, sequencing and expression of the gene encoding the extracellular metalloprotease of Aeromonas caviae.

    Science.gov (United States)

    Kawakami, K; Toma, C; Honma, Y

    2000-01-01

    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.

  16. Cloning, sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene.

    Science.gov (United States)

    De Zoysa, P A; Connerton, I F; Watson, D C; Johnston, J R

    1991-08-01

    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.

  17. 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;

    2003-01-01

    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...

  18. Rat serum amyloid P component. Analysis of cDNA sequence and gene expression.

    Science.gov (United States)

    Dowton, S B; McGrew, S D

    1990-09-01

    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.

  19. 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

    2014-09-01

    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

  20. Next generation sequencing-based expression profiling identifies signatures from benign stromal proliferations that define stromal components of breast cancer

    Science.gov (United States)

    2013-01-01

    Introduction Multiple studies have shown that the tumor microenvironment (TME) of carcinomas can play an important role in the initiation, progression, and metastasis of cancer. Here we test the hypothesis that specific benign fibrous soft tissue tumor gene expression profiles may represent distinct stromal fibroblastic reaction types that occur in different breast cancers. The discovered stromal profiles could classify breast cancer based on the type of stromal reaction patterns in the TME. Methods Next generation sequencing-based gene expression profiling (3SEQ) was performed on formalin fixed, paraffin embedded (FFPE) samples of 10 types of fibrous soft tissue tumors. We determined the extent to which these signatures could identify distinct subsets of breast cancers in four publicly available breast cancer datasets. Results A total of 53 fibrous tumors were sequenced by 3SEQ with an average of 29 million reads per sample. Both the gene signatures derived from elastofibroma (EF) and fibroma of tendon sheath (FOTS) demonstrated robust outcome results for survival in the four breast cancer datasets. The breast cancers positive for the EF signature (20-33% of the cohort) demonstrated significantly better outcome for survival. In contrast, the FOTS signature-positive breast cancers (11-35% of the cohort) had a worse outcome. Conclusions We defined and validated two new stromal signatures in breast cancer (EF and FOTS), which are significantly associated with prognosis. Our group has previously identified novel cancer stromal gene expression signatures associated with outcome differences in breast cancer by gene expression profiling of three soft tissue tumors, desmoid-type fibromatosis (DTF), solitary fibrous tumor (SFT), and tenosynovial giant cell tumor (TGCT/CSF1), as surrogates for stromal expression patterns. By combining the stromal signatures of EF and FOTS, with our previously identified DTF and TGCT/CSF1 signatures we can now characterize clinically

  1. Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

    Science.gov (United States)

    Poidevin, Laetitia; Andreeva, Kalina; Khachatoorian, Careen; Judelson, Howard S

    2015-01-01

    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.

  2. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat.

    Science.gov (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

    2016-01-01

    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.

  3. 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

    2016-08-01

    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

  4. SSH Analysis of Endosperm Transcripts and Characterization of Heat Stress Regulated Expressed Sequence Tags in Bread Wheat

    Science.gov (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.

    2016-01-01

    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. 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

    2004-01-01

    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.

  6. 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

    2009-01-01

    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.

  7. 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.

    1998-01-01

    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

  8. Update of the Gene Discovery Program in Schistosoma mansoni with the Expressed Sequence Tag Approach

    Directory of Open Access Journals (Sweden)

    Élida ML Rabelo

    1997-09-01

    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

  9. Molecular Cloning and Expression of Sequence Variants of Manganese Superoxide Dismutase Genes from Wheat

    Science.gov (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...

  10. The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

    Science.gov (United States)

    2010-01-01

    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. 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

    2015-09-01

    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 (http://www.ncbi.nlm.nih.gov/sra. 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

  12. 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: zc83good@126.com [Department of Pharmacy of Luzhou Medical College, Luzhou, Sichuan (China)

    2015-03-27

    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.

  13. Cloning, sequencing and functional expression of cytosolic malate dehydrogenase from Taenia solium: Purification and characterization of the recombinant enzyme.

    Science.gov (United States)

    Nava, Gabriela; Laclette, Juan P; Bobes, Raúl; Carrero, Julio C; Reyes-Vivas, Horacio; Enriquez-Flores, Sergio; Mendoza-Hernández, Guillermo; Plancarte, Agustín

    2011-07-01

    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.

  14. Bioinformatic prediction, deep sequencing of microRNAs and expression analysis during phenotypic plasticity in the pea aphid, Acyrthosiphon pisum

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    Leterme Nathalie

    2010-05-01

    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: http://microrna.sanger.ac.uk/sequences/ Conclusions Our study has identified candidates as putative regulators involved in reproductive polyphenism in aphids and opens new avenues for further functional analyses.

  15. Sequence, evolution and tissue expression patterns of an epidermal type I keratin from the shark Scyliorhinus stellaris.

    Science.gov (United States)

    Schaffeld, Michael; Höffling, Simon; Jürgen, Markl

    2004-08-01

    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. Sequence analysis and gene expression of putative exo- and endo-glucanases from oil palm (Elaeis guineensis) during fungal infection.

    Science.gov (United States)

    Yeoh, Keat-Ai; Othman, Abrizah; Meon, Sariah; Abdullah, Faridah; Ho, Chai-Ling

    2012-10-15

    Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum. Copyright © 2012 Elsevier GmbH. All rights reserved.

  17. 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.

  18. 23个葡萄砧木的抗寒性比较与评价%Comparison and evaluation of the cold resistance of twenty-three rootstock varieties

    Institute of Scientific and Technical Information of China (English)

    郝停停; 李妍琪; 徐炎; 张剑侠

    2016-01-01

    UsingVitis amurensis accession Zuoshan75097 andV. vinifera cv. Red Globe as cold-resistance control (CK1) and cold-sensitive control (CK2) respectively, the relative conductivity, proline, malondialdehyde, soluble protein and soluble sugars of one-year old branches of twenty-three rootstock varieties after appropriate freezing temperature treatment was measured. Comprehensive evaluation of the cold-resistance level of rootstocks was performed with subordinative function method. The results showed that Beta, Zuoshan 75097, Yanshan-1, 101-14, 5A and 3309C belonged to high resistance; 5BB, 188-08, Heshan-1,V. Riparia (♀), 110R and 1103P belonged to cold resistance; Kangzhen 3, Freedom, YH56, SO4, 225Ru, Dog Ridge, Borner and 1-1-6 belonged to middle resistance; 6-12-1, 6-12-4 and 6-12-6 belonged to low resistance; Salt Creek and Red Globe present susceptible. Cold-resistant varieties could be used in grape production in cold area. Hybrids of Heshan-1, YH56 and 1-1-6 can be used for further screening of adversity resistance rootstock.%本文以山葡萄左山75097和欧洲葡萄红地球分别作为抗寒对照(CK1)和不抗寒对照(CK2),通过人工低温胁迫处理一年生休眠枝条的方法,测定了23个葡萄砧木品种和杂种优株的相对电导率、脯氨酸、丙二醛、可溶性糖及可溶性蛋白等5项生理指标,然后利用隶属函数法综合评价其抗寒性,为生产应用和砧木育种提供参考依据。结果表明:贝达、山葡萄左山75097、燕山-1、101-14、5A和3309C属于高抗类型;5BB、188-08、河山-1、河岸(♀)、110R和1103P属于抗寒类型;抗砧3号、Freedom、YH56、SO4、225Ru、狗脊、Borner和1-1-6属于中抗类型;6-12-4、6-12-1和6-12-6属于低抗类型;Salt Creek和红地球属于不抗类型。高抗和抗寒品种可用作寒地葡萄栽培的砧木,抗寒杂种优株河山-1和中抗杂种优株YH56、1-1-6可用作进一步的抗逆性鉴定以选育多抗砧木。

  19. 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

    2006-01-01

    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.

  20. 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

    2009-01-01

    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.

  1. Comprehensive profiling of EBV gene expression in nasopharyngeal carcinoma through paired-end transcriptome sequencing.

    Science.gov (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

    2016-03-01

    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.

  2. Nucleotide sequence and expression of relaxin-like gonad-stimulating peptide gene in starfish Asterina pectinifera.

    Science.gov (United States)

    Haraguchi, Shogo; Ikeda, Narumi; Abe, Michiko; Tsutsui, Kazuyoshi; Mita, Masatoshi

    2016-02-01

    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.

  3. 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

    2012-02-01

    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.

  4. Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

    Science.gov (United States)

    Lau, Yun-Fai; Kan, Yuet Wai

    1983-09-01

    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.

  5. Gene Expression Profiles in Paired Gingival Biopsies from Periodontitis-Affected and Healthy Tissues Revealed by Massively Parallel Sequencing

    Science.gov (United States)

    Båge, Tove; Lagervall, Maria; Jansson, Leif; Lundeberg, Joakim; Yucel-Lindberg, Tülay

    2012-01-01

    Periodontitis is a chronic inflammatory disease affecting the soft tissue and bone that surrounds the teeth. Despite extensive research, distinctive genes responsible for the disease have not been identified. The objective of this study was to elucidate transcriptome changes in periodontitis, by investigating gene expression profiles in gingival tissue obtained from periodontitis-affected and healthy gingiva from the same patient, using RNA-sequencing. Gingival biopsies were obtained from a disease-affected and a healthy site from each of 10 individuals diagnosed with periodontitis. Enrichment analysis performed among uniquely expressed genes for the periodontitis-affected and healthy tissues revealed several regulated pathways indicative of inflammation for the periodontitis-affected condition. Hierarchical clustering of the sequenced biopsies demonstrated clustering according to the degree of inflammation, as observed histologically in the biopsies, rather than clustering at the individual level. Among the top 50 upregulated genes in periodontitis-affected tissues, we investigated two genes which have not previously been demonstrated to be involved in periodontitis. These included interferon regulatory factor 4 and chemokine (C-C motif) ligand 18, which were also expressed at the protein level in gingival biopsies from patients with periodontitis. In conclusion, this study provides a first step towards a quantitative comprehensive insight into the transcriptome changes in periodontitis. We demonstrate for the first time site-specific local variation in gene expression profiles of periodontitis-affected and healthy tissues obtained from patients with periodontitis, using RNA-seq. Further, we have identified novel genes expressed in periodontitis tissues, which may constitute potential therapeutic targets for future treatment strategies of periodontitis. PMID:23029519

  6. 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

    2014-01-01

    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.

  7. Prediction of G gene epitopes of viral hemorrhagic septicemia virus and eukaryotic expression of major antigen determinant sequence.

    Science.gov (United States)

    Sun, T; Yin, W-L; Fang, B-H; Wang, Q; Liang, C-Z; Yue, Z-Q

    2017-08-15

    This study aims to express fish Viral hemorrhagic septicemia virus (VHSV) G main antigen domain by using Bac-to-bac expression system. Using bioinformatics tools, B cell epitope of VHSV G gene was predicted, and G main antigen domain was optimized. GM gene was inserted into pFastBac1 vector, then transferred recombinant plasmid into DH10Bac to get recombinant rBacmid-GM. Obtained shuttle plasmid rBacmid-GM was transfected into sf9 cells. GM expression was examined using by PCR and western-blot. Results indicated that G main antigen domain gene of VHSV was successfully cloned and sequenced which contains 1209 bp. PCR proved that shuttle plasmid rBacmid-GM was constructed correctly. SDS-PAGE electrophoresis analysis detected a band of protein about 45kD in expression product of G gene. Obtained recombinant G protein reacted with VHSV-positive serum that was substantiated by western-blot analysis. In conclusion, the main antigen domain of VHSV G was successfully expressed in the Bac-to-Bac baculovirus system.

  8. RNA Sequence Analysis of Human Huntington Disease Brain Reveals an Extensive Increase in Inflammatory and Developmental Gene Expression.

    Directory of Open Access Journals (Sweden)

    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.

  9. ESTPiper – a web-based analysis pipeline for expressed sequence tags

    Directory of Open Access Journals (Sweden)

    Tang Zuojian

    2009-04-01

    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 http://estpiper.cgb.indiana.edu/ to now support projects of all size. The software is also freely available from the authors for

  10. Structure, sequence and expression of the hepatitis delta (δ) viral genome

    Science.gov (United States)

    Wang, Kang-Sheng; Choo, Qui-Lim; Weiner, Amy J.; Ou, Jing-Hsiung; Najarian, Richard C.; Thayer, Richard M.; Mullenbach, Guy T.; Denniston, Katherine J.; Gerin, John L.; Houghton, Michael

    1986-10-01

    Biochemical and electron microscopic data indicate that the human hepatitis δ viral agent contains a covalently closed circular and single-stranded RNA genome that has certain similarities with viroid-like agents from plants. The sequence of the viral genome (1,678 nucleotides) has been determined and an open reading frame within the complementary strand has been shown to encode an antigen that binds specifically to antisera from patients with chronic hepatitis δ viral infections.

  11. Transient and Stable GFP Expression in Germ Cells by the vasa Regulatory Sequences from the Red Seabream (Pagrus major

    Directory of Open Access Journals (Sweden)

    Fan Lin, Qinghua Liu, Mingyou Li, Zhendong Li, Ni Hong, Jun Li, Yunhan Hong

    2012-01-01

    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.

  12. Natural sequence variants of yeast environmental sensors confer cell-to-cell expression variability.

    Science.gov (United States)

    Fehrmann, Steffen; Bottin-Duplus, Hélène; Leonidou, Andri; Mollereau, Esther; Barthelaix, Audrey; Wei, Wu; Steinmetz, Lars M; Yvert, Gaël

    2013-10-08

    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.

  13. Development and characterization of 1,827 expressed sequence tag-derived simple sequence repeat markers for ramie (Boehmeria nivea L. Gaud.

    Directory of Open Access Journals (Sweden)

    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.

  14. Candidate genes revealed by a genome scan for mosquito resistance to a bacterial insecticide: sequence and gene expression variations

    Directory of Open Access Journals (Sweden)

    David Jean-Philippe

    2009-11-01

    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

  15. RNA-sequence analysis of gene expression from honeybees (Apis mellifera) infected with Nosema ceranae.

    Science.gov (United States)

    Badaoui, Bouabid; 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

    2017-01-01

    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.

  16. Language trees support the express-train sequence of Austronesian expansion.

    Science.gov (United States)

    Gray, R D; Jordan, F M

    2000-06-29

    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.

  17. RNA-sequence analysis of gene expression from honeybees (Apis mellifera) infected with Nosema ceranae

    Science.gov (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

    2017-01-01

    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

  18. Characterization of high-level expression and sequencing of the Escherichia coli K-12 cynS gene encoding cyanase.

    OpenAIRE

    Sung, Y C; Anderson, P. M.; Fuchs, J A

    1987-01-01

    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...

  19. Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing

    Directory of Open Access Journals (Sweden)

    Dorothee Pflueger

    2013-11-01

    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.

  20. Geometric Feature-Based Facial Expression Recognition in Image Sequences Using Multi-Class AdaBoost and Support Vector Machines

    Directory of Open Access Journals (Sweden)

    Joonwhoan Lee

    2013-06-01

    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.

  1. 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

    1986-01-01

    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...

  2. 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...

  3. Laccase Gene Family in Cerrena sp. HYB07: Sequences, Heterologous Expression and Transcriptional Analysis

    Directory of Open Access Journals (Sweden)

    Jie Yang

    2016-08-01

    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.

  4. 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;

    2013-01-01

    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...

  5. Identification of expressed sequences in the coffee genome potentially associated with somatic embryogenesis.

    Science.gov (United States)

    Silva, A T; Paiva, L V; Andrade, A C; Barduche, D

    2013-05-21

    Brazil possesses the most modern and productive coffee growing farms in the world, but technological development is desired to cope with the increasing world demand. One way to increase Brazilian coffee growing productivity is wide scale production of clones with superior genotypes, which can be obtained with in vitro propagation technique, or from tissue culture. These procedures can generate thousands of clones. However, the methodologies for in vitro cultivation are genotype-dependent, which leads to an almost empirical development of specific protocols for each species. Therefore, molecular markers linked to the biochemical events of somatic embryogenesis would greatly facilitate the development of such protocols. In this context, sequences potentially involved in embryogenesis processes in the coffee plant were identified in silico from libraries generated by the Brazilian Coffee Genome Project. Through these in silico analyses, we identified 15 EST-contigs related to the embryogenesis process. Among these, 5 EST-contigs (3605, 9850, 13686, 17240, and 17265) could readily be associated with plant embryogenesis. Sequence analysis of EST-contig 3605, 9850, and 17265 revealed similarity to a polygalacturonase, to a cysteine-proteinase, and to an allergenine, respectively. Results also show that EST-contig 17265 sequences presented similarity to an expansin. Finally, analysis of EST-contig 17240 revealed similarity to a protein of unknown function, but it grouped in the similarity dendrogram with the WUSCHEL transcription factor. The data suggest that these EST-contigs are related to the embryogenic process and have potential as molecular markers to increase methodological efficiency in obtaining coffee plant embryogenic materials.

  6. 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

    2009-06-01

    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.

  7. 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

    1996-02-01

    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.

  8. 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

    2015-01-01

    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.

  9. Spider web glue: two proteins expressed from opposite strands of the same DNA sequence.

    Science.gov (United States)

    Choresh, Omer; Bayarmagnai, Battuya; Lewis, Randolph V

    2009-10-12

    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.

  10. 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

    2017-03-01

    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.

  11. Comparison of RNAi Sequences in Insect-Resistant Plants to Expressed Sequences of a Beneficial Lady Beetle: A Closer Look at Off-Target Considerations.

    Science.gov (United States)

    Allen, Margaret L

    2017-03-01

    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.

  12. High-frequency Oligonucleotides in Watermelon Expressed Sequenced Tag-unigenes Are Useful in Producing Polymorphic Polymerase Chain Reaction Markers among Watermelon Genotypes

    Science.gov (United States)

    In this study, we report a simple procedure for developing gene targeted primers, named high-frequency gene sequence (HFGS) primers, based on oligonucleotides that exist in high frequency in watermelon expressed sequence tag (EST)-unigenes. These HFGS primers were constructed by first screening for...

  13. Survey of glycine-rich proteins (GRPs in the Eucalyptus expressed sequence tag database (ForEST

    Directory of Open Access Journals (Sweden)

    Silvia Nora Bocca

    2005-01-01

    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.

  14. Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence.

    Science.gov (United States)

    Gromek, Katarzyna A; Meddaugh, Hannah R; Wrobel, Russell L; Suchy, Fabian P; Bingman, Craig A; Primm, John G; Fox, Brian G

    2013-06-01

    Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Expression

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    Wang-Xia Wang

    2014-02-01

    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.

  16. Identification of novel and differentially expressed MicroRNAs of dairy goat mammary gland tissues using solexa sequencing and bioinformatics.

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    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.

  17. Regulation of gene expression in Mycoplasmas: contribution from Mycoplasma hyopneumoniae and Mycoplasma synoviae genome sequences

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    Humberto Maciel França Madeira

    2007-01-01

    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.

  18. [Genomic DNA sequences and functional expression, purification of BmalphaTX14 neurotoxin from scorpion Buthus martensii Karsch].

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    Lü, Meng; Wang, Kun; Cao, Zhi-Jian; Jiang, Da-He; Mao, Xin; Li, Wen-Xin

    2005-11-01

    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.

  19. Comparative analysis of expressed sequence tags (ESTs) between drought-tolerant and -susceptible genotypes of chickpea under terminal drought stress

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    2011-01-01

    Background Chickpea (Cicer arietinum L.) is an important grain-legume crop that is mainly grown in rainfed areas, where terminal drought is a major constraint to its productivity. We generated expressed sequence tags (ESTs) by suppression subtraction hybridization (SSH) to identify differentially expressed genes in drought-tolerant and -susceptible genotypes in chickpea. Results EST libraries were generated by SSH from root and shoot tissues of IC4958 (drought tolerant) and ICC 1882 (drought resistant) exposed to terminal drought conditions by the dry down method. SSH libraries were also constructed by using 2 sets of bulks prepared from the RNA of root tissues from selected recombinant inbred lines (RILs) (10 each) for the extreme high and low root biomass phenotype. A total of 3062 unigenes (638 contigs and 2424 singletons), 51.4% of which were novel in chickpea, were derived by cluster assembly and sequence alignment of 5949 ESTs. Only 2185 (71%) unigenes showed significant BLASTX similarity (chickpea for the first time. This study not only serves as resource for marker discovery, but can provide a better insight into the selection of candidate genes (both up- and downregulated) associated with drought tolerance. These results can be used to identify suitable targets for manipulating the drought-tolerance trait in chickpea. PMID:21513527

  20. Molecular cloning, sequence identification and expression profile of domestic guinea pig (Cavia porcellus UGT1A1 gene

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    Yang Deming

    2016-01-01

    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.

  1. 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

    2011-02-01

    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.

  2. Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

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    Bendahmane Abdelhafid

    2011-05-01

    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

  3. Characterization of expressed sequence tags obtained by SSH during somatic embryogenesis in Cichorium intybus L

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    Quillet Marie-Christine

    2007-06-01

    Full Text Available Abstract Background Somatic embryogenesis (SE is an asexual propagation pathway requiring a somatic-to-embryonic transition of differentiated somatic cells toward embryogenic cells capable of producing embryos in a process resembling zygotic embryogenesis. In chicory, genetic variability with respect to the formation of somatic embryos was detected between plants from a population of Cichorium intybus L. landrace Koospol. Though all plants from this population were self incompatible, we managed by repeated selfing to obtain a few seeds from one highly embryogenic (E plant, K59. Among the plants grown from these seeds, one plant, C15, was found to be non-embryogenic (NE under our SE-inducing conditions. Being closely related, we decided to exploit the difference in SE capacity between K59 and its descendant C15 to study gene expression during the early stages of SE in chicory. Results Cytological analysis indicated that in K59 leaf explants the first cell divisions leading to SE were observed at day 4 of culture. In contrast, in C15 explants no cell divisions were observed and SE development seemed arrested before cell reactivation. Using mRNAs isolated from leaf explants from both genotypes after 4 days of culture under SE-inducing conditions, an E and a NE cDNA-library were generated by SSH. A total of 3,348 ESTs from both libraries turned out to represent a maximum of 2,077 genes. In silico subtraction analysis sorted only 33 genes as differentially expressed in the E or NE genotype, indicating that SSH had resulted in an effective normalisation. Real-time RT-PCR was used to verify the expression levels of 48 genes represented by ESTs from either library. The results showed preferential expression of genes related to protein synthesis and cell division in the E genotype, and related to defence in the NE genotype. Conclusion In accordance with the cytological observations, mRNA levels in explants from K59 and C15 collected at day 4 of SE

  4. 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

    2008-10-01

    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

  5. Expressed sequence tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers

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    Peterson Katherine

    2009-09-01

    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

  6. Molecular cloning, sequence identification and tissue expression profile of three novel sheep (Ovis aries) genes - BCKDHA, NAGA and HEXA.

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    Liu, G Y; Gao, S Z

    2009-01-01

    The complete coding sequences of three sheep genes- BCKDHA, NAGA and HEXA were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR), based on the conserved sequence information of the mouse or other mammals. The nucleotide sequences of these three genes revealed that the sheep BCKDHA gene encodes a protein of 313 amino acids which has high homology with the BCKDHA gene that encodes a protein of 447 amino acids that has high homology with the Branched chain keto acid dehydrogenase El, alpha polypeptide (BCKDHA) of five species chimpanzee (93%), human (96%), crab-eating macaque (93%), bovine (98%) and mouse (91%). The sheep NAGA gene encodes a protein of 411 amino acids that has high homology with the alpha-N-acetylgalactosaminidase (NAGA) of five species human (85%), bovine (94%), mouse (91%), rat (83%) and chicken (74%). The sheep HEXA gene encodes a protein of 529 amino acids that has high homology with the hexosaminidase A(HEXA) of five species bovine (98%), human (84%), Bornean orangután (84%), rat (80%) and mouse (81%). Finally these three novel sheep genes were assigned to GenelDs: 100145857, 100145858 and 100145856. The phylogenetic tree analysis revealed that the sheep BCKDHA, NAGA, and HEXA all have closer genetic relationships to the BCKDHA, NAGA, and HEXA of bovine. Tissue expression profile analysis was also carried out and results revealed that sheep BCKDHA, NAGA and HEXA genes were differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, small and large intestine. Our experiment is the first to establish the primary foundation for further research on these three sheep genes.

  7. Cloning, sequence identification and expression profile analysis of α-L-fucosidase gene from the Mediterranean fruit fly Ceratitis capitata.

    Science.gov (United States)

    Intra, Jari; Perotti, Maria-Elisa; Pasini, Maria Enrica

    2011-04-01

    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. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Transcriptome sequencing and profiling of expressed genes in phloem and xylem of ramie (Boehmeria nivea L. Gaud.

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    Jianrong Chen

    Full Text Available Ramie (Boehmeria nivea L. Gaud is a highly versatile herbaceous plant which is widely cropped in southern China. The success of this herbaceous plant relies on wide use in modern industry. Understanding the profiling of expressed genes in phloem and xylem of ramie is crucial for improving its industrial performance. Herein, we uncover the transcriptome profile in phloem and xylem in present study. Using Illumina paired-end sequencing technology, 57 million high quality reads were generated. De novo assembly yielded 87,144 unigenes with an average length of 635 bp. By sequence similarity searching for public databases, a total of 32,541 (41.77% unigenes were annotated for their function. Among these genes, 57,873 (66.4% and 28,678 (32.9% unigenes were assigned to categories of Gene Ontology and Orthologous Groups database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG, 18,331 (21.0% unigenes were mapped to 125 pathways. The metabolic pathways were assigned the most unigene (4,793, 26.2%. Furthermore, Pol II and Pol III subunits as well as the genes of Galactose metabolism pathway had higher expression in phloem compared to xylem. In addition, fatty acid metabolism pathway genes showed more abundant in xylem than phloem. These results suggest that high activities of RNA synthesis and Galactose metabolism pathway promises fiber synthesis in phloem. The present study is the initial exploration to uncover the fiber biosynthesis difference between phloem and xylem in ramie through the analysis of deep sequencing data.

  9. Dissection of two soybean QTL conferring partial resistance to Phytophthora sojae through sequence and gene expression analysis

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    Wang Hehe

    2012-08-01

    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 soybean QTL conferring resistance to P. sojae. SNP markers derived from these candidate genes can contribute to fine mapping of QTL and marker assisted breeding for

  10. Transcriptome sequencing and profiling of expressed genes in phloem and xylem of ramie (Boehmeria nivea L. Gaud).

    Science.gov (United States)

    Chen, Jianrong; Liu, Fang; Tang, Yinghong; Yuan, Youmei; Guo, Qingquan

    2014-01-01

    Ramie (Boehmeria nivea L. Gaud) is a highly versatile herbaceous plant which is widely cropped in southern China. The success of this herbaceous plant relies on wide use in modern industry. Understanding the profiling of expressed genes in phloem and xylem of ramie is crucial for improving its industrial performance. Herein, we uncover the transcriptome profile in phloem and xylem in present study. Using Illumina paired-end sequencing technology, 57 million high quality reads were generated. De novo assembly yielded 87,144 unigenes with an average length of 635 bp. By sequence similarity searching for public databases, a total of 32,541 (41.77%) unigenes were annotated for their function. Among these genes, 57,873 (66.4%) and 28,678 (32.9%) unigenes were assigned to categories of Gene Ontology and Orthologous Groups database, respectively. By searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG), 18,331 (21.0%) unigenes were mapped to 125 pathways. The metabolic pathways were assigned the most unigene (4,793, 26.2%). Furthermore, Pol II and Pol III subunits as well as the genes of Galactose metabolism pathway had higher expression in phloem compared to xylem. In addition, fatty acid metabolism pathway genes showed more abundant in xylem than phloem. These results suggest that high activities of RNA synthesis and Galactose metabolism pathway promises fiber synthesis in phloem. The present study is the initial exploration to uncover the fiber biosynthesis difference between phloem and xylem in ramie through the analysis of deep sequencing data.

  11. A spider tRNA(Ala) requires a far upstream sequence element for expression.

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    Cintrón, I; Capó, L; Plazaola, A; Arroyo, G; Candelas, G C

    1999-04-29

    Within the series of timed differential accumulations of small RNAs we have shown to prelude the synthesis of fibroin in the large ampullate glands of Nephila clavipes (Nc), we are currently directing our attention to the alanine tRNAs. This work reports the subcloning of the members of a tRNAAla gene cluster and the optimization of their transcription in a heterologous cell-free system derived from Bombyx mori (Bm) silkglands. Our data show that the heterologous cell-free system supports the faithful and differential transcription of the individual spider alanine tRNA genes. We are thus making use of the extract to characterize the individual genes with respect to flank-contained regulatory elements through cell-free transcription of gene derivatives. The work has been initiated with pNTA3 because of its high transcriptional activity. Interestingly, the transcription of this gene requires a far upstream sequence, an uncommon modality in tRNA genes.

  12. Characterization of high-level expression and sequencing of the Escherichia coli K-12 cynS gene encoding cyanase.

    Science.gov (United States)

    Sung, Y C; Anderson, P M; Fuchs, J A

    1987-11-01

    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 DNA fragment of pSJ105 was transferred into pUC18 derivatives that contain a hybrid tac promoter, instead of the lac promoter, and a strong terminator to generate pSJ124. Stationary-phase cultures of JM101 containing plasmid pSJ124 overexpressed a similar level of cyanase. In JM101(pSJ124), maximum production of cyanase could be obtained either by induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 h or by growth without IPTG into late stationary phase. The latter conditions resulted in a 10- to 20-fold increase in plasmid content and presumably titration of the lac repressor. The nucleotide sequence of the cloned cynS gene from Escherichia coli K-12 was determined. The predicted amino acid sequence differed from the known amino acid sequence of cyanase isolated from a B strain by four residues. However, overexpressed cyanase was purified to homogeneity, and a comparison of the enzymes from the two sources indicated that they did not differ with respect to physical and kinetic properties. The cynS gene was located next to the lac operon, and the direction of cynS transcription was opposite that of lac.

  13. Generation of a Transcriptome in a Model Lepidopteran Pest, Heliothis virescens, Using Multiple Sequencing Strategies for Profiling Midgut Gene Expression.

    Science.gov (United States)

    Perera, Omaththage P; Shelby, Kent S; Popham, Holly J R; Gould, Fred; Adang, Michael J; Jurat-Fuentes, Juan Luis

    2015-01-01

    Heliothine pests such as the tobacco budworm, Heliothis virescens (F.), pose a significant threat to production of a variety of crops and ornamental plants and are models for developmental and physiological studies. The efforts to develop new control measures for H. virescens, as well as its use as a relevant biological model, are hampered by a lack of molecular resources. The present work demonstrates the utility of next-generation sequencing technologies for rapid molecular resource generation from this species for which lacks a sequenced genome. In order to amass a de novo transcriptome for this moth, transcript sequences generated from Illumina, Roche 454, and Sanger sequencing platforms were merged into a single de novo transcriptome assembly. This pooling strategy allowed a thorough sampling of transcripts produced under diverse environmental conditions, developmental stages, tissues, and infections with entomopathogens used for biological control, to provide the most complete transcriptome to date for this species. Over 138 million reads from the three platforms were assembled into the final set of 63,648 contigs. Of these, 29,978 had significant BLAST scores indicating orthologous relationships to transcripts of other insect species, with the top-hit species being the monarch butterfly (Danaus plexippus) and silkworm (Bombyx mori). Among identified H. virescens orthologs were immune effectors, signal transduction pathways, olfactory receptors, hormone biosynthetic pathways, peptide hormones and their receptors, digestive enzymes, and insecticide resistance enzymes. As an example, we demonstrate the utility of this transcriptomic resource to study gene expression profiling of larval midguts and detect transcripts of putative Bacillus thuringiensis (Bt) Cry toxin receptors. The substantial molecular resources described in this study will facilitate development of H. virescens as a relevant biological model for functional genomics and for new biological

  14. O-demethylase from Acetobacterium dehalogenans--cloning, sequencing, and active expression of the gene encoding the corrinoid protein.

    Science.gov (United States)

    Kaufmann, F; Wohlfarth, G; Diekert, G

    1998-10-15

    The ether-cleaving O-demethylase from the strictly anaerobic homoacetogen Acetobacterium dehalogenans catalyses the methyltransfer from 4-hydroxy-3-methoxy-benzoate (vanillate) to tetrahydrofolate. In the first step a vanillate :corrinoid protein methyltransferase (methyltransferase I) mediates the methylation of a 25-kDa corrinoid protein with the cofactor reduced to cob(I)alamin. The methyl group is then transferred to tetrahydrofolate by the action of a methylcorrinoid protein:tetrahydrofolate methyltransferase (methyltransferase II). Using primers derived from the amino-terminal sequences of the corrinoid protein and the vanillate:corrinoid protein methyltransferase (methyltransferase I), a 723-bp fragment was amplified by PCR, which contained the gene odmA encoding the corrinoid protein of O-demethylase. Downstream of odmA, part of the odmB gene encoding methyltransferase I was identified. The amino acid sequence deduced from odmA showed about 60% similarity to the cobalamin-binding domain of methionine synthase from Escherichia coli (MetH) and to corrinoid proteins of methyltransferase systems involved in methanogenesis from methanol and methylamines. The sequence contained the DXHXXG consensus sequence typical for displacement of the dimethylbenzimidazole base of the corrinoid cofactor by a histidine from the protein. Heterologous expression of odmA in E. coli yielded a colourless, oxygen-insensitive apoprotein, which was able to bind one mol cobalamin or methylcobalamin/mol protein. Both of these reconstituted forms of the protein were active in the overall O-demethylation reaction. OdmA reconstituted with hydroxocobalamin and reduced by titanium(III) citrate to the cob(I)alamin form was methylated with vanillate by methyltransferase I in an irreversible reaction. Methylcobalamin carrying OdmA served as methyl group donor for the methylation of tetrahydrofolate by methyltransferase II. This reaction was found to be reversible, since methyltranSferase II

  15. Generation of a Transcriptome in a Model Lepidopteran Pest, Heliothis virescens, Using Multiple Sequencing Strategies for Profiling Midgut Gene Expression.

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    Omaththage P Perera

    Full Text Available Heliothine pests such as the tobacco budworm, Heliothis virescens (F., pose a significant threat to production of a variety of crops and ornamental plants and are models for developmental and physiological studies. The efforts to develop new control measures for H. virescens, as well as its use as a relevant biological model, are hampered by a lack of molecular resources. The present work demonstrates the utility of next-generation sequencing technologies for rapid molecular resource generation from this species for which lacks a sequenced genome. In order to amass a de novo transcriptome for this moth, transcript sequences generated from Illumina, Roche 454, and Sanger sequencing platforms were merged into a single de novo transcriptome assembly. This pooling strategy allowed a thorough sampling of transcripts produced under diverse environmental conditions, developmental stages, tissues, and infections with entomopathogens used for biological control, to provide the most complete transcriptome to date for this species. Over 138 million reads from the three platforms were assembled into the final set of 63,648 contigs. Of these, 29,978 had significant BLAST scores indicating orthologous relationships to transcripts of other insect species, with the top-hit species being the monarch butterfly (Danaus plexippus and silkworm (Bombyx mori. Among identified H. virescens orthologs were immune effectors, signal transduction pathways, olfactory receptors, hormone biosynthetic pathways, peptide hormones and their receptors, digestive enzymes, and insecticide resistance enzymes. As an example, we demonstrate the utility of this transcriptomic resource to study gene expression profiling of larval midguts and detect transcripts of putative Bacillus thuringiensis (Bt Cry toxin receptors. The substantial molecular resources described in this study will facilitate development of H. virescens as a relevant biological model for functional genomics and for new

  16. Sequence analysis and expression pattern of MGTA1 gene in rice blast pathogen Magnaporthe grisea

    Institute of Scientific and Technical Information of China (English)

    WANG Jiao-yu; LIU Xiao-hong; LU Jian-ping; LIN Fu-cheng

    2005-01-01

    MGTA1, a putative fungal Zn(Ⅱ)2Cys6 transcriptional activator-encoding gene, was isolated from rice blast pathogen Magnaporthe grisea, which is homologous to CLTA1 from Colletotrichum lindemuthianum with 51% identity at protein level.MGTA1 cassette contains a 2370 bp open reading frame, consisting of 6 exons, coding a 790 amino acid peptide. MGTA1 gene exists as a single copy in genomes of 7 strains of M. grisea, and is expressed in tip hyphae, conidia, and mature appressoria of strain Guy 11.

  17. Sequence analysis and expression pattern of MGTA1 gene in rice blast pathogen Magnaporthe grisea *

    Science.gov (United States)

    Wang, Jiao-yu; Liu, Xiao-hong; Lu, Jian-ping; Lin, Fu-cheng

    2005-01-01

    MGTA1, a putative fungal Zn(II)2Cys6 transcriptional activator-encoding gene, was isolated from rice blast pathogen Magnaporthe grisea, which is homologous to CLTA1 from Colletotrichum lindemuthianum with 51% identity at protein level. MGTA1 cassette contains a 2370 bp open reading frame, consisting of 6 exons, coding a 790 amino acid peptide. MGTA1 gene exists as a single copy in genomes of 7 strains of M. grisea, and is expressed in tip hyphae, conidia, and mature appressoria of strain Guy11. PMID:16052717

  18. Sequence and Spatiotemporal Expression Analysis of CLE-Motif Containing Genes from the Reniform Nematode (Rotylenchulus reniformis Linford & Oliveira).

    Science.gov (United States)

    Wubben, Martin J; Gavilano, Lily; Baum, Thomas J; Davis, Eric L

    2015-06-01

    The reniform nematode, Rotylenchulus reniformis, is a sedentary semi-endoparasitic species with a host range that encompasses more than 77 plant families. Nematode effector proteins containing plant-ligand motifs similar to CLAVATA3/ESR (CLE) peptides have been identified in the Heterodera, Globodera, and Meloidogyne genera of sedentary endoparasites. Here, we describe the isolation, sequence analysis, and spatiotemporal expression of three R. reniformis genes encoding putative CLE motifs named Rr-cle-1, Rr-cle-2, and Rr-cle-3. The Rr-cle cDNAs showed >98% identity with each other and the predicted peptides were identical with the exception of a short stretch of residues at the carboxy(C)-terminus of the variable domain (VD). Each RrCLE peptide possessed an amino-terminal signal peptide for secretion and a single C-terminal CLE motif that was most similar to Heterodera CLE motifs. Aligning the Rr-cle cDNAs with their corresponding genomic sequences showed three exons with an intron separating the signal peptide from the VD and a second intron separating the VD from the CLE motif. An alignment of the RrCLE1 peptide with Heterodera glycines and Heterodera schachtii CLE proteins revealed a high level of homology within the VD region associated with regulating in planta trafficking of the processed CLE peptide. Quantitative RT-PCR (qRT-PCR) showed similar expression profiles for each Rr-cle transcript across the R. reniformis life-cycle with the greatest transcript abundance being in sedentary parasitic female nematodes. In situ hybridization showed specific Rr-cle expression within the dorsal esophageal gland cell of sedentary parasitic females.

  19. RNA deep sequencing reveals differential microRNA expression during development of sea urchin and sea star.

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    Sabah Kadri

    Full Text Available microRNAs (miRNAs are small (20-23 nt, non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin and Patiria miniata (sea star are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc. to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads. Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common. We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html.

  20. Expression differentiation of CYC-like floral symmetry genes correlated with their protein sequence divergence in Chirita heterotricha (Gesneriaceae).

    Science.gov (United States)

    Gao, Qiu; Tao, Ju-Hong; Yan, Dan; Wang, Yin-Zheng; Li, Zhen-Yu

    2008-07-01

    CYCLOIDIEA (CYC) and its homologues have been studied intensively in the model organism Antirrhinum majus and related species regarding their function in controlling floral dorsoventral (adaxial-abaxial) asymmetry, including aborting the adaxial and lateral stamens. This raises the question whether the same mechanism underlies the great morphological diversity of zygomorphy in angiosperms, especially in Lamiales sensu lato, a major clade predominantly with zygomorphic flowers. To address this, we selected a representative in Gesneriaceae, the sister to the remainder of Lamiales s.l., to isolate CYC homologues and further investigate their expression patterns using locus-specific semiquantitative reverse transcriptase polymerase chain reaction. Our results showed that four CYC homologues in Chirita heterotricha differentiated spatially and temporally in expression, in which ChCYC1D was only expressed in the adaxial regions, and transcripts of ChCYC1C were distributed in both the adaxial and lateral regions, while ChCYC2A and ChCYC2B transcripts were only detected in the young inflorescences. ChCYC1C expression in the lateral regions correlated with abortion of the lateral stamens in C. heterotricha hinted at its gain of function, i.e., expanding from the adaxial to the lateral regions in expression. Correlatively, the protein sequences of ChCYC genes exhibited remarkable divergences, in which some lineage-specific amino acids between GCYC1 and GCYC2 in conserved functional domains and two sublineage-specific motifs between GCYC1C and GCYC1D in GCYC1 genes had further been identified. Our results indicated that ChCYC genes had probably undergone an expressional differentiation and specialization in establishing the floral dorsoventral asymmetry in C. heterotricha responding to different selective pressure after gene duplication.

  1. Mathematical modeling of translation initiation for the estimation of its efficiency to computationally design mRNA sequences with desired expression levels in prokaryotes

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    Lee Sunjae

    2010-05-01

    Full Text Available Abstract Background Within the emerging field of synthetic biology, engineering paradigms have recently been used to design biological systems with novel functionalities. One of the essential challenges hampering the construction of such systems is the need to precisely optimize protein expression levels for robust operation. However, it is difficult to design mRNA sequences for expression at targeted protein levels, since even a few nucleotide modifications around the start codon may alter translational efficiency and dramatically (up to 250-fold change protein expression. Previous studies have used ad hoc approaches (e.g., random mutagenesis to obtain the desired translational efficiencies for mRNA sequences. Hence, the development of a mathematical methodology capable of estimating translational efficiency would greatly facilitate the future design of mRNA sequences aimed at yielding desired protein expression levels. Results We herein propose a mathematical model that focuses on translation initiation, which is the rate-limiting step in translation. The model uses mRNA-folding dynamics and ribosome-binding dynamics to estimate translational efficiencies solely from mRNA sequence information. We confirmed the feasibility of our model using previously reported expression data on the MS2 coat protein. For further confirmation, we used our model to design 22 luxR mRNA sequences predicted to have diverse translation efficiencies ranging from 10-5 to 1. The expression levels of these sequences were measured in Escherichia coli and found to be highly correlated (R2 = 0.87 with their estimated translational efficiencies. Moreover, we used our computational method to successfully transform a low-expressing DsRed2 mRNA sequence into a high-expressing mRNA sequence by maximizing its translational efficiency through the modification of only eight nucleotides upstream of the start codon. Conclusions We herein describe a mathematical model that uses m

  2. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    Science.gov (United States)

    Irigoyen, Nerea; Firth, Andrew E; Jones, Joshua D; Chung, Betty Y-W; Siddell, Stuart G; Brierley, Ian

    2016-02-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  3. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

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    Nerea Irigoyen

    2016-02-01

    Full Text Available Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV and Middle East respiratory syndrome-related coronavirus (MERS-CoV, are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59, a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the

  4. Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags

    DEFF Research Database (Denmark)

    Gododkin, Jan; Cirera, Susanna; Hedegaard, Jakob

    2007-01-01

    Background: Knowledge of the structure of gene expression is essential for mammalian transcriptomics research. We analyzed a collection of more than one million porcine expressed sequence tags (ESTs), of which two-thirds were generated in the Sino-Danish Pig Genome Project and one-third are from ...

  5. Sequencing, characterization, and gene expression analysis of the histidine decarboxylase gene cluster of Morganella morganii.

    Science.gov (United States)

    Ferrario, Chiara; Borgo, Francesca; de Las Rivas, Blanca; Muñoz, Rosario; Ricci, Giovanni; Fortina, Maria Grazia

    2014-03-01

    The histidine decarboxylase gene cluster of Morganella morganii DSM30146(T) was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 °C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods.

  6. Integrative analyses of RNA editing, alternative splicing, and expression of young genes in human brain transcriptome by deep RNA sequencing.

    Science.gov (United States)

    Wu, Dong-Dong; Ye, Ling-Qun; Li, Yan; Sun, Yan-Bo; Shao, Yi; Chen, Chunyan; Zhu, Zhu; Zhong, Li; Wang, Lu; Irwin, David M; Zhang, Yong E; Zhang, Ya-Ping

    2015-08-01

    Next-generation RNA sequencing has been successfully used for identification of transcript assembly, evaluation of gene expression levels, and detection of post-transcriptional modifications. Despite these large-scale studies, additional comprehensive RNA-seq data from different subregions of the human brain are required to fully evaluate the evolutionary patterns experienced by the human brain transcriptome. Here, we provide a total of 6.5 billion RNA-seq reads from different subregions of the human brain. A significant correlation was observed between the levels of alternative splicing and RNA editing, which might be explained by a competition between the molecular machineries responsible for the splicing and editing of RNA. Young human protein-coding genes demonstrate biased expression to the neocortical and non-neocortical regions during evolution on the lineage leading to humans. We also found that a significantly greater number of young human protein-coding genes are expressed in the putamen, a tissue that was also observed to have the highest level of RNA-editing activity. The putamen, which previously received little attention, plays an important role in cognitive ability, and our data suggest a potential contribution of the putamen to human evolution. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  7. Cathepsin B from the white shrimp Litopenaeus vannamei: cDNA sequence analysis, tissues-specific expression and biological activity.

    Science.gov (United States)

    Stephens, A; Rojo, L; Araujo-Bernal, S; Garcia-Carreño, F; Muhlia-Almazan, A

    2012-01-01

    Cathepsin B is a cystein proteinase scarcely studied in crustaceans. Its function has not been clearly described in shrimp species belonging to the sub-order Dendrobranchiata, which includes the white shrimp Litopenaeus vannamei and other species from the Penaeidae family. Studies on vertebrates suggest that these lysosomal enzymes intracellularly hydrolize protein, as other cystein proteinases. However, the expression of the gene encoding the shrimp cathepsin B in the midgut gland was affected by starvation in a similar way as other digestive proteinases which extracellularly hydrolyze food protein. In this study the white shrimp L. vannamei cathepsin B (LvCathB) cDNA was sequenced, and characterized. Its gene expression was evaluated in various shrimp tissues, and changes in the mRNA amounts were compared with those observed on other digestive proteinases from the midgut gland during starvation. By using qRT-PCR it was found that LvCathB is expressed in most shrimp tissues except in pleopods and eye stalk. Changes on LvCathB mRNA during starvation suggest that the enzyme participates during intracellular protein hydrolysis but also, after food ingestion, it participates in hydrolyzing food proteins extracellularly as confirmed by the high activity levels we found in the gastric juice and midgut gland of the white shrimp.

  8. RNA-Sequencing Analysis of 5' Capped RNAs Identifies Many New Differentially Expressed Genes in Acute Hepatitis C Virus Infection

    Directory of Open Access Journals (Sweden)

    Bret S. E. Heale

    2012-04-01

    Full Text Available We describe the first report of RNA sequencing of 5' capped (Pol II RNAs isolated from acutely hepatitis C virus (HCV infected Huh 7.5 cells that provides a general approach to identifying differentially expressed annotated and unannotated genes that participate in viral-host interactions. We identified 100, 684, and 1,844 significantly differentially expressed annotated genes in acutely infected proliferative Huh 7.5 cells at 6, 48, and 72 hours, respectively (fold change ≥ 1.5 and Bonferroni adjusted p-values < 0.05. Most of the differentially expressed genes (>80% and biological pathways (such as adipocytokine, Notch, Hedgehog and NOD-like receptor signaling were not identified by previous gene array studies. These genes are critical components of host immune, inflammatory and oncogenic pathways and provide new information regarding changes that may benefit the virus or mediate HCV induced pathology. RNAi knockdown studies of newly identified highly upregulated FUT1 and KLHDC7B genes provide evidence that their gene products regulate and facilitate HCV replication in hepatocytes. Our approach also identified novel Pol II unannotated transcripts that were upregulated. Results further identify new pathways that regulate HCV replication in hepatocytes and suggest that our approach will have general applications in studying viral-host interactions in model systems and clinical biospecimens.

  9. A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia.

    Science.gov (United States)

    Yang, Jiang-Ke; Chen, Fang-Yuan; Yan, Xiang-Xiang; Miao, Li-Hong; Dai, Jiang-Hong

    2012-01-01

    In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE) was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp) and Aspergillus niger phytase gene phyA (1404 bp). Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.

  10. A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia.

    Directory of Open Access Journals (Sweden)

    Jiang-Ke Yang

    Full Text Available In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp and Aspergillus niger phytase gene phyA (1404 bp. Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.

  11. Sequencing and expression analysis of CD3γ/δ and CD3εchains in mandarin fish, Siniperca chuatsi

    Institute of Scientific and Technical Information of China (English)

    GUO Zheng; NIE Pin

    2013-01-01

    The genomic and cDNA sequences of the CD3γ/δ and CD3ε homologues in the mandarin fish,Siniperca chuatsi,were determined.As in other vertebrate CD3 molecules,the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ε contained conserved residues and motifs,such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs.However,mandarin fish CD3γ/δ and CD3ε showed some differences to their mammalian counterparts,specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ.Additionally,while an N-glycosylation site was present in CD3ε,the site was not observed in CD3γ/δ.The CD3γ/δ and CD3ε subunit sequences contain six and five exons,respectively,consistent with homologues from Atlantic salmon,Salmo salar.Phylogenetic analysis also revealed that CD3γ/δ and CD3ε in mandarin fish are closely related to their counterparts in Acanthopterygian fish.Real-time PCR showed CD3γ/δ and CD3ε were expressed mainly in the thymus and spleen in normal healthy fish and,to a lesser extent,in mucosal-associated lymphoid tissues,such as the intestine and gills.When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin,concanavalin,and polyriboinosinic polyribocytidylic acid,mRNA expression levels of CD3γ/δ and CD3ε were significantly elevated within 12 h of treatment.This indicated the presence of T lymphocytes in the head kidney of teleost fish,and also the recognition of mitogens by the lymphocytes.Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ε mRNA,indicating that CD3γ/δ and CD3ε lymphocytes are involved in the immune response of this species.

  12. Sequencing and expression analysis of CD3γ/δ and CD3ɛ chains in mandarin fish, Siniperca chuatsi

    Science.gov (United States)

    Guo, Zheng; Nie, Pin

    2013-01-01

    The genomic and cDNA sequences of the CD3γ/δ and CD3ɛ homologues in the mandarin fish, Siniperca chuats i, were determined. As in other vertebrate CD3 molecules, the deduced amino acid sequences of mandarin fish CD3γ/δ and CD3ɛ contained conserved residues and motifs, such as cysteine residues and CXXC and immunoreceptor tyrosine-based activation motifs. However, mandarin fish CD3γ/δ and CD3ɛ showed some differences to their mammalian counterparts, specifically the absence of a negatively charged residue in the transmembrane region of CD3γ/δ. Additionally, while an N -glycosylation site was present in CD3ɛ, the site was not observed in CD3γ/δ. The CD3γ/δ and CD3ɛ subunit sequences contain six and five exons, respectively, consistent with homologues from Atlantic salmon, Salmo salar. Phylogenetic analysis also revealed that CD3γ/δ and CD3ɛ in mandarin fish are closely related to their counterparts in Acanthopterygian fish. Real-time PCR showed CD3γ/δ and CD3ɛ were expressed mainly in the thymus and spleen in normal healthy fish and, to a lesser extent, in mucosal-associated lymphoid tissues, such as the intestine and gills. When lymphocytes isolated from head kidney were treated with the mitogens phytohemagglutinin, concanavalin, and polyriboinosinic polyribocytidylic acid, mRNA expression levels of CD3γ/δ and CD3ɛ were significantly elevated within 12 h of treatment. This indicated the presence of T lymphocytes in the head kidney of teleost fish, and also the recognition of mitogens by the lymphocytes. Mandarin fish infected with the bacterial pathogen Flavobacterium columnare also showed an increase in the expression of CD3γ/δ and CD3ɛ mRNA, indicating that CD3γ/δ and CD3ɛ lymphocytes are involved in the immune response of this species.

  13. Analysis and functional annotation of expressed sequence tags (ESTs from multiple tissues of oil palm (Elaeis guineensis Jacq.

    Directory of Open Access Journals (Sweden)

    Lee Weng-Wah

    2007-10-01

    Full Text Available Abstract Background Oil palm is the second largest source of edible oil which contributes to approximately 20% of the world's production of oils and fats. In order to understand the molecular biology involved in in vitro propagation, flowering, efficient utilization of nitrogen sources and root diseases, we have initiated an expressed sequence tag (EST analysis on oil palm. Results In this study, six cDNA libraries from oil palm zygotic embryos, suspension cells, shoot apical meristems, young flowers, mature flowers and roots, were constructed. We have generated a total of 14537 expressed sequence tags (ESTs from these libraries, from which 6464 tentative unique contigs (TUCs and 2129 singletons were obtained. Approximately 6008 of these tentative unique genes (TUGs have significant matches to the non-redundant protein database, from which 2361 were assigned to one or more Gene Ontology categories. Predominant transcripts and differentially expressed genes were identified in multiple oil palm tissues. Homologues of genes involved in many aspects of flower development were also identified among the EST collection, such as CONSTANS-like, AGAMOUS-like (AGL2, AGL20, LFY-like, SQUAMOSA, SQUAMOSA binding protein (SBP etc. Majority of them are the first representatives in oil palm, providing opportunities to explore the cause of epigenetic homeotic flowering abnormality in oil palm, given the importance of flowering in fruit production. The transcript levels of two flowering-related genes, EgSBP and EgSEP were analysed in the flower tissues of various developmental stages. Gene homologues for enzymes involved in oil biosynthesis, utilization of nitrogen sources, and scavenging of oxygen radicals, were also uncovered among the oil palm ESTs. Conclusion The EST sequences generated will allow comparative genomic studies between oil palm and other monocotyledonous and dicotyledonous plants, development of gene-targeted markers for the reference genetic map

  14. Expressed sequence tags (ESTs) based identification of genes and expression analysis of leukocyte cell-derived chemotaxin-2 (LECT2) from Epinephelus bruneus.

    Science.gov (United States)

    Harikrishnan, Ramasamy; Kim, Ju-Sang; Kim, Man-Chul; Balasundaram, Chellam; Heo, Moon-Soo

    2012-01-01

    The kelp grouper, Epinephelus bruneus, is an economically important intensively cultured species in Southeast Asia. Despite the insatiable demand its large-scale production has been hindered by problems associated with water quality, nutrition, and diseases especially due to increased rearing density. It is generally accepted that in fish both innate and adaptive immune system provide protection from diseases. In the present study a cDNA library of Streptococcus iniae-challenged kelp grouper was constructed to identify the genes that reveal molecular mechanism, physiological functions, and gene expression in different tissues using expressed sequence tags (ESTs) and RT-PCR strategy. Of a total of 2170 ESTs examined 279 (12.9%) were identified as contig and 860 (39.6%) as singletons. A total of 190 important immune and enzyme related genes (16.7%) were identified in both contig and singletons. The key immune molecules identified comprise complement factors, chemotaxin, chemokine, Fas ligand, ferritins, hepcidin, lysozyme c, MHC, and TLR which are involved in the innate or adaptive immune system. Among the genes a full-length cDNA of leukocyte cell-derived chemotaxin-2 (EbLECT2) with 540 base pair (bp) was identified; it consists of a 5'-untranslated region (UTR) of 17 bp, a 3'-UTR of 76 bp, and a stop codon TAA in 3'-UTR. The EbLECT2 is an important molecule in the innate immunity. It is a multifunctional protein involved in cell growth, differentiation, and autoimmunity. The open reading frame (ORF) of the EbLECT2 encodes with 155 amino acid (aa) residues with a predicted molecular weight and isoelectric point (pI) of 17 kDa and 9, respectively. The close phylogenetic relationship of EbLECT2 shares the highest similarity with the already reported LECT2 from Epinephelus coioides (96%) and Epinephelus akaara (94%). EbLECT2 mRNA was expressed predominantly in liver, spleen, and kidney while the expression was moderate in gills, heart, and muscle in E. bruneus after

  15. Genome Sequencing

    DEFF Research Database (Denmark)

    Sato, Shusei; Andersen, Stig Uggerhøj

    2014-01-01

    The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based on transcr......The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based...

  16. Sequence Conservation and Sexually Dimorphic Expression of the Ftz-F1 Gene in the Crustacean Daphnia magna.

    Directory of Open Access Journals (Sweden)

    Nur Syafiqah Mohamad Ishak

    Full Text Available Identifying the genes required for environmental sex determination is important for understanding the evolution of diverse sex determination mechanisms in animals. Orthologs of Drosophila orphan receptor Fushi tarazu factor-1 (Ftz-F1 are known to function in genetic sex determination. In contrast, their roles in environmental sex determination remain unknown. In this study, we have cloned and characterized the Ftz-F1 ortholog in the branchiopod crustacean Daphnia magna, which produces males in response to environmental stimuli. Similar to that observed in Drosophila, D. magna Ftz-F1 (DapmaFtz-F1 produces two splicing variants, αFtz-F1 and βFtz-F1, which encode 699 and 777 amino acids, respectively. Both isoforms share a DNA-binding domain, a ligand-binding domain, and an AF-2 activation domain and differ only at the A/B domain. The phylogenetic position and genomic structure of DapmaFtz-F1 suggested that this gene has diverged from an ancestral gene common to branchiopod crustacean and insect Ftz-F1 genes. qRT-PCR showed that at the one cell and gastrulation stages, both DapmaFtz-F1 isoforms are two-fold more abundant in males than in females. In addition, in later stages, their sexual dimorphic expressions were maintained in spite of reduced expression. Time-lapse imaging of DapmaFtz-F1 RNAi embryos was performed in H2B-GFP expressing transgenic Daphnia, demonstrating that development of the RNAi embryos slowed down after the gastrulation stage and stopped at 30-48 h after ovulation. DapmaFtz-F1 shows high homology to insect Ftz-F1 orthologs based on its amino acid sequence and exon-intron organization. The sexually dimorphic expression of DapmaFtz-F1 suggests that it plays a role in environmental sex determination of D. magna.

  17. Sequence Conservation and Sexually Dimorphic Expression of the Ftz-F1 Gene in the Crustacean Daphnia magna.

    Science.gov (United States)

    Mohamad Ishak, Nur Syafiqah; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2016-01-01

    Identifying the genes required for environmental sex determination is important for understanding the evolution of diverse sex determination mechanisms in animals. Orthologs of Drosophila orphan receptor Fushi tarazu factor-1 (Ftz-F1) are known to function in genetic sex determination. In contrast, their roles in environmental sex determination remain unknown. In this study, we have cloned and characterized the Ftz-F1 ortholog in the branchiopod crustacean Daphnia magna, which produces males in response to environmental stimuli. Similar to that observed in Drosophila, D. magna Ftz-F1 (DapmaFtz-F1) produces two splicing variants, αFtz-F1 and βFtz-F1, which encode 699 and 777 amino acids, respectively. Both isoforms share a DNA-binding domain, a ligand-binding domain, and an AF-2 activation domain and differ only at the A/B domain. The phylogenetic position and genomic structure of DapmaFtz-F1 suggested that this gene has diverged from an ancestral gene common to branchiopod crustacean and insect Ftz-F1 genes. qRT-PCR showed that at the one cell and gastrulation stages, both DapmaFtz-F1 isoforms are two-fold more abundant in males than in females. In addition, in later stages, their sexual dimorphic expressions were maintained in spite of reduced expression. Time-lapse imaging of DapmaFtz-F1 RNAi embryos was performed in H2B-GFP expressing transgenic Daphnia, demonstrating that development of the RNAi embryos slowed down after the gastrulation stage and stopped at 30-48 h after ovulation. DapmaFtz-F1 shows high homology to insect Ftz-F1 orthologs based on its amino acid sequence and exon-intron organization. The sexually dimorphic expression of DapmaFtz-F1 suggests that it plays a role in environmental sex determination of D. magna.

  18. Seasonal variation of urinary microRNA expression in male goats (Capra hircus) as assessed by next generation sequencing.

    Science.gov (United States)

    Longpre, Kristy M; Kinstlinger, Noah S; Mead, Edward A; Wang, Yongping; Thekkumthala, Austin P; Carreno, Katherine A; Hot, Azra; Keefer, Jennifer M; Tully, Luke; Katz, Larry S; Pietrzykowski, Andrzej Z

    2014-04-01

    Testosterone plays a key role in preparation of a male domesticated goat (Capra hircus) to breeding season including changes in the urogenital tract of a male goat (buck). microRNAs are important regulators of cellular metabolism, differentiation and function. They are powerful intermediaries of hormonal activity in the body, including the urogenital tract. We investigated seasonal changes in expression of microRNAs in goat buck urine and their potential consequences using next generation sequencing (microRNA-Seq). We determined the location of each microRNA gene in the goat genome. Testosterone was measured by radioimmunoassay and the androgen receptor binding sites (ARBS) in the promoters of the microRNA genes were determined by MatInspector. The overall impact of regulated microRNAs on cellular physiology was assessed by mirPath. We observed high testosterone levels during the breeding season and changes in the expression of forty microRNAs. Nineteen microRNAs were upregulated, while twenty-one were downregulated. We identified several ARBS in the promoters of regulated microRNAs. Notably, the mostly inhibited microRNA, miR-1246, has a unique set of several gene copy variants associated with a cluster of androgen receptor binding sites. Concomitant changes in regulated microRNA expression could promote transcription, proliferation and differentiation of urogenital tract cells. Together, these findings indicate that in a domesticated goat (Capra hircus), there are specific changes in the microRNA expression profile in buck urine during breeding season, which could be attributable to high testosterone levels during breeding, and could help in preparation of the urogenital tract for high metabolic demands of that season.

  19. A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing

    Science.gov (United States)

    Bermudez, Jaclyn Y.; Webber, Hannah C.; Brown, Bartley; Braun, Terry A.; Clark, Abbot F.; Mao, Weiming

    2017-01-01

    The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student’s t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs. PMID:28068412

  20. Avian resistance to Campylobacter jejuni colonization is associated with an intestinal immunogene expression signature identified by mRNA sequencing.

    Directory of Open Access Journals (Sweden)

    Sarah Connell

    Full Text Available Campylobacter jejuni is the most common cause of human bacterial gastroenteritis and is associated with several post-infectious manifestations, including onset of the autoimmune neuropathy Guillain-Barré syndrome, causing significant morbidity and mortality. Poorly-cooked chicken meat is the most frequent source of infection as C. jejuni colonizes the avian intestine in a commensal relationship. However, not all chickens are equally colonized and resistance seems to be genetically determined. We hypothesize that differences in immune response may contribute to variation in colonization levels between susceptible and resistant birds. Using high-throughput sequencing in an avian infection model, we investigate gene expression associated with resistance or susceptibility to colonization of the gastrointestinal tract with C. jejuni and find that gut related immune mechanisms are critical for regulating colonization. Amongst a single population of 300 4-week old chickens, there was clear segregation in levels of C. jejuni colonization 48 hours post-exposure. RNAseq analysis of caecal tissue from 14 C. jejuni-susceptible and 14 C. jejuni-resistant birds generated over 363 million short mRNA sequences which were investigated to identify 219 differentially expressed genes. Significantly higher expression of genes involved in the innate immune response, cytokine signaling, B cell and T cell activation and immunoglobulin production, as well as the renin-angiotensin system was observed in resistant birds, suggesting an early active immune response to C. jejuni. Lower expression of these genes in colonized birds suggests suppression or inhibition of a clearing immune response thus facilitating commensal colonization and generating vectors for zoonotic transmission. This study describes biological processes regulating C. jejuni colonization of the avian intestine and gives insight into the differential immune mechanisms incited in response to commensal

  1. Differential display-mediated identification of three drought-responsive expressed sequence tags in tea [Camellia sinensis (L.) O. Kuntze

    Indian Academy of Sciences (India)

    Priti Sharma; Sanjay Kumar

    2005-03-01

    There is no information on drought-modulated gene(s) in tea [Camellia sinensis (L.) O. Kuntze], a woody and perennial plant of commercial importance. Using differential display of mRNA, three drought-modulated expressed sequence tags (ESTs) were identified. Northern and BLAST analysis revealed that clone dr1 (droughtresponsive), induced only by drought but not by ABA, showed significant scores with PR-5 (pathogenesis related) family of PR-protein gene. Another clone dr2, repressed by drought but not by ABA, had nucleotide repeats for polyasparate that are also present in chicken calsequestrin-like mRNA. Clone dr3, responded similarly to clone dr2 but did not show significant homology with the reported genes, hence appears to be novel. Identification of these ESTs is an initial step to clone the full length genes and their promoters.

  2. RNA-sequencing reveals previously unannotated protein- and microRNA-coding genes expressed in aleurone cells of rice seeds.

    Science.gov (United States)

    Watanabe, Kenneth A; Ringler, Patricia; Gu, Lingkun; Shen, Qingxi J

    2014-01-01

    The rice genome annotation has been greatly improved in recent years, largely due to the availability of full length cDNA sequences derived from many tissues. Among those yet to be studied is the aleurone layer, which produces hydrolases for mobilization of seed storage reserves during seed germination and post germination growth. Herein, we report transcriptomes of aleurone cells treated with the hormones abscisic acid, gibberellic acid, or both. Using a comprehensive approach, we identified hundreds of novel genes. To minimize the number of false positives, only transcripts that did not overlap with existing annotations, had a high level of expression, and showed a high level of uniqueness within the rice genome were considered to be novel genes. This approach led to the identification of 553 novel genes that encode proteins and/or microRNAs. The transcriptome data reported here will help to further improve the annotation of the rice genome.

  3. Rediscovering medicinal plants' potential with OMICS: microsatellite survey in expressed sequence tags of eleven traditional plants with potent antidiabetic properties.

    Science.gov (United States)

    Sahu, Jagajjit; Sen, Priyabrata; Choudhury, Manabendra Dutta; Dehury, Budheswar; Barooah, Madhumita; Modi, Mahendra Kumar; Talukdar, Anupam Das

    2014-05-01

    Herbal medicines and traditionally used medicinal plants present an untapped potential for novel molecular target discovery using systems science and OMICS biotechnology driven strategies. Since up to 40% of the world's poor people have no access to government health services, traditional and folk medicines are often the only therapeutics available to them. In this vein, North East (NE) India is recognized for its rich bioresources. As part of the Indo-Burma hotspot, it is regarded as an epicenter of biodiversity for several plants having myriad traditional uses, including medicinal use. However, the improvement of these valuable bioresources through molecular breeding strategies, for example, using genic microsatellites or Simple Sequence Repeats (SSRs) or Expressed Sequence Tags (ESTs)-derived SSRs has not been fully utilized in large scale to date. In this study, we identified a total of 47,700 microsatellites from 109,609 ESTs of 11 medicinal plants (pineapple, papaya, noyontara, bitter orange, bermuda brass, ratalu, barbados nut, mango, mulberry, lotus, and guduchi) having proven antidiabetic properties. A total of 58,159 primer pairs were designed for the non-redundant 8060 SSR-positive ESTs and putative functions were assigned to 4483 unique contigs. Among the identified microsatellites, excluding mononucleotide repeats, di-/trinucleotides are predominant, among which repeat motifs of AG/CT and AAG/CTT were most abundant. Similarity search of SSR containing ESTs and antidiabetic gene sequences revealed 11 microsatellites linked to antidiabetic genes in five plants. GO term enrichment analysis revealed a total of 80 enriched GO terms widely distributed in 53 biological processes, 17 molecular functions, and 10 cellular components associated with the 11 markers. The present study therefore provides concrete insights into the frequency and distribution of SSRs in important medicinal resources. The microsatellite markers reported here markedly add to the genetic

  4. An expressed sequence tag (EST library for Drosophila serrata, a model system for sexual selection and climatic adaptation studies

    Directory of Open Access Journals (Sweden)

    McGraw Elizabeth A

    2009-01-01

    Full Text Available Abstract Background The native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup. Results A normalized cDNA library was constructed from whole fly bodies at several developmental stages, including larvae and adults. Assembly of 11,616 clones sequenced from the 3' end allowed us to identify 6,607 unique contigs, of which at least 90% encoded peptides. Partial transcripts were discovered from a variety of genes of evolutionary interest by BLASTing contigs against the 12 Drosophila genomes currently sequenced. By incorporating into the cDNA library multiple individuals from populations spanning a large portion of the geographical range of D. serrata, we were able to identify 11,057 putative single nucleotide polymorphisms (SNPs, with 278 different contigs having at least one "double hit" SNP that is highly likely to be a real polymorphism. At least 394 EST-associated microsatellite markers, representing 355 different contigs, were also found, providing an additional set of genetic markers. The assembled EST library is available online at http://www.chenowethlab.org/serrata/index.cgi. Conclusion We have provided the first gene collection and largest set of polymorphic genetic markers, to date, for the fly D. serrata. The EST

  5. Annotation of expressed sequence tags for the East African cichlid fish Astatotilapia burtoni and evolutionary analyses of cichlid ORFs

    Directory of Open Access Journals (Sweden)

    Braasch Ingo

    2008-02-01

    Full Text Available Abstract Background The cichlid fishes in general, and the exceptionally diverse East African haplochromine cichlids in particular, are famous examples of adaptive radiation and explosive speciation. Here we report the collection and annotation of more than 12,000 expressed sequence tags (ESTs generated from three different cDNA libraries obtained from the East African haplochromine cichlid species Astatotilapia burtoni and Metriaclima zebra. Results We first annotated more than 12,000 newly generated cichlid ESTs using the Gene Ontology classification system. For evolutionary analyses, we combined these ESTs with all available sequence data for haplochromine cichlids, which resulted in a total of more than 45,000 ESTs. The ESTs represent a broad range of molecular functions and biological processes. We compared the haplochromine ESTs to sequence data from those available for other fish model systems such as pufferfish (Takifugu rubripes and Tetraodon nigroviridis, trout, and zebrafish. We characterized genes that show a faster or slower rate of base substitutions in haplochromine cichlids compared to other fish species, as this is indicative of a relaxed or reinforced selection regime. Four of these genes showed the signature of positive selection as revealed by calculating Ka/Ks ratios. Conclusion About 22% of the surveyed ESTs were found to have cichlid specific rate differences suggesting that these genes might play a role in lineage specific characteristics of cichlids. We also conclude that the four genes with a Ka/Ks ratio greater than one appear as good candidate genes for further work on the genetic basis of evolutionary success of haplochromine cichlid fishes.

  6. Transcriptomic analysis of the venom gland of the red-headed krait (Bungarus flaviceps using expressed sequence tags

    Directory of Open Access Journals (Sweden)

    Vonk Freek J

    2010-03-01

    Full Text Available Abstract Background The Red-headed krait (Bungarus flaviceps, Squamata: Serpentes: Elapidae is a medically important venomous snake that inhabits South-East Asia. Although the venoms of most species of the snake genus Bungarus have been well characterized, a detailed compositional analysis of B. flaviceps is currently lacking. Results Here, we have sequenced 845 expressed sequence tags (ESTs from the venom gland of a B. flaviceps. Of the transcripts, 74.8% were putative toxins; 20.6% were cellular; and 4.6% were unknown. The main venom protein families identified were three-finger toxins (3FTxs, Kunitz-type serine protease inhibitors (including chain B of β-bungarotoxin, phospholipase A2 (including chain A of β-bungarotoxin, natriuretic peptide (NP, CRISPs, and C-type lectin. Conclusion The 3FTxs were found to be the major component of the venom (39%. We found eight groups of unique 3FTxs and most of them were different from the well-characterized 3FTxs. We found three groups of Kunitz-type serine protease inhibitors (SPIs; one group was comparable to the classical SPIs and the other two groups to chain B of β-bungarotoxins (with or without the extra cysteine based on sequence identity. The latter group may be functional equivalents of dendrotoxins in Bungarus venoms. The natriuretic peptide (NP found is the first NP for any Asian elapid, and distantly related to Australian elapid NPs. Our study identifies several unique toxins in B. flaviceps venom, which may help in understanding the evolution of venom toxins and the pathophysiological symptoms induced after envenomation.

  7. Comparative analysis of expressed sequence tags (ESTs between drought-tolerant and -susceptible genotypes of chickpea under terminal drought stress

    Directory of Open Access Journals (Sweden)

    Raju N L

    2011-04-01

    Full Text Available Abstract Background Chickpea (Cicer arietinum L. is an important grain-legume crop that is mainly grown in rainfed areas, where terminal drought is a major constraint to its productivity. We generated expressed sequence tags (ESTs by suppression subtraction hybridization (SSH to identify differentially expressed genes in drought-tolerant and -susceptible genotypes in chickpea. Results EST libraries were generated by SSH from root and shoot tissues of IC4958 (drought tolerant and ICC 1882 (drought resistant exposed to terminal drought conditions by the dry down method. SSH libraries were also constructed by using 2 sets of bulks prepared from the RNA of root tissues from selected recombinant inbred lines (RILs (10 each for the extreme high and low root biomass phenotype. A total of 3062 unigenes (638 contigs and 2424 singletons, 51.4% of which were novel in chickpea, were derived by cluster assembly and sequence alignment of 5949 ESTs. Only 2185 (71% unigenes showed significant BLASTX similarity ( Conclusion Our study compares not only genes that are up- and down-regulated in a drought-tolerant genotype under terminal drought stress and a drought susceptible genotype but also between the bulks of the selected RILs exhibiting extreme phenotypes. More than 50% of the genes identified have been shown to be associated with drought stress in chickpea for the first time. This study not only serves as resource for marker discovery, but can provide a better insight into the selection of candidate genes (both up- and downregulated associated with drought tolerance. These results can be used to identify suitable targets for manipulating the drought-tolerance trait in chickpea.

  8. Identification and Characterization of 43 Microsatellite Markers Derived from Expressed Sequence Tags of the Sea Cucumber (Apostichopusjaponicus)

    Institute of Scientific and Technical Information of China (English)

    JIANG Qun; LI Qi; YU Hong; KONG Lingfeng

    2011-01-01

    The sea cucumber Apostichopus japonicus is a commercially and ecologically important species in China. A total of 3056 potential unigenes were generated after assembling 7597 A. japonicus expressed sequence tags (ESTs) downloaded from GenBank. Two hundred and fifty microsatellite-containing ESTs (8.18%) and 299 simple sequence repeats (SSRs) were detected. The average density of SSRs was 1 per 7.403 kb of EST after redundancy elimination. Di-nucleotide repeat motifs appeared to be the most abundant type with a percentage of 69.90%. Of the 126 primer pairs designed, 90 amplified the expected products and 43 showed polymorphism in 30 individuals tested. The number of alleles per locus ranged from 2 to 26 with an average of 7.0 alleles,and the observed and expected heterozygosities varied from 0.067 to 1.000 and from 0.066 to 0.959, respectively. These new EST-derived microsatellite markers would provide sufficient polymorphism for population genetic studies and genome mapping of this sea cucumber species.

  9. Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing.

    Science.gov (United States)

    Paul, Sujay; Kundu, Anirban; Pal, Amita

    2014-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformatic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resources for better understanding of miRNA mediated post-transcriptional gene regulation.

  10. De Novo Assembly of Bitter Gourd Transcriptomes: Gene Expression and Sequence Variations in Gynoecious and Monoecious Lines.

    Science.gov (United States)

    Shukla, Anjali; Singh, V K; Bharadwaj, D R; Kumar, Rajesh; Rai, Ashutosh; Rai, A K; Mugasimangalam, Raja; Parameswaran, Sriram; Singh, Major; Naik, P S

    2015-01-01

    Bitter gourd (Momordica charantia L.) is a nutritious vegetable crop of Asian origin, used as a medicinal herb in Indian and Chinese traditional medicine. Molecular breeding in bitter gourd is in its infancy, due to limited molecular resources, particularly on functional markers for traits such as gynoecy. We performed de novo transcriptome sequencing of bitter gourd using Illumina next-generation sequencer, from root, flower buds, stem and leaf samples of gynoecious line (Gy323) and a monoecious line (DRAR1). A total of 65,540 transcripts for Gy323 and 61,490 for DRAR1 were obtained. Comparisons revealed SNP and SSR variations between these lines and, identification of gene classes. Based on available transcripts we identified 80 WRKY transcription factors, several reported in responses to biotic and abiotic stresses; 56 ARF genes which play a pivotal role in auxin-regulated gene expression and development. The data presented will be useful in both functions studies and breeding programs in bitter gourd.

  11. Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing

    Institute of Scientific and Technical Information of China (English)

    Sujay Paul; Anirban Kundu; Amita Pal

    2014-01-01

    MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that play a crucial role in post-transcriptional gene regulation. Several conserved and species-specific miRNAs have been characterized to date, predominantly from the plant species whose genome is well characterized. However, information on the variability of these regulatory RNAs in economically important but genetically less characterized crop species are limited. Vigna mungo is an important grain legume, which is grown primarily for its protein-rich edible seeds. miRNAs from this species have not been identified to date due to lack of genome sequence information. To identify miRNAs from V. mungo, a small RNA library was constructed from young leaves. High-throughput Illumina sequencing technology and bioinformat-ic analysis of the small RNA reads led to the identification of 66 miRNA loci represented by 45 conserved miRNAs belonging to 19 families and eight non-conserved miRNAs belonging to seven families. Besides, 13 novel miRNA candidates in V. mungo were also identified. Expression patterns of selected conserved, non-conserved, and novel miRNA candidates have been demonstrated in leaf, stem, and root tissues by quantitative polymerase chain reaction, and potential target genes were predicted for most of the conserved miRNAs. This information offers genomic resour-ces for better understanding of miRNA mediated post-transcriptional gene regulation.

  12. Identification of differentially expressed genes in longissimus muscle of pigs with high and low intramuscular fat content using RNA sequencing.

    Science.gov (United States)

    Lim, K S; Lee, K T; Park, J E; Chung, W H; Jang, G W; Choi, B H; Hong, K C; Kim, T H

    2017-04-01

    Intramuscular fat (IMF) content in pork is an important element of consumer preference and is positively correlated with meat quality, including tenderness and juiciness. With advances in RNA sequencing technologies, transcriptome-related differences can be associated with specific traits in animals. The objective of this study was to investigate differentially expressed genes (DEGs) closely related to IMF content in porcine longissimus muscle using RNA sequencing. A total of 107 Berkshire pigs were used for IMF content measurements, and significant differences between extremely high (H, n = 3) and low (L, n = 3) IMF content groups were found (P change ≥2). Functional analyses with DEGs revealed that lipid metabolism (SCD and FASN) was one of the significant biological processes related to IMF content determination. In addition, we found that DEGs related to muscle regeneration (MYOG and VEGFA) and extracellular matrix (COL1A1, COL1A2, COL5A1, COL14A1 and COL15A1) were changed among individuals with extreme IMF contents. These results will aid in understanding the regulation of IMF content in pigs.

  13. Gene Sequence, Soluble Expression and Homologous Comparison of a D-Hydantoinase from Pseudomonas putida YZ-26

    Institute of Scientific and Technical Information of China (English)

    SHI Ya-wei; ZHAO Li-xia; NIU Li-xi; FENG Xia; YUAN Jing-ming

    2005-01-01

    A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced(GenBank AY387829). The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/E.coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure: ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.

  14. SEQUENCES OF PLANT TRANSFORMATION: THE RESULT OF THE INSERTED FOREIGN GENE EXPRESSION OR THE STRESS REACTION?

    Directory of Open Access Journals (Sweden)

    Enikeev A.G.

    2012-08-01

    take into account that observed effects may not result from action of the invected gene only. Conclusions: 1. To consider the transgenesis to be completed solely if expression of transferred gene is present – is methodologically incorrect way. The transferred genes could be silenced because of the response defense reaction likely as under a “pathogen attack”. So the absence of transferred gene expression doesn’t mean the absence of transformation as fact. Moreover the deletion of inserted construction could take place but physiological trace of the insertion nevertheless can be noticeable.2. The assessment of physiological consequences of transgenesis when using the plants transformed by disarmed constructions and the plants transformed by constructions including foreign geterological genes should be carried out carefully because of these systems are different. The process of transformation by disarmed constructions is very similar with natural agrobacterial infection where plants and bacteria have been coadapted during evolution, so the transformation by insertion of foreign genes leads to forming much more unstable systems.

  15. Identification and differential expression of microRNAs in ovaries of laying and Broody geese (Anser cygnoides by Solexa sequencing.

    Directory of Open Access Journals (Sweden)

    Qi Xu

    Full Text Available BACKGROUND: Recent functional studies have demonstrated that the microRNAs (miRNAs play critical roles in ovarian gonadal development, steroidogenesis, apoptosis, and ovulation in mammals. However, little is known about the involvement of miRNAs in the ovarian function of fowl. The goose (Anas cygnoides is a commercially important food that is cultivated widely in China but the goose industry has been hampered by high broodiness and poor egg laying performance, which are influenced by ovarian function. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the miRNA transcriptomes of ovaries from laying and broody geese were profiled using Solexa deep sequencing and bioinformatics was used to determine differential expression of the miRNAs. As a result, 11,350,396 and 9,890,887 clean reads were obtained in laying and broodiness goose, respectively, and 1,328 conserved known miRNAs and 22 novel potential miRNA candidates were identified. A total of 353 conserved microRNAs were significantly differentially expressed between laying and broody ovaries. Compared with miRNA expression in the laying ovary, 127 miRNAs were up-regulated and 126 miRNAs were down-regulated in the ovary of broody birds. A subset of the differentially expressed miRNAs (G-miR-320, G-miR-202, G-miR-146, and G-miR-143* were validated using real-time quantitative PCR. In addition, 130,458 annotated mRNA transcripts were identified as putative target genes. Gene ontology annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the differentially expressed miRNAs are involved in ovarian function, including hormone secretion, reproduction processes and so on. CONCLUSIONS: The present study provides the first global miRNA transcriptome data in A. cygnoides and identifies novel and known miRNAs that are differentially expressed between the ovaries of laying and broody geese. These findings contribute to our understanding of the functional involvement of mi

  16. Analyses of expressed sequence tags from the maize foliar pathogen Cercospora zeae-maydis identify novel genes expressed during vegetative, infectious, and reproductive growth

    Directory of Open Access Journals (Sweden)

    Kema Gert HJ

    2008-11-01

    Full Text Available Abstract Background The ascomycete fungus Cercospora zeae-maydis is an aggressive foliar pathogen of maize that causes substantial losses annually throughout the Western Hemisphere. Despite its impact on maize production, little is known about the regulation of pathogenesis in C. zeae-maydis at the molecular level. The objectives of this study were to generate a collection of expressed sequence tags (ESTs from C. zeae-maydis and evaluate their expression during vegetative, infectious, and reproductive growth. Results A total of 27,551 ESTs was obtained from five cDNA libraries constructed from vegetative and sporulating cultures of C. zeae-maydis. The ESTs, grouped into 4088 clusters and 531 singlets, represented 4619 putative unique genes. Of these, 36% encoded proteins similar (E value ≤ 10-05 to characterized or annotated proteins from the NCBI non-redundant database representing diverse molecular functions and biological processes based on Gene Ontology (GO classification. We identified numerous, previously undescribed genes with potential roles in photoreception, pathogenesis, and the regulation of development as well as Zephyr, a novel, actively transcribed transposable element. Differential expression of selected genes was demonstrated by real-time PCR, supporting their proposed roles in vegetative, infectious, and reproductive growth. Conclusion Novel genes that are potentially involved in regulating growth, development, and pathogenesis were identified in C. zeae-maydis, providing specific targets for characterization by molecular genetics and functional genomics. The EST data establish a foundation for future studies in evolutionary and comparative genomics among species of Cercospora and other groups of plant pathogenic fungi.

  17. Predicting the expression of recombinant monoclonal antibodies in Chinese hamster ovary cells based on sequence features of the CDR3 domain.

    Science.gov (United States)

    Pybus, Leon P; James, David C; Dean, Greg; Slidel, Tim; Hardman, Colin; Smith, Andrew; Daramola, Olalekan; Field, Ray

    2014-01-01

    Despite the development of high-titer bioprocesses capable of producing >10 g L(-1) of recombinant monoclonal antibody (MAb), some so called "difficult-to-express" (DTE) MAbs only reach much lower process titers. For widely utilized "platform" processes the only discrete variable is the protein coding sequence of the recombinant product. However, there has been little systematic study to identify the sequence parameters that affect expression. This information is vital, as it would allow us to rationally design genetic sequence and engineering strategies for optimal bioprocessing. We have therefore developed a new computational tool that enables prediction of MAb titer in Chinese hamster ovary (CHO) cells based on the recombinant coding sequence of the expressed MAb. Model construction utilized a panel of MAbs, which following a 10-day fed-batch transient production process varied in titer 5.6-fold, allowing analysis of the sequence features that impact expression over a range of high and low MAb productivity. The model identified 18 light chain (LC)-specific sequence features within complementarity determining region 3 (CDR3) capable of predicting MAb titer with a root mean square error of 0.585 relative expression units. Furthermore, we identify that CDR3 variation influences the rate of LC-HC dimerization during MAb synthesis, which could be exploited to improve the production of DTE MAb variants via increasing the transfected LC:HC gene ratio. Taken together these data suggest that engineering intervention strategies to improve the expression of DTE recombinant products can be rationally implemented based on an identification of the sequence motifs that render a recombinant product DTE.

  18. Cloning,sequence analysis and expression in E.coli of the group 3 allergen of Dermatophagoides farinae

    Institute of Scientific and Technical Information of China (English)

    CUI Yu-bao; CAI Hong-xing; LI Li; ZHOU Ying; GAO Cui-xiang; SHI Wei-hong; YU Ming

    2009-01-01

    Background The dust mites,which are mostly represented by Dermatophagoides spp.(Acari:Pyroglyphidae),are the major sources of indoor allergens.Identification and characterization of these mite allergen molecules are an important step in the development of new effective diagnostic procedures and possible therapeutic strategies for allergic disorders associated with dust mites.Methods Total RNA was extracted from Derrnatophagoides farinae.The gene coding for Der f 3 was amplified by RT-PCR with the primers designed based on previous sequence published in GenBank.The target gene was cloned intermediately into pMD19-T plasmid and finally into plasmid pET28a(+),expressed in E.coli BL21 at the aid of the inducer isopropyI-D-thiogalactopyranoside(IPTG).The physicochemical properties,spatial structure of the allergen were analyzed with bioinformatics software.Results The cDNA coding for group 3 allergen of Dermatophagoides farinae from China was cloned and expressed successfully.Sequencing analysis showed that there were nineteen mismatched nucleotides in five Der f 3 cDNA clones in comparison with the reference(GenBank Accession No.AY283291),which resulted in deduced amino acid sequence incompatibility in eleven residues.Bioinformatics analysis revealed that the Der f 3 pro-protein was an extracellular hydrophobic protein,consisting of 259 amino acids with a 16 amino acid signal peptide.The protein was deduced to have three chymotrypsin active sites(53-68 AA,108-122 AA and 205-217 hA),one N-glycosylation site,one cAMP-and cGMP-dependent protein kinase phosphorylation site,four protein kinase C phosphorylation sites,two casein kinase Ⅱ phosphorylation sites,and five N-myristoylation sites.Conclusions Der f 3 is an extracellular hydrophobic protein which possesses multiple activation and phosphorylation sites.Polymorphism may exist in the Der f3 gene but this needs to be further confirmed in the future.

  19. Acetylcholinesterase of the sand fly, Phlebotomus papatasi (Scopoli): cDNA sequence, baculovirus expression, and biochemical properties.

    Science.gov (United States)

    Temeyer, Kevin B; Brake, Danett K; Tuckow, Alexander P; Li, Andrew Y; Pérez de León, Adalberto A

    2013-02-04

    Millions of people and domestic animals around the world are affected by leishmaniasis, a disease caused by various species of flagellated protozoans in the genus Leishmania that are transmitted by several sand fly species. Insecticides are widely used for sand fly population control to try to reduce or interrupt Leishmania transmission. Zoonotic cutaneous leishmaniasis caused by L. major is vectored mainly by Phlebotomus papatasi (Scopoli) in Asia and Africa. Organophosphates comprise a class of insecticides used for sand fly control, which act through the inhibition of acetylcholinesterase (AChE) in the central nervous system. Point mutations producing an altered, insensitive AChE are a major mechanism of organophosphate resistance in insects and preliminary evidence for organophosphate-insensitive AChE has been reported in sand flies. This report describes the identification of complementary DNA for an AChE in P. papatasi and the biochemical characterization of recombinant P. papatasi AChE. A P. papatasi Israeli strain laboratory colony was utilized to prepare total RNA utilized as template for RT-PCR amplification and sequencing of cDNA encoding acetylcholinesterase 1 using gene specific primers and 3'-5'-RACE. The cDNA was cloned into pBlueBac4.5/V5-His TOPO, and expressed by baculovirus in Sf21 insect cells in serum-free medium. Recombinant P. papatasi acetylcholinesterase was biochemically characterized using a modified Ellman's assay in microplates. A 2309 nucleotide sequence of PpAChE1 cDNA [GenBank: JQ922267] of P. papatasi from a laboratory colony susceptible to insecticides is reported with 73-83% nucleotide identity to acetylcholinesterase mRNA sequences of Culex tritaeniorhynchus and Lutzomyia longipalpis, respectively. The P. papatasi cDNA ORF encoded a 710-amino acid protein [GenBank: AFP20868] exhibiting 85% amino acid identity with acetylcholinesterases of Cx. pipiens, Aedes aegypti, and 92% amino acid identity for L. longipalpis. Recombinant P

  20. Hyperlipidemia-associated gene variations and expression patterns revealed by whole-genome and transcriptome sequencing of rabbit models

    Science.gov (United States)

    Wang, Zhen; Zhang, Jifeng; Li, Hong; Li, Junyi; Niimi, Manabu; Ding, Guohui; Chen, Haifeng; Xu, Jie; Zhang, Hongjiu; Xu, Ze; Dai, Yulin; Gui, Tuantuan; Li, Shengdi; Liu, Zhi; Wu, Sujuan; Cao, Mushui; Zhou, Lu; Lu, Xingyu; Wang, Junxia; Yang, Jing; Fu, Yunhe; Yang, Dongshan; Song, Jun; Zhu, Tianqing; Li, Shen; Ning, Bo; Wang, Ziyun; Koike, Tomonari; Shiomi, Masashi; Liu, Enqi; Chen, Luonan; Fan, Jianglin; Chen, Y. Eugene; Li, Yixue

    2016-01-01

    The rabbit (Oryctolagus cuniculus) is an important experimental animal for studying human diseases, such as hypercholesterolemia and atherosclerosis. Despite this, genetic information and RNA expression profiling of laboratory rabbits are lacking. Here, we characterized the whole-genome variants of three breeds of the most popular experimental rabbits, New Zealand White (NZW), Japanese White (JW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Although the genetic diversity of WHHL rabbits was relatively low, they accumulated a large proportion of high-frequency deleterious mutations due to the small population size. Some of the deleterious mutations were associated with the pathophysiology of WHHL rabbits in addition to the LDLR deficiency. Furthermore, we conducted transcriptome sequencing of different organs of both WHHL and cholesterol-rich diet (Chol)-fed NZW rabbits. We found that gene expression profiles of the two rabbit models were essentially similar in the aorta, even though they exhibited different types of hypercholesterolemia. In contrast, Chol-fed rabbits, but not WHHL rabbits, exhibited pronounced inflammatory responses and abnormal lipid metabolism in the liver. These results provide valuable insights into identifying therapeutic targets of hypercholesterolemia and atherosclerosis with rabbit models. PMID:27245873

  1. Stress gene (hsp70) sequences and quantitative expression in Milnesium tardigradum (Tardigrada) during active and cryptobiotic stages.

    Science.gov (United States)

    Schill, Ralph O; Steinbrück, Günther H B; Köhler, Heinz-R

    2004-04-01

    The eutardigrade Milnesium tardigradum can undergo cryptobiosis, i.e. entry into a reversible ametabolic stage induced by dehydration, cooling and, probably, osmotic and anoxic stress. For the first time in tardigrades, we described partial sequences of three heat-shock protein (hsp70 family) genes and examined gene expression on the way from an active to a cryptobiotic and back to an active stage again. Results showed different patterns of gene expression in the hsp70 isoforms. All three isoforms seem to be true heat-shock proteins since transcription could be clearly enhanced by temperature elevation. Isoform 1 and, at a lower level, isoform 3 do not seem to have a specific function for cryptobiosis. By contrast, transcription of isoform 2 is significantly induced in the transitional stage between the active and the cryptobiotic stage, resulting in a comparatively high mRNA copy number also during cryptobiosis. This pattern of induction implies that isoform 2 is the most relevant hsp70 gene for M. tardigradum individuals entering the cryptobiotic stage.

  2. Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli.

    Science.gov (United States)

    Nakanishi, Shusuke; Tanaka, Mamoru

    2010-06-01

    A small cryptic plasmid, namely, pCBM588, was obtained from Clostridium butyricum MIYAIRI 588 (CBM588)--a bacterium used in probiotics. The complete sequence of pCBM588 was determined. The size of pCBM588 was 8060 bp and the G + C content was 24.3%. Nine open reading frames (ORFs) were predicted, and ORF3 showed significant homologies with a structural bacteriocin gene of Clostridium tyrobutyricum. The putative bacteriocin gene was inserted into the pET21d expression vector in frame; it was expressed as a His-tagged recombinant protein in Escherichia coli BL21 (DE3). A total of 10240AU of the recombinant bacteriocin were purified from 100 ml of E. coli culture. The bacteriocin was cleaved into 2 portions, and the small C-terminal polypeptide consisting of 83 amino acids possessed bactericidal activity. These results demonstrated that the ORF3 of pCBM588 encoded a bacteriocin, which is identical or very similar to the previously reported butyricin 7423.

  3. Simultaneous removal of phosphorus and nitrogen in a sequencing batch biofilm reactor with transgenic bacteria expressing polyphosphate kinase.

    Science.gov (United States)

    Du, Hongwei; Yang, Liuyan; Wu, Jun; Xiao, Lin; Wang, Xiaolin; Jiang, Lijuan

    2012-10-01

    To improve phosphorus removal from wastewater, we constructed a high-phosphate-accumulating microorganism, KTPPK, using Pseudomonas putida KT2440 as a host. The expression plasmid was constructed by inserting and expressing polyphosphate kinase gene (ppk) from Microcystis aeruginosa NIES-843 into broad-host-range plasmid, pBBR1MCS-2. KTPPK was then added to a sequencing batch biofilm reactor (SBBFR) using lava as a biological carrier. The results showed that SBBFR with KTPPK not only efficiently removed COD, NH(3)-N, and NO(3)(-)-N but also had a high removal capacity for PO(4)(3-)-P, resulting in a low phosphorus concentration remaining in the outflow of the SBBFR. The biofilm increased by 30-53% on the lava in the SBBFR that contained KTPPK after 11 days when compared with the reactor that contained P. putida KT2440. Real-time quantitative polymerase chain reaction confirmed that the copy of ppk was maintained at about 3.5 × 10(10) copies per μL general DNA in the biofilm after 20 days. Thus, the transgenic bacteria KTPPK could maintain a high density and promote phosphorus removal in the SBBFR. In summary, this study indicates that the use of SBBFR with transgenic bacteria has the potential to remove phosphorus and nitrogen from wastewater.

  4. Sequence and expression of the CAPA/CAP2b gene in the tobacco hawkmoth, Manduca sexta.

    Science.gov (United States)

    Loi, Poh Kheng; Tublitz, Nathan J

    2004-10-01

    The gene coding for cardioacceleratory peptide 2b (CAP2b; pELYAFPRV) has been isolated and sequenced from the moth Manduca sexta (GenBank accession #AY649544). Because of its significant homology to the CAPA gene in Drosophila melanogaster, this gene is called the Manduca CAPA gene. The Manduca CAPA gene is 958 nucleotides long with 29 untranslated nucleotides from the beginning of the sequence to the putative start initiation site. The CAPA gene has a single open reading frame, 441 nucleotides long, that codes for a predicted precursor protein of 147 amino acids. The predicted prepropeptide encodes a single copy of each of three deduced propeptides, a CAP2b propeptide, with a Q substituted for an E at the N-terminus (QLYAFPRVa), and two novel CAP2b-related propeptides (DGVLNLYPFPRVa and TEGPGMWFGPRLa). To reduce confusion and to adopt a more standardized nomenclature, we rename pELYAFPRVa as Mas-CAPA-1 and assign the names of Mas-CAPA-2 to DGVLNLYPFPRVa and Mas-PK-1 (Pyrokinin-1) to TEGPGMWFGPRLa. The spatial and temporal expression pattern of the CAPA gene in the Manduca central nervous system (CNS) was determined in all major post-embryonic stages using in situ hybridization techniques. The CAPA gene is expressed in a total of 27 pairs of neurons in the post-embryonic Manduca CNS. A total of 16 pairs of cells is observed in the brain, two pairs in the sub-esophageal ganglion (SEG), one pair in the third thoracic ganglion (T3), one pair in each unfused abdominal ganglion (A1-A6) and two pairs in the fused terminal ganglion. The mRNA from the CAPA gene is present in nearly every ganglion in each post-embryonic stage. The number of cells expressing the CAPA gene varies during post-embryonic life, starting at 54 cells in first-instar larvae and declining to a minimum of 14 cells midway through adult development.

  5. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Korfhagen, T.R.; Glasser, S.W.; Wert, S.E.; Bruno, M.D.; Daugherty, C.C.; McNeish, J.D.; Stock, J.L.; Potter, S.S.; Whitsett, J.A. (Cincinnati College of Medicine, OH (USA))

    1990-08-01

    Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5{prime}-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulm