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Sample records for hybridizations parent-of-origin-specific gene

  1. Identification of imprinted genes subject to parent-of-origin specific expression in Arabidopsis thaliana seeds

    LENUS (Irish Health Repository)

    McKeown, Peter C

    2011-08-12

    Abstract Background Epigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants. Results cDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs) displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination). We identified these MEGs by developing a bioinformatics tool (GenFrag) which can directly determine the identities of transcript-derived fragments from (i) their size and (ii) which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1 seeds was

  2. ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Riso, Vincenzo; Cammisa, Marco; Kukreja, Harpreet

    2016-01-01

    ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi......-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS...... the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet...

  3. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche.

    Science.gov (United States)

    Perry, John R B; Day, Felix; Elks, Cathy E; Sulem, Patrick; Thompson, Deborah J; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I; Esko, Tõnu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q; Corre, Tanguy; Cousminer, Diana L; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D; Kjellqvist, Sanela; Lunetta, Kathryn L; McMahon, George; Nolte, Ilja M; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Tšernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K; Amin, Najaf; Bierut, Laura J; Byrne, Enda M; Hottenga, Jouke-Jan; Koller, Daniel L; Mangino, Massimo; Pers, Tune H; Yerges-Armstrong, Laura M; Hua Zhao, Jing; Andrulis, Irene L; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E; Bolla, Manjeet K; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collée, J Margriet; Couch, Fergus J; Couper, David; Coviello, Andrea D; Cox, Angela; Czene, Kamila; D'adamo, Adamo Pio; Davey Smith, George; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K; Dunning, Alison M; Eiriksdottir, Gudny; Eriksson, Johan G; Fasching, Peter A; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E; García-Closas, Montserrat; Geller, Frank; de Geus, Eco E J; Giles, Graham G; Gudbjartsson, Daniel F; Gudnason, Vilmundur; Guénel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A; Heath, Andrew C; Hofman, Albert; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Hunter, David J; Karasik, David; Kiel, Douglas P; Knight, Julia A; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Mägi, Reedik; Magnusson, Patrik K; Mannermaa, Arto; Martin, Nicholas G; Masson, Gisli; McArdle, Patrick F; McArdle, Wendy L; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A; Oldehinkel, Albertine J; Oostra, Ben A; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul D P; Postma, Dirkje S; Pouta, Anneli; Pylkäs, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K; Southey, Mellissa C; Sovio, Ulla; Stampfer, Meir J; Stöckl, Doris; Storniolo, Anna M; Timpson, Nicholas J; Tyrer, Jonathan; Visser, Jenny A; Vollenweider, Peter; Völzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce H R; Wright, Margaret J; Boomsma, Dorret I; Econs, Michael J; Khaw, Kay-Tee; Loos, Ruth J F; McCarthy, Mark I; Montgomery, Grant W; Rice, John P; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B; Ingelsson, Erik; Järvelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E; Ridker, Paul M; Snieder, Harold; Sørensen, Thorkild I A; Spector, Tim D; Strachan, David P; Uitterlinden, André G; Wareham, Nicholas J; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F; Stefansson, Kari; Murabito, Joanne M; Ong, Ken K

    2014-10-02

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition.

  4. Parent-of-origin specific allelic associations among 106 genomic loci for age at menarche

    Science.gov (United States)

    Thompson, Deborah J; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I; Esko, Tõnu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q; Corre, Tanguy; Cousminer, Diana L; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D; Kjellqvist, Sanela; Lunetta, Kathryn L; McMahon, George; Nolte, Ilja M; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Tšernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K; Amin, Najaf; Bierut, Laura J; Byrne, Enda M; Hottenga, Jouke-Jan; Koller, Daniel L; Mangino, Massimo; Pers, Tune H; Yerges-Armstrong, Laura M; Zhao, Jing Hua; Andrulis, Irene L; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E; Bolla, Manjeet K; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collée, J. Margriet; Couch, Fergus J; Couper, David; Coveillo, Andrea D; Cox, Angela; Czene, Kamila; D’adamo, Adamo Pio; Smith, George Davey; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K; Dunning, Alison M; Eiriksdottir, Gudny; Eriksson, Johan G; Fasching, Peter A; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E; García-Closas, Montserrat; Geller, Frank; de Geus, Eco EJ; Giles, Graham G; Gudbjartsson, Daniel F; Gudnason, Vilmundur; Guénel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A; Heath, Andrew C; Hofman, Albert; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Hunter, David J; Karasik, David; Kiel, Douglas P; Knight, Julia A; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Mägi, Reedik; Magnusson, Patrik K; Mannermaa, Arto; Martin, Nicholas G; Masson, Gisli; McArdle, Patrick F; McArdle, Wendy L; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A; Oldehinkel, Albertine J; Oostra, Ben A; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul DP; Postma, Dirkje S; Pouta, Anneli; Pylkäs, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K; Southey, Mellissa C; Sovio, Ulla; Stampfer, Meir J; Stöckl, Doris; Storniolo, Anna M; Timpson, Nicholas J; Tyrer, Jonathan; Visser, Jenny A; Vollenweider, Peter; Völzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce HR; Wright, Margaret J; Boomsma, Dorret I; Econs, Michael J; Khaw, Kay-Tee; Loos, Ruth JF; McCarthy, Mark I; Montgomery, Grant W; Rice, John P; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B; Ingelsson, Erik; Järvelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E; Ridker, Paul M; Snieder, Harold; Sørensen, Thorkild IA; Spector, Tim D; Strachan, David P; Uitterlinden, André G; Wareham, Nicholas J; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F

    2014-01-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality1. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation2,3, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P<5×10−8) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1/WDR25, MKRN3/MAGEL2 and KCNK9) demonstrating parent-of-origin specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and gamma-aminobutyric acid-B2 receptor signaling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition. PMID:25231870

  5. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche

    DEFF Research Database (Denmark)

    Perry, John R. B.; Day, Felix; Elks, Cathy E.

    2014-01-01

    . Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and gamma...

  6. Analysis of IMGSAC autism susceptibility loci: evidence for sex limited and parent of origin specific effects

    Science.gov (United States)

    Lamb, J; Barnby, G; Bonora, E; Sykes, N; Bacchelli, E; Blasi, F; Maestrini, E; Broxholme, J; Tzenova, J; Weeks, D; Bailey, A; Monaco, A; the, I

    2005-01-01

    Background and methods: Autism is a severe neurodevelopmental disorder, which has a complex genetic predisposition. The ratio of males to females affected by autism is approximately 4:1, suggesting that sex specific factors are involved in its development. We reported previously the results of a genomewide screen for autism susceptibility loci in 83 affected sibling pairs (ASP), and follow up analysis in 152 ASP. Here, we report analysis of an expanded sample of 219 ASP, using sex and parent of origin linkage modelling at loci on chromosomes 2, 7, 9, 15, and 16. Results: The results suggest that linkage to chromosomes 7q and 16p is contributed largely by the male–male ASP (MLS = 2.55 v 0.12, and MLS = 2.48 v 0.00, for the 145 male–male and 74 male–female/female–female ASP on chromosomes 7 and 16 respectively). Conversely linkage to chromosome 15q appears to be attributable to the male–female/female–female ASP (MLS = 2.62 v 0.00, for non-male and male–male ASP respectively). On chromosomes 2 and 9, all ASP contribute to linkage. These data, supported by permutation, suggest a possible sex limited effect of susceptibility loci on chromosomes 7, 15, and 16. Parent of origin linkage modelling indicates two distinct regions of paternal and maternal identity by descent sharing on chromosome 7 (paternal MLS = 1.46 at ∼112 cM, and maternal MLS = 1.83 at ∼135 cM; corresponding maternal and paternal MLS = 0.53 and 0.28 respectively), and maternal specific sharing on chromosome 9 (maternal MLS = 1.99 at ∼30 cM; paternal MLS = 0.02). Conclusion: These data support the possibility of two discrete loci underlying linkage of autism to chromosome 7, and implicate possible parent of origin specific effects in the aetiology of autism. PMID:15689451

  7. No evidence for a parent of origin specific differentially methylated region linked to RASGRF1

    Directory of Open Access Journals (Sweden)

    Punita ePitamber

    2012-03-01

    Full Text Available Abstract Imprinted genes are expressed from one parental allele in a parent-of-origin manner. This mono-allelic behavior is regulated by allele-specific DNA methylation that is confined to differentially methylated regions (DMRs. To date there are over 80 known human imprinted genes of which only three are known to have paternally methylated DMRs. In mice there exists an additional paternally methylated DMR associated with Rasgrf1. The Rasgrf1 gene forms part of the MAPK signaling pathway and plays a role in long-term memory formation and growth control. The presence of a RASGRF1-associated DMR in humans and its methylation status has not been explored. The primary aim of this study was to determine whether the human RASGRF1 gene contains a DMR and whether this DMR is paternally methylated. Computational assessments were done to identify putative CTCF binding sites, CpG islands (CGIs that could serve as potential RASGRF1 DMRs and tandem repeats within or adjacent to these CGIs. The methylation status of three putative CGIs was assessed using quantitative pyrosequencing technology. None of the putative CTCF binding sites were found to occur in the predicted CGIs. The three putative CGIs linked to RASGRF1 did not display allele-specific methylation in blood DNA nor unique paternal methylation in sperm DNA. While one of the three CGIs was found to be hypomethylated in both blood DNA and sperm DNA, the other two were found to be hypermethylated. The tandem repeats that were identified were associated with only one of the putative CGIs. This CGI is located at the promoter region of RASGRF1 and was found to be hypomethylated both in the peripheral blood and in the sperm samples. The CGIs evaluated in this study did not fit the criteria of being a DMR. The human RASGRF1-associated CpG rich regions do not exhibit differential methylation in a parent-of-origin manner. However, expression studies would need to be performed to confirm the imprint status of

  8. Hybrid dysfunction and physiological compensation in gene expression.

    Science.gov (United States)

    Barreto, Felipe S; Pereira, Ricardo J; Burton, Ronald S

    2015-03-01

    The formation of new species is often a consequence of genetic incompatibilities accumulated between populations during allopatric divergence. When divergent taxa interbreed, these incompatibilities impact physiology and have a direct cost resulting in reduced hybrid fitness. Recent surveys of gene regulation in interspecific hybrids have revealed anomalous expression across large proportions of the genome, with 30-70% of all genes exhibiting transgressive expression (i.e., higher or lower levels compared with both parental taxa), and these were mostly in the direction of downregulation. However, as most of these studies have focused on pairs of species exhibiting high degrees of reproductive isolation, the association between regulatory disruption and reduced hybrid fitness prior to species formation remains unclear. Within the copepod species Tigriopus californicus, interpopulation hybrids at F2 or later generations show reduced fitness associated with mitochondrial dysfunction. Here we show that in contrast to studies of interspecific hybrids, only 1.2% of the transcriptome is transgressively expressed in F3+ interpopulation hybrids of T. californicus, and nearly 80% of these genes are overexpressed rather than underexpressed; remarkably, none of these genes are among those showing divergent expression between parentals, nor is magnitude of transgressive gene expression in hybrids dependent on levels of protein sequence divergence. Moreover, many genes with transgressive expression are components of functional pathways impacted by mitonuclear incompatibilities in hybrid T. californicus (e.g., oxidative phosphorylation and antioxidant response). Our results suggest that hybrid breakdown at early stages of speciation may result from initial incompatibilities amplified by the cost of compensatory physiological responses.

  9. Radiation hybrid mapping of cataract genes in the dog

    NARCIS (Netherlands)

    Hunter, L; Sidjanin, D; Johnson, J; Zangerl, B; Galibert, F; Andre, C; Kirkness, E; Talamas, E; Acland, G; Aguirre, G

    2006-01-01

    Purpose: To facilitate the molecular characterization of naturally occurring cataracts in dogs by providing the radiation hybrid location of 21 cataract-associated genes along with their closely associated polymorphic markers. These can be used for segregation testing of the candidate genes in canin

  10. GPAW optimized for Blue Gene/P using hybrid programming

    DEFF Research Database (Denmark)

    Kristensen, Mads Ruben Burgdorff; Happe, Hans Henrik; Vinter, Brian

    2009-01-01

    In this work we present optimizations of a Grid-based projector-augmented wave method software, GPAW for the Blue Gene/P architecture. The improvements are achieved by exploring the advantage of shared and distributed memory programming also known as hybrid programming. The work focuses on optimi......In this work we present optimizations of a Grid-based projector-augmented wave method software, GPAW for the Blue Gene/P architecture. The improvements are achieved by exploring the advantage of shared and distributed memory programming also known as hybrid programming. The work focuses...

  11. Atypical haemolytic uraemic syndrome associated with a hybrid complement gene.

    Directory of Open Access Journals (Sweden)

    Julian P Venables

    2006-10-01

    Full Text Available BACKGROUND: Sequence analysis of the regulators of complement activation (RCA cluster of genes at chromosome position 1q32 shows evidence of several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for factor H (CFH and the genes for the five factor H-related proteins (CFHL1-5; aliases CFHR1-5. CFH mutations have been described in association with atypical haemolytic uraemic syndrome (aHUS. The majority of the mutations are missense changes that cluster in the C-terminal region and impair the ability of factor H to regulate surface-bound C3b. Some have arisen as a result of gene conversion between CFH and CFHL1. In this study we tested the hypothesis that nonallelic homologous recombination between low-copy repeats in the RCA cluster could result in the formation of a hybrid CFH/CFHL1 gene that predisposes to the development of aHUS. METHODS AND FINDINGS: In a family with many cases of aHUS that segregate with the RCA cluster we used cDNA analysis, gene sequencing, and Southern blotting to show that affected individuals carry a heterozygous CFH/CFHL1 hybrid gene in which exons 1-21 are derived from CFH and exons 22/23 from CFHL1. This hybrid encodes a protein product identical to a functionally significant CFH mutant (c.3572C>T, S1191L and c.3590T>C, V1197A that has been previously described in association with aHUS. CONCLUSIONS: CFH mutation screening is recommended in all aHUS patients prior to renal transplantation because of the high risk of disease recurrence post-transplant in those known to have a CFH mutation. Because of our finding it will be necessary to implement additional screening strategies that will detect a hybrid CFH/CFHL1 gene.

  12. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, Karen E.

    2005-10-14

    A. ABSTRACT The importance of lateral gene transfer (LGT) in the evolution of microbial species has become increasingly evident with each completed microbial genome sequence. Most significantly, the genome of Thermotoga maritima MSB8, a hyperthermophilic bacterium isolated by Karl Stetter and workers from Vulcano Italy in 1986, and sequenced at The Institute for Genomic Research (TIGR) in Rockville Maryland in 1999, revealed extensive LGT between % . this bacterium and members of the archaeal domain (in particular Archaeoglobus fulgidus, and Pyracoccus frcriosus species). Based on whole genome comparisons, it was estimated that 24% of the genetic information in this organism was acquired by genetic exchange with archaeal species, Independent analyses including periodicity analysis of the T. maritimu genomic DNA sequence, phylogenetic reconstruction based on genes that appear archaeal-like, and codon and amino acid usage, have provided additional evidence for LGT between T. maritima and the archaea. More recently, DiRuggiero and workers have identified a very recent LGT event between two genera of hyperthermophilic archaea, where a nearly identical DNA fragment of 16 kb in length flanked by insertion sequence (IS) elements, exists. Undoubtedly, additional examples of LGT will be identified as more microbial genomes are completed. For the present moment however, the genome sequence of T. maritima and other hyperthermophiles including P. furiosus, Pyrococcus horikoshii, Pyrococcus abyssi, A. fulgidus, and Aquifex aeolicus, have significantly increased out awareness of evolution being a web of life rather than a tree of life, as suggested by single gene phylogenies. In this proposal, we will aim to determine the extent of LGT across the hyperthemophiles, employing iY maritima as the model organism. A variety of biochemical techniques and phylogenetic reconstructions will allow for a detailed and thorough characterization of the extent of LGT in this species. The

  13. Hybrid Nanomaterial Complexes for Advanced Phage-guided Gene Delivery

    Directory of Open Access Journals (Sweden)

    Teerapong Yata

    2014-01-01

    Full Text Available Developing nanomaterials that are effective, safe, and selective for gene transfer applications is challenging. Bacteriophages (phage, viruses that infect bacteria only, have shown promise for targeted gene transfer applications. Unfortunately, limited progress has been achieved in improving their potential to overcome mammalian cellular barriers. We hypothesized that chemical modification of the bacteriophage capsid could be applied to improve targeted gene delivery by phage vectors into mammalian cells. Here, we introduce a novel hybrid system consisting of two classes of nanomaterial systems, cationic polymers and M13 bacteriophage virus particles genetically engineered to display a tumor-targeting ligand and carry a transgene cassette. We demonstrate that the phage complex with cationic polymers generates positively charged phage and large aggregates that show enhanced cell surface attachment, buffering capacity, and improved transgene expression while retaining cell type specificity. Moreover, phage/polymer complexes carrying a therapeutic gene achieve greater cancer cell killing than phage alone. This new class of hybrid nanomaterial platform can advance targeted gene delivery applications by bacteriophage.

  14. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche

    NARCIS (Netherlands)

    Perry, J.R.; Day, F.; Elks, C.E.; Sulem, P.; Thompson, D.J.; Ferreira, T.; He, C.; Chasman, D.I.; Esko, T.; Thorleifsson, G.; Albrecht, E.; Ang, W.Q.; Corre, T.; Cousminer, D.L.; Feenstra, B.; Franceschini, N.; Ganna, A.; Johnson, A.D.; Kjellqvist, S.; Lunetta, K.L.; McMahon, G.; Nolte, I.M.; Paternoster, L.; Porcu, E.; Smith, A.V.; Stolk, L.; Teumer, A.; Tsernikova, N.; Tikkanen, E.; Ulivi, S.; Wagner, E.K.; Amin, N.; Bierut, L.J.; Byrne, E.M.; Hottenga, J.J.; Koller, D.L.; Mangino, M.; Pers, T.H.; Yerges-Armstrong, L.M.; Zhao, J.; Andrulis, I.L.; Anton-Culver, H.; Atsma, F.; Bandinelli, S.; Beckmann, M.W.; Benitez, J.; Blomqvist, C.; Bojesen, S.E.; Bolla, M.K.; Bonanni, B.; Brauch, H.; Brenner, H.; Buring, J.E.; Chang-Claude, J.; Chanock, S.; Chen, J.; Chenevix-Trench, G.; Collee, J.M.; Couch, F.J.; Couper, D.; Coviello, A.D.; Cox, A; Czene, K.; D'Adamo, P.; Smith, G.; Vivo, I. De; Demerath, E.W.; Dennis, J.; Devilee, P.; Dieffenbach, A.K.; Dunning, A.M.; Eiriksdottir, G.; Eriksson, J.G.; Fasching, P.A.; Ferrucci, L.; Flesch-Janys, D.; Flyger, H.; Foroud, T.; Franke, L.; Garcia, M.E.; Garcia-Closas, M.; Geller, F.; Geus, E.E. de; Giles, G.G.; Gudbjartsson, D.F.; Gudnason, V.; Guenel, P.; Guo, S.; Hall, P.; Hamann, U.; Haring, R.; Hartman, C.A.; Heath, A.C.; Hofman, A.; Hooning, M.J.; Hopper, J.L.; Hu, F.B.; Hunter, D.J.; Karasik, D.; Kiel, D.P.

    2014-01-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal

  15. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche

    NARCIS (Netherlands)

    J.R.B. Perry (John); F.R. Day (Felix); C.E. Elks (Cathy); P. Sulem (Patrick); D. Thompson (Deborah); T. Ferreira (Teresa); C. He (Chunyan); D.I. Chasman (Daniel); T. Esko (Tõnu); G. Thorleifsson (Gudmar); E. Albrecht (Eva); W.Q. Ang (Wei); T. Corre (Tanguy); D.L. Cousminer (Diana); B. Feenstra (Bjarke); N. Franceschini (Nora); A. Ganna (Andrea); A.D. Johnson (Andrew); S. Kjellqvist (Sanela); K.L. Lunetta (Kathryn); G. Mcmahon (George); I.M. Nolte (Ilja); L. Paternoster (Lavinia); E. Porcu (Eleonora); G.D. Smith; L. Stolk (Lisette); A. Teumer (Alexander); N. Tsernikova (Natalia); E. Tikkanen (Emmi); S. Ulivi (Shelia); E.K. Wagner (Erin K.); N. Amin (Najaf); L.J. Bierut (Laura); E.M. Byrne (Enda); J.J. Hottenga (Jouke Jan); D.L. Koller (Daniel); M. Mangino (Massimo); T.H. Pers (Tune); L.M. Yerges-Armstrong (Laura); J.H. Zhao; I.L. Andrulis (Irene); H. Anton-Culver (Hoda); F. Atsma (Femke); S. Bandinelli (Stefania); M.W. Beckmann (Matthias W.); J. Benítez (Javier); C. Blomqvist (Carl); S.E. Bojesen (Stig); M.K. Bolla (Manjeet); B. Bonnani (Bernardo); H. Brauch (Hiltrud); H. Brenner (Hermann); J.E. Buring (Julie); J. Chang-Claude (Jenny); S.J. Chanock (Stephen); J. Chen (Jinhui); G. Chenevix-Trench (Georgia); J.M. Collee (Margriet); F.J. Couch (Fergus); D.J. Couper (David); A.D. Coviello (Andrea); A. Cox (Angela); K. Czene (Kamila); P. d' Adamo (Pio); G.D. Smith; I. de Vivo (Immaculata); E.W. Demerath (Ellen); J. Dennis (Joe); P. Devilee (Peter); A.K. Dieffenbach (Aida Karina); A.M. Dunning (Alison); G. Eiriksdottir (Gudny); K. Hagen (Knut); P.A. Fasching (Peter); L. Ferrucci (Luigi); D. Flesch-Janys (Dieter); H. Flyger (Henrik); T. Foroud (Tatiana); L. Franke (Lude); M. Garcia (Melissa); M. García-Closas (Montserrat); F. Geller (Frank); E.E.J. De Geus (Eco E. J.); G.G. Giles (Graham); D.F. Gudbjartsson (Daniel); V. Gudnason (Vilmundur); P. Guénel (Pascal); S. Guo (Suiqun); P. Hall (Per); U. Hamann (Ute); R. Haring (Robin); C.A. Hartman (Catharina); A.C. Heath (Andrew); A. Hofman (Albert); M.J. Hooning (Maartje); J.L. Hopper (John); F.B. Hu (Frank); D. Hunter (David); D. Karasik (David); D.P. Kiel (Douglas P.); J.A. Knight (Julia); V-M. Kosma (Veli-Matti); Z. Kutalik (Zoltán); S. Lai (Sandra); D. Lambrechts (Diether); A. Lindblom (Annika); R. Mägi (Reedik); P.K. Magnusson (Patrik); A. Mannermaa (Arto); N.G. Martin (Nicholas); G. Masson (Gisli); P.F. McArdle (Patrick); W.L. McArdle (Wendy); M. Melbye (Mads); K. Michailidou (Kyriaki); E. Mihailov (Evelin); L. Milani (Lili); R.L. Milne (Roger L); H. Nevanlinna (Heli); P. Neven (Patrick); C. Nohr (Christian); A.J. Oldehinkel (Albertine); B.A. Oostra (Ben); A. Palotie (Aarno); M. Peacock (Munro); N.L. Pedersen (Nancy L.); P. Peterlongo (Paolo); J. Peto (Julian); P.D.P. Pharoah (Paul); D.S. Postma (Dirkje); A. Pouta (Anneli); K. Pykäs (Katri); P. Radice (Paolo); S.M. Ring (Susan); F. Rivadeneira Ramirez (Fernando); A. Robino (Antonietta); L.M. Rose (Lynda); A. Rudolph (Anja); V. Salomaa (Veikko); S. Sanna (Serena); D. Schlessinger (David); M.K. Schmidt (Marjanka); M.C. Southey (Mellissa C.); U. Sovio (Ulla); M.J. Stampfer (Meir J.); D. Stöckl (Doris); A.M. Storniolo (Anna M.); N. Timpson (Nicholas); J.P. Tyrer (Jonathan); J.A. Visser (Jenny); P. Vollenweider (Peter); H. Völzke (Henry); G. Waeber (Gérard); M. Waldenberger (Melanie); H. Wallaschofski (Henri); Q. Wang (Qing); G.A.H.M. Willemsen (Gonneke); R. Winqvist (Robert); B.H.R. Wolffenbuttel (Bruce H. R.); M.J. Wright (Margaret); D.I. Boomsma (Dorret); M.J. Econs (Michael); K.T. Khaw; R.J.F. Loos (Ruth); M.I. McCarthy (Mark); G.W. Montgomery (Grant); J.P. Rice (John); E.A. Streeten (Elizabeth); U. Thorsteinsdottir (Unnur); C.M. van Duijn (Cock); B.Z. Alizadeh (Behrooz); S.M. Bergmann (Sven); E.A. Boerwinkle (Eric); H.A. Boyd (Heather); L. Crisponi (Laura); P. Gasparini (Paolo); C. Gieger (Christian); T.B. Harris (Tamara); E. Ingelsson (Erik); M.-R. Jarvelin (Marjo-Riitta); P. Kraft (Peter); D.A. Lawlor (Debbie); A. Metspalu (Andres); C.E. Pennell (Craig); P.M. Ridker (Paul); H. Snieder (Harold); T.I.A. Sørensen; T.D. Spector (Timothy); D.P. Strachan (David P.); A.G. Uitterlinden (André); N.J. Wareham (Nick); E. Widen (Elisabeth); M. Zygmunt (Marek); A. Murray (Anna); D.F. Easton (Douglas); J-A. Zwart (John-Anker); J. Murabito (Joanne); K.K. Ong (Ken K.)

    2014-01-01

    textabstractAge at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and

  16. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche

    NARCIS (Netherlands)

    Perry, John R. B.; Day, Felix; Elks, Cathy E.; Sulem, Patrick; Thompson, Deborah J.; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I.; Esko, Toenu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q.; Corre, Tanguy; Cousminer, Diana L.; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D.; Kjellqvist, Sanela; Lunetta, Kathryn L.; McMahon, George; Nolte, Ilja M.; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V.; Stolk, Lisette; Teumer, Alexander; Tsernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K.; Amin, Najaf; Bierut, Laura J.; Byrne, Enda M.; Hottenga, Jouke-Jan; Koller, Daniel L.; Mangino, Massimo; Pers, Tune H.; Yerges-Armstrong, Laura M.; Zhao, Jing Hua; Andrulis, Irene L.; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W.; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E.; Bolla, Manjeet K.; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E.; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collee, J. Margriet; Couch, Fergus J.; Couper, David; Coviello, Andrea D.; Cox, Angela; Czene, Kamila; D'adamo, Adamo Pio; Smith, George Davey; De Vivo, Immaculata; Demerath, Ellen W.; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K.; Dunning, Alison M.; Eiriksdottir, Gudny; Eriksson, Johan G.; Fasching, Peter A.; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E.; Garcia-Closas, Montserrat; Geller, Frank; de Geus, Eco E. J.; Giles, Graham G.; Gudbjartsson, Daniel F.; Gudnason, Vilmundur; Guenel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A.; Heath, AndrewC.; Hofman, Albert; Hooning, Maartje J.; Hopper, John L.; Hu, Frank B.; Hunter, David J.; Karasik, David; Kiel, Douglas P.; Knight, Julia A.; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Maegi, Reedik; Magnusson, Patrik K.; Mannermaa, Arto; Martin, Nicholas G.; Masson, Gisli; McArdle, Patrick F.; McArdle, Wendy L.; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L.; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A.; Oldehinkel, Albertine J.; Oostra, Ben A.; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L.; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul D. P.; Postma, Dirkje S.; Pouta, Anneli; Pylkaes, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M.; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K.; Southey, Mellissa C.; Sovio, Ulla; Stampfer, Meir J.; Stoeckl, Doris; Storniolo, Anna M.; Timpson, Nicholas J.; Tyrer, Jonathan; Visser, Jenny A.; Vollenweider, Peter; Voelzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce H. R.; Wright, Margaret J.; Boomsma, Dorret I.; Econs, Michael J.; Khaw, Kay-Tee; Loos, Ruth J. F.; McCarthy, Mark I.; Montgomery, Grant W.; Rice, John P.; Streeten, Elizabeth A.; Thorsteinsdottir, Unnur; van Duijn, Cornelia M.; Alizadeh, Behrooz Z.; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A.; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B.; Ingelsson, Erik; Jaervelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E.; Ridker, Paul M.; Snieder, Harold; Sorensen, Thorkild I. A.; Spector, Tim D.; Strachan, David P.; Uitterlinden, Andre G.; Wareham, Nicholas J.; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F.; Stefansson, Kari; Murabito, Joanne M.; Ong, Ken K.

    2014-01-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-causemortality(1). Studies of rare human disorders of puberty and animal mo

  17. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche

    NARCIS (Netherlands)

    Perry, John R. B.; Day, Felix; Elks, Cathy E.; Sulem, Patrick; Thompson, Deborah J.; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I.; Esko, Toenu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q.; Corre, Tanguy; Cousminer, Diana L.; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D.; Kjellqvist, Sanela; Lunetta, Kathryn L.; McMahon, George; Nolte, Ilja M.; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V.; Stolk, Lisette; Teumer, Alexander; Tsernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K.; Amin, Najaf; Bierut, Laura J.; Byrne, Enda M.; Hottenga, Jouke-Jan; Koller, Daniel L.; Mangino, Massimo; Pers, Tune H.; Yerges-Armstrong, Laura M.; Zhao, Jing Hua; Andrulis, Irene L.; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W.; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E.; Bolla, Manjeet K.; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E.; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collee, J. Margriet; Couch, Fergus J.; Couper, David; Coviello, Andrea D.; Cox, Angela; Czene, Kamila; D'adamo, Adamo Pio; Smith, George Davey; De Vivo, Immaculata; Demerath, Ellen W.; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K.; Dunning, Alison M.; Eiriksdottir, Gudny; Eriksson, Johan G.; Fasching, Peter A.; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E.; Garcia-Closas, Montserrat; Geller, Frank; de Geus, Eco E. J.; Giles, Graham G.; Gudbjartsson, Daniel F.; Gudnason, Vilmundur; Guenel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A.; Heath, AndrewC.; Hofman, Albert; Hooning, Maartje J.; Hopper, John L.; Hu, Frank B.; Hunter, David J.; Karasik, David; Kiel, Douglas P.; Knight, Julia A.; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Maegi, Reedik; Magnusson, Patrik K.; Mannermaa, Arto; Martin, Nicholas G.; Masson, Gisli; McArdle, Patrick F.; McArdle, Wendy L.; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L.; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A.; Oldehinkel, Albertine J.; Oostra, Ben A.; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L.; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul D. P.; Postma, Dirkje S.; Pouta, Anneli; Pylkaes, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M.; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K.; Southey, Mellissa C.; Sovio, Ulla; Stampfer, Meir J.; Stoeckl, Doris; Storniolo, Anna M.; Timpson, Nicholas J.; Tyrer, Jonathan; Visser, Jenny A.; Vollenweider, Peter; Voelzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce H. R.; Wright, Margaret J.; Boomsma, Dorret I.; Econs, Michael J.; Khaw, Kay-Tee; Loos, Ruth J. F.; McCarthy, Mark I.; Montgomery, Grant W.; Rice, John P.; Streeten, Elizabeth A.; Thorsteinsdottir, Unnur; van Duijn, Cornelia M.; Alizadeh, Behrooz Z.; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A.; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B.; Ingelsson, Erik; Jaervelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E.; Ridker, Paul M.; Snieder, Harold; Sorensen, Thorkild I. A.; Spector, Tim D.; Strachan, David P.; Uitterlinden, Andre G.; Wareham, Nicholas J.; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F.; Stefansson, Kari; Murabito, Joanne M.; Ong, Ken K.

    2014-01-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-causemortality(1). Studies of rare human disorders of puberty and animal

  18. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche

    NARCIS (Netherlands)

    J.R.B. Perry (John); F.R. Day (Felix); C.E. Elks (Cathy); P. Sulem (Patrick); D. Thompson (Deborah); T. Ferreira (Teresa); C. He (Chunyan); D.I. Chasman (Daniel); T. Esko (Tõnu); G. Thorleifsson (Gudmar); E. Albrecht (Eva); W.Q. Ang (Wei); T. Corre (Tanguy); D.L. Cousminer (Diana); B. Feenstra (Bjarke); N. Franceschini (Nora); A. Ganna (Andrea); A.D. Johnson (Andrew); S. Kjellqvist (Sanela); K.L. Lunetta (Kathryn); G. Mcmahon (George); I.M. Nolte (Ilja); L. Paternoster (Lavinia); E. Porcu (Eleonora); G.D. Smith; L. Stolk (Lisette); A. Teumer (Alexander); N. Tsernikova (Natalia); E. Tikkanen (Emmi); S. Ulivi (Shelia); E.K. Wagner (Erin K.); N. Amin (Najaf); L.J. Bierut (Laura); E.M. Byrne (Enda); J.J. Hottenga (Jouke Jan); D.L. Koller (Daniel); M. Mangino (Massimo); T.H. Pers (Tune); L.M. Yerges-Armstrong (Laura); J.H. Zhao; I.L. Andrulis (Irene); H. Anton-Culver (Hoda); F. Atsma (Femke); S. Bandinelli (Stefania); M.W. Beckmann (Matthias W.); J. Benítez (Javier); C. Blomqvist (Carl); S.E. Bojesen (Stig); M.K. Bolla (Manjeet); B. Bonnani (Bernardo); H. Brauch (Hiltrud); H. Brenner (Hermann); J.E. Buring (Julie); J. Chang-Claude (Jenny); S.J. Chanock (Stephen); J. Chen (Jinhui); G. Chenevix-Trench (Georgia); J.M. Collee (Margriet); F.J. Couch (Fergus); D.J. Couper (David); A.D. Coviello (Andrea); A. Cox (Angela); K. Czene (Kamila); P. d' Adamo (Pio); G.D. Smith; I. de Vivo (Immaculata); E.W. Demerath (Ellen); J. Dennis (Joe); P. Devilee (Peter); A.K. Dieffenbach (Aida Karina); A.M. Dunning (Alison); G. Eiriksdottir (Gudny); K. Hagen (Knut); P.A. Fasching (Peter); L. Ferrucci (Luigi); D. Flesch-Janys (Dieter); H. Flyger (Henrik); T. Foroud (Tatiana); L. Franke (Lude); M. Garcia (Melissa); M. García-Closas (Montserrat); F. Geller (Frank); E.E.J. De Geus (Eco E. J.); G.G. Giles (Graham); D.F. Gudbjartsson (Daniel); V. Gudnason (Vilmundur); P. Guénel (Pascal); S. Guo (Suiqun); P. Hall (Per); U. Hamann (Ute); R. Haring (Robin); C.A. Hartman (Catharina); A.C. Heath (Andrew); A. Hofman (Albert); M.J. Hooning (Maartje); J.L. Hopper (John); F.B. Hu (Frank); D. Hunter (David); D. Karasik (David); D.P. Kiel (Douglas P.); J.A. Knight (Julia); V-M. Kosma (Veli-Matti); Z. Kutalik (Zoltán); S. Lai (Sandra); D. Lambrechts (Diether); A. Lindblom (Annika); R. Mägi (Reedik); P.K. Magnusson (Patrik); A. Mannermaa (Arto); N.G. Martin (Nicholas); G. Masson (Gisli); P.F. McArdle (Patrick); W.L. McArdle (Wendy); M. Melbye (Mads); K. Michailidou (Kyriaki); E. Mihailov (Evelin); L. Milani (Lili); R.L. Milne (Roger L); H. Nevanlinna (Heli); P. Neven (Patrick); C. Nohr (Christian); A.J. Oldehinkel (Albertine); B.A. Oostra (Ben); A. Palotie (Aarno); M. Peacock (Munro); N.L. Pedersen (Nancy L.); P. Peterlongo (Paolo); J. Peto (Julian); P.D.P. Pharoah (Paul); D.S. Postma (Dirkje); A. Pouta (Anneli); K. Pykäs (Katri); P. Radice (Paolo); S.M. Ring (Susan); F. Rivadeneira Ramirez (Fernando); A. Robino (Antonietta); L.M. Rose (Lynda); A. Rudolph (Anja); V. Salomaa (Veikko); S. Sanna (Serena); D. Schlessinger (David); M.K. Schmidt (Marjanka); M.C. Southey (Mellissa C.); U. Sovio (Ulla); M.J. Stampfer (Meir J.); D. Stöckl (Doris); A.M. Storniolo (Anna M.); N. Timpson (Nicholas); J.P. Tyrer (Jonathan); J.A. Visser (Jenny); P. Vollenweider (Peter); H. Völzke (Henry); G. Waeber (Gérard); M. Waldenberger (Melanie); H. Wallaschofski (Henri); Q. Wang (Qing); G.A.H.M. Willemsen (Gonneke); R. Winqvist (Robert); B.H.R. Wolffenbuttel (Bruce H. R.); M.J. Wright (Margaret); D.I. Boomsma (Dorret); M.J. Econs (Michael); K.T. Khaw; R.J.F. Loos (Ruth); M.I. McCarthy (Mark); G.W. Montgomery (Grant); J.P. Rice (John); E.A. Streeten (Elizabeth); U. Thorsteinsdottir (Unnur); C.M. van Duijn (Cock); B.Z. Alizadeh (Behrooz); S.M. Bergmann (Sven); E.A. Boerwinkle (Eric); H.A. Boyd (Heather); L. Crisponi (Laura); P. Gasparini (Paolo); C. Gieger (Christian); T.B. Harris (Tamara); E. Ingelsson (Erik); M.-R. Jarvelin (Marjo-Riitta); P. Kraft (Peter); D.A. Lawlor (Debbie); A. Metspalu (Andres); C.E. Pennell (Craig); P.M. Ridker (Paul); H. Snieder (Harold); T.I.A. Sørensen; T.D. Spector (Timothy); D.P. Strachan (David P.); A.G. Uitterlinden (André); N.J. Wareham (Nick); E. Widen (Elisabeth); M. Zygmunt (Marek); A. Murray (Anna); D.F. Easton (Douglas); J-A. Zwart (John-Anker); J. Murabito (Joanne); K.K. Ong (Ken K.)

    2014-01-01

    textabstractAge at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and

  19. Breeding for Parents of Hybrid Rice with Maize pepc Gene

    Institute of Scientific and Technical Information of China (English)

    WANG De-zheng; WANG Xiu-feng; JIAO De-mao; WU Shuang; LI Xia; LI Li; CHI Wei; WANG Shou-hai; LI Cheng-quan; LUO Yan-chang

    2002-01-01

    The results of the investigation on transgenic rice with maize C4-specific phosphoenolpyruvate carboxylase (pepc) gene showed that the transgenic rice plants with the maize pepc gene expressed at high level and the maize PEPC expression was inherited in the progenies in a Mendelian manner. The transgenic plants had PEPC activity of more than 10-fold higher than untransformed plants. As compared with untransformed plants, the panicle per plant, spikelet per panicle, 1000-grain weight and grain-weight per plant for transgenic plants increased by 14.9 %, 5.7%, 1.3 % and 13.9 %, respectively. By crossing the maize pepc gene was incorporated into the parents of hybrid rice, which were the photo-sensitive genic male sterile (PGMS)lines of two-line hybrid rice such as Peiai64s, 7001s, 2302s, 2304s and 2306s-1, and the BT type of cytoplasmic male sterile (CMS) line of three-line hybrid rice such as Shuangjiu A, and restorer lines 5129, Wanjing97 in the spring of 1998. The following progresses were made: (1) The inheritance of the high-level expression of the maize PEPC was stable in different genetic background of rice; (2) PEPC activity of F1 hybrid was the mean of the two parents. Its saturated photosynthetic rate (Pn) rose to 50 % higher than that of the receptor parent. These results demonstrated that it is possible to increase the vigor of the rice plant by transgenic approach with maize pepc gene; (3) The activity of PEPC in leaf could be considered as the major physiological index because the correlation coefficient between PEPC activity and Pn was 0.6470 * *; (4) We have developed three rice lines with maize pepc gene; (5) The selection method of high photosynthetic efficiency rice has been established, which includes soaking seeds into solution of hygromycin phosphotransferase to germinate, tracing the pepc gene by PCR analysis, evaluating the performance of the rice plants in the field and examining PEPC activities and Pn of rice plants with maize pepc gene.

  20. Gene expression analysis of the ovary of hybrid females of Xenopus laevis and X. muelleri

    Directory of Open Access Journals (Sweden)

    Malone John H

    2008-03-01

    Full Text Available Abstract Background Interspecific hybrids of frogs of the genus Xenopus result in sterile hybrid males and fertile hybrid females. Previous work has demonstrated a dramatic asymmetrical pattern of misexpression in hybrid males compared to the two parental species with relatively few genes misexpressed in comparisons of hybrids and the maternal species (X. laevis and dramatically more genes misexpressed in hybrids compared to the paternal species (X. muelleri. In this work, we examine the gene expression pattern in hybrid females of X. laevis × X. muelleri to determine if this asymmetrical pattern of expression also occurs in hybrid females. Results We find a similar pattern of asymmetry in expression compared to males in that there were more genes differentially expressed between hybrids and X. muelleri compared to hybrids and X. laevis. We also found a dramatic increase in the number of misexpressed genes with hybrid females having about 20 times more genes misexpressed in ovaries compared to testes of hybrid males and therefore the match between phenotype and expression pattern is not supported. Conclusion We discuss these intriguing findings in the context of reproductive isolation and suggest that divergence in female expression may be involved in sterility of hybrid males due to the inherent sensitivity of spermatogenesis as defined by the faster male evolution hypothesis for Haldane's rule.

  1. Stacked -gene hybrids were not found to be superior to glyphosate resistant or Non-GMO corn hybrids

    Science.gov (United States)

    Seed costs of modern corn hybrids genetically modified with multiple traits for insect and herbicide resistance “stacked-gene” are in excess of $100.00 US per acre. Yields and net returns per acre along with yield component data were determined for ten hybrids, four stacked-gene, four glyphosate re...

  2. Comparative gene expression profiles between heterotic and non-heterotic hybrids of tetraploid Medicago sativa

    Directory of Open Access Journals (Sweden)

    Nettleton Dan

    2009-08-01

    Full Text Available Abstract Background Heterosis, the superior performance of hybrids relative to parents, has clear agricultural value, but its genetic control is unknown. Our objective was to test the hypotheses that hybrids expressing heterosis for biomass yield would show more gene expression levels that were different from midparental values and outside the range of parental values than hybrids that do not exhibit heterosis. Results We tested these hypotheses in three Medicago sativa (alfalfa genotypes and their three hybrids, two of which expressed heterosis for biomass yield and a third that did not, using Affymetrix M. truncatula GeneChip arrays. Alfalfa hybridized to approximately 47% of the M. truncatula probe sets. Probe set signal intensities were analyzed using MicroArray Suite v.5.0 (MAS and robust multi-array average (RMA algorithms. Based on MAS analysis, the two heterotic hybrids performed similarly, with about 27% of genes showing differential expression among the parents and their hybrid compared to 12.5% for the non-heterotic hybrid. At a false discovery rate of 0.15, 4.7% of differentially expressed genes in hybrids (~300 genes showed nonadditive expression compared to only 0.5% (16 genes in the non-heterotic hybrid. Of the nonadditively expressed genes, approximately 50% showed expression levels that fell outside the parental range in heterotic hybrids, but only one of 16 showed a similar profile in the non-heterotic hybrid. Genes whose expression differed in the parents were three times more likely to show nonadditive expression than genes whose parental transcript levels were equal. Conclusion The higher proportions of probe sets with expression level that differed from the parental midparent value and that were more extreme than either parental value in the heterotic hybrids compared to a non-heterotic hybrid were also found using RMA. We conclude that nonadditive expression of transcript levels may contribute to heterosis for biomass

  3. Regulation of a metallothionein-growth hormone hybrid gene in bovine papilloma virus.

    OpenAIRE

    Pavlakis, G N; Hamer, D H

    1983-01-01

    We have constructed bovine papilloma virus recombinants carrying a hybrid gene in which human growth hormone structural sequences are fused to the promoter and presumptive control region of the mouse metallothionein-I gene. Mouse cells transformed with the recombinants synthesize metallothionein-growth hormone hybrid mRNA with the same 5' end as metallothionein mRNA. Hybrid mRNA is inducible by cadmium but not by dexamethasone, whereas the chromosomal metallothionein genes in the same cells a...

  4. Rapid male-specific regulatory divergence and down regulation of spermatogenesis genes in Drosophila species hybrids.

    Directory of Open Access Journals (Sweden)

    Jennifer Ferguson

    Full Text Available In most crosses between closely related species of Drosophila, the male hybrids are sterile and show postmeiotic abnormalities. A series of gene expression studies using genomic approaches have found significant down regulation of postmeiotic spermatogenesis genes in sterile male hybrids. These results have led some to suggest a direct relationship between down regulation in gene expression and hybrid sterility. An alternative explanation to a cause-and-effect relationship between misregulation of gene expression and male sterility is rapid divergence of male sex regulatory elements leading to incompatible interactions in an interspecies hybrid genome. To test the effect of regulatory divergence in spermatogenesis gene expression, we isolated 35 fertile D. simulans strains with D. mauritiana introgressions in either the X, second or third chromosome. We analyzed gene expression in these fertile hybrid strains for a subset of spermatogenesis genes previously reported as significantly under expressed in sterile hybrids relative to D. simulans. We found that fertile autosomal introgressions can cause levels of gene down regulation similar to that of sterile hybrids. We also found that X chromosome heterospecific introgressions cause significantly less gene down regulation than autosomal introgressions. Our results provide evidence that rapid male sex gene regulatory divergence can explain misexpression of spermatogenesis genes in hybrids.

  5. Rapid male-specific regulatory divergence and down regulation of spermatogenesis genes in Drosophila species hybrids.

    Science.gov (United States)

    Ferguson, Jennifer; Gomes, Suzanne; Civetta, Alberto

    2013-01-01

    In most crosses between closely related species of Drosophila, the male hybrids are sterile and show postmeiotic abnormalities. A series of gene expression studies using genomic approaches have found significant down regulation of postmeiotic spermatogenesis genes in sterile male hybrids. These results have led some to suggest a direct relationship between down regulation in gene expression and hybrid sterility. An alternative explanation to a cause-and-effect relationship between misregulation of gene expression and male sterility is rapid divergence of male sex regulatory elements leading to incompatible interactions in an interspecies hybrid genome. To test the effect of regulatory divergence in spermatogenesis gene expression, we isolated 35 fertile D. simulans strains with D. mauritiana introgressions in either the X, second or third chromosome. We analyzed gene expression in these fertile hybrid strains for a subset of spermatogenesis genes previously reported as significantly under expressed in sterile hybrids relative to D. simulans. We found that fertile autosomal introgressions can cause levels of gene down regulation similar to that of sterile hybrids. We also found that X chromosome heterospecific introgressions cause significantly less gene down regulation than autosomal introgressions. Our results provide evidence that rapid male sex gene regulatory divergence can explain misexpression of spermatogenesis genes in hybrids.

  6. Ploidy and Hybridity Effects on Growth Vigor and Gene Expression in Arabidopsis thaliana Hybrids and Their Parents.

    Science.gov (United States)

    Miller, Marisa; Zhang, Changqing; Chen, Z Jeffrey

    2012-04-01

    Both ploidy and hybridity affect cell size and growth vigor in plants and animals, but the relative effects of genome dosage and hybridization on biomass, fitness, and gene expression changes have not been systematically examined. Here we performed the first comparative analysis of seed, cell, and flower sizes, starch and chlorophyll content, biomass, and gene expression changes in diploid, triploid, and tetraploid hybrids and their respective parents in three Arabidopsis thaliana ecotypes: Columbia, C24, and Landsberg erecta (Ler). Ploidy affects many morphological and fitness traits, including stomatal size, flower size, and seed weight, whereas hybridization between the ecotypes leads to altered expression of central circadian clock genes and increased starch and chlorophyll content, biomass, and seed weight. However, varying ploidy levels has subtle effects on biomass, circadian clock gene expression, and chlorophyll and starch content. Interestingly, biomass, starch content, and seed weight are significantly different between the reciprocal hybrids at all ploidy levels tested, with the lowest and highest levels found in the reciprocal triploid hybrids, suggesting parent-of-origin effects on biomass, starch content, and seed weight. These findings provide new insights into molecular events of polyploidy and heterosis, as well as complex agronomic traits that are important to biomass and seed production in hybrid and polyploid crops.

  7. Hybrid Lethal Systems in the Drosophila Melanogaster Species Complex. I. the Maternal Hybrid Rescue (Mhr) Gene of Drosophila Simulans

    Science.gov (United States)

    Sawamura, K.; Taira, T.; Watanabe, T. K.

    1993-01-01

    Hybrid females from Drosophila simulans females X Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as late larvae. The other two classes are sterile adults. Letting C, X, and Y designate egg cytoplasm, X, and Y chromosomes, respectively, and subscripts m and s stand for melanogaster and simulans, C(m)X(m)Y(s) males are lethal in the larval stage and are rescued by the previously reported genes, Lhr (Lethal hybrid rescue) in simulans or Hmr (Hybrid male rescue) in melanogaster. We report here another rescue gene located on the second chromosome of simulans, mhr (maternal hybrid rescue) that, when present in the mother, rescues C(s)X(m)X(s) females from embryonic lethality. It has been postulated that the hybrids not carrying the X(s) like C(m)X(m)Y(s) males are larval lethal and that the hybrids carrying both the C(s) and the X(m) like C(s)X(m)X(s) females are embryonic lethal. According to these postulates C(s)X(m)Y(s) males (obtained by mating attached-X simulans females to melanogaster males) should be doubly lethal, at both embryo and larval stages. When both rescuing genes are present, Hmr in the father and mhr in the mother, males of this genotype are fully viable, as predicted. PMID:8436276

  8. Breeding lines with neutral genes to improve fertility of intersubspecific hybrid rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Since the sterility neutral allele S5n has been incorporated into indica or japonica varieties, many intersubspecific hybrids have been released commercially. These hybrids showed high heterosis, but some of them exhibited unstable seed setting rate under low temperature.When the hybrids flowered at low temperature, the fertility of female gamete was normal but the pollen showed sterile. To improve the stability of fertility, the effect of pollen sterility neutral gene was studied for intersubspecific hybrids.

  9. An essential cell cycle regulation gene causes hybrid inviability in Drosophila.

    Science.gov (United States)

    Phadnis, Nitin; Baker, EmilyClare P; Cooper, Jacob C; Frizzell, Kimberly A; Hsieh, Emily; de la Cruz, Aida Flor A; Shendure, Jay; Kitzman, Jacob O; Malik, Harmit S

    2015-12-18

    Speciation, the process by which new biological species arise, involves the evolution of reproductive barriers, such as hybrid sterility or inviability between populations. However, identifying hybrid incompatibility genes remains a key obstacle in understanding the molecular basis of reproductive isolation. We devised a genomic screen, which identified a cell cycle-regulation gene as the cause of male inviability in hybrids resulting from a cross between Drosophila melanogaster and D. simulans. Ablation of the D. simulans allele of this gene is sufficient to rescue the adult viability of hybrid males. This dominantly acting cell cycle regulator causes mitotic arrest and, thereby, inviability of male hybrid larvae. Our genomic method provides a facile means to accelerate the identification of hybrid incompatibility genes in other model and nonmodel systems.

  10. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    Science.gov (United States)

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

    2016-06-01

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

  11. Genes of both parental origins are differentially involved in early embryogenesis of a tobacco interspecies hybrid.

    Directory of Open Access Journals (Sweden)

    Jun-E Zhang

    Full Text Available BACKGROUND: In animals, early embryonic development is largely dependent on maternal transcripts synthesized during gametogenesis. However, in higher plants, the extent of maternal control over zygote development and early embryogenesis is not fully understood yet. Nothing is known about the activity of the parental genomes during seed formation of interspecies hybrids. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that an interspecies hybridization system between SR1 (Nicotiana tabacum and Hamayan (N. rustica has been successfully established. Based on the system we selected 58 genes that have polymorphic sites between SR1 and Hamayan, and analyzed the allele-specific expression of 28 genes in their hybrid zygotes (Hamayan x SR1. Finally the allele-specific expressions of 8 genes in hybrid zygotes were repeatedly confirmed. Among them, 4 genes were of paternal origin, 1 gene was of maternal origin and 3 genes were of biparental origin. These results revealed obvious biparental involvement and differentially contribution of parental-origin genes to zygote development in the interspecies hybrid. We further detected the expression pattern of the genes at 8-celled embryo stage found that the involvement of the parental-origin genes may change at different stages of embryogenesis. CONCLUSIONS/SIGNIFICANCE: We reveal that genes of both parental origins are differentially involved in early embryogenesis of a tobacco interspecies hybrid and functions in a developmental stage-dependent manner. This finding may open a window to seek for the possible molecular mechanism of hybrid vigor.

  12. The Use of Whole-Mount "in Situ" Hybridization to Illustrate Gene Expression Regulation

    Science.gov (United States)

    Llamusí, Beatriz; Muñoz-Soriano, Verónica; Paricio, Nuria; Artero, Rubén

    2014-01-01

    "In situ" hybridization is a widely used technique for studying gene expression. Here, we describe two experiments addressed to postgraduate genetics students in which the effect of transcription factors on gene expression is analyzed in "Drosophila embryos of different genotypes by whole-mount in situ hybridization. In one of the…

  13. Targeted Gene Capture by Hybridization to Illuminate Ecosystem Functioning.

    Science.gov (United States)

    Ribière, Céline; Beugnot, Réjane; Parisot, Nicolas; Gasc, Cyrielle; Defois, Clémence; Denonfoux, Jérémie; Boucher, Delphine; Peyretaillade, Eric; Peyret, Pierre

    2016-01-01

    Microbial communities are extremely abundant and diverse on earth surface and play key role in the ecosystem functioning. Thus, although next-generation sequencing (NGS) technologies have greatly improved knowledge on microbial diversity, it is necessary to reduce the biological complexity to better understand the microorganism functions. To achieve this goal, we describe a promising approach, based on the solution hybrid selection (SHS) method for the selective enrichment in a target-specific biomarker from metagenomic and metatranscriptomic samples. The success of this method strongly depends on the determination of sensitive, specific, and explorative probes to assess the complete targeted gene repertoire. Indeed, in this method, RNA probes were used to capture large DNA or RNA fragments harboring biomarkers of interest that potentially allow to link structure and function of communities of interest.

  14. Sterility and gene expression in hybrid males of Xenopus laevis and X. muelleri.

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    John H Malone

    Full Text Available BACKGROUND: Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri. METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. CONCLUSIONS/SIGNIFICANCE: Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly

  15. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository.

    Science.gov (United States)

    Edgar, Ron; Domrachev, Michael; Lash, Alex E

    2002-01-01

    The Gene Expression Omnibus (GEO) project was initiated in response to the growing demand for a public repository for high-throughput gene expression data. GEO provides a flexible and open design that facilitates submission, storage and retrieval of heterogeneous data sets from high-throughput gene expression and genomic hybridization experiments. GEO is not intended to replace in house gene expression databases that benefit from coherent data sets, and which are constructed to facilitate a particular analytic method, but rather complement these by acting as a tertiary, central data distribution hub. The three central data entities of GEO are platforms, samples and series, and were designed with gene expression and genomic hybridization experiments in mind. A platform is, essentially, a list of probes that define what set of molecules may be detected. A sample describes the set of molecules that are being probed and references a single platform used to generate its molecular abundance data. A series organizes samples into the meaningful data sets which make up an experiment. The GEO repository is publicly accessible through the World Wide Web at http://www.ncbi.nlm.nih.gov/geo.

  16. Male sex interspecies divergence and down regulation of expression of spermatogenesis genes in Drosophila sterile hybrids.

    Science.gov (United States)

    Sundararajan, Vignesh; Civetta, Alberto

    2011-01-01

    Male sex genes have shown a pattern of rapid interspecies divergence at both the coding and gene expression level. A common outcome from crosses between closely-related species is hybrid male sterility. Phenotypic and genetic studies in Drosophila sterile hybrid males have shown that spermatogenesis arrest is postmeiotic with few exceptions, and that most misregulated genes are involved in late stages of spermatogenesis. Comparative studies of gene regulation in sterile hybrids and parental species have mainly used microarrays providing a whole genome representation of regulatory problems in sterile hybrids. Real-time PCR studies can reject or reveal differences not observed in microarray assays. Moreover, differences in gene expression between samples can be dependant on the source of RNA (e.g., whole body vs. tissue). Here we survey expression in D. simulans, D. mauritiana and both intra and interspecies hybrids using a real-time PCR approach for eight genes expressed at the four main stages of sperm development. We find that all genes show a trend toward under expression in the testes of sterile hybrids relative to parental species with only the two proliferation genes (bam and bgcn) and the two meiotic class genes (can and sa) showing significant down regulation. The observed pattern of down regulation for the genes tested can not fully explain hybrid male sterility. We discuss the down regulation of spermatogenesis genes in hybrids between closely-related species within the contest of rapid divergence experienced by the male genome, hybrid sterility and possible allometric changes due to subtle testes-specific developmental abnormalities.

  17. An Epigenetic Role for Disrupted Paternal Gene Expression in Postzygotic Seed Abortion in Arabidopsis Interspecific Hybrids.

    Science.gov (United States)

    Kirkbride, Ryan C; Yu, Helen Hong; Nah, Gyoungju; Zhang, Changqing; Shi, Xiaoli; Chen, Z Jeffrey

    2015-12-07

    Interspecific hybrids often increase the levels of heterozygosity and hybrid vigor, but some interspecific hybrid seeds are aborted shortly after fertilization. The mechanism behind this postzygotic seed abortion is poorly understood. Here, we report genome-wide analysis of allelic expression changes in developing siliques and seeds in three F1 interspecific crosses between Arabidopsis thaliana (Col, Ler, or C24) and Arabidopsis arenosa. The majority of maternally expressed genes (MEGs) were shared among all three F1 interspecific crosses, whereas ∼90% of 272 paternally expressed genes (PEGs) were found only in one or two F1 crosses, suggesting a role for disrupted paternal gene expression in seed abortion that varies in different crosses. Consistent with this notion, 12 PEGs in the infertile interspecific hybrids matched MEGs in fertile intraspecific hybrids. This disruption of PEGs in the interspecific hybrids was consistent with the upregulation of the genes in the paternal-excess interploidy cross (2X6) between a diploid mother and a hexaploid father, leading to the seed abortion. Moreover, a subset of PEGs in the interspecific crosses were also upregulated in the intraspecific hybrid met1XWT or meaXWT, in which the mutant of MET1 (DNA METHYLTRANSFERASE1) or MEDEA, a Polycomb Repressive Complex2 gene, was used as the maternal parent. These data suggest that maternal epigenetic factors and paternal gene expression play important roles in the postzygotic seed abortion in interspecific hybrids or neo-allopolyploids.

  18. A Six Nuclear Gene Phylogeny of Citrus (Rutaceae) Taking into Account Hybridization and Lineage Sorting

    Science.gov (United States)

    Keremane, Manjunath L.; Lee, Richard F.; Maureira-Butler, Ivan J.; Roose, Mikeal L.

    2013-01-01

    Background Genus Citrus (Rutaceae) comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4–12 Ma), incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species. Methodology and Principal Findings (1) We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2) Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3) To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation). Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test. Conclusions We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches–gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence simulations. We

  19. A six nuclear gene phylogeny of Citrus (Rutaceae taking into account hybridization and lineage sorting.

    Directory of Open Access Journals (Sweden)

    Chandrika Ramadugu

    Full Text Available BACKGROUND: Genus Citrus (Rutaceae comprises many important cultivated species that generally hybridize easily. Phylogenetic study of a group showing extensive hybridization is challenging. Since the genus Citrus has diverged recently (4-12 Ma, incomplete lineage sorting of ancestral polymorphisms is also likely to cause discrepancies among genes in phylogenetic inferences. Incongruence of gene trees is observed and it is essential to unravel the processes that cause inconsistencies in order to understand the phylogenetic relationships among the species. METHODOLOGY AND PRINCIPAL FINDINGS: (1 We generated phylogenetic trees using haplotype sequences of six low copy nuclear genes. (2 Published simple sequence repeat data were re-analyzed to study population structure and the results were compared with the phylogenetic trees constructed using sequence data and coalescence simulations. (3 To distinguish between hybridization and incomplete lineage sorting, we developed and utilized a coalescence simulation approach. In other studies, species trees have been inferred despite the possibility of hybridization having occurred and used to generate null distributions of the effect of lineage sorting alone (by coalescent simulation. Since this is problematic, we instead generate these distributions directly from observed gene trees. Of the six trees generated, we used the most resolved three to detect hybrids. We found that 11 of 33 samples appear to be affected by historical hybridization. Analysis of the remaining three genes supported the conclusions from the hybrid detection test. CONCLUSIONS: We have identified or confirmed probable hybrid origins for several Citrus cultivars using three different approaches-gene phylogenies, population structure analysis and coalescence simulation. Hybridization and incomplete lineage sorting were identified primarily based on differences among gene phylogenies with reference to null expectations via coalescence

  20. Genome-wide misexpression of X-linked versus autosomal genes associated with hybrid male sterility.

    Science.gov (United States)

    Lu, Xuemei; Shapiro, Joshua A; Ting, Chau-Ti; Li, Yan; Li, Chunyan; Xu, Jin; Huang, Huanwei; Cheng, Ya-Jen; Greenberg, Anthony J; Li, Shou-Hsien; Wu, Mao-Lien; Shen, Yang; Wu, Chung-I

    2010-08-01

    Postmating reproductive isolation is often manifested as hybrid male sterility, for which X-linked genes are overrepresented (the so-called large X effect). In contrast, X-linked genes are significantly under-represented among testis-expressing genes. This seeming contradiction may be germane to the X:autosome imbalance hypothesis on hybrid sterility, in which the X-linked effect is mediated mainly through the misexpression of autosomal genes. In this study, we compared gene expression in fertile and sterile males in the hybrids between two Drosophila species. These hybrid males differ only in a small region of the X chromosome containing the Ods-site homeobox (OdsH) (also known as Odysseus) locus of hybrid sterility. Of genes expressed in the testis, autosomal genes were, indeed, more likely to be misexpressed than X-linked genes under the sterilizing action of OdsH. Since this mechanism of X:autosome interaction is only associated with spermatogenesis, a connection between X:autosome imbalance and the high rate of hybrid male sterility seems plausible.

  1. Hybrid SPR algorithm to select predictive genes for effectual cancer classification

    OpenAIRE

    2012-01-01

    Designing an automated system for classifying DNA microarray data is an extremely challenging problem because of its high dimension and low amount of sample data. In this paper, a hybrid statistical pattern recognition algorithm is proposed to reduce the dimensionality and select the predictive genes for the classification of cancer. Colon cancer gene expression profiles having 62 samples of 2000 genes were used for the experiment. A gene subset of 6 highly informative genes was selecte...

  2. Genome-wide disruption of gene expression in allopolyploids but not hybrids of rice subspecies.

    Science.gov (United States)

    Xu, Chunming; Bai, Yan; Lin, Xiuyun; Zhao, Na; Hu, Lanjuan; Gong, Zhiyun; Wendel, Jonathan F; Liu, Bao

    2014-05-01

    Hybridization and polyploidization are prominent processes in plant evolution. Hybrids and allopolyploids typically exhibit radically altered gene expression patterns relative to their parents, a phenomenon termed "transcriptomic shock." To distinguish the effects of hybridization from polyploidization on coregulation of divergent alleles, we analyzed expression of parental copies (homoeologs) of 11,608 genes using RNA-seq-based transcriptome profiling in reciprocal hybrids and tetraploids constructed from subspecies japonica and indica of Asian rice (Oryza sativa L.). The diploid hybrids and their derived allopolyploids differ dramatically in morphology, despite having the same suite of genes and genic proportions. Allelic and homoeolog-specific transcripts were unequivocally diagnosed in the hybrids and tetraploids based on parent-specific SNPs. Compared with the in silico hybrid (parental mix), the range of progenitor expression divergence was significantly reduced in both reciprocally generated F1 hybrids, presumably due to the ameliorating effects of a common trans environment on divergent cis-factors. In contrast, parental expression differences were greatly elaborated at the polyploid level, which we propose is a consequence of stoichiometric disruptions associated with the numerous chromosomal packaging and volumetric changes accompanying nascent polyploidy. We speculate that the emergent property of "whole genome doubling" has repercussions that reverberate throughout the transcriptome and downstream, ultimately generating altered phenotypes. This perspective may yield insight into the nature of adaptation and the origin of evolutionary novelty accompanying polyploidy.

  3. Transcriptome Analysis Identifies Key Candidate Genes Mediating Purple Ovary Coloration in Asiatic Hybrid Lilies

    Science.gov (United States)

    Xu, Leifeng; Yang, Panpan; Yuan, Suxia; Feng, Yayan; Xu, Hua; Cao, Yuwei; Ming, Jun

    2016-01-01

    Lily tepals have a short lifespan. Once the tepals senesce, the ornamental value of the flower is lost. Some cultivars have attractive purple ovaries and fruits which greatly enhance the ornamental value of Asiatic hybrid lilies. However, little is known about the molecular mechanisms of anthocyanin biosynthesis in Asiatic hybrid lily ovaries. To investigate the transcriptional network that governs purple ovary coloration in Asiatic hybrid lilies, we obtained transcriptome data from green ovaries (S1) and purple ovaries (S2) of Asiatic “Tiny Padhye”. Comparative transcriptome analysis revealed 4228 differentially expressed genes. Differential expression analysis revealed that ten unigenes including four CHS genes, one CHI gene, one F3H gene, one F3′H gene, one DFR gene, one UFGT gene, and one 3RT gene were significantly up-regulated in purple ovaries. One MYB gene, LhMYB12-Lat, was identified as a key transcription factor determining the distribution of anthocyanins in Asiatic hybrid lily ovaries. Further qPCR results showed unigenes related to anthocyanin biosynthesis were highly expressed in purple ovaries of three purple-ovaried Asiatic hybrid lilies at stages 2 and 3, while they showed an extremely low level of expression in ovaries of three green-ovaried Asiatic hybrid lilies during all developmental stages. In addition, shading treatment significantly decreased pigment accumulation by suppressing the expression of several unigenes related to anthocyanin biosynthesis in ovaries of Asiatic “Tiny Padhye”. Lastly, a total of 15,048 Simple Sequence Repeats (SSRs) were identified in 13,710 sequences, and primer pairs for SSRs were designed. The results could further our understanding of the molecular mechanisms of anthocyanin biosynthesis in Asiatic hybrid lily ovaries. PMID:27879624

  4. Regulatory divergence of X-linked genes and hybrid male sterility in mice.

    Science.gov (United States)

    Oka, Ayako; Shiroishi, Toshihiko

    2014-01-01

    Postzygotic reproductive isolation is the reduction of fertility or viability in hybrids between genetically diverged populations. One example of reproductive isolation, hybrid male sterility, may be caused by genetic incompatibility between diverged genetic factors in two distinct populations. Genetic factors involved in hybrid male sterility are disproportionately located on the X chromosome. Recent studies showing the evolutionary divergence in gene regulatory networks or epigenetic effects suggest that the genetic incompatibilities occur at much broader levels than had previously been thought (e.g., incompatibility of protein-protein interactions). The latest studies suggest that evolutionary divergence of transcriptional regulation causes genetic incompatibilities in hybrid animals, and that such incompatibilities preferentially involve X-linked genes. In this review, we focus on recent progress in understanding hybrid sterility in mice, including our studies, and we discuss the evolutionary significance of regulatory divergence for speciation.

  5. Elucidation of the Mechanism of Gene Silencing using Small Interferin RNA: DNA Hybrid Molecules

    Energy Technology Data Exchange (ETDEWEB)

    Dugan, L

    2006-02-08

    The recent discovery that short hybrid RNA:DNA molecules (siHybrids) induce long-term silencing of gene expression in mammalian cells conflicts with the currently hypothesized mechanisms explaining the action of small, interfering RNA (siRNA). As a first step to elucidating the mechanism for this effect, we set out to quantify the delivery of siHybrids and determine their cellular localization in mammalian cells. We then tracked the segregation of the siHybrids into daughter cells after cell division. Markers for siHybrid delivery were shown to enter cells with and without the use of a transfection agent. Furthermore, delivery without transfection agent only occurred after a delay of 2-4 hours, suggesting a degradation process occurring in the cell culture media. Therefore, we studied the effects of nucleases and backbone modifications on the stability of siHybrids under cell culture conditions.

  6. Asymmetrical gene flow in a hybrid zone of Hawaiian Schiedea (Caryophyllaceae species with contrasting mating systems.

    Directory of Open Access Journals (Sweden)

    Lisa E Wallace

    Full Text Available Asymmetrical gene flow, which has frequently been documented in naturally occurring hybrid zones, can result from various genetic and demographic factors. Understanding these factors is important for determining the ecological conditions that permitted hybridization and the evolutionary potential inherent in hybrids. Here, we characterized morphological, nuclear, and chloroplast variation in a putative hybrid zone between Schiedea menziesii and S. salicaria, endemic Hawaiian species with contrasting breeding systems. Schiedea menziesii is hermaphroditic with moderate selfing; S. salicaria is gynodioecious and wind-pollinated, with partially selfing hermaphrodites and largely outcrossed females. We tested three hypotheses: 1 putative hybrids were derived from natural crosses between S. menziesii and S. salicaria, 2 gene flow via pollen is unidirectional from S. salicaria to S. menziesii and 3 in the hybrid zone, traits associated with wind pollination would be favored as a result of pollen-swamping by S. salicaria. Schiedea menziesii and S. salicaria have distinct morphologies and chloroplast genomes but are less differentiated at the nuclear loci. Hybrids are most similar to S. menziesii at chloroplast loci, exhibit nuclear allele frequencies in common with both parental species, and resemble S. salicaria in pollen production and pollen size, traits important to wind pollination. Additionally, unlike S. menziesii, the hybrid zone contains many females, suggesting that the nuclear gene responsible for male sterility in S. salicaria has been transferred to hybrid plants. Continued selection of nuclear genes in the hybrid zone may result in a population that resembles S. salicaria, but retains chloroplast lineage(s of S. menziesii.

  7. HybridGO-Loc: mining hybrid features on gene ontology for predicting subcellular localization of multi-location proteins.

    Directory of Open Access Journals (Sweden)

    Shibiao Wan

    Full Text Available Protein subcellular localization prediction, as an essential step to elucidate the functions in vivo of proteins and identify drugs targets, has been extensively studied in previous decades. Instead of only determining subcellular localization of single-label proteins, recent studies have focused on predicting both single- and multi-location proteins. Computational methods based on Gene Ontology (GO have been demonstrated to be superior to methods based on other features. However, existing GO-based methods focus on the occurrences of GO terms and disregard their relationships. This paper proposes a multi-label subcellular-localization predictor, namely HybridGO-Loc, that leverages not only the GO term occurrences but also the inter-term relationships. This is achieved by hybridizing the GO frequencies of occurrences and the semantic similarity between GO terms. Given a protein, a set of GO terms are retrieved by searching against the gene ontology database, using the accession numbers of homologous proteins obtained via BLAST search as the keys. The frequency of GO occurrences and semantic similarity (SS between GO terms are used to formulate frequency vectors and semantic similarity vectors, respectively, which are subsequently hybridized to construct fusion vectors. An adaptive-decision based multi-label support vector machine (SVM classifier is proposed to classify the fusion vectors. Experimental results based on recent benchmark datasets and a new dataset containing novel proteins show that the proposed hybrid-feature predictor significantly outperforms predictors based on individual GO features as well as other state-of-the-art predictors. For readers' convenience, the HybridGO-Loc server, which is for predicting virus or plant proteins, is available online at http://bioinfo.eie.polyu.edu.hk/HybridGoServer/.

  8. HybridGO-Loc: Mining Hybrid Features on Gene Ontology for Predicting Subcellular Localization of Multi-Location Proteins

    Science.gov (United States)

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2014-01-01

    Protein subcellular localization prediction, as an essential step to elucidate the functions in vivo of proteins and identify drugs targets, has been extensively studied in previous decades. Instead of only determining subcellular localization of single-label proteins, recent studies have focused on predicting both single- and multi-location proteins. Computational methods based on Gene Ontology (GO) have been demonstrated to be superior to methods based on other features. However, existing GO-based methods focus on the occurrences of GO terms and disregard their relationships. This paper proposes a multi-label subcellular-localization predictor, namely HybridGO-Loc, that leverages not only the GO term occurrences but also the inter-term relationships. This is achieved by hybridizing the GO frequencies of occurrences and the semantic similarity between GO terms. Given a protein, a set of GO terms are retrieved by searching against the gene ontology database, using the accession numbers of homologous proteins obtained via BLAST search as the keys. The frequency of GO occurrences and semantic similarity (SS) between GO terms are used to formulate frequency vectors and semantic similarity vectors, respectively, which are subsequently hybridized to construct fusion vectors. An adaptive-decision based multi-label support vector machine (SVM) classifier is proposed to classify the fusion vectors. Experimental results based on recent benchmark datasets and a new dataset containing novel proteins show that the proposed hybrid-feature predictor significantly outperforms predictors based on individual GO features as well as other state-of-the-art predictors. For readers' convenience, the HybridGO-Loc server, which is for predicting virus or plant proteins, is available online at http://bioinfo.eie.polyu.edu.hk/HybridGoServer/. PMID:24647341

  9. Split-gene system for hybrid wheat seed production.

    Science.gov (United States)

    Kempe, Katja; Rubtsova, Myroslava; Gils, Mario

    2014-06-24

    Hybrid wheat plants are superior in yield and growth characteristics compared with their homozygous parents. The commercial production of wheat hybrids is difficult because of the inbreeding nature of wheat and the lack of a practical fertility control that enforces outcrossing. We describe a hybrid wheat system that relies on the expression of a phytotoxic barnase and provides for male sterility. The barnase coding information is divided and distributed at two loci that are located on allelic positions of the host chromosome and are therefore "linked in repulsion." Functional complementation of the loci is achieved through coexpression of the barnase fragments and intein-mediated ligation of the barnase protein fragments. This system allows for growth and maintenance of male-sterile female crossing partners, whereas the hybrids are fertile. The technology does not require fertility restorers and is based solely on the genetic modification of the female crossing partner.

  10. Detection of biomarkers for Hepatocellular Carcinoma using a hybrid univariate gene selection methods

    Directory of Open Access Journals (Sweden)

    Abdel Samee Nagwan M

    2012-08-01

    Full Text Available Abstract Background Discovering new biomarkers has a great role in improving early diagnosis of Hepatocellular carcinoma (HCC. The experimental determination of biomarkers needs a lot of time and money. This motivates this work to use in-silico prediction of biomarkers to reduce the number of experiments required for detecting new ones. This is achieved by extracting the most representative genes in microarrays of HCC. Results In this work, we provide a method for extracting the differential expressed genes, up regulated ones, that can be considered candidate biomarkers in high throughput microarrays of HCC. We examine the power of several gene selection methods (such as Pearson’s correlation coefficient, Cosine coefficient, Euclidean distance, Mutual information and Entropy with different estimators in selecting informative genes. A biological interpretation of the highly ranked genes is done using KEGG (Kyoto Encyclopedia of Genes and Genomes pathways, ENTREZ and DAVID (Database for Annotation, Visualization, and Integrated Discovery databases. The top ten genes selected using Pearson’s correlation coefficient and Cosine coefficient contained six genes that have been implicated in cancer (often multiple cancers genesis in previous studies. A fewer number of genes were obtained by the other methods (4 genes using Mutual information, 3genes using Euclidean distance and only one gene using Entropy. A better result was obtained by the utilization of a hybrid approach based on intersecting the highly ranked genes in the output of all investigated methods. This hybrid combination yielded seven genes (2 genes for HCC and 5 genes in different types of cancer in the top ten genes of the list of intersected genes. Conclusions To strengthen the effectiveness of the univariate selection methods, we propose a hybrid approach by intersecting several of these methods in a cascaded manner. This approach surpasses all of univariate selection methods when

  11. Fine Mapping and Transcriptome Analysis Reveal Candidate Genes Associated with Hybrid Lethality in Cabbage (Brassica Oleracea).

    Science.gov (United States)

    Xiao, Zhiliang; Hu, Yang; Zhang, Xiaoli; Xue, Yuqian; Fang, Zhiyuan; Yang, Limei; Zhang, Yangyong; Liu, Yumei; Li, Zhansheng; Liu, Xing; Liu, Zezhou; Lv, Honghao; Zhuang, Mu

    2017-06-05

    Hybrid lethality is a deleterious phenotype that is vital to species evolution. We previously reported hybrid lethality in cabbage (Brassica oleracea) and performed preliminary mapping of related genes. In the present study, the fine mapping of hybrid lethal genes revealed that BoHL1 was located on chromosome C1 between BoHLTO124 and BoHLTO130, with an interval of 101 kb. BoHL2 was confirmed to be between insertion-deletion (InDels) markers HL234 and HL235 on C4, with a marker interval of 70 kb. Twenty-eight and nine annotated genes were found within the two intervals of BoHL1 and BoHL2, respectively. We also applied RNA-Seq to analyze hybrid lethality in cabbage. In the region of BoHL1, seven differentially expressed genes (DEGs) and five resistance (R)-related genes (two in common, i.e., Bo1g153320 and Bo1g153380) were found, whereas in the region of BoHL2, two DEGs and four R-related genes (two in common, i.e., Bo4g173780 and Bo4g173810) were found. Along with studies in which R genes were frequently involved in hybrid lethality in other plants, these interesting R-DEGs may be good candidates associated with hybrid lethality. We also used SNP/InDel analyses and quantitative real-time PCR to confirm the results. This work provides new insight into the mechanisms of hybrid lethality in cabbage.

  12. Identification by Suppression Subtractive Hybridization of Frankia Genes Induced under Nitrogen-Fixing Conditions▿ †

    OpenAIRE

    Yamaura, Masatoshi; UCHIUMI, Toshiki; Higashi, Shiro; Abe, Mikiko; Kucho, Ken-ichi

    2010-01-01

    Frankia is an actinobacterium that fixes nitrogen under both symbiotic and free-living conditions. We identified genes upregulated in free-living nitrogen-fixing cells by using suppression subtractive hybridization. They included genes with predicted functions related to nitrogen fixation, as well as with unknown function. Their upregulation was a novel finding in Frankia.

  13. Characterization of R genes involved in resistance to Cherry leaf roll virus in paradox hybrids

    Science.gov (United States)

    A single dominant ‘R’ gene (clrvR), in black walnuts (Juglans hindsii) or ‘paradox’ hybrids (J. hindsii x J. regia) confers resistance to Cherry leaf roll virus (CLRV), the causal agent of blackline disease. The identification and cloning of the ‘R’ gene is expected to aid the walnut breeding progra...

  14. Misregulation of Gene Expression and Sterility in Interspecies Hybrids: Causal Links and Alternative Hypotheses.

    Science.gov (United States)

    Civetta, Alberto

    2016-05-01

    Understanding the origin of species is of interest to biologist in general and evolutionary biologist in particular. Hybrid male sterility (HMS) has been a focus in studies of speciation because sterility imposes a barrier to free gene flow between organisms, thus effectively isolating them as distinct species. In this review, I focus on the role of differential gene expression in HMS and speciation. Microarray and qPCR assays have established associations between misregulation of gene expression and sterility in hybrids between closely related species. These studies originally proposed disrupted expression of spermatogenesis genes as a causative of sterility. Alternatively, rapid genetic divergence of regulatory elements, particularly as they relate to the male sex (fast-male evolution), can drive the misregulation of sperm developmental genes in the absence of sterility. The use of fertile hybrids (both backcross and F1 progeny) as controls has lent support to this alternative explanation. Differences in gene expression between fertile and sterile hybrids can also be influenced by a pattern of faster evolution of the sex chromosome (fast-X evolution) than autosomes. In particular, it would be desirable to establish whether known X-chromosome sterility factors can act as trans-regulatory drivers of genome-wide patterns of misregulation. Genome-wide expression studies coupled with assays of proxies of sterility in F1 and BC progeny have identified candidate HMS genes but functional assays, and a better phenotypic characterization of sterility phenotypes, are needed to rigorously test how these genes might contribute to HMS.

  15. Gene expression disruptions of organism versus organ in Drosophila species hybrids.

    Directory of Open Access Journals (Sweden)

    Daniel J Catron

    Full Text Available Hybrid dysfunctions, such as sterility, may result in part from disruptions in the regulation of gene expression. Studies of hybrids within the Drosophila simulans clade have reported genes expressed above or below the expression observed in their parent species, and such misexpression is associated with male sterility in multigenerational backcross hybrids. However, these studies often examined whole bodies rather than testes or had limited replication using less-sensitive but global techniques. Here, we use a new RNA isolation technique to re-examine hybrid gene expression disruptions in both testes and whole bodies from single Drosophila males by real-time quantitative RT-PCR. We find two early-spermatogenesis transcripts are underexpressed in hybrid whole-bodies but not in assays of testes alone, while two late-spermatogenesis transcripts seem to be underexpressed in both whole-bodies and testes alone. Although the number of transcripts surveyed is limited, these results provide some support for a previous hypothesis that the spermatogenesis pathway in these sterile hybrids may be disrupted sometime after the expression of the early meiotic arrest genes.

  16. The probability of a gene tree topology within a phylogenetic network with applications to hybridization detection.

    Directory of Open Access Journals (Sweden)

    Yun Yu

    Full Text Available Gene tree topologies have proven a powerful data source for various tasks, including species tree inference and species delimitation. Consequently, methods for computing probabilities of gene trees within species trees have been developed and widely used in probabilistic inference frameworks. All these methods assume an underlying multispecies coalescent model. However, when reticulate evolutionary events such as hybridization occur, these methods are inadequate, as they do not account for such events. Methods that account for both hybridization and deep coalescence in computing the probability of a gene tree topology currently exist for very limited cases. However, no such methods exist for general cases, owing primarily to the fact that it is currently unknown how to compute the probability of a gene tree topology within the branches of a phylogenetic network. Here we present a novel method for computing the probability of gene tree topologies on phylogenetic networks and demonstrate its application to the inference of hybridization in the presence of incomplete lineage sorting. We reanalyze a Saccharomyces species data set for which multiple analyses had converged on a species tree candidate. Using our method, though, we show that an evolutionary hypothesis involving hybridization in this group has better support than one of strict divergence. A similar reanalysis on a group of three Drosophila species shows that the data is consistent with hybridization. Further, using extensive simulation studies, we demonstrate the power of gene tree topologies at obtaining accurate estimates of branch lengths and hybridization probabilities of a given phylogenetic network. Finally, we discuss identifiability issues with detecting hybridization, particularly in cases that involve extinction or incomplete sampling of taxa.

  17. Transfer of Lysozyme Gene into indica Parents of Hybrid Rice by Backcrossing

    Institute of Scientific and Technical Information of China (English)

    YI Zi-li; WANG Zi-xuan; QIN Jing-ping; JIANG Jian-xiong; TAN Yan-ning; ZHOU Qing-ming

    2006-01-01

    Alysozyme gene resistant to rice blast was transferred from the donor transgenic japonica rice Zhonghua 9 (D2-1-2) into a sterile line Pei'ai 64S(PA 64S) and restorer line 9311 of the two-line hybrid rice Liangyoupeijiu, and the restorer line Minghui 63 (MH63) of three-line hybrid rice Shanyou 63 by successive backcrossing. The PCR analysis confirmed that foreign lysozyme gene was B2F2 9311, B2F2 MH63 and B1F2 PA64S, indicating that the foreign gene was stably inherited over successive generations as a dominant single copy gene. The resistance against rice blast in backcross or selfed generations and corresponding testcross combinations were investigated in 2003 and 2004. The results showed that the resistance of the transgenic rice to blast had a greater improvement than that of the corresponding recurrent parents or the corresponding check hybrid combinations. The resistance of the advanced backcross and selfed generations to rice blast is much stronger than that of the early generations. The study confirmed thattransferring the lysozyme gene into hybrid parents by backcrossing was a simple and effective approach to develop new hybrid rice resistant to rice blast.

  18. Hybrid weakness in a rice interspecific hybrid is nitrogen-dependent, and accompanied by changes in gene expression at both total transcript level and parental allele partitioning

    Science.gov (United States)

    Lin, Xiuyun; Wang, Jie; Yu, Jiamiao; Sun, Yue; Miao, Yiling; Li, Qiuping; Sanguinet, Karen A.; Liu, Bao

    2017-01-01

    Background Hybrid weakness, a phenomenon opposite to heterosis, refers to inferior growth and development in a hybrid relative to its pure-line parents. Little attention has been paid to the phenomenological or mechanistic aspect of hybrid weakness, probably due to its rare occurrence. Methodology/Principal findings Here, using a set of interspecific triploid F1 hybrids between Oryza sativa, ssp. japonica (genome AA) and a tetraploid wild rice species, O. alta (genome, CCDD), we investigated the phenotypic and physiological differences between the F1 hybrids and their parents under normal and nitrogen-limiting conditions. We quantified the expression levels of 21 key genes involved in three important pathways pertinent to the assayed phenotypic and physiological traits by real-time qRT-PCR. Further, we assayed expression partitioning of parental alleles for eight genes in the F1 hybrids relative to the in silico “hybrids” (parental cDNA mixture) under both normal and N-limiting conditions by using locus-specific cDNA pyrosequencing. Conclusions/Significance We report that the F1 hybrids showed weakness in several phenotypic traits at the final seedling-stage compared with their corresponding mid-parent values (MPVs). Nine of the 21 studied genes showed contrasted expression levels between hybrids and parents (MPVs) under normal vs. N-limiting conditions. Interestingly, under N-limiting conditions, the overtly enhanced partitioning of maternal allele expression in the hybrids for eight assayed genes echo their attenuated hybrid weakness in phenotypes, an observation further bolstered by more resemblance of hybrids to the maternal parent under N-limiting conditions compared to normal conditions in a suite of measured physiological traits. Our observations suggest that both overall expression level and differential partitioning of parental alleles of critical genes contribute to condition-specific hybrid weakness. PMID:28248994

  19. Hybridization of cloned Rhodopseudomonas capsulata photosynthesis genes with DNA from other photosynthetic bacteria.

    OpenAIRE

    Beatty, J T; Cohen, S N

    1983-01-01

    The homology of Rhodopseudomonas capsulata DNA segments carrying photosynthesis genes with sequences present in total DNA from certain other photosynthetic and non-photosynthetic bacterial species was determined by hybridization. R. capsulata DNA fragments that carry loci for production of peptide components of the photosynthetic reaction center and light-harvesting I antenna complex were found to hybridize to DNA from some photosynthetic species. However, fragments that carry carotenoid or b...

  20. Identification of up-regulated genes in human uterine leiomyoma by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.

  1. Formation of diploid and triploid hybrid groupers (hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂) and their 5S gene analysis.

    Science.gov (United States)

    Huang, Wen; Qin, Qinbo; Yang, Huirong; Li, Shuisheng; Hu, Chaoqun; Wang, Yude; Zhang, Yong; Liu, Shaojun; Lin, Haoran

    2016-10-07

    Interspecies hybridization is widely used to achieve heterosis or hybrid vigor, which has been observed and harnessed by breeders for centuries. Natural allopolyploid hybrids generally exhibit more superior heterosis than both the diploid progenies and their parental species. However, polyploid formation processes have been long ignored, the genetic basis of heterosis in polyploids remains elusive. In the present study, triploid hybrids had been demonstrated to contain two sets of chromosomes from mother species and one set from father species. Cellular polyploidization process in the embryos had been traced. The triploid hybrids might be formed by failure formation of the second polarized genome during the second meiosis stage. Four spindle centers were observed in anaphase stage of the first cell division. Three spindle centers were observed in side of cell plate after the first cell division. The 5S rDNA genes of four types of groupers were cloned and analyzed. The diploid and triploid hybrids had been proved to contain the tandem chimera structures which were recombined by maternal and paternal monomer units. The results indicated that genome re-fusion had occurred in the hybrid progenies. To further elucidate the genetic patterns of diploid and triploid hybrids, fluorescence chromosome location had been carried out, maternal 5S gene (M-386) were used as the probe. The triploid hybrids contained fewer fluorescence loci numbers than the maternal species. The results indicated that participation of paternal 5S gene in the triploid hybrid genome had degraded the match rates of M-386 probe. Our study is the first to investigate the cellular formation processes of natural allopolyploids in hybrid fish, the cellular polyploidization process may be caused by failure formation of the second polarized genome during the meiosis, and our results will provide the molecular basis of hybrid vigor in interspecies hybridization.

  2. Limited gene misregulation is exacerbated by allele-specific upregulation in lethal hybrids between Drosophila melanogaster and Drosophila simulans.

    Science.gov (United States)

    Wei, Kevin H-C; Clark, Andrew G; Barbash, Daniel A

    2014-07-01

    Misregulation of gene expression is often observed in interspecific hybrids and is generally attributed to regulatory incompatibilities caused by divergence between the two genomes. However, it has been challenging to distinguish effects of regulatory divergence from secondary effects including developmental and physiological defects common to hybrids. Here, we use RNA-Seq to profile gene expression in F1 hybrid male larvae from crosses of Drosophila melanogaster to its sibling species D. simulans. We analyze lethal and viable hybrid males, the latter produced using a mutation in the X-linked D. melanogaster Hybrid male rescue (Hmr) gene and compare them with their parental species and to public data sets of gene expression across development. We find that Hmr has drastically different effects on the parental and hybrid genomes, demonstrating that hybrid incompatibility genes can exhibit novel properties in the hybrid genetic background. Additionally, we find that D. melanogaster alleles are preferentially affected between lethal and viable hybrids. We further determine that many of the differences between the hybrids result from developmental delay in the Hmr(+) hybrids. Finally, we find surprisingly modest expression differences in hybrids when compared with the parents, with only 9% and 4% of genes deviating from additivity or expressed outside of the parental range, respectively. Most of these differences can be attributed to developmental delay and differences in tissue types. Overall, our study suggests that hybrid gene misexpression is prone to overestimation and that even between species separated by approximately 2.5 Ma, regulatory incompatibilities are not widespread in hybrids. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Allelic asymmetry of the Lethal hybrid rescue (Lhr) gene expression in the hybrid between Drosophila melanogaster and D. simulans: confirmation by using genetic variations of D. melanogaster.

    Science.gov (United States)

    Shirata, Mika; Araye, Quenta; Maehara, Kazunori; Enya, Sora; Takano-Shimizu, Toshiyuki; Sawamura, Kyoichi

    2014-02-01

    In the cross between Drosophila melanogaster females and D. simulans males, hybrid males die at the late larval stage, and the sibling females also die at later stages at high temperatures. Removing the D. simulans allele of the Lethal hybrid rescue gene (Lhr (sim) ) improves the hybrid incompatibility phenotypes. However, the loss-of-function mutation of Lhr (sim) (Lhr (sim0) ) does not rescue the hybrid males in crosses with several D. melanogaster strains. We first describe the genetic factor possessed by the D. melanogaster strains. It has been suggested that removing the D. melanogaster allele of Lhr (Lhr (mel) ), that is Lhr (mel0) , does not have the hybrid male rescue effect, contrasting to Lhr (sim0) . Because the expression level of the Lhr gene is known to be Lhr (sim) > Lhr (mel) in the hybrid, Lhr (mel0) may not lead to enough of a reduction in total Lhr expression. Then, there is a possibility that the D. melanogaster factor changes the expression level to Lhr (sim) Lhr (mel) in the hybrid irrespectively of the presence of the factor. At last, we showed that Lhr (mel0) slightly improves the viability of hybrid females, which was not realized previously. All of the present results are consistent with the allelic asymmetry model of the Lhr gene expression in the hybrid.

  4. Global gene expression of Poncirus trifoliata, Citrus sunki and their hybrids under infection of Phytophthora parasitica

    Directory of Open Access Journals (Sweden)

    Takita Marco A

    2011-01-01

    Full Text Available Abstract Background Gummosis and root rot caused by Phytophthora are among the most economically important diseases in citrus. Four F1 resistant hybrids (Pool R, and four F1 susceptible hybrids (Pool S to P. parasitica, were selected from a cross between susceptible Citrus sunki and resistant Poncirus trifoliata cv. Rubidoux. We investigated gene expression in pools of four resistant and four susceptible hybrids in comparison with their parents 48 hours after P. parasitica inoculation. We proposed that genes differentially expressed between resistant and susceptible parents and between their resistant and susceptible hybrids provide promising candidates for identifying transcripts involved in disease resistance. A microarray containing 62,876 UniGene transcripts selected from the CitEST database and prepared by NimbleGen Systems was used for analyzing global gene expression 48 hours after infection with P. parasitica. Results Three pairs of data comparisons (P. trifoliata/C. sunki, Pool R/C. sunki and Pool R/Pool S were performed. With a filter of false-discovery rate less than 0.05 and fold change greater than 3.0, 21 UniGene transcripts common to the three pairwise comparative were found to be up-regulated, and 3 UniGene transcripts were down-regulated. Among them, our results indicated that the selected transcripts were probably involved in the whole process of plant defense responses to pathogen attack, including transcriptional regulation, signaling, activation of defense genes participating in HR, single dominant genes (R gene such as TIR-NBS-LRR and RPS4 and switch of defense-related metabolism pathway. Differentially expressed genes were validated by RT-qPCR in susceptible and resistant plants and between inoculated and uninoculated control plants Conclusions Twenty four UniGene transcripts were identified as candidate genes for Citrus response to P. parasitica. UniGene transcripts were likely to be involved in disease resistance, such

  5. High-fat diet disrupts metabolism in two generations of rats in a parent-of-origin specific manner

    Science.gov (United States)

    Chambers, T. J. G.; Morgan, M. D.; Heger, A. H.; Sharpe, R. M.; Drake, A. J.

    2016-01-01

    Experimental and epidemiological evidence demonstrate that ancestral diet might contribute towards offspring health. This suggests that nutrition may be able to modify genetic or epigenetic information carried by germ cells (GCs). To examine if a parental high fat diet (HFD) influences metabolic health in two generations of offspring, GC-eGFP Sprague Dawley rats were weaned onto HFD (45% fat) or Control Diet (CD; 10% fat). At 19 weeks, founders (F0) were bred with controls, establishing the F1 generation. HFD resulted in 9.7% and 14.7% increased weight gain in male and female F0 respectively. F1 offspring of HFD mothers and F1 daughters of HFD-fed fathers had increased weight gain compared to controls. F1 rats were bred with controls at 19 weeks to generate F2 offspring. F2 male offspring derived from HFD-fed maternal grandfathers exhibited increased adiposity, plasma leptin and luteinising hormone to testosterone ratio. Despite transmission via the founding male germline, we did not find significant changes in the F0 intra-testicular GC transcriptome. Thus, HFD consumption by maternal grandfathers results in a disrupted metabolic and reproductive hormone phenotype in grandsons in the absence of detectable changes in the intra-testicular GC transcriptome. PMID:27550193

  6. Modulation of tyrosine hydroxylase gene expression in the central nervous system visualized by in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Berod, A.; Biguet, N.F.; Dumas, S.; Bloch, B.; Mallet, J.

    1987-03-01

    cDNA probe was used for in situ hybridization studies on histological sections through the locus coeruleus, substantia nigra, and the ventral tegmental area of the rat brain. Experimental conditions were established that yielded no background and no signal when pBR322 was used as control probe. Using the tyrosine hydroxylase probe, the authors ascertained the specificity of the labeling over catecholaminergic cells by denervation experiments and comparison of the hybridization pattern with that of immunoreactivity. The use of /sup 35/S-labeled probe enabled the hybridization signal to be resolved at the cellular level. A single injection of reserpine into the rat led to an increase of the intensity of the autoradiographic signal over the locus coeruleus area, confirming an RNA gel blot analysis. The potential of in situ hybridization to analyze patterns of modulation of gene activity as a result of nervous activity is discussed.

  7. NOVEL HYBRID GENE VECTOR STABILIZED BY CROSS-LINKING WITH GOLD NANOPARTICLES

    Institute of Scientific and Technical Information of China (English)

    You-xiang Wang; Ying Zhu; Jia-cong Shen

    2008-01-01

    Enhanced stability of polyplexes in physiological condition was an important prerequisite for successful systemic gene delivery. Herein novel method was reported to develop stable gene vector by nanotechnology. Thiolated polyplexes were constructed and then cross-linked with gold nanoparticles (AuNPs) by gold-thiol interactions. TEM pictures showed that AuNPs were attached to the shell of spherical polyplexes. The hybrid gene vector was stable enough in physiological condition and maintained efficient transfection, which showed great potential in gene delivery research and application.

  8. Expression profiling of sucrose metabolizing genes in Saccharum, Sorghum and their hybrids.

    Science.gov (United States)

    Ramalashmi, K; Prathima, P T; Mohanraj, K; Nair, N V

    2014-10-01

    Sucrose phosphate synthase (SPS; EC 2.4.1.14), sucrose synthase (SuSy; EC 2.4.1.13) and soluble acid invertase (SAI; EC 3.2.1.26) are key enzymes that regulate sucrose fluxes in sink tissues for sucrose accumulation in sugarcane and sorghum. In this study, the expression profiling of sucrose-related genes, i.e. SPS, SuSy and SAI in two sets of hybrids viz., one from a Sorghum × Saccharum cross and the other from a Saccharum × Sorghum cross, high- and low-sucrose varieties, sweet and grain sorghum lines was carried out using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) at monthly intervals. The results indicated differential expression of the three genes in high- and low-sucrose forms. Expression of SPS and SuSy genes was high in high-sucrose varieties, Saccharum × Sorghum hybrids and sweet sorghum and lower in low-sucrose varieties, Sorghum × Saccharum hybrids and grain sorghum. SAI showed a lower expression in high-sucrose varieties, Saccharum × Sorghum hybrids and sweet sorghum and higher expression in low-sucrose varieties, Sorghum × Saccharum hybrids and the grain sorghum. This study describes the positive association of SPS and SuSy and negative association of SAI on sucrose accumulation. This is the first report of differential expression profiling of SPS, SuSy and SAI in intergeneric hybrids involving sugarcane and sorghum, which opens the possibility for production of novel hybrids with improved sucrose content and with early maturity.

  9. Neighboring genes for DNA-binding proteins rescue male sterility in Drosophila hybrids.

    Science.gov (United States)

    Liénard, Marjorie A; Araripe, Luciana O; Hartl, Daniel L

    2016-07-19

    Crosses between closely related animal species often result in male hybrids that are sterile, and the molecular and functional basis of genetic factors for hybrid male sterility is of great interest. Here, we report a molecular and functional analysis of HMS1, a region of 9.2 kb in chromosome 3 of Drosophila mauritiana, which results in virtually complete hybrid male sterility when homozygous in the genetic background of sibling species Drosophila simulans. The HMS1 region contains two strong candidate genes for the genetic incompatibility, agt and Taf1 Both encode unrelated DNA-binding proteins, agt for an alkyl-cysteine-S-alkyltransferase and Taf1 for a subunit of transcription factor TFIID that serves as a multifunctional transcriptional regulator. The contribution of each gene to hybrid male sterility was assessed by means of germ-line transformation, with constructs containing complete agt and Taf1 genomic sequences as well as various chimeric constructs. Both agt and Taf1 contribute about equally to HMS1 hybrid male sterility. Transgenes containing either locus rescue sterility in about one-half of the males, and among fertile males the number of offspring is in the normal range. This finding suggests compensatory proliferation of the rescued, nondysfunctional germ cells. Results with chimeric transgenes imply that the hybrid incompatibilities result from interactions among nucleotide differences residing along both agt and Taf1 Our results challenge a number of preliminary generalizations about the molecular and functional basis of hybrid male sterility, and strongly reinforce the role of DNA-binding proteins as a class of genes contributing to the maintenance of postzygotic reproductive isolation.

  10. Identification of differentially expressed genes after partial rat liver ischemia/reperfusion by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    Christine Fallsehr; Christina Zapletal; Michael Kremer; Resit Demir; Magnus von Knebel Doeberitz; Ernst Klar

    2005-01-01

    AIM: To identify potential diagnostic target genes in early reperfusion periods following warm liver ischemia before irreversible liver damage occurs.METHODS: We used two strategies (SSH suppression subtractive hybridization and hybridization of cDNA arrays)to determine early changes in gene expression profiles in a rat model of partial WI/R, comparing postischemic and adjacent nonischemic liver lobes. Differential gene expression was verified (WT/R; 1 h/2 h) and analyzed in more detail after warm ischemia (1 h) in a reperfusion time kinetics (0, 1, 2 and 6 h) and compared to untreated livers by Northern blot hybridizations. Protein expression was examined on Western blots and by immunohistochemistry for four differentially expressed target genes (Hsp70,Hsp27, Gadd45a and IL-1rl).RESULTS: Thirty-two individual WI/R target genes showing altered RNA levels after confirmation by Northern blot analyzes were identified. Among them, six functionally uncharacteristic expressed sequences and 26 known genes (12 induced in postischemic liver lobes, 14 with higher transcriptional expression in adjacent nonischemic liver lobes). Functional categories of the verified marker genes indicate on the one hand cellular stress and tissue damage but otherwise activation of protective cellular reactions (AP-1 transcription factors, apoptosis related genes, heat shock genes). In order to assign the transcriptional status to the biological relevant protein level we demonstrated that Hsp70, Hsp27, Gadd45a and IL-1rI were clearly up-regulated comparing postischemic and untreated rat livers, suggesting their involvement in the WI/R context.CONCLUSION: This study unveils a WI/R response gene set that will help to explore molecular pathways involved in the tissue damage after WI/R. In addition, these genes especially Hsp70and Gadd45a might represent promising new candidates indicating WI/R liver damage.

  11. Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries

    DEFF Research Database (Denmark)

    Alvaro, D.; Sunjevaric, I.; Reid, R. J.

    2006-01-01

    We have developed a new method, systematic hybrid loss of heterozygosity, to facilitate genomic screens utilizing the yeast gene deletion library. Screening is performed using hybrid diploid strains produced through mating the library haploids with strains from a different genetic background...... mating a library gene deletion haploid to such a conditional centromere strain, which corresponds to the chromosome carrying the gene deletion, loss of heterozygosity (LOH) at the gene deletion locus can be generated in these otherwise hybrid diploids. The use of hybrid diploid strains permits...... complementation of any spurious recessive mutations in the library strain, facilitating attribution of the observed phenotype to the documented gene deletion and dramatically reducing false positive results commonly obtained in library screens. The systematic hybrid LOH method can be applied to virtually any...

  12. Identification of a pheA gene associated with Streptococcus mitis by using suppression subtractive hybridization.

    Science.gov (United States)

    Park, Hee Kuk; Dang, Hien Thanh; Myung, Soon Chul; Kim, Wonyong

    2012-04-01

    We performed suppression subtractive hybridization to identify genomic differences between Streptococcus mitis and Streptococcus pneumoniae. Based on the pheA gene, a primer set specific to S. mitis detection was found in 18 out of 103 S. mitis-specific clones. Our findings would be useful for discrimination of S. mitis from other closely related cocci in the oral environment.

  13. Identification of a pheA Gene Associated with Streptococcus mitis by Using Suppression Subtractive Hybridization

    OpenAIRE

    Park, Hee Kuk; Dang, Hien Thanh; Myung, Soon Chul; Kim, Wonyong

    2012-01-01

    We performed suppression subtractive hybridization to identify genomic differences between Streptococcus mitis and Streptococcus pneumoniae. Based on the pheA gene, a primer set specific to S. mitis detection was found in 18 out of 103 S. mitis-specific clones. Our findings would be useful for discrimination of S. mitis from other closely related cocci in the oral environment.

  14. Hybrid Male Sterility in Rice Controlled by Interaction between Divergent Alleles of Two Adjacent Genes

    National Research Council Canada - National Science Library

    Yunming Long; Lifeng Zhao; Baixiao Niu; Jing Su; Hao Wu; Yuanling Chen; Qunyu Zhang; Jingxin Guo; Chuxiong Zhuang; Mantong Mei; Jixing Xia; Lan Wang; Haibin Wu; Yao-Guang Liu

    2008-01-01

    .... Here we show that a locus for indica-japonica hybrid male sterility, Sa, comprises two adjacent genes, SaM and SaF, encoding a small ubiquitin-like modifier E3 ligase-like protein and an F-box protein, respectively...

  15. Hybrid Deterministic Views about Genes in Biology Textbooks: A Key Problem in Genetics Teaching

    Science.gov (United States)

    dos Santos, Vanessa Carvalho; Joaquim, Leyla Mariane; El-Hani, Charbel Nino

    2012-01-01

    A major source of difficulties in promoting students' understanding of genetics lies in the presentation of gene concepts and models in an inconsistent and largely ahistorical manner, merely amalgamated in hybrid views, as if they constituted linear developments, instead of being built for different purposes and employed in specific contexts. In…

  16. Hybrid Deterministic Views about Genes in Biology Textbooks: A Key Problem in Genetics Teaching

    Science.gov (United States)

    dos Santos, Vanessa Carvalho; Joaquim, Leyla Mariane; El-Hani, Charbel Nino

    2012-01-01

    A major source of difficulties in promoting students' understanding of genetics lies in the presentation of gene concepts and models in an inconsistent and largely ahistorical manner, merely amalgamated in hybrid views, as if they constituted linear developments, instead of being built for different purposes and employed in specific contexts. In…

  17. Use of suppression subtractive hybridization to identify downy mildew genes expressed during infection of Arabidopsis thaliana.

    Science.gov (United States)

    Bittner-Eddy, Peter D; Allen, Rebecca L; Rehmany, Anne P; Birch, Paul; Beynon, Jim L

    2003-11-01

    SUMMARY Peronospora parasitica is an obligate biotrophic oomycete that causes downy mildew in Arabidopsis thaliana and Brassica species. Our goal is to identify P. parasitica (At) genes that are involved in pathogenicity. We used suppression subtractive hybridization (SSH) to generate cDNA libraries enriched for in planta-expressed parasite genes and up-regulated host genes. A total of 1345 clones were sequenced representing cDNA fragments from 25 putative P. parasitica (At) genes (Ppat 1-25) and 618 Arabidopsis genes. Analyses of expression patterns showed that 15 Ppats were expressed only in planta. Eleven Ppats encoded peptides with homology (BlastP values planta-expressed genes from P. parasitica (At) that complements other gene discovery approaches such as EST sequencing.

  18. An in situ hybridization-based screen for heterogeneously expressed genes in mouse ES cells.

    Science.gov (United States)

    Carter, Mark G; Stagg, Carole A; Falco, Geppino; Yoshikawa, Toshiyuki; Bassey, Uwem C; Aiba, Kazuhiro; Sharova, Lioudmila V; Shaik, Nabeebi; Ko, Minoru S H

    2008-02-01

    We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.

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

    Directory of Open Access Journals (Sweden)

    Keng-Hoong Ng

    Full Text Available BACKGROUND: Clustering is a key step in the processing of Expressed Sequence Tags (ESTs. The primary goal of clustering is to put ESTs from the same transcript of a single gene into a unique cluster. Recent EST clustering algorithms mostly adopt the alignment-free distance measures, where they tend to yield acceptable clustering accuracies with reasonable computational time. Despite the fact that these clustering methods work satisfactorily on a majority of the EST datasets, they have a common weakness. They are prone to deliver unsatisfactory clustering results when dealing with ESTs from the genes derived from the same family. The root cause is the distance measures applied on them are not sensitive enough to separate these closely related genes. METHODOLOGY/PRINCIPAL FINDINGS: We propose a hybrid distance measure that combines the global and local features extracted from ESTs, with the aim to address the clustering problem faced by ESTs derived from the same gene family. The clustering process is implemented using the DBSCAN algorithm. We test the hybrid distance measure on the ten EST datasets, and the clustering results are compared with the two alignment-free EST clustering tools, i.e. wcd and PEACE. The clustering results indicate that the proposed hybrid distance measure performs relatively better (in terms of clustering accuracy than both EST clustering tools. CONCLUSIONS/SIGNIFICANCE: The clustering results provide support for the effectiveness of the proposed hybrid distance measure in solving the clustering problem for ESTs that originate from the same gene family. The improvement of clustering accuracies on the experimental datasets has supported the claim that the sensitivity of the hybrid distance measure is sufficient to solve the clustering problem.

  20. Identifying Gene Regulatory Networks in Arabidopsis by In Silico Prediction, Yeast-1-Hybrid, and Inducible Gene Profiling Assays.

    Science.gov (United States)

    Sparks, Erin E; Benfey, Philip N

    2016-01-01

    A system-wide understanding of gene regulation will provide deep insights into plant development and physiology. In this chapter we describe a threefold approach to identify the gene regulatory networks in Arabidopsis thaliana that function in a specific tissue or biological process. Since no single method is sufficient to establish comprehensive and high-confidence gene regulatory networks, we focus on the integration of three approaches. First, we describe an in silico prediction method of transcription factor-DNA binding, then an in vivo assay of transcription factor-DNA binding by yeast-1-hybrid and lastly the identification of co-expression clusters by transcription factor induction in planta. Each of these methods provides a unique tool to advance our understanding of gene regulation, and together provide a robust model for the generation of gene regulatory networks.

  1. Identification of differentially expressed radiation-induced genes in cervix carcinoma cells using suppression subtractive hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jun Sang; Lee, Young Sook; Lee, Jeung Hoon; Lee, Woong Hee; Seo, Eun Young; Cho, Moon June [Chungnam National University, Daejeon (Korea, Republic of)

    2005-03-15

    A number of genes and their products are induced early or late following exposure of cells to ionizing radiation. These radiation-induced genes have various effects of irradiated cells and tissues. Suppression subtractive hybridization (SSH) based on PCR was used to identify the differentially expressed genes by radiation in cervix carcinoma cells. Total RNA and poly (A){sup +} mRNA were isolated from irradiated and non-irradiated HeLa cells. Forward-and reverse-subtracted cDNA libraries were constructed using SSH. Eighty-eight clones of each were used to randomly select differentially expressed genes using reverse Northern blotting (dot blot analysis). Northern blotting was used to verify the screened genes. Of the 176 clones, 10 genes in the forward-subtracted library and 9 genes in the reverse-subtracted library were identified as differentially expressed radiation-induced genes by PCR-select differential screening. Three clones from the forward-subtracted library were confirmed by Northern blotting, and showed increased expression in a dose-dependent manner, including a telomerase catalytic subunit and sodium channel-like protein gene, and an ESTs (expressed sequence tags) gene. We identified differentially expressed radiation-induced genes with low-abundance genes with SSH, but further characterization of theses genes are necessary to clarify the biological functions of them.

  2. Cloning and characterization of an up-regulated GA 20-oxidase gene in hybrid maize

    Institute of Scientific and Technical Information of China (English)

    Jinkun Du; Yingyin Yao; Zhongfu Ni; Qixin Sun

    2009-01-01

    Previous studies revealed that GA content and metabolism are positively correlated with a faster shoot growth rate of hybrid, and recently, genes participating in both GA biosynthesis and GA response pathways were also found to be differentially expressed between wheat hybrid and its parental inbreds. In this study, an up-regulated GA 20-oxidase gene in a maize hybrid, designated ZmGA20, was cloned. ZmGA20 contains an open reading frame (ORF) encoding 391 amino acid residues. BLASTX searches in GenBank revealed that the ZmGA20 is homologous to the sequences of GA20ox proteins from different species, and analysis also indicated that ZmGA20 had typical features of GA 20-oxidase proteins, including a "LPWKET" sequence. Semi-quantitative RT-PCR analysis showed that ZmGA20 was expressed in different tissues and/or organs. The expression level of ZmGA20 in the hybrid was higher than that in two parents (in roots, leaves, stems and embryo, and ears). The abundance of ZmGA20 transcript was equal to that of the highly expressed parents, which provided molecular evidence for the observed GA content heterosis in maize hybrids.

  3. Ephemeral association between gene CG5762 and hybrid male sterility in Drosophila sibling species.

    Science.gov (United States)

    Ma, Daina; Michalak, Pawel

    2011-10-01

    Interspecies divergence in regulatory pathways may result in hybrid male sterility (HMS) when dominance and epistatic interactions between alleles that are functional within one genome are disrupted in hybrid genomes. The identification of genes contributing to HMS and other hybrid dysfunctions is essential for understanding the origin of new species (speciation). Previously, we identified a panel of male-specific loci misexpressed in sterile male hybrids of Drosophila simulans and D. mauritiana relative to parental species. In the current work, we attempt to dissect the genetic associations between HMS and one of the genes, CG5762, a Drosophila-unique locus characterized by rapid sequence divergence within the genus, presumably driven by positive natural selection. CG5762 is underexpressed in sterile backcross males compared with their fertile brothers. In CG5762 heterozygotes, the D. mauritiana allele is consistently overexpressed on both the D. simulans and D. mauritiana backcross genomic background, suggesting a cis-acting regulation factor. There is a significant association between heterozygosity and HMS in hybrid males from early but not later backcross generations. Microsatellite markers spanning CG5762 fail to associate with HMS. These observations lead to a conclusion that CG5762 is not a causative factor of HMS. Although genetic linkage between CG5762 and a neighboring causative introgression cannot be ruled out, it seems that the pattern is most consistent with CG5762 participating in epistatic interactions that are disrupted in flies with HMS.

  4. Hybrid male sterility in rice controlled by interaction between divergent alleles of two adjacent genes.

    Science.gov (United States)

    Long, Yunming; Zhao, Lifeng; Niu, Baixiao; Su, Jing; Wu, Hao; Chen, Yuanling; Zhang, Qunyu; Guo, Jingxin; Zhuang, Chuxiong; Mei, Mantong; Xia, Jixing; Wang, Lan; Wu, Haibin; Liu, Yao-Guang

    2008-12-01

    Sterility is common in hybrids between divergent populations, such as the indica and japonica subspecies of Asian cultivated rice (Oryza sativa). Although multiple loci for plant hybrid sterility have been identified, it remains unknown how alleles of the loci interact at the molecular level. Here we show that a locus for indica-japonica hybrid male sterility, Sa, comprises two adjacent genes, SaM and SaF, encoding a small ubiquitin-like modifier E3 ligase-like protein and an F-box protein, respectively. Most indica cultivars contain a haplotype SaM(+)SaF(+), whereas all japonica cultivars have SaM(-)SaF(-) that diverged by nucleotide variations in wild rice. Male semi-sterility in this heterozygous complex locus is caused by abortion of pollen carrying SaM(-). This allele-specific gamete elimination results from a selective interaction of SaF(+) with SaM(-), a truncated protein, but not with SaM(+) because of the presence of an inhibitory domain, although SaM(+) is required for this male sterility. Lack of any one of the three alleles in recombinant plants does not produce male sterility. We propose a two-gene/three-component interaction model for this hybrid male sterility system. The findings have implications for overcoming male sterility in inter-subspecific hybrid rice breeding.

  5. Super-distant molecular hybridization of plant seeds by ion beam-mediated gene cluster

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The N beam-mediated distant molecular hybridization between Ginkgo biloba I and watermelon was studied. The results showed that the ester gene of Ginkgo biloba L was successfully expressed in two varieties of watermelon. 3-16 and SR2-14-2, in both of which the ester quantities were measured as 17.0756 μg/g and 45.9998 μg/g respectively. Meanwhile, superoxide dismutase (SOD) activity in leaves of the watennelon expressing ester gene was increased twofold as compared to that of the control, showing that ion beam could mediate distant and/or super-distant donor gene expression in the cells of a receptor. Furthermore, the molecular nechanism of distant hybridization was analyzed.

  6. [Analysis of allelic content of genes responsible for baking properties in allocytoplasmic wheat hybrids].

    Science.gov (United States)

    Klimushina, M V; Divashuk, M G; Mukhammed, T A K; Semenov, O G; Karlov, G I

    2013-05-01

    A collection comprised of allocytoplasmic hybrids of mild wheat (ACPH) was screened for the allelic state of genes responsible for baking properties (high-molecular glutenins, puroindolines, and Waxy). The possibility of the introgression of the Waxy gene of T. timopheevii into the mild wheat genome was demonstrated in several ACPH samples using the set of molecular markers. Allelic gene variants responsible for the baking properties were revealed for 22 ACPH samples, which make it possible to detect the most challenging samples for both molecular-genetic research and applied science.

  7. Identification of Differentially Expressed Genes During Ethylene Climacteric of Melon Fruit by Suppression Subtractive Hybridization

    Institute of Scientific and Technical Information of China (English)

    GAO Feng; NIU Yi-ding; HAO Jin-feng; BADE Rengui; ZHANG Li-quan; HASI Agula

    2013-01-01

    Melon (Cucumis melo L.) is an important horticultural crop worldwide. Ethylene regulates the ripening process and affects the ripening rate. To screen genes that are differentially expressed at the burst of ethylene climacteric in melon fruit, we performed suppression subtractive hybridization (SSH) to generate forward and reverse libraries, for which we sequenced 439 and 445 clones, respectively. Our BLAST analysis showed that the genes from the 2 libraries were involved in metabolism, signal transduction, cell structure, transcription, translation, and defense. Six genes were analyzed by qRT-PCR during the differential developmental stage of melon fruit. Our results provide new insight into the understanding of climacteric ripening of melon fruit.

  8. 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.

  9. Interallelic and intergenic incompatibilities of the Prdm9 (Hst1 gene in mouse hybrid sterility.

    Directory of Open Access Journals (Sweden)

    Petr Flachs

    Full Text Available The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect epistatic interactions. Although the mechanisms of speciation are of great interest, no incompatibility has been characterized at the gene level in mammals. The Hybrid sterility 1 gene (Hst1 participates in the arrest of meiosis in F(1 males of certain strains from two Mus musculus subspecies, e.g., PWD from M. m. musculus and C57BL/6J (henceforth B6 from M. m. domesticus. Hst1 has been identified as a meiotic PR-domain gene (Prdm9 encoding histone 3 methyltransferase in the male offspring of PWD females and B6 males, (PWD×B6F(1. To characterize the incompatibilities underlying hybrid sterility, we phenotyped reproductive and meiotic markers in males with altered copy numbers of Prdm9. A partial rescue of fertility was observed upon removal of the B6 allele of Prdm9 from the azoospermic (PWD×B6F(1 hybrids, whereas removing one of the two Prdm9 copies in PWD or B6 background had no effect on male reproduction. Incompatibility(ies not involving Prdm9(B6 also acts in the (PWD×B6F(1 hybrids, since the correction of hybrid sterility by Prdm9(B6 deletion was not complete. Additions and subtractions of Prdm9 copies, as well as allelic replacements, improved meiotic progression and fecundity also in the progeny-producing reciprocal (B6×PWDF(1 males. Moreover, an increased dosage of Prdm9 and reciprocal cross enhanced fertility of other sperm-carrying male hybrids, (PWD×B6-C3H.Prdm9F(1, harboring another Prdm9 allele of M. m. domesticus origin. The levels of Prdm9 mRNA isoforms were similar in the prepubertal testes of all types of F(1 hybrids of PWD with B6 and B6-C3H.Prdm9 despite their different prospective fertility, but decreased to 53% after removal of Prdm9(B6. Therefore, the Prdm9(B6 allele probably takes part in posttranscriptional dominant-negative hybrid interaction(s absent in the parental strains.

  10. Interallelic and intergenic incompatibilities of the Prdm9 (Hst1) gene in mouse hybrid sterility.

    Science.gov (United States)

    Flachs, Petr; Mihola, Ondřej; Simeček, Petr; Gregorová, Soňa; Schimenti, John C; Matsui, Yasuhisa; Baudat, Frédéric; de Massy, Bernard; Piálek, Jaroslav; Forejt, Jiří; Trachtulec, Zdenek

    2012-01-01

    The Dobzhansky-Muller model of incompatibilities explains reproductive isolation between species by incorrect epistatic interactions. Although the mechanisms of speciation are of great interest, no incompatibility has been characterized at the gene level in mammals. The Hybrid sterility 1 gene (Hst1) participates in the arrest of meiosis in F(1) males of certain strains from two Mus musculus subspecies, e.g., PWD from M. m. musculus and C57BL/6J (henceforth B6) from M. m. domesticus. Hst1 has been identified as a meiotic PR-domain gene (Prdm9) encoding histone 3 methyltransferase in the male offspring of PWD females and B6 males, (PWD×B6)F(1). To characterize the incompatibilities underlying hybrid sterility, we phenotyped reproductive and meiotic markers in males with altered copy numbers of Prdm9. A partial rescue of fertility was observed upon removal of the B6 allele of Prdm9 from the azoospermic (PWD×B6)F(1) hybrids, whereas removing one of the two Prdm9 copies in PWD or B6 background had no effect on male reproduction. Incompatibility(ies) not involving Prdm9(B6) also acts in the (PWD×B6)F(1) hybrids, since the correction of hybrid sterility by Prdm9(B6) deletion was not complete. Additions and subtractions of Prdm9 copies, as well as allelic replacements, improved meiotic progression and fecundity also in the progeny-producing reciprocal (B6×PWD)F(1) males. Moreover, an increased dosage of Prdm9 and reciprocal cross enhanced fertility of other sperm-carrying male hybrids, (PWD×B6-C3H.Prdm9)F(1), harboring another Prdm9 allele of M. m. domesticus origin. The levels of Prdm9 mRNA isoforms were similar in the prepubertal testes of all types of F(1) hybrids of PWD with B6 and B6-C3H.Prdm9 despite their different prospective fertility, but decreased to 53% after removal of Prdm9(B6). Therefore, the Prdm9(B6) allele probably takes part in posttranscriptional dominant-negative hybrid interaction(s) absent in the parental strains.

  11. Clinical Application of Fluorescence in Situ Hybridization (FISH) to Detect HER-2 Gene in Breast Cancer

    Institute of Scientific and Technical Information of China (English)

    Maurie Buehler; Ellie Guardino; Jung Sik Park; Eun Jeong Jang

    2014-01-01

    Objective: To investigate the clinical application of the detection of HER-2 gene by lfuorescence in situ hybridization (FISH) in breast cancer and the correlation between HER-2 gene ampliifcation and clinicopathology of breast cancer. Methods:Parafifn-embedded breast inifltrating ductal carcinoma from 48 patients were detected by FISH and immunohistochemistry (IHC) respectively for comparing the results of two methods. Results: HER-2 protein expressions were classified into three groups (3+/2+/1+ or 0) and the positive rates of HER-2 gene ampliifcation by FISH were 77.8%, 57.1% and 10.5%, respectively. Of the 29 cases with positive axillary lymph node, 12 were with HER-2 gene ampliifcation (P0.05). Conclusion:The false positive and negative rates are higher in HER-2 protein expression by IHC. Compared with IHC, FISH, being more effective and precise, can be applied extensively in clinic. HER-2 gene ampliifcation is concerned with axillary nodes metastases.

  12. Genetics of hybrid inviability and sterility in Drosophila: dissection of introgression of D. simulans genes in D. melanogaster genome.

    Science.gov (United States)

    Sawamura, Kyoichi; Karr, Timothy L; Yamamoto, Masa-Toshi

    2004-03-01

    Interspecific crosses between Drosophila melanogaster and Drosophila simulans usually produce sterile unisexual hybrids. The barrier preventing genetic analysis of hybrid inviability and sterility has been taken away by the discovery of a D. simulans strain which produces fertile female hybrids. D. simulans genes in the cytological locations of 21A1 to 22C1-23B1 and 30F3-31C5 to 36A2-7 have been introgressed into the D. melanogaster genetic background by consecutive backcrosses. Flies heterozygous for the introgression are fertile, while homozygotes are sterile both in females and males. The genes responsible for the sterility have been mapped in the introgression. The male sterility is caused by the synergistic effect of multiple genes, while the female sterility genes have been localized to a 170 kb region (32D2 to 32E4) containing 20 open reading frames. Thus, the female sterility might be attributed to a single gene with a large effect. We have also found that the Lethal hybrid rescue mutation which prevents the inviability of male hybrids from the cross of D. melanogaster females and D. simulans males cannot rescue those carrying the introgression, suggesting that D. simulans genes maybe non-functional in this hybrid genotype. The genes responsible for the inviability have not been separated from the female sterility genes by recombination.

  13. Suppression subtractive hybridization and comparative expression analysis to identify developmentally regulated genes in filamentous fungi.

    Science.gov (United States)

    Gesing, Stefan; Schindler, Daniel; Nowrousian, Minou

    2013-09-01

    Ascomycetes differentiate four major morphological types of fruiting bodies (apothecia, perithecia, pseudothecia and cleistothecia) that are derived from an ancestral fruiting body. Thus, fruiting body differentiation is most likely controlled by a set of common core genes. One way to identify such genes is to search for genes with evolutionary conserved expression patterns. Using suppression subtractive hybridization (SSH), we selected differentially expressed transcripts in Pyronema confluens (Pezizales) by comparing two cDNA libraries specific for sexual and for vegetative development, respectively. The expression patterns of selected genes from both libraries were verified by quantitative real time PCR. Expression of several corresponding homologous genes was found to be conserved in two members of the Sordariales (Sordaria macrospora and Neurospora crassa), a derived group of ascomycetes that is only distantly related to the Pezizales. Knockout studies with N. crassa orthologues of differentially regulated genes revealed a functional role during fruiting body development for the gene NCU05079, encoding a putative MFS peptide transporter. These data indicate conserved gene expression patterns and a functional role of the corresponding genes during fruiting body development; such genes are candidates of choice for further functional analysis.

  14. Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

    DEFF Research Database (Denmark)

    Ronander, Elena; Bengtsson, Dominique C; Joergensen, Louise;

    2012-01-01

    and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA...... fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE(1). Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human...... erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System(2) (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription...

  15. Differential Gene Expression Between Hybrids and Their Parents During the Four Crucial Stages of Cotton Growth and Development

    Institute of Scientific and Technical Information of China (English)

    ZHAO Yun-lei; YU Shu-xun; XING Chao-zhu; FAN Shu-li; SONG Mei-zhen; YE Wu-wei

    2009-01-01

    The study aims to clarify the differential gene expression between cotton hybrids and their parents in order to better understand the molecular basis of cotton heterosis. The research focused on cotton heterotic and lower heterotic hybrids and their parents during the four crucial stages, which were analyzed using a differential display technique. The results indicated that there were both quantitative and qualitative differences in gene expression amongst them. The quantitative differences include over- and under-expression of parental genes and the dominant expression of highly-expressed parental genes in hybrids. In contrast, the qualitative differences are the following: (i) Bands were observed in both parents but not in the F1 hybrid (BPnF1); (ii) bands occurred in either of the parents but not in the F1 hybrid (UPnF1); (iii) bands presented only in the F1 hybrid but not in either of the parents (UF1nP); and (iv) bands were detected in either of the parents and the F1 hybrid (UPF1). Overall, the major differences of gene expression occurred in the qualitative level and four related differential patterns were observed. Furthermore, the amount of differential patterns during the flowering stage was relatively higher than those of other stages. At this juncture, both the amount of hybrid-specific expression patterns at flowering stage and the silenced expression patterns at boll-forming stage in highly heterotic hybrids were found higher than those in the lower heterotic ones. It was concluded that significant differences of gene expression in leaves were present between cotton hybrid and its parents during the whole growing stages. Hence, these differences might be responsible for the observed cotton heterosis.

  16. Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene

    DEFF Research Database (Denmark)

    Efrat, S; Linde, S; Kofod, Hans

    1988-01-01

    Three pancreatic beta-cell lines have been established from insulinomas derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene. The beta tumor cell (beta TC) lines maintain the features of differentiated beta cells for about 50 passages in culture. The ...... both to immortalize a rare cell type and to provide a selection for the maintenance of its differentiated phenotype....

  17. Localization of the human OB gene (OBS) to chromosome 7q32 by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Geffroy, S.; Duban, B.; Martinville, B. de [Universitaire de Lille (France)] [and others

    1995-08-10

    An important gene involved in the pathogenesis of obesity is the product of the human homologue of the murine obese gene (gene symbol OBS). Using fluorescence in situ hybridization (FISH), we have localized the human OB gene to human chromosome 7, specifically to region 7q32.1. The FISH data of human OBS provide a gene-associated marker for genetic mapping. 8 refs., 1 fig.

  18. Suppression subtractive hybridization identified differentially expressed genes in lung adenocarcinoma: ERGIC3 as a novel lung cancer-related gene

    Directory of Open Access Journals (Sweden)

    Wu Mingsong

    2013-02-01

    Full Text Available Abstract Background To understand the carcinogenesis caused by accumulated genetic and epigenetic alterations and seek novel biomarkers for various cancers, studying differentially expressed genes between cancerous and normal tissues is crucial. In the study, two cDNA libraries of lung cancer were constructed and screened for identification of differentially expressed genes. Methods Two cDNA libraries of differentially expressed genes were constructed using lung adenocarcinoma tissue and adjacent nonmalignant lung tissue by suppression subtractive hybridization. The data of the cDNA libraries were then analyzed and compared using bioinformatics analysis. Levels of mRNA and protein were measured by quantitative real-time polymerase chain reaction (q-RT-PCR and western blot respectively, as well as expression and localization of proteins were determined by immunostaining. Gene functions were investigated using proliferation and migration assays after gene silencing and gene over-expression. Results Two libraries of differentially expressed genes were obtained. The forward-subtracted library (FSL and the reverse-subtracted library (RSL contained 177 and 59 genes, respectively. Bioinformatic analysis demonstrated that these genes were involved in a wide range of cellular functions. The vast majority of these genes were newly identified to be abnormally expressed in lung cancer. In the first stage of the screening for 16 genes, we compared lung cancer tissues with their adjacent non-malignant tissues at the mRNA level, and found six genes (ERGIC3, DDR1, HSP90B1, SDC1, RPSA, and LPCAT1 from the FSL were significantly up-regulated while two genes (GPX3 and TIMP3 from the RSL were significantly down-regulated (P  Conclusions The two libraries of differentially expressed genes may provide the basis for new insights or clues for finding novel lung cancer-related genes; several genes were newly found in lung cancer with ERGIC3 seeming a novel lung cancer

  19. Hybrid Binary Imperialist Competition Algorithm and Tabu Search Approach for Feature Selection Using Gene Expression Data

    Science.gov (United States)

    Aorigele; Zeng, Weiming; Hong, Xiaomin

    2016-01-01

    Gene expression data composed of thousands of genes play an important role in classification platforms and disease diagnosis. Hence, it is vital to select a small subset of salient features over a large number of gene expression data. Lately, many researchers devote themselves to feature selection using diverse computational intelligence methods. However, in the progress of selecting informative genes, many computational methods face difficulties in selecting small subsets for cancer classification due to the huge number of genes (high dimension) compared to the small number of samples, noisy genes, and irrelevant genes. In this paper, we propose a new hybrid algorithm HICATS incorporating imperialist competition algorithm (ICA) which performs global search and tabu search (TS) that conducts fine-tuned search. In order to verify the performance of the proposed algorithm HICATS, we have tested it on 10 well-known benchmark gene expression classification datasets with dimensions varying from 2308 to 12600. The performance of our proposed method proved to be superior to other related works including the conventional version of binary optimization algorithm in terms of classification accuracy and the number of selected genes. PMID:27579323

  20. [Physical localization of ribosomal genes and chromosome DAPI banding by in situ hybridization in Medicago sativa L].

    Science.gov (United States)

    Chen, Jian-Min; Hong, Yi-Huan; Wang, You-Ping; Bowley, Steve; Wan, Jian-Min

    2006-02-01

    Physical localization of ribosomal genes in diploid and tetraploid alfalfa (Medicago. sativa) was studied using fluorescent in situ hybridization (FISH). It was revealed that 45s gene was only located at nucleolus organizer region (NOR) with a single locus in both diploid and tetraploid alfalfa, while 5s gene had 2-3 loci on chromosomes. Using the genomic DNA from M. coerulea and M. falcata as probe to hybridize with tetraploid species in alfalfa, both diploid species were successfully hybridized with tetraploid chromosomes, only showing the difference in hybridization signals in different numbers of chromosomes. Chromosomes of alfalfa exhibited DAPI banding by FISH analysis. In general, the patterns of distribution of DAPI banding were consistent with C-banding for M. coerulea. The possible origination of tetraploid alfalfa was discussed based on DAPI banding patterns and FISH analysis for ribosomal genes .

  1. Identification of differentially expressed genes in rat silicosis model by suppression subtractive hybridization analysis

    Institute of Scientific and Technical Information of China (English)

    Zhongyuan Jin; Chunyan Fu; Jifang Wen; Baoan Liu; Deyun Feng; Chen Chen; Xiang Li; Yongbin Hu; Jinwu Peng; Yu Liu; Jing Du

    2008-01-01

    The critical molecular mechanism in the development of the pulmonary fibrosis remains unknown, leaving diagnosed patients with a poor prognosis. To isolate the genes specifi-cally up-regulated in pulmonary fibrosis, we established a rat silicosis model 360 d after treatment with crystalline silica suspension. Radiographs of chests showed that some scattered high-density shadows appeared in the lung field.Typical microscopic fibrosing silicotic nodules formed in the lung,alveolar epithelial cells and bronchial epithelial cells,particularly around the partial fibrosing silicotic nodules;some of them showed atypical hyperplasia that suggested a correlation between silicosis and lung cancer.Suppression subtractive hybridization analysis was performed to compare gene expression in lung tissue with silicosis and normal lung tissue.Reverse transcription-polymerase chain rection showed that the expressions of seven novel cDNA sequences identified by suppression subtractive hybridization in lung tissue with silicosis differed from normal lung tissue. Bioinformatics analysis showed that 47 positive clones rep-resented 35 genes containing two putative proteins and four predicted similar proteins.The analysis also showed that some screened genes in silicosis,such as prolyl 4-hydroxylases,actin-related protein-2/3 complex and acidic mammalian chitinase,have not been previously reported.These genes may provide new clues for investigating the molecular mechanisms in the development of pulmonary fibrosis.

  2. Isolation of differentially expressed sex genes in garden asparagus using suppression subtractive hybridization.

    Science.gov (United States)

    Deng, Chuan-liang; Wang, Ning-na; Li, Shu-fen; Dong, Tian-yu; Zhao, Xin-peng; Wang, Shao-jing; Gao, Wu-jun; Lu, Long-dou

    2015-09-01

    Garden asparagus (Asparagus officinalis L.) is a dioecious species whose male and female flowers are found in separate unisexual individuals. A region called the M-locus, located on a pair of homomorphic sex chromosomes, controls sexual dimorphism in asparagus. To date, no sex determining gene has been isolated from asparagus. To identify more genes involved in flower development in asparagus, subtractive hybridization library of male flowers in asparagus was constructed by suppression subtraction hybridization. A total of 107 expressed sequence tags (ESTs) were identified. BLASTX analysis showed that the library contained several genes that could be related to flower development. The expression patterns of seven selected genes believed to be involved in the development of asparagus male flower were further analyzed by semi-quantitative or real-time reverse-transcription polymerase chain reaction (RT-PCR). Results showed that AOEST4-5, AOEST12-40, and AOEST13-38 were strongly expressed in the male flower stage, whereas no transcript level of AOEST13-38 was detected in the female flower stage. The expression levels of AOEST13-87, AOEST13-92, AOEST13-40, and AOEST18-87 in the male flower stage were also higher than those in the female flower stage, although these transcripts were also expressed in other tissues. The identified genes can provide a strong starting point for further studies on the underlying molecular differences between the male and female flowers of asparagus.

  3. Identification of differentially expressed genes in uveal melanoma using suppressive subtractive hybridization

    Science.gov (United States)

    Landreville, Solange; Lupien, Caroline B.; Vigneault, Francois; Gaudreault, Manon; Mathieu, Mélissa; Rousseau, Alain P.; Guérin, Sylvain L.

    2011-01-01

    Purpose Uveal melanoma (UM) is the most common primary cancer of the eye, resulting not only in vision loss, but also in metastatic death. This study attempts to identify changes in the patterns of gene expression that lead to malignant transformation and proliferation of normal uveal melanocytes (UVM) using the Suppressive Subtractive Hybridization (SSH) technique. Methods The SSH technique was used to isolate genes that are differentially expressed in the TP31 cell line derived from a primary UM compared to UVM. The expression level of selected genes was further validated by microarray, semi-quantitative RT–PCR and western blot analyses. Results Analysis of the subtracted libraries revealed that 37 and 36 genes were, respectively, up- and downregulated in TP31 cells compared to UVM. Differential expression of the majority of these genes was confirmed by comparing UM cells with UVM by microarray. The expression pattern of selected genes was analyzed by semi-quantitative RT–PCR and western blot, and was found to be consistent with the SSH findings. Conclusions We demonstrated that the SSH technique is efficient to detect differentially expressed genes in UM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor. PMID:21647268

  4. Identification of Genes Associated with Morphology in Aspergillus Niger by Using Suppression Subtractive Hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Ziyu; Mao, Xingxue; Magnuson, Jon K.; Lasure, Linda L.

    2004-04-01

    The morphology of citric acid production strains of Aspergillus niger is sensitive to a variety of factors including the concentration of manganese (Mn2+). Upon increasing the Mn2+ concentration in A. niger (ATCC 11414) cultures to 14 ppb or higher, the morphology switches from pelleted to filamentous, accompanied by a rapid decline in citric acid production. Molecular mechanisms through which Mn2+ exerts effects on morphology and citric acid production in A. niger have not been well defined, but our use of suppression subtractive hybridization has identified 22 genes responsive to Mn2+. Fifteen genes were differentially expressed when A. niger was grown in media containing 1000 ppb Mn2+ (filamentous form) and seven genes in 10 ppb Mn2+ (pelleted form). Of the fifteen filamentous-associated genes, seven are novel and eight share 47-100% identity to genes from other organisms. Five of the pellet-associated genes are novel, and the other two genes encode a pepsin-type protease and polyubiquitin. All ten genes with deduced functions are either involved in amino acid metabolism/protein catabolism or cell regulatory processes. Northern-blot analysis showed that the transcripts of all 22 genes were rapidly enhanced or suppressed by Mn2+. Steady-state mRNA levels of six selected filamentous associated genes remained high during five days of culture in a filamentous state and low under pelleted growth conditions. The opposite behavior was observed for four selected pellet-associated genes. The full-length cDNA of the filamentous-associated clone, Brsa-25 was isolated. Antisense expression of Brsa-25 permitted pelleted growth and increased citrate production at higher concentrations of Mn2+ than could be tolerated by the parent strain. The results suggest the involvement of the newly isolated genes in regulation of A. niger morphology.

  5. Modeling Hybridization Kinetics of Gene Probes in a DNA Biochip Using FEMLAB

    Directory of Open Access Journals (Sweden)

    Ahsan Munir

    2017-05-01

    Full Text Available Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids-based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand present in the sample are of interest. To achieve optimal sensitivity with minimum sample size and rapid hybridization, ability to predict the kinetics of hybridization based on the thermodynamic characteristics of the probe is crucial. In this study, a computer aided numerical model for the design and optimization of a flow-through biochip was developed using a finite element technique packaged software tool (FEMLAB; package included in COMSOL Multiphysics to simulate the transport of DNA through a microfluidic chamber to the reaction surface. The model accounts for fluid flow, convection and diffusion in the channel and on the reaction surface. Concentration, association rate constant, dissociation rate constant, recirculation flow rate, and temperature were key parameters affecting the rate of hybridization. The model predicted the kinetic profile and signal intensities of eighteen 20-mer probes targeting vancomycin resistance genes (VRGs. Predicted signal intensities and hybridization kinetics strongly correlated with experimental data in the biochip (R2 = 0.8131.

  6. Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization

    Directory of Open Access Journals (Sweden)

    Chetta M

    2008-01-01

    Full Text Available Background: Standard methods of mutation detection are time consuming in Hemophilia A (HA rendering their application unavailable in some analysis such as prenatal diagnosis. Objectives: To evaluate the feasibility of combinatorial sequencing-by-hybridization (cSBH as an alternative and reliable tool for mutation detection in FVIII gene. Patients/Methods: We have applied a new method of cSBH that uses two different colors for detection of multiple point mutations in the FVIII gene. The 26 exons encompassing the HA gene were analyzed in 7 newly diagnosed Italian patients and in 19 previously characterized individuals with FVIII deficiency. Results: Data show that, when solution-phase TAMRA and QUASAR labeled 5-mer oligonucleotide sets mixed with unlabeled target PCR templates are co-hybridized in the presence of DNA ligase to universal 6-mer oligonucleotide probe-based arrays, a number of mutations can be successfully detected. The technique was reliable also in identifying a mutant FVIII allele in an obligate heterozygote. A novel missense mutation (Leu1843Thr in exon 16 and three novel neutral polymorphisms are presented with an updated protocol for 2-color cSBH. Conclusions: cSBH is a reliable tool for mutation detection in FVIII gene and may represent a complementary method for the genetic screening of HA patients.

  7. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    asy gene is a novel apoptosis-inducing gene,but its mechanism is unclear.To investigate the mechanism of asy inducing apoptosis,a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system.The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels.Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end,and share a common open reading frame encoding a polypeptide of 236 amino acids.Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence.Two highly hydrophobic regions encoding potentially two transmembrane domains are present.The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu).Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells.Compared with asy gene,overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.

  8. Identification of cold tolerance genes from leaves of mangrove plant Kandelia obovata by suppression subtractive hybridization.

    Science.gov (United States)

    Fei, Jiao; Wang, You-Shao; Jiang, Zhao-Yu; Cheng, Hao; Zhang, Jian-Dong

    2015-10-01

    Low temperature is a major abiotic stress that seriously limits mangrove productivity and distribution, the molecular mechanisms of cold tolerance involved in mangroves are still poorly understood at present. It was used to identify the potential cold-related genes in Kandelia obovata (K. obovata) by suppression subtractive hybridization. 334 cold-related expressed sequence tags (ESTs) out of 670 clones were isolated and sequenced. Among these ESTs, 143 unique cDNAs were identified and classified into ten groups, such as metabolism, energy, cell rescue and defense, transcription and photosynthesis according to NCBI blast. Based on bioinformatics analysis, these ESTs were mainly related to response to stimulus and metabolic process, and were included to 72 KEGG pathways. Two selected genes (e.g., aquaporin gene and zinc family protein gene) from the library were further analyzed by quantitative real-time PCR analysis. Both the two genes were found to be transcriptionally up-regulated under cold stress, which partly approve the construction of the subtractive cDNA library. The diversity of the putative functions of these genes indicated that cold stress resulted in a complex response in K. obovata. Further investigation on the functions and potential pathways of these genes will facilitate the understanding of the molecular adaptations to cold tolerance in mangrove plants.

  9. Identification of immune response-related genes in the Chinese oak silkworm, Antheraea pernyi by suppression subtractive hybridization.

    Science.gov (United States)

    Liu, Qiu-Ning; Zhu, Bao-Jian; Wang, Lei; Wei, Guo-Qing; Dai, Li-Shang; Lin, Kun-Zhang; Sun, Yu; Qiu, Jian-Feng; Fu, Wei-Wei; Liu, Chao-Liang

    2013-11-01

    Insects possess an innate immune system that responds to invading microorganisms. In this study, a subtractive cDNA library was constructed to screen for immune response-related genes in the fat bodies of Antheraea pernyi (Lepidoptera: Saturniidae) pupa challenged with Escherichia coli. Four hundred putative EST clones were identified by suppression subtractive hybridization (SSH), including 50 immune response-related genes, three cytoskeleton genes, eight cell cycle and apoptosis genes, five respiration and energy metabolism genes, five transport genes, 40 metabolism genes, ten stress response genes, four transcription and translation regulation genes and 77 unknown genes. To verify the reliability of the SSH data, the transcription of a set of randomly selected immune response-related genes were confirmed by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time quantitative reverse transcription-PCR (qRT-PCR). These identified immune response-related genes provide insight into understanding the innate immunity in A. pernyi.

  10. Isolation and identification of genes expressed differentially in rice inflorescence meristem with suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A subtracted cDNA library of rice (Oryza sativa L.) inflorescence meristem (IM) was constructed using the sup-pression subtractive hybridization (SSH) method. The cDNAs of the rice shoot apical meristem (SAM) were used as "driver" and inflorescence meristem (IM) as "tester" in the experiment, respectively. Forty of 250 randomly chosen cDNA clones were identified by differential screening, which were IM-specific or IM-highly expressed. Most of the rice IM cDNAs cloned by SSH appear to represent rare transcripts, 40% of which were derived from truly differentially ex-pressed genes. Of all the forty sequenced cDNA inserts, eleven contain the regions with 60%-90% identity to their homolog in GenBank, eighteen are expected to be new genes, only two correspond to published rice genes.

  11. Isolating Soil Drought-Induced Genes from Maize Seedling Leaves Through Suppression Subtractive Hybridization

    Institute of Scientific and Technical Information of China (English)

    LI Hui-yong; HUANG Su-hua; SHI Yun-su2; SONG Yan-chun; ZHAO Jiu-ran; WANG Feng-ge; WANG Tian-yu; LI Yu

    2007-01-01

    In this study, a forward cDNA library was constructed by suppression subtractive hybridization using seedling leaves of CN165, a drought-tolerant maize inbred line. In the suppression subtractive hybridization (SSH) library, 672 positive clones were picked up randomly. After polymerase chain reaction (PCR) of each clone, all the single clones were sequenced. Totally 598 available sequences were obtained. After cluster analysis of the EST sequences, 80 uniESTs were obtained, among which 57 uniESTs were contigs and 23 uniESTs were singlets. The results of BLASTN showed that all the uniESTs had homologous sequences in the nr database. The BLASTX results indicated that 68 uniESTs had significant protein homology, 8 uniESTs with homology of unknown proteins and putative proteins, and 4 uniESTs without protein homology. Those drought stress-induced genes were involved in many metabolism pathways to regulate plant growth and development under drought stress.

  12. Analysis of genome-wide gene expression in root of wheat hybrid and its parents using Barley1 GeneChip

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yinhong; NI Zhongfu; YAO Yingyin; ZHAO Jun; SUN Qixin

    2006-01-01

    Previous studies indicated that differential gene expression between hybrids and their parents is responsible for the heterosis. In this paper, we report genome-wide gene expression analysis in roots of a highly heterotic inter-specific hybrid 3338/2463 and its parental inbreds using Barley GeneChip. A total of 1187 genes displayed difference in gene expressions between hybrid 3338/2463 and its parents, and they can be clustered into eight differential expression patterns. Further analysis revealed that among these 1187 genes, 975genes showed high sequence similarity to the GenBank entries, and represented diverse functional categories, such as metabolism, cell growth and maintenance, signal transduction, response to stress, transcription regulation and others. Fourteen genes were selected for RT-PCR analysis and expression patterns of 9 (64.29%) genes were confirmed. Remarkably, 380 differentially expressed genes could be mapped on the Chinese Spring deletion bins, and with the number of genes in seven homoeologous groups being 158, 148, 121, 140,132, 94 and 127 respectively. It is concluded that a combination of systematic identification of differentially expressed genes with comparative mapping would provide further insight into understanding of molecular basis of heterosis.

  13. Identification of differentially expressed genes in Mongolian sheep ovaries by suppression subtractive hybridization.

    Science.gov (United States)

    He, Xiaolong; Li, Bei; Wang, Feng; Tian, Chunying; Rong, Weiheng; Liu, Yongbin

    2012-07-01

    Fecundity is an important trait in sheep. Because it is directly related to production costs and efficiency, it has great economic impact in sheep husbandry. Because Mongolian sheep are a longstanding, indigenous breed, they are genetically related to most other breeds of sheep in China. The study of genes related to reproductive traits is essential to improving the fecundity of Mongolian sheep. In the present study, suppression subtractive hybridization (SSH) was performed using forward and reverse nested primers on cDNA libraries from ovarian tissue of single-bearing (S) and biparous (B) Mongolian sheep (MS). This yielded 768 clones. The length of the inserted fragments ranged from 150 to 1000 bp. From these, dot blot hybridization followed by sequencing and homology blast search in GenBank resolved 373 differentially expressed clones, representing 185 gene sequences (homology >85% and length >200 bp), 10 expressed sequence tags (ESTs; homology >95% and length >100 bp), and 4 unknown ESTs. The analysis of the differentially expressed gene functions allowed these genes to be categorized into seven groups: cell/body or immune defense, metabolism, transportation, nucleic acid modification, cell development, signal transduction, and cell structure. Four differentially expressed genes, a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), inhibitor of DNA binding 3 (ID3), bone morphogenetic protein 6 (BMP6), and integrin beta 1 (ITGB1), were randomly selected and verified using relative quantitative real-time polymerase chain reaction (RQ-PCR). The expression of these genes in BMS ovaries was 30.06, 11.55, 0.82, and 1.12-fold that of SMS ovaries, respectively.

  14. Knockdown of antiapoptotic genes in breast cancer cells by siRNA loaded into hybrid nanoparticles

    Science.gov (United States)

    João de Mello, Leônidas, Jr.; Rosa Souza, Gabriela Regina; Winter, Evelyn; Silva, Adny Henrique; Pittella, Frederico; Creczynski-Pasa, Tânia Beatriz

    2017-04-01

    Tumorigenesis is related to an imbalance in controlling mechanisms of apoptosis. Expression of the genes BCL-2 and BCL-xL results in the promotion of cell survival by inhibiting apoptosis. Thus, a novel approach to suppress antiapoptotic genes is the use of small interfering RNA (siRNA) in cancer cells. However, there are some limitations for the application of siRNA such as the need for vectors to pass the cell membrane and deliver the nucleic acid. In this study CaP-siRNA-PEG-polyanion hybrid nanoparticles were developed to promote siRNA delivery to cultured human breast cancer cells (MCF-7) in order to evaluate whether the silencing of antiapoptotic genes BCL-2 and BCL-xL by siRNA would increase cancer cell death. After 48 h of incubation the expression of BCL-2 and BCL-xL genes decreased to 49% and 23%, respectively. The siRNA sequence used induced cancer cell death at a concentration of 200 nM siRNA after 72 h of incubation. As the targeted proteins are related to the resistance to chemotherapeutic drugs, the nanocarriers systems were also tested in the presence of doxorubicin (DOX). The results showed a significant reduction in the CC50 of the DOX, after silencing the antiapoptotic genes. In addition, an increase in apoptotic cell counts for both incubations conditions was observed as well. In conclusion, silencing antiapoptotic genes such as BCL-2 and BCL-xL through the use of siRNA carried by hybrid nanoparticles showed to be effective in vitro, and presents a promising strategy for pre-clinical analysis, especially when combined with DOX against breast cancer.

  15. Profiling of differentially expressed genes using suppression subtractive hybridization in an equine model of chronic asthma.

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Lavoie

    Full Text Available BACKGROUND: Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma. OBJECTIVE: To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition. METHODS: Eleven adult horses (6 heaves-affected and 5 controls were studied while horses with heaves were in clinical remission (Pasture, and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge. Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH, lung cDNAs of controls (Pasture and Challenge and asymptomatic heaves-affected horses (Pasture were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge. The differential expression of selected genes of interest was confirmed using quantitative PCR assay. RESULTS: Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways. CONCLUSIONS: Pathways representing new possible targets for anti-inflammatory and anti

  16. A top-down approach for construction of hybrid polymer-virus gene delivery vectors.

    Science.gov (United States)

    Ramsey, Joshua D; Vu, Halong N; Pack, Daniel W

    2010-05-21

    Safe and efficient delivery of therapeutic nucleic acids remains the primary hurdle for human gene therapy. While many researchers have attempted to re-engineer viruses to be suited for gene delivery, others have sought to develop non-viral alternatives. We have developed a complementary approach in which viral and synthetic components are combined to form hybrid nanoparticulate vectors. In particular, we complexed non-infectious retrovirus-like particles lacking a viral envelope protein, from Moloney murine leukemia virus (M-VLP) or human immunodeficiency virus (H-VLP), with poly-L-lysine (PLL) or polyethylenimine (PEI) over a range of polymer/VLP ratios. At appropriate stoichiometry (75-250 microg polymer/10(6) VLP), the polymers replace the function of the viral envelope protein and interact with the target cell membrane, initiate cellular uptake and facilitate escape from endocytic vesicles. The viral particle, once in the cytosol, efficiently completes its normal infection process including integration of viral genes with the host genome as demonstrated by long-term (at least 5 weeks) transgene expression. In addition, hybrid vectors comprising H-VLP were shown to be capable of infecting non-dividing cells.

  17. Identification of mycosis-related genes in the eastern subterranean termite by suppression subtractive hybridization.

    Science.gov (United States)

    Gao, Qi; Tancredi, Sarah E; Thompson, Graham J

    2012-07-01

    The Eastern subterranean termite Reticulitermes flavipes (Isoptera, Rhinotermitidae) is a cosmopolitan, structural pest that is the target of research into termite innate immunity. In this study, we use suppression subtractive hybridization to construct a normalized cDNA library of genes excessively expressed upon fungal infection. At 24 h postinfection with Metarhizium anisopliae, the library revealed 182 expressed sequence tag (EST) clones that potentially represent immune responsive genes. The nucleotide sequence from a majority (97%) of ESTs assembled into a small number (n = 13) of contiguous sequences, with the remainder (n = 6) representing singletons. Our screen therefore captured as many as 19 different mRNAs highly expressed in response to the fungal pathogen at this time. Primary sequencing of all loci revealed that approximately half (n = 10) contained open reading frames with significant similarity to known proteins. These clones represent nuclear and mitochondrial coding genes, as well as putative long noncoding RNA genes. Quantitative polymerase chain reaction analysis of coding genes on independently infected groups of worker termites confirms in each case that the transcripts identified from the library are up-regulated postfungal infection. The genes identified here are relevant to future studies on termite biocontrol and social insect immunity.

  18. Identification of novel genes involved in gastric carcinogenesis by suppression subtractive hybridization.

    Science.gov (United States)

    Mottaghi-Dastjerdi, N; Soltany-Rezaee-Rad, M; Sepehrizadeh, Z; Roshandel, G; Ebrahimifard, F; Setayesh, N

    2015-01-01

    Gastric cancer (GC) is one of the most common and life-threatening types of malignancies. Identification of the differentially expressed genes in GC is one of the best approaches for establishing new diagnostic and therapeutic targets. Furthermore, these investigations could advance our knowledge about molecular biology and the carcinogenesis of this cancer. To screen for the overexpressed genes in gastric adenocarcinoma, we performed suppression subtractive hybridization (SSH) on gastric adenocarcinoma tissue and the corresponding normal gastric tissue, and eight genes were found to be overexpressed in the tumor compared with those of the normal tissue. The genes were ribosomal protein L18A, RNase H2 subunit B, SEC13, eukaryotic translation initiation factor 4A1, tetraspanin 8, cytochrome c oxidase subunit 2, NADH dehydrogenase subunit 4, and mitochondrially encoded ATP synthase 6. The common functions among the identified genes include involvement in protein synthesis, involvement in genomic stability maintenance, metastasis, metabolic improvement, cell signaling pathways, and chemoresistance. Our results provide new insights into the molecular biology of GC and drug discovery: each of the identified genes could be further investigated as targets for prognosis evaluation, diagnosis, treatment, evaluation of the response to new anticancer drugs, and determination of the molecular pathogenesis of GC.

  19. Suppression subtractive hybridization reveals differential gene expression in sunflower grown in high P.

    Science.gov (United States)

    Padmanabhan, Priya; Sahi, Shivendra V

    2011-06-01

    Sunflower (Helianthus annuus L.) is a commercially important oilseed crop. Previous studies proved that this crop is a promising plant species for phytoextraction of excess soil phosphorus (P) because of its superior P accumulating characteristics. Suppression subtractive hybridization (SSH) strategy was employed to isolate and characterize genes that are induced in response to high P in this crop. SSH library was prepared using cDNA generated from plants treated with high P as the 'tester'. Based on the results of dot blot analysis, 360 positive cDNA clones were selected from the SSH library for sequencing. A total of 89 non-redundant expressed sequence tags (ESTs) were identified as high P-responsive genes and they were classified into 6 functional groups. Several genes involved in metabolism showed markedly preferential expression in the library. For further confirmation, thirteen of the representative ESTs were selected from all categories for RT-PCR analysis and the results showed up-regulation of these genes in response to high P-treatment. The gene expression data derived from this study suggested that several of the up-regulated genes identified under high P-treatment might be involved in P-accumulation and tolerance in this plant.

  20. Cloning and characterization of the gene for L-amino acid oxidase in hybrid tilapia.

    Science.gov (United States)

    Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

    2015-12-01

    Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P tilapia. The investigation of relationship between polymorphism of LAO gene and disease resistance and growth in tilapia showed that one SNP was associated significantly with body length. Further experiments on whether SNPs in the LAO gene are associated with growth in tilapia and other populations could be useful in understanding more functions of the LAO gene.

  1. High-resolution comparative genomic hybridization of inflammatory breast cancer and identification of candidate genes.

    Directory of Open Access Journals (Sweden)

    Ismahane Bekhouche

    Full Text Available BACKGROUND: Inflammatory breast cancer (IBC is an aggressive form of BC poorly defined at the molecular level. We compared the molecular portraits of 63 IBC and 134 non-IBC (nIBC clinical samples. METHODOLOGY/FINDINGS: Genomic imbalances of 49 IBCs and 124 nIBCs were determined using high-resolution array-comparative genomic hybridization, and mRNA expression profiles of 197 samples using whole-genome microarrays. Genomic profiles of IBCs were as heterogeneous as those of nIBCs, and globally relatively close. However, IBCs showed more frequent "complex" patterns and a higher percentage of genes with CNAs per sample. The number of altered regions was similar in both types, although some regions were altered more frequently and/or with higher amplitude in IBCs. Many genes were similarly altered in both types; however, more genes displayed recurrent amplifications in IBCs. The percentage of genes whose mRNA expression correlated with CNAs was similar in both types for the gained genes, but ∼7-fold lower in IBCs for the lost genes. Integrated analysis identified 24 potential candidate IBC-specific genes. Their combined expression accurately distinguished IBCs and nIBCS in an independent validation set, and retained an independent prognostic value in a series of 1,781 nIBCs, reinforcing the hypothesis for a link with IBC aggressiveness. Consistent with the hyperproliferative and invasive phenotype of IBC these genes are notably involved in protein translation, cell cycle, RNA processing and transcription, metabolism, and cell migration. CONCLUSIONS: Our results suggest a higher genomic instability of IBC. We established the first repertory of DNA copy number alterations in this tumor, and provided a list of genes that may contribute to its aggressiveness and represent novel therapeutic targets.

  2. Multicolor in situ hybridization and linkage analysis order Charcot-Marie-Tooth type I (CMTIA) gene-region markers

    Energy Technology Data Exchange (ETDEWEB)

    Lebo, R.V.; Lynch, E.D.; Golbus, M.S. (Univ. of California, San Francisco (United States)); Bird, T.D. (Univ. of Washington, Seattle (United States)); Barker, D.F.; O' Connell, P.; Chance, P.F. (Univ. of Utah, Salt Lake City (United States))

    1992-01-01

    This study demonstrates a clear and current role for multicolor in situ hybridization in expediting positional cloning studies of unknown disease genes. Nine polymorphic DNA cosmids have been mapped to eight ordered locations spanning the Charcot-Marie-Tooth type 1 (CMT1A) disease gene region in distal band 17p11.2, by multicolor in situ hybridization. When used with linkage analysis, these methods have generated a fine physical map and have firmly assigned the CMT1A gene to distal band 17p11.2. Linkage analysis with four CMT1A pedigrees mapped the CMT1A gene with respect to two flanking markers. Additional loci were physically mapped and ordered by in situ hybridization and analysis of phase-known recombinants in CMT1A pedigrees. These data demonstrate the ability of in situ hybridization to resolve loci within 0.5 Mb on early-metaphase chromosomes. Multicolor in situ hybridization also excluded the possibility of pericentric inversions in two unrelated patients with CMT1 and neurofibromatosis type 1. When used with pulsed-field gel electrophoresis, multicolor in situ hybridization can establish physical location, order, and distance in closely spaced chromosome loci.

  3. Identification of warm day and cool night conditions induced flowering-related genes in a Phalaenopsis orchid hybrid by suppression subtractive hybridization.

    Science.gov (United States)

    Li, D M; Lü, F B; Zhu, G F; Sun, Y B; Xu, Y C; Jiang, M D; Liu, J W; Wang, Z

    2014-02-14

    The influence of warm day and cool night conditions on induction of spikes in Phalaenopsis orchids has been studied with respect to photosynthetic efficiency, metabolic cycles and physiology. However, molecular events involved in spike emergence induced by warm day and cool night conditions are not clearly understood. We examined gene expression induced by warm day and cool night conditions in the Phalaenopsis hybrid Fortune Saltzman through suppression subtractive hybridization, which allowed identification of flowering-related genes in warm day and cool night conditions in spikes and leaves at vegetative phase grown under warm daily temperatures. In total, 450 presumably regulated expressed sequence tags (ESTs) were identified and classified into functional categories, including metabolism, development, transcription factor, signal transduction, transportation, cell defense, and stress. Furthermore, database comparisons revealed a notable number of Phalaenopsis hybrid Fortune Saltzman ESTs that matched genes with unknown function. The expression profiles of 24 genes (from different functional categories) have been confirmed by quantitative real-time PCR in induced spikes and juvenile apical leaves. The results of the real-time PCR showed that, compared to the vegetative apical leaves, the transcripts of genes encoding flowering locus T, AP1, AP2, KNOX1, knotted1-like homeobox protein, R2R3-like MYB, adenosine kinase 2, S-adenosylmethionine synthetase, dihydroflavonol 4-reductase, and naringenin 3-dioxygenase accumulated significantly higher levels, and genes encoding FCA, retrotransposon protein Ty3 and C3HC4-type RING finger protein accumulated remarkably lower levels in spikes of early developmental stages. These results suggested that the genes of two expression changing trends may play positive and negative roles in the early floral transition of Phalaenopsis orchids. In conclusion, spikes induced by warm day and cool night conditions were complex in

  4. Genome-wide gene expression of a natural hybrid between Saccharomyces cerevisiae and S. kudriavzevii under enological conditions.

    Science.gov (United States)

    Combina, Mariana; Pérez-Torrado, Roberto; Tronchoni, Jordi; Belloch, Carmela; Querol, Amparo

    2012-07-16

    The species Saccharomyces cerevisiae plays a predominant role in the wine making process. However, other species have been associated with must fermentation, such as Saccharomyces uvarum (Saccharomyces bayanus var. uvarum) or Saccharomyces paradoxus. Recently, yeast hybrids of different Saccharomyces species have also been reported as responsible for wine production. Yeast hybrids between the species S. cerevisiae×S. kudriavzevii isolated in wine fermentations show enhanced performance in low temperature enological conditions and increased production of interesting aroma compounds. In this work, we have studied the transcriptomic response in enological conditions of a S. cerevisiae×S. kudriavzevii hybrid strain and compared it with the reference species of S. cerevisiae and S. kudriavzevii. The results show that the hybrid strain presents an up-regulation of genes belonging to functional group translation and amino-acid metabolism. Moreover, key genes related to cold stress and production of glycerol and aroma compounds were also up-regulated. While some genes inherited regulation patterns from one of the parents, most of the up-regulated genes presented a new gene expression pattern, probably generated during the hybridization and adaptation process.

  5. Isolation and characterization of a human apoptosis-inducing gene with yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    齐兵; 齐义鹏; Masuo; Yutsudo; 刘青珍

    2000-01-01

    asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5’ end and different 3’ end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of

  6. Identification of Differentially Expressed Genes Associated with Apple Fruit Ripening and Softening by Suppression Subtractive Hybridization.

    Directory of Open Access Journals (Sweden)

    Zongying Zhang

    Full Text Available Apple is one of the most economically important horticultural fruit crops worldwide. It is critical to gain insights into fruit ripening and softening to improve apple fruit quality and extend shelf life. In this study, forward and reverse suppression subtractive hybridization libraries were generated from 'Taishanzaoxia' apple fruits sampled around the ethylene climacteric to isolate ripening- and softening-related genes. A set of 648 unigenes were derived from sequence alignment and cluster assembly of 918 expressed sequence tags. According to gene ontology functional classification, 390 out of 443 unigenes (88% were assigned to the biological process category, 356 unigenes (80% were classified in the molecular function category, and 381 unigenes (86% were allocated to the cellular component category. A total of 26 unigenes differentially expressed during fruit development period were analyzed by quantitative RT-PCR. These genes were involved in cell wall modification, anthocyanin biosynthesis, aroma production, stress response, metabolism, transcription, or were non-annotated. Some genes associated with cell wall modification, anthocyanin biosynthesis and aroma production were up-regulated and significantly correlated with ethylene production, suggesting that fruit texture, coloration and aroma may be regulated by ethylene in 'Taishanzaoxia'. Some of the identified unigenes associated with fruit ripening and softening have not been characterized in public databases. The results contribute to an improved characterization of changes in gene expression during apple fruit ripening and softening.

  7. Identification of Differentially Expressed Genes Associated with Apple Fruit Ripening and Softening by Suppression Subtractive Hybridization.

    Science.gov (United States)

    Zhang, Zongying; Jiang, Shenghui; Wang, Nan; Li, Min; Ji, Xiaohao; Sun, Shasha; Liu, Jingxuan; Wang, Deyun; Xu, Haifeng; Qi, Sumin; Wu, Shujing; Fei, Zhangjun; Feng, Shouqian; Chen, Xuesen

    2015-01-01

    Apple is one of the most economically important horticultural fruit crops worldwide. It is critical to gain insights into fruit ripening and softening to improve apple fruit quality and extend shelf life. In this study, forward and reverse suppression subtractive hybridization libraries were generated from 'Taishanzaoxia' apple fruits sampled around the ethylene climacteric to isolate ripening- and softening-related genes. A set of 648 unigenes were derived from sequence alignment and cluster assembly of 918 expressed sequence tags. According to gene ontology functional classification, 390 out of 443 unigenes (88%) were assigned to the biological process category, 356 unigenes (80%) were classified in the molecular function category, and 381 unigenes (86%) were allocated to the cellular component category. A total of 26 unigenes differentially expressed during fruit development period were analyzed by quantitative RT-PCR. These genes were involved in cell wall modification, anthocyanin biosynthesis, aroma production, stress response, metabolism, transcription, or were non-annotated. Some genes associated with cell wall modification, anthocyanin biosynthesis and aroma production were up-regulated and significantly correlated with ethylene production, suggesting that fruit texture, coloration and aroma may be regulated by ethylene in 'Taishanzaoxia'. Some of the identified unigenes associated with fruit ripening and softening have not been characterized in public databases. The results contribute to an improved characterization of changes in gene expression during apple fruit ripening and softening.

  8. Identification of differentially expressed genes in parasitic phase Miamiensis avidus (Ciliophora: Scuticociliatia) using suppression subtractive hybridization.

    Science.gov (United States)

    Lee, Eun Hye; Kim, Ki Hong

    2011-04-06

    Miamiensis avidus, a causative agent of scuticociliatosis in cultured marine fish, can live not only in seawater as a free-living organism but also in fish as a parasite. In this study, a cDNA library of representative mRNAs more specific to parasitic phase M. avidus was generated using suppression subtractive hybridization (SSH), and 520 clones selected from the SSH library were single-run sequenced. The differential gene expression patterns were confirmed by semi-quantitative reverse-transcription PCR. Of the 510 SSH clones, 21 clones of 6 putative genes did not match sequences in the public database. The expectation values (E-values) of 117 clones encoding 9 putative genes were greater than 1 x 10(-5). The other 372 clones that met the criterion of E value <1 x 10-5 were matched to 26 known sequences in the database. Genes associated with signal transduction, cell proliferation, membrane transportation, protein translocation, and transcription regulation were preferentially expressed in parasitic phase M. avidus. The differential gene expression may be needed for the ciliates to survive in the host fish, and the corresponding proteins might be used as antigen candidates for development of scuticociliatosis vaccines.

  9. Analysis of gene expression in granulosa cells of ovine antral growing follicles using suppressive subtractive hybridization.

    Science.gov (United States)

    Chen, A Qin; Wang, Zheng Guang; Xu, Zi Rong; Yu, Song Dong; Yang, Zhi Gang

    2009-10-01

    Follicular growth, development and ovulation are highly ordered processes that involve the expression of many genes under precise temporal and spatial regulation. However, information on stage-specific gene expression during the antral follicle phase in sheep is not well understood. In the present study, suppressive subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the granulosa cells between large follicles (LF, >5mm) and small follicles (SF, 3-5mm), and subtractive cDNA library was constructed. Furthermore, with dot-blot analysis, a total of 90 clones randomly selected from the library were proven to be differentially expressed in the granulosa cells. Among these, 38 exhibited high homology to known genes, 14 sequences were corresponding to novel expressed sequence tags (ESTs). Four ESTs, LAPTM4A, SERPINE2, GSTA1, and INHBA, were further examined the reproducibility of the SSH data by the real-time quantitative PCR. Results confirmed an increase expression of respective mRNA in granulosa cells of large follicles compared with that of small follicles. It is concluded that we have identified several genes (known or unknown) that may effect follicular growth, dominance or ovulation in ewes.

  10. Streptococcus pneumoniae Supragenome Hybridization Arrays for Profiling of Genetic Content and Gene Expression.

    Science.gov (United States)

    Kadam, Anagha; Janto, Benjamin; Eutsey, Rory; Earl, Joshua P; Powell, Evan; Dahlgren, Margaret E; Hu, Fen Z; Ehrlich, Garth D; Hiller, N Luisa

    2015-02-02

    There is extensive genomic diversity among Streptococcus pneumoniae isolates. Approximately half of the comprehensive set of genes in the species (the supragenome or pangenome) is present in all the isolates (core set), and the remaining is unevenly distributed among strains (distributed set). The Streptococcus pneumoniae Supragenome Hybridization (SpSGH) array provides coverage for an extensive set of genes and polymorphisms encountered within this species, capturing this genomic diversity. Further, the capture is quantitative. In this manner, the SpSGH array allows for both genomic and transcriptomic analyses of diverse S. pneumoniae isolates on a single platform. In this unit, we present the SpSGH array, and describe in detail its design and implementation for both genomic and transcriptomic analyses. The methodology can be applied to construction and modification of SpSGH array platforms, as well to other bacterial species as long as multiple whole-genome sequences are available that collectively capture the vast majority of the species supragenome.

  11. A Hybrid CFHR3-1 Gene Causes Familial C3 Glomerulopathy.

    LENUS (Irish Health Repository)

    Malik, Talat H

    2012-07-01

    Controlled activation of the complement system, a key component of innate immunity, enables destruction of pathogens with minimal damage to host tissue. Complement factor H (CFH), which inhibits complement activation, and five CFH-related proteins (CFHR1-5) compose a family of structurally related molecules. Combined deletion of CFHR3 and CFHR1 is common and confers a protective effect in IgA nephropathy. Here, we report an autosomal dominant complement-mediated GN associated with abnormal increases in copy number across the CFHR3 and CFHR1 loci. In addition to normal copies of these genes, affected individuals carry a unique hybrid CFHR3-1 gene. In addition to identifying an association between these genetic observations and complement-mediated kidney disease, these results provide insight into the protective role of the combined deletion of CFHR3 and CFHR1 in IgA nephropathy.

  12. Non-additive gene regulation in a citrus allotetraploid somatic hybrid between C. reticulata Blanco and C. limon (L.) Burm.

    Science.gov (United States)

    Bassene, J B; Froelicher, Y; Dubois, C; Ferrer, R M; Navarro, L; Ollitrault, P; Ancillo, G

    2010-09-01

    Polyploid plants often produce new phenotypes, exceeding the range of variability existing in the diploid gene pool. Several hundred citrus allotetraploid hybrids have been created by somatic hybridization. These genotypes are interesting models to study the immediate effects of allopolyploidization on the regulation of gene expression. Here, we report genome-wide gene expression analysis in fruit pulp of a Citrus interspecific somatic allotetraploid between C. reticulata cv 'Willowleaf mandarin'+C. limon cv 'Eureka lemon', using a Citrus 20K cDNA microarray. Around 4% transcriptome divergence was observed between the two parental species, and 212 and 160 genes were more highly expressed in C. reticulata and C. limon, respectively. Differential expression of certain genes was confirmed by quantitative real-time RT-PCR. A global downregulation of the allotetraploid hybrid transcriptome was observed, as compared with a theoretical mid parent, for the genes displaying interspecific expression divergence between C. reticulata and C. limon. The genes underexpressed in mandarin, as compared with lemon, were also systematically repressed in the allotetraploid. When genes were overexpressed in C. reticulata compared with C. limon, the distribution of allotetraploid gene expression was far more balanced. Cluster analysis on the basis of gene expression clearly indicated the hybrid was much closer to C. reticulata than to C. limon. These results suggest there is a global dominance of the mandarin transcriptome, in consistence with our previous studies on aromatic compounds and proteomics. Interspecific differentiation of gene expression and non-additive gene regulation involved various biological pathways and different cellular components.

  13. Maize x Teosinte Hybrid Cobs Do Not Prevent Crop Gene Introgression.

    Science.gov (United States)

    Chavez, Nancy B; Flores, Jose J; Martin, Joseph; Ellstrand, Norman C; Guadagnuolo, Roberto; Heredia, Sylvia; Welles, Shana R

    2012-06-01

    Maize x Teosinte Hybrid Cobs Do Not Prevent Crop Gene Introgression. Whether introgression from crops to wild relatives can occur is an important component of transgene risk assessment. In the case of maize, which co-occurs with its wild relative teosinte in Mexico, the possibility of introgression has been controversial. Maize is cross-compatible with teosinte, and spontaneous hybridization is known to occur. Some scientists have hypothesized that the maize x teosinte cob infructescence will prevent progeny dispersal, thus preventing introgression. Motivated by a prior study where we found maize x teosinte hybrid fruits naturally dispersed under field conditions, we tested whether hybrid cobs hold their fruits as tightly as maize cobs. We found the force required to detach hybrid fruits was substantially and significantly less than that for maize. Consequently, we expect that introgression of transgenes from maize into teosinte in Mexico should occur largely unimpeded by the hybrid cob.La mazorca o elote híbrido de maíz x teocintle no impide la introgresión de genes transgénicos provenientes del cultivo. La introgresión entre el maíz cultivado y el maíz silvestre, o teocintle, es un componente importante en la evaluación ambiental relacionada con los riesgos de la introducción de genes transgénicos. La posibilidad de introgresión entre el maíz domesticado y el teocintle ha sido un tema controversial, en particular en México, donde maíz y teocintle coexisten. El maíz es compatible con el teocintle y la hibridización espontánea ocurre entre ellos. Algunos científicos han planteado como hipótesis que al cruzar el maíz con teocintle, la estructura interna de la infrutescencia que sujeta los frutos conocida como la mazorca de maíz o el elote, impide la dispersión de la progenie evitando que la introgresión ocurra. Los resultados de un estudio previo evidencian la dispersión de los frutos híbridos del maíz x teocintle en condiciones naturales

  14. Identification of cold responsive genes in Pacific white shrimp (Litopenaeus vannamei) by suppression subtractive hybridization.

    Science.gov (United States)

    Peng, Jinxia; Wei, Pinyuan; Chen, Xiuli; Zeng, Digang; Chen, Xiaohan

    2016-01-10

    The Pacific white shrimp (Litopenaeus vannamei) is one of the most widely cultured shrimp species in the world. Despite L. vannamei having tropical origins, it is being reared subtropically, with low temperature stress being one of the most severe threats to its growth, survival and distribution. To unravel the molecular basis of cold tolerance in L. vannamei, the suppression subtractive hybridization (SSH) platform was employed to identify cold responsive genes in the hepatopancreas of L. vannamei. Both forward and reverse cDNA libraries were constructed, followed by dot blot hybridization, cloning, sequence analysis and quantitative real-time PCR. These approaches identified 92 cold induced and 48 cold inhibited ESTs to give a total of 37 cold induced and 17 cold inhibited contigs. Some of the identified genes related to stress response or cell defense, such as tetraspanins (TSPANs), DEAD-box helicase, heat shock proteins (HSPs) and metallothionein (MT), which were more abundant in the forward SSH library than in the reverse SSH library. The most abundant Est was a tetraspanin-8 (TSPAN8) homolog dubbed LvTSPAN8. A multiple sequence alignment and transmembrane domain prediction was also performed for LvTSPAN8. LvTSPAN8 expression was also examined in the gills, muscle, heart and hepatopancreas following cold exposure and showed the highest expression levels in the hepatopancreas. Overall, this study was able to identify several known genes and novel genes via SSH that appear to be associated with cold stress and will help to provide further insights into the molecular mechanisms regulating cold tolerance in L. vannamei.

  15. Genome-Wide Gene Expressions Respond Differently to A-subgenome Origins in Brassica napus Synthetic Hybrids and Natural Allotetraploid

    Science.gov (United States)

    Zhang, Dawei; Pan, Qi; Tan, Chen; Zhu, Bin; Ge, Xianhong; Shao, Yujiao; Li, Zaiyun

    2016-01-01

    The young allotetraploid Brassica napus (2n = 38, AACC) is one of models to study genomic responses to allopolyploidization. The extraction of AA component from natural B. napus and then restitution of progenitor B. rapa should provide a unique opportunity to reveal the genome interplay for gene expressions during the evolution. Herein, B. napus hybrids (2n = 19, AC) between the extracted and extant B. rapa (2n = 20, AA) and the same B. oleracea genotype (2n = 18, CC) were studied by RNA-seq and compared with natural B. napus donor, to reveal the gene expression changes from hybridization and domestication and the effects of A genome with different origins. Upon the initial merger of two diploid genomes, additive gene expression was prevalent in these two hybrids, for non-additively expressed genes only represented a small portion of total expressed genes. A high proportion of genes exhibited expression level dominance, with no preference to either of the parental genomes. Comparison of homoeolog expressions also showed no bias toward any genomes and the parental expression patterns were often maintained in the hybrids and natural allotetraploids. Although, the overall patterns of gene expression were highly conserved between two hybrids, the extracted B. rapa responded less and appeared more compatible for hybridization than the extant B. rapa. Our results suggested that expression level dominance and homoeolog expressions bias were balanced at the initial stage of genome merger, and such balance were largely maintained during the domestication of B. napus, despite the increased extent over time. PMID:27790227

  16. Glyphosate drift promotes changes in fitness and transgene gene flow in canola (Brassica napus) and hybrids

    Science.gov (United States)

    Londo, Jason P.; Bautista, Nonnatus S.; Sagers, Cynthia L.; Lee, E. Henry; Watrud, Lidia S.

    2010-01-01

    Background and Aims With the advent of transgenic crops, genetically modified, herbicide-resistant Brassica napus has become a model system for examining the risks and potential ecological consequences of escape of transgenes from cultivation into wild compatible species. Escaped transgenic feral B. napus and hybrids with compatible weedy species have been identified outside of agriculture and without the apparent selection for herbicide resistance. However, herbicide (glyphosate) exposure can extend beyond crop field boundaries, and a drift-level of herbicide could function as a selective agent contributing to increased persistence of transgenes in the environment. Methods The effects of a drift level (0·1 × the field application rate) of glyphosate herbicide and varied levels of plant competition were examined on plant fitness-associated traits and gene flow in a simulated field plot, common garden experiment. Plants included transgenic, glyphosate-resistant B. napus, its weedy ancestor B. rapa, and hybrid and advanced generations derived from them. Key Results The results of this experiment demonstrate reductions in reproductive fitness for non-transgenic genotypes and a contrasting increase in plant fitness for transgenic genotypes as a result of glyphosate-drift treatments. Results also suggest that a drift level of glyphosate spray may influence the movement of transgenes among transgenic crops and weeds and alter the processes of hybridization and introgression in non-agronomic habitats by impacting flowering phenology and pollen availability within the community. Conclusions The results of this study demonstrate the potential for persistence of glyphosate resistance transgenes in weedy plant communities due to the effect of glyphosate spray drift on plant fitness. Additionally, glyphosate drift has the potential to change the gene-flow dynamics between compatible transgenic crops and weeds, simultaneously reducing direct introgression into weedy species

  17. 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.

  18. Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

    Institute of Scientific and Technical Information of China (English)

    Li Liang; Yan-Qing Ding; Xin Li; Guang-Zhi Yang; Jun Xiao; Li-Chun Lu; Jin-Hua Zhang

    2004-01-01

    AIM: To construct a differentially-expressed gene subtracted cDNA library from two colorectal carcinoma (CRC) cell lines with different metastatic phenotypes by suppression subtractive hybridization.METHODS: Two cell lines of human CRC from the same patient were used. SW620 cell line showing highly metastatic potential was regarded as tester in the forward subtractive hybridization, while SW480 cell line with lowly metastatic potential was treated as tester in the reverse hybridization. Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentially expressed genes for the metastasis of CRC. These fragments were ligated with T vectors, screened through the bluewhite screening system to establish cDNA library.RESULTS: After the blue-white screening, 235 white clones were picked out from the positive-going hybridization and 232 from the reverse. PCR results showed that 200-700 bp inserts were seen in 98% and 91% clones from the forward and reverse hybridizations, respectively.CONCLUSIONS: A subtractive cDNA library of differentially expressed genes specific for metastasis of CRC can be constructed with SSH and T/A cloning techniques.

  19. A critical assessment of cross-species detection of gene duplicates using comparative genomic hybridization

    Directory of Open Access Journals (Sweden)

    Renn Suzy CP

    2010-05-01

    Full Text Available Abstract Background Comparison of genomic DNA among closely related strains or species is a powerful approach for identifying variation in evolutionary processes. One potent source of genomic variation is gene duplication, which is prevalent among individuals and species. Array comparative genomic hybridization (aCGH has been successfully utilized to detect this variation among lineages. Here, beyond the demonstration that gene duplicates among species can be quantified with aCGH, we consider the effect of sequence divergence on the ability to detect gene duplicates. Results Using the X chromosome genomic content difference between male D. melanogaster and female D. yakuba and D. simulans, we describe a decrease in the ability to accurately measure genomic content (copy number for orthologs that are only 90% identical. We demonstrate that genome characteristics (e.g. chromatin environment and non-orthologous sequence similarity can also affect the ability to accurately measure genomic content. We describe a normalization strategy and statistical criteria to be used for the identification of gene duplicates among any species group for which an array platform is available from a closely related species. Conclusions Array CGH can be used to effectively identify gene duplication and genome content; however, certain biases are present due to sequence divergence and other genome characteristics resulting from the divergence between lineages. Highly conserved gene duplicates will be more readily recovered by aCGH. Duplicates that have been retained for a selective advantage due to directional selection acting on many loci in one or both gene copies are likely to be under-represented. The results of this study should inform the interpretation of both previously published and future work that employs this powerful technique.

  20. Differential expression of genes identified by suppression subtractive hybridization in petals of opening carnation flowers.

    Science.gov (United States)

    Harada, Taro; Torii, Yuka; Morita, Shigeto; Masumura, Takehiro; Satoh, Shigeru

    2010-05-01

    Flower opening is an event accompanied by morphological changes in petals which include elongation, expansion, and outward-curving. Petal cell growth is a fundamental process that underlies such phenomena, but its molecular mechanism remains largely unknown. Suppression subtractive hybridization was performed between petals during the early elongation period (stage 1) and during the opening period (stage 5) in carnation flowers and a pair of subtraction libraries abundant in differentially expressed genes was constructed at each stage. 393 cDNA clones picked up by differential screening out of 1728 clones were sequenced and 235 different cDNA fragments were identified, among which 211 did not match any known nucleotide sequence of carnation genes in the databases. BLASTX search of nucleotide sequences revealed that putative functions of the translational products can be classified into several categories including transcription, signalling, cell wall modification, lipid metabolism, and transport. Open reading frames of 15 selected genes were successfully determined by rapid amplification of cDNA ends (RACE). Time-course analysis of these genes by real-time RT-PCR showed that transcript levels of several genes correlatively fluctuate in petals of opening carnation flowers, suggesting an association with the morphological changes by elongation or curving. Based on the results, it is suggested that the growth of carnation petals is controlled by co-ordinated gene expression during the progress of flower opening. In addition, the possible roles of some key genes in the initiation of cell growth, the construction of the cell wall and cuticle, and transport across membranes were discussed.

  1. Effect of Thyrotropin Releasing Hormone (TRH on Gene Expressions in Rat Pancreas: Approach by Microarray Hybridization

    Directory of Open Access Journals (Sweden)

    Luo LG

    2004-07-01

    Full Text Available CONTEXT: Thyrotropin releasing hormone (TRH, originally identified as a hypothalamic hormone, expresses in the pancreas. The effects of TRH such as, inhibiting amylase secretion in rats through a direct effect on acinar cells, enhancing basal glucagon secretion from isolated perfused rat pancreas, and potentiating glucose-stimulated insulin secretion in perfused rat islets and insulin-secreting clonal beta-cell lines, suggest that TRH may play a role in pancreas. TRH also enlarged pancreas and increased pancreatic DNA content but deletion of TRH gene expression caused hyperglycemia in mice, suggesting that TRH may play a critical role in pancreatic development; however, the biological mechanisms of TRH in the adult pancreas remains unclear. OBJECTIVES: This study explored the effect of TRH on rat pancreas. SUBJECTS: Four male-Sprague-Dawley-rats (200-250 g were given 10 microg/kg BW of TRH intraperitoneally on 1st and 3rd day and sacrificed on 7th day. Four same-strain rats without TRH injection served as controls. MAIN OUTCOME MEASURES: Wet pancreatic weights were measured. Pancreatic tissues were homogenized and extracted. The insulin levels of the extracts were measured by ELISA. Total RNA from the pancreases were fluorescently labeled and hybridized to microarray with 1,081 spot genes. RESULTS: TRH increased pancreatic wet weight and insulin contents. About 75% of the 1,081 genes were detected in the pancreas. TRH regulated up 99 genes and down 76 genes. The administration of TRH induced various types of gene expressions, such as G-protein coupled receptors (GPCR and signal transduction related genes (GPCR kinase 4, transducin beta subunit 5, arrestin beta1MAPK3, MAPK5, c-Src kinase, PKCs, PI3 kinase, growth factors (PDGF-B, IGF-2, IL-18, IGF-1, IL-2, IL-6, endothelin-1 and apoptotic factors (Bcl2, BAD, Bax. CONCLUSION: Reprogramming of transcriptome may be a way for TRH-regulation of pancreatic cellular functions.

  2. Screening and cloning of differentially expressed genes in placentas from patients of pregnancy-induced hypertension by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    尹国武; 姜锋; 李东红; 姚元庆

    2003-01-01

    Suppresssion subtractive hybridization (SSH) was preformed to compare gene expression profiles of PIH patients and normal pregnancy placentas. The subtractive cDNA library of PIH placenta was set up and screedned. Differential cDNAs were cloned, and sequenced by T 7 primer methodology. One hundred and three differential cDNAs were isolated by SSH. Sequencing and BLAST analysis showed 90 inserts shared more than 95% homolog with sequences in the GenBank/EMBL database. We identified 36 putative genes including pregnancy-specific glycoprotein gene (BC005924), serine protease inhibitor gene(BC012868), VEGFR-1 gene(AF063657, etc.

  3. Progress on Transferring Elite Genes from Non-AA Genome Wild Rice into Oryza sativa through Interspecific Hybridization

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The progress of research on transferring elite genes from non-AA genome wild rice into Oryza sativa through interspecific hybridization are in three respects,that is,breeding monosomic alien addition lines (MAALs),constructing introgression lines (ILs) and analyzing the heredity of the characters and mapping the related genes.There are serious reproductive barriers,mainly incrossability and hybrid sterility,in the interspecific hybridization of O.sativa with non-AA genome wild rice.These are the 'bottleneck' for transferring elite genes from wild rice to O.sativa.Combining traditional crossing method with biotechnique is a reliable way to overcome the reproductive barriers and to improve the utilizing efficiency of non-AA genome wild rice.

  4. HyDRA: gene prioritization via hybrid distance-score rank aggregation.

    Science.gov (United States)

    Kim, Minji; Farnoud, Farzad; Milenkovic, Olgica

    2015-04-01

    Gene prioritization refers to a family of computational techniques for inferring disease genes through a set of training genes and carefully chosen similarity criteria. Test genes are scored based on their average similarity to the training set, and the rankings of genes under various similarity criteria are aggregated via statistical methods. The contributions of our work are threefold: (i) first, based on the realization that there is no unique way to define an optimal aggregate for rankings, we investigate the predictive quality of a number of new aggregation methods and known fusion techniques from machine learning and social choice theory. Within this context, we quantify the influence of the number of training genes and similarity criteria on the diagnostic quality of the aggregate and perform in-depth cross-validation studies; (ii) second, we propose a new approach to genomic data aggregation, termed HyDRA (Hybrid Distance-score Rank Aggregation), which combines the advantages of score-based and combinatorial aggregation techniques. We also propose incorporating a new top-versus-bottom (TvB) weighting feature into the hybrid schemes. The TvB feature ensures that aggregates are more reliable at the top of the list, rather than at the bottom, since only top candidates are tested experimentally; (iii) third, we propose an iterative procedure for gene discovery that operates via successful augmentation of the set of training genes by genes discovered in previous rounds, checked for consistency. Fundamental results from social choice theory, political and computer sciences, and statistics have shown that there exists no consistent, fair and unique way to aggregate rankings. Instead, one has to decide on an aggregation approach using predefined set of desirable properties for the aggregate. The aggregation methods fall into two categories, score- and distance-based approaches, each of which has its own drawbacks and advantages. This work is motivated by the

  5. Identification of immunity-related genes in the burying beetle Nicrophorus vespilloides by suppression subtractive hybridization.

    Science.gov (United States)

    Vogel, H; Badapanda, C; Vilcinskas, A

    2011-12-01

    Burying beetles reproduce on small vertebrate cadavers which they bury in the soil after localization through volatiles emitted from the carcass. They then chemically preserve the carcass and prepare it as a diet for the adults and their offspring. It is predicted that exposure to high loads of soil and/or carrion-associated microbes necessitates an effective immune system. In the present paper, we report experimental screening for immunity-related genes in the burying beetle Nicrophorus vespilloides using the suppression subtractive hybridization approach. A total of 1179 putative gene objects were identified in the Nicrophorus cDNA library, which was enriched for transcripts differentially expressed upon challenge with heat-inactivated bacteria. In addition to genes known to be involved in immunity-related recognition and signalling, we found transcripts encoding for antimicrobial peptides and for an array of enzymes that can be linked to immunity or to stress-induced pathways. We also determined proteins that may contribute to detoxification of toxins produced by microbial competitors. In addition, factors involved in mRNA stability determination and central components of the RNA interference machinery were identified, implying transcriptional reprogramming and potential stress-induced retrotransposon elimination. The identified candidate immune effector and stress-related genes may provide important information about the unusual ecology and evolution of the burying beetles.

  6. Identification of cadmium-induced Agaricus blazei genes through suppression subtractive hybridization.

    Science.gov (United States)

    Wang, Liling; Li, Haibo; Wei, Hailong; Wu, Xueqian; Ke, Leqin

    2014-01-01

    Cadmium (Cd) is one of the most serious environmental pollutants. Filamentous fungi are very promising organisms for controlling and reducing the amount of heavy metals released by human and industrial activities. However, the molecular mechanisms involved in Cd accumulation and tolerance of filamentous fungi are not fully understood. Agaricus blazei Murrill, an edible mushroom with medicinal properties, demonstrates high tolerance for heavy metals, especially Cd. To investigate the molecular mechanisms underlying the response of A. blazei after Cd exposure, we constructed a forward subtractive library that represents cadmium-induced genes in A. blazei under 4 ppm Cd stress for 14 days using suppression subtractive hybridization combined with mirror orientation selection. Differential screening allowed us to identify 39 upregulated genes, 26 of which are involved in metabolism, protein fate, cellular transport, transport facilitation and transport routes, cell rescue, defense and virulence, transcription, and the action of proteins with a binding function, and 13 are encoding hypothetical proteins with unknown functions. Induction of six A. blazei genes after Cd exposure was further confirmed by RT-qPCR. The cDNAs isolated in this study contribute to our understanding of genes involved in the biochemical pathways that participate in the response of filamentous fungi to Cd exposure.

  7. Differentially expressed genes of virulent and nonvirulent Entamoeba histolytica strains identified by suppression subtractive hybridization.

    Science.gov (United States)

    Freitas, Michelle A R; Alvarenga, Ângela C; Fernandes, Helen C; Gil, Frederico F; Melo, Maria N; Pesquero, Jorge L; Gomes, Maria A

    2014-01-01

    Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH) technique, differential gene expressions of two E. histolytica strains, one virulent (EGG) and one nonvirulent (452), have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

  8. Differentially Expressed Genes of Virulent and Nonvirulent Entamoeba histolytica Strains Identified by Suppression Subtractive Hybridization

    Directory of Open Access Journals (Sweden)

    Michelle A. R. Freitas

    2014-01-01

    Full Text Available Entamoeba histolytica is a parasite which presents capacity to degrade tissues and therefore has a pathogenic behavior. As this behavior is not shown by all strains, there have been several studies investigating molecular basis of the cytotoxicity process. Using the suppression subtractive hybridization (SSH technique, differential gene expressions of two E. histolytica strains, one virulent (EGG and one nonvirulent (452, have been analyzed with the purpose of isolating genes which may be involved with amoebic virulence. Nine cDNA fragments presenting high homology with E. histolytica previously sequenced genes were subtracted. Of these, four genes were confirmed by RT-PCR. Two coding for hypothetical proteins, one for a cysteine-rich protein, expressed only in the virulent strain, EGG and another one, coding for grainin 2 protein, exclusive from 452 strain. This study provided new insight into the proteins differences in the virulent and nonvirulent E. histolytica strains. We believe that further studies with these proteins may prove association of them with tissue damage, providing new perceptions to improve treatment or diagnosis of the invasive disease.

  9. Differentially expressed genes implicated in embryo abortion of mango identified by suppression subtractive hybridization.

    Science.gov (United States)

    He, J H; Ma, F W; Chen, Y Y; Shu, H R

    2012-11-14

    Embryo abortion in mango severely damages mango production worldwide. The mechanisms by which the mango embryos abort have long been an intriguing question. We used subtractive suppression hybridization to investigate the differentially expressed genes involved in this process. We generated 2 cDNA libraries from normal seed and aborted seed embryos of mango cultivar 'Jinhuang'. One thousand five hundred and seventy-two high-quality expressed sequence tags (ESTs) were obtained, with 1092 from the normal seed tester library and 480 from the aborted seed tester library. These ESTs were assembled into 783 unigenes, including 147 contigs and 636 singletons in contigs; 297 singletons in gene ontology (GO) indicated coverage of a broad range of GO categories. Seven candidate genes from different categories were selected for semi-quantitative PCR analysis, and their possible functions in embryo abortion are discussed. These data provide new insight into the genetic regulation of embryo abortion in mango and may aid in further identification of novel genes and their functions.

  10. Screening for metronidazole-resistance associated gene fragments of H pylori by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    Ai-Qing Li; Ning Dai; Jie Yan; Yong-Liang Zhu

    2007-01-01

    AIM:To screen for metronidazole (MTZ)-resistance associated gene fragments of H pylori by suppression subtractive hybridization (SSH).METHODS:Five MTZ-resistant (tester,T) and 1 MTZ-susceptible (driver,D) clinical H pylori isolates were selected. Genomic DMAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5'-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting.RESULTS:Among the randomly selected 120 subtractive colonies,37 DNA fragments had a different number of DNA copies (≥2 times) in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies (≥3 times). Among the sequences obtained from the 17 DNA fragments,new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments,representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein (dppA),permease protein (dppB),ribosomal protein S4 (rps4),ribonuclease in (rnc),protease (pqqE),diaminopimelate epimerase (dapF),acetatekinase (ackA),H pylori plasmid pHPSl and H pylori gene 1334.CONCLUSION:Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant H pylori.

  11. Comparison of gene expression profiles and responses to zinc chloride among inter- and intraspecific hybrids with growth abnormalities in wheat and its relatives.

    Science.gov (United States)

    Takamatsu, Kiyofumi; Iehisa, Julio C M; Nishijima, Ryo; Takumi, Shigeo

    2015-07-01

    Hybrid necrosis is a well-known reproductive isolation mechanism in plant species, and an autoimmune response is generally considered to trigger hybrid necrosis through epistatic interaction between disease resistance-related genes in hybrids. In common wheat, the complementary Ne1 and Ne2 genes control hybrid necrosis, defined as type I necrosis. Two other types of hybrid necrosis (type II and type III) have been observed in interspecific hybrids between tetraploid wheat and Aegilops tauschii. Another type of hybrid necrosis, defined here as type IV necrosis, has been reported in F1 hybrids between Triticum urartu and some accessions of Triticum monococcum ssp. aegilopoides. In types I, III and IV, cell death occurs gradually starting in older tissues, whereas type II necrosis symptoms occur only under low temperature. To compare comprehensive gene expression patterns of hybrids showing growth abnormalities, transcriptome analysis of type I and type IV necrosis was performed using a wheat 38k oligo-DNA microarray. Defense-related genes including many WRKY transcription factor genes were dramatically up-regulated in plants showing type I and type IV necrosis, similarly to other known hybrid abnormalities, suggesting an association with an autoimmune response. Reactive oxygen species generation and necrotic cell death were effectively inhibited by ZnCl2 treatment in types I, III and IV necrosis, suggesting a significant association of Ca(2+) influx in upstream signaling of necrotic cell death in wheat hybrid necrosis.

  12. 'Specific' oligonucleotides often recognize more than one gene: the limits of in situ hybridization applied to GABA receptors.

    Science.gov (United States)

    Mladinic, M; Didelon, F; Cherubini, E; Bradbury, A

    2000-05-15

    As exquisite probes for gene sequences, oligonucleotides are one of the most powerful tools of recombinant molecular biology. In studying the GABA receptor subunits in the neonatal hippocampus we have used oligonucleotide probes in in situ hybridization and cloning techniques. The oligonucleotides used and assumed to be specific for the target gene, actually recognized more than one gene, leading to surprising and contradictory results. In particular, we found that a GABA(A)-rho specific oligonucleotide recognized an abundant, previously unknown, transcription factor in both in situ and library screening, while oligos 'specific' for GABA(A) subunits were able to recognize 30 additional unrelated genes in library screening. This suggests that positive results obtained with oligonucleotides should be interpreted with caution unless confirmed by identical results with oligonucleotides from different parts of the same gene, or cDNA library screening excludes the presence of other hybridizing species.

  13. Identification of transcriptome SNPs for assessing allele-specific gene expression in a super-hybrid rice Xieyou9308.

    Directory of Open Access Journals (Sweden)

    Rongrong Zhai

    Full Text Available Hybridization, a common process in nature, can give rise to a vast reservoir of allelic variants. Combination of these allelic variants may result in novel patterns of gene action and is thought to contribute to heterosis. In this study, we analyzed genome-wide allele-specific gene expression (ASGE in the super-hybrid rice variety Xieyou9308 using RNA sequencing technology (RNA-Seq. We identified 9325 reliable single nucleotide polymorphisms (SNPs distributed throughout the genome. Nearly 68% of the identified polymorphisms were CT and GA SNPs between R9308 and Xieqingzao B, suggesting the existence of DNA methylation, a heritable epigenetic mark, in the parents and their F1 hybrid. Of 2793 identified transcripts with consistent allelic biases, only 480 (17% showed significant allelic biases during tillering and/or heading stages, implying that trans effects may mediate most transcriptional differences in hybrid offspring. Approximately 67% and 62% of the 480 transcripts showed R9308 allelic expression biases at tillering and heading stages, respectively. Transcripts with higher levels of gene expression in R9308 also exhibited R9308 allelic biases in the hybrid. In addition, 125 transcripts were identified with significant allelic expression biases at both stages, of which 74% showed R9308 allelic expression biases. R9308 alleles may tend to preserve their characteristic states of activity in the hybrid and may play important roles in hybrid vigor at both stages. The allelic expression of 355 transcripts was highly stage-specific, with divergent allelic expression patterns observed at different developmental stages. Many transcripts associated with stress resistance were differently regulated in the F1 hybrid. The results of this study may provide valuable insights into molecular mechanisms of heterosis.

  14. Evolution of Ig- and T-cell receptor gene configuration in a Ph1+ hybrid leukemia patient

    DEFF Research Database (Denmark)

    Jensen, Arne Willy; Hokland, Peter; Kristensen, J S

    1992-01-01

    In a longitudinal study of a 32-year-old male with Ph1+ hybrid leukemia we have followed the immunophenotype and configuration of Ig- and TCR genes during the course of different chemotherapy regimens directed first against the myeloid and later against the lymphoid components of the disease. We...... by location of the translocation-breakpoint on chromosome 22, our data clearly indicate that Ig- and TCR gene rearrangements might prove a valuable addition in monitoring Ph1+ hybrid leukemias, providing guidelines for optimizing chemotherapy. Udgivelsesdato: 1992-Feb...

  15. Identifying genes related to choriogenesis in insect panoistic ovaries by Suppression Subtractive Hybridization

    Directory of Open Access Journals (Sweden)

    Bellés Xavier

    2009-04-01

    Full Text Available Abstract Background Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH library to identify genes involved in panoistic choriogenesis, using the cockroach Blattella germanica as model. Results We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in B. germanica. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis. Conclusion The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in B. germanica. For

  16. Dorsal horn-enriched genes identified by DNA microarray, in situ hybridization and immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Koblan Kenneth S

    2002-08-01

    Full Text Available Abstract Background Neurons in the dorsal spinal cord play important roles in nociception and pain. These neurons receive input from peripheral sensory neurons and then transmit the signals to the brain, as well as receive and integrate descending control signals from the brain. Many molecules important for pain transmission have been demonstrated to be localized to the dorsal horn of the spinal cord. Further understanding of the molecular interactions and signaling pathways in the dorsal horn neurons will require a better knowledge of the molecular neuroanatomy in the dorsal spinal cord. Results A large scale screening was conducted for genes with enriched expression in the dorsal spinal cord using DNA microarray and quantitative real-time PCR. In addition to genes known to be specifically expressed in the dorsal spinal cord, other neuropeptides, receptors, ion channels, and signaling molecules were also found enriched in the dorsal spinal cord. In situ hybridization and immunohistochemistry revealed the cellular expression of a subset of these genes. The regulation of a subset of the genes was also studied in the spinal nerve ligation (SNL neuropathic pain model. In general, we found that the genes that are enriched in the dorsal spinal cord were not among those found to be up-regulated in the spinal nerve ligation model of neuropathic pain. This study also provides a level of validation of the use of DNA microarrays in conjunction with our novel analysis algorithm (SAFER for the identification of differences in gene expression. Conclusion This study identified molecules that are enriched in the dorsal horn of the spinal cord and provided a molecular neuroanatomy in the spinal cord, which will aid in the understanding of the molecular mechanisms important in nociception and pain.

  17. Gene expression regulation and lineage evolution: the North and South tale of the hybrid polyploid Squalius alburnoides complex

    Science.gov (United States)

    Pala, Irene; Schartl, Manfred; Brito, Miguel; Vacas, Joana Malta; Coelho, Maria Manuela

    2010-01-01

    The evolution of hybrid polyploid vertebrates, their viability and their perpetuation over evolutionary time have always been questions of great interest. However, little is known about the impact of hybridization and polyploidization on the regulatory networks that guarantee the appropriate quantitative and qualitative gene expression programme. The Squalius alburnoides complex of hybrid fish is an attractive system to address these questions, as it includes a wide variety of diploid and polyploid forms, and intricate systems of genetic exchange. Through the study of genome-specific allele expression of seven housekeeping and tissue-specific genes, we found that a gene copy silencing mechanism of dosage compensation exists throughout the distribution range of the complex. Here we show that the allele-specific patterns of silencing vary within the complex, according to the geographical origin and the type of genome involved in the hybridization process. In southern populations, triploids of S. alburnoides show an overall tendency for silencing the allele from the minority genome, while northern population polyploids exhibit preferential biallelic gene expression patterns, irrespective of genomic composition. The present findings further suggest that gene copy silencing and variable expression of specific allele combinations may be important processes in vertebrate polyploid evolution. PMID:20554543

  18. Identification and characterization of low temperature stress responsive genes in Poncirus trifoliata by suppression subtractive hybridization.

    Science.gov (United States)

    Peng, T; Zhu, X F; Fan, Q J; Sun, P P; Liu, J H

    2012-01-15

    Trifoliate orange (Poncirus trifoliata (L.) Raf.) is extremely cold hardy when fully acclimated, but knowledge relevant to the molecular events underlying the acclimation is still limited so far. In this study, forward (4 °C over 25 °C) and reverse (25 °C over 4 °C) suppression subtractive hybridization (SSH) libraries were constructed in order to identify the genes involved in cold acclimation in trifoliate orange. After reverse northern blotting analysis and sequencing, a total of 105 and 117 non-redundant differentially expressed sequence tags (ESTs) were obtained from the forward and reverse libraries, respectively. Blast2go analysis revealed that 91 ESTs, 31 from the forward library and 60 from the reverse library, displayed significant sequence homology to the genes with known or putative functions. They were categorized into various functional groups, including catalytic activity, binding protein, structural molecule, enzyme regulator, molecular transducer, electron carrier, and transport activity/transcription regulation. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction was consistent with the results of differential screening. In addition, time-course expression patterns of the genes further confirmed that they were responsive to low temperature treatment. Among the genes of known functions, many are related to maintenance of cell wall integrity, adjustment of osmotic potential and maintenance of reactive oxygen species homeostasis, implying that these physiological processes might be of paramount significance in rendering protective mechanisms against the low temperature stress. The data presented here gain an insight into the molecular changes underlying the cold acclimation of trifoliate orange, and the results can be of reference for unraveling candidate genes that hold great potential for genetic engineering in an effort to create novel germplasms with enhanced cold stress tolerance.

  19. Identification of two novel nodule-specific genes from Astragalus sinicus L. by suppressive subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To identify the genes involved in nodule formation and to increase usable molecular probes, a cDNA library of Astragalus sinicus genes specifically expressed in infected roots by Mesorhizobium huakuii 7653R is generated using a PCR-based suppressive subtractive hybridization (SSH) technique with two mRNA populations of infected and uninfected control roots. Two nodule-specific genes, AsIIC259 and AsG2511, are identified from infected roots of A. sinicus. The amino acid sequences deduced from the open reading frames (ORFs) reveal that AsIIC259 and AsG2511 encodes a polypeptide with 134 and 58 amino acids, respectively. A signal peptide sequence is predicted with high probability at the N-termini of the AsIIC259 and AsG2511. The motif searches show that the deduced polypeptide of AsIIC259 contains two N-glycosylation sites, a cAMP- and cGMP-dependent protein kinase phosphorylation site and a casein kinase II phosphorylation site. BLASTP searches reveal that AsIIC259 putative protein displays a low degree of similarity to a unique nodulin from Lupinus luteus nodules. No significant identity is displayed over the predicted polypeptides of AsG2511 with any published sequences. Virtual Northern blot and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicate that the two genes are expressed exclusively in inoculated roots and that their expression is 2-4 d later than that of the leghaemoglobin (Lb) gene during nodule development.

  20. The impact of quantitative optimization of hybridization conditions on gene expression analysis

    Directory of Open Access Journals (Sweden)

    Auburn Richard P

    2011-03-01

    Full Text Available Abstract Background With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. Results As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of up to 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays. For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We can ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirmed an unbiased determination of generally optimal experimental conditions. Conclusions Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive pro filing of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks.

  1. Radiation hybrid mapping of a cytokine gene cluster located in the proximal region of 5q

    Energy Technology Data Exchange (ETDEWEB)

    Segal, A.L.; McPherson, J.D.; Wasmuth, J.J. [Univ. of California, Irvine (United States)

    1994-09-01

    The long (q) arm of chromosome 5 has been shown to contain a large number of genes encoding growth factors, growth factor receptors, hormone receptors and neurotransmitter receptors. IL-3, IL-4, IL-5, IL-9, IL-13, GM-CSF and IRF-1 are located in the 5q22-31.1 interval, while three GABA receptors map to 5q33-34. A number of receptors, including the prolactin and growth hormone receptors, the IL-7 receptor and the leukemia inhibitory factor receptor, map to proximal 5p. Genes encoding three of the complement components, C6, C7 and C9, are also located in the same region. YAC data indicates that C6 and C7 lie within 170 kb of each other. We have used a panel of 180 Chinese hamster-human radiation hybrids possessing fragments of human chromosome 5 to construct a physical map of this region of 5q. Two-point and multi-point analyses were done on the data and significant LOD scores (from 3 to 30) were observed. LIFR, PRLR, GHR, IL-7R, C6, C7, C9, TARS, and a number of CEPH-Genethon dinucleotide repeat markers were ordered and mapped. Yeast artificial chromosomes and cosmids have been isolated and inter-Alu PCR products from them are being used to construct a contig and to improve the physical map. The long term goal of this work is to identify and characterize new genes in the region.

  2. Identification of calcium stress induced genes in amaranth leaves through suppression subtractive hybridization.

    Science.gov (United States)

    Aguilar-Hernández, Hugo S; Santos, Leticia; León-Galván, Fabiola; Barrera-Pacheco, Alberto; Espitia-Rangel, Eduardo; De León-Rodríguez, Antonio; Guevara-González, Ramón G; Barba de la Rosa, Ana P

    2011-11-15

    Calcium (Ca(2+)) is a critical ion for the growth and development of plants and plays an important role in signal transduction pathways in response to biotic and abiotic stresses. We investigated the Ca(2+) stress responsive-genes in amaranth leaves by using the suppression subtractive hybridization technique. Screening of the libraries generated 420 up-regulated transcripts and 199 down-regulated transcripts. The differentially expressed transcripts were associated with general stress response, transcription factors, gene regulation, signal transduction, and some other with unknown function. Selected genes were used to study their differential regulation by sqRT-PCR. Among the up-regulated transcripts, a fragment containing the motif of C3HC4-type RING-Zinc family was further characterized. The ORF of amaranth zinc finger protein (AhZnf) has a closer relationship with its ortholog from Ricinus communis while is distantly related to the Arabidopsis thaliana C3HC4-type ortholog. We have identified a novel putative zinc finger protein along with other novel proteins such as the wall associated kinase, phosphoinositide binding protein, and rhomboid protease involved in response to Ca(2+) stress in amaranth leaves.

  3. Enhancing disease resistances of Super Hybrid Rice with four antifungal genes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A plant expression vector harboring four antifungal genes was delivered into the embryogenic calli of ‘9311’, an indica restorer line of Super Hybrid Rice, via modified biolistic particle bombardment. Southern blot analysis indicated that in the regenerated hygromycin-resistant plants, all the four anti-fungal genes, including RCH10, RAC22, β-Glu and B-RIP, were integrated into the genome of ‘9311’, co-transmitted altogether with the marker gene hpt in a Mendelian pattern. Some transgenic R1 and R2 progenies, with all transgenes displaying a normal expression level in the Northern blot analysis, showed high resistance to Magnaporthe grisea when tested in the typical blast nurseries located in Yanxi and Sanya respectively. Furthermore, transgenic F1 plants, resulting from a cross of R2 homo-zygous lines with high resistance to rice blast with the non-transgenic male sterile line Peiai 64S, showed not only high resistance to M. grisea but also enhanced resistance to rice false smut (a disease caused by Ustilaginoidea virens) and rice kernel smut (another disease caused by Tilletia barclayana).

  4. Identifying New Candidate Genes and Chemicals Related to Prostate Cancer Using a Hybrid Network and Shortest Path Approach

    Science.gov (United States)

    Yuan, Fei; Zhou, You; Wang, Meng; Yang, Jing; Wu, Kai; Lu, Changhong; Kong, Xiangyin; Cai, Yu-Dong

    2015-01-01

    Prostate cancer is a type of cancer that occurs in the male prostate, a gland in the male reproductive system. Because prostate cancer cells may spread to other parts of the body and can influence human reproduction, understanding the mechanisms underlying this disease is critical for designing effective treatments. The identification of as many genes and chemicals related to prostate cancer as possible will enhance our understanding of this disease. In this study, we proposed a computational method to identify new candidate genes and chemicals based on currently known genes and chemicals related to prostate cancer by applying a shortest path approach in a hybrid network. The hybrid network was constructed according to information concerning chemical-chemical interactions, chemical-protein interactions, and protein-protein interactions. Many of the obtained genes and chemicals are associated with prostate cancer. PMID:26504486

  5. Allele distributions at hybrid incompatibility loci facilitate the potential for gene flow between cultivated and weedy rice in the US.

    Science.gov (United States)

    Craig, Stephanie M; Reagon, Michael; Resnick, Lauren E; Caicedo, Ana L

    2014-01-01

    The accumulation of independent mutations over time in two populations often leads to reproductive isolation. Reproductive isolation between diverging populations may be reinforced by barriers that occur either pre- or postzygotically. Hybrid sterility is the most common form of postzygotic isolation in plants. Four postzygotic sterility loci, comprising three hybrid sterility systems (Sa, s5, DPL), have been recently identified in Oryza sativa. These loci explain, in part, the limited hybridization that occurs between the domesticated cultivated rice varieties, O. sativa spp. japonica and O. sativa spp. indica. In the United States, cultivated fields of japonica rice are often invaded by conspecific weeds that have been shown to be of indica origin. Crop-weed hybrids have been identified in crop fields, but at low frequencies. Here we examined the possible role of these hybrid incompatibility loci in the interaction between cultivated and weedy rice. We identified a novel allele at Sa that seemingly prevents loss of fertility in hybrids. Additionally, we found wide-compatibility type alleles at strikingly high frequencies at the Sa and s5 loci in weed groups, and a general lack of incompatible alleles between crops and weeds at the DPL loci. Our results suggest that weedy individuals, particularly those of the SH and BRH groups, should be able to freely hybridize with the local japonica crop, and that prezygotic factors, such as differences in flowering time, have been more important in limiting weed-crop gene flow in the past. As the selective landscape for weedy rice changes due to increased use of herbicide resistant strains of cultivated rice, the genetic barriers that hinder indica-japonica hybridization cannot be counted on to limit the flow of favorable crop genes into weeds.

  6. Allele distributions at hybrid incompatibility loci facilitate the potential for gene flow between cultivated and weedy rice in the US.

    Directory of Open Access Journals (Sweden)

    Stephanie M Craig

    Full Text Available The accumulation of independent mutations over time in two populations often leads to reproductive isolation. Reproductive isolation between diverging populations may be reinforced by barriers that occur either pre- or postzygotically. Hybrid sterility is the most common form of postzygotic isolation in plants. Four postzygotic sterility loci, comprising three hybrid sterility systems (Sa, s5, DPL, have been recently identified in Oryza sativa. These loci explain, in part, the limited hybridization that occurs between the domesticated cultivated rice varieties, O. sativa spp. japonica and O. sativa spp. indica. In the United States, cultivated fields of japonica rice are often invaded by conspecific weeds that have been shown to be of indica origin. Crop-weed hybrids have been identified in crop fields, but at low frequencies. Here we examined the possible role of these hybrid incompatibility loci in the interaction between cultivated and weedy rice. We identified a novel allele at Sa that seemingly prevents loss of fertility in hybrids. Additionally, we found wide-compatibility type alleles at strikingly high frequencies at the Sa and s5 loci in weed groups, and a general lack of incompatible alleles between crops and weeds at the DPL loci. Our results suggest that weedy individuals, particularly those of the SH and BRH groups, should be able to freely hybridize with the local japonica crop, and that prezygotic factors, such as differences in flowering time, have been more important in limiting weed-crop gene flow in the past. As the selective landscape for weedy rice changes due to increased use of herbicide resistant strains of cultivated rice, the genetic barriers that hinder indica-japonica hybridization cannot be counted on to limit the flow of favorable crop genes into weeds.

  7. Differential gene expression in an elite hybrid rice cultivar (Oryza sativa, L and its parental lines based on SAGE data

    Directory of Open Access Journals (Sweden)

    Chen Chen

    2007-09-01

    Full Text Available Abstract Background It was proposed that differentially-expressed genes, aside from genetic variations affecting protein processing and functioning, between hybrid and its parents provide essential candidates for studying heterosis or hybrid vigor. Based our serial analysis of gene expression (SAGE data from an elite Chinese super-hybrid rice (LYP9 and its parental cultivars (93-11 and PA64s in three major tissue types (leaves, roots and panicles at different developmental stages, we analyzed the transcriptome and looked for candidate genes related to rice heterosis. Results By using an improved strategy of tag-to-gene mapping and two recently annotated genome assemblies (93-11 and PA64s, we identified 10,268 additional high-quality tags, reaching a grand total of 20,595 together with our previous result. We further detected 8.5% and 5.9% physically-mapped genes that are differentially-expressed among the triad (in at least one of the three stages with P-values less than 0.05 and 0.01, respectively. These genes distributed in 12 major gene expression patterns; among them, 406 up-regulated and 469 down-regulated genes (P Conclusion We improved tag-to-gene mapping strategy by combining information from transcript sequences and rice genome annotation, and obtained a more comprehensive view on genes that related to rice heterosis. The candidates for heterosis-related genes among different genotypes provided new avenue for exploring the molecular mechanism underlying heterosis.

  8. 1-Mb resolution array-based comparative genomic hybridization using a BAC clone set optimized for cancer gene analysis

    NARCIS (Netherlands)

    Greshock, J; Naylor, TL; Margolin, A; Diskin, S; Cleaver, SH; Futreal, PA; deJong, PJ; Zhao, SY; Liebman, M; Weber, BL

    2004-01-01

    Array-based comparative genomic hybridization (aCGH) is a recently developed tool for genome-wide determination of DNA copy number alterations. This technology has tremendous potential for disease-gene discovery in cancer and developmental disorders as well as numerous other applications. However, w

  9. A hybrid Bacillus thuringiensis delta-endotoxin gene gives resistance against a coleopteran and a lepidopteran pest in transgenic potato

    NARCIS (Netherlands)

    Naimov, S.; Dukiandjiev, S.; Maagd, de R.A.

    2003-01-01

    Expression of Bacillus thuringiensis delta-endotoxins has proven to be a successful strategy for obtaining insect resistance in transgenic plants. Drawbacks of expression of a single resistance gene are the limited target spectrum and the potential for rapid adaptation of the pest. Hybrid toxins wit

  10. Hybridization between Yellowstone cutthroat trout and rainbow trout alters the expression of muscle growth-related genes and their relationships with growth patterns

    Science.gov (United States)

    Ostberg, Carl O.; Chase, Dorothy M.; Hauser, Lorenz

    2015-01-01

    Hybridization creates novel gene combinations that may generate important evolutionary novelty, but may also reduce existing adaptation by interrupting inherent biological processes, such as genotype-environment interactions. Hybridization often causes substantial change in patterns of gene expression, which, in turn, may cause phenotypic change. Rainbow trout (Oncorhynchus mykiss) and cutthroat trout (O. clarkii) produce viable hybrids in the wild, and introgressive hybridization with introduced rainbow trout is a major conservation concern for native cutthroat trout. The two species differ in body shape, which is likely an evolutionary adaptation to their native environments, and their hybrids tend to show intermediate morphology. The characterization of gene expression patterns may provide insights on the genetic basis of hybrid and parental morphologies, as well as on the ecological performance of hybrids in the wild. Here, we evaluated the expression of eight growth-related genes (MSTN-1a, MSTN-1b, MyoD1a, MyoD1b, MRF-4, IGF-1, IGF-2, and CAST-L) and the relationship of these genes with growth traits (length, weight, and condition factor) in six line crosses: both parental species, both reciprocal F1 hybrids, and both first-generation backcrosses (F1 x rainbow trout and F1 x cutthroat trout). Four of these genes were differentially expressed among rainbow, cutthroat, and their hybrids. Transcript abundance was significantly correlated with growth traits across the parent species, but not across hybrids. Our findings suggest that rainbow and cutthroat trout exhibit differences in muscle growth regulation, that transcriptional networks may be modified by hybridization, and that hybridization disrupts intrinsic relationships between gene expression and growth patterns that may be functionally important for phenotypic adaptations.

  11. Hybridization between Yellowstone Cutthroat Trout and Rainbow Trout Alters the Expression of Muscle Growth-Related Genes and Their Relationships with Growth Patterns.

    Directory of Open Access Journals (Sweden)

    Carl O Ostberg

    Full Text Available Hybridization creates novel gene combinations that may generate important evolutionary novelty, but may also reduce existing adaptation by interrupting inherent biological processes, such as genotype-environment interactions. Hybridization often causes substantial change in patterns of gene expression, which, in turn, may cause phenotypic change. Rainbow trout (Oncorhynchus mykiss and cutthroat trout (O. clarkii produce viable hybrids in the wild, and introgressive hybridization with introduced rainbow trout is a major conservation concern for native cutthroat trout. The two species differ in body shape, which is likely an evolutionary adaptation to their native environments, and their hybrids tend to show intermediate morphology. The characterization of gene expression patterns may provide insights on the genetic basis of hybrid and parental morphologies, as well as on the ecological performance of hybrids in the wild. Here, we evaluated the expression of eight growth-related genes (MSTN-1a, MSTN-1b, MyoD1a, MyoD1b, MRF-4, IGF-1, IGF-2, and CAST-L and the relationship of these genes with growth traits (length, weight, and condition factor in six line crosses: both parental species, both reciprocal F1 hybrids, and both first-generation backcrosses (F1 x rainbow trout and F1 x cutthroat trout. Four of these genes were differentially expressed among rainbow, cutthroat, and their hybrids. Transcript abundance was significantly correlated with growth traits across the parent species, but not across hybrids. Our findings suggest that rainbow and cutthroat trout exhibit differences in muscle growth regulation, that transcriptional networks may be modified by hybridization, and that hybridization disrupts intrinsic relationships between gene expression and growth patterns that may be functionally important for phenotypic adaptations.

  12. Hybridization between Yellowstone Cutthroat Trout and Rainbow Trout Alters the Expression of Muscle Growth-Related Genes and Their Relationships with Growth Patterns.

    Science.gov (United States)

    Ostberg, Carl O; Chase, Dorothy M; Hauser, Lorenz

    2015-01-01

    Hybridization creates novel gene combinations that may generate important evolutionary novelty, but may also reduce existing adaptation by interrupting inherent biological processes, such as genotype-environment interactions. Hybridization often causes substantial change in patterns of gene expression, which, in turn, may cause phenotypic change. Rainbow trout (Oncorhynchus mykiss) and cutthroat trout (O. clarkii) produce viable hybrids in the wild, and introgressive hybridization with introduced rainbow trout is a major conservation concern for native cutthroat trout. The two species differ in body shape, which is likely an evolutionary adaptation to their native environments, and their hybrids tend to show intermediate morphology. The characterization of gene expression patterns may provide insights on the genetic basis of hybrid and parental morphologies, as well as on the ecological performance of hybrids in the wild. Here, we evaluated the expression of eight growth-related genes (MSTN-1a, MSTN-1b, MyoD1a, MyoD1b, MRF-4, IGF-1, IGF-2, and CAST-L) and the relationship of these genes with growth traits (length, weight, and condition factor) in six line crosses: both parental species, both reciprocal F1 hybrids, and both first-generation backcrosses (F1 x rainbow trout and F1 x cutthroat trout). Four of these genes were differentially expressed among rainbow, cutthroat, and their hybrids. Transcript abundance was significantly correlated with growth traits across the parent species, but not across hybrids. Our findings suggest that rainbow and cutthroat trout exhibit differences in muscle growth regulation, that transcriptional networks may be modified by hybridization, and that hybridization disrupts intrinsic relationships between gene expression and growth patterns that may be functionally important for phenotypic adaptations.

  13. Suppression subtractive hybridization reveals differentially expressed genes in supraspinous ligaments of patients with ankylosing spondylitis.

    Science.gov (United States)

    Zhang, Ying; Hu, Xu; Zhang, Chao; Zhou, Yue; Chu, Tong-Wei

    2015-06-01

    Ankylosing spondylitis (AS) is a severe chronic inflammatory disease that may ultimately result in the development of a 'bamboo‑like' spine. Although the pathological changes that occur in AS have been extensively investigated, the mechanism underlying spinal fusion during AS remains elusive. Differentially expressed genes (DEGs) in paraspinal tissues from patients with AS compared with those from healthy controls were therefore investigated. Polymerase chain reaction (PCR)‑based suppression subtractive hybridization was performed using total mRNA from the supraspinal ligaments of three patients with AS and three patients with spinal fractures as controls. From this, 27 genes were identified in all of the three independent forward libraries, which were defined as DEGs associated with AS. Reverse transcription‑quantitative PCR demonstrated that six DEGs were overexpressed in the tissues from patients with AS compared with those from individuals in the control group, including those encoding transforming growth factor β types I and III receptor, vascular endothelial growth factor, matrix metalloproteinase‑3, core‑binding factor α1 and bone morphogenetic protein 2. Western blot analysis showed increased expression in all six of these proteins in the samples from patients with AS compared with those in the control groups. These findings suggested that changes in the expression of these genes and proteins are associated with the development of spinal fusion during the pathogenesis of AS. Furthermore, these genes may be novel markers of the risk of developing AS, in addition to being targets for the treatment of this disease.

  14. The role of fluorescence in situ hybridization and gene expression profiling in myeloma risk stratification.

    Science.gov (United States)

    Hose, Dirk; Seckinger, Anja; Jauch, Anna; Rème, Thierry; Moreaux, Jérôme; Bertsch, Uta; Neben, Kai; Klein, Bernard; Goldschmidt, Hartmut

    2011-12-01

    Multiple myeloma patients' survival under treatment varies from a few months to more than 15 years. Clinical prognostic factors, especially beta2-microglobulin (B2M) and the international staging system (ISS), allow risk assessment to a certain extent, but do not identify patients at very high risk. As malignant plasma cells are characterized by a variety of chromosomal aberrations and changes in gene expression, a molecular characterization ofCD138-purified myeloma cells by interphase fluorescence in situ hybridization (iFISH) and gene expression profiling (GEP) can be used for improved risk assessment, iFISH allows a risk stratification with presence of a translocation t(4;14) and/or deletion of 17p13 being the best documented adverse prognostic factors. A deletion of 13q14 is no longer considered to define adverse risk. Patients harbouring a t(4;14) seems to benefit from a bortezomib- or lenalidomide containing regimen, whereas patients with deletion 17p13 seem only to benefit from a high dose therapy approach using long term bortezomib (in induction and maintenance) and autologous tandem-transplantation as used in the GMMG-HD4 trial, or the total therapy 3 concept. Gene expression profiling allows the assessment of high risk scores (IFM, UAMS), remaining prognostic despite treatment with novel agents, and prognostic surrogates of biological factors (e.g. proliferation) and (prognostic) target gene expression (e.g. Aurora-kinase A). Thus, assessment of B2M and ISS-stage, iFISH, and GEP is considered extended routine diagnostics in therapy requiring multiple myeloma patients for risk assessment and, even now, to a certain extent selection of treatment.

  15. Imprinting of the polycomb group gene MEDEA serves as a ploidy sensor in Arabidopsis.

    Science.gov (United States)

    Erilova, Aleksandra; Brownfield, Lynette; Exner, Vivien; Rosa, Marisa; Twell, David; Mittelsten Scheid, Ortrun; Hennig, Lars; Köhler, Claudia

    2009-09-01

    Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed abortion, a phenomenon that has been termed "triploid block." Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions. We identified a mutant forming unreduced pollen that enabled us to investigate direct effects of unbalanced parental genome contributions on seed development and to reveal the underlying molecular mechanism of dosage sensitivity. We provide evidence that parent-of-origin-specific expression of the Polycomb group (PcG) gene MEDEA is causally responsible for seed developmental aberrations in Arabidopsis seeds with increased paternal genome contributions. We propose that imprinted expression of PcG genes is an evolutionary conserved mechanism to balance parental genome contributions in embryo nourishing tissues.

  16. Microsatellite-Aided Screening for Fertility Restoration Genes (Rf Facilitates Hybrid Improvement

    Directory of Open Access Journals (Sweden)

    Raafat El-Namaky

    2016-05-01

    Full Text Available DNA markers enabled to determine the chromosomal locations of the two Rf genes (Rf3 and Rf4 in the wild-abortive cytoplasmic male sterility (WA-CMS system. Four simple sequence repeats (SSRs RM171, RM258, RM315 and RM443 were used to detect the allelic status with respect to the fertility restoration genes (Rf3 and Rf4 in 300 rice cultivars or breeding lines. The results revealed that out of 300 lines, 90 lines screened had Rf3, 65 lines had Rf4, and 45 lines had Rf3 and Rf4 alleles. Furthermore, 45 lines selected using SSR markers were mated with a CMS line (IR58025A to analyze their restoring ability. Offspring of all the test lines except HHZ8-SAL9DT1-Y1, HHZ5-SAL9-Y3-1 and IDSA77 exhibited higher pollen and spikelet fertility (> 80%, thus confirming they bear the Rf alleles. The hybrid offspring of ARH12-6-1-1-B-3-1, IR32307-10-3-2-1 and Sahel 329 had the highest pollen fertility (97.39%, 98.30% and 97.10%, respectively and spikelet fertility (95.10%, 97.07% and 96.10%, respectively.

  17. Microsatellite-Aided Screening for Fertility Restoration Genes (Rf) Facilitates Hybrid Improvement

    Institute of Scientific and Technical Information of China (English)

    RAAFAT El-Namaky; SABER Sedeek; YONNELLE Dea Moukoumbi; RODOMIRO Ortiz; BABOUCARR Manneh

    2016-01-01

    DNA markers enabled to determine the chromosomal locations of the twoRf genes (Rf3 andRf4) in the wild-abortive cytoplasmic male sterility (WA-CMS) system. Four simple sequence repeats (SSRs) RM171, RM258, RM315 and RM443 were used to detect the alelic status with respect to the fertility restoration genes (Rf3and Rf4) in 300 rice cultivars or breeding lines. The results revealed that out of 300 lines, 90 lines screened hadRf3, 65 lines hadRf4, and 45 lines hadRf3 andRf4 aleles. Furthermore, 45 lines selected using SSR markers were mated with a CMS line (IR58025A) to analyze their restoring ability. Offspring of al the test lines except HHZ8-SAL9DT1-Y1, HHZ5-SAL9-Y3-1 and IDSA77 exhibited higher polen and spikelet fertility (> 80%), thus confirming they bear theRfaleles. The hybrid offspring of ARH12-6-1-1-B-3-1, IR32307-10-3-2-1 and Sahel 329 had the highest polen fertility (97.39%, 98.30% and 97.10%, respectively) and spikelet fertility (95.10%, 97.07% and 96.10%, respectively).

  18. Comparative gene mapping in cattle, Indian muntjac, and Chinese muntjac by fluorescence in situ hybridization.

    Science.gov (United States)

    Murmann, Andrea E; Mincheva, Antoaneta; Scheuermann, Markus O; Gautier, Mathieu; Yang, Fentang; Buitkamp, Johannes; Strissel, Pamela L; Strick, Reiner; Rowley, Janet D; Lichter, Peter

    2008-11-01

    The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n = 6 in the female and 2n = 7 in the male. The karyotypic evolution of Indian muntjac via extensive tandem fusions and several centric fusions are well documented by molecular cytogenetic studies mainly utilizing chromosome paints. To achieve higher resolution mapping, a set of 42 different genomic clones coding for 37 genes and the nucleolar organizer region were used to examine homologies between the cattle (2n = 60), human (2n = 46), Indian muntjac (2n = 6/7) and Chinese muntjac (2n = 46) karyotypes. These genomic clones were mapped by fluorescence in situ hybridization (FISH). Localization of genes on all three pairs of M. m. vaginalis chromosomes and on the acrocentric chromosomes of M. reevesi allowed not only the analysis of the evolution of syntenic regions within the muntjac genus but also allowed a broader comparison of synteny with more distantly related species, such as cattle and human, to shed more light onto the evolving genome organization.

  19. Effect of ploidy increase on transgene expression: example from Citrus diploid cybrid and allotetraploid somatic hybrid expressing the EGFP gene.

    Science.gov (United States)

    Xu, Shi-Xiao; Cai, Xiao-Dong; Tan, Bin; Li, Ding-Li; Guo, Wen-Wu

    2011-07-01

    Polyploidization is an important speciation mechanism for all eukaryotes, and it has profound impacts on biodiversity dynamics and ecosystem functioning. Green fluorescent protein (GFP) has been used as an effective marker to visually screen somatic hybrids at an early stage in protoplast fusion. We have previously reported that the intensity of GFP fluorescence of regenerated embryoids was also an early indicator of ploidy level. However, little is known concerning the effects of ploidy increase on the GFP expression in citrus somatic hybrids at the plant level. Herein, allotetraploid and diploid cybrid plants with enhanced GFP (EGFP) expression were regenerated from the fusion of embryogenic callus protoplasts from 'Murcott' tangor (Citrus reticulata Blanco × Citrus sinensis (L.) Osbeck) and mesophyll protoplasts from transgenic 'Valencia' orange (C. sinensis (L.) Osbeck) expressing the EGFP gene, via electrofusion. Subsequent simple sequence repeat (SSR), chloroplast simple sequence repeat and cleaved amplified polymorphic sequence analysis revealed that the two regenerated tetraploid plants were true allotetraploid somatic hybrids possessing nuclear genomic DNA of both parents and cytoplasmic DNA from the callus parent, while the five regenerated diploid plants were cybrids containing nuclear DNA of the leaf parent and with complex segregation of cytoplasmic DNA. Furthermore, EGFP expression was compared in cells and protoplasts from mature leaves of these diploid cybrids and allotetraploid somatic hybrids. Results showed that the intensity of GFP fluorescence per cell or protoplast in diploid was generally brighter than in allotetraploid. Moreover, same hybridization signal was detected on allotetraploid and diploid plants by Southern blot analysis. By real-time RT-PCR and Western blot analysis, GFP expression level of the diploid cybrid was revealed significantly higher than that of the allotetraploid somatic hybrid. These results suggest that ploidy

  20. Misregulation of spermatogenesis genes in Drosophila hybrids is lineage-specific and driven by the combined effects of sterility and fast male regulatory divergence.

    Science.gov (United States)

    Gomes, S; Civetta, A

    2014-09-01

    Hybrid male sterility is a common outcome of crosses between different species. Gene expression studies have found that a number of spermatogenesis genes are differentially expressed in sterile hybrid males, compared with parental species. Late-stage sperm development genes are particularly likely to be misexpressed, with fewer early-stage genes affected. Thus, a link has been posited between misexpression and sterility. A more recent alternative explanation for hybrid gene misexpression has been that it is independent of sterility and driven by divergent evolution of male-specific regulatory elements between species (faster male hypothesis). The faster male hypothesis predicts that misregulation of spermatogenesis genes should be independent of sterility and approximately the same in both hybrids, whereas sterility should only affect gene expression in sterile hybrids. To test the faster male hypothesis vs. the effect of sterility on gene misexpression, we analyse spermatogenesis gene expression in different species pairs of the Drosophila phylogeny, where hybrid male sterility occurs in only one direction of the interspecies cross (i.e. unidirectional sterility). We find significant differences among genes in misexpression with effects that are lineage-specific and caused by sterility or fast male regulatory divergence. © 2014 The Authors. Journal of Evolutionary Biology © 2014 European Society For Evolutionary Biology.

  1. Comparing mRNA levels using in situ hybridization of a target gene and co-stain.

    Science.gov (United States)

    Wunderlich, Zeba; Bragdon, Meghan D; DePace, Angela H

    2014-06-15

    In situ hybridization is an important technique for measuring the spatial expression patterns of mRNA in cells, tissues, and whole animals. However, mRNA levels cannot be compared across experiments using typical protocols. Here we present a semi-quantitative method to compare mRNA levels of a gene across multiple samples. This method yields an estimate of the error in the measurement to allow statistical comparison. Our method uses a typical in situ hybridization protocol to stain for a target gene and an internal standard, which we refer to as a co-stain. As a proof of concept, we apply this method to multiple lines of transgenic Drosophila embryos, harboring constructs that express reporter genes to different levels. We generated this test set by mutating enhancer sequences to contain different numbers of binding sites for Zelda, a transcriptional activator. We demonstrate that using a co-stain with in situ hybridization is an effective method to compare mRNA levels across samples. This method requires only minor modifications to existing in situ hybridization protocols and uses straightforward analysis techniques. This strategy can be broadly applied to detect quantitative, spatially resolved changes in mRNA levels.

  2. Cytoplasmic-genetic male sterility gene provides direct evidence for some hybrid rice recently evolving into weedy rice.

    Science.gov (United States)

    Zhang, Jingxu; Lu, Zuomei; Dai, Weimin; Song, Xiaoling; Peng, Yufa; Valverde, Bernal E; Qiang, Sheng

    2015-05-27

    Weedy rice infests paddy fields worldwide at an alarmingly increasing rate. There is substantial evidence indicating that many weedy rice forms originated from or are closely related to cultivated rice. There is suspicion that the outbreak of weedy rice in China may be related to widely grown hybrid rice due to its heterosis and the diversity of its progeny, but this notion remains unsupported by direct evidence. We screened weedy rice accessions by both genetic and molecular marker tests for the cytoplasmic male sterility (CMS) genes (Wild abortive, WA, and Boro type, BT) most widely used in the production of indica and japonica three-line hybrid rice as a diagnostic trait of direct parenthood. Sixteen weedy rice accessions of the 358 tested (4.5%) contained the CMS-WA gene; none contained the CMS-BT gene. These 16 accessions represent weedy rices recently evolved from maternal hybrid rice derivatives, given the primarily maternal inheritance of this trait. Our results provide key direct evidence that hybrid rice can be involved in the evolution of some weedy rice accessions, but is not a primary factor in the recent outbreak of weedy rice in China.

  3. What, if anything, are hybrids: enduring truths and challenges associated with population structure and gene flow

    OpenAIRE

    Gompert, Zachariah; Buerkle, C Alex

    2016-01-01

    Abstract Hybridization is a potent evolutionary process that can affect the origin, maintenance, and loss of biodiversity. Because of its ecological and evolutionary consequences, an understanding of hybridization is important for basic and applied sciences, including conservation biology and agriculture. Herein, we review and discuss ideas that are relevant to the recognition of hybrids and hybridization. We supplement this discussion with simulations. The ideas we present have a long histor...

  4. Comparing mRNA levels using in situ hybridization of a target gene and co-stain

    OpenAIRE

    Wunderlich, Zeba; Bragdon, Meghan D.; Angela H. DePace

    2014-01-01

    In situ hybridization is an important technique for measuring the spatial expression patterns of mRNA in cells, tissues, and whole animals. However, mRNA levels cannot be compared across experiments using typical protocols. Here we present a semi-quantitative method to compare mRNA levels of a gene across multiple samples. This method yields an estimate of the error in the measurement to allow statistical comparison. Our method uses a typical in situ hybridization protocol to stain for a targ...

  5. Hybridizing Daphnia communities from ten neighbouring lakes: spatio-temporal dynamics, local processes, gene flow and invasiveness.

    Science.gov (United States)

    Yin, Mingbo; Gießler, Sabine; Griebel, Johanna; Wolinska, Justyna

    2014-04-12

    In natural communities of cyclical parthenogens, rapid response to environmental change is enabled by switching between two reproduction modes. While long periods of asexual reproduction allow some clones to outcompete others, and may result in "clonal erosion", sexual reproduction restores genetic variation in such systems. Moreover, sexual reproduction may result in the formation of interspecific hybrids. These hybrids can then reach high abundances, through asexual clonal reproduction. In the present study, we explored genetic variation in water fleas of the genus Daphnia. The focus was on the short-term dynamics within several clonal assemblages from the hybridizing Daphnia longispina complex and the impact of gene flow at small spatial scales. Daphnia individuals belonged either to the parental species D. galeata and D. longispina, or to different hybrid classes, as identified by 15 microsatellite markers. The distribution and genotypic structure of parental species, but not hybrids, corresponded well with the geographical positions of the lakes. Within parental species, the genetic distance among populations of D. galeata was lower than among populations of D. longispina. Moreover, D. galeata dominance was associated with higher phosphorous load. Finally, there was no evidence for clonal erosion. Our results suggest that the contemporary structure of hybridizing Daphnia communities from ten nearby lakes is influenced by colonization events from neighbouring habitats as well as by environmental factors. Unlike the parental species, however, there was little evidence for successful dispersal of hybrids, which seem to be produced locally. Finally, in contrast to temporary Daphnia populations, in which a decrease in clonal diversity was sometimes detectable over a single growing season, the high clonal diversity and lack of clonal erosion observed here might result from repeated hatching of sexually produced offspring. Overall, our study provides insights into

  6. A hybrid photoreceptor expressing both rod and cone genes in a mouse model of enhanced S-cone syndrome.

    Directory of Open Access Journals (Sweden)

    Joseph C Corbo

    2005-08-01

    Full Text Available Rod and cone photoreceptors subserve vision under dim and bright light conditions, respectively. The differences in their function are thought to stem from their different gene expression patterns, morphologies, and synaptic connectivities. In this study, we have examined the photoreceptor cells of the retinal degeneration 7(rd7 mutant mouse, a model for the human enhanced S-cone syndrome (ESCS. This mutant carries a spontaneous deletion in the mouse ortholog of NR2E3, an orphan nuclear receptor transcription factor mutated in ESCS. Employing microarray and in situ hybridization analysis we have found that the rd7 retina contains a modestly increased number of S-opsin-expressing cells that ultrastructurally appear to be normal cones. Strikingly, the majority of the photoreceptors in the rd7 retina represent a morphologically hybrid cell type that expresses both rod- and cone-specific genes. In addition, in situ hybridization screening of genes shown to be up-regulated in the rd7 mutant retina by microarray identified ten new cone-specific or cone-enriched genes with a wide range of biochemical functions, including two genes specifically involved in glucose/glycogen metabolism. We suggest that the abnormal electroretinograms, slow retinal degeneration, and retinal dysmorphology seen in humans with ESCS may, in part, be attributable to the aberrant function of a hybrid photoreceptor cell type similar to that identified in this study. The functional diversity of the novel cone-specific genes identified here indicates molecular differences between rods and cones extending far beyond those previously discovered.

  7. Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F₁ interspecies hybrids.

    Science.gov (United States)

    Shen, Yingjia; Catchen, Julian; Garcia, Tzintzuni; Amores, Angel; Beldorth, Ion; Wagner, Jonathan; Zhang, Ziping; Postlethwait, John; Warren, Wes; Schartl, Manfred; Walter, Ronald B

    2012-01-01

    Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, Xiphophorus maculatus and Xiphophorus couchianus, and their F(1) interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈5-10 million years ago, were identified about every 700 bp. Using 6524 transcripts with identified SNPs between the two parental species (X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F(1) interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the in silico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F(1) interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression.

  8. Applications of Multiple Nuclear Genes to the Molecular Phylogeny, Population Genetics and Hybrid Identification in the Mangrove Genus Rhizophora.

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    Yongmei Chen

    Full Text Available The genus Rhizophora is one of the most important components of mangrove forests. It is an ideal system for studying biogeography, molecular evolution, population genetics, hybridization and conservation genetics of mangroves. However, there are no sufficient molecular markers to address these topics. Here, we developed 77 pairs of nuclear gene primers, which showed successful PCR amplifications across all five Rhizophora species and sequencing in R. apiculata. Here, we present three tentative applications using a subset of the developed nuclear genes to (I reconstruct the phylogeny, (II examine the genetic structure and (III identify natural hybridization in Rhizophora. Phylogenetic analyses support the hypothesis that Rhizophora had disappeared in the Atlantic-East Pacific (AEP region and was re-colonized from the IWP region approximately 12.7 Mya. Population genetics analyses in four natural populations of R. apiculata in Hainan, China, revealed extremely low genetic diversity, strong population differentiation and extensive admixture, suggesting that the Pleistocene glaciations, particularly the last glacial maximum, greatly influenced the population dynamics of R. apiculata in Hainan. We also verified the hybrid status of a morphologically intermediate individual between R. apiculata and R. stylosa in Hainan. Based on the sequences of five nuclear genes and one chloroplast intergenic spacer, this individual is likely to be an F1 hybrid, with R. stylosa as its maternal parent. The nuclear gene markers developed in this study should be of great value for characterizing the hybridization and introgression patterns in other cases of this genus and testing the role of natural selection using population genomics approaches.

  9. Over-expressed Genes Detected by Suppression Subtractive Hybridization in Carcinoma Derived From Transformed 16HBE Cells Induced by BPDE

    Institute of Scientific and Technical Information of China (English)

    SHE-JUAN AN; JIA-KUN CHEN; LI-LI LIU; YAN-FENG ZHAO; XUE-MIN CHEN

    2005-01-01

    Objective To screen the over differentially expressed genes in carcinoma induced by BPDE-transformed 16HBE cells (16HBE-C cells). Methods The suppression subtractive hybridization (SSH) method was performed to profile differentially expressed genes between 16HBE-C cells and 16HBE cells. The cDNA fragments of differentially expressed genes were inserted into TA cloning vector and transformed competent E. coli strain. Positive clones were randomly picked up and identified by the colony PCR method. Dot blot was used to test the same source with the tester. The differentially expressed cDNA fragments were sequenced and compared with known genes and EST database in Genbank. Results Eight known genes were over-expressed in 16HBE-C cells including eukaryotic translation elongation factor 1 alpha 1, HIF-1 responsive RTP801, ribosomal protein L10 (RPL10), ribosomal protein S29 (RPS29), mitochondrion related genes, and laminin receptor 1. Three differentially expressed cDNA fragments could not be matched to the known genes but to the EST database. Conclusion The SSH method can detect differentially expressed genes between 16HBE-C and 16HBE cells. BPDE-induced carcinogenesis may be related to alteration of at least eight known genes and three unknown genes. These expression data provide a clue to further cloning novel genes and studying functions in BPDE-induced carcinoma.

  10. Gene transcript accumulation and in situ mRNA hybridization of two putative glutamate dehydrogenase genes in etiolated Glycine max seedlings.

    Science.gov (United States)

    Dimou, M; Tsaniklidis, G; Aivalakis, G; Katinakis, P

    2015-01-01

    Glutamate dehydrogenase (EC 1.4.1.2) is a multimeric enzyme that catalyzes the reversible amination of α-ketoglutarate to form glutamate. We characterized cDNA clones of two Glycine max sequences, GmGDH1 and GmGDH2, that code for putative α- and β-subunits, respectively, of the NADH dependent enzyme. Temporal and spatial gene transcript accumulation studies using semiquantitative RT-PCR and in situ hybridization have shown an overlapping gene transcript accumulation pattern with differences in relative gene transcript accumulation in the organs examined. Detection of NADH-dependent glutamate dehydrogenase activity in situ using a histochemical method showed concordance with the spatial gene transcript accumulation patterns. Our findings suggest that although the two gene transcripts are co-localized in roots of etiolated soybean seedlings, the ratio of the two subunits of the active holoenzyme may vary among tissues.

  11. Ribosomal RNA gene silencing in interpopulation hybrids of Tigriopus californicus: nucleolar dominance in the absence of intergenic spacer subrepeats.

    Science.gov (United States)

    Flowers, Jonathan M; Burton, Ronald S

    2006-07-01

    A common feature of interspecific animal and plant hybrids is the uniparental silencing of ribosomal RNA gene transcription, or nucleolar dominance. A leading explanation for the genetic basis of nucleolar dominance in animal hybrids is the enhancer-imbalance model. The model proposes that limiting transcription factors are titrated by a greater number of enhancer-bearing subrepeat elements in the intergenic spacer (IGS) of the dominant cluster of genes. The importance of subrepeats for nucleolar dominance has repeatedly been supported in competition assays between Xenopus laevis and X. borealis minigene constructs injected into oocytes. However, a more general test of the importance of IGS subrepeats for nuclear dominance in vivo has not been conducted. In this report, rRNA gene expression was examined in interpopulation hybrids of the marine copepod Tigriopus californicus. This species offers a rare opportunity to test the role of IGS subrepeats in nucleolar dominance because the internal subrepeat structure, found in the IGS of virtually all animal and plant species, is absent in T. californicus. Our results clearly establish that nucleolar dominance occurs in F1 and F2 interpopulation hybrids of this species. In the F2 generation, nucleolar dominance appears to break down in some hybrids in a fashion that is inconsistent with a transcription factor titration model. These results are significant because they indicate that nucleolar dominance can be established and maintained without enhancer-bearing repeat elements in the IGS. This challenges the generality of the enhancer-imbalance model for nucleolar dominance and suggests that dominance of rRNA transcription in animals may be determined by epigenetic factors as has been established in plants.

  12. Spatial gene expression quantification: a tool for analysis of in situ hybridizations in sea anemone Nematostella vectensis

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    Botman Daniel

    2012-10-01

    Full Text Available Abstract Background Spatial gene expression quantification is required for modeling gene regulation in developing organisms. The fruit fly Drosophila melanogaster is the model system most widely applied for spatial gene expression analysis due to its unique embryonic properties: the shape does not change significantly during its early cleavage cycles and most genes are differentially expressed along a straight axis. This system of development is quite exceptional in the animal kingdom. In the sea anemone Nematostella vectensis the embryo changes its shape during early development; there are cell divisions and cell movement, like in most other metazoans. Nematostella is an attractive case study for spatial gene expression since its transparent body wall makes it accessible to various imaging techniques. Findings Our new quantification method produces standardized gene expression profiles from raw or annotated Nematostella in situ hybridizations by measuring the expression intensity along its cell layer. The procedure is based on digital morphologies derived from high-resolution fluorescence pictures. Additionally, complete descriptions of nonsymmetric expression patterns have been constructed by transforming the gene expression images into a three-dimensional representation. Conclusions We created a standard format for gene expression data, which enables quantitative analysis of in situ hybridizations from embryos with various shapes in different developmental stages. The obtained expression profiles are suitable as input for optimization of gene regulatory network models, and for correlation analysis of genes from dissimilar Nematostella morphologies. This approach is potentially applicable to many other metazoan model organisms and may also be suitable for processing data from three-dimensional imaging techniques.

  13. Toxic effects of octylphenol on the expression of genes in liver identified by suppression subtractive hybridization of Rana chensinensis.

    Science.gov (United States)

    Li, Xin-Yi; Xiao, Ning; Zhang, Yu-Hui

    2014-01-01

    Octylphenol (OP) is the degradative product of alkylphenol ethoxylates that are widely used to produce rubber, pesticides, and paints. It is chemically stable substance and demonstrates estrogenic effects, toxicity and carcinogenic effects in the environment. The toxin accumulates rapidly in the liver where it exerts most of its damage, but the molecular mechanisms behind its toxicity remain unclear. Due to limited information concerning the effect of OP on liver, this study investigates how OP causes hepatotoxicity in liver. Here, suppression subtractive hybridization was used to identify the alterations in gene transcription of the frog (Rana chensinensis) after exposure to OP. After hybridization and cloning, the subtractive cDNA libraries were obtained. At random, 207 positive clones were selected and sequenced from the subtractive libraries, which gave a total of 75 gene fragment sequences. The screening identified numerous genes involved in apoptosis, signal transduction, cytoskeletal remodeling, innate immunity, material and energy metabolism, translation and transcription which were extensively discussed. Two sequenced genes were analyzed further using real time quantitative PCR. The two genes from the library were found to be transcriptionally up-regulated. These results confirmed the successful construction of the subtractive cDNA library that was enriched for the genes that were differentially transcribed in the amphibian liver challenged with OP, and for the first time present the basic data on toxicity effect of OP on liver.

  14. Molecular mapping of the hybrid necrosis gene NetJingY176 in Aegilops tauschii using microsatellite markers

    Directory of Open Access Journals (Sweden)

    Fengbo Xue

    2015-08-01

    Full Text Available The rich genetic variation preserved in collections of Aegilops tauschii can be readily exploited to improve common wheat using synthetic hexaploid wheat lines. However, hybrid necrosis, which is characterized by progressive death of leaves or plants, has been observed in certain interspecific crosses between tetraploid wheat and Ae. tauschii. The aim of this study was to construct a fine genetic map of a gene (temporarily named NetJingY176 conferring hybrid necrosis in Ae. tauschii accession Jing Y176. A triploid F1 population derived from distant hybridization between Ae. tauschii and tetraploid wheat was used to map the gene with microsatellite markers. The newly developed markers XsdauK539 and XsdauK561 co-segregated with NetJingY176 on chromosome arm 2DS. The tightly linked markers developed in this study were used to genotype 91 Ae. tauschii accessions. The marker genotype analysis suggested that 49.45% of the Ae. tauschii accessions carry NetJingY176. Interestingly, hybrid necrosis genotypes tended to appear more commonly in Ae. tauschii ssp. tauschii than in Ae. tauschii ssp. strangulata.

  15. Molecular mapping of the hybrid necrosis gene NetJingY176 in Aegilops tauschii using microsatellite markers

    Institute of Scientific and Technical Information of China (English)

    Fengbo; Xue; Jun; Guo; Changying; Guan; Hongwei; Wang; Anfei; Li; Lingrang; Kong

    2015-01-01

    The rich genetic variation preserved in collections of Aegilops tauschii can be readily exploited to improve common wheat using synthetic hexaploid wheat lines. However,hybrid necrosis, which is characterized by progressive death of leaves or plants, has been observed in certain interspecific crosses between tetraploid wheat and Ae. tauschii. The aim of this study was to construct a fine genetic map of a gene(temporarily named Net Jing Y176)conferring hybrid necrosis in Ae. tauschii accession Jing Y176. A triploid F1 population derived from distant hybridization between Ae. tauschii and tetraploid wheat was used to map the gene with microsatellite markers. The newly developed markers Xsdau K539 and Xsdau K561 co-segregated with Net Jing Y176 on chromosome arm 2DS. The tightly linked markers developed in this study were used to genotype 91 Ae. tauschii accessions. The marker genotype analysis suggested that 49.45% of the Ae. tauschii accessions carry Net Jing Y176. Interestingly, hybrid necrosis genotypes tended to appear more commonly in Ae. tauschii ssp. tauschii than in Ae. tauschii ssp. strangulata.

  16. Molecular mapping of the hybrid necrosis gene NetJingY176 in Aegilops tauschii using microsatellite markers

    Institute of Scientific and Technical Information of China (English)

    Fengbo Xue; Jun Guo; Changying Guan; Hongwei Wang; Anfei Li; Lingrang Kong

    2015-01-01

    The rich genetic variation preserved in collections of Aegilops tauschii can be readily exploited to improve common wheat using synthetic hexaploid wheat lines. However, hybrid necrosis, which is characterized by progressive death of leaves or plants, has been observed in certain interspecific crosses between tetraploid wheat and Ae. tauschii. The aim of this study was to construct a fine genetic map of a gene (temporarily named NetJingY176) conferring hybrid necrosis in Ae. tauschii accession Jing Y176. A triploid F1 population derived from distant hybridization between Ae. tauschii and tetraploid wheat was used to map the gene with microsatellite markers. The newly developed markers XsdauK539 and XsdauK561 co-segregated with NetJingY176 on chromosome arm 2DS. The tightly linked markers developed in this study were used to genotype 91 Ae. tauschii accessions. The marker genotype analysis suggested that 49.45% of the Ae. tauschii accessions carry NetJingY176. Interestingly, hybrid necrosis genotypes tended to appear more commonly in Ae. tauschii ssp. tauschii than in Ae. tauschii ssp. strangulata.

  17. Construction and analysis of a suppression subtractive hybridization library of regeneration-related genes in soybean.

    Science.gov (United States)

    Sun, J; Li, J; Liu, M; Zhang, B B; Li, D M; Wang, M; Zhang, C; Li, W B; Su, A Y; Wu, X X

    2015-01-30

    The development of a genetic transformation system is needed to address the problem of the low efficiency associated with soybean regeneration. To contribute to the enhancement of the soybean regenerative capacity, we explored the developmental mechanisms of soybean regeneration at the molecular level using a suppression subtractive hybridization cDNA library constructed from cotyledonary nodes of soybean cultivar DN50. A total of 918 positive clones were identified and screened, with most inserted fragments ranging from 100 to 750 bp. Of these, 411 differentially expressed functional expressed sequence tags were identified and annotated based on their similarity to orthologs and paralogs detected in GenBank using the nucleotide and translated nucleotide Basic Local Alignment Search Tools. Functional analysis revealed that the associated genes were involved in signal transduction, synthesis, and metabolism of macromolecules, glucose and protein synthesis and metabolism, light and leaf morphogenesis, regulation of apoptosis, cell defense, cell wall differentiation, and a variety of hormone and cytokinin-mediated signaling pathways. The information uncovered in our study should serve as a foundation for the establishment of an efficient and stable genetic transformation system for soybean regeneration.

  18. Analysis of cycle gene expression in Aedes aegypti brains by in situ hybridization.

    Directory of Open Access Journals (Sweden)

    Samira Chahad-Ehlers

    Full Text Available Even though the blood-sucking mosquito Aedes aegypti is one of the most important disease vectors, relatively little is known about the molecular mechanisms underlying processes involved in the temporal pattern of its activity and host seeking behavior. In this study, we analyzed the expression of the cycle (cyc gene, one of the core components of the circadian clock, in Ae. aegypti brains by in situ hybridization at two different time points in light-dark conditions and compared the results with those obtained using a quantitative PCR assay (qPCR. Within the brain, differential labeling was detected according to distinct areas empirically pre-defined. Six out of seven of these areas showed significantly higher staining at ZT3 (three hours after light-on compared to ZT11 (one before light-off, which is consistent with the qPCR data. Predominant staining was observed in three of those areas which correspond to positions of the optical and antennal lobes, as well as the region where the neurons controlling activity rhythms are presumably localized.

  19. A microarray gene expression data classification using hybrid back propagation neural network

    Directory of Open Access Journals (Sweden)

    Vimaladevi M.

    2014-01-01

    Full Text Available Classification of cancer establishes appropriate treatment and helps to decide the diagnosis. Cancer expands progressively from an alteration in a cell's genetic structure. This change (mutation results in cells with uncontrolled growth patterns. In cancer classification, the approach, Back propagation is sufficient and also it is a universal technique of training artificial neural networks. It is also called supervised learning method. It needs many dataset for input and output for making up the training set. The back propagation method may execute the function of collaborate multiple parties. In existing method, collaborative learning is limited and it considers only two parties. The proposed collaborative function can perform well and problems can be solved by utilizing the power of cloud computing. This technical note applies hybrid models of Back Propagation Neural networks (BPN and fast Genetic Algorithms (GA to estimate the feature selection in gene expression data. The proposed research work examines many feature selection algorithms which are “fragile”; that is, the superiority of their results varies broadly over data sets. By this research, it is suggested that this is due to higherorder interactions between features causing restricted minima in search space in which the algorithm becomes attentive. GAs may escape from such minima by chance, because works are highly stochastic. A neural net classifier with a genetic algorithm, using the GA to select features for classification by the neural net and incorporating the net as part of the objective function of the GA.

  20. Trypanosoma cruzi genotypes of insect vectors and patients with Chagas of Chile studied by means of cytochrome b gene sequencing, minicircle hybridization, and nuclear gene polymorphisms.

    Science.gov (United States)

    Arenas, Marco; Campos, Ricardo; Coronado, Ximena; Ortiz, Sylvia; Solari, Aldo

    2012-03-01

    Fifty-six Trypanosoma cruzi stocks from Chile and neighboring countries and different hosts, humans, and Triatoma infestans and Mepraia sp., vectors of domiciliary and natural environments were characterized by using three molecular markers. These were cytochrome b (Cyt b) gene sequencing, minicircle DNA blotting, and hybridization with five genotype-specific DNA probes and nuclear analysis of 1f8 and gp72 by polymerase chain reaction-restriction fragment length polymorphism. The results with all three molecular markers are concordant, with minor limitations, grouping T. cruzi stocks into four discrete typing units (DTUs) (TcI, TcII, TcV, and TcVI). TcI and TcII stocks were heterogeneous. TcI and TcII stocks were clustered in two main subgroups determined by Cyt b gene sequencing and minicircle hybridization. However, TcV and TcVI stocks were homogeneous and not differentiated by Cyt b gene sequencing or minicircle DNA hybridization. The discriminatory power and limitations of the molecular markers are discussed, as well as the distribution of the four DTUs in the domiciliary and sylvatic transmission cycles of Chile and the limitations of each marker for molecular epidemiological studies performed with T. cruzi stocks rather than the analysis of direct T. cruzi samples from natural hosts.

  1. Hybridization study of developmental plastid gene expression in mustard (Sinapsis alba L.) with cloned probes for most plastid DNA regions.

    Science.gov (United States)

    Link, G

    1984-07-01

    An approach to assess the extent of developmental gene expression of various regions of plastid (pt)DNA in mustard (Sinapis alba L.) is described. It involves cloning of most ptDNA regions. The cloned regions then serve as hybridization probes to detect and assess the abundance of complementary RNA sequences represented in total plastid RNA. By comparison of the hybridization pattern observed with plastid RNA from either dark-grown or light-grown plants it was found that many ptDNA regions are constitutively expressed, while several 'inducible' regions account for much higher transcript levels in the chloroplast than in the etioplast stage. The reverse situation, i.e. 'repressed' regions which would account for higher transcript levels in the etioplast, was not observed. The hybridization results obtained with RNA from 'intermediatetype' plastids suggest that transient gene expression is a common feature during light-induced chloroplast development. The time-course of gene expression differs for various ptDNA regions.

  2. Lambda exonuclease-based subtractive hybridization approach to isolate differentially expressed genes from leaf cultures of Paulownia kawakamii.

    Science.gov (United States)

    Low, R K; Prakash, A P; Swarup, S; Goh, C J; Kumar, P P

    2001-08-15

    Genes that are preferentially expressed in a particular developmental pathway can be isolated by subtractive hybridization (SH). We developed a PCR-based approach coupled with lambda exonuclease digestion that allows for generating single-stranded tester and driver nucleic acids suitable for SH starting from cDNA libraries. An efficient subtraction strategy was developed to overcome some of the problems in the previously described SH protocols, such as the need for large amounts of experimental tissue, RNase contamination during solution hybridization, and postsubtraction recovery of nucleic acids. We used this method to obtain cDNA corresponding to genes expressed during adventitious shoot regeneration from excised leaf cultures of the fast-growing tree Paulownia kawakamii. Over 36 cDNA clones were isolated and 1 of the differentially expressed clones codes for a leucine zipper transcription factor. This clone showed about sixfold higher level of expression in the shoot-forming tissues (tester) compared to that in the callus-forming tissues (driver) of Paulownia, suggesting that differentially expressed genes can be efficiently isolated using this simple lambda exonuclease-based subtractive hybridization method.

  3. Gene expression profiles in squamous cell cervical carcinoma using array-based comparative genomic hybridization analysis.

    Science.gov (United States)

    Choi, Y-W; Bae, S M; Kim, Y-W; Lee, H N; Kim, Y W; Park, T C; Ro, D Y; Shin, J C; Shin, S J; Seo, J-S; Ahn, W S

    2007-01-01

    Our aim was to identify novel genomic regions of interest and provide highly dynamic range information on correlation between squamous cell cervical carcinoma and its related gene expression patterns by a genome-wide array-based comparative genomic hybridization (array-CGH). We analyzed 15 cases of cervical cancer from KangNam St Mary's Hospital of the Catholic University of Korea. Microdissection assay was performed to obtain DNA samples from paraffin-embedded cervical tissues of cancer as well as of the adjacent normal tissues. The bacterial artificial chromosome (BAC) array used in this study consisted of 1440 human BACs and the space among the clones was 2.08 Mb. All the 15 cases of cervical cancer showed the differential changes of the cervical cancer-associated genetic alterations. The analysis limit of average gains and losses was 53%. A significant positive correlation was found in 8q24.3, 1p36.32, 3q27.1, 7p21.1, 11q13.1, and 3p14.2 changes through the cervical carcinogenesis. The regions of high level of gain were 1p36.33-1p36.32, 8q24.3, 16p13.3, 1p36.33, 3q27.1, and 7p21.1. And the regions of homozygous loss were 2q12.1, 22q11.21, 3p14.2, 6q24.3, 7p15.2, and 11q25. In the high level of gain regions, GSDMDC1, RECQL4, TP73, ABCF3, ALG3, HDAC9, ESRRA, and RPS6KA4 were significantly correlated with cervical cancer. The genes encoded by frequently lost clones were PTPRG, GRM7, ZDHHC3, EXOSC7, LRP1B, and NR3C2. Therefore, array-CGH analyses showed that specific genomic alterations were maintained in cervical cancer that were critical to the malignant phenotype and may give a chance to find out possible target genes present in the gained or lost clones.

  4. The mining of pearl formation genes in pearl oyster Pinctada fucata by cDNA suppression subtractive hybridization.

    Science.gov (United States)

    Wang, Ning; Kinoshita, Shigeharu; Nomura, Naoko; Riho, Chihiro; Maeyama, Kaoru; Nagai, Kiyohito; Watabe, Shugo

    2012-04-01

    Recent researches revealed the regional preference of biomineralization gene transcription in the pearl oyster Pinctada fucata: it transcribed mainly the genes responsible for nacre secretion in mantle pallial, whereas the ones regulating calcite shells expressed in mantle edge. This study took use of this character and constructed the forward and reverse suppression subtractive hybridization (SSH) cDNA libraries. A total of 669 cDNA clones were sequenced and 360 expressed sequence tags (ESTs) greater than 100 bp were generated. Functional annotation associated 95 ESTs with specific functions, and 79 among them were identified from P. fucata at the first time. In the forward SSH cDNA library, it recognized mass amount of nacre protein genes, biomineralization genes dominantly expressed in the mantle pallial, calcium-ion-binding genes, and other biomineralization-related genes important for pearl formation. Real-time PCR showed that all the examined genes were distributed in oyster mantle tissues with a consistence to the SSH design. The detection of their RNA transcripts in pearl sac confirmed that the identified genes were certainly involved in pearl formation. Therefore, the data from this work will initiate a new round of pearl formation gene study and shed new insights into molluscan biomineralization.

  5. Identification of differentially expressed genes in lung tissues of nickel-exposed rats using suppression subtractive hybridization.

    Science.gov (United States)

    Zhang, Jing; Zhang, Jun; Fan, Yingying; Liu, Lihong; Li, Mengjie; Zhou, Yang; Shao, Zhihua; Shi, Hongjun; Wang, Ying

    2011-11-01

    Occupational exposure to nickel compound, such as nickel refining, electroplating, and in conjunction with other metals, is harmful to the health, causing respiratory distress, and lung and nasal cancer. In this work, the different gene expression patterns of lung tissues from nickel-exposed rats and controls were investigated. The suppression subtractive hybridization (SSH) method was used to generate two subtracted cDNA libraries with gene transcripts differentially expressed after nickel inducing. Dot-blot hybridizations were used to confirm differential ratios of expression of obtained SSH clones. Out of 768 unique SSH clones, which were chosen randomly from the two subtraction libraries (384 of each), 319 could be verified as differentially expressed. According to blast screening and functional annotation, 28% genes in nickel-induced cDNA library were related to cell differentiation, whereas 21% in driver library were related to oxygen transport. Two novel expressed sequence tags (ESTs; NCBI Accession No. FC809414 and No. FC809411) in nickel-induced cDNA library were obtained. The genes detected in the present study are probably important genes associated with nickel-induced lung cancer.

  6. Identification of differentially expressed genes in Tetrahymena thermophila in response to dichlorodiphenyltrichloroethane (DDT) by suppression subtractive hybridization.

    Science.gov (United States)

    Miao, Wei; Yu, Ting; Orias, Eduardo; Wan, Mingliang; Fu, Chenjie

    2006-06-01

    The insecticide dichlorodiphenyltrichloroethane (DDT) is persistent in the environment, and continues to cause health problems. Tetrahymena has potential as a model organism for assaying low levels of DDT and for analysing the mechanisms of its toxicity. We constructed the suppression subtractive hybridization library of T. thermophila exposed to DDT, and screened out 90 Expressed Sequence Tags whose expressions were significantly up- or downregulated with DDT treatment. From this, a series of important genes related to the DDT metabolism and detoxification were discovered, such as P450 gene, glutathione S-transferase gene and sterol carrier protein 2 gene. Furthermore, their expressions under different concentrations of DDT treatment were detected by real-time fluorescent quantitative PCR. The results show that Tetrahymena is a relevant and useful model organism for detecting DDT in the environment and for discovering biomarkers that can be used to develop specific bio-reporters at the molecular and genomic levels.

  7. Localization of introduced genes on the chromosomes of transgenic barley, wheat and triticale by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Pedersen, C.; Zimny, J.; Becker, D.

    1997-01-01

    transformant showed a totally different integration pattern. Southern analysis confirmed that the inserted genes were segregating independently, resulting in different integration patterns among the progeny lines. The application of the FISH technique for the analysis of transgenic plants is discussed.......Using fluorescence in situ hybridization (FISH) we localized introduced genes on metaphase chromosomes of barley, wheat, and triticale transformed by microprojectile bombardment of microspores and scutellar tissue with the pDB1 plasmid containing the uidA and bar genes. Thirteen integration sites...... of single-copy integrations. There was a slight tendency towards the localization of transgenes in distal chromosome regions. Using the GAA-satellite sequence for chromosome banding, the chromosomes containing the inserted genes were identified in most cases. Two barley lines derived from the same...

  8. Two Tightly Linked Genes at the hsa1 Locus Cause Both F1 and F2 Hybrid Sterility in Rice.

    Science.gov (United States)

    Kubo, Takahiko; Takashi, Tomonori; Ashikari, Motoyuki; Yoshimura, Atsushi; Kurata, Nori

    2016-02-01

    Molecular mechanisms of hybrid breakdown associated with sterility (F2 sterility) are poorly understood as compared with those of F1 hybrid sterility. Previously, we characterized three unlinked epistatic loci, hybrid sterility-a1 (hsa1), hsa2, and hsa3, responsible for the F2 sterility in a cross between Oryza sativa ssp. indica and japonica. In this study, we identified that the hsa1 locus contains two interacting genes, HSA1a and HSA1b, within a 30-kb region. HSA1a-j (japonica allele) encodes a highly conserved plant-specific domain of unknown function protein (DUF1618), whereas the indica allele (HSA1a-i(s)) has two deletion mutations that cause disruption of domain structure. The second gene, HSA1b-i(s), encodes an uncharacterized protein with some similarity to a nucleotide-binding protein. Homozygous introgression of indica HSA1a-i(s)-HSA1b-i(s) alleles into japonica showed female gamete abortion at an early mitotic stage. The fact that the recombinant haplotype HSA1a-j-HSA1b-i(s) caused semi-sterility in the heterozygous state with the HSA1a-i(s)-HSA1b-i(s) haplotype suggests that variation in the hsa1 locus is a possible cause of the wide-spectrum sterility barriers seen in F1 hybrids and successive generations in rice. We propose a simple genetic model to explain how a single causal mechanism can drive both F1 and F2 hybrid sterility.

  9. Fine-scale genetic mapping of a hybrid sterility factor between Drosophila simulans and D. mauritiana: the varied and elusive functions of "speciation genes"

    Directory of Open Access Journals (Sweden)

    Lemos Bernardo

    2010-12-01

    Full Text Available Abstract Background Hybrid male sterility (HMS is a usual outcome of hybridization between closely related animal species. It arises because interactions between alleles that are functional within one species may be disrupted in hybrids. The identification of genes leading to hybrid sterility is of great interest for understanding the evolutionary process of speciation. In the current work we used marked P-element insertions as dominant markers to efficiently locate one genetic factor causing a severe reduction in fertility in hybrid males of Drosophila simulans and D. mauritiana. Results Our mapping effort identified a region of 9 kb on chromosome 3, containing three complete and one partial coding sequences. Within this region, two annotated genes are suggested as candidates for the HMS factor, based on the comparative molecular characterization and public-source information. Gene Taf1 is partially contained in the region, but yet shows high polymorphism with four fixed non-synonymous substitutions between the two species. Its molecular functions involve sequence-specific DNA binding and transcription factor activity. Gene agt is a small, intronless gene, whose molecular function is annotated as methylated-DNA-protein-cysteine S-methyltransferase activity. High polymorphism and one fixed non-synonymous substitution suggest this is a fast evolving gene. The gene trees of both genes perfectly separate D. simulans and D. mauritiana into monophyletic groups. Analysis of gene expression using microarray revealed trends that were similar to those previously found in comparisons between whole-genome hybrids and parental species. Conclusions The identification following confirmation of the HMS candidate gene will add another case study leading to understanding the evolutionary process of hybrid incompatibility.

  10. A new method to identify and improve the purity of hybrid rice with herbicide resistant gene

    Institute of Scientific and Technical Information of China (English)

    HUANGDanian; ZHANGShunqing; XUERui; HUAZhihua; XIEXiaobo; WANGXiaoling

    1998-01-01

    There is a dose relationship between the hybrid rice production and seed purity, Two-line bybrid rice with bigher beterosis is produced through the hybridization between a photothermo sensitive genetic male sterile (GMS) rice line and a paternat variety, But the fertilety of photo-thermo sensitive GMS rice line is not stable because it changes with the total climate.

  11. Screening of aplastic anaemia-related genes in bone marrow CD4+ T cells by suppressive subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    ZHENG Miao; LIU Wen-li; FU Jin-rong; SUN Han-ying; ZHOU Jian-feng; XU Hui-zhen

    2007-01-01

    Background CD4+ T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4+ T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4+ T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia.Methods The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4+T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library.Results PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4+T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4+ T cells in aplastic anaemia.Conclusions Screening and cloning genes, which regulate functions of CD4+ T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4+ T cells in

  12. Neuronal gene expression in aluminum-induced neurofibrillary pathology: an in situ hybridization study.

    Science.gov (United States)

    Chambers, C B; Muma, N A

    1997-01-01

    Alterations in cytoskeletal proteins such as the perikaryal accumulation of neurofilaments (NFs) occur in a number of human neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis and may contribute to their debilitating effects. The administration of aluminum salts to rabbits induces the aberrant accumulation of NFs within the proximal axons and perikarya of vulnerable neurons and is one animal model which has been extensively studied in an attempt to gain insight into the mechanism(s) of NF perturbations in human disease. Previous studies using Northern blotting techniques to examine mRNA levels in the aluminum-induced neuropathy model have led to seemingly contradictory results. We have used in situ hybridization which provides the cellular resolution needed to: 1) determine whether there are generalized decreases in the levels of mRNA expression or decreases in mRNA encoding specific proteins; 2) determine whether alterations in mRNA levels occur specifically in neurons with NF accumulations; and 3) begin to resolve some of the apparent contradictions in the literature. A moderate dose of aluminum lactate administered on two consecutive days produced neurofibrillary tangles in spinal cord neurons seven days after the first dose. Polyadenylated mRNA levels were not altered in spinal cord neurons in aluminum-treated compared to saline-treated control animals or in tangle-bearing compared to non tangle-bearing neurons in aluminum-treated animals. Middle and high NF subunit (NFH) mRNA levels were not significantly different from polyadenylated mRNA levels in spinal cord neurons in aluminum-treated/control animals. NFH mRNA levels were decreased in neurons containing aluminum-induced NF accumulations. These results suggest that NFH gene expression may be down regulated by an inhibitory feedback mechanism induced by perikaryal accumulations of NFs. This inhibitory feedback regulation for NFH may have

  13. [The determination of the genotype of natural reassortant influenza A viruses according to the core protein genes by the methods of competitive dot hybridization and sequencing].

    Science.gov (United States)

    Grinbaum, E B; Zolotarev, F N; Petrov, N A; Litvinova, O M; Konovalenko, I B; Luzianina, T Ia; Golubev, D B

    1992-01-01

    Simultaneous circulation of different subtypes of influenza A viruses provides conditions for reassortant strains formation. A comparative investigation of genome of 47 influenza A virus strains (H1N1, H2N2, and H3N2) was carried out by competitive dot hybridization technique and sequence analysis of some of cDNA-copies of the virus genes. All the genes of 43 strains encoding nonglycolysed proteins corresponded to the serum subtype of surface glycoproteins. The reassortant pattern of genome for some genes of core proteins was revealed in 4 viruses. All the dot hybridization data were completely confirmed by sequence analysis of the genes.

  14. Identification of male gametogenesis expressed genes from the scallop Nodipecten subnodosus by suppressive subtraction hybridization and pyrosequencing.

    Science.gov (United States)

    Llera-Herrera, Raúl; García-Gasca, Alejandra; Abreu-Goodger, Cei; Huvet, Arnaud; Ibarra, Ana M

    2013-01-01

    Despite the great advances in sequencing technologies, genomic and transcriptomic information for marine non-model species with ecological, evolutionary, and economical interest is still scarce. In this work we aimed to identify genes expressed during spermatogenesis in the functional hermaphrodite scallop Nodipecten subnodosus (Mollusca: Bivalvia: Pectinidae), with the purpose of obtaining a panel of genes that would allow for the study of differentially transcribed genes between diploid and triploid scallops in the context of meiotic arrest and reproductive sterility. Because our aim was to isolate genes involved in meiosis and other testis maturation-related processes, we generated suppressive subtractive hybridization libraries of testis vs. inactive gonad. We obtained 352 and 177 ESTs by clone sequencing, and using pyrosequencing (454-Roche) we maximized the identified ESTs to 34,276 reads. A total of 1,153 genes from the testis library had a blastx hit and GO annotation, including genes specific for meiosis, spermatogenesis, sex-differentiation, and transposable elements. Some of the identified meiosis genes function in chromosome pairing (scp2, scp3), recombination and DNA repair (dmc1, rad51, ccnb1ip1/hei10), and meiotic checkpoints (rad1, hormad1, dtl/cdt2). Gene expression analyses in different gametogenic stages in both sexual regions of the gonad of meiosis genes confirmed that the expression was specific or increased towards the maturing testis. Spermatogenesis genes included known testis-specific ones (kelch-10, shippo1, adad1), with some of these known to be associated to sterility. Sex differentiation genes included one of the most conserved genes at the bottom of the sex-determination cascade (dmrt1). Transcript from transposable elements, reverse transcriptase, and transposases in this library evidenced that transposition is an active process during spermatogenesis in N. subnodosus. In relation to the inactive library, we identified 833

  15. Identification of male gametogenesis expressed genes from the scallop Nodipecten subnodosus by suppressive subtraction hybridization and pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Raúl Llera-Herrera

    Full Text Available Despite the great advances in sequencing technologies, genomic and transcriptomic information for marine non-model species with ecological, evolutionary, and economical interest is still scarce. In this work we aimed to identify genes expressed during spermatogenesis in the functional hermaphrodite scallop Nodipecten subnodosus (Mollusca: Bivalvia: Pectinidae, with the purpose of obtaining a panel of genes that would allow for the study of differentially transcribed genes between diploid and triploid scallops in the context of meiotic arrest and reproductive sterility. Because our aim was to isolate genes involved in meiosis and other testis maturation-related processes, we generated suppressive subtractive hybridization libraries of testis vs. inactive gonad. We obtained 352 and 177 ESTs by clone sequencing, and using pyrosequencing (454-Roche we maximized the identified ESTs to 34,276 reads. A total of 1,153 genes from the testis library had a blastx hit and GO annotation, including genes specific for meiosis, spermatogenesis, sex-differentiation, and transposable elements. Some of the identified meiosis genes function in chromosome pairing (scp2, scp3, recombination and DNA repair (dmc1, rad51, ccnb1ip1/hei10, and meiotic checkpoints (rad1, hormad1, dtl/cdt2. Gene expression analyses in different gametogenic stages in both sexual regions of the gonad of meiosis genes confirmed that the expression was specific or increased towards the maturing testis. Spermatogenesis genes included known testis-specific ones (kelch-10, shippo1, adad1, with some of these known to be associated to sterility. Sex differentiation genes included one of the most conserved genes at the bottom of the sex-determination cascade (dmrt1. Transcript from transposable elements, reverse transcriptase, and transposases in this library evidenced that transposition is an active process during spermatogenesis in N. subnodosus. In relation to the inactive library, we identified

  16. Nucleolar Dominance and Repression of 45S Ribosomal RNA Genes in Hybrids between Xenopus borealis and X. muelleri (2n = 36).

    Science.gov (United States)

    Maciak, Sebastian; Michalak, Katarzyna; Kale, Shiv D; Michalak, Pawel

    2016-01-01

    Nucleolar dominance is a dramatic disruption in the formation of nucleoli and the expression of ribosomal RNA (rRNA) genes, characteristic of some plant and animal hybrids. Here, we report that F1 hybrids produced from reciprocal crosses between 2 sister species of Xenopus clawed frogs, X. muelleri and X. borealis, undergo nucleolar dominance somewhat distinct from a pattern previously reported in hybrids between phylogenetically more distant Xenopus species. Patterns of nucleolar development, 45S rRNA expression, and gene copy inheritance were investigated using a combination of immunostaining, pyrosequencing, droplet digital PCR, flow cytometry, and epigenetic inhibition. In X. muelleri × X. borealis hybrids, typically only 1 nucleolus is formed, and 45S rRNA genes are predominantly expressed from 1 progenitor's alleles, X. muelleri, regardless of the cross-direction. These changes are accompanied by an extensive (∼80%) loss of rRNA gene copies in the hybrids relative to their parents, with the transcriptionally underdominant variant (X. borealis) being preferentially lost. Chemical treatment of hybrid larvae with a histone deacetylase inhibitor resulted in a partial derepression of the underdominant variant. Together, these observations shed light on the genetic and epigenetic basis of nucleolar dominance as an underappreciated manifestation of genetic conflicts within a hybrid genome.

  17. Self-assembled cationic micelles based on PEG-PLL-PLLeu hybrid polypeptides as highly effective gene vectors.

    Science.gov (United States)

    Deng, Jizhe; Gao, Ningning; Wang, Yanan; Yi, Huqiang; Fang, Shengtao; Ma, Yifan; Cai, Lintao

    2012-11-12

    Developing safe and effective nonviral gene vector is highly crucial for successful gene therapy. In the present study, we designed a series of biodegradable micelles based on hybrid polypeptide copolymers of poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-leucine) (PEG-PLL-PLLeu) for efficient gene delivery. A group of amphiphilic PEG-PLL-PLLeu hybrid polypeptide copolymers were synthesized by ring-opening polymerization of N-carboxyanhydride, and the chemical structure of each copolymer was characterized by (1)H NMR and FT-IR spectroscopy measurement. The PEG-PLL-PLLeu micelles were positively charged with tunable sizes ranging from 40 to 90 nm depending on the length of PLL and PLLeu segment. Compared with PEG-PLL copolymers, PEG-PLL-PLLeu micelles demonstrated significantly higher transfection efficiency and less cytotoxicity. Furthermore, the transfection efficiency and biocompatibility of the micelles can be simultaneously improved by tuning the length of PLL and PLLeu segments. The transfection efficiency of PEG-PLL-PLLeu micelles in vivo was two to three times higher than that of PEI(25k), which was attributable to their capability of promoting DNA condensation and cell internalization as well as successful lysosome escape. Hence well-defined PEG-PLL-PLLeu micelles would serve as highly effective nonviral vectors for in vivo gene delivery.

  18. Diversification of the plant-specific hybrid glycine-rich protein (HyGRP genes in cereals

    Directory of Open Access Journals (Sweden)

    Kenji eFujino

    2014-09-01

    Full Text Available Plant-specific hybrid proline- or glycine-rich proteins (HyP/GRPs are involved in diverse gene functions including plant development and responses to biotic and abiotic stresses. The quantitative trait locus, qLTG3-1, enhances seed germination in rice under low-temperature conditions and encodes a member with a glycine-rich motif of the HyP/GRP family. The function of this gene may be related to the weakening of tissue covering the embryo during seed germination. In the present study, the diversification of the HyP/GRP gene family was elucidated in rice based on phylogenetic relationships and gene expression levels. At least 21 members of the HyP/GRP family have been identified in the rice genome and clustered in five regions on four chromosomes by tandem and chromosomal duplications. Of these, OsHyPRP05 (qLTG3-1 and its paralogous gene, OsHyPRP21, had a glycine-rich motif. Furthermore, orthologous genes with a glycine-rich motif and the HyP/GRP gene family were detected in four genome-sequenced monocots: 12 in barley, 10 in Brachypodium, 20 in maize, and 28 in sorghum, using a BLAST search of qLTG3-1 as the query. All members of the HyP/GRP family in these five species were classified into seven main groups, which were clustered together in these species. These results suggested that the HyP/GRP gene family was formed in the ancestral genome before the divergence of these species. The collinearity of chromosomal regions around qLTG3-1 and its orthologous genes were conserved among rice, Brachypodium, sorghum, and maize, indicating that qLTG3-1 and orthologous genes conserve gene function during seed germination.

  19. Characterization of two novel gadd45a genes in hybrid tilapia and their responses to the infection of Streptococcus agalactiae.

    Science.gov (United States)

    Shen, Yubang; Ma, Keyi; Liu, Feng; Yue, Gen Hua

    2016-07-01

    Diseases are one of the major challenges in tilapia aquaculture. Identification of DNA markers associated with disease resistance may facilitate the acceleration of the selection for disease resistance. Gadd45a (growth arrest and DNA damage 45 A), a stress-inducible gene in humans and mice, has not been studied in fish. We characterized the two prologues of Gadd45a genes in hybrid tilapia. Gadd45a1 and Gadd45a2 shared an identical gene structure and showed an amino acid sequence identity of 73.8%. Their expressions were detected in all 10 tissues examined, with the kidney and gill having high transcriptional expressions. The expression levels of Gadd45a1 were significantly lower than those of Gadd45a2 in all examined tissues. After a challenge with a bacterial pathogen Streptococcus agalactiae, the expressions of the two genes were up-regulated significantly in the spleen, kidney, liver and intestine. These findings suggest that the two Gadd45a genes play an important role in the resistance to S. agalactiae in tilapia. We identified 10 SNPs in the two genes. The SNP markers in the two Gadd45a genes could be used to examine whether they are associated with resistance to S. agalactiae.

  20. Selection of genes associated with variations in the Circle of Willis in gerbils using suppression subtractive hybridization.

    Science.gov (United States)

    Li, Zhenkun; Huo, Xueyun; Zhang, Shuangyue; Lu, Jing; Li, Changlong; Guo, Meng; Fu, Rui; He, Zhengming; Du, Xiaoyan; Chen, Zhenwen

    2015-01-01

    Deformities in the Circle of Willis (CoW) can significantly increase the risk of cerebrovascular disease in humans. However, the molecular mechanisms underlying these deformities have not been understood. Based on our previous studies, variations in the CoW of gerbils are hereditary. A normal CoW is observed in approximately 60% of gerbils, a percentage that also applies to humans. Thus, gerbil is an ideal experimental model for studying variations in the CoW. To study the mechanisms underlying these variations, we selected genes associated with different types of the CoW using suppression subtractive hybridization (SSH). After evaluating the efficiency of SSH using quantitative real-time polymerase chain reaction (qPCR) on subtracted and unsubtracted cDNA and Southern blotting on SSH PCR products, 12 SSH libraries were established. We identified 4 genes (CST3, GNAS, GPx4 and PFN2) associated with variations in the CoW. These genes were identified with qPCR and Western blotting using 70 expressed sequence tags from the SSH libraries. Cloning and sequencing allowed us to demonstrate that the 4 genes were closely related to mouse genes. We may assume that these 4 genes play an important role in the development of variations in the CoW. This study provides a foundation for further research of genes related to development of variations in the CoW and the mechanisms of dysmorphosis of cerebral vessels.

  1. Selection of genes associated with variations in the Circle of Willis in gerbils using suppression subtractive hybridization.

    Directory of Open Access Journals (Sweden)

    Zhenkun Li

    Full Text Available Deformities in the Circle of Willis (CoW can significantly increase the risk of cerebrovascular disease in humans. However, the molecular mechanisms underlying these deformities have not been understood. Based on our previous studies, variations in the CoW of gerbils are hereditary. A normal CoW is observed in approximately 60% of gerbils, a percentage that also applies to humans. Thus, gerbil is an ideal experimental model for studying variations in the CoW. To study the mechanisms underlying these variations, we selected genes associated with different types of the CoW using suppression subtractive hybridization (SSH. After evaluating the efficiency of SSH using quantitative real-time polymerase chain reaction (qPCR on subtracted and unsubtracted cDNA and Southern blotting on SSH PCR products, 12 SSH libraries were established. We identified 4 genes (CST3, GNAS, GPx4 and PFN2 associated with variations in the CoW. These genes were identified with qPCR and Western blotting using 70 expressed sequence tags from the SSH libraries. Cloning and sequencing allowed us to demonstrate that the 4 genes were closely related to mouse genes. We may assume that these 4 genes play an important role in the development of variations in the CoW. This study provides a foundation for further research of genes related to development of variations in the CoW and the mechanisms of dysmorphosis of cerebral vessels.

  2. Gene flow and hybridization between numerically imbalanced populations of two duck species in the Falkland Islands.

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    Kevin G McCracken

    Full Text Available Interspecific hybridization is common in plants and animals, particularly in waterfowl (Anatidae. One factor shown to contribute to hybridization is restricted mate choice, which can occur when two species occur in sympatry but one is rare. The Hubbs principle, or "desperation hypothesis," states that under such circumstances the rarer species is more likely to mate with heterospecifics. Here we report interspecific hybridization between two waterfowl species that coexist in broad sympatry and mixed flocks throughout southern South America. Speckled teal (Anas flavirostris and yellow-billed pintails (Anas georgica are abundant in continental South America, but in the Falkland Islands speckled teal outnumber yellow-billed pintails approximately ten to one. Using eight genetic loci (mtDNA and 7 nuclear introns coupled with Bayesian assignment tests and relatedness analysis, we identified a speckled teal x yellow-billed pintail F(1 hybrid female and her duckling sired by a male speckled teal. Although our sample in the Falkland Islands was small, we failed to identify unequivocal evidence of hybridization or introgression in a much larger sample from Argentina using a three-population "isolation with migration" coalescent analysis. While additional data are needed to determine if this event in the Falkland Islands was a rare singular occurrence, our results provide further support for the "desperation hypothesis," which states that scarcity in one population and abundance of another will often lead to hybridization.

  3. Identification of differentially expressed genes in SHSY5Y cells exposed to okadaic acid by suppression subtractive hybridization

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    Valdiglesias Vanessa

    2012-01-01

    Full Text Available Abstract Background Okadaic acid (OA, a toxin produced by several dinoflagellate species is responsible for frequent food poisonings associated to shellfish consumption. Although several studies have documented the OA effects on different processes such as cell transformation, apoptosis, DNA repair or embryogenesis, the molecular mechanistic basis for these and other effects is not completely understood and the number of controversial data on OA is increasing in the literature. Results In this study, we used suppression subtractive hybridization in SHSY5Y cells to identify genes that are differentially expressed after OA exposure for different times (3, 24 and 48 h. A total of 247 subtracted clones which shared high homology with known genes were isolated. Among these, 5 specific genes associated with cytoskeleton and neurotransmission processes (NEFM, TUBB, SEPT7, SYT4 and NPY were selected to confirm their expression levels by real-time PCR. Significant down-regulation of these genes was obtained at the short term (3 and 24 h OA exposure, excepting for NEFM, but their expression was similar to the controls at 48 h. Conclusions From all the obtained genes, 114 genes were up-regulated and 133 were down-regulated. Based on the NCBI GenBank and Gene Ontology databases, most of these genes are involved in relevant cell functions such as metabolism, transport, translation, signal transduction and cell cycle. After quantitative PCR analysis, the observed underexpression of the selected genes could underlie the previously reported OA-induced cytoskeleton disruption, neurotransmission alterations and in vivo neurotoxic effects. The basal expression levels obtained at 48 h suggested that surviving cells were able to recover from OA-caused gene expression alterations.

  4. Specific down-regulation of spermatogenesis genes targeted by 22G RNAs in hybrid sterile males associated with an X-Chromosome introgression.

    Science.gov (United States)

    Li, Runsheng; Ren, Xiaoliang; Bi, Yu; Ho, Vincy Wing Sze; Hsieh, Chia-Ling; Young, Amanda; Zhang, Zhihong; Lin, Tingting; Zhao, Yanmei; Miao, Long; Sarkies, Peter; Zhao, Zhongying

    2016-09-01

    Hybrid incompatibility (HI) prevents gene flow between species, thus lying at the heart of speciation genetics. One of the most common HIs is male sterility. Two superficially contradictory observations exist for hybrid male sterility. First, an introgression on the X Chromosome is more likely to produce male sterility than on autosome (so-called large-X theory); second, spermatogenesis genes are enriched on the autosomes but depleted on the X Chromosome (demasculinization of X Chromosome). Analysis of gene expression in Drosophila hybrids suggests a genetic interaction between the X Chromosome and autosomes that is essential for male fertility. However, the prevalence of such an interaction and its underlying mechanism remain largely unknown. Here we examine the interaction in nematode species by contrasting the expression of both coding genes and transposable elements (TEs) between hybrid sterile males and its parental nematode males. We use two lines of hybrid sterile males, each carrying an independent introgression fragment from Caenorhabditis briggsae X Chromosome in an otherwise Caenorhabditis nigoni background, which demonstrate similar defects in spermatogenesis. We observe a similar pattern of down-regulated genes that are specific for spermatogenesis between the two hybrids. Importantly, the down-regulated genes caused by the X Chromosome introgressions show a significant enrichment on the autosomes, supporting an epistatic interaction between the X Chromosome and autosomes. We investigate the underlying mechanism of the interaction by measuring small RNAs and find that a subset of 22G RNAs specifically targeting the down-regulated spermatogenesis genes is significantly up-regulated in hybrids, suggesting that perturbation of small RNA-mediated regulation may contribute to the X-autosome interaction.

  5. Screening differentially expressed genes in an amphipod (Hyalella azteca) exposed to fungicide vinclozolin by suppression subtractive hybridization.

    Science.gov (United States)

    Wu, Yun H; Wu, Tsung M; Hong, Chwan Y; Wang, Yei S; Yen, Jui H

    2014-01-01

    Vinclozolin, a dicarboximide fungicide, is an endocrine disrupting chemical that competes with an androgenic endocrine disruptor compound. Most research has focused on the epigenetic effect of vinclozolin in humans. In terms of ecotoxicology, understanding the effect of vinclozolin on non-target organisms is important. The expression profile of a comprehensive set of genes in the amphipod Hyalella azteca exposed to vinclozolin was examined. The expressed sequence tags in low-dose vinclozolin-treated and -untreated amphipods were isolated and identified by suppression subtractive hybridization. DNA dot blotting was used to confirm the results and establish a subtracted cDNA library for comparing all differentially expressed sequences with and without vinclozolin treatment. In total, 494 differentially expressed genes, including hemocyanin, heatshock protein, cytochrome, cytochrome oxidase and NADH dehydrogenase were detected. Hemocyanin was the most abundant gene. DNA dot blotting revealed 55 genes with significant differential expression. These genes included larval serum protein 1 alpha, E3 ubiquitin-protein ligase, mitochondrial cytochrome c oxidase, mitochondrial protein, proteasome inhibitor, hemocyanin, zinc-finger-containing protein, mitochondrial NADH-ubiquinone oxidoreductase and epididymal sperm-binding protein. Vinclozolin appears to upregulate stress-related genes and hemocyanin, related to immunity. Moreover, vinclozolin downregulated NADH dehydrogenase, related to respiration. Thus, even a non-lethal concentration of vinclozolin still has an effect at the genetic level in H. azteca and presents a potential risk, especially as it would affect non-target organism hormone metabolism.

  6. Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization.

    Science.gov (United States)

    Henke, R T; Maitra, A; Paik, S; Wellstein, A

    2005-01-01

    Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.

  7. Characterization of the duodenase-1 gene and its associations with resistance to Streptococuus agalactiae in hybrid tilapia (Oreochromis spp.).

    Science.gov (United States)

    Shen, Yubang; Fu, Gui Hong; Liu, Feng; Yue, Gen Hua

    2015-08-01

    Tilapia is a group of cultured teleost fishes whose production is threatened by some diseases. Identification of DNA markers associated with disease resistance in candidate genes may facilitate to accelerate the selection of disease resistance. The gene encoding a duodenase, which can trigger immune response, has not been studied in fish. We characterized the cDNA of duodenase-1 gene of hybrid tilapia. Its ORF is 759 bp, encoding a serine protease of 252 amino acids. This gene consisted of five exons and four introns. Its expression was detected in all 10 tissues examined, and it was highly expressed in the intestine and kidney. After a challenge with the bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the intestine, liver and spleen. We identified seven SNPs in the gene and found that four of them were significantly associated with the resistance to S. agalactiae (P tilapia. The SNP markers in the duodenase-1 gene associated with resistance to the bacterial pathogen, may facilitate the selection of tilapia resistant to the bacterial disease.

  8. Identification of Differentially Expressed Genes During Anther Abortion of Taigu Genic Male Sterile Wheat by Combining Suppression Subtractive Hybridization and cDNA Array

    Institute of Scientific and Technical Information of China (English)

    Qing-Shan Chang; Rong-Hua Zhou; Xiu-Ying Kong; Zeng-Liang Yu; Ji-Zeng Jia

    2006-01-01

    Taigu Genic Male Sterile Wheat (TGMSW; Triticum aestivum L.), a dominant genic male sterile germplasm, is of considerable value in the genetic improvement of wheat because of its stable inherence, complete male abortion, and high cross-fertilization rate. To identify specially transcribed genes in sterile anther, a suppression subtractive hybridization (SSH) library was constructed with sterile anther as the tester and fertile anther as the driver. A total of 2 304 SSH inserts amplified by polymerase chain reaction were arrayed using robotic printing. The cDNA arrays were hybridized with 32P-labeled probes prepared from the RNA of forward- and reverse-subtracted anthers. Ninety-six clones were scored as upregulated in sterile anthers compared with the corresponding fertile anthers and some clones were selected for sequencing and analysis in GenBank. Based on their putative functions, 87 non-redundant clones were classified into the following groups: (i) eight genes involved in metabolic processes; (ii) four material transportation genes;(iii) three signal transduction-associated genes; (iv) four stress response and senescence-associated protein genes; (v) seven other functional protein genes; (vi) five genes with no known function; and (vii)another 56 genes with no match to the databases. To test the hybridization efficiency, eight genes were selected and analyzed by Northern blot. The results of the present study provide a comprehensive overview of the genes and gene products involved in anther abortion in TGMSW.

  9. Transcriptional profiles of hybrid Eucalyptus genotypes with contrasting lignin content reveal that monolignol biosynthesis-related genes regulate wood composition

    Directory of Open Access Journals (Sweden)

    Tomotaka eShinya

    2016-04-01

    Full Text Available Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected three-year old hybrid Eucalyptus (Eucalyptus urophylla x E. grandis genotypes (AM063 and AM380 that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0% and 48.2%, -cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA and sucrose synthase (SUSY were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase (UGP and xyloglucan endotransglucoxylase (XTH than those in AM380. Most monolignol biosynthesis- related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase (PAL, cinnamate-4-hydroxylase (C4H and 4-coumarate-CoA ligase (4CL. Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents

  10. VACCINATION OF HYBRID STRIPED BASS: GROWTH, IMMUNE REACTION AND GENE EXPRESSION

    Directory of Open Access Journals (Sweden)

    Mark Westerman

    2012-10-01

    Full Text Available Hybrid striped bass (42.6+4.9 g wet wt; 139.3+6.1 mm length, were randomly stocked into one of 6 tanks (n=6 tank-1 of a custom designed, recirculating life support system (RLSS. Water quality was as follows: DO2 (6.50.6 mg l-1, pH (7.70.5, TAN (0.06-1.31 mg l-1, nitrite (0.06-0.60 mg l-1 and nitrate (2.0-32.1 mg l-1, salinity 5 ppt, temperature 28±1 oC. A 12:12 photophase:scotophase was used, with a 30 min. dusk-dawn dimming of lights. Fish were fed at 4% body wt d-1 as two separate feedings (08.00 and 16.00 h. Dietary crude protein and lipid levels were 40% and 10% respectively. Tanks were randomly paired and fish either left untreated, vaccinated, or sham injected. The vaccine employed was an experimental formalin killed Streptococcus iniae oil-in-water adjuvanted bacterin. Fish were weighed and measured bimonthly for 8 wk with group weights being employed to adjust feeding rates. At trial termination, all animals were weighed and measured, and their condition factor (CF and feed conversion ratios (FCR calculated. Visceral somatic (VSI and hepatosomatic (HSI indices and intraperitoneal fat (IPF and muscle ratios (MR were also assessed. Blood was taken from anaesthetized fish and hematocrit recorded. Blood was allowed to clot overnight at 5 oC after which serum protein levels were recorded and lysozyme activity measured. Livers were prepared for microarray evaluation using standard techniques using a Danio rerio genechip to assess global changes in gene expression. No differences were observed with respect to final weights, lengths, CF, FCR, or HSI although differences (P < 0.001 were determined for VSI, which was higher in control animals. Packed cell volume and serum protein levels were similar across groups (P > 0.05. Time-course of changes in serum lysozyme activity described an initial reduction in lysozyme activity followed by a rebounding in activity which peaked 25 days post-treatments. Evaluation of lysozyme activity among time

  11. Genetic expression and effects of the rolled leaf gene Rl(t)in hybrid rice (Oryza sativa L.)

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    We constructed a near isogenic line of rolled leaf gene Rl(t), which expressed incompletely dominance for the character of rolled leaf(RL), with genetic background of Zhenshan 97B. Using RL Zhenshan 97B and the original Zhenshan 97B as the female parents,and Minghui 63 and Yanhui 559 as the male parents, crosses of RL Shanyou 63 (RS63) and Shanyou 63(S63), RL Shanyou 559 ( RS559) and Shanyou 559 (S559) were made. Inheritance and effects of Rl(t) in hybrid rice were studied at the flowering and at the 20 d after flowering, respectively. Results were as follows:

  12. Localization of 18S + 28S and 5S ribosomal RNA genes in the dog by fluorescence in situ hybridization.

    Science.gov (United States)

    Mäkinen, A; Zijlstra, C; de Haan, N A; Mellink, C H; Bosma, A A

    1997-01-01

    The gene clusters encoding 18S + 28S and 5S rRNA in the dog (Canis familiaris) have been localized by using GTG-banding and fluorescence in situ hybridization. The 18S + 28S rDNA maps to chromosome regions 7q2.5-->q2.7, 17q1.7, qter of a medium-sized, not yet numbered autosome, and Yq1.2-->q1.3. Our data show that there is one cluster of 5S rDNA in the dog, which maps to chromosome region 4q1.4.

  13. Epidermal growth factor receptor and AKT1 gene copy numbers by multi-gene fluorescence in situ hybridization impact on prognosis in breast cancer.

    Science.gov (United States)

    Li, Jiao; Su, Wei; Zhang, Sheng; Hu, Yunhui; Liu, Jingjing; Zhang, Xiaobei; Bai, Jingchao; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lei, Zhenmin; Zhang, Jin

    2015-05-01

    The epidermal growth factor receptor (EGFR)/PI3K/AKT signaling pathway aberrations play significant roles in breast cancer occurrence and development. However, the status of EGFR and AKT1 gene copy numbers remains unclear. In this study, we showed that the rates of EGFR and AKT1 gene copy number alterations were associated with the prognosis of breast cancer. Among 205 patients, high EGFR and AKT1 gene copy numbers were observed in 34.6% and 27.8% of cases by multi-gene fluorescence in situ hybridization, respectively. Co-heightened EGFR/AKT1 gene copy numbers were identified in 11.7% cases. No changes were found in 49.3% of patients. Although changes in EGFR and AKT1 gene copy numbers had no correlation with patients' age, tumor stage, histological grade and the expression status of other molecular makers, high EGFR (P = 0.0002) but not AKT1 (P = 0.1177) gene copy numbers correlated with poor 5-year overall survival. The patients with co-heightened EGFR/AKT1 gene copy numbers displayed a poorer prognosis than those with tumors with only high EGFR gene copy numbers (P = 0.0383). Both Univariate (U) and COX multivariate (C) analyses revealed that high EGFR and AKT1 gene copy numbers (P = 0.000 [U], P = 0.0001 [C]), similar to histological grade (P = 0.001 [U], P = 0.012 [C]) and lymph node metastasis (P = 0.046 [U], P = 0.158 [C]), were independent prognostic indicators of 5-year overall survival. These results indicate that high EGFR and AKT1 gene copy numbers were relatively frequent in breast cancer. Co-heightened EGFR/AKT1 gene copy numbers had a worse outcome than those with only high EGFR gene copy numbers, suggesting that evaluation of these two genes together may be useful for selecting patients for anti-EGFR-targeted therapy or anti-EGFR/AKT1-targeted therapy and for predicting outcomes. © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  14. Analysis of a plant complex resistance gene locus underlying immune-related hybrid incompatibility and its occurrence in nature.

    Directory of Open Access Journals (Sweden)

    Rubén Alcázar

    2014-12-01

    Full Text Available Mechanisms underlying speciation in plants include detrimental (incompatible genetic interactions between parental alleles that incur a fitness cost in hybrids. We reported on recessive hybrid incompatibility between an Arabidopsis thaliana strain from Poland, Landsberg erecta (Ler, and many Central Asian A. thaliana strains. The incompatible interaction is determined by a polymorphic cluster of Toll/interleukin-1 receptor-nucleotide binding-leucine rich repeat (TNL RPP1 (Recognition of Peronospora parasitica1-like genes in Ler and alleles of the receptor-like kinase Strubbelig Receptor Family 3 (SRF3 in Central Asian strains Kas-2 or Kond, causing temperature-dependent autoimmunity and loss of growth and reproductive fitness. Here, we genetically dissected the RPP1-like Ler locus to determine contributions of individual RPP1-like Ler (R1-R8 genes to the incompatibility. In a neutral background, expression of most RPP1-like Ler genes, except R3, has no effect on growth or pathogen resistance. Incompatibility involves increased R3 expression and engineered R3 overexpression in a neutral background induces dwarfism and sterility. However, no individual RPP1-like Ler gene is sufficient for incompatibility between Ler and Kas-2 or Kond, suggesting that co-action of at least two RPP1-like members underlies this epistatic interaction. We find that the RPP1-like Ler haplotype is frequent and occurs with other Ler RPP1-like alleles in a local population in Gorzów Wielkopolski (Poland. Only Gorzów individuals carrying the RPP1-like Ler haplotype are incompatible with Kas-2 and Kond, whereas other RPP1-like alleles in the population are compatible. Therefore, the RPP1-like Ler haplotype has been maintained in genetically different individuals at a single site, allowing exploration of forces shaping the evolution of RPP1-like genes at local and regional population scales.

  15. Further mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

    Science.gov (United States)

    Lewis, J B; Anderson, C W; Atkins, J F

    1977-09-01

    RNA isolated from the cytoplasm of human cells at late times after infection by adenovirus type 2 (Ad2) has been fractionated by hybridization to fragments of Ad2 DNA which were produced by digestion with the restriction endonucleases Hpa I, Eco RI, Bam HI and Hind III. Cell-free translation of these partially purified mRNAs indicates that the genes for the late Ad2 proteins lie within the following intervals on the conventional Ad2 map: 15K (4.4-17.0 map units), IX and IVa2 (7.5-17.0), IIIa (29.1-40.9), III and V (29.1-57.0), pVIII (40.9-57.0), pVI and II (40.9-70.7), 100K (59.0-83.4), pVIII (70.7-83.4) and IV (85.0-100). In addition to the primary hybridization of the late Ad2 mRNAs to the regions indicated above, most late Ad2 mRNAs (except those for 15K, IX and IVa2) exhibited some hybridization to a secondary site between 17.0 and 29.1 map units.

  16. Preliminary study on the genes related to the fertility of indica/japonica hybrid rice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@Studying the sterility of indica/japonica hybrid was a prerequisite for the utilization of heterosis in high yield rice breeding. In 1986, Ikehashi et al found that wide compatibility varieties, which possessed S5-n allele, couldovercome the sterility of indica/japonica hybrids, and thesterilitv was mainly caused by allelic interaction at S-5 lo-cus. Further studies indicated that the sterility could notbe completely overcome just by S5-n. There were must beother loci responsible for the sterility of indica/ japonicahybrids.

  17. Analysis and characterization of differential gene expression during rapid trophoblastic elongation in the pig using suppression subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Malayer Jerry R

    2003-02-01

    Full Text Available Abstract During late peri-implantation development, porcine conceptuses undergo a rapid (2–3 hrs morphological transformation from a 10 mm sphere to a thin filamentous form greater than 150 mm in length. Elongation of the conceptus is important for establishing adequate placental surface area needed for embryo and fetal survival throughout gestation. Genes involved with triggering this unique transition in conceptus development are not well defined. Objective of the present study was to utilize suppression subtractive hybridization (SSH to characterize the change in gene expression during conceptus transformation from spherical (8–9 mm to tubular (15–40 mm to early filamentous (>150 mm morphology. Spherical, tubular, and filamentous conceptuses were collected from pregnant gilts and subjected to SSH. Forward and reverse subtractions were performed to identify candidate genes differentially expressed during spherical to tubular and tubular to filamentous transition. A total of 384 transcripts were differentially screened to ensure unique expression. Of the transcripts screened, sequences were obtained for 142 that were confirmed to be differentially expressed among the various morphologies. Gene expression profiles during rapid trophoblastic elongation were generated for selected mRNAs using quantitative real-time PCR. During the transition from tubular to early filamentous conceptuses, s-adenosylhomocysteine hydrolase and heat shock cognate 70 kDa expression were significantly enhanced. A novel unknown gene was isolated and shown to be significantly up-regulated at the onset of rapid trophoblastic elongation and further enhanced in filamentous conceptuses.

  18. Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in virus infected cells

    Directory of Open Access Journals (Sweden)

    Munir Shirin

    2004-01-01

    Full Text Available High throughput detection of differential expression of genes is an efficient means of identifying genes and pathways that may play a role in biological systems under certain experimental conditions. There exist a variety of approaches that could be used to identify groups of genes that change in expression in response to a particular stimulus or environment. We here describe the application of suppression subtractive hybridization (SSH coupled with cDNA microarray analysis for isolation and identification of chicken transcripts that change in expression on infection of host cells with a paramyxovirus. SSH was used for initial isolation of differentially expressed transcripts, a large-scale validation of which was accomplished by microarray analysis. The data reveals a large group of regulated genes constituting many biochemical pathways that could serve as targets for future investigations to explore their role in paramyxovirus pathogenesis. The detailed methods described herein could be useful and adaptable to any biological system for studying changes in gene expression.

  19. Gene expression profiling of osteoclast differentiation by combined suppression subtractive hybridization (SSH) and cDNA microarray analysis.

    Science.gov (United States)

    Rho, Jaerang; Altmann, Curtis R; Socci, Nicholas D; Merkov, Lubomir; Kim, Nacksung; So, Hongseob; Lee, Okbok; Takami, Masamichi; Brivanlou, Ali H; Choi, Yongwon

    2002-08-01

    Bone homeostasis is maintained by the balanced action of bone-forming osteoblasts and bone-resorbing osteoclasts. Multinucleated, mature osteoclasts develop from hematopoietic stem cells via the monocyte-macrophage lineage, which also give rise to macrophages and dendritic cells. Despite their distinct physiologic roles in bone and the immune system, these cell types share many molecular and biochemical features. To provide insights into how osteoclasts differentiate and function to control bone metabolism, we employed a systematic approach to profile patterns of osteoclast-specific gene expression by combining suppression subtractive hybridization (SSH) and cDNA microarray analysis. Here we examined how gene expression profiles of mature osteoclast differ from macrophage or dendritic cells, how gene expression profiles change during osteoclast differentiation, and how Mitf, a transcription factor critical for osteoclast maturation, affects the gene expression profile. This approach revealed a set of genes coordinately regulated for osteoclast function, some of which have previously been implicated in several bone diseases in humans.

  20. Gene expression profiling related to powdery mildew resistance in wheat with the method of suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    "Bainong 3217×Mardler" BC5F4 wheat line at the initial stage of inoculation with powdery mildew pathogen (Erysiphe graminis DC) was used to construct a suppression subtractive hybridization (SSH) cDNA library. Totally 760 ESTs were obtained through sequencing. Similarity analysis of ESTs based on BLASTn and BLASTx with the sequences in GenBank, in combination with macroarray differential screening, revealed that 199 ESTs of 65 kinds were known to be functionally disease resistance related. Based on the gene expression profiling in the present study, it is postulated that salicylic acid (SA) and MAP-related signal transduction pathways were involved in powdery mildew resistance in wheat. System acquired resistance genes were predominant in terms of kinds and quantity. With the initiation of cell defense reaction, the genes conferring anti-oxida- tion substances were largely expressed and thus cell protection mechanism was activated. Much evidence revealed that phenylpropanes metabolic pathway was involved in phytoalexin synthesis in wheat powdery mildew resistance. Genes conferring some enzymes of structural modification of cell walls and proteinase inhibitors inhibiting pathogen growth were also detected. The genes controlling a few proteinases (mainly cysteine proteinase) had a considerable redundancy of expression.

  1. Identification of differentially expressed genes in pistils from self-incompatible Citrus reticulata by suppression subtractive hybridization.

    Science.gov (United States)

    Miao, Hongxia; Qin, Yonghua; da Silva, Jaime A Teixeira; Ye, Zixing; Hu, Guibing

    2013-01-01

    Self-incompatibility (SI) is one important factor that can result in Citrus seedlessness. However, the molecular mechanism of SI in Citrus is not clear yet. To isolate the pistil's SI-related genes, a suppression subtractive hybridization library was constructed using mature pistils of 'Wuzishatangju' mandarin (SI) as the tester and mature pistils of 'Shatangju' mandarin (self-compatibility, SC) as the driver. 229 differentially expressed cDNA clones from 967 positive clones were sequenced and identified. Differentially expressed ESTs are possibly involved in the SI reaction of 'Wuzishatangju' through a regulating signaling pathway, serine/threonine phosphatase activity, receptor kinase, embryonic development, gibberellin stimulus, or transcription. 11 out of 36 SI candidate genes displayed different expression patterns in various tissues and stages after self- and cross-pollination of 'Wuzishatangju'. The expression of CaBP (WY65), a senescence-protease (WY372), an unknown gene (WY283), and a WRKY (WY17) were up-regulated in the styles of 'Wuzishatangju' while higher expression of WY190 was observed in styles of 'Shatangju'. Highest expression levels of WY65, WY372, an annexin (WY598), the zinc-finger protein (WY376), a C2-protein (WY291), and an unknown gene (WY318) were detected in styles at 3 days after self-pollination of 'Wuzishatangju' while lowest levels were observed in styles at 3 days after cross-pollination of 'Wuzishatangju' × 'Shatangju'. The potential involvement of these genes in the SI reaction is discussed.

  2. Identification of genes differentially expressed by Metarhizium anisopliae growing on Locusta migratoria wings using suppression subtractive hybridization.

    Science.gov (United States)

    Zhang, Chuanbo; Xia, Yuxian; Li, Zhongyuan

    2011-05-01

    Insect-pathogenic fungi penetrate their hosts directly through the cuticle. To better understand this process, we identified genes that were up-regulated by Metarhizium anisopliae germinating and differentiating on Locusta migratoria wings using suppression subtractive hybridization (SSH). A total of 78 unique expressed sequence tags (ESTs) up-regulated more than twofold during fungal growth on locust wings were identified. Among these 78 ESTs, 30 (38.5%) shared significant similarity with NCBI annotated hypothetical proteins, 16 (20.5%) shared low similarity to known or predicted genes, might represent novel genes, and 32 (41.0%) shared significant similarity with known proteins that are involved in various cell and molecular processes such as cell metabolism, protein metabolism, stress response and defense, and cell structure and function. Semi-quantitative RT-PCR analysis of six randomly selected genes confirmed the SSH results, verifying the fidelity of the SSH data. The results of this study provide novel information on genes expressed during early stages of infection with M. anisopliae, and improve current understanding of fungal pathogenesis.

  3. Identification and Cloning of Differentially Expressed SOUL and ELIP Genes in Saffron Stigmas Using a Subtractive Hybridization Approach

    Science.gov (United States)

    Ahrazem, Oussama; Argandoña, Javier; Castillo, Raquel; Rubio-Moraga, Ángela

    2016-01-01

    Using a subtractive hybridization approach, differentially expressed genes involved in the light response in saffron stigmas were identified. Twenty-two differentially expressed transcript-derived fragments were cloned and sequenced. Two of them were highly induced by light and had sequence similarity to early inducible proteins (ELIP) and SOUL heme-binding proteins. Using these sequences, we searched for other family members expressed in saffron stigma. ELIP and SOUL are represented by small gene families in saffron, with four and five members, respectively. The expression of these genes was analyzed during the development of the stigma and in light and dark conditions. ELIP transcripts were detected in all the developmental stages showing much higher expression levels in the developed stigmas of saffron and all were up-regulated by light but at different levels. By contrast, only one SOUL gene was up-regulated by light and was highly expressed in the stigma at anthesis. Both the ELIP and SOUL genes induced by light in saffron stigmas might be associated with the structural changes affecting the chromoplast of the stigma, as a result of light exposure, which promotes the development and increases the number of plastoglobules, specialized in the recruitment of specific proteins, which enables them to act in metabolite synthesis and disposal under changing environmental conditions and developmental stages. PMID:28030614

  4. p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing.

    Directory of Open Access Journals (Sweden)

    Frank Herrmann

    Full Text Available The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs. We adapted a commercially-available yeast one-hybrid (Y1H selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation 'hotspots'. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci.

  5. p53 Gene Repair with Zinc Finger Nucleases Optimised by Yeast 1-Hybrid and Validated by Solexa Sequencing

    Science.gov (United States)

    Herrmann, Frank; Garriga-Canut, Mireia; Baumstark, Rebecca; Fajardo-Sanchez, Emmanuel; Cotterell, James; Minoche, André; Himmelbauer, Heinz; Isalan, Mark

    2011-01-01

    The tumor suppressor gene p53 is mutated or deleted in over 50% of human tumors. As functional p53 plays a pivotal role in protecting against cancer development, several strategies for restoring wild-type (wt) p53 function have been investigated. In this study, we applied an approach using gene repair with zinc finger nucleases (ZFNs). We adapted a commercially-available yeast one-hybrid (Y1H) selection kit to allow rapid building and optimization of 4-finger constructs from randomized PCR libraries. We thus generated novel functional zinc finger nucleases against two DNA sites in the human p53 gene, near cancer mutation ‘hotspots’. The ZFNs were first validated using in vitro cleavage assays and in vivo episomal gene repair assays in HEK293T cells. Subsequently, the ZFNs were used to restore wt-p53 status in the SF268 human cancer cell line, via ZFN-induced homologous recombination. The frequency of gene repair and mutation by non-homologous end-joining was then ascertained in several cancer cell lines, using a deep sequencing strategy. Our Y1H system facilitates the generation and optimisation of novel, sequence-specific four- to six-finger peptides, and the p53-specific ZFN described here can be used to mutate or repair p53 in genomic loci. PMID:21695267

  6. A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes.

    Science.gov (United States)

    Maxwell, E S; Sarge, K D

    1988-11-30

    We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

  7. From amplification to gene in thyroid cancer: A high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X.N.; Gonsky, R.; Korenberg, J.R. [UCLA School of Medicine, Los Angeles, CA (United States). Cedars-Sinai Research Inst.; Knauf, J.A.; Fagin, J.A. [Univ. of Cincinnati, OH (United States). Div. of Endocrinology/Metabolism; Wang, M.; Lai, E.H. [Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Pharmacology; Chissoe, S. [Washington Univ. School of Medicine, St. Louis, MO (United States). Genome Sequencing

    1998-08-01

    Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. The authors now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. They used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3--6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKC{epsilon}), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKC{epsilon} as a previously unmapped candidate gene involved in thyroid tumorigenesis.

  8. Adaptive traits are maintained on steep selective gradients despite gene flow and hybridization in the intertidal zone.

    Directory of Open Access Journals (Sweden)

    Gerardo I Zardi

    Full Text Available Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency. Congeneric, externally-fertilizing fucoid brown algae occur as distinct morphotypes along intertidal exposure gradients despite gene flow. Combining analyses of genetic and phenotypic traits, we investigate the potential for physiological resilience to emersion stressors to act as an isolating mechanism in the face of gene flow. Along vertical exposure gradients in the intertidal zone of Northern Portugal and Northwest France, the mid-low shore species Fucus vesiculosus, the upper shore species Fucus spiralis, and an intermediate distinctive morphotype of F. spiralis var. platycarpus were morphologically characterized. Two diagnostic microsatellite loci recovered 3 genetic clusters consistent with prior morphological assignment. Phylogenetic analysis based on single nucleotide polymorphisms in 14 protein coding regions unambiguously resolved 3 clades; sympatric F. vesiculosus, F. spiralis, and the allopatric (in southern Iberia population of F. spiralis var. platycarpus. In contrast, the sympatric F. spiralis var. platycarpus (from Northern Portugal was distributed across the 3 clades, strongly suggesting hybridization/introgression with both other entities. Common garden experiments showed that physiological resilience following exposure to desiccation/heat stress differed significantly between the 3 sympatric genetic taxa; consistent with their respective vertical distribution on steep environmental clines in exposure time. Phylogenetic analyses indicate that F. spiralis var. platycarpus is a distinct entity in allopatry, but that extensive gene flow occurs with both higher and lower shore species in sympatry. Experimental results suggest that strong selection on physiological traits across steep intertidal exposure gradients

  9. Adaptive Traits Are Maintained on Steep Selective Gradients despite Gene Flow and Hybridization in the Intertidal Zone

    Science.gov (United States)

    Canovas, Fernando; Ferreira Costa, Joana; Serrão, Ester A.; Pearson, Gareth A.

    2011-01-01

    Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency. Congeneric, externally-fertilizing fucoid brown algae occur as distinct morphotypes along intertidal exposure gradients despite gene flow. Combining analyses of genetic and phenotypic traits, we investigate the potential for physiological resilience to emersion stressors to act as an isolating mechanism in the face of gene flow. Along vertical exposure gradients in the intertidal zone of Northern Portugal and Northwest France, the mid-low shore species Fucus vesiculosus, the upper shore species Fucus spiralis, and an intermediate distinctive morphotype of F. spiralis var. platycarpus were morphologically characterized. Two diagnostic microsatellite loci recovered 3 genetic clusters consistent with prior morphological assignment. Phylogenetic analysis based on single nucleotide polymorphisms in 14 protein coding regions unambiguously resolved 3 clades; sympatric F. vesiculosus, F. spiralis, and the allopatric (in southern Iberia) population of F. spiralis var. platycarpus. In contrast, the sympatric F. spiralis var. platycarpus (from Northern Portugal) was distributed across the 3 clades, strongly suggesting hybridization/introgression with both other entities. Common garden experiments showed that physiological resilience following exposure to desiccation/heat stress differed significantly between the 3 sympatric genetic taxa; consistent with their respective vertical distribution on steep environmental clines in exposure time. Phylogenetic analyses indicate that F. spiralis var. platycarpus is a distinct entity in allopatry, but that extensive gene flow occurs with both higher and lower shore species in sympatry. Experimental results suggest that strong selection on physiological traits across steep intertidal exposure gradients acts to maintain

  10. Adaptive traits are maintained on steep selective gradients despite gene flow and hybridization in the intertidal zone.

    Science.gov (United States)

    Zardi, Gerardo I; Nicastro, Katy R; Canovas, Fernando; Costa, Joana Ferreira; Serrão, Ester A; Pearson, Gareth A

    2011-01-01

    Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency. Congeneric, externally-fertilizing fucoid brown algae occur as distinct morphotypes along intertidal exposure gradients despite gene flow. Combining analyses of genetic and phenotypic traits, we investigate the potential for physiological resilience to emersion stressors to act as an isolating mechanism in the face of gene flow. Along vertical exposure gradients in the intertidal zone of Northern Portugal and Northwest France, the mid-low shore species Fucus vesiculosus, the upper shore species Fucus spiralis, and an intermediate distinctive morphotype of F. spiralis var. platycarpus were morphologically characterized. Two diagnostic microsatellite loci recovered 3 genetic clusters consistent with prior morphological assignment. Phylogenetic analysis based on single nucleotide polymorphisms in 14 protein coding regions unambiguously resolved 3 clades; sympatric F. vesiculosus, F. spiralis, and the allopatric (in southern Iberia) population of F. spiralis var. platycarpus. In contrast, the sympatric F. spiralis var. platycarpus (from Northern Portugal) was distributed across the 3 clades, strongly suggesting hybridization/introgression with both other entities. Common garden experiments showed that physiological resilience following exposure to desiccation/heat stress differed significantly between the 3 sympatric genetic taxa; consistent with their respective vertical distribution on steep environmental clines in exposure time. Phylogenetic analyses indicate that F. spiralis var. platycarpus is a distinct entity in allopatry, but that extensive gene flow occurs with both higher and lower shore species in sympatry. Experimental results suggest that strong selection on physiological traits across steep intertidal exposure gradients acts to maintain

  11. Screening of genes related to ovarian development in the swimming crab, Portunus trituberculatus, by suppression subtractive hybridization.

    Science.gov (United States)

    Yu, Z B; Mu, C K; Song, W W; Li, R H; Chen, Y E; Wang, C L

    2015-12-29

    The swimming crab, Portunus trituberculatus, is an important marine animal and is widely cultured in China. In the present study, suppression subtractive hybridization was applied to identify the differentially expressed genes in the ovaries of mature and immature P. trituberculatus. One hundred and seventy six expressed sequence tag (ESTs) were identified, of which 100 were down-regulated, and 76 up-regulated. BLAST analysis identified 51 unigenes, of which 27 were down-regulated, and 24 up-regulated. Quantitative real-time reverse transcriptase polymerase chain reaction results indicated that the SSH technique is valuable in screening genes related to ovarian development. Genes identified in this study encoded proteins corresponding to a wide range of functions and included immune response protein, transcription initiation factor, metabolic proteins, chromosome, histone h3, ovarian development-related protein, and vitellogenin. In addition, 64 metabolic pathways were annotated in differentially expressed ESTs by using the Kyoto Encyclopedia of Genes and Genomes pathway. Four annotated pathways (oxidative phosphorylation, carbon metabolism, fatty acid degradation, and protein digestion and absorption) appeared to be involved in ovarian development. In ontology analysis, 5.83% of the cellular process genes in reverse subtraction cDNA library are involved in reproduction, and 5.88% involved in developmental process. In up-regulated genes, myosin II-expressed polehole-like protein; histone h3; ovigerous-hair stripping substance; peritrophin 48; and ovarian development-related protein appeared to be involved in ovarian development. Identification of differentially expressed genes in the mature and immature ovary of the swimming crab provides new insights for further studies on the mechanism underlying ovarian development in this species.

  12. Exploring Mycobacterium tuberculosis infection-induced alterations in gene expression in macrophage by microarray hybridization

    Institute of Scientific and Technical Information of China (English)

    XIE; Jianping; (谢建平); LI; Yao; (李; 瑶); YUE; Jun; (乐; 军); XU; Yongzhong; (徐永忠); HUANG; Daqiang; (黄达蔷); LIANG; Li; (梁; 莉); WANG; Honghai; (王洪海)

    2003-01-01

    Tuberculosis remains a serious threat to public health. Its causative agent Mycobacte- rium tuberculosis is an intracellular pathogen which survives and replicates within cells of the host immune system, primarily macrophages. Knowledge of the bacteria-macrophage interaction can help to develop novel measures to combat the disease. The global gene expression of macro- phage following invasion by and growth of M. tuberculosis was studied by cDNA microarray. Of the 12800 human genes analyzed, totally 473 (3.7%) macrophage genes were differentially expressed after being infected by M. tuberculosis, among which, only 25 (5.2%, corresponding to less than 0.2% of the 12800 genes) genes were up-regulated, while others (94.8%) were down-regulated against the control. Of the 473 genes, 376 genes are registered in the GenBank, and 97 are novel genes. Expression of 5 up-regulated genes has been induced by more than 3-fold. 25 genes were down-regulated by more than 3-fold. Syndecan binding protein has been down-regu- lated up to 12.5-fold. The data gave an insight into the early gene expression in macrophage ensuing M. tuberculosis infection and a basis for further study.

  13. Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain

    Directory of Open Access Journals (Sweden)

    Hauptmann Giselbert

    2011-04-01

    Full Text Available Abstract Background In recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization. Despite the benefits of this technique, visualization of overlapping transcript distributions by differently colored precipitates remains difficult because of masking of lighter signals by darker color precipitates and lack of three-dimensional visualization properties. Fluorescent detection of transcript distributions may be able to solve these issues. However, despite the use of signal amplification systems for increasing sensitivity, fluorescent detection in whole-mounts suffers from rapid quenching of peroxidase (POD activity compared to alkaline phosphatase chromogenic reactions. Thus, less strongly expressed genes cannot be efficiently detected. Results We developed an optimized procedure for fluorescent detection of transcript distribution in whole-mount zebrafish embryos using tyramide signal amplification (TSA. Conditions for hybridization and POD-TSA reaction were optimized by the application of the viscosity-increasing polymer dextran sulfate and the use of the substituted phenol compounds 4-iodophenol and vanillin as enhancers of POD activity. In combination with highly effective bench-made tyramide substrates, these improvements resulted in dramatically increased signal-to-noise ratios. The strongly enhanced signal intensities permitted fluorescent visualization of less abundant transcripts of tissue-specific regulatory genes. When performing multicolor fluorescent in situ hybridization (FISH experiments, the highly sensitive POD reaction conditions required effective POD inactivation after each detection cycle by glycine-hydrochloric acid treatment. This optimized FISH procedure permitted the simultaneous fluorescent visualization of up to three unique transcripts

  14. Novel RNA hybridization method for the in situ detection of ETV1, ETV4, and ETV5 gene fusions in prostate cancer.

    Science.gov (United States)

    Kunju, Lakshmi P; Carskadon, Shannon; Siddiqui, Javed; Tomlins, Scott A; Chinnaiyan, Arul M; Palanisamy, Nallasivam

    2014-09-01

    The genetic basis of 50% to 60% of prostate cancer (PCa) is attributable to rearrangements in E26 transformation-specific (ETS) (ERG, ETV1, ETV4, and ETV5), BRAF, and RAF1 genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of PCa for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERG rearrangement status by fluorescence in situ hybridization. However, specific antibodies for ETV1, ETV4, and ETV5 are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization-based assay for the in situ detection of ETV1, ETV4, and ETV5 in formalin-fixed paraffin-embedded tissues from prostate needle biopsies, prostatectomy, and metastatic PCa specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of PCa with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright-field in situ detection of ETS gene aberrations in routine clinically available PCa specimens.

  15. Identification of genes involved in the response of haemocytes of Penaeus japonicus by suppression subtractive hybridization (SSH) following microbial challenge.

    Science.gov (United States)

    He, Nanhai; Liu, Haipeng; Xu, Xun

    2004-08-01

    Penaeus japonicus were injected with a heat-killed microorganism suspension and 291 randomly selected cDNA fragments generated by suppression subtractive hybridization (SSH) were sequenced. A total of 71 cDNA clones corresponding to 25 genes were found to have enhanced expression, of which eight are found for the first time in shrimp. The most abundant gene in the subtractive library was Kunitz-type protease inhibitor, clearly indicating this protease inhibitor in the response. A number of genes encoding signaling molecules, such as Ras-related nuclear protein (Ran), growth factor receptor bound protein (Grb), TGF-beta receptor interacting protein, integrin binding protein and interferon receptor bound protein were found for the first time in the shrimp, and they may be involved in the regulation of the host defense against the injected microbes. Furthermore, cDNAs of chaperonin, proteasome, antioxidant as well as genes associated with actin reorganization, which may be necessary for phagocytosis and encapsulation, were also expressed at a higher level after the challenge. These results may facilitate the understanding of shrimp immune responses.

  16. Suppression subtraction hybridization (SSH) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus.

    Science.gov (United States)

    Dios, S; Poisa-Beiro, L; Figueras, A; Novoa, B

    2007-03-01

    Despite of the impact that viruses have on aquatic organisms, relatively little is known on how fish fight against these infections. In this work, the brain gene expression pattern of sea bream (Sparus aurata) in response to nodavirus infection was investigated. We used the suppression subtractive hybridization (SSH) method to generate a subtracted cDNA library enriched with gene transcripts differentially expressed after 1 day post-infection. Some of the ESTs from the infected tissues fell in gene categories related to stress and immune responses. For the reverse library (ESTs expressed in controls compared with infected tissues) the most abundant transcripts were of ribosomal and mitochondrial nature. Several ESTs potentially induced by virus exposure were selected for in vivo expression studies. We observed a clear difference in expression between infected and control samples for two candidate genes, ubiquitin conjugating enzyme 7 interacting protein, which seems to play an important role in apoptosis and the interferon induced protein with helicase C domain 1 (mda-5) that contributes to apoptosis and regulates the type I IFN production, a key molecule of the antiviral innate response in most organisms.

  17. Differentially-expressed genes identified by suppression subtractive hybridization in the bone marrow hematopoietic stem cells of patients with psoriasis.

    Science.gov (United States)

    Zhang, Zhenying; Yu, Zhen; Tian, Pan; Hou, Suchun; Han, Shixin; Tan, Xuejing; Piao, Yongjun; Liu, Xiaoming

    2014-07-01

    Psoriasis is a T cell-mediated, chronic, relapsing and inflammatory cutaneous disorder. The dysfunctional activity of T cells in patients with psoriasis is attributed to bone marrow hematopoietic stem cells (BMHSCs). To understand the pathogenic roles of BMHSCs in psoriasis, a differential gene expression analysis was performed using suppression subtractive hybridization of the BMHSCs from a patient with psoriasis and a healthy control. Using a cDNA array dot blot screening to screen 600 genes from forward- and reverse-subtracted cDNA libraries, 17 differentially-expressed sequence tags (ESTs) were identified. The genes within the ESTs were observed to be the homologs of genes that are involved in various cellular processes, including hormone signaling, RNA catabolism, protein ADP DNA base melting, transcriptional regulation, cell cycle regulation and metabolism. CD45, which was overexpressed in the psoriatic BMHSCs, was further analyzed using relative quantitative polymerase chain reaction. In addition, the levels of CD45 in the peripheral blood cells (PBCs) of the patients with psoriasis were markedly increased and closely associated with disease severity. An abnormality of hematopoietic progenitor cells, e.g., CD45 overexpression, may be transferred to PBCs via hematopoiesis, and may account for the psoriasis-inducing properties of activated T cells.

  18. An extensive candidate gene approach to speciation: diversity, divergence and linkage disequilibrium in candidate pigmentation genes across the European crow hybrid zone.

    Science.gov (United States)

    Poelstra, J W; Ellegren, H; Wolf, J B W

    2013-12-01

    Colouration patterns have an important role in adaptation and speciation. The European crow system, in which all-black carrion crows and grey-coated hooded crows meet in a narrow hybrid zone, is a prominent example. The marked phenotypic difference is maintained by assortative mating in the absence of neutral genetic divergence, suggesting the presence of few pigmentation genes of major effect. We made use of the rich phenotypic and genetic resources in mammals and identified a comprehensive panel of 95 candidate pigmentation genes for birds. Based on functional annotation, we chose a subset of the most promising 37 candidates, for which we developed a marker system that demonstrably works across the avian phylogeny. In total, we sequenced 107 amplicons (∼3 loci per gene, totalling 60 kb) in population samples of crows (n=23 for each taxon). Tajima's D, Fu's FS, DHEW and HKA (Hudson-Kreitman-Aguade) statistics revealed several amplicons that deviated from neutrality; however, none of these showed significantly elevated differentiation between the two taxa. Hence, colour divergence in this system may be mediated by uncharacterized pigmentation genes or regulatory regions outside genes. Alternatively, the observed high population recombination rate (4Ner∼0.03), with overall linkage disequilibrium dropping rapidly within the order of few 100 bp, may compromise the power to detect causal loci with nearby markers. Our results add to the debate as to the utility of candidate gene approaches in relation to genomic features and the genetic architecture of the phenotypic trait in question.

  19. An interspecific fungal hybrid reveals cross-kingdom rules for allopolyploid gene expression patterns.

    Directory of Open Access Journals (Sweden)

    Murray P Cox

    2014-03-01

    Full Text Available Polyploidy, a state in which the chromosome complement has undergone an increase, is a major force in evolution. Understanding the consequences of polyploidy has received much attention, and allopolyploids, which result from the union of two different parental genomes, are of particular interest because they must overcome a suite of biological responses to this merger, known as "genome shock." A key question is what happens to gene expression of the two gene copies following allopolyploidization, but until recently the tools to answer this question on a genome-wide basis were lacking. Here we utilize high throughput transcriptome sequencing to produce the first genome-wide picture of gene expression response to allopolyploidy in fungi. A novel pipeline for assigning sequence reads to the gene copies was used to quantify their expression in a fungal allopolyploid. We find that the transcriptional response to allopolyploidy is predominantly conservative: both copies of most genes are retained; over half the genes inherit parental gene expression patterns; and parental differential expression is often lost in the allopolyploid. Strikingly, the patterns of gene expression change are highly concordant with the genome-wide expression results of a cotton allopolyploid. The very different nature of these two allopolyploids implies a conserved, eukaryote-wide transcriptional response to genome merger. We provide evidence that the transcriptional responses we observe are mostly driven by intrinsic differences between the regulatory systems in the parent species, and from this propose a mechanistic model in which the cross-kingdom conservation in transcriptional response reflects conservation of the mutational processes underlying eukaryotic gene regulatory evolution. This work provides a platform to develop a universal understanding of gene expression response to allopolyploidy and suggests that allopolyploids are an exceptional system to investigate gene

  20. Fine mapping of a gene causing hybrid pollen sterility between Yunnan weedy rice and cultivated rice (Oryza sativa L.) and phylogenetic analysis of Yunnan weedy rice.

    Science.gov (United States)

    Wang, Yong; Zhong, Zheng Zheng; Zhao, Zhi Gang; Jiang, Ling; Bian, Xiao Feng; Zhang, Wen Wei; Liu, Ling Long; Ikehashi, H; Wan, Jian Min

    2010-02-01

    Weedy rice represents an important resource for rice improvement. The F(1) hybrid between the japonica wide compatibility rice cultivar 02428 and a weedy rice accession from Yunnan province (SW China) suffered from pollen sterility. Pollen abortion in the hybrid occurred at the early bicellular pollen stage, as a result of mitotic failure in the microspore, although the tapetum developed normally. Genetic mapping in a BC(1)F(1) population (02428//Yunnan weedy rice (YWR)/02428) showed that a major QTL for hybrid pollen sterility (qPS-1) was present on chromosome 1. qPS-1 was fine-mapped to a 110 kb region known to contain the hybrid pollen sterility gene Sa, making it likely that qPS-1 is either identical to, or allelic with Sa. Interestingly, F(1) hybrid indicated that Dular and IR36 were assumed to carry the sterility-neutral allele, Sa ( n ). Re-sequencing SaM and SaF, the two component genes present at Sa, suggested that variation for IR36 and Dular may be responsible for the loss of male sterility, and the qPS-1 sequence might be derived from wild rice or indica cultivars. A phylogenetic analysis based on microsatellite genotyping suggested that the YWR accession is more closely related to wild rice and indica type cultivars than to japonica types. Thus it is probable that the YWR accession evolved from a spontaneous hybrid between wild rice and an ancient cultivated strain of domesticated rice.

  1. Astrocyte Cultures Mimicking Brain Astrocytes in Gene Expression, Signaling, Metabolism and K(+) Uptake and Showing Astrocytic Gene Expression Overlooked by Immunohistochemistry and In Situ Hybridization.

    Science.gov (United States)

    Hertz, Leif; Chen, Ye; Song, Dan

    2017-01-01

    Based on differences in gene expression between cultured astrocytes and freshly isolated brain astrocytes it has been claimed that cultured astrocytes poorly reflect the characteristics of their in vivo counterparts. This paper shows that this is not the case with the cultures of mouse astrocytes we have used since 1978. The culture is prepared following guidelines provided by Drs. Monique Sensenbrenner and John Booher, with the difference that dibutyryl cyclic AMP is added to the culture medium from the beginning of the third week. This addition has only minor effects on glucose and glutamate metabolism, but it is crucial for effects by elevated K(+) concentrations and for Ca(2+) homeostasis, important aspects of astrocyte function. Work by Liang Peng and her colleagues has shown identity between not only gene expression but also drug-induced gene upregulations and editings in astrocytes cultured by this method and astrocytes freshly isolated from brains of drug-treated animals. Dr. Norenberg's laboratory has demonstrated identical upregulation of the cotransporter NKCC1 in ammonia-exposed astrocytes and rats with liver failure. Similarity between cultured and freshly isolated astrocytes has also been shown in metabolism, K(+) uptake and several aspects of signaling. However, others have shown that the gene for the glutamate transporter GLT1 is not expressed, and rat cultures show some abnormalities in K(+) effects. Nevertheless, the overall reliability of the cultured cells is important because immunohistochemistry and in situ hybridization poorly demonstrate many astrocytic genes, e.g., those of nucleoside transporters, and even microarray analysis of isolated cells can be misleading.

  2. [Gene expression profiling by suppression subtractive hybridization (SSH): a example for its application to the study of lymphomas].

    Science.gov (United States)

    Villalva, C; Trempat, P; Zenou, R C; Delsol, G; Brousset, P

    2001-03-01

    Suppression subtractive hybridization (SSH) was used to isolate genes that were differentially expressed in anaplastic lymphoma kinase (ALK)-positive and ALK-negative anaplastic large cell lymphoma. In addition, this approach was applied to Hodgkin's disease cases with different clinical outcomes. SSH combines a normalization step that equalizes the abundance of cDNAs within the sequences to be tested and a subtraction step that excludes the common sequences between the target and the control. In a model system, the SSH technique enriches for rare sequences up to 5,000-fold in one round. We have isolated several genes whose expression varied significantly with regard to the tumour subtypes. There were different genes with known or unknown functions. We aim to compare the results of the SSH approach with those obtained with high density filters. In a near future, we would like to design DNA chips specific of each pathology that could be used for clinical purposes (evaluation of prognosis and therapeutic response).

  3. Isolation and Expression Analysis of Novel Silicon Absorption Gene from Roots of Mangrove (Rhizophora apiculata via Suppression Subtractive Hybridization

    Directory of Open Access Journals (Sweden)

    Mahbod Sahebi

    2014-01-01

    Full Text Available Silicon (Si is the second most abundant element in soil after oxygen. It is not an essential element for plant growth and formation but plays an important role in increasing plant tolerance towards different kinds of abiotic and biotic stresses. The molecular mechanism of Si absorption and accumulation may differ between plants, such as monocotyledons and dicotyledons. Silicon absorption and accumulation in mangrove plants are affected indirectly by some proteins rich in serine and proline amino acids. The expression level of the genes responsible for Si absorption varies in different parts of plants. In this study, Si is mainly observed in the epidermal roots’ cell walls of mangrove plants compared to other parts. The present work was carried out to discover further information on Si stress responsive genes in Rhizophora apiculata, using the suppression subtractive hybridization technique. To construct the cDNA library, two-month-old seedlings were exposed to 0.5, 1, and 1.5 mM SiO2 for 15 hrs and for 1 to 6 days resulting in a total of 360 high quality ESTs gained. Further examination by RT-PCR and real-time qRT-PCR showed the expression of a candidate gene of serine-rich protein.

  4. Isolation and expression analysis of novel silicon absorption gene from roots of mangrove (Rhizophora apiculata) via suppression subtractive hybridization.

    Science.gov (United States)

    Sahebi, Mahbod; Hanafi, Mohamed M; Abdullah, Siti Nor Akmar; Rafii, Mohd Y; Azizi, Parisa; Nejat, Naghmeh; Idris, Abu Seman

    2014-01-01

    Silicon (Si) is the second most abundant element in soil after oxygen. It is not an essential element for plant growth and formation but plays an important role in increasing plant tolerance towards different kinds of abiotic and biotic stresses. The molecular mechanism of Si absorption and accumulation may differ between plants, such as monocotyledons and dicotyledons. Silicon absorption and accumulation in mangrove plants are affected indirectly by some proteins rich in serine and proline amino acids. The expression level of the genes responsible for Si absorption varies in different parts of plants. In this study, Si is mainly observed in the epidermal roots' cell walls of mangrove plants compared to other parts. The present work was carried out to discover further information on Si stress responsive genes in Rhizophora apiculata, using the suppression subtractive hybridization technique. To construct the cDNA library, two-month-old seedlings were exposed to 0.5, 1, and 1.5 mM SiO2 for 15 hrs and for 1 to 6 days resulting in a total of 360 high quality ESTs gained. Further examination by RT-PCR and real-time qRT-PCR showed the expression of a candidate gene of serine-rich protein.

  5. Identification of genes escaping X inactivation by allelic expression analysis in a novel hybrid mouse model.

    Science.gov (United States)

    Berletch, Joel B; Ma, Wenxiu; Yang, Fan; Shendure, Jay; Noble, William S; Disteche, Christine M; Deng, Xinxian

    2015-12-01

    X chromosome inactivation (XCI) is a female-specific mechanism that serves to balance gene dosage between the sexes whereby one X chromosome in females is inactivated during early development. Despite this silencing, a small portion of genes escape inactivation and remain expressed from the inactive X (Xi). Little is known about the distribution of escape from XCI in different tissues in vivo and about the mechanisms that control tissue-specific differences. Using a new binomial model in conjunction with a mouse model with identifiable alleles and skewed X inactivation we are able to survey genes that escape XCI in vivo. We show that escape from X inactivation can be a common feature of some genes, whereas others escape in a tissue specific manner. Furthermore, we characterize the chromatin environment of escape genes and show that expression from the Xi correlates with factors associated with open chromatin and that CTCF co-localizes with escape genes. Here, we provide a detailed description of the experimental design and data analysis pipeline we used to assay allele-specific expression and epigenetic characteristics of genes escaping X inactivation. The data is publicly available through the GEO database under ascension numbers GSM1014171, GSE44255, and GSE59779. Interpretation and discussion of these data are included in a previously published study (Berletch et al., 2015) [1].

  6. Isolation of novel non-HLA gene fragments from the hemochromatosis region (6p21. 3) by cDNA hybridization selection

    Energy Technology Data Exchange (ETDEWEB)

    Goei, V.L.; Capossela, A.; Gruen, J.R.; Parimoo, S.; Chu, T.W. (Yale Univ. School of Medicine, New Haven, CT (United States))

    1994-02-01

    It has previously been shown that cDNA hybridization selection can identify and recover novel genes from large cloned genomic DNA such as cosmids or YACs. In an effort to identify candidate genes for hemochromatosis, this technique was applied to a 320-kb YAC containing the HLA-A gene. A short fragment cDNA library derived from human duodenum was selected with the YAC DNA. Ten novel gene fragments were isolated, characterized, and localized on the physical map of the YAC. 39 refs., 4 figs., 3 tabs.

  7. Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus.

    Science.gov (United States)

    McCarthy, S A; DePaola, A; Kaysner, C A; Hill, W E; Cook, D W

    2000-12-01

    Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene. The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V. parahaemolyticus. The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V. parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources. The probes were specific for detection of the V. parahaemolyticus tdh gene.

  8. Hybrid models identified a 12-gene signature for lung cancer prognosis and chemoresponse prediction.

    Directory of Open Access Journals (Sweden)

    Ying-Wooi Wan

    Full Text Available Lung cancer remains the leading cause of cancer-related deaths worldwide. The recurrence rate ranges from 35-50% among early stage non-small cell lung cancer patients. To date, there is no fully-validated and clinically applied prognostic gene signature for personalized treatment.From genome-wide mRNA expression profiles generated on 256 lung adenocarcinoma patients, a 12-gene signature was identified using combinatorial gene selection methods, and a risk score algorithm was developed with Naïve Bayes. The 12-gene model generates significant patient stratification in the training cohort HLM & UM (n = 256; log-rank P = 6.96e-7 and two independent validation sets, MSK (n = 104; log-rank P = 9.88e-4 and DFCI (n = 82; log-rank P = 2.57e-4, using Kaplan-Meier analyses. This gene signature also stratifies stage I and IB lung adenocarcinoma patients into two distinct survival groups (log-rank P<0.04. The 12-gene risk score is more significant (hazard ratio = 4.19, 95% CI: [2.08, 8.46] than other commonly used clinical factors except tumor stage (III vs. I in multivariate Cox analyses. The 12-gene model is more accurate than previously published lung cancer gene signatures on the same datasets. Furthermore, this signature accurately predicts chemoresistance/chemosensitivity to Cisplatin, Carboplatin, Paclitaxel, Etoposide, Erlotinib, and Gefitinib in NCI-60 cancer cell lines (P<0.017. The identified 12 genes exhibit curated interactions with major lung cancer signaling hallmarks in functional pathway analysis. The expression patterns of the signature genes have been confirmed in RT-PCR analyses of independent tumor samples.The results demonstrate the clinical utility of the identified gene signature in prognostic categorization. With this 12-gene risk score algorithm, early stage patients at high risk for tumor recurrence could be identified for adjuvant chemotherapy; whereas stage I and II patients at low risk could be spared the toxic side effects of

  9. Chromosomal localization of silkworm (Bombyx mori) sericin gene 1 and chymotrypsin inhibitor 13 using fluorescence in situ hybridization

    Institute of Scientific and Technical Information of China (English)

    SONG FangZhou; CHANG PingAn; ZHANG PingBo; YI FaPing; MA YongPing; LU Cheng; Yutaka BANNO; Hiroshi FUJII

    2008-01-01

    The chromosomal locations of two single-copy genes, Ser-1 and C1-13, in silkworm (Bombyx mori)were detected at the molecular cytogenetics level by fluorescence in situ hybridization in the study. The resuits showed that Ser-1 is located near the distal end of the 11th linkage group, relatively st the 12.5±1.4position in pachytene; and that C1-13 has been mapped near the distal end of the 2nd linkage group,relatively at the 8.2±1.2 position in pachytene. Furthermore, their location model map-FISH map on silkworm chromosome was drawn. The FISH technique and its application to silkworm are also discussed in this paper.

  10. Chromosomal localization of silkworm (Bombyx mori) sericin gene 1 and chymotrypsin inhibitor 13 using fluorescence in situ hybridization

    Institute of Scientific and Technical Information of China (English)

    Yutaka; BANNO; Hiroshi; FUJII

    2008-01-01

    The chromosomal locations of two single-copy genes, Ser-1 and CI-13, in silkworm (Bombyx mori) were detected at the molecular cytogenetics level by fluorescence in situ hybridization in the study. The results showed that Ser-1 is located near the distal end of the 11th linkage group, relatively at the 12.5±1.4 position in pachytene; and that CI-13 has been mapped near the distal end of the 2nd linkage group, relatively at the 8.2±1.2 position in pachytene. Furthermore, their location model map-FISH map on silkworm chromosome was drawn. The FISH technique and its application to silkworm are also discussed in this paper.

  11. Inhibitory effect of pironetin analogue/colchicine hybrids on the expression of the VEGF, hTERT and c-Myc genes.

    Science.gov (United States)

    Vilanova, Concepción; Díaz-Oltra, Santiago; Murga, Juan; Falomir, Eva; Carda, Miguel; Marco, J Alberto

    2015-08-15

    A small group of hybrid molecules composed of a colchicine moiety and a pironetin analogue fragment have been investigated for their ability to inhibit the expression of the VEGF, hTERT and c-Myc genes. The VEGF gene is involved in the generation of the vascular endothelial growth factor (VEGF) and thus in the angiogenic process whereas the two latter ones are related to the activation of telomerase. All three genes therefore may be of paramount importance in the cancer generation process. It has been found that colchicine and some of its derivatives display a measurable ability to inhibit the expression of the VEGF and the two other telomerase-related genes. In the case of some of the hybrids, the available data point to the colchicine fragment being responsible for the observed biological activities. It is the first time that the last biological feature has been reported for colchicine or derivatives thereof. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Identification of Differentially Expressed Genes in Metastatic and Non-Metastatic Nasopharyngeal Carcinoma Cells by Suppression Subtractive Hybridization

    Directory of Open Access Journals (Sweden)

    Xu-Yu Yang

    2005-01-01

    Full Text Available Background & Objective: Nasopharyngeal carcinoma (NPC is an epithelial neoplasm with high occurrence rates in southern China. The disease often metastasizes to regional lymphnodes at a very early stage. Local recurrences and metastasis occur frequently in patients with NPC and are a leading cause of death, despite improvements on treatment modalities. The molecular mechanism underlying the metastasis of nasopharyngeal carcinoma remains poorly understood, however, and requires additional elucidation. The aim of this study was to explore possible NPC gene candidates that may play key roles in NPC metastasis. Methods: Subtractive suppression hybridization (SSH was performed to isolate differentially expressed clones between the metastatic 5-8F and non-metastatic 6-10B nasopharyngeal carcinoma cell lines. Differentially expressed clones were screened and confirmed by reverse Northern blotting. The sequences of cDNA fragments were subsequently analyzed and compared to known sequences in Genbank. Results & Discussion: The SSH library contained thousands of positive clones. Random analysis of 300 clones by PCR demonstrated that 269 clones contained inserted fragments. Reverse Northern blot confirmed that 20 out of 192 clones examined were significantly up-regulated in the 5-8F cell line. Among these 20 clones, 16 were previously identified genes (flotilin-2, ezrin, pim-3, fli-1, mel, neugrin, znf216, ASB1, raly, UBE2A, keratin6A, TMED7, EIF3S9, FTL, two ribosomal proteins RPL21 and RPL16, two were predicted genes (c9orf74 and MDS006, and two sequences shared no homology with known genes listed in GenBank and may represent novel genes. The proposed functions of the genes identified in this study include cell signal transduction, cell survival, transcription regulation, cell mobility, protein synthesis, and DNA damage repair. Flotillin-2, fli-1, pim-3 and ezrin have previously been reported to be associated with tumor metastasis and progression. The

  13. A Hybrid One-Way ANOVA Approach for the Robust and Efficient Estimation of Differential Gene Expression with Multiple Patterns.

    Directory of Open Access Journals (Sweden)

    Mohammad Manir Hossain Mollah

    Full Text Available Identifying genes that are differentially expressed (DE between two or more conditions with multiple patterns of expression is one of the primary objectives of gene expression data analysis. Several statistical approaches, including one-way analysis of variance (ANOVA, are used to identify DE genes. However, most of these methods provide misleading results for two or more conditions with multiple patterns of expression in the presence of outlying genes. In this paper, an attempt is made to develop a hybrid one-way ANOVA approach that unifies the robustness and efficiency of estimation using the minimum β-divergence method to overcome some problems that arise in the existing robust methods for both small- and large-sample cases with multiple patterns of expression.The proposed method relies on a β-weight function, which produces values between 0 and 1. The β-weight function with β = 0.2 is used as a measure of outlier detection. It assigns smaller weights (≥ 0 to outlying expressions and larger weights (≤ 1 to typical expressions. The distribution of the β-weights is used to calculate the cut-off point, which is compared to the observed β-weight of an expression to determine whether that gene expression is an outlier. This weight function plays a key role in unifying the robustness and efficiency of estimation in one-way ANOVA.Analyses of simulated gene expression profiles revealed that all eight methods (ANOVA, SAM, LIMMA, EBarrays, eLNN, KW, robust BetaEB and proposed perform almost identically for m = 2 conditions in the absence of outliers. However, the robust BetaEB method and the proposed method exhibited considerably better performance than the other six methods in the presence of outliers. In this case, the BetaEB method exhibited slightly better performance than the proposed method for the small-sample cases, but the the proposed method exhibited much better performance than the BetaEB method for both the small- and large

  14. Search for genes responsible for the remarkably high acetic acid tolerance of a Zygosaccharomyces bailii-derived interspecies hybrid strain.

    Science.gov (United States)

    Palma, Margarida; Roque, Filipa de Canaveira; Guerreiro, Joana Fernandes; Mira, Nuno Pereira; Queiroz, Lise; Sá-Correia, Isabel

    2015-12-16

    Zygosaccharomyces bailii is considered the most problematic acidic food spoilage yeast species due to its exceptional capacity to tolerate high concentrations of weak acids used as fungistatic preservatives at low pH. However, the mechanisms underlying its intrinsic remarkable tolerance to weak acids remain poorly understood. The identification of genes and mechanisms involved in Z. bailii acetic acid tolerance was on the focus of this study. For this, a genomic library from the highly acetic acid tolerant hybrid strain ISA1307, derived from Z. bailii and a closely related species and isolated from a sparkling wine production plant, was screened for acetic acid tolerance genes. This screen was based on the transformation of an acetic acid susceptible Saccharomyces cerevisiae mutant deleted for the gene encoding the acetic acid resistance determinant transcription factor Haa1. The expression of 31 different DNA inserts from ISA1307 strain genome was found to significantly increase the host cell tolerance to acetic acid. The in silico analysis of these inserts was facilitated by the recently available genome sequence of this strain. In total, 65 complete or truncated ORFs were identified as putative determinants of acetic acid tolerance and an S. cerevisiae gene homologous to most of them was found. These include genes involved in cellular transport and transport routes, protein fate, protein synthesis, amino acid metabolism and transcription. The role of strong candidates in Z. bailii and S. cerevisiae acetic acid tolerance was confirmed based on homologous and heterologous expression analyses. ISA1307 genes homologous to S. cerevisiae genes GYP8, WSC4, PMT1, KTR7, RKR1, TIF3, ILV3 and MSN4 are proposed as strong candidate determinants of acetic acid tolerance. The ORF ZBAI_02295 that contains a functional domain associated to the uncharacterised integral membrane proteins of unknown function of the DUP family is also suggested as a relevant tolerance determinant

  15. Molecular Breeding of Rice Restorer Lines and Hybrids for Brown Planthopper (BPH) Resistance Using the Bph14 and Bph15 Genes.

    Science.gov (United States)

    Wang, Hongbo; Ye, Shengtuo; Mou, Tongmin

    2016-12-01

    The development of hybrid rice is a practical approach for increasing rice production. However, the brown planthopper (BPH), Nilaparvata lugens Stål, causes severe yield loss of rice (Oryza sativa L.) and can threaten food security. Therefore, breeding hybrid rice resistant to BPH is the most effective and economical strategy to maintain high and stable production. Fortunately, numerous BPH resistance genes have been identified, and abundant linkage markers are available for molecular marker-assisted selection (MAS) in breeding programs. Hence, we pyramided two BPH resistance genes, Bph14 and Bph15, into a susceptive CMS restorer line Huahui938 and its derived hybrids using MAS to improve the BPH resistance of hybrid rice. Three near-isogenic lines (NILs) with pyramided Bph14 and Bph15 were obtained by molecular marker-assisted backcross (MAB) and phenotypic selection. The genomic components of these NILs were detected using the whole-genome SNP (Single nucleotide polymorphism) array, RICE6K, suggesting that the recurrent parent genome (RPG) recovery of the NILs was 87.88, 87.70 and 86.62 %, respectively. BPH bioassays showed that the improved NILs and their derived hybrids carrying homozygous Bph14 and Bph15 were resistant to BPH. However, the hybrids with heterozygous Bph14 and Bph15 remained susceptible to BPH. The developed NILs showed no significant differences in major agronomic traits and rice qualities compared with the recurrent parent. Moreover, the improved hybrids derived from the NILs exhibited better agronomic performance and rice quality compared with the controls under natural field conditions. This study demonstrates that it is essential to stack Bph14 and Bph15 into both the maternal and paternal parents for developing BPH-resistant hybrid rice varieties. The SNP array with abundant DNA markers is an efficient tool for analyzing the RPG recovery of progenies and can be used to monitor the donor segments in NILs, thus being extremely important

  16. A Hybrid SOM-SVM Approach for the Zebrafish Gene Expression Analysis

    Institute of Scientific and Technical Information of China (English)

    Wei Wu; Xin Liu; Min Xu; Jin-Rong Peng; Rudy Setiono

    2005-01-01

    Microarray technology can be employed to quantitatively measure the expression of thousands of genes in a single experiment. It has become one of the main tools for global gene expression analysis in molecular biology research in recent years. The large amount of expression data generated by this technology makes the study of certain complex biological problems possible, and machine learning methods are expected to play a crucial role in the analysis process. In this paper,we present our results from integrating the self-organizing map (SOM) and the support vector machine (SVM) for the analysis of the various functions of zebrafish genes based on their expression. The most distinctive characteristic of our zebrafish gene expression is that the number of samples of different classes is imbalanced. We discuss how SOM can be used as a data-filtering tool to improve the classification performance of the SVM on this data set.

  17. ANALYSIS OF SLG GENE – THE MOLECULAR MARKER IN HYBRID BREEDING OF OIL SEED RAPE

    Directory of Open Access Journals (Sweden)

    L DOLANSKÁ

    2004-07-01

    Full Text Available Oil seed rape (Brassica napus L. cultivars, donors of quality (SC and self-incompatible (SI lines have been analysed using identification of S-locus. In several Brassica napus cultivars one S-locus SLG gene was detected as dominant and the second S-locus as recessive. Amplification class II SLG gene screened recessive gene in all analysed samples (SC and SI. The DNA fragment of recessive gene corresponded to SLG gene W found in cv. Westar. S-haplotypes were analysed by PCR-RFLP. Different Brassica napus cultivars had an identical electrophoretic profile conforming with nonfunctional A10 allele in B. campestris. In B. napus A10 allele is localised in genome A. The functional recessive SLG gene is probably localised in genome C. Model of their segregation was suggested. SC and SI plants segregated in F2 generation at the ratio of approximately 3:1. This indicates a recessive monogenic disposition of SI in the experimental population.

  18. Suppression subtractive hybridization (SSH) for isolation and characterization of genes related to testicular development in the giant tiger shrimp Penaeus monodon.

    Science.gov (United States)

    Leelatanawit, Rungnapa; Klinbunga, Sirawut; Aoki, Takashi; Hirono, Ikuo; Valyasevi, Rudd; Menasveta, Piamsak

    2008-11-30

    Suppression subtractive hybridization (SSH) cDNA libraries of the giant tiger shrimp, Penaeus monodon, were constructed. In total, 178 and 187 clones from the forward and reverse SSH libraries, respectively, of P. monodon were unidirectionally sequenced. From these, 37.1% and 53.5% Expressed Sequence Tags (ESTs) significantly matched known genes (E-value 0.05).

  19. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH)

    NARCIS (Netherlands)

    Leveau, J.H.J.; Gerards, S.; De Boer, W.; Van Veen, J.A.

    2004-01-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia

  20. Assignment of the gene for human tetranectin (TNA) to chromosome 3p22-->p21.3 by somatic cell hybrid mapping

    DEFF Research Database (Denmark)

    Durkin, M E; Naylor, S L; Albrechtsen, R

    1997-01-01

    Tetranectin is a plasminogen-binding protein that is induced during the mineralization phase of osteogenesis. By screening a human chromosome 3 somatic cell hybrid mapping panel, we have localized the human tetranectin gene (TNA) to 3p22-->p21.3, which is distinct from the loci of two human conne...

  1. Evidence for introduction bottleneck and extensive inter-gene pool (Mesoamerica x Andes) hybridization in the European common bean (Phaseolus vulgaris L.) germplasm.

    Science.gov (United States)

    Gioia, Tania; Logozzo, Giuseppina; Attene, Giovanna; Bellucci, Elisa; Benedettelli, Stefano; Negri, Valeria; Papa, Roberto; Spagnoletti Zeuli, Pierluigi

    2013-01-01

    Common bean diversity within and between Mesoamerican and Andean gene pools was compared in 89 landraces from America and 256 landraces from Europe, to elucidate the effects of bottleneck of introduction and selection for adaptation during the expansion of common bean (Phaseolus vulgaris L.) in Europe. Thirteen highly polymorphic nuclear microsatellite markers (nuSSRs) were used to complement chloroplast microsatellite (cpSSRs) and nuclear markers (phaseolin and Pv-shatterproof1) data from previous studies. To verify the extent of the introduction bottleneck, inter-gene pool hybrids were distinguished from "pure" accessions. Hybrids were identified on the basis of recombination of gene pool specific cpSSR, phaseolin and Pv-shatterproof1 markers with a Bayesian assignments based on nuSSRs, and with STRUCTURE admixture analysis. More hybrids were detected than previously, and their frequency was almost four times larger in Europe (40.2%) than in America (12.3%). The genetic bottleneck following the introduction into Europe was not evidenced in the analysis including all the accessions, but it was significant when estimated only with "pure" accessions, and five times larger for Mesoamerican than for Andean germplasm. The extensive inter-gene pool hybridization generated a large amount of genotypic diversity that mitigated the effects of the bottleneck that occurred when common bean was introduced in Europe. The implication for evolution and the advantages for common bean breeding are discussed.

  2. Evidence for introduction bottleneck and extensive inter-gene pool (Mesoamerica x Andes hybridization in the European common bean (Phaseolus vulgaris L. germplasm.

    Directory of Open Access Journals (Sweden)

    Tania Gioia

    Full Text Available Common bean diversity within and between Mesoamerican and Andean gene pools was compared in 89 landraces from America and 256 landraces from Europe, to elucidate the effects of bottleneck of introduction and selection for adaptation during the expansion of common bean (Phaseolus vulgaris L. in Europe. Thirteen highly polymorphic nuclear microsatellite markers (nuSSRs were used to complement chloroplast microsatellite (cpSSRs and nuclear markers (phaseolin and Pv-shatterproof1 data from previous studies. To verify the extent of the introduction bottleneck, inter-gene pool hybrids were distinguished from "pure" accessions. Hybrids were identified on the basis of recombination of gene pool specific cpSSR, phaseolin and Pv-shatterproof1 markers with a Bayesian assignments based on nuSSRs, and with STRUCTURE admixture analysis. More hybrids were detected than previously, and their frequency was almost four times larger in Europe (40.2% than in America (12.3%. The genetic bottleneck following the introduction into Europe was not evidenced in the analysis including all the accessions, but it was significant when estimated only with "pure" accessions, and five times larger for Mesoamerican than for Andean germplasm. The extensive inter-gene pool hybridization generated a large amount of genotypic diversity that mitigated the effects of the bottleneck that occurred when common bean was introduced in Europe. The implication for evolution and the advantages for common bean breeding are discussed.

  3. Synergistic effects in gene delivery-a structure-activity approach to the optimisation of hybrid dendritic-lipidic transfection agents.

    Science.gov (United States)

    Jones, Simon P; Gabrielson, Nathan P; Pack, Daniel W; Smith, David K

    2008-10-21

    Novel gene delivery agents based on combining cholesterol units with spermine-functionalised dendrons exhibit enhanced transfection ability-we report significant synergistic effects in mixed (hybrid) systems which combine aspects of both main classes of synthetic vectors, i.e., cationic polymers and lipids.

  4. A systems-level approach to parental genomic imprinting: the imprinted gene network includes extracellular matrix genes and regulates cell cycle exit and differentiation.

    Science.gov (United States)

    Al Adhami, Hala; Evano, Brendan; Le Digarcher, Anne; Gueydan, Charlotte; Dubois, Emeric; Parrinello, Hugues; Dantec, Christelle; Bouschet, Tristan; Varrault, Annie; Journot, Laurent

    2015-03-01

    Genomic imprinting is an epigenetic mechanism that restrains the expression of ∼ 100 eutherian genes in a parent-of-origin-specific manner. The reason for this selective targeting of genes with seemingly disparate molecular functions is unclear. In the present work, we show that imprinted genes are coexpressed in a network that is regulated at the transition from proliferation to quiescence and differentiation during fibroblast cell cycle withdrawal, adipogenesis in vitro, and muscle regeneration in vivo. Imprinted gene regulation is not linked to alteration of DNA methylation or to perturbation of monoallelic, parent-of-origin-dependent expression. Overexpression and knockdown of imprinted gene expression alters the sensitivity of preadipocytes to contact inhibition and adipogenic differentiation. In silico and in cellulo experiments showed that the imprinted gene network includes biallelically expressed, nonimprinted genes. These control the extracellular matrix composition, cell adhesion, cell junction, and extracellular matrix-activated and growth factor-activated signaling. These observations show that imprinted genes share a common biological process that may account for their seemingly diverse roles in embryonic development, obesity, diabetes, muscle physiology, and neoplasm.

  5. Identification of floral genes for sex determination in Calamus palustris Griff. by using suppression subtractive hybridization.

    Science.gov (United States)

    Ng, C Y; Wickneswari, R; Choong, C Y

    2014-08-07

    Calamus palustris Griff. is an economically important dioecious rattan species in Southeast Asia. However, dioecy and onset of flowering at 3-4 years old render uncertainties in desired female:male seedling ratios to establish a productive seed orchard for this rattan species. We constructed a subtractive library for male floral tissue to understand the genetic mechanism for gender determination in C. palustris. The subtractive library produced 1536 clones with 1419 clones of high quality. Reverse Northern screening showed 313 clones with differential expression, and sequence analyses clustered them into 205 unigenes, including 32 contigs and 173 singletons. The subtractive library was further validated with reverse transcription-quantitative polymerase chain reaction analysis. Homology identification classified the unigenes into 12 putative functional proteins with 83% unigenes showing significant match to proteins in databases. Functional annotations of these unigenes revealed genes involved in male flower development, including MADS-box genes, pollen-related genes, phytohormones for flower development, and male flower organ development. Our results showed that the male floral genes may play a vital role in sex determination in C. palustris. The identified genes can be exploited to understand the molecular basis of sex determination in C. palustris.

  6. Duplication and Loss of Function of Genes Encoding RNA Polymerase III Subunit C4 Causes Hybrid Incompatibility in Rice

    Directory of Open Access Journals (Sweden)

    Giao Ngoc Nguyen

    2017-08-01

    Full Text Available Reproductive barriers are commonly observed in both animals and plants, in which they maintain species integrity and contribute to speciation. This report shows that a combination of loss-of-function alleles at two duplicated loci, DUPLICATED GAMETOPHYTIC STERILITY 1 (DGS1 on chromosome 4 and DGS2 on chromosome 7, causes pollen sterility in hybrid progeny derived from an interspecific cross between cultivated rice, Oryza sativa, and an Asian annual wild rice, O. nivara. Male gametes carrying the DGS1 allele from O. nivara (DGS1-nivaras and the DGS2 allele from O. sativa (DGS2-T65s were sterile, but female gametes carrying the same genotype were fertile. We isolated the causal gene, which encodes a protein homologous to DNA-dependent RNA polymerase (RNAP III subunit C4 (RPC4. RPC4 facilitates the transcription of 5S rRNAs and tRNAs. The loss-of-function alleles at DGS1-nivaras and DGS2-T65s were caused by weak or nonexpression of RPC4 and an absence of RPC4, respectively. Phylogenetic analysis demonstrated that gene duplication of RPC4 at DGS1 and DGS2 was a recent event that occurred after divergence of the ancestral population of Oryza from other Poaceae or during diversification of AA-genome species.

  7. Detection of MYCN Gene Amplification in Neuroblastoma by Fluorescence In Situ Hybridization: A Pediatric Oncology Group Study

    Directory of Open Access Journals (Sweden)

    Prasad Mathew

    2001-01-01

    Full Text Available To assess the utility of fluorescence in situ hybridization (FISH for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (≤30% replacement in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

  8. Identification and analysis of differentially expressed genes in differentiating xylem of Chinese fir (Cunninghamia lanceolata) by suppression subtractive hybridization.

    Science.gov (United States)

    Wang, Guifeng; Gao, Yan; Yang, Liwei; Shi, Jisen

    2007-12-01

    Wood is an important raw material for global industries with rapidly increasing demand. To isolate the genes differentially expressed during xylogenesis of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.), we used a novel system. Forward and reverse subtracted cDNA libraries were constructed using the suppression subtractive hybridization method; for the forward library we used cDNA from the mutant Dugansha as the tester and cDNA from the wild-type clone Jurong 0 as the driver, and for the reverse library we used Jurong 0 cDNA as the tester and Dugansha cDNA as the driver. Transcriptional profiling was performed using a macroarray with 4 digoxigenin-labeled probes. We obtained 618 and 409 clones from the forward and the reverse subtracted library, respectively. A total of 405 unique expressed sequence tags (ESTs) were obtained. Forty percent of the ESTs exhibited homologies with proteins of known function and fell into 4 major classes: metabolism, cell wall biogenesis and remodeling, signal transduction, and stress. Real-time PCR was performed to confirm the results. The expression levels of 11 selected ESTs were consistent with both macroarray and real-time PCR results. The systematic analysis of genes involved in wood formation in Chinese fir provides valuable insights into the molecular mechanisms involved in xylem differentiation and is an important resource for forest research that can be directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

  9. Genome-wide comparative analysis reveals similar types of NBS genes in hybrid Citrus sinensis genome and original Citrus clementine genome and provides new insights into non-TIR NBS genes.

    Directory of Open Access Journals (Sweden)

    Yunsheng Wang

    Full Text Available In this study, we identified and compared nucleotide-binding site (NBS domain-containing genes from three Citrus genomes (C. clementina, C. sinensis from USA and C. sinensis from China. Phylogenetic analysis of all Citrus NBS genes across these three genomes revealed that there are three approximately evenly numbered groups: one group contains the Toll-Interleukin receptor (TIR domain and two different Non-TIR groups in which most of proteins contain the Coiled Coil (CC domain. Motif analysis confirmed that the two groups of CC-containing NBS genes are from different evolutionary origins. We partitioned NBS genes into clades using NBS domain sequence distances and found most clades include NBS genes from all three Citrus genomes. This suggests that three Citrus genomes have similar numbers and types of NBS genes. We also mapped the re-sequenced reads of three pomelo and three mandarin genomes onto the C. sinensis genome. We found that most NBS genes of the hybrid C. sinensis genome have corresponding homologous genes in both pomelo and mandarin genomes. The homologous NBS genes in pomelo and mandarin suggest that the parental species of C. sinensis may contain similar types of NBS genes. This explains why the hybrid C. sinensis and original C. clementina have similar types of NBS genes in this study. Furthermore, we found that sequence variation amongst Citrus NBS genes were shaped by multiple independent and shared accelerated mutation accumulation events among different groups of NBS genes and in different Citrus genomes. Our comparative analyses yield valuable insight into the structure, organization and evolution of NBS genes in Citrus genomes. Furthermore, our comprehensive analysis showed that the non-TIR NBS genes can be divided into two groups that come from different evolutionary origins. This provides new insights into non-TIR genes, which have not received much attention.

  10. Genome-wide comparative analysis reveals similar types of NBS genes in hybrid Citrus sinensis genome and original Citrus clementine genome and provides new insights into non-TIR NBS genes.

    Science.gov (United States)

    Wang, Yunsheng; Zhou, Lijuan; Li, Dazhi; Dai, Liangying; Lawton-Rauh, Amy; Srimani, Pradip K; Duan, Yongping; Luo, Feng

    2015-01-01

    In this study, we identified and compared nucleotide-binding site (NBS) domain-containing genes from three Citrus genomes (C. clementina, C. sinensis from USA and C. sinensis from China). Phylogenetic analysis of all Citrus NBS genes across these three genomes revealed that there are three approximately evenly numbered groups: one group contains the Toll-Interleukin receptor (TIR) domain and two different Non-TIR groups in which most of proteins contain the Coiled Coil (CC) domain. Motif analysis confirmed that the two groups of CC-containing NBS genes are from different evolutionary origins. We partitioned NBS genes into clades using NBS domain sequence distances and found most clades include NBS genes from all three Citrus genomes. This suggests that three Citrus genomes have similar numbers and types of NBS genes. We also mapped the re-sequenced reads of three pomelo and three mandarin genomes onto the C. sinensis genome. We found that most NBS genes of the hybrid C. sinensis genome have corresponding homologous genes in both pomelo and mandarin genomes. The homologous NBS genes in pomelo and mandarin suggest that the parental species of C. sinensis may contain similar types of NBS genes. This explains why the hybrid C. sinensis and original C. clementina have similar types of NBS genes in this study. Furthermore, we found that sequence variation amongst Citrus NBS genes were shaped by multiple independent and shared accelerated mutation accumulation events among different groups of NBS genes and in different Citrus genomes. Our comparative analyses yield valuable insight into the structure, organization and evolution of NBS genes in Citrus genomes. Furthermore, our comprehensive analysis showed that the non-TIR NBS genes can be divided into two groups that come from different evolutionary origins. This provides new insights into non-TIR genes, which have not received much attention.

  11. Facilitating genome navigation : survey sequencing and dense radiation-hybrid gene mapping

    NARCIS (Netherlands)

    Hitte, C; Madeoy, J; Kirkness, EF; Priat, C; Lorentzen, TD; Senger, F; Thomas, D; Derrien, T; Ramirez, C; Scott, C; Evanno, G; Pullar, B; Cadieu, E; Oza, [No Value; Lourgant, K; Jaffe, DB; Tacher, S; Dreano, S; Berkova, N; Andre, C; Deloukas, P; Fraser, C; Lindblad-Toh, K; Ostrander, EA; Galibert, F

    2005-01-01

    Accurate and comprehensive sequence coverage for large genomes has been restricted to only a few species of specific interest. Lower sequence coverage (survey sequencing) of related species can yield a wealth of information about gene content and putative regulatory elements. But survey sequences la

  12. Transcript analysis of 1003 novel yeast genes using high-throughput northern hybridizations.

    Science.gov (United States)

    Brown, A J; Planta, R J; Restuhadi, F; Bailey, D A; Butler, P R; Cadahia, J L; Cerdan, M E; De Jonge, M; Gardner, D C; Gent, M E; Hayes, A; Kolen, C P; Lombardia, L J; Murad, A M; Oliver, R A; Sefton, M; Thevelein, J M; Tournu, H; van Delft, Y J; Verbart, D J; Winderickx, J; Oliver, S G

    2001-06-15

    The expression of 1008 open reading frames (ORFs) from the yeast Saccharomyces cerevisiae has been examined under eight different physiological conditions, using classical northern analysis. These northern data have been compared with publicly available data from a microarray analysis of the diauxic transition in S.cerevisiae. The results demonstrate the importance of comparing biologically equivalent situations and of the standardization of data normalization procedures. We have also used our northern data to identify co-regulated gene clusters and define the putative target sites of transcriptional activators responsible for their control. Clusters containing genes of known function identify target sites of known activators. In contrast, clusters comprised solely of genes of unknown function usually define novel putative target sites. Finally, we have examined possible global controls on gene expression. It was discovered that ORFs that are highly expressed following a nutritional upshift tend to employ favoured codons, whereas those overexpressed in starvation conditions do not. These results are interpreted in terms of a model in which competition between mRNA molecules for translational capacity selects for codons translated by abundant tRNAs.

  13. The male-determining gene SRY is a hybrid of DGCR8 and SOX3, and is regulated by the transcription factor CP2.

    Science.gov (United States)

    Sato, Youichi; Shinka, Toshikatsu; Sakamoto, Kozue; Ewis, Ashraf A; Nakahori, Yutaka

    2010-04-01

    In mammals, sex is determined by the presence or absence of the Y chromosome that bears a male-dominant sex-determining gene SRY, which switches the differentiation of gonads into male testes. The molecular signaling mechanism turning on the switch, however, has remained unclear for 18 years since the identification of the gene. Here, we describe how this gene emerged and started to work. From amino acid homology, we realized that SRY is a hybrid gene between a portion of the first exon of DiGeorge syndrome critical region gene 8 (DGCR8) and the high-mobility group (HMG) box of SRY box-3 (SOX3) gene. We identified the regulatory sequence in the SRY promotor region by searching for a common motif shared with DGCR8 mRNA. From the motif search between DGCR8 mRNA and the SRY upstream sequence, we found that the transcription factor CP2 (TFCP2) binding motif is present in both. TFCP2 overexpression did not show a significant increase of SRY mRNA expression, and TFCP2 suppression by RNA interference (RNAi) significantly reduced SRY mRNA expression. Furthermore, electrophoretic mobility shift assay (EMSA) demonstrated that TFCP2 acts as a regulator by directly binding to the SRY promoter. We conclude that SRY is a hybrid gene composed of two genes, DGCR8 and SOX3; and TFCP2 is an essential transcription factor for SRY expression regulation.

  14. Characterization and localization of a hybrid non-ribosomal peptide synthetase and polyketide synthase gene from the toxic dinoflagellate Karenia brevis.

    Science.gov (United States)

    López-Legentil, Susanna; Song, Bongkeun; DeTure, Michael; Baden, Daniel G

    2010-02-01

    The toxic dinoflagellate Karenia brevis, a causative agent of the red tides in Florida, produces a series of toxic compounds known as brevetoxins and their derivatives. Recently, several putative genes encoding polyketide synthase (PKS) were identified from K. brevis in an effort to elucidate the genetic systems involved in brevetoxin production. In this study, novel PKS sequences were isolated from three clones of K. brevis. Eighteen unique sequences were obtained for the PKS ketosynthase (KS) domain of K. brevis. Phylogenetic comparison with closely related PKS genes revealed that 16 grouped with cyanobacteria sequences, while the remaining two grouped with Apicomplexa and previously reported sequences for K. brevis. A fosmid library was also constructed to further characterize PKS genes detected in K. brevis Wilson clone. Several fosmid clones were positive for the presence of PKS genes, and one was fully sequenced to determine the full structure of the PKS cluster. A hybrid non ribosomal peptide synthetase and PKS (NRPS-PKS) gene cluster of 16,061 bp was isolated. In addition, we assessed whether the isolated gene was being actively expressed using reverse transcription polymerase chain reaction (RT-PCR) and determined its localization at the cellular level by chloroplast isolation. RT-PCR analyses revealed that this gene was actively expressed in K. brevis cultures. The hybrid NRPS-PKS gene cluster was located in the chloroplast, suggesting that K. brevis acquired the ability to produce some of its secondary metabolites through endosymbiosis with ancestral cyanobacteria. Further work is needed to determine the compound produced by the NRPS-PKS hybrid, to find other PKS gene sequences, and to assess their role in K. brevis toxin biosynthetic pathway.

  15. [Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing recA and NLS-recA-licBM3 genes].

    Science.gov (United States)

    Komakhin, R A; Komakhina, V V; Miliukova, N A; Zhuchenko, A A

    2012-01-01

    To study and induce meiotic recombination in plants, we generated and analyzed transgenic tomato hybrids F1-RecA and F1-NLS-recA-LicBM3 expressing, respectively, the recA gene of Escherichia coli and the NLS-recA-licBM3 gene. It was found that the recA and NLS-recA-licBM3 genes are inherited through the maternal and paternal lineages, they have no selective influence on the pollen and are contained in tomato F1-RecA and F1-NLS-RecA-LicBM3 hybrids outside the second chromosome in the hemizygous state. The comparative analysis of the meiotic recombination frequency (rf) in the progenies of the transgenic and nontransgenic hybrids showed that only the expression of the recA gene of E. coli in cells of the F1-RecA plants produced a 1.2-1.5-fold increase in the frequency of recombination between some linked marker genes of the second chromosome of tomato.

  16. Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7

    Energy Technology Data Exchange (ETDEWEB)

    Flores, J.; Sears, J.; Schael, I.P.; White, L.; Garcia, D.; Lanata, C.; Kapikian, A.Z. (National Institutes of Health, Bethesda, MD (USA))

    1990-08-01

    We have synthesized {sup 32}P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

  17. Microarray and suppression subtractive hybridization analyses of gene expression in hybrid poplar (Populus alba × Populus tremula var. glandulosa) cell suspension cultures after exposure to NaCl.

    Science.gov (United States)

    Bae, Eun-Kyung; Lee, Hyoshin; Lee, Jae-Soon; Noh, Eun-Woon; Choi, Young-Im; Lee, Byung-Hyun; Choi, Dong-Woog

    2012-09-01

    The gene expression profiles of hybrid poplar (Populus alba × Populus tremula var. glandulosa) cells in suspension culture after exposure to salinity (NaCl) induced stress were examined by constructing two suppression subtractive hybridization (SSH) libraries. cDNA from non-treated cells was used as a driver and cDNA samples from cell suspension cultures exposed to 150 mM NaCl for 2 or 10 h were used as testers. Randomly selected clones from each SSH library were sequenced and 727 high-quality expressed sequence tags (ESTs) were obtained and analyzed. Four novel ESTs were identified. Between the two libraries, 542 unique SSH clones were selected for placement on a cDNA microarray. In total, 18 differentially expressed genes were identified with 4 and 12 genes being significantly differentially expressed 2 and 10 h after the treatment, respectively. Genes related to metabolism and protein synthesis and several genes whose protein products are implicated in salt or other abiotic stress-related responses were expressed in the salt-stressed cells.

  18. Improving the measurement of semantic similarity between gene ontology terms and gene products: insights from an edge- and IC-based hybrid method.

    Directory of Open Access Journals (Sweden)

    Xiaomei Wu

    Full Text Available BACKGROUND: Explicit comparisons based on the semantic similarity of Gene Ontology terms provide a quantitative way to measure the functional similarity between gene products and are widely applied in large-scale genomic research via integration with other models. Previously, we presented an edge-based method, Relative Specificity Similarity (RSS, which takes the global position of relevant terms into account. However, edge-based semantic similarity metrics are sensitive to the intrinsic structure of GO and simply consider terms at the same level in the ontology to be equally specific nodes, revealing the weaknesses that could be complemented using information content (IC. RESULTS AND CONCLUSIONS: Here, we used the IC-based nodes to improve RSS and proposed a new method, Hybrid Relative Specificity Similarity (HRSS. HRSS outperformed other methods in distinguishing true protein-protein interactions from false. HRSS values were divided into four different levels of confidence for protein interactions. In addition, HRSS was statistically the best at obtaining the highest average functional similarity among human-mouse orthologs. Both HRSS and the groupwise measure, simGIC, are superior in correlation with sequence and Pfam similarities. Because different measures are best suited for different circumstances, we compared two pairwise strategies, the maximum and the best-match average, in the evaluation. The former was more effective at inferring physical protein-protein interactions, and the latter at estimating the functional conservation of orthologs and analyzing the CESSM datasets. In conclusion, HRSS can be applied to different biological problems by quantifying the functional similarity between gene products. The algorithm HRSS was implemented in the C programming language, which is freely available from http://cmb.bnu.edu.cn/hrss.

  19. Hybridization of different antisense oligonucleotides on the surface of gold nanoparticles to silence zinc metalloproteinase gene after uptake by Leishmania major.

    Science.gov (United States)

    Jebali, Ali; Anvari-Tafti, Mohammad Hosssein

    2015-05-01

    The use of antisense oligonucleotides is a novel strategy to treat infectious diseases. In this approach, vital mRNAs are targeted by antisense oligonucleotides. The aim of this study was to evaluate the effects of gold nanoparticles hybridized with different antisense oligonucleotides on Leishmania (L) major. In this project, gold nanoparticles were first synthesized, and then conjugated with primary oligonucleotides, 3'-AAA-5'. Next, conjugated gold nanoparticles (NP1) were separately hybridized with three types of antisense oligonucleotide from coding reign of GP63 gene (NP2), non-coding reign of GP63 gene (NP3), and both coding and non-coding reigns of GP63 (NP4). Then, 1mL of L. major suspension was separately added to 1mL of different hybridized gold nanoparticles at serial concentrations (1-200μg/mL), and incubated for 24, 48, and 72h at 37°C. Next, the uptake of each nanoparticle was separately measured by atomic absorption spectroscopy. After incubation, the cell viability was separately evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Also, the expression of GP63 gene was read out by quantitative-real-time PCR. This study showed that NP2 and NP3 had higher (5-fold) uptake than NP1 and NP4. Moreover, NP2 and NP3 led to less cell viability and gene expression, compared with NP1 and NP4. It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major. Importantly, these antisense oligonucleotides can be obtained from both coding and non-coding reign of GP63 gene. Moreover, hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L. major. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Gene expression pattern in transmitochondrial cytoplasmic hybrid cells harboring type 2 diabetes-associated mitochondrial DNA haplogroups.

    Directory of Open Access Journals (Sweden)

    Seungwoo Hwang

    Full Text Available Decreased mitochondrial function plays a pivotal role in the pathogenesis of type 2 diabetes mellitus (T2DM. Recently, it was reported that mitochondrial DNA (mtDNA haplogroups confer genetic susceptibility to T2DM in Koreans and Japanese. Particularly, mtDNA haplogroup N9a is associated with a decreased risk of T2DM, whereas haplogroups D5 and F are associated with an increased risk. To examine functional consequences of these haplogroups without being confounded by the heterogeneous nuclear genomic backgrounds of different subjects, we constructed transmitochondrial cytoplasmic hybrid (cybrid cells harboring each of the three haplogroups (N9a, D5, and F in a background of a shared nuclear genome. We compared the functional consequences of the three haplogroups using cell-based assays and gene expression microarrays. Cell-based assays did not detect differences in mitochondrial functions among the haplogroups in terms of ATP generation, reactive oxygen species production, mitochondrial membrane potential, and cellular dehydrogenase activity. However, differential expression and clustering analyses of microarray data revealed that the three haplogroups exhibit a distinctive nuclear gene expression pattern that correlates with their susceptibility to T2DM. Pathway analysis of microarray data identified several differentially regulated metabolic pathways. Notably, compared to the T2DM-resistant haplogroup N9a, the T2DM-susceptible haplogroup F showed down-regulation of oxidative phosphorylation and up-regulation of glycolysis. These results suggest that variations in mtDNA can affect the expression of nuclear genes regulating mitochondrial functions or cellular energetics. Given that impaired mitochondrial function caused by T2DM-associated mtDNA haplogroups is compensated by the nuclear genome, we speculate that defective nuclear compensation, under certain circumstances, might lead to the development of T2DM.

  1. Hybrid polymer-grafted multiwalled carbon nanotubes for in vitro gene delivery.

    Science.gov (United States)

    Nunes, Antonio; Amsharov, Nadja; Guo, Chang; Van den Bossche, Jeroen; Santhosh, Padmanabhan; Karachalios, Theodoros K; Nitodas, Stephanos F; Burghard, Marko; Kostarelos, Kostas; Al-Jamal, Khuloud T

    2010-10-18

    Carbon nanotubes (CNTs) consist of carbon atoms arranged in sheets of graphene rolled up into cylindrical shapes. This class of nanomaterials has attracted attention because of their extraordinary properties, such as high electrical and thermal conductivity. In addition, development in CNT functionalization chemistry has led to an enhanced dispersibility in aqueous physiological media which indeed broadens the spectrum for their potential biological applications including gene delivery. The aim of this study is to determine the capability of different cationic polymer-grafted multiwalled carbon nanotubes (MWNTs) (polymer-g-MWNTs) to efficiently complex and transfer plasmid DNA (pCMV-βGal) in vitro without promoting cytotoxicity. Carboxylated MWNT is chemically conjugated to the cationic polymers polyethylenimine (PEI), polyallylamine (PAA), or a mixture of the two polymers. In order to explore the potential of these polymer-g-MWNTs as gene delivery systems, we first study their capacity to complex plasmid DNA (pDNA) using agarose gel electrophoresis. Gel migration studies confirm pDNA binding to polymer-g-MWNT with different affinities, highest for PEI-g-MWNT and PEI/PAA-g-CNT constructs. β-galactosidase expression is assessed in human lung epithelial (A549) cells, and the cytotoxicity is determined by modified LDH assay after 24 h incubation period. Additionally, PEI-g-MWNT and/or PEI/PAA-g-MWNT reveal an improvement in gene expression when compared to the naked pDNA or to the equivalent amounts of PEI polymer alone. Mechanistically, pDNA was delivered by the polymer-g-MWNT constructs via a different pathway compared to those used by polyplexes. In conclusion, polymer-g-MWNTs may be considered in the future as a versatile tool for efficient gene transfer in cancer cells in vitro, provided their toxicological profile is established.

  2. Analysis of immune system gene expression in small rheumatoid arthritis biopsies using a combination of subtractive hybridization and high-density cDNA arrays.

    Science.gov (United States)

    Zanders, E D; Goulden, M G; Kennedy, T C; Kempsell, K E

    2000-01-13

    Subtractive hybridization of cDNAs generated from synovial RNA which had been isolated from patients with rheumatoid arthritis (RA) or normal controls was used in conjunction with high-density array hybridization to identify genes of immunological interest. The method was designed to detect gene expression in small needle biopsy specimens by means of a prior amplification of nanogram amounts of total RNA to full-length cDNA using PCR. The latter was cut with Rsa I, ligated with adapters, hybridized with unmodified driver cDNA, and subjected to suppression subtraction PCR. Differentially expressed products were cloned into E. coli and picked into 384 well plates. Inserts were obtained by PCR across the multiple cloning site, and the products arrayed at high density on nylon filters. The subtracted cDNAs were also labelled by random priming for use as probes for library screening. The libraries chosen were the subtracted one described above and a set of 45,000 ESTs from the I.M. A.G.E consortium. Clones showing positive hybridization were identified by sequence analysis and homology searching. The results showed that the subtracted hybridization approach could identify many gene fragments expressed at different levels, the most abundant being immunoglobulins and HLA-DR. The expression profile was characteristic of macrophage, B cell and plasma cell infiltration with evidence of interferon induction. In addition, a significant number of sequences without matches in the nucleotide databases were obtained, this demonstrates the utility of the method in finding novel gene fragments for further characterisation as potential members of the immune system. Although RA was studied here, the technology is applicable to any disease process even in cases where amounts of tissue may be limited.

  3. Transcriptome analysis of Gerbera hybrid: including in silico confirmation of defence genes found

    Directory of Open Access Journals (Sweden)

    Yiqian eFu

    2016-03-01

    Full Text Available For the ornamental crop Gerbera hybrida, breeding at the moment is done using conventional methods. As this has drawbacks in breeding speed and efficiency, especially for complex traits like disease resistance, we set out to develop genomic resources. The leaf and flower bud transcriptomes of four parents, used to generate two gerbera populations, were sequenced using Illumina paired-end sequencing. In total, 36,770 contigs with an average length of 1397 bp were generated and these have been the starting point for SNP identification and annotation. The consensus contig sequences were used to map reads of individual parents, to identify genotype specific SNPs, and to assess the presence of common SNPs between genotypes.Comparison with the non-redundant protein database (nr showed that 29,146 contigs gave BLAST hits. Of sequences with blast results, 73.3% obtained a clear gene ontology (GO annotation. EST contigs coding for enzymes were found in Kyoto Encyclopedia of Genes and Genomes maps (KEGG. Through these annotated data and KEGG molecular interaction network, transcripts associated with the phenylpropanoid metabolism, other secondary metabolite biosynthesis pathways, phytohormone biosynthesis and signal transduction were analysed in more detail. Identifying genes involved in these processes could provide genetic and genomic resources for studying the mechanism of disease resistance in gerbera.

  4. [Interspecific hybridization in the genus Paeonia (Paeoniaceae): polymorphic sites in transcribed spacers of the 45S rRNA genes as indicators of natural and artificial peony hybrids].

    Science.gov (United States)

    Punina, E O; Machs, E M; Krapivskaia, E E; Kim, E S; Mordak, E V; Miakoshina, Iu A; Rodionov, A V

    2012-07-01

    The ITS1-5.8S rDNA-ITS2 regions of 33 accessions belonging to 16 species and five natural and garden interspecific hybrids of the genus Paeonia L. were sequenced. Chromatograms of the peony hybrids demonstrated the presence of the signals, corresponding to two different nucleotides at the positions differing in the parents, indicating that in the hybrids, no rDNA isogenization usually occurred, and they preserved rDNA of both parents. Analysis of these polymorphic sites (PS) showed that P. x majkoae was interspecific hybrid between P. tenuifolia and P. caucasica. The ITS of P. hybrida differs from ITS of P. x majkoae in 19 mutations. Because of this, P. x majkoae is definitely not synonymous to P. hybrida. Comparative analysis of ITS 1-5.8S rDNA-ITS2 showed that species diversity in section Paeonia was based on recombination as a result of intraspecific hybridization of three haplotype families. Specifically, haplotypes A, typical of the P. tenuifolia and P. anomala genomes, haplotypes B, typical of P. mlokosewitschii and P. obovata, and haplotypes of family C, currently represented in rDNA of diploid and tetraploid forms of some Caucasian and Mediterranean species. The ITS regions many diploid peonies contain no dimorphic sites, while P. oreogeton, P. cambessedesii, P. rhodia, and P. daurica carry from ten to 17 PS, and supposed to be the interspecific hybrids. Most of the tetraploid peonies contain from six to 18 PS in the ITS regions. These are alloploids with one of the parental genomes similar to that of P. mlokosewitschii (B1), or P. obovata (B3). The second parental genome in P. banatica, P. peregrina, and P. russii is represented by the genome, close to that of P. tenuifolia (A). P. macrophylla, P. mascula, P. coriacea, P. wittmanniana, and P. tomentosa carry genome of series B and genome of series C, which slightly resembles genome A.

  5. Fertility Expression of TGMS-Genes in the Backgrounds of indica CMS-lines, B-lines and R-lines of Hybrid Rice

    Institute of Scientific and Technical Information of China (English)

    WANG Ji-feng; LU Zuo-mei

    2006-01-01

    The generation fertility of 51 F1, 19 F2 and 6 BC1 between 3 thermo-sensitive genic male sterile lines (TGMS-lines) Pei'ai64S, 6311S and 360S, and the three lines of hybrid rice including 7 indica cytoplasmic male sterile lines (CMS-lines) and their corresponding maintainer lines (B-lines) and 3 indica restorer lines (R-lines) were investigated to study the expression of TGMS-genes in the backgrounds of the three lines of hybrid rice. Pei'ai 64S has stronger fertility restoring (Rf) genes for CMS-lines and its TGMS trait is governed by 2 pairs of independent recessive genes; The TGMS trait of 6311S is governed by a single recessive gene with weaker Rf-gene in 6311S and the TGMS trait of 360S is governed by a single recessive gene with no Rf-gene in 360S. The investigation on the fertility of F1 plants between 5 CMS-lines and 4 TGMS generations selected from F2 plants of 4 CMS-lines ×6311S confirmed that the expression of TGMS-gene was controlled by Rf-gene in the genetic background of cytoplasm of CMS-lines,but not affected by Rf-gene in the genetic background of normal fertile cytoplasm. The potential breeding strategies of TGMS-lines with cytoplasm of CMS-lines and CMS-lines with the nucleus of TGMS-genes were discussed.

  6. Identification of a deletion in the mismatch repair gene, MSH2, using mouse-human cell hybrids monosomal for chromosome 2.

    Science.gov (United States)

    Pyatt, R E; Nakagawa, H; Hampel, H; Sedra, M; Fuchik, M B; Comeras, I; de la Chapelle, A; Prior, T W

    2003-03-01

    Hereditary non-polyposis colorectal cancer is characterized by mutations in one of the DNA mismatch repair genes, primarily MLH1, MSH2, or MSH6. We report here the identification of a genomic deletion of approximately 11.4 kb encompassing the first two exons of the MSH2 gene in two generations of an Ohio family. By Southern blot analysis, using a cDNA probe spanning the first seven exons of MSH2, an alteration in each of three different enzyme digests (including a unique 13-kb band on HindIII digests) was observed, which suggested the presence of a large alteration in the 5' region of this gene. Mouse-human cell hybrids from a mutation carrier were then generated which contained a single copy each of human chromosome 2 on which the MSH2 gene resides. Southern blots on DNA from the cell hybrids demonstrated the same, unique 13-kb band from one MSH2 allele, as seen in the diploid DNA. DNA from this same monosomal cell hybrid failed to amplify in polymerase chain reactions (PCRs) using primers to exons 1 and 2, demonstrating the deletion of these sequences in one MSH2 allele, and the breakpoints involving Alu repeats were identified by PCR amplification and sequence analysis.

  7. Nucleotide divergence vs. gene expression differentiation: comparative transcriptome sequencing in natural isolates from the carrion crow and its hybrid zone with the hooded crow.

    Science.gov (United States)

    Wolf, Jochen B W; Bayer, Till; Haubold, Bernhard; Schilhabel, Markus; Rosenstiel, Philip; Tautz, Diethard

    2010-03-01

    Recent advances in sequencing technology promise to provide new strategies for studying population differentiation and speciation phenomena in their earliest phases. We focus here on the black carrion crow (Corvus [corone] corone), which forms a zone of hybridization and overlap with the grey coated hooded crow (Corvus [corone] cornix). However, although these semispecies are taxonomically distinct, previous analyses based on several types of genetic markers did not reveal significant molecular differentiation between them. We here corroborate this result with sequence data obtained from a set of 25 nuclear intronic loci. Thus, the system represents a case of a very early phase of species divergence that requires new molecular approaches for its description. We have therefore generated RNAseq expression profiles using barcoded massively parallel pyrosequencing of brain mRNA from six individuals of the carrion crow and five individuals from a hybrid zone with the hooded crow. We obtained 856 675 reads from two runs, with average read length of 270 nt and coverage of 8.44. Reads were assembled de novo into 19 552 contigs, 70% of which could be assigned to annotated genes in chicken and zebra finch. This resulted in a total of 7637 orthologous genes and a core set of 1301 genes that could be compared across all individuals. We find a clear clustering of expression profiles for the pure carrion crow animals and disperse profiles for the animals from the hybrid zone. These results suggest that gene expression differences may indeed be a sensitive indicator of initial species divergence.

  8. Preliminary comparing the toxicities of the hybrid cry1Acs fused with different heterogenous genes provided guidance for the fusion expression of Cry proteins.

    Science.gov (United States)

    Tang, Ying; Tong, Jinying; Zhang, Yunlei; Wang, Lei; Hu, Shengbiao; Li, Wenping; Lv, Yuan

    2012-01-01

    In order to provide guidance for selecting suitable heterogenous gene that can efficiently enhance toxicity or broaden insecticidal spectrum of Cry1Ac through fusion expression, two hybrid cry1Acs fused with chitinase-encoding gene tchiB and neurotoxin gene hwtx-1 respectively were constructed and their toxicities were compared. A Bacillus thuringiensis strain harboring the cry1Ac gene in vector pHT315 was used as control. Bioassay revealed that LC(50) (after 72 h) of Cry1Ac protoxin was 41.01 μg mL(-1), while the hybrid cry1Acs fused with tchiB and hwtx-1 were 4.89 and 23.14 μg mL(-1), which were 8.23- and 1.77-fold higher than Cry1Ac protoxin in terms of relative toxicity respectively. Both fusion crystals had a higher toxicity than the original Cry1Ac protein and the toxicity of hybrid cry1Acs fused with hwtx-1 experienced a more significant increase than that fused with tchiB.

  9. Molecular characterization of a cellulose synthase gene (AaxmCesA1) isolated from an Acacia auriculiformis x Acacia mangium hybrid.

    Science.gov (United States)

    Yong, Seok Yien Christina; Wickneswari, Ratnam

    2013-01-01

    Cellulose is the major component of plant cell walls, providing mechanical strength to the structural framework of plants. In association with lignin, hemicellulose, protein and pectin, cellulose forms the strong yet flexible bio-composite tissue of wood. Wood formation is an essential biological process and is of significant importance to the cellulosic private sector industry. Cellulose synthase genes encode the catalytic subunits of a large protein complex responsible for the biogenesis of cellulose in higher plants. The hybrid Acacia auriculiformis x Acacia mangium represents an important source of tree cellulose for forest-based product manufacturing, with enormous economic potential. In this work, we isolate the first cellulose synthase gene, designated AaxmCesA1, from this species. The isolated full-length AaxmCesA1 cDNA encodes a polypeptide of 1,064 amino acids. Sequence analyses revealed that AaxmCesA1 cDNA possesses the key motif characteristics of a CesA protein. AaxmCesA1 shares more than 75 % amino acid sequence identity with CesA proteins from other plant species. Subsequently, the full-length AaxmCesA1 gene of 7,389 bp with partial regulatory and 13 intron regions was also isolated. Relative gene expression analysis by quantitative PCR in different tissues of the Acacia hybrid, suggests the involvement of the AaxmCesA1 gene in primary cell wall synthesis of rapidly dividing young root cells. Similarity analyses using Blast algorithms also suggests a role in primary cell wall deposition in the Acacia hybrid. Southern analysis predicts that AaxmCesA1 is a member of a multigene family with at least two isoforms in the genome of the Acacia hybrid.

  10. A hybrid dynamic Bayesian network approach for modelling temporal associations of gene expressions for hypertension diagnosis.

    Science.gov (United States)

    Akutekwe, Arinze; Seker, Huseyin

    2014-01-01

    Computational and machine learning techniques have been applied in identifying biomarkers and constructing predictive models for diagnosis of hypertension. Strategies such as improved classification rules based on decision trees have been proposed. Other techniques such as Fuzzy Expert Systems (FES) and Neuro-Fuzzy Systems (NFS) have recently been applied. However, these methods lack the ability to detect temporal relationships among biomarker genes that will aid better understanding of the mechanism of hypertension disease. In this paper we apply a proposed two-stage bio-network construction approach that combines the power and computational efficiency of classification methods with the well-established predictive ability of Dynamic Bayesian Network. We demonstrate our method using the analysis of male young-onset hypertension microarray dataset. Four key genes were identified by the Least Angle Shrinkage and Selection Operator (LASSO) and three Support Vector Machine Recursive Feature Elimination (SVM-RFE) methods. Results show that cell regulation FOXQ1 may inhibit the expression of focusyltransferase-6 (FUT6) and that ABCG1 ATP-binding cassette sub-family G may also play inhibitory role against NR2E3 nuclear receptor sub-family 2 and CGB2 Chromatin Gonadotrophin.

  11. Skin Barrier Function Is Not Impaired and Kallikrein 7 Gene Polymorphism Is Frequently Observed in Korean X-linked Ichthyosis Patients Diagnosed by Fluorescence in Situ Hybridization and Array Comparative Genomic Hybridization.

    Science.gov (United States)

    Lee, Noo Ri; Yoon, Na Young; Jung, Minyoung; Kim, Ji-Yun; Seo, Seong Jun; Wang, Hye-Young; Lee, Hyeyoung; Sohn, Young Bae; Choi, Eung Ho

    2016-08-01

    X-linked ichthyosis (XLI) is a recessively inherited ichthyosis. Skin barrier function of XLI patients reported in Western countries presented minimally abnormal or normal. Here, we evaluated the skin barrier properties and a skin barrier-related gene mutation in 16 Korean XLI patients who were diagnosed by fluorescence in situ hybridization and array comparative genomic hybridization analysis. Skin barrier properties were measured, cytokine expression levels in the stratum corneum (SC) were evaluated with the tape stripped specimen from skin surface, and a genetic test was done on blood. XLI patients showed significantly lower SC hydration, but normal basal trans-epidermal water loss and skin surface pH as compared to a healthy control group. Histopathology of ichthyosis epidermis showed no acanthosis, and levels of the pro-inflammatory cytokines in the corneal layer did not differ between control and lesional/non-lesional skin of XLI patients. Among the mutations in filaggrin (FLG), kallikrein 7 (KLK7), and SPINK5 genes, the prevalence of KLK7 gene mutations was significantly higher in XLI patients (50%) than in controls (0%), whereas FLG and SPINK5 prevalence was comparable. Korean XLI patients exhibited unimpaired skin barrier function and frequent association with the KLK7 gene polymorphism, which may differentiate them from Western XLI patients.

  12. Molecular characterization of enterohemorrhagic Escherichia coli hemolysin gene (EHEC-hlyA)-harboring isolates from cattle reveals a diverse origin and hybrid diarrheagenic strains.

    Science.gov (United States)

    Askari Badouei, Mahdi; Morabito, Stefano; Najafifar, Arash; Mazandarani, Emad

    2016-04-01

    In the present study we investigated the occurrence of Escherichia coli strains harboring the gene encoding enterohemorrhagic E. coli hemolysin (EHEC-HlyA) in cattle and the association of this gene with various diarrheagenic E. coli (DEC) pathotypes. First, the bovine E. coli isolates were screened for EHEC-hlyA gene by PCR, and then they were characterized for the phylogenetic groups and the presence of the major virulence genes of different DEC pathotypes. In total, 25 virulence gene profiles were observed in 54 EHEC-hlyA+ isolates that reflect a considerable heterogeneity. The EHEC-hlyA+ strains were mostly associated with EHEC (72%), while only 7.4% were enteropathogenic E. coli (EPEC). We also showed the presence of estA gene of enterotoxigenic E. coli (ETEC) in 6 isolates (11.1%). Interestingly, two of the estA+ strains showed hybrid pathotypes with one carrying eae/estA (EPEC/ETEC), and the other one stx2/astA/estA (EHEC/ETEC). None of the isolates were related to enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and necrotoxigenic E. coli (NTEC). The EHEC-plasmid encoded genes occurred in seven different combinations with EHEC-hlyA/saa/subA/espP being the most prevalent (46.3%). All stx-/eae+ strains carried O island 57 (OI-57) molecular marker(s) that may indicate these to be the progenitors of EHEC or strains losing stx. The most prevalent phylogroup was B1 (61.1%), but the most heterogeneous strains including the hybrid strains belonged to A phylogroup. Overall, our results indicate that cattle EHEC-hlyA encoding E. coli isolates consist of diverse diarrheagenic strains with the possible existence of hybrid pathotypes. Future studies are required to clarify the evolutionary aspects and clinical significance of these strains in humans and domestic animals.

  13. Survivin antiapoptotic gene expression as a prognostic factor in non-small cell lung cancer: in situ hybridization study.

    Directory of Open Access Journals (Sweden)

    A Jabłonka

    2005-12-01

    Full Text Available Survivin is an inhibitor of apoptosis that plays a significant role in cell cycle regulation and is important for survival prognosis in many neoplasms. Survivin expression was assessed by in situ hybridization (ISH in 60 consecutive patients (54 males and 4 females with NSCLC treated between 1993 and 1997. The examined patients had IIB and IIIA stage according to TNM system. In all cases the chemotherapy with cisplatin and etoposide (2 cycles was administered prior the surgery; in patients responding to the therapy one more cycle was applied. Survivin gene overexpression was observed in 35 patients (58.3%. There was no correlation between survivin mRNA level and histological type of tumor, stage of cell differentiation, stage of disease according to TNM classification, performance status according to WHO and number of chemotherapy regimens administered (p > 0.05. However, the correlation between survivin gene expression and response to the chemotherapy was statistically significant (p = 0.04. Statistical analysis showed that median survival in patients with survivin gene overexpression was shorter (14.0 months as compared to patients with no expression (60.0 months; p = 0.00002. In survival assessment by means of Kaplan-Meier test, 14.3% of five-year survival was achieved in the former group versus 60% in the latter (p = 0.00003. Univariate analysis (log-rank test showed that significant independent prognostic factors in NSCLC included: stage of the disease according to TNM classification (p = 0.006, response to chemotherapy (p = 0.005 and pattern of survivin gene expression (p = 0.00003. Multivariate analysis utilizing Cox's model showed that for survival assessment the stage according to TNM, response to the chemotherapy and survivin expression estimated by means of ISH are of statistical significance (p=0.00001. The calculated predictive values showed that ISH technique was quite accurate in assessment of five-year survival. Our data show

  14. Fine mapping of Hch1, the causal D-genome gene for hybrid chlorosis in interspecific crosses between tetraploid wheat and Aegilops tauschii.

    Science.gov (United States)

    Hirao, Kana; Nishijima, Ryo; Sakaguchi, Kohei; Takumi, Shigeo

    2015-01-01

    Hybrid chlorosis, one of the reproductive barriers between tetraploid wheat and its D-genome progenitor, Aegilops tauschii, inhibits normal growth of synthetic wheat hexaploids. Hybrid chlorosis appears to be due to an epistatic interaction of two loci from the AB and D wheat genomes. Our previous study assigned the causal D-genome gene for hybrid chlorosis, Hch1, to the short arm of chromosome 7D. Here, we constructed a fine map of 7DS near Hch1 using 280 F2 individuals from a cross of two wheat synthetic lines, one showing normal growth and the other showing hybrid chlorosis. The hybrid chlorosis phenotype was controlled by a single dominant allele of the Hch1 locus in the synthetic hexaploids. Hch1 was closely linked to four new markers within 0.2 cM, and may be localized near or within the two Ae. tauschii scaffolds containing the linked markers on 7DS. Comparative analysis of the Hch1 chromosomal region for Ae. tauschii, barley and Brachypodium showed that a local inversion occurred in the region proximal to Hch1 during the divergence between barley and Ae. tauschii, and that the Hch1 region on wheat 7DS is syntenic to Brachypodium chromosome 1. These observations provide useful information for further studies toward map-based cloning of Hch1.

  15. Performance Characteristics of Hybrid MPI/OpenMP Scientific Applications on a Large-Scale Multithreaded BlueGene/Q Supercomputer

    KAUST Repository

    Wu, Xingfu

    2013-07-01

    In this paper, we investigate the performance characteristics of five hybrid MPI/OpenMP scientific applications (two NAS Parallel benchmarks Multi-Zone SP-MZ and BT-MZ, an earthquake simulation PEQdyna, an aerospace application PMLB and a 3D particle-in-cell application GTC) on a large-scale multithreaded Blue Gene/Q supercomputer at Argonne National laboratory, and quantify the performance gap resulting from using different number of threads per node. We use performance tools and MPI profile and trace libraries available on the supercomputer to analyze and compare the performance of these hybrid scientific applications with increasing the number OpenMP threads per node, and find that increasing the number of threads to some extent saturates or worsens performance of these hybrid applications. For the strong-scaling hybrid scientific applications such as SP-MZ, BT-MZ, PEQdyna and PLMB, using 32 threads per node results in much better application efficiency than using 64 threads per node, and as increasing the number of threads per node, the FPU (Floating Point Unit) percentage decreases, and the MPI percentage (except PMLB) and IPC (Instructions per cycle) per core (except BT-MZ) increase. For the weak-scaling hybrid scientific application such as GTC, the performance trend (relative speedup) is very similar with increasing number of threads per node no matter how many nodes (32, 128, 512) are used. © 2013 IEEE.

  16. [Whole cDNA sequence cloning and expression of chicken L-FABP gene and its relationship with lipid deposition of hybrid chickens].

    Science.gov (United States)

    Yu, Ying; Wang, Dong; Sun, Dong-Xiao; Xu, Gui-Yun; Li, Jun-Ying; Zhang, Yuan

    2011-07-01

    Liver fatty acid-binding protein (L-FABP) is closely related to intracellular transportation and deposition of lipids. A positive differential displayed fragment was found in the liver tissue among Silkie (CC), CAU-brown chicken (CD), and their reciprocal hybrids (CD and DC) at 8 weeks-old using differential display RT-PCR techniques (DDRT-PCR). Through recycling, sequencing, and alignment analysis, the fragment was identified as chicken liver fatty acid-binding protein gene (L-FABP, GenBank accession number AY321365). Reverse Northern dot blot and semi-quantitative RT-PCR revealed that the avian L-FABP gene was over-expressed in the liver tissue of the reciprocal hybrids (CD and DC) compared to their parental lines (CC and DD), which was consistent with the fact that higher abdomen fat weight and wider inter-muscular fat width observed in the reciprocal hybrids. Considering the higher expression of L-FABP may contribute to the increased lipid deposition in the hybrid chickens, the functional study of avian L-FABP is warranted in future.

  17. Development of resistant tomato population with bacterial canker resistance genes from interspecific hybrids by the support of embryo rescue

    Directory of Open Access Journals (Sweden)

    Aylin KABAŞ

    2016-06-01

    Full Text Available Bacterial canker is one of the most important diseases causing economic yield loss in tomato production areas in the world. The best way to control for this disease is to use resistant varieties. However, there are few studies on variety breeding studies of this disease compared with other disease resistant breeding studies. In this study we aimed to improve inbred lines carrying bacterial canker resistance genes to use in the breeding of resistant varieties. Susceptible inbred line AK1 (S. esculentum and resistant LA2157 (S. peruvianum were crossed. Embryo rescue and ovule culture techniques were applied in 30 fruits to get F1 hybrids. Rescued embryos and immature ovules were cultured in petri dishes containing solidified MS medium without hormone. 30 healty embryos were excised and cultured from 30 fruits 27-61 day old (1 embryo fruit-1 in embryo rescue method. The two surviving plants from acclimatization were transferred to the greenhouse to get their BC1 progenies. Resistance tests were performed according to the stem inoculation method in the BC1 and BC2 progenies. The mixture of 14 aggressive Turkish Cmm strains were used to confirm the resistance. The plants were valued by 0-4 scale. Plants with 0 and 1 scale values were used to obtain next progenies. A total of 80 BC3 resistant progenies were transferred to our variety breeding programme.

  18. Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridization and RT-PCR.

    Science.gov (United States)

    Barthe, G A; Ceccardi, T L; Manjunath, K L; Derrick, K S

    1998-06-01

    Citrus psorosis virus (CPV) is a multicomponent ssRNA virus with a coat protein of approximately 48 kDa. The viral genome is encapsidated in short and long particles that are readily separated by sucrose density-gradient centrifugation. CPV particles are spiral filaments that are referred to as spiroviruses (SV). A cDNA library of purified short particles from isolate CPV-4 was prepared in a Lambda vector and screened for expression of the coat protein gene (CPG) with a monoclonal antibody to the coat protein. Sequencing of immunopositive clones indicated a single ORF encoding a 49 kDa protein. This ORF, when expressed in E. coli, gave a protein identical in size and immunoreactivity to the CPV coat protein. A full-length clone of the CPG was transcribed and used in Northern hybridization assays to establish that short particle RNA of CPV is negative sense and contains the CPG. Moreover, the CPG was not found on RNA extracted from long particles or on the sedimentable dsRNA from CPV infected tissue. RT-PCR assays were developed for the amplification of a 600 bp fragment of CPG and for the complete CPG (1317 bp). The 600 bp fragment from a biologically and serologically different isolate, CPV-6, was cloned, sequenced and found to share 86% (nucleotide) and 96% (amino acid) identity with CPV-4. BLAST analysis of sequences from CPV-4 and CPV-6 detected no significant nucleic acid or protein similarity with any known viral sequences.

  19. Identification and characterization of genes differentially expressed in X and Y sperm using suppression subtractive hybridization and cDNA microarray.

    Science.gov (United States)

    Chen, Xiaoli; Yue, Yang; He, Yanan; Zhu, Huabin; Hao, Haisheng; Zhao, Xueming; Qin, Tong; Wang, Dong

    2014-10-01

    Differential expression of genes leads to variations in the phenotypes of X and Y sperm, although some differentially expressed gene products are shared through intercellular bridges. Genes differentially expressed in bovine X and Y sperm were identified by a combination of suppression subtractive hybridization (SSH), cDNA microarray, and sequence-homology analysis. Microarray data and Significance Analysis of Microarrays software were used to identify 31 differentially expressed genes, only four of which were previously identified. These genes are involved in fundamental life processes of mature sperm, and may be associated with the differences between X and Y sperm since 27 versus 4 were upregulated in X versus Y sperm, respectively. The levels of expression of seven genes-including the known genes UTY, DPH3, CYTB, and ISCU, and the unknown genes X + Y contig 41, X + Y contig 18, and Y + X contig 16-were validated by quantitative real-time PCR, and some genes were clearly differentially expressed by X and Y sperm, despite the presence of intercellular bridges among spermatids. These results provide a theoretical basis for research on gene expression during sperm development, as well as on sex control at the level of sperm.

  20. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    Science.gov (United States)

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

  1. Annotation of a hybrid partial genome of the coffee rust (Hemileia vastatrix) contributes to the gene repertoire catalog of the Pucciniales.

    Science.gov (United States)

    Cristancho, Marco A; Botero-Rozo, David Octavio; Giraldo, William; Tabima, Javier; Riaño-Pachón, Diego Mauricio; Escobar, Carolina; Rozo, Yomara; Rivera, Luis F; Durán, Andrés; Restrepo, Silvia; Eilam, Tamar; Anikster, Yehoshua; Gaitán, Alvaro L

    2014-01-01

    Coffee leaf rust caused by the fungus Hemileia vastatrix is the most damaging disease to coffee worldwide. The pathogen has recently appeared in multiple outbreaks in coffee producing countries resulting in significant yield losses and increases in costs related to its control. New races/isolates are constantly emerging as evidenced by the presence of the fungus in plants that were previously resistant. Genomic studies are opening new avenues for the study of the evolution of pathogens, the detailed description of plant-pathogen interactions and the development of molecular techniques for the identification of individual isolates. For this purpose we sequenced 8 different H. vastatrix isolates using NGS technologies and gathered partial genome assemblies due to the large repetitive content in the coffee rust hybrid genome; 74.4% of the assembled contigs harbor repetitive sequences. A hybrid assembly of 333 Mb was built based on the 8 isolates; this assembly was used for subsequent analyses. Analysis of the conserved gene space showed that the hybrid H. vastatrix genome, though highly fragmented, had a satisfactory level of completion with 91.94% of core protein-coding orthologous genes present. RNA-Seq from urediniospores was used to guide the de novo annotation of the H. vastatrix gene complement. In total, 14,445 genes organized in 3921 families were uncovered; a considerable proportion of the predicted proteins (73.8%) were homologous to other Pucciniales species genomes. Several gene families related to the fungal lifestyle were identified, particularly 483 predicted secreted proteins that represent candidate effector genes and will provide interesting hints to decipher virulence in the coffee rust fungus. The genome sequence of Hva will serve as a template to understand the molecular mechanisms used by this fungus to attack the coffee plant, to study the diversity of this species and for the development of molecular markers to distinguish races/isolates.

  2. Annotation of a hybrid partial genome of the Coffee Rust (Hemileia vastatrix contributes to the gene repertoire catalogue of the Pucciniales

    Directory of Open Access Journals (Sweden)

    Marco Aurelio Cristancho

    2014-10-01

    Full Text Available Coffee leaf rust caused by the fungus Hemileia vastatrix is the most damaging disease to coffee worldwide. The pathogen has recently appeared in multiple outbreaks in coffee producing countries resulting in significant yield losses and increases in costs related to its control. New races/isolates are constantly emerging as evidenced by the presence of the fungus in plants that were previously resistant. Genomic studies are opening new avenues for the study of the evolution of pathogens, the detailed description of plant-pathogen interactions and the development of molecular techniques for the identification of individual isolates. For this purpose we sequenced 8 different H. vastatrix isolates using NGS technologies and gathered partial genome assemblies due to the large repetitive content in the coffee rust hybrid genome; 74.4% of the assembled contigs harbor repetitive sequences. A hybrid assembly of 333Mb was built based on the 8 isolates; this assembly was used for subsequent analyses.Analysis of the conserved gene space showed that the hybrid H. vastatrix genome, though highly fragmented, had a satisfactory level of completion with 91.94% of core protein-coding orthologous genes present. RNA-Seq from urediniospores was used to guide the de novo annotation of the H. vastatrix gene complement. In total, 14,445 genes organized in 3,921 families were uncovered; a considerable proportion of the predicted proteins (73.8% were homologous to other Pucciniales species genomes. Several gene families related to the fungal lifestyle were identified, particularly 483 predicted secreted proteins that represent candidate effector genes and will provide interesting hints to decipher virulence in the coffee rust fungus. The genome sequence of Hva will serve as a template to understand the molecular mechanisms used by this fungus to attack the coffee plant, to study the diversity of this species and for the development of molecular markers to distinguish

  3. Evolutionary analysis of allotetraploid hybrids of red crucian carp × common carp,based on ISSR,AFLP molecular markers and cloning of cyclins genes

    Institute of Scientific and Technical Information of China (English)

    LIU LiangGuo; YAN JinPeng; LIU ShaoJun; LIU Dong; YOU CuiPing; ZHONG Huan; TAO Min; LIU Yun

    2009-01-01

    The allotetraploid hybrids of red crucian carp × common carp are the first reported artificially cultured polyploid fish with bisexual fertility and stable inheritance in vertebrate.Using ISSR and AFLP markers and the cyclins genes,the genomes and cyclin gene sequence changes were analyzed between the allotetraploid hybrids and their parents.The results indicated that the allotetraploids inherited many genetic characteristics from their parents and the genetic characteristics were stable after 15 generations.However,the allotetraploids had a closer genetic relationship with their original female parents and represented a bias toward the maternal progenitor.DNA fingerprinting analysis showed that the allotetraploids had undergone sequences deletion from their original parents and that the deleted sequences were mostly from the male parent's genome.Some non-parental bands were found in the allotetraploid hybrids.Sequences analysis of the cyclin A1 and B1 genes showed nonsynonymous substitutions of single nucleotides in codons that were different from their original parents,leading to non-parental amino acid loci.We speculate that the non-additivity in the allotetraploids,compared with their progenitors,could be an adjustment to the genomic shock from heterozygosity and polyploidy, allowing maintenance of genetic stability.

  4. Chromosomal localization of the [delta] opioid receptor gene to human 1p34. 3-36. 1 and mouse 4D bands by in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Befort, K.; Kieffer, B. (Ecole Superieure de Biotechnologie de Strasbourg (France)); Mattei, M.G.; Roeckel, N. (Hopital d' Enfants de la Timone, Marseille (France))

    1994-03-01

    The aim of the present study is to determine the precise localization of this gene in the murine as well as human genome by in situ hybridization. Southern analysis, using the noncoding part of the cDNA as a probe (NotI-BamHI fragment, 1040 bp) under high-stringency conditions, shows the existence of a single-copy gene in the murine genome. In a similar analysis performed on human genomic DNA, the coding part of the cDNA (PstI-NotI fragment, 976 bp) clearly detected a single gene in the human genome (not shown). The authors therefore used these two probes for the chromosomal localization of the murine gene and its human counterpart, respectively. Chromosome spread preparations from concanavalin A (mouse) or phytohemagglutinin (human)-stimulated lymphocytes were hybridized with tritium-labeled cDNAs, exposed for 15 days, and developed, as described previously. For the murine assignment, a total of 100 metaphase cells were examined, and 149 silver grains were found. Forty-two grains were associated with chromosome 4, and 73.8% of them mapped to the D1-D3 region of the chromosome. In the 120 metaphase human cells analyzed, 197 silver grains were found, 25.8% of which were located on chromosome 1. The distribution of grains allowed mapping of the human [delta] opioid receptor gene to the p34.3-p36.1 region of the short arm with a maximum in the p35 band.

  5. Isolation and characterization of CCoAOMT in interspecific hybrid of Acacia auriculiformis x Acacia mangium--a key gene in lignin biosynthesis.

    Science.gov (United States)

    Pang, S L; Ong, S S; Lee, H H; Zamri, Z; Kandasamy, K I; Choong, C Y; Wickneswari, R

    2014-09-05

    This study was directed at the understanding of the function of CCoAOMT isolated from Acacia auriculiformis x Acacia mangium. Full length cDNA of the Acacia hybrid CCoAOMT (AhCCoAOMT) was 1024-bp long, containing 750-bp coding regions, with one major open reading frame of 249 amino acids. On the other hand, full length genomic sequence of the CCoAOMT (AhgflCCoAOMT) was 2548 bp long, containing three introns and four exons with a 5' untranslated region (5'UTR) of 391 bp in length. The 5'UTR of the characterized CCoAOMT gene contains various regulatory elements. Southern analysis revealed that the Acacia hybrid has more than three copies of the CCoAOMT gene. Real-time PCR showed that this gene was expressed in root, inner bark, leaf, flower and seed pod of the Acacia hybrid. Downregulation of the homologous CCoAOMT gene in tobacco by antisense (AS) and intron-containing hairpin (IHP) constructs containing partial AhCCoAOMT led to reduction in lignin content. Expression of the CCoAOMT in AS line (pART-HAS78-03) and IHP line (pART-HIHP78-06) was reduced respectively by 37 and 75% compared to the control, resulting in a decrease in the estimated lignin content by 24 and 56%, respectively. AhCCoAOMT was found to have altered not only S and G units but also total lignin content, which is of economic value to the pulp industry. Subsequent polymorphism analysis of this gene across eight different genetic backgrounds each of A. mangium and A. auriculiformis revealed 47 single nucleotide polymorphisms (SNPs) in A. auriculiformis CCoAOMT and 30 SNPs in A. mangium CCoAOMT.

  6. Electrochemical detection of avian influenza virus H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode

    Energy Technology Data Exchange (ETDEWEB)

    Liu Xianggang [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Cheng Ziqiang, E-mail: czqsd@126.com [College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, Shandong (China); Fan Hai [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Ai Shiyun, E-mail: ashy@sdau.edu.cn [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China); Han Ruixia [College of Chemistry and Material Science, Shandong Agricultural University, Taian 271018, Shandong (China)

    2011-07-15

    Highlights: > A sensitive electrochemical biosensor for the detection of gene sequence was developed. > The biosensor was assembled by MWNT, polypyrrole nanowires and gold nanoparticles. > The hybrid nanomaterials could provide a porous structure with good properties. > The biosensor has highly selectivity and sensitivity. > The design strategy is expected to have extensive applications in other biosensors - Abstract: A sensitive electrochemical method for the detection of avian influenza virus (AIV) H5N1 gene sequence using a DNA aptamer immobilized onto a hybrid nanomaterial-modified electrode was developed. To enhance the selectivity and sensitivity, the modified electrode was assembled with multi-wall carbon nanotubes (MWNT), polypyrrole nanowires (PPNWs) and gold nanoparticles (GNPs). This electrode offered a porous structure with a large effective surface area, highly electrocatalytic activities and electronic conductivity. Therefore, the amount of DNA aptamer immobilized onto the electrode was increased while the accessibility of the detection target was maintained. The biosensor is based on the hybridization and preferred orientation of a DNA aptamer immobilized onto a modified electrode surface with its target (H5N1 specific sequence) present in solution. It is selective for the H5N1 specific sequence, and the signal of the indicator was approximately linear to log(concentration) of the H5N1 specific sequence from 5.0 x 10{sup -12} to 1.0 x 10{sup -9} M (R = 0.9863) with a detection limit of 4.3 x 10{sup -13} M. These studies showed that the new hybrid nanomaterial (MWNT/PPNWs/GNPs) and the DNA aptamer could be used to fabricate an electrochemical biosensor for gene sequence detection. Furthermore, this design strategy is expected to have extensive applications in other biosensors.

  7. Phylogeny-function analysis of (meta)genomic libraries: screening for expression of ribosomal RNA genes by large-insert library fluorescent in situ hybridization (LIL-FISH).

    Science.gov (United States)

    Leveau, Johan H J; Gerards, Saskia; de Boer, Wietse; van Veen, Johannes A

    2004-09-01

    We assessed the utility of fluorescent in situ hybridization (FISH) in the screening of clone libraries of (meta)genomic or environmental DNA for the presence and expression of bacterial ribosomal RNA (rRNA) genes. To establish proof-of-principle, we constructed a fosmid-based library in Escherichia coli of large-sized genomic DNA fragments of the mycophagous soil bacterium Collimonas fungivorans, and hybridized 768 library clones with the Collimonas-specific fluorescent probe CTE998-1015. Critical to the success of this approach (which we refer to as large-insert library FISH or LIL-FISH) was the ability to induce fosmid copy number, the exponential growth status of library clones in the FISH assay and the use of a simple pooling strategy to reduce the number of hybridizations. Twelve out of 768 E. coli clones were suspected to harbour and express Collimonas 16S rRNA genes based on their hybridization to CTE998-1015. This was confirmed by the finding that all 12 clones were also identified in an independent polymerase chain reaction-based screening of the same 768 clones using a primer set for the specific detection of Collimonas 16S ribosomal DNA (rDNA). Fosmids isolated from these clones were grouped by restriction analysis into two distinct contigs, confirming that C. fungivorans harbours at least two 16S rRNA genes. For one contig, representing 1-2% of the genome, the nucleotide sequence was determined, providing us with a narrow but informative view of Collimonas genome structure and content.

  8. Identification of Botrytis cinerea genes up-regulated during infection and controlled by the Galpha subunit BCG1 using suppression subtractive hybridization (SSH).

    Science.gov (United States)

    Schulze Gronover, Christian; Schorn, Corinna; Tudzynski, Bettina

    2004-05-01

    The Galpha subunit BCG1 plays an important role during the infection of host plants by Botrytis cinerea. Delta bcg1 mutants are able to conidiate, penetrate host leaves, and produce small primary lesions. However, in contrast to the wild type, the mutants completely stop invasion of plant tissue at this stage; secondary lesions have never been observed. Suppression subtractive hybridization (SSH) was used to identify fungal genes whose expression on the host plant is specifically affected in bcg1 mutants. Among the 22 differentially expressed genes, we found those which were predicted to encode proteases, enzymes involved in secondary metabolism, and others encoding cell wall-degrading enzymes. All these genes are highly expressed during infection in the wild type but not in the mutant. However, the genes are expressed in both the wild type and the mutant under certain conditions in vitro. Most of the BCG1-controlled genes are still expressed in adenylate cyclase (bac) mutants in planta, suggesting that BCG1 is involved in at least one additional signaling cascade in addition to the cAMP-depending pathway. In a second SSH approach, 1,500 clones were screened for those that are specifically induced by the wild type during the infection of bean leaves. Of the 22 BCG1-controlled genes, 11 also were found in the in planta SSH library. Therefore, SSH technology can be successfully applied to identify target genes of signaling pathways and differentially expressed genes in planta.

  9. Cationic lipid-nanoceria hybrids, a novel nonviral vector-mediated gene delivery into mammalian cells: investigation of the cellular uptake mechanism.

    Science.gov (United States)

    Das, Joydeep; Han, Jae Woong; Choi, Yun-Jung; Song, Hyuk; Cho, Ssang-Goo; Park, Chankyu; Seo, Han Geuk; Kim, Jin-Hoi

    2016-07-06

    Gene therapy is a promising technique for the treatment of various diseases. The development of minimally toxic and highly efficient non-viral gene delivery vectors is the most challenging undertaking in the field of gene therapy. Here, we developed dimethyldioctadecylammonium bromide (DODAB)-nanoceria (CeO2) hybrids as a new class of non-viral gene delivery vectors. These DODAB-modified CeO2 nanoparticles (CeO2/DODAB) could effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines. The CeO2/DODAB nanovectors were also found to be non-toxic and did not induce ROS formation as well as any stress responsive and pro-survival signaling pathways. The overall vector performance of CeO2/DODAB nanohybrids was comparable with lipofectamine and DOTAP, and higher than calcium phosphate and DEAE-dextran for transfecting small plasmids. The increased cellular uptake of the nanovector/DNA complexes through clathrin- and caveolae-mediated endocytosis and subsequent release from the endosomes further support the increased gene transfection efficiency of the CeO2/DODAB vectors. Besides, CeO2/DODAB nanovectors could transfect genes in vivo without any sign of toxicity. Taken together, this new nano-vector has the potential to be used for gene delivery in biomedical applications.

  10. Reduced expression of the SHORT-ROOT gene increases the rates of growth and development in hybrid poplar and Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Jiehua Wang

    Full Text Available SHORT-ROOT (SHR is a well characterized regulator of cell division and cell fate determination in the Arabidopsis primary root. However, much less is known about the functions of SHR in the aerial parts of the plant. In this work, we cloned SHR gene from Populus trichocarpa (PtSHR1 as an AtSHR ortholog and down-regulated its expression in hybrid poplar (Populus tremula×P. tremuloides Michx-clone T89 in order to determine its physiological functions in shoot development. Sharing a 90% similarity to AtSHR at amino acid level, PtSHR1 was able to complement the Arabidopsis shr mutant. Down regulation of PtSHR1 led to a strong enhancement of primary (height and secondary (girth growth rates in the transgenic poplars. A similar approach in Arabidopsis showed a comparable accelerated growth and development phenotype. Our results suggest that the response to SHR could be dose-dependent and that a partial down-regulation of SHR could lead to enhanced meristem activity and a coordinated acceleration of plant growth in woody species. Therefore, SHR functions in plant growth and development as a regulator of cell division and meristem activity not only in the roots but also in the shoots. Reducing SHR expression in transgenic poplar was shown to lead to significant increases in primary and secondary growth rates. Given the current interest in bioenergy crops, SHR has a broader role as a key regulator of whole plant growth and development and SHR suppression has considerable potential for accelerating biomass accumulation in a variety of species.

  11. The low fertility of Chinese white poplar: dynamic changes in anatomical structure, endogenous hormone concentrations, and key gene expression in the reproduction of a naturally occurring hybrid.

    Science.gov (United States)

    Ma, Kaifeng; Song, Yuepeng; Huang, Zhen; Lin, Liyuan; Zhang, Zhiyi; Zhang, Deqiang

    2013-03-01

    KEY MESSAGE : We report that low fertility during intraspecific hybridization in Chinese white poplar was caused by prefertilization barriers, reduced ovules, and embryonic abortion. Hormone concentrations and gene expression patterns were also evaluated during the fertilization process. Hybrid vigor holds tremendous potential for yield increases and trait improvement; however, some hybridization combinations within Populus show very low fertility. To explore the causes of this low fertility in intraspecific hybridization of Chinese white poplar, we examined anatomical structure, hormone levels and expression of key genes in two unique crossing combinations of Populus × tomentosa "Pt02" × P. × tomentosa "LM50", and (P. × tomentosa × P. alba cv. bolleana "Ptb") × P. × tomentosa "LM50". The seed set potential in the intraspecific hybridization P. × tomentosa "Pt02" × P. × tomentosa "LM50" was quite low, which was likely caused by prefertilization barriers, reduced ovule numbers, and embryonic abortion in ovaries. During intraspecific hybridization, we found reduced indoleacetic acid (IAA) in pistils, which may cause pollen tube deformations and increased IAA in heart-stage embryos, which may affect embryo development. Gibberellin A3 (GA3) decreased from the zygote dormancy stage to globular-stage embryos, which may be caused by failure of fertilization in specific embryos. The maximum zeatin (Z) concentration was found in heart-stage embryos, but Z concentrations quickly decreased, which may affect endosperm development. Increasing concentrations of abscisic acid (ABA) during zygote dormancy and eight-cell proembryo stages likely induced abscission of the infructescence. High ABA concentrations also regulated embryo maturity. Measurement of genes expression showed that high expression of SRK and/or SLG may result in rejection of pollen by stigmatic papillae through a mechanism, reminiscent of self-incompatibility. Also, low expression of

  12. Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

    Science.gov (United States)

    Guiliano, D; Ganatra, M; Ware, J; Parrot, J; Daub, J; Moran, L; Brennecke, H; Foster, J M; Supali, T; Blaxter, M; Scott, A L; Williams, S A; Slatko, B E

    1999-07-01

    A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.

  13. Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene.

    Science.gov (United States)

    Cunha, Karin Soares; Oliveira, Nathalia Silva; Fausto, Anna Karoline; de Souza, Carolina Cruz; Gros, Audrey; Bandres, Thomas; Idrissi, Yamina; Merlio, Jean-Philippe; de Moura Neto, Rodrigo Soares; Silva, Rosane; Geller, Mauro; Cappellen, David

    2016-12-17

    Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations.

  14. Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene

    Science.gov (United States)

    Cunha, Karin Soares; Oliveira, Nathalia Silva; Fausto, Anna Karoline; de Souza, Carolina Cruz; Gros, Audrey; Bandres, Thomas; Idrissi, Yamina; Merlio, Jean-Philippe; de Moura Neto, Rodrigo Soares; Silva, Rosane; Geller, Mauro; Cappellen, David

    2016-01-01

    Neurofibromatosis 1 (NF1) is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns) from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11). We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G). Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns) for different types of pathogenic variations, including the deep intronic splicing mutations. PMID:27999334

  15. Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene

    Directory of Open Access Journals (Sweden)

    Karin Soares Cunha

    2016-12-01

    Full Text Available Neurofibromatosis 1 (NF1 is one of the most common genetic disorders and is caused by mutations in the NF1 gene. NF1 gene mutational analysis presents a considerable challenge because of its large size, existence of highly homologous pseudogenes located throughout the human genome, absence of mutational hotspots, and diversity of mutations types, including deep intronic splicing mutations. We aimed to evaluate the use of hybridization capture-based next-generation sequencing to screen coding and noncoding NF1 regions. Hybridization capture-based next-generation sequencing, with genomic DNA as starting material, was used to sequence the whole NF1 gene (exons and introns from 11 unrelated individuals and 1 relative, who all had NF1. All of them met the NF1 clinical diagnostic criteria. We showed a mutation detection rate of 91% (10 out of 11. We identified eight recurrent and two novel mutations, which were all confirmed by Sanger methodology. In the Sanger sequencing confirmation, we also included another three relatives with NF1. Splicing alterations accounted for 50% of the mutations. One of them was caused by a deep intronic mutation (c.1260 + 1604A > G. Frameshift truncation and missense mutations corresponded to 30% and 20% of the pathogenic variants, respectively. In conclusion, we show the use of a simple and fast approach to screen, at once, the entire NF1 gene (exons and introns for different types of pathogenic variations, including the deep intronic splicing mutations.

  16. Microarray hybridization analysis of light-dependent gene expression in Penicillium chrysogenum identifies bZIP transcription factor PcAtfA.

    Science.gov (United States)

    Wolfers, Simon; Kamerewerd, Jens; Nowrousian, Minou; Sigl, Claudia; Zadra, Ivo; Kürnsteiner, Hubert; Kück, Ulrich; Bloemendal, Sandra

    2015-04-01

    The fungal velvet complex is a light-dependent master regulator of secondary metabolism and development in the major penicillin producer, Penicillium chrysogenum. However, the light-dependent mechanism is unclear. To identify velvet-dependent transcriptional regulators that show light-regulated expression, we performed microarray hybridizations with RNA isolated from P. chrysogenum ΔPcku70 cultures grown under 13 different long-term, light-dependent growth conditions. We compared these expression data to data from two velvet complex deletion mutants; one lacked a subunit of the velvet complex (ΔPcvelA), and the other lacked a velvet-associated protein (ΔPclaeA). We sought to identify genes that were up-regulated in light, but down-regulated in ΔPcvelA and ΔPclaeA. We identified 148 co-regulated genes that displayed this regulatory pattern. In silico analyses of the co-regulated genes identified six proteins with fungal-specific transcription factor domains. Among these, we selected the bZIP transcription factor, PcAtfA, for functional characterization in deletion and complementation strains. Our data clearly indicates that PcAtfA governs spore germination. This comparative analysis of different microarray hybridization data sets provided results that may be useful for identifying genes for future functional analyses.

  17. Analysis of Differential Gene Expression Pattern in Brassica napus Hybrid Huayouza 6 and Its Parents Using Arabidopsis cDNA Microarray

    Institute of Scientific and Technical Information of China (English)

    SHEN Jun-ru; WU Jian-yong; ZHANG Jian; LIU Ping-wu; YANG Guang-sheng

    2006-01-01

    Huayouza 6, a new semi-winter Brassica napus variety with high-yield, good quality, prematurity and extensive adaptability, was derived from the cross between the female parent 8086A and male parent 7-5. Two cDNA-based Arabidopisis microarray were used to analyze gene differential expression in bud of an elite B. napus hybrid Huayouza6 and its parents,in which there were 83 over-expression transcripts and 331 under-expression transcripts between Huayouza 6 and its female parent 8086A and 94 over-expression transcripts, and 423 under-expression transcripts were demonstrated betweenHuayouza 6 and its male parent 7-5. Further analysis showed that there were significant number of genes responsible for photosynthesis, and its implication for heterosis was discussed. Northern analysis of phosphoribulokinase coincided with its expression pattern derived from hybridization of Arabidopsis cDNA microarray and B. napus mRNA, this system of heterologous hybridization analysis should be applicable to other close relatives of Arabidopsis thaliana.

  18. Ecological differentiation, lack of hybrids involving diploids, and asymmetric gene flow between polyploids in narrow contact zones of Senecio carniolicus (syn. Jacobaea carniolica, Asteraceae).

    Science.gov (United States)

    Hülber, Karl; Sonnleitner, Michaela; Suda, Jan; Krejčíková, Jana; Schönswetter, Peter; Schneeweiss, Gerald M; Winkler, Manuela

    2015-03-01

    Areas of immediate contact of different cytotypes offer a unique opportunity to study evolutionary dynamics within heteroploid species and to assess isolation mechanisms governing coexistence of cytotypes of different ploidy. The degree of reproductive isolation of cytotypes, that is, the frequency of heteroploid crosses and subsequent formation of viable and (partly) fertile hybrids, plays a crucial role for the long-term integrity of lineages in contact zones. Here, we assessed fine-scale distribution, spatial clustering, and ecological niches as well as patterns of gene flow in parental and hybrid cytotypes in zones of immediate contact of di-, tetra-, and hexaploid Senecio carniolicus (Asteraceae) in the Eastern Alps. Cytotypes were spatially separated also at the investigated microscale; the strongest spatial separation was observed for the fully interfertile tetra- and hexaploids. The three main cytotypes showed highly significant niche differences, which were, however, weaker than across their entire distribution ranges in the Eastern Alps. Individuals with intermediate ploidy levels were found neither in the diploid/tetraploid nor in the diploid/hexaploid contact zones indicating strong reproductive barriers. In contrast, pentaploid individuals were frequent in the tetraploid/hexaploid contact zone, albeit limited to a narrow strip in the immediate contact zone of their parental cytotypes. AFLP fingerprinting data revealed introgressive gene flow mediated by pentaploid hybrids from tetra- to hexaploid individuals, but not vice versa. The ecological niche of pentaploids differed significantly from that of tetraploids but not from hexaploids.

  19. Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing.

    Science.gov (United States)

    Walther, Charles; Hofvander, Jakob; Nilsson, Jenny; Magnusson, Linda; Domanski, Henryk A; Gisselsson, David; Tayebwa, Johnbosco; Doyle, Leona A; Fletcher, Christopher D M; Mertens, Fredrik

    2015-09-01

    Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNA-Seq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues.

  20. Suppression subtractive hybridization (SSH) combined with bioinformatics method: an integrated functional annotation approach for analysis of differentially expressed immune-genes in insects.

    Science.gov (United States)

    Badapanda, Chandan

    2013-01-01

    The suppression subtractive hybridization (SSH) approach, a PCR based approach which amplifies differentially expressed cDNAs (complementary DNAs), while simultaneously suppressing amplification of common cDNAs, was employed to identify immuneinducible genes in insects. This technique has been used as a suitable tool for experimental identification of novel genes in eukaryotes as well as prokaryotes; whose genomes have been sequenced, or the species whose genomes have yet to be sequenced. In this article, I have proposed a method for in silico functional characterization of immune-inducible genes from insects. Apart from immune-inducible genes from insects, this method can be applied for the analysis of genes from other species, starting from bacteria to plants and animals. This article is provided with a background of SSH-based method taking specific examples from innate immune-inducible genes in insects, and subsequently a bioinformatics pipeline is proposed for functional characterization of newly sequenced genes. The proposed workflow presented here, can also be applied for any newly sequenced species generated from Next Generation Sequencing (NGS) platforms.

  1. Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer: Fluorescence in situ hybridization analysis of a tissue microarray.

    Science.gov (United States)

    Hu, Xiaoyu; Yang, Zhu; Zeng, Manman; Liu, Y I; Yang, Xiaotao; Li, Yanan; Li, X U; Yu, Qiubo

    2016-07-01

    The aim of the present study was to investigate the status of speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) gene located on chromosome 17q21 in ovarian cancer (OC). The present study evaluated a tissue microarray, which contained 90 samples of ovarian cancer and 10 samples of normal ovarian tissue, using fluorescence in situ hybridization (FISH). FISH is a method where a SPOP-specific DNA red fluorescence probe was used for the experimental group and a centromere-specific DNA green fluorescence probe for chromosome 17 was used for the control group. The present study demonstrated that a deletion of the SPOP gene was observed in 52.27% (46/88) of the ovarian cancer tissues, but was not identified in normal ovarian tissues. Simultaneously, monosomy 17 was frequently identified in the ovarian cancer tissues, but not in the normal ovarian tissues. Furthermore, the present data revealed that the ovarian cancer histological subtype and grade were significantly associated with a deletion of the SPOP gene, which was assessed by the appearance of monosomy 17 in the ovarian cancer samples; the deletion of the SPOP gene was observed in a large proportion of serous epithelial ovarian cancer (41/61; 67.21%), particularly in grade 3 (31/37; 83.78%). In conclusion, deletion of the SPOP gene on chromosome 17 in ovarian cancer samples, which results from monosomy 17, indicates that the SPOP gene may serve as a tumor suppressor gene in ovarian cancer.

  2. Screening of candidate genes encoding proteins expressed in pectoral fins of fugu Takifugu rubripes, in relation to habitat site of parasitic copepod Caligus fugu, using suppression subtractive hybridization.

    Science.gov (United States)

    Tasumi, Satoshi; Norshida, Ismail; Boxshall, Geoffrey A; Kikuchi, Kiyoshi; Suzuki, Yuzuru; Ohtsuka, Susumu

    2015-05-01

    Caligus fugu is a parasitic copepod specific to the tetraodontid genus Takifugu including the commercially important Takifugu rubripes. Despite the rapid accumulation of knowledge on other aspects of its biology, the host and settlement-site recognition mechanisms of this parasite are not yet well understood. Since the infective copepodid stage shows preferential site selection in attaching to the fins, we considered it likely that the copepodid recognizes chemical cues released or leaking from the fins, and/or transmembrane protein present on the fins. To isolate molecules potentially related to attachment site specificity, we applied suppression subtractive hybridization (SSH) PCR by identifying genes expressed more highly in pectoral fins of T. rubripes than in the body surface skin. We sequenced plasmid DNA from 392 clones in a SSH library. The number of non-redundant sequences was 276, which included 135 sequences located on 117 annotated genes and 141 located in positions where no genes had been annotated. We characterized those annotated genes on the basis of gene ontology terms, and found that 46 of the identified genes encode secreted proteins, enzymes or membrane proteins. Among them nine showed higher expression in the pectoral fins than in the skin. These could be candidate genes for involvement in behavioral mechanisms related to the site specificity shown by the infective copepodids of C. fugu.

  3. A duplicated coxI gene is associated with cytoplasmic male sterility in an alloplasmic Brassica juncea line derived from somatic hybridization with Diplotaxis catholica

    Indian Academy of Sciences (India)

    Aruna Pathania; Rajesh Kumar; V. Dinesh Kumar; Ashutosh; K. K. Dwivedi; P. B. Kirti; P. Prakash; V. L. Chopra; S. R. Bhat

    2007-08-01

    A cytoplasmic male sterile (CMS) line of Brassica juncea was derived by repeated backcrossing of the somatic hybrid (Diplotaxis catholica + B. juncea) to B. juncea. The new CMS line is comparable to euplasmic lines for almost all characters, except for flowers which bear slender, needle-like anthers with aborted pollen. Detailed Southern analysis revealed two copies of coxI gene in the CMS line. One copy, coxI-1 is similar to the coxI gene of B. juncea, whereas the second copy, coxI-2 is present in a novel rearranged region. Northern analysis with eight mitochondrial gene probes showed altered transcript pattern only for the coxI gene. Two transcripts of 2.0 and 2.4 kb, respectively, were detected in the CMS line. The novel 2.4 kb transcript was present in floral bud tissue but absent in the leaf tissue. In plants where male sterility broke down under high temperature during the later part of the growing season, the 2.4 kb coxI transcript was absent, which suggested its association with the CMS. The two coxI genes from the CMS line showed two amino acid changes in the coding region. The novel coxI gene showed unique repeats in the 5′ region suggesting recombination of mitochondrial genomes of the two species. The possible role of the duplicated coxI gene in causing male sterility is discussed.

  4. Recombinant hybrid infectious hematopoietic necrosis virus (IHNV) carrying viral haemorrhagic septicaemia virus (VHSV) G or NV genes show different virulence properities

    DEFF Research Database (Denmark)

    Einer-Jensen, Katja; Biacchesi, S.; Stegmann, Anders

    Viral haemorrhagic septicaemia virus (VHSV) is the economically most important viral disease in European rainbow trout farming. The virus was introduced to fresh water farms in the 1950ies from a reservoir of VHSV in the marine environment. Isolates from wild marine fish and fresh water farms....... By a reverse genetics approach using the related novirrhabdovirus infectious hematopoietic necrosis virus (IHNV) as basis, four hybrid IHNV-VHSV variants were generated. These chimeric variants included substitution of the IHNV glyco(G) or nonstrutrual (Nv) protein with the corresponding G or Nv-protein from...... or fresh water VHSV. Recombinant IHNV gained higher virulence following substitution of the homologous G gene with the VHSV G gene, while the opposite was the case following substitution of the Nv gene. These findings suggest that higher virulence of VHSV compared to IHNV might be related to the G protein...

  5. Temporal shift of circadian-mediated gene expression and carbon fixation contributes to biomass heterosis in maize hybrids

    Science.gov (United States)

    Heterosis has been widely used in agriculture, but the molecular mechanism for this remains largely elusive. In Arabidopsis hybrids and allopolyploids, increased photosynthetic and metabolic activities are linked to altered expression of circadian clock regulators, including CIRCADIAN CLOCK ASSOCIAT...

  6. Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, D.R.; Fazio, M.J.; Shamban, A.T.; Rosenbloom, J.; Uitto, J.

    1988-05-15

    Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approx. 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutix laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

  7. The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization

    Directory of Open Access Journals (Sweden)

    Lo Fang-Yi

    2012-06-01

    Full Text Available Abstract Background Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis. Methods Array-comparative genomic hybridization (array-CGH was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR, chromogenic in situ hybridization (CISH, reverse transcriptase-qPCR (RT-qPCR, and immunohistochemistry (IHC in more patients. Results We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1 functioning in Rho activity control, FRAT2 (10q24.1 involved in Wnt signaling, PAFAH1B1 (17p13.3 functioning in motility control, and ZNF322A (6p22.1 involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (PP=0.06. In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of

  8. Climate-Driven Reshuffling of Species and Genes: Potential Conservation Roles for Species Translocations and Recombinant Hybrid Genotypes

    Directory of Open Access Journals (Sweden)

    Jon Mark Scriber

    2013-12-01

    Full Text Available Comprising 50%–75% of the world’s fauna, insects are a prominent part of biodiversity in communities and ecosystems globally. Biodiversity across all levels of biological classifications is fundamentally based on genetic diversity. However, the integration of genomics and phylogenetics into conservation management may not be as rapid as climate change. The genetics of hybrid introgression as a source of novel variation for ecological divergence and evolutionary speciation (and resilience may generate adaptive potential and diversity fast enough to respond to locally-altered environmental conditions. Major plant and herbivore hybrid zones with associated communities deserve conservation consideration. This review addresses functional genetics across multi-trophic-level interactions including “invasive species” in various ecosystems as they may become disrupted in different ways by rapid climate change. “Invasive genes” (into new species and populations need to be recognized for their positive creative potential and addressed in conservation programs. “Genetic rescue” via hybrid translocations may provide needed adaptive flexibility for rapid adaptation to environmental change. While concerns persist for some conservationists, this review emphasizes the positive aspects of hybrids and hybridization. Specific implications of natural genetic introgression are addressed with a few examples from butterflies, including transgressive phenotypes and climate-driven homoploid recombinant hybrid speciation. Some specific examples illustrate these points using the swallowtail butterflies (Papilionidae with their long-term historical data base (phylogeographical diversity changes and recent (3-decade climate-driven temporal and genetic divergence in recombinant homoploid hybrids and relatively recent hybrid speciation of Papilio appalachiensis in North America. Climate-induced “reshuffling” (recombinations of species composition, genotypes

  9. Identification of gene products suppressed by human immunodeficiency virus type 1 infection or gp120 exposure of primary human astrocytes by rapid subtraction hybridization.

    Science.gov (United States)

    Su, Zao-Zhong; Kang, Dong-Chul; Chen, Yinming; Pekarskaya, Olga; Chao, Wei; Volsky, David J; Fisher, Paul B

    2003-06-01

    Neurodegeneration and human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are the major disease manifestations of HIV-1 colonization of the central nervous system (CNS). In the brain, HIV-1 replicates in microglial cells and infiltrating macrophages and it persists in a low-productive, noncytolytic state in astrocytes. Astrocytes play critical roles in the maintenance of the brain microenvironment, responses to injury, and in neuronal signal transmission, and disruption of these functions by HIV-1 could contribute to HAD. To better understand the potential effects of HIV-1 on astrocyte biology, the authors investigated changes in gene expression using an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue, low-passage cells were infected with HIV-1 or mock infected, and total cellular RNAs were isolated at multiple time points over a period of 1 week. This approach is designed to identify gene products modulated early and late after HIV-1 infection and limits the cloning of genes displaying normal cell-cycle fluctuations in astrocytes. By subtracting temporal cDNAs derived from HIV-1-infected astrocytes from temporal cDNAs made from uninfected cells, 10 genes displaying reduced expression in infected cells, termed astrocyte suppressed genes (ASGs), were identified and their suppression was confirmed by Northern blot hybridization. Both known and novel ASGs, not reported in current DNA databases, that are down-regulated by HIV-1 infection are described. Northern blotting confirms suppression of the same panel of ASGs by treatment of astrocytes with recombinant HIV-1 envelope glycoprotein, gp120. These results extend our previous analysis of astrocyte genes induced or enhanced by HIV-1 infection and together they suggest that HIV-1 and viral proteins have profound effects on astrocyte physiology, which may influence their function in the CNS.

  10. Suppression subtractive hybridization library construction and identification of epidermal bladder cell related genes in the common ice plant, Mesembryanthemum crystallinum L.

    Directory of Open Access Journals (Sweden)

    Siranet Roeurn

    2016-10-01

    Full Text Available Mesembryanthemum crystallinum L., a halophytic species, displays modified trichomes, epidermal bladder cells (EBC, on the surfaces of its aerial organs. EBCs serve to sequester excessive salt from underlying metabolically active tissues. To elucidate the molecular determinants governing EBC development in the common ice plant, we constructed a cDNA-based suppression subtractive hybridization library and identified genes differentially expressed between the wild-type and the EBC-less mutant. After hybridization, 38 clones were obtained. Among them, 24 clones had homology with plant genes of known functions, whose roles might not be directly related to EBC-morphology, while 14 clones were homologous to genes of unknown functions. After confirmation by northern blot analysis, 12 out of 14 clones of unknown functions were chosen for semi-quantitative RT-PCR analysis, and the results revealed that three clones designated as MW3, MW21, and MW31 preferentially expressed in the EBC-less mutant, whereas the other two designated as WM10 and WM28 preferentially expressed in the wild type. Among these genes, the expression of a putative jasmonate-induced gene, designated as WM28 was completely suppressed in the EBC-mutant. In addition, the deletion of C-box cis-acting element was found in the promoter region of WM28 in the EBC-less mutant. Overexpression of WM28 in Arabidopsis resulted in increased trichome number due to the upregulation of key trichome-related genes GLABRA1 (GL1, and GLABRA3 (GL3. These results demonstrate that WM28 can be an important factor responsible for EBC formation, and also suggest the similarity of developmental mechanism between trichome in Arabidopsis and EBC in common ice plant.

  11. Microarray analysis of gene expression patterns in the leaf during potato tuberization in the potato somatic hybrid Solanum tuberosum and Solanum etuberosum.

    Science.gov (United States)

    Tiwari, Jagesh Kumar; Devi, Sapna; Sundaresha, S; Chandel, Poonam; Ali, Nilofer; Singh, Brajesh; Bhardwaj, Vinay; Singh, Bir Pal

    2015-06-01

    Genes involved in photoassimilate partitioning and changes in hormonal balance are important for potato tuberization. In the present study, we investigated gene expression patterns in the tuber-bearing potato somatic hybrid (E1-3) and control non-tuberous wild species Solanum etuberosum (Etb) by microarray. Plants were grown under controlled conditions and leaves were collected at eight tuber developmental stages for microarray analysis. A t-test analysis identified a total of 468 genes (94 up-regulated and 374 down-regulated) that were statistically significant (p ≤ 0.05) and differentially expressed in E1-3 and Etb. Gene Ontology (GO) characterization of the 468 genes revealed that 145 were annotated and 323 were of unknown function. Further, these 145 genes were grouped based on GO biological processes followed by molecular function and (or) PGSC description into 15 gene sets, namely (1) transport, (2) metabolic process, (3) biological process, (4) photosynthesis, (5) oxidation-reduction, (6) transcription, (7) translation, (8) binding, (9) protein phosphorylation, (10) protein folding, (11) ubiquitin-dependent protein catabolic process, (12) RNA processing, (13) negative regulation of protein, (14) methylation, and (15) mitosis. RT-PCR analysis of 10 selected highly significant genes (p ≤ 0.01) confirmed the microarray results. Overall, we show that candidate genes induced in leaves of E1-3 were implicated in tuberization processes such as transport, carbohydrate metabolism, phytohormones, and transcription/translation/binding functions. Hence, our results provide an insight into the candidate genes induced in leaf tissues during tuberization in E1-3.

  12. The oxytocin receptor gene (OXTR) localizes to human chromosome 3p25 by fluorescence in situ hybridization and PCR analysis of somatic cell hybrids

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, C.F. Jr.; Clancy, T.E.; Quan, R. [Children`s Hospital, Boston, MA (United States)] [and others

    1995-04-10

    The human oxytocin receptor regulates parturition and myometrial contractility, breast milk let-down, and reproductive behaviors in the mammalian central nervous system. Kimura et al. recently identified a human oxytocin receptor cDNA by means of expression cloning from a human myometrial cDNA library. To elucidate further the molecular mechanisms that regulate oxytocin receptor gene expression and to define the expected Mendelian inheritance of possible human disease states, we must determine the number of genes, their localization, and their organization and structure. We summarize below our data indicating that the human oxytocin receptor gene is localized to 3p25 and exists as a single copy in the haploid genome. 9 refs., 2 figs.

  13. The correlation between dual-color chromogenic in situ hybridization and fluorescence in situ hybridization in assessing HER2 gene amplification in breast cancer

    DEFF Research Database (Denmark)

    Pedersen, Marianne; Rasmussen, Birgitte Bruun

    2009-01-01

    obtained by the 2 methods showed excellent correlation (correlation coefficient 0.95). In conclusion, it is possible by dual-color CISH method to demonstrate HER2 genes and chromosome 17 genes, in the same tissue section and reliably assess HER2 status. The CISH method is a very promising alternative...... invasive breast carcinomas, both FISH and CISH detected HER2 amplification in 24 cases and nonamplification was detected in 47 cases. One case showed a discrepancy between FISH and CISH. The concordance between CISH and FISH was found to be almost perfect (98.6%). The correlation between the HER2 ratios...

  14. Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization.

    Science.gov (United States)

    Nasopoulou, Constantina; Pohjanen, Johanna; Koskimäki, Janne J; Zabetakis, Ioannis; Pirttilä, Anna Maria

    2014-08-15

    Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF.

  15. Identification of a Differentially Expressed Gene PPP1CB between Porcine Longissimus dorsi of Meishan and Large White×Meishan Hybrids

    Institute of Scientific and Technical Information of China (English)

    Tao HUANG; Yuan-Zhu XIONG; Ming-Gang LEI; De-Quan XU; Chang-Yan DENG

    2006-01-01

    To study the molecular basis of heterosis, suppression subtractive hybridization was used to investigate the differences in gene expression between porcine Longissimus dorsi of F1 hybrids Large White×Meishan and their female parents Meishan. From two specific subtractive cDNA libraries, the clones selected by reverse Northern high-density blot screening were chosen to clone full-length cDNA by rapid amplification of cDNA ends. An expression-upregulated gene for Meishan skeletal muscle, designated protein phosphatase 1, catalytic subunit, beta isoform (PPP1CB), was identified. Porcine PPP1CB contains an open reading frame encoding 327 amino acid residues with 13 and 1763 nucleotides in the 5′ and 3′ untranslated regions, respectively. A DNA fragment of 721 nucleotides was amplified and a mutation that creates/disrupts a restriction site for endonuclease RsaI was found. The derived amino acid sequence of PPP1CB has high homology with the PPP1CB of three species, Mus musculus (99%), human (99%) and mouse (100%). The tissue expression analysis indicated that the swine PPP1CB gene is generally expressed in most tissues. The possible role of PPP1CB and its relation to porcine heterosis are discussed.

  16. Comparative analysis of gene expression at early seedling stage between a rice hybrid and its parents using a cDNA microarray of 9198 uni-sequences

    Institute of Scientific and Technical Information of China (English)

    HUANG; Yi; LI; Lihua; CHEN; Ying; LI; Xianghua; XU; Caiguo; WANG; Shiping; ZHANG; Qifa

    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.

  17. Analysis of differentially expressed genes in the precocious line of Eimeria maxima and its parent strain using suppression subtractive hybridization and cDNA microarrays.

    Science.gov (United States)

    Dong, Hui; Lin, Jiaojiao; Han, Hongyu; Jiang, Lianlian; Zhao, Qiping; Zhu, Shunhai; Huang, Bing

    2011-04-01

    The precocious line of Eimeria spp., obtained by repeated passages of oocysts initially collected from feces of previously infected chickens, has unique phenotypes and plays an important role in immunizing chickens against coccidiosis. However, the genetic basis of precocious phenotype in Eimeria is still poorly understood. To investigate gene expression changes in sporulated oocysts between the precocious line of E. maxima and its parent strain, subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH). A total of 3,164 cDNA fragments were selected from the SSH cDNA libraries to fabricate cDNA microarrays and further identify the differentially expressed genes. The credibility of the microarray data was verified by real-time PCR. A total of 360 valid expressed sequence tags (ESTs) were obtained, which represented 32 unique sequences. Twenty-one genes were validated as downregulated and 11 genes as upregulated in the precocious line. Homology searching of the public sequence database showed that six genes encoded proteins homologous with previously reported proteins, including rhomboid-like protein and transhydrogenase of E. tenella, serpin, and cation-transporting ATPase of E. acervulina, a heat-shock protein of E. maxima, and a conserved hypothetical protein of Toxoplasma gondii. Thus, the remaining 26 ESTs have not been previously reported. Further characterization of these differentially expressed genes will be useful in understanding the genetic basis for the precocious phenotype in Eimeria spp.

  18. Identification of differentially expressed genes in the oviduct of two rabbit lines divergently selected for uterine capacity using suppression subtractive hybridization.

    Science.gov (United States)

    Ballester, M; Castelló, A; Peiró, R; Argente, M J; Santacreu, M A; Folch, J M

    2013-06-01

    Suppressive subtractive hybridization libraries from oviduct at 62 h post-mating of two lines of rabbits divergently selected for uterine capacity were generated to identify differentially expressed genes. A total of 438 singletons and 126 contigs were obtained by cluster assembly and sequence alignment of 704 expressed sequence tags (ESTs), of which 54% showed homology to known proteins of the non-redundant NCBI databases. Differential screening by dot blot validated 71 ESTs, of which 47 showed similarity to known genes. Transcripts of genes were functionally annotated in the molecular function and the biological process gene ontology categories using the BLAST2GO software and were assigned to reproductive developmental process, immune response, amino acid metabolism and degradation, response to stress and apoptosis terms. Finally, three interesting genes, PGR, HSD17B4 and ERO1L, were identified as overexpressed in the low line using RT-qPCR. Our study provides a list of candidate genes that can be useful to understanding the molecular mechanisms underlying the phenotypic differences observed in early embryo survival and development traits.

  19. Analysis of gene expression patterns by microarray hybridization in blood mononuclear cells of SLA-DRB1 defined Canadian Yorkshire pigs.

    Science.gov (United States)

    Nino-Soto, Maria I; Jozani, Razi Jafari; Bridle, Byram; Mallard, Bonnie A

    2008-06-23

    The Swine Leukocyte Antigen (SLA) system encodes molecules for self-nonself discrimination and is associated with immune responses and disease resistance. Three lines of pigs defined by their SLA-DRB1 alleles were developed at the University of Guelph for xenotransplantation and immune response studies. The aim of this project was to explore the potential association between defined SLA-DRB1 alleles and gene transcriptional patterns of other immune-related genes in blood mononuclear cells. Three SLA-DRB1 alleles were characterized using a RT-PCR-based sequencing method. The loci represented included a new allele, DRB1*04ns01. Next, microarray heterologous (bovine-porcine) hybridization together with qPCR were used to explore differential gene expression between SLA-DRB1-defined groups. Microarray analysis showed significant (p SLA-DRB1 allele was characterized. A potential association was found between SLA-DRB1 alleles and inflammation-related genes. However, the influence of other genes cannot be ruled out. These preliminary findings agree with other studies linking MHC haplotypes and inflammation processes, including autoimmune disease. The study provides an initial view of the biological interactions between the SLA complex and other immune-related genes. Future studies will focus on characterization of SLA-haplotypes associated with these particular alleles and the dynamics of the immune response to antigenic challenges.

  20. Genes with Restricted Introgression in a Field Cricket (Gryllus firmus/Gryllus pennsylvanicus) Hybrid Zone Are Concentrated on the X Chromosome and a Single Autosome.

    Science.gov (United States)

    Maroja, Luana S; Larson, Erica L; Bogdanowicz, Steven M; Harrison, Richard G

    2015-08-26

    Characterizing the extent of genomic differentiation between recently diverged lineages provides an important context for understanding the early stages of speciation. When such lineages form discrete hybrid zones, patterns of differential introgression allow direct estimates of which genome regions are likely involved in speciation and local adaptation. Here we use a backcross experimental design to construct a genetic linkage map for the field crickets Gryllus firmus and Gryllus pennsylvanicus, which interact in a well-characterized hybrid zone in eastern North America. We demonstrate that loci with major allele frequency differences between allopatric populations are not randomly distributed across the genome. Instead, most are either X-linked or map to a few small autosomal regions. Furthermore, the subset of those highly differentiated markers that exhibit restricted introgression across the cricket hybrid zone are also concentrated on the X chromosome (39 of 50 loci) and in a single 7-cM region of one autosome. Although the accumulation on the sex chromosome of genes responsible for postzygotic barriers is a well-known phenomenon, less attention has been given to the genomic distribution of genes responsible for prezygotic barriers. We discuss the implications of our results for speciation, both in the context of the role of sex chromosomes and also with respect to the likely causes of heterogeneous genomic divergence. Although we do not yet have direct evidence for the accumulation of ecological, behavioral, or fertilization prezygotic barrier genes on the X chromosome, faster-X evolution could make these barriers more likely to be X-linked.

  1. A fragmented adeno-associated viral dual vector strategy for treatment of diseases caused by mutations in large genes leads to expression of hybrid transcripts

    Science.gov (United States)

    McClements, Michelle E.; Charbel Issa, Peter; Blouin, Véronique; MacLaren, Robert E.

    2017-01-01

    Objective Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems. Methods Oversized (>8kb) transgene constructs containing ABCA4 coding sequence were packaged as truncated fragments <5kb in size into various AAV serotypes. In vitro transductions with these fAAV vector preparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and western blot techniques. Results Transductions with fAAV vector preparations yielded ABCA4 mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full length ABCA4 CDS with additional hybrid ABCA4 variants, indicating truncated transgenes without regions of overlap were joining and forming stable hybrid transgenes. In contrast, an ABCA4 overlapping dual vector system (OV) with a defined complementary region generated only full length mRNA transcripts plus detectable ABCA4 protein. Conclusion Despite previous success shown with the fAAV approach, the lack of repeatability and identification of stable hybrid transcripts capable of protein production suggests there is more refinement required before considering this approach in a clinical setting. PMID:28239514

  2. Exclusion of APC and VHL gene deletions by array-based comparative hybridization in two patients with microscopically visible chromosomal aberrations.

    Science.gov (United States)

    Wallerstein, Robert J; Brooks, Susan Sklower; Streck, Deanna L; Kurvathi, Rohini; Toruner, Gokce A

    2007-10-15

    Karyotyping is a major component of the genetic work-up of patients with dysmorphism. Cytogenetic aberrations close to a known tumor suppressor gene raise important clinical issues because deletion of that tumor suppressor gene can cause genetic predisposition to cancer. We present two cancer-free dysmorphic patients with karyotypes of 46,XX,del(5)(q15q22.3) and 46,XX,del(3)(p25.2~pter). These deletions are close to the APC and VHL genes that confer susceptibility to familial Adenomatous polyposis (OMIM #17510) and von-Hippel-Lindau syndrome (OMIM #193300), respectively. The array-based comparative genomic hybridization (array-CGH) analysis using a custom Agilent 44K oligonucleotide array demonstrated an interstitial 20.7-megabase (Mb) deletion on 5q (chr5: 89,725,638-110,491,345) and a terminal 9.45-Mb deletion on 3p (chr3:pter-9,450,984). According to the March 2006 human reference sequence, the APC gene is located at chr5: 112,101,483-112,209,835 and the VHL gene is located at chr3: 10,158,319-10,168,746. These results indicate that the APC gene is 2,300 kilobases (kb) and the VHL gene is 700 kb away from deleted regions. Southern blot analysis for APC and VHL genes were negative, consistent with array-CGH findings. These results demonstrate the power of array-CCH to assess potential tumor suppressor gene involvement and cancer risk in patients with microscopically visible deletions in areas near tumor suppressors.

  3. Construction of a repeat-free dual color fluorescent in situ hybridization probe for ROS1 gene in non-small cell lung cancer diagnosis

    Institute of Scientific and Technical Information of China (English)

    程弘夏

    2014-01-01

    Objective To establish a repeat-free ROS1 gene fluorescence in situ hybridization(FISH)probe,and to compare its efficacy with those of commercial FISH probes in non-small cell lung cancer.Methods The probe was constructed by combining human Cot-1 DNA genome into double-stranded sequence,and then digested by duples specific nuclease to establish a repeat-free sequene.The final repeat-free ROS1 FISH probe was labeled by red and green fluoresceins.Results Compared

  4. A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis

    Directory of Open Access Journals (Sweden)

    Tsalavouta Matina

    2007-02-01

    Full Text Available Abstract Background Comparative teleost studies are of great interest since they are important in aquaculture and in evolutionary issues. Comparing genomes of fully sequenced model fish species with those of farmed fish species through comparative mapping offers shortcuts for quantitative trait loci (QTL detections and for studying genome evolution through the identification of regions of conserved synteny in teleosts. Here a comparative mapping study is presented by radiation hybrid (RH mapping genes of the gilthead sea bream Sparus aurata, a non-model teleost fish of commercial and evolutionary interest, as it represents the worldwide distributed species-rich family of Sparidae. Results An additional 74 microsatellite markers and 428 gene-based markers appropriate for comparative mapping studies were mapped on the existing RH map of Sparus aurata. The anchoring of the RH map to the genetic linkage map resulted in 24 groups matching the karyotype of Sparus aurata. Homologous sequences to Tetraodon were identified for 301 of the gene-based markers positioned on the RH map of Sparus aurata. Comparison between Sparus aurata RH groups and Tetraodon chromosomes (karyotype of Tetraodon consists of 21 chromosomes in this study reveals an unambiguous one-to-one relationship suggesting that three Tetraodon chromosomes correspond to six Sparus aurata radiation hybrid groups. The exploitation of this conserved synteny relationship is furthermore demonstrated by in silico mapping of gilthead sea bream expressed sequence tags (EST that give a significant similarity hit to Tetraodon. Conclusion The addition of primarily gene-based markers increased substantially the density of the existing RH map and facilitated comparative analysis. The anchoring of this gene-based radiation hybrid map to the genome maps of model species broadened the pool of candidate genes that mainly control growth, disease resistance, sex determination and reversal, reproduction as well

  5. HybHyp--hybridizing the host: the long reach of parasite genes. A new hypothesis to explain host-parasite interrelationships in plant hybrid complexes.

    Science.gov (United States)

    Wissemann, Volker

    2010-12-01

    Ever since existence of sexuality in plants was accepted in around 1700, questions centred about the role and maintenance of sexual reproduction in general, leading to a number of hypotheses like the Vicar of Bray, the Ratchet or the Hitch-hiker theory. Bell (The masterpiece of nature. The evolution and genetics of sexuality. University of California Press, Berkeley, LA, 1982) formulated the Red Queen Hypothesis (RQH) which explains the persistence of sexual reproduction as an outcome of a coevolutionary arms race between hosts and parasites. By sexual recombination and genetic diversification hosts minimize the risk of pathogen infection. Since virulence of pathogens is genetically determined and often species specific, parasites are mostly adapted to common host genotypes, whereas rare and divergent genotypes are less infected and therefore have a selective advantage. Employing Dawkins (The extended phenotype. The long reach of the gene, 1999) central theorem of the extended phenotype to the RQH, mating systems in hosts might be a result of the long reach of the parasites genes. Here now the hypothesis is proposed, that evolution by hybridisation and polyploidy in host plants is an extended phenotype of parasites, a response of hosts triggered by the parasites genes to slow down the effects of the Red Queen strategy of plants. Thus, hybridisation and polyploidy might have evolved by parasite pressure and not by host strategy. This hypothesis is called the "hybridisation-of-the-host-hypothesis".

  6. Polymerase chain reaction-hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation.

    Science.gov (United States)

    Xu, Chen; Zhang, Nan; Huo, Qianyu; Chen, Minghui; Wang, Rengfeng; Liu, Zhili; Li, Xue; Liu, Yunde; Bao, Huijing

    2016-04-15

    In this article, we discuss the polymerase chain reaction (PCR)-hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA-BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase-streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR-hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR-hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.

  7. [Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach].

    Science.gov (United States)

    Zhenlong, Cao; Jiangye, Hao; Yanfen, Zhou; Zhe, Zhang; Zhihua, Ni; Yuanxiang, Hu; Weili, Liu; Yongchao, Li; Daniel, R Storm; Runlin, Z Ma; Zhenshan, Wang

    2014-06-01

    Adenylate cyclase 3 (AC3) is one of the major players in the olfactory signaling within the main olfactory epithelium (MOE) of mice. However, we are not ascertained whether deficiency of AC3 will lead to the differential expression of related genes in the MOE. Forward and reverse subtractive libraries were constructed by suppression subtractive hybridization (SSH) approach, with MOEs from AC3(-/-) and AC3(+/+) mice. These two libraries were primarily screened by Dot blot, differential expressed clones were sequenced and analyzed by bioinformatics, and differential expressed genes were verified by qRT-PCR. A total of 386 differentially expressed clones were picked out after Dot blot. The DNA sequences of 80 clones randomly selected were determined, and 62 clones were identified by blasting in GenBank. We found that 24 up-regulated clones were corresponded to genes of kcnk3, mapk7, megf11, and 38 down-regulated clones were corresponded to tmem88b, c-mip, skp1a, mlycd, etc. Their functions were annotated with Gene Ontology (GO) and found to be mainly focused on molecular binding, cell cycle, processes of biology and cells. Five genes (kcnk3, c-mip, mlycd, tmem88b and trappc5) were verified by qRT-PCR with individuals of AC3(+/+) and AC3(-/-) mice. The data indicate that kcnk3 gene is up-regulated significantly, increasing 1.27 folds compared to control mice, whereas c-mip, mlycd, tmem88b and trappc5 are down-regulated significantly, decreasing 20%, 7%, 32% and 29% compared to the AC3(+/+)mice. The functions of these genes are closely related with K(+) channels, cell differentiation, metabolism of fats, membrane transportation, and so on. It is tempting to speculate that these genes might work together with AC3 to orchestrate the olfactory transduction signaling in the MOE.

  8. Introduction of cryIB-cryIAa Hybrid Gene Into Rice (Oryza sativa Genom cv. Rojolele using Agrobacterium-Mediated Transformation

    Directory of Open Access Journals (Sweden)

    SYAMSIDAH RAHMAWATI

    2006-03-01

    Full Text Available Rojolele is one of Indonesian local variety from Javanica group that susceptible to yellow stem borer (Scirpophaga incertulas. Previous study showed that Rojolele can be cultured and regenerated in vitro. Two cry genes, cryIB-cryIAa were fused and introduced into rice cv. Rojolele in an attempt to improve resistance and to obtain durable resistance rice against the yellow stem borer. Two-week old embryogenic calli of Rojolele rice were inoculated with Agrobacterium tumefaciens harbored with binary vector pCAMBIA 1301, 1303, or 1304 carrying cryIB-cryIAa hybrid gene, hygromycin resistant gene (hpt, and -glucuronidase (gus gene interrupted with an intron. The transformed calli were selected gradually on medium containing hygromycin (50, 100 mg/l and regenerated on medium containing 0.5 mg/l IAA and 0.3 mg/l BAP. GUS activity in infected calli was detected by histochemical assay 3 days after inoculation. The highest (100% transformation efficiency were obtained from calli transformed with pCAMBIA 1303 and 1304. Thirty four out of 77 transformed shoots were tested positive for the cryIB-cryIAa gene using PCR analysis. These shoots were grown in the soil to maturity and to collect the seeds. PCR analysis of the T1 progeny revealed that two out of six lines tested showed a Mendelian segregation pattern. These two lines were also potentially resistant to yellow stem borer based on bioassay in planta.

  9. Differentially expressed genes in a flock of Chinese local-breed chickens infected with a subgroup J avian leukosis virus using suppression subtractive hybridization

    Directory of Open Access Journals (Sweden)

    Guiping Zhao

    2010-01-01

    Full Text Available Avian leukosis virus subgroup J (ALV-J is a new type of virus that mainly induces myeloid leukosis (ML in chickens. To further elucidate the pathogenesis of ALV-J infection and tumor development, expression profiles from the bone marrow tissue of 15 infected and 18 non-infected birds from a local-breed poultry-farm under naturally infected conditions, were analyzed by suppression-subtractive hybridization. The birds were diagnosed as ML+ (or ML- by specific ALV-J detection methods, involving serological tests for antigens and antibodies, and RT-PCR to detect viral RNA. A total of 59 partial gene sequences were revealed by differential screening of 496 forward and 384 reverse subtracted cDNA clones. Of these, 22 identified genes, including 8 up-regulated and 14 down-regulated, were related to immune functions, these genes being, MHC B-G antigen, translationally-controlled tumor protein (TPT1/TPTC, transferrin and ferritin, hemoglobin and Carbonic anhydrase. Four of the down-regulated genes were selected for further analysis, in view of their predicted roles in infection and immunity by real-time qRT-PCR, using RNA collected from the same birds as those used for SSH. The four genes were expressed at significantly lower levels (p < 0.001 in ALV-J infected birds than in non-infected ones.

  10. Differentially expressed genes in a flock of Chinese local-breed chickens infected with a subgroup J avian leukosis virus using suppression subtractive hybridization.

    Science.gov (United States)

    Zhao, Guiping; Zheng, Maiqing; Chen, Jilan; Wen, Jie; Wu, Chunmei; Li, Wenjuan; Liu, Libo; Zhang, Yuan

    2010-01-01

    Avian leukosis virus subgroup J (ALV-J) is a new type of virus that mainly induces myeloid leukosis (ML) in chickens. To further elucidate the pathogenesis of ALV-J infection and tumor development, expression profiles from the bone marrow tissue of 15 infected and 18 non-infected birds from a local-breed poultry-farm under naturally infected conditions, were analyzed by suppression-subtractive hybridization. The birds were diagnosed as ML+ (or ML-) by specific ALV-J detection methods, involving serological tests for antigens and antibodies, and RT-PCR to detect viral RNA. A total of 59 partial gene sequences were revealed by differential screening of 496 forward and 384 reverse subtracted cDNA clones. Of these, 22 identified genes, including 8 up-regulated and 14 down-regulated, were related to immune functions, these genes being, MHC B-G antigen, translationally-controlled tumor protein (TPT1/TPTC), transferrin and ferritin, hemoglobin and Carbonic anhydrase. Four of the down-regulated genes were selected for further analysis, in view of their predicted roles in infection and immunity by real-time qRT-PCR, using RNA collected from the same birds as those used for SSH. The four genes were expressed at significantly lower levels (p < 0.001) in ALV-J infected birds than in non-infected ones.

  11. Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura, Urechidae) using suppression subtractive hybridization and cDNA microarray analysis.

    Science.gov (United States)

    Shi, Xiaoli; Shao, Mingyu; Zhang, Litao; Ma, Yubin; Zhang, Zhifeng

    2012-09-01

    Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50 μM sulfide for 24 h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism.

  12. Isolation and identification of novel genes involved in artemisinin production from flowers of Artemisia annua using suppression subtractive hybridization and metabolite analysis.

    Science.gov (United States)

    Liu, Shuoqian; Tian, Na; Li, Juan; Huang, Jianan; Liu, Zhonghua

    2009-11-01

    Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Artemisinin is highly effective against multidrug-resistant Plasmodium falciparum and it has been widely used as part of the artemisinin-based combination therapies against malaria. To elucidate the biosynthetic pathway of artemisinin and to clone related genes in Artemisia annua, differentially expressed genes between blooming flowers and flower buds were isolated and characterized by a combined approach of suppression subtractive hybridization (SSH) and metabolite analysis. A total of 350 cDNA clones from a subtractive cDNA library were randomly picked, sequenced and analyzed and 253 high-quality sequences were obtained. BLASTX comparisons indicated that about 9.9 % of the clones encoded enzymes involved in isoprenoid (including artemisinin) biosynthesis. The expression of 4 gene transcripts involved in artemisinin biosynthesis was examined by RT-PCR and the results confirmed the higher expression of these transcripts in blooming flowers than in flower buds. In addition, 2 putative transcript factors transparenta testa glabra 1 (TTG1) and ENHANCER OF GLABRA3 (GL3), which promote trichome initiation, were presented in the library. Finally, this study demonstrated that the increase of expression level of the putative TTG1 gene correlated with the improvement of glandular trichome density and artemisinin production in A. annua leaves. The subtractive cDNA library described in the present study provides important candidate genes for future research in order to increase the artemisinin content in A. annua.

  13. Transactivating effect of complete S protein of hepatitis B virus and cloning of genes transactivated by complete S protein using suppression subtractive hybridization technique

    Institute of Scientific and Technical Information of China (English)

    Gui-Qin Bai; Yan Liu; Jun Cheng; Shu-Lin Zhang; Ya-Fei Yue; Yan-Ping Huang; Li-Ying Zhang

    2005-01-01

    AIM: To investigate the transactivating effect of complete S protein of hepatitis B virus (HBV) and to construct a subtractive cDNA library of genes transactivated by complete S protein of HBV by suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivation activity, and to pave the way for elucidating the pathogenesis of hepatitis B virus infection.METHODS: pcDNA3.1(-)-complete S containing full-length HBV S gene was constructed by insertion of HBV complete S gene into BarmH-I/Kpn I sites. HepG2 cells were cotransfected with pcDNA3.1(-)-complete S and pSV-lacZ.After 48 h, cells were collected and detected for the expression of β-galactosidase (β-gal). Suppression subtractive hybridization and bioinformatics techniques were used.The mRNA of HepG2 cells transfected with pcDNA3.1(-)-complete S and pcDNA3.1(-) empty vector was isolated,and detected for the expression of complete S protein by reverse transcription polymerase chain reaction (RT-PCR)method, and cDNA was synthesized. After digestion with restriction enzyme RcaI, cDNA fragments were obtained.Tester cDNA was then divided into two groups and ligated to the specific adaptors 1 and 2, respectively. After tester cDNA had been hybridized with driver cDNA twice and underwent nested PCR twice, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. Amplification of the library was carried out within E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) amplification.RESULTS: The complete S mRNA could be detected by RT-PCR in HepG2 cells transfected with the pcDNA3.1(-)-complete S. The activity of β-gal in HepG2 cells transfected with the pcDNA3.1(-)-complete S was 6.9 times higher than that of control plasmid. The subtractive library of genes transactivated by HBV complete S protein was constructed successfully. The amplified library contains 86

  14. Suppression subtractive hybridization (SSH)-based method for estimating Cd-induced differences in gene expression at cultivar level and identification of genes induced by Cd in two water spinach cultivars.

    Science.gov (United States)

    Huang, Baifei; Xin, Junliang; Yang, Zhongyi; Zhou, Yihui; Yuan, Jiangang; Gong, Yulian

    2009-10-14

    The abilities to accumulate cadmium (Cd) are different among cultivars (cv.) in many species. The characteristic of Cd concentration among cultivars is heritable and is probably controlled by genes, but rather limited information about the relevant genes in vegetable crops has been published. In the present study, a suppression subtractive hybridization (SSH) approach was used to identify genes induced by Cd in two water spinach (an important vegetable in southern China) cultivars that differ in Cd accumulation in their edible parts. The two cultivars were cv. Qiangkunqinggu (QK), a low Cd accumulative cultivar and cv. Taiwan 308 (TW), a high Cd accumulative cultivar. In the construction of QK and TW libraries, the plants without Cd treatment were taken as drivers and the plants exposed to 6 mg L(-1) Cd for 24 h as testers. Four hundred clones were sequenced, and 164 nonrepeated sequences (112 from the QK library and 52 from the TW library) were assigned to being functional genes or proteins. A tremendous difference in Cd-induced gene expressions between the two libraries was observed. In the QK library, genes implicated in disease/defense comprised one of the largest sets (20.6%), whereas the proportion was only 8.8% in the TW library. An MT3 gene (Q5), a wound inductive gene (Q22), an antioxidation relevant gene (Q34), a lectin gene (Q45), an f-box family protein gene (Q319), a 20S proteasome subunit gene (T17), a multidrug resistance associated protein gene (T156), and a cationic amino acid transporter gene (T218) were selected to compare semiquantitatively their expression between cv. QK and cv. TW using the RT-PCR method, and obvious differences were detected. The relationships between the identified differences in the expressions of the genes and the Cd accumulation of the two cultivars were discussed, and it was concluded that the SSH approach is useful for finding the difference in expression of Cd-induced gene even at the cultivar level and is applicable

  15. Attomolar electrochemical detection of the BCR/ABL fusion gene based on an amplifying self-signal metal nanoparticle-conducting polymer hybrid composite.

    Science.gov (United States)

    Avelino, Karen Y P S; Frias, Isaac A M; Lucena-Silva, Norma; Gomes, Renan G; de Melo, Celso P; Oliveira, Maria D L; Andrade, César A S

    2016-12-01

    In the last ten years, conjugated polymers started to be used in the immobilization of nucleic acids via non-covalent interactions. In the present study, we describe the construction and use of an electrochemical DNA biosensor based on a nanostructured polyaniline-gold composite, specifically developed for the detection of the BCR/ABL chimeric oncogene. This chromosome translocation is used as a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). The working principle of the biosensor rests on measuring the conductivity resulting from the non-covalent interactions between the hybrid nanocomposite and the DNA probe. The nanostructured platform exhibits a large surface area that enhances the conductivity. Positive cases, which result from the hybridization between DNA probe and targeted gene, induce changes in the amperometric current and in the charge transfer resistance (RCT) responses. Atomic force microscopy (AFM) images showed changes in the genosensor surface after exposure to cDNA sample of patient with leukemia, evidencing the hybridization process. This new hybrid sensing-platform displayed high specificity and selectivity, and its detection limit is estimated to be as low as 69.4 aM. The biosensor showed excellent analytical performance for the detection of the BCR/ABL oncogene in clinical samples of patients with leukemia. Hence, this electrochemical sensor appears as a simple and attractive tool for the molecular diagnosis of the BCR/ABL oncogene even in early-stage cases of leukemia and for the monitoring of minimum levels of residual disease.

  16. A Hybrid One-Way ANOVA Approach for the Robust and Efficient Estimation of Differential Gene Expression with Multiple Patterns

    OpenAIRE

    Mohammad Manir Hossain Mollah; Rahman Jamal; Norfilza Mohd Mokhtar; Roslan Harun; Md. Nurul Haque Mollah

    2015-01-01

    Background Identifying genes that are differentially expressed (DE) between two or more conditions with multiple patterns of expression is one of the primary objectives of gene expression data analysis. Several statistical approaches, including one-way analysis of variance (ANOVA), are used to identify DE genes. However, most of these methods provide misleading results for two or more conditions with multiple patterns of expression in the presence of outlying genes. In this paper, an attempt ...

  17. Gene flow and hybridization between numerically imbalanced populations of two duck species on the subantarctic island of South Georgia.

    Directory of Open Access Journals (Sweden)

    Kevin G McCracken

    Full Text Available Hybridization is common between species of animals, particularly in waterfowl (Anatidae. One factor shown to promote hybridization is restricted mate choice, which can occur when 2 species occur in sympatry but one is rare. According to the Hubbs principle, or "desperation hypothesis," the rarer species is more likely to mate with heterospecifics. We report the second of 2 independent examples of hybridization between 2 species of ducks inhabiting island ecosystems in the Subantarctic and South Atlantic Ocean. Yellow-billed pintails (Anas georgica and speckled teal (Anas flavirostris are abundant in continental South America, where they are sympatric and coexist in mixed flocks. But on South Georgia, an isolated island in the Subantarctic, the pintail population of approximately 6000 pairs outnumbers a small breeding population of speckled teal 300∶1. Using 6 genetic loci (mtDNA and 5 nuclear introns and Bayesian assignment tests coupled with coalescent analyses, we identified hybrid-origin speckled teal alleles in 2 pintails on South Georgia. While it is unclear whether introgression has also occurred into the speckled teal population, our data suggest that this hybridization was not a recent event, but occurred some time ago. We also failed to identify unequivocal evidence of introgression in a much larger sample of pintails and speckled teal from Argentina using a 3-population "Isolation-with-Migration" coalescent analysis. Combined with parallel findings of hybridization between these same 2 duck species in the Falkland Islands, where population ratios are reversed and pintails are outnumbered by speckled teal 1:10, our results provide further support for the desperation hypothesis, which predicts that scarcity in one population and abundance of another will often lead to hybridization. While the South Georgia pintail population appears to be thriving, it's possible that low density of conspecific mates and inverse density dependence

  18. A genome-wide screen for ethylene-induced ethylene response factors (ERFs) in hybrid aspen stem identifies ERF genes that modify stem growth and wood properties.

    Science.gov (United States)

    Vahala, Jorma; Felten, Judith; Love, Jonathan; Gorzsás, András; Gerber, Lorenz; Lamminmäki, Airi; Kangasjärvi, Jaakko; Sundberg, Björn

    2013-10-01

    Ethylene Response Factors (ERFs) are a large family of transcription factors that mediate responses to ethylene. Ethylene affects many aspects of wood development and is involved in tension wood formation. Thus ERFs could be key players connecting ethylene action to wood development. We identified 170 gene models encoding ERFs in the Populus trichocarpa genome. The transcriptional responses of ERF genes to ethylene treatments were determined in stem tissues of hybrid aspen (Populus tremula × tremuloides) by qPCR. Selected ethylene-responsive ERFs were overexpressed in wood-forming tissues and characterized for growth and wood chemotypes by FT-IR. Fifty ERFs in Populus showed more than five-fold increased transcript accumulation in response to ethylene treatments. Twenty-six ERFs were selected for further analyses. A majority of these were induced during tension wood formation. Overexpression of ERFs 18, 21, 30, 85 and 139 in wood-forming tissues of hybrid aspen modified the wood chemotype. Moreover, overexpression of ERF139 caused a dwarf-phenotype with altered wood development, and overexpression of ERF18, 34 and 35 slightly increased stem diameter. We identified ethylene-induced ERFs that respond to tension wood formation, and modify wood formation when overexpressed. This provides support for their role in ethylene-mediated regulation of wood development.

  19. A Novel Hybrid Dimension Reduction Technique for Undersized High Dimensional Gene Expression Data Sets Using Information Complexity Criterion for Cancer Classification

    Directory of Open Access Journals (Sweden)

    Esra Pamukçu

    2015-01-01

    Full Text Available Gene expression data typically are large, complex, and highly noisy. Their dimension is high with several thousand genes (i.e., features but with only a limited number of observations (i.e., samples. Although the classical principal component analysis (PCA method is widely used as a first standard step in dimension reduction and in supervised and unsupervised classification, it suffers from several shortcomings in the case of data sets involving undersized samples, since the sample covariance matrix degenerates and becomes singular. In this paper we address these limitations within the context of probabilistic PCA (PPCA by introducing and developing a new and novel approach using maximum entropy covariance matrix and its hybridized smoothed covariance estimators. To reduce the dimensionality of the data and to choose the number of probabilistic PCs (PPCs to be retained, we further introduce and develop celebrated Akaike’s information criterion (AIC, consistent Akaike’s information criterion (CAIC, and the information theoretic measure of complexity (ICOMP criterion of Bozdogan. Six publicly available undersized benchmark data sets were analyzed to show the utility, flexibility, and versatility of our approach with hybridized smoothed covariance matrix estimators, which do not degenerate to perform the PPCA to reduce the dimension and to carry out supervised classification of cancer groups in high dimensions.

  20. Spatial gene expression quantification: a tool for analysis of in situ hybridizations in sea anemone Nematostella vectensis

    NARCIS (Netherlands)

    Botman, D.; Kaandorp, J.A.

    2012-01-01

    Background Spatial gene expression quantification is required for modeling gene regulation in developing organisms. The fruit fly Drosophila melanogaster is the model system most widely applied for spatial gene expression analysis due to its unique embryonic properties: the shape does not change sig

  1. Homologous genes for mouse 4.5S hybRNA are found in all eukaryotes and their low molecular weight RNA transcripts intermolecularly hybridize with eukaryotic 18S ribosomal RNAs.

    Science.gov (United States)

    Trinh-Rohlik, Q; Maxwell, E S

    1988-07-11

    Previous work has reported the isolation and sequencing of a mouse low molecular weight RNA species designated 4.5S hybridizing RNA or hybRNA because of its ability to intermolecularly hybridize with mouse mRNA and 18S rRNA sequences. Using synthetic DNA oligonucleotide probes we have examined the conservation of this gene sequence and its expression as a lmwRNA transcript across evolution. Southern blot analysis has shown that homologous genes of single or low copy number are found in all eukaryotes examined as well as in E. coli. Northern blot analysis has demonstrated 4.5S hybRNA transcription in all mouse tissues as well as expression in yeast and Xenopus laevis as lmwRNAs of approximately 130 and 100 nucleotides, respectively, as compared with mouse/rat/hamster species of approximately 87 nucleotides. Yeast and X. laevis 4.5S hybRNA homologs, isolated by hybrid-selection, were shown by Northern blot analysis to intermolecularly hybridize with homologous as well as heterologous 18S rRNA sequences. The conservation of 4.5S hybRNA homologous genes and their expression as lmwRNA transcripts with common intermolecular RNA:RNA hybridization capabilities in fungi, amphibians, and mammals argues for a common, conserved and required biological function for this lmwRNA in all eukaryotes and potential utilization of its intermolecular RNA:RNA hybridization capabilities to carry out this function.

  2. Suppression Subtractive Hybridization Analysis of Genes Regulated by Application of Exogenous Abscisic Acid in Pepper Plant (Capsicum annuum L. Leaves under Chilling Stress.

    Directory of Open Access Journals (Sweden)

    Wei-Li Guo

    Full Text Available Low temperature is one of the major factors limiting pepper (Capsicum annuum L. production during winter and early spring in non-tropical regions. Application of exogenous abscisic acid (ABA effectively alleviates the symptoms of chilling injury, such as wilting and formation of necrotic lesions on pepper leaves; however, the underlying molecular mechanism is not understood. The aim of this study was to identify genes that are differentially up- or downregulated in ABA-pretreated hot pepper seedlings incubated at 6°C for 48 h, using a suppression subtractive hybridization (SSH method. A total of 235 high-quality ESTs were isolated, clustered and assembled into a collection of 73 unigenes including 18 contigs and 55 singletons. A total of 37 unigenes (50.68% showed similarities to genes with known functions in the non-redundant database; the other 36 unigenes (49.32% showed low similarities or unknown functions. Gene ontology analysis revealed that the 37 unigenes could be classified into nine functional categories. The expression profiles of 18 selected genes were analyzed using quantitative RT-PCR; the expression levels of 10 of these genes were at least two-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated (control plants under chilling stress. In contrast, the other eight genes were downregulated in ABA-pretreated seedlings under chilling stress, with expression levels that were one-third or less of the levels observed in control seedlings under chilling stress. These results suggest that ABA can positively and negatively regulate genes in pepper plants under chilling stress.

  3. Identiifcation of genes upregulated by recombinant inter feron-alpha in HepG2 cells by suppressive subtractive hybridization analysis

    Institute of Scientific and Technical Information of China (English)

    Jian-Hui Qu; Jun Cheng; Ling-Xia Zhang; Li-Ying Zhang; Yan-Wei Zhong; Yan Liu; Lin Wang; Jiu-Zeng Dai; Dong-Ping Xu

    2007-01-01

    BACKGROUND:Interferon-alpha (IFN-α) is an important cytokine with multiple functions, but the target genes transactivated by IFN-αremain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-α. To isolate the gene transcripts speciifcally upregulated by IFN-α in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-α protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-α (rIFN-α, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-αas driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed signiifcant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-α was constructed successfully. rIFN-α upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc ifnger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-α can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-α.

  4. Suppression Subtractive Hybridization Analysis of Genes Regulated by Application of Exogenous Abscisic Acid in Pepper Plant (Capsicum annuum L.) Leaves under Chilling Stress.

    Science.gov (United States)

    Guo, Wei-Li; Chen, Ru-Gang; Gong, Zhen-Hui; Yin, Yan-Xu; Li, Da-Wei

    2013-01-01

    Low temperature is one of the major factors limiting pepper (Capsicum annuum L.) production during winter and early spring in non-tropical regions. Application of exogenous abscisic acid (ABA) effectively alleviates the symptoms of chilling injury, such as wilting and formation of necrotic lesions on pepper leaves; however, the underlying molecular mechanism is not understood. The aim of this study was to identify genes that are differentially up- or downregulated in ABA-pretreated hot pepper seedlings incubated at 6°C for 48 h, using a suppression subtractive hybridization (SSH) method. A total of 235 high-quality ESTs were isolated, clustered and assembled into a collection of 73 unigenes including 18 contigs and 55 singletons. A total of 37 unigenes (50.68%) showed similarities to genes with known functions in the non-redundant database; the other 36 unigenes (49.32%) showed low similarities or unknown functions. Gene ontology analysis revealed that the 37 unigenes could be classified into nine functional categories. The expression profiles of 18 selected genes were analyzed using quantitative RT-PCR; the expression levels of 10 of these genes were at least two-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated (control) plants under chilling stress. In contrast, the other eight genes were downregulated in ABA-pretreated seedlings under chilling stress, with expression levels that were one-third or less of the levels observed in control seedlings under chilling stress. These results suggest that ABA can positively and negatively regulate genes in pepper plants under chilling stress.

  5. Meta-analysis of studies using suppression subtractive hybridization and microarrays to investigate the effects of environmental stress on gene transcription in oysters.

    Directory of Open Access Journals (Sweden)

    Kelli Anderson

    Full Text Available Many microarray and suppression subtractive hybridization (SSH studies have analyzed the effects of environmental stress on gene transcription in marine species. However, there have been no unifying analyses of these data to identify common stress response pathways. To address this shortfall, we conducted a meta-analysis of 14 studies that investigated the effects of different environmental stressors on gene expression in oysters. The stressors tested included chemical contamination, hypoxia and infection, as well as extremes of temperature, pH and turbidity. We found that the expression of over 400 genes in a range of oyster species changed significantly after exposure to environmental stress. A repeating pattern was evident in these transcriptional responses, regardless of the type of stress applied. Many of the genes that responded to environmental stress encoded proteins involved in translation and protein processing (including molecular chaperones, the mitochondrial electron transport chain, anti-oxidant activity and the cytoskeleton. In light of these findings, we put forward a consensus model of sub-cellular stress responses in oysters.

  6. Identification of genes involved in indole-3-butyric acid-induced adventitious root formation in nodal cuttings of Camellia sinensis (L.) by suppression subtractive hybridization.

    Science.gov (United States)

    Wei, Kang; Wang, Liyuan; Cheng, Hao; Zhang, Chengcai; Ma, Chunlei; Zhang, Liqun; Gong, Wuyun; Wu, Liyun

    2013-02-10

    The plant hormone auxin plays a key role in adventitious rooting. To increase our understanding of genes involved in adventitious root formation, we identified transcripts differentially expressed in single nodal cuttings of Camellia sinensis treated with or without indole-3-butyric acid (IBA) by suppressive subtractive hybridization (SSH). A total of 77 differentially expressed transcripts, including 70 up-regulated and 7 down-regulated sequences, were identified in tea cuttings under IBA treatment. Seven candidate transcripts were selected and analyzed for their response to IBA, and IAA by real time RT-PCR. All these transcripts were up regulated by at least two folds one day after IBA treatment. Meanwhile, IAA showed less positive effects on the expression of candidate transcripts. The full-length cDNA of a F-box/kelch gene was also isolated and found to be similar to a group of At1g23390 like genes. These unigenes provided a new source for mining genes related to adventitious root formation, which facilitate our understanding of relative fundamental metabolism.

  7. Meta-analysis of studies using suppression subtractive hybridization and microarrays to investigate the effects of environmental stress on gene transcription in oysters.

    Science.gov (United States)

    Anderson, Kelli; Taylor, Daisy A; Thompson, Emma L; Melwani, Aroon R; Nair, Sham V; Raftos, David A

    2015-01-01

    Many microarray and suppression subtractive hybridization (SSH) studies have analyzed the effects of environmental stress on gene transcription in marine species. However, there have been no unifying analyses of these data to identify common stress response pathways. To address this shortfall, we conducted a meta-analysis of 14 studies that investigated the effects of different environmental stressors on gene expression in oysters. The stressors tested included chemical contamination, hypoxia and infection, as well as extremes of temperature, pH and turbidity. We found that the expression of over 400 genes in a range of oyster species changed significantly after exposure to environmental stress. A repeating pattern was evident in these transcriptional responses, regardless of the type of stress applied. Many of the genes that responded to environmental stress encoded proteins involved in translation and protein processing (including molecular chaperones), the mitochondrial electron transport chain, anti-oxidant activity and the cytoskeleton. In light of these findings, we put forward a consensus model of sub-cellular stress responses in oysters.

  8. Differentially expressed genes of human microvascular endothelial cells in response to anti-dengue virus NS1 antibodies by suppression subtractive hybridization.

    Science.gov (United States)

    Yin, Yue; Jiang, Lan; Fang, Danyun; Jiang, Lifang; Zhou, Junmei

    2013-06-01

    It has been previously shown that anti-dengue virus (DENV) nonstructural protein NS1 antibodies could act as autoantibodies that direct against one or more of the host's own proteins, which has potential implications for dengue hemorrhagic fever pathogenesis. In the present study, we have employed suppression subtractive hybridization (SSH) to identify the differentially expressed genes from human microvascular endothelial cells (HMEC-1) in response to anti-dengue virus type 2 NS1 antibodies (anti-DENV2 NS1 Abs). A total of 35 clones from the SSH cDNA library were randomly selected for further analysis using bioinformatics tools after vector screening. After searching for sequence homology in NCBI GenBank database with BLASTN and BLASTX programs, 23 obtained sequences with significant matches (E-values <1×10(-4)) in the SSH library. The predicted genes in the subtracted library include immune response molecules (CD59 antigen preproprotein preproprotein, MURR1), signal transduction molecules (Nuclear casein kinase and cyclin-dependent kinase substrate 1), calcium-binding proteins (S100A6, Annexin A2 isoform 1/2), and cell-membrane component (Yip1 domain family). From these clones, 5 upregulated genes were selected for differential expression profiling by real-time RT-PCR to confirm their upregulated status. The results confirmed their differential upregulation, and thus verified the success of SSHs and the likely involvement of these genes in dengue pathogenesis.

  9. Expression profile analysis of genes involved in horizontal gravitropism bending growth in the creeping shoots of ground-cover chrysanthemum by suppression subtractive hybridization.

    Science.gov (United States)

    Xia, Shengjun; Chen, Yu; Jiang, Jiafu; Chen, Sumei; Guan, Zhiyong; Fang, Weimin; Chen, Fadi

    2013-01-01

    The molecular mechanisms underlying gravitropic bending of shoots are poorly understood and how genes related with this growing progress is still unclear. To identify genes related to asymmetric growth in the creeping shoots of chrysanthemum, suppression subtractive hybridization was used to visualize differential gene expression in the upper and lower halves of creeping shoots of ground-cover chrysanthemum under gravistimulation. Sequencing of 43 selected clones produced 41 unigenes (40 singletons and 1 unigenes), which were classifiable into 9 functional categories. A notable frequency of genes involve in cell wall biosynthesis up-regulated during gravistimulation in the upper side or lower side were found, such as beta tubulin (TUB), subtilisin-like protease (SBT), Glutathione S-transferase (GST), and expensing-like protein (EXP), lipid transfer proteins (LTPs), glycine-rich protein (GRP) and membrane proteins. Our findings also highlighted the function of some metal transporter during asymmetric growth, including the boron transporter (BT) and ZIP transporter (ZT), which were thought primarily for maintaining the integrity of cell walls and played important roles in cellulose biosynthesis. CmTUB (beta tubulin) was cloned, and the expression profile and phylogeny was examined, because the cytoskeleton of plant cells involved in the plant gravitropic bending growth is well known.

  10. Influence of Genetic Drift of Restoring Gene (Rf on Seed Purity of Yuetai A, a Honglian-Type Cytoplasmic Male Sterile Line in Hybrid Rice

    Directory of Open Access Journals (Sweden)

    Ji-feng Wang

    2008-06-01

    Full Text Available The seed samples of Yuetai A, a Honglian (HL type cytoplasmic male sterile (CMS line in hybrid rice were investigated to assess the seed purity and to analyze the cause of off-type plants by imitating the biological contaminant to Yuetai A in Nanjing, Jiangsu Province and Lingshui, Hainan Province during 2004–2006. The seed impurity of Yuetai A mainly resulted from the genetic drift of restoring fertility gene (Rf after biological contamination but not from its sterility unstability. All of the ten maintainer lines, five restorer lines and three thermo-sensitive genic male sterile lines used in the study could pollinate Yuetai A and Yuetai B to produce F1 plants, directly or indirectly resulting in Rf-gene drifting into Yuetai A and generating ‘iso-cytoplasm restoring-generations’. Furthermore, high outcrossing rate and similar heading date of Yuetai A with many varieties used in rice production might easily result in the biological contamination. After removing all plants with Rf-gene mixed in Yuetai A and preventing Rf-gene drifting into Yuetai A, the seed purity of Yuetai A and Yuetai B had been raised to 100%.

  11. Differentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR.

    Science.gov (United States)

    Feng, Lifang; Miao, Wei; Wu, Yuxuan

    2007-02-15

    Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T. thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system.

  12. Analyses of numerical aberrations of chromosome 17 and tp53 gene deletion/amplification in human oral squamous cell carcinoma using dual-color fluorescence in situ hybridization

    Directory of Open Access Journals (Sweden)

    Noemi MESZAROS

    2010-05-01

    Full Text Available In Romania, oral and facial cancers represent approximately 5% of all cancers. Deactivation and unregulated expression of oncogenes and tumor suppressor genes may be involved in the pathogenesis of oral squamous cell carcinoma. The genomic change results in numerical and structural chromosomal alterations, particularly in chromosomes 3, 9, 11 and 17. The aim of our study was to identify numerical aberrations of chromosome 17, deletion or amplification of p53 gene and to reveal correlations between abnormalities of chromosome 17and of p53 gene with TNM status and grading in 15 subjects with oral squamous cell carcinoma. 80 % of cases presented chromosome 17 polysomy and only 20% of cases had chromosome 17 monosomy. 46.6 % of samples revealed p53 gene amplification and 33.3 % of them p53 deletion. Polysomy of chromosome 17 was also detected in tumor-adjacent epithelia. The degree of the cytogenetic abnormality did not correlate with the stage of the disease, the histological differentiation of oral squamous cell carcinoma and lymph node metastasis. Molecular cytogenetic techniques, using fluorescence in situ hybridization with chromosome-specific DNA probes, facilitate the confirmation of presumed chromosomal aberrations with high sensitivity and specificity.

  13. Influence of Genetic Drift of Restoring Gene (Rf) on Seed Purity of Yuetai A, a Honglian-Type Cytoplasmic Male Sterile Line in Hybrid Rice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The seed samples of Yuetai A,a Honglian (HL) type cytoplasmic male sterile (CMS) line in hybrid rice were investigated to assess the seed purity and to analyze the cause of off-type plants by imitating the biological contaminant to Yuetai A in Nanjing,Jiangsu Province and Lingshui,Hainan Province during 2004-2006.The seed impurity of Yuotai A mainly resulted from the genetic drift of restoring fertility gene (Rt) after biological contamination but not from its sterility unstability.All of the ten maintainer lines,five restorer lines and three thermo-sensitive genic male sterile lines used in the study could pollinate Yuetai A and Yuetai B to produce F1 plants,directly or indirectly resulting in Rf-gene drifting into Yuetai A and generating 'iso-cytoplasm restoring-generations'.Furthermore,high outcrossing rate and similar heading date of Yuetai A with many varieties used in rice production might easily result in the biological contamination.After removing all plants with Rf-gene mixed in Yuetai A and preventing Rf-gene drifting into Yuetai A,the seed purity of Yuetai A and Yuetai B had been raised to 100%.

  14. Targeted array comparative genomic hybridization--a new diagnostic tool for the detection of large copy number variations in nemaline myopathy-causing genes.

    Science.gov (United States)

    Kiiski, K; Laari, L; Lehtokari, V-L; Lunkka-Hytönen, M; Angelini, C; Petty, R; Hackman, P; Wallgren-Pettersson, C; Pelin, K

    2013-01-01

    Nemaline myopathy (NM) constitutes a heterogeneous group of congenital myopathies. Mutations in the nebulin gene (NEB) are the main cause of recessively inherited NM. NEB is one of the most largest genes in human. To date, 68 NEB mutations, mainly small deletions or point mutations have been published. The only large mutation characterized is the 2.5 kb deletion of exon 55 in the Ashkenazi Jewish population. To investigate any copy number variations in this enormous gene, we designed a novel custom comparative genomic hybridization microarray, NM-CGH, targeted towards the seven known genes causative for NM. During the validation of the NM-CGH array we identified two novel deletions in two different families. The first is the largest deletion characterized in NEB to date, (∼53 kb) encompassing 24 exons. The second deletion (1 kb) covers two exons. In both families, the copy number change was the second mutation to be characterized and shown to have been inherited from one of the healthy carrier parents. In addition to these novel mutations, copy number variation was identified in four samples in three families in the triplicate region of NEB. We conclude that this method appears promising for the detection of copy number variations in NEB. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Isolation of Related Genes of Cardiac Hypertrophy in Pressure overloaded Rat Models with Subtractive Hybridization%压力负荷型心肌肥厚大鼠心肌组织相关基因的分离

    Institute of Scientific and Technical Information of China (English)

    田靫; 潘德思; 陈兰英

    2000-01-01

    Cardiac hypertrophy is intensively related with the morbidity of heart failure,atherosclerosis and stroke. The rat models of left ventricular hypertrophy caused by abdominal aorta constriction and the method of subtractive hybridization to isolate genes expressing differently were used during cardiac hypertrophy. 24 cDNA segments were isolated and identified by colony and dot hybridization. Sequence homogenous comparison showed that some of them were very similar to known genes or segments, others were limitedly homogenous and the rest were not found to be significantly homogenous.

  16. Identification of differentially expressed genes in American cockroach ovaries and testes by suppression subtractive hybridization and the prediction of its miRNAs.

    Science.gov (United States)

    Chen, Wan; Jiang, Guo-Fang; Sun, Shu-Hong; Lu, Yong; Ma, Fei; Li, Bin

    2013-11-01

    Studies on the cockroach have contributed to our understanding of several important developmental processes, especially those that can be easily studied in the embryo. However, our knowledge on late events such as gonad differentiation in the cockroach is still limited. The major aim of the present study was to identify sex-specific genes between adult female and male Periplaneta americana. Two cDNA libraries were constructed using the suppression subtractive hybridization method; a total of 433 and 599 unique sequences were obtained from the forward library and the reverse library, respectively, by cluster assembly, and sequence alignment of 1,032 expressed sequence tags. The analysis of the differentially expressed gene functions allowed these genes to be categorized into three groups: biological process, molecular function, and cellular component. The differentially expressed genes were suggested to be related to the development of the gonads of P. americana. Twelve differentially expressed genes were randomly selected and verified using relative quantitative real-time polymerase chain reaction (qRT-PCR). Meanwhile, by adopting a range of filtering criteria, we predicted two potential microRNA sequences for P. americana, pam-miR100-3p and pam-miR7. To confirm the expression of potential microRNAs (miRNAs) in American cockroach, a qRT-PCR approach was also employed. The data presented here offer the insights into the molecular foundation of sex differences in American cockroach, and the first report for the miRNAs in this species. In addition, the results can be used as a reference for unraveling candidate genes associated with the sex and reproduction of cockroaches.

  17. Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes.

    Science.gov (United States)

    Salvemini, Marco; D'Amato, Rocco; Petrella, Valeria; Ippolito, Domenica; Ventre, Giuseppe; Zhang, Ying; Saccone, Giuseppe

    2014-01-01

    The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) anddifferential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

  18. Identification of heat stress-responsive genes in heat-adapted thermal Agrostis scabra by suppression subtractive hybridization.

    Science.gov (United States)

    Tian, Jiang; Belanger, Faith C; Huang, Bingru

    2009-04-01

    To gain insights into molecular mechanisms of grass tolerance to heat stress, we constructed a suppression subtractive cDNA library to identify heat-responsive genes for a C(3) grass species, thermal Agrostis scabra adapted to heat stress in geothermal areas in Yellowstone National Park. Plants were exposed to 20 degrees C (control) or 35 degrees C for 12d. The SSH analysis was performed with control samples as the driver and heat-stressed samples as the tester. Differentially expressed cDNA fragments were cloned to screen the heat up-regulated library. The SSH analysis identified 120 non-redundant putative heat-responsive cDNAs out of 1180 clones. Genes with homology to known proteins were categorized into six functional groups, with the largest group of genes involved in stress/defense, followed by the group of genes related to protein metabolism. Immunoblot analysis confirmed increases in transcripts of selected genes under heat stress. Transcripts of seven and eight genes were strongly enhanced or induced in shoots and roots, respectively, while two genes were only induced in roots under heat stress. The heat up-regulated genes in thermal A. scabra adapted to long-term heat stress are potential candidate genes for engineering stress-tolerant grasses and for revealing molecular mechanisms of grass adaptation to heat stress.

  19. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    Science.gov (United States)

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells.

  20. SPR Detection and Discrimination of the Oligonucleotides Related to the Normal and the Hybrid bcr-abl Genes by Two Stringency Control Strategies

    Science.gov (United States)

    Matsishin, M. J.; Ushenin, Iu. V.; Rachkov, A. E.; Solatkin, A. P.

    2016-01-01

    In this study, we applied two stringency control strategies for surface plasmon resonance (SPR) detection of DNA hybridization and discrimination of completely and partially complementary 24-mer sequences. These sequences are specific to the human normal bcr and the hybrid bcr-abl genes, protein products of which are responsible for some leukemia. SPR sensors based on resonance phenomena in nanoscale gold films are well suited for label-free, real-time investigations of the macromolecule interactions. Thermodynamic parameters obtained using the web server DINAMelt allowed supposing the possibility for realization (a) stringency control based on the ionic strength of the hybridization buffer and (b) stringency control based on the temperature elevation. The first one resulted in that the discrimination index of completely complementary and partially complementary oligonucleotides depending on the target concentration varied from 1.3 to 1.8 in 2 × SSC and from 2.0 to 2.9 in 0.5 × SSC. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~50 °C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 °C. Thus, the absolutely effective thermodiscrimination of this pair of oligonucleotides was achieved in this temperature range (40-50 °C).

  1. Isolation and analysis of differentially expressed genes during asexual sporulation in liquid static culture of Ganoderma lucidum by suppression subtractive hybridization.

    Science.gov (United States)

    Xu, Jun-Wei; Zhao, Wei; Xu, Yi-Ning; Zhong, Jian-Jiang

    2012-04-01

    Ganoderma lucidum differentiates in liquid static culture by forming aerial mycelia and asexual spores, and this differentiation process is accompanied by higher production of anti-tumor compounds ganoderic acids. To gain an insight into the molecular events during asexual sporulation of G. lucidum, comparative transcriptome analysis using suppression subtractive hybridization (SSH) technique was performed to identify preferentially expressed genes in liquid static culture vs. in traditional shaking culture. After macroarray analysis of 1920 cDNAs from SSH library, 147 unigenes which exhibited high expression in static culture were identified. Among these sequences, putative translations of 88 unigenes possessed much similarity to known proteins involved in cell organization, signal transduction, cell metabolism, protein biosynthesis and transcription regulation; 13 had significant similarity to hypothetical proteins; the remaining 46 showed little or no similarity to GenBank sequences. RT-qPCR analysis confirmed increases in transcripts of selected genes under liquid static culture condition. The results of this study present the useful application of EST analysis on G. lucidum and provide preliminary indication of gene expression putatively involved in asexual sporulation process.

  2. Suppression subtractive hybridization coupled with microarray analysis to examine differential expression of genes in Japanese flounder Paralichthys olivaceus leucocytes during Edwardsiella tarda and viral hemorrhagic septicemia virus infection.

    Science.gov (United States)

    Matsuyama, Tomomasa; Fujiwara, Atushi; Takano, Tomokazu; Nakayasu, Chihaya

    2011-10-01

    Transcriptional changes in the peripheral blood leucocytes (PBL) of Japanese flounder Paralichthys olivaceus challenged by Edwardsiella tarda and viral hemorrhagic septicemia virus (VHSV) were investigated using suppression subtractive hybridization (SSH) coupled with cDNA microarray analysis. First, we constructed an SSH cDNA library using mRNA samples isolated from PBL of P. olivaceus that had been experimentally infected with E. tarda. We then examined the transcriptional changes occurring in the PBL due to E. tarda and VHSV infection using a cDNA microarray produced using clones produced from the SSH library. A total of 565 and 180 cDNA sequences corresponding to mRNA species that are either up- or down-regulated by E. tarda infection were isolated by SSH. While host gene expression responses in response to E. tarda and VHSV infection share several response elements, distinct patterns of gene expression were also observed. Specifically, E. tarda infection enhanced the expression of cell adhesion molecules while VHSV enhanced the expression of interferon and proteasome-related genes. In challenge trials of the two infectious agents, expression profiles of chemokines were also observed to differ. The results indicated that distinguishing between viral and bacterial infection is possible based on the RNA expression profiles of PBL from infected fish.

  3. Molecular characterization, expression patterns, and polymorphism of a differentially expressed porcine gene (PYGM) isolated by suppression subtractive hybridization and two-dimensional gel electrophoresis analysis.

    Science.gov (United States)

    Xu, Yongjie; Yu, Wenmin; Feng, Xiaoting; Xie, Hongtao; Xiong, Yuanzhu

    2012-01-01

    Suppression subtractive hybridization was performed to detect the differences in gene expression of porcine longissimus dorsi muscles between Large White and Chinese Meishan pigs. An upregulated gene in Large White that shared high homology with human muscle glycogen phosphorylase (PYGM) was identified. The porcine PYGM gene contains an open reading frame encoding 842 amino acid residues with 26 and 283 nucleotides in the 5' and 3' untranslated regions, respectively. Tissue distribution analysis indicated that porcine PYGM mRNAs are highly expressed in all tissues. Expression pattern of PYGM was similar in the two breeds. Both breeds had the highest expression levels when 120 days old (p<0.01), and PYGM was upregulated during skeletal muscle development. A similar expression pattern of PYGM in protein level was also observed by differential proteome analysis of skeletal muscle development using two-dimensional gel electrophoresis and mass spectroscopy. The mRNA abundance of PYGM in Large White was higher than Meishan at all four stages (p<0.05). Moreover, a G/T mutation in exon 8 was identified and association analysis with meat quality traits showed that it was significantly associated with lean meat percentage (p<0.05). Our data may provide further insight into the molecular mechanisms responsible for breed-specific differences in porcine growth and meat quality.

  4. c-myc Gene Copy Number Variation in Cervical Exfoliated Cells Detected on Fluorescence in situ Hybridization for Cervical Cancer Screening.

    Science.gov (United States)

    Zhao, Wei-Hong; Hao, Min; Cheng, Xiao-Tong; Yang, Xin; Wang, Zhi-Lian; Cheng, Ke-Yan; Liu, Fang-Li; Bai, Yao-Xian

    2016-01-01

    This study assessed the clinical significance of c-myc gene copy number gain detected by fluorescence in situ hybridization (FISH) in the prediction of cervical intraepithelial neoplasia (CIN) progression. We retrospectively investigated 140 Thinprep cytologic test (TCT) specimens that were histopathologically diagnosed with various stages of cervical neoplasia or malignancy. The specimens were subjected to TCT, human papillomavirus (HPV) testing, and FISH analysis with a c-myc-specific probe. The diagnostic reliability of these methods in determining progression was assessed according to sensitivity, specificity, and κ coefficients. The gene copy number gain of c-myc was significantly higher in the cervical lesion of advanced histologic grade (p c-myc gene test and either the TCT or the HPV DNA test were 0.538 and 0.399, respectively (p c-myc oncogene could be a useful adjunct screening method for the early diagnosis of high-grade cervical lesions. Moreover, c-myc may be a new molecular biomarker for the early diagnosis of cervical lesion progression. © 2016 S. Karger AG, Basel.

  5. GhHyPRP4, a cotton gene encoding putative hybrid proline-rich protein, is preferentially expressed in leaves and involved in plant response to cold stress

    Institute of Scientific and Technical Information of China (English)

    Gengqing Huang; Siying Gong; Wenliang Xu; Peng Li; Dejing Zhang; Lixia Qin; Wen Li; Xuebao Li

    2011-01-01

    Plant hybrid proline-rich proteins (HyPRPs) usually consist of an N-terminal signal peptide, a central prolinerich domain, and a conserved eight-cysteine motif C-terminal domain. In this study, one gene (designated as GhHyPRP4) encoding putative HyPRP was isolated from cotton cDNA library. Northern blot and quantitative reverse transcriptase-polymerase chain reaction analyses revealed that GhHyPRP4 was preferentially expressed in leaves. Under cold stress, GhHyPRP4 expression was significantly up-regulated in leaves of cotton seedlings.Using the genome walking approach, a promoter fragment of GhHyPRP4 gene was isolated from cotton genome.GUS (β-glucuronidase) gene driven by GhHyPRP4 promoter was specifically expressed in leaves and cotyledons of the transgenic 4rabidopsis thaliana. Furthermore,GUS expression in leaves was remarkably induced by cold stress. Overexpression of GhHyPRP4 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival rate upon treatment under -20℃ for 60 h.These data suggested that GhHyPRP4 may be involved in plant response to cold stress during seedling development of cotton.

  6. Comparative transcript profiling of gene expression between seedless Ponkan mandarin and its seedy wild type during floral organ development by suppression subtractive hybridization and cDNA microarray.

    Science.gov (United States)

    Qiu, Wen-Ming; Zhu, An-Dan; Wang, Yao; Chai, Li-Jun; Ge, Xiao-Xia; Deng, Xiu-Xin; Guo, Wen-Wu

    2012-08-16

    Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored. An integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated. Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future.

  7. Identification of genes differentially expressed in vivo by Metarhizium anisopliae in the hemolymph of Locusta migratoria using suppression-subtractive hybridization.

    Science.gov (United States)

    Zhang, Chuanbo; Xia, Yuxian

    2009-08-01

    Metarhizium anisopliae is an important insect pathogenic fungus widely used in biological pest control. The aim of this study was to identify genes differentially expressed in vivo by M. anisopliae CQMa102 in the hemolymph of infected Locusta migratoria. Suppression-subtractive hybridization was performed using cDNA generated from hyphal bodies purified from hemolymph and the fungus germinating and differentiating on locust wings. A total of 350/1,600 random clones screened by cDNA array dot blotting were sequenced, resulting in 120 uniquely expressed sequence tags (ESTs) that were up-regulated during colonization of hemolymph. Among these 120 ESTs, 42 (35.0%) had matches in the NR protein database, and 29 (24.2%) were significantly similar to known proteins involved in various cellular processes, including general metabolism, cell wall remodeling, protein synthesis, signal transduction and stress responses. In contrast, the remaining 78 ESTs (65.0%) either had low similarity in the NR database or represented novel genes. Semi-quantitative RT-PCR analysis of five randomly selected genes revealed that all were highly expressed in the host hemolymph. These results provide new insight into the underlying molecular mechanisms of adaptation to host hemolymph and may increase understanding of host-pathogen interactions.

  8. Rapid Genotyping of the Human Renin (REN Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

    Directory of Open Access Journals (Sweden)

    Line Wee

    2010-01-01

    Full Text Available Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm. Ninety-two mother-father-child triads (n=276 from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs in REN. All three htSNPs (rs5705, rs1464816 and rs3795575 were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041 were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

  9. Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis.

    Science.gov (United States)

    Paungfoo, Chanyarat; Prasertsan, Poonsuk; Burrell, Paul C; Intrasungkha, Nugul; Blackall, Linda L

    2007-07-01

    Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.

  10. Detection of Helicobacter Pylori Genome with an Optical Biosensor Based on Hybridization of Urease Gene with a Gold Nanoparticles-Labeled Probe

    Science.gov (United States)

    Shahrashoob, M.; Mohsenifar, A.; Tabatabaei, M.; Rahmani-Cherati, T.; Mobaraki, M.; Mota, A.; Shojaei, T. R.

    2016-05-01

    A novel optics-based nanobiosensor for sensitive determination of the Helicobacter pylori genome using a gold nanoparticles (AuNPs)-labeled probe is reported. Two specific thiol-modified capture and signal probes were designed based on a single-stranded complementary DNA (cDNA) region of the urease gene. The capture probe was immobilized on AuNPs, which were previously immobilized on an APTES-activated glass, and the signal probe was conjugated to different AuNPs as well. The presence of the cDNA in the reaction mixture led to the hybridization of the AuNPs-labeled capture probe and the signal probe with the cDNA, and consequently the optical density of the reaction mixture (AuNPs) was reduced proportionally to the cDNA concentration. The limit of detection was measured at 0.5 nM.

  11. Rapid identification of Candida spp. frequently involved in invasive mycoses by using flow-through hybridization and Gene Chip (FHGC) technology.

    Science.gov (United States)

    Li, Chen; Ding, Xiurong; Liu, Zhizhong; Zhu, Juanjuan

    2017-01-01

    The incidence of invasive fungal infections in immunocompromised patients has increased in recent decades. Rapid and accurate identification of these pathogenic fungi is crucial for initiating a timely, safe, and effective antifungal therapy. Here we developed a microarray based on flow-through hybridization gene chip technology. The microarray was tested for its specificity using a panel of reference and blinded clinical isolates. The results proved that this microarray was highly discriminative, leading to the unequivocal identification of each species, including Candida famata and the highly related species Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis. This new system represents a reliable method that is of potential use in clinical laboratories for the simultaneous detection and identification of the most common pathogenic fungi. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Development of SNP-based dCAPS markers for identifying male sterile gene tms5 in two-line hybrid rice.

    Science.gov (United States)

    Song, F S; Ni, J L; Qian, Y L; Li, L; Ni, D H; Yang, J B

    2016-08-29

    Molecular markers can increase both the efficiency and speed of breeding programs. Functional markers that detect the functional mutations causing phenotypic changes offer a precise method for genetic identification. In this study, we used newly derived cleaved amplified polymorphic sequence markers to detect the functional mutations of tms5, which is a male sterile gene that is widely used in rice production in China. In addition, restriction cutting sites were designed to specifically digest amplicons of tms5 but not wild type (TMS5), in order to avoid the risk of false positive results. By optimizing the condition of the polymerase chain reaction amplifications and restriction enzyme digestions, the newly designed markers could accurately distinguish between tms5 and TMS5. These markers can be applied in marker-assisted selection for breeding novel thermo-sensitive genic male sterile (TGMS) lines, as well as to rapidly identify the TGMS hybrid seed purity.

  13. Indications for potential parent-of-origin effects within the FTO gene.

    Directory of Open Access Journals (Sweden)

    Xuanshi Liu

    Full Text Available Genome-Wide Association Studies (GWAS were successfully applied to discover associations with obesity. However, the GWAS design is usually based on unrelated individuals and inheritance information on the parental origin of the alleles is missing. Taking into account parent-of-origin may provide further insights into the genetic mechanisms contributing to obesity. We hypothesized that there may be variants within the robustly replicated fat mass and obesity associated (FTO gene that may confer different risk for obesity depending on transmission from mother or father. Genome-wide genotypes and pedigree information from the Sorbs population were used. Phased genotypes among 525 individuals were generated by AlphaImpute. Subsequently, 22 SNPs within FTO introns 1 to 3 were selected and parent-of-origin specific association analyses were performed using PLINK. Interestingly, we identified several SNPs conferring different genetic effects (P≤0.05 depending on parental origin--among them, rs1861868, rs1121980 and rs9939973 (all in intron 1. To confirm our findings, we investigated the selected variants in 705 German trios comprising an (extremely obese child or adolescent and both parents. Again, we observed evidence for POE effects in intron 2 and 3 (P≤0.05 as indicated by the parental asymmetry test. Our results suggest that the obesity risk transmitted by several FTO variants may depend on the parental origin of the allele. Larger family-based studies are warranted to replicate our findings.

  14. Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    AcNPV(Autographa californica nuclear polyhedrosis virus)and BmNPV(Bombyx mori nuclear polyhedrosis virus)are two principal insect-baculovirus expression systems,each having different characteristics.AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression,but the small size of the host insect,A.californica,makes AcNPV less suitable for large scale protein synthesis.In contrast,BmNPV can only infect the silkworm,Bornbyx rnori,which is well-known for its easy rearing and large size.These characteristics make the BmNPV system especially suitable for large-scale industrial expression.To utilize the advantages of both AcNPV and BmNPV,we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV,designated as HyNPV.The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV.Taking the human basic fibroblast growth factor(Bfgf)gene as an application example,we constructed a recombinant,HyNPV-Bfgf.This construct is able to express the Bfgf protein both in silkworm larvae and in common-use cell lines,sf21,sf9 and High-five.Moreover,to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus,we knocked out the cysteinase gene coding for one of the most important baculovirus proteases.This knockout mutation improves the production efficiency of the Bfgf recombinant protein.

  15. Identification of differentially expressed genes associated with bud dormancy release in tree peony (Paeonia suffruticosa) by suppression subtractive hybridization

    Institute of Scientific and Technical Information of China (English)

    HUANG Xin; ZHENG Guo-sheng; DAI Si-lan; GAI Shu-peng

    2008-01-01

    A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies. In order to identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening. Of these, 185 clones were selected to be sequenced. A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database. Semi-quantitative RT-PCR was used to confirm further the expression profiles for 12 transcripts identified within the subtraetive cDNA library. Gene ontology analyses indicated that many of the different genes identified have unknown or hypothetical functions while it is speculated that other genes play different molecular roles. In our study, genes involved in bud dormancy release were growth-related or stress-responsive, while low-temperature-induced ribosomal proteins may also play a role in bud dormancy release. Our results provide interesting information for further understanding of the molecular mechanism of bud dormancy release in tree peonies.

  16. Identification of a male meiosis-specific gene, Tcte2, which is differentially spliced in species that form sterile hybrids with laboratory mice and deleted in t chromosomes showing meiotic drive.

    Science.gov (United States)

    Braidotti, G; Barlow, D P

    1997-06-01

    Tcte2 (t complex testes expressed 2) is a male meiosis-specific gene that maps to band 3.3 of mouse chromosome 17. Two distinct male fertility defects, hybrid sterility and transmission ratio distortion, have previously been mapped to this region. Hybrid sterility arises in crosses between different mouse species and the F1 generation males have defects in the first meiotic division and are sterile. Transmission ratio distortion is shown by males heterozygous for the t haplotype form of chromosome 17 and is a type of meiotic drive in which male gametes function unequally at fertilization. The Tcte2 gene expresses a coding mRNA and a number of putative non-ORF transcripts in meiosis I. A deletion of the 5' part of the locus abolishes Tcte2 expression on the t haplotype form of chromosome 17. Additionally, the series of putative non-ORF RNAs at the Tcte2 locus are differentially spliced in species that show hybrid sterility when crossed to laboratory mice. The identification of polymorphisms in t haplotypes and in different mouse species allows alleles of Tcte2 to be proposed as candidates for loci which contribute to both meiotic drive and hybrid sterility phenotypes. While theoretical considerations have previously been used to propose that speciation and meiotic drive involve alleles of the same genes, Tcte2 is the first cloned candidate gene to support this link at a molecular level.

  17. Hybrid Baryons

    CERN Document Server

    Page, P R

    2003-01-01

    We review the status of hybrid baryons. The only known way to study hybrids rigorously is via excited adiabatic potentials. Hybrids can be modelled by both the bag and flux-tube models. The low-lying hybrid baryon is N 1/2^+ with a mass of 1.5-1.8 GeV. Hybrid baryons can be produced in the glue-rich processes of diffractive gamma N and pi N production, Psi decays and p pbar annihilation.

  18. Identification of differentially-expressed genes potentially implicated in drought response in pitaya (Hylocereus undatus) by suppression subtractive hybridization and cDNA microarray analysis.

    Science.gov (United States)

    Fan, Qing-Jie; Yan, Feng-Xia; Qiao, Guang; Zhang, Bing-Xue; Wen, Xiao-Peng

    2014-01-01

    Drought is one of the most severe threats to the growth, development and yield of plant. In order to unravel the molecular basis underlying the high tolerance of pitaya (Hylocereus undatus) to drought stress, suppression subtractive hybridization (SSH) and cDNA microarray approaches were firstly combined to identify the potential important or novel genes involved in the plant responses to drought stress. The forward (drought over drought-free) and reverse (drought-free over drought) suppression subtractive cDNA libraries were constructed using in vitro shoots of cultivar 'Zihonglong' exposed to drought stress and drought-free (control). A total of 2112 clones, among which half were from either forward or reverse SSH library, were randomly picked up to construct a pitaya cDNA microarray. Microarray analysis was carried out to verify the expression fluctuations of this set of clones upon drought treatment compared with the controls. A total of 309 expressed sequence tags (ESTs), 153 from forward library and 156 from reverse library, were obtained, and 138 unique ESTs were identified after sequencing by clustering and blast analyses, which included genes that had been previously reported as responsive to water stress as well as some functionally unknown genes. Thirty six genes were mapped to 47 KEGG pathways, including carbohydrate metabolism, lipid metabolism, energy metabolism, nucleotide metabolism, and amino acid metabolism of pitaya. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction (RT-PCR) corroborated the results of differential screening. Moreover, time-course expression patterns of these selected ESTs further confirmed that they were closely responsive to drought treatment. Among the differentially expressed genes (DEGs), many are related to stress tolerances including drought tolerance. Thereby, the mechanism of drought tolerance of this pitaya genotype is a very complex physiological and biochemical process, in

  19. Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization

    Institute of Scientific and Technical Information of China (English)

    XUE QIONG WU; YANG LU; JIAN QIN LIANG; JUN XIAN ZHANG; GUANG YU ZHANG; CUI HUAN L(U); HONG MIN LI; BEI CHUAN DING

    2006-01-01

    The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol (EMB) resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization (RDBH) in addition to evaluating the clinical value with application of PCR-RD-BH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates (65.9%) showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation (38/91,41.8% ) and the ATA mutation ( 16/91, 17.6% ) were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing for one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.

  20. Transcription profiles of boron-deficiency-responsive genes in citrus rootstock root by suppression subtractive hybridization and cDNA microarray.

    Science.gov (United States)

    Zhou, Gao-Feng; Liu, Yong-Zhong; Sheng, Ou; Wei, Qing-Jiang; Yang, Cheng-Quan; Peng, Shu-Ang

    2014-01-01

    Boron (B) deficiency has seriously negative effect on citrus production. Carrizo citrange (CC) has been reported as a B-deficiency tolerant rootstock. However, the molecular mechanism of its B-deficiency tolerance remained not well-explored. To understand the molecular basis of citrus rootstock to B-deficiency, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes responsive to B-deficiency. Firstly four SSH libraries were constructed for the root tissue of two citrus rootstocks CC and Trifoliate orange (TO) to compare B-deficiency treated and non-treated plants. Then 7680 clones from these SSH libraries were used to construct a cDNA array and microarray analysis was carried out to verify the expression changes of these clones upon B-deficiency treatment at various time points compared to the corresponding controls. A total of 139 unigenes that were differentially expressed upon B-deficiency stress either in CC or TO were identified from microarray analysis, some of these genes have not previously been reported to be associated with B-deficiency stress. In this work, several genes involved in cell wall metabolism and transmembrane transport were identified to be highly regulated under B-deficiency stress, and a total of 23 metabolic pathways were affected by B-deficiency, especially the lignin biosynthesis pathway, nitrogen metabolism, and glycolytic pathway. All these results indicated that CC was more tolerant than TO to B-deficiency stress. The B-deficiency responsive genes identified in this study could provide further information for understanding the mechanisms of B-deficiency tolerance in citrus.

  1. High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

    Directory of Open Access Journals (Sweden)

    Clark Taane G

    2010-04-01

    Full Text Available Abstract Background Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. Results Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%. Conclusions Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes

  2. Karyotyping and single-gene detection using fluorescence in situ hybridization on chromosomes of Hydra magnipapillata (Cnidaria: Hydrozoa

    Directory of Open Access Journals (Sweden)

    B Anokhin

    2010-12-01

    Full Text Available The fresh water polyp Hydra L., 1758 (Cnidaria, Hydrozoa plays a key role as a model organism in modern evolutionary and developmental biology. A complete genome sequence has been published recently for Hydra magnipapillata Ito, 1947 and molecular data are rapidly accumulating in the literature, but little information is available on its chromosomes. In this study, an efficient fluorescence in situ hybridization (FISH method is described for H. magnipapillata which not only allows identification of the chromosomes but also visualization of the location of individual genetic loci. Together with cDNA and genomic sequencing this may provide the foundation for increasingly precise genetic and physical mapping in this basal metazoan model organism.

  3. Mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments.

    Science.gov (United States)

    Lewis, J B; Atkins, J F; Anderson, C W; Baum, P R; Gesteland, R F

    1975-04-01

    Cytoplasmic RNA, isolated from cells late after infection by adenovirus type 2 and fractionated by hybridization to specific fragments of adenovirus DNA produced by cleavage with the endonuclease R-EcoRI, was used as template for protein synthesis in cell-free mammalian extracts. Each of the R-EcoRI fragments of DNA selects RNA that encodes specific subsets of the viral polypeptides. From the known order of the R-EcoRI fragments, the following partial map is deduced: (III, IIIa, IVa2, V, P-VII, IX), (II, P-VI), 100K, IV-where the relative order of the components enclosed in parentheses has not yet been determined.

  4. Toxic effect on tissues and differentially expressed genes in hepatopancreas identified by suppression subtractive hybridization of freshwater pearl mussel (Hyriopsis cumingii) following microcystin-LR challenge.

    Science.gov (United States)

    Yang, Ziyan; Wu, Hongjuan; Li, Yuan

    2012-07-01

    Microcystins are a family of potent hepatotoxins produced by freshwater cyanobacteria and can cause animal intoxications and human diseases. In this study, the effect of microcystin-LR (MC-LR) on the tissues of freshwater pearl mussel (Hyriopsis cumingii) was evaluated and differentially expressed genes in the hepatopancreas of the mussel exposed to MC-LR were identified. HPLC analysis of cell extracts from various tissues of the mussel indicated that the hepatopancreas had the highest MC-LR levels (55.78 ± 6.73 μg g⁻¹ DW) after 15-day exposure. The MC-LR concentration in gill or muscle was an order of magnitude less than in hepatopancreas or gonad. Subtractive cDNA library was constructed by suppression subtractive hybridization (SSH), and ∼400 positive clones were sequenced, from which 98 high quality sequences were obtained by BLAST analysis. The screening identified numerous genes involved in apoptosis, signal transduction, cytoskeletal remodel, innate immunity, material and energy metabolism, translation and transcription which were extensively discussed. The results of this study add large amount of information to the mussel genome data, and for the first time present the basic data on toxicity effect of MC-LR on mussel.

  5. Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes.

    Science.gov (United States)

    Ye, Y W; Ling, N; Han, Y J; Wu, Q P

    2014-11-01

    Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10(2)cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28 °C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.

  6. Clinical application of modified bag-of-features coupled with hybrid neural-based classifier in dengue fever classification using gene expression data.

    Science.gov (United States)

    Chatterjee, Sankhadeep; Dey, Nilanjan; Shi, Fuqian; Ashour, Amira S; Fong, Simon James; Sen, Soumya

    2017-09-11

    Dengue fever detection and classification have a vital role due to the recent outbreaks of different kinds of dengue fever. Recently, the advancement in the microarray technology can be employed for such classification process. Several studies have established that the gene selection phase takes a significant role in the classifier performance. Subsequently, the current study focused on detecting two different variations, namely, dengue fever (DF) and dengue hemorrhagic fever (DHF). A modified bag-of-features method has been proposed to select the most promising genes in the classification process. Afterward, a modified cuckoo search optimization algorithm has been engaged to support the artificial neural (ANN-MCS) to classify the unknown subjects into three different classes namely, DF, DHF, and another class containing convalescent and normal cases. The proposed method has been compared with other three well-known classifiers, namely, multilayer perceptron feed-forward network (MLP-FFN), artificial neural network (ANN) trained with cuckoo search (ANN-CS), and ANN trained with PSO (ANN-PSO). Experiments have been carried out with different number of clusters for the initial bag-of-features-based feature selection phase. After obtaining the reduced dataset, the hybrid ANN-MCS model has been employed for the classification process. The results have been compared in terms of the confusion matrix-based performance measuring metrics. The experimental results indicated a highly statistically significant improvement with the proposed classifier over the traditional ANN-CS model.

  7. Human carbonyl reductase (CBR) localized to band 21q22. 1 by high-resolution fluorescence in situ hybridization displays gene dosage effects in trisomy 21 cells

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    Lemieux, N. (Universite de Montreal (Canada)); Malfoy, B. (Institut Curie Section de Biologie, Paris (France)); Forrest, G.L. (Beckman Research Institute at the City of Hope, Duarte, CA (United States))

    1993-01-01

    Human carbonyl reductase (CBR) belongs to a group of NADPH-dependent enzymes called aldo-keto reductases. The enzyme can function as an aldo-keto reductase or as a quinone reductase with potential for modulating quinone-mediated oxygen free radicals. The CBR gene was mapped by high-resolution fluorescence in situ hybridization to band 21q22.12, very close to the SOD1 locus at position 2lq22.11. CBR displayed gene dosage effects in trisomy 21 human lymphoblasts at the DNA and mRNA levels. Lymphoblasts with increasing chromosome 21 ploidy also showed increased aldo-keto reductase activity and increased quinone reductase activity. Both aldo-keto reductase activity and quinone reductase activity have been shown to be associated with carbonyl reductase. The location of CBR near SOD1 and the increased enzyme activity and potential for free radical modulation in trisomy 21 cells implicate CBR as a candidate for contributing to the pathology of certain diseases such as Down syndrome and Alzheimer disease. 28 refs., 1 fig., 1 tab.

  8. Identification of potential marker genes for Trichoderma harzianum strains with high antagonistic potential against Rhizoctonia solani by a rapid subtraction hybridization approach.

    Science.gov (United States)

    Scherm, Barbara; Schmoll, Monika; Balmas, Virgilio; Kubicek, Christian P; Migheli, Quirico

    2009-02-01

    A rapid subtraction hybridization approach was used to isolate genes differentially expressed during mycelial contact between Trichoderma harzianum (Hypocrea lixii) and Rhizoctonia solani, and could serve as marker genes for selection of superior biocontrol strains. Putatively positive clones were evaluated by transcription analysis during mycelial contact with R. solani versus growth on glucose, and for their differential transcription between two strains with either strong or poor biocontrol capability before, at, and after contact with R. solani. Besides four clones, which had similarity to putative but as yet uncharacterized proteins, they comprised ribosomal proteins, proteins involved in transcriptional switch and regulation, amino acid and energy catabolism, multidrug resistance, and degradation of proteins and glucans. Transcription of three clones was evaluated in five T. harzianum strains under confrontation conditions with R. solani. Two clones-acetyl-xylane esterase AXE1 and endoglucanase Cel61b-showed significant upregulation during in vivo confrontation of a T. harzianum strain that successively demonstrated a very high antagonistic capability towards R. solani, while expression was progressively lower in a series of T. harzianum strains with intermedi