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Sample records for cell-lineage gene highly

  1. Heritable Gene Regulation in the CD4:CD8 T Cell Lineage Choice

    PubMed Central

    Issuree, Priya D. A.; Ng, Charles P.; Littman, Dan R.

    2017-01-01

    The adaptive immune system is dependent on functionally distinct lineages of T cell antigen receptor αβ-expressing T cells that differentiate from a common progenitor in the thymus. CD4+CD8+ progenitor thymocytes undergo selection following interaction with MHC class I and class II molecules bearing peptide self-antigens, giving rise to CD8+ cytotoxic and CD4+ helper or regulatory T cell lineages, respectively. The strict correspondence of CD4 and CD8 expression with distinct cellular phenotypes has made their genes useful surrogates for investigating molecular mechanisms of lineage commitment. Studies of Cd4 and Cd8 transcriptional regulation have uncovered cis-regulatory elements that are critical for mediating epigenetic modifications at distinct stages of development to establish heritable transcriptional programs. In this review, we examine the epigenetic mechanisms involved in Cd4 and Cd8 gene regulation during T cell lineage specification and highlight the features that make this an attractive system for uncovering molecular mechanisms of heritability. PMID:28382035

  2. Extracting Fluorescent Reporter Time Courses of Cell Lineages from High-Throughput Microscopy at Low Temporal Resolution

    PubMed Central

    Downey, Mike J.; Jeziorska, Danuta M.; Ott, Sascha; Tamai, T. Katherine; Koentges, Georgy; Vance, Keith W.; Bretschneider, Till

    2011-01-01

    The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell

  3. Snai2 and Snai3 transcriptionally regulate cellular fitness and functionality of T cell lineages through distinct gene programs.

    PubMed

    Pioli, Peter D; Whiteside, Sarah K; Weis, Janis J; Weis, John H

    2016-05-01

    T lymphocytes are essential contributors to the adaptive immune system and consist of multiple lineages that serve various effector and regulatory roles. As such, precise control of gene expression is essential to the proper development and function of these cells. Previously, we identified Snai2 and Snai3 as being essential regulators of immune tolerance partly due to the impaired function of CD4(+) regulatory T cells in Snai2/3 conditional double knockout mice. Here we extend those previous findings using a bone marrow transplantation model to provide an environmentally unbiased view of the molecular changes imparted onto various T lymphocyte populations once Snai2 and Snai3 are deleted. The data presented here demonstrate that Snai2 and Snai3 transcriptionally regulate the cellular fitness and functionality of not only CD4(+) regulatory T cells but effector CD8(α+) and CD4(+) conventional T cells as well. This is achieved through the modulation of gene sets unique to each cell type and includes transcriptional targets relevant to the survival and function of each T cell lineage. As such, Snai2 and Snai3 are essential regulators of T cell immunobiology.

  4. Identification of a cell lineage-specific gene coding for a sea urchin alpha 2(IV)-like collagen chain.

    PubMed

    Exposito, J Y; Suzuki, H; Geourjon, C; Garrone, R; Solursh, M; Ramirez, F

    1994-05-06

    We report the isolation of several overlapping cDNAs from an embryonic library of Strongylocentrotus purpuratus coding for a novel sea urchin collagen chain. The conceptual amino acid translation of the cDNAs indicated that the protein displays the structural features of a vertebrate type IV-like collagen alpha chain. In addition to a putative 31-residue signal peptide, the sea urchin molecule contains a 14-residue amino-terminal non-collagenous segment, a discontinuous 1,477-amino acid triple helical domain, and a 225-residue carboxyl-terminal domain rich in cysteines. The amino- and carboxyl-terminal non-collagenous regions of the echinoid molecule are remarkably similar to the 7 S and carboxyl-terminal non-collagenous (NC1) domains of the alpha 1 and alpha 2 chains of vertebrate type IV collagen. The sequence similarity and distinct structural features of the 7 S and NC1 domains strongly suggest that the sea urchin polypeptide is evolutionarily related to the alpha 2(IV) class of collagen chains. Finally, in situ hybridizations revealed that expression of this collagen gene is restricted to the mesenchyme cell lineage of the developing sea urchin embryo.

  5. Dysregulation of granulocyte, erythrocyte, and NK cell lineages in Fli-1 gene-targeted mice.

    PubMed

    Masuya, Masahiro; Moussa, Omar; Abe, Takanori; Deguchi, Takao; Higuchi, Tsukasa; Ebihara, Yasuhiro; Spyropoulos, Demetri D; Watson, Dennis K; Ogawa, Makio

    2005-01-01

    Targeted disruption of the Friend leukemia integration 1 (Fli-1) proto-oncogene results in severe dysmegakaryopoiesis and embryonic lethality. We used morula-stage aggregation as a strategy to further clarify the hematopoietic defects of the Fli-1 gene-targeted mice. Analyses of lineage expression of Fli-1(+/-) and Fli-1(-/-) cells in the peripheral blood and bone marrow of chimeric mice consistently demonstrated reduced numbers of neutrophilic granulocytes and monocytes and increased numbers of natural killer (NK) cells. Transplantation studies using sorted Fli-1 mutant cells produced similar findings. Clonal culture studies of bone marrow cells revealed increased numbers of granulocytic and early erythroid progenitors in the Fli-1(+/-) cells. The sorted Fli-1(-/-) bone marrow cells revealed specific down-regulation of CCAAT/enhancer binding protein-alpha (C/EBPalpha) and C/EBPepsilon, and the receptors for granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF), consistent with their critical roles in granulopoiesis. Collectively, these observations suggest previously unknown physiologic roles for Fli-1 in granulocytic, erythroid, and NK cell proliferation and differentiation. Production of chimeras by morula-stage embryo aggregation is an effective way to unravel cell-autonomous hematopoietic defects in gene-targeted mice.

  6. Digital development: a database of cell lineage differentiation in C. elegans with lineage phenotypes, cell-specific gene functions and a multiscale model

    PubMed Central

    Santella, Anthony; Kovacevic, Ismar; Herndon, Laura A.; Hall, David H.; Du, Zhuo; Bao, Zhirong

    2016-01-01

    Developmental systems biology is poised to exploit large-scale data from two approaches: genomics and live imaging. The combination of the two offers the opportunity to map gene functions and gene networks in vivo at single-cell resolution using cell tracking and quantification of cellular phenotypes. Here we present Digital Development (http://www.digital-development.org), a database of cell lineage differentiation with curated phenotypes, cell-specific gene functions and a multiscale model. The database stores data from recent systematic studies of cell lineage differentiation in the C. elegans embryo containing ∼200 conserved genes, 1400 perturbed cell lineages and 600 000 digitized single cells. Users can conveniently browse, search and download four categories of phenotypic and functional information from an intuitive web interface. This information includes lineage differentiation phenotypes, cell-specific gene functions, differentiation landscapes and fate choices, and a multiscale model of lineage differentiation. Digital Development provides a comprehensive, curated, multidimensional database for developmental biology. The scale, resolution and richness of biological information presented here facilitate exploration of gene-specific and systems-level mechanisms of lineage differentiation in Metazoans. PMID:26503254

  7. Cloning and characterization of a vasa-like gene in rainbow trout and its expression in the germ cell lineage.

    PubMed

    Yoshizaki, G; Sakatani, S; Tominaga, H; Takeuchi, T

    2000-04-01

    The origin of germ cells and the molecular mechanisms of primordial germ cell (PGC) determination in teleosts are unclear. Vasa is a member of the DEAD protein family and plays an indispensable role in germ cell determination in Drosophila and Xenopus species. In this study, we isolated and characterized a rainbow trout vasa cDNA as a first step towards understanding the molecular mechanisms of PGC determination and development and to develop a molecular marker to identify the PGCs in rainbow trout. Cloning of vasa cDNA was performed by degenerate- and RACE-PCR. The predicted amino acid sequence of rainbow trout Vasa contained eight consensus sequences for the DEAD protein family and five arginine-glycine-glycine repeats, a common character of known Vasa homologues. Overall amino acid similarity to the Vasa of Drosophila was 79.2%. Whole-mount in situ hybridization of eyed stage embryos (eighty somite stage) revealed that signals were localized to the putative PGCs. In adult rainbow trout tissues, both ovaries and testes contained large amounts of vasa gene transcripts. A reverse transcription-polymerase chain reaction analysis of unfertilized eggs proved that trout vasa is a maternal factor. Although we have not determined whether rainbow trout vasa functions as a germ cell determinant, its limited expression in the germ cell lineage proved that rainbow trout vasa can be used as a marker molecule for PGCs. This marker will make it possible to identify the PGCs or presumptive PGCs in early trout embryos whose germ cells can not be distinguished by morphological characteristics.

  8. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage

    PubMed Central

    Lu, Chenggang; Fuller, Margaret T.

    2015-01-01

    Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages. PMID:26624996

  9. Analysis of GzmbCre as a Model System for Gene Deletion in the Natural Killer Cell Lineage.

    PubMed

    Xu, Yiying; Evaristo, Cesar; Alegre, Maria-Luisa; Gurbuxani, Sandeep; Kee, Barbara L

    2015-01-01

    The analysis of gene function in mature and activated natural killer cells has been hampered by the lack of model systems for Cre-mediated recombination in these cells. Here we have investigated the utility of GzmbCre for recombination of loxp sequences in these cells predicated on the observation that Gzmb mRNA is highly expressed in mature and activated natural killer cells. Using two different reporter strains we determined that gene function could be investigated in mature natural killer cells after GzmbCre mediated recombination in vitro in conditions that lead to natural killer cell activation such as in the cytokine combination of interleukin 2 and interleukin 12. We demonstrated the utility of this model by creating GzmbCre;Rosa26IKKbca mice in which Cre-mediated recombination resulted in expression of constitutively active IKKβ, which results in activation of the NFκB transcription factor. In vivo and in vitro activation of IKKβ in natural killer cells revealed that constitutive activation of this pathway leads to natural killer cell hyper-activation and altered morphology. As a caveat to the use of GzmbCre we found that this transgene can lead to recombination in all hematopoietic cells the extent of which varies with the particular loxp flanked allele under investigation. We conclude that GzmbCre can be used under some conditions to investigate gene function in mature and activated natural killer cells.

  10. Down-Regulating the Expression of 53 Soybean Transcription Factor Genes Uncovers a Role for SPEECHLESS in Initiating Stomatal Cell Lineages during Embryo Development.

    PubMed

    Danzer, John; Mellott, Eric; Bui, Anhthu Q; Le, Brandon H; Martin, Patrick; Hashimoto, Meryl; Perez-Lesher, Jeanett; Chen, Min; Pelletier, Julie M; Somers, David A; Goldberg, Robert B; Harada, John J

    2015-07-01

    We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and inducer of C-repeat/dehydration responsive element-binding factor expression1/scream2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development.

  11. Down-Regulating the Expression of 53 Soybean Transcription Factor Genes Uncovers a Role for SPEECHLESS in Initiating Stomatal Cell Lineages during Embryo Development1[OPEN

    PubMed Central

    Danzer, John; Mellott, Eric; Bui, Anhthu Q.; Le, Brandon H.; Martin, Patrick; Hashimoto, Meryl; Perez-Lesher, Jeanett; Chen, Min; Pelletier, Julie M.; Somers, David A.; Goldberg, Robert B.; Harada, John J.

    2015-01-01

    We used an RNA interference screen to assay the function of 53 transcription factor messenger RNAs (mRNAs) that accumulate specifically within soybean (Glycine max) seed regions, subregions, and tissues during development. We show that basic helix-loop-helix (bHLH) transcription factor genes represented by Glyma04g41710 and its paralogs are required for the formation of stoma in leaves and stomatal precursor complexes in mature embryo cotyledons. Phylogenetic analysis indicates that these bHLH transcription factor genes are orthologous to Arabidopsis (Arabidopsis thaliana) SPEECHLESS (SPCH) that initiate asymmetric cell divisions in the leaf protoderm layer and establish stomatal cell lineages. Soybean SPCH (GmSPCH) mRNAs accumulate primarily in embryo, seedling, and leaf epidermal layers. Expression of Glyma04g41710 under the control of the SPCH promoter rescues the Arabidopsis spch mutant, indicating that Glyma04g41710 is a functional ortholog of SPCH. Developing soybean embryos do not form mature stoma, and stomatal differentiation is arrested at the guard mother cell stage. We analyzed the accumulation of GmSPCH mRNAs during soybean seed development and mRNAs orthologous to MUTE, FAMA, and INDUCER OF C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT-BINDING FACTOR EXPRESSION1/SCREAM2 that are required for stoma formation in Arabidopsis. The mRNA accumulation patterns provide a potential explanation for guard mother cell dormancy in soybean embryos. Our results suggest that variation in the timing of bHLH transcription factor gene expression can explain the diversity of stomatal forms observed during plant development. PMID:25963149

  12. BMP Signaling and Its pSMAD1/5 Target Genes Differentially Regulate Hair Follicle Stem Cell Lineages

    PubMed Central

    Genander, Maria; Cook, Peter J.; Ramsköld, Daniel; Keyes, Brice E.; Mertz, Aaron F.; Sandberg, Rickard; Fuchs, Elaine

    2014-01-01

    Hair follicle stem cells (HFSCs) and their transit amplifying cell (TAC) progeny sense BMPs at defined stages of the hair cycle to control their proliferation and differentiation. Here, we exploit the distinct spatial and temporal localizations of these cells to selectively ablate BMP signaling in each compartment and examine its functional role. We find that BMP signaling is required for HFSC quiescence and to promote TAC differentiation along different lineages as the hair cycle progresses. We also combine in vivo genome-wide chromatin immunoprecipitation and deep-sequencing, transcriptional profiling, and loss-of-function genetics to define BMP-regulated genes. We show that some pSMAD1/5 targets, like Gata3, function specifically in TAC lineage-progression. Others, like Id1 and Id3, function in both HFSCs and TACs, but in distinct ways. Our study therefore illustrates the complex differential roles that a key signaling pathway can play in regulation of closely-related stem/progenitor cells within the context of their overall niche. PMID:25312496

  13. Characterization of cytarabine-resistant leukemic cell lines established from five different blood cell lineages using gene expression and proteomic analyses.

    PubMed

    Negoro, Eiju; Yamauchi, Takahiro; Urasaki, Yoshimasa; Nishi, Rie; Hori, Hiroki; Ueda, Takanori

    2011-04-01

    Cytarabine (ara-C) is the key drug for treatment of acute myeloid leukemia. Since intracellular cytarabine triphosphate (ara-CTP) is an active metabolite of ara-C, factors that reduce the amount of ara-CTP are known to induce drug resistance. However, these factors do not fully explain the development of resistance to ara-C. The present study was conducted to search for new candidate ara-C resistance factors, including those that are unrelated to ara-CTP production. For this purpose, we newly established five ara-C-resistant leukemic clones from different blood cell lineage leukemic cell lines (HL-60, K562, CEM, THP1 and U937). The resistant subclones were 5-58-fold more ara-C-resistant than their parental counterparts. All of the ara-C-resistant subclones, except for ara-C-resistant CEM cells, displayed alteration of ara-CTP-related factors such as ara-C membrane transport capacity, deoxycytidine kinase activity or cytosolic nucleotidase II activity. To identify new candidate factors, we used two comprehensive approaches: DNA microarray and proteome analyses. The DNA microarray analysis revealed eight genes (C19orf2, HSPA8, LGALS1, POU4F3, PSAP, AKT1, MBC2 and CACNA2D3) that were altered in all five ara-C-resistant lines compared to parental cells. Both proteome and DNA microarray analyses further detected a reduced protein level of stathmin1 in the ara-C-resistant CEM subclone compared to its parental line. Thus, the present findings suggested the involvement of novel multiple mechanisms in mediating the ara-C resistance of leukemic cells. The role of some of these molecules in resistance is still unclear.

  14. Evolution of two prototypic T cell lineages

    PubMed Central

    Das, Sabyasachi; Li, Jianxu; Hirano, Masayuki; Sutoh, Yoichi; Herrin, Brantley R.; Cooper, Max D.

    2015-01-01

    Jawless vertebrates, which occupy a unique position in chordate phylogeny, employ leucine-rich repeat (LRR)-based variable lymphocyte receptors (VLR) for antigen recognition. During the assembly of the VLR genes (VLRA, VLRB and VLRC), donor LRR-encoding sequences are copied in a step-wise manner into the incomplete germ-line genes. The assembled VLR genes are differentially expressed by discrete lymphocyte lineages: VLRA- and VLRC-producing cells are T-cell like, whereas VLRB-producing cells are B-cell like. VLRA+ and VLRC+ lymphocytes resemble the two principal T-cell lineages of jawed vertebrates that express the αβ or γδ T-cell receptors (TCR). Reminiscent of the interspersed nature of the TCRα/TCRδ locus in jawed vertebrates, the close proximity of the VLRA and VLRC loci facilitates sharing of donor LRR sequences during VLRA and VLRC assembly. Here we discuss the insight these findings provide into vertebrate T- and B-cell evolution, and the alternative types of anticipatory receptors they use for adaptive immunity. PMID:25958271

  15. In vivo cell lineage analysis during chemical hepatocarcinogenesis in rats using retroviral-mediated gene transfer: evidence for dedifferentiation of mature hepatocytes.

    PubMed

    Gournay, Jérôme; Auvigne, Isabelle; Pichard, Virginie; Ligeza, Catherine; Bralet, Marie-Pierre; Ferry, Nicolas

    2002-06-01

    Feeding adult rats with a diet containing 2-acetylaminofluorene (2-AAF) results in suppression of hepatocyte proliferation and stimulation of oval cell proliferation. Although oval cells may be facultative liver stem cells, the actual relationship between oval cells and liver cancer has not been clearly established in vivo. Our goal was to label hepatic cells in vivo using retroviral vectors and follow their fate during the early steps of chemically induced hepatocarcinogenesis. Oval cell proliferation was induced by continuous feeding with a carcinogenic diet containing 2-AAF. We used two different strategies to genetically label hepatic cells: (a) labeling of proliferating cells in rats fed 2-AAF by injecting recombinant retroviral vectors containing the beta-galactosidase gene either in a peripheral vein or in the common bile duct at the peak of oval cell proliferation and (b) prelabeling of hepatocytes by intravenously injecting recombinant vectors 1 day after partial hepatectomy and 1 week before subsequent administration of 2-AAF. Using the first strategy, transgene expression occurred in both oval cells and hepatocytes. Using the second strategy, we could selectively label, and hence study the fate of, differentiated hepatocytes. In the latter case, we observed clusters of beta-galactosidase-positive hepatocytes, some of them also expressing preneoplastic markers such as gamma-glutamyl transpeptidase as well as the placental form of glutathione-S-transferase. These results demonstrate that preneoplastic foci can originate from mature hepatocytes and are consistent with the hypothesis that dedifferentiation of mature hepatocytes may occur during the course of carcinogenic regimen.

  16. Ezh2 represses the basal cell lineage during lung endoderm development.

    PubMed

    Snitow, Melinda E; Li, Shanru; Morley, Michael P; Rathi, Komal; Lu, Min Min; Kadzik, Rachel S; Stewart, Kathleen M; Morrisey, Edward E

    2015-01-01

    The development of the lung epithelium is regulated in a stepwise fashion to generate numerous differentiated and stem cell lineages in the adult lung. How these different lineages are generated in a spatially and temporally restricted fashion remains poorly understood, although epigenetic regulation probably plays an important role. We show that the Polycomb repressive complex 2 component Ezh2 is highly expressed in early lung development but is gradually downregulated by late gestation. Deletion of Ezh2 in early lung endoderm progenitors leads to the ectopic and premature appearance of Trp63+ basal cells that extend the entire length of the airway. Loss of Ezh2 also leads to reduced secretory cell differentiation. In their place, morphologically similar cells develop that express a subset of basal cell genes, including keratin 5, but no longer express high levels of either Trp63 or of standard secretory cell markers. This suggests that Ezh2 regulates the phenotypic switch between basal cells and secretory cells. Together, these findings show that Ezh2 restricts the basal cell lineage during normal lung endoderm development to allow the proper patterning of epithelial lineages during lung formation.

  17. Post-transplantation lymphoproliferative disease of natural killer cell lineage: a clinicopathological and molecular analysis.

    PubMed

    Kwong, Y L; Lam, C C; Chan, T M

    2000-07-01

    Post-transplantation lymphoproliferative disorders (PTLD) occur after solid organ and bone marrow transplantation. They are predominantly of B-cell and occasionally of T-cell lineage. We report a case of PTLD of natural killer (NK) cell lineage. A renal allograft recipient developed progressive pancytopenia 1 year after transplantation. Serial bone marrow biopsies showed an increasing infiltration by large granular lymphoid cells. A subsequent leukaemic phase also developed with systemic infiltration of other organs. Immunophenotyping showed that these cells were CD2+, CD3-, CD3epsilon+, CD56+, CD94+, CD158a- and CD158b-. In situ hybridization showed Epstein-Barr virus (EBV) infection of the neoplastic cells. Genotypical analysis showed the T-cell receptor gene in germline configuration and clonal EBV episomal integration. The overall features were consistent with NK cell lymphoma/leukaemia. The patient did not respond to cessation of immunosuppression or anti-EBV treatment. Combination chemotherapy was given, but the patient died ultimately of disseminated fungal infection. In conclusion, we have demonstrated that NK cell lymphoma is another rare type of PTLD that appears to be highly aggressive and therefore may require early chemotherapy to improve treatment outcome.

  18. High-content image informatics of the structural nuclear protein NuMA parses trajectories for stem/progenitor cell lineages and oncogenic transformation.

    PubMed

    Vega, Sebastián L; Liu, Er; Arvind, Varun; Bushman, Jared; Sung, Hak-Joon; Becker, Matthew L; Lelièvre, Sophie; Kohn, Joachim; Vidi, Pierre-Alexandre; Moghe, Prabhas V

    2017-02-01

    Stem and progenitor cells that exhibit significant regenerative potential and critical roles in cancer initiation and progression remain difficult to characterize. Cell fates are determined by reciprocal signaling between the cell microenvironment and the nucleus; hence parameters derived from nuclear remodeling are ideal candidates for stem/progenitor cell characterization. Here we applied high-content, single cell analysis of nuclear shape and organization to examine stem and progenitor cells destined to distinct differentiation endpoints, yet undistinguishable by conventional methods. Nuclear descriptors defined through image informatics classified mesenchymal stem cells poised to either adipogenic or osteogenic differentiation, and oligodendrocyte precursors isolated from different regions of the brain and destined to distinct astrocyte subtypes. Nuclear descriptors also revealed early changes in stem cells after chemical oncogenesis, allowing the identification of a class of cancer-mitigating biomaterials. To capture the metrology of nuclear changes, we developed a simple and quantitative "imaging-derived" parsing index, which reflects the dynamic evolution of the high-dimensional space of nuclear organizational features. A comparative analysis of parsing outcomes via either nuclear shape or textural metrics of the nuclear structural protein NuMA indicates the nuclear shape alone is a weak phenotypic predictor. In contrast, variations in the NuMA organization parsed emergent cell phenotypes and discerned emergent stages of stem cell transformation, supporting a prognosticating role for this protein in the outcomes of nuclear functions.

  19. The Drosophila cyst stem cell lineage

    PubMed Central

    Zoller, Richard; Schulz, Cordula

    2012-01-01

    In all animals, germline cells differentiate in intimate contact with somatic cells and interactions between germline and soma are particularly important for germline development and function. In the male gonad of Drosophila melanogaster, the developing germline cells are enclosed by somatic cyst cells. The cyst cells are derived from cyst stem cells (CySCs) of somatic origin and codifferentiate with the germline cells. The fast generation cycle and the genetic tractability of Drosophila has made the Drosophila testis an excellent model for studying both the roles of somatic cells in guiding germline development and the interdependence of two separate stem cell lineages. This review focuses on our current understanding of CySC specification, CySC self-renewing divisions, cyst cell differentiation, and soma-germline interactions. Many of the mechanisms guiding these processes in Drosophila testes are similarly essential for the development and function of tissues in other organisms, most importantly for gametogenesis in mammals. PMID:23087834

  20. Matrix elasticity directs stem cell lineage specification

    NASA Astrophysics Data System (ADS)

    Discher, Dennis

    2010-03-01

    Adhesion of stem cells - like most cells - is not just a membrane phenomenon. Most tissue cells need to adhere to a ``solid'' for viability, and over the last decade it has become increasingly clear that the physical ``elasticity'' of that solid is literally ``felt'' by cells. Here we show that Mesenchymal Stem Cells (MSCs) specify lineage and commit to phenotypes with extreme sensitivity to the elasticity typical of tissues [1]. In serum only media, soft matrices that mimic brain appear neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. Inhibition of nonmuscle myosin II activity blocks all elasticity directed lineage specification, which indicates that the cytoskeleton pulls on matrix through adhesive attachments. Results have significant implications for `therapeutic' stem cells and have motivated development of a proteomic-scale method to identify mechano-responsive protein structures [2] as well as deeper physical studies of matrix physics [3] and growth factor pathways [4]. [4pt] [1] A. Engler, et al. Matrix elasticity directs stem cell lineage specification. Cell (2006).[0pt] [2] C.P. Johnson, et al. Forced unfolding of proteins within cells. Science (2007).[0pt] [3] A.E.X. Brown, et al. Multiscale mechanics of fibrin polymer: Gel stretching with protein unfolding and loss of water. Science (2009).[0pt] [4] D.E. Discher, et al. Growth factors, matrices, and forces combine and control stem cells. Science (2009).

  1. Cre-mediated recombination in cell lineages that express the progesterone receptor.

    PubMed

    Soyal, Selma M; Mukherjee, Atish; Lee, Kevin Y-S; Li, Jie; Li, Huaiguang; DeMayo, Francesco J; Lydon, John P

    2005-02-01

    Using gene-targeting methods, a progesterone receptor Cre knockin (PR-Cre) mouse was generated in which Cre recombinase was inserted into exon 1 of the PR gene. The insertion positions the Cre gene downstream (and under the specific control) of the endogenous PR promoter. As for heterozygotes for the progesterone receptor knockout (PRKO) mutation, mice heterozygous for the Cre knockin insertion are phenotypically indistinguishable from wildtype. Crossing the PR-Cre with the ROSA26R reporter revealed that Cre excision activity is restricted to cells that express PR in progesterone-responsive tissues such as the uterus, ovary, oviduct, pituitary gland, and mammary gland. Initial characterization of the PR-Cre mouse underscores the utility of this model to precisely ablate floxed target genes specifically in cell lineages that express the PR. In the wider context of female reproductive tissue ontology, this model will be indispensable in tracing the developmental fate of cell lineages that descend from PR positive progenitors.

  2. A new way to build cell lineages

    PubMed Central

    Zhang, Xiuwei

    2017-01-01

    A combination of single-cell techniques and computational analysis enables the simultaneous discovery of cell states, lineage relationships and the genes that control developmental decisions. PMID:28332977

  3. Related pituitary cell lineages develop into interdigitated 3D cell networks.

    PubMed

    Budry, Lionel; Lafont, Chrystel; El Yandouzi, Taoufik; Chauvet, Norbert; Conéjero, Geneviève; Drouin, Jacques; Mollard, Patrice

    2011-07-26

    The pituitary gland has long been considered to be a random patchwork of hormone-producing cells. By using pituitary-scale tridimensional imaging for two of the least abundant cell lineages, the corticotropes and gonadotropes, we have now uncovered highly organized and interdigitated cell networks that reflect homotypic and heterotypic interactions between cells. Although newly differentiated corticotrope cells appear on the ventral surface of the gland, they rapidly form homotypic strands of cells that extend from the lateral tips of the anterior pituitary along its ventral surface and into the medial gland. As the corticotrope network is established away from the microvasculature, cell morphology changes from rounded, to polygonal, and finally to cells with long cytoplasmic processes or cytonemes that connect corticotropes to the perivascular space. Gonadotropes differentiate later and are positioned in close proximity to corticotropes and capillaries. Blockade of corticotrope terminal differentiation produced by knockout of the gene encoding the transcription factor Tpit results in smaller gonadotropes within an expanded cell network, particularly in the lateral gland. Thus, pituitary-scale tridimensional imaging reveals highly structured cell networks of unique topology for each pituitary lineage. The sequential development of interdigitated cell networks during organogenesis indicate that extensive cell:cell interactions lead to a highly ordered cell positioning rather than random patchwork.

  4. Use of transgenic mice to map cis-acting elements in the intestinal fatty acid binding protein gene (Fabpi) that control its cell lineage- specific and regional patterns of expression along the duodenal-colonic and crypt-villus axes of the gut epithelium

    PubMed Central

    1992-01-01

    The mouse intestinal epithelium is able to establish and maintain complex lineage-specific, spatial, and temporal patterns of gene expression despite its rapid and continuous renewal. A multipotent stem cell located near the base of each intestinal crypt gives rise to progeny which undergo amplification and allocation to either enterocytic, Paneth cell, goblet cell, or enteroendocrine cell lineages. Differentiation of these four lineages occurs during their geographically ordered migration along the crypt-villus axis. Gut stem cells appear to have a "positional address" which is manifested by differences in the differentiation programs of their lineal descendants along the duodenal-colonic (cephalocaudal) axis. We have used the intestinal fatty acid binding protein gene (Fabpi) as a model to identify cis-acting elements which regulate cell- and region-specific patterns of gene expression in the gut. Nucleotides -1178 to +28 of rat Fabpi direct a pattern of expression of a reporter (human growth hormone [hGH]) which mimics that of mouse Fabpi (a) steady-state levels of hGH mRNA are highest in the distal jejunum of adult transgenic mice and fall progressively toward both the duodenum and the mid-colon; and (b) hGH is confined to the enterocytic lineage and first appears as postmitotic, differentiating cells exit the crypt and migrate to the base of small intestinal villi or their colonic homologs, the surface epithelial cuffs. Nucleotides -103 to +28, which are highly conserved in rat, mouse and human Fabpi, are able to correctly initiate transgene expression in late fetal life, restrict hGH to the enterocytic lineage, and establish an appropriate cephalocaudal gradient of reporter expression. This cephalocaudal gradient is also influenced by cis- acting elements located between nucleotides -1178 and -278, and -277 and -185 that enhance and suppress (respectively) expression in the ileum and colon and by element(s) located upstream of nucleotide -277 that are needed

  5. Fibroblast growth factor receptor-3 (FGFR-3) regulates expression of paneth cell lineage-specific genes in intestinal epithelial cells through both TCF4/beta-catenin-dependent and -independent signaling pathways.

    PubMed

    Brodrick, Brooks; Vidrich, Alda; Porter, Edith; Bradley, Leigh; Buzan, Jenny M; Cohn, Steven M

    2011-05-27

    Fibroblast growth factor receptor-3 (FGFR-3) expression in the developing intestine is restricted to the undifferentiated epithelial cells within the lower portion of the crypt. We previously showed that mice lacking functional FGFR-3 have a significant decrease in the number of Paneth cells in the small intestine. Here, we used Caco2 cells to investigate whether FGFR-3 signaling can directly modulate expression of Paneth cell differentiation markers through its effects on TCF4/β-catenin or through other signaling pathways downstream of this receptor. Caco2 cells treated with FGFR-3 ligands or expressing FGFR-3(K650E), a constitutively active mutant, resulted in a significantly increased expression of genes characteristic of mature Paneth cells, including human α-defensins 5 and 6 (HD5 and HD6) and Paneth cell lysozyme, whereas enterocytic differentiation markers were reduced. Activation of FGFR-3 signaling sustained high levels of β-catenin mRNA expression, leading to increased TCF4/β-catenin-regulated transcriptional activity in Caco2 cells. Sustained activity of the TCF4/β-catenin pathway was required for the induction of Paneth cell markers. Activation of the MAPK pathway by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on TCF4/β-catenin activity. These studies suggest that coordinate activation of multiple independent signaling pathways downstream of FGFR-3 is involved in regulation of Paneth cell differentiation.

  6. Cell Lineage of the Ilyanassa Embryo: Evolutionary Acceleration of Regional Differentiation during Early Development

    PubMed Central

    Goulding, Morgan Q.

    2009-01-01

    Cell lineage studies in mollusk embryos have documented numerous variations on the lophotrochozoan theme of spiral cleavage. In the experimentally tractable embryo of the mud snail Ilyanassa, cell lineage has previously been described only up to the 29-cell stage. Here I provide a chronology of cell divisions in Ilyanassa to the stage of 84 cells (about 16 hours after first cleavage at 23°C), and show spatial arrangements of identified nuclei at stages ranging from 27 to 84 cells. During this period the spiral cleavage pattern gives way to a bilaterally symmetric, dorsoventrally polarized pattern of mitotic timing and geometry. At the same time, the mesentoblast cell 4d rapidly proliferates to form twelve cells lying deep to the dorsal ectoderm. The onset of epiboly coincides with a period of mitotic quiescence throughout the ectoderm. As in other gastropod embryos, cell cycle lengths vary widely and predictably according to cell identity, and many of the longest cell cycles occur in small daughters of highly asymmetric divisions. While Ilyanassa shares many features of embryonic cell lineage with two other caenogastropod genera, Crepidula and Bithynia, it is distinguished by a general tendency toward earlier and more pronounced diversification of cell division pattern along axes of later differential growth. PMID:19430530

  7. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells

    PubMed Central

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.

    2009-01-01

    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  8. Identification and characterization of Xenopus tropicalis common progenitors of Sertoli and peritubular myoid cell lineages

    PubMed Central

    Tlapakova, Tereza; Nguyen, Thi Minh Xuan; Vegrichtova, Marketa; Sidova, Monika; Strnadova, Karolina; Blahova, Monika

    2016-01-01

    ABSTRACT The origin of somatic cell lineages during testicular development is controversial in mammals. Employing basal amphibian tetrapod Xenopus tropicalis we established a cell culture derived from testes of juvenile male. Expression analysis showed transcription of some pluripotency genes and Sertoli cell, peritubular myoid cell and mesenchymal cell markers. Transcription of germline-specific genes was downregulated. Immunocytochemistry revealed that a majority of cells express vimentin and co-express Sox9 and smooth muscle α-actin (Sma), indicating the existence of a common progenitor of Sertoli and peritubular myoid cell lineages. Microinjection of transgenic, red fluorescent protein (RFP)-positive somatic testicular cells into the peritoneal cavity of X. tropicalis tadpoles resulted in cell deposits in heart, pronephros and intestine, and later in a strong proliferation and formation of cell-to-cell net growing through the tadpole body. Immunohistochemistry analysis of transplanted tadpoles showed a strong expression of vimentin in RFP-positive cells. No co-localization of Sox9 and Sma signals was observed during the first three weeks indicating their dedifferentiation to migratory-active mesenchymal cells recently described in human testicular biopsies. PMID:27464670

  9. Does Cell Lineage in the Developing Cerebral Cortex Contribute to its Columnar Organization?

    PubMed Central

    Costa, Marcos R.; Hedin-Pereira, Cecilia

    2010-01-01

    Since the pioneer work of Lorente de Nó, Ramón y Cajal, Brodmann, Mountcastle, Hubel and Wiesel and others, the cerebral cortex has been seen as a jigsaw of anatomic and functional modules involved in the processing of different sets of information. In fact, a columnar distribution of neurons displaying similar functional properties throughout the cerebral cortex has been observed by many researchers. Although it has been suggested that much of the anatomical substrate for such organization would be already specified at early developmental stages, before activity-dependent mechanisms could take place, it is still unclear whether gene expression in the ventricular zone (VZ) could play a role in the development of discrete functional units, such as minicolumns or columns. Cell lineage experiments using replication-incompetent retroviral vectors have shown that the progeny of a single neuroepithelial/radial glial cell in the dorsal telencephalon is organized into discrete radial clusters of sibling excitatory neurons, which have a higher propensity for developing chemical synapses with each other rather than with neighboring non-siblings. Here, we will discuss the possibility that the cell lineage of single neuroepithelial/radial glia cells could contribute for the columnar organization of the neocortex by generating radial columns of sibling, interconnected neurons. Borrowing some concepts from the studies on cell–cell recognition and transcription factor networks, we will also touch upon the potential molecular mechanisms involved in the establishment of sibling-neuron circuits. PMID:20676384

  10. Cell lineage-specific expression of modulo, a dose-dependent modifier of variegation in Drosophila.

    PubMed Central

    Garzino, V; Pereira, A; Laurenti, P; Graba, Y; Levis, R W; Le Parco, Y; Pradel, J

    1992-01-01

    Variegation in Drosophila is a manifest illustration of the important role played by chromatin structure in gene expression. We have isolated mutants of modulo (mod) and shown that this gene is a dominant suppressor of variegation. Null mutants are recessive lethal with a melanotic tumour phenotype. The mod protein directly binds DNA, which indicates that it may serve to anchor multimeric complexes promoting chromatin compaction and silencing. Using a specific monoclonal antibody we examined by immunocytochemistry the accumulation pattern of mod protein during embryogenesis. The protein is first detected before the blastoderm cellularization in all somatic nuclei, precisely when pericentromeric heterochromatin becomes visible. After the first cell division, mod protein is expressed in lineages of specific embryonic primordia. Based on its dominant phenotype, expression pattern and DNA-binding activity of its product, we propose that mod regulates chromatin structure and activity in specific cell lineages. Images PMID:1425581

  11. Transmissible [corrected] dog cancer genome reveals the origin and history of an ancient cell lineage.

    PubMed

    Murchison, Elizabeth P; Wedge, David C; Alexandrov, Ludmil B; Fu, Beiyuan; Martincorena, Inigo; Ning, Zemin; Tubio, Jose M C; Werner, Emma I; Allen, Jan; De Nardi, Andrigo Barboza; Donelan, Edward M; Marino, Gabriele; Fassati, Ariberto; Campbell, Peter J; Yang, Fengtang; Burt, Austin; Weiss, Robin A; Stratton, Michael R

    2014-01-24

    Canine transmissible venereal tumor (CTVT) is the oldest known somatic cell lineage. It is a transmissible cancer that propagates naturally in dogs. We sequenced the genomes of two CTVT tumors and found that CTVT has acquired 1.9 million somatic substitution mutations and bears evidence of exposure to ultraviolet light. CTVT is remarkably stable and lacks subclonal heterogeneity despite thousands of rearrangements, copy-number changes, and retrotransposon insertions. More than 10,000 genes carry nonsynonymous variants, and 646 genes have been lost. CTVT first arose in a dog with low genomic heterozygosity that may have lived about 11,000 years ago. The cancer spawned by this individual dispersed across continents about 500 years ago. Our results provide a genetic identikit of an ancient dog and demonstrate the robustness of mammalian somatic cells to survive for millennia despite a massive mutation burden.

  12. Clonal analysis of the cell lineages in the male flower of maize

    SciTech Connect

    Dawe, R.K.; Freeling, M. )

    1990-11-01

    The cell lineages in the male flower of maize were characterized using X-rays and transposable elements to produce clonal sectors differing in anthocyanin pigmentation. Less than 50% of the somatic tassel mutations (caused by reversion of unstable color mutations) that were visible on the anther wall were sexually transmitted by the male gametes, unless the sectors were larger than half the tassel circumference. This result is explained by showing that: (a) both the outer (LI) and inner (LII) lineages of the shoot apical meristem form a cell layer in the bilayered anther wall, and that anther pigmentation can be derived from either cell layer; and that (b) the male germ cells are derived almost exclusively from the LII. Therefore, while reversion events in either the LI or LII are visible on the anther, only the LII events are heritable. Reversion events that occur prior to the organization of the shoot apical meristem however, produce large (usually more than one-half tassel) sectors that include both the outer and inner lineages. In contrast to the high level of cell layer invasion previously reported during leaf development, during anther development less than 10(-3) cells in the LI invade the LII to form male gametes. The strong correlation between cell lineage and cell fate in the maize anther has implications for studies on plant evolution and the genetic improvement of cereals by DNA transformation.

  13. Cell lineage analysis demonstrates an endodermal origin of the distal urethra and perineum.

    PubMed

    Seifert, Ashley W; Harfe, Brian D; Cohn, Martin J

    2008-06-01

    Congenital malformations of anorectal and genitourinary (collectively, anogenital) organs occur at a high frequency in humans, however the lineage of cells that gives rise to anogenital organs remains poorly understood. The penile urethra has been reported to develop from two cell populations, with the proximal urethra developing from endoderm and the distal urethra forming from an apical ectodermal invagination, however this has never been tested by direct analysis of cell lineage. During gut development, endodermal cells express Sonic hedgehog (Shh), which is required for normal patterning of digestive and genitourinary organs. We have taken advantage of the properties of Shh expression to genetically label and follow the fate of posterior gut endoderm during anogenital development. We report that the entire urethra, including the distal (glandar) region, is derived from endoderm. Cloacal endoderm also gives rise to the epithelial linings of the bladder, rectum and anterior region of the anus. Surprisingly, the lineage map also revealed an endodermal origin of the perineum, which is the first demonstration that endoderm differentiates into skin. In addition, we fate mapped genital tubercle ectoderm and show that it makes no detectable contribution to the urethra. In males, formation of the urethral tube involves septation of the urethral plate by continued growth of the urorectal septum. Analysis of cell lineage following disruption of androgen signaling revealed that the urethral plate of flutamide-treated males does not undergo this septation event. Instead, urethral plate cells persist to the ventral margin of the tubercle, mimicking the pattern seen in females. Based on these spatial and temporal fate maps, we present a new model for anogenital development and suggest that disruptions at specific developmental time points can account for the association between anorectal and genitourinary defects.

  14. Co-localization of Cell Lineage Markers and the Tomato Signal.

    PubMed

    Jing, Yan; Hinton, Robert J; Chan, Kevin S; Feng, Jian Q

    2016-12-28

    The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-Cre(ERT2) (Agg-Cre(ERT2)), crossed with Rosa26(tdTomato). Both Col10 and aggrecan are well-recognized markers for chondrocytes. On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

  15. Enobosarm (GTx-024) Modulates Adult Skeletal Muscle Mass Independently of the Androgen Receptor in the Satellite Cell Lineage.

    PubMed

    Dubois, Vanessa; Simitsidellis, Ioannis; Laurent, Michaël R; Jardi, Ferran; Saunders, Philippa T K; Vanderschueren, Dirk; Claessens, Frank

    2015-12-01

    Androgens increase skeletal muscle mass, but their clinical use is hampered by a lack of tissue selectivity and subsequent side effects. Selective androgen receptor modulators elicit muscle-anabolic effects while only sparingly affecting reproductive tissues. The selective androgen receptor modulator, GTx-024 (enobosarm), is being investigated for cancer cachexia, sarcopenia, and muscle wasting diseases. Here we investigate the role of muscle androgen receptor (AR) in the anabolic effect of GTx-024. In mice lacking AR in the satellite cell lineage (satARKO), the weight of the androgen-sensitive levator ani muscle was lower but was decreased further upon orchidectomy. GTx-024 was as effective as DHT in restoring levator ani weights to sham levels. Expression of the muscle-specific, androgen-responsive genes S-adenosylmethionine decarboxylase and myostatin was decreased by orchidectomy and restored by GTx-024 and DHT in control mice, whereas the expression was low and unaffected by androgen status in satARKO. In contrast, insulin-like growth factor 1Ea expression was not different between satARKO and control muscle, decreased upon castration, and was restored by DHT and GTx-024 in both genotypes. These data indicate that GTx-024 does not selectively modulate AR in the satellite cell lineage and that cells outside this lineage remain androgen responsive in satARKO muscle. Indeed, residual AR-positive cells were present in satARKO muscle, coexpressing the fibroblast-lineage marker vimentin. AR positive, muscle-resident fibroblasts could therefore be involved in the indirect effects of androgens on muscle. In conclusion, both DHT and GTx-024 target AR pathways in the satellite cell lineage, but cells outside this lineage also contribute to the anabolic effects of androgens.

  16. Thymopentin enhances the generation of T-cell lineage derived from human embryonic stem cells in vitro.

    PubMed

    Zhu, Ming-Xia; Wan, Wen-Li; Li, Hai-Shen; Wang, Jing; Chen, Gui-An; Ke, Xiao-Yan

    2015-02-15

    Thymopentin is a group of biologically active peptide secreted mainly by the epithelial cells of thymic cortex and medulla. Whether it promotes T cells production from human embryonic stem cells(hESCs) in vitro remains an elusive issue. In the present study, we develop a novel strategy that enhances T-cell lineage differentiation of hESCs in collagen matrix culture by sequential cytokine cocktails treatment combined with thymopentin stimulation. We observed that approximately 30.75% cells expressed CD34 on day 14 of the cultures and expressed the surface markers of erythroid, lymphoid and myeloid lineages. The results of colony assays and gene expressions by RT-PCR analysis also demonstrated that hematopoietic progenitor cells (HPCs) derived from hESCs were capable of multi-lineage differentiation. Further study revealed that culturing with thymopentin treatment, the CD34(+)CD45RA(+)CD7(+) cells sorted from HPCs expressed T-cell-related genes, IKAROS, DNTT, TCRγ and TCRβ, and T-cell surface markers, CD3, cytoplasmic CD3, CD5, CD27, TCRγδ, CD4 and CD8. The differentiated cells produced the cytokines including IFN-γ, IL-2 and TNF-α in response to stimulation, providing the evidence for T-cell function of these cells. In conclusion, thymopentin enhances T-cell lineage differentiation from hESCs in vitro by mimicking thymus peptide environment in vivo.

  17. Pox neuro control of cell lineages that give rise to larval poly-innervated external sensory organs in Drosophila.

    PubMed

    Jiang, Yanrui; Boll, Werner; Noll, Markus

    2015-01-15

    The Pox neuro (Poxn) gene of Drosophila plays a crucial role in the development of poly-innervated external sensory (p-es) organs. However, how Poxn exerts this role has remained elusive. In this study, we have analyzed the cell lineages of all larval p-es organs, namely of the kölbchen, papilla 6, and hair 3. Surprisingly, these lineages are distinct from any previously reported cell lineages of sensory organs. Unlike the well-established lineage of mono-innervated external sensory (m-es) organs and a previously proposed model of the p-es lineage, we demonstrate that all wild-type p-es lineages exhibit the following features: the secondary precursor, pIIa, gives rise to all three support cells-socket, shaft, and sheath, whereas the other secondary precursor, pIIb, is neuronal and gives rise to all neurons. We further show that in one of the p-es lineages, that of papilla 6, one cell undergoes apoptosis. By contrast in Poxn null mutants, all p-es lineages have a reduced number of cells and their pattern of cell divisions is changed to that of an m-es organ, with the exception of a lineage in a minority of mutant kölbchen that retains a second bipolar neuron. Indeed, the role of Poxn in p-es lineages is consistent with the specification of the developmental potential of secondary precursors and the regulation of cell division but not apoptosis.

  18. Prethymic CD34+ progenitors capable of developing into T cells are not committed to the T cell lineage.

    PubMed

    Blom, B; Res, P; Noteboom, E; Weijer, K; Spits, H

    1997-04-15

    Progenitor cells that seed the fetal thymus are derived from the fetal liver and the bone marrow. These cells migrate through the fetal blood to the thymus. In this work, we address which peripheral progenitor cells have the potential to become T cells and whether these progenitor cells are already committed to the T cell lineage. All CD34+CD38- precursor cells, regardless of their origin, are able to develop into T cells in a hybrid human/mouse fetal thymic organ culture. Previously, we found that the more differentiated CD34+CD38+ progenitor cells from fetal liver cannot develop into T cells. In this work, we show that CD34+CD38+ cells from fetal bone marrow and cord blood are capable of T cell development. In spite of the T cell-developing potential, we did not detect rearrangements of TCR-delta or TCR-beta loci in any of the CD34+ peripheral precursors. CD34+ fetal bone marrow cell subpopulations express pre-TCR-alpha. However, we could not detect expression of pT alpha or of recombination-activating gene 1 in CD34+ cord blood cells. Since cord blood CD34+ cells should contain the direct progenitors of the CD34+ thymocytes, our data do not support the notion that in humans commitment to the T cell lineage occurs before the cells migrate into the thymus.

  19. Cell lineage tracing in the developing enteric nervous system: superstars revealed by experiment and simulation

    PubMed Central

    Cheeseman, Bevan L.; Zhang, Dongcheng; Binder, Benjamin J.; Newgreen, Donald F.; Landman, Kerry A.

    2014-01-01

    Cell lineage tracing is a powerful tool for understanding how proliferation and differentiation of individual cells contribute to population behaviour. In the developing enteric nervous system (ENS), enteric neural crest (ENC) cells move and undergo massive population expansion by cell division within self-growing mesenchymal tissue. We show that single ENC cells labelled to follow clonality in the intestine reveal extraordinary and unpredictable variation in number and position of descendant cells, even though ENS development is highly predictable at the population level. We use an agent-based model to simulate ENC colonization and obtain agent lineage tracing data, which we analyse using econometric data analysis tools. In all realizations, a small proportion of identical initial agents accounts for a substantial proportion of the total final agent population. We term these individuals superstars. Their existence is consistent across individual realizations and is robust to changes in model parameters. This inequality of outcome is amplified at elevated proliferation rate. The experiments and model suggest that stochastic competition for resources is an important concept when understanding biological processes which feature high levels of cell proliferation. The results have implications for cell-fate processes in the ENS. PMID:24501272

  20. Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy.

    PubMed

    Amat, Fernando; Keller, Philipp J

    2013-05-01

    Understanding the development of complex multicellular organisms as a function of the underlying cell behavior is one of the most fundamental goals of developmental biology. The ability to quantitatively follow cell dynamics in entire developing embryos is an indispensable step towards such a system-level understanding. In recent years, light-sheet fluorescence microscopy has emerged as a particularly promising strategy for recording the in vivo data required to realize this goal. Using light-sheet fluorescence microscopy, entire complex organisms can be rapidly imaged in three dimensions at sub-cellular resolution, achieving high temporal sampling and excellent signal-to-noise ratio without damaging the living specimen or bleaching fluorescent markers. The resulting datasets allow following individual cells in vertebrate and higher invertebrate embryos over up to several days of development. However, the complexity and size of these multi-terabyte recordings typically preclude comprehensive manual analyses. Thus, new computational approaches are required to automatically segment cell morphologies, accurately track cell identities and systematically analyze cell behavior throughout embryonic development. We review current efforts in light-sheet microscopy and bioimage informatics towards this goal, and argue that comprehensive cell lineage reconstructions are finally within reach for many key model organisms, including fruit fly, zebrafish and mouse.

  1. Cell lineage and cis-regulation for a unique GABAergic/glycinergic neuron type in the larval nerve cord of the ascidian Ciona intestinalis.

    PubMed

    Nishitsuji, Koki; Horie, Takeo; Ichinose, Aoi; Sasakura, Yasunori; Yasuo, Hitoyoshi; Kusakabe, Takehiro G

    2012-02-01

    The tunicate Ciona intestinalis larva has a simple central nervous system (CNS), consisting of fewer than 400 cells, which is homologous to the vertebrate CNS. Recent studies have revealed neuronal types and networks in the larval CNS of C. intestinalis, yet their cell lineage and the molecular mechanism by which particular types of neurons are specified and differentiate remain poorly understood. Here, we report cell lineage origin and a cis-regulatory module for the anterior caudal inhibitory neurons (ACINs), a putative component of the central pattern generator regulating swimming locomotion. The vesicular GABA⁄ glycine transporter gene Ci-VGAT, a specific marker for GABAergic ⁄ glycinergic neurons, is expressed in distinct sets of neurons, including ACINs of the tail nerve cord and others in the brain vesicle and motor ganglion. Comparative genomics analysis between C. intestinalis and Ciona savignyi and functional analysis in vivo identified the cis-regulatory module responsible for Ci-VGAT expression in ACINs. Our cell lineage analyses inferred that ACINs derive from A11.116 cells, which have been thought to solely give rise to glial ependymal cells of the lateral wall of the nerve cord. The present findings will provide a solid basis for future studies addressing the molecular mechanism underlying specification of ACINs, which play a critical role in controlling larval locomotion

  2. Cell lineage analysis of the mandibular segment of the amphipod Orchestia cavimana reveals that the crustacean paragnaths are sternal outgrowths and not limbs

    PubMed Central

    Wolff, Carsten; Scholtz, Gerhard

    2006-01-01

    The question of arthropod head segmentation has become one of the central issues in Evolutionary Developmental Biology. The number of theories pertaining to head segments progressively enlarges, old concepts have been revitalized, and nearly every conceivable composition of the arthropod head has at some point received discussion. One contentious issue involves a characteristic mouthpart in crustaceans – the lower lips or the so-called paragnaths. The paragnaths build the posterior border of the mouth region antagonistic to the upper lip – the labrum. We show here the development of the appendage-like structures in the mandibular region of the amphipod crustacean Orchestia cavimana at a high level of cellular resolution. The embryos are examined during development of the mouthparts using in vivo labeling. An invariant cell division pattern of the mandibular segment was detected by 4D-microscopy and a preliminary model for pattern of the first cleavages in the mandibular region created. With this indispensable precondition single ectodermal cells of the grid-like pattern were labeled with DiI – a lipophilic fluorescent dye – to trace cell lineages and determine the clonal composition of the developing mouthparts, especially the mandibular segment. From our data it is evident that the paragnaths are sternal outgrowths of the mandible segment. The assumption of the limb nature of paragnaths and the presence of an additional head segment between the mandibular and the second antennal segments are clearly refuted by our data. Our results show the power of cell lineage and clonal analyses for inferences on the nature, origin and thus homology of morphological structures. With this kind of investigation morphological and gene expression data can be complemented. We discuss notable similarities of paragnath anlagen to those of the hypopharynx complex in myriapods and hexapods. The fact that both structures grow out as two lateral buds in the same region of the

  3. Cell lineage analysis of the mandibular segment of the amphipod Orchestia cavimana reveals that the crustacean paragnaths are sternal outgrowths and not limbs.

    PubMed

    Wolff, Carsten; Scholtz, Gerhard

    2006-12-04

    The question of arthropod head segmentation has become one of the central issues in Evolutionary Developmental Biology. The number of theories pertaining to head segments progressively enlarges, old concepts have been revitalized, and nearly every conceivable composition of the arthropod head has at some point received discussion. One contentious issue involves a characteristic mouthpart in crustaceans--the lower lips or the so-called paragnaths. The paragnaths build the posterior border of the mouth region antagonistic to the upper lip--the labrum. We show here the development of the appendage-like structures in the mandibular region of the amphipod crustacean Orchestia cavimana at a high level of cellular resolution. The embryos are examined during development of the mouthparts using in vivo labeling. An invariant cell division pattern of the mandibular segment was detected by 4D-microscopy and a preliminary model for pattern of the first cleavages in the mandibular region created. With this indispensable precondition single ectodermal cells of the grid-like pattern were labeled with DiI--a lipophilic fluorescent dye--to trace cell lineages and determine the clonal composition of the developing mouthparts, especially the mandibular segment. From our data it is evident that the paragnaths are sternal outgrowths of the mandible segment. The assumption of the limb nature of paragnaths and the presence of an additional head segment between the mandibular and the second antennal segments are clearly refuted by our data. Our results show the power of cell lineage and clonal analyses for inferences on the nature, origin and thus homology of morphological structures. With this kind of investigation morphological and gene expression data can be complemented. We discuss notable similarities of paragnath anlagen to those of the hypopharynx complex in myriapods and hexapods. The fact that both structures grow out as two lateral buds in the same region of the mandibular

  4. The potential of dental stem cells differentiating into neurogenic cell lineage after cultivation in different modes in vitro.

    PubMed

    Yang, Chao; Sun, Liang; Li, Xinghan; Xie, Li; Yu, Mei; Feng, Lian; Jiang, Zongting; Guo, Weihua; Tian, Weidong

    2014-10-01

    Trauma or degenerative diseases of the central nervous system (CNS) cause the loss of neurons or glial cells. Stem cell transplantation has become a vital strategy for CNS regeneration. It is necessary to effectively induce nonneurogenic stem cells to differentiate into neurogenic cell lineages because of the limited source of neurogenic stem cells, relatively difficult cultivation, and ethical issues. Previous studies have found that dental stem cells can be used for transplantation therapy. The aim of this study was to explore a better inductive mode and time point for dental stem cells to differentiate into neural-like cells and evaluate a better candidate cell. In this study, dental follicle stem cells (DFSCs), dental papilla stem cells (DPSCs), and stem cells from apical papilla (SCAPs) were cultivated in five different modes. The proliferation ability, morphology, and expression of neural marker genes were analyzed. Results showed that DFSCs showed a higher proliferation potential. The proliferation was decreased after cultivation in chemical inductive medium as cultivation modes 3 and 5. The cells could present neural-like cell morphology after cultivation with human epidermal growth factor (EGF) and fibroblast growth factor-basic (bFGF) as cultivation modes 4 and 5. The vast majority of DFSCs gene expression levels in mode 4 on the third day was upregulated significantly. In conclusion, our data suggested that different dental stem cells exhibited different neural differentiation potentials. DFSCs might be the better candidate cell type. Furthermore, cultivation mode 4 and timing of the third day may promote differentiation into neurogenic cell lineages more effectively before transplantation to treat neurological diseases.

  5. Human haematopoietic stem cell lineage commitment is a continuous process.

    PubMed

    Velten, Lars; Haas, Simon F; Raffel, Simon; Blaszkiewicz, Sandra; Islam, Saiful; Hennig, Bianca P; Hirche, Christoph; Lutz, Christoph; Buss, Eike C; Nowak, Daniel; Boch, Tobias; Hofmann, Wolf-Karsten; Ho, Anthony D; Huber, Wolfgang; Trumpp, Andreas; Essers, Marieke A G; Steinmetz, Lars M

    2017-03-20

    Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a 'continuum of low-primed undifferentiated haematopoietic stem and progenitor cells' (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies.

  6. Cell lineage tracing reveals a biliary origin of intrahepatic cholangiocarcinoma

    PubMed Central

    Guest, Rachel V; Boulter, Luke; Kendall, Timothy J; Minnis-Lyons, Sarah E; Walker, Robert; Wigmore, Stephen J; Sansom, Owen J; Forbes, Stuart J

    2014-01-01

    Intrahepatic cholangiocarcinoma (ICC) is a treatment refractory malignancy with a high mortality and an increasing incidence worldwide. Recent studies have observed that activation of Notch and AKT signalling within mature hepatocytes is able to induce the formation of tumours displaying biliary lineage markers, thereby raising the suggestion that it is hepatocytes, rather than cholangiocytes or hepatic progenitor cells that represent the cell of origin of this tumour. Here we utilise a cholangiocyte-lineage tracing system to target p53 loss to biliary epithelia and observe the appearance of labelled biliary lineage tumours in response to chronic injury. Consequent to this, up-regulation of native functional Notch signalling is observed to occur spontaneously within cholangiocytes and hepatocytes in this model as well as in human ICC. These data prove that in the context of chronic inflammation and p53 loss, frequent occurrences in human disease, biliary epithelia are a target of transformation and an origin of ICC. PMID:24310400

  7. Down-Regulation of Human Enteric Antimicrobial Peptides by NOD2 during Differentiation of the Paneth Cell Lineage

    PubMed Central

    Tan, Gao; Li, Run-hua; Li, Chen; Wu, Fang; Zhao, Xin-mei; Ma, Jia-yi; Lei, Shan; Zhang, Wen-di; Zhi, Fa-chao

    2015-01-01

    Ileal Crohn's disease (CD) arising from the alteration of intestinal homeostasis is characterized by two features, namely a decrease in Paneth cell-produced antimicrobial peptides that play a key role in maintaining this balance and an increase in NOD2, an intracellular sensor. Although mutations in NOD2 are highly correlated with the incidence of CD, the physiological role of NOD2 in intestinal immunity remains elusive. Here, we show that NOD2 can down-regulate the expression of human enteric antimicrobial peptides during differentiation of the Paneth cell lineage. This finding, which links the decrease of human enteric antimicrobial peptides to increased NOD2 in ileal CD patients, provides a new view into the pathogenesis of ileal CD. PMID:25670499

  8. Decoding the DNA Methylome of Mantle Cell Lymphoma in the Light of the Entire B Cell Lineage.

    PubMed

    Queirós, Ana C; Beekman, Renée; Vilarrasa-Blasi, Roser; Duran-Ferrer, Martí; Clot, Guillem; Merkel, Angelika; Raineri, Emanuele; Russiñol, Nuria; Castellano, Giancarlo; Beà, Sílvia; Navarro, Alba; Kulis, Marta; Verdaguer-Dot, Núria; Jares, Pedro; Enjuanes, Anna; Calasanz, María José; Bergmann, Anke; Vater, Inga; Salaverría, Itziar; van de Werken, Harmen J G; Wilson, Wyndham H; Datta, Avik; Flicek, Paul; Royo, Romina; Martens, Joost; Giné, Eva; Lopez-Guillermo, Armando; Stunnenberg, Hendrik G; Klapper, Wolfram; Pott, Christiane; Heath, Simon; Gut, Ivo G; Siebert, Reiner; Campo, Elías; Martín-Subero, José I

    2016-11-14

    We analyzed the in silico purified DNA methylation signatures of 82 mantle cell lymphomas (MCL) in comparison with cell subpopulations spanning the entire B cell lineage. We identified two MCL subgroups, respectively carrying epigenetic imprints of germinal-center-inexperienced and germinal-center-experienced B cells, and we found that DNA methylation profiles during lymphomagenesis are largely influenced by the methylation dynamics in normal B cells. An integrative epigenomic approach revealed 10,504 differentially methylated regions in regulatory elements marked by H3K27ac in MCL primary cases, including a distant enhancer showing de novo looping to the MCL oncogene SOX11. Finally, we observed that the magnitude of DNA methylation changes per case is highly variable and serves as an independent prognostic factor for MCL outcome.

  9. Cell lineage, axis formation, and the origin of germ layers in the amphipod crustacean Orchestia cavimana.

    PubMed

    Wolff, Carsten; Scholtz, Gerhard

    2002-10-01

    Embryos of the amphipod crustacean Orchestia cavimana are examined during cleavage, gastrulation, and segmentation by using in vivo labelling. Single blastomeres of the 8- and 16-cell stages were labelled with DiI to trace cell lineages. Early cleavage follows a distinct pattern and the a/p and d/v body axes are already determined at the 4- and 8-cell stages, respectively. In these stages, the germinal rudiment and the naupliar mesoderm can be traced back to a single blastomere each. In addition, the ectoderm and the postnaupliar mesoderm are separated into right and left components. At the16-cell stage, naupliar ectoderm is divided from the postnaupliar ectoderm, and extraembryonic lineages are separated from postnaupliar mesoderm and endoderm. From our investigation, it is evident that the cleavage pattern and cell lineage of Orchestia cavimana are not of the spiral type. Furthermore, the results of the labelling show many differences to cleavage patterns and cell lineages in other crustaceans, in particular, other Malacostraca. The cleavage and cell lineage patterns of the amphipod Orchestia are certainly derived within Malacostraca, whose ancestral cleavage mode was most likely of the superficial type. On the other hand, Orchestia exhibits a stereotyped cell division pattern during formation and differentiation of the germ band that is typical for malacostracans. Hence, a derived (apomorphic) early cleavage pattern is the ontogenetic basis for an evolutionarily older cell division pattern of advanced developmental stages. O. cavimana offers the possibility to trace the lineages and the fates of cells from early developmental stages up to the formation of segmental structures, including neurogenesis at a level of resolution that is not matched by any other arthropod system.

  10. Regulatory effects on the population dynamics and wave propagation in a cell lineage model.

    PubMed

    Wang, Mao-Xiang; Ma, Yu-Qiang; Lai, Pik-Yin

    2016-03-21

    We consider the interplay of cell proliferation, cell differentiation (and de-differentiation), cell movement, and the effect of feedback regulations on the population and propagation dynamics of different cell types in a cell lineage model. Cells are assumed to secrete and respond to negative feedback molecules which act as a control on the cell lineage. The cell densities are described by coupled reaction-diffusion partial differential equations, and the propagating wave front solutions in one dimension are investigated analytically and by numerical solutions. In particular, wavefront propagation speeds are obtained analytically and verified by numerical solutions of the equations. The emphasis is on the effects of the feedback regulations on different stages in the cell lineage. It is found that when the progenitor cell is negatively regulated, the populations of the cell lineage are strongly down-regulated with the steady growth rate of the progenitor cell being driven to zero beyond a critical regulatory strength. An analytic expression for the critical regulation strength in terms of the model parameters is derived and verified by numerical solutions. On the other hand, if the inhibition is acting on the differentiated cells, the change in the population dynamics and wave propagation speed is small. In addition, it is found that only the propagating speed of the progenitor cells is affected by the regulation when the diffusion of the differentiated cells is large. In the presence of de-differentiation, the effect on down-regulating the progenitor population is weakened and there is no effect on the propagation speed due to regulation, suggesting that the effect of regulatory control is diminished by de-differentiation pathways.

  11. Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies.

    PubMed

    Gao, Feng; Bonsignori, Mattia; Liao, Hua-Xin; Kumar, Amit; Xia, Shi-Mao; Lu, Xiaozhi; Cai, Fangping; Hwang, Kwan-Ki; Song, Hongshuo; Zhou, Tongqing; Lynch, Rebecca M; Alam, S Munir; Moody, M Anthony; Ferrari, Guido; Berrong, Mark; Kelsoe, Garnett; Shaw, George M; Hahn, Beatrice H; Montefiori, David C; Kamanga, Gift; Cohen, Myron S; Hraber, Peter; Kwong, Peter D; Korber, Bette T; Mascola, John R; Kepler, Thomas B; Haynes, Barton F

    2014-07-31

    Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization-traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.

  12. EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells.

    PubMed

    Wang, Chenhui; Guo, Xingting; Xi, Rongwen

    2014-05-01

    Quiescent, multipotent gastric stem cells (GSSCs) in the copper cell region of adult Drosophila midgut can produce all epithelial cell lineages found in the region, including acid-secreting copper cells, interstitial cells and enteroendocrine cells, but mechanisms controlling their quiescence and the ternary lineage differentiation are unknown. By using cell ablation or damage-induced regeneration assays combined with cell lineage tracing and genetic analysis, here we demonstrate that Delta (Dl)-expressing cells in the copper cell region are the authentic GSSCs that can self-renew and continuously regenerate the gastric epithelium after a sustained damage. Lineage tracing analysis reveals that the committed GSSC daughter with activated Notch will invariably differentiate into either a copper cell or an interstitial cell, but not the enteroendocrine cell lineage, and loss-of-function and gain-of-function studies revealed that Notch signaling is both necessary and sufficient for copper cell/interstitial cell differentiation. We also demonstrate that elevated epidermal growth factor receptor (EGFR) signaling, which is achieved by the activation of ligand Vein from the surrounding muscle cells and ligand Spitz from progenitor cells, mediates the regenerative proliferation of GSSCs following damage. Taken together, we demonstrate that Dl is a specific marker for Drosophila GSSCs, whose cell cycle status is dependent on the levels of EGFR signaling activity, and the Notch signaling has a central role in controlling cell lineage differentiation from GSSCs by separating copper/interstitial cell lineage from enteroendocrine cell lineage.

  13. Blood cells of Drosophila: cell lineages and role in host defence.

    PubMed

    Meister, Marie

    2004-02-01

    Drosophila haemopoiesis gives rise to three independent cell lineages: plasmatocytes, crystal cells and lamellocytes. The regulation of Drosophila stem cell proliferation and lineage specification involves transactivators and signalling pathways, many of which have mammalian counterparts that control haemopoietic processes. Drosophila plasmatocytes are professional phagocytes that resemble the monocyte/macrophage lineage, crystal cells play a critical role in defence-related melanisation, and lamellocytes encapsulate large invaders. Crystal cells and lamellocytes have no clear mammalian homologues. Research into the molecular mechanisms that underlie the various immune functions of Drosophila blood cells, such as non-self recognition, is now taking wing.

  14. Core Transcription Factors, MicroRNAs, and Small Molecules Drive Transdifferentiation of Human Fibroblasts Towards The Cardiac Cell Lineage

    PubMed Central

    Christoforou, Nicolas; Chakraborty, Syandan; Kirkton, Robert D.; Adler, Andrew F.; Addis, Russell C.; Leong, Kam W.

    2017-01-01

    Transdifferentiation has been described as a novel method for converting human fibroblasts into induced cardiomyocyte-like cells. Such an approach can produce differentiated cells to study physiology or pathophysiology, examine drug interactions or toxicities, and engineer cardiac tissues. Here we describe the transdifferentiation of human dermal fibroblasts towards the cardiac cell lineage via the induced expression of transcription factors GATA4, TBX5, MEF2C, MYOCD, NKX2–5, and delivery of microRNAs miR-1 and miR-133a. Cells undergoing transdifferentiation expressed ACTN2 and TNNT2 and partially organized their cytoskeleton in a cross-striated manner. The conversion process was associated with significant upregulation of a cohort of cardiac-specific genes, activation of pathways associated with muscle contraction and physiology, and downregulation of fibroblastic markers. We used a genetically encoded calcium indicator and readily detected active calcium transients although no spontaneous contractions were observed in transdifferentiated cells. Finally, we determined that inhibition of Janus kinase 1, inhibition of Glycogen synthase kinase 3, or addition of NRG1 significantly enhanced the efficiency of transdifferentiation. Overall, we describe a method for achieving transdifferentiation of human dermal fibroblasts into induced cardiomyocyte-like cells via transcription factor overexpression, microRNA delivery, and molecular pathway manipulation. PMID:28071742

  15. Colon Stem Cell and Crypt Dynamics Exposed by Cell Lineage Reconstruction

    PubMed Central

    Itzkovitz, Shalev; Elbaz, Judith; Maruvka, Yosef E.; Segev, Elad; Shlush, Liran I.; Dekel, Nava; Shapiro, Ehud

    2011-01-01

    Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems. PMID:21829376

  16. Early cell lineage specification in a marsupial: a case for diverse mechanisms among mammals.

    PubMed

    Frankenberg, Stephen; Shaw, Geoff; Freyer, Claudia; Pask, Andrew J; Renfree, Marilyn B

    2013-03-01

    Early cell lineage specification in eutherian mammals results in the formation of a pluripotent inner cell mass (ICM) and trophoblast. By contrast, marsupials have no ICM. Here, we present the first molecular analysis of mechanisms of early cell lineage specification in a marsupial, the tammar wallaby. There was no overt differential localisation of key lineage-specific transcription factors in cleavage and early unilaminar blastocyst stages. Pluriblast cells (equivalent to the ICM) became distinguishable from trophoblast cells by differential expression of POU5F1 and, to a greater extent, POU2, a paralogue of POU5F1. Unlike in the mouse, pluriblast-trophoblast differentiation coincided with a global nuclear-to-cytoplasmic transition of CDX2 localisation. Also unlike in the mouse, Hippo pathway factors YAP and WWTR1 showed mutually distinct localisation patterns that suggest non-redundant roles. NANOG and GATA6 were conserved as markers of epiblast and hypoblast, respectively, but some differences to the mouse were found in their mode of differentiation. Our results suggest that there is considerable evolutionary plasticity in the mechanisms regulating early lineage specification in mammals.

  17. The role of single-cell analyses in understanding cell lineage commitment.

    PubMed

    Gibson, Tyler M; Gersbach, Charles A

    2013-04-01

    The study of cell lineage commitment is critical for improving our understanding of tissue development and regeneration, and for realizing stem cell-based therapies and engineered tissue replacements. Recently, the discovery of an unanticipated degree of variability in fundamental biological processes, including divergent responses of genetically identical cells to various stimuli, has provided mechanistic insight into cellular decision making and the collective behavior of cell populations. Therefore, the study of lineage commitment with single-cell resolution could provide greater knowledge of cellular differentiation mechanisms and the influence of noise on cellular processes. This will require the adoption of new technologies for single-cell analysis as traditional methods typically measure average values of bulk population behavior. This review discusses the recent developments in methods for analyzing the behavior of individual cells, and how these approaches are leading to a deeper understanding and better control of cellular decision making.

  18. Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution

    PubMed Central

    Spiewak, Jessica E.

    2014-01-01

    Summary Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage and mate choice and have played important roles in speciation. Here, we review recent studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns. PMID:25421288

  19. Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans.

    PubMed

    Mold, Jeff E; Venkatasubrahmanyam, Shivkumar; Burt, Trevor D; Michaëlsson, Jakob; Rivera, Jose M; Galkina, Sofiya A; Weinberg, Kenneth; Stoddart, Cheryl A; McCune, Joseph M

    2010-12-17

    Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or "layering") of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development. We also provide evidence that the fetal T cell lineage is biased toward immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.

  20. Primordial germ cells: the first cell lineage or the last cells standing?

    PubMed Central

    Johnson, Andrew D.; Alberio, Ramiro

    2015-01-01

    Embryos of many animal models express germ line determinants that suppress transcription and mediate early germ line commitment, which occurs before the somatic cell lineages are established. However, not all animals segregate their germ line in this manner. The ‘last cell standing’ model describes primordial germ cell (PGC) development in axolotls, in which PGCs are maintained by an extracellular signalling niche, and germ line commitment occurs after gastrulation. Here, we propose that this ‘stochastic’ mode of PGC specification is conserved in vertebrates, including non-rodent mammals. We postulate that early germ line segregation liberates genetic regulatory networks for somatic development to evolve, and that it therefore emerged repeatedly in the animal kingdom in response to natural selection. PMID:26286941

  1. Differential development of progenitor activity for three B-cell lineages.

    PubMed Central

    Kantor, A B; Stall, A M; Adams, S; Herzenberg, L A; Herzenberg, L A

    1992-01-01

    Cell-transfer studies presented here distinguish three murine B cell lineages: conventional B cells, which develop late and are continually replenished from progenitors in adult bone marrow; Ly-1 B cells (B-1a), which develop early and maintain their numbers by self-replenishment; and Ly-1B "sister" (B-1b) cells, which share many of the properties of Ly-1 B cells, including self-replenishment and feedback regulation of development but can also readily develop from progenitors in adult bone marrow. The sequential emergence of these lineages, the time at which their progenitors function during ontogeny, and the distinctions among their repertoires and functions suggest that evolution has created a layered immune system in which the immune response potential of each successive lineage is adapted to its particular niche. Images PMID:1565622

  2. Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila.

    PubMed

    Biteau, Benoît; Jasper, Heinrich

    2014-06-26

    In order to maintain tissue homeostasis, cell fate decisions within stem cell lineages have to respond to the needs of the tissue. This coordination of lineage choices with regenerative demand remains poorly characterized. Here, we identify a signal from enteroendocrine cells (EEs) that controls lineage specification in the Drosophila intestine. We find that EEs secrete Slit, a ligand for the Robo2 receptor in intestinal stem cells (ISCs) that limits ISC commitment to the endocrine lineage, establishing negative feedback control of EE regeneration. Furthermore, we show that this lineage decision is made within ISCs and requires induction of the transcription factor Prospero in ISCs. Our work identifies a function for the conserved Slit/Robo pathway in the regulation of adult stem cells, establishing negative feedback control of ISC lineage specification as a critical strategy to preserve tissue homeostasis. Our results further amend the current understanding of cell fate commitment within the Drosophila ISC lineage.

  3. Building a lineage from single cells: genetic techniques for cell lineage tracking.

    PubMed

    Woodworth, Mollie B; Girskis, Kelly M; Walsh, Christopher A

    2017-04-01

    Resolving lineage relationships between cells in an organism is a fundamental interest of developmental biology. Furthermore, investigating lineage can drive understanding of pathological states, including cancer, as well as understanding of developmental pathways that are amenable to manipulation by directed differentiation. Although lineage tracking through the injection of retroviral libraries has long been the state of the art, a recent explosion of methodological advances in exogenous labelling and single-cell sequencing have enabled lineage tracking at larger scales, in more detail, and in a wider range of species than was previously considered possible. In this Review, we discuss these techniques for cell lineage tracking, with attention both to those that trace lineage forwards from experimental labelling, and those that trace backwards across the life history of an organism.

  4. Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution.

    PubMed

    Parichy, David M; Spiewak, Jessica E

    2015-01-01

    Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage, and mate choice and have played important roles in speciation. Here, we review studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve-associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns.

  5. Mechanical modulation of nascent stem cell lineage commitment in tissue engineering scaffolds.

    PubMed

    Song, Min Jae; Dean, David; Knothe Tate, Melissa L

    2013-07-01

    Taking inspiration from tissue morphogenesis in utero, this study tests the concept of using tissue engineering scaffolds as delivery devices to modulate emergent structure-function relationships at early stages of tissue genesis. We report on the use of a combined computational fluid dynamics (CFD) modeling, advanced manufacturing methods, and experimental fluid mechanics (micro-piv and strain mapping) for the prospective design of tissue engineering scaffold geometries that deliver spatially resolved mechanical cues to stem cells seeded within. When subjected to a constant magnitude global flow regime, the local scaffold geometry dictates the magnitudes of mechanical stresses and strains experienced by a given cell, and in a spatially resolved fashion, similar to patterning during morphogenesis. In addition, early markers of mesenchymal stem cell lineage commitment relate significantly to the local mechanical environment of the cell. Finally, by plotting the range of stress-strain states for all data corresponding to nascent cell lineage commitment (95% CI), we begin to "map the mechanome", defining stress-strain states most conducive to targeted cell fates. In sum, we provide a library of reference mechanical cues that can be delivered to cells seeded on tissue engineering scaffolds to guide target tissue phenotypes in a temporally and spatially resolved manner. Knowledge of these effects allows for prospective scaffold design optimization using virtual models prior to prototyping and clinical implementation. Finally, this approach enables the development of next generation scaffolds cum delivery devices for genesis of complex tissues with heterogenous properties, e.g., organs, joints or interface tissues such as growth plates.

  6. Mechanical Modulation of Nascent Stem Cell Lineage Commitment in Tissue Engineering Scaffolds

    PubMed Central

    Song, Min Jae; Dean, David; Tate, Melissa L. Knothe

    2013-01-01

    Taking inspiration from tissue morphogenesis in utero, this study tests the concept of using tissue engineering scaffolds as delivery devices to modulate emergent structure-function relationships at early stages of tissue genesis. We report on the use of a combined computational fluid dynamics (CFD) modeling, advanced manufacturing methods, and experimental fluid mechanics (micro-piv and strain mapping) for the prospective design of tissue engineering scaffold geometries that deliver spatially resolved mechanical cues to cells seeded within. When subjected to a constant magnitude global flow regime, the local scaffold geometry dictates the magnitudes of mechanical stresses and strains experienced by a given cell, and in a spatially resolved fashion, similar to patterning during morphogenesis. In addition, early markers of mesenchymal stem cell lineage commitment relate significantly to the local mechanical environment of the cell. Finally, by plotting the range of stress-strain states for all data corresponding to nascent cell lineage commitment (95% CI), we begin to “map the mechanome”, defining stress-strain states most conducive to targeted cell fates. In sum, we provide a library of reference mechanical cues that can be delivered to cells seeded on tissue engineering scaffolds to guide target tissue phenotypes in a temporally and spatially resolved manner. Knowledge of these effects allows for prospective scaffold design optimization using virtual models prior to prototyping and clinical implementation. Finally, this approach enables the development of next generation scaffolds cum delivery devices for genesis of complex tissues with heterogenous properties, e.g., organs, joints or interface tissues such as growth plates. PMID:23660249

  7. FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans

    PubMed Central

    Minor, Paul J.; He, Ting-Fang; Sohn, Chang Ho; Asthagiri, Anand R.; Sternberg, Paul W.

    2013-01-01

    The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/β-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans. PMID:23946444

  8. FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans.

    PubMed

    Minor, Paul J; He, Ting-Fang; Sohn, Chang Ho; Asthagiri, Anand R; Sternberg, Paul W

    2013-09-01

    The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/β-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans.

  9. Three RNA binding proteins form a complex to promote differentiation of germline stem cell lineage in Drosophila.

    PubMed

    Chen, Di; Wu, Chan; Zhao, Shaowei; Geng, Qing; Gao, Yu; Li, Xin; Zhang, Yang; Wang, Zhaohui

    2014-11-01

    In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3'UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage.

  10. Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila

    PubMed Central

    Zhao, Shaowei; Geng, Qing; Gao, Yu; Li, Xin; Zhang, Yang; Wang, Zhaohui

    2014-01-01

    In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3′UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage. PMID:25412508

  11. Human natural killer cell committed thymocytes and their relation to the T cell lineage

    PubMed Central

    1993-01-01

    identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway. PMID:7504051

  12. Spatial and temporal dynamics of cell generations within an invasion wave: a link to cell lineage tracing.

    PubMed

    Cheeseman, Bevan L; Newgreen, Donald F; Landman, Kerry A

    2014-12-21

    Mathematical models of a cell invasion wave have included both continuum partial differential equation (PDE) approaches and discrete agent-based cellular automata (CA) approaches. Here we are interested in modelling the spatial and temporal dynamics of the number of divisions (generation number) that cells have undergone by any time point within an invasion wave. In the CA framework this is performed from agent lineage tracings, while in the PDE approach a multi-species generalized Fisher equation is derived for the cell density within each generation. Both paradigms exhibit qualitatively similar cell generation densities that are spatially organized, with agents of low generation number rapidly attaining a steady state (with average generation number increasing linearly with distance) behind the moving wave and with evolving high generation number at the wavefront. This regularity in the generation spatial distributions is in contrast to the highly stochastic nature of the underlying lineage dynamics of the population. In addition, we construct a method for determining the lineage tracings of all agents without labelling and tracking the agents, but through either a knowledge of the spatial distribution of the generations or the number of agents in each generation. This involves determining generation-dependent proliferation probabilities and using these to define a generation-dependent Galton-Watson (GDGW) process. Monte-Carlo simulations of the GDGW process are used to determine the individual lineage tracings. The lineages of the GDGW process are analyzed using Lorenz curves and found to be similar to outcomes generated by direct lineage tracing in CA realizations. This analysis provides the basis for a potentially useful technique for deducing cell lineage data when imaging every cell is not feasible.

  13. Cell lineage patterns in the shoot meristem of the sunflower embryo in the dry seed

    SciTech Connect

    Jegla, D.E.; Sussex, I.M.

    1989-01-01

    We mapped the fate of cells in the shoot meristem of the dry-seed embryo of sunflower, Helianthus annuus L. cv. Peredovic, using irradiation-induced somatic sectors. We analyzed 249 chlorophyll-deficient or glabrous (hairless) sectors generated in 236 plants. Most sectors observed in the inflorescence extended into vegetative nodes. Thus cell lineages that ultimately gave rise to reproductive structures also contributed to vegetative structures. No single sector extended the entire length of the shoot. Thus the shoot is not derived from one or a few apical initials. Rather, the position, vertical extent, and width of the sectors at different levels of the shoot suggest that the shoot is derived from three to four circumferential populations of cells in each of three cell layers of the embryo meristem. Sectors had no common boundaries even in plants with two or three independent sectors, but varied in extent and overlapped along the length of the shoot. Thus individual cells in a single circumferential population behaved independently to contribute lineages of different vertical extents to the growing shoot. The predicted number of circumferential populations of cells as well as the apparent cell number in each population was consistent with the actual number of cells in the embryo meristem observed in histological sections.

  14. Candidate genes and pathways downstream of PAX8 involved in ovarian high-grade serous carcinoma

    PubMed Central

    Soriano, Amata Amy; Monticelli, Antonella; Affinito, Ornella; Cocozza, Sergio; Zannini, Mariastella

    2016-01-01

    Understanding the biology and molecular pathogenesis of ovarian epithelial cancer (EOC) is key to developing improved diagnostic and prognostic indicators and effective therapies. Although research has traditionally focused on the hypothesis that high-grade serous carcinoma (HGSC) arises from the ovarian surface epithelium (OSE), recent studies suggest that additional sites of origin exist and a substantial proportion of cases may arise from precursor lesions located in the Fallopian tubal epithelium (FTE). In FTE cells, the transcription factor PAX8 is a marker of the secretory cell lineage and its expression is retained in 96% of EOC. We have recently reported that PAX8 is involved in the tumorigenic phenotype of ovarian cancer cells. In this study, to uncover genes and pathways downstream of PAX8 involved in ovarian carcinoma we have determined the molecular profiles of ovarian cancer cells and in parallel of Fallopian tube epithelial cells by means of a silencing approach followed by an RNA-seq analysis. Interestingly, we highlighted the involvement of pathways like WNT signaling, epithelial-mesenchymal transition, p53 and apoptosis. We believe that our analysis has led to the identification of candidate genes and pathways regulated by PAX8 that could be additional targets for the therapy of ovarian carcinoma. PMID:27259239

  15. MYSM1-dependent checkpoints in B cell lineage differentiation and B cell-mediated immune response.

    PubMed

    Förster, Michael; Farrington, Kyo; Petrov, Jessica C; Belle, Jad I; Mindt, Barbara C; Witalis, Mariko; Duerr, Claudia U; Fritz, Jörg H; Nijnik, Anastasia

    2017-03-01

    MYSM1 is a chromatin-binding histone deubiquitinase. MYSM1 mutations in humans result in lymphopenia whereas loss of Mysm1 in mice causes severe hematopoietic abnormalities, including an early arrest in B cell development. However, it remains unknown whether MYSM1 is required at later checkpoints in B cell development or for B cell-mediated immune responses. We analyzed conditional mouse models Mysm1(fl/fl)Tg.mb1-cre, Mysm1(fl/fl)Tg.CD19-cre, and Mysm1(fl/fl)Tg.CD21-cre with inactivation of Mysm1 at prepro-B, pre-B, and follicular B cell stages of development. We show that loss of Mysm1 at the prepro-B cell stage in Mysm1(fl/fl)Tg.mb1-cre mice results in impaired B cell differentiation, with an ∼2-fold reduction in B cell numbers in the lymphoid organs. Mysm1(fl/fl)Tg.mb1-cre B cells also showed increased expression of activation markers and impaired survival and proliferation. In contrast, Mysm1 was largely dispensable from the pre-B cell stage onward, with Mysm1(fl/fl)Tg.CD19-cre and Mysm1(fl/fl)Tg.CD21-cre mice showing no alterations in B cell numbers and largely normal responses to stimulation. MYSM1, therefore, has an essential role in B cell lineage specification but is dispensable at later stages of development. Importantly, MYSM1 activity at the prepro-B cell stage of development is important for the normal programming of B cell responses to stimulation once they complete their maturation process.

  16. NtDRP is necessary for accurate zygotic division orientation and differentiation of basal cell lineage toward suspensor formation.

    PubMed

    Zhao, Jing; Xin, Haiping; Cao, Lingyan; Huang, Xiaorong; Shi, Ce; Zhao, Peng; Fu, Ying; Sun, Meng-Xiang

    2016-11-01

    Plant embryogenesis begins with an asymmetric division of the zygote, producing apical and basal cells with distinct cell fates. The asymmetric zygote division is thought to be critical for embryo pattern formation; however, the molecular mechanisms regulating this process, especially maintaining the accurate position and proper orientation of cell division plane, remain poorly understood. Here, we report that a dynamin-related protein in Nicotiana tabacum, NtDRP, plays a critical role in maintaining orientation of zygotic division plane. Down-regulation of NtDRP caused zygotic cell division to occur in different, incorrect orientations and resulted in disruption of suspensor formation, and even development of twin embryos. The basal cell lineage totally integrated with the apical cell lineage into an embryo-like structure, suggesting that NtDRP is essential to accurate zygotic division orientation and differentiation of basal cell lineage toward suspensor formation. We also reveal that NtDRP plays its role by modulating microtubule spatial organization and spindle orientation during early embryogenesis. Thus, we revealed that NtDRP is involved in orientation of the asymmetric zygotic division and differentiation of distinct suspensor and embryo domains, as well as subsequent embryo pattern formation.

  17. Antibody-based therapies in B-cell lineage acute lymphoblastic leukaemia.

    PubMed

    Le Jeune, Caroline; Thomas, Xavier

    2015-02-01

    Targeted therapies represent a major breakthrough in the treatment of adult acute lymphoblastic leukaemia (ALL). Because lymphoblastic leukaemia cells express a variety of specific antigens, those ones can serve as targets for monoclonal antibodies (MoAbs). Anti-CD20 (rituximab), anti-CD19 (blinatumomab, SAR3419), anti-CD22 (epratuzumab, inotuzumab ozogamicin) and anti-CD52 (alemtuzumab) have therefore been developed. Possible strategies even include recruitment of CD3 cytotoxic T cells (blinatumomab) or adoptive T-cell therapy by gene transfer of CD19-chimeric antigen receptors (CD19-CARs). Recent data show that antibody-based therapy is a highly promising treatment approach. However, optimal treatment approach still needs to be defined.

  18. Epithelial stem cell repertoire in the gut: clues to the origin of cell lineages, proliferative units and cancer

    PubMed Central

    Wright, Nicholas A

    2000-01-01

    Gastrointestinal stem cells are shown to be pluripotential and to give rise to all cell lineages in the epithelium. After damage, gut stem cells produce reparative cell lineages that produce a wide range of peptides with important actions on cell proliferation and migration, and promote regeneration and healing. Increase in stem cell number is considered to induce crypt fission, and lead to increases in the number of crypts, even in the adult; it is also the mode of spread of mutated clones in the colorectal mucosa. Stem cell repertoire is defined by both intrinsic programming of the stem cell itself, but signalling from the mesenchyme is also vitally important for defining both stem cell progeny and proliferation. Carcinogenesis in the colon occurs through sequential mutations, possibly occurring in a single cell. A case is made for this being the stem cell, but recent studies indicate that several stem cells may need to be so involved, since early lesions appear to be polyclonal in derivation. PMID:10762441

  19. Genetic Ablation of Parietal Cells in Transgenic Mice: A New Model for Analyzing Cell Lineage Relationships in the Gastric Mucosa

    NASA Astrophysics Data System (ADS)

    Canfield, Victor; West, A. Brian; Goldenring, James R.; Levenson, Robert

    1996-03-01

    The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.

  20. Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa).

    PubMed

    Barfield, Sarah; Aglyamova, Galina V; Matz, Mikhail V

    2016-01-13

    The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions.

  1. Evolutionary origins of germline segregation in Metazoa: evidence for a germ stem cell lineage in the coral Orbicella faveolata (Cnidaria, Anthozoa)

    PubMed Central

    Barfield, Sarah; Aglyamova, Galina V.; Matz, Mikhail V.

    2016-01-01

    The ability to segregate a committed germ stem cell (GSC) lineage distinct from somatic cell lineages is a characteristic of bilaterian Metazoans. However, the occurrence of GSC lineage specification in basally branching Metazoan phyla, such as Cnidaria, is uncertain. Without an independently segregated GSC lineage, germ cells and their precursors must be specified throughout adulthood from continuously dividing somatic stem cells, generating the risk of propagating somatic mutations within the individual and its gametes. To address the potential for existence of a GSC lineage in Anthozoa, the sister-group to all remaining Cnidaria, we identified moderate- to high-frequency somatic mutations and their potential for gametic transfer in the long-lived coral Orbicella faveolata (Anthozoa, Cnidaria) using a 2b-RAD sequencing approach. Our results demonstrate that somatic mutations can drift to high frequencies (up to 50%) and can also generate substantial intracolonial genetic diversity. However, these somatic mutations are not transferable to gametes, signifying the potential for an independently segregated GSC lineage in O. faveolata. In conjunction with previous research on germ cell development in other basally branching Metazoan species, our results suggest that the GSC system may be a Eumetazoan characteristic that evolved in association with the emergence of greater complexity in animal body plan organization and greater specificity of stem cell functions. PMID:26763699

  2. Chromatin Dynamics Regulate Mesenchymal Stem Cell Lineage Specification and Differentiation to Osteogenesis

    PubMed Central

    Wu, Hai; Gordon, Jonathan A.R.; Whitfield, Troy W.; Tai, Phillip W.L.; van Wijnen, Andre J.; Stein, Janet L.; Stein, Gary S.; Lian, Jane B.

    2017-01-01

    Multipotent mesenchymal stromal cells (MSCs) are critical for regeneration of multiple tissues. Epigenetic mechanisms are fundamental regulators of lineage specification and cell fate, and as such, we addressed the question of which epigenetic modifications characterize the transition of nascent MSCs to a tissue specific MSC-derived phenotype. By profiling the temporal changes of seven histone marks correlated to gene expression during proliferation, early commitment, matrix deposition, and mineralization stages, we identified distinct epigenetic mechanisms that regulate transcriptional programs necessary for tissue-specific phenotype development. Patterns of stage-specific enrichment of histone modifications revealed distinct modes of repression and activation of gene expression that would not be detected using single endpoint analysis. We discovered that at commitment, H3K27me3 is removed from genes that are upregulated and is not acquired on downregulated genes. Additionally, we found that the absence of H3K4me3 modification at promoters defined a subset of osteoblast-specific upregulated genes, indicating acquisition of acetyl modifications drive activation of these genes. Significantly, loss or gain of H3K36me3 was the primary predictor of dynamic changes in temporal gene expression. Using unsupervised pattern discovery analysis the signature of osteogenic-related histone modifications identified novel functional cis regulatory modules associated with enhancer regions that control tissue-specific genes. Our work provides a cornerstone to understand the epigenetic regulation of transcriptional programs that are important for MSC lineage commitment and lineage, as well as insights to facilitate MSC-based therapeutic interventions. PMID:28077316

  3. An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium.

    PubMed Central

    Falk, P; Roth, K A; Borén, T; Westblom, T U; Gordon, J I; Normark, S

    1993-01-01

    Helicobacter pylori is a microaerophilic bacterium found in the stomach of asymptomatic humans as well as patients with acid peptic disease and gastric adenocarcinoma. We have developed an in situ adherence assay to examine the cell lineage-specific nature of binding of this organism and to characterize the nature of cell surface receptors that recognize its adhesin. Fluorescein isothiocyanate-labeled H. pylori strains were bound to surface mucous cells present in the pit region of human and rat gastric units but not to mucous neck, parietal, or chief cell lineages present in the glandular domains of these units. Binding was abolished by proteinase K treatment of tissue sections and by pretreatment of the bacteria with bovine submaxillary gland mucin, a rich source of fucosylated and sialylated carbohydrates. Several lines of evidence suggest that binding to surface mucous cells is not dependent upon terminal nonsubstituted alpha 2,3- and alpha 2,6-linked sialic acids in the adhesin receptor: (i) binding was not inhibited by incubating H. pylori strains with sialylated glycoconjugates such as fetuin and free sialyllactose; (ii) immunohistochemical stainings using the sialic acid-specific Sambucus nigra and Maackia amurensis lectins and the cholera toxin B subunit did not detect any sialylated glycoconjugates in these epithelial cells; and (iii) binding was not sensitive to metaperiodate under conditions that selectively cleaved carbons 8 and 9 of terminal nonmodified sialic acids. A role for fucosylated epitopes in the glycoprotein(s) that mediate binding of H. pylori to surface mucous cells was suggested by the facts that this lineage coexpresses the adhesin receptor and major fucosylated histo-blood group antigens, that monoclonal antibodies specific for histo-blood group antigens H, B, and Leb block binding, and that the lectin Ulex europaeus type 1 agglutinin, which is specific for alpha-L-fucose, also bound to the same cells that bound the bacteria

  4. β8 integrin expression and activation of TGF-β by intestinal dendritic cells is determined by both tissue microenvironment and cell lineage

    PubMed Central

    Boucard-Jourdin, Mathilde; Kugler, David; Endale Ahanda, Marie-Laure; This, Sébastien; De Calisto, Jaime; Zhang, Ailiang; Mora, J. Rodrigo; Stuart, Lynda M.; Savill, John; Lacy-Hulbert, Adam; Paidassi, Helena

    2016-01-01

    Activation of TGF-β by dendritic cells (DCs) expressing αvβ8 integrin is essential for the generation of intestinal regulatory T cells (Tregs) that in turn promote tolerance to intestinal antigens. We have recently shown that αvβ8 integrin is preferentially expressed by CD103+ DCs, and confers their ability to activate TGF-β and generate Tregs. However, how these DCs become specialized for this vital function is unknown. Here we show that β8 expression is controlled by a combination of factors that include DC lineage, and signals derived from the tissue microenvironment and microbiota. Specifically, our data demonstrate that TGF-β itself, along with retinoic acid (RA) and Toll-like receptor (TLR) signaling, drive expression of αvβ8 in DCs. However, these signals only result in high levels of β8 expression in cells of the cDC1 lineage, CD8α+ or CD103+CD11b- DCs, and this is associated with epigenetic changes in the Itgb8 locus. Together, these data provide a key illustrative example of how microenvironmental factors and cell lineage drive the generation of regulatory αvβ8-expressing DCs specialized for activation of TGF-β to facilitate Treg generation. PMID:27481847

  5. Transcription factor KLF7 regulates differentiation of neuroectodermal and mesodermal cell lineages

    SciTech Connect

    Caiazzo, Massimiliano; Colucci-D'Amato, Luca; Esposito, Maria T.; Parisi, Silvia; Stifani, Stefano; Ramirez, Francesco; Porzio, Umberto di

    2010-08-15

    Previous gene targeting studies in mice have implicated the nuclear protein Krueppel-like factor 7 (KLF7) in nervous system development while cell culture assays have documented its involvement in cell cycle regulation. By employing short hairpin RNA (shRNA)-mediated gene silencing, here we demonstrate that murine Klf7 gene expression is required for in vitro differentiation of neuroectodermal and mesodermal cells. Specifically, we show a correlation of Klf7 silencing with down-regulation of the neuronal marker microtubule-associated protein 2 (Map2) and the nerve growth factor (NGF) tyrosine kinase receptor A (TrkA) using the PC12 neuronal cell line. Similarly, KLF7 inactivation in Klf7-null mice decreases the expression of the neurogenic marker brain lipid-binding protein/fatty acid-binding protein 7 (BLBP/FABP7) in neural stem cells (NSCs). We also report that Klf7 silencing is detrimental to neuronal and cardiomyocytic differentiation of embryonic stem cells (ESCs), in addition to altering the adipogenic and osteogenic potential of mouse embryonic fibroblasts (MEFs). Finally, our results suggest that genes that are key for self-renewal of undifferentiated ESCs repress Klf7 expression in ESCs. Together with previous findings, these results provide evidence that KLF7 has a broad spectrum of regulatory functions, which reflect the discrete cellular and molecular contexts in which this transcription factor operates.

  6. Rewiring of human lung cell lineage and mitotic networks in lung adenocarcinomas

    PubMed Central

    Kim, Il-Jin; Quigley, David; To, Minh D.; Pham, Patrick; Lin, Kevin; Jo, Brian; Jen, Kuang-Yu; Raz, Dan; Kim, Jae; Mao, Jian-Hua; Jablons, David; Balmain, Allan

    2015-01-01

    Analysis of gene expression patterns in normal tissues and their perturbations in tumors can help to identify the functional roles of oncogenes or tumor suppressors and identify potential new therapeutic targets. Here, gene expression correlation networks were derived from 92 normal human lung samples and patient-matched adenocarcinomas. The networks from normal lung show that NKX2-1 is linked to the alveolar type 2 lineage, and identify PEBP4 as a novel marker expressed in alveolar type 2 cells. Differential correlation analysis shows that the NKX2-1 network in tumors includes pathways associated with glutamate metabolism, and identifies Vaccinia-related kinase (VRK1) as a potential drug target in a tumor-specific mitotic network. We show that VRK1 inhibition cooperates with inhibition of PARP signaling to inhibit growth of lung tumor cells. Targeting of genes that are recruited into tumor mitotic networks may provide a wider therapeutic window than that seen by inhibition of known mitotic genes. PMID:23591868

  7. Whole-brain neural network analysis (connectomics) using cell lineage-based neuron-labeling method.

    PubMed

    Ito, Kei; Ito, Masayoshi

    2014-11-01

    The brain is a computing machine that receives input signals from sensory neurons, calculates best responses to changing environments, and sends output signals to motor muscles. How such computation is materialized remains largely unknown. Understanding the entire wiring network of neural connections in the brain, which is recently called the connectomics (connection + omics), should provide indispensable insights on this problem.To resolve the circuit diagram from the tangled thickets of neural fibers, only a small subset of neurons should be visualized at one time. Previous studies visualized such selective cells by injecting dyes or by detecting specific molecules or gene expression patterns using antibodies and expression driver strains. These approaches were unfortunately not efficient enough for identifying all the brain cells in a comprehensive and systematic manner.Neurons are generated by neural stem cells. The entire neural population can therefore be divided into a finite number of families - or clones - of the cells that are the progeny of each single stem cell. The central brain of the fruit fly Drosophila melanogaster consists of about 15,000 neurons per side and is made by utmost 100 stem cells. By genetically labeling one of such stem cells and tracing the projection patterns of its progeny in the adult brain, we were able to identify the neural projections of almost all the clonal cell groups.To visualize these neural projections, we made serial optical sections of the fly brain using laser confocal microscopy. Because of its relatively small size (0.6-mm wide and less than 0.3-mm thick), the entire fly brain can be imaged using high-resolution objectives with n.a. 1.2. Neuronal fibers are visualized by ectopically expressed cytoplasmic and membrane-bound fluorescent proteins, and the output synaptic sites are visualized with ectopically expressed tag proteins that are fused with the proteins associated with synaptic vesicles. In addition, density

  8. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq

    PubMed Central

    Blakeley, Paul; Fogarty, Norah M. E.; del Valle, Ignacio; Wamaitha, Sissy E.; Hu, Tim Xiaoming; Elder, Kay; Snell, Philip; Christie, Leila; Robson, Paul; Niakan, Kathy K.

    2015-01-01

    Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells. PMID:26293300

  9. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.

    PubMed

    Blakeley, Paul; Fogarty, Norah M E; del Valle, Ignacio; Wamaitha, Sissy E; Hu, Tim Xiaoming; Elder, Kay; Snell, Philip; Christie, Leila; Robson, Paul; Niakan, Kathy K

    2015-09-15

    Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells.

  10. Functional studies of regulatory genes in the sea urchin embryo.

    PubMed

    Cavalieri, Vincenzo; Di Bernardo, Maria; Spinelli, Giovanni

    2009-01-01

    Sea urchin embryos are characterized by an extremely simple mode of development, rapid cleavage, high transparency, and well-defined cell lineage. Although they are not suitable for genetic studies, other approaches are successfully used to unravel mechanisms and molecules involved in cell fate specification and morphogenesis. Microinjection is the elective method to study gene function in sea urchin embryos. It is used to deliver precise amounts of DNA, RNA, oligonucleotides, peptides, or antibodies into the eggs or even into blastomeres. Here we describe microinjection as it is currently applied in our laboratory and show how it has been used in gene perturbation analyses and dissection of cis-regulatory DNA elements.

  11. ADAR1 is vital for B cell lineage development in the mouse bone marrow

    PubMed Central

    Marcu-Malina, Victoria; Goldberg, Sanja; Vax, Einav; Amariglio, Ninette

    2016-01-01

    Adenosine deaminase acting on RNA (ADAR) 1 is the master editor of the transcriptome, catalyzing the conversion of adenosine to inosine (A-to-I). RNA transcripts fold into a variety of secondary structures including long intramolecular RNA duplexes that are the major substrate of ADAR1. Most A-to-I editing sites occur within RNA duplexes formed by complementary pairing of inverted retrotransposable elements interspersed within noncoding regions of transcripts. This catalytic activity of ADAR1 most likely prevents the abnormal activation of cytosolic nucleic acid sensors by self-dsRNAs. Homozygous disruption of mouse Adar is embryonic lethal due to a toxic type-I interferons response and correspondingly biallelic missense mutations in human ADAR1 cause a severe congenital interferonopathy. Here, we report that Cd19-Cre-mediated Adar gene ablation in the mouse causes a significant defect in the final stages of B cell development with an almost complete absence of newly formed immature and CD23+ mature recirculating B cells in the BM. Adar ablation in pre-B cells induced upregulation of typical interferon-stimulated genes (ISGs) and apoptosis upon further maturation. ADAR1 deficiency also inhibited the in vitro, IL-7-mediated, differentiation of BM-derived B cell precursors. In summary, ADAR1 is required, non-redundantly, for normal B lymphopoiesis in the BM and peripheral maintenance. PMID:27494846

  12. Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineage divergence

    PubMed Central

    Rodríguez-Seguel, Elisa; Mah, Nancy; Naumann, Heike; Pongrac, Igor M.; Cerdá-Esteban, Nuria; Fontaine, Jean-Fred; Wang, Yongbo; Chen, Wei; Andrade-Navarro, Miguel A.; Spagnoli, Francesca M.

    2013-01-01

    Understanding how distinct cell types arise from multipotent progenitor cells is a major quest in stem cell biology. The liver and pancreas share many aspects of their early development and possibly originate from a common progenitor. However, how liver and pancreas cells diverge from a common endoderm progenitor population and adopt specific fates remains elusive. Using RNA sequencing (RNA-seq), we defined the molecular identity of liver and pancreas progenitors that were isolated from the mouse embryo at two time points, spanning the period when the lineage decision is made. The integration of temporal and spatial gene expression profiles unveiled mutually exclusive signaling signatures in hepatic and pancreatic progenitors. Importantly, we identified the noncanonical Wnt pathway as a potential developmental regulator of this fate decision and capable of inducing the pancreas program in endoderm and liver cells. Our study offers an unprecedented view of gene expression programs in liver and pancreas progenitors and forms the basis for formulating lineage-reprogramming strategies to convert adult hepatic cells into pancreatic cells. PMID:24013505

  13. Maternal exposure to 3,3'-iminodipropionitrile targets late-stage differentiation of hippocampal granule cell lineages to affect brain-derived neurotrophic factor signaling and interneuron subpopulations in rat offspring.

    PubMed

    Itahashi, Megu; Abe, Hajime; Tanaka, Takeshi; Mizukami, Sayaka; Kikuchihara, Yoh; Yoshida, Toshinori; Shibutani, Makoto

    2015-08-01

    3,3'-Iminodipropionitrile (IDPN) causes neurofilament (NF)-filled swellings in the proximal segments of many large-caliber myelinated axons. This study investigated the effect of maternal exposure to IDPN on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 67 or 200 ppm IDPN in drinking water from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, female offspring subjected to analysis had decreased parvalbumin(+), reelin(+) and phospho-TrkB(+) interneurons in the dentate hilus at 200 ppm and increased granule cell populations expressing immediate-early gene products, Arc or c-Fos, at ≥  67 ppm. mRNA expression in the dentate gyrus examined at 200 ppm decreased with brain-derived neurotrophic factor (Bdnf) and very low density lipoprotein receptor. Immunoreactivity for phosphorylated NF heavy polypeptide decreased in the molecular layer of the dentate gyrus and the stratum radiatum of the cornu ammonis (CA) 3, portions showing axonal projections from mossy cells and pyramidal neurons, at 200 ppm on PND 21, whereas immunoreactivity for synaptophysin was unchanged in the dentate gyrus. Observed changes all disappeared on PND 77. There were no fluctuations in the numbers of apoptotic cells, proliferating cells and subpopulations of granule cell lineage in the subgranular zone on PND 21 and PND 77. Thus, maternal IDPN exposure may reversibly affect late-stage differentiation of granule cell lineages involving neuronal plasticity as evident by immediate-early gene responses to cause BDNF downregulation resulting in a reduction in parvalbumin(+) or reelin(+) interneurons and suppression of axonal plasticity in the mossy cells and CA3 pyramidal neurons.

  14. A planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages.

    PubMed

    Pearson, Bret J; Sánchez Alvarado, Alejandro

    2010-01-01

    The functions of adult stem cells and tumor suppressor genes are known to intersect. However, when and how tumor suppressors function in the lineages produced by adult stem cells is unknown. With a large population of stem cells that can be manipulated and studied in vivo, the freshwater planarian is an ideal system with which to investigate these questions. Here, we focus on the tumor suppressor p53, homologs of which have no known role in stem cell biology in any invertebrate examined thus far. Planaria have a single p53 family member, Smed-p53, which is predominantly expressed in newly made stem cell progeny. When Smed-p53 is targeted by RNAi, the stem cell population increases at the expense of progeny, resulting in hyper-proliferation. However, ultimately the stem cell population fails to self-renew. Our results suggest that prior to the vertebrates, an ancestral p53-like molecule already had functions in stem cell proliferation control and self-renewal.

  15. A mex3 homolog is required for differentiation during planarian stem cell lineage development.

    PubMed

    Zhu, Shu Jun; Hallows, Stephanie E; Currie, Ko W; Xu, ChangJiang; Pearson, Bret J

    2015-06-26

    Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment.

  16. A mex3 homolog is required for differentiation during planarian stem cell lineage development

    PubMed Central

    Zhu, Shu Jun; Hallows, Stephanie E; Currie, Ko W; Xu, ChangJiang; Pearson, Bret J

    2015-01-01

    Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment. DOI: http://dx.doi.org/10.7554/eLife.07025.001 PMID:26114597

  17. Stage-specific differentiation of iPSCs toward retinal ganglion cell lineage

    PubMed Central

    Deng, Fei; Chen, Mengfei; Liu, Ying; Hu, Huiling; Xiong, Yunfan; Xu, Chaochao; Liu, Yuchun; Li, Kangjun; Zhuang, Jing

    2016-01-01

    Purpose As an alternative and desirable approach for regenerative medicine, human induced pluripotent stem cell (hiPSC) technology raises the possibility of developing patient-tailored cell therapies to treat intractable degenerative diseases in the future. This study was undertaken to guide human Tenon’s capsule fibroblasts-derived iPSCs (TiPSCs) to differentiate along the retinal ganglion cell (RGC) lineage, aiming at producing appropriate cellular material for RGC regeneration. Methods By mimicking RGC genesis, we deliberately administered the whole differentiation process and directed the stage-specific differentiation of human TiPSCs toward an RGC fate via manipulation of the retinal inducers (DKK1+Noggin+Lefty A) alongside master gene (Atoh7) sequentially. Throughout this stepwise differentiation process, changes in primitive neuroectodermal, eye field, and RGC marker expression were monitored with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and/or flow cytometry. Results Upon retinal differentiation, a large fraction of the cells developed characteristics of retinal progenitor cells (RPCs) in response to simulated environment signaling (DKK1+Noggin+Lefty A), which was selectively recovered with manual isolation approaches and then maintained in the presence of mitogen for multiple passages. Thereafter, overexpression of ATOH7 further promoted RGC specification in TiPSC-derived RPCs. A subset of transfected cells displayed RGC-specific expression patterns, including Brn3b, iSlet1, calretinin, and Tuj, and approximately 23% of Brn3b-positive RGC-like cells were obtained finally. Conclusions Our DKK1+Noggin+Lefty A/Atoh7-based RGC-induction regime could efficiently direct TiPSCs to differentiate along RGC lineage in a stage-specific manner, which may provide a benefit to develop possible cell therapies to treat retinal degenerative diseases such as glaucoma. PMID:27293372

  18. A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage

    PubMed Central

    Faure, Emmanuel; Savy, Thierry; Rizzi, Barbara; Melani, Camilo; Stašová, Olga; Fabrèges, Dimitri; Špir, Róbert; Hammons, Mark; Čúnderlík, Róbert; Recher, Gaëlle; Lombardot, Benoît; Duloquin, Louise; Colin, Ingrid; Kollár, Jozef; Desnoulez, Sophie; Affaticati, Pierre; Maury, Benoît; Boyreau, Adeline; Nief, Jean-Yves; Calvat, Pascal; Vernier, Philippe; Frain, Monique; Lutfalla, Georges; Kergosien, Yannick; Suret, Pierre; Remešíková, Mariana; Doursat, René; Sarti, Alessandro; Mikula, Karol; Peyriéras, Nadine; Bourgine, Paul

    2016-01-01

    The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology. PMID:26912388

  19. Characterization of the platelet-derived growth factor receptor-α-positive cell lineage during murine late lung development.

    PubMed

    Ntokou, Aglaia; Klein, Friederike; Dontireddy, Daria; Becker, Sven; Bellusci, Saverio; Richardson, William D; Szibor, Marten; Braun, Thomas; Morty, Rory E; Seeger, Werner; Voswinckel, Robert; Ahlbrecht, Katrin

    2015-11-01

    A reduced number of alveoli is the structural hallmark of diseases of the neonatal and adult lung, where alveoli either fail to develop (as in bronchopulmonary dysplasia), or are progressively destroyed (as in chronic obstructive pulmonary disease). To correct the loss of alveolar septa through therapeutic regeneration, the mechanisms of septa formation must first be understood. The present study characterized platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cell populations during late lung development in mice. PDGFRα(+) cells (detected using a PDGFRα(GFP) reporter line) were noted around the proximal airways during the pseudoglandular stage. In the canalicular stage, PDGFRα(+) cells appeared in the more distal mesenchyme, and labeled α-smooth muscle actin-positive tip cells in the secondary crests and lipofibroblasts in the primary septa during alveolarization. Some PDGFRα(+) cells appeared in the mesenchyme of the adult lung. Over the course of late lung development, PDGFRα(+) cells consistently expressed collagen I, and transiently expressed markers of mesenchymal stem cells. With the use of both, a constitutive and a conditional PDGFRα(Cre) line, it was observed that PDGFRα(+) cells generated alveolar myofibroblasts including tip cells of the secondary crests, and lipofibroblasts. These lineages were committed before secondary septation. The present study provides new insights into the time-dependent commitment of the PDGFRα(+) cell lineage to lipofibroblasts and myofibroblasts during late lung development that is needed to better understand the cellular contribution to the process of alveolarization.

  20. Plasticity within the αβ+CD4+ T-cell lineage: when, how and what for?

    PubMed Central

    Coomes, Stephanie M.; Pelly, Victoria S.; Wilson, Mark S.

    2013-01-01

    Following thymic output, αβ+CD4+ T cells become activated in the periphery when they encounter peptide–major histocompatibility complex. A combination of cytokine and co-stimulatory signals instructs the differentiation of T cells into various lineages and subsequent expansion and contraction during an appropriate and protective immune response. Our understanding of the events leading to T-cell lineage commitment has been dominated by a single fate model describing the commitment of T cells to one of several helper (TH), follicular helper (TFH) or regulatory (TREG) phenotypes. Although a single lineage-committed and dedicated T cell may best execute a single function, the view of a single fate for T cells has recently been challenged. A relatively new paradigm in αβ+CD4+ T-cell biology indicates that T cells are much more flexible than previously appreciated, with the ability to change between helper phenotypes, between helper and follicular helper, or, most extremely, between helper and regulatory functions. In this review, we comprehensively summarize the recent literature identifying when TH or TREG cell plasticity occurs, provide potential mechanisms of plasticity and ask if T-cell plasticity is beneficial or detrimental to immunity. PMID:23345540

  1. Gene therapy for high-grade glioma

    PubMed Central

    Natsume, Atsushi

    2008-01-01

    The treatment of high-grade gliomas remains difficult despite recent advances in surgery, radiotherapy and chemotherapy. True advances may emerge from the increasing understanding in molecular biology and discovery of novel mechanisms for the delivery of tumoricidal agents. In an attempt to overcome this formidable neoplasm, molecular approaches using gene therapy have been investigated clinically since 1992. The clinical trials have mainly been classified into three approaches: suicide gene therapy, immune gene therapy and oncolytic viral therapy. In this article, we review these approaches, which have been studied in previous and ongoing clinical trials. PMID:19262115

  2. Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage.

    PubMed

    Zhang, Qiao; Shalaby, Nevine A; Buszczak, Michael

    2014-01-17

    Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process within specific lineages remain poorly understood. Here, we identify a Drosophila RNA polymerase I (Pol I) regulatory complex composed of Under-developed (Udd), TAF1B, and a TAF1C-like factor. Disruption of udd or TAF1B results in reduced ovarian germline stem cell (GSC) proliferation. Female GSCs display high levels of ribosomal RNA (rRNA) transcription, and Udd becomes enriched in GSCs relative to their differentiating daughters. Increasing Pol I transcription delays differentiation, whereas reducing rRNA production induces both morphological changes that accompany multicellular cyst formation and specific decreased expression of the bone morphogenetic protein (BMP) pathway component Mad. These findings demonstrate that modulating rRNA synthesis fosters changes in the cell fate, growth, and proliferation of female Drosophila GSCs and their daughters.

  3. The Drosophila female germline stem cell lineage acts to spatially restrict DPP function within the niche.

    PubMed

    Liu, Ming; Lim, Tit Meng; Cai, Yu

    2010-07-27

    Maintenance of stem cells requires spatially restricted, niche-associated signals. In the Drosophila female germline stem cell (GSC) niche, Decapentaplegic (DPP) is the primary niche-associated factor and functions over a short range to promote GSC self-renewal rather than differentiation. Here, we show that the GSC lineage and, more specifically, the stem cells themselves participate in the spatial restriction of DPP function by activating epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) signaling in the surrounding somatic cells. EGFR-MAPK signaling in somatic cells repressed the expression of dally, which encodes a glypican required for DPP movement and stability. Consequently, only GSCs close to the DPP source (the somatic cells in the niche) showed high signal activation and were maintained as stem cells, whereas cystoblasts outside the niche showed low signal activation and initiated differentiation. Thus, our data reveal that the reciprocal crosstalk between the GSCs and the somatic cells defines the spatial limits of DPP action and therefore the extent of the GSC niche.

  4. Distinct mechanisms define murine B cell lineage immunoglobulin heavy chain (IgH) repertoires.

    PubMed

    Yang, Yang; Wang, Chunlin; Yang, Qunying; Kantor, Aaron B; Chu, Hiutung; Ghosn, Eliver Eb; Qin, Guang; Mazmanian, Sarkis K; Han, Jian; Herzenberg, Leonore A

    2015-09-30

    Processes that define immunoglobulin repertoires are commonly presumed to be the same for all murine B cells. However, studies here that couple high-dimensional FACS sorting with large-scale quantitative IgH deep-sequencing demonstrate that B-1a IgH repertoire differs dramatically from the follicular and marginal zone B cells repertoires and is defined by distinct mechanisms. We track B-1a cells from their early appearance in neonatal spleen to their long-term residence in adult peritoneum and spleen. We show that de novo B-1a IgH rearrangement mainly occurs during the first few weeks of life, after which their repertoire continues to evolve profoundly, including convergent selection of certain V(D)J rearrangements encoding specific CDR3 peptides in all adults and progressive introduction of hypermutation and class-switching as animals age. This V(D)J selection and AID-mediated diversification operate comparably in germ-free and conventional mice, indicating these unique B-1a repertoire-defining mechanisms are driven by antigens that are not derived from microbiota.

  5. Lentivirally overexpressed T-bet regulates T-helper cell lineage commitment in chronic hepatitis B patients.

    PubMed

    Liu, Xueni; Tang, Zhenghao; Zhang, Yi; Hu, Jianjun; Li, Dan; Zang, Guoqing; Yu, Yongsheng

    2012-08-01

    Chronic hepatitis B virus (HBV) infection is commonly considered to occur as a result of disturbance of the immune system. T-box expressed in T cells (T-bet) is an essential transcription factor for T helper (Th) cell differentiation and function. The aim of this study was to investigate the effect of T-bet overexpression on Th cell differentiation and the possible mechanism in chronic hepatitis B (CHB) patients. CD4+ T cells from the peripheral blood of 23 CHB patients, 8 acute hepatitis B (AHB) patients and 10 healthy controls were isolated. T-bet mRNA expression of CD4+ T cells was detected by quantitative real-time polymerase chain reaction (PCR). The T-bet DNA fragment was subcloned into the pGC-FU vector containing GFP to generate a recombinant lentiviral vector, pGC-FU-T-bet, while a no-load pGC-FU vector was used as the negative control. After transduction into CD4+ T cells from another 22 CHB patients, the induction of Th1- and Th2-type cytokines was assayed by an enzyme-linked immunosorbent assay (ELISA), and RT-PCR and western blot analysis were used to measure the mRNA and transcription levels of H2.0-like homeobox (HLX1), GATA-3 and STAT-6. T-bet mRNA expression in CD4+ T cells from AHB patients was enhanced compared with CHB patients and healthy controls. Th1-type cytokines and HLX1 expression was upregulated, while Th2-type cytokines and GATA-3 and STAT-6 expression was repressed after lentiviral introduction of T-bet. In conclusion, lentivirally overexpressed T-bet regulates Th cell lineage commitment in CHB patients, which may be mediated by regulating HLX1, GATA-3 and STAT-6 expression.

  6. Immunohistochemical study of melanocyte-melanocyte stem cell lineage in vitiligo; a clue to interfollicular melanocyte stem cell reservoir.

    PubMed

    Seleit, Iman; Bakry, Ola Ahmed; Abdou, Asmaa Gaber; Dawoud, Noha Mohammed

    2014-05-01

    There has been a long lasting controversy over whether melanocytes (MCs) in vitiligo are actually lost or still present but functionally inactive. We aimed to evaluate the MC cell lineage in follicular and interfollicular vitiliginous epidermis through immunohistochemical localization of Human Melanoma Black-45 (HMB-45) and Tyrosinase Related Protein 2 (TRP2) and to correlate it with clinicopathologic parameters. Using immunohistochemical techniques, skin biopsies from 50 vitiligo patients and 20 age- and gender-matched healthy subjects were examined. Differentiated active MCs were detected in 44% of interfollicular epidermis (IFE) and 46.7% of follicular epidermis (FE) in lesional skin. Melanocyte precursors/stem cells were detected in 54% of IFE and 63.3% of FE in lesional skin. Melanocyte precursors/stem cells of IFE were significantly associated with residual melanin pigment (p = 0.007) and with absence of angiogenesis (p = 0.05). HMB-45 percentage of expression in IFE was positively correlated with MC precursors/stem cells percentage in FE (r = +0.65, p < 0.001) and IFE (r = +0.33, p = 0.01). Melanocyte precursors/stem cells positivity (p < 0.001) was progressively decreasing with advanced histopathologic grading. There was no significant association between interfollicular or follicular expression of HMB-45, TRP2 or MC precursors/stem cells and the clinical type of vitiligo or its duration. In conclusion, functioning MCs may exist in vitiligo. The presence of MC precursors/stem cells in IFE may provide an additional reservoir needed for repigmentation.

  7. Generalist genes and high cognitive abilities.

    PubMed

    Haworth, Claire M A; Dale, Philip S; Plomin, Robert

    2009-07-01

    The concept of generalist genes operating across diverse domains of cognitive abilities is now widely accepted. Much less is known about the etiology of the high extreme of performance. Is there more specialization at the high extreme? Using a representative sample of 4,000 12-year-old twin pairs from the UK Twins Early Development Study, we investigated the genetic and environmental overlap between web-based tests of general cognitive ability, reading, mathematics and language performance for the top 15% of the distribution using DF extremes analysis. Generalist genes are just as evident at the high extremes of performance as they are for the entire distribution of abilities and for cognitive disabilities. However, a smaller proportion of the phenotypic intercorrelations appears to be explained by genetic influences for high abilities.

  8. Clustering of High Throughput Gene Expression Data

    PubMed Central

    Pirim, Harun; Ekşioğlu, Burak; Perkins, Andy; Yüceer, Çetin

    2012-01-01

    High throughput biological data need to be processed, analyzed, and interpreted to address problems in life sciences. Bioinformatics, computational biology, and systems biology deal with biological problems using computational methods. Clustering is one of the methods used to gain insight into biological processes, particularly at the genomics level. Clearly, clustering can be used in many areas of biological data analysis. However, this paper presents a review of the current clustering algorithms designed especially for analyzing gene expression data. It is also intended to introduce one of the main problems in bioinformatics - clustering gene expression data - to the operations research community. PMID:23144527

  9. Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine.

    PubMed

    Hey, Fiona; Giblett, Susan; Forrest, Stephanie; Herbert, Chelsea; Pritchard, Catrin

    2016-01-01

    The WNT signalling pathway controls many developmental processes and plays a key role in maintenance of intestine renewal and homeostasis. Glycogen Synthase Kinase 3 (GSK3) is an important component of the WNT pathway and is involved in regulating β-catenin stability and expression of WNT target genes. The mechanisms underpinning GSK3 regulation in this context are not completely understood, with some evidence suggesting this occurs through inhibitory N-terminal serine phosphorylation in a similar way to GSK3 inactivation in insulin signaling. To investigate this in a physiologically relevant context, we have analysed the intestinal phenotype of GSK3 knockin mice in which N-terminal serines 21/9 of GSK3α/β have been mutated to non-phosphorylatable alanine residues. We show that these knockin mutations have very little effect on overall intestinal integrity, cell lineage commitment, β-catenin localization or WNT target gene expression although a small increase in apoptosis at villi tips is observed. Our results provide in vivo evidence that GSK3 is regulated through mechanisms independent of N-terminal serine phosphorylation in order for β-catenin to be stabilised.

  10. The Molecular Signature of HIV-1-Associated Lipomatosis Reveals Differential Involvement of Brown and Beige/Brite Adipocyte Cell Lineages.

    PubMed

    Cereijo, Rubén; Gallego-Escuredo, José Miguel; Moure, Ricardo; Villarroya, Joan; Domingo, Joan Carles; Fontdevila, Joan; Martínez, Esteban; Gutiérrez, Maria del Mar; Mateo, María Gracia; Giralt, Marta; Domingo, Pere; Villarroya, Francesc

    2015-01-01

    Highly active antiretroviral therapy has remarkably improved quality of life of HIV-1-infected patients. However, this treatment has been associated with the so-called lipodystrophic syndrome, which conveys a number of adverse metabolic effects and morphological alterations. Among them, lipoatrophy of subcutaneous fat in certain anatomical areas and hypertrophy of visceral depots are the most common. Less frequently, lipomatous enlargements of subcutaneous fat at distinct anatomic areas occur. Lipomatous adipose tissue in the dorso-cervical area ("buffalo hump") has been associated with a partial white-to-brown phenotype transition and with increased cell proliferation, but, to date, lipomatous enlargements arising in other parts of the body have not been characterized. In order to establish the main molecular events associated with the appearance of lipomatosis in HIV-1 patients, we analyzed biopsies of lipomatous tissue from "buffalo hump" and from other anatomical areas in patients, in comparison with healthy subcutaneous adipose tissue, using a marker gene expression approach. Both buffalo-hump and non-buffalo-hump lipomatous adipose tissues exhibited similar patterns of non-compromised adipogenesis, unaltered inflammation, non-fibrotic phenotype and proliferative activity. Shorter telomere length, prelamin A accumulation and SA-β-Gal induction, reminiscent of adipocyte senescence, were also common to both types of lipomatous tissues. Buffalo hump biopsies showed expression of marker genes of brown adipose tissue (e.g. UCP1) and, specifically, of "classical" brown adipocytes (e.g. ZIC1) but not of beige/brite adipocytes. No such brown fat-related gene expression occurred in lipomatous tissues at other anatomical sites. In conclusion, buffalo hump and other subcutaneous adipose tissue enlargements from HIV-1-infected patients share a similar lipomatous character. However, a distorted induction of white-to-"classical brown adipocyte" phenotype appears unique of

  11. Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage.

    PubMed

    Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W; Burt, David W; Kaiser, Pete; Hume, David A; Sang, Helen M

    2014-08-01

    We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens.

  12. Visualisation of chicken macrophages using transgenic reporter genes: insights into the development of the avian macrophage lineage

    PubMed Central

    Balic, Adam; Garcia-Morales, Carla; Vervelde, Lonneke; Gilhooley, Hazel; Sherman, Adrian; Garceau, Valerie; Gutowska, Maria W.; Burt, David W.; Kaiser, Pete; Hume, David A.; Sang, Helen M.

    2014-01-01

    We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens. PMID:25063453

  13. The Molecular Signature of HIV-1-Associated Lipomatosis Reveals Differential Involvement of Brown and Beige/Brite Adipocyte Cell Lineages

    PubMed Central

    Cereijo, Rubén; Gallego-Escuredo, José Miguel; Moure, Ricardo; Villarroya, Joan; Domingo, Joan Carles; Fontdevila, Joan; Martínez, Esteban; Gutiérrez, Maria del Mar; Mateo, María Gracia; Giralt, Marta; Domingo, Pere; Villarroya, Francesc

    2015-01-01

    Highly active antiretroviral therapy has remarkably improved quality of life of HIV-1-infected patients. However, this treatment has been associated with the so-called lipodystrophic syndrome, which conveys a number of adverse metabolic effects and morphological alterations. Among them, lipoatrophy of subcutaneous fat in certain anatomical areas and hypertrophy of visceral depots are the most common. Less frequently, lipomatous enlargements of subcutaneous fat at distinct anatomic areas occur. Lipomatous adipose tissue in the dorso-cervical area (“buffalo hump”) has been associated with a partial white-to-brown phenotype transition and with increased cell proliferation, but, to date, lipomatous enlargements arising in other parts of the body have not been characterized. In order to establish the main molecular events associated with the appearance of lipomatosis in HIV-1 patients, we analyzed biopsies of lipomatous tissue from “buffalo hump” and from other anatomical areas in patients, in comparison with healthy subcutaneous adipose tissue, using a marker gene expression approach. Both buffalo-hump and non-buffalo-hump lipomatous adipose tissues exhibited similar patterns of non-compromised adipogenesis, unaltered inflammation, non-fibrotic phenotype and proliferative activity. Shorter telomere length, prelamin A accumulation and SA-β-Gal induction, reminiscent of adipocyte senescence, were also common to both types of lipomatous tissues. Buffalo hump biopsies showed expression of marker genes of brown adipose tissue (e.g. UCP1) and, specifically, of “classical” brown adipocytes (e.g. ZIC1) but not of beige/brite adipocytes. No such brown fat-related gene expression occurred in lipomatous tissues at other anatomical sites. In conclusion, buffalo hump and other subcutaneous adipose tissue enlargements from HIV-1-infected patients share a similar lipomatous character. However, a distorted induction of white-to-“classical brown adipocyte” phenotype

  14. Tyrosine kinase receptor c-ros-oncogene 1 mediates TWIST-1 regulation of human mesenchymal stem cell lineage commitment.

    PubMed

    Camp, Esther; Anderson, Peter J; Zannettino, Andrew C W; Gronthos, Stan

    2017-01-01

    The TWIST-1 gene encodes a basic helix-loop-helix (bHLH) transcription factor important in mediating skeletal and head mesodermal tissue development. Bone marrow-derived mesenchymal stem/stromal cells (BMSC), express high levels of TWIST-1, which is down regulated during ex vivo expansion. Cultured BMSC over-expressing TWIST-1 display decreased capacity for osteogenic differentiation and an enhanced capacity to undergo adipogenesis, suggesting that TWIST-1 is a mediator of lineage commitment. However, little is known regarding the mechanism(s) by which TWIST-1 mediates cell fate determination. In this study, microarray analysis was used to identify a novel downstream TWIST-1 target, tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1), which was down regulated in TWIST-1 over-expressing BMSC. Chromatin immunoprecipitation analysis showed that TWIST-1 directly bound to two E-box binding sites on the proximal C-ROS-1 promoter. Knock-down of C-ROS-1 in human BMSC and cranial bone cells resulted in a decreased capacity for osteogenic differentiation in vitro. Conversely, suppression of C-ROS-1 in BMSC resulted in an enhanced capacity to undergo adipogenesis. Furthermore, reduced C-ROS-1 levels led to activation of different components of the PI3K/AKT/mTORC1 signalling pathway during osteogenic and adipogenic differentiation. Collectively, these data suggest that C-ROS-1 is involved in BMSC fate switching between osteogenesis and adipogenesis, mediated via PI3K/AKT/mTORC1 signalling.

  15. FOG-1-mediated recruitment of NuRD is required for cell lineage re-enforcement during haematopoiesis.

    PubMed

    Gao, Zhiguang; Huang, Zan; Olivey, Harold E; Gurbuxani, Sandeep; Crispino, John D; Svensson, Eric C

    2010-01-20

    The transcriptional co-factor Friend of GATA1 (FOG-1) has been shown to interact with subunits of the nucleosome remodelling and histone deacetylase (NuRD) complex through a specific motif located at its N-terminus. To test the importance of FOG-1/NuRD interaction for haematopoiesis in vivo, we generated mice with a mutation that specifically disrupts FOG-1/NuRD interaction (FOG-1(R3K5A)). Homozygous FOG-1(R3K5A) mice were found to have splenomegaly, extramedullary erythropoiesis, granulocytosis and thrombocytopaenia secondary to a block in megakaryocyte maturation. FOG-1(R3K5A/R3K5A) megakaryocytes and erythroid progenitors expressed increased levels of GATA2, showing that FOG-1/NuRD interaction is required for the earlier described 'GATA Switch'. In addition, ablation of FOG-1/NuRD interaction led to inappropriate expression of mast cell and eosinophil-specific genes in the megakaryocyte and erythroid lineages. Chromatin immunoprecipitation experiments revealed that the NuRD complex was not properly recruited to a mast cell gene promoter in FOG-1(R3K5A/R3K5A) megakaryocytes, suggesting that FOG-1/NuRD interaction is required for the direct suppression of mast cell gene expression. Taken together, these results underscore the importance of the FOG-1/NuRD interaction for the re-enforcement of lineage commitment during erythropoiesis and megakaryopoiesis in vivo.

  16. Loss of CD44dim Expression from Early Progenitor Cells Marks T-Cell Lineage Commitment in the Human Thymus

    PubMed Central

    Canté-Barrett, Kirsten; Mendes, Rui D.; Li, Yunlei; Vroegindeweij, Eric; Pike-Overzet, Karin; Wabeke, Tamara; Langerak, Anton W.; Pieters, Rob; Staal, Frank J. T.; Meijerink, Jules P. P.

    2017-01-01

    Human T-cell development is less well studied than its murine counterpart due to the lack of genetic tools and the difficulty of obtaining cells and tissues. Here, we report the transcriptional landscape of 11 immature, consecutive human T-cell developmental stages. The changes in gene expression of cultured stem cells on OP9-DL1 match those of ex vivo isolated murine and human thymocytes. These analyses led us to define evolutionary conserved gene signatures that represent pre- and post-αβ T-cell commitment stages. We found that loss of dim expression of CD44 marks human T-cell commitment in early CD7+CD5+CD45dim cells, before the acquisition of CD1a surface expression. The CD44−CD1a− post-committed thymocytes have initiated in frame T-cell receptor rearrangements that are accompanied by loss of capacity to differentiate toward myeloid, B- and NK-lineages, unlike uncommitted CD44dimCD1a− thymocytes. Therefore, loss of CD44 represents a previously unrecognized human thymocyte stage that defines the earliest committed T-cell population in the thymus. PMID:28163708

  17. Ttk69 acts as a master repressor of enteroendocrine cell specification in Drosophila intestinal stem cell lineages.

    PubMed

    Wang, Chenhui; Guo, Xingting; Dou, Kun; Chen, Hongyan; Xi, Rongwen

    2015-10-01

    In adult Drosophila midgut, intestinal stem cells (ISCs) periodically produce progenitor cells that undergo a binary fate choice determined primarily by the levels of Notch activity that they receive, before terminally differentiating into enterocytes (ECs) or enteroendocrine (EE) cells. Here we identified Ttk69, a BTB domain-containing transcriptional repressor, as a master repressor of EE cell specification in the ISC lineages. Depletion of ttk69 in progenitor cells induced ISC proliferation and caused all committed progenitor cells to adopt EE fate, leading to the production of supernumerary EE cells in the intestinal epithelium. Conversely, forced expression of Ttk69 in progenitor cells was sufficient to prevent EE cell specification. The expression of Ttk69 was not regulated by Notch signaling, and forced activation of Notch, which is sufficient to induce EC specification of normal progenitor cells, failed to prevent EE cell specification of Ttk69-depleted progenitors. Loss of Ttk69 led to derepression of the acheate-scute complex (AS-C) genes scute and asense, which then induced prospero expression to promote EE cell specification. These studies suggest that Ttk69 functions in parallel with Notch signaling and acts as a master repressor of EE cell specification in Drosophila ISC lineages primarily by suppressing AS-C genes.

  18. Transcriptional establishment of cell-type identity: dynamics and causal mechanisms of T-cell lineage commitment

    PubMed Central

    Rothenberg, Ellen V.; Champhekar, Ameya; Damle, Sagar; Del Real, Marissa Morales; Kueh, Hao Yuan; Li, Long; Yui, Mary A.

    2014-01-01

    Precursor cell entry into the T-cell developmental pathway can be divided into two phases by the closure of T-lineage commitment. As cells decide against the last alternative options to the T-cell fate, they turn on the transcription factor Bcl11b and silence expression of a group of multipotent progenitor regulatory factors that include hematopoietic transcription factor PU.1. Functional perturbation tests show that Bcl11b is needed for commitment while PU.1 actively participates in keeping open access to alternative fates, until it is silenced; however, PU.1 as well as Bcl11b contributes positively to T-cell development. Our recent work reviewed here sheds light on the transcriptional regulatory network that determines the timing and irreversibility of Bcl11b activation, the ways that Notch signaling from the thymic microenvironment restricts the action of PU.1 to prevent it from diverting cells to non-T fates, and the target genes that PU.1 still regulates under the influence of Notch signaling to contribute to T-cell generation. We argue that T-cell development depends on the sequential operation of two interlaced, but mutually antagonistic, gene regulatory networks, one initially supporting expansion before commitment and the other imposing a “terminal” differentiation process on committed cells. PMID:24135716

  19. Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer

    PubMed Central

    2012-01-01

    Background Cancers arise through an evolutionary process in which cell populations are subjected to selection; however, to date, the process of bladder cancer, which is one of the most common cancers in the world, remains unknown at a single-cell level. Results We carried out single-cell exome sequencing of 66 individual tumor cells from a muscle-invasive bladder transitional cell carcinoma (TCC). Analyses of the somatic mutant allele frequency spectrum and clonal structure revealed that the tumor cells were derived from a single ancestral cell, but that subsequent evolution occurred, leading to two distinct tumor cell subpopulations. By analyzing recurrently mutant genes in an additional cohort of 99 TCC tumors, we identified genes that might play roles in the maintenance of the ancestral clone and in the muscle-invasive capability of subclones of this bladder cancer, respectively. Conclusions This work provides a new approach of investigating the genetic details of bladder tumoral changes at the single-cell level and a new method for assessing bladder cancer evolution at a cell-population level. PMID:23587365

  20. Genetic mapping of Foxb1-cell lineage shows migration from caudal diencephalon to telencephalon and lateral hypothalamus

    PubMed Central

    Zhao, Tianyu; Szabó, Nora; Ma, Jun; Luo, Lingfei; Zhou, Xunlei; Alvarez-Bolado, Gonzalo

    2008-01-01

    The hypothalamus is a brain region with vital functions, and alterations in its development can cause human disease. However, we still do not have a complete description of how this complex structure is put together during embryonic and early postnatal stages. Radially oriented, outside-in migration of cells is prevalent in the developing hypothalamus. In spite of this, cell contingents from outside the hypothalamus as well as tangential hypothalamic migrations also have an important role. Here we study migrations in the hypothalamic primordium by genetically labeling the Foxb1 diencephalic lineage. Foxb1 is a transcription factor gene expressed in the neuroepithelium of the developing neural tube with a rostral expression boundary between caudal and rostral diencephalon, and therefore appropriate for marking migrations from caudal levels into the hypothalamus. We have found a large, longitudinally oriented migration stream apparently originating in the thalamic region and following an axonal bundle to end in the anterior portion of the lateral hypothalamic area. Additionally, we have mapped a specific expansion of the neuroepithelium into the rostral diencephalon. The expanded neuroepithelium generates abundant neurons for the medial hypothalamus at the tuberal level. Finally, we have uncovered novel diencephalon-to-telencephalon migrations into septum, piriform cortex and amygdala. PMID:19046377

  1. Dicer Regulates the Balance of Short-Lived Effector and Long-Lived Memory CD8 T Cell Lineages

    PubMed Central

    Baumann, Florian M.; Yuzefpolskiy, Yevgeniy; Sarkar, Surojit; Kalia, Vandana

    2016-01-01

    MicroRNAs constitute a major post-transcriptional mechanism for controlling protein expression, and are emerging as key regulators during T cell development and function. Recent reports of augmented CD8 T cell activation and effector differentiation, and aberrant migratory properties upon ablation of Dicer/miRNAs in naïve cells have established a regulatory role of miRNAs during priming. Whether miRNAs continue to exert similar functions or are dispensable during later stages of CD8 T cell expansion and memory differentiation remains unclear. Here, we report a critical role of Dicer/miRNAs in regulating the balance of long-lived memory and short-lived terminal effector fates during the post-priming stages when CD8 T cells undergo clonal expansion to generate a large cytotoxic T lymphocyte (CTL) pool and subsequently differentiate into a quiescent memory state. Conditional ablation of Dicer/miRNAs in early effector CD8 T cells following optimal activation and expression of granzyme B, using unique dicerfl/fl gzmb-cre mice, led to a strikingly diminished peak effector size relative to wild-type antigen-specific cells in the same infectious milieu. Diminished expansion of Dicer-ablated CD8 T cells was associated with lack of sustained antigen-driven proliferation and reduced accumulation of short-lived effector cells. Additionally, Dicer-ablated CD8 T cells exhibited more pronounced contraction after pathogen clearance and comprised a significantly smaller proportion of the memory pool, despite significantly higher proportions of CD127Hi memory precursors at the effector peak. Combined with previous reports of dynamic changes in miRNA expression as CD8 T cells differentiate from naïve to effector and memory states, these findings support distinct stage-specific roles of miRNA-dependent gene regulation during CD8 T cell differentiation. PMID:27627450

  2. Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages.

    PubMed

    Casero, David; Sandoval, Salemiz; Seet, Christopher S; Scholes, Jessica; Zhu, Yuhua; Ha, Vi Luan; Luong, Annie; Parekh, Chintan; Crooks, Gay M

    2015-12-01

    To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.

  3. Gene regulation: hacking the network on a sugar high.

    PubMed

    Ellis, Tom; Wang, Xiao; Collins, James J

    2008-04-11

    In a recent issue of Molecular Cell, Kaplan et al. (2008) determine the input functions for 19 E. coli sugar-utilization genes by using a two-dimensional high-throughput approach. The resulting input-function map reveals that gene network regulation follows non-Boolean, and often nonmonotonic, logic.

  4. Generation of a Nkx2.2(Cre) knock-in mouse line: Analysis of cell lineages in the central nervous system.

    PubMed

    Jarrar, Wassan; Vauti, Franz; Arnold, Hans-Henning; Holz, Andreas

    2015-01-01

    A Nkx2.2(cre) knock-in mutant mouse line was generated that on the appropriate reporter strain enables cell fate analysis of the Nkx2.2 cell lineage in the central nervous system and elsewhere. We here demonstrate that Nkx2.2 lineage-marked cells reside in the ventral p3 region along the entire length of the CNS and also in pancreas of mouse embryos. Nkx2.2(+) progenitor cells develop into V3 interneurons in spinal cord and generate the branchio-visceral motor nuclei of cranial nerves in hindbrain. Nkx2.2(+) cells in hindbrain also form serotonergic neurons and oligodendrocytes during later developmental stages. In mouse mutants lacking Nkx2.2 protein the neuronal progenitor cells in spinal cord are transformed to the distinct fate of somatic motor neurons including their axonal projections that exit the CNS ventrally and no longer cross the midline at the commissure. These data identify Nkx2.2 as key regulator to determine neuronal subtypes in the p3 domain of the central nervous system.

  5. Life-long in vivo cell-lineage tracing shows that no oogenesis originates from putative germline stem cells in adult mice.

    PubMed

    Zhang, Hua; Liu, Lian; Li, Xin; Busayavalasa, Kiran; Shen, Yan; Hovatta, Outi; Gustafsson, Jan-Åke; Liu, Kui

    2014-12-16

    Whether or not oocyte regeneration occurs in adult life has been the subject of much debate. In this study, we have traced germ-cell lineages over the life spans of three genetically modified mouse models and provide direct evidence that oogenesis does not originate from any germline stem cells (GSCs) in adult mice. By selective ablation of all existing oocytes in a Gdf9-Cre;iDTR mouse model, we have demonstrated that no new germ cells were ever regenerated under pathological conditions. By in vivo tracing of oocytes and follicles in the Sohlh1-CreER(T2);R26R and Foxl2-CreER(T2);mT/mG mouse models, respectively, we have shown that the initial pool of oocytes is the only source of germ cells throughout the life span of the mice and that no adult oogenesis ever occurs under physiological conditions. Our findings clearly show that there are no GSCs that contribute to adult oogenesis in mice and that the initial pool of oocytes formed in early life is the only source of germ cells throughout the entire reproductive life span.

  6. Thymic and Postthymic Regulation of Naïve CD4+ T-Cell Lineage Fates in Humans and Mice Models

    PubMed Central

    Belizário, José E.; Brandão, Wesley; Rossato, Cristiano; Peron, Jean Pierre

    2016-01-01

    Our understanding of how thymocytes differentiate into many subtypes has been increased progressively in its complexity. At early life, the thymus provides a suitable microenvironment with specific combination of stromal cells, growth factors, cytokines, and chemokines to induce the bone marrow lymphoid progenitor T-cell precursors into single-positive CD4+ and CD8+ T effectors and CD4+CD25+ T-regulatory cells (Tregs). At postthymic compartments, the CD4+ T-cells acquire distinct phenotypes which include the classical T-helper 1 (Th1), T-helper 2 (Th2), T-helper 9 (Th9), T-helper 17 (Th17), follicular helper T-cell (Tfh), and induced T-regulatory cells (iTregs), such as the regulatory type 1 cells (Tr1) and transforming growth factor-β- (TGF-β-) producing CD4+ T-cells (Th3). Tregs represent only a small fraction, 5–10% in mice and 1-2% in humans, of the overall CD4+ T-cells in lymphoid tissues but are essential for immunoregulatory circuits mediating the inhibition and expansion of all lineages of T-cells. In this paper, we first provide an overview of the major cell-intrinsic developmental programs that regulate T-cell lineage fates in thymus and periphery. Next, we introduce the SV40 immortomouse as a relevant mice model for implementation of new approaches to investigate thymus organogenesis, CD4 and CD8 development, and thymus cells tumorogenesis. PMID:27313405

  7. Nonsyntenic Genes Drive Highly Dynamic Complementation of Gene Expression in Maize Hybrids[W

    PubMed Central

    Larson, Nick B.; Marcon, Caroline; Schnable, James C.; Yeh, Cheng-Ting; Lanz, Christa; Nettleton, Dan; Piepho, Hans-Peter; Schnable, Patrick S.

    2014-01-01

    Maize (Zea mays) displays an exceptional level of structural genomic diversity, which is likely unique among higher eukaryotes. In this study, we surveyed how the genetic divergence of two maize inbred lines affects the transcriptomic landscape in four different primary root tissues of their F1-hybrid progeny. An extreme instance of complementation was frequently observed: genes that were expressed in only one parent but in both reciprocal hybrids. This single-parent expression (SPE) pattern was detected for 2341 genes with up to 1287 SPE patterns per tissue. As a consequence, the number of active genes in hybrids exceeded that of their parents in each tissue by >400. SPE patterns are highly dynamic, as illustrated by their excessive degree of tissue specificity (80%). The biological significance of this type of complementation is underpinned by the observation that a disproportionally high number of SPE genes (75 to 82%) is nonsyntenic, as opposed to all expressed genes (36%). These genes likely evolved after the last whole-genome duplication and are therefore younger than the syntenic genes. In summary, SPE genes shape the remarkable gene expression plasticity between root tissues and complementation in maize hybrids, resulting in a tissue-specific increase of active genes in F1-hybrids compared with their inbred parents. PMID:25315323

  8. Gene activation and cell fate control in plants: a chromatin perspective.

    PubMed

    Engelhorn, Julia; Blanvillain, Robert; Carles, Cristel C

    2014-08-01

    In plants, environment-adaptable organogenesis extends throughout the lifespan, and iterative development requires repetitive rounds of activation and repression of several sets of genes. Eukaryotic genome compaction into chromatin forms a physical barrier for transcription; therefore, induction of gene expression requires alteration in chromatin structure. One of the present great challenges in molecular and developmental biology is to understand how chromatin is brought from a repressive to permissive state on specific loci and in a very specific cluster of cells, as well as how this state is further maintained and propagated through time and cell division in a cell lineage. In this review, we report recent discoveries implementing our knowledge on chromatin dynamics that modulate developmental gene expression. We also discuss how new data sets highlight plant specificities, likely reflecting requirement for a highly dynamic chromatin.

  9. Collagen scaffold microenvironments modulate cell lineage commitment for differentiation of bone marrow cells into regulatory dendritic cells

    PubMed Central

    Fang, Yongxiang; Wang, Bin; Zhao, Yannan; Xiao, Zhifeng; Li, Jing; Cui, Yi; Han, Sufang; Wei, Jianshu; Chen, Bing; Han, Jin; Meng, Qingyuan; Hou, Xianglin; Luo, Jianxun; Dai, Jianwu; Jing, Zhizhong

    2017-01-01

    The microenvironment plays a pivotal role for cell survival and functional regulation, and directs the cell fate determination. The biological functions of DCs have been extensively investigated to date. However, the influences of the microenvironment on the differentiation of bone marrow cells (BMCs) into dendritic cells (DCs) are not well defined. Here, we established a 3D collagen scaffold microenvironment to investigate whether such 3D collagen scaffolds could provide a favourable niche for BMCs to differentiate into specialised DCs. We found that BMCs embedded in the 3D collagen scaffold differentiated into a distinct subset of DC, exhibiting high expression of CD11b and low expression of CD11c, co-stimulator (CD40, CD80, CD83, and CD86) and MHC-II molecules compared to those grown in 2D culture. DCs cultured in the 3D collagen scaffold possessed weak antigen uptake ability and inhibited T-cell proliferation in vitro; in addition, they exhibited potent immunoregulatory function to alleviate allo-delay type hypersensitivity when transferred in vivo. Thus, DCs differentiated in the 3D collagen scaffold were defined as regulatory DCs, indicating that collagen scaffold microenvironments probably play an important role in modulating the lineage commitment of DCs and therefore might be applied as a promising tool for generation of specialised DCs. PMID:28169322

  10. Piwi is required in multiple cell types to control germline stem cell lineage development in the Drosophila ovary.

    PubMed

    Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

    2014-01-01

    The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary.

  11. Body composition changes and inhibition of fat development in vivo implicates androgen in regulation of stem cell lineage allocation

    PubMed Central

    Semirale, Anthony A.; Zhang, Xiaowei; Wiren, Kristine M.

    2011-01-01

    Androgens regulate body composition in youth and declining testosterone that occurs with aging is associated with muscle wasting, increased fat mass and osteopenia. Transgenic mice with targeted androgen receptor (AR) overexpression in mesenchymal stem cells (MSC) were generated to explore the role of androgen signaling in the regulation of body composition. Transgenic males, but not females, were shorter and have reduced body weight and visceral fat accumulation. Dual energy x-ray absorptiometry (DXA) revealed significant reductions in fat mass with a reciprocal increase in lean mass, yet no difference in food consumption or locomotor activity was observed. Adipose tissue weight was normal in brown fat but reduced in both gonadal and perirenal depots, and reduced hyperplasia was observed with smaller adipocyte size in visceral and subcutaneous white adipose tissue. Although serum leptin, adiponectin, triglyceride, and insulin levels were no different between the genotypes, intraperitoneal glucose tolerance testing showed improved glucose clearance in transgenic males. High levels of the AR transgene are detected in MSCs but not in mature fat tissue. Reduced fibroblast colony forming units indicate fewer progenitor cells resident in the marrow in vivo. Precocious expression of GLUT4, PPARγ and C/EBPα was observed in proliferating precursor cultures from transgenic mice compared to controls. In more mature cultures, there was little difference between the genotypes. We propose a mechanism where enhanced androgen sensitivity can alter lineage commitment in vivo to reduce progenitor number and fat development, while increasing the expression of key factors to promote smaller adipocytes with improved glucose clearance. PMID:21381083

  12. Regional and Stage-Specific Effects of Prospectively Purified Vascular Cells on the Adult V-SVZ Neural Stem Cell Lineage

    PubMed Central

    Crouch, Elizabeth E.; Liu, Chang; Silva-Vargas, Violeta

    2015-01-01

    Adult neural stem cells reside in specialized niches. In the ventricular-subventricular zone (V-SVZ), quiescent neural stem cells (qNSCs) become activated (aNSCs), and generate transit amplifying cells (TACs), which give rise to neuroblasts that migrate to the olfactory bulb. The vasculature is an important component of the adult neural stem cell niche, but whether vascular cells in neurogenic areas are intrinsically different from those elsewhere in the brain is unknown. Moreover, the contribution of pericytes to the neural stem cell niche has not been defined. Here, we describe a rapid FACS purification strategy to simultaneously isolate primary endothelial cells and pericytes from brain microregions of nontransgenic mice using CD31 and CD13 as surface markers. We compared the effect of purified vascular cells from a neurogenic (V-SVZ) and non-neurogenic brain region (cortex) on the V-SVZ stem cell lineage in vitro. Endothelial and pericyte diffusible signals from both regions differentially promote the proliferation and neuronal differentiation of qNSCs, aNSCs, and TACs. Unexpectedly, diffusible cortical signals had the most potent effects on V-SVZ proliferation and neurogenesis, highlighting the intrinsic capacity of non-neurogenic vasculature to support stem cell behavior. Finally, we identify PlGF-2 as an endothelial-derived mitogen that promotes V-SVZ cell proliferation. This purification strategy provides a platform to define the functional and molecular contribution of vascular cells to stem cell niches and other brain regions under different physiological and pathological states. PMID:25788671

  13. A Comparative Analysis of the In Vitro Effects of Pulsed Electromagnetic Field Treatment on Osteogenic Differentiation of Two Different Mesenchymal Cell Lineages

    PubMed Central

    Ceccarelli, Gabriele; Bloise, Nora; Mantelli, Melissa; Gastaldi, Giulia; Fassina, Lorenzo; De Angelis, Maria Gabriella Cusella; Ferrari, Davide; Imbriani, Marcello

    2013-01-01

    Abstract Human mesenchymal stem cells (MSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications. Exposure of MSCs to physical stimuli favors early and rapid activation of the tissue repair process. In this study we investigated the in vitro effects of pulsed electromagnetic field (PEMF) treatment on the proliferation and osteogenic differentiation of bone marrow MSCs (BM-MSCs) and adipose-tissue MSCs (ASCs), to assess if both types of MSCs could be indifferently used in combination with PEMF exposure for bone tissue healing. We compared the cell viability, cell matrix distribution, and calcified matrix production in unstimulated and PEMF-stimulated (magnetic field: 2 mT, amplitude: 5 mV) mesenchymal cell lineages. After PEMF exposure, in comparison with ASCs, BM-MSCs showed an increase in cell proliferation (p<0.05) and an enhanced deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p<0.05). Calcium deposition was 1.5-fold greater in BM-MSC–derived osteoblasts (p<0.05). The immunofluorescence related to the deposition of bone matrix proteins and calcium showed their colocalization to the cell-rich areas for both types of MSC-derived osteoblast. Alkaline phosphatase activity increased nearly 2-fold (p<0.001) and its protein content was 1.2-fold higher in osteoblasts derived from BM-MSCs. The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed up-regulated transcription specific for bone sialoprotein, osteopontin, osteonectin, and Runx2, but at a higher level for cells differentiated from BM-MSCs. All together these results suggest that PEMF promotion of bone extracellular matrix deposition is more efficient in osteoblasts differentiated from BM-MSCs. PMID:23914335

  14. OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica

    PubMed Central

    Cota, Ignacio; Bunk, Boyke; Spröer, Cathrin; Overmann, Jörg; König, Christoph; Casadesús, Josep

    2016-01-01

    Phase variation of the Salmonella enterica opvAB operon generates a bacterial lineage with standard lipopolysaccharide structure (OpvABOFF) and a lineage with shorter O-antigen chains (OpvABON). Regulation of OpvAB lineage formation is transcriptional, and is controlled by the LysR-type factor OxyR and by DNA adenine methylation. The opvAB regulatory region contains four sites for OxyR binding (OBSA-D), and four methylatable GATC motifs (GATC1–4). OpvABOFF and OpvABON cell lineages display opposite DNA methylation patterns in the opvAB regulatory region: (i) in the OpvABOFF state, GATC1 and GATC3 are non-methylated, whereas GATC2 and GATC4 are methylated; (ii) in the OpvABON state, GATC2 and GATC4 are non-methylated, whereas GATC1 and GATC3 are methylated. We provide evidence that such DNA methylation patterns are generated by OxyR binding. The higher stability of the OpvABOFF lineage may be caused by binding of OxyR to sites that are identical to the consensus (OBSA and OBSc), while the sites bound by OxyR in OpvABON cells (OBSB and OBSD) are not. In support of this view, amelioration of either OBSB or OBSD locks the system in the ON state. We also show that the GATC-binding protein SeqA and the nucleoid protein HU are ancillary factors in opvAB control. PMID:26687718

  15. [Distribution of abnormal cell clone with deletion of chromosome 20q in marrow cell lineages and apoptosis cells in myelodysplastic syndrome].

    PubMed

    Qin, Ling; Wang, Chun; Qin, You-Wen; Xie, Kuang-Cheng; Yan, Shi-Ke; Gao, Yan-Rong; Wang, Xiao-Rui; Zhao, Chu-Xian

    2008-06-01

    This study was aimed to investigate the distribution of abnormal clone in marrow cell lineages and apoptosis cells in myelodysplastic syndrome (MDS) with deletion of chromosome 20q. Monoclonal antibodies recognizing myeloid precursors (CD15), erythroid precursors (GPA), T cells (CD3(+)CD56(-)CD16(-)), B cells (CD19), NK cells (CD3(-)CD56(+)CD16(+)) were used to sort bone marrow cells in a MDS patient with del (20q) by fluorescence activated cell sorting (FACS). Annexin V-FITC and PI were used to sort bone marrow Annexin V(+)PI(-) and Annexin V(-)PI(-) cells by FACS. The sorted positive cells were detected by interphase dual-color fluorescence in situ hybridization (D-FISH) using a LSI D20S108 probe (Spectrum Orange) and a Telvysion TM 20p probe (Spectrum Green). FACS and FISH analysis were also performed on the samples from 4 cases with normal karyotype. The results showed that the proportions of MDS clone in the myeloid and erythroid precursors were 70.50% and 93.33% respectively, in the RAEB-1 patient with del (20q) and were obviously higher than that in control group (5.39% and 6.17%). The proportions of abnormal clone in T, B and NK cells were 3.23%, 4.32% and 5.77% respectively and were less than that in control group (5.76%, 4.85%, 6.36%). The percentage of apoptotic cells in the bone marrow nucleated cells was 16.09%. The proportions of MDS clone in Annexin V(+)PI(-) and Annexin V(-)PI(-) cells were 32.48% and 70.11%, respectively. It is concluded that most myeloid and erythroid precursors are originated from the abnormal clone in MDS with del (20q). A little part of apoptotic cells are derived from the abnormal clone.

  16. Protruding disordered loop of gC1qR is specifically exposed and related to antiapoptotic property in germ cell lineage.

    PubMed

    Kitazawa, Sohei; Takenaka, Atsushi; Kondo, Takeshi; Mizoguchi, Akira; Kitazawa, Riko

    2006-12-01

    We established a monoclonal antibody (MAb), 5G9, with the use of a fixed seminoma tissue from an archival paraffin-embedded specimen, as an immunogen. Without antigen retrieval, positive 5G9-immunohistochemical staining was confined mostly to primordial germ cells, spermatogonia and various germ cell tumors. 5G9 recognized a mitochondrial 32-kD protein with an isoelectric point of pH 4.2, identified as a multifunctional ubiquitous protein, receptor for globular head of C1q (gC1qR), whose epitope was mapped in a disordered loop connecting the beta3 and the beta4 strands. Reflecting the ubiquitous distribution of gC1qR, with antigen retrieval, 5G9 was found reactive to a wide range of normal and tumor tissues. Since several co-precipitated and phosphorylated bands were observed in various human cell lines but not in germ cell tumor cell lines by in vitro phosphorylation assay, we speculate that the epitope of gC1qR is specifically unmasked in the germ cell lineage. By reducing gC1qR by siRNA, a significant increase was observed in the number of apoptotic cells in ITO-II and TCam-2 cell lines, but to a lesser extent in the Colo201 colon cancer cell line, showing an antiapoptotic property of gC1qR in the germ cells. Since protein-protein interaction is partially preserved by fixation, archival paraffin-embedded specimens can be a valuable source of immunogens for generating monoclonal antibodies (MAbs) that recognize tissue-specific protein conformation.

  17. Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

    PubMed

    McCulloch, Daniel R; Le Goff, Carine; Bhatt, Sumantha; Dixon, Laura J; Sandy, John D; Apte, Suneel S

    2009-06-01

    The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. beta-Galactosidase (beta-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E 9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5-15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

  18. Identification of co-expressed gene signatures in mouse B1, marginal zone and B2 B-cell populations

    PubMed Central

    Mabbott, Neil A; Gray, David

    2014-01-01

    In mice, three major B-cell subsets have been identified with distinct functionalities: B1 B cells, marginal zone B cells and follicular B2 B cells. Here, we used the growing body of publicly available transcriptomics data to create an expression atlas of 84 gene expression microarray data sets of distinct mouse B-cell subsets. These data were subjected to network-based cluster analysis using BioLayout Express3D. Using this analysis tool, genes with related functions clustered together in discrete regions of the network graph and enabled the identification of transcriptional networks that underpinned the functional activity of distinct cell populations. Some gene clusters were expressed highly by most of the cell populations included in this analysis (such as those with activity related to house-keeping functions). Others contained genes with expression patterns specific to distinct B-cell subsets. While these clusters contained many genes typically associated with the activity of the cells they were specifically expressed in, many novel B-cell-subset-specific candidate genes were identified. A large number of uncharacterized genes were also represented in these B-cell lineage-specific clusters. Further analysis of the activities of these uncharacterized candidate genes will lead to the identification of novel B-cell lineage-specific transcription factors and regulators of B-cell function. We also analysed 36 microarray data sets from distinct human B-cell populations. These data showed that mouse and human germinal centre B cells shared similar transcriptional features, whereas mouse B1 B cells were distinct from proposed human B1 B cells. PMID:24032749

  19. High-throughput, high-sensitivity analysis of gene expression in Arabidopsis.

    PubMed

    Kris, Richard Martin; Felder, Stephen; Deyholos, Michael; Lambert, Georgina M; Hinton, James; Botros, Ihab; Martel, Ralph; Seligmann, Bruce; Galbraith, David W

    2007-07-01

    High-throughput gene expression analysis of genes expressed during salt stress was performed using a novel multiplexed quantitative nuclease protection assay that involves customized DNA microarrays printed within the individual wells of 96-well plates. The levels of expression of the transcripts from 16 different genes were quantified within crude homogenates prepared from Arabidopsis (Arabidopsis thaliana) plants also grown in a 96-well plate format. Examples are provided of the high degree of reproducibility of quantitative dose-response data and of the sensitivity of detection of changes in gene expression within limiting amounts of tissue. The lack of requirement for RNA purification renders the assay particularly suited for high-throughput gene expression analysis and for the discovery of novel chemical compounds that specifically modulate the expression of endogenous target genes.

  20. Gene expression profiling in peanut using high density oligonucleotide microarrays

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. PMID:19523230

  1. High frequency of horizontal gene transfer in the oceans.

    PubMed

    McDaniel, Lauren D; Young, Elizabeth; Delaney, Jennifer; Ruhnau, Fabian; Ritchie, Kim B; Paul, John H

    2010-10-01

    Oceanic bacteria perform many environmental functions, including biogeochemical cycling of many elements, metabolizing of greenhouse gases, functioning in oceanic food webs (microbial loop), and producing valuable natural products and viruses. We demonstrate that the widespread capability of marine bacteria to participate in horizontal gene transfer (HGT) in coastal and oceanic environments may be the result of gene transfer agents (GTAs), viral-like particles produced by α-Proteobacteria. We documented GTA-mediated gene transfer frequencies a thousand to a hundred million times higher than prior estimates of HGT in the oceans, with as high as 47% of the culturable natural microbial community confirmed as gene recipients. These findings suggest a plausible mechanism by which marine bacteria acquire novel traits, thus ensuring resilience in the face of environmental change.

  2. Muscle plasticity and high throughput gene expression studies.

    PubMed

    Reggiani, Carlo; Kronnie, Geertruuy Te

    2004-01-01

    Changes in gene expression are known to contribute to muscle plasticity. Until recently most studies have described differences of one or few genes at a time, in the last few years, however, the development of new technology of high throughput mRNA expression analysis has allowed the study of a large part if not all transcripts in the same experiment. Knowledge on any muscle adaptive response has already gained from the application of this novel approach, but the most important new findings have come from studies on muscle atrophy. A new and unexpected groups of genes, which increase their expression during atrophy and are, therefore, designated as atrogins, have been discovered. In spite of the impressive power of the new technology many problems are still to be resolved to optimize the experimental design and to extract all information which are provided by the outcome of the global mRNA assessment.

  3. Identifying the genes of unconventional high temperature superconductors.

    PubMed

    Hu, Jiangping

    We elucidate a recently emergent framework in unifying the two families of high temperature (high [Formula: see text]) superconductors, cuprates and iron-based superconductors. The unification suggests that the latter is simply the counterpart of the former to realize robust extended s-wave pairing symmetries in a square lattice. The unification identifies that the key ingredients (gene) of high [Formula: see text] superconductors is a quasi two dimensional electronic environment in which the d-orbitals of cations that participate in strong in-plane couplings to the p-orbitals of anions are isolated near Fermi energy. With this gene, the superexchange magnetic interactions mediated by anions could maximize their contributions to superconductivity. Creating the gene requires special arrangements between local electronic structures and crystal lattice structures. The speciality explains why high [Formula: see text] superconductors are so rare. An explicit prediction is made to realize high [Formula: see text] superconductivity in Co/Ni-based materials with a quasi two dimensional hexagonal lattice structure formed by trigonal bipyramidal complexes.

  4. HOXB13 and other high penetrant genes for prostate cancer

    PubMed Central

    Pilie, Patrick G; Giri, Veda N; Cooney, Kathleen A

    2016-01-01

    Cancer initiation and progression is the result of an accumulation of mutations in key tumor suppressor genes, mismatch repair genes, or oncogenes, which impact cancer cell growth, death, and differentiation. Mutations occurring in cancer tissue are termed somatic; whereas, heritable mutations that may be passed onto subsequent generations occur in germline DNA. It is these germline mutations that can lead to cancer family syndromes whereby family members carrying a deleterious germline mutation have an increased susceptibility to certain cancer phenotypes. Common features of hereditary cancer syndromes include early age-of-onset, multiple affected generations, rare tumor types, and/or multiple primary malignancies. Approximately, 5%–10% of all common cancers, including prostate cancer, have a hereditary component and are attributable to highly penetrant germline mutations.1 Across all cancer types, known cancer susceptibility syndromes number >100; however, it is important to note that mutations in high-penetrance genes explain only a fraction of heritable cancers.2 Well-known examples of hereditary cancer syndromes include Lynch (HNPCC), Cowden (PHTS), Li-Fraumeni, and Hereditary Breast and Ovarian Cancer (HBOC) syndromes, which are attributable to mutations in mismatch repair genes, PTEN, p53, and BRCA1/2, respectively.3 PMID:27034017

  5. High-throughput gene mapping in Caenorhabditis elegans.

    PubMed

    Swan, Kathryn A; Curtis, Damian E; McKusick, Kathleen B; Voinov, Alexander V; Mapa, Felipa A; Cancilla, Michael R

    2002-07-01

    Positional cloning of mutations in model genetic systems is a powerful method for the identification of targets of medical and agricultural importance. To facilitate the high-throughput mapping of mutations in Caenorhabditis elegans, we have identified a further 9602 putative new single nucleotide polymorphisms (SNPs) between two C. elegans strains, Bristol N2 and the Hawaiian mapping strain CB4856, by sequencing inserts from a CB4856 genomic DNA library and using an informatics pipeline to compare sequences with the canonical N2 genomic sequence. When combined with data from other laboratories, our marker set of 17,189 SNPs provides even coverage of the complete worm genome. To date, we have confirmed >1099 evenly spaced SNPs (one every 91 +/- 56 kb) across the six chromosomes and validated the utility of our SNP marker set and new fluorescence polarization-based genotyping methods for systematic and high-throughput identification of genes in C. elegans by cloning several proprietary genes. We illustrate our approach by recombination mapping and confirmation of the mutation in the cloned gene, dpy-18.

  6. Robust lineage reconstruction from high-dimensional single-cell data

    PubMed Central

    Giecold, Gregory; Marco, Eugenio; Garcia, Sara P.; Trippa, Lorenzo; Yuan, Guo-Cheng

    2016-01-01

    Single-cell gene expression data provide invaluable resources for systematic characterization of cellular hierarchy in multi-cellular organisms. However, cell lineage reconstruction is still often associated with significant uncertainty due to technological constraints. Such uncertainties have not been taken into account in current methods. We present ECLAIR (Ensemble Cell Lineage Analysis with Improved Robustness), a novel computational method for the statistical inference of cell lineage relationships from single-cell gene expression data. ECLAIR uses an ensemble approach to improve the robustness of lineage predictions, and provides a quantitative estimate of the uncertainty of lineage branchings. We show that the application of ECLAIR to published datasets successfully reconstructs known lineage relationships and significantly improves the robustness of predictions. ECLAIR is a powerful bioinformatics tool for single-cell data analysis. It can be used for robust lineage reconstruction with quantitative estimate of prediction accuracy. PMID:27207878

  7. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection.

    PubMed

    Nguyen Thi, Le Thuy; Sarmiento, Maria Elena; Calero, Romel; Camacho, Frank; Reyes, Fatima; Hossain, Md Murad; Gonzalez, Gustavo Sierra; Norazmi, Mohd Nor; Acosta, Armando

    2014-09-01

    The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.

  8. A Highly Efficient Gene-Targeting System for Aspergillus parasiticus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gene targeting via homologous recombination is often used to elucidate gene function. For filamentous fungi, the majority of transforming DNA integrates ectopically. Deletion of Aspergillus parasiticus ku70, a gene of the non-homologous end-joining pathway, drastically increased the gene targeting...

  9. Transgenic zebrafish reporter lines reveal conserved Toll-like receptor signaling potential in embryonic myeloid leukocytes and adult immune cell lineages.

    PubMed

    Hall, Chris; Flores, Maria Vega; Chien, Annie; Davidson, Alan; Crosier, Kathryn; Crosier, Phil

    2009-05-01

    The immune response of a host to an invading pathogen is dependent on the capacity of its immune cell compartment to recognize highly conserved pathogen components using an ancient class of pattern recognition receptors known as Toll-like receptors (TLRs). Initiation of TLR-mediated signaling results in the induction of proinflammatory cytokines that help govern the scale and duration of any ensuing response. Specificity for TLR signaling is, in part, a result of the differential recruitment of intracellular adaptor molecules. Of these, MyD88 is required for the majority of TLR signaling. Zebrafish have been shown to possess TLRs and adaptor molecules throughout early development, including MyD88, strongly suggesting conservation of this ancient defense mechanism. However, information about which embryonic cells/tissues possess this conserved signaling potential is lacking. To help define which embryonic cells, in particular, those of the innate immune system, have the potential for MyD88-dependent, TLR-mediated signaling, we generated transgenic reporter lines using regulatory elements of the myd88 gene to drive the fluorescent reporters enhanced GFP and Discosoma red fluorescent protein 2 within live zebrafish. These lines possess fluorescently marked cells/tissues consistent with endogenous myd88 expression, including a subset of myeloid leukocytes. These innate immune cells were confirmed to express other TLR adaptors including Mal, trif, and Sarm. Live wound-healing and infection assays validated the potential of these myd88-expressing leukocytes to participate in immune responses. These lines will provide a valuable resource for further resolving the contribution of MyD88 to early vertebrate immunity.

  10. Expression and comparative genomics of two serum response factor genes in zebrafish.

    PubMed

    Davis, Jody L; Long, Xiaochun; Georger, Mary A; Scott, Ian C; Rich, Adam; Miano, Joseph M

    2008-01-01

    Serum response factor (SRF) is a single copy, highly conserved transcription factor that governs the expression of hundreds of genes involved with actin cytoskeletal organization, cellular growth and signaling, neuronal circuitry and muscle differentiation. Zebrafish have emerged as a facile and inexpensive vertebrate model to delineate gene expression, regulation, and function, and yet the study of SRF in this animal has been virtually unexplored. Here, we report the existence of two srf genes in zebrafish, with partially overlapping patterns of expression in 3 and 7 day old developing animals. The mammalian ortholog (srf1) encodes for a 520 amino acid protein expressed in adult vascular and visceral smooth muscle cells, cardiac and skeletal muscle, as well as neuronal cells. The second zebrafish srf gene (srf2), encoding for a presumptive protein of only 314 amino acids, is transcribed at lower levels and appears to be less widely expressed across adult tissues. Both srf genes are induced by the SRF coactivator myocardin and attenuated with a short hairpin RNA to mammalian SRF. Promoter studies with srf1 reveal conserved CArG boxes that are the targets of SRF-myocardin in embryonic zebrafish cells. These results reveal that SRF was duplicated in the zebrafish genome and that its protein expression in all three muscle cell types is highly conserved across vertebrate animals suggesting an ancient code for transcriptional regulation of genes unique to muscle cell lineages.

  11. Deciphering the onychophoran 'segmentation gene cascade': Gene expression reveals limited involvement of pair rule gene orthologs in segmentation, but a highly conserved segment polarity gene network.

    PubMed

    Janssen, Ralf; Budd, Graham E

    2013-10-01

    The hallmark of the arthropods is their segmented body, although origin of segmentation, however, is unresolved. In order to shed light on the origin of segmentation we investigated orthologs of pair rule genes (PRGs) and segment polarity genes (SPGs) in a member of the closest related sister-group to the arthropods, the onychophorans. Our gene expression data analysis suggests that most of the onychophoran PRGs do not play a role in segmentation. One possible exception is the even-skipped (eve) gene that is expressed in the posterior end of the onychophoran where new segments are likely patterned, and is also expressed in segmentation-gene typical transverse stripes in at least a number of newly formed segments. Other onychophoran PRGs such as runt (run), hairy/Hes (h/Hes) and odd-skipped (odd) do not appear to have a function in segmentation at all. Onychophoran PRGs that act low in the segmentation gene cascade in insects, however, are potentially involved in segment-patterning. Most obvious is that from the expression of the pairberry (pby) gene ortholog that is expressed in a typical SPG-pattern. Since this result suggested possible conservation of the SPG-network we further investigated SPGs (and associated factors) such as Notum in the onychophoran. We find that the expression patterns of SPGs in arthropods and the onychophoran are highly conserved, suggesting a conserved SPG-network in these two clades, and indeed also in an annelid. This may suggest that the common ancestor of lophotrochozoans and ecdysozoans was already segmented utilising the same SPG-network, or that the SPG-network was recruited independently in annelids and onychophorans/arthropods.

  12. Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms[OPEN

    PubMed Central

    Li, Zhen; Van de Peer, Yves; De Smet, Riet

    2016-01-01

    Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of “gene duplicability” is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes. PMID:26744215

  13. Gene Duplicability of Core Genes Is Highly Consistent across All Angiosperms.

    PubMed

    Li, Zhen; Defoort, Jonas; Tasdighian, Setareh; Maere, Steven; Van de Peer, Yves; De Smet, Riet

    2016-02-01

    Gene duplication is an important mechanism for adding to genomic novelty. Hence, which genes undergo duplication and are preserved following duplication is an important question. It has been observed that gene duplicability, or the ability of genes to be retained following duplication, is a nonrandom process, with certain genes being more amenable to survive duplication events than others. Primarily, gene essentiality and the type of duplication (small-scale versus large-scale) have been shown in different species to influence the (long-term) survival of novel genes. However, an overarching view of "gene duplicability" is lacking, mainly due to the fact that previous studies usually focused on individual species and did not account for the influence of genomic context and the time of duplication. Here, we present a large-scale study in which we investigated duplicate retention for 9178 gene families shared between 37 flowering plant species, referred to as angiosperm core gene families. For most gene families, we observe a strikingly consistent pattern of gene duplicability across species, with gene families being either primarily single-copy or multicopy in all species. An intermediate class contains gene families that are often retained in duplicate for periods extending to tens of millions of years after whole-genome duplication, but ultimately appear to be largely restored to singleton status, suggesting that these genes may be dosage balance sensitive. The distinction between single-copy and multicopy gene families is reflected in their functional annotation, with single-copy genes being mainly involved in the maintenance of genome stability and organelle function and multicopy genes in signaling, transport, and metabolism. The intermediate class was overrepresented in regulatory genes, further suggesting that these represent putative dosage-balance-sensitive genes.

  14. Sources of Variability in a Synthetic Gene Oscillator

    PubMed Central

    Veliz-Cuba, Alan; Hirning, Andrew J.; Atanas, Adam A.; Hussain, Faiza; Vancia, Flavia; Josić, Krešimir; Bennett, Matthew R.

    2015-01-01

    Synthetic gene oscillators are small, engineered genetic circuits that produce periodic variations in target protein expression. Like other gene circuits, synthetic gene oscillators are noisy and exhibit fluctuations in amplitude and period. Understanding the origins of such variability is key to building predictive models that can guide the rational design of synthetic circuits. Here, we developed a method for determining the impact of different sources of noise in genetic oscillators by measuring the variability in oscillation amplitude and correlations between sister cells. We first used a combination of microfluidic devices and time-lapse fluorescence microscopy to track oscillations in cell lineages across many generations. We found that oscillation amplitude exhibited high cell-to-cell variability, while sister cells remained strongly correlated for many minutes after cell division. To understand how such variability arises, we constructed a computational model that identified the impact of various noise sources across the lineage of an initial cell. When each source of noise was appropriately tuned the model reproduced the experimentally observed amplitude variability and correlations, and accurately predicted outcomes under novel experimental conditions. Our combination of computational modeling and time-lapse data analysis provides a general way to examine the sources of variability in dynamic gene circuits. PMID:26693906

  15. High intensity ultrasound transducer used in gene transfection

    NASA Astrophysics Data System (ADS)

    Morrison, Kyle P.; Keilman, George W.; Noble, Misty L.; Brayman, Andrew A.; Miao, Carol H.

    2012-11-01

    This paper describes a novel therapeutic high intensity non-focused ultrasound (HIU) transducer designed with uniform pressure distribution to aid in accelerated gene transfer in large animal liver tissues in vivo. The underlying HIU transducer was used to initiate homogeneous cavitation throughout the tissue while delivering up to 2.7 MPa at 1.1 MHz across its radiating surface. The HIU transducer was built into a 6 cm diameter x 1.3 cm tall housing ergonomically designed to avoid collateral damage to the surrounding anatomy during dynamic motion. The ultrasound (US) radiation was applied in a 'paintbrush-like' manner to the surface of the liver. The layers and geometry of the transducer were carefully selected to maximize the active diameter (5.74 cm), maximize the electrical to acoustic conversion efficiency (85%) to achieve 2.7 MPa of peak negative pressure, maximize the frequency operating band at the fundamental resonance to within a power transfer delta of 1 dB, and reduce the pressure delta to within 2 dB across the radiating surface. For maximum peak voltage into the transducer, a high performance piezoceramic was chosen and a DC bias circuit was built integral to the system. An apodized two element annular pattern was made from a single piezoceramic element, resulting in significant pressure uniformity enhancement. In addition to using apodization for pressure uniformity, a proprietary multi-layered structure was used to improve efficiency while sustaining an operating band from 900 kHz to 1.3 MHz. The resultant operating band allowed for dithering techniques using frequency modulation. The underlying HIU transducer for use in large animals enhances gene expression up to 6300-fold.

  16. Regulation of photoreceptor gene transcription via a highly conserved transcriptional regulatory element by vsx gene products

    PubMed Central

    Pan, Yi; Comiskey, Daniel F.; Kelly, Lisa E.; Chandler, Dawn S.

    2016-01-01

    Purpose The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site. Methods X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos. Results We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities. Conclusions vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system. PMID:28003732

  17. Identification of a five B cell-associated gene prognostic and predictive signature for advanced glioma patients harboring immunosuppressive subtype preference

    PubMed Central

    Wang, Haoyuan; Song, Sonya Wei

    2016-01-01

    High grade gliomas contribute to most brain tumor mortality. A few studies reported that the immune system affected glioma development, and immune biomarkers helped understand the disease and formulate effective immunotherapy for patients. Currently, no B lymphocyte-based prognostic signature was reported in gliomas. By applying 78 B cell lineage-specific genes, we conducted a whole-genome gene expression analysis in 782 high grade gliomas derived from three independent datasets by Cox regression analysis and risk score method for signature identification, and then used Gene Ontology, Gene Set Enrichment Analysis, and other statistical methods for functional annotations of the signature-defined differences. We developed a five B cell-associated gene signature for prognosis of high grade glioma patients, which is independent of clinicopathological and genetic features. The signature identified high risk patients suitable for chemoradiotherapy, whereas low risk patients should rule out chemotherapy with radiotherapy only. We found that tumors of TCGA Mesenchymal subtype and wild type IDH1 were preferentially stratified to the high risk group, which bore strong immunosuppressive microenvironment, while tumors of TCGA Proneural subtype and mutated IDH1 were significantly accumulated to the low risk group, which exhibited less immunosuppressive state. The five B cell-associated gene signature predicts poor survival of high risk patients bearing strong immunosuppression and helps select optimal therapeutic regimens for glioma patients. PMID:27738332

  18. Methods for High-Density Admixture Mapping of Disease Genes

    PubMed Central

    Patterson, Nick; Hattangadi, Neil; Lane, Barton; Lohmueller, Kirk E.; Hafler, David A.; Oksenberg, Jorge R.; Hauser, Stephen L.; Smith, Michael W.; O’Brien, Stephen J.; Altshuler, David; Daly, Mark J.; Reich, David

    2004-01-01

    Admixture mapping (also known as “mapping by admixture linkage disequilibrium,” or MALD) has been proposed as an efficient approach to localizing disease-causing variants that differ in frequency (because of either drift or selection) between two historically separated populations. Near a disease gene, patient populations descended from the recent mixing of two or more ethnic groups should have an increased probability of inheriting the alleles derived from the ethnic group that carries more disease-susceptibility alleles. The central attraction of admixture mapping is that, since gene flow has occurred recently in modern populations (e.g., in African and Hispanic Americans in the past 20 generations), it is expected that admixture-generated linkage disequilibrium should extend for many centimorgans. High-resolution marker sets are now becoming available to test this approach, but progress will require (a) computational methods to infer ancestral origin at each point in the genome and (b) empirical characterization of the general properties of linkage disequilibrium due to admixture. Here we describe statistical methods to estimate the ancestral origin of a locus on the basis of the composite genotypes of linked markers, and we show that this approach accurately estimates states of ancestral origin along the genome. We apply this approach to show that strong admixture linkage disequilibrium extends, on average, for 17 cM in African Americans. Finally, we present power calculations under varying models of disease risk, sample size, and proportions of ancestry. Studying ∼2,500 markers in ∼2,500 patients should provide power to detect many regions contributing to common disease. A particularly important result is that the power of an admixture mapping study to detect a locus will be nearly the same for a wide range of mixture scenarios: the mixture proportion should be 10%–90% from both ancestral populations. PMID:15088269

  19. Differential activity of Drosophila Hox genes induces myosin expression and can maintain compartment boundaries.

    PubMed

    Curt, Jesús R; de Navas, Luis F; Sánchez-Herrero, Ernesto

    2013-01-01

    Compartments are units of cell lineage that subdivide territories with different developmental potential. In Drosophila, the wing and haltere discs are subdivided into anterior and posterior (A/P) compartments, which require the activity of Hedgehog, and into dorsal and ventral (D/V) compartments, needing Notch signaling. There is enrichment in actomyosin proteins at the compartment boundaries, suggesting a role for these proteins in their maintenance. Compartments also develop in the mouse hindbrain rhombomeres, which are characterized by the expression of different Hox genes, a group of genes specifying different structures along their main axis of bilaterians. We show here that the Drosophila Hox gene Ultrabithorax can maintain the A/P and D/V compartment boundaries when Hedgehog or Notch signaling is compromised, and that the interaction of cells with and without Ultrabithorax expression induces high levels of non-muscle myosin II. In the absence of Ultrabithorax there is occasional mixing of cells from different segments. We also show a similar role in cell segregation for the Abdominal-B Hox gene. Our results suggest that the juxtaposition of cells with different Hox gene expression leads to their sorting out, probably through the accumulation of non-muscle myosin II at the boundary of the different cell territories. The increase in myosin expression seems to be a general mechanism used by Hox genes or signaling pathways to maintain the segregation of different groups of cells.

  20. Identification of novel genes and networks governing hematopoietic stem cell development.

    PubMed

    Han, Tianxu; Yang, Chao-Shun; Chang, Kung-Yen; Zhang, Danhua; Imam, Farhad B; Rana, Tariq M

    2016-12-01

    Hematopoietic stem cells (HSCs) are capable of giving rise to all blood cell lineages throughout adulthood, and the generation of engraftable HSCs from human pluripotent stem cells is a major goal for regenerative medicine. Here, we describe a functional genome-wide RNAi screen to identify genes required for the differentiation of embryonic stem cell (ESC) into hematopoietic stem/progenitor cells (HSPCs) in vitro We report the discovery of novel genes important for the endothelial-to-hematopoietic transition and subsequently for HSPC specification. High-throughput sequencing and bioinformatic analyses identified twelve groups of genes, including a set of 351 novel genes required for HSPC specification. As in vivo proof of concept, four of these genes, Ap2a1, Mettl22, Lrsam1, and Hal, are selected for validation, confirmed to be essential for HSPC development in zebrafish and for maintenance of human HSCs. Taken together, our results not only identify a number of novel regulatory genes and pathways essential for HSPC development but also serve as valuable resource for directed differentiation of therapy grade HSPCs using human pluripotent stem cells.

  1. Too Few High-Risk Women Tested for Breast Cancer Gene: Survey

    MedlinePlus

    ... html Too Few High-Risk Women Tested for Breast Cancer Gene: Survey Only half got BRCA screen, and ... News) -- Though testing for two genes that raise breast cancer risk has been around for decades, a new ...

  2. High-throughput comparison of gene fitness among related bacteria

    PubMed Central

    2012-01-01

    Background The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella strains and thereby reveal differences not apparent at the gene sequence level. Results A transposon Tn5 derivative was used to construct mutants in Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 isolate (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of transposon insertions or had very few. For three of these nine genes, part of the annotated gene lacked transposon integrations (yejM, ftsN and murB). At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes (STM14_1498, STM14_2872, STM14_3360, and STM14_5442) are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential. Conclusions Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene contributions to fitness among syntenic homologs. Further differences in

  3. High Gene Family Turnover Rates and Gene Space Adaptation in the Compact Genome of the Carnivorous Plant Utricularia gibba.

    PubMed

    Carretero-Paulet, Lorenzo; Librado, Pablo; Chang, Tien-Hao; Ibarra-Laclette, Enrique; Herrera-Estrella, Luis; Rozas, Julio; Albert, Victor A

    2015-05-01

    Utricularia gibba is an aquatic carnivorous plant with highly specialized morphology, featuring fibrous floating networks of branches and leaf-like organs, no recognizable roots, and bladder traps that capture and digest prey. We recently described the compressed genome of U. gibba as sufficient to control the development and reproduction of a complex organism. We hypothesized intense deletion pressure as a mechanism whereby most noncoding DNA was deleted, despite evidence for three independent whole-genome duplications (WGDs). Here, we explore the impact of intense genome fractionation in the evolutionary dynamics of U. gibba's functional gene space. We analyze U. gibba gene family turnover by modeling gene gain/death rates under a maximum-likelihood statistical framework. In accord with our deletion pressure hypothesis, we show that the U. gibba gene death rate is significantly higher than those of four other eudicot species. Interestingly, the gene gain rate is also significantly higher, likely reflecting the occurrence of multiple WGDs and possibly also small-scale genome duplications. Gene ontology enrichment analyses of U. gibba-specific two-gene orthogroups, multigene orthogroups, and singletons highlight functions that may represent adaptations in an aquatic carnivorous plant. We further discuss two homeodomain transcription factor gene families (WOX and HDG/HDZIP-IV) showing conspicuous differential expansions and contractions in U. gibba. Our results 1) reconcile the compactness of the U. gibba genome with its accommodation of a typical number of genes for a plant genome, and 2) highlight the role of high gene family turnover in the evolutionary diversification of U. gibba's functional gene space and adaptations to its unique lifestyle and highly specialized body plan.

  4. Cell types differ in global coordination of splicing and proportion of highly expressed genes

    PubMed Central

    Trakhtenberg, Ephraim F.; Pho, Nam; Holton, Kristina M.; Chittenden, Thomas W.; Goldberg, Jeffrey L.; Dong, Lingsheng

    2016-01-01

    Balance in the transcriptome is regulated by coordinated synthesis and degradation of RNA molecules. Here we investigated whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. Our findings show that cell types differ in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, which represent distinct properties of the transcriptome and may reflect intrinsic differences in global coordination of synthesis, splicing, and degradation of RNA molecules. PMID:27577089

  5. Application of local gene induction by infrared laser-mediated microscope and temperature stimulator to amphibian regeneration study.

    PubMed

    Kawasumi-Kita, Aiko; Hayashi, Toshinori; Kobayashi, Takuya; Nagayama, Chikashi; Hayashi, Shinichi; Kamei, Yasuhiro; Morishita, Yoshihiro; Takeuchi, Takashi; Tamura, Koji; Yokoyama, Hitoshi

    2015-12-01

    Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three-dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the single-cell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR-LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature-controlled metal probe (temperature stimulator). The local gene manipulation systems, the IR-LEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre-LoxP system as well as to analyze gene function in a target region or cells with less off-target effects in the study of amphibian regeneration.

  6. Double-filter identification of vascular-expressed genes using Arabidopsis plants with vascular hypertrophy and hypotrophy.

    PubMed

    Ckurshumova, Wenzislava; Scarpella, Enrico; Goldstein, Rochelle S; Berleth, Thomas

    2011-08-01

    Genes expressed in vascular tissues have been identified by several strategies, usually with a focus on mature vascular cells. In this study, we explored the possibility of using two opposite types of altered tissue compositions in combination with a double-filter selection to identify genes with a high probability of vascular expression in early organ primordia. Specifically, we generated full-transcriptome microarray profiles of plants with (a) genetically strongly reduced and (b) pharmacologically vastly increased vascular tissues and identified a reproducible cohort of 158 transcripts that fulfilled the dual requirement of being underrepresented in (a) and overrepresented in (b). In order to assess the predictive value of our identification scheme for vascular gene expression, we determined the expression patterns of genes in two unbiased subsamples. First, we assessed the expression patterns of all twenty annotated transcription factor genes from the cohort of 158 genes and found that seventeen of the twenty genes were preferentially expressed in leaf vascular cells. Remarkably, fifteen of these seventeen vascular genes were clearly expressed already very early in leaf vein development. Twelve genes with published leaf expression patterns served as a second subsample to monitor the representation of vascular genes in our cohort. Of those twelve genes, eleven were preferentially expressed in leaf vascular tissues. Based on these results we propose that our compendium of 158 genes represents a sample that is highly enriched for genes expressed in vascular tissues and that our approach is particularly suited to detect genes expressed in vascular cell lineages at early stages of their inception.

  7. Transgenic mice demonstrate that epithelial homing of gamma/delta T cells is determined by cell lineages independent of T cell receptor specificity

    PubMed Central

    1990-01-01

    gamma/delta T cells with different TCR repertoires are compartmentalized in different epithelia. This raises the possibility that the TCR-gamma/delta directs homing of T cells to these epithelia. Alternatively, the signals that induce TCR-gamma/delta expression in developing T cells may also induce homing properties in such cells, presumably in the form of cell surface receptors. We have examined this issue by studying the homing of gamma/delta T cells in transgenic mice constructed with specific pairs of rearranged gamma and delta genes. In such mice, most gamma/delta T cells express the transgene-encoded TCR. We find that homing to both skin and gut epithelia is a property of T cells and is not determined by the type of gamma and delta genes used to encode their TCR. We also studied the effect of TCR replacement on the expression of Thy-1 and CD8 proteins on the gamma/delta T cells associated with gut epithelia. Our results show that the expression of the appropriate type of TCR-gamma/delta is not required for the Thy-1 expression by these T cells, suggesting that Thy-1 is not an activation marker. In contrast, CD8 expression by gut gamma/delta T cells seems to depend on the expression of the appropriate type of TCR. PMID:2109035

  8. A Flexible Approach for Highly Multiplexed Candidate Gene Targeted Resequencing

    PubMed Central

    Natsoulis, Georges; Bell, John M.; Xu, Hua; Buenrostro, Jason D.; Ordonez, Heather; Grimes, Susan; Newburger, Daniel; Jensen, Michael; Zahn, Jacob M.; Zhang, Nancy; Ji, Hanlee P.

    2011-01-01

    We have developed an integrated strategy for targeted resequencing and analysis of gene subsets from the human exome for variants. Our capture technology is geared towards resequencing gene subsets substantially larger than can be done efficiently with simplex or multiplex PCR but smaller in scale than exome sequencing. We describe all the steps from the initial capture assay to single nucleotide variant (SNV) discovery. The capture methodology uses in-solution 80-mer oligonucleotides. To provide optimal flexibility in choosing human gene targets, we designed an in silico set of oligonucleotides, the Human OligoExome, that covers the gene exons annotated by the Consensus Coding Sequencing Project (CCDS). This resource is openly available as an Internet accessible database where one can download capture oligonucleotides sequences for any CCDS gene and design custom capture assays. Using this resource, we demonstrated the flexibility of this assay by custom designing capture assays ranging from 10 to over 100 gene targets with total capture sizes from over 100 Kilobases to nearly one Megabase. We established a method to reduce capture variability and incorporated indexing schemes to increase sample throughput. Our approach has multiple applications that include but are not limited to population targeted resequencing studies of specific gene subsets, validation of variants discovered in whole genome sequencing surveys and possible diagnostic analysis of disease gene subsets. We also present a cost analysis demonstrating its cost-effectiveness for large population studies. PMID:21738606

  9. High or low correlation between co-occuring gene clusters and 16S rRNA gene phylogeny.

    PubMed

    Rudi, Knut; Sekelja, Monika

    2013-02-01

    Ribosomal RNA (rRNA) genes are universal for all living organisms. Yet, the correspondence between genome composition and rRNA phylogeny remains poorly known. The aim of this study was to use the information from genome sequence databases to address the correlation between rRNA gene phylogeny and total gene composition in bacteria. This was done by analysing 327 genomes with TIGRFAM functional gene annotations. Our approach consisted of two steps. First, we searched for discriminatory clusters of co-occurring genes. Using a multivariate statistical approach, we identified 11 such clusters which contain genes that were co-occurring only in a subset of genomes and contributed to explain the gene content differences between genome subsets. Second, we mapped the discovered clusters to 16S rRNA-based phylogeny and calculated the correlation between co-occuring genes and phylogeny. Six of the 11 clusters exhibited significant correlation with 16S rRNA gene phylogeny. The most distinct phylogenetic finding was a high correlation between iron-sulfur oxidoreductases in combination with carbon nitrogen ligases and Chlorobium. The other correlations identified covered relatively large phylogroups: Actinobacteria were positively associated with kinases, while Gammaproteobacteria were positively associated with methylases and acyltransferases. The suggested functional differences between higher phylogroups, however, need experimental verification.

  10. Cell type-selective disease-association of genes under high regulatory load.

    PubMed

    Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

    2015-10-15

    We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3' UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner.

  11. Cell type-selective disease-association of genes under high regulatory load

    PubMed Central

    Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

    2015-01-01

    We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3′ UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. PMID:26338775

  12. High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila.

    PubMed

    Zhu, Jun-Yi; Fu, Yulong; Nettleton, Margaret; Richman, Adam; Han, Zhe

    2017-01-20

    Genomic sequencing has implicated large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system is needed to validate gene associations with pathology. We developed a Drosophila-based functional system to screen candidate disease genes identified from Congenital Heart Disease (CHD) patients. 134 genes were tested in the Drosophila heart using RNAi-based gene silencing. Quantitative analyses of multiple cardiac phenotypes demonstrated essential structural, functional, and developmental roles for more than 70 genes, including a subgroup encoding histone H3K4 modifying proteins. We also demonstrated the use of Drosophila to evaluate cardiac phenotypes resulting from specific, patient-derived alleles of candidate disease genes. We describe the first high throughput in vivo validation system to screen candidate disease genes identified from patients. This approach has the potential to facilitate development of precision medicine approaches for CHD and other diseases associated with genetic factors.

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

    PubMed

    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.

  14. New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

    DOE PAGES

    Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; ...

    2008-01-01

    A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline inmore » strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

  15. A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage.

    PubMed Central

    Schulz, Cordula; Kiger, Amy A; Tazuke, Salli I; Yamashita, Yukiko M; Pantalena-Filho, Luiz C; Jones, D Leanne; Wood, Cricket G; Fuller, Margaret T

    2004-01-01

    Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. Here we utilize the Drosophila system to identify genes that cause defects in the male GSC lineage when forcibly expressed. We conducted a gain-of-function screen using a collection of 2050 EP lines and found 55 EP lines that caused defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, our analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGF beta signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGF beta signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation. PMID:15238523

  16. Elimination of Tumorigenic Stem Cells from Differentiated Progeny and Selection of Definitive Endoderm Reveals a Pdx1+ Foregut Endoderm Stem Cell Lineage

    PubMed Central

    Kahan, Brenda; Magliocca, Joseph; Merriam, Fabiola; Treff, Nathan; Budde, Melisa; Nelson, Jeffrey; Browning, Victoria; Ziehr, Benjamin; Odorico, Jon

    2011-01-01

    Embryonic stem cell (ESC) derivatives offer promise for generating clinically useful tissues for transplantation, yet the specter of producing tumors in patients remains a significant concern. We have developed a simple method that eliminates the tumorigenic potential from differentiated ESC cultures of murine and human origin while purifying lineage-restricted, definitive endoderm-committed cells. A three-stage scheme utilizing magnetic bead sorting and specific antibodies to remove undifferentiated ESCs and extraembryonic endoderm cells, followed by positive selection of definitive endoderm cells on the basis of epithelial cell adhesion molecule (EpCAM) expression, was used to isolate a population of EpCAM+SSEA1−SSEA3− cells. Sorted cells do not form teratomas after transplantation into immunodeficient mice, but display gene and protein expression profiles indicative of definitive endoderm cells. Sorted cells could be subsequently expanded in vitro and further differentiated to express key pancreas specification proteins. In vivo transplantation of sorted cells resulted in small, benign tissues that uniformly express PDX1. These studies describe a straightforward method without genetic manipulation that eliminates the risk of teratoma formation from ESC differentiated derivatives. Significantly, enriched populations isolated by this method appear to be lineage-restricted definitive endoderm cells with limited proliferation capacity. PMID:21130058

  17. Role of hypoxia and HIF2α in development of the sympathoadrenal cell lineage and chromaffin cell tumours with distinct catecholamine phenotypic features

    PubMed Central

    Richter, Susan; Qin, Nan; Pacak, Karel; Eisenhofer, Graeme

    2013-01-01

    Hypoxia has wide-ranging impact in normal physiology and disease processes. This stimulus evokes changes in gene expression mediated by transcription factors termed hypoxia-inducible factors (HIFs) that affect numerous processes: angiogenesis, cell survival, cellular metabolism, stem cell self- renewal and multipotency, migration, invasiveness and metastatic progression in tumour cells. Over the past decade increasing numbers of reports have emerged documenting differential roles of HIF1α and HIF2α in these processes. In cells of the sympathoadrenal lineage both HIFs differentially mediate influences of hypoxia on catecholamine synthesis and secretion, but HIF2α signalling has particularly prominent functions in regulating developmental processes of growth and differentiation. This article discusses the role of HIF2α and HIF1α in the context of the development, phenotypic features and functions of chromaffin cells. Moreover, current knowledge about tumour formation in cells of the sympathoadrenal lineage, leading to catecholamine producing pheochromocytomas and paragangliomas, is analysed in the light of the HIF2α signalling network. PMID:24054150

  18. VEGFA-dependent and -independent pathways synergise to drive Scl expression and initiate programming of the blood stem cell lineage in Xenopus

    PubMed Central

    Ciau-Uitz, Aldo; Pinheiro, Philip; Kirmizitas, Arif; Zuo, Jie; Patient, Roger

    2013-01-01

    The first haematopoietic stem cells share a common origin with the dorsal aorta and derive from putative adult haemangioblasts in the dorsal lateral plate (DLP) mesoderm. Here we show that the transcription factor (TF) stem cell leukaemia (Scl/Tal1) is crucial for development of these adult haemangioblasts in Xenopus and establish the regulatory cascade controlling its expression. We show that VEGFA produced in the somites is required to initiate adult haemangioblast programming in the adjacent DLP by establishing endogenous VEGFA signalling. This response depends on expression of the VEGF receptor Flk1, driven by Fli1 and Gata2. Scl activation requires synergy between this VEGFA-controlled pathway and a VEGFA-independent pathway controlled by Fli1, Gata2 and Etv2/Etsrp/ER71, which also drives expression of the Scl partner Lmo2. Thus, the two ETS factors Fli1 and Etv6, which drives the VEGFA expression in both somites and the DLP, sit at the top of the adult haemangioblast gene regulatory network (GRN). Furthermore, Gata2 is initially activated by Fli1 but later maintained by another ETS factor, Etv2. We also establish that Flk1 and Etv2 act independently in the two pathways to Scl activation. Thus, detailed temporal, epistatic measurements of key TFs and VEGFA plus its receptor have enabled us to build a Xenopus adult haemangioblast GRN. PMID:23637333

  19. CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast

    PubMed Central

    Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota; Nødvig, Christina Spuur; Mølgaard, Louise; Halkier, Barbara Ann; Mortensen, Uffe Hasbro

    2017-01-01

    Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside. PMID:28134264

  20. Structural Relationships between Highly Conserved Elements and Genes in Vertebrate Genomes

    PubMed Central

    Sun, Hong; Skogerbø, Geir; Wang, Zhen; Liu, Wei; Li, Yixue

    2008-01-01

    Large numbers of sequence elements have been identified to be highly conserved among vertebrate genomes. These highly conserved elements (HCEs) are often located in or around genes that are involved in transcription regulation and early development. They have been shown to be involved in cis-regulatory activities through both in vivo and additional computational studies. We have investigated the structural relationships between such elements and genes in six vertebrate genomes human, mouse, rat, chicken, zebrafish and tetraodon and detected several thousand cases of conserved HCE-gene associations, and also cases of HCEs with no common target genes. A few examples underscore the potential significance of our findings about several individual genes. We found that the conserved association between HCE/HCEs and gene/genes are not restricted to elements by their absolute distance on the genome. Notably, long-range associations were identified and the molecular functions of the associated genes do not show any particular overrepresentation of the functional categories previously reported. HCEs in close proximity are found to be linked with different set of gene/genes. The results reflect the highly complex correlation between HCEs and their putative target genes. PMID:19008958

  1. Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny

    PubMed Central

    Sato, Sachiko; Rancourt, Ann; Sato, Yukiko; Satoh, Masahiko S.

    2016-01-01

    Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells. PMID:27003384

  2. Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates.

    PubMed

    Spring-Pearson, Senanu M; Stone, Joshua K; Doyle, Adina; Allender, Christopher J; Okinaka, Richard T; Mayo, Mark; Broomall, Stacey M; Hill, Jessica M; Karavis, Mark A; Hubbard, Kyle S; Insalaco, Joseph M; McNew, Lauren A; Rosenzweig, C Nicole; Gibbons, Henry S; Currie, Bart J; Wagner, David M; Keim, Paul; Tuanyok, Apichai

    2015-01-01

    The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is 'open', with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.

  3. Heterogeneity of natural Foxp3+ T cells: a committed regulatory T-cell lineage and an uncommitted minor population retaining plasticity.

    PubMed

    Komatsu, Noriko; Mariotti-Ferrandiz, Maria Encarnita; Wang, Ying; Malissen, Bernard; Waldmann, Herman; Hori, Shohei

    2009-02-10

    Natural regulatory T cells (T(reg)) represent a distinct lineage of T lymphocytes committed to suppressive functions, and expression of the transcription factor Foxp3 is thought to identify this lineage specifically. Here we report that, whereas the majority of natural CD4(+)Foxp3(+) T cells maintain stable Foxp3 expression after adoptive transfer to lymphopenic or lymphoreplete recipients, a minor fraction enriched within the CD25(-) subset actually lose it. Some of those Foxp3(-) T cells adopt effector helper T cell (T(h)) functions, whereas some retain "memory" of previous Foxp3 expression, reacquiring Foxp3 upon activation. This minority "unstable" population exhibits flexible responses to cytokine signals, relying on transforming growth factor-beta to maintain Foxp3 expression and responding to other cytokines by differentiating into effector T(h) in vitro. In contrast, CD4(+)Foxp3(+)CD25(high) T cells are resistant to such conversion to effector T(h) even after many rounds of cell division. These results demonstrate that natural Foxp3(+) T cells are a heterogeneous population consisting of a committed T(reg) lineage and an uncommitted subpopulation with developmental plasticity.

  4. Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli.

    PubMed

    Nielsen, Morten T; Madsen, Karina M; Seppälä, Susanna; Christensen, Ulla; Riisberg, Lone; Harrison, Scott J; Møller, Birger Lindberg; Nørholm, Morten H H

    2015-03-20

    Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing, and collaborative efforts such as the iGEM competition. All of these attributes facilitate exploitation of the wealth of genetic information made available by genome and RNA sequencing. Standardization also comes at the cost of reduced flexibility. We addressed this paradox by formulating a set of design principles aimed at maximizing standardization while maintaining high flexibility in choice of cloning technique and minimizing the impact of standard sequences. The design principles were applied to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable and simplifies the experimental design of projects aimed at biosynthetic pathway construction or engineering.

  5. High fitness costs and instability of gene duplications reduce rates of evolution of new genes by duplication-divergence mechanisms.

    PubMed

    Adler, Marlen; Anjum, Mehreen; Berg, Otto G; Andersson, Dan I; Sandegren, Linus

    2014-06-01

    An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different submodels, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kilo base pairs of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modeling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be offset by positive selection for novel beneficial functions.

  6. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

    PubMed

    Kosuri, Sriram; Eroshenko, Nikolai; Leproust, Emily M; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M

    2010-12-01

    Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

  7. Gene Expression Profile of High IFN-γ Producers Stimulated with Leishmania braziliensis Identifies Genes Associated with Cutaneous Leishmaniasis

    PubMed Central

    Carneiro, Marcia W.; Fukutani, Kiyoshi F.; Andrade, Bruno B.; Curvelo, Rebecca P.; Cristal, Juqueline R.; Carvalho, Augusto M.; Barral, Aldina

    2016-01-01

    Background The initial response to Leishmania parasites is essential in determining disease development or resistance. In vitro, a divergent response to Leishmania, characterized by high or low IFN-γ production has been described as a potential tool to predict both vaccine response and disease susceptibility in vivo. Methods and findings We identified uninfected and healthy individuals that were shown to be either high- or low IFN-γ producers (HPs and LPs, respectively) following stimulation of peripheral blood cells with Leishmania braziliensis. Following stimulation, RNA was processed for gene expression analysis using immune gene arrays. Both HPs and LPs were shown to upregulate the expression of CXCL10, IFI27, IL6 and LTA. Genes expressed in HPs only (CCL7, IL8, IFI44L and IL1B) were associated with pathways related to IL17 and TREM 1 signaling. In LPs, uniquely expressed genes (for example IL9, IFI44, IFIT1 and IL2RA) were associated with pathways related to pattern recognition receptors and interferon signaling. We then investigated whether the unique gene expression profiles described here could be recapitulated in vivo, in individuals with active Cutaneous Leishmaniasis or with subclinical infection. Indeed, using a set of six genes (TLR2, JAK2, IFI27, IFIT1, IRF1 and IL6) modulated in HPs and LPs, we could successfully discriminate these two clinical groups. Finally, we demonstrate that these six genes are significantly overexpressed in CL lesions. Conclusion Upon interrogation of the peripheral response of naive individuals with diverging IFN-γ production to L. braziliensis, we identified differences in the innate response to the parasite that are recapitulated in vivo and that discriminate CL patients from individuals presenting a subclinical infection. PMID:27870860

  8. Gene interaction network analysis suggests differences between high and low doses of acetaminophen

    SciTech Connect

    Toyoshiba, Hiroyoshi . E-mail: toyoshiba.hiroyoshi@nies.go.jp; Sone, Hideko; Yamanaka, Takeharu; Parham, Frederick M.; Irwin, Richard D.; Boorman, Gary A.; Portier, Christopher J.

    2006-09-15

    Bayesian networks for quantifying linkages between genes were applied to detect differences in gene expression interaction networks between multiple doses of acetaminophen at multiple time points. Seventeen (17) genes were selected from the gene expression profiles from livers of rats orally exposed to 50, 150 and 1500 mg/kg acetaminophen (APAP) at 6, 24 and 48 h after exposure using a variety of statistical and bioinformatics approaches. The selected genes are related to three biological categories: apoptosis, oxidative stress and other. Gene interaction networks between all 17 genes were identified for the nine dose-time observation points by the TAO-Gen algorithm. Using k-means clustering analysis, the estimated nine networks could be clustered into two consensus networks, the first consisting of the low and middle dose groups, and the second consisting of the high dose. The analysis suggests that the networks could be segregated by doses and were consistent in structure over time of observation within grouped doses. The consensus networks were quantified to calculate the probability distribution for the strength of the linkage between genes connected in the networks. The quantifying analysis showed that, at lower doses, the genes related to the oxidative stress signaling pathway did not interact with the apoptosis-related genes. In contrast, the high-dose network demonstrated significant interactions between the oxidative stress genes and the apoptosis genes and also demonstrated a different network between genes in the oxidative stress pathway. The approaches shown here could provide predictive information to understand high- versus low-dose mechanisms of toxicity.

  9. Gene Model Annotations for Drosophila melanogaster: Impact of High-Throughput Data

    PubMed Central

    Matthews, Beverley B.; dos Santos, Gilberto; Crosby, Madeline A.; Emmert, David B.; St. Pierre, Susan E.; Gramates, L. Sian; Zhou, Pinglei; Schroeder, Andrew J.; Falls, Kathleen; Strelets, Victor; Russo, Susan M.; Gelbart, William M.

    2015-01-01

    We report the current status of the FlyBase annotated gene set for Drosophila melanogaster and highlight improvements based on high-throughput data. The FlyBase annotated gene set consists entirely of manually annotated gene models, with the exception of some classes of small non-coding RNAs. All gene models have been reviewed using evidence from high-throughput datasets, primarily from the modENCODE project. These datasets include RNA-Seq coverage data, RNA-Seq junction data, transcription start site profiles, and translation stop-codon read-through predictions. New annotation guidelines were developed to take into account the use of the high-throughput data. We describe how this flood of new data was incorporated into thousands of new and revised annotations. FlyBase has adopted a philosophy of excluding low-confidence and low-frequency data from gene model annotations; we also do not attempt to represent all possible permutations for complex and modularly organized genes. This has allowed us to produce a high-confidence, manageable gene annotation dataset that is available at FlyBase (http://flybase.org). Interesting aspects of new annotations include new genes (coding, non-coding, and antisense), many genes with alternative transcripts with very long 3′ UTRs (up to 15–18 kb), and a stunning mismatch in the number of male-specific genes (approximately 13% of all annotated gene models) vs. female-specific genes (less than 1%). The number of identified pseudogenes and mutations in the sequenced strain also increased significantly. We discuss remaining challenges, for instance, identification of functional small polypeptides and detection of alternative translation starts. PMID:26109357

  10. Gene Model Annotations for Drosophila melanogaster: Impact of High-Throughput Data.

    PubMed

    Matthews, Beverley B; Dos Santos, Gilberto; Crosby, Madeline A; Emmert, David B; St Pierre, Susan E; Gramates, L Sian; Zhou, Pinglei; Schroeder, Andrew J; Falls, Kathleen; Strelets, Victor; Russo, Susan M; Gelbart, William M

    2015-06-24

    We report the current status of the FlyBase annotated gene set for Drosophila melanogaster and highlight improvements based on high-throughput data. The FlyBase annotated gene set consists entirely of manually annotated gene models, with the exception of some classes of small non-coding RNAs. All gene models have been reviewed using evidence from high-throughput datasets, primarily from the modENCODE project. These datasets include RNA-Seq coverage data, RNA-Seq junction data, transcription start site profiles, and translation stop-codon read-through predictions. New annotation guidelines were developed to take into account the use of the high-throughput data. We describe how this flood of new data was incorporated into thousands of new and revised annotations. FlyBase has adopted a philosophy of excluding low-confidence and low-frequency data from gene model annotations; we also do not attempt to represent all possible permutations for complex and modularly organized genes. This has allowed us to produce a high-confidence, manageable gene annotation dataset that is available at FlyBase (http://flybase.org). Interesting aspects of new annotations include new genes (coding, non-coding, and antisense), many genes with alternative transcripts with very long 3' UTRs (up to 15-18 kb), and a stunning mismatch in the number of male-specific genes (approximately 13% of all annotated gene models) vs. female-specific genes (less than 1%). The number of identified pseudogenes and mutations in the sequenced strain also increased significantly. We discuss remaining challenges, for instance, identification of functional small polypeptides and detection of alternative translation starts.

  11. Horizontal transfer of a large and highly toxic secondary metabolic gene cluster between fungi.

    PubMed

    Slot, Jason C; Rokas, Antonis

    2011-01-25

    Genes involved in intermediary and secondary metabolism in fungi are frequently physically linked or clustered. For example, in Aspergillus nidulans the entire pathway for the production of sterigmatocystin (ST), a highly toxic secondary metabolite and a precursor to the aflatoxins (AF), is located in a ∼54 kb, 23 gene cluster. We discovered that a complete ST gene cluster in Podospora anserina was horizontally transferred from Aspergillus. Phylogenetic analysis shows that most Podospora cluster genes are adjacent to or nested within Aspergillus cluster genes, although the two genera belong to different taxonomic classes. Furthermore, the Podospora cluster is highly conserved in content, sequence, and microsynteny with the Aspergillus ST/AF clusters and its intergenic regions contain 14 putative binding sites for AflR, the transcription factor required for activation of the ST/AF biosynthetic genes. Examination of ∼52,000 Podospora expressed sequence tags identified transcripts for 14 genes in the cluster, with several expressed at multiple life cycle stages. The presence of putative AflR-binding sites and the expression evidence for several cluster genes, coupled with the recent independent discovery of ST production in Podospora [1], suggest that this HGT event probably resulted in a functional cluster. Given the abundance of metabolic gene clusters in fungi, our finding that one of the largest known metabolic gene clusters moved intact between species suggests that such transfers might have significantly contributed to fungal metabolic diversity. PAPERFLICK:

  12. A Bayesian clustering approach for detecting gene-gene interactions in high-dimensional genotype data.

    PubMed

    Chen, Sui-Pi; Huang, Guan-Hua

    2014-06-01

    This paper uses a Bayesian formulation of a clustering procedure to identify gene-gene interactions under case-control studies, called the Algorithm via Bayesian Clustering to Detect Epistasis (ABCDE). The ABCDE uses Dirichlet process mixtures to model SNP marker partitions, and uses the Gibbs weighted Chinese restaurant sampling to simulate posterior distributions of these partitions. Unlike the representative Bayesian epistasis detection algorithm BEAM, which partitions markers into three groups, the ABCDE can be evaluated at any given partition, regardless of the number of groups. This study also develops permutation tests to validate the disease association for SNP subsets identified by the ABCDE, which can yield results that are more robust to model specification and prior assumptions. This study examines the performance of the ABCDE and compares it with the BEAM using various simulated data and a schizophrenia SNP dataset.

  13. Cell Lineage Analysis of Mouse Prostate Carcinogenesis

    DTIC Science & Technology

    2014-07-01

    tamoxifen to induce genetic combination to mark luminal epithelial cells (month 24-30) Status: We are expecting the first experimental animals to be...born within the next 90 days. These animals will be ready for intraperitoneal injection of tamoxifen at 6 weeks of age. Expected revised timeline...clippings are taken for genotyping analyses at 21 days of age at weaning. C. Intraperitoneal injection of tamoxifen to induce genetic

  14. Cell Lineage Analysis of Mouse Prostate Carcinogenesis

    DTIC Science & Technology

    2015-09-01

    epithelial stem cells are efficient targets for prostate cancer initiation. Proceedings of the National Academy of Sciences of the United States of...America 2010, 107(6):2610-2615. 4. Dor Y, Brown J, Martinez OI, Melton DA: Adult pancreatic beta- cells are formed by self- duplication rather than stem ...Shen C, Shen MM: A luminal epithelial stem cell that is a cell of origin for prostate cancer. Nature 2009, 461(7263):495-500. 6. Liu J, Pascal LE

  15. Gene expression profiling in undifferentiated thyroid carcinoma induced by high-dose radiation

    PubMed Central

    Bang, Hyun Soon; Choi, Moo Hyun; Kim, Cha Soon; Choi, Seung Jin

    2016-01-01

    Published gene expression studies for radiation-induced thyroid carcinogenesis have used various methodologies. In this study, we identified differential gene expression in a human thyroid epithelial cell line after exposure to high-dose γ-radiation. HTori-3 cells were exposed to 5 or 10 Gy of ionizing radiation using two dose rates (high-dose rate: 4.68 Gy/min, and low-dose rate: 40 mGy/h) and then implanted into the backs of BALB/c nude mice after 4 (10 Gy) or 5 weeks (5 Gy). Decreases in cell viability, increases in giant cell frequency, anchorage-independent growth in vitro, and tumorigenicity in vivo were observed. Particularly, the cells irradiated with 5 Gy at the high-dose rate or 10 Gy at the low-dose rate demonstrated more prominent tumorigenicity. Gene expression profiling was analyzed via microarray. Numerous genes that were significantly altered by a fold-change of >50% following irradiation were identified in each group. Gene expression analysis identified six commonly misregulated genes, including CRYAB, IL-18, ZNF845, CYP24A1, OR4N4 and VN1R4, at all doses. These genes involve apoptosis, the immune response, regulation of transcription, and receptor signaling pathways. Overall, the altered genes in high-dose rate (HDR) 5 Gy and low-dose rate (LDR) 10 Gy were more than those of LDR 5 Gy and HDR 10 Gy. Thus, we investigated genes associated with aggressive tumor development using the two dosage treatments. In this study, the identified gene expression profiles reflect the molecular response following high doses of external radiation exposure and may provide helpful information about radiation-induced thyroid tumors in the high-dose range. PMID:27006382

  16. The novel C. elegans gene sop-3 modulates Wnt signaling to regulate Hox gene expression.

    PubMed

    Zhang, H; Emmons, S W

    2001-03-01

    We describe the properties of a new gene, sop-3, that is required for the regulated expression of a C. elegans Hox gene, egl-5, in a postembryonic neuroectodermal cell lineage. Regulated expression of egl-5 in this cell lineage is necessary for development of the sensory rays of the male tail. sop-3 encodes a predicted novel protein of 1475 amino acids without clear homologs in other organisms. However, the sequence contains motifs consisting of homopolymeric runs of amino acids found in several other transcriptional regulators, some of which also act in Hox gene regulatory pathways. The genetic properties of sop-3 are very similar to those of sop-1, which encodes a component of the transcriptional Mediator complex, and mutations in the two genes are synthetic lethal. This suggests that SOP-3 may act at the level of the Mediator complex in regulating transcription initiation. In a sop-3 loss-of-function background, egl-5 is expressed ectopically in lineage branches that normally do not express this gene. Such expression is dependent on the Hox gene mab-5, as it is in branches where egl-5 is normally expressed. Ectopic egl-5 expression is also dependent on the Wnt pathway. Thus, sop-3 contributes to the combinatorial control of egl-5 by blocking egl-5 activation by MAB-5 and the Wnt pathway in inappropriate lineage branches.

  17. Recombinant cells that highly express chromosomally-integrated heterologous gene

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2007-03-20

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  18. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, L.O.; Ohta, Kazuyoshi; Wood, B.E.

    1998-10-13

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol. 13 figs.

  19. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    2000-08-22

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  20. Recombinant cells that highly express chromosomally-integrated heterologous genes

    DOEpatents

    Ingram, Lonnie O.; Ohta, Kazuyoshi; Wood, Brent E.

    1998-01-01

    Recombinant host cells are obtained that comprise (A) a heterologous, polypeptide-encoding polynucleotide segment, stably integrated into a chromosome, which is under transcriptional control of an endogenous promoter and (B) a mutation that effects increased expression of the heterologous segment, resulting in enhanced production by the host cells of each polypeptide encoded by that segment, relative to production of each polypeptide by the host cells in the absence of the mutation. The increased expression thus achieved is retained in the absence of conditions that select for cells displaying such increased expression. When the integrated segment comprises, for example, ethanol-production genes from an efficient ethanol producer like Zymomonas mobilis, recombinant Escherichia coli and other enteric bacterial cells within the present invention are capable of converting a wide range of biomass-derived sugars efficiently to ethanol.

  1. Searching for the Genes of Unconventional High Temperature Superconductors

    NASA Astrophysics Data System (ADS)

    Hu, Jiangping

    In the past, both curates and iron-based superconductors were discovered accidentally. Lacking of successful predictions on new high Tc materials is one of major obstacles to reach a consensus on the high Tc mechanism. In this talk, we discuss two emergent principles, which are called as the correspondence principle and the selective magnetic pairing rule, to unify the understanding of both cuprates and iron-based superconductors. These two principles provide an unified explanation why the d-wave pairing symmetry and the s-wave pairing symmetry are robust respectively in cuprates and iron-based superconductors. In the meanwhile, the above two principles explain the rareness of unconventional high Tc superconductivity, identify necessary electronic environments required for high Tc superconductivity and finally serve as direct guiding rules to search new high Tc materials. We predict that the third family of unconventional high Tc superconductors exist in the compounds which carry two dimensional hexagonal lattices formed by cation-anion trigonal bipyramidal complexes with a d filling configuration on the cation ions. Their superconducting states are expected to be dominated by the d+id pairing symmetry and their maximum Tc should be higher than those of iron-based superconductors. Verifying the prediction can convincingly establish the high Tc superconducting mechanism and pave a way to design new high Tc superconductors

  2. Highly and moderately aggressive mouse ovarian cancer cell lines exhibit differential gene expression

    PubMed Central

    Zhang, Wensheng; Kale, Shubha P.; McFerrin, Harris; Davenport, Ian; Wang, Guangdi; Skripnikova, Elena; Li, Xiao-Lin; Bowen, Nathan J.; McDaniels, Leticia B; Meng, Yuan-Xiang; Polk, Paula; Liu, Yong-Yu; Zhang, Qian-Jin

    2017-01-01

    Patients with advanced epithelial ovarian cancer often experience disease recurrence after standard therapies, a critical factor in determining their five-year survival rate. Recent reports indicated that long-term or short-term survival is associated with varied gene expression of cancer cells. Thus, identification of novel prognostic biomarkers should be considered. Since the mouse genome is similar to the human genome, we explored potential prognostic biomarkers using two groups of mouse ovarian cancer cell lines (group 1: IG-10, IG-10pw, and IG-10pw/agar; group 2: IG-10 clones 2, 3, and 11) which display highly and moderately aggressive phenotypes in vivo. Mice injected with these cell lines have different survival time and rates, capacities of tumor, and ascites formations, reflecting different prognostic potentials. Using an Affymetrix Mouse Genome 430 2.0 Array, a total of 181 genes were differentially expressed (P<0.01) by at least twofold between two groups of the cell lines. Of the 181 genes, 109 and 72 genes were overexpressed in highly and moderately aggressive cell lines, respectively. Analysis of the 109 and 72 genes using Ingenuity Pathway Analysis (IPA) tool revealed two cancer-related gene networks. One was associated with the highly aggressive cell lines and affiliated with MYC gene, and another was associated with the moderately aggressive cell lines and affiliated with the androgen receptor (AR). Finally, the gene enrichment analysis indicated that the overexpressed 89 genes (out of 109 genes) in highly aggressive cell lines had a function annotation in the David database. The cancer-relevant significant gene ontology (GO) terms included Cell cycle, DNA metabolic process, and Programmed cell death. None of the genes from a set of the 72 genes overexpressed in the moderately aggressive cell lines had a function annotation in the David database. Our results suggested that the overexpressed MYC and 109 gene set represented highly aggressive ovarian

  3. Highly conserved low-copy nuclear genes as effective markers for phylogenetic analyses in angiosperms.

    PubMed

    Zhang, Ning; Zeng, Liping; Shan, Hongyan; Ma, Hong

    2012-09-01

    Organismal phylogeny provides a crucial evolutionary framework for many studies and the angiosperm phylogeny has been greatly improved recently, largely using organellar and rDNA genes. However, low-copy protein-coding nuclear genes have not been widely used on a large scale in spite of the advantages of their biparental inheritance and vast number of choices. Here, we identified 1083 highly conserved low-copy nuclear genes by genome comparison. Furthermore, we demonstrated the use of five nuclear genes in 91 angiosperms representing 46 orders (73% of orders) and three gymnosperms as outgroups for a highly resolved phylogeny. These nuclear genes are easy to clone and align, and more phylogenetically informative than widely used organellar genes. The angiosperm phylogeny reconstructed using these genes was largely congruent with previous ones mainly inferred from organellar genes. Intriguingly, several new placements were uncovered for some groups, including those among the rosids, the asterids, and between the eudicots and several basal angiosperm groups. These conserved universal nuclear genes have several inherent qualities enabling them to be good markers for reconstructing angiosperm phylogeny, even eukaryotic relationships, further providing new insights into the evolutionary history of angiosperms.

  4. Genetic control of eosinophilia in mice: gene(s) expressed in bone marrow-derived cells control high responsiveness

    SciTech Connect

    Vadas, M.A.

    1982-02-01

    A heterogeneity in the capacity of strains of mice to mount eosinophilia is described. BALB/c and C3H are eosinophil high responder strains (EO-HR) and CBA and A/J are eosinophil low responder strains (EO-LR), judged by the response of blood eosinophils to Ascaris suum, and the response of blood, bone marrow, and spleen eosinophils to keyhole limpet hemocyanin given 2 days after 150 mg/kg cyclophosphamide. Some of the gene(s) for high responsiveness appear to be dominant because (EO-HR x EO-LR)F/sub 1/ mice were intermediate to high responders. This gene is expressed in bone marrow-derived cells because radiation chimeras of the type EO-HR..-->..F/sub 1/ were high responders and EO-LR..-->..F/sub 1/ were low responders. This description of a genetic control of eosinophilia in mice may be useful in understanding the role of this cell in parasite immunity and allergy.

  5. High-Resolution Chromosome Ideogram Representation of Currently Recognized Genes for Autism Spectrum Disorders

    PubMed Central

    Butler, Merlin G.; Rafi, Syed K.; Manzardo, Ann M.

    2015-01-01

    Recently, autism-related research has focused on the identification of various genes and disturbed pathways causing the genetically heterogeneous group of autism spectrum disorders (ASD). The list of autism-related genes has significantly increased due to better awareness with advances in genetic technology and expanding searchable genomic databases. We compiled a master list of known and clinically relevant autism spectrum disorder genes identified with supporting evidence from peer-reviewed medical literature sources by searching key words related to autism and genetics and from authoritative autism-related public access websites, such as the Simons Foundation Autism Research Institute autism genomic database dedicated to gene discovery and characterization. Our list consists of 792 genes arranged in alphabetical order in tabular form with gene symbols placed on high-resolution human chromosome ideograms, thereby enabling clinical and laboratory geneticists and genetic counsellors to access convenient visual images of the location and distribution of ASD genes. Meaningful correlations of the observed phenotype in patients with suspected/confirmed ASD gene(s) at the chromosome region or breakpoint band site can be made to inform diagnosis and gene-based personalized care and provide genetic counselling for families. PMID:25803107

  6. High-performance web services for querying gene and variant annotation.

    PubMed

    Xin, Jiwen; Mark, Adam; Afrasiabi, Cyrus; Tsueng, Ginger; Juchler, Moritz; Gopal, Nikhil; Stupp, Gregory S; Putman, Timothy E; Ainscough, Benjamin J; Griffith, Obi L; Torkamani, Ali; Whetzel, Patricia L; Mungall, Christopher J; Mooney, Sean D; Su, Andrew I; Wu, Chunlei

    2016-05-06

    Efficient tools for data management and integration are essential for many aspects of high-throughput biology. In particular, annotations of genes and human genetic variants are commonly used but highly fragmented across many resources. Here, we describe MyGene.info and MyVariant.info, high-performance web services for querying gene and variant annotation information. These web services are currently accessed more than three million times permonth. They also demonstrate a generalizable cloud-based model for organizing and querying biological annotation information. MyGene.info and MyVariant.info are provided as high-performance web services, accessible at http://mygene.info and http://myvariant.info . Both are offered free of charge to the research community.

  7. Identifying reproducible cancer-associated highly expressed genes with important functional significances using multiple datasets

    PubMed Central

    Huang, Haiyan; Li, Xiangyu; Guo, You; Zhang, Yuncong; Deng, Xusheng; Chen, Lufei; Zhang, Jiahui; Guo, Zheng; Ao, Lu

    2016-01-01

    Identifying differentially expressed (DE) genes between cancer and normal tissues is of basic importance for studying cancer mechanisms. However, current methods, such as the commonly used Significance Analysis of Microarrays (SAM), are biased to genes with low expression levels. Recently, we proposed an algorithm, named the pairwise difference (PD) algorithm, to identify highly expressed DE genes based on reproducibility evaluation of top-ranked expression differences between paired technical replicates of cells under two experimental conditions. In this study, we extended the application of the algorithm to the identification of DE genes between two types of tissue samples (biological replicates) based on several independent datasets or sub-datasets of a dataset, by constructing multiple paired average gene expression profiles for the two types of samples. Using multiple datasets for lung and esophageal cancers, we demonstrated that PD could identify many DE genes highly expressed in both cancer and normal tissues that tended to be missed by the commonly used SAM. These highly expressed DE genes, including many housekeeping genes, were significantly enriched in many conservative pathways, such as ribosome, proteasome, phagosome and TNF signaling pathways with important functional significances in oncogenesis. PMID:27796338

  8. Characterization of Squamate Olfactory Receptor Genes and Their Transcripts by the High-Throughput Sequencing Approach

    PubMed Central

    Dehara, Yuki; Hashiguchi, Yasuyuki; Matsubara, Kazumi; Yanai, Tokuma; Kubo, Masahito; Kumazawa, Yoshinori

    2012-01-01

    The olfactory receptor (OR) genes represent the largest multigene family in the genome of terrestrial vertebrates. Here, the high-throughput next-generation sequencing (NGS) approach was applied to characterization of OR gene repertoires in the green anole lizard Anolis carolinensis and the Japanese four-lined ratsnake Elaphe quadrivirgata. Tagged polymerase chain reaction (PCR) products amplified from either genomic DNA or cDNA of the two species were used for parallel pyrosequencing, assembling, and screening for errors in PCR and pyrosequencing. Starting from the lizard genomic DNA, we accurately identified 56 of 136 OR genes that were identified from its draft genome sequence. These recovered genes were broadly distributed in the phylogenetic tree of vertebrate OR genes without severe biases toward particular OR families. Ninety-six OR genes were identified from the ratsnake genomic DNA, implying that the snake has more OR gene loci than the anole lizard in response to an increased need for the acuity of olfaction. This view is supported by the estimated number of OR genes in the Burmese python's draft genome (∼280), although squamates may generally have fewer OR genes than terrestrial mammals and amphibians. The OR gene repertoire of the python seems unique in that many class I OR genes are retained. The NGS approach also allowed us to identify candidates of highly expressed and silent OR gene copies in the lizard's olfactory epithelium. The approach will facilitate efficient and parallel characterization of considerable unbiased proportions of multigene family members and their transcripts from nonmodel organisms. PMID:22511035

  9. Searching for resistance genes to Bursaphelenchus xylophilus using high throughput screening

    PubMed Central

    2012-01-01

    Background Pine wilt disease (PWD), caused by the pinewood nematode (PWN; Bursaphelenchus xylophilus), damages and kills pine trees and is causing serious economic damage worldwide. Although the ecological mechanism of infestation is well described, the plant’s molecular response to the pathogen is not well known. This is due mainly to the lack of genomic information and the complexity of the disease. High throughput sequencing is now an efficient approach for detecting the expression of genes in non-model organisms, thus providing valuable information in spite of the lack of the genome sequence. In an attempt to unravel genes potentially involved in the pine defense against the pathogen, we hereby report the high throughput comparative sequence analysis of infested and non-infested stems of Pinus pinaster (very susceptible to PWN) and Pinus pinea (less susceptible to PWN). Results Four cDNA libraries from infested and non-infested stems of P. pinaster and P. pinea were sequenced in a full 454 GS FLX run, producing a total of 2,083,698 reads. The putative amino acid sequences encoded by the assembled transcripts were annotated according to Gene Ontology, to assign Pinus contigs into Biological Processes, Cellular Components and Molecular Functions categories. Most of the annotated transcripts corresponded to Picea genes-25.4-39.7%, whereas a smaller percentage, matched Pinus genes, 1.8-12.8%, probably a consequence of more public genomic information available for Picea than for Pinus. The comparative transcriptome analysis showed that when P. pinaster was infested with PWN, the genes malate dehydrogenase, ABA, water deficit stress related genes and PAR1 were highly expressed, while in PWN-infested P. pinea, the highly expressed genes were ricin B-related lectin, and genes belonging to the SNARE and high mobility group families. Quantitative PCR experiments confirmed the differential gene expression between the two pine species. Conclusions Defense-related genes

  10. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae

    PubMed Central

    Azvolinsky, Anna; Giresi, Paul G.; Lieb, Jason D.; Zakian, Virginia A.

    2009-01-01

    SUMMARY Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. PMID:19560424

  11. Mitogenomic analyses propose positive selection in mitochondrial genes for high-altitude adaptation in galliform birds.

    PubMed

    Zhou, Taicheng; Shen, Xuejuan; Irwin, David M; Shen, Yongyi; Zhang, Yaping

    2014-09-01

    Galliform birds inhabit very diverse habitats, including plateaus that are above 3000 m in altitude. At high altitude, lower temperature and hypoxia are two important factors influencing survival. Mitochondria, as the ultimate oxygen transductor, play an important role in aerobic respiration through oxidative phosphorylation (OXPHOS). We analyzed the mitochondrial genomes of six high-altitude phasianidae birds and sixteen low-altitude relatives in an attempt to determine the role of mitochondrial genes in high-altitude adaptation. We reconstructed the phylogenetic relationships of these phasianidae birds and relatives and found at least four lineages that independently occupied this high-altitude habitat. Selective analyses revealed significant evidence for positive selection in the genes ND2, ND4, and ATP6 in three of the high-altitude lineages. This result strongly suggests that adaptive evolution of mitochondrial genes played a critical role during the independent acclimatization to high altitude by galliform birds.

  12. High throughput in vivo functional validation of candidate congenital heart disease genes in Drosophila

    PubMed Central

    Zhu, Jun-yi; Fu, Yulong; Nettleton, Margaret; Richman, Adam; Han, Zhe

    2017-01-01

    Genomic sequencing has implicated large numbers of genes and de novo mutations as potential disease risk factors. A high throughput in vivo model system is needed to validate gene associations with pathology. We developed a Drosophila-based functional system to screen candidate disease genes identified from Congenital Heart Disease (CHD) patients. 134 genes were tested in the Drosophila heart using RNAi-based gene silencing. Quantitative analyses of multiple cardiac phenotypes demonstrated essential structural, functional, and developmental roles for more than 70 genes, including a subgroup encoding histone H3K4 modifying proteins. We also demonstrated the use of Drosophila to evaluate cardiac phenotypes resulting from specific, patient-derived alleles of candidate disease genes. We describe the first high throughput in vivo validation system to screen candidate disease genes identified from patients. This approach has the potential to facilitate development of precision medicine approaches for CHD and other diseases associated with genetic factors. DOI: http://dx.doi.org/10.7554/eLife.22617.001 PMID:28084990

  13. High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

    PubMed Central

    Brommage, Robert; Liu, Jeff; Hansen, Gwenn M; Kirkpatrick, Laura L; Potter, David G; Sands, Arthur T; Zambrowicz, Brian; Powell, David R; Vogel, Peter

    2014-01-01

    Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening (HTS) data for 3 762 distinct global gene knockout (KO) mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type (WT) cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass: 23 previously reported genes (Calcr, Cebpb, Crtap, Dcstamp, Dkk1, Duoxa2, Enpp1, Fgf23, Kiss1/Kiss1r, Kl (Klotho), Lrp5, Mstn, Neo1, Npr2, Ostm1, Postn, Sfrp4, Slc30a5, Slc39a13, Sost, Sumf1, Src, Wnt10b), five novel genes extensively characterized (Cldn18, Fam20c, Lrrk1, Sgpl1, Wnt16), five novel genes with preliminary characterization (Agpat2, Rassf5, Slc10a7, Slc26a7, Slc30a10) and three novel undisclosed genes coding for potential osteoporosis drug targets. PMID:26273529

  14. A highly divergent gene cluster in honey bees encodes a novel silk family.

    PubMed

    Sutherland, Tara D; Campbell, Peter M; Weisman, Sarah; Trueman, Holly E; Sriskantha, Alagacone; Wanjura, Wolfgang J; Haritos, Victoria S

    2006-11-01

    The pupal cocoon of the domesticated silk moth Bombyx mori is the best known and most extensively studied insect silk. It is not widely known that Apis mellifera larvae also produce silk. We have used a combination of genomic and proteomic techniques to identify four honey bee fiber genes (AmelFibroin1-4) and two silk-associated genes (AmelSA1 and 2). The four fiber genes are small, comprise a single exon each, and are clustered on a short genomic region where the open reading frames are GC-rich amid low GC intergenic regions. The genes encode similar proteins that are highly helical and predicted to form unusually tight coiled coils. Despite the similarity in size, structure, and composition of the encoded proteins, the genes have low primary sequence identity. We propose that the four fiber genes have arisen from gene duplication events but have subsequently diverged significantly. The silk-associated genes encode proteins likely to act as a glue (AmelSA1) and involved in silk processing (AmelSA2). Although the silks of honey bees and silkmoths both originate in larval labial glands, the silk proteins are completely different in their primary, secondary, and tertiary structures as well as the genomic arrangement of the genes encoding them. This implies independent evolutionary origins for these functionally related proteins.

  15. Global gene analysis of oocytes from early stages in human folliculogenesis shows high expression of novel genes in reproduction.

    PubMed

    Markholt, S; Grøndahl, M L; Ernst, E H; Andersen, C Yding; Ernst, E; Lykke-Hartmann, K

    2012-02-01

    The pool of primordial follicles in humans is laid down during embryonic development and follicles can remain dormant for prolonged intervals, often decades, until individual follicles resume growth. The mechanisms that induce growth and maturation of primordial follicles are poorly understood but follicles once activated either continue growth or undergo atresia. We have isolated pure populations of oocytes from human primordial, intermediate and primary follicles using laser capture micro-dissection microscopy and evaluated the global gene expression profiles by whole-genome microarray analysis. The array data were confirmed by qPCR for selected genes. A total of 6301 unique genes were identified as significantly expressed representing enriched specific functional categories such as 'RNA binding', 'translation initiation' and 'structural molecule activity'. Several genes, some not previously known to be associated with early oocyte development, were identified with exceptionally high expression levels, such as the anti-proliferative transmembrane protein with an epidermal growth factor-like and two follistatin-like domains (TMEFF2), the Rho-GTPase-activating protein oligophrenin 1 (OPHN1) and the mitochondrial-encoded ATPase6 (ATP6). Thus, the present study provides not only a technique to capture and perform transcriptome analysis of the sparse material of human oocytes from the earliest follicle stages but further includes a comprehensive basis for our understanding of the regulatory factors and pathways present during early human folliculogenesis.

  16. High polymorphism in big defensin gene expression reveals presence-absence gene variability (PAV) in the oyster Crassostrea gigas.

    PubMed

    Rosa, Rafael D; Alonso, Pascal; Santini, Adrien; Vergnes, Agnès; Bachère, Evelyne

    2015-04-01

    We report here the first evidence in an invertebrate, the oyster Crassostrea gigas, of a phenomenon of Presence-Absence Variation (PAV) affecting immune-related genes. We previously evidenced an extraordinary interindividual variability in the basal mRNA abundances of oyster immune genes including those coding for a family of antimicrobial peptides, the big defensins (Cg-BigDef). Cg-BigDef is a diverse family composed of three members: Cg-BigDef1 to -3. Here, we show that besides a high polymorphism in Cg-BigDef mRNA expression, not all individual oysters express simultaneously the three Cg-BigDefs. Moreover, in numerous individuals, no expression of Cg-BigDefs could be detected. Further investigation at the genomic level revealed that in individuals in which the transcription of one or all Cg-BigDefs was absent the corresponding Cg-bigdef gene was missing. In our experiments, no correlation was found between Cg-bigdef PAV and oyster capacity to survive Vibrio infections. The discovery of P-A immune genes in oysters leads to reconsider the role that the immune system plays in the individual adaptation to survive environmental, biotic and abiotic stresses.

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

    PubMed

    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.

  18. A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii.

    PubMed

    Xu, Chuan; Zhang, Xing; Qian, Ying; Chen, Xiaoxuan; Liu, Ran; Zeng, Guohong; Zhao, Hong; Fang, Weiguo

    2014-01-01

    Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the "model" fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of

  19. High frequency vector-mediated transformation and gene replacement in Tetrahymena.

    PubMed Central

    Gaertig, J; Gu, L; Hai, B; Gorovsky, M A

    1994-01-01

    Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene

  20. Gene expression signatures defining fundamental biological processes in pluripotent, early, and late differentiated embryonic stem cells.

    PubMed

    Gaspar, John Antonydas; Doss, Michael Xavier; Winkler, Johannes; Wagh, Vilas; Hescheler, Jürgen; Kolde, Raivo; Vilo, Jaak; Schulz, Herbert; Sachinidis, Agapios

    2012-09-01

    Investigating the molecular mechanisms controlling the in vivo developmental program postembryogenesis is challenging and time consuming. However, the developmental program can be partly recapitulated in vitro by the use of cultured embryonic stem cells (ESCs). Similar to the totipotent cells of the inner cell mass, gene expression and morphological changes in cultured ESCs occur hierarchically during their differentiation, with epiblast cells developing first, followed by germ layers and finally somatic cells. Combination of high throughput -omics technologies with murine ESCs offers an alternative approach for studying developmental processes toward organ-specific cell phenotypes. We have made an attempt to understand differentiation networks controlling embryogenesis in vivo using a time kinetic, by identifying molecules defining fundamental biological processes in the pluripotent state as well as in early and the late differentiation stages of ESCs. Our microarray data of the differentiation of the ESCs clearly demonstrate that the most critical early differentiation processes occur at days 2 and 3 of differentiation. Besides monitoring well-annotated markers pertinent to both self-renewal and potency (capacity to differentiate to different cell lineage), we have identified candidate molecules for relevant signaling pathways. These molecules can be further investigated in gain and loss-of-function studies to elucidate their role for pluripotency and differentiation. As an example, siRNA knockdown of MageB16, a gene highly expressed in the pluripotent state, has proven its influence in inducing differentiation when its function is repressed.

  1. Neuromodulin (GAP43): a neuronal protein kinase C substrate is also present in 0-2A glial cell lineage. Characterization of neuromodulin in secondary cultures of oligodendrocytes and comparison with the neuronal antigen.

    PubMed

    Deloulme, J C; Janet, T; Au, D; Storm, D R; Sensenbrenner, M; Baudier, J

    1990-10-01

    Neuromodulin (also called GAP43, G50, F1, pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellate-shaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of neuromodulin in secondary cultures of newborn rat oligodendrocytes and its absence in type 1 astrocytes was confirmed by Northern blot analysis of isolated total RNA from these different types of cells using a cDNA probe for the neuromodulin mRNA and by Western blot analysis of the cell extracts using polyclonal antibodies against neuromodulin. The properties of the neuromodulin protein in cultured oligodendrocytes and neuronal cells have been compared. Although neuromodulin in oligodendrocytes is soluble in 2.5% perchloric acid like the neuronal counterpart it migrates essentially as a single protein spot on two-dimensional gel electrophoresis whereas the neuronal antigen can be resolved into at least three distinct protein spots. To obtain precise alignments of the different neuromodulin spots from these two cell types, oligodendrocyte and neuronal cell extracts were mixed together and run on the same two-dimensional gel electrophoresis system. Oligodendroglial neuromodulin migrates with a pI identical to the basic forms of the neuronal protein in isoelectric focusing gel. However, the glial neuromodulin shows a slightly lower mobility in the second

  2. Mating-type genes and MAT switching in Saccharomyces cerevisiae.

    PubMed

    Haber, James E

    2012-05-01

    Mating type in Saccharomyces cerevisiae is determined by two nonhomologous alleles, MATa and MATα. These sequences encode regulators of the two different haploid mating types and of the diploids formed by their conjugation. Analysis of the MATa1, MATα1, and MATα2 alleles provided one of the earliest models of cell-type specification by transcriptional activators and repressors. Remarkably, homothallic yeast cells can switch their mating type as often as every generation by a highly choreographed, site-specific homologous recombination event that replaces one MAT allele with different DNA sequences encoding the opposite MAT allele. This replacement process involves the participation of two intact but unexpressed copies of mating-type information at the heterochromatic loci, HMLα and HMRa, which are located at opposite ends of the same chromosome-encoding MAT. The study of MAT switching has yielded important insights into the control of cell lineage, the silencing of gene expression, the formation of heterochromatin, and the regulation of accessibility of the donor sequences. Real-time analysis of MAT switching has provided the most detailed description of the molecular events that occur during the homologous recombinational repair of a programmed double-strand chromosome break.

  3. Lung Gene Therapy with Highly Compacted DNA Nanoparticles that Overcome the Mucus Barrier

    PubMed Central

    Suk, Jung Soo; Kim, Anthony J.; Trehan, Kanika; Schneider, Craig S.; Cebotaru, Liudmila; Woodward, Owen M.; Boylan, Nicholas J.; Boyle, Michael P.; Lai, Samuel K.; Guggino, William B.; Hanes, Justin

    2014-01-01

    Inhaled gene carriers must penetrate the highly viscoelastic and adhesive mucus barrier in the airway in order to overcome rapid mucociliary clearance and reach the underlying epithelium; however, even the most widely used viral gene carriers are unable to efficiently do so. We developed two polymeric gene carriers that compact plasmid DNA into small and highly stable nanoparticles with dense polyethylene glycol (PEG) surface coatings. These highly compacted, densely PEG-coated DNA nanoparticles rapidly penetrate human cystic fibrosis (CF) mucus ex vivo and mouse airway mucus ex situ. Intranasal administration of the mucus penetrating DNA nanoparticles greatly enhanced particle distribution, retention and gene transfer in the mouse lung airways compared to conventional gene carriers. Successful delivery of a full-length plasmid encoding the cystic fibrosis transmembrane conductance regulator protein was achieved in mouse lungs and airway cells, including a primary culture of mucus-covered human airway epithelium grown at air-liquid interface, without causing acute inflammation or toxicity. Highly compacted mucus penetrating DNA nanoparticles hold promise for lung gene therapy. PMID:24440664

  4. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

    PubMed Central

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-01

    Summary CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing. PMID:26771356

  5. High-Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells.

    PubMed

    Carlson-Stevermer, Jared; Goedland, Madelyn; Steyer, Benjamin; Movaghar, Arezoo; Lou, Meng; Kohlenberg, Lucille; Prestil, Ryan; Saha, Krishanu

    2016-01-12

    CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.

  6. High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.

    PubMed

    Griffin, Jennifer E; Gawronski, Jeffrey D; Dejesus, Michael A; Ioerger, Thomas R; Akerley, Brian J; Sassetti, Christopher M

    2011-09-01

    The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets.

  7. High-throughput quantification of antibiotic resistance genes from an urban wastewater treatment plant.

    PubMed

    Karkman, Antti; Johnson, Timothy A; Lyra, Christina; Stedtfeld, Robert D; Tamminen, Manu; Tiedje, James M; Virta, Marko

    2016-03-01

    Antibiotic resistance among bacteria is a growing problem worldwide, and wastewater treatment plants have been considered as one of the major contributors to the dissemination of antibiotic resistance to the environment. There is a lack of comprehensive quantitative molecular data on extensive numbers of antibiotic resistance genes (ARGs) in different seasons with a sampling strategy that would cover both incoming and outgoing water together with the excess sludge that is removed from the process. In order to fill that gap we present a highly parallel quantitative analysis of ARGs and horizontal gene transfer potential over four seasons at an urban wastewater treatment plant using a high-throughput qPCR array. All analysed transposases and two-thirds of primer sets targeting ARGs were detected in the wastewater. The relative abundance of most of the genes was highest in influent and lower in effluent water and sludge. The resistance profiles of the samples cluster by sample location with a shift from raw influent through the final effluents and dried sludge to the sediments. Wastewater discharge enriched only a few genes, namely Tn25 type transposase gene and clinical class 1 integrons, in the sediment near the discharge pipe, but those enriched genes may indicate a potential for horizontal gene transfer.

  8. High-Resolution Melting Analysis of the TPMT Gene: A Study in the Polish Population

    PubMed Central

    Borun, Pawel; Milanowska, Katarzyna; Jakubowska-Burek, Ludwika; Zakerska, Oliwia; Dobrowolska-Zachwieja, Agnieszka; Plawski, Andrzej; Froster, Ursula G.; Szalata, Marlena; Slomski, Ryszard

    2013-01-01

    The thiopurine S-methyltransferase (TPMT) gene encoding thiopurine methyltransferase is a crucial enzyme in metabolism of thiopurine drugs: azathioprine and 6-mercoptopurine, which are used in the treatment of leukemia or inflammatory bowel diseases. Genetic polymorphism of the TPMT gene correlates with activity of this enzyme, individual reaction, and dosing of thiopurines. Thirty-one variants of the TPMT gene with low enzymatic activity have been described with three major alleles: TPMT*2 (c.238G>C), *3A (c.460 G>A, c.719A>G), and *3C (c.719A>G), accounting for 80% to 95% of inherited TPMT deficiency in different populations in the world. The aim of the study was to establish a rapid and highly sensitive method of analysis for the complete coding sequence of the TPMT gene and to determine the spectrum and prevalence of the TPMT gene sequence variations in the Polish population. Recently, high-resolution melting analysis (HRMA) has become a highly sensitive, automated, and economical technique for mutation screening or genotyping. We applied HRMA for the first time to TPMT gene scanning. In total, we analyzed 548 alleles of the Polish population. We found 11 different sequence variations, where two are novel changes: c.200T>C (p.P67S, TPMT*30) and c.595G>A (p.V199I, TPMT*31). Detection of these new rare alleles TPMT*30 and *31 in the Polish population suggests the need to analyze the whole TPMT gene and maybe also the extension of routinely used tests containing three major alleles, TPMT*2, *3A, and *3C. Identification of sequence variants using HRMA is highly sensitive and less time consuming compared to standard sequencing. We conclude that HRMA can be easy integrated into genetic testing of the TPMT gene in patients treated with thiopurines. PMID:23252704

  9. High-resolution melting analysis of the TPMT gene: a study in the Polish population.

    PubMed

    Skrzypczak-Zielinska, Marzena; Borun, Pawel; Milanowska, Katarzyna; Jakubowska-Burek, Ludwika; Zakerska, Oliwia; Dobrowolska-Zachwieja, Agnieszka; Plawski, Andrzej; Froster, Ursula G; Szalata, Marlena; Slomski, Ryszard

    2013-02-01

    The thiopurine S-methyltransferase (TPMT) gene encoding thiopurine methyltransferase is a crucial enzyme in metabolism of thiopurine drugs: azathioprine and 6-mercoptopurine, which are used in the treatment of leukemia or inflammatory bowel diseases. Genetic polymorphism of the TPMT gene correlates with activity of this enzyme, individual reaction, and dosing of thiopurines. Thirty-one variants of the TPMT gene with low enzymatic activity have been described with three major alleles: TPMT*2 (c.238G>C), *3A (c.460 G>A, c.719A>G), and *3C (c.719A>G), accounting for 80% to 95% of inherited TPMT deficiency in different populations in the world. The aim of the study was to establish a rapid and highly sensitive method of analysis for the complete coding sequence of the TPMT gene and to determine the spectrum and prevalence of the TPMT gene sequence variations in the Polish population. Recently, high-resolution melting analysis (HRMA) has become a highly sensitive, automated, and economical technique for mutation screening or genotyping. We applied HRMA for the first time to TPMT gene scanning. In total, we analyzed 548 alleles of the Polish population. We found 11 different sequence variations, where two are novel changes: c.200T>C (p.P67S, TPMT*30) and c.595G>A (p.V199I, TPMT*31). Detection of these new rare alleles TPMT*30 and *31 in the Polish population suggests the need to analyze the whole TPMT gene and maybe also the extension of routinely used tests containing three major alleles, TPMT*2, *3A, and *3C. Identification of sequence variants using HRMA is highly sensitive and less time consuming compared to standard sequencing. We conclude that HRMA can be easy integrated into genetic testing of the TPMT gene in patients treated with thiopurines.

  10. Hypoxia: adapting to high altitude by mutating EPAS-1, the gene encoding HIF-2α.

    PubMed

    van Patot, Martha C Tissot; Gassmann, Max

    2011-01-01

    Living at high altitude is demanding and thus drives adaptational mechanisms. The Tibetan population has had a longer evolutionary period to adapt to high altitude than other mountain populations such as Andeans. As a result, some Tibetans living at high altitudes do not show markedly elevated red blood cell production as compared to South American high altitude natives such as Quechuas or Aymaras, thereby avoiding high blood viscosity creating cardiovascular risk. Unexpectedly, the responsible mutation(s) reducing red blood cell production do not involve either the gene encoding the blood hormone erythropoietin (Epo), or the corresponding regulatory sequences flanking the Epo gene. Similarly, functional mutations in the hypoxia-inducible transcription factor 1α (HIF-1α) gene that represents the oxygen-dependent subunit of the HIF-1 heterodimer, the latter being the main regulator of over 100 hypoxia-inducible genes, have not been described so far. It was not until very recently that three independent groups showed that the gene encoding HIF-2α, EPAS-1 (Wenger et al. 1997), represents a key gene mutated in Tibetan populations adapted to living at high altitudes (Beall et al. 2010 , Yi et al. 2010 , Simonson et al. 2010). Hypoxia-inducible transcription factors were first identified by the description of HIF-1 (Semenza et al. 1991 , 1992), which was subsequently found to enhance transcription of multiple genes that encode proteins necessary for rescuing from hypoxic exposure, including erythropoietic, angiogenic and glycolytic proteins. Then HIF-2 was identified (Ema et al. 1997 ; Flamme et al. 1997 ; Hogenesch et al. 1997 ; and Tian et al. 1997) and although it is highly similar to HIF-1 and has the potential to bind (Camenisch et al. 2001) and mediate (Mole et al. 2009) many of the same genes as HIF-1, its biological actions in response to hypoxia are distinct from those of HIF-1 (reviewed by Loboda et al. 2010). By now, several of these HIF-2 mediated

  11. Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells.

    PubMed

    Page, Andrew F; Minocha, Rakesh; Minocha, Subhash C

    2012-01-01

    Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied changes in the expression of all genes of the Orn/Arg biosynthetic pathway in response to up-regulation [via transgenic expression of mouse Orn decarboxylase (mODC)] of PA biosynthesis in poplar (Populus nigra × maximowiczii) cells grown in culture. Cloning and sequencing of poplar genes involved in the Orn/Arg biosynthetic pathway showed that they have high homology with similar genes in other plants. The expression of the genes of Orn, Arg and PA biosynthetic pathway fell into two hierarchical clusters; expression of one did not change in response to high putrescine, while members of the other cluster showed a shift in expression pattern during the 7-day culture cycle. Gene expression of branch point enzymes (N-acetyl-Glu synthase, Orn aminotransferase, Arg decarboxylase, and spermidine synthase) in the sub-pathways, constituted a separate cluster from those involved in intermediary reactions of the pathway (N-acetyl-Glu kinase, N-acetyl-Glu-5-P reductase, N-acetyl-Orn aminotransferase, N (2)-acetylOrn:N-acetyl-Glu acetyltransferase, N (2)-acetyl-Orn deacetylase, Orn transcarbamylase, argininosuccinate synthase, carbamoylphosphate synthetase, argininosuccinate lyase, S-adenosylmethionine decarboxylase, spermine synthase). We postulate that expression of all genes of the Glu-Orn-Arg pathway is constitutively coordinated and is not influenced by the increase in flux rate through this pathway in response to increased utilization of Orn by mODC; thus the pathway involves mostly biochemical regulation rather than changes in gene expression. We further suggest that Orn itself plays a major role in the

  12. Highly compacted biodegradable DNA nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy.

    PubMed

    Mastorakos, Panagiotis; da Silva, Adriana L; Chisholm, Jane; Song, Eric; Choi, Won Kyu; Boyle, Michael P; Morales, Marcelo M; Hanes, Justin; Suk, Jung Soo

    2015-07-14

    Gene therapy has emerged as an alternative for the treatment of diseases refractory to conventional therapeutics. Synthetic nanoparticle-based gene delivery systems offer highly tunable platforms for the delivery of therapeutic genes. However, the inability to achieve sustained, high-level transgene expression in vivo presents a significant hurdle. The respiratory system, although readily accessible, remains a challenging target, as effective gene therapy mandates colloidal stability in physiological fluids and the ability to overcome biological barriers found in the lung. We formulated highly stable DNA nanoparticles based on state-of-the-art biodegradable polymers, poly(β-amino esters) (PBAEs), possessing a dense corona of polyethylene glycol. We found that these nanoparticles efficiently penetrated the nanoporous and highly adhesive human mucus gel layer that constitutes a primary barrier to reaching the underlying epithelium. We also discovered that these PBAE-based mucus-penetrating DNA nanoparticles (PBAE-MPPs) provided uniform and high-level transgene expression throughout the mouse lungs, superior to several gold standard gene delivery systems. PBAE-MPPs achieved robust transgene expression over at least 4 mo following a single administration, and their transfection efficiency was not attenuated by repeated administrations, underscoring their clinical relevance. Importantly, PBAE-MPPs demonstrated a favorable safety profile with no signs of toxicity following intratracheal administration.

  13. Characterization of Promoter Elements Regulating the Expression of the Human Neurotensin/Neuromedin N Gene*

    PubMed Central

    Wang, Xiaofu; Gulhati, Pat; Li, Jing; Dobner, Paul R.; Weiss, Heidi; Townsend, Courtney M.; Evers, B. Mark

    2011-01-01

    Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine N cells in the distal small intestine. We have identified key regulatory elements in the promoter region that are involved in human NT/N (hNT/N) gene expression in the novel human endocrine cell line, BON, which resembles intestinal N cells in several important aspects including NT/N precursor protein processing, ratios of different NT/N mRNA isoforms, and high levels of constitutive expression of the NT/N gene. In this study, we demonstrated multiple cis-regulatory elements including a proximal region containing a cAMP-responsive element (CRE)/AP-1-like element that binds both the AP-1 and CRE-binding protein (CREB)/ATF proteins (c-Jun, ATF-1, ATF-2, JunD, and CREB). Similar to the rat NT/N gene, this region is critical for constitutive hNT/N gene expression. Moreover, we identified a novel region that binds the orphan hormone receptor, NR2F2. We have demonstrated that the C terminus of NR2F2 strongly represses hNT/N transcription, whereas an N-terminal domain antagonizes this repressive effect. Regulation of NT/N expression by NR2F2 may have important consequences for lipid metabolism. We speculate that a complex interplay between the proximal CRE/AP-1-like motif and NR2F2 binding region exists to regulate hNT/N expression, which is critical for the high level of constitutive expression of NT/N in enteroendocrine cells. Finally, the BON cell line provides a unique model to characterize the factors regulating expression of the hNT/N gene and to better understand the mechanisms responsible for terminal differentiation of the N cell lineage in the gut. PMID:21030593

  14. Currently recognized genes for schizophrenia: High-resolution chromosome ideogram representation.

    PubMed

    Butler, Merlin G; McGuire, Austen B; Masoud, Humaira; Manzardo, Ann M

    2016-03-01

    A large body of genetic data from schizophrenia-related research has identified an assortment of genes and disturbed pathways supporting involvement of complex genetic components for schizophrenia spectrum and other psychotic disorders. Advances in genetic technology and expanding studies with searchable genomic databases have led to multiple published reports, allowing us to compile a master list of known, clinically relevant, or susceptibility genes contributing to schizophrenia. We searched key words related to schizophrenia and genetics from peer-reviewed medical literature sources, authoritative public access psychiatric websites and genomic databases dedicated to gene discovery and characterization of schizophrenia. Our list of 560 genes were arranged in alphabetical order in tabular form with gene symbols placed on high-resolution human chromosome ideograms. Genome wide pathway analysis using GeneAnalytics was carried out on the resulting list of genes to assess the underlying genetic architecture for schizophrenia. Recognized genes of clinical relevance, susceptibility or causation impact a broad range of biological pathways and mechanisms including ion channels (e.g., CACNA1B, CACNA1C, CACNA1H), metabolism (e.g., CYP1A2, CYP2C19, CYP2D6), multiple targets of neurotransmitter pathways impacting dopamine, GABA, glutamate, and serotonin function, brain development (e.g., NRG1, RELN), signaling peptides (e.g., PIK3CA, PIK4CA) and immune function (e.g., HLA-DRB1, HLA-DQA1) and interleukins (e.g., IL1A, IL10, IL6). This summary will enable clinical and laboratory geneticists, genetic counselors, and other clinicians to access convenient pictorial images of the distribution and location of contributing genes to inform diagnosis and gene-based treatment as well as provide risk estimates for genetic counseling of families with affected relatives.

  15. Supramolecular Aggregate as a High-Efficiency Gene Carrier Mediated with Optimized Assembly Structure.

    PubMed

    Zhang, Yi; Duan, Junkun; Cai, Lingguang; Ma, Dong; Xue, Wei

    2016-11-02

    For cancer gene therapy, a safe and high-efficient gene carrier is a must. To resolve the contradiction between gene transfection efficiency and cytotoxicity, many polymers with complex topological structures have been synthesized, although their synthesis processes and structure control are difficult as well as the high molecular weight also bring high cytotoxicity. We proposed an alternative strategy that uses supramolecular inclusion to construct the aggregate from the small molecules for gene delivery, and to further explore the relationship between the topological assembly structure and their ability to deliver gene. Herein, PEI-1.8k-conjugating β-CD through 6-hydroxyl (PEI-6-CD) and 2-hydroxyl (PEI-2-CD) have been synthesized respectively and then assembled with diferrocene (Fc)-ended polyethylene glycol (PEG-Fc). The obtained aggregates were then used to deliver MMP-9 shRNA plasmid for MCF-7 cancer therapy. It was found that the higher gene transfection efficiency can be obtained by selecting PEI-2-CD as the host and tuning the host/guest molar ratios. With the rational modulation of supramolecular architectures, the aggregate played the functions similar to macromolecules which exhibit higher transfection efficiency than PEI-25k, but show much lower cytotoxicity because of the nature of small/low molecules. In vitro and in vivo assays confirmed that the aggregate could deliver MMP-9 shRNA plasmid effectively into MCF-7 cells and then downregulate MMP-9 expression, which induced the significant MCF-7 cell apoptosis, as well inhibit MCF-7 tumor growth with low toxicity. The supramolecular aggregates maybe become a promising carrier for cancer gene therapy and also provided an alternative strategy for designing new gene carriers.

  16. Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco.

    PubMed

    Ye, G N; Hajdukiewicz, P T; Broyles, D; Rodriguez, D; Xu, C W; Nehra, N; Staub, J M

    2001-02-01

    Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

  17. A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips

    PubMed Central

    Kosuri, Sriram; Eroshenko, Nikolai; LeProust, Emily; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M.

    2010-01-01

    Development of cheap, high-throughput, and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology1. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis2. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude3,4,5, yet efforts to scale their use have been largely unsuccessful due to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols, and enzymatic error correction to develop a highly parallel gene synthesis platform. We tested our platform by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ~35 kilo-basepairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ~2.5 megabases of DNA, which is at least 50 times larger than previously published attempts. PMID:21113165

  18. Parallel Gene Expression Differences between Low and High Latitude Populations of Drosophila melanogaster and D. simulans.

    PubMed

    Zhao, Li; Wit, Janneke; Svetec, Nicolas; Begun, David J

    2015-05-01

    Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3' UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes.

  19. Regularized logistic regression with adjusted adaptive elastic net for gene selection in high dimensional cancer classification.

    PubMed

    Algamal, Zakariya Yahya; Lee, Muhammad Hisyam

    2015-12-01

    Cancer classification and gene selection in high-dimensional data have been popular research topics in genetics and molecular biology. Recently, adaptive regularized logistic regression using the elastic net regularization, which is called the adaptive elastic net, has been successfully applied in high-dimensional cancer classification to tackle both estimating the gene coefficients and performing gene selection simultaneously. The adaptive elastic net originally used elastic net estimates as the initial weight, however, using this weight may not be preferable for certain reasons: First, the elastic net estimator is biased in selecting genes. Second, it does not perform well when the pairwise correlations between variables are not high. Adjusted adaptive regularized logistic regression (AAElastic) is proposed to address these issues and encourage grouping effects simultaneously. The real data results indicate that AAElastic is significantly consistent in selecting genes compared to the other three competitor regularization methods. Additionally, the classification performance of AAElastic is comparable to the adaptive elastic net and better than other regularization methods. Thus, we can conclude that AAElastic is a reliable adaptive regularized logistic regression method in the field of high-dimensional cancer classification.

  20. Parallel Gene Expression Differences between Low and High Latitude Populations of Drosophila melanogaster and D. simulans

    PubMed Central

    Zhao, Li; Wit, Janneke; Svetec, Nicolas; Begun, David J.

    2015-01-01

    Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3’ UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes. PMID:25950438

  1. Transcriptional profiling of arbuscular mycorrhizal roots exposed to high levels of phosphate reveals the repression of cell cycle-related genes and secreted protein genes in Rhizophagus irregularis.

    PubMed

    Sugimura, Yusaku; Saito, Katsuharu

    2017-02-01

    The development of arbuscular mycorrhiza (AM) is strongly suppressed under high-phosphate (Pi) conditions. To investigate AM fungal responses during the suppression of AM by high Pi, we performed an RNA-seq analysis of Rhizophagus irregularis colonizing Lotus japonicus roots at different levels of Pi (20, 100, 300, and 500 μM). AM fungal colonization decreased markedly under high-Pi conditions. In total, 163 fungal genes were differentially expressed among the four Pi treatments. Among these genes, a cell cycle-regulatory gene, cyclin-dependent kinase CDK1, and several DNA replication- and mitosis-related genes were repressed under high-Pi conditions. More than 20 genes encoding secreted proteins were also downregulated by high-Pi conditions, including the strigolactone-induced putative secreted protein 1 gene that enhances AM fungal colonization. In contrast, the expression of genes related to aerobic respiration and transport in R. irregularis were largely unaffected. Our data suggest that high Pi suppresses the expression of genes associated with fungal cell cycle progression or that encode secreted proteins that may be required for intercellular hyphal growth and arbuscule formation. However, high Pi has little effect on the transcriptional regulation of the primary metabolism or transport in preformed fungal structures.

  2. Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity

    NASA Astrophysics Data System (ADS)

    Corcoran, Martin M.; Phad, Ganesh E.; Bernat, Néstor Vázquez; Stahl-Hennig, Christiane; Sumida, Noriyuki; Persson, Mats A. A.; Martin, Marcel; Hedestam, Gunilla B. Karlsson

    2016-12-01

    Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool.

  3. Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity

    PubMed Central

    Corcoran, Martin M.; Phad, Ganesh E.; Bernat, Néstor Vázquez; Stahl-Hennig, Christiane; Sumida, Noriyuki; Persson, Mats A.A.; Martin, Marcel; Hedestam, Gunilla B. Karlsson

    2016-01-01

    Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool. PMID:27995928

  4. Prediction of highly expressed genes in microbes based on chromatin accessibility

    PubMed Central

    Willenbrock, Hanni; Ussery, David W

    2007-01-01

    Background It is well known that gene expression is dependent on chromatin structure in eukaryotes and it is likely that chromatin can play a role in bacterial gene expression as well. Here, we use a nucleosomal position preference measure of anisotropic DNA flexibility to predict highly expressed genes in microbial genomes. We compare these predictions with those based on codon adaptation index (CAI) values, and also with experimental data for 6 different microbial genomes, with a particular interest in experimental data from Escherichia coli. Moreover, position preference is examined further in 328 sequenced microbial genomes. Results We find that absolute gene expression levels are correlated with the position preference in many microbial genomes. It is postulated that in these regions, the DNA may be more accessible to the transcriptional machinery. Moreover, ribosomal proteins and ribosomal RNA are encoded by DNA having significantly lower position preference values than other genes in fast-replicating microbes. Conclusion This insight into DNA structure-dependent gene expression in microbes may be exploited for predicting the expression of non-translated genes such as non-coding RNAs that may not be predicted by any of the conventional codon usage bias approaches. PMID:17295928

  5. High-throughput platform for the discovery of elicitors of silent bacterial gene clusters

    PubMed Central

    Seyedsayamdost, Mohammad R.

    2014-01-01

    Over the past decade, bacterial genome sequences have revealed an immense reservoir of biosynthetic gene clusters, sets of contiguous genes that have the potential to produce drugs or drug-like molecules. However, the majority of these gene clusters appear to be inactive for unknown reasons prompting terms such as “cryptic” or “silent” to describe them. Because natural products have been a major source of therapeutic molecules, methods that rationally activate these silent clusters would have a profound impact on drug discovery. Herein, a new strategy is outlined for awakening silent gene clusters using small molecule elicitors. In this method, a genetic reporter construct affords a facile read-out for activation of the silent cluster of interest, while high-throughput screening of small molecule libraries provides potential inducers. This approach was applied to two cryptic gene clusters in the pathogenic model Burkholderia thailandensis. The results not only demonstrate a prominent activation of these two clusters, but also reveal that the majority of elicitors are themselves antibiotics, most in common clinical use. Antibiotics, which kill B. thailandensis at high concentrations, act as inducers of secondary metabolism at low concentrations. One of these antibiotics, trimethoprim, served as a global activator of secondary metabolism by inducing at least five biosynthetic pathways. Further application of this strategy promises to uncover the regulatory networks that activate silent gene clusters while at the same time providing access to the vast array of cryptic molecules found in bacteria. PMID:24808135

  6. A fast and high performance multiple data integration algorithm for identifying human disease genes

    PubMed Central

    2015-01-01

    Background Integrating multiple data sources is indispensable in improving disease gene identification. It is not only due to the fact that disease genes associated with similar genetic diseases tend to lie close with each other in various biological networks, but also due to the fact that gene-disease associations are complex. Although various algorithms have been proposed to identify disease genes, their prediction performances and the computational time still should be further improved. Results In this study, we propose a fast and high performance multiple data integration algorithm for identifying human disease genes. A posterior probability of each candidate gene associated with individual diseases is calculated by using a Bayesian analysis method and a binary logistic regression model. Two prior probability estimation strategies and two feature vector construction methods are developed to test the performance of the proposed algorithm. Conclusions The proposed algorithm is not only generated predictions with high AUC scores, but also runs very fast. When only a single PPI network is employed, the AUC score is 0.769 by using F2 as feature vectors. The average running time for each leave-one-out experiment is only around 1.5 seconds. When three biological networks are integrated, the AUC score using F3 as feature vectors increases to 0.830, and the average running time for each leave-one-out experiment takes only about 12.54 seconds. It is better than many existing algorithms. PMID:26399620

  7. Evolution of high cellulolytic activity in symbiotic Streptomyces through selection of expanded gene content and coordinated gene expression

    DOE PAGES

    Book, Adam J.; Lewin, Gina R.; McDonald, Bradon R.; ...

    2016-06-08

    In this study, the evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil andmore » symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.« less

  8. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression.

    PubMed

    Book, Adam J; Lewin, Gina R; McDonald, Bradon R; Takasuka, Taichi E; Wendt-Pienkowski, Evelyn; Doering, Drew T; Suh, Steven; Raffa, Kenneth F; Fox, Brian G; Currie, Cameron R

    2016-06-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology.

  9. Evolution of High Cellulolytic Activity in Symbiotic Streptomyces through Selection of Expanded Gene Content and Coordinated Gene Expression

    PubMed Central

    McDonald, Bradon R.; Takasuka, Taichi E.; Wendt-Pienkowski, Evelyn; Doering, Drew T.; Raffa, Kenneth F.; Fox, Brian G.; Currie, Cameron R.

    2016-01-01

    The evolution of cellulose degradation was a defining event in the history of life. Without efficient decomposition and recycling, dead plant biomass would quickly accumulate and become inaccessible to terrestrial food webs and the global carbon cycle. On land, the primary drivers of plant biomass deconstruction are fungi and bacteria in the soil or associated with herbivorous eukaryotes. While the ecological importance of plant-decomposing microbes is well established, little is known about the distribution or evolution of cellulolytic activity in any bacterial genus. Here we show that in Streptomyces, a genus of Actinobacteria abundant in soil and symbiotic niches, the ability to rapidly degrade cellulose is largely restricted to two clades of host-associated strains and is not a conserved characteristic of the Streptomyces genus or host-associated strains. Our comparative genomics identify that while plant biomass degrading genes (CAZy) are widespread in Streptomyces, key enzyme families are enriched in highly cellulolytic strains. Transcriptomic analyses demonstrate that cellulolytic strains express a suite of multi-domain CAZy enzymes that are coregulated by the CebR transcriptional regulator. Using targeted gene deletions, we verify the importance of a highly expressed cellulase (GH6 family cellobiohydrolase) and the CebR transcriptional repressor to the cellulolytic phenotype. Evolutionary analyses identify complex genomic modifications that drive plant biomass deconstruction in Streptomyces, including acquisition and selective retention of CAZy genes and transcriptional regulators. Our results suggest that host-associated niches have selected some symbiotic Streptomyces for increased cellulose degrading activity and that symbiotic bacteria are a rich biochemical and enzymatic resource for biotechnology. PMID:27276034

  10. Express primer tool for high-throughput gene cloning and expression.

    SciTech Connect

    Yoon, J. R.; Laible, P. D.; Gu, M.; Scott, H. N.; Collart, F. R.; Biosciences Division

    2002-12-01

    High-throughput approaches for gene cloning and expression require the development of new nonstandard tools for molecular biologists and biochemists. We introduce a Web-based tool to design primers specifically for the generation of expression clones for both laboratory-scale and high-throughput projects. The application is designed not only to allow the user complete flexibility to specify primer design parameters but also to minimize the amount of manual intervention needed to generate a large number of primers for the simultaneous amplification of multiple target genes.

  11. Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection

    PubMed Central

    Watkins-Chow, Dawn E.; Varshney, Gaurav K.; Garrett, Lisa J.; Chen, Zelin; Jimenez, Erin A.; Rivas, Cecilia; Bishop, Kevin S.; Sood, Raman; Harper, Ursula L.; Pavan, William J.; Burgess, Shawn M.

    2016-01-01

    Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity. PMID:28040780

  12. Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection.

    PubMed

    Watkins-Chow, Dawn E; Varshney, Gaurav K; Garrett, Lisa J; Chen, Zelin; Jimenez, Erin A; Rivas, Cecilia; Bishop, Kevin S; Sood, Raman; Harper, Ursula L; Pavan, William J; Burgess, Shawn M

    2017-02-09

    Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.

  13. Cyclen-Based Cationic Lipids for Highly Efficient Gene Delivery towards Tumor Cells

    PubMed Central

    Zhang, Yang; Ren, Jiang; Fu, Yun; Zhang, Ji; Zhu, Wen; Yu, Xiao-Qi

    2011-01-01

    Background Gene therapy has tremendous potential for both inherited and acquired diseases. However, delivery problems limited their clinical application, and new gene delivery vehicles with low cytotoxicity and high transfection efficiency are greatly required. Methods In this report, we designed and synthesized three amphiphilic molecules (L1–L3) with the structures involving 1, 4, 7, 10-tetraazacyclododecane (cyclen), imidazolium and a hydrophobic dodecyl chain. Their interactions with plasmid DNA were studied via electrophoretic gel retardation assays, fluorescent quenching experiments, dynamic light scattering and transmission electron microscopy. The in vitro gene transfection assay and cytotoxicity assay were conducted in four cell lines. Results Results indicated that L1 and L3-formed liposomes could effectively bind to DNA to form well-shaped nanoparticles. Combining with neutral lipid DOPE, L3 was found with high efficiency in gene transfer in three tumor cell lines including A549, HepG2 and H460. The optimized gene transfection efficacy of L3 was nearly 5.5 times more efficient than that of the popular commercially available gene delivery agent Lipofectamine 2000™ in human lung carcinoma cells A549. In addition, since L1 and L3 had nearly no gene transfection performance in normal cells HEK293, these cationic lipids showed tumor cell-targeting property to a certain extent. No significant cytotoxicity was found for the lipoplexes formed by L1–L3, and their cytotoxicities were similar to or slightly lower than the lipoplexes prepared from Lipofectamine 2000™. Conclusion Novel cyclen-based cationic lipids for effective in vitro gene transfection were founded, and these studies here may extend the application areas of macrocyclic polyamines, especially for cyclen. PMID:21887233

  14. Molecular Recognition Enables Nanosubstrate-Mediated Delivery of Gene-Encapsulated Nanoparticles with High Efficiency

    PubMed Central

    2015-01-01

    Substrate-mediated gene delivery is a promising method due to its unique ability to preconcentrate exogenous genes onto designated substrates. However, many challenges remain to enable continuous and multiround delivery of the gene using the same substrates without depositing payloads and immobilizing cells in each round of delivery. Herein we introduce a gene delivery system, nanosubstrate-mediated delivery (NSMD) platform, based on two functional components with nanoscale features, including (1) DNA⊂SNPs, supramolecular nanoparticle (SNP) vectors for gene encapsulation, and (2) Ad-SiNWS, adamantane (Ad)-grafted silicon nanowire substrates. The multivalent molecular recognition between the Ad motifs on Ad-SiNWS and the β-cyclodextrin (CD) motifs on DNA⊂SNPs leads to dynamic assembly and local enrichment of DNA⊂SNPs from the surrounding medium onto Ad-SiNWS. Subsequently, once cells settled on the substrate, DNA⊂SNPs enriched on Ad-SiNWS were introduced through the cell membranes by intimate contact with individual nanowires on Ad-SiNWS, resulting in a highly efficient delivery of exogenous genes. Most importantly, sequential delivery of multiple batches of exogenous genes on the same batch cells settled on Ad-SiNWS was realized by sequential additions of the corresponding DNA⊂SNPs with equivalent efficiency. Moreover, using the NSMD platform in vivo, cells recruited on subcutaneously transplanted Ad-SiNWS were also efficiently transfected with exogenous genes loaded into SNPs, validating the in vivo feasibility of this system. We believe that this nanosubstrate-mediated delivery platform will provide a superior system for in vitro and in vivo gene delivery and can be further used for the encapsulation and delivery of other biomolecules. PMID:24708312

  15. Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic

    PubMed Central

    Beauchemin, Mathieu; Roy, Sougata; Daoust, Philippe; Dagenais-Bellefeuille, Steve; Bertomeu, Thierry; Letourneau, Louis; Lang, B. Franz; Morse, David

    2012-01-01

    Dinoflagellates are an important component of the marine biota, but a large genome with high–copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae. PMID:23019363

  16. High-resolution chromosome ideogram representation of recognized genes for bipolar disorder.

    PubMed

    Douglas, Lindsay N; McGuire, Austen B; Manzardo, Ann M; Butler, Merlin G

    2016-07-15

    Bipolar disorder (BPD) is genetically heterogeneous with a growing list of BPD associated genes reported in recent years resulting from increased genetic testing using advanced genetic technology, expanded genomic databases, and better awareness of the disorder. We compiled a master list of recognized susceptibility and genes associated with BPD identified from peer-reviewed medical literature sources using PubMed and by searching online databases, such as OMIM. Searched keywords were related to bipolar disorder and genetics. Our compiled list consisted of 290 genes with gene names arranged in alphabetical order in tabular form with source documents and their chromosome location and gene symbols plotted on high-resolution human chromosome ideograms. The identified genes impacted a broad range of biological pathways and processes including cellular signaling pathways particularly cAMP and calcium (e.g., CACNA1C, CAMK2A, CAMK2D, ADCY1, ADCY2); glutamatergic (e.g., GRIK1, GRM3, GRM7), dopaminergic (e.g., DRD2, DRD4, COMT, MAOA) and serotonergic (e.g., HTR1A, HTR2A, HTR3B) neurotransmission; molecular transporters (e.g., SLC39A3, SLC6A3, SLC8A1); and neuronal growth (e.g., BDNF, IGFBP1, NRG1, NRG3). The increasing prevalence of BPD calls for better understanding of the genetic etiology of this disorder and associations between the observed BPD phenotype and genes. Visual representation of genes for bipolar disorder becomes a tool enabling clinical and laboratory geneticists, genetic counselors, and other health care providers and researchers easy access to the location and distribution of currently recognized BPD associated genes. Our study may also help inform diagnosis and advance treatment developments for those affected with this disorder and improve genetic counseling for families.

  17. Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation.

    PubMed

    Beck, Michaël; Rombouts, Charlotte; Moreels, Marjan; Aerts, An; Quintens, Roel; Tabury, Kevin; Michaux, Arlette; Janssen, Ann; Neefs, Mieke; Ernst, Eric; Dieriks, Birger; Lee, Ryonfa; De Vos, Winnok H; Lambert, Charles; Van Oostveldt, Patrick; Baatout, Sarah

    2014-10-01

    Ionizing radiation can elicit harmful effects on the cardiovascular system at high doses. Endothelial cells are critical targets in radiation-induced cardiovascular damage. Astronauts performing a long-term deep space mission are exposed to consistently higher fluences of ionizing radiation that may accumulate to reach high effective doses. In addition, cosmic radiation contains high linear energy transfer (LET) radiation that is known to produce high values of relative biological effectiveness (RBE). The aim of this study was to broaden the understanding of the molecular response to high LET radiation by investigating the changes in gene expression in endothelial cells. For this purpose, a human endothelial cell line (EA.hy926) was irradiated with accelerated nickel ions (Ni) (LET, 183 keV/µm) at doses of 0.5, 2 and 5 Gy. DNA damage was measured 2 and 24 h following irradiation by γ-H2AX foci detection by fluorescence microscopy and gene expression changes were measured by microarrays at 8 and 24 h following irradiation. We found that exposure to accelerated nickel particles induced a persistent DNA damage response up to 24 h after treatment. This was accompanied by a downregulation in the expression of a multitude of genes involved in the regulation of the cell cycle and an upregulation in the expression of genes involved in cell cycle checkpoints. In addition, genes involved in DNA damage response, oxidative stress, apoptosis and cell-cell signaling (cytokines) were found to be upregulated. An in silico analysis of the involved genes suggested that the transcription factors, E2F and nuclear factor (NF)-κB, may be involved in these cellular responses.

  18. Spread of a New Parasitic B Chromosome Variant Is Facilitated by High Gene Flow

    PubMed Central

    Manrique-Poyato, María Inmaculada; López-León, María Dolores; Cabrero, Josefa; Perfectti, Francisco; Camacho, Juan Pedro M.

    2013-01-01

    The B24 chromosome variant emerged several decades ago in a Spanish population of the grasshopper Eyprepocnemis plorans and is currently reaching adjacent populations. Here we report, for the first time, how a parasitic B chromosome (a strictly vertically transmitted parasite) expands its geographical range aided by high gene flow in the host species. For six years we analyzed B frequency in several populations to the east and west of the original population and found extensive spatial variation, but only a slight temporal trend. The highest B24 frequency was found in its original population (Torrox) and it decreased closer to both the eastern and the western populations. The analysis of Inter Simple Sequence Repeat (ISSR) markers showed the existence of a low but significant degree of population subdivision, as well as significant isolation by distance (IBD). Pairwise Nem estimates suggested the existence of high gene flow between the four populations located in the Torrox area, with higher values towards the east. No significant barriers to gene flow were found among these four populations, and we conclude that high gene flow is facilitating B24 diffusion both eastward and westward, with minor role for B24 drive due to the arrival of drive suppressor genes which are also frequent in the donor population. PMID:24386259

  19. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes

    PubMed Central

    Ayoub, Nadia A.; Garb, Jessica E.; Tinghitella, Robin M.; Collin, Matthew A.; Hayashi, Cheryl Y.

    2007-01-01

    Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers. PMID:17565367

  20. Meta-analysis of clinical data using human meiotic genes identifies a novel cohort of highly restricted cancer-specific marker genes.

    PubMed

    Feichtinger, Julia; Aldeailej, Ibrahim; Anderson, Rebecca; Almutairi, Mikhlid; Almatrafi, Ahmed; Alsiwiehri, Naif; Griffiths, Keith; Stuart, Nicholas; Wakeman, Jane A; Larcombe, Lee; McFarlane, Ramsay J

    2012-08-01

    Identifying cancer-specific biomarkers represents an ongoing challenge to the development of novel cancer diagnostic, prognostic and therapeutic strategies. Cancer/testis (CT) genes are an important gene family with expression tightly restricted to the testis in normal individuals but which can also be activated in cancers. Here we develop a pipeline to identify new CT genes. We analysed and validated expression profiles of human meiotic genes in normal and cancerous tissue followed by meta-analyses of clinical data sets from a range of tumour types resulting in the identification of a large cohort of highly specific cancer biomarker genes, including the recombination hot spot activator PRDM9 and the meiotic cohesin genes SMC1beta and RAD21L. These genes not only provide excellent cancer biomarkers for diagnostics and prognostics, but may serve as oncogenes and have excellent drug targeting potential.

  1. High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets.

    PubMed

    Chan, H F; Ma, S; Tian, J; Leong, K W

    2017-03-09

    The rapid advances in synthetic biology and biotechnology are increasingly demanding high-throughput screening technology, such as screening of the functionalities of synthetic genes for optimization of protein expression. Compartmentalization of single cells in water-in-oil (W/O) emulsion droplets allows screening of a vast number of individualized assays, and recent advances in automated microfluidic devices further help realize the potential of droplet technology for high-throughput screening. However these single-emulsion droplets are incompatible with aqueous phase analysis and the inner droplet environment cannot easily communicate with the external phase. We present a high-throughput, miniaturized screening platform for microchip-synthesized genes using microfluidics-generated water-in-oil-in-water (W/O/W) double emulsion (DE) droplets that overcome these limitations. Synthetic gene variants of fluorescent proteins are synthesized with a custom-built microarray inkjet synthesizer, which are then screened for expression in Escherichia coli (E. coli) cells. Bacteria bearing individual fluorescent gene variants are encapsulated as single cells into DE droplets where fluorescence signals are enhanced by 100 times within 24 h of proliferation. Enrichment of functionally-correct genes by employing an error correction method is demonstrated by screening DE droplets containing fluorescent clones of bacteria with the red fluorescent protein (rfp) gene. Permeation of isopropyl β-d-1-thiogalactopyranoside (IPTG) through the thin oil layer from the external solution initiates target gene expression. The induced expression of the synthetic fluorescent proteins from at least ∼100 bacteria per droplet generates detectable fluorescence signals to enable fluorescence-activated cell sorting (FACS) of the intact droplets. This technology obviates time- and labor-intensive cell culture typically required in conventional bulk experiment.

  2. High EGFR gene copy number predicts poor outcome in triple-negative breast cancer.

    PubMed

    Park, Heae Surng; Jang, Min Hye; Kim, Eun Joo; Kim, Hyun Jeong; Lee, Hee Jin; Kim, Yu Jung; Kim, Jee Hyun; Kang, Eunyoung; Kim, Sung-Won; Kim, In Ah; Park, So Yeon

    2014-09-01

    Epidermal growth factor receptor (EGFR) is frequently overexpressed in triple-negative breast cancer and is emerging as a therapeutic target. EGFR gene copy number alteration and mutation are highly variable and scientists have been challenged to define their prognostic significance in triple-negative breast cancer. We examined EGFR protein expression, EGFR gene copy number alteration and mutation of exon 18 to 21 in 151 cases of triple-negative breast cancer and correlated these findings with clinical outcomes. In addition, intratumoral agreement of EGFR protein overexpression and gene copy number alteration was evaluated. EGFR overexpression was found in 97 of 151 cases (64%) and high EGFR gene copy number was detected in 50 cases (33%), including 3 gene amplification (2%) and 47 high polysomy (31%). Five EGFR mutations were detected in 4 of 151 cases (3%) and included G719A in exon 18 (n=1), V786M in exon 20 (n=1), and L858R in exon 21 (n=3). One case had two mutations (G719A and L858R). High EGFR copy number, but not EGFR mutation, correlated with EGFR protein overexpression. Intratumoral heterogeneity of EGFR protein overexpression and EGFR copy number alteration was not significant. In survival analyses, high EGFR copy number was found to be an independent prognostic factor for poor disease-free survival in patients with triple-negative breast cancer. Our findings showed that EGFR mutation was a rare event, but high EGFR copy number was relatively frequent and correlated with EGFR overexpression in triple-negative breast cancer. Moreover, high EGFR copy number was associated with poor clinical outcome in triple-negative breast cancer, suggesting that evaluation of EGFR copy number may be useful for predicting outcomes in patients with triple-negative breast cancer and for selecting patients for anti-EGFR-targeted therapy.

  3. A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish.

    PubMed

    Liu, Yun; Luo, Daji; Lei, Yong; Hu, Wei; Zhao, Hui; Cheng, Christopher H K

    2014-08-15

    Transcription activator like effector nucleases (TALENs) is a promising approach to disrupt intended genomic loci. The assembly of highly effective TALENs is critical for successful genome editing. Recently we reported a convenient and robust platform to construct customized TALENs. The TALENs generated by this platform have been proven to be highly effective for gene disruption in Xenopus tropicalis and zebrafish as well as large genomic deletions in zebrafish. The one-time success rate of targeted gene disruption is about 90% for more than 100 genomic loci tested, with the mutation frequencies often reaching above 50%. Here we describe the validated protocol for TALEN assembly, methods for generating gene knockout animals in X. tropicalis and zebrafish, as well as the protocol for engineering large genomic deletions in zebrafish.

  4. High-throughput genomic mapping of vector integration sites in gene therapy studies.

    PubMed

    Beard, Brian C; Adair, Jennifer E; Trobridge, Grant D; Kiem, Hans-Peter

    2014-01-01

    Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed: linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods

  5. Screening of upregulated genes induced by high density in the vetch aphid Megoura crassicauda.

    PubMed

    Ishikawa, Asano; Ishikawa, Yuki; Okada, Yasukazu; Miyazaki, Satoshi; Miyakawa, Hitoshi; Koshikawa, Shigeyuki; Brisson, Jennifer A; Miura, Toru

    2012-03-01

    Aphids exhibit several polyphenisms in which discontinuous, alternative phenotypes are produced depending on environmental conditions. One representative example is the wing polyphenism, where winged and wingless females are produced through parthenogenesis. Previous work has shown that, in some aphid species, the density condition sensed by the mother aphid determines the developmental fate of embryos in her ovary, with high densities leading to winged progeny and low densities to wingless progeny. However, little is known about the molecular and physiological mechanisms underlying the wing polyphenism. To identify genes involved in the wing-morph determination in the vetch aphid, Megoura crassicauda, we compared maternal and embryonic transcripts between high- and low-density conditions using differential display, followed by quantitative real-time PCR (qRT-PCR). Under the high-density condition, two genes (Uba1 and Naca) were found to be upregulated in maternal tissues without ovaries, while one gene (ClpP) was upregulated in ovaries containing embryos. Uba1 and Naca encode factors that function in protein modification or transcriptional/translational regulation, respectively. In addition to differential display, candidate gene approaches focusing on morphogenetic and endocrine genes, i.e., wg, dpp, ap, hh, InR, IRS, Foxo, EcR, and USP, were also carried out. We found that wg was upregulated in maternal tissues under the high-density condition. The identified genes from both approaches are candidates for further study of their involvement in the transduction of density signals in mother aphids and/or the initial process of wing differentiation in embryos.

  6. Gene expression profiles in testis of pigs with extreme high and low levels of androstenone

    PubMed Central

    Moe, Maren; Meuwissen, Theo; Lien, Sigbjørn; Bendixen, Christian; Wang, Xuefei; Conley, Lene Nagstrup; Berget, Ingunn; Tajet, Håvard; Grindflek, Eli

    2007-01-01

    Background: Boar taint is a major obstacle when using uncastrated male pigs for swine production. One of the main compounds causing this taint is androstenone, a pheromone produced in porcine testis. Here we use microarrays to study the expression of thousands of genes simultaneously in testis of high and low androstenone boars. The study allows identification of genes and pathways associated with elevated androstenone levels, which is essential for recognising potential molecular markers for breeding purposes. Results: Testicular tissue was collected from 60 boars, 30 with extreme high and 30 with extreme low levels of androstenone, from each of the two breeds Duroc and Norwegian Landrace. The samples were hybridised to porcine arrays containing 26,877 cDNA clones, detecting 563 and 160 genes that were differentially expressed (p < 0.01) in Duroc and Norwegian Landrace, respectively. Of these significantly up- and down-regulated clones, 72 were found to be common for the two breeds, suggesting the possibility of both general and breed specific mechanisms in regulation of, or response to androstenone levels in boars. Ten genes were chosen for verification of expression patterns by quantitative real competitive PCR and real-time PCR. As expected, our results point towards steroid hormone metabolism and biosynthesis as important biological processes for the androstenone levels, but other potential pathways were identified as well. Among these were oxidoreductase activity, ferric iron binding, iron ion binding and electron transport activities. Genes belonging to the cytochrome P450 and hydroxysteroid dehydrogenase families were highly up-regulated, in addition to several genes encoding different families of conjugation enzymes. Furthermore, a number of genes encoding transcription factors were found both up- and down-regulated. The high number of clones belonging to ferric iron and iron ion binding suggests an importance of these genes, and the association between

  7. High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake

    PubMed Central

    DeMaere, Matthew Z.; Williams, Timothy J.; Allen, Michelle A.; Brown, Mark V.; Gibson, John A. E.; Rich, John; Lauro, Federico M.; Dyall-Smith, Michael; Davenport, Karen W.; Woyke, Tanja; Kyrpides, Nikos C.; Tringe, Susannah G.; Cavicchioli, Ricardo

    2013-01-01

    Deep Lake in Antarctica is a globally isolated, hypersaline system that remains liquid at temperatures down to −20 °C. By analyzing metagenome data and genomes of four isolates we assessed genome variation and patterns of gene exchange to learn how the lake community evolved. The lake is completely and uniformly dominated by haloarchaea, comprising a hierarchically structured, low-complexity community that differs greatly to temperate and tropical hypersaline environments. The four Deep Lake isolates represent distinct genera (∼85% 16S rRNA gene similarity and ∼73% genome average nucleotide identity) with genomic characteristics indicative of niche adaptation, and collectively account for ∼72% of the cellular community. Network analysis revealed a remarkable level of intergenera gene exchange, including the sharing of long contiguous regions (up to 35 kb) of high identity (∼100%). Although the genomes of closely related Halobacterium, Haloquadratum, and Haloarcula (>90% average nucleotide identity) shared regions of high identity between species or strains, the four Deep Lake isolates were the only distantly related haloarchaea to share long high-identity regions. Moreover, the Deep Lake high-identity regions did not match to any other hypersaline environment metagenome data. The most abundant species, tADL, appears to play a central role in the exchange of insertion sequences, but not the exchange of high-identity regions. The genomic characteristics of the four haloarchaea are consistent with a lake ecosystem that sustains a high level of intergenera gene exchange while selecting for ecotypes that maintain sympatric speciation. The peculiarities of this polar system restrict which species can grow and provide a tempo and mode for accentuating gene exchange. PMID:24082106

  8. High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake.

    PubMed

    DeMaere, Matthew Z; Williams, Timothy J; Allen, Michelle A; Brown, Mark V; Gibson, John A E; Rich, John; Lauro, Federico M; Dyall-Smith, Michael; Davenport, Karen W; Woyke, Tanja; Kyrpides, Nikos C; Tringe, Susannah G; Cavicchioli, Ricardo

    2013-10-15

    Deep Lake in Antarctica is a globally isolated, hypersaline system that remains liquid at temperatures down to -20 °C. By analyzing metagenome data and genomes of four isolates we assessed genome variation and patterns of gene exchange to learn how the lake community evolved. The lake is completely and uniformly dominated by haloarchaea, comprising a hierarchically structured, low-complexity community that differs greatly to temperate and tropical hypersaline environments. The four Deep Lake isolates represent distinct genera (∼85% 16S rRNA gene similarity and ∼73% genome average nucleotide identity) with genomic characteristics indicative of niche adaptation, and collectively account for ∼72% of the cellular community. Network analysis revealed a remarkable level of intergenera gene exchange, including the sharing of long contiguous regions (up to 35 kb) of high identity (∼100%). Although the genomes of closely related Halobacterium, Haloquadratum, and Haloarcula (>90% average nucleotide identity) shared regions of high identity between species or strains, the four Deep Lake isolates were the only distantly related haloarchaea to share long high-identity regions. Moreover, the Deep Lake high-identity regions did not match to any other hypersaline environment metagenome data. The most abundant species, tADL, appears to play a central role in the exchange of insertion sequences, but not the exchange of high-identity regions. The genomic characteristics of the four haloarchaea are consistent with a lake ecosystem that sustains a high level of intergenera gene exchange while selecting for ecotypes that maintain sympatric speciation. The peculiarities of this polar system restrict which species can grow and provide a tempo and mode for accentuating gene exchange.

  9. DeGNServer: deciphering genome-scale gene networks through high performance reverse engineering analysis.

    PubMed

    Li, Jun; Wei, Hairong; Zhao, Patrick Xuechun

    2013-01-01

    Analysis of genome-scale gene networks (GNs) using large-scale gene expression data provides unprecedented opportunities to uncover gene interactions and regulatory networks involved in various biological processes and developmental programs, leading to accelerated discovery of novel knowledge of various biological processes, pathways and systems. The widely used context likelihood of relatedness (CLR) method based on the mutual information (MI) for scoring the similarity of gene pairs is one of the accurate methods currently available for inferring GNs. However, the MI-based reverse engineering method can achieve satisfactory performance only when sample size exceeds one hundred. This in turn limits their applications for GN construction from expression data set with small sample size. We developed a high performance web server, DeGNServer, to reverse engineering and decipher genome-scale networks. It extended the CLR method by integration of different correlation methods that are suitable for analyzing data sets ranging from moderate to large scale such as expression profiles with tens to hundreds of microarray hybridizations, and implemented all analysis algorithms using parallel computing techniques to infer gene-gene association at extraordinary speed. In addition, we integrated the SNBuilder and GeNa algorithms for subnetwork extraction and functional module discovery. DeGNServer is publicly and freely available online.

  10. Identification of Genes Preferentially Expressed by Highly Virulent Piscine Streptococcus agalactiae upon Interaction with Macrophages

    PubMed Central

    Guo, Chang-Ming; Chen, Rong-Rong; Kalhoro, Dildar Hussain; Wang, Zhao-Fei; Liu, Guang-Jin; Lu, Cheng-Ping; Liu, Yong-Jie

    2014-01-01

    Streptococcus agalactiae, long recognized as a mammalian pathogen, is an emerging concern with regard to fish. In this study, we used a mouse model and in vitro cell infection to evaluate the pathogenetic characteristics of S. agalactiae GD201008-001, isolated from tilapia in China. This bacterium was found to be highly virulent and capable of inducing brain damage by migrating into the brain by crossing the blood–brain barrier (BBB). The phagocytosis assays indicated that this bacterium could be internalized by murine macrophages and survive intracellularly for more than 24 h, inducing injury to macrophages. Further, selective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 6 functional categories. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria, and four showed no significant similarities to any other previously described genes. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cross the BBB. PMID:24498419

  11. Mammalian ets-1 and ets-2 genes encode highly conserved proteins.

    PubMed Central

    Watson, D K; McWilliams, M J; Lapis, P; Lautenberger, J A; Schweinfest, C W; Papas, T S

    1988-01-01

    Cellular ets sequences homologous to v-ets of the avian leukemia virus E26 are highly conserved. In mammals the ets sequences are dispersed on two separate chromosomal loci, called ets-1 and ets-2. To determine the structure of these two genes and identify the open reading frames that code for the putative proteins, we have sequenced human ets-1 cDNAs and ets-2 cDNA clones obtained from both human and mouse. The human ETS1 gene is capable of encoding a protein of 441 amino acids. This protein is greater than 95% identical to the chicken c-ets-1 gene product. Thus, the human ETS1 gene is homologous to the chicken c-ets-1 gene, the protooncogene that the E26 virus transduced. Human and mouse ets-2 cDNA clones are closely related and contain open reading frames capable of encoding proteins of 469 and 468 residues, respectively. Direct comparison of these data with previously published findings indicates that ets is a family of genes whose members share distinct domains. PMID:2847145

  12. Preventing High Fat Diet-induced Obesity and Improving Insulin Sensitivity through Neuregulin 4 Gene Transfer

    PubMed Central

    Ma, Yongjie; Gao, Mingming; Liu, Dexi

    2016-01-01

    Neuregulin 4 (NRG4), an epidermal growth factor-like signaling molecule, plays an important role in cell-to-cell communication during tissue development. Its function to regulate energy metabolism has recently been reported. This current study was designed to assess the preventive and therapeutic effects of NRG4 overexpression on high fat diet (HFD)-induced obesity. Using the hydrodynamic gene transfer method, we demonstrate that Nrg4 gene transfer in mice suppressed the development of diet-induced obesity, but did not affect pre-existing adiposity and body weight in obese mice. Nrg4 gene transfer curbed HFD-induced hepatic steatosis by inhibiting lipogenesis and PPARγ-mediated lipid storage. Concurrently, overexpression of NRG4 reduced chronic inflammation in both preventive and treatment studies, evidenced by lower mRNA levels of macrophage marker genes including F4/80, Cd68, Cd11b, Cd11c, and macrophage chemokine Mcp1, resulting in improved insulin sensitivity. Collectively, these results demonstrate that overexpression of the Nrg4 gene by hydrodynamic gene delivery prevents HFD-induced weight gain and fatty liver, alleviates obesity-induced chronic inflammation and insulin resistance, and supports the health benefits of NRG4 in managing obesity and obesity-associated metabolic disorders. PMID:27184920

  13. A Benefit of High Temperature: Increased Effectiveness of a Rice Bacterial Blight Disease Resistance Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High temperatures promote development of many plant diseases and reduce effectiveness of disease resistance (R) genes. In many rice producing countries, two crops of rice are produced, with more disease occurring in the season with higher day/night temperatures. While studying the factors that influ...

  14. High School Students' Understanding of Chromosome/Gene Behavior during Meiosis.

    ERIC Educational Resources Information Center

    Stewart, Jim; Dale, Michael

    1989-01-01

    Investigates high school students' understanding of the physical relationship of chromosomes and genes as expressed in their conceptual models and in their ability to manipulate the models to explain solutions to dihybrid cross problems. Describes three typical models and three students' reasoning processes. Discusses four implications. (YP)

  15. Turning (Ir gene) low responders into high responders by antibody manipulation of the developing immune system.

    PubMed Central

    Martinz, C; Marcos, M A; Pereira, P; Marquez, C; Toribio, M; de la Hera, A; Cazenave, P A; Coutinho, A

    1987-01-01

    The ability of helper T cells directed against trinitrophenyl-modified syngeneic spleen cells to recognize low-hapten densities on target cells is under major histocompatibility complex-linked Ir gene control. Thus, BALB/c (H-2d) mice are low responders while H-2 congenic BALB.C3H (H-2k) mice are high responders. Immunization of adult BALB/c mice with the monoclonal antibody F6(51), directed to shared idiotopes by anti-trinitrophenyl antibodies and clonal receptors on anti-trinitrophenyl-self helper T cells, leads to the production of high titers of circulating idiotype, has no influence on helper T cell idiotypic profiles, but shifts to a high-responder phenotype the ability of helper T cells to recognize low-hapten densities. These effects on Ir gene phenotype are even more striking in untreated progenies from F6(51)-immunized BALB/c females, which are better responders than genetically high-responder BALB.C3H mice, although completely different in the expression of the F6(51)-defined clonotype. The general significance of these findings on Ir gene-directed T-cell repertoire selection is discussed, for they constitute formal evidence against antigen-presentation as a mechanism of Ir gene effects and strong support for the importance of maternal influences on the development of T-cell repertoires. PMID:2954161

  16. Antibiotic Resistance Genes in Freshwater Biofilms May Reflect Influences from High-Intensity Agriculture.

    PubMed

    Winkworth-Lawrence, Cynthia; Lange, Katharina

    2016-11-01

    Antibiotic resistance is a major public health concern with growing evidence of environmental gene reservoirs, especially in freshwater. However, the presence of antibiotic resistance genes in freshwater, in addition to the wide spectrum of land use contaminants like nitrogen and phosphate, that waterways are subjected to is inconclusive. Using molecular analyses, freshwater benthic rock biofilms were screened for genes conferring resistance to antibiotics used in both humans and farmed animals (aacA-aphD to aminoglycosides; mecA to ß-lactams; ermA and ermB to macrolides; tetA, tetB, tetK, and tetM to tetracyclines; vanA and vanB to glycopeptides). We detected widespread low levels of antibiotic resistance genes from 20 waterways across southern New Zealand throughout the year (1.3 % overall detection rate; 480 samples from three rocks per site, 20 sites, eight occasions; July 2010-May 2011). Three of the ten genes, ermB, tetK, and tetM, were detected in 62 of the 4800 individual screens; representatives confirmed using Sanger sequencing. No distinction could be made between human and agricultural land use contamination sources based on gene presence distribution alone. However, land use pressures are suggested by moderate correlations between antibiotic resistance genes and high-intensity farming in winter. The detection of antibiotic resistance genes at several sites not subject to known agricultural pressures suggests human sources of resistance, like waterway contamination resulting from unsatisfactory toilet facilities at recreational sites.

  17. Putative cis-regulatory elements in genes highly expressed in rice sperm cells

    PubMed Central

    2011-01-01

    Background The male germ line in flowering plants is initiated within developing pollen grains via asymmetric division. The smaller cell then becomes totally encased within a much larger vegetative cell, forming a unique "cell within a cell structure". The generative cell subsequently divides to give rise to two non-motile diminutive sperm cells, which take part in double fertilization and lead to the seed set. Sperm cells are difficult to investigate because of their presence within the confines of the larger vegetative cell. However, recently developed techniques for the isolation of rice sperm cells and the fully annotated rice genome sequence have allowed for the characterization of the transcriptional repertoire of sperm cells. Microarray gene expression data has identified a subset of rice genes that show unique or highly preferential expression in sperm cells. This information has led to the identification of cis-regulatory elements (CREs), which are conserved in sperm-expressed genes and are putatively associated with the control of cell-specific expression. Findings We aimed to identify the CREs associated with rice sperm cell-specific gene expression data using in silico prediction tools. We analyzed 1-kb upstream regions of the top 40 sperm cell co-expressed genes for over-represented conserved and novel motifs. Analysis of upstream regions with the SIGNALSCAN program with the PLACE database, MEME and the Mclip tool helped to find combinatorial sets of known transcriptional factor-binding sites along with two novel motifs putatively associated with the co-expression of sperm cell-specific genes. Conclusions Our data shows the occurrence of novel motifs, which are putative CREs and are likely targets of transcriptional factors regulating sperm cell gene expression. These motifs can be used to design the experimental verification of regulatory elements and the identification of transcriptional factors that regulate sperm cell-specific gene expression. PMID

  18. Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

    PubMed

    Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

    2013-07-01

    Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

  19. Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression.

    PubMed

    Rautio, Jari J; Huuskonen, Anne; Vuokko, Heikki; Vidgren, Virve; Londesborough, John

    2007-09-01

    Brewer's yeast experiences constantly changing environmental conditions during wort fermentation. Cells can rapidly adapt to changing surroundings by transcriptional regulation. Changes in genomic expression can indicate the physiological condition of yeast in the brewing process. We monitored, using the transcript analysis with aid of affinity capture (TRAC) method, the expression of some 70 selected genes relevant to wort fermentation at high frequency through 9-10 day fermentations of very high gravity wort (25 degrees P) by an industrial lager strain. Rapid changes in expression occurred during the first hours of fermentations for several genes, e.g. genes involved in maltose metabolism, glycolysis and ergosterol synthesis were strongly upregulated 2-6 h after pitching. By the time yeast growth had stopped (72 h) and total sugars had dropped by about 50%, most selected genes had passed their highest expression levels and total mRNA was less than half the levels during growth. There was an unexpected upregulation of some genes of oxygen-requiring pathways during the final fermentation stages. For five genes, expression of both the Saccharomyces cerevisiae and S. bayanus components of the hybrid lager strain were determined. Expression profiles were either markedly different (ADH1, ERG3) or very similar (MALx1, ILV5, ATF1) between these two components. By frequent analysis of a chosen set of genes, TRAC provided a detailed and dynamic picture of the physiological state of the fermenting yeast. This approach offers a possible way to monitor and optimize the performance of yeast in a complex process environment.

  20. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases.

    PubMed

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-06-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

  1. Two homologous low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins.

    PubMed Central

    Capel, J; Jarillo, J A; Salinas, J; Martínez-Zapater, J M

    1997-01-01

    We have characterized two related cDNAs (RCI2A and RCI2B) corresponding to genes from Arabidopsis thaliana, the expression of which is transiently induced by low, nonfreezing temperatures. RCI2A and RCI2B encode small (54 amino acids), highly hydrophobic proteins that bear two potential transmembrane domains. They show similarity to proteins encoded by genes from barley (Hordeum vulgare L.) and wheatgrass (Lophophyrum elongatum) that are regulated by different stress conditions. Their high level of sequence homology (78%) and their genomic location in a single restriction fragment suggest that both genes originated as a result of a tandem duplication. However, their regulatory sequences have diverged enough to confer on them different expression patterns. Like most of the cold-inducible plant genes characterized, the expression of RCI2A and RCI2B is also promoted by abscisic acid (ABA) and dehydration but is not a general response to stress conditions, since it is not induced by salt stress or by anaerobiosis. Furthermore, low temperatures are able to induce RCI2A and RCI2B expression in ABA-deficient and -insensitive genetic backgrounds, indicating that both ABA-dependent and -independent pathways regulate the low-temperature responsiveness of these two genes. PMID:9342870

  2. Different gene expressions between cattle and yak provide insights into high-altitude adaptation.

    PubMed

    Wang, K; Yang, Y; Wang, L; Ma, T; Shang, H; Ding, L; Han, J; Qiu, Q

    2016-02-01

    DNA sequence variation has been widely reported as the genetic basis for adaptation, in both humans and other animals, to the hypoxic environment experienced at high altitudes. However, little is known about the patterns of gene expression underlying such hypoxic adaptations. In this study, we examined the differences in the transcriptomes of four organs (heart, kidney, liver and lung) between yak and cattle, a pair of closely related species distributed at high and low altitudes respectively. Of the four organs examined, heart shows the greatest differentiation between the two species in terms of gene expression profiles. Detailed analyses demonstrated that some genes associated with the oxygen supply system and the defense systems that respond to threats of hypoxia are differentially expressed. In addition, genes with significantly differentiated patterns of expression in all organs exhibited an unexpected uniformity of regulation along with an elevated frequency of nonsynonymous substitutions. This co-evolution of protein sequences and gene expression patterns is likely to be correlated with the optimization of the yak metabolic system to resist hypoxia.

  3. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases

    PubMed Central

    Qiu, Zhongwei; Liu, Meizhen; Chen, Zhaohua; Shao, Yanjiao; Pan, Hongjie; Wei, Gaigai; Yu, Chao; Zhang, Long; Li, Xia; Wang, Ping; Fan, Heng-Yu; Du, Bing; Liu, Bin; Liu, Mingyao; Li, Dali

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ∼40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background. PMID:23630316

  4. Reverse engineering and analysis of genome-wide gene regulatory networks from gene expression profiles using high-performance computing.

    PubMed

    Belcastro, Vincenzo; Gregoretti, Francesco; Siciliano, Velia; Santoro, Michele; D'Angelo, Giovanni; Oliva, Gennaro; di Bernardo, Diego

    2012-01-01

    Regulation of gene expression is a carefully regulated phenomenon in the cell. “Reverse-engineering” algorithms try to reconstruct the regulatory interactions among genes from genome-scale measurements of gene expression profiles (microarrays). Mammalian cells express tens of thousands of genes; hence, hundreds of gene expression profiles are necessary in order to have acceptable statistical evidence of interactions between genes. As the number of profiles to be analyzed increases, so do computational costs and memory requirements. In this work, we designed and developed a parallel computing algorithm to reverse-engineer genome-scale gene regulatory networks from thousands of gene expression profiles. The algorithm is based on computing pairwise Mutual Information between each gene-pair. We successfully tested it to reverse engineer the Mus Musculus (mouse) gene regulatory network in liver from gene expression profiles collected from a public repository. A parallel hierarchical clustering algorithm was implemented to discover “communities” within the gene network. Network communities are enriched for genes involved in the same biological functions. The inferred network was used to identify two mitochondrial proteins.

  5. Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia.

    PubMed

    Li, Chun Ge; Wang, Hui; Chen, Hong Ju; Zhao, Yan; Fu, Pei Sheng; Ji, Xiang Shan

    2014-01-01

    Nowadays, high temperature effects on the molecular pathways during sex differentiation in teleosts need to be deciphered. In this study, a systematic differential expression analysis of genes involved in high temperature-induced sex differentiation was done in the Nile tilapia gonad and brain. Our results showed that high temperature caused significant down-regulation of CYP19A1A in the gonad of both sexes in induction group, and FOXL2 in the ovary of the induction group. The expressions of GTHα, LHβ and ERα were also significantly down-regulated in the brain of both sexes in the induction and recovery groups. On the contrary, the expression of CYP11B2 was significantly up-regulated in the ovary, but not in the testis in both groups. Spearman rank correlation analysis showed that there are significant correlations between the expressions of CYP19A1A, FOXL2, or DMRT1 in the gonads and the expression of some genes in the brain. Another result in this study showed that high temperature up-regulated the expression level of DNMT1 in the testis of the induction group, and DNMT1 and DNMT3A in the female brain of both groups. The expression and correlation analysis of HSPs showed that high temperature action on tilapia HSPs might indirectly induce the expression changes of sex differentiation genes in the gonads. These findings provide new insights on TSD and suggest that sex differentiation related genes, heat shock proteins, and DNA methylation genes are new candidates for studying TSD in fish species.

  6. SNP-based high density genetic map and mapping of btwd1 dwarfing gene in barley

    PubMed Central

    Ren, Xifeng; Wang, Jibin; Liu, Lipan; Sun, Genlou; Li, Chengdao; Luo, Hong; Sun, Dongfa

    2016-01-01

    A high-density linkage map is a valuable tool for functional genomics and breeding. A newly developed sequence-based marker technology, restriction site associated DNA (RAD) sequencing, has been proven to be powerful for the rapid discovery and genotyping of genome-wide single nucleotide polymorphism (SNP) markers and for the high-density genetic map construction. The objective of this research was to construct a high-density genetic map of barley using RAD sequencing. 1894 high-quality SNP markers were developed and mapped onto all seven chromosomes together with 68 SSR markers. These 1962 markers constituted a total genetic length of 1375.8 cM and an average of 0.7 cM between adjacent loci. The number of markers within each linkage group ranged from 209 to 396. The new recessive dwarfing gene btwd1 in Huaai 11 was mapped onto the high density linkage maps. The result showed that the btwd1 is positioned between SNP marks 7HL_6335336 and 7_249275418 with a genetic distance of 0.9 cM and 0.7 cM on chromosome 7H, respectively. The SNP-based high-density genetic map developed and the dwarfing gene btwd1 mapped in this study provide critical information for position cloning of the btwd1 gene and molecular breeding of barley. PMID:27530597

  7. High occurrence of functional new chimeric genes in survey of rice chromosome 3 short arm genome sequences.

    PubMed

    Zhang, Chengjun; Wang, Jun; Marowsky, Nicholas C; Long, Manyuan; Wing, Rod A; Fan, Chuanzhu

    2013-01-01

    In an effort to identify newly evolved genes in rice, we searched the genomes of Asian-cultivated rice Oryza sativa ssp. japonica and its wild progenitors, looking for lineage-specific genes. Using genome pairwise comparison of approximately 20-Mb DNA sequences from the chromosome 3 short arm (Chr3s) in six rice species, O. sativa, O. nivara, O. rufipogon, O. glaberrima, O. barthii, and O. punctata, combined with synonymous substitution rate tests and other evidence, we were able to identify potential recently duplicated genes, which evolved within the last 1 Myr. We identified 28 functional O. sativa genes, which likely originated after O. sativa diverged from O. glaberrima. These genes account for around 1% (28/3,176) of all annotated genes on O. sativa's Chr3s. Among the 28 new genes, two recently duplicated segments contained eight genes. Fourteen of the 28 new genes consist of chimeric gene structure derived from one or multiple parental genes and flanking targeting sequences. Although the majority of these 28 new genes were formed by single or segmental DNA-based gene duplication and recombination, we found two genes that were likely originated partially through exon shuffling. Sequence divergence tests between new genes and their putative progenitors indicated that new genes were most likely evolving under natural selection. We showed all 28 new genes appeared to be functional, as suggested by Ka/Ks analysis and the presence of RNA-seq, cDNA, expressed sequence tag, massively parallel signature sequencing, and/or small RNA data. The high rate of new gene origination and of chimeric gene formation in rice may demonstrate rice's broad diversification, domestication, its environmental adaptation, and the role of new genes in rice speciation.

  8. High-resolution timing of cell cycle-regulated gene expression

    PubMed Central

    Rowicka, Maga; Kudlicki, Andrzej; Tu, Benjamin P.; Otwinowski, Zbyszek

    2007-01-01

    The eukaryotic cell division cycle depends on an intricate sequence of transcriptional events. Using an algorithm based on maximum-entropy deconvolution, and expression data from a highly synchronized yeast culture, we have timed the peaks of expression of transcriptionally regulated cell cycle genes to an accuracy of 2 min (≈1% of the cell cycle time). The set of 1,129 cell cycle-regulated genes was identified by a comprehensive analysis encompassing all available cell cycle yeast data sets. Our results reveal distinct subphases of the cell cycle undetectable by morphological observation, as well as the precise timeline of macromolecular complex assembly during key cell cycle events. PMID:17827275

  9. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  10. A high-resolution map of the regulator of the complement activation gene cluster on 1q32 that integrates new genes and markers.

    PubMed

    Heine-Suñer, D; Díaz-Guillén, M A; de Villena, F P; Robledo, M; Benítez, J; Rodríguez de Córdoba, S

    1997-01-01

    Sixteen microsatellite markers, including two described here, were used to construct a high-resolution map of the 1q32 region encompassing the regulator of the complement activation (RCA) gene cluster. The RCA genes are a group of related genes coding for plasma and membrane associated proteins that collectively control activation of the complement component C3. We provide here the location of two new genes within the RCA gene cluster. These genes are PFKFB2 that maps 15 kilobases (kb) upstream of the C4BPB gene, and a gene located 4 kb downstream of C4BPA, which seems to code for the 72 000 Mr component of the signal recognition particle (SRP72). Neither of these two genes is related structurally or functionally to the RCA genes. In addition, our map shows the centromere-telomere orientation of the C4BPB/MCP linkage group, which is: centromere-PFKFB2-C4BPB-C4BPA-SRP72-C4BPAL1++ +-C4BPAL2-telomere, and outlines an interval with a significant female-male recombination difference which suggests the presence of a female-specific hotspot(s) of recombination.

  11. High initiation rates at the ribosomal gene promoter do not depend upon spacer transcription.

    PubMed Central

    Labhart, P; Reeder, R H

    1989-01-01

    We report experiments that test the model that in Xenopus laevis, RNA polymerase I is "handed over" in a conservative fashion from the T3 terminator to the adjacent gene promoter. We have introduced transcription-terminating lesions into the ribosomal DNA repeat by irradiating cultured cells with ultraviolet light. We used isolated nuclei to measure the effect of such lesions on transcription. UV damage sufficient to prevent all elongating RNA polymerase from reaching T3 from upstream had no adverse effect on the density of RNA polymerase at the very 5' end of the gene. We conclude that high rates of transcription initiation at the gene promoter do not depend upon polymerase passing from one repeat to the next or on polymerase initiating at the spacer promoters. Images PMID:2470092

  12. Gene Signature of High White Blood Cell Count in B-Precursor Acute Lymphoblastic Leukemia.

    PubMed

    Edwards, Holly; Rubenstein, Mara; Dombkowski, Alan A; Caldwell, J Timothy; Chu, Roland; Xavier, Ana C; Thummel, Ryan; Neely, Melody; Matherly, Larry H; Ge, Yubin; Taub, Jeffrey W

    2016-01-01

    In this study we sought to identify genetic factors associated with the presenting white blood cell (WBC) count in B-precursor acute lymphoblastic leukemia (BP-ALL). Using ETV6-RUNX1-positive BP-ALL patient samples, a homogeneous subtype, we identified 16 differentially expressed genes based on the presenting WBC count (< 50,000/cumm vs > 50,000). We further confirmed that IL1R1, BCAR3, KCNH2, PIR, and ZDHHC23 were differentially expressed in a larger cohort of ETV6-RUNX1-negative BP-ALL patient samples. Statistical analysis demonstrated that expression levels of these genes could accurately categorize high and low WBC count subjects using two independent patient sets, representing positive and negative ETV6-RUNX1 cases. Further studies in leukemia cell line models will better delineate the role of these genes in regulating the white blood cell count and potentially identify new therapeutic targets.

  13. Diet-dependent gene expression in honey bees: honey vs. sucrose or high fructose corn syrup

    PubMed Central

    Wheeler, Marsha M.; Robinson, Gene E.

    2014-01-01

    Severe declines in honey bee populations have made it imperative to understand key factors impacting honey bee health. Of major concern is nutrition, as malnutrition in honey bees is associated with immune system impairment and increased pesticide susceptibility. Beekeepers often feed high fructose corn syrup (HFCS) or sucrose after harvesting honey or during periods of nectar dearth. We report that, relative to honey, chronic feeding of either of these two alternative carbohydrate sources elicited hundreds of differences in gene expression in the fat body, a peripheral nutrient-sensing tissue analogous to vertebrate liver and adipose tissues. These expression differences included genes involved in protein metabolism and oxidation-reduction, including some involved in tyrosine and phenylalanine metabolism. Differences between HFCS and sucrose diets were much more subtle and included a few genes involved in carbohydrate and lipid metabolism. Our results suggest that bees receive nutritional components from honey that are not provided by alternative food sources widely used in apiculture. PMID:25034029

  14. Diet-dependent gene expression in honey bees: honey vs. sucrose or high fructose corn syrup.

    PubMed

    Wheeler, Marsha M; Robinson, Gene E

    2014-07-17

    Severe declines in honey bee populations have made it imperative to understand key factors impacting honey bee health. Of major concern is nutrition, as malnutrition in honey bees is associated with immune system impairment and increased pesticide susceptibility. Beekeepers often feed high fructose corn syrup (HFCS) or sucrose after harvesting honey or during periods of nectar dearth. We report that, relative to honey, chronic feeding of either of these two alternative carbohydrate sources elicited hundreds of differences in gene expression in the fat body, a peripheral nutrient-sensing tissue analogous to vertebrate liver and adipose tissues. These expression differences included genes involved in protein metabolism and oxidation-reduction, including some involved in tyrosine and phenylalanine metabolism. Differences between HFCS and sucrose diets were much more subtle and included a few genes involved in carbohydrate and lipid metabolism. Our results suggest that bees receive nutritional components from honey that are not provided by alternative food sources widely used in apiculture.

  15. Analysis on differential expressed genes of ovarian tissue between high- and low-yield laying hen.

    PubMed

    Chen, Wei; Song, Ling-Jun; Zeng, Yong-Qing; Yang, Yun; Wang, Hui

    2013-01-01

    In order to elucidate molecular genetic mechanism of laying hen reproduction at the transcriptional level and the structure of significantly differential genes, the mRNA differential display and reverse northern dot-blot were used to detect the differential expression of genes in the ovary tissue of low-yield laying hens and high-yield laying hens in the present study. Sixteen 32-week-old CAU-pink laying hens divided into two groups were used and the laying performance was measured. The results showed that only the egg numbers were significantly different between the two groups; and from 15 primer pairs, a total of 336 bands were displayed of which 59 cDNA bands were found to be differentially expressed in both high-yield and low-yield laying hen. The sequence analysis indicated that the expression of such bands as H-AP5, H-P5, and H-P4 was significantly potentiated in high-yield laying hen using primer pairs AP5/HT11G, P5/HT11G and P4/HT11G and these transcripts had high homology (98%) to HoxDb, HoxCa, and HoxBa, respectively. The differentially expressed gene fragments may be relevant to the progression of the high-yield hens to the egg-laying stage. And further study is required to elucidate the molecular function to improve the productivity of laying hens.

  16. Putative extremely high rate of proteome innovation in lancelets might be explained by high rate of gene prediction errors.

    PubMed

    Bányai, László; Patthy, László

    2016-08-01

    A recent analysis of the genomes of Chinese and Florida lancelets has concluded that the rate of creation of novel protein domain combinations is orders of magnitude greater in lancelets than in other metazoa and it was suggested that continuous activity of transposable elements in lancelets is responsible for this increased rate of protein innovation. Since morphologically Chinese and Florida lancelets are highly conserved, this finding would contradict the observation that high rates of protein innovation are usually associated with major evolutionary innovations. Here we show that the conclusion that the rate of proteome innovation is exceptionally high in lancelets may be unjustified: the differences observed in domain architectures of orthologous proteins of different amphioxus species probably reflect high rates of gene prediction errors rather than true innovation.

  17. Putative extremely high rate of proteome innovation in lancelets might be explained by high rate of gene prediction errors

    PubMed Central

    Bányai, László; Patthy, László

    2016-01-01

    A recent analysis of the genomes of Chinese and Florida lancelets has concluded that the rate of creation of novel protein domain combinations is orders of magnitude greater in lancelets than in other metazoa and it was suggested that continuous activity of transposable elements in lancelets is responsible for this increased rate of protein innovation. Since morphologically Chinese and Florida lancelets are highly conserved, this finding would contradict the observation that high rates of protein innovation are usually associated with major evolutionary innovations. Here we show that the conclusion that the rate of proteome innovation is exceptionally high in lancelets may be unjustified: the differences observed in domain architectures of orthologous proteins of different amphioxus species probably reflect high rates of gene prediction errors rather than true innovation. PMID:27476717

  18. Reduced Set of Virulence Genes Allows High Accuracy Prediction of Bacterial Pathogenicity in Humans

    PubMed Central

    Iraola, Gregorio; Vazquez, Gustavo; Spangenberg, Lucía; Naya, Hugo

    2012-01-01

    Although there have been great advances in understanding bacterial pathogenesis, there is still a lack of integrative information about what makes a bacterium a human pathogen. The advent of high-throughput sequencing technologies has dramatically increased the amount of completed bacterial genomes, for both known human pathogenic and non-pathogenic strains; this information is now available to investigate genetic features that determine pathogenic phenotypes in bacteria. In this work we determined presence/absence patterns of different virulence-related genes among more than finished bacterial genomes from both human pathogenic and non-pathogenic strains, belonging to different taxonomic groups (i.e: Actinobacteria, Gammaproteobacteria, Firmicutes, etc.). An accuracy of 95% using a cross-fold validation scheme with in-fold feature selection is obtained when classifying human pathogens and non-pathogens. A reduced subset of highly informative genes () is presented and applied to an external validation set. The statistical model was implemented in the BacFier v1.0 software (freely available at ), that displays not only the prediction (pathogen/non-pathogen) and an associated probability for pathogenicity, but also the presence/absence vector for the analyzed genes, so it is possible to decipher the subset of virulence genes responsible for the classification on the analyzed genome. Furthermore, we discuss the biological relevance for bacterial pathogenesis of the core set of genes, corresponding to eight functional categories, all with evident and documented association with the phenotypes of interest. Also, we analyze which functional categories of virulence genes were more distinctive for pathogenicity in each taxonomic group, which seems to be a completely new kind of information and could lead to important evolutionary conclusions. PMID:22916122

  19. Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing.

    PubMed

    Vannette, Rachel L; Mohamed, Abbas; Johnson, Brian R

    2015-11-09

    Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging.

  20. High intensity focused ultrasound-induced gene activation in sublethally injured tumor cells in vitro

    NASA Astrophysics Data System (ADS)

    Liu, Yunbo; Kon, Takashi; Li, Chuanyuan; Zhong, Pei

    2005-11-01

    Cultured human cervical cancer (HeLa) and rat mammary carcinoma (R3230Ac) cells were transfected with vectors encoding green fluorescent protein (GFP) under the control of hsp70B promoter. Aliquots of 10-μl transfected cells (5×107 cells/ml) were placed in 0.2-ml thin-wall polymerase chain reaction tubes and exposed to 1.1-MHz high intensity focused ultrasound (HIFU) at a peak negative pressure P-=2.68 MPa. By adjusting the duty cycle of the HIFU transducer, the cell suspensions were heated to a peak temperature from 50 to 70 °C in 1-10 s. Exposure dependent cell viability and gene activation were evaluated. For a 5-s HIFU exposure, cell viability dropped from 95% at 50 °C to 13% at 70 °C. Concomitantly, gene activation in sublethally injured tumor cells increased from 4% at 50 °C to 41% at 70 °C. A similar trend was observed at 60 °C peak temperature as the exposure time increased from 1 to 5 s. Further increase of exposure duration to 10 s led to significantly reduced cell viability and lower overall gene activation in exposed cells. Altogether, maximum HIFU-induced gene activation was achieved at 60 °C in 5 s. Under these experimental conditions, HIFU-induced gene activation was found to be produced primarily by thermal rather than mechanical stresses.

  1. High rates of antimicrobial drug resistance gene acquisition after international travel, The Netherlands.

    PubMed

    von Wintersdorff, Christian J H; Penders, John; Stobberingh, Ellen E; Oude Lashof, Astrid M L; Hoebe, Christian J P A; Savelkoul, Paul H M; Wolffs, Petra F G

    2014-04-01

    We investigated the effect of international travel on the gut resistome of 122 healthy travelers from the Netherlands by using a targeted metagenomic approach. Our results confirm high acquisition rates of the extended-spectrum β-lactamase encoding gene blaCTX-M, documenting a rise in prevalence from 9.0% before travel to 33.6% after travel (p<0.001). The prevalence of quinolone resistance encoding genes qnrB and qnrS increased from 6.6% and 8.2% before travel to 36.9% and 55.7% after travel, respectively (both p<0.001). Travel to Southeast Asia and the Indian subcontinent was associated with the highest acquisition rates of qnrS and both blaCTX-M and qnrS, respectively. Investigation of the associations between the acquisitions of the blaCTX-M and qnr genes showed that acquisition of a blaCTX-M gene was not associated with that of a qnrB (p = 0.305) or qnrS (p = 0.080) gene. These findings support the increasing evidence that travelers contribute to the spread of antimicrobial drug resistance.

  2. Forager bees (Apis mellifera) highly express immune and detoxification genes in tissues associated with nectar processing

    PubMed Central

    Vannette, Rachel L.; Mohamed, Abbas; Johnson, Brian R.

    2015-01-01

    Pollinators, including honey bees, routinely encounter potentially harmful microorganisms and phytochemicals during foraging. However, the mechanisms by which honey bees manage these potential threats are poorly understood. In this study, we examine the expression of antimicrobial, immune and detoxification genes in Apis mellifera and compare between forager and nurse bees using tissue-specific RNA-seq and qPCR. Our analysis revealed extensive tissue-specific expression of antimicrobial, immune signaling, and detoxification genes. Variation in gene expression between worker stages was pronounced in the mandibular and hypopharyngeal gland (HPG), where foragers were enriched in transcripts that encode antimicrobial peptides (AMPs) and immune response. Additionally, forager HPGs and mandibular glands were enriched in transcripts encoding detoxification enzymes, including some associated with xenobiotic metabolism. Using qPCR on an independent dataset, we verified differential expression of three AMP and three P450 genes between foragers and nurses. High expression of AMP genes in nectar-processing tissues suggests that these peptides may contribute to antimicrobial properties of honey or to honey bee defense against environmentally-acquired microorganisms. Together, these results suggest that worker role and tissue-specific expression of AMPs, and immune and detoxification enzymes may contribute to defense against microorganisms and xenobiotic compounds acquired while foraging. PMID:26549293

  3. High Rates of Antimicrobial Drug Resistance Gene Acquisition after International Travel, the Netherlands

    PubMed Central

    von Wintersdorff, Christian J.H.; Penders, John; Stobberingh, Ellen E.; Lashof, Astrid M.L. Oude; Hoebe, Christian J.P.A.; Savelkoul, Paul H.M.

    2014-01-01

    We investigated the effect of international travel on the gut resistome of 122 healthy travelers from the Netherlands by using a targeted metagenomic approach. Our results confirm high acquisition rates of the extended-spectrum β-lactamase encoding gene blaCTX-M, documenting a rise in prevalence from 9.0% before travel to 33.6% after travel (p<0.001). The prevalence of quinolone resistance encoding genes qnrB and qnrS increased from 6.6% and 8.2% before travel to 36.9% and 55.7% after travel, respectively (both p<0.001). Travel to Southeast Asia and the Indian subcontinent was associated with the highest acquisition rates of qnrS and both blaCTX-M and qnrS, respectively. Investigation of the associations between the acquisitions of the blaCTX-M and qnr genes showed that acquisition of a blaCTX-M gene was not associated with that of a qnrB (p = 0.305) or qnrS (p = 0.080) gene. These findings support the increasing evidence that travelers contribute to the spread of antimicrobial drug resistance. PMID:24655888

  4. Cellular Stress Response Gene Expression During Upper and Lower Body High Intensity Exercises

    PubMed Central

    Kochanowicz, Andrzej; Sawczyn, Stanisław; Niespodziński, Bartłomiej; Mieszkowski, Jan; Kochanowicz, Kazimierz

    2017-01-01

    Objectives The aim was to compare the effect of upper and lower body high-intensity exercise on chosen genes expression in athletes and non-athletes. Method Fourteen elite male artistic gymnasts (EAG) aged 20.6 ± 3.3 years and 14 physically active men (PAM) aged 19.9 ± 1.0 years performed lower and upper body 30 s Wingate Tests. Blood samples were collected before, 5 and 30 minutes after each effort to assess gene expression via PCR. Results Significantly higher mechanical parameters after lower body exercise was observed in both groups, for relative power (8.7 ± 1.2 W/kg in gymnasts, 7.2 ± 1.2 W/kg in controls, p = 0.01) and mean power (6.7 ± 0.7 W/kg in gymnasts, 5.4 ± 0.8 W/kg in controls, p = 0.01). No differences in lower versus upper body gene expression were detected for all tested genes as well as between gymnasts and physical active man. For IL-6 m-RNA time-dependent effect was observed. Conclusions Because of no significant differences in expression of genes associated with cellular stress response the similar adaptive effect to exercise may be obtained so by lower and upper body exercise. PMID:28141870

  5. Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

    USGS Publications Warehouse

    Pearson, T.; Giffard, P.; Beckstrom-Sternberg, S.; Auerbach, R.; Hornstra, H.; Tuanyok, A.; Price, E.P.; Glass, M.B.; Leadem, B.; Beckstrom-Sternberg, J. S.; Allan, G.J.; Foster, J.T.; Wagner, D.M.; Okinaka, R.T.; Sim, S.H.; Pearson, O.; Wu, Z.; Chang, J.; Kaul, R.; Hoffmaster, A.R.; Brettin, T.S.; Robison, R.A.; Mayo, M.; Gee, J.E.; Tan, P.; Currie, B.J.; Keim, P.

    2009-01-01

    Background: Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results: Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion: We describe an

  6. Phylogeographic reconstruction of a bacterial species with high levels of lateral gene transfer

    PubMed Central

    2009-01-01

    Background Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction. Results Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia. Conclusion We describe an

  7. A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family.

    PubMed Central

    Braun, E L; Fuge, E K; Padilla, P A; Werner-Washburne, M

    1996-01-01

    The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest. PMID:8955308

  8. Gene therapy to improve high-density lipoprotein metabolism and function.

    PubMed

    Van Craeyveld, Eline; Gordts, Stephanie; Jacobs, Frank; De Geest, Bart

    2010-05-01

    Plasma levels of high-density lipoprotein (HDL) cholesterol and its major apolipoprotein (apo), apo A-I, are inversely correlated with the incidence of ischemic cardiovascular diseases. Till now, evaluation of the hypothesis that elevation of HDL cholesterol reduces atherosclerotic burden and/or decreases ischemic cardiovascular events in humans has been hampered by the lack of drugs that selectively increase HDL cholesterol. In contrast to the lack of clinical data, evidence for a direct causal role of HDL in modulating atherogenesis in experimental models has been provided by investigations in human apo A-I transgenic mice and rabbits. The development of gene transfer technologies with a sufficiently high therapeutic index may pave the road for a selective and effective HDL raising therapeutic intervention. The goal of a therapeutic strategy that modulates HDL metabolism is not an increase of HDL cholesterol as such, but an enhancement of HDL function. The value of HDL cholesterol as a surrogate end-point to predict reduced atherosclerosis or a decrease in clinical events may be highly dependent on the mechanism leading to an increased level of HDL cholesterol. In the case of gene transfer, this implies that beneficial effects of increasing HDL cholesterol will be dependent on the transgene that is expressed. Here, we critically review HDL metabolism and HDL function in relation to the development of HDL raising gene transfer, advances and drawbacks of different gene transfer technologies, and experimental gene transfer studies evaluating the effect of raised HDL on histological and functional outcomes in animal models.

  9. High Rates of Gene Flow by Pollen and Seed in Oak Populations across Europe

    PubMed Central

    Gerber, Sophie; Chadœuf, Joël; Gugerli, Felix; Lascoux, Martin; Buiteveld, Joukje; Cottrell, Joan; Dounavi, Aikaterini; Fineschi, Silvia; Forrest, Laura L.; Fogelqvist, Johan; Goicoechea, Pablo G.; Jensen, Jan Svejgaard; Salvini, Daniela; Vendramin, Giovanni G.; Kremer, Antoine

    2014-01-01

    Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3], [47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5–8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21–88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20–66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands. PMID:24454802

  10. High rates of gene flow by pollen and seed in oak populations across Europe.

    PubMed

    Gerber, Sophie; Chadœuf, Joël; Gugerli, Felix; Lascoux, Martin; Buiteveld, Joukje; Cottrell, Joan; Dounavi, Aikaterini; Fineschi, Silvia; Forrest, Laura L; Fogelqvist, Johan; Goicoechea, Pablo G; Jensen, Jan Svejgaard; Salvini, Daniela; Vendramin, Giovanni G; Kremer, Antoine

    2014-01-01

    Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3], [47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5-8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21-88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20-66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands.

  11. Selecting highly affine and well-expressed TCRs for gene therapy of melanoma.

    PubMed

    Jorritsma, Annelies; Gomez-Eerland, Raquel; Dokter, Maarten; van de Kasteele, Willeke; Zoet, Yvonne M; Doxiadis, Ilias I N; Rufer, Nathalie; Romero, Pedro; Morgan, Richard A; Schumacher, Ton N M; Haanen, John B A G

    2007-11-15

    A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

  12. High mobility group (HMG-box) genes in the honeybee fungal pathogen Ascosphaera apis.

    PubMed

    Aronstein, K A; Murray, K D; de León, J H; Qin, X; Weinstock, G M

    2007-01-01

    The genome of the honeybee fungal pathogen Ascosphaera apis (Maassen) encodes three putative high mobility group (HMG-box) transcription factors. The predicted proteins (MAT1-2, STE11 and HTF), each of which contain a single strongly conserved HMG-box, exhibit high similarity to mating type proteins and STE11-like transcription factors previously identified in other ascomycete fungi, some of them important plant and human pathogens. In this study we characterized the A. apis HMG-box containing genes and analyzed the structure of the mating type locus (MAT1-2) and its flanking regions. The MAT1-2 locus contains a single gene encoding a protein with an HMG-box. We also have determined the transcriptional patterns of all three HMG-box containing genes in both mating type idiomorphs and discuss a potential role of these transcription factors in A. apis development and reproduction. A multiplex PCR method with primers amplifying mat1-2-1 and Ste11 gene fragments is described. This new method allows for identification of a single mating type idiomorph and might become an essential tool for applied and basic research of chalkbrood disease in honeybees.

  13. High Throughput Gene Expression Analysis Identifies Reliable Expression Markers of Human Corneal Endothelial Cells

    PubMed Central

    Chng, Zhenzhi; Peh, Gary S. L.; Herath, Wishva B.; Cheng, Terence Y. D.; Ang, Heng-Pei; Toh, Kah-Peng; Robson, Paul; Mehta, Jodhbir S.; Colman, Alan

    2013-01-01

    Considerable interest has been generated for the development of suitable corneal endothelial graft alternatives through cell-tissue engineering, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types was also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type. PMID:23844023

  14. High amino acid diversity and positive selection at a putative coral immunity gene (tachylectin-2)

    PubMed Central

    2010-01-01

    Background Genes involved in immune functions, including pathogen recognition and the activation of innate defense pathways, are among the most genetically variable known, and the proteins that they encode are often characterized by high rates of amino acid substitutions, a hallmark of positive selection. The high levels of variation characteristic of immunity genes make them useful tools for conservation genetics. To date, highly variable immunity genes have yet to be found in corals, keystone organisms of the world's most diverse marine ecosystem, the coral reef. Here, we examine variation in and selection on a putative innate immunity gene from Oculina, a coral genus previously used as a model for studies of coral disease and bleaching. Results In a survey of 244 Oculina alleles, we find high nonsynonymous variation and a signature of positive selection, consistent with a putative role in immunity. Using computational protein structure prediction, we generate a structural model of the Oculina protein that closely matches the known structure of tachylectin-2 from the Japanese horseshoe crab (Tachypleus tridentatus), a protein with demonstrated function in microbial recognition and agglutination. We also demonstrate that at least three other genera of anthozoan cnidarians (Acropora, Montastrea and Nematostella) possess proteins structurally similar to tachylectin-2. Conclusions Taken together, the evidence of high amino acid diversity, positive selection and structural correspondence to the horseshoe crab tachylectin-2 suggests that this protein is 1) part of Oculina's innate immunity repertoire, and 2) evolving adaptively, possibly under selective pressure from coral-associated microorganisms. Tachylectin-2 may serve as a candidate locus to screen coral populations for their capacity to respond adaptively to future environmental change. PMID:20482872

  15. High-level protein expression following single and dual gene cloning of infectious bronchitis virus N and S genes using baculovirus systems.

    PubMed

    Abdel-Moneim, Ahmed S; Giesow, Katrin; Keil, Günther M

    2014-03-01

    Baculovirus is an efficient system for the gene expression that can be used for gene transfer to both insect and different vertebrate hosts. The nucleocapsid gene (N) of the infectious bronchitis virus was cloned in a baculovirus expression system for insect cell expression. Dual expression vectors containing IBV N and spike (S) proteins of the avian infectious bronchitis virus were engineered under the control of human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in both vertebrate and insect cells. Transduction of the N gene in the insect Sf9 cells revealed a high level of protein expression. The expressed protein, used in ELISA, effectively detected chicken anti-IBV antibodies with high specificity. Transduction of mammalian and avian cells with BacMam viruses revealed that dual expression cassettes yielded high levels of protein from both transcription units.

  16. Testing candidate genes for attention-deficit/hyperactivity disorder in fruit flies using a high throughput assay for complex behavior.

    PubMed

    Rohde, Palle Duun; Madsen, Lisbeth Strøm; Neumann Arvidson, Sandra Marie; Loeschcke, Volker; Demontis, Ditte; Kristensen, Torsten Nygaard

    2016-01-02

    Fruit flies are important model organisms for functional testing of candidate genes in multiple disciplines, including the study of human diseases. Here we use a high-throughput locomotor activity assay to test the response on activity behavior of gene disruption in Drosophila melanogaster. The aim was to investigate the impact of disruption of 14 candidate genes for human attention-deficit/hyperactivity disorder (ADHD) on fly behavior. By obtaining a range of correlated measures describing the space of variables for behavioral activity we show, that some mutants display similar phenotypic responses, and furthermore, that the genes disrupted in those mutants had common molecular functions; namely processes related to cGMP activity, cation channels and serotonin receptors. All but one of the candidate genes resulted in aberrant behavioral activity, suggesting involvement of these genes in behavioral activity in fruit flies. Results provide additional support for the investigated genes being risk candidate genes for ADHD in humans.

  17. High-throughput genome sequencing of lichenizing fungi to assess gene loss in the ammonium transporter/ammonia permease gene family

    PubMed Central

    2013-01-01

    Background Horizontal gene transfer has shaped the evolution of the ammonium transporter/ammonia permease gene family. Horizontal transfers of ammonium transporter/ammonia permease genes into the fungi include one transfer from archaea to the filamentous ascomycetes associated with the adaptive radiation of the leotiomyceta. The horizontally transferred gene has subsequently been lost in most of the group but has been selectively retained in lichenizing fungi. However, some groups of lichens appear to have secondarily lost the archaeal ammonium transporter. Definitive assessment of gene loss can only be made via whole genome sequencing. Results Ammonium transporter/ammonia permease gene sequences were recovered from the assembled genomes of eight lichenizing fungi in key clades including the Caliciales, the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacterium as a primary photobiont or lichens living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the lichens from the Acarosporomycetidae, Chaetotheriomycetes and Arthoniomycetes. In addition, the four ammonium transporter/ammonia permease genes from Cladonia grayi were shown to be functional by expressing the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source. Conclusions Given sufficient coverage, next-generation sequencing technology can definitively address the loss of a gene in a genome when using environmental DNA isolated from lichen thalli collected from their natural habitats. Lichen-forming fungi have been losing ammonium transporters

  18. High capacity nanoporous silicon carrier for systemic delivery of gene silencing therapeutics.

    PubMed

    Shen, Jianliang; Xu, Rong; Mai, Junhua; Kim, Han-Cheon; Guo, Xiaojing; Qin, Guoting; Yang, Yong; Wolfram, Joy; Mu, Chaofeng; Xia, Xiaojun; Gu, Jianhua; Liu, Xuewu; Mao, Zong-Wan; Ferrari, Mauro; Shen, Haifa

    2013-11-26

    Gene silencing agents such as small interfering RNA (siRNA) and microRNA offer the promise to modulate expression of almost every gene for the treatment of human diseases including cancer. However, lack of vehicles for effective systemic delivery to the disease organs has greatly limited their in vivo applications. In this study, we developed a high capacity polycation-functionalized nanoporous silicon (PCPS) platform comprised of nanoporous silicon microparticles functionalized with arginine-polyethyleneimine inside the nanopores for effective delivery of gene silencing agents. Incubation of MDA-MB-231 human breast cancer cells with PCPS loaded with STAT3 siRNA (PCPS/STAT3) or GRP78 siRNA (PCPS/GRP78) resulted in 91 and 83% reduction of STAT3 and GRP78 gene expression in vitro. Treatment of cells with a microRNA-18a mimic in PCPS (PCPS/miR-18) knocked down 90% expression of the microRNA-18a target gene ATM. Systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells, causing significant reduction of cancer stem cells in the residual tumor tissue. At the therapeutic dosage, PCPS/STAT3 siRNA did not trigger acute immune response in FVB mice, including changes in serum cytokines, chemokines, and colony-stimulating factors. In addition, weekly dosing of PCPS/STAT3 siRNA for four weeks did not cause signs of subacute toxicity based on changes in body weight, hematology, blood chemistry, and major organ histology. Collectively, the results suggest that we have developed a safe vehicle for effective delivery of gene silencing agents.

  19. Metagenomic analysis of stress genes in microbial mat communities from Antarctica and the High Arctic.

    PubMed

    Varin, Thibault; Lovejoy, Connie; Jungblut, Anne D; Vincent, Warwick F; Corbeil, Jacques

    2012-01-01

    Polar and alpine microbial communities experience a variety of environmental stresses, including perennial cold and freezing; however, knowledge of genomic responses to such conditions is still rudimentary. We analyzed the metagenomes of cyanobacterial mats from Arctic and Antarctic ice shelves, using high-throughput pyrosequencing to test the hypotheses that consortia from these extreme polar habitats were similar in terms of major phyla and subphyla and consequently in their potential responses to environmental stresses. Statistical comparisons of the protein-coding genes showed similarities between the mats from the two poles, with the majority of genes derived from Proteobacteria and Cyanobacteria; however, the relative proportions differed, with cyanobacterial genes more prevalent in the Antarctic mat metagenome. Other differences included a higher representation of Actinobacteria and Alphaproteobacteria in the Arctic metagenomes, which may reflect the greater access to diasporas from both adjacent ice-free lands and the open ocean. Genes coding for functional responses to environmental stress (exopolysaccharides, cold shock proteins, and membrane modifications) were found in all of the metagenomes. However, in keeping with the greater exposure of the Arctic to long-range pollutants, sequences assigned to copper homeostasis genes were statistically (30%) more abundant in the Arctic samples. In contrast, more reads matching the sigma B genes were identified in the Antarctic mat, likely reflecting the more severe osmotic stress during freeze-up of the Antarctic ponds. This study underscores the presence of diverse mechanisms of adaptation to cold and other stresses in polar mats, consistent with the proportional representation of major bacterial groups.

  20. A gateway cloning vector set for high-throughput functional analysis of genes in planta.

    PubMed

    Curtis, Mark D; Grossniklaus, Ueli

    2003-10-01

    The current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes. In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence. To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation. With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp. binary vector system that facilitates fast and reliable DNA cloning. This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly. The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the beta-glucuronidase protein so that the subcellular localization of a protein can be identified. They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments. All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.

  1. Impact of high predation risk on genome-wide hippocampal gene expression in snowshoe hares.

    PubMed

    Lavergne, Sophia G; McGowan, Patrick O; Krebs, Charles J; Boonstra, Rudy

    2014-11-01

    The population dynamics of snowshoe hares (Lepus americanus) are fundamental to the ecosystem dynamics of Canada's boreal forest. During the 8- to 11-year population cycle, hare densities can fluctuate up to 40-fold. Predators in this system (lynx, coyotes, great-horned owls) affect population numbers not only through direct mortality but also through sublethal effects. The chronic stress hypothesis posits that high predation risk during the decline severely stresses hares, leading to greater stress responses, heightened ability to mobilize cortisol and energy, and a poorer body condition. These effects may result in, or be mediated by, differential gene expression. We used an oligonucleotide microarray designed for a closely-related species, the European rabbit (Oryctolagus cuniculus), to characterize differences in genome-wide hippocampal RNA transcript abundance in wild hares from the Yukon during peak and decline phases of a single cycle. A total of 106 genes were differentially regulated between phases. Array results were validated with quantitative real-time PCR, and mammalian protein sequence similarity was used to infer gene function. In comparison to hares from the peak, decline phase hares showed increased expression of genes involved in metabolic processes and hormone response, and decreased expression of immune response and blood cell formation genes. We found evidence for predation risk effects on the expression of genes whose putative functions correspond with physiological impacts known to be induced by predation risk in snowshoe hares. This study shows, for the first time, a link between changes in demography and alterations in neural RNA transcript abundance in a natural population.

  2. Cyclophosphamide Alters the Gene Expression Profile in Patients Treated with High Doses Prior to Stem Cell Transplantation

    PubMed Central

    El-Serafi, Ibrahim; Abedi-Valugerdi, Manuchehr; Potácová, Zuzana; Afsharian, Parvaneh; Mattsson, Jonas; Moshfegh, Ali; Hassan, Moustapha

    2014-01-01

    Background Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. Methods We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. Results Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. Conclusion This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression. PMID:24466173

  3. The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome.

    PubMed

    Fu, H; Park, W; Yan, X; Zheng, Z; Shen, B; Dooner, H K

    2001-07-17

    The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3' untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

  4. Novel Bifidobacterium promoters selected through microarray analysis lead to constitutive high-level gene expression.

    PubMed

    Wang, Yan; Kim, Jin Yong; Park, Myeong Soo; Ji, Geun Eog

    2012-08-01

    For the development of a food-grade expression system for Bifidobacterium, a strong promoter leading to high-level expression of cloned gene is a prerequisite. For this purpose, a promoter screening host-vector system for Bifidobacterium has been established using β-glucosidase from Bifidobacterium lactis as a reporter and Bifidobacterium bifidum BGN4 as a host, which is β-glucosidase negative strain. Seven putative promoters showing constitutive high-level expression were selected through microarray analysis based on the genome sequence of B. bifidum BGN4. They were cloned into upstream of β-glucosidase gene and transformed into Escherichia coli DH5α and B. bifidum BGN4. Promoter activities were analyzed both in E. coli and B. bifidum BGN4 by measuring β-glucosidase activity. β-Glucosidase activities in all of the transformants showed growth-associated characteristics. Among them, P919 was the strongest in B. bifidum BGN4 and showed maximum activity at 18 h, while P895 was the strongest in E. coli DH5α at 7 h. This study shows that novel strong promoters such as P919 can be used for high-level expression of foreign genes in Bifidobacterium and will be useful for the construction of an efficient food-grade expression system.

  5. Effects of High Fat Feeding on Liver Gene Expression in Diabetic Goto-Kakizaki Rats

    PubMed Central

    Almon, Richard R.; DuBois, Debra C.; Sukumaran, Siddharth; Wang, Xi; Xue, Bai; Nie, Jing; Jusko, William J.

    2012-01-01

    Effects of high fat diet (HFD) on obesity and, subsequently, on diabetes are highly variable and modulated by genetics in both humans and rodents. In this report, we characterized the response of Goto-Kakizaki (GK) rats, a spontaneous polygenic model for lean diabetes and healthy Wistar-Kyoto (WKY) controls, to high fat feeding from weaning to 20 weeks of age. Animals fed either normal diet or HFD were sacrificed at 4, 8, 12, 16 and 20 weeks of age and a wide array of physiological measurements were made along with gene expression profiling using Affymetrix gene array chips. Mining of the microarray data identified differentially regulated genes (involved in inflammation, metabolism, transcription regulation, and signaling) in diabetic animals, as well as the response of both strains to HFD. Functional annotation suggested that HFD increased inflammatory differences between the two strains. Chronic inflammation driven by heightened innate immune response was identified to be present in GK animals regardless of diet. In addition, compensatory mechanisms by which WKY animals on HFD resisted the development of diabetes were identified, thus illustrating the complexity of diabetes disease progression. PMID:23236253

  6. Gene expression of the ericoid mycorrhizal fungus Oidiodendron maius in the presence of high zinc concentrations.

    PubMed

    Vallino, Marta; Drogo, Vanessa; Abba', Simona; Perotto, Silvia

    2005-07-01

    A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from roots of Vaccinium myrtillus growing in soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated the genetic basis of this fungal strain tolerance to high zinc concentrations by using an untargeted approach. From a cDNA library constructed by using mRNA from Zn-treated O. maius mycelia, 444 clones were randomly selected and 318 were sequenced. Sequence analysis identified 219 unique clones: 117 showed homology to previously identified genes, 26 matched unknown protein coding regions found in other organisms, and 76 were novel. Variation in the gene expression level after a 20-day treatment with high concentrations of Zn was monitored on 130 unigenes by reverse northern blot hybridisation. Sixteen unigenes were shown to be either up- (9) or down- (7) regulated. The putative function of these genes and their involvement in stress tolerance is discussed.

  7. High sequence turnover in the regulatory regions of the developmental gene hunchback in insects.

    PubMed

    Hancock, J M; Shaw, P J; Bonneton, F; Dover, G A

    1999-02-01

    Extensive sequence analysis of the developmental gene hunchback and its 5' and 3' regulatory regions in Drosophila melanogaster, Drosophila virilis, Musca domestica, and Tribolium castaneum, using a variety of computer algorithms, reveals regions of high sequence simplicity probably generated by slippage-like mechanisms of turnover. No regions are entirely refractory to the action of slippage, although the density and composition of simple sequence motifs varies from region to region. Interestingly, the 5' and 3' flanking regions share short repetitive motifs despite their separation by the gene itself, and the motifs are different in composition from those in the exons and introns. Furthermore, there are high levels of conservation of motifs in equivalent orthologous regions. Detailed sequence analysis of the P2 promoter and DNA footprinting assays reveal that the number, orientation, sequence, spacing, and protein-binding affinities of the BICOID-binding sites varies between species and that the 'P2' promoter, the nanos response element in the 3' untranslated region, and several conserved boxes of sequence in the gene (e.g., the two zinc-finger regions) are surrounded by cryptically-simple-sequence DNA. We argue that high sequence turnover and genetic redundancy permit both the general maintenance of promoter functions through the establishment of coevolutionary (compensatory) changes in cis- and trans-acting genetic elements and, at the same time, the possibility of subtle changes in the regulation of hunchback in the different species.

  8. A highly conserved SOX6 double binding site mediates SOX6 gene downregulation in erythroid cells

    PubMed Central

    Cantu', Claudio; Grande, Vito; Alborelli, Ilaria; Cassinelli, Letizia; Cantu’, Ileana; Colzani, Maria Teresa; Ierardi, Rossella; Ronzoni, Luisa; Cappellini, Maria Domenica; Ferrari, Giuliana; Ottolenghi, Sergio; Ronchi, Antonella

    2011-01-01

    The Sox6 transcription factor plays critical roles in various cell types, including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells, we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar, evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation, mediated by the double Sox6 binding site within its own promoter, may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation. PMID:20852263

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

    PubMed

    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.

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

    PubMed Central

    Brown, Alistair J.P.; Planta, Rudi J.; Restuhadi, Fajar; Bailey, David A.; Butler, Philip R.; Cadahia, Jose L.; Cerdan, M.Esperanza; De Jonge, Martine; Gardner, David C.J.; Gent, Manda E.; Hayes, Andrew; Kolen, Carin P.A.M.; Lombardia, Luis J.; Murad, Abdul Munir Abdul; Oliver, Rachel A.; Sefton, Mark; Thevelein, Johan M.; Tournu, Helene; van Delft, Yvon J.; Verbart, Dennis J.; Winderickx, Joris; Oliver, Stephen G.

    2001-01-01

    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. PMID:11406594

  11. The myostatin gene of Mytilus chilensis evidences a high level of polymorphism and ubiquitous transcript expression.

    PubMed

    Núñez-Acuña, Gustavo; Gallardo-Escárate, Cristian

    2014-02-15

    Myostatin (MSTN) is a protein of the Transforming Growth Factor-β (TGF-β) superfamily and plays a crucial role in muscular development for higher vertebrates. However, its biological function in marine invertebrates remains undiscovered. This study characterizes the full-length sequence of the Mytilus chilensis myostatin gene (Mc-MSTN). Furthermore, tissue transcription patterns and putative single nucleotide polymorphisms (SNPs) were also identified. The Mc-MSTN cDNA sequence showed 3528 base pairs (bp), consisting of 161 bp of 5' UTR, 2,110 bp of 3' UTR, and an open reading frame of 1,257 bp encoding for 418 amino acids and with an RXXR proteolytic site and nine cysteine-conserved residues. Gene transcription analysis revealed that the Mc-MSTN has ubiquitous expression among several tissues, with higher expression in the gonads and mantle than in the digestive gland, gills, and hemolymph. Furthermore, high levels of polymorphisms were detected (28 SNPs in 3'-UTR and 9 SNPs in the coding region). Two SNPs were non-synonymous and involved amino acid changes between Glu/Asp and Thr/Ile. Until now, the MSTN gene has been mainly related to muscle growth in marine bivalves. However, the present study suggests a putative biological function not entirely associated to muscle tissue and contributes molecular evidence to the current debate about the function of the MSTN gene in marine invertebrates.

  12. A high-fat diet coordinately downregulates genes required for mitochondrial oxidative phosphorylation in skeletal muscle.

    PubMed

    Sparks, Lauren M; Xie, Hui; Koza, Robert A; Mynatt, Randall; Hulver, Matthew W; Bray, George A; Smith, Steven R

    2005-07-01

    Obesity and type 2 diabetes have been associated with a high-fat diet (HFD) and reduced mitochondrial mass and function. We hypothesized a HFD may affect expression of genes involved in mitochondrial function and biogenesis. To test this hypothesis, we fed 10 insulin-sensitive males an isoenergetic HFD for 3 days with muscle biopsies before and after intervention. Oligonucleotide microarray analysis revealed 297 genes were differentially regulated by the HFD (Bonferonni adjusted P < 0.001). Six genes involved in oxidative phosphorylation (OXPHOS) decreased. Four were members of mitochondrial complex I: NDUFB3, NDUFB5, NDUFS1, and NDUFV1; one was SDHB in complex II and a mitochondrial carrier protein SLC25A12. Peroxisome proliferator-activated receptor gamma coactivator-1 (PGC1) alpha and PGC1beta mRNA were decreased by -20%, P < 0.01, and -25%, P < 0.01, respectively. In a separate experiment, we fed C57Bl/6J mice a HFD for 3 weeks and found that the same OXPHOS and PGC1 mRNAs were downregulated by approximately 90%, cytochrome C and PGC1alpha protein by approximately 40%. Combined, these results suggest a mechanism whereby HFD downregulates genes necessary for OXPHOS and mitochondrial biogenesis. These changes mimic those observed in diabetes and insulin resistance and, if sustained, may result in mitochondrial dysfunction in the prediabetic/insulin-resistant state.

  13. Predictive models of gene regulation from high-throughput epigenomics data.

    PubMed

    Althammer, Sonja; Pagès, Amadís; Eyras, Eduardo

    2012-01-01

    The epigenetic regulation of gene expression involves multiple factors. The synergistic or antagonistic action of these factors has suggested the existence of an epigenetic code for gene regulation. Highthroughput sequencing (HTS) provides an opportunity to explore this code and to build quantitative models of gene regulation based on epigenetic differences between specific cellular conditions. We describe a new computational framework that facilitates the systematic integration of HTS epigenetic data. Our method relates epigenetic signals to expression by comparing two conditions. We show its effectiveness by building a model that predicts with high accuracy significant expression differences between two cell lines, using epigenetic data from the ENCODE project. Our analyses provide evidence for a degenerate epigenetic code, which involves multiple genic regions. In particular, signal changes at the 1st exon, 1st intron, and downstream of the polyadenylation site are found to associate strongly with expression regulation. Our analyses also show a different epigenetic code for intron-less and intron-containing genes. Our work provides a general methodology to do integrative analysis of epigenetic differences between cellular conditions that can be applied to other studies, like cell differentiation or carcinogenesis.

  14. Restricted isotype, distinct variable gene usage, and high rate of gp120 specificity of HIV-1 envelope-specific B cells in colostrum compared with those in blood of HIV-1-infected, lactating African women.

    PubMed

    Sacha, C R; Vandergrift, N; Jeffries, T L; McGuire, E; Fouda, G G; Liebl, B; Marshall, D J; Gurley, T C; Stiegel, L; Whitesides, J F; Friedman, J; Badiabo, A; Foulger, A; Yates, N L; Tomaras, G D; Kepler, T B; Liao, H X; Haynes, B F; Moody, M A; Permar, S R

    2015-03-01

    A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1(∼)69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

  15. Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

    PubMed

    De Meyer, Sofie E; Briscoe, Leah; Martínez-Hidalgo, Pilar; Agapakis, Christina M; de-Los Santos, Paulina Estrada; Seshadri, Rekha; Reeve, Wayne; Weinstock, George; O'Hara, Graham; Howieson, John G; Hirsch, Ann M

    2016-08-01

    Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

  16. Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

    PubMed Central

    Taghian, D G; Nickoloff, J A

    1997-01-01

    Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion. PMID:9343400

  17. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.

    PubMed

    Gil-Humanes, Javier; Wang, Yanpeng; Liang, Zhen; Shan, Qiwei; Ozuna, Carmen V; Sánchez-León, Susana; Baltes, Nicholas J; Starker, Colby; Barro, Francisco; Gao, Caixia; Voytas, Daniel F

    2017-03-01

    The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

  18. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    SciTech Connect

    Jett, J.

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  19. Comparison of low and high dose ionising radiation using topological analysis of gene coexpression networks

    PubMed Central

    2012-01-01

    Background The growing use of imaging procedures in medicine has raised concerns about exposure to low-dose ionising radiation (LDIR). While the disastrous effects of high dose ionising radiation (HDIR) is well documented, the detrimental effects of LDIR is not well understood and has been a topic of much debate. Since little is known about the effects of LDIR, various kinds of wet-lab and computational analyses are required to advance knowledge in this domain. In this paper we carry out an “upside-down pyramid” form of systems biology analysis of microarray data. We characterised the global genomic response following 10 cGy (low dose) and 100 cGy (high dose) doses of X-ray ionising radiation at four time points by analysing the topology of gene coexpression networks. This study includes a rich experimental design and state-of-the-art computational systems biology methods of analysis to study the differences in the transcriptional response of skin cells exposed to low and high doses of radiation. Results Using this method we found important genes that have been linked to immune response, cell survival and apoptosis. Furthermore, we also were able to identify genes such as BRCA1, ABCA1, TNFRSF1B, MLLT11 that have been associated with various types of cancers. We were also able to detect many genes known to be associated with various medical conditions. Conclusions Our method of applying network topological differences can aid in identifying the differences among similar (eg: radiation effect) yet very different biological conditions (eg: different dose and time) to generate testable hypotheses. This is the first study where a network level analysis was performed across two different radiation doses at various time points, thereby illustrating changes in the cellular response over time. PMID:22594378

  20. GeneChip Expression Profiling Reveals the Alterations of Energy Metabolism Related Genes in Osteocytes under Large Gradient High Magnetic Fields

    PubMed Central

    Wang, Yang; Chen, Zhi-Hao; Yin, Chun; Ma, Jian-Hua; Li, Di-Jie; Zhao, Fan; Sun, Yu-Long; Hu, Li-Fang; Shang, Peng; Qian, Ai-Rong

    2015-01-01

    The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has recently been applied in life science research. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. Osteocyte, as the most important mechanosensor in bone, takes a pivotal position in mediating the mechano-induced bone remodeling. In this study, the effects of LG-HMF on gene expression profiling of osteocyte-like cell line MLO-Y4 were investigated by Affymetrix DNA microarray. LG-HMF affected osteocyte gene expression profiling. Differentially expressed genes (DEGs) and data mining were further analyzed by using bioinfomatic tools, such as DAVID, iReport. 12 energy metabolism related genes (PFKL, AK4, ALDOC, COX7A1, STC1, ADM, CA9, CA12, P4HA1, APLN, GPR35 and GPR84) were further confirmed by real-time PCR. An integrated gene interaction network of 12 DEGs was constructed. Bio-data mining showed that genes involved in glucose metabolic process and apoptosis changed notablly. Our results demostrated that LG-HMF affected the expression of energy metabolism related genes in osteocyte. The identification of sensitive genes to special environments may provide some potential targets for preventing and treating bone loss or osteoporosis. PMID:25635858

  1. Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes

    PubMed Central

    Park, Wonkeun; Zhai, Jixian; Lee, Jung-Youn

    2009-01-01

    Gene silencing is a useful technique for elucidating biological function of genes by knocking down their expression. A recently developed artificial microRNAs (amiRNAs) exploits an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs that target transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed such that they have a few mismatching nucleotides with respect to their target sites as well as within mature amiRNA duplexes. In this study, we performed an analysis in which the conventional and modified form of an amiRNA was compared side by side. We showed that the amiRNA containing 5′ mismatch with its amiRNA* and perfect complementarity to its target gene acted as a highly potent gene silencing agent against AP1, achieving a desired null mutation effect. In addition, a simultaneous silencing of two independent genes, AP1 and CAL1 wastested by employing a multimeric form of amiRNAs. Advantages and potential disadvantages of using amiRNAs with perfect complementarity to the target gene are discussed. The results presented here should be helpful in designing more specific and effective gene silencing agents. PMID:19066901

  2. GeneChip expression profiling reveals the alterations of energy metabolism related genes in osteocytes under large gradient high magnetic fields.

    PubMed

    Wang, Yang; Chen, Zhi-Hao; Yin, Chun; Ma, Jian-Hua; Li, Di-Jie; Zhao, Fan; Sun, Yu-Long; Hu, Li-Fang; Shang, Peng; Qian, Ai-Rong

    2015-01-01

    The diamagnetic levitation as a novel ground-based model for simulating a reduced gravity environment has recently been applied in life science research. In this study a specially designed superconducting magnet with a large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels (μ-g, 1-g, and 2-g), was used to simulate a space-like gravity environment. Osteocyte, as the most important mechanosensor in bone, takes a pivotal position in mediating the mechano-induced bone remodeling. In this study, the effects of LG-HMF on gene expression profiling of osteocyte-like cell line MLO-Y4 were investigated by Affymetrix DNA microarray. LG-HMF affected osteocyte gene expression profiling. Differentially expressed genes (DEGs) and data mining were further analyzed by using bioinfomatic tools, such as DAVID, iReport. 12 energy metabolism related genes (PFKL, AK4, ALDOC, COX7A1, STC1, ADM, CA9, CA12, P4HA1, APLN, GPR35 and GPR84) were further confirmed by real-time PCR. An integrated gene interaction network of 12 DEGs was constructed. Bio-data mining showed that genes involved in glucose metabolic process and apoptosis changed notablly. Our results demostrated that LG-HMF affected the expression of energy metabolism related genes in osteocyte. The identification of sensitive genes to special environments may provide some potential targets for preventing and treating bone loss or osteoporosis.

  3. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    PubMed

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-02

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  4. The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli.

    PubMed

    Lee, C Y; Szittner, R B; Meighen, E A

    1991-10-01

    The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5-kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty-acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured. Determination of the 7.3-kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty-acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem-loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.

  5. EST mining for structure and expression of genes in the region of the wheat high-molecular-weight glutenin loci.

    PubMed

    Anderson, O D

    2009-08-01

    An in-depth analysis was carried out with expressed sequence tags (ESTs) for genes in and near the HMW-GS loci. Considerations for using ESTs are discussed, including the occurrence of chimeric and aberrant HMW-GS ESTs. Complete gene sequences demonstrated the feasibility of constructing accurate full-length coding regions from EST assemblies and found, or supported, errors in several previously reported HMW-GS gene sequences. New complete HMW-GS gene sequences are reported for the cultivars Chinese Spring and Glenlea. The Ay subunit gene, which is considered null in cultivated wheats, was shown to transcribe in at least two germplasms. Analyses support the conclusion that of the five known genes within this genomic region, the two HMW-GS genes and the globulin gene are highly expressed. The other two genes, encoding a receptor kinase and a protein kinase, have one and no identifiable wheat EST, respectively, although ESTs are found for the orthologous genes in barley. The ESTs of all five genes within the HMW-GS region are either definitely associated with the endosperm or possibly originate from imbibed seed, suggesting the four distinct gene classes in this region are part of a seed or endosperm chromatin domain. EST resources were also used to determine relative abundance of ESTs for all classes of wheat prolamines and indicated differential levels of expression both among germplasms and among the three genomes of hexaploid wheats.

  6. Reconciling extremely strong barriers with high levels of gene exchange in annual sunflowers.

    PubMed

    Sambatti, Julianno B M; Strasburg, Jared L; Ortiz-Barrientos, Daniel; Baack, Eric J; Rieseberg, Loren H

    2012-05-01

    In several cases, estimates of gene flow between species appear to be higher than we might predict given the strength of interspecific barriers separating these species pairs. However, as far as we are aware, detailed measurements of reproductive isolation have not previously been compared with a coalescent-based assessment of gene flow. Here, we contrast these two measures in two species of sunflower, Helianthus annuus and H. petiolaris. We quantified the total reproductive barrier strength between these species by compounding the contributions of the following prezygotic and postzygotic barriers: ecogeographic isolation, reproductive asynchrony, niche differentiation, pollen competition, hybrid seed formation, hybrid seed germination, hybrid fertility, and extrinsic postzygotic isolation. From this estimate, we calculated the probability that a reproductively successful hybrid is produced: estimates of P(hyb) range from 10(-4) to 10(-6) depending on the direction of the cross and the degree of independence among reproductive barriers. We then compared this probability with population genetic estimates of the per generation migration rate (m). We showed that the relatively high levels of gene flow estimated between these sunflower species (N(e) m= 0.34-0.76) are mainly due to their large effective population sizes (N(e) > 10(6)). The interspecific migration rate (m) is very small (<10(-7)) and an order of magnitude lower than that expected based on our reproductive barrier strength estimates. Thus, even high levels of reproductive isolation (>0.999) may produce genomic mosaics.

  7. Different pattern of Galleria mellonella jhbp gene expression in high five and Sf9 cells.

    PubMed

    Andruszewska, Grażyna; Ożyhar, Andrzej; Kochman, Marian; Schmidt, Marcin

    2013-03-01

    Juvenile hormone binding protein (JHBP) is the key element of the system that transmits hormone signals to target tissues. Recently, we found that the core promoter of the jhbp gene is strongly under the control of the TATA box and the transcription start site. In this report, we have shown that the jhbp promoter contains distal regulatory elements whose functionality clearly depends on the particular cell environment and that the scope of research from one cell line is insufficient to generalize the conclusions of the analysis. Cf1/Usp (where Usp is ultraspiracle protein previously known as Cf1, chorion factor 1) elements suppressed transcription of the reporter gene in the High Five cell line but not in the Sf9 cell line. However, upstream from all three Cf1/Usp elements there is a DNA sequence, containing the Zeste element, which activates jhbp in both systems. We found that juvenile hormone strongly inhibited the activity of the jhbp promoter in the Sf9 cell line, whereas it did not have an effect in the High Five cell line. A second key hormone that controls insect development--20-hydroxyecdysone, was also found to suppress the transcription of jhbp. This is the first report describing how these two hormones affect jhbp gene expression in different cell lines.

  8. Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation

    PubMed Central

    Horlbeck, Max A; Gilbert, Luke A; Villalta, Jacqueline E; Adamson, Britt; Pak, Ryan A; Chen, Yuwen; Fields, Alexander P; Park, Chong Yon; Corn, Jacob E; Kampmann, Martin; Weissman, Jonathan S

    2016-01-01

    We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites. DOI: http://dx.doi.org/10.7554/eLife.19760.001 PMID:27661255

  9. A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system.

    PubMed

    Eroglu, Erdal; Tiwari, Pooja M; Waffo, Alain B; Miller, Michael E; Vig, Komal; Dennis, Vida A; Singh, Shree R

    2013-01-01

    The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs), which consisted of poly(2-hydroxyethyl methacrylate) nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV)-F gene construct (a model for a DNA vaccine). The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM), fluorescence activated cell sorting (FACS), and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR), we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo.

  10. A nonviral pHEMA+chitosan nanosphere-mediated high-efficiency gene delivery system

    PubMed Central

    Eroglu, Erdal; Tiwari, Pooja M; Waffo, Alain B; Miller, Michael E; Vig, Komal; Dennis, Vida A; Singh, Shree R

    2013-01-01

    The transport of DNA into eukaryotic cells is minimal because of the cell membrane barrier, and this limits the application of DNA vaccines, gene silencing, and gene therapy. Several available transfection reagents and techniques have been used to circumvent this problem. Alternatively, nonviral nanoscale vectors have been shown to bypass the eukaryotic cell membrane. In the present work, we developed a unique nanomaterial, pHEMA+chitosan nanospheres (PCNSs), which consisted of poly(2-hydroxyethyl methacrylate) nanospheres surrounded by a chitosan cationic shell, and we used this for encapsulation of a respiratory syncytial virus (RSV)-F gene construct (a model for a DNA vaccine). The new nanomaterial was capable of transfecting various eukaryotic cell lines without the use of a commercial transfection reagent. Using transmission electron microscopy, (TEM), fluorescence activated cell sorting (FACS), and immunofluorescence, we clearly demonstrated that the positively charged PCNSs were able to bind to the negatively charged cell membrane and were taken up by endocytosis, in Cos-7 cells. Using quantitative polymerase chain reaction (qPCR), we also evaluated the efficiency of transfection achieved with PCNSs and without the use of a liposomal-based transfection mediator, in Cos-7, HEp-2, and Vero cells. To assess the transfection efficiency of the PCNSs in vivo, these novel nanomaterials containing RSV-F gene were injected intramuscularly into BALB/c mice, resulting in high copy number of the transgene. In this study, we report, for the first time, the application of the PCNSs as a nanovehicle for gene delivery in vitro and in vivo. PMID:23610520

  11. High-affinity L-arabinose transport operon. Nucleotide sequence and analysis of gene products.

    PubMed

    Scripture, J B; Voelker, C; Miller, S; O'Donnell, R T; Polgar, L; Rade, J; Horazdovsky, B F; Hogg, R W

    1987-09-05

    The nucleotide sequence of the "high-affinity" L-arabinose transport operon has been determined 3' from the regulatory region and found to contain three open reading frames designated araF, araG and araH. The first gene 3' to the regulatory region, araF, encodes the 23-residue signal peptide and the 306-residue mature form of the L-arabinose binding protein (33,200 Mr). The binding protein, which has been described elsewhere, is hydrophilic, soluble and found in the periplasm of Escherichia coli. This gene is followed by an intragenic space of 72 nucleotides, which contains a region of dyad symmetry 23 nucleotides long capable of forming an 11-member stem-loop. The second gene, designated araG, contains an open reading frame capable of encoding an equally hydrophilic protein containing 504 residues (55,000 Mr). Following a 14-nucleotide spacer, which does not appear to have any secondary structure, the third open reading frame, herein designated araH, is capable of encoding a hydrophobic protein containing 329 residues (34,000 Mr) that can only be envisioned as having an integral membrane location. 3' to araH there is a T-rich region containing a 24-nucleotide area of dyad symmetry centered 55 nucleotides from the termination codon. Analysis of the derived primary sequences of the araG and araH products indicates the nature and potential features of these components. The araG protein was found to possess internal homology between its amino and carboxyl-terminal halves, suggesting a common origin. The araG gene product has been shown to be homologous to the rbsA gene product, the hisP product, the ptsB product and the malK product, all of which presumably play similar roles in their respective transport systems. Putative ATP binding sites are observed within the regions of homology. The araH gene product has been shown to be homologous to the rbsC gene product, which is the first observed homology between two purported membrane proteins.

  12. Innate-like functions of natural killer T cell subsets result from highly divergent gene programs.

    PubMed

    Engel, Isaac; Seumois, Grégory; Chavez, Lukas; Samaniego-Castruita, Daniela; White, Brandie; Chawla, Ashu; Mock, Dennis; Vijayanand, Pandurangan; Kronenberg, Mitchell

    2016-06-01

    Natural killer T cells (NKT cells) have stimulatory or inhibitory effects on the immune response that can be attributed in part to the existence of functional subsets of NKT cells. These subsets have been characterized only on the basis of the differential expression of a few transcription factors and cell-surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic level and epigenomic level and by single-cell RNA sequencing. Our data indicated that despite their similar antigen specificity, the functional NKT cell subsets were highly divergent populations with many gene-expression and epigenetic differences. Therefore, the thymus 'imprints' distinct gene programs on subsets of innate-like NKT cells that probably impart differences in proliferative capacity, homing, and effector functions.

  13. Innate-like functions of natural killer T cell subsets result from highly divergent gene programs

    PubMed Central

    Engel, Isaac; Seumois, Grégory; Chavez, Lukas; Samaniego-Castruita, Daniela; White, Brandie; Chawla, Ashu; Mock, Dennis; Vijayanand, Pandurangan; Kronenberg, Mitchell

    2016-01-01

    Natural killer T cells (NKT cells) have stimulatory or inhibitory effects on the immune response that can be attributed in part to the existence of functional subsets of NKT cells. These subsets have been characterized only on the basis of the differential expression of a few transcription factors and cell-surface molecules. Here we have analyzed purified populations of thymic NKT cell subsets at both the transcriptomic level and epigenomic level and by single-cell RNA sequencing. Our data indicated that despite their similar antigen specificity, the functional NKT cell subsets were highly divergent populations with many gene-expression and epigenetic differences. Therefore, the thymus ‘imprints’ distinct gene programs on subsets of innate-like NKT cells that probably impart differences in proliferative capacity, homing, and effector functions. PMID:27089380

  14. High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

    PubMed Central

    Boast, S; La Mantia, G; Lania, L; Blasi, F

    1983-01-01

    Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells. Images Fig. 2. Figure 3. PMID:6321161

  15. A high-density gene map of loblolly pine (Pinus taeda L.) based on exome sequence capture genotyping.

    PubMed

    Neves, Leandro Gomide; Davis, John M; Barbazuk, William B; Kirst, Matias

    2014-01-10

    Loblolly pine (Pinus taeda L.) is an economically and ecologically important conifer for which a suite of genomic resources is being generated. Despite recent attempts to sequence the large genome of conifers, their assembly and the positioning of genes remains largely incomplete. The interspecific synteny in pines suggests that a gene-based map would be useful to support genome assemblies and analysis of conifers. To establish a reference gene-based genetic map, we performed exome sequencing of 14729 genes on a mapping population of 72 haploid samples, generating a resource of 7434 sequence variants segregating for 3787 genes. Most markers are single-nucleotide polymorphisms, although short insertions/deletions and multiple nucleotide polymorphisms also were used. Marker segregation in the population was used to generate a high-density, gene-based genetic map. A total of 2841 genes were mapped to pine's 12 linkage groups with an average of one marker every 0.58 cM. Capture data were used to detect gene presence/absence variations and position 65 genes on the map. We compared the marker order of genes previously mapped in loblolly pine and found high agreement. We estimated that 4123 genes had enough sequencing depth for reliable detection of markers, suggesting a high marker conversation rate of 92% (3787/4123). This is possible because a significant portion of the gene is captured and sequenced, increasing the chances of identifying a polymorphic site for characterization and mapping. This sub-centiMorgan genetic map provides a valuable resource for gene positioning on chromosomes and guide for the assembly of a reference pine genome.

  16. Strategies for achieving high-level expression of genes in Escherichia coli.

    PubMed Central

    Makrides, S C

    1996-01-01

    Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression. There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression. New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation. Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium. Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport. Negative results derived from such studies are also instructive in formulating different strategies. The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins. Codon usage is known to present a potential impediment to high-level gene expression in E. coli. Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels. Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes. Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E. coli. PMID:8840785

  17. Overexpression of Cotton RAV1 Gene in Arabidopsis Confers Transgenic Plants High Salinity and Drought Sensitivity

    PubMed Central

    Li, Xiao-Jie; Li, Mo; Zhou, Ying; Hu, Shan; Hu, Rong; Chen, Yun; Li, Xue-Bao

    2015-01-01

    RAV (related to ABI3/VP1) protein containing an AP2 domain in the N-terminal region and a B3 domain in the C-terminal region, which belongs to AP2 transcription factor family, is unique in higher plants. In this study, a gene (GhRAV1) encoding a RAV protein of 357 amino acids was identified in cotton (Gossypium hirsutum). Transient expression analysis of the eGFP:GhRAV1 fusion genes in tobacco (Nicotiana tabacum) epidermal cells revealed that GhRAV1 protein was localized in the cell nucleus. Quantitative RT-PCR analysis indicated that expression of GhRAV1 in cotton is induced by abscisic acid (ABA), NaCl and polyethylene glycol (PEG). Overexpression of GhRAV1 in Arabidopsis resulted in plant sensitive to ABA, NaCl and PEG. With abscisic acid (ABA) treatment, seed germination and green seedling rates of the GhRAV1 transgenic plants were remarkably lower than those of wild type. In the presence of NaCl, the seed germination and seedling growth of the GhRAV1 transgenic lines were inhibited greater than those of wild type. And chlorophyll content and maximum photochemical efficiency of the transgenic plants were significantly lower than those of wild type. Under drought stress, the GhRAV1 transgenic plants displayed more severe wilting than wild type. Furthermore, expressions of the stress-related genes were altered in the GhRAV1 transgenic Arabidopsis plants under high salinity and drought stresses. Collectively, our data suggested that GhRAV1 may be involved in response to high salinity and drought stresses through regulating expressions of the stress-related genes during cotton development. PMID:25710493

  18. High-throughput profiling of antibiotic resistance genes in drinking water treatment plants and distribution systems.

    PubMed

    Xu, Like; Ouyang, Weiying; Qian, Yanyun; Su, Chao; Su, Jianqiang; Chen, Hong

    2016-06-01

    Antibiotic resistance genes (ARGs) are present in surface water and often cannot be completely eliminated by drinking water treatment plants (DWTPs). Improper elimination of the ARG-harboring microorganisms contaminates the water supply and would lead to animal and human disease. Therefore, it is of utmost importance to determine the most effective ways by which DWTPs can eliminate ARGs. Here, we tested water samples from two DWTPs and distribution systems and detected the presence of 285 ARGs, 8 transposases, and intI-1 by utilizing high-throughput qPCR. The prevalence of ARGs differed in the two DWTPs, one of which employed conventional water treatments while the other had advanced treatment processes. The relative abundance of ARGs increased significantly after the treatment with biological activated carbon (BAC), raising the number of detected ARGs from 76 to 150. Furthermore, the final chlorination step enhanced the relative abundance of ARGs in the finished water generated from both DWTPs. The total enrichment of ARGs varied from 6.4-to 109.2-fold in tap water compared to finished water, among which beta-lactam resistance genes displayed the highest enrichment. Six transposase genes were detected in tap water samples, with the transposase gene TnpA-04 showing the greatest enrichment (up to 124.9-fold). We observed significant positive correlations between ARGs and mobile genetic elements (MGEs) during the distribution systems, indicating that transposases and intI-1 may contribute to antibiotic resistance in drinking water. To our knowledge, this is the first study to investigate the diversity and abundance of ARGs in drinking water treatment systems utilizing high-throughput qPCR techniques in China.

  19. High expression of genes for calcification-regulating proteins in human atherosclerotic plaques.

    PubMed Central

    Shanahan, C M; Cary, N R; Metcalfe, J C; Weissberg, P L

    1994-01-01

    Calcification is common in atheromatous plaques and may contribute to plaque rupture and subsequent thrombosis. However, little is known about the mechanisms which regulate the calcification process. Using in situ hybridization and immunohistochemistry we show that two bone-associated proteins, osteopontin (OP) and matrix Gla protein (MGP), are highly expressed in human atheromatous plaques. High levels of OP mRNA and protein were found in association with necrotic lipid cores and areas of calcification. The predominant cell type in these areas was the macrophage-derived foam cell, although some smooth muscle cells could also be identified. MGP was expressed uniformly by smooth muscle cells in the normal media and at high levels in parts of the atheromatous intima. Highest levels of this matrix-associated protein were found in lipid-rich areas of the plaque. The pattern of expression of these two genes contrasted markedly with that of calponin and SM22 alpha, genes expressed predominantly by differentiated smooth muscle cells and whose expression was generally confined to the media of the vessel. The postulated function of OP and MGP as regulators of calcification in bone and the high levels and colocalization of both in atheromatous plaques suggest they have an important role in plaque pathogenesis and stability. Images PMID:8200973

  20. Fish Oil Decreases Hepatic Lipogenic Genes in Rats Fasted and Refed on a High Fructose Diet

    PubMed Central

    de Castro, Gabriela S.; Cardoso, João Felipe R.; Calder, Philip C.; Jordão, Alceu A.; Vannucchi, Helio

    2015-01-01

    Fasting and then refeeding on a high-carbohydrate diet increases serum and hepatic triacylglycerol (TAG) concentrations compared to standard diets. Fructose is a lipogenic monosaccharide which stimulates de novo fatty acid synthesis. Omega-3 (n-3) fatty acids stimulate hepatic β-oxidation, partitioning fatty acids away from TAG synthesis. This study investigated whether dietary n-3 fatty acids from fish oil (FO) improve the hepatic lipid metabolic response seen in rats fasted and then refed on a high-fructose diet. During the post-prandial (fed) period, rats fed a FO rich diet showed an increase in hepatic peroxisome proliferator-activated receptor α (PPAR-α) gene expression and decreased expression of carbohydrate responsive element binding protein (ChREBP), fatty acid synthase (FAS) and microsomal triglyceride transfer protein (MTTP). Feeding a FO rich diet for 7 days prior to 48 h of fasting resulted in lower hepatic TAG, lower PPAR-α expression and maintenance of hepatic n-3 fatty acid content. Refeeding on a high fructose diet promoted an increase in hepatic and serum TAG and in hepatic PPAR-α, ChREBP and MTTP expression. FO did not prevent the increase in serum and hepatic TAG after fructose refeeding, but did decrease hepatic expression of lipogenic genes and increased the n-3 fatty acid content of the liver. n-3 Fatty acids can modify some components of the hepatic lipid metabolic response to later feeding with a high fructose diet. PMID:25751821

  1. Fish oil decreases hepatic lipogenic genes in rats fasted and refed on a high fructose diet.

    PubMed

    de Castro, Gabriela S; Cardoso, João Felipe R; Calder, Philip C; Jordão, Alceu A; Vannucchi, Helio

    2015-03-05

    Fasting and then refeeding on a high-carbohydrate diet increases serum and hepatic triacylglycerol (TAG) concentrations compared to standard diets. Fructose is a lipogenic monosaccharide which stimulates de novo fatty acid synthesis. Omega-3 (n-3) fatty acids stimulate hepatic β-oxidation, partitioning fatty acids away from TAG synthesis. This study investigated whether dietary n-3 fatty acids from fish oil (FO) improve the hepatic lipid metabolic response seen in rats fasted and then refed on a high-fructose diet. During the post-prandial (fed) period, rats fed a FO rich diet showed an increase in hepatic peroxisome proliferator-activated receptor α (PPAR-α) gene expression and decreased expression of carbohydrate responsive element binding protein (ChREBP), fatty acid synthase (FAS) and microsomal triglyceride transfer protein (MTTP). Feeding a FO rich diet for 7 days prior to 48 h of fasting resulted in lower hepatic TAG, lower PPAR-α expression and maintenance of hepatic n-3 fatty acid content. Refeeding on a high fructose diet promoted an increase in hepatic and serum TAG and in hepatic PPAR-α, ChREBP and MTTP expression. FO did not prevent the increase in serum and hepatic TAG after fructose refeeding, but did decrease hepatic expression of lipogenic genes and increased the n-3 fatty acid content of the liver. n-3 Fatty acids can modify some components of the hepatic lipid metabolic response to later feeding with a high fructose diet.

  2. Analysis of KLLN as a high-penetrance breast cancer predisposition gene.

    PubMed

    Thompson, Ella R; Gorringe, Kylie L; Choong, David Y H; Eccles, Diana M; Mitchell, Gillian; Campbell, Ian G

    2012-07-01

    KLLN is a p53 target gene with DNA binding function and represents a highly plausible candidate breast cancer predisposition gene. We screened for predisposing variants in 860 high-risk breast cancer families using high resolution melt analysis. A germline c.339_340delAG variant predicted to cause premature termination of the protein after 57 alternative amino acid residues was identified in 3/860 families who tested negative for BRCA1 and BRCA2 mutations and in 1/84 sporadic breast cancer cases. However, the variant was also detected in 2/182 families with known BRCA1 or BRCA2 mutations and in 2/464 non-cancer controls. Furthermore, loss of the mutant allele was detected in 2/2 breast tumors. Our data suggest that pathogenic mutations in KLLN are rare in breast cancer families and the c.339_340delAG variant does not represent a high-penetrance breast cancer risk allele.

  3. BRILIA: Integrated Tool for High-Throughput Annotation and Lineage Tree Assembly of B-Cell Repertoires

    PubMed Central

    Lee, Donald W.; Khavrutskii, Ilja V.; Wallqvist, Anders; Bavari, Sina; Cooper, Christopher L.; Chaudhury, Sidhartha

    2017-01-01

    The somatic diversity of antigen-recognizing B-cell receptors (BCRs) arises from Variable (V), Diversity (D), and Joining (J) (VDJ) recombination and somatic hypermutation (SHM) during B-cell development and affinity maturation. The VDJ junction of the BCR heavy chain forms the highly variable complementarity determining region 3 (CDR3), which plays a critical role in antigen specificity and binding affinity. Tracking the selection and mutation of the CDR3 can be useful in characterizing humoral responses to infection and vaccination. Although tens to hundreds of thousands of unique BCR genes within an expressed B-cell repertoire can now be resolved with high-throughput sequencing, tracking SHMs is still challenging because existing annotation methods are often limited by poor annotation coverage, inconsistent SHM identification across the VDJ junction, or lack of B-cell lineage data. Here, we present B-cell repertoire inductive lineage and immunosequence annotator (BRILIA), an algorithm that leverages repertoire-wide sequencing data to globally improve the VDJ annotation coverage, lineage tree assembly, and SHM identification. On benchmark tests against simulated human and mouse BCR repertoires, BRILIA correctly annotated germline and clonally expanded sequences with 94 and 70% accuracy, respectively, and it has a 90% SHM-positive prediction rate in the CDR3 of heavily mutated sequences; these are substantial improvements over existing methods. We used BRILIA to process BCR sequences obtained from splenic germinal center B cells extracted from C57BL/6 mice. BRILIA returned robust B-cell lineage trees and yielded SHM patterns that are consistent across the VDJ junction and agree with known biological mechanisms of SHM. By contrast, existing BCR annotation tools, which do not account for repertoire-wide clonal relationships, systematically underestimated both the size of clonally related B-cell clusters and yielded inconsistent SHM frequencies. We demonstrate

  4. A high efficiency gene disruption strategy using a positive-negative split selection marker and electroporation for Fusarium oxysporum.

    PubMed

    Liang, Liqin; Li, Jianqiang; Cheng, Lin; Ling, Jian; Luo, Zhongqin; Bai, Miao; Xie, Bingyan

    2014-11-01

    The Fusarium oxysporum species complex consists of fungal pathogens that cause serial vascular wilt disease on more than 100 cultivated species throughout the world. Gene function analysis is rapidly becoming more and more important as the whole-genome sequences of various F. oxysporum strains are being completed. Gene-disruption techniques are a common molecular tool for studying gene function, yet are often a limiting step in gene function identification. In this study we have developed a F. oxysporum high-efficiency gene-disruption strategy based on split-marker homologous recombination cassettes with dual selection and electroporation transformation. The method was efficiently used to delete three RNA-dependent RNA polymerase (RdRP) genes. The gene-disruption cassettes of three genes can be constructed simultaneously within a short time using this technique. The optimal condition for electroporation is 10μF capacitance, 300Ω resistance, 4kV/cm field strength, with 1μg of DNA (gene-disruption cassettes). Under these optimal conditions, we were able to obtain 95 transformants per μg DNA. And after positive-negative selection, the transformants were efficiently screened by PCR, screening efficiency averaged 85%: 90% (RdRP1), 85% (RdRP2) and 77% (RdRP3). This gene-disruption strategy should pave the way for high throughout genetic analysis in F. oxysporum.

  5. In situ Expression of Functional Genes Reveals Nitrogen Cycling at High Temperatures in Terrestrial Hydrothermal Systems

    NASA Astrophysics Data System (ADS)

    Loiacono, S. T.; Meyer-Dombard, D. R.

    2011-12-01

    An essential element for life, nitrogen occurs in all living organisms and is critical for the synthesis of amino acids, proteins, nucleic acids, and other forms of biomass. Thus, nitrogen cycling likely plays a vital role in microbial metabolic processes as well as nutrient availability. For microorganisms in "extreme" environments, this means developing adaptations that allow them to survive in harsh conditions and still perform the metabolisms essential to sustain life. Recent studies have screened biofilms and thermal sediments of Yellowstone National Park (YNP) thermal features for the presence of nifH genes, which code for a key enzyme in the nitrogen fixation process [1-4]. Furthermore, analysis of nitrogen isotopes in biofilms across a temperature and chemical gradient revealed that nitrogen fixation likely varies across the chemosynthetic/photosynthetic ecotone [5]. Although research has evaluated and confirmed the presence of nifH genes in various thermophilic microbial communities, the existence of a gene in the DNA of an organism does not verify its use. Instead, other methods, such as culturing, isotope tracer assays, and gene expression studies are required to provide direct evidence of biological nitrogen fixation. Culturing and isotope tracer approaches have successfully revealed high-temperature biological nitrogen fixation in both marine hydrothermal vent microbial communities [6] and in acidic, terrestrial hydrothermal sediment [3]. Transcriptomics-based techniques (using mRNA extracted from samples to confirm in situ expression of targeted genes) have been much more limited in number, and only a few studies have, to date, investigated in situ expression of the nifH gene in thermophilic microbial communities [2, 7]. This study explores the presence and expression of nifH genes in several features of the Lower Geyser Basin (LGB) of YNP. Nucleic acids from chemosynthetic and photosynthetic microbial communities were extracted and then amplified

  6. High-resolution melt analysis to detect sequence variations in highly homologous gene regions: application to CYP2B6.

    PubMed

    Twist, Greyson P; Gaedigk, Roger; Leeder, J Steven; Gaedigk, Andrea

    2013-06-01

    High-resolution melt (HRM) analysis using 'release-on-demand' dyes, such as EvaGreen(®) has the potential to resolve complex genotypes in situations where genotype interpretation is complicated by the presence of pseudogenes or allelic variants in close proximity to the locus of interest. We explored the utility of HRM to genotype a SNP (785A>G, K262R, rs2279343) that is located within exon 5 of the CYP2B6 gene, which contributes to the metabolism of a number of clinically used drugs. Testing of 785A>G is challenging, but crucial for accurate genotype determination. This SNP is part of multiple known CYP2B6 haplotypes and located in a region that is identical to CYP2B7, a nonfunctional pseudogene. Because small CYP2B6-specific PCR amplicons bracketing 785A>G cannot be generated, we simultaneously amplified both genes. A panel of 235 liver tissue DNAs and five Coriell samples were assessed. Eight CYP2B6/CYP2B7 diplotype combinations were found and a novel variant 769G>A (D257N) was discovered. The frequency of 785G corresponded to those reported for Caucasians and African-Americans. Assay performance was confirmed by CYP2B6 and/or CYP2B7 sequence analysis in a subset of samples, using a preamplified CYP2B6-specific long-range-PCR amplicon as HRM template. Inclusion rather than exclusion of a homologous pseudogene allowed us to devise a sensitive, reliable and affordable assay to test this CYP2B6 SNP. This assay design may be utilized to overcome the challenges and limitations of other methods. Owing to the flexibility of HRM, this assay design can easily be adapted to other gene loci of interest.

  7. Differential gene expression profiling of mouse skin after sulfur mustard exposure: Extended time response and inhibitor effect

    SciTech Connect

    Gerecke, Donald R. Chen Minjun; Isukapalli, Sastry S.; Gordon, Marion K.; Chang, Y.-C.; Tong Weida; Androulakis, Ioannis P.; Georgopoulos, Panos G.

    2009-01-15

    Sulfur mustard (HD, SM), is a chemical warfare agent that within hours causes extensive blistering at the dermal-epidermal junction of skin. To better understand the progression of SM-induced blistering, gene expression profiling for mouse skin was performed after a single high dose of SM exposure. Punch biopsies of mouse ears were collected at both early and late time periods following SM exposure (previous studies only considered early time periods). The biopsies were examined for pathological disturbances and the samples further assayed for gene expression profiling using the Affymetrix microarray analysis system. Principal component analysis and hierarchical cluster analysis of the differently expressed genes, performed with ArrayTrack showed clear separation of the various groups. Pathway analysis employing the KEGG library and Ingenuity Pathway Analysis (IPA) indicated that cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), and hematopoietic cell lineage are common pathways affected at different time points. Gene ontology analysis identified the most significantly altered biological processes as the immune response, inflammatory response, and chemotaxis; these findings are consistent with other reported results for shorter time periods. Selected genes were chosen for RT-PCR verification and showed correlations in the general trends for the microarrays. Interleukin 1 beta was checked for biological analysis to confirm the presence of protein correlated to the corresponding microarray data. The impact of a matrix metalloproteinase inhibitor, MMP-2/MMP-9 inhibitor I, against SM exposure was assessed. These results can help in understanding the molecular mechanism of SM-induced blistering, as well as to test the efficacy of different inhibitors.

  8. Two ras genes in Dictyostelium minutum show high sequence homology, but different developmental regulation from Dictyostelium discoideum rasD and rasG genes.

    PubMed

    van Es, S; Kooistra, R A; Schaap, P

    1997-03-10

    The social amoeba Dictyostelium discoideum expresses five ras genes at different stages of development. One of them, DdrasD is expressed during postaggregative development and transcription is induced by extracellular cAMP. A homologue of DdrasD, the DdrasG gene, is expressed exclusively during vegetative growth. We cloned two ras homologues Dmras1 and Dmras2 from the primitive species D. minutum, which show high homology to DdrasD and DdrasG and less homology to the other Ddras genes. In contrast to the DdrasD and DdrasG genes, both the Dmras1 and Dmras2 genes are expressed during the entire course of development. The expression levels are low during growth, increase at the onset of starvation and do not decrease until fruiting bodies have formed. Expression of neither Dmras1 or Dmras2 is regulated by cAMP. So even though the high degree of homology between the ras genes of different species suggests conservation of function, this function is apparently not associated with a specific developmental stage.

  9. Heterogeneous high throughput scientific computing with APM X-Gene and Intel Xeon Phi

    DOE PAGES

    Abdurachmanov, David; Bockelman, Brian; Elmer, Peter; ...

    2015-05-22

    Electrical power requirements will be a constraint on the future growth of Distributed High Throughput Computing (DHTC) as used by High Energy Physics. Performance-per-watt is a critical metric for the evaluation of computer architectures for cost- efficient computing. Additionally, future performance growth will come from heterogeneous, many-core, and high computing density platforms with specialized processors. In this paper, we examine the Intel Xeon Phi Many Integrated Cores (MIC) co-processor and Applied Micro X-Gene ARMv8 64-bit low-power server system-on-a-chip (SoC) solutions for scientific computing applications. As a result, we report our experience on software porting, performance and energy efficiency and evaluatemore » the potential for use of such technologies in the context of distributed computing systems such as the Worldwide LHC Computing Grid (WLCG).« less

  10. Heterogeneous high throughput scientific computing with APM X-Gene and Intel Xeon Phi

    SciTech Connect

    Abdurachmanov, David; Bockelman, Brian; Elmer, Peter; Eulisse, Giulio; Knight, Robert; Muzaffar, Shahzad

    2015-05-22

    Electrical power requirements will be a constraint on the future growth of Distributed High Throughput Computing (DHTC) as used by High Energy Physics. Performance-per-watt is a critical metric for the evaluation of computer architectures for cost- efficient computing. Additionally, future performance growth will come from heterogeneous, many-core, and high computing density platforms with specialized processors. In this paper, we examine the Intel Xeon Phi Many Integrated Cores (MIC) co-processor and Applied Micro X-Gene ARMv8 64-bit low-power server system-on-a-chip (SoC) solutions for scientific computing applications. As a result, we report our experience on software porting, performance and energy efficiency and evaluate the potential for use of such technologies in the context of distributed computing systems such as the Worldwide LHC Computing Grid (WLCG).

  11. Heterogeneous High Throughput Scientific Computing with APM X-Gene and Intel Xeon Phi

    NASA Astrophysics Data System (ADS)

    Abdurachmanov, David; Bockelman, Brian; Elmer, Peter; Eulisse, Giulio; Knight, Robert; Muzaffar, Shahzad

    2015-05-01

    Electrical power requirements will be a constraint on the future growth of Distributed High Throughput Computing (DHTC) as used by High Energy Physics. Performance-per-watt is a critical metric for the evaluation of computer architectures for cost- efficient computing. Additionally, future performance growth will come from heterogeneous, many-core, and high computing density platforms with specialized processors. In this paper, we examine the Intel Xeon Phi Many Integrated Cores (MIC) co-processor and Applied Micro X-Gene ARMv8 64-bit low-power server system-on-a-chip (SoC) solutions for scientific computing applications. We report our experience on software porting, performance and energy efficiency and evaluate the potential for use of such technologies in the context of distributed computing systems such as the Worldwide LHC Computing Grid (WLCG).

  12. Genes that are involved in high hydrostatic pressure treatments in a Listeria monocytogenes Scott A ctsR deletion mutant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Listeria monocytogenes is a food-borne pathogen of significant threat to public health. High Hydrostatic Pressure (HHP) treatment can be used to control L. monocytogenes in food. The CtsR (class three stress gene repressor) protein negatively regulates the expression of class III heat shock genes....

  13. Predicted highly expressed genes in Nocardia farcinica and the implication to its primary metabolism and nocardial virulence

    SciTech Connect

    Wu, Gang; Nie, Lei; Zhang, Weiwen

    2006-02-23

    Nocardia farcinica is a gram positive, filamentous bacterium, and is considered an opportunistic pathogen. In this study, the highly expressed genes in N. farcinica were predicted using the codon adaptation index (CAI) as a numerical estimator of gene expressivity. Using ribosomal protein (RP) genes as references, the top {approx}10% of the genes were predicted to be the predicted highly expressed (PHX) genes in N. farcinica using a CAI cutoff of greater than 0.73. Consistent with early analysis in Streptomyces genomes, most of the PHX genes in N. farcinica were involved in various ''house-keeping'' functions important for cell growth. However, fifteen genes putatively involved in no cardial virulence were predicted as PHX in N. farcinica, which included genes encoding four Mce virulence proteins, cyclopropane fatty acid synthase which is involved in the modification of cell wall important for nocardia virulence, polyketide synthase PKS13 for mycolic acid synthesis and non-ribosomal peptide synthetase involved in biosynthesis of a mycobactin-related siderophore. In addition, multiple genes involved in defense against reactive oxygen species (ROS) produced by the phagocyte were predicted with high expressivity, which included alkylhydroperoxide reductase (ahpC), catalase (katG), superoxide dismutase (sodF), thioredoxin, thioredoxin reductase, glutathione peroxidase, and peptide methionine sulfoxide reductase, suggesting that combating against ROS was essential for survival of N. farcinica in host cells. The study also showed that the distribution of PHX genes in the N. farcinica circular chromosome was uneven, with more PHX genes located in the regions close to replication initiation site. The results provided the first approximates of global gene expression patterns in N. farcinica, which will be useful in guiding experimental design for further investigation.

  14. Comparative analysis of Mycobacterium tuberculosis pe and ppe genes reveals high sequence variation and an apparent absence of selective constraints.

    PubMed

    McEvoy, Christopher R E; Cloete, Ruben; Müller, Borna; Schürch, Anita C; van Helden, Paul D; Gagneux, Sebastien; Warren, Robin M; Gey van Pittius, Nicolaas C

    2012-01-01

    Mycobacterium tuberculosis complex (MTBC) genomes contain 2 large gene families termed pe and ppe. The function of pe/ppe proteins remains enigmatic but studies suggest that they are secreted or cell surface associated and are involved in bacterial virulence. Previous studies have also shown that some pe/ppe genes are polymorphic, a finding that suggests involvement in antigenic variation. Using comparative sequence analysis of 18 publicly available MTBC whole genome sequences, we have performed alignments of 33 pe (excluding pe_pgrs) and 66 ppe genes in order to detect the frequency and nature of genetic variation. This work has been supplemented by whole gene sequencing of 14 pe/ppe (including 5 pe_pgrs) genes in a cohort of 40 diverse and well defined clinical isolates covering all the main lineages of the M. tuberculosis phylogenetic tree. We show that nsSNP's in pe (excluding pgrs) and ppe genes are 3.0 and 3.3 times higher than in non-pe/ppe genes respectively and that numerous other mutation types are also present at a high frequency. It has previously been shown that non-pe/ppe M. tuberculosis genes display a remarkably low level of purifying selection. Here, we also show that compared to these genes those of the pe/ppe families show a further reduction of selection pressure that suggests neutral evolution. This is inconsistent with the positive selection pressure of "classical" antigenic variation. Finally, by analyzing such a large number of genes we were able to detect large differences in mutation type and frequency between both individual genes and gene sub-families. The high variation rates and absence of selective constraints provides valuable insights into potential pe/ppe function. Since pe/ppe proteins are highly antigenic and have been studied as potential vaccine components these results should also prove informative for aspects of M. tuberculosis vaccine design.

  15. High-Throughput Retina-Array for Screening 93 Genes Involved in Inherited Retinal Dystrophy

    PubMed Central

    Song, Jin; Smaoui, Nizar; Ayyagari, Radha; Stiles, David; Benhamed, Sonia; MacDonald, Ian M.; Daiger, Stephen P.; Tumminia, Santa J.; Hejtmancik, Fielding

    2011-01-01

    Purpose. Retinal dystrophy (RD) is a broad group of hereditary disorders with heterogeneous genotypes and phenotypes. Current available genetic testing for these diseases is complicated, time consuming, and expensive. This study was conducted to develop and apply a microarray-based, high-throughput resequencing system to detect sequence alterations in genes related to inherited RD. Methods. A customized 300-kb resequencing chip, Retina-Array, was developed to detect sequence alterations of 267,550 bases of both sense and antisense sequence in 1470 exons spanning 93 genes involved in inherited RD. Retina-Array was evaluated in 19 patient samples with inherited RD provided by the eyeGENE repository and four Centre d'Etudes du Polymorphisme Humaine reference samples through a high-throughput experimental approach that included an automated PCR assay setup and quantification, efficient post-quantification data processing, optimized pooling and fragmentation, and standardized chip processing. Results. The performance of the chips demonstrated that the average base pair call rate and accuracy were 93.56% and 99.86%, respectively. In total, 304 candidate variations were identified using a series of customized screening filters. Among 174 selected variations, 123 (70.7%) were further confirmed by dideoxy sequencing. Analysis of patient samples using Retina-Array resulted in the identification of 10 known mutations and 12 novel variations with high probability of deleterious effects. Conclusions. This study suggests that Retina-Array might be a valuable tool for the detection of disease-causing mutations and disease severity modifiers in a single experiment. Retinal-Array may provide a powerful and feasible approach through which to study genetic heterogeneity in retinal diseases. PMID:22025579

  16. Aspergillus glaucus Aquaglyceroporin Gene glpF Confers High Osmosis Tolerance in Heterologous Organisms.

    PubMed

    Liu, Xiao-Dan; Wei, Yi; Zhou, Xiao-Yang; Pei, Xue; Zhang, Shi-Hong

    2015-10-01

    Aquaglyceroporins (GlpFs) that transport glycerol along with water and other uncharged solutes are involved in osmoregulation in myriad species. Fungal species form a large group of eukaryotic organisms, and their GlpFs may be diverse, exhibiting various activities. However, few filamentous fungal GlpFs have been biologically investigated. Here, a glpF gene from the halophilic fungus Aspergillus glaucus (AgglpF) was verified to be a channel of water or glycerol in Xenopus laevis oocytes and was further functionally analyzed in three heterologous systems. In Saccharomyces cerevisiae, cells overexpressing AgglpF possessed significant tolerance of drought, salt, and certain metal ions. AgglpF was then characterized in the filamentous fungus of Neurospora crassa. Based on the N. crassa aquaporin gene (NcAQP) disruption mutant (the Δaqp mutant), a series of complementary strains carrying NcAQP and AgglpF and three asparagine-proline-alanine-gene (NPA)-deleted AgglpF fragments were created. As revealed by salt resistance analysis, the AgglpF complementary strain possessed the highest salt resistance among the tested strains. In addition, the intracellular glycerol content in the AgglpF complementary strain was markedly higher than that in the other strains. The AgGlpF-green fluorescent protein (GFP) fusion protein was subcellularly localized in the plasma membrane of onion epidermal cells, suggesting that AgglpF functions in plants. Indeed, when AgglpF was expressed in Arabidopsis thaliana, transgenic lines survived under conditions of high osmotic stress and under conditions of drought stress in particular. Overall, our results revealed that AgGlpF as a water/glycerol transporter is required for survival of both fungi and plants under conditions of high osmotic stress and may have value in applications in genetic engineering for generating high salt and drought resistance.

  17. [Recent progress in gene mapping through high-throughput sequencing technology and forward genetic approaches].

    PubMed

    Lu, Cairui; Zou, Changsong; Song, Guoli

    2015-08-01

    Traditional gene mapping using forward genetic approaches is conducted primarily through construction of a genetic linkage map, the process of which is tedious and time-consuming, and often results in low accuracy of mapping and large mapping intervals. With the rapid development of high-throughput sequencing technology and decreasing cost of sequencing, a variety of simple and quick methods of gene mapping through sequencing have been developed, including direct sequencing of the mutant genome, sequencing of selective mutant DNA pooling, genetic map construction through sequencing of individuals in population, as well as sequencing of transcriptome and partial genome. These methods can be used to identify mutations at the nucleotide level and has been applied in complex genetic background. Recent reports have shown that sequencing mapping could be even done without the reference of genome sequence, hybridization, and genetic linkage information, which made it possible to perform forward genetic study in many non-model species. In this review, we summarized these new technologies and their application in gene mapping.

  18. High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool.

    PubMed

    Winfield, Mark O; Allen, Alexandra M; Burridge, Amanda J; Barker, Gary L A; Benbow, Harriet R; Wilkinson, Paul A; Coghill, Jane; Waterfall, Christy; Davassi, Alessandro; Scopes, Geoff; Pirani, Ali; Webster, Teresa; Brew, Fiona; Bloor, Claire; King, Julie; West, Claire; Griffiths, Simon; King, Ian; Bentley, Alison R; Edwards, Keith J

    2016-05-01

    In wheat, a lack of genetic diversity between breeding lines has been recognized as a significant block to future yield increases. Species belonging to bread wheat's secondary and tertiary gene pools harbour a much greater level of genetic variability, and are an important source of genes to broaden its genetic base. Introgression of novel genes from progenitors and related species has been widely employed to improve the agronomic characteristics of hexaploid wheat, but this approach has been hampered by a lack of markers that can be used to track introduced chromosome segments. Here, we describe the identification of a large number of single nucleotide polymorphisms that can be used to genotype hexaploid wheat and to identify and track introgressions from a variety of sources. We have validated these markers using an ultra-high-density Axiom(®) genotyping array to characterize a range of diploid, tetraploid and hexaploid wheat accessions and wheat relatives. To facilitate the use of these, both the markers and the associated sequence and genotype information have been made available through an interactive web site.

  19. Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

    PubMed Central

    TerMaat, Joel R.; Pienaar, Elsje; Whitney, Scott E.; Mamedov, Tarlan G.; Subramanian, Anuradha

    2013-01-01

    Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653 bp) and pUC19 β-lactamase (929 bp) in under 20 min. However, rapid integrated PCA–PCR was found to be problematic when attempted with the TM-1 gene (1509 bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers. PMID:19799938

  20. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

    PubMed

    Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A; Tabor, Kathryn M; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A

    2015-04-20

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

  1. High-Throughput and Combinatorial Gene Expression on a Chip for Metabolism-Induced Toxicology Screening

    PubMed Central

    Kwon, Seok Joon; Lee, Dong Woo; Shah, Dhiral A.; Ku, Bosung; Jeon, Sang Youl; Solanki, Kusum; Ryan, Jessica D.; Clark, Douglas S.; Dordick, Jonathan S.; Lee, Moo-Yeal

    2014-01-01

    Differential expression of various drug-metabolizing enzymes in the human liver may cause deviations of pharmacokinetic profiles, resulting in inter-individual variability of drug toxicity and/or efficacy. Here we present the “Transfected Enzyme and Metabolism Chip” (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip. The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CYP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray. Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner. PMID:24799042

  2. Genomic Integration of High-Risk HPV Alters Gene Expression in Oropharyngeal Squamous Cell Carcinoma.

    PubMed

    Walline, Heather M; Komarck, Christine M; McHugh, Jonathan B; Bellile, Emily L; Brenner, J Chad; Prince, Mark E; McKean, Erin L; Chepeha, Douglas B; Wolf, Gregory T; Worden, Francis P; Bradford, Carol R; Carey, Thomas E

    2016-10-01

    High-risk HPV (hrHPV) is the leading etiologic factor in oropharyngeal cancer. HPV-positive oropharyngeal tumors generally respond well to therapy, with complete recovery in approximately 80% of patients. However, it remains unclear why some patients are nonresponsive to treatment, with 20% of patients recurring within 5 years. In this study, viral factors were examined for possible clues to differences in tumor behavior. Oropharynx tumors that responded well to therapy were compared with those that persisted and recurred. Viral oncogene alternate transcripts were assessed, and cellular sites of viral integration were mapped and sequenced. Effects of integration on gene expression were assessed by transcript analysis at the integration sites. All of the tumors demonstrated active viral oncogenesis, indicated by expression of HPV E6 and E7 oncogenes and alternate E6 splicing. In the responsive tumors, HPV integration occurred exclusively in intergenic chromosome regions, except for one tumor with viral integration into TP63. Each recurrent tumor exhibited complex HPV integration patterns into cancer-associated genes, including TNFRSF13B, SCN2A, SH2B1, UBE2V2, SMOC1, NFIA, and SEMA6D Disrupted cellular transcripts were identified in the region of integration in four of the seven affected genes.

  3. High throughput profiling of antibiotic resistance genes in urban park soils with reclaimed water irrigation.

    PubMed

    Wang, Feng-Hua; Qiao, Min; Su, Jian-Qiang; Chen, Zheng; Zhou, Xue; Zhu, Yong-Guan

    2014-08-19

    Reclaimed water irrigation (RWI) in urban environments is becoming popular, due to rapid urbanization and water shortage. The continuous release of residual antibiotics and antibiotic resistance genes (ARGs) from reclaimed water could result in the dissemination of ARGs in the downstream environment. This study provides a comprehensive profile of ARGs in park soils exposed to RWI through a high-throughput quantitative PCR approach. 147 ARGs encoding for resistance to a broad-spectrum of antibiotics were detected among all park soil samples. Aminoglycoside and beta-lactam were the two most dominant types of ARGs, and antibiotic deactivation and efflux pump were the two most dominant mechanisms in these RWI samples. The total enrichment of ARGs varied from 99.3-fold to 8655.3-fold compared to respective controls. Six to 60 ARGs were statistically enriched among these RWI samples. Four transposase genes were detected in RWI samples. TnpA-04 was the most enriched transposase gene with an enrichment was up to 2501.3-fold in Urumqi RWI samples compared with control soil samples. Furthermore, significantly positive correlation was found between ARGs and transposase abundances, indicating that transposase might be involved in the propagation of ARGs. This study demonstrated that RWI resulted in the enrichment of ARGs in urban park soils.

  4. High Expression of Endogenous Retroviral Envelope Gene in the Equine Fetal Part of the Placenta

    PubMed Central

    Stefanetti, Valentina; Marenzoni, Maria Luisa; Passamonti, Fabrizio; Cappelli, Katia; Garcia-Etxebarria, Koldo; Coletti, Mauro; Capomaccio, Stefano

    2016-01-01

    Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses that have co-evolved with vertebrate genomes for millions of years. Previous studies have identified the envelope (env) protein genes of retroviral origin preferentially expressed in the placenta which suggests a role in placentation based on their membrane fusogenic capacity and therefore they have been named syncytins. Until now, all the characterized syncytins have been associated with three invasive placentation types: the endotheliochorial (Carnivora), the synepitheliochorial (Ruminantia), and the hemochorial placentation (human, mouse) where they play a role in the syncytiotrophoblast formation. The purpose of the present study was to evaluate whether EqERV env RNA is expressed in horse tissues as well and investigate if the horse, possessing an epitheliochorial placenta, has “captured” a common retroviral env gene with syncytin-like properties in placental tissues. Interestingly, although in the equine placenta there is no syncytiotrophoblast layer at the maternal-fetal interface, our results showed that EqERV env RNA is highly expressed at that level, as expected for a candidate syncytin-like gene but with reduced abundance in the other somatic tissues (nearly 30-fold lower) thus suggesting a possible role in the placental tissue. Although the horse is one of the few domestic animals with a sequenced genome, few studies have been conducted about the EqERV and their expression in placental tissue has never been investigated. PMID:27176223

  5. High blood pressure in transgenic mice carrying the rat angiotensinogen gene.

    PubMed Central

    Kimura, S; Mullins, J J; Bunnemann, B; Metzger, R; Hilgenfeldt, U; Zimmermann, F; Jacob, H; Fuxe, K; Ganten, D; Kaling, M

    1992-01-01

    Transgenic mice were generated by injecting the entire rat angiotensinogen gene into the germline of NMRI mice. The resulting transgenic animals were characterized with respect to hemodynamics, parameters of the renin angiotension system, and expression of the transgene. The transgenic line TGM(rAOGEN)123 developed hypertension with a mean arterial blood pressure of 158 mmHg in males and 132 mmHg in females. In contrast, the transgenic line TGM(rAOGEN)92 was not hypertensive. Rat angiotensinogen was detectable only in plasma of animals of line 123. Total plasma angiotensinogen and plasma angiotensin II concentrations were about three times as high as those of negative control mice. In TGM(rAOGEN)123 the transgene was highly expressed in liver and brain. Transcripts were also detected in heart, kidney and testis. In TGM(rAOGEN)92 the brain was the main expressing organ. In situ hybridization revealed an mRNA distribution in the brain of TGM(rAOGEN)123 similar to the one in rat. In TGM(rAOGEN)92 the expression pattern in the brain was aberrant. These data indicate that overexpression of the angiotensinogen gene in liver and brain leads to the development of hypertension in transgenic mice. The TGM(rAOGEN)123 constitutes a high angiotensin II type of hypertension and may provide a new experimental animal model to study the kinetics and function of the renin angiotensin system. Images PMID:1547785

  6. High rates of virus-induced gene silencing by tobacco rattle virus in Populus.

    PubMed

    Shen, Zedan; Sun, Jian; Yao, Jun; Wang, Shaojie; Ding, Mingquan; Zhang, Huilong; Qian, Zeyong; Zhao, Nan; Sa, Gang; Zhao, Rui; Shen, Xin; Polle, Andrea; Chen, Shaoliang

    2015-09-01

    Virus-induced gene silencing (VIGS) has been shown to be an effective tool for investigating gene functions in herbaceous plant species, but has rarely been tested in trees. The establishment of a fast and reliable transformation system is especially important for woody plants, many of which are recalcitrant to transformation. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for two Populus species, Populus euphratica and P. × canescens. Here, TRV constructs carrying a 266 bp or a 558 bp fragment of the phytoene desaturase (PDS) gene were Agrobacterium-infiltrated into leaves of the two poplar species. Agrobacterium-mediated delivery of the shorter insert, TRV2-PePDS266, into the host poplars resulted in expected photobleaching in both tree species, but not the longer insert, PePDS558. The efficiency of VIGS was temperature-dependent, increasing by raising the temperature from 18 to 28 °C. The optimized TRV-VIGS system at 28 °C resulted in a high silencing frequency and efficiency up to 65-73 and 83-94%, respectively, in the two tested poplars. Moreover, syringe inoculation of Agrobacterium in 100 mM acetosyringone induced a more efficient silencing in the two poplar species, compared with other agroinfiltration methods, e.g., direct injection, misting and agrodrench. There were plant species-related differences in the response to VIGS because the photobleaching symptoms were more severe in P. × canescens than in P. euphratica. Furthermore, VIGS-treated P. euphratica exhibited a higher recovery rate (50%) after several weeks of the virus infection, compared with TRV-infected P. × canescens plants (20%). Expression stability of reference genes was screened to assess the relative abundance of PePDS mRNA in VIGS-treated P. euphratica and P. × canescens. PeACT7 was stably expressed in P. euphratica and UBQ-L was selected as the most suitable reference gene for P. × canescens using three different

  7. High diversity and no significant selection signal of human ADH1B gene in Tibet

    PubMed Central

    2012-01-01

    Background ADH1B is one of the most studied human genes with many polymorphic sites. One of the single nucleotide polymorphism (SNP), rs1229984, coding for the Arg48His substitution, have been associated with many serious diseases including alcoholism and cancers of the digestive system. The derived allele, ADH1B*48His, reaches high frequency only in East Asia and Southwest Asia, and is highly associated with agriculture. Micro-evolutionary study has defined seven haplogroups for ADH1B based on seven SNPs encompassing the gene. Three of those haplogroups, H5, H6, and H7, contain the ADH1B*48His allele. H5 occurs in Southwest Asia and the other two are found in East Asia. H7 is derived from H6 by the derived allele of rs3811801. The H7 haplotype has been shown to have undergone significant positive selection in Han Chinese, Hmong, Koreans, Japanese, Khazak, Mongols, and so on. Methods In the present study, we tested whether Tibetans also showed evidence for selection by typing 23 SNPs in the region covering the ADH1B gene in 1,175 individuals from 12 Tibetan populations representing all districts of the Tibet Autonomous Region. Multiple statistics were estimated to examine the gene diversities and positive selection signals among the Tibetans and other populations in East Asia. Results The larger Tibetan populations (Qamdo, Lhasa, Nagqu, Nyingchi, Shannan, and Shigatse) comprised mostly farmers, have around 12% of H7, and 2% of H6. The smaller populations, living on hunting or recently switched to farming, have lower H7 frequencies (Tingri 9%, Gongbo 8%, Monba and Sherpa 6%). Luoba (2%) and Deng (0%) have even lower frequencies. Long-range haplotype analyses revealed very weak signals of positive selection for H7 among Tibetans. Interestingly, the haplotype diversity of H7 is higher in Tibetans than in any other populations studied, indicating a longer diversification history for that haplogroup in Tibetans. Network analysis on the long-range haplotypes revealed

  8. Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions.

    PubMed

    Cassan-Wang, Hua; Soler, Marçal; Yu, Hong; Camargo, Eduardo Leal O; Carocha, Victor; Ladouce, Nathalie; Savelli, Bruno; Paiva, Jorge A P; Leplé, Jean-Charles; Grima-Pettenati, Jacqueline

    2012-12-01

    Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.

  9. High frequencies of antibiotic resistance genes in infants' meconium and early fecal samples.

    PubMed

    Gosalbes, M J; Vallès, Y; Jiménez-Hernández, N; Balle, C; Riva, P; Miravet-Verde, S; de Vries, L E; Llop, S; Agersø, Y; Sørensen, S J; Ballester, F; Francino, M P

    2016-02-01

    The gastrointestinal tract (GIT) microbiota has been identified as an important reservoir of antibiotic resistance genes (ARGs) that can be horizontally transferred to pathogenic species. Maternal GIT microbes can be transmitted to the offspring, and recent work indicates that such transfer starts before birth. We have used culture-independent genetic screenings to explore whether ARGs are already present in the meconium accumulated in the GIT during fetal life and in feces of 1-week-old infants. We have analyzed resistance to β-lactam antibiotics (BLr) and tetracycline (Tcr), screening for a variety of genes conferring each. To evaluate whether ARGs could have been inherited by maternal transmission, we have screened perinatal fecal samples of the 1-week-old babies' mothers, as well as a mother-infant series including meconium, fecal samples collected through the infant's 1st year, maternal fecal samples and colostrum. Our results reveal a high prevalence of BLr and Tcr in both meconium and early fecal samples, implying that the GIT resistance reservoir starts to accumulate even before birth. We show that ARGs present in the mother may reach the meconium and colostrum and establish in the infant GIT, but also that some ARGs were likely acquired from other sources. Alarmingly, we identified in both meconium and 1-week-olds' samples a particularly elevated prevalence of mecA (>45%), six-fold higher than that detected in the mothers. The mecA gene confers BLr to methicillin-resistant Staphylococcus aureus, and although its detection does not imply the presence of this pathogen, it does implicate the young infant's GIT as a noteworthy reservoir of this gene.

  10. Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

    PubMed

    Shmueli, Ron B; Bhise, Nupura S; Green, Jordan J

    2013-03-01

    Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi

  11. The MAR-binding protein SATB1 orchestrates temporal and spatial expression of multiple genes during T-cell development

    PubMed Central

    Alvarez, John D.; Yasui, Dag H.; Niida, Hiroyuki; Joh, Tadashi; Loh, Dennis Y.; Kohwi-Shigematsu, Terumi

    2000-01-01

    SATB1 is expressed primarily in thymocytes and can act as a transcriptional repressor. SATB1 binds in vivo to the matrix attachment regions (MARs) of DNA, which are implicated in the loop domain organization of chromatin. The role of MAR-binding proteins in specific cell lineages is unknown. We generated SATB1-null mice to determine how SATB1 functions in the T-cell lineage. SATB1-null mice are small in size, have disproportionately small thymi and spleens, and die at 3 weeks of age. At the cellular level, multiple defects in T-cell development were observed. Immature CD3−CD4−CD8− triple negative (TN) thymocytes were greatly reduced in number, and thymocyte development was blocked mainly at the DP stage. The few peripheral CD4+ single positive (SP) cells underwent apoptosis and failed to proliferate in response to activating stimuli. At the molecular level, among 589 genes examined, at least 2% of genes including a proto-oncogene, cytokine receptor genes, and apoptosis-related genes were derepressed at inappropriate stages of T-cell development in SATB1-null mice. For example, IL-2Rα and IL-7Rα genes were ectopically transcribed in CD4+CD8+ double positive (DP) thymocytes. SATB1 appears to orchestrate the temporal and spatial expression of genes during T-cell development, thereby ensuring the proper development of this lineage. Our data provide the first evidence that MAR-binding proteins can act as global regulators of cell function in specific cell lineages. PMID:10716941

  12. Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

    SciTech Connect

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng . E-mail: Dongfeng_pan@yahoo.com

    2005-09-09

    PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10{sup 9} PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.

  13. Metagenomic analysis revealed highly diverse microbial arsenic metabolism genes in paddy soils with low-arsenic contents.

    PubMed

    Xiao, Ke-Qing; Li, Li-Guan; Ma, Li-Ping; Zhang, Si-Yu; Bao, Peng; Zhang, Tong; Zhu, Yong-Guan

    2016-04-01

    Microbe-mediated arsenic (As) metabolism plays a critical role in global As cycle, and As metabolism involves different types of genes encoding proteins facilitating its biotransformation and transportation processes. Here, we used metagenomic analysis based on high-throughput sequencing and constructed As metabolism protein databases to analyze As metabolism genes in five paddy soils with low-As contents. The results showed that highly diverse As metabolism genes were present in these paddy soils, with varied abundances and distribution for different types and subtypes of these genes. Arsenate reduction genes (ars) dominated in all soil samples, and significant correlation existed between the abundance of arr (arsenate respiration), aio (arsenite oxidation), and arsM (arsenite methylation) genes, indicating the co-existence and close-relation of different As resistance systems of microbes in wetland environments similar to these paddy soils after long-term evolution. Among all soil parameters, pH was an important factor controlling the distribution of As metabolism gene in five paddy soils (p = 0.018). To the best of our knowledge, this is the first study using high-throughput sequencing and metagenomics approach in characterizing As metabolism genes in the five paddy soil, showing their great potential in As biotransformation, and therefore in mitigating arsenic risk to humans.

  14. Conserved cis-regulatory modules in promoters of genes encoding wheat high-molecular-weight glutenin subunits

    PubMed Central

    Ravel, Catherine; Fiquet, Samuel; Boudet, Julie; Dardevet, Mireille; Vincent, Jonathan; Merlino, Marielle; Michard, Robin; Martre, Pierre

    2014-01-01

    The concentration and composition of the gliadin and glutenin seed storage proteins (SSPs) in wheat flour are the most important determinants of its end-use value. In cereals, the synthesis of SSPs is predominantly regulated at the transcriptional level by a complex network involving at least five cis-elements in gene promoters. The high-molecular-weight glutenin subunits (HMW-GS) are encoded by two tightly linked genes located on the long arms of group 1 chromosomes. Here, we sequenced and annotated the HMW-GS gene promoters of 22 electrophoretic wheat alleles to identify putative cis-regulatory motifs. We focused on 24 motifs known to be involved in SSP gene regulation. Most of them were identified in at least one HMW-GS gene promoter sequence. A common regulatory framework was observed in all the HMW-GS gene promoters, as they shared conserved cis-regulatory modules (CCRMs) including all the five motifs known to regulate the transcription of SSP genes. This common regulatory framework comprises a composite box made of the GATA motifs and GCN4-like Motifs (GLMs) and was shown to be functional as the GLMs are able to bind a bZIP transcriptional factor SPA (Storage Protein Activator). In addition to this regulatory framework, each HMW-GS gene promoter had additional motifs organized differently. The promoters of most highly expressed x-type HMW-GS genes contain an additional box predicted to bind R2R3-MYB transcriptional factors. However, the differences in annotation between promoter alleles could not be related to their level of expression. In summary, we identified a common modular organization of HMW-GS gene promoters but the lack of correlation between the cis-motifs of each HMW-GS gene promoter and their level of expression suggests that other cis-elements or other mechanisms regulate HMW-GS gene expression. PMID:25429295

  15. Development of a facile method for high throughput screening with reporter gene assays.

    PubMed

    Goetz, A S; Andrews, J L; Littleton, T R; Ignar, D M

    2000-10-01

    This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

  16. Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage.

    PubMed

    Shiraki, Toshiyuki; Kondo, Shinji; Katayama, Shintaro; Waki, Kazunori; Kasukawa, Takeya; Kawaji, Hideya; Kodzius, Rimantas; Watahiki, Akira; Nakamura, Mari; Arakawa, Takahiro; Fukuda, Shiro; Sasaki, Daisuke; Podhajska, Anna; Harbers, Matthias; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide

    2003-12-23

    We introduce cap analysis gene expression (CAGE), which is based on preparation and sequencing of concatamers of DNA tags deriving from the initial 20 nucleotides from 5' end mRNAs. CAGE allows high-throughout gene expression analysis and the profiling of transcriptional start points (TSP), including promoter usage analysis. By analyzing four libraries (brain, cortex, hippocampus, and cerebellum), we redefined more accurately the TSPs of 11-27% of the analyzed transcriptional units that were hit. The frequency of CAGE tags correlates well with results from other analyses, such as serial analysis of gene expression, and furthermore maps the TSPs more accurately, including in tissue-specific cases. The high-throughput nature of this technology paves the way for understanding gene networks via correlation of promoter usage and gene transcriptional factor expression.

  17. A blood-based gene expression and signaling pathway analysis to differentiate between high and low grade gliomas

    PubMed Central

    Ponnampalam, Stephen N.; Kamaluddin, Nor Rizan; Zakaria, Zubaidah; Matheneswaran, Vickneswaran; Ganesan, Dharmendra; Haspani, Mohammed Saffari; Ryten, Mina; Hardy, John A.

    2016-01-01

    The aims of the present study were to undertake gene expression profiling of the blood of glioma patients to determine key genetic components of signaling pathways and to develop a panel of genes that could be used as a potential blood-based biomarker to differentiate between high and low grade gliomas, non-gliomas and control samples. In this study, blood samples were obtained from glioma patients, non-glioma and control subjects. Ten samples each were obtained from patients with high and low grade tumours, respectively, ten samples from non-glioma patients and twenty samples from control subjects. Total RNA was isolated from each sample after which first and second strand synthesis was performed. The resulting cRNA was then hybridized with the Agilent Whole Human Genome (4×44K) microarray chip according to the manufacturer's instructions. Universal Human Reference RNA and samples were labeled with Cy3 CTP and Cy5 CTP, respectively. Microarray data were analyzed by the Agilent Gene Spring 12.1V software using stringent criteria which included at least a 2-fold difference in gene expression between samples. Statistical analysis was performed using the unpaired Student's t-test with a P<0.01. Pathway enrichment was also performed, with key genes selected for validation using droplet digital polymerase chain reaction (ddPCR). The gene expression profiling indicated that were a substantial number of genes that were differentially expressed with more than a 2-fold change (P<0.01) between each of the four different conditions. We selected key genes within significant pathways that were analyzed through pathway enrichment. These key genes included regulators of cell proliferation, transcription factors, cytokines and tumour suppressor genes. In the present study, we showed that key genes involved in significant and well established pathways, could possibly be used as a potential blood-based biomarker to differentiate between high and low grade gliomas, non-gliomas and

  18. A blood-based gene expression and signaling pathway analysis to differentiate between high and low grade gliomas.

    PubMed

    Ponnampalam, Stephen N; Kamaluddin, Nor Rizan; Zakaria, Zubaidah; Matheneswaran, Vickneswaran; Ganesan, Dharmendra; Haspani, Mohammed Saffari; Ryten, Mina; Hardy, John A

    2017-01-01

    The aims of the present study were to undertake gene expression profiling of the blood of glioma patients to determine key genetic components of signaling pathways and to develop a panel of genes that could be used as a potential blood-based biomarker to differentiate between high and low grade gliomas, non-gliomas and control samples. In this study, blood samples were obtained from glioma patients, non-glioma and control subjects. Ten samples each were obtained from patients with high and low grade tumours, respectively, ten samples from non-glioma patients and twenty samples from control subjects. Total RNA was isolated from each sample after which first and second strand synthesis was performed. The resulting cRNA was then hybridized with the Agilent Whole Human Genome (4x44K) microarray chip according to the manufacturer's instructions. Universal Human Reference RNA and samples were labeled with Cy3 CTP and Cy5 CTP, respectively. Microarray data were analyzed by the Agilent Gene Spring 12.1V software using stringent criteria which included at least a 2-fold difference in gene expression between samples. Statistical analysis was performed using the unpaired Student's t-test with a p<0.01. Pathway enrichment was also performed, with key genes selected for validation using droplet digital polymerase chain reaction (ddPCR). The gene expression profiling indicated that were a substantial number of genes that were differentially expressed with more than a 2-fold change (p<0.01) between each of the four different conditions. We selected key genes within significant pathways that were analyzed through pathway enrichment. These key genes included regulators of cell proliferation, transcription factors, cytokines and tumour suppressor genes. In the present study, we showed that key genes involved in significant and well established pathways, could possibly be used as a potential blood-based biomarker to differentiate between high and low grade gliomas, non-gliomas and

  19. [Effects of intergenic interaction of the high pigmentation gene hp-2(dg) (high pigment-2 dark green) with the gene B (beta-carotene) in tomato].

    PubMed

    Kuzemenskiĭ, A V

    2008-01-01

    It was shown that during intergenic interaction of genes hp-2(dg) and B in dihomozygote an additive factor is formed activating biogenesis of beta-carotene in tomato fruits. In the genotype B/B//hp-2(dg)/hp-2(dg) there is preserved the positive effects of the gene hp-2(dg) on the content of ascorbic acid and the negative one on the content of titrated acids. With this stabilization of the gene hp-2(dg) genetic depression is observed, which is manifested in the increased productivity of B/B//hp-2(dg)/hp-2(dg)-genotypes.

  20. High-throughput chemiluminometric genotyping of single nucleotide polymorphisms of histamine, serotonin, and adrenergic receptor genes.

    PubMed

    Toubanaki, Dimitra K; Christopoulos, Theodore K; Ioannou, Penelope C; Flordellis, Christodoulos S

    2009-02-01

    Several pharmacogenetic studies are focused on the investigation of the relation between the efficacy of various antipsychotic agents (e.g., clozapine) and the genetic profile of the patient with an emphasis on genes that code for neurotransmitter receptors such as histamine, serotonin, and adrenergic receptors. We report a high-throughput method for genotyping of single nucleotide polymorphisms (SNPs) within the genes of histamine H2 receptor (HRH2), serotonin receptor (HTR2A1 and HTR2A2), and beta(3) adrenergic receptor (ADRB3). The method combines the high specificity of allele discrimination by oligonucleotide ligation reaction (OLR) and the superior sensitivity and simplicity of chemiluminometric detection in a microtiter well assay configuration. The genomic region that spans the locus of interest is first amplified by polymerase chain reaction (PCR). Subsequently, an oligonucleotide ligation reaction is performed using a biotinylated common probe and two allele-specific probes that are labeled at the 3' end with digoxigenin and fluorescein. The ligation products are immobilized in polystyrene wells via biotin-streptavidin interaction, and the hybrids are denatured. Detection is accomplished by the addition of alkaline phosphatase-conjugated anti-digoxigenin or anti-fluorescein antibodies in combination with a chemiluminogenic substrate. The ratio of the luminescence signals obtained from digoxigenin and fluorescein indicates the genotype of the sample. The method was applied successfully to the genotyping of 23 blood samples for all four SNPs. The results were in concordance with both PCR-restriction fragment length polymorphism analysis and sequencing.

  1. High-Throughput, Motility-Based Sorter for Microswimmers and Gene Discovery Platform

    NASA Astrophysics Data System (ADS)

    Yuan, Jinzhou; Raizen, David; Bau, Haim

    2015-11-01

    Animal motility varies with genotype, disease progression, aging, and environmental conditions. In many studies, it is desirable to carry out high throughput motility-based sorting to isolate rare animals for, among other things, forward genetic screens to identify genetic pathways that regulate phenotypes of interest. Many commonly used screening processes are labor-intensive, lack sensitivity, and require extensive investigator training. Here, we describe a sensitive, high throughput, automated, motility-based method for sorting nematodes. Our method was implemented in a simple microfluidic device capable of sorting many thousands of animals per hour per module, and is amenable to parallelism. The device successfully enriched for known C. elegans motility mutants. Furthermore, using this device, we isolated low-abundance mutants capable of suppressing the somnogenic effects of the flp-13 gene, which regulates sleep-like quiescence in C. elegans. Subsequent genomic sequencing led to the identification of a flp-13-suppressor gene. This research was supported, in part, by NIH NIA Grant 5R03AG042690-02.

  2. Rapid identification of Borrelia by high resolution melting analysis of the groEL gene.

    PubMed

    Koś, Władysław; Wodecka, Beata; Anklewicz, Marek; Skotarczak, Bogumiła

    2013-01-01

    This study examined the possibility of applying a new diagnostic method, high resolution analysis of DNA denaturation curve (high resolution melting - HRM), for identification of Borrelia species. DNA samples were obtained from Ixodes ricinus ticks collected from vegetation and removed from hunted roe deer. For differentiation of Borrelia species, the HRM protocol based on the analysis of the groEL gene was applied. A product characteristic for Borrelia was obtained in 19/123 samples (15.4%). The studied isolates were classified as four species: B. garinii, B. valaisiana, B. afzelii and B. miyamotoi. Two separate groups of isolates within the B. afzelii species were also found. The results show that the groEL gene is useful for rapid differentiation of B. burgdorferi sensu lato with the HRM method from different extracts of DNA and it also allows precise differentiation of Borrelia species and strains. The HRM method shortened and simplified detection and differentiation of Borrelia species from different biological sources.

  3. High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

    PubMed Central

    Mendonça, Mônica Sawan; Peraçolli, Terezinha S; Silva-Vergara, Mário León; Ribeiro, Sílvio C; Oliveira, Rafael Faria; Mendes, Rinaldo Poncio; Rodrigues, Virmondes

    2015-01-01

    Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4 gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp. (p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4 promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp. infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4 gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described. PMID:26517657

  4. High-resolution mapping of the gene for cystinosis, using combined biochemical and linkage analysis

    SciTech Connect

    Jean, G.; Fuchshuber, A.; Gribouval, O.

    1996-03-01

    Infantile nephropathic cystinosis is an autosomal recessive disorder characterized biochemically by an abnormally high intracellular content of free cystine in different organs and tissues due to a transport defect of cystine through the lysosomal membrane. Affected children present with the Fanconi syndrome and usually develop progressive renal failure within the 1st decade of life. Measurement of free cystine in purified polymorphonuclear leukocytes provides an accurate method for diagnosis and detection of heterozygous carriers previously determined by their leukocyte cystine content in the linkage analysis. This approach allowed us to obtain highly significant results, confirming the localization of the cystinosis gene locus recently mapped to the short arm of chromosome 17 by the Cystinosis Collaborative Research Group. Crucial recombination events allowed us to refine the interval of the cystinosis gene to a genetic distance of 1 cM. No evidence of genetic heterogeneity was found. Our results demonstrate that the use of the previously determined phenotypes of heterozygous carriers in linkage analysis provides a reliable method for the investigation of simplex families in autosomal recessive traits. 25 refs., 4 figs., 1 tab.

  5. Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus.

    PubMed

    Umelo-Njaka, E; Nomellini, J F; Yim, H; Smit, J

    2001-07-01

    Caulobacter crescentus is a bacterium with a distinctive life cycle and so it is studied as a cell development model. In addition, we have adapted this bacterium for recombinant protein production and display based on the crystalline surface protein (S)-layer and its C-terminal secretion signal. We report here the development of small, high-copy-number plasmid vectors and methods for producing an obligate expression host. The vectors are based on a narrow-host-range colE1-replicon-based plasmid commonly used in Escherichia coli, to which was added the replication origin of the IncQ plasmid RSF1010. C. crescentus strains were modified to enable plasmid replication by introduction of the RSF1010 repBAC genes at the recA locus. The small (4.0-4.5 kb) plasmids were in high copy numbers in both C. crescentus and E. coli and amenable to rapid methods for plasmid isolation and DNA sequencing. The method for introducing repBAC is suitable for other C. crescentus strains or any bacterium with an adequately homologous recA gene. Application of the vector for protein expression, based on the type I secretion system of the S-layer protein, when compared to constructs in broad-host-range plasmids, resulted in reduced time and steps required from clone construction to recombinant protein recovery and increased protein yield.

  6. Lineage-specific regulation of epigenetic modifier genes in human liver and brain.

    PubMed

    Weng, Matthias K; Natarajan, Karthick; Scholz, Diana; Ivanova, Violeta N; Sachinidis, Agapios; Hengstler, Jan G; Waldmann, Tanja; Leist, Marcel

    2014-01-01

    Despite an abundance of studies on chromatin states and dynamics, there is an astonishing dearth of information on the expression of genes responsible for regulating histone and DNA modifications. We used here a set of 156 defined epigenetic modifier genes (EMG) and profiled their expression pattern in cells of different lineages. As reference value, expression data from human embryonic stem cells (hESC) were used. Hepatocyte-like cells were generated from hESC, and their EMG expression was compared to primary human liver cells. In parallel, we generated postmitotic human neurons (Lu d6), and compared their relative EMG expression to human cortex (Ctx). Clustering analysis of all cell types showed that neuronal lineage samples grouped together (94 similarly regulated EMG), as did liver cells (61 similarly-regulated), while the two lineages were clearly distinct. The general classification was followed by detailed comparison of the major EMG groups; genes that were higher expressed in differentiated cells than in hESC included the acetyltransferase KAT2B and the methyltransferase SETD7. Neuro-specific EMGs were the histone deacetylases HDAC5 and HDAC7, and the arginine-methyltransferase PRMT8. Comparison of young (Lu d6) and more aged (Ctx) neuronal samples suggested a maturation-dependent switch in the expression of functionally homologous proteins. For instance, the ratio of the histone H3 K27 methyltransfereases, EZH1 to EZH2, was high in Ctx and low in Lu d6. The same was observed for the polycomb repressive complex 1 (PRC1) subunits CBX7 and CBX8. A large proportion of EMGs in differentiated cells was very differently expressed than in hESC, and absolute levels were significantly higher in neuronal samples than in hepatic cells. Thus, there seem to be distinct qualitative and quantitative differences in EMG expression between cell lineages.

  7. Lognormality and oscillations in the coverage of high-throughput transcriptomic data towards gene ends

    NASA Astrophysics Data System (ADS)

    Innocenti, Nicolas; Aurell, Erik

    2013-10-01

    High-throughput transcriptomics experiments have reached the stage where the count of the number of reads alignable to a given position can be treated as an almost-continuous signal. This allows us to ask questions of biophysical/biotechnical nature, but which may still have biological implications. Here we show that when sequencing RNA fragments from one end, as is the case on most platforms, an oscillation in the read count is observed at the other end. We further show that these oscillations can be well described by Kolmogorov’s 1941 broken stick model. We investigate how the model can be used to improve predictions of gene ends (3‧ transcript ends), but conclude that with present data the improvement is only marginal. The results highlight subtle effects in high-throughput transcriptomics experiments which do not have a biological origin, but which may still be used to obtain biological information.

  8. Improve carbon metabolic flux in Saccharomyces cerevisiae at high temperature by overexpressed TSL1 gene.

    PubMed

    Ge, Xiang-Yang; Xu, Yan; Chen, Xiang

    2013-04-01

    This study describes a novel strategy to improve the glycolysis flux of Saccharomyces cerevisiae at high temperature. The TSL1 gene-encoding regulatory subunit of the trehalose synthase complex was overexpressed in S. cerevisiae Z-06, which increased levels of trehalose synthase activity in extracts, enhanced stress tolerance and glucose consuming rate of the yeast cells. As a consequence, the final ethanol concentration of 185.5 g/L was obtained at 38 °C for 36 h (with productivity up to 5.2 g/L/h) in 7-L fermentor, and the ethanol productivity was 92.7 % higher than that of the parent strain. The results presented here provide a novel way to enhance the carbon metabolic flux at high temperature, which will be available for the purposes of producing other primary metabolites of commercial interest using S. cerevisiae as a host.

  9. High-sensitivity array analysis of gene expression for the early detection of disseminated breast tumor cells in peripheral blood

    PubMed Central

    Martin, Katherine J.; Graner, Edgard; Li, Yi; Price, Laura M.; Kritzman, Brian M.; Fournier, Marcia V.; Rhei, Esther; Pardee, Arthur B.

    2001-01-01

    Early detection is an effective means of reducing cancer mortality. Here, we describe a highly sensitive high-throughput screen that can identify panels of markers for the early detection of solid tumor cells disseminated in peripheral blood. The method is a two-step combination of differential display and high-sensitivity cDNA arrays. In a primary screen, differential display identified 170 candidate marker genes differentially expressed between breast tumor cells and normal breast epithelial cells. In a secondary screen, high-sensitivity arrays assessed expression levels of these genes in 48 blood samples, 22 from healthy volunteers and 26 from breast cancer patients. Cluster analysis identified a group of 12 genes that were elevated in the blood of cancer patients. Permutation analysis of individual genes defined five core genes (P ≤ 0.05, permax test). As a group, the 12 genes generally distinguished accurately between healthy volunteers and patients with breast cancer. Mean expression levels of the 12 genes were elevated in 77% (10 of 13) untreated invasive cancer patients, whereas cluster analysis correctly classified volunteers and patients (P = 0.0022, Fisher's exact test). Quantitative real-time PCR confirmed array results and indicated that the sensitivity of the assay (1:2 × 108 transcripts) was sufficient to detect disseminated solid tumor cells in blood. Expression-based blood assays developed with the screening approach described here have the potential to detect and classify solid tumor cells originating from virtually any primary site in the body. PMID:11226293

  10. Identification of Transcription Factor Genes and Their Correlation with the High Diversity of Stramenopiles

    PubMed Central

    Buitrago-Flórez, Francisco Javier; Restrepo, Silvia; Riaño-Pachón, Diego Mauricio

    2014-01-01

    The biological diversity among Stramenopiles is striking; they range from large multicellular seaweeds to tiny unicellular species, they embrace many ecologically important autothrophic (e.g., diatoms, brown algae), and heterotrophic (e.g., oomycetes) groups. Transcription factors (TFs) and other transcription regulators (TRs) regulate spatial and temporal gene expression. A plethora of transcriptional regulatory proteins have been identified and classified into families on the basis of sequence similarity. The purpose of this work is to identify the TF and TR complement in diverse species belonging to Stramenopiles in order to understand how these regulators may contribute to their observed diversity. We identified and classified 63 TF and TR families in 11 species of Stramenopiles. In some species we found gene families with high relative importance. Taking into account the 63 TF and TR families identified, 28 TF and TR families were established to be positively correlated with specific traits like number of predicted proteins, number of flagella and number of cell types during the life cycle. Additionally, we found gains and losses in TF and TR families specific to some species and clades, as well as, two families with high abundance specific to the autotrophic species and three families with high abundance specific to the heterotropic species. For the first time, there is a systematic search of TF and TR families in Stramenopiles. The attempts to uncover relationships between these families and the complexity of this group may be of great impact, considering that there are several important pathogens of plants and animals, as well as, important species involved in carbon cycling. Specific TF and TR families identified in this work appear to be correlated with particular traits in the Stramenopiles group and may be correlated with the high complexity and diversity in Stramenopiles. PMID:25375671

  11. Identification of transcription factor genes and their correlation with the high diversity of stramenopiles.

    PubMed

    Buitrago-Flórez, Francisco Javier; Restrepo, Silvia; Riaño-Pachón, Diego Mauricio

    2014-01-01

    The biological diversity among Stramenopiles is striking; they range from large multicellular seaweeds to tiny unicellular species, they embrace many ecologically important autothrophic (e.g., diatoms, brown algae), and heterotrophic (e.g., oomycetes) groups. Transcription factors (TFs) and other transcription regulators (TRs) regulate spatial and temporal gene expression. A plethora of transcriptional regulatory proteins have been identified and classified into families on the basis of sequence similarity. The purpose of this work is to identify the TF and TR complement in diverse species belonging to Stramenopiles in order to understand how these regulators may contribute to their observed diversity. We identified and classified 63 TF and TR families in 11 species of Stramenopiles. In some species we found gene families with high relative importance. Taking into account the 63 TF and TR families identified, 28 TF and TR families were established to be positively correlated with specific traits like number of predicted proteins, number of flagella and number of cell types during the life cycle. Additionally, we found gains and losses in TF and TR families specific to some species and clades, as well as, two families with high abundance specific to the autotrophic species and three families with high abundance specific to the heterotropic species. For the first time, there is a systematic search of TF and TR families in Stramenopiles. The attempts to uncover relationships between these families and the complexity of this group may be of great impact, considering that there are several important pathogens of plants and animals, as well as, important species involved in carbon cycling. Specific TF and TR families identified in this work appear to be correlated with particular traits in the Stramenopiles group and may be correlated with the high complexity and diversity in Stramenopiles.

  12. GeneLab for High Schools: Data Mining for the Next Generation

    NASA Technical Reports Server (NTRS)

    Blaber, Elizabeth A.; Ly, Diana; Sato, Kevin Y.; Taylor, Elizabeth

    2016-01-01

    Modern biological sciences have become increasingly based on molecular biology and high-throughput molecular techniques, such as genomics, transcriptomics, and proteomics. NASA Scientists and the NASA Space Biology Program have aimed to examine the fundamental building blocks of life (RNA, DNA and protein) in order to understand the response of living organisms to space and aid in fundamental research discoveries on Earth. In an effort to enable NASA funded science to be available to everyone, NASA has collected the data from omics studies and curated them in a data system called GeneLab. Whilst most college-level interns, academics and other scientists have had some interaction with omics data sets and analysis tools, high school students often have not. Therefore, the Space Biology Program is implementing a new Summer Program for high-school students that aims to inspire the next generation of scientists to learn about and get involved in space research using GeneLabs Data System. The program consists of three main components core learning modules, focused on developing students knowledge on the Space Biology Program and Space Biology research, Genelab and the data system, and previous research conducted on model organisms in space; networking and team work, enabling students to interact with guest lecturers from local universities and their fellow peers, and also enabling them to visit local universities and genomics centers around the Bay area; and finally an independent learning project, whereby students will be required to form small groups, analyze a dataset on the Genelab platform, generate a hypothesis and develop a research plan to test their hypothesis. This program will not only help inspire high-school students to become involved in space-based research but will also help them develop key critical thinking and bioinformatics skills required for most college degrees and furthermore, will enable them to establish networks with their peers and connections

  13. High-Stearic and High-Oleic Cottonseed Oils Produced by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing1

    PubMed Central

    Liu, Qing; Singh, Surinder P.; Green, Allan G.

    2002-01-01

    We have genetically modified the fatty acid composition of cottonseed oil using the recently developed technique of hairpin RNA-mediated gene silencing to down-regulate the seed expression of two key fatty acid desaturase genes, ghSAD-1-encoding stearoyl-acyl-carrier protein Δ9-desaturase and ghFAD2-1-encoding oleoyl-phosphatidylcholine ω6-desaturase. Hairpin RNA-encoding gene constructs (HP) targeted against either ghSAD-1 or ghFAD2-1 were transformed into cotton (Gossypium hirsutum cv Coker 315). The resulting down-regulation of the ghSAD-1 gene substantially increased stearic acid from the normal levels of 2% to 3% up to as high as 40%, and silencing of the ghFAD2-1 gene resulted in greatly elevated oleic acid content, up to 77% compared with about 15% in seeds of untransformed plants. In addition, palmitic acid was significantly lowered in both high-stearic and high-oleic lines. Similar fatty acid composition phenotypes were also achieved by transformation with conventional antisense constructs targeted against the same genes, but at much lower frequencies than were achieved with the HP constructs. By intercrossing the high-stearic and high-oleic genotypes, it was possible to simultaneously down-regulate both ghSAD-1 and ghFAD2-1 to the same degree as observed in the individually silenced parental lines, demonstrating for the first time, to our knowledge, that duplex RNA-induced posttranslational gene silencing in independent genes can be stacked without any diminution in the degree of silencing. The silencing of ghSAD-1 and/or ghFAD2-1 to various degrees enables the development of cottonseed oils having novel combinations of palmitic, stearic, oleic, and linoleic contents that can be used in margarines and deep frying without hydrogenation and also potentially in high-value confectionery applications. PMID:12177486

  14. Applications of myeloid-specific promoters in transgenic mice support in vivo imaging and functional genomics but do not support the concept of distinct macrophage and dendritic cell lineages or roles in immunity.

    PubMed

    Hume, David A

    2011-04-01

    Myeloid lineage cells contribute to innate and acquired immunity, homeostasis, wound repair, and inflammation. There is considerable interest in manipulation of their function in transgenic mice using myeloid-specific promoters. This review considers the applications and specificity of some of the most widely studied transgenes, driven by promoter elements of the lysM, csf1r, CD11c, CD68, macrophage SRA, and CD11b genes, as well as several others. Transgenes have been used in mice to generate myeloid lineage-specific cell ablation, expression of genes of interest, including fluorescent reporters, or deletion via recombination. In general, the specificity of such transgenes has been overinterpreted, and none of them provide well-documented, reliable, differential expression in any specific myeloid cell subset, macrophages, granulocytes, or myeloid DCs. Nevertheless, they have proved valuable in cell isolation, functional genomics, and live imaging of myeloid cell behavior in many different pathologies.

  15. High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation

    PubMed Central

    Clowney, E. Josephine; Magklara, Angeliki; Colquitt, Bradley M.; Pathak, Nidhi; Lane, Robert P.; Lomvardas, Stavros

    2011-01-01

    The olfactory receptor (OR) genes are the largest mammalian gene family and are expressed in a monogenic and monoallelic fashion in olfactory neurons. Using a high-throughput approach, we mapped the transcription start sites of 1085 of the 1400 murine OR genes and performed computational analysis that revealed potential transcription factor binding sites shared by the majority of these promoters. Our analysis produced a hierarchical model for OR promoter recognition in which unusually high AT content, a unique epigenetic signature, and a stereotypically positioned O/E site distinguish OR promoters from the rest of the murine promoters. Our computations revealed an intriguing correlation between promoter AT content and evolutionary plasticity, as the most AT-rich promoters regulate rapidly evolving gene families. Within the AT-rich promoter category the position of the TATA-box does not correlate with the transcription start site. Instead, a spike in GC composition might define the exact location of the TSS, introducing the concept of “genomic contrast” in transcriptional regulation. Finally, our experiments show that genomic neighborhood rather than promoter sequence correlates with the probability of different OR genes to be expressed in the same olfactory cell. PMID:21705439

  16. Effects of high-sulphur water on hepatic gene expression of steers fed fibre-based diets.

    PubMed

    Kessler, K L; Olson, K C; Wright, C L; Austin, K J; McInnerney, K; Johnson, P S; Cockrum, R R; Jons, A M; Cammack, K M

    2013-10-01

    Sulphur-induced polioencephalomalacia (sPEM), a neurological disorder affecting ruminants, is frequently associated with the consumption of high-sulphur (S) water and subsequent poor performance. Currently, there is no economical method for S removal from surface water sources, and alternative water sources are typically neither readily available nor cost-effective. Determination of genes differentially expressed in response to high-S water consumption may provide a better understanding of the physiology corresponding to high dietary S and ultimately lead to the development of treatment and prevention strategies. The objective of this study was to determine changes in gene expression in the liver, an organ important for S metabolism, of fibre-fed steers consuming high-S water. For this study, liver tissues were collected on the final day of a trial from yearling steers randomly assigned to low-S water control (566 mg/kg SO4 ; n = 24), high-S water (3651 mg/kg SO4 ; n = 24) or high-S water plus clinoptilolite supplemented at either 2.5% (n = 24) or 5.0% (n = 24) of diet dry matter (DM). Microarray analyses on randomly selected healthy low-S control (n = 4) and high-S (n = 4; no clinoptilolite) steers using the Affymetrix GeneChip Bovine Genome Array revealed 488 genes upregulated (p < 0.05) and 154 genes downregulated (p < 0.05) in response to the high- vs. low-S water consumption. Real-time RT-PCR confirmed the upregulation (p < 0.10) of seven genes involved in inflammatory response and immune functions. Changes in such genes suggest that ruminant animals administered high-S water may be undergoing an inflammation or immune response, even if signs of sPEM or compromised health are not readily observed. Further study of these, and other affected genes, may deliver new insights into the physiology underlying the response to high dietary S, ultimately leading to the development of treatments for high S-affected ruminant

  17. Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

    PubMed Central

    Zhang, Tingting; Stilwell, Jackie L.; Gerion, Daniele; Ding, Lianghao; Elboudwarej, Omeed; Cooke, Patrick A.; Gray, Joe W.; Alivisatos, A. Paul; Chen, Fanqing Frank

    2009-01-01

    Quantum dots (Qdots) are now used extensively for labeling in biomedical research, and this use is predicted to grow because of their many advantages over alternative labeling methods. Uncoated Qdots made of core/shell CdSe/ZnS are toxic to cells because of the release of Cd2+ ions into the cellular environment. This problem has been partially overcome by coating Qdots with polymers, poly(ethylene glycol) (PEG), or other inert molecules. The most promising coating to date, for reducing toxicity, appears to be PEG. When PEG-coated silanized Qdots (PEG-silane-Qdots) are used to treat cells, toxicity is not observed, even at dosages above 10–20 nM, a concentration inducing death when cells are treated with polymer or mercaptoacid coated Qdots. Because of the importance of Qdots in current and future biomedical and clinical applications, we believe it is essential to more completely understand and verify this negative global response from cells treated with PEG-silane-Qdots. Consequently, we examined the molecular and cellular response of cells treated with two different dosages of PEG-silane-Qdots. Human fibroblasts were exposed to 8 and 80 nM of these Qdots, and both phenotypic as well as whole genome expression measurements were made. PEG-silane-Qdots did not induce any statistically significant cell cycle changes and minimal apoptosis/necrosis in lung fibroblasts (IMR-90) as measured by high content image analysis, regardless of the treatment dosage. A slight increase in apoptosis/necrosis was observed in treated human skin fibroblasts (HSF-42) at both the low and the high dosages. We performed genome-wide expression array analysis of HSF-42 exposed to doses 8 and 80 nM to link the global cell response to a molecular and genetic phenotype. We used a gene array containing ~22,000 total probe sets, containing 18,400 probe sets from known genes. Only ~50 genes (~0.2% of all the genes tested) exhibited a statistically significant change in expression level of greater

  18. Methionine synthase: high-resolution mapping of the human gene and evaluation as a candidate locus for neural tube defects.

    PubMed

    Brody, L C; Baker, P J; Chines, P S; Musick, A; Molloy, A M; Swanson, D A; Kirke, P N; Ghosh, S; Scott, J M; Mills, J L

    1999-08-01

    Periconceptual folate supplementation has been found to prevent the occurrence of many neural tube defects (NTDs). Consequently, genetic variation in folate metabolism genes is expected to contribute to the risk for neural tube defects. Methionine synthase catalyzes the vitamin B(12)-dependent conversion of homocysteine and 5-methyltetrahydrofolate to methionine and tetrahydrofolate. The observation that homocysteine and vitamin B(12) levels are independent predictors of NTD risk suggested that methionine synthase could be a candidate gene for NTDs. To assess the role of the MS gene in NTDs, we performed high-resolution physical mapping of the MS locus, isolated highly polymorphic markers linked to the MS gene, and tested for an association between specific MS alleles and NTDs. We mapped the MS gene to a position between 909 and 913 cR(10000) on chromosome 1 by radiation hybrid mapping. Polymorphic markers D1S1567 and D1S1568 map to locations no more than 900 and 194 kb from the MS gene, respectively. The segregation of these polymorphic markers was measured in 85 Irish NTD families. No allele of either marker showed a significant association with NTDs using the transmission disequilibrium test. A lack of association was also observed for the D1919G missense mutation within the gene. Our results suggest that inherited variation in the MS gene does not contribute to NTD risk in this population.

  19. Genes for psychosis and creativity: a promoter polymorphism of the neuregulin 1 gene is related to creativity in people with high intellectual achievement.

    PubMed

    Kéri, Szabolcs

    2009-09-01

    Why are genetic polymorphisms related to severe mental disorders retained in the gene pool of a population? A possible answer is that these genetic variations may have a positive impact on psychological functions. Here, I show that a biologically relevant polymorphism of the promoter region of the neuregulin 1 gene (SNP8NRG243177/rs6994992) is associated with creativity in people with high intellectual and academic performance. Intriguingly, the highest creative achievements and creative-thinking scores were found in people who carried the T/T genotype, which was previously shown to be related to psychosis risk and altered prefrontal activation.

  20. The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

    PubMed Central

    Szafron, Lukasz Michal; Balcerak, Anna; Grzybowska, Ewa Anna; Pienkowska-Grela, Barbara; Felisiak-Golabek, Anna; Podgorska, Agnieszka; Kulesza, Magdalena; Nowak, Natalia; Pomorski, Pawel; Wysocki, Juliusz; Rubel, Tymon; Dansonka-Mieszkowska, Agnieszka; Konopka, Bozena; Lukasik, Martyna; Kupryjanczyk, Jolanta

    2015-01-01

    CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene. PMID:25978564

  1. High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes

    PubMed Central

    Alout, H; Labbé, P; Berthomieu, A; Makoundou, P; Fort, P; Pasteur, N; Weill, M

    2016-01-01

    We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l−1 and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1R allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1R and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1V or the duplicated ace-1D allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects. PMID:26463842

  2. High chlorpyrifos resistance in Culex pipiens mosquitoes: strong synergy between resistance genes.

    PubMed

    Alout, H; Labbé, P; Berthomieu, A; Makoundou, P; Fort, P; Pasteur, N; Weill, M

    2016-02-01

    We investigated the genetic determinism of high chlorpyrifos resistance (HCR), a phenotype first described in 1999 in Culex pipiens mosquitoes surviving chlorpyrifos doses ⩾1 mg l(-1) and more recently found in field samples from Tunisia, Israel or Indian Ocean islands. Through chlorpyrifos selection, we selected several HCR strains that displayed over 10 000-fold resistance. All strains were homozygous for resistant alleles at two main loci: the ace-1 gene, with the resistant ace-1(R) allele expressing the insensitive G119S acetylcholinesterase, and a resistant allele of an unknown gene (named T) linked to the sex and ace-2 genes. We constructed a strain carrying only the T-resistant allele and studied its resistance characteristics. By crossing this strain with strains harboring different alleles at the ace-1 locus, we showed that the resistant ace-1(R) and the T alleles act in strong synergy, as they elicited a resistance 100 times higher than expected from a simple multiplicative effect. This effect was specific to chlorpyrifos and parathion and was not affected by synergists. We also examined how HCR was expressed in strains carrying other ace-1-resistant alleles, such as ace-1(V) or the duplicated ace-1(D) allele, currently spreading worldwide. We identified two major parameters that influenced the level of resistance: the number and the nature of the ace-1-resistant alleles and the number of T alleles. Our data fit a model that predicts that the T allele acts by decreasing chlorpyrifos concentration in the compartment targeted in insects.

  3. Fasting and sampling time affect liver gene expression of high-fat diet-fed mice.

    PubMed

    Lee, C Y

    2010-05-01

    Several physiological and biological variables are known to affect peroxisome proliferator-activated receptor (PPAR)-α-dependent signaling pathway and plasma biochemical profiles. However, less is known about the effect of these variables on high-fat diet-fed mice. In a 5-week study, C57BL/6 mice were divided into control (C) and high-fat diet-fed (H) groups, whereby before dissection, each group was subdivided into non-fasted (nC and nH) and a 15-h fasted mice (fC and fH) killed in the early light cycle, and a 15-h fasted mice (eC and eH) killed in the late phase of the light cycle. Liver and blood from the vena cava were collected. Non-fasted nC and nH mice have a marginal difference in their body weight gain, whereas significant differences were found for fasted mice. In nH mice, PPAR-α, acyl-CoA oxidase and insulin-like growth factor-binding protein expressions were significantly elevated, in contrast to fatty acid synthase (Fasn), stearoyl CoA-desaturase (SCD)-1, and elongase (ELOVL)-6 expressions. Fasn was profoundly induced in fH mice, while decreased sterol regulatory-binding protein-1 and SCD-1 were found only in eH mice. Different from the gene expression profiles, plasma total cholesterol level of the eH mice was higher than controls, whereas nH mice have increased plasma non-esterified fatty acids. Only glucose level of the fH mice was higher than that observed for controls. Results showed that fasting and sampling time have significantly affected liver gene expression and plasma biochemical indices of the high-fat diet-treated mice. An overlook in these aspects can cause serious discrepancies in the experimental data and their interpretations.

  4. Gene expression in breast muscle and duodenum from low and high feed efficient broilers.

    PubMed

    Ojano-Dirain, C; Toyomizu, M; Wing, T; Cooper, M; Bottje, W G

    2007-02-01

    This study was conducted to evaluate messenger RNA (mRNA) expression of genes that are involved in energy metabolism and mitochondrial biogenesis: avian adenine nucleotide translocator (avANT), cytochrome oxidase III (COX III), inducible nitric oxide synthase (iNOS), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), avian PPAR-gamma coactivator-1alpha (avPGC-1alpha), and avian uncoupling protein in breast muscle and duodenum of broilers with low and high feed efficiency (FE). Total RNA was extracted from snap-frozen tissues from male broilers with low (0.55 +/- 0.01) and high (0.72 +/- 0.01) FE (n = 8 per group). Total RNA was reverse-transcribed using oligo(dT), random primers, or both followed by real-time reverse transcription-PCR. Protein oxidation, measured as protein carbonyls, was also evaluated in duodenal mucosa. Protein carbonyls were higher in low FE mucosa in tissue homogenate and mitochondrial fraction. The mRNA expression of iNOS and PPAR-gamma in the duodenum was lower in the low FE broilers, with no differences in avANT, COX III, and avPGC-1alpha. In contrast, expression of avANT and COX III mRNA in breast muscle was lower in low FE broilers with no differences in iNOS, PPAR-gamma, and avPGC-1alpha. The avian uncoupling protein in breast muscle was higher in low FE birds (P = 0.068). These results indicate that there are differences in the expression of mRNA encoding for mitochondrial transcription factors and proteins in breast muscle and duodenal tissue between low and high FE birds. The differences that were observed may also reflect inherent metabolic and gene regulation differences between tissues.

  5. Gene sequence variations and expression patterns of mitochondrial genes are associated with the adaptive evolution of two Gynaephora species (Lepidoptera: Lymantriinae) living in different high-elevation environments.

    PubMed

    Zhang, Qi-Lin; Zhang, Li; Zhao, Tian-Xuan; Wang, Juan; Zhu, Qian-Hua; Chen, Jun-Yuan; Yuan, Ming-Long

    2017-04-30

    The adaptive evolution of animals to high-elevation environments has been extensively studied in vertebrates, while few studies have focused on insects. Gynaephora species (Lepidoptera: Lymantriinae) are endemic to the Qinghai-Tibetan Plateau (QTP) and represent an important insect pest of alpine meadows. Here, we present a detailed comparative analysis of the mitochondrial genomes (mitogenomes) of two Gynaephora species inhabiting different high-elevation environments: G. alpherakii and G. menyuanensis. The results indicated that the general mitogenomic features (genome size, nucleotide composition, codon usage and secondary structures of tRNAs) were well conserved between the two species. All of mitochondrial protein-coding genes were evolving under purifying selection, suggesting that selection constraints may play a role in ensuring adequate energy production. However, a number of substitutions and indels were identified that altered the protein conformations of ATP8 and NAD1, which may be the result of adaptive evolution of the two Gynaephora species to different high-elevation environments. Levels of gene expression for nine mitochondrial genes in nine different developmental stages were significantly suppressed in G. alpherakii, which lives at the higher elevation (~4800m above sea level), suggesting that gene expression patterns could be modulated by atmospheric oxygen content and environmental temperature. These results enhance our understanding of the genetic bases for the adaptive evolution of insects endemic to the QTP.

  6. Understanding the differential nitrogen sensing mechanism in rice genotypes through expression analysis of high and low affinity ammonium transporter genes.

    PubMed

    Gaur, Vikram Singh; Singh, U S; Gupta, Atul K; Kumar, Anil

    2012-03-01

    Two rice genotypes, Kalanamak 3119 (KN3119) and Pusa Basmati 1(PB1) differing in their optimum nitrogen requirements (30 and 120 kg/ha, respectively) were undertaken to study the expression of both high and low affinity ammonium transporter genes responsible for ammonium uptake. Exposing the roots of the seedlings of both the genotypes to increasing (NH(4))(2)SO(4) concentrations revealed that all the three families of rice AMT genes are expressed, some of which get altered in a genotype and concentration specific manner. This indicates that individual ammonium transporter genes have defined contributions for ammonium uptake and plant growth. Interestingly, in response to increasing nitrogen concentrations, a root specific high affinity gene, AMT1;3, was repressed in the roots of KN3119 but not in PB1 indicating the existence of a differential ammonium sensing mechanism. This also indicates that not only AMT1;3 is involved not only in ammonium uptake but may also in ammonium sensing. Further, if it can differentiate and could be used as a biomarker for nitrogen responsiveness. Expression analysis of low affinity AMT genes showed that, both AMT2;1 and AMT2;2 have high levels of expression in both roots and shoots and in KN3119 are induced at low ammonium concentrations. Expressions of AMT3 family genes were higher shoots than in the roots indicating that these genes are probably involved in the translocation and distribution of ammonium ions in leaves. The expression of the only high affinity AMT gene, AMT1;1, along with six low affinity AMT genes in the shoots suggests that low affinity AMTs in the shoots leaves are involved in supporting AMT1;1 to carry out its activities/function efficiently.

  7. The transcription factor Spn1 regulates gene expression via a highly conserved novel structural motif

    PubMed Central

    Pujari, Venugopal; Radebaugh, Catherine A.; Chodaparambil, Jayanth V.; Muthurajan, Uma M.; Almeida, Adam R.; Fischbeck, Julie A.; Luger, Karolin; Stargell, Laurie A.

    2010-01-01

    Spn1 plays essential roles in the regulation of gene expression by RNA Polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP, TFIIS and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells. Here we report the high-resolution (1.85Å) crystal structure of the conserved central domain of Saccharomyces cerevisiae Spn1. The central domain is comprised of eight alpha-helices in a right handed super helical arrangement, and exhibits structural similarity to domain I of TFIIS. A unique structural feature of Spn1 is a highly conserved loop, which defines one side of a pronounced cavity. The loop and the other residues forming the cavity are highly conserved at the amino acid level among all Spn1 family members, suggesting that this is a signature motif for Spn1 orthologs. The locations and the molecular characterization of temperature-sensitive mutations in Spn1 indicate that the cavity is a key attribute of Spn1 that is critical for its regulatory functions during RNAPII-mediated transcriptional activity. PMID:20875428

  8. The transcription factor Spn1 regulates gene expression via a highly conserved novel structural motif.

    PubMed

    Pujari, Venugopal; Radebaugh, Catherine A; Chodaparambil, Jayanth V; Muthurajan, Uma M; Almeida, Adam R; Fischbeck, Julie A; Luger, Karolin; Stargell, Laurie A

    2010-11-19

    Spn1/Iws1 plays essential roles in the regulation of gene expression by RNA polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP (TATA-binding protein), TFIIS (transcription factor IIS), and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells. Here, we report the high-resolution (1.85 Å) crystal structure of the conserved central domain of Saccharomyces cerevisiae Spn1. The central domain is composed of eight α-helices in a right-handed superhelical arrangement and exhibits structural similarity to domain I of TFIIS. A unique structural feature of Spn1 is a highly conserved loop, which defines one side of a pronounced cavity. The loop and the other residues forming the cavity are highly conserved at the amino acid level among all Spn1 family members, suggesting that this is a signature motif for Spn1 orthologs. The locations and the molecular characterization of temperature-sensitive mutations in Spn1 indicate that the cavity is a key attribute of Spn1 that is critical for its regulatory functions during RNAPII-mediated transcriptional activity.

  9. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-07-15

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical ev