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Sample records for endogenous gene expression

  1. Transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans

    CSIR Research Space (South Africa)

    Crampton, Michael C

    2010-07-01

    Full Text Available This presentation focused on the transcriptional analysis of heterologous gene expression using the endogenous sD promoter from Bacillus halodurans. It concludes to a successful implementation of a high throughput mRNA sandwich hybridisation...

  2. Gene expression analysis in prostate cancer: the importance of the endogenous control.

    LENUS (Irish Health Repository)

    Vajda, Alice

    2013-03-01

    Aberrant gene expression is a hallmark of cancer. Quantitative reverse-transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and commonly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer.

  3. Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

    Science.gov (United States)

    Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

    2014-10-17

    The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

  4. Effects of in ovo electroporation on endogenous gene expression: genome-wide analysis

    Directory of Open Access Journals (Sweden)

    Chambers David

    2011-04-01

    Full Text Available Abstract Background In ovo electroporation is a widely used technique to study gene function in developmental biology. Despite the widespread acceptance of this technique, no genome-wide analysis of the effects of in ovo electroporation, principally the current applied across the tissue and exogenous vector DNA introduced, on endogenous gene expression has been undertaken. Here, the effects of electric current and expression of a GFP-containing construct, via electroporation into the midbrain of Hamburger-Hamilton stage 10 chicken embryos, are analysed by microarray. Results Both current alone and in combination with exogenous DNA expression have a small but reproducible effect on endogenous gene expression, changing the expression of the genes represented on the array by less than 0.1% (current and less than 0.5% (current + DNA, respectively. The subset of genes regulated by electric current and exogenous DNA span a disparate set of cellular functions. However, no genes involved in the regional identity were affected. In sharp contrast to this, electroporation of a known transcription factor, Dmrt5, caused a much greater change in gene expression. Conclusions These findings represent the first systematic genome-wide analysis of the effects of in ovo electroporation on gene expression during embryonic development. The analysis reveals that this process has minimal impact on the genetic basis of cell fate specification. Thus, the study demonstrates the validity of the in ovo electroporation technique to study gene function and expression during development. Furthermore, the data presented here can be used as a resource to refine the set of transcriptional responders in future in ovo electroporation studies of specific gene function.

  5. Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

    Directory of Open Access Journals (Sweden)

    Turner Renee J

    2009-08-01

    Full Text Available Abstract Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT, 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder. Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

  6. Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms.

    Science.gov (United States)

    Svingen, T; Jørgensen, A; Rajpert-De Meyts, E

    2014-08-01

    The measurement of gene expression levels in cells and tissues typically depends on a suitable point of reference for inferring biological relevance. For quantitative (or real-time) RT-PCR assays, the method of choice is often to normalize gene expression data to an endogenous gene that is stably expressed across the samples analysed: a so-called normalizing or housekeeping gene. Although this is a valid strategy, the identification of stable normalizing genes has proved challenging and a gene showing stable expression across all cells or tissues is unlikely to exist. Therefore, it is necessary to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further recommend that such studies should be accompanied by additional assessment of histology and cellularity of each sample. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Floral pathway integrator gene expression mediates gradual transmission of environmental and endogenous cues to flowering time.

    Science.gov (United States)

    van Dijk, Aalt D J; Molenaar, Jaap

    2017-01-01

    The appropriate timing of flowering is crucial for the reproductive success of plants. Hence, intricate genetic networks integrate various environmental and endogenous cues such as temperature or hormonal statues. These signals integrate into a network of floral pathway integrator genes. At a quantitative level, it is currently unclear how the impact of genetic variation in signaling pathways on flowering time is mediated by floral pathway integrator genes. Here, using datasets available from literature, we connect Arabidopsis thaliana flowering time in genetic backgrounds varying in upstream signalling components with the expression levels of floral pathway integrator genes in these genetic backgrounds. Our modelling results indicate that flowering time depends in a quite linear way on expression levels of floral pathway integrator genes. This gradual, proportional response of flowering time to upstream changes enables a gradual adaptation to changing environmental factors such as temperature and light.

  8. Floral pathway integrator gene expression mediates gradual transmission of environmental and endogenous cues to flowering time

    Directory of Open Access Journals (Sweden)

    Aalt D.J. van Dijk

    2017-04-01

    Full Text Available The appropriate timing of flowering is crucial for the reproductive success of plants. Hence, intricate genetic networks integrate various environmental and endogenous cues such as temperature or hormonal statues. These signals integrate into a network of floral pathway integrator genes. At a quantitative level, it is currently unclear how the impact of genetic variation in signaling pathways on flowering time is mediated by floral pathway integrator genes. Here, using datasets available from literature, we connect Arabidopsis thaliana flowering time in genetic backgrounds varying in upstream signalling components with the expression levels of floral pathway integrator genes in these genetic backgrounds. Our modelling results indicate that flowering time depends in a quite linear way on expression levels of floral pathway integrator genes. This gradual, proportional response of flowering time to upstream changes enables a gradual adaptation to changing environmental factors such as temperature and light.

  9. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

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    Chapman, Joanne R; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

  10. Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

    International Nuclear Information System (INIS)

    Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

    2005-01-01

    In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian

  11. Competition between virus-derived and endogenous small RNAs regulates gene expression in Caenorhabditis elegans.

    Science.gov (United States)

    Sarkies, Peter; Ashe, Alyson; Le Pen, Jérémie; McKie, Mikel A; Miska, Eric A

    2013-08-01

    Positive-strand RNA viruses encompass more than one-third of known virus genera and include many medically and agriculturally relevant human, animal, and plant pathogens. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay, have recently emerged as a new animal model to understand the mechanisms and evolution of innate immune responses. In particular, the RNA interference (RNAi) pathway is required for C. elegans resistance to viral infection. Here we report the first genome-wide analyses of gene expression upon viral infection in C. elegans. Using the laboratory strain N2, we identify a novel C. elegans innate immune response specific to viral infection. A subset of these changes is driven by the RNAi response to the virus, which redirects the Argonaute protein RDE-1 from its endogenous small RNA cofactors, leading to loss of repression of endogenous RDE-1 targets. Additionally, we show that a C. elegans wild isolate, JU1580, has a distinct gene expression signature in response to viral infection. This is associated with a reduction in microRNA (miRNA) levels and an up-regulation of their target genes. Intriguingly, alterations in miRNA levels upon JU1580 infection are associated with a transformation of the antiviral transcriptional response into an antibacterial-like response. Together our data support a model whereby antiviral RNAi competes with endogenous small RNA pathways, causing widespread transcriptional changes. This provides an elegant mechanism for C. elegans to orchestrate its antiviral response, which may have significance for the relationship between small RNA pathways and immune regulation in other organisms.

  12. Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo

    International Nuclear Information System (INIS)

    Vaiphei, S. Thangminlal; Keppen, Joshua; Nongrum, Saibadaiahun; Sharan, R.N.; Chaubey, R.C.; Kma, L.

    2015-01-01

    In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving 60 Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of 60 Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min -1 at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples. (author)

  13. Evaluation of endogenous control gene(s) for gene expression studies in human blood exposed to 60Co γ-rays ex vivo.

    Science.gov (United States)

    Vaiphei, S Thangminlal; Keppen, Joshua; Nongrum, Saibadaiahun; Chaubey, R C; Kma, L; Sharan, R N

    2015-01-01

    In gene expression studies, it is critical to normalize data using a stably expressed endogenous control gene in order to obtain accurate and reliable results. However, we currently do not have a universally applied endogenous control gene for normalization of data for gene expression studies, particularly those involving (60)Co γ-ray-exposed human blood samples. In this study, a comparative assessment of the gene expression of six widely used housekeeping endogenous control genes, namely 18S, ACTB, B2M, GAPDH, MT-ATP6 and CDKN1A, was undertaken for a range of (60)Co γ-ray doses (0.5, 1.0, 2.0 and 4.0 Gy) at 8.4 Gy min(-1) at 0 and 24 h post-irradiation time intervals. Using the NormFinder algorithm, real-time PCR data obtained from six individuals (three males and three females) were analyzed with respect to the threshold cycle (Ct) value and abundance, ΔCt pair-wise comparison, intra- and inter-group variability assessments, etc. GAPDH, either alone or in combination with 18S, was found to be the most suitable endogenous control gene and should be used in gene expression studies, especially those involving qPCR of γ-ray-exposed human blood samples. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  14. Expression of the pol gene of human endogenous retroviruses HERV-K and -W in leukemia patients.

    Science.gov (United States)

    Bergallo, Massimiliano; Montanari, Paola; Mareschi, Katia; Merlino, Chiara; Berger, Massimo; Bini, Ilaria; Daprà, Valentina; Galliano, Ilaria; Fagioli, Franca

    2017-12-01

    The human endogenous retroviruses (HERVs) are a family of endogenous retroviruses that integrated into the germ cell DNA of primates over 30 million years ago. HERV expression seems impaired in several diseases, ranging from autoimmune to neoplastic disorders. The purpose of this study was to evaluate the overall endogenous retroviral transcription profile in bone marrow (BM) samples. A total of 30 paediatric high-risk leukaemia patients (lymphoid and myeloid malignancies) were tested for HERVs virus gene expression. Our findings show that HERV-K expression was significantly higher in leukaemia patients when compared to healthy donors of a similar median age. We observed a significantly high expression of HERV-K in acute lymphoblastic leukemia (ALL) patients. In this study, we also found a relative overexpression of the endogenous retrovirus HERV-K in BM cells from the majority of leukemia samples analyzed, in particular in ALL. This overexpression might be related to lymphatic leukemogenesis and it warrants further investigations.

  15. The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition.

    Science.gov (United States)

    Schneider, P M; Witzel-Schlömp, K; Rittner, C; Zhang, L

    2001-02-01

    Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.

  16. Activation of endogenous opioid gene expression in human keratinocytes and fibroblasts by pulsed radiofrequency energy fields

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    Moffett J

    2012-09-01

    Full Text Available John Moffett,1 Linley M Fray,1 Nicole J Kubat21Life Science Department, 2Independent Consultant, Regenesis Biomedical Inc, Scottsdale, AZ, USABackground: Pulsed radiofrequency energy (PRFE fields are being used increasingly for the treatment of pain arising from dermal trauma. However, despite their increased use, little is known about the biological and molecular mechanism(s responsible for PRFE-mediated analgesia. In general, current therapeutics used for analgesia target either endogenous factors involved in inflammation, or act on endogenous opioid pathways.Methods and Results: Using cultured human dermal fibroblasts (HDF and human epidermal keratinocytes (HEK, we investigated the effect of PRFE treatment on factors, which are involved in modulating peripheral analgesia in vivo. We found that PRFE treatment did not inhibit cyclooxygenase enzyme activity, but instead had a positive effect on levels of endogenous opioid precursor mRNA (proenkephalin, pro-opiomelanocortin, prodynorphin and corresponding opioid peptide. In HEK cells, increases in opioid mRNA were dependent, at least in part, on endothelin-1. In HDF cells, additional pathways also appear to be involved. PRFE treatment was also followed by changes in endogenous expression of several cytokines, including increased levels of interleukin-10 mRNA and decreased levels of interleukin-1β mRNA in both cell types.Conclusion: These findings provide a new insight into the molecular mechanism underlying PRFE-mediated analgesia reported in the clinical setting.Keywords: peripheral analgesia, endogenous opioids, endothelin-1, endothelin receptor A, endothelin receptor B, pulsed radiofrequency energy field, cyclooxygenase

  17. Validation of endogenous normalizing genes for expression analyses in adult human testis and germ cell neoplasms

    DEFF Research Database (Denmark)

    Svingen, T; Jørgensen, Anne; Rajpert-De Meyts, E

    2014-01-01

    to define suitable normalizing genes for specific cells and tissues. Here, we report on the performance of a panel of nine commonly employed normalizing genes in adult human testis and testicular pathologies. Our analyses revealed significant variability in transcript abundance for commonly used normalizers......, highlighting the importance of selecting appropriate normalizing genes as comparative measurements can yield variable results when different normalizing genes are employed. Based on our results, we recommend using RPS20, RPS29 or SRSF4 when analysing relative gene expression levels in human testis...... and associated testicular pathologies. OCT4 and SALL4 can be used with caution as second-tier normalizers when determining changes in gene expression in germ cells and germ cell tumour components, but the relative transcript abundance appears variable between different germ cell tumour types. We further...

  18. Identification of a set of endogenous reference genes for miRNA expression studies in Parkinson's disease blood samples.

    Science.gov (United States)

    Serafin, Alice; Foco, Luisa; Blankenburg, Hagen; Picard, Anne; Zanigni, Stefano; Zanon, Alessandra; Pramstaller, Peter P; Hicks, Andrew A; Schwienbacher, Christine

    2014-10-10

    Research on microRNAs (miRNAs) is becoming an increasingly attractive field, as these small RNA molecules are involved in several physiological functions and diseases. To date, only few studies have assessed the expression of blood miRNAs related to Parkinson's disease (PD) using microarray and quantitative real-time PCR (qRT-PCR). Measuring miRNA expression involves normalization of qRT-PCR data using endogenous reference genes for calibration, but their choice remains a delicate problem with serious impact on the resulting expression levels. The aim of the present study was to evaluate the suitability of a set of commonly used small RNAs as normalizers and to identify which of these miRNAs might be considered reliable reference genes in qRT-PCR expression analyses on PD blood samples. Commonly used reference genes snoRNA RNU24, snRNA RNU6B, snoRNA Z30 and miR-103a-3p were selected from the literature. We then analyzed the effect of using these genes as reference, alone or in any possible combination, on the measured expression levels of the target genes miR-30b-5p and miR-29a-3p, which have been previously reported to be deregulated in PD blood samples. We identified RNU24 and Z30 as a reliable and stable pair of reference genes in PD blood samples.

  19. The Sporothrix schenckii Gene Encoding for the Ribosomal Protein L6 Has Constitutive and Stable Expression and Works as an Endogenous Control in Gene Expression Analysis

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    Elías Trujillo-Esquivel

    2017-09-01

    Full Text Available Sporothrix schenckii is one of the causative agents of sporotrichosis, a worldwide-distributed mycosis that affects humans and other mammals. The interest in basic and clinical features of this organism has significantly increased in the last years, yet little progress in molecular aspects has been reported. Gene expression analysis is a set of powerful tools that helps to assess the cell response to changes in the extracellular environment, the genetic networks controlling metabolic pathways, and the adaptation to different growth conditions. Most of the quantitative methodologies used nowadays require data normalization, and this is achieved measuring the expression of endogenous control genes. Reference genes, whose expression is assumed to suffer minimal changes regardless the cell morphology, the stage of the cell cycle or the presence of harsh extracellular conditions are commonly used as controls in Northern blotting assays, microarrays, and semi-quantitative or quantitative RT-PCR. Since the biology of the organisms is usually species specific, it is difficult to find a reliable group of universal genes that can be used as controls for data normalization in experiments addressing the gene expression, regardless the taxonomic classification of the organism under study. Here, we compared the transcriptional stability of the genes encoding for elongation factor 1A, Tfc1, a protein involved in transcription initiation on Pol III promoters, ribosomal protein L6, histone H2A, β-actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase, UAF30, the upstream activating factor 30, and the transcription initiation factor TFIID subunit 10, during the fungal growth in different culture media and cell morphologies. Our results indicated that only the gene encoding for the ribosomal protein L6 showed a stable and constant expression. Furthermore, it displayed not transcriptional changes when S. schenckii infected larvae of Galleria mellonella or

  20. Effect of PTTG on endogenous gene expression in HEK 293 cells

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    Panguluri Siva K

    2009-12-01

    Full Text Available Abstract Background Pituitary tumor transforming gene (PTTG, also known as securin, is highly expressed in various tumors including pituitary, thyroid, colon, ovary, testis, lung, and breast. An overexpression of PTTG enhances cell proliferation, induces cellular transformation in vitro, and promotes tumor development in nude mice. PTTG also inhibits separation of sister chromatids leading to aneuploidy and genetic instability. A great amount of work has been undertaken to understand the biology of PTTG and its expression in various tumors. However, mechanisms by which PTTG mediates its tumorigenic function are not fully understood. To utilize this gene for cancer therapy, identification of the downstream signaling genes regulated by PTTG in mediation of its tumorigenic function is necessary. For this purpose, we expressed PTTG in human embryonic kidney (HEK293 cells that do not express PTTG and analyzed the downstream genes using microarray analysis. Results A total of 22,277 genes printed on an Affymetrix HG-U133A 2.0 GeneChip™ array were screened with labeled cRNA prepared from HEK293 cells infected with adenovirus vector expressing PTTG cDNA (AdPTTG cDNA and compared with labeled cRNA prepared from HEK293 cells infected with control adenovirus (control Ad or adenovirus vector expressing GFP (AdGFP. Out of 22,277 genes, 71 genes were down-regulated and 35 genes were up-regulated with an FDR corrected p-value of ≤ 0.05 and a fold change of ≥2. Most of the altered genes identified are involved in the cell cycle and cell apoptosis; a few are involved in mRNA processing and nitrogen metabolism. Most of the up-regulated genes belong to the histone protein family. Conclusion PTTG is a well-studied oncogene for its role in tumorigenesis. In addition to its importance in regulation of the cell cycle, this gene has also been recently shown to play a role in the induction of cell apoptosis. The microarray analysis in the present study

  1. Diminished humoral responses against and reduced gene expression levels of human endogenous retrovirus-K (HERV-K) in psoriasis.

    Science.gov (United States)

    Gupta, Rashmi; Michaud, Henri-Alexandre; Zeng, Xue; Debbaneh, Maya; Arron, Sarah T; Jones, R Brad; Ormsby, Christopher E; Nixon, Douglas F; Liao, Wilson

    2014-09-16

    Psoriasis is a multifactorial, chronic disease of skin affecting 2-3% of the world's population. Genetic studies of psoriasis have identified a number of susceptibility genes that are involved in anti-viral immunity. Furthermore, physiological studies have also found an increase in anti-viral proteins in psoriatic skin. These findings suggest the presence of an anti-viral state in psoriatic skin. However, the triggers for this anti-viral cascade and its consequences for host immunity are not known. Endogenous retroviruses have previously been described in many autoimmune diseases including psoriasis. In the present study we examined the humoral immune response against human endogenous retrovirus-K (HERV-K) proteins and the cutaneous expression levels of multiple HERV-K genes in psoriasis patients and healthy controls. In psoriatic sera we observed a significant decrease in IgM response against three HERV-K proteins: Env surface unit (SU), Env transmembrane protein (TM), and Gag capsid (CA) in comparison to sera obtained from blood bank healthy controls. A decrease in IgG response was also observed against CA. Furthermore, using quantitative RT-PCR we observed a decrease in the expression of HERV-K Env, Gag, Pol and Rec as well as ERV-9 genes in lesional psoriatic skin as compared to healthy skin. Together, our results suggest that the pro-inflammatory, anti-viral state in psoriasis is associated with diminished expression of HERV-K gene transcripts and a concomitant decrease in humoral responses to HERV-K. Our results indicate that a simple model where continuous, minimally changing HERV-K expression serves as an antigenic trigger in psoriasis might not be correct and further studies are needed to decipher the possible relationship between psoriasis and HERVs.

  2. The expression of petunia strigolactone pathway genes is altered as part of the endogenous developmental program

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    Revel S M Drummond

    2012-01-01

    Full Text Available Analysis of mutants with increased branching has revealed the strigolactone synthesis/perception pathway which regulates branching in plants. However, whether variation in this well conserved developmental signalling system contributes to the unique plant architectures of different species is yet to be determined. We examined petunia orthologues of the Arabidopsis MAX1 and MAX2 genes to characterise their role in petunia architecture. A single orthologue of MAX1, PhMAX1 which encodes a cytochrome P450, was identified and was able to complement the max1 mutant of Arabidopsis. Petunia has two copies of the MAX2 gene, PhMAX2A and PhMAX2B which encode F-Box proteins. Differences in the transcript levels of these two MAX2-like genes suggest diverging functions. Unlike PhMAX2B, PhMAX2A mRNA levels increase as leaves age. Nonetheless, this gene functionally complements the Arabidopsis max2 mutant indicating that the biochemical activity of the PhMAX2A protein is not significantly different from MAX2. The expression of the petunia strigolactone pathway genes (PhCCD7, PhCCD8, PhMAX1, PhMAX2A, and PhMAX2B was then further investigated throughout the development of wild-type petunia plants. Three of these genes showed changes in mRNA levels over the development series. Alterations to the expression of these genes over time, or in different regions of the plant, may influence the branching growth habit of the plant. Alterations to strigolactone production and/or sensitivity could allow both subtle and dramatic changes to branching within and between species.

  3. Robust heat-inducible gene expression by two endogenous hsp70-derived promoters in transgenic Aedes aegypti

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    Carpenetti, Tiffany L. G.; Aryan, Azadeh; Myles, Kevin M.; Adelman, Zach N.

    2011-01-01

    Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. Reverse genetic approaches to the study of gene function in this mosquito have been limited by the lack of a robust inducible promoter to allow precise temporal control over a protein-encoding or hairpin RNA transgene. Likewise, investigations into the molecular and biochemical basis of vector competence would benefit from the ability to activate an anti-pathogen molecule at specific times during infection. We have characterized the ability of genomic sequences derived from two Ae. aegypti hsp70 genes to drive heat-inducible expression of a reporter in both transient and germline transformation contexts. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Luciferase expression in transgenic Ae. aegypti increased by ∼25-50 fold in whole adults by four hours after heat-shock, with significant activity (∼20 fold) remaining at 24 hr. Heat-induced expression was even more dramatic in midgut tissues, with one strain showing a ∼2500-fold increase in luciferase activity. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. PMID:22142225

  4. Vanillylacetone up-regulates anthocyanin accumulation and expression of anthocyanin biosynthetic genes by inducing endogenous abscisic acid in grapevine tissues.

    Science.gov (United States)

    Enoki, Shinichi; Hattori, Tomoki; Ishiai, Shiho; Tanaka, Sayumi; Mikami, Masachika; Arita, Kayo; Nagasaka, Shu; Suzuki, Shunji

    2017-12-01

    We investigated the effect of vanillylacetone (VA) on anthocyanin accumulation with aim of improving grape berry coloration. Spraying Vitis vinifera cv. Muscat Bailey A berries with VA at veraison increased sugar/acid ratio, an indicator of maturation and total anthocyanin accumulation. To elucidate the molecular mechanism underlying the effect of VA on anthocyanin accumulation, in vitro VA treatment of a grapevine cell culture was carried out. Endogenous abscisic acid (ABA) content was higher in the VA-treated cell cultures than in control at 3h after treatment. Consistent with this, the relative expression levels of anthocyanin-synthesis-related genes, including DFR, LDOX, MybA1 and UFGT, in VA-treated cell cultures were much higher than those in control, and high total anthocyanin accumulation was noted in the VA-treated cell cultures as well. These results suggest that VA up-regulates the expression of genes leading to anthocyanin accumulation by inducing endogenous ABA. In addition, VA increased total anthocyanin content in a dose-dependent manner. Although VA treatment in combination with exogenous ABA did not exhibit any synergistic effect, treatment with VA alone showed an equivalent effect to that with exogenous ABA alone on total anthocyanin accumulation. These findings point to the possibility of using VA for improving grape berry coloration. Copyright © 2017 Elsevier GmbH. All rights reserved.

  5. Regulation and patterns of endogenous and exogenous gene expression during differentiation of embryonal carcinoma cells

    International Nuclear Information System (INIS)

    Astigiano, S.; Sherman, M.I.; Abarzua, P.

    1989-01-01

    Embryonal carcinoma (EC) cells offer an interesting model system for evaluating differentiation because the cells are pluripotent, thus resembling germ cells and embryonic stem cells, and because a number of agents have been defined that are capable of promoting the differentiation of these cells. This chapter examines how EC cells might be triggered to differentiate, with emphasis on retinoic acid because this compound is a potent, naturally occurring inducer that has been studied extensively in this system. The nature of alterations in gene expression during EC cell differentiation is reviewed from the perspective of evaluating whether these changes are likely to be responsible for, or a result of, the differentiation event. Finally, the authors consider in molecular terms why EC cells, but not their differentiated derivatives, are refractory to the expression of many viral genomes following infection. Based upon these studies, they propose that fundamental changes in gene expression that are observed when differentiation is triggered in EC cells are likely to be due to the disappearance or neutralization of strong repressor elements

  6. Acute modulation of cytokine gene expression in bovine peripheral blood mononuclear cells (PBMCs) by endogenous cortisol

    Science.gov (United States)

    Cortisol suppresses many aspects of immune function. However, recent publications suggest acute cortisol exposure may actually enhance immune function (Dhabhar. 2009. Neuroimmunomod. 16:300). The objective of this study was to determine the influence of acute increases in endogenous cortisol on expr...

  7. Acute modulation of cytokine gene expression in bovine PBMCs by endogenous cortisol

    Science.gov (United States)

    Cortisol suppresses many aspects of immune function. However, recent publications suggest acute cortisol exposure may actually enhance immune function (Dhabhar, Neuroimmunomod 2009;16:300). The objective of this study was to determine the influence of acute increases in endogenous cortisol on expres...

  8. Endogenous Cortisol: Acute Modulation of Cytokine Gene Expression in Bovine PBMCs

    Science.gov (United States)

    Cortisol suppresses many aspects of immune function. However, recent publications suggest acute cortisol exposure may actually enhance immune function (Dhabhar, Neuroimmunomod 2009;16:300). The objective of this study was to determine the influence of acute increases in endogenous cortisol on expres...

  9. Safety assessment considerations for food and feed derived from plants with genetic modifications that modulate endogenous gene expression and pathways.

    Science.gov (United States)

    Kier, Larry D; Petrick, Jay S

    2008-08-01

    The current globally recognized comparative food and feed safety assessment paradigm for biotechnology-derived crops is a robust and comprehensive approach for evaluating the safety of both the inserted gene product and the resulting crop. Incorporating many basic concepts from food safety, toxicology, nutrition, molecular biology, and plant breeding, this approach has been used effectively by scientists and regulatory agencies for 10-15 years. Current and future challenges in agriculture include the need for improved yields, tolerance to biotic and abiotic stresses, and improved nutrition. The next generation of biotechnology-derived crops may utilize regulatory proteins, such as transcription factors that modulate gene expression and/or endogenous plant pathways. In this review, we discuss the applicability of the current safety assessment paradigm to biotechnology-derived crops developed using modifications involving regulatory proteins. The growing literature describing the molecular biology underlying plant domestication and conventional breeding demonstrates the naturally occurring genetic variation found in plants, including significant variation in the classes, expression, and activity of regulatory proteins. Specific examples of plant modifications involving insertion or altered expression of regulatory proteins are discussed as illustrative case studies supporting the conclusion that the current comparative safety assessment process is appropriate for these types of biotechnology-developed crops.

  10. Endogenous CO2 may inhibit bacterial growth and induce virulence gene expression in enteropathogenic Escherichia coli.

    Science.gov (United States)

    Martínez, Haydee; Buhse, Thomas; Rivera, Marco; Parmananda, P; Ayala, Guadalupe; Sánchez, Joaquín

    2012-07-01

    Analysis of the growth kinetics of enteropathogenic Escherichia coli (EPEC) revealed that growth was directly proportional to the ratio between the exposed surface area and the liquid culture volume (SA/V). It was hypothesized that this bacterial behavior was caused by the accumulation of an endogenous volatile growth inhibitor metabolite whose escape from the medium directly depended on the SA/V. The results of this work support the theory that an inhibitor is produced and indicate that it is CO(2). We also report that concomitant to the accumulation of CO(2), there is secretion of the virulence-related EspB and EspC proteins from EPEC. We therefore postulate that endogenous CO(2) may have an effect on both bacterial growth and virulence. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression.

    Science.gov (United States)

    Denzler, Rémy; McGeary, Sean E; Title, Alexandra C; Agarwal, Vikram; Bartel, David P; Stoffel, Markus

    2016-11-03

    Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Endogenous interferon-β-inducible gene expression and interferon-β-treatment are associated with reduced T cell responses to myelin basic protein in multiple sclerosis

    DEFF Research Database (Denmark)

    Börnsen, Lars; Christensen, Jeppe Romme; Ratzer, Rikke

    2015-01-01

    Autoreactive CD4+ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used for......-induced CD4+ T-cell autoreactivity in interferon-β-treated multiple sclerosis patients may be mediated by monocyte-derived interleukin-10.......Autoreactive CD4+ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used...... for treatment of multiple sclerosis, and some untreated multiple sclerosis patients have increased expression levels of type I interferon-inducible genes in immune cells. The role of endogenous type I interferons in multiple sclerosis is controversial: some studies found an association of high expression levels...

  13. Induced pluripotency with endogenous and inducible genes

    International Nuclear Information System (INIS)

    Duinsbergen, Dirk; Eriksson, Malin; Hoen, Peter A.C. 't; Frisen, Jonas; Mikkers, Harald

    2008-01-01

    The recent discovery that two partly overlapping sets of four genes induce nuclear reprogramming of mouse and even human cells has opened up new possibilities for cell replacement therapies. Although the combination of genes that induce pluripotency differs to some extent, Oct4 and Sox2 appear to be a prerequisite. The introduction of four genes, several of which been linked with cancer, using retroviral approaches is however unlikely to be suitable for future clinical applications. Towards developing a safer reprogramming protocol, we investigated whether cell types that express one of the most critical reprogramming genes endogenously are predisposed to reprogramming. We show here that three of the original four pluripotency transcription factors (Oct4, Klf4 and c-Myc or MYCER TAM ) induced reprogramming of mouse neural stem (NS) cells exploiting endogenous SoxB1 protein levels in these cells. The reprogrammed neural stem cells differentiated into cells of each germ layer in vitro and in vivo, and contributed to mouse development in vivo. Thus a combinatorial approach taking advantage of endogenously expressed genes and inducible transgenes may contribute to the development of improved reprogramming protocols

  14. Epigenetic editing using programmable zinc ginger proteins : inherited silencing of endogenous gene expression by targeted DNA methylation

    NARCIS (Netherlands)

    Stolzenburg, Sabine

    2014-01-01

    Cancer development is not only the result of genetic mutations but also stems from modifications in the epigenetic code leading to an aberrant expression of genes relevant for cancer. The most studied epigenetic mark is DNA methylation of cytosines in the promoters of genes, which is associated with

  15. Target gene expression levels and competition between transfected and endogenous microRNAs are strong confounding factors in microRNA high-throughput experiments

    Science.gov (United States)

    2012-01-01

    Background MicroRNA (miRNA) target genes tend to have relatively long and conserved 3' untranslated regions (UTRs), but to what degree these characteristics contribute to miRNA targeting is poorly understood. Different high-throughput experiments have, for example, shown that miRNAs preferentially regulate genes with both short and long 3' UTRs and that target site conservation is both important and irrelevant for miRNA targeting. Results We have analyzed several gene context-dependent features, including 3' UTR length, 3' UTR conservation, and messenger RNA (mRNA) expression levels, reported to have conflicting influence on miRNA regulation. By taking into account confounding factors such as technology-dependent experimental bias and competition between transfected and endogenous miRNAs, we show that two factors - target gene expression and competition - could explain most of the previously reported experimental differences. Moreover, we find that these and other target site-independent features explain about the same amount of variation in target gene expression as the target site-dependent features included in the TargetScan model. Conclusions Our results show that it is important to consider confounding factors when interpreting miRNA high throughput experiments and urge special caution when using microarray data to compare average regulatory effects between groups of genes that have different average gene expression levels. PMID:22325809

  16. Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

    Science.gov (United States)

    Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

    2010-09-01

    Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

  17. Engineering high Zn in tomato shoots through expression of AtHMA4 involves tissue-specific modification of endogenous genes.

    Science.gov (United States)

    Kendziorek, Maria; Klimecka, Maria; Barabasz, Anna; Borg, Sören; Rudzka, Justyna; Szczęsny, Paweł; Antosiewicz, Danuta Maria

    2016-08-12

    To increase the Zn level in shoots, AtHMA4 was ectopically expressed in tomato under the constitutive CaMV 35S promoter. However, the Zn concentration in the shoots of transgenic plants failed to increase at all tested Zn levels in the medium. Modification of Zn root/shoot distribution in tomato expressing 35S::AtHMA4 depended on the concentration of Zn in the medium, thus indicating involvement of unknown endogenous metal-homeostasis mechanisms. To determine these mechanisms, those metal-homeostasis genes that were expressed differently in transgenic and wild-type plants were identified by microarray and RT-qPCR analysis using laser-assisted microdissected RNA isolated from two root sectors: (epidermis + cortex and stele), and leaf sectors (upper epidermis + palisade parenchyma and lower epidermis + spongy parenchyma). Zn-supply-dependent modification of Zn root/shoot distribution in AtHMA4-tomato (increase at 5 μM Zn, no change at 0.5 μM Zn) involved tissue-specific, distinct from that in the wild type, expression of tomato endogenous genes. First, it is suggested that an ethylene-dependent pathway underlies the detected changes in Zn root/shoot partitioning, as it was induced in transgenic plants in a distinct way depending on Zn exposure. Upon exposure to 5 or 0.5 μM Zn, in the epidermis + cortex of the transgenics' roots the expression of the Strategy I Fe-uptake system (ethylene-dependent LeIRT1 and LeFER) was respectively lower or higher than in the wild type and was accompanied by respectively lower or higher expression of the identified ethylene genes (LeNR, LeACO4, LeACO5) and of LeChln. Second, the contribution of LeNRAMP2 expression in the stele is shown to be distinct for wild-type and transgenic plants at both Zn exposures. Ethylene was also suggested as an important factor in a pathway induced in the leaves of transgenic plants by high Zn in the apoplast, which results in the initiation of loading of the excess Zn into the

  18. TCDD and a putative endogenous AhR ligand, ITE, elicit the same immediate changes in gene expression in mouse lung fibroblasts.

    Science.gov (United States)

    Henry, Ellen C; Welle, Stephen L; Gasiewicz, Thomas A

    2010-03-01

    The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, mediates toxicity of several classes of xenobiotics and also has important physiological roles in differentiation, reproduction, and immunity, although the endogenous ligand(s) mediating these functions is/are as yet unidentified. One candidate endogenous ligand, 2-(1'H-indolo-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), is a potent AhR agonist in vitro, activates the murine AhR in vivo, but does not induce toxicity. We hypothesized that ITE and the toxic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may modify transcription of different sets of genes to account for their different toxicity. To test this hypothesis, primary mouse lung fibroblasts were exposed to 0.5muM ITE, 0.2nM TCDD, or vehicle for 4 h, and total gene expression was evaluated using microarrays. After this short-term and low-dose treatment, several hundred genes were changed significantly, and the response to ITE and TCDD was remarkably similar, both qualitatively and quantitatively. Induced gene sets included the expected battery of AhR-dependent xenobiotic-metabolizing enzymes, as well as several sets that reflect the inflammatory role of lung fibroblasts. Real time quantitative RT-qPCR assay of several selected genes confirmed these microarray data and further suggested that there may be kinetic differences in expression between ligands. These data suggest that ITE and TCDD elicit an analogous change in AhR conformation such that the initial transcription response is the same. Furthermore, if the difference in toxicity between TCDD and ITE is mediated by differences in gene expression, then it is likely that secondary changes enabled by the persistent TCDD, but not by the shorter lived ITE, are responsible.

  19. Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig.

    Science.gov (United States)

    Gu, Y R; Li, M Z; Zhang, K; Chen, L; Jiang, A A; Wang, J Y; Li, X W

    2011-08-01

    To normalize a set of quantitative real-time PCR (q-PCR) data, it is essential to determine an optimal number/set of housekeeping genes, as the abundance of housekeeping genes can vary across tissues or cells during different developmental stages, or even under certain environmental conditions. In this study, of the 20 commonly used endogenous control genes, 13, 18 and 17 genes exhibited credible stability in 56 different tissues, 10 types of adipose tissue and five types of muscle tissue, respectively. Our analysis clearly showed that three optimal housekeeping genes are adequate for an accurate normalization, which correlated well with the theoretical optimal number (r ≥ 0.94). In terms of economical and experimental feasibility, we recommend the use of the three most stable housekeeping genes for calculating the normalization factor. Based on our results, the three most stable housekeeping genes in all analysed samples (TOP2B, HSPCB and YWHAZ) are recommended for accurate normalization of q-PCR data. We also suggest that two different sets of housekeeping genes are appropriate for 10 types of adipose tissue (the HSPCB, ALDOA and GAPDH genes) and five types of muscle tissue (the TOP2B, HSPCB and YWHAZ genes), respectively. Our report will serve as a valuable reference for other studies aimed at measuring tissue-specific mRNA abundance in porcine samples. © 2011 Blackwell Verlag GmbH.

  20. Identification of valid endogenous control genes for determining gene expression in C6 glioma cell line treated with conditioned medium from adipose-derived stem cell.

    Science.gov (United States)

    Iser, I C; de Campos, R P; Bertoni, A P S; Wink, M R

    2015-10-01

    There is growing evidence that mesenchymal stem cells (MSCs) can be important players in the tumor microenvironment. They can affect the glioma progression through the modulation of different genes. This modulation can be evaluated through a very useful model, treating the tumor cells with MSC-conditioned medium. However, for an accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is a prerequisite. We performed a systematic review in an attempt to find a reference gene to use when analyzing gene expression in C6 glioma cells lines. Considering that we were not able to find a reference gene originated by an appropriate validation, in this study we evaluated candidate genes to be used as reference gene in C6 cells under different treatments with adipose-derived stem cells conditioned medium (CM-ADSCs). β-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine-guanine phosphoribosyltransferase I (HPRT-1); TATA box binding protein (TBP) and beta-2-microglobulin (B2M) were evaluated by real-time reverse transcription PCR (RT-qPCR). The mean Cq, the maximum fold change (MFC) and NormFinder software were used for reference gene evaluation and selection. The GAPDH and ACTB genes have been the most widely used reference genes to normalize among the different investigated genes in our review, however, controversially these genes underwent a substantial variability among the genes evaluated in the present work. Individually, TBP gene was more stable when compared with other genes analyzed and the combination of TBP and HPRT-1 was even more stable. These results evidence the importance of appropriate validation of reference genes before performing qPCR experiments. Besides, our data will contribute with researchers that work analyzing the role of ADSCs in glioma microenvironment through gene expression. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  1. Low-Dose Gene Therapy for Murine PKU Using Episomal Naked DNA Vectors Expressing PAH from Its Endogenous Liver Promoter

    Directory of Open Access Journals (Sweden)

    Hiu Man Grisch-Chan

    2017-06-01

    Full Text Available Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH by delivery of naked DNA/minicircle (MC-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU. Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of 95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.

  2. Endogenous retrovirus sequences expressed in male mammalian ...

    African Journals Online (AJOL)

    In humans, one ERV family, human endogenous retrovirus- K (HERV-K) is abundantly expressed, and is associated with germ cell tumours, while ERV3 env is expressed in normal human testis. Conclusion: The expression of ERVs in male reproductive tissues suggests a possible role in normal and disease conditions ...

  3. Human endogenous retrovirus expression is inversely related with the up-regulation of interferon-inducible genes in the skin of patients with lichen planus.

    Science.gov (United States)

    Nogueira, Marcelle Almeida de Sousa; Gavioli, Camila Fátima Biancardi; Pereira, Nátalli Zanete; de Carvalho, Gabriel Costa; Domingues, Rosana; Aoki, Valéria; Sato, Maria Notomi

    2015-04-01

    Lichen planus (LP) is a common inflammatory skin disease of unknown etiology. Reports of a common transactivation of quiescent human endogenous retroviruses (HERVs) support the connection of viruses to the disease. HERVs are ancient retroviral sequences in the human genome and their transcription is often deregulated in cancer and autoimmune diseases. We explored the transcriptional activity of HERV sequences as well as the antiviral restriction factor and interferon-inducible genes in the skin from LP patients and healthy control (HC) donors. The study included 13 skin biopsies from patients with LP and 12 controls. Real-time PCR assay identified significant decrease in the HERV-K gag and env mRNA expression levels in LP subjects, when compared to control group. The expressions of HERV-K18 and HERV-W env were also inhibited in the skin of LP patients. We observed a strong correlation between HERV-K gag with other HERV sequences, regardless the down-modulation of transcripts levels in LP group. In contrast, a significant up-regulation of the cytidine deaminase APOBEC 3G (apolipoprotein B mRNA-editing), and the GTPase MxA (Myxovirus resistance A) mRNA expression level was identified in the LP skin specimens. Other transcript expressions, such as the master regulator of type I interferon-dependent immune responses, STING (stimulator of interferon genes) and IRF-7 (interferon regulatory factor 7), IFN-β and the inflammassome NALP3, had increased levels in LP, when compared to HC group. Our study suggests that interferon-inducible factors, in addition to their role in innate immunity against exogenous pathogens, contribute to the immune control of HERVs. Evaluation of the balance between HERV and interferon-inducible factor expression could possibly contribute to surveillance of inflammatory/malignant status of skin diseases.

  4. Gene expression

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

    1983-01-01

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  5. Endogenous retrovirus sequences expressed in male mammalian ...

    African Journals Online (AJOL)

    Objectives: To review the research findings on the expression of endogenous retroviruses and retroviral-related particles in male mammalian reproductive tissues, and to discuss their possible role in normal cellular events and association with disease conditions in male reproductive tissues. Data sources: Published ...

  6. Positive feedback regulation of a Lycium chinense-derived VDE gene by drought-induced endogenous ABA, and over-expression of this VDE gene improve drought-induced photo-damage in Arabidopsis.

    Science.gov (United States)

    Guan, Chunfeng; Ji, Jing; Zhang, Xuqiang; Li, Xiaozhou; Jin, Chao; Guan, Wenzhu; Wang, Gang

    2015-03-01

    Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under light stress. We have cloned a VDE gene (LcVDE) from Lycium chinense, a deciduous woody perennial halophyte, which can grow in a large variety of soil types. The amino acid sequence of LcVDE has high homology with VDEs in other plants. Under drought stress, relative expression of LcVDE and the de-epoxidation ratio (Z+0.5A)/(V+A+Z) increased rapidly, and non-photochemical quenching (NPQ) also rose. Interestingly, these elevations induced by drought stress were reduced by the topical administration of abamine SG, a potent ABA inhibitor via inhibition of NCED in the ABA synthesis pathway. Until now, little has been done to explore the relationship between endogenous ABA and the expression of VDE genes. Since V serves as a common precursor for ABA, these data support the possible involvement of endogenous ABA in the positive feedback regulation of LcVDE gene expression in L. chinense under drought stress. Moreover, the LcVDE may be involved in modulating the level of photosynthesis damage caused by drought stress. Furthermore, the ratio of (Z+0.5A)/(V+A+Z) and NPQ increased more in transgenic Arabidopsis over-expressing LcVDE gene than the wild types under drought stress. The maximum quantum yield of primary photochemistry of PSII (Fv/Fm) in transgenic Arabidopsis decreased more slowly during the stressed period than that in wild types under the same conditions. Furthermore, transgenic Arabidopsis over-expressing LcVDE showed increased tolerance to drought stress. Copyright © 2014 Elsevier GmbH. All rights reserved.

  7. Chronic nicotine-induced changes in gene expression of delta and kappa-opioid receptors and their endogenous ligands in the mesocorticolimbic system of the rat.

    Science.gov (United States)

    Ugur, Muzeyyen; Kaya, Egemen; Gozen, Oguz; Koylu, Ersin O; Kanit, Lutfiye; Keser, Aysegul; Balkan, Burcu

    2017-09-01

    Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)-derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real-time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied. © 2017 Wiley Periodicals, Inc.

  8. Engineering high Zn in tomato shoots through expression of AtHMA4 involves tissue-specific modification of endogenous genes

    OpenAIRE

    Kendziorek, Maria; Klimecka, Maria; Barabasz, Anna; Borg, S?ren; Rudzka, Justyna; Szcz?sny, Pawe?; Antosiewicz, Danuta Maria

    2016-01-01

    Background To increase the Zn level in shoots, AtHMA4 was ectopically expressed in tomato under the constitutive CaMV 35S promoter. However, the Zn concentration in the shoots of transgenic plants failed to increase at all tested Zn levels in the medium. Modification of Zn root/shoot distribution in tomato expressing 35S::AtHMA4 depended on the concentration of Zn in the medium, thus indicating involvement of unknown endogenous metal-homeostasis mechanisms. To determine these mechanisms, thos...

  9. Endogenous Methanol Regulates Mammalian Gene Activity

    Science.gov (United States)

    Komarova, Tatiana V.; Petrunia, Igor V.; Shindyapina, Anastasia V.; Silachev, Denis N.; Sheshukova, Ekaterina V.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis. PMID:24587296

  10. Endogenous methanol regulates mammalian gene activity.

    Directory of Open Access Journals (Sweden)

    Tatiana V Komarova

    Full Text Available We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.

  11. Gene expression of hematoregulatory cytokines is elevated endogenously after sublethal gamma irradiation and is differentially enhanced by therapeutic administration of biologic response modifiers

    International Nuclear Information System (INIS)

    Peterson, V.M.; Adamovicz, J.J.; Madonna, G.S.; Gause, W.C.; Elliott, T.B.; Moore, M.M.; Ledney, G.D.; Jackson, W.E. III

    1994-01-01

    Prompt, cytokine-mediated restoration of hematopoiesis is a prerequisite for survival after irradiation. Therapy with biologic response modifiers (BRMs), such as LPS, 3D monophosphoryl lipid A (MPL), and synthetic trehalose dicrynomycolate (S-TDCM) presumably accelerates hematopoietic recovery after irradiation are poorly defined. One hour after sublethal (7.0 Gy) 60 Co gamma irradiation, B6D2F1/J female mice received a single i.p. injection of LPS, MPL, S-TDCM, an extract from Serratia marcescens (Sm-BRM), or Tween 80 in saline (TS). Five hours later, a quantitative reverse transcription-PCR assay demonstrated marked splenic gene expression for IL-1β, IL-3, IL-6, and granulocyte-CSF (G-CSF). Enhanced gene expression for TNF-α, macrophage-CSF (M-CSF), and stem cell factor (SCF) was not detected. Injection of any BRM further enhanced cytokine gene expression and plasma levels of CSF activity within 24 h after irradiation and hastened bone marrow recovery. Mice injected with S-TDCM or Sm-BRM sustained expression of the IL-6 gene for at least 24 h after irradiation. Sm-BRM-treated mice exhibited greater gene expression for IL-1β, IL-3, TNF-α, and G-CSF at day 1 than any other BRM. When challenged with 2 LD 50/30 of Klebsiella pneumoniae 4 days after irradiation, 100% of Sm-BRM-treated mice and 70% of S-TDCM-treated mice survived, whereas ≤30% of mice treated with LPS, MPL, or TS survived. Thus, sublethal irradiation induces transient, splenic cytokine gene expression that can be differentially amplified and prolonged by BRMs. BRMs that sustained and/or enhanced irradiation-induced expression of specific cytokine genes improved survival after experimental infection. 67 refs., 7 figs., 1 tab

  12. Co-factors necessary for PPAR mediated transactivation of endogenous target genes

    DEFF Research Database (Denmark)

    Grøntved, Lars; Nielsen, Ronni; Stunnenberg, Henk

    of endogenous target gene in different cell types are elusive. To mutually compare the ability of the PPAR subtypes to activate endogenous target genes in a given cell, PPARa, PPARb/d and PPARg2 were HA tagged and rapidly, equally and synchronously expressed using adenoviral delivery. Within a few hours after...... subtype specific activation of target genes. Accumulating evidence suggests that transcriptional co-factors can function as master regulators for nuclear receptors and impose promoter selectivity. To study co-factor necessity for PPAR mediated transactivation of endogenous target genes, specific co...

  13. Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.

    Science.gov (United States)

    Pichery, Mélanie; Mirey, Emilie; Mercier, Pascale; Lefrancais, Emma; Dujardin, Arnaud; Ortega, Nathalie; Girard, Jean-Philippe

    2012-04-01

    IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.

  14. Fowlpoxvirus recombinants coding for the CIITA gene increase the expression of endogenous MHC-II and Fowlpox Gag/Pro and Env SIV transgenes.

    Science.gov (United States)

    Bissa, Massimiliano; Forlani, Greta; Zanotto, Carlo; Tosi, Giovanna; De Giuli Morghen, Carlo; Accolla, Roberto S; Radaelli, Antonia

    2018-01-01

    A complete eradication of an HIV infection has never been achieved by vaccination and the search for new immunogens that can induce long-lasting protective responses is ongoing. Avipoxvirus recombinants are host-restricted for replication to avian species and they do not have the undesired side effects induced by vaccinia recombinants. In particular, Fowlpox (FP) recombinants can express transgenes over long periods and can induce protective immunity in mammals, mainly due to CD4-dependent CD8+ T cells. In this context, the class II transactivator (CIITA) has a pivotal role in triggering the adaptive immune response through induction of the expression of class-II major histocompatibility complex molecule (MHC-II), that can present antigens to CD4+ T helper cells. Here, we report on construction of novel FPgp and FPenv recombinants that express the highly immunogenic SIV Gag-pro and Env structural antigens. Several FP-based recombinants, with single or dual genes, were also developed that express CIITA, driven from H6 or SP promoters. These recombinants were used to infect CEF and Vero cells in vitro and determine transgene expression, which was evaluated by real-time PCR and Western blotting. Subcellular localisation of the different proteins was evaluated by confocal microscopy, whereas HLA-DR or MHC-II expression was measured by flow cytometry. Fowlpox recombinants were also used to infect syngeneic T/SA tumour cells, then injected into Balb/c mice to elicit MHC-II immune response and define the presentation of the SIV transgene products in the presence or absence of FPCIITA. Antibodies to Env were measured by ELISA. Our data show that the H6 promoter was more efficient than SP to drive CIITA expression and that CIITA can enhance the levels of the gag/pro and env gene products only when infection is performed by FP single recombinants. Also, CIITA expression is higher when carried by FP single recombinants than when combined with FPgp or FPenv constructs and can

  15. Epigenetic interplay between mouse endogenous retroviruses and host genes.

    Science.gov (United States)

    Rebollo, Rita; Miceli-Royer, Katharine; Zhang, Ying; Farivar, Sharareh; Gagnier, Liane; Mager, Dixie L

    2012-10-03

    Transposable elements are often the targets of repressive epigenetic modifications such as DNA methylation that, in theory, have the potential to spread toward nearby genes and induce epigenetic silencing. To better understand the role of DNA methylation in the relationship between transposable elements and genes, we assessed the methylation state of mouse endogenous retroviruses (ERVs) located near genes. We found that ERVs of the ETn/MusD family show decreased DNA methylation when near transcription start sites in tissues where the nearby gene is expressed. ERVs belonging to the IAP family, however, are generally heavily methylated, regardless of the genomic environment and the tissue studied. Furthermore, we found full-length ETn and IAP copies that display differential DNA methylation between their two long terminal repeats (LTRs), suggesting that the environment surrounding gene promoters can prevent methylation of the nearby LTR. Spreading from methylated ERV copies to nearby genes was rarely observed, with the regions between the ERVs and genes apparently acting as a boundary, enriched in H3K4me3 and CTCF, which possibly protects the unmethylated gene promoter. Furthermore, the flanking regions of unmethylated ERV copies harbor H3K4me3, consistent with spreading of euchromatin from the host gene toward ERV insertions. We have shown that spreading of DNA methylation from ERV copies toward active gene promoters is rare. We provide evidence that genes can be protected from ERV-induced heterochromatin spreading by either blocking the invasion of repressive marks or by spreading euchromatin toward the ERV copy.

  16. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

    Directory of Open Access Journals (Sweden)

    Lin Yuan

    Full Text Available Three hundred one-day-old male broiler chickens (Ross-308 were fed corn-soybean basal diets containing non-starch polysaccharide (NSP enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI and average daily gain (ADG were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05. Feed-to-gain ratio (FGR was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05. Apparent digestibility of crude protein (ADCP was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05. Cholecystokinin (CCK level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05, but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05, respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05. However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05. The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05. Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  17. Unexpected Diversity and Expression of Avian Endogenous Retroviruses

    Science.gov (United States)

    Bolisetty, Mohan; Blomberg, Jonas; Benachenhou, Farid; Sperber, Göran; Beemon, Karen

    2012-01-01

    ABSTRACT Endogenous retroviruses (ERVs) were identified and characterized in three avian genomes to gain insight into early retroviral evolution. Using the computer program RetroTector to detect relatively intact ERVs, we identified 500 ERVs in the chicken genome, 150 in the turkey genome, and 1,200 in the zebra finch genome. Previous studies suggested that endogenous alpharetroviruses were present in chicken genomes. In this analysis, a small number of alpharetroviruses were seen in the chicken and turkey genomes; however, these were greatly outnumbered by beta-like, gamma-like, and alphabeta proviruses. While the avian ERVs belonged to the same major groups as mammalian ERVs, they were more heterogeneous. In particular, the beta-like viruses revealed an evolutionary continuum with the gradual acquisition and loss of betaretroviral markers and a transition from beta to alphabeta and then to alpharetroviruses. Thus, it appears that birds may resemble a melting pot for early ERV evolution. Many of the ERVs were integrated in clusters on chromosomes, often near centromeres. About 25% of the chicken ERVs were in or near cellular transcription units; this is nearly random. The majority of these integrations were in the sense orientation in introns. A higher-than-random number of integrations were >100 kb from the nearest gene. Deep-sequencing studies of chicken embryo fibroblasts revealed that about 20% of the 500 ERVs were transcribed and translated. A subset of these were also transcribed in vivo in chickens, showing tissue-specific patterns of expression. PMID:23073767

  18. Avian endogenous provirus (ev-3) env gene sequencing: implication for pathogenic retrovirus origination.

    Science.gov (United States)

    Tikhonenko, A T; Lomovskaya, O L

    1990-02-01

    The avian endogenous env gene product blocks the surface receptor and, as a result, cells become immune to related exogenous retroviruses. On the other hand, the same sequence can be included in the pathogenic retrovirus genome, as shown by oligonucleotide mapping. However, since the complete env gene sequence was not known, the comparison of genomic nucleotide sequences was not possible. Therefore an avian endogenous provirus with an intact env gene was cloned from a chicken gene bank and the regions coding for the C terminus of the gp85 and gp37 proteins were sequenced. Comparison of this sequence with those of other retroviruses proved that one of the pathogenic viruses associated with osteopetrosis is a cross between avian endogenous virus and Rous sarcoma virus. Retroviruses and, especially, endogenous retroviruses are traditionally of the most developed models of viral carcinogenesis. Many endogenous retroviruses are implicated in neoplastic transformation of the cell. For instance, endogenous mouse mammary tumor virus of some inbred lines appears to be the only causative agent in these mammary cancers. Other even nonpathogenic murine endogenous retroviruses are involved in the origination of MCF-type recombinant acute leukosis viruses. Some endogenous retroviruses are implicated in the transduction or activation of cellular protooncogenes. Our interest in endogenous viruses is based on their ability to make cells resistant to exogenous retroviruses. Expression of their major envelope glycoprotein leads to cellular surface receptor blockage and imparts immunity to infection by the related leukemia retroviruses. This problem is quite elaborated for chicken endogenous virus RAV-O (7-9).(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Electroacupuncture-Induced Dynamic Processes of Gene Expression Levels of Endogenous Opioid Peptide Precursors and Opioid Receptors in the CNS of Goats

    Directory of Open Access Journals (Sweden)

    Li-Li Cheng

    2013-01-01

    Full Text Available In order to investigate the dynamic processes of mRNA levels of proenkephalin, proopiomelanocortin, prodynorphin, and opioid receptors (δ-, μ-, and κ-receptor induced by electroacupuncture (EA in the central nerve system, goats were stimulated by EA of 60 Hz for 0.5 h at a set of Baihui, Santai, Ergen, and Sanyangluo points. The pain threshold was measured using the method of potassium iontophoresis. The mRNA levels of the three opioid peptide precursors and three opioid receptors were determined with quantitative real-time PCR and the levels of Met-enkephalin with SABC immunohistochemistry at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA. The results showed that the pain threshold correlated (P<0.01 with Met-enkephalin immunoactivities in the measured nuclei and areas of goats. The analgesic aftereffect lasted for 12 h at least. The mRNA levels of the three opioid peptide precursors and three opioid receptors began to increase at 0 h, reached the peak during the time from 4 h to 6 h or at 12 h, and remained higher at 24 h after EA was discontinued. These results suggested that the initiation of gene expression of opioid peptides and the three receptors may be associated with EA-induced analgesic aftereffect.

  20. [Mechanisms of endogenous drug resistance acquisition by spontaneous chromosomal gene mutation].

    Science.gov (United States)

    Fukuda, H; Hiramatsu, K

    1997-05-01

    Endogenous resistance in bacteria is caused by a change or loss of function and generally genetically recessive. However, this type of resistance acquisition are now prevalent in clinical setting. Chromosomal genes that afford endogenous resistance are the genes correlated with the target of the drug, the drug inactivating enzymes, and permeability of the molecules including the antibacterial agents. Endogenous alteration of the drug target are mediated by the spontaneous mutation of their structural gene. This mutation provides much lower affinity of the drugs for the target. Gene expression of the inactivating enzymes, such as class C beta-lactamase, is generally regulated by regulatory genes. Spontaneous mutations in the regulatory genes cause constitutive enzyme production and provides the resistant to the agent which is usually stable for such enzymes. Spontaneous mutation in the structural gene gives the enzyme extra-spectrum substrate specificity, like ESBL (Extra-Spectrum-beta-Lactamase). Expression of structural genes encoding the permeability systems are also regulated by some regulatory genes. The spontaneous mutation of the regulatory genes reduce an amount of porin protein. This mutation causes much lower influx of the drug in the cell. Spontaneous mutation in promoter region of the structural gene of efflux protein was observed. This mutation raised the gene transcription and overproduced efflux protein. This protein progresses the drug efflux from the cell.

  1. Expression and function of endogenous retroviruses in the placenta.

    Science.gov (United States)

    Denner, Joachim

    2016-01-01

    Although the expression of endogenous retroviruses in the placenta of numerous species was observed a long time ago, their physiological function during gestation was demonstrated only very recently. Expression of retroviral envelope proteins, also called syncytins, in the placenta allows generation of the multinuclear syncytiotrophoblast as an outer cellular layer of the placenta by fusion of the trophoblast cells. This fusion process is crucial for the development of the placenta and for successful pregnancy. It is still unclear whether the immunosuppressive properties of the transmembrane envelope protein of the endogenous retroviruses expressed in the placenta contribute to immunosuppression to prevent the rejection of the semiallotransplant embryo. The presence of placenta cells expressing retroviral envelope proteins surrounded by immune cells deep in the maternal tissue supports an immunosuppressive function. It is important to emphasize that during evolution different species utilized ('enslaved') different endogenous retroviruses and that two or more endogenous retroviruses are involved in placentogenesis in each species. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  2. Temporal repression of endogenous pluripotency genes during reprogramming of porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane; Christensen, Marianne; Rasmussen, Mikkel Aabech

    2012-01-01

    Porcine induced pluripotent stem cells (piPSCs) have the capacity to differentiate in vitro and in vivo and form chimeras. However, the lack of transgene silencing of exogenous DNA integrated into the genome and the inability of cells to proliferate in the absence of transgene expression...... pluripotency in the pig. This may help to explain the difficulties in producing stable piPSCs and bona fide embryonic stem cell lines in this species....... transgenes on the expression of the porcine endogenous pluripotency machinery. Endogenous and exogenous gene expression of OCT4, NANOG, SOX2, KLF4, and cMYC was determined at passages 5, 10, 15, and 20, both in cells cultured at 1¿µg/mL doxycycline or 4¿µg/mL doxycycline. Our results revealed that endogenous...

  3. Impact of cell culture process changes on endogenous retrovirus expression.

    Science.gov (United States)

    Brorson, Kurt; De Wit, Christina; Hamilton, Elizabeth; Mustafa, Mehnaz; Swann, Patrick G; Kiss, Robert; Taticek, Ron; Polastri, Gian; Stein, Kathryn E; Xu, Yuan

    2002-11-05

    Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other

  4. Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane

    Directory of Open Access Journals (Sweden)

    Bantong Xue

    2014-05-01

    Full Text Available Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM crops by quantitative real-time PCR (qPCR or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

  5. Lipofection indirectly increases expression of endogenous major histocompatibility complex class I molecules on tumor cells.

    Science.gov (United States)

    Fox, B A; Drury, M; Hu, H M; Cao, Z; Huntzicker, E G; Qie, W; Urba, W J

    1998-01-01

    Direct intratumoral injection of a lipid/DNA complex encoding an allogeneic major histocompatibility complex (MHC) class I molecule leads to regression of both an immunogenic murine tumor and also melanoma lesions in some patients. We have sought to understand the mechanism(s) for this augmentation of antitumor activity. While optimizing parameters for in vitro gene transfer into the D5 subclone of B16BL6, it was noted that lipofected tumors not only expressed the new alloantigen but also exhibited increased expression of endogenous MHC class I, both H-2 Kb and H-2 Db. This increase in expression was not restricted to the small percentage of cells that expressed the transfected gene, but appeared to affect the majority of cells in culture. Class I expression was not increased by lipopolysaccharide, DNA alone, lipid, or lipid/lipopolysaccharide mixtures. Enhanced class I expression required a DNA/lipid complex and was greatest when parameters optimized for gene transfer of the alloantigen were used. All DNA plasmids tested had this effect, including one plasmid whose DNA was not transcribed because it lacked an expression cassette. Because of the critical role that MHC class I antigens play in immune recognition, we propose that lipid complex-mediated gene transfer may provide immunological advantages beyond those that are attributable to expression of the specific gene transferred.

  6. Commensal bacteria drive endogenous transformation and tumour stem cell marker expression through a bystander effect.

    Science.gov (United States)

    Wang, Xingmin; Yang, Yonghong; Huycke, Mark M

    2015-03-01

    Commensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect. Primary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)-an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis. Primary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice. These results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  7. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  8. Endogenous Locus Reporter Assays.

    Science.gov (United States)

    Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

    2018-01-01

    Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

  9. Novel endogenous retrovirus-derived transcript expressed in the bovine placenta is regulated by WNT signaling.

    Science.gov (United States)

    Sakurai, Toshihiro; Nakagawa, So; Bai, Hanako; Bai, Rulan; Kusama, Kazuya; Ideta, Atsushi; Aoyagi, Yoshito; Kaneko, Kazuyuki; Iga, Kosuke; Yasuda, Jiro; Miyazawa, Takayuki; Imakawa, Kazuhiko

    2017-10-10

    Endogenous retroviruses (ERVs) are involved in placentation; perhaps, the most well-known ERV s are the syncytins, actively transcribed env genes involved in cell-cell fusion and possible morphological variations. However, ERVs other than syncytins that play an important role in placental development have not been well characterized. To identify ERV genes expressed during the onset of placentation in the bovine species, we characterized the expression profiles of bovine conceptus transcripts during the peri-attachment period using RNA-seq analysis, and confirming some candidates through real-time PCR. Using in silico and PCR analyses, we identified a novel ERV proviral sequence derived from a gag region, designated bovine endogenous retroviruses (BERV)-K3, containing Gag _p10 and Gag _p24, zinc finger domain. Initial expression of this ERV in bovine conceptuses was on day 20 (day 0 = day of estrus), soon after conceptus attachment to the endometrial epithelium, and its high placental expression was maintained up to the middle of pregnancy. The BERV-K3 transcript was also found in the uterine luminal and glandular epithelia, liver, kidney, intestine, and skin. BERV-K3 is located on chromosome 7 and integrated within LOC100848658 , from which noncoding RNA could be transcribed. Furthermore, the expression of endogenous BERV-K3 in bovine trophoblast cell lines was induced by a WNT agonist, a signaling system common to genes expressed in placentas. These data support the argument that during the evolutionary process, mammals incorporated not only similar ERV sequences, but also ERV s unique to individual species. BERV-K3 is in the latter case, likely providing functions unique to ruminant gestation. © 2017 The Author(s).

  10. RNA interference and retinoblastoma-related genes are required for repression of endogenous siRNA targets in Caenorhabditis elegans.

    Science.gov (United States)

    Grishok, Alla; Hoersch, Sebastian; Sharp, Phillip A

    2008-12-23

    In Caenorhabditis elegans, a vast number of endogenous short RNAs corresponding to thousands of genes have been discovered recently. This finding suggests that these short interfering RNAs (siRNAs) may contribute to regulation of many developmental and other signaling pathways in addition to silencing viruses and transposons. Here, we present a microarray analysis of gene expression in RNA interference (RNAi)-related mutants rde-4, zfp-1, and alg-1 and the retinoblastoma (Rb) mutant lin-35. We found that a component of Dicer complex RDE-4 and a chromatin-related zinc finger protein ZFP-1, not implicated in endogenous RNAi, regulate overlapping sets of genes. Notably, genes a) up-regulated in the rde-4 and zfp-1 mutants and b) up-regulated in the lin-35(Rb) mutant, but not the down-regulated genes are highly represented in the set of genes with corresponding endogenous siRNAs (endo-siRNAs). Our study suggests that endogenous siRNAs cooperate with chromatin factors, either C. elegans ortholog of acute lymphoblastic leukemia-1 (ALL-1)-fused gene from chromosome 10 (AF10), ZFP-1, or tumor suppressor Rb, to regulate overlapping sets of genes and predicts a large role for RNAi-based chromatin silencing in control of gene expression in C. elegans.

  11. Genome polymorphism markers and stress genes expression for ...

    African Journals Online (AJOL)

    SAM

    2014-06-11

    Jun 11, 2014 ... RNA extraction and purification for SOD and PAL gene expression. Fresh leaf tissues (100 mg), from ... Data analysis. Gelquant program for quantification of protein, DNA and RNA gel. (version 1.8.2) was used for .... by reprogramming the expression of endogenous genes. Higher level of these antioxidant ...

  12. Sensory Neuropeptides and Endogenous Opioids Expression in Human Dental Pulp with Asymptomatic Inflammation: In Vivo Study

    Directory of Open Access Journals (Sweden)

    Daniel Chavarria-Bolaños

    2015-01-01

    Full Text Available Purpose. This study quantified the expression of substance P (SP, calcitonin gene-related peptide (CGRP, β-endorphins (β-End, and methionine-enkephalin (Met-Enk in human dental pulp following orthodontic intrusion. Methods. Eight patients were selected according to preestablished inclusion criteria. From each patient, two premolars (indicated for extraction due to orthodontic reasons were randomly assigned to two different groups: the asymptomatic inflammation group (EXPg, which would undergo controlled intrusive force for seven days, and the control group (CTRg, which was used to determine the basal levels of each substance. Once extracted, dental pulp tissue was prepared to determine the expression levels of both neuropeptides and endogenous opioids by radioimmunoassay (RIA. Results. All samples from the CTRg exhibited basal levels of both neuropeptides and endogenous opioids. By day seven, all patients were asymptomatic, even when all orthodontic-intrusive devices were still active. In the EXPg, the SP and CGRP exhibited statistically significant different levels. Although none of the endogenous opioids showed statistically significant differences, they all expressed increasing trends in the EXPg. Conclusions. SP and CGRP were identified in dental pulp after seven days of controlled orthodontic intrusion movement, even in the absence of pain.

  13. Gene expression and gene therapy imaging

    International Nuclear Information System (INIS)

    Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

    2007-01-01

    The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

  14. Challenges in testing genetically modified crops for potential increases in endogenous allergen expression for safety.

    Science.gov (United States)

    Panda, R; Ariyarathna, H; Amnuaycheewa, P; Tetteh, A; Pramod, S N; Taylor, S L; Ballmer-Weber, B K; Goodman, R E

    2013-02-01

    Premarket, genetically modified (GM) plants are assessed for potential risks of food allergy. The major risk would be transfer of a gene encoding an allergen or protein nearly identical to an allergen into a different food source, which can be assessed by specific serum testing. The potential that a newly expressed protein might become an allergen is evaluated based on resistance to digestion in pepsin and abundance in food fractions. If the modified plant is a common allergenic source (e.g. soybean), regulatory guidelines suggest testing for increases in the expression of endogenous allergens. Some regulators request evaluating endogenous allergens for rarely allergenic plants (e.g. maize and rice). Since allergic individuals must avoid foods containing their allergen (e.g. peanut, soybean, maize, or rice), the relevance of the tests is unclear. Furthermore, no acceptance criteria are established and little is known about the natural variation in allergen concentrations in these crops. Our results demonstrate a 15-fold difference in the major maize allergen, lipid transfer protein between nine varieties, and complex variation in IgE binding to various soybean varieties. We question the value of evaluating endogenous allergens in GM plants unless the intent of the modification was production of a hypoallergenic crop. © 2012 John Wiley & Sons A/S.

  15. Evaluation and validation of candidate endogenous control genes for real-time quantitative PCR studies of breast cancer

    Directory of Open Access Journals (Sweden)

    Miller Nicola

    2007-11-01

    Full Text Available Abstract Background Real-time quantitative PCR (RQ-PCR forms the basis of many breast cancer biomarker studies and novel prognostic assays, paving the way towards personalised cancer treatments. Normalisation of relative RQ-PCR data is required to control for non-biological variation introduced during sample preparation. Endogenous control (EC genes, used in this context, should ideally be expressed constitutively and uniformly across treatments in all test samples. Despite widespread recognition that the accuracy of the normalised data is largely dependent on the reliability of the EC, there are no reports of the systematic validation of genes commonly used for this purpose in the analysis of gene expression by RQ-PCR in primary breast cancer tissues. The aim of this study was to identify the most suitable endogenous control genes for RQ-PCR analysis of primary breast tissue from a panel of eleven candidates in current use. Oestrogen receptor alpha (ESR1 was used a target gene to compare the effect of choice of EC on the estimate of gene quantity. Results The expression and validity of candidate ECs (GAPDH, TFRC, ABL, PPIA, HPRT1, RPLP0, B2M, GUSB, MRPL19, PUM1 and PSMC4 was determined in 6 benign and 21 malignant primary breast cancer tissues. Gene expression data was analysed using two different statistical models. MRPL19 and PPIA were identified as the most stable and reliable EC genes, while GUSB, RPLP0 and ABL were least stable. There was a highly significant difference in variance between ECs. ESR1 expression was appreciably higher in malignant compared to benign tissues and there was a significant effect of EC on the magnitude of the error associated with the relative quantity of ESR1. Conclusion We have validated two endogenous control genes, MRPL19 and PPIA, for RQ-PCR analysis of gene expression in primary breast tissue. Of the genes in current use in this field, the above combination offers increased accuracy and resolution in the

  16. Identification of endogenous control genes for normalisation of real-time quantitative PCR data in colorectal cancer.

    LENUS (Irish Health Repository)

    Kheirelseid, Elrasheid A H

    2010-01-01

    BACKGROUND: Gene expression analysis has many applications in cancer diagnosis, prognosis and therapeutic care. Relative quantification is the most widely adopted approach whereby quantification of gene expression is normalised relative to an endogenously expressed control (EC) gene. Central to the reliable determination of gene expression is the choice of control gene. The purpose of this study was to evaluate a panel of candidate EC genes from which to identify the most stably expressed gene(s) to normalise RQ-PCR data derived from primary colorectal cancer tissue. RESULTS: The expression of thirteen candidate EC genes: B2M, HPRT, GAPDH, ACTB, PPIA, HCRT, SLC25A23, DTX3, APOC4, RTDR1, KRTAP12-3, CHRNB4 and MRPL19 were analysed in a cohort of 64 colorectal tumours and tumour associated normal specimens. CXCL12, FABP1, MUC2 and PDCD4 genes were chosen as target genes against which a comparison of the effect of each EC gene on gene expression could be determined. Data analysis using descriptive statistics, geNorm, NormFinder and qBasePlus indicated significant difference in variances between candidate EC genes. We determined that two genes were required for optimal normalisation and identified B2M and PPIA as the most stably expressed and reliable EC genes. CONCLUSION: This study identified that the combination of two EC genes (B2M and PPIA) more accurately normalised RQ-PCR data in colorectal tissue. Although these control genes might not be optimal for use in other cancer studies, the approach described herein could serve as a template for the identification of valid ECs in other cancer types.

  17. MED1 independent activation of endogenous target genes by PPARα

    DEFF Research Database (Denmark)

    Grøntved, Lars; Bugge, Anne K.; Roeder, Robert G.

    The mediator complex serves as a transcriptional co-activator complex by acting as a bridge between promoter-bound transcription factors and the preinitiation complex. Genetic and biochemical studies indicate that nuclear receptors recruit the mediator complex through direct interaction with the ......The mediator complex serves as a transcriptional co-activator complex by acting as a bridge between promoter-bound transcription factors and the preinitiation complex. Genetic and biochemical studies indicate that nuclear receptors recruit the mediator complex through direct interaction...... derived from TRAP220 KO mice. Interestingly, rescue experiments in confluent TRAP220 KO MEFs with different versions of MED1 indicate that the LXXLL motif is not necessary for PPARgamma mediated gene activation (Ge et al, MCB published online ahead of print 2007). By analogy, we show here that MED1...... is dispensable for PPARalpha transcriptional activity in proliferating but is necessary in confluent AML-12 cells and TRAP220 KO MEFs. Collectively this indicates that the PPARs might have adopted an alternative mediator recruitment mechanism that is dispensable of direct interaction with MED1 on endogenous...

  18. Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription.

    Science.gov (United States)

    Johnston, Stephen; Gallaher, Zachary; Czaja, Krzysztof

    2012-05-15

    Quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2(-∆∆Ct) normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxin selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference β-III tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

  19. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

    International Nuclear Information System (INIS)

    Sorensen, Brita Singers; Alsner, Jan; Overgaard, Jens; Horsman, Michael R.

    2007-01-01

    Background: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression. The current study is a detailed investigation using a range of pH values from 6.3 to 7.5 in two human cell lines to establish the pH dependency of hypoxia induced gene expression. Methods: Human tumour cell lines (uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma [FaDu DD ]) were used. Hypoxia was induced by gassing cells in airtight chambers with various oxygen concentrations (21%, 1%, 0.1%, 0.01% and 0%) for up to 24 h. The media were titrated to a range of pH values (7.5, 7.0, 6.7, 6.5 and 6.3). Gene expression was determined by real-time PCR. Results: In both SiHa and FaDu DD cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu DD cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu DD . Glut1 and LDH-A had a similar expression pattern to each other, with a maximum expression at 0.01% oxygen, in both cell lines. For these genes pH 6.5 and 6.3 changed the expression pattern in SiHa cells. OPN was up regulated at low oxygen in SiHa cells, but was not induced by hypoxia in FaDu DD cells. Conclusion: As tumour hypoxia occurs in a deprived microenvironment, other environmental factors, for example low pH, might interact with the effect of low oxygen concentration on gene expression. This study shows that pH in two cell lines has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers

  20. Imaging gene expression in gene therapy

    International Nuclear Information System (INIS)

    Wiebe, Leonard I.

    1997-01-01

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on 'suicide gene therapy' of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k + ) has been use for 'suicide' in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k + gene expression where the H S V-1 t k + gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([ 18 F]F H P G; [ 18 F]-A C V), and pyrimidine- ([ 123 / 131 I]I V R F U; [ 124 / 131I ]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [ 123 / 131I ]I V R F U imaging with the H S V-1 t k + reporter gene will be presented

  1. Imaging gene expression in gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Wiebe, Leonard I. [Alberta Univ., Edmonton (Canada). Noujaim Institute for Pharmaceutical Oncology Research

    1997-12-31

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on `suicide gene therapy` of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k{sup +}) has been use for `suicide` in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k{sup +} gene expression where the H S V-1 t k{sup +} gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([{sup 18} F]F H P G; [{sup 18} F]-A C V), and pyrimidine- ([{sup 123}/{sup 131} I]I V R F U; [{sup 124}/{sup 131I}]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [{sup 123}/{sup 131I}]I V R F U imaging with the H S V-1 t k{sup +} reporter gene will be presented

  2. Association of murine lupus and thymic full-length endogenous retroviral expression maps to a bone marrow stem cell

    International Nuclear Information System (INIS)

    Krieg, A.M.; Gourley, M.F.; Steinberg, A.D.

    1991-01-01

    Recent studies of thymic gene expression in murine lupus have demonstrated 8.4-kb (full-length size) modified polytropic (Mpmv) endogenous retroviral RNA. In contrast, normal control mouse strains do not produce detectable amounts of such RNA in their thymuses. Prior studies have attributed a defect in experimental tolerance in murine lupus to a bone marrow stem cell rather than to the thymic epithelium; in contrast, infectious retroviral expression has been associated with the thymic epithelium, rather than with the bone marrow stem cell. The present study was designed to determine whether the abnormal Mpmv expression associated with murine lupus mapped to thymic epithelium or to a marrow precursor. Lethally irradiated control and lupus-prone mice were reconstituted with T cell depleted bone marrow; one month later their thymuses were studied for endogenous retroviral RNA and protein expression. Recipients of bone marrow from nonautoimmune donors expressed neither 8.4-kb Mpmv RNA nor surface MCF gp70 in their thymuses. In contrast, recipients of bone marrow from autoimmune NZB or BXSB donors expressed thymic 8.4-kb Mpmv RNA and mink cell focus-forming gp70. These studies demonstrate that lupus-associated 8.4-kb Mpmv endogenous retroviral expression is determined by bone marrow stem cells

  3. Decreased expression of endogenous feline leukemia virus in cat lymphomas: a case control study.

    Science.gov (United States)

    Krunic, Milica; Ertl, Reinhard; Hagen, Benedikt; Sedlazeck, Fritz J; Hofmann-Lehmann, Regina; von Haeseler, Arndt; Klein, Dieter

    2015-04-10

    Cats infected with exogenous feline leukemia virus (exFeLV) have a higher chance of lymphoma development than uninfected cats. Furthermore, an increased exFeLV transcription has been detected in lymphomas compared to non-malignant tissues. The possible mechanisms of lymphoma development by exFeLV are insertional mutagenesis or persistent stimulation of host immune cells by viral antigens, bringing them at risk for malignant transformation. Vaccination of cats against exFeLV has in recent years decreased the overall infection rate in most countries. Nevertheless, an increasing number of lymphomas have been diagnosed among exFeLV-negative cats. Endogenous feline leukemia virus (enFeLV) is another retrovirus for which transcription has been observed in cat lymphomas. EnFeLV provirus elements are present in the germline of various cat species and share a high sequence similarity with exFeLV but, due to mutations, are incapable of producing infectious viral particles. However, recombination between exFeLV and enFeLV could produce infectious particles. We examined the FeLV expression in cats that have developed malignant lymphomas and discussed the possible mechanisms that could have induced malignant transformation. For expression analysis we used next-generation RNA-sequencing (RNA-Seq) and for validation reverse transcription quantitative PCR (RT-qPCR). First, we showed that there was no expression of exFeLV in all samples, which eliminates the possibility of recombination between exFeLV and enFeLV. Next, we analyzed the difference in expression of three enFeLV genes between control and lymphoma samples. Our analysis showed an average of 3.40-fold decreased viral expression for the three genes in lymphoma compared to control samples. The results were confirmed by RT-qPCR. There is a decreased expression of enFeLV genes in lymphomas versus control samples, which contradicts previous observations for the exFeLV. Our results suggest that a persistent stimulation of host

  4. Endogenous α-crystallin inhibits expression of caspase-3 induced by hypoxia in retinal neurons.

    Science.gov (United States)

    Ying, Xi; Peng, Yanli; Zhang, Jiaping; Wang, Xingli; Wu, Nan; Zeng, Yuxiao; Wang, Yi

    2014-08-28

    To investigate the expression of endogenous, hypoxic stress-induced α-crystallin and caspase-3 in rat retinal neurons in vitro. Retinal neurons were cultured from Long-Evans rats. The expression of endogenous α-crystallin was analyzed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Furthermore, hypoxic exposure was performed in cultured cells, and the expression of endogenous α-crystallin and caspase-3 was assayed by Western blotting. Positive α-crystallin staining was observed in cultured retinal neurons, and expression of endogenous α-crystallin mRNA peaked 3-5d after inoculation (Pendogenous, hypoxic stress-induced α-crystallin expression increased gradually, peaking 6h after hypoxia. The expression was more abundant compared to the control (Pendogenous α-crystallin in retinal neurons, especially over-expression induced by hypoxic stress, results in the down regulation of caspase-3. The data suggest that endogenous α-crystallin may act as an endogenous neuroprotective factor in retinal neurons. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Regulation of eucaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Brent, R.; Ptashne, M.S

    1989-05-23

    This patent describes a method of regulating the expression of a gene in a eucaryotic cell. The method consists of: providing in the eucaryotic cell, a peptide, derived from or substantially similar to a peptide of a procaryotic cell able to bind to DNA upstream from or within the gene, the amount of the peptide being sufficient to bind to the gene and thereby control expression of the gene.

  6. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  7. The essential role of endogenous ghrelin in growth hormone expression during zebrafish adenohypophysis development.

    Science.gov (United States)

    Li, Xi; He, Jiangyan; Hu, Wei; Yin, Zhan

    2009-06-01

    Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development.

  8. Differential Gene Expression and Aging

    Directory of Open Access Journals (Sweden)

    Laurent Seroude

    2002-01-01

    Full Text Available It has been established that an intricate program of gene expression controls progression through the different stages in development. The equally complex biological phenomenon known as aging is genetically determined and environmentally modulated. This review focuses on the genetic component of aging, with a special emphasis on differential gene expression. At least two genetic pathways regulating organism longevity act by modifying gene expression. Many genes are also subjected to age-dependent transcriptional regulation. Some age-related gene expression changes are prevented by caloric restriction, the most robust intervention that slows down the aging process. Manipulating the expression of some age-regulated genes can extend an organism's life span. Remarkably, the activity of many transcription regulatory elements is linked to physiological age as opposed to chronological age, indicating that orderly and tightly controlled regulatory pathways are active during aging.

  9. Endogenous Retrovirus ev21 Dose Not Recombine with ALV-J and Induces the Expression of ISGs in the Host.

    Science.gov (United States)

    Feng, Min; Tan, Yan; Dai, Manman; Li, Yuanfang; Xie, Tingting; Li, Hongmei; Shi, Meiqing; Zhang, Xiquan

    2016-01-01

    Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression. Endogenous viruses integrate into host genomes and can recombine with exogenous avian leukosis virus (ALV). In this study, we analyzed the interaction of endogenous retrovirus 21 ( ev21 ) with the ALV-J in late-feathering Chinese yellow chicken. Two ALV-J strains M180 and K243 were isolated from late-feathering and fast-feathering Chinese yellow chicken flocks, respectively. The env gene of the two strains showed 94.2-94.8% nucleotide identity with reference ALV-J strains. Compared with the env gene and the LTR of ev21 and M180, the nucleotide identity of LTR was 69.7% and env gene was 58.4%, respectively, especially the amino acid identity of env gene as low as 14.2%. Phylogenetic analysis of the nucleotide sequence of the env gene and the 3'LTR showed that M180 was closely related to ALV-J, and was located in a distinct group with ev21 in the phylogenetic tree. Using co-immunoprecipitation (co-IP), we next demonstrate that the envelope protein of ev21 does not interact with the M180 envelope protein. We further show that the envelope protein of ev21 cannot activate ALV-J LTR promoter activity using luciferase-reporter assays. qPCR and western blot analysis revealed that envelope protein of endogenous ev21 can facilitate the expression of PKR at 6h post ALV-J infection (hpi) and facilitate the expression of ISG12 and CH25H at 24 hpi. However, the expression of the env gene of M180 strain was not significantly at 6 and 24 hpi. We conclude that there is no evidence of recombination between endogenous retrovirus ev21 and ALV-J strain M180 in late-feathering Chinese yellow chicken, and envelope protein of ev21 can affect the expression of host ISGs, but appears not to influence the replication of ALV-J strain M180. This is the first report of interaction among the endogenous retrovirus ev21, ALV-J and the late-feathering chicken.

  10. Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle.

    Science.gov (United States)

    Bowles, D K; Maddali, K K; Ganjam, V K; Rubin, L J; Tharp, D L; Turk, J R; Heaps, C L

    2004-11-01

    Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current (ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7-8 mo; 35-45 kg) were obtained from the breeder. Males were left intact (IM, n=8), castrated (CM, n=8), or castrated with testosterone replacement (CMT, n=8; 10 mg/day Androgel). Females remained gonad intact (n=8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.

  11. Quantitative tissue-specific dynamics of in vivo GILZ mRNA expression and regulation by endogenous and exogenous glucocorticoids.

    Science.gov (United States)

    Ayyar, Vivaswath S; Almon, Richard R; Jusko, William J; DuBois, Debra C

    2015-06-01

    Glucocorticoids (GC) are steroid hormones, which regulate metabolism and immune function. Synthetic GCs, or corticosteroids (CS), have appreciable clinical utility via their ability to suppress inflammation in immune-mediated diseases like asthma and rheumatoid arthritis. Recent work has provided insight to novel GC-induced genes that mediate their anti-inflammatory effects, including glucocorticoid-induced leucine zipper (GILZ). Since GILZ comprises an important part of GC action, its regulation by both drug and hormone will influence CS therapy. In addition, GILZ expression is often employed as a biomarker of GC action, which requires judicious selection of sampling time. Understanding the in vivo regulation of GILZ mRNA expression over time will provide insight into both the physiological regulation of GILZ by endogenous GC and the dynamics of its enhancement by CS. A highly quantitative qRT-PCR assay was developed for measuring GILZ mRNA expression in tissues obtained from normal and CS-treated rats. This assay was applied to measure GILZ mRNA expression in eight tissues; to determine its endogenous regulation over time; and to characterize its dynamics in adipose tissue, muscle, and liver following treatment with CS. We demonstrate that GILZ mRNA is expressed in several tissues. GILZ mRNA expression in adipose tissue displayed a robust circadian rhythm that was entrained with the circadian oscillation of endogenous corticosterone; and is strongly enhanced by acute and chronic dosing. Single dosing also enhanced GILZ mRNA in muscle and liver, but the dynamics varied. In conclusion, GILZ is widely expressed in the rat and highly regulated by endogenous and exogenous GCs. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  12. Cerebellum-specific and age-dependent expression of an endogenous retrovirus with intact coding potential

    Directory of Open Access Journals (Sweden)

    Itoh Takayuki

    2011-10-01

    Full Text Available Abstract Background Endogenous retroviruses (ERVs, including murine leukemia virus (MuLV type-ERVs (MuLV-ERVs, are presumed to occupy ~10% of the mouse genome. In this study, following the identification of a full-length MuLV-ERV by in silico survey of the C57BL/6J mouse genome, its distribution in different mouse strains and expression characteristics were investigated. Results Application of a set of ERV mining protocols identified a MuLV-ERV locus with full coding potential on chromosome 8 (named ERVmch8. It appears that ERVmch8 shares the same genomic locus with a replication-incompetent MuLV-ERV, called Emv2; however, it was not confirmed due to a lack of relevant annotation and Emv2 sequence information. The ERVmch8 sequence was more prevalent in laboratory strains compared to wild-derived strains. Among 16 different tissues of ~12 week-old female C57BL/6J mice, brain homogenate was the only tissue with evident expression of ERVmch8. Further ERVmch8 expression analysis in six different brain compartments and four peripheral neuronal tissues of C57BL/6J mice revealed no significant expression except for the cerebellum in which the ERVmch8 locus' low methylation status was unique compared to the other brain compartments. The ERVmch8 locus was found to be surrounded by genes associated with neuronal development and/or inflammation. Interestingly, cerebellum-specific ERVmch8 expression was age-dependent with almost no expression at 2 weeks and a plateau at 6 weeks. Conclusions The ecotropic ERVmch8 locus on the C57BL/6J mouse genome was relatively undermethylated in the cerebellum, and its expression was cerebellum-specific and age-dependent.

  13. Identification of a chicken (Gallus gallus) endogenous reference gene (Actb) and its application in meat adulteration.

    Science.gov (United States)

    Xiang, Wenjin; Shang, Ying; Wang, Qin; Xu, Yuancong; Zhu, Pengyu; Huang, Kunlun; Xu, Wentao

    2017-11-01

    The genes commonly used to determine meat species are mainly mitochondrial, but the copy numbers of such genes are high, meaning they cannot be accurately quantified. In this paper, for the first time, the chromosomal gene Actb was selected as an endogenous reference gene for chicken species. It was assayed in four different chicken varieties and 16 other species using both qualitative and quantitative PCR. No amplification of the Actb gene was found in species other than chicken and no allelic variations were detected in chicken. Southern blot and digital-PCR confirmed the Actb gene was present as a single copy in the chicken genome. The quantitative detection limit was 10pg of DNA, which is equivalent to eight copies. All experiments indicated that the Actb gene is a useful endogenous reference gene for chicken, and provides a convenient and accurate approach for detection of chicken in feed and food. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Endogenous TasiRNAs mediate non-cell autonomous effects on gene regulation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rebecca Schwab

    Full Text Available BACKGROUND: Different classes of small RNAs (sRNAs refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs, which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs, and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells. PRINCIPAL FINDINGS: We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities. CONCLUSIONS/SIGNIFICANCE: Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.

  15. Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula.

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    Wun S Chao

    Full Text Available Quantitative real-time polymerase chain reaction (qRT-PCR is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4 were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100 identified from the analyses of leafy spurge microarray data; (2 3 orthologs of Arabidopsis "general purpose" traditional reference genes (GAPDH_1, GAPDH_2, and UBC; and (3 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the C(T values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ΔC(T method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes

  16. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226...

  17. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies....... For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce...

  18. Endogenous Antimicrobial Peptide Expression in Response to Bacterial Epidermal Colonization

    Directory of Open Access Journals (Sweden)

    Michael Brandwein

    2017-11-01

    Full Text Available Bacterial commensal colonization of human skin is vital for the training and maintenance of the skin’s innate and adaptive immune functions. In addition to its physical barrier against pathogen colonization, the skin expresses a variety of antimicrobial peptides (AMPs which are expressed constitutively and induced in response to pathogenic microbial stimuli. These AMPs are differentially effective against a suite of microbial skin colonizers, including both bacterial and fungal residents of the skin. We review the breadth of microorganism-induced cutaneous AMP expression studies and their complementary findings on the efficacy of skin AMPs against different bacterial and fungal species. We suggest further directions for skin AMP research based on emerging skin microbiome knowledge in an effort to advance our understanding of the nuanced host–microbe balance on human skin. Such advances should enable the scientific community to bridge the gap between descriptive disease-state AMP studies and experimental single-species in vitro studies, thereby enabling research endeavors that more closely mimic the natural skin environs.

  19. Expression patterns of endogenous avian retrovirus ALVE1 and its response to infection with exogenous avian tumour viruses.

    Science.gov (United States)

    Hu, Xuming; Zhu, Wenqi; Chen, Shihao; Liu, Yangyang; Sun, Zhen; Geng, Tuoyu; Song, Chengyi; Gao, Bo; Wang, Xiaoyan; Qin, Aijian; Cui, Hengmi

    2017-01-01

    Endogenous retroviruses (ERVs) are genomic elements that are present in a wide range of vertebrates and have been implicated in a variety of human diseases, including cancer. However, the characteristic expression patterns of ERVs, particularly in virus-induced tumours, is not fully clear. DNA methylation was analysed by bisulfite pyrosequencing, and gene expression was analysed by RT-qPCR. In this study, we first found that the endogenous avian retrovirus ALVE1 was highly expressed in some chicken tissues (including the heart, bursa, thymus, and spleen) at 2 days of age, but its expression was markedly decreased at 35 days of age. In contrast, the CpG methylation level of ALVE1 was significantly lower in heart and bursa at 2 days than at 35 days of age. Moreover, we found that the expression of ALVE1 was significantly inhibited in chicken embryo fibroblast cells (CEFs) and MSB1 cells infected with avian leukosis virus subgroup J (ALVJ) and reticuloendotheliosis virus (REV) at the early stages of infection. In contrast, the expression of the ALVE1 env gene was significantly induced in CEFs and MSB1 cells infected with Marek's disease virus (MDV). However, the methylation and expression levels of the ALVE1 long terminal repeat (LTR) did not show obvious alterations in response to viral infection. The present study revealed the expression patterns of ALVE1 in a variety of chicken organs and tissues and in chicken cells in response to avian tumour virus infection. These findings may be of significance for understanding the role and function of ERVs that are present in the host genome.

  20. Abnormal trafficking of endogenously expressed BMPR2 mutant allelic products in patients with heritable pulmonary arterial hypertension.

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    Andrea L Frump

    Full Text Available More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH. More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations. These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2 in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+ mice. The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+ mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

  1. Exogenous short-term silicon application regulates macro-nutrients, endogenous phytohormones, and protein expression in Oryza sativa L.

    Science.gov (United States)

    Jang, Soo-Won; Kim, Yoonha; Khan, Abdul Latif; Na, Chae-In; Lee, In-Jung

    2018-01-04

    Silicon (Si) has been known to regulate plant growth; however, the underlying mechanisms of short-term exogenous Si application on the regulation of calcium (Ca) and nitrogen (N), endogenous phytohormones, and expression of essential proteins have been little understood. Exogenous Si application significantly increased Si content as compared to the control. Among Si treatments, 1.0 mM Si application showed increased phosphorus content as compared to other Si treatments (0.5, 2.0, and 4.0 mM). However, Ca accumulation was significantly reduced (1.8- to 2.0-fold) at the third-leaf stage in the control, whereas all Si treatments exhibited a dose-dependent increase in Ca as determined by radioisotope 45 Ca analysis. Similarly, the radioisotope 15 N for nitrogen localization and uptake showed a varying but reduced response (ranging from 1.03-10.8%) to different Si concentrations as compared to 15 N application alone. Physiologically active endogenous gibberellin (GA 1 ) was also significantly higher with exogenous Si (1.0 mM) as compared to GA 20 and the control plants. A similar response was noted for endogenous jasmonic and salicylic acid synthesis in rice plants with Si application. Proteomic analysis revealed the activation of several essential proteins, such as Fe-S precursor protein, putative thioredoxin, Ser/Thr phosphatase, glucose-6-phosphate isomerase (G6P), and importin alpha-1b (Imp3), with Si application. Among the most-expressed proteins, confirmatory gene expression analysis for G6P and Imp3 showed a similar response to those of the Si treatments. In conclusion, the current results suggest that short-term exogenous Si can significantly regulate rice plant physiology by influencing Ca, N, endogenous phytohormones, and proteins, and that 1.0 mM Si application is more beneficial to plants than higher concentrations.

  2. Endogenous Opioid Function and Responses to Morphine: The Moderating Effects of Anger Expressiveness.

    Science.gov (United States)

    Burns, John W; Bruehl, Stephen; France, Christopher R; Schuster, Erik; Orlowska, Daria; Chont, Melissa; Gupta, Rajnish K; Buvanendran, Asokumar

    2017-08-01

    Long-term use of opioid analgesics may be ineffective or associated with significant negative side effects for some people. At present, there is no sound method of identifying optimal opioid candidates. Individuals with chronic low back pain (n = 89) and healthy control individuals (n = 102) underwent ischemic pain induction with placebo, opioid blockade (naloxone), and morphine in counterbalanced order. They completed the Spielberger Anger-Out subscale. Endogenous opioid function × Anger-out × Pain status (chronic pain, healthy control) interactions were tested for morphine responses to ischemic threshold, tolerance, and pain intensity (McGill Sensory and Affective subscales) and side effects. For individuals with chronic pain and healthy control participants, those with low endogenous opioid function and low anger-out scores exhibited the largest morphine analgesic responses, whereas those with high anger-out and low endogenous opioid function showed relatively weaker morphine analgesic responses. Further, individuals with chronic pain with low endogenous opioid function and low anger-out scores also reported the fewest negative effects to morphine, whereas those with low endogenous opioid function and high anger-out reported the most. Findings point toward individuals with chronic pain who may strike a favorable balance of good analgesia with few side effects, as well as those who have an unfavorable balance of poor analgesia and many side effects. We sought to identify optimal candidates for opioid pain management. Low back pain patients who express anger and also have deficient endogenous opioid function may be poor candidates for opioid therapy. In contrast, low back patients who tend not to express anger and who also have deficient endogenous opioid function may make optimal candidates for opioid therapy. Copyright © 2017 American Pain Society. Published by Elsevier Inc. All rights reserved.

  3. Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

    DEFF Research Database (Denmark)

    Domanski, Michal; Molloy, Kelly; Jiang, Hua

    2012-01-01

    An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous...

  4. The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum.

    Directory of Open Access Journals (Sweden)

    Dashuai Mu

    Full Text Available Ganoderma lucidum is one of the most important medicinal mushrooms; however, molecular genetics research on this species has been limited due to a lack of reliable reverse genetic tools. In this study, the endogenous orotidine 5'-monophosphate decarboxylase gene (URA3 was cloned as a silencing reporter, and four gene-silencing methods using hairpin, sense, antisense, and dual promoter constructs, were introduced into G. lucidum through a simple electroporation procedure. A comparison and evaluation of silencing efficiency demonstrated that all of the four methods differentially suppressed the expression of URA3. Our data unequivocally indicate that the dual promoter silencing vector yields the highest rate of URA3 silencing compared with other vectors (up to 81.9%. To highlight the advantages of the dual promoter system, we constructed a co-silencing system based on the dual promoter method and succeeded in co-silencing URA3 and laccase in G. lucidum. The reduction of the mRNA levels of the two genes were correlated. Thus, the screening efficiency for RNAi knockdown of multiple genes may be improved by the co-silencing of an endogenous reporter gene. The molecular tools developed in this study should facilitate the isolation of genes and the characterization of the functions of multiple genes in this pharmaceutically important species, and these tools should be highly useful for the study of other basidiomycetes.

  5. Efficient disruption of endogenous Bombyx gene by TAL effector nucleases

    Czech Academy of Sciences Publication Activity Database

    Sajwan, Suresh; Takasu, Y.; Tamura, T.; Uchino, K.; Sezutsu, H.; Žurovec, Michal

    2013-01-01

    Roč. 43, č. 1 (2013), s. 17-23 ISSN 0965-1748 R&D Projects: GA ČR GAP305/10/2406 Grant - others:Japan Society for the Promotion of Science(JP) 23580083 Institutional support: RVO:60077344 Keywords : gene targeting * ZFN * nonhomologous end joining Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.420, year: 2013 http://www.sciencedirect.com/science/article/pii/S0965174812001622#

  6. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  7. Human papillomavirus gene expression

    International Nuclear Information System (INIS)

    Chow, L.T.; Hirochika, H.; Nasseri, M.; Stoler, M.H.; Wolinsky, S.M.; Chin, M.T.; Hirochika, R.; Arvan, D.S.; Broker, T.R.

    1987-01-01

    To determine the role of tissue differentiation on expression of each of the papillomavirus mRNA species identified by electron microscopy, the authors prepared exon-specific RNA probes that could distinguish the alternatively spliced mRNA species. Radioactively labeled single-stranded RNA probes were generated from a dual promoter vector system and individually hybridized to adjacent serial sections of formalin-fixed, paraffin-embedded biopsies of condylomata. Autoradiography showed that each of the message species had a characteristic tissue distribution and relative abundance. The authors have characterized a portion of the regulatory network of the HPVs by showing that the E2 ORF encodes a trans-acting enhancer-stimulating protein, as it does in BPV-1 (Spalholz et al. 1985). The HPV-11 enhancer was mapped to a 150-bp tract near the 3' end of the URR. Portions of this region are duplicated in some aggressive strains of HPV-6 (Boshart and zur Hausen 1986; Rando et al. 1986). To test the possible biological relevance of these duplications, they cloned tandem arrays of the enhancer and demonstrated, using a chloramphenicol acetyltransferase (CAT) assay, that they led to dramatically increased transcription proportional to copy number. Using the CAT assays, the authors found that the E2 proteins of several papillomavirus types can cross-stimulate the enhancers of most other types. This suggests that prior infection of a tissue with one papillomavirus type may provide a helper effect for superinfection and might account fo the HPV-6/HPV-16 coinfections in condylomata that they have observed

  8. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

    Directory of Open Access Journals (Sweden)

    Lucie Kosinová

    Full Text Available The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3 in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information

  9. Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells

    International Nuclear Information System (INIS)

    Sharungbam, Geeta D; Schwager, Christian; Chiblak, Sara; Brons, Stephan; Hlatky, Lynn; Haberer, Thomas; Debus, Jürgen; Abdollahi, Amir

    2012-01-01

    Quantitative analysis of transcriptional regulation of genes is a prerequisite for a better understanding of the molecular mechanisms of action of different radiation qualities such as photon, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR (qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpreting these results it is critical to normalize the expression levels of the target genes by that of appropriately selected endogenous control genes (ECGs) or housekeeping genes. We sought to systematically investigate common ECG candidates for their stability after different radiation modalities in different human cell lines by qRT-PCR. We aimed to identify the most robust set of ECGs or housekeeping genes for transcriptional analysis in irradiation studies. We tested the expression stability of 32 ECGs in three human cancer cell lines. The epidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and the pancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbon ions. Expression Heat maps, clustering and statistic algorithms were employed using SUMO software package. The expression stability was evaluated by computing: mean, standard deviation, ANOVA, coefficient of variation and the stability measure (M) given by the geNorm algorithm. Expression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs (p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function of different radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, one of the most frequently used ECG, was differentially regulated as the function of different radiation qualities (p ≤ 0.01). A comprehensive search for the most stable ECGs using the geNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization. In contrast, 6 ECGs were required to properly normalize expression data in the more

  10. Spatiotemporal expression of endogenous opioid processing enzymes in mouse uterus at peri-implantation.

    Science.gov (United States)

    Wu, Weiwei; Kong, Shuangbo; Wang, Bingyan; Chen, Yongjie; Wang, Haibin

    2016-02-01

    Successful implantation requires intimate interactions between a competent blastocyst and a receptive uterus. We recently demonstrated that the aberrant activation of opioid signaling by exogenous ligands adversely affects preimplantation embryonic development and subsequent implantation in mice. However, the underlying machinery governing the dynamic homeostasis of the endogenous opioid system in the uterus during early pregnancy remains elusive. We now show that all three major endogenous opioid precursors are spatiotemporally expressed in the uterus during early pregnancy. Moreover, we observe the well-coordinated expression of the synthetic enzyme prohormone convertases 1/3 (PC1/3) at lower levels and of its inhibitor proprotein convertase subtilisin/kexin type 1 inhibitor (Pcsk1n) and the degrading enzyme membrane metallo-endopeptidase (MME) at higher levels in the receptive uterus. Both estrogen and progestin tend to reduce the uterine levels of opioid ligand precursors in the ovariectomized mouse model. This tight regulation of the endogenous opioid system by PC1/3, Pcsk1n and MME has been further confirmed in physiologically related pseudopregnancy and delayed implantation mouse models. The coordinated regulation of opioid precursor biosynthesis and metabolism helps to create appropriate opioid signaling ensuring uterine receptivity for implantation. Thus, endogenous uterine opioid levels are primarily determined by the coordinated expressions of PC1/3, Pcsk1n and MME under the influence of ovarian progestin and estrogen. Our findings raise an additional cautionary note regarding the effects of opioid abuse on early pregnancy events.

  11. Functional characterization of endogenous siRNA target genes in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Heikkinen Liisa

    2008-06-01

    Full Text Available Abstract Background Small interfering RNA (siRNA molecules mediate sequence specific silencing in RNA interference (RNAi, a gene regulatory phenomenon observed in almost all organisms. Large scale sequencing of small RNA libraries obtained from C. elegans has revealed that a broad spectrum of siRNAs is endogenously transcribed from genomic sequences. The biological role and molecular diversity of C. elegans endogenous siRNA (endo-siRNA molecules, nonetheless, remain poorly understood. In order to gain insight into their biological function, we annotated two large libraries of endo-siRNA sequences, identified their cognate targets, and performed gene ontology analysis to identify enriched functional categories. Results Systematic trends in categorization of target genes according to the specific length of siRNA sequences were observed: 18- to 22-mer siRNAs were associated with genes required for embryonic development; 23-mers were associated uniquely with post-embryonic development; 24–26-mers were associated with phosphorus metabolism or protein modification. Moreover, we observe that some argonaute related genes associate with siRNAs with multiple reads. Sequence frequency graphs suggest that different lengths of siRNAs share similarities in overall sequence structure: the 5' end begins with G, while the body predominates with U and C. Conclusion These results suggest that the lengths of endogenous siRNA molecules are consequential to their biological functions since the gene ontology categories for their cognate mRNA targets vary depending upon their lengths.

  12. Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

    Science.gov (United States)

    Reeves, R H; O'Brien, S J

    1984-01-01

    RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114. Images PMID:6090693

  13. Stably Expressed Genes Involved in Basic Cellular Functions.

    Directory of Open Access Journals (Sweden)

    Kejian Wang

    Full Text Available Stably Expressed Genes (SEGs whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age in both sexes of F344 rats (n = 4/group; 320 samples. Expression changes (calculated as the maximum expression / minimum expression for each gene of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination, RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics or exogenous agents (e.g., drugs, environmental factors may cause serious adverse effects.

  14. POD-1/Tcf21 overexpression reduces endogenous SF-1 and StAR expression in rat adrenal cells

    Directory of Open Access Journals (Sweden)

    M. M. França

    2015-12-01

    Full Text Available During gonad and adrenal development, the POD-1/capsulin/TCF21transcription factor negatively regulates SF-1/NR5A1expression, with higher SF-1 levels being associated with increased adrenal cell proliferation and tumorigenesis. In adrenocortical tumor cells, POD-1 binds to the SF-1 E-box promoter region, decreasing SF-1 expression. However, the modulation of SF-1 expression by POD-1 has not previously been described in normal adrenal cells. Here, we analyzed the basal expression of Pod-1 and Sf-1 in primary cultures of glomerulosa (G and fasciculata/reticularis (F/R cells isolated from male Sprague-Dawley rats, and investigated whether POD-1 overexpression modulates the expression of endogenous Sf-1 and its target genes in these cells. POD-1 overexpression, following the transfection of pCMVMycPod-1, significantly decreased the endogenous levels of Sf-1 mRNA and protein in F/R cells, but not in G cells, and also decreased the expression of the SF-1 target StAR in F/R cells. In G cells overexpressing POD-1, no modulation of the expression of SF-1 targets, StAR and CYP11B2, was observed. Our data showing that G and F/R cells respond differently to ectopic POD-1 expression emphasize the functional differences between the outer and inner zones of the adrenal cortex, and support the hypothesis that SF-1 is regulated by POD-1/Tcf21 in normal adrenocortical cells lacking the alterations in cellular physiology found in tumor cells.

  15. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    (ectoderm) specification with co-opted functions in notochord formation in chordates and left/right determination in ambulacrarians and vertebrates. The caudal ortholog, TtrCdx, is first expressed in the ectoderm of the gastrulating embryo in the posterior region of the blastopore. Its expression stays......The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa...... completion of larval development, which is marked by a three-lobed body with larval setae. Expression starts at gastrulation in two areas lateral to the blastopore and subsequently extends over the animal pole of the gastrula. With elongation of the gastrula, expression at the animal pole narrows to a small...

  16. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  17. Robust, synergistic regulation of human gene expression using TALE activators.

    Science.gov (United States)

    Maeder, Morgan L; Linder, Samantha J; Reyon, Deepak; Angstman, James F; Fu, Yanfang; Sander, Jeffry D; Joung, J Keith

    2013-03-01

    Artificial activators designed using transcription activator-like effector (TALE) technology have broad utility, but previous studies suggest that these monomeric proteins often exhibit low activities. Here we demonstrate that TALE activators can robustly function individually or in synergistic combinations to increase expression of endogenous human genes over wide dynamic ranges. These findings will encourage applications of TALE activators for research and therapy, and guide design of monomeric TALE-based fusion proteins.

  18. Endogenous Jaagsiekte Sheep Retrovirus RNA is expressed by different cell types in ovine foetus and placenta

    Directory of Open Access Journals (Sweden)

    E Sanna

    2010-01-01

    Full Text Available The endogenous retroviruses are inherited elements transmitted trough the germline of most animal species and their biological role is still controversial. Ovine Pulmonary Carcinoma (OPC represents a good model for studying the interactions of endogenous retroviruses with their exogenous counterparts. The type D exogenous retrovirus known as Jaagsiekte Sheep Retro-Virus (JSRV is necessary and sufficient to cause OPC in domestic and wild sheep, but both affected and unaffected animals host in their genome 15 to 20 copies of related endogenous retroviruses named endogenous JSRV (enJSRV. In this study we evaluated the expression of enJSRV gag sequences in ovine foetal and placental tissues. RNA in situ hybridisation was performed on tissue sections of thymi, lymph nodes and lungs from ovine foetuses and related placentas, taken at a late stage of development. Reverse transcriptase- in situ polymerase chain reactions were also carried out on placental samples to better define the involved cells. In foetal tissues, specific signals were observed in the thymus medulla, lymph nodes and, at a lesser extent, in foetal bronchiolar cells. In the placental tissues, positive areas were detected in various cell types in the sincythium-and cyto-trophoblast. These data demonstrate that en JSRV RNA is largely expressed in a broad spectrum of cells including tissues which are critical for the development of the immune system.

  19. Neighboring Genes Show Correlated Evolution in Gene Expression

    Science.gov (United States)

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  20. Interdependence of cell growth and gene expression: origins and consequences.

    Science.gov (United States)

    Scott, Matthew; Gunderson, Carl W; Mateescu, Eduard M; Zhang, Zhongge; Hwa, Terence

    2010-11-19

    In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of cell growth. A theory incorporating these constraints can accurately predict how cell proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated.

  1. Gene expression profile of pulpitis.

    Science.gov (United States)

    Galicia, J C; Henson, B R; Parker, J S; Khan, A A

    2016-06-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology.

  2. Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

    Directory of Open Access Journals (Sweden)

    Hackett Perry B

    2006-06-01

    Full Text Available Abstract Background Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Results Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA system that is capable of activating the expression of genes under control of a Tet response element (TRE promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Conclusion Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene

  3. Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression.

    Science.gov (United States)

    Layé, S; Gheusi, G; Cremona, S; Combe, C; Kelley, K; Dantzer, R; Parnet, P

    2000-07-01

    The present study was designed to determine the role of endogenous brain interleukin (IL)-1 in the anorexic response to lipopolysaccharide (LPS). Intraperitoneal administration of LPS (5-10 microgram/mouse) induced a dramatic, but transient, decrease in food intake, associated with an enhanced expression of proinflammatory cytokine mRNA (IL-1beta, IL-6, and tumor necrosis factor-alpha) in the hypothalamus. This dose of LPS also increased plasma levels of IL-1beta. Intracerebroventricular pretreatment with IL-1 receptor antagonist (4 microgram/mouse) attenuated LPS-induced depression of food intake and totally blocked the LPS-induced enhanced expression of proinflammatory cytokine mRNA measured in the hypothalamus 1 h after treatment. In contrast, LPS-induced increases in plasma levels of IL-1beta were not altered. These findings indicate that endogenous brain IL-1 plays a pivotal role in the development of the hypothalamic cytokine response to a systemic inflammatory stimulus.

  4. A comparative study of three different gene expression analysis methods.

    Science.gov (United States)

    Choe, Jae Young; Han, Hyung Soo; Lee, Seon Duk; Lee, Hanna; Lee, Dong Eun; Ahn, Jae Yun; Ryoo, Hyun Wook; Seo, Kang Suk; Kim, Jong Kun

    2017-12-04

    TNF-α regulates immune cells and acts as an endogenous pyrogen. Reverse transcription polymerase chain reaction (RT-PCR) is one of the most commonly used methods for gene expression analysis. Among the alternatives to PCR, loop-mediated isothermal amplification (LAMP) shows good potential in terms of specificity and sensitivity. However, few studies have compared RT-PCR and LAMP for human gene expression analysis. Therefore, in the present study, we compared one-step RT-PCR, two-step RT-LAMP and one-step RT-LAMP for human gene expression analysis. We compared three gene expression analysis methods using the human TNF-α gene as a biomarker from peripheral blood cells. Total RNA from the three selected febrile patients were subjected to the three different methods of gene expression analysis. In the comparison of three gene expression analysis methods, the detection limit of both one-step RT-PCR and one-step RT-LAMP were the same, while that of two-step RT-LAMP was inferior. One-step RT-LAMP takes less time, and the experimental result is easy to determine. One-step RT-LAMP is a potentially useful and complementary tool that is fast and reasonably sensitive. In addition, one-step RT-LAMP could be useful in environments lacking specialized equipment or expertise.

  5. An active role for endogenous beta-1,3-glucanase genes in transgene-mediated co-suppression in tobacco.

    Science.gov (United States)

    Sanders, Matthew; Maddelein, Wendy; Depicker, Anna; Van Montagu, Marc; Cornelissen, Marc; Jacobs, John

    2002-11-01

    Post-transcriptional gene silencing (PTGS) is characterized by the accumulation of short interfering RNAs that are proposed to mediate sequence-specific degradation of cognate and secondary target mRNAs. In plants, it is unclear to what extent endogenous genes contribute to this process. Here, we address the role of the endogenous target genes in transgene-mediated PTGS of beta-1,3-glucanases in tobacco. We found that mRNA sequences of the endogenous glucanase glb gene with varying degrees of homology to the Nicotiana plumbaginifolia gn1 transgene are targeted by the silencing machinery, although less efficiently than corresponding transgene regions. Importantly, we show that endogene-specific nucleotides in the glb sequence provide specificity to the silencing process. Consistent with this finding, small sense and antisense 21- to 23-nucleotide RNAs homologous to the endogenous glb gene were detected. Combined, these data demonstrate that a co-suppressed endogenous glucan ase gene is involved in signal amplification and selection of homologous targets, and show that endogenous genes can actively participate in PTGS in plants. The findings are introduced as a further sophistication of the post-transciptional silencing model.

  6. Endogenous oxytocin levels are associated with the perception of emotion in dynamic body expressions in schizophrenia.

    Science.gov (United States)

    Strauss, Gregory P; Keller, William R; Koenig, James I; Sullivan, Sara K; Gold, James M; Buchanan, Robert W

    2015-03-01

    Lower endogenous oxytocin levels have been associated with impaired social cognition in schizophrenia, particularly facial affect identification. Little is known about the relationship between oxytocin and other forms of emotion perception. In the current study, 41 individuals with schizophrenia (SZ) and 22 demographically matched healthy controls (CN) completed a forced-choice affective body expression classification task. Stimuli included dynamic videos of male and female actors portraying 4 discrete emotions: happiness, sadness, anger, and neutral. Plasma oxytocin levels were determined via radioimmunoassay. Results indicated that SZ had significantly higher plasma oxytocin concentrations than CN. SZ were also less accurate at identifying expressions of happiness and sadness; however, there were no group differences for anger or neutral stimuli. A group×sex interaction was also present, such that female CN were more accurate than male CN, whereas male SZ were more accurate than female SZ. Higher endogenous oxytocin levels were associated with better total recognition in both SZ and CN; this association was specific to females in SZ. Findings indicate that sex plays an important role in identifying emotional expressions in body gestures in SZ, and that individual differences in endogenous oxytocin predict emotion perception accuracy. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR

    Directory of Open Access Journals (Sweden)

    Kristiansen Glen

    2007-06-01

    Full Text Available Abstract Background Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in various studies were examined with regard to their usefulness as reference genes. Results The expression of the potential reference genes was examined in matched malignant and non-malignant tissue specimens from 25 patients with clear cell renal cell carcinoma. Quality assessment of isolated RNA performed with a 2100 Agilent Bioanalyzer showed a mean RNA integrity number of 8.7 for all samples. The between-run variations related to the crossing points of PCR reactions of a control material ranged from 0.17% to 0.38%. The expression of all genes did not depend on age, sex, and tumour stage. Except the genes TATA box binding protein (TBP and peptidylprolyl isomerase A (PPIA, all genes showed significant differences in expression between malignant and non-malignant pairs. The expression stability of the candidate reference genes was additionally controlled using the software programs geNorm and NormFinder. TBP and PPIA were validated as suitable reference genes by normalizing the target gene ADAM9 using these two most stably expressed genes in comparison with up- and down-regulated housekeeping genes of the panel. Conclusion Our study demonstrated the suitability of the two housekeeping genes PPIA and TBP as endogenous reference genes when comparing malignant tissue samples with adjacent normal tissue samples from clear cell renal cell carcinoma. Both genes are recommended as reference genes for relative gene quantification in gene profiling studies either as single gene or preferably in combination.

  8. Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events

    Directory of Open Access Journals (Sweden)

    Tigst Demeke

    2018-05-01

    Full Text Available Droplet digital PCR (ddPCR has been used for absolute quantification of genetically engineered (GE events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences (HMG-I/Y, FatA(A, CruA and Ccf for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A, reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes. Keywords: Canola, Digital PCR, DNA extraction, GMO, Reference genes

  9. Expression of endogenous proteins in maize hybrids in a multi-location field trial in India.

    Science.gov (United States)

    Gutha, Linga R; Purushottam, Divakar; Veeramachaneni, Aruna; Tigulla, Sarita; Kodappully, Vikas; Enjala, Chandana; Rajput, Hitendrasinh; Anderson, Jennifer; Hong, Bonnie; Schmidt, Jean; Bagga, Shveta

    2018-05-17

    Genetically modified (GM) crops undergo large scale multi-location field trials to characterize agronomics, composition, and the concentration of newly expressed protein(s) [herein referred to as transgenic protein(s)]. The concentration of transgenic proteins in different plant tissues and across the developmental stages of the plant is considered in the safety assessment of GM crops. Reference or housekeeping proteins are expected to maintain a relatively stable expression pattern in healthy plants given their role in cellular functions. Understanding the effects of genotype, growth stage and location on the concentration of endogenous housekeeping proteins may provide insight into the contribution these factors could have on transgenic protein concentrations in GM crops. The concentrations of three endogenous proteins (actin, elongation factor 1-alpha, and glyceraldehyde 3-phosphate dehydrogenase) were measured in several different maize hybrids grown across multiple field locations over 2 years. Leaf samples were collected from healthy plants at three developmental stages across the growing seasons, and protein concentrations were quantified by indirect enzyme-linked immunosorbent assay (ELISA) for each protein. In general, the concentrations of these three endogenous proteins were relatively consistent across hybrid backgrounds, when compared within one growth stage and location (2-26%CV), whereas the concentrations of proteins in the same hybrid and growth stage across different locations were more variable (12-64%CV). In general, the protein concentrations in 2013 and 2014 show similar trends in variability. Some degree of variability in protein concentrations should be expected for both transgenic and endogenous plant-expressed proteins. In the case of GM crops, the potential variation in protein concentrations due to location effects is captured in the current model of multi-location field testing.

  10. Using gene expression noise to understand gene regulation

    NARCIS (Netherlands)

    Munsky, B.; Neuert, G.; van Oudenaarden, A.

    2012-01-01

    Phenotypic variation is ubiquitous in biology and is often traceable to underlying genetic and environmental variation. However, even genetically identical cells in identical environments display variable phenotypes. Stochastic gene expression, or gene expression "noise," has been suggested as a

  11. Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events.

    Science.gov (United States)

    Demeke, Tigst; Eng, Monika

    2018-05-01

    Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences ( HMG-I/Y , FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.

  12. Novel MeCP2 isoform-specific antibody reveals the endogenous MeCP2E1 expression in murine brain, primary neurons and astrocytes.

    Directory of Open Access Journals (Sweden)

    Robby M Zachariah

    Full Text Available Rett Syndrome (RTT is a severe neurological disorder in young females, and is caused by mutations in the X-linked MECP2 gene. MECP2/Mecp2 gene encodes for two protein isoforms; MeCP2E1 and MeCP2E2 that are identical except for the N-terminus region of the protein. In brain, MECP2E1 transcripts are 10X higher, and MeCP2E1 is suggested to be the relevant isoform for RTT. However, due to the unavailability of MeCP2 isoform-specific antibodies, the endogenous expression pattern of MeCP2E1 is unknown. To gain insight into the expression of MeCP2E1 in brain, we have developed an anti-MeCP2E1 antibody and validated its specificity in cells exogenously expressing individual MeCP2 isoforms. This antibody does not show any cross-reactivity with MeCP2E2 and detects endogenous MeCP2E1 in mice brain, with no signal in Mecp2(tm1.1Bird y/- null mice. Additionally, we show the endogenous MeCP2E1 expression throughout different brain regions in adult mice, and demonstrate its highest expression in the brain cortex. Our results also indicate that MeCP2E1 is highly expressed in primary neurons, as compared to primary astrocytes. This is the first report of the endogenous MeCP2E1 expression at the protein levels, providing novel avenues for understanding different aspects of MeCP2 function.

  13. A constructive approach to gene expression dynamics

    International Nuclear Information System (INIS)

    Ochiai, T.; Nacher, J.C.; Akutsu, T.

    2004-01-01

    Recently, experiments on mRNA abundance (gene expression) have revealed that gene expression shows a stationary organization described by a scale-free distribution. Here we propose a constructive approach to gene expression dynamics which restores the scale-free exponent and describes the intermediate state dynamics. This approach requires only one assumption: Markov property

  14. Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

    DEFF Research Database (Denmark)

    Khan, Aly A; Betel, Doron; Miller, Martin L

    2009-01-01

    Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect--that is, competiti...

  15. Endogenous CNS Expression of Neurotensin and Neurotensin Receptors Is Altered during the Postpartum Period in Outbred Mice

    Science.gov (United States)

    Driessen, Terri M.; Zhao, Changjiu; Whittlinger, Anna; Williams, Horecia; Gammie, Stephen C.

    2014-01-01

    Neurotensin (NT) is a neuropeptide identical in mice and humans that is produced and released in many CNS regions associated with maternal behavior. NT has been linked to aspects of maternal care and previous studies have indirectly suggested that endogenous NT signaling is altered in the postpartum period. In the present study, we directly examine whether NT and its receptors exhibit altered gene expression in maternal relative to virgin outbred mice using real time quantitative PCR (qPCR) across multiple brain regions. We also examine NT protein levels using anti-NT antibodies and immunohistochemistry in specific brain regions. In the medial preoptic area (MPOA), which is critical for maternal behaviors, mRNA of NT and NT receptor 3 (Sort1) were significantly up-regulated in postpartum mice compared to virgins. NT mRNA was also elevated in postpartum females in the bed nucleus of the stria terminalis dorsal. However, in the lateral septum, NT mRNA was down-regulated in postpartum females. In the paraventricular nucleus of the hypothalamus (PVN), Ntsr1 expression was down-regulated in postpartum females. Neurotensin receptor 2 (Ntsr2) expression was not altered in any brain region tested. In terms of protein expression, NT immunohistochemistry results indicated that NT labeling was elevated in the postpartum brain in the MPOA, lateral hypothalamus, and two subregions of PVN. Together, these findings indicate that endogenous changes occur in NT and its receptors across multiple brain regions, and these likely support the emergence of some maternal behaviors. PMID:24416154

  16. Modulation of gene expression made easy

    DEFF Research Database (Denmark)

    Solem, Christian; Jensen, Peter Ruhdal

    2002-01-01

    A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected...

  17. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can......The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...

  18. Expression of human gamma-globin genes in human erythroleukemia (K562) cells.

    Science.gov (United States)

    Donovan-Peluso, M; Acuto, S; Swanson, M; Dobkin, C; Bank, A

    1987-12-15

    K562 cells express embryonic (epsilon) and fetal (gamma) globins and hemoglobins but not adult (beta) globin. To define the cis acting regulatory elements involved in the discrimination between gamma and beta genes, we have constructed chimeric genes composed of portions of gamma and beta and evaluated their expression in stable K562 transfectants. A gamma beta fusion gene containing gamma 5' sequences to the EcoRI site in exon 3 and beta sequences 3' is expressed at 10-40% that of the endogenous gamma level. In 50% of the lines, this fusion gene appropriately increases its expression in response to hemin, an inducer of endogenous globin gene expression in K562 cells. In contrast, a beta gamma fusion gene, containing beta sequences 5' to the EcoRI site in exon 3 and gamma sequences 3', is neither expressed nor correctly initiated. A beta gene containing gamma-intervening sequence (IVS) 2 accumulates an mRNA transcript when analyzed with a 3' beta probe. However, no correctly initiated beta mRNA is observed. A gamma gene with beta-IVS 2 is only inducible in one of six expressing clones. All the results are consistent with the presence of stage-specific trans acting factors in K562 cells that stimulate expression of gamma genes and suggest a significant role for gamma-IVS 2 in gamma gene expression.

  19. An endogenous reference gene of common and durum wheat for detection of genetically modified wheat.

    Science.gov (United States)

    Imai, Shinjiro; Tanaka, Keiko; Nishitsuji, Yasuyuki; Kikuchi, Yosuke; Matsuoka, Yasuyuki; Arami, Shin-Ichiro; Sato, Megumi; Haraguchi, Hiroyuki; Kurimoto, Youichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

    2012-01-01

    To develop a method for detecting GM wheat that may be marketed in the near future, we evaluated the proline-rich protein (PRP) gene as an endogenous reference gene of common wheat (Triticum aestivum L.) and durum wheat (Triticum durum L.). Real-time PCR analysis showed that only DNA of wheat was amplified and no amplification product was observed for phylogenetically related cereals, indicating that the PRP detection system is specific to wheat. The intensities of the amplification products and Ct values among all wheat samples used in this study were very similar, with no nonspecific or additional amplification, indicating that the PRP detection system has high sequence stability. The limit of detection was estimated at 5 haploid genome copies. The PRP region was demonstrated to be present as a single or double copy in the common wheat haploid genome. Furthermore, the PRP detection system showed a highly linear relationship between Ct values and the amount of plasmid DNA, indicating that an appropriate calibration curve could be constructed for quantitative detection of GM wheat. All these results indicate that the PRP gene is a suitable endogenous reference gene for PCR-based detection of GM wheat.

  20. Investigation of Endogenous Retrovirus Sequences in the Neighborhood of Genes Up-regulated in a Neuroblastoma Model after Treatment with Hypoxia-Mimetic Cobalt Chloride.

    Science.gov (United States)

    Brütting, Christine; Narasimhan, Harini; Hoffmann, Frank; Kornhuber, Malte E; Staege, Martin S; Emmer, Alexander

    2018-01-01

    Human endogenous retroviruses (ERVs) have been found to be associated with different diseases, e.g., multiple sclerosis (MS). Most human ERVs integrated in our genome are not competent to replicate and these sequences are presumably silent. However, transcription of human ERVs can be reactivated, e.g., by hypoxia. Interestingly, MS has been linked to hypoxia since decades. As some patterns of demyelination are similar to white matter ischemia, hypoxic damage is discussed. Therefore, we are interested in the association between hypoxia and ERVs. As a model, we used human SH-SY5Y neuroblastoma cells after treatment with the hypoxia-mimetic cobalt chloride and analyzed differences in the gene expression profiles in comparison to untreated cells. The vicinity of up-regulated genes was scanned for endogenous retrovirus-derived sequences. Five genes were found to be strongly up-regulated in SH-SY5Y cells after treatment with cobalt chloride: clusterin, glutathione peroxidase 3, insulin-like growth factor 2, solute carrier family 7 member 11, and neural precursor cell expressed developmentally down-regulated protein 9. In the vicinity of these genes we identified large (>1,000 bp) open reading frames (ORFs). Most of these ORFs showed only low similarities to proteins from retro-transcribing viruses. However, we found very high similarity between retrovirus envelope sequences and a sequence in the vicinity of neural precursor cell expressed developmentally down-regulated protein 9. This sequence encodes the human endogenous retrovirus group FRD member 1, the encoded protein product is called syncytin 2. Transfection of syncytin 2 into the well-characterized Ewing sarcoma cell line A673 was not able to modulate the low immunostimulatory activity of this cell line. Future research is needed to determine whether the identified genes and the human endogenous retrovirus group FRD member 1 might play a role in the etiology of MS.

  1. Gene expression of manganese superoxide dismutase in human glioma cells

    Directory of Open Access Journals (Sweden)

    Novi S. Hardiany

    2010-02-01

    Full Text Available Aim This study analyze the MnSOD gene expression as endogenous antioxidant in human glioma cells compared with leucocyte cells as control.Methods MnSOD gene expression of 20 glioma patients was analyzed by measuring the relative expression of mRNA and enzyme activity of MnSOD in brain and leucocyte cells. The relative expression of mRNA MnSOD was determined by using quantitative Real Time RT-PCR and the enzyme activity of MnSOD using biochemical kit assay (xantine oxidase inhibition. Statistic analysis for mRNA and enzyme activity of MnSOD was performed using Kruskal Wallis test.Results mRNA of MnSOD in glioma cells of 70% sample was 0.015–0.627 lower, 10% was 1.002-1.059 and 20% was 1.409-6.915 higher than in leucocyte cells. Also the specific activity of MnSOD enzyme in glioma cells of 80% sample showed 0,064-0,506 lower and 20% sample was 1.249-2.718 higher than in leucocyte cells.Conclusion MnSOD gene expression in human glioma cells are significantly lower than its expression in leucocytes cells. (Med J Indones 2010; 19:21-5Keywords : MnSOD, glioma, gene expression

  2. Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.

    Science.gov (United States)

    Mehta, Rohini; Birerdinc, Aybike; Hossain, Noreen; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair; Baranova, Ancha

    2010-05-21

    Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

  3. Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

    Directory of Open Access Journals (Sweden)

    Afendy Arian

    2010-05-01

    Full Text Available Abstract Background Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Results Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. Conclusions We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

  4. Adaptive Evolution of Gene Expression in Drosophila

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    Armita Nourmohammad

    2017-08-01

    Full Text Available Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis.

  5. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  6. Over-expressed maltose transporters in laboratory and lager yeasts: localization and competition with endogenous transporters.

    Science.gov (United States)

    Vidgren, Virve; Londesborough, John

    2018-05-31

    Plain and fluorescently tagged versions of Agt1, Mtt1 and Malx1 maltose transporters were over-expressed in two laboratory yeasts and one lager yeast. The plain and tagged versions of each transporter supported similar transport activities, indicating that they are similarly trafficked and have similar catalytic activities. When they were expressed under the control of the strong constitutive PGK1 promoter only minor proportions of the fluorescent transporters were associated with the plasma membrane, the rest being found in intracellular structures. Transport activity of each tagged transporter in each host was roughly proportional to the plasma membrane-associated fluorescence. All three transporters were subject to glucose-triggered inactivation when the medium glucose concentration was abruptly raised. Results also suggest competition between endogenous and over-expressed transporters for access to the plasma membrane. This article is protected by copyright. All rights reserved.

  7. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    OpenAIRE

    Ezer, Daphne; Moignard, Victoria; G?ttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete ...

  8. The evolution of gene expression in primates

    OpenAIRE

    Tashakkori Ghanbarian, Avazeh

    2015-01-01

    The evolution of a gene’s expression profile is commonly assumed to be independent of its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between expression of neighboring genes in extant taxa. Indeed, in all eukaryotic genomes, genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their e...

  9. Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Nishimura, Kohji [Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Jisaka, Mitsuo; Nagaya, Tsutomu [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan); Shono, Fumiaki [Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima 770-8514 (Japan); Yokota, Kazushige, E-mail: yokotaka@life.shimane-u.ac.jp [Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504 (Japan)

    2012-02-15

    Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD{sub 2} and its non-enzymatic dehydration products, PGJ{sub 2} series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD{sub 2} and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD{sub 2} from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE{sub 2} remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize {Delta}{sup 12}-PGJ{sub 2} increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists including troglitazone and {Delta}{sup 12}-PGJ{sub 2}. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPAR{gamma} signaling pathway without the contribution of endogenous pro-adipogenic prostanoids

  10. Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

    Science.gov (United States)

    Guelke, Eileen; Bucan, Vesna; Liebsch, Christina; Lazaridis, Andrea; Radtke, Christine; Vogt, Peter M; Reimers, Kerstin

    2015-04-10

    For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Degradation of endogenous and exogenous genes of genetically modified rice with Cry1Ab during food processing.

    Science.gov (United States)

    Zhang, Wei; Xing, Fuguo; Selvaraj, Jonathan Nimal; Liu, Yang

    2014-05-01

    In order to assess the degradation of endogenous and exogenous genes during food processing, genetically modified rice with Cry1Ab was used as raw material to produce 4 processed foods: steamed rice, rice noodles, rice crackers, and sweet rice wine. The results showed various processing procedures caused different degrees of degradation of both endogenous and exogenous genes. During the processing of steamed rice and rice noodles, the procedures were so mild that only genes larger than 1500 bp were degraded, and no degradation of NOS terminator and Hpt gene was detected. For rice crackers, frying was the most severe procedure, followed by microwaving, baking, boiling, 1st drying, and 2nd drying. For sweet rice wine, fermentation had more impact on degradation of genes than the other processing procedures. All procedures in this study did not lead to degradation of genes to below 200 bp, except for NOS terminator. In the case of stability of the genes studied during processing of rice crackers and sweet rice wine, SPS gene was the most, followed by the Cry1Ab gene, Hpt gene, Pubi promoter, and NOS terminator. In our study, we gained some information about the degradation of endogenous and exogenous genes during 4 foods processing, compared the different stabilities between endogenous and exogenous genes, and analyzed different effects of procedure on degradation of genes. In addition, the fragments of endogenous and exogenous genes about 200 bp could be detected in final products, except NOS terminator. As a result, we provided some base information about risk assessment of genetically modified (GM) food and appropriate length of fragment to detect GM component in processed foods. © 2014 Institute of Food Technologists®

  12. Combination of the Endogenous lhcsr1 Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Manuel Hiss

    2017-10-01

    Full Text Available The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.

  13. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    Science.gov (United States)

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

  14. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    Directory of Open Access Journals (Sweden)

    Huali Huang

    Full Text Available Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L. DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

  15. Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection

    Science.gov (United States)

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

  16. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo.

    Directory of Open Access Journals (Sweden)

    Sebastian Frese

    Full Text Available Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human

  17. A transgenic approach to study argininosuccinate synthetase gene expression

    Science.gov (United States)

    2014-01-01

    Background Argininosuccinate synthetase (ASS) participates in urea, nitric oxide and arginine production. Besides transcriptional regulation, a post-transcriptional regulation affecting nuclear precursor RNA stability has been reported. To study whether such post-transcriptional regulation underlines particular temporal and spatial ASS expression, and to investigate how human ASS gene behaves in a mouse background, a transgenic mouse system using a modified bacterial artificial chromosome carrying the human ASS gene tagged with EGFP was employed. Results Two lines of ASS-EGFP transgenic mice were generated: one with EGFP under transcriptional control similar to that of the endogenous ASS gene, another with EGFP under both transcriptional and post-transcriptional regulation as that of the endogenous ASS mRNA. EGFP expression in the liver, the organ for urea production, and in the intestine and kidney that are responsible for arginine biosynthesis, was examined. Organs taken from embryos E14.5 stage to young adult were examined under a fluorescence microscope either directly or after cryosectioning. The levels of EGFP and endogenous mouse Ass mRNAs were also quantified by S1 nuclease mapping. EGFP fluorescence and EGFP mRNA levels in both the liver and kidney were found to increase progressively from embryonic stage toward birth. In contrast, EGFP expression in the intestine was higher in neonates and started to decline at about 3 weeks after birth. Comparison between the EGFP profiles of the two transgenic lines indicated the developmental and tissue-specific regulation was mainly controlled at the transcriptional level. The ASS transgene was of human origin. EGFP expression in the liver followed essentially the mouse Ass pattern as evidenced by zonation distribution of fluorescence and the level of EGFP mRNA at birth. However, in the small intestine, Ass mRNA level declined sharply at 3 week of age, and yet substantial EGFP mRNA was still detectable at this stage

  18. Expression of endogenous ALV antigens and susceptibility to subgroup E ALV in three strains of chickens (Endogenous avian C. type virus)

    International Nuclear Information System (INIS)

    Robinson, H.L.; Lamoreux, W.F.

    1976-01-01

    Cells from three strains of Kimber Farms chickens, K16, K18, and K28, have been characterized for the expression of endogenous avian C-type virus (ALV) antigens and for susceptibility to subgroup E ALV. In K16 the coordinate dominant expression of chick helper factor (chf ), the type specific antigen of endogenous subgroup E ALV, and the group specific (gs) antigen of ALV was observed. The expression of chf and gs antigen in K16 chf + gs + cells was similar to that observed in SPAFAS and H and N gs + cells. In K18 chf but not gs antigen was expressed. The expression of chf in K18 chf + gs + cells was distinct from that observed for the chf + gs + pedigrees but similar to that found in SPAFAS chf + gs - helper extremely high (h/sub E/) cells. Cells from K16 and K18 chickens were uniformly resistant to subgroup E ALV. In cells from K28 chickens, susceptibility to subgroup B virus correlated with susceptibility to subgroup E virus. The efficiency of plating of subgroup E virus on susceptible K28 cells with 10 3 --10 4 -fold lower on chf + cells than on chf - cells

  19. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy [Davis, CA; Bachkirova, Elena [Davis, CA; Rey, Michael [Davis, CA

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  20. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  1. FOLATE CYCLE GENE POLYMORPHISM AND ENDOGENOUS PEPTIDES IN CHILDREN WITH COW’S MILK PROTEIN ALLERGY

    Directory of Open Access Journals (Sweden)

    T. A. Shumatova

    2016-01-01

    Full Text Available Folate cycle gene polymorphisms and the levels of endogenous antimicrobial peptides and proteins in the blood and coprofiltrates were studied in 45 children aged 3 to 12 months with cow’s milk protein allergy. The polymorphic variants of the MTHFR, MTRR, and MTR genes were shown to be considered as a risk factor for the development of allergy. There was a significant increase in the levels of zonulin, β-defensin 2, transthyretin, and eosinophil cationic protein in the coprofiltrates and in those of eotaxin, fatty acidbinding proteins, and membrane permeability-increasing protein in the serum (p<0.05. The finding can improve the diagnosis of the disease for a predictive purpose for the evaluation of the efficiency of performed therapy.

  2. cis sequence effects on gene expression

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    Jacobs Kevin

    2007-08-01

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

  3. Visualization of the Dynamics of Gene Expression in the Living Mouse

    Directory of Open Access Journals (Sweden)

    Amy Ryan

    2004-01-01

    Full Text Available Reporter genes can monitor the status and activity of recombinant genomes in a diverse array of organisms, from bacteria and yeast to plants and animals. We have combined luciferase reporter genes with a conditional gene expression system based on regulatory elements from the lac Operon of Escherichia coli to visualize the dynamics of gene expression in realtime in the living mouse. Using this technology, we have determined the rate of gene induction and repression, the level of target gene activity in response to different doses of inducer, and the schedule of induction during early embryogenesis of both the endogenous and the experimentally manipulated programs of mammalian gene expression associated with the HD/Hdh locus. The combination of in vivo imaging and lac regulation is a powerful tool for generating conditional transgenic mice that can be screened rapidly for optimal regulation and expression patterns, and for monitoring the induction and repression of regulated genes noninvasively in the living animal.

  4. Automatic Control of Gene Expression in Mammalian Cells.

    Science.gov (United States)

    Fracassi, Chiara; Postiglione, Lorena; Fiore, Gianfranco; di Bernardo, Diego

    2016-04-15

    Automatic control of gene expression in living cells is paramount importance to characterize both endogenous gene regulatory networks and synthetic circuits. In addition, such a technology can be used to maintain the expression of synthetic circuit components in an optimal range in order to ensure reliable performance. Here we present a microfluidics-based method to automatically control gene expression from the tetracycline-inducible promoter in mammalian cells in real time. Our approach is based on the negative-feedback control engineering paradigm. We validated our method in a monoclonal population of cells constitutively expressing a fluorescent reporter protein (d2EYFP) downstream of a minimal CMV promoter with seven tet-responsive operator motifs (CMV-TET). These cells also constitutively express the tetracycline transactivator protein (tTA). In cells grown in standard growth medium, tTA is able to bind the CMV-TET promoter, causing d2EYFP to be maximally expressed. Upon addition of tetracycline to the culture medium, tTA detaches from the CMV-TET promoter, thus preventing d2EYFP expression. We tested two different model-independent control algorithms (relay and proportional-integral (PI)) to force a monoclonal population of cells to express an intermediate level of d2EYFP equal to 50% of its maximum expression level for up to 3500 min. The control input is either tetracycline-rich or standard growth medium. We demonstrated that both the relay and PI controllers can regulate gene expression at the desired level, despite oscillations (dampened in the case of the PI controller) around the chosen set point.

  5. Gene expression inference with deep learning.

    Science.gov (United States)

    Chen, Yifei; Li, Yi; Narayan, Rajiv; Subramanian, Aravind; Xie, Xiaohui

    2016-06-15

    Large-scale gene expression profiling has been widely used to characterize cellular states in response to various disease conditions, genetic perturbations, etc. Although the cost of whole-genome expression profiles has been dropping steadily, generating a compendium of expression profiling over thousands of samples is still very expensive. Recognizing that gene expressions are often highly correlated, researchers from the NIH LINCS program have developed a cost-effective strategy of profiling only ∼1000 carefully selected landmark genes and relying on computational methods to infer the expression of remaining target genes. However, the computational approach adopted by the LINCS program is currently based on linear regression (LR), limiting its accuracy since it does not capture complex nonlinear relationship between expressions of genes. We present a deep learning method (abbreviated as D-GEX) to infer the expression of target genes from the expression of landmark genes. We used the microarray-based Gene Expression Omnibus dataset, consisting of 111K expression profiles, to train our model and compare its performance to those from other methods. In terms of mean absolute error averaged across all genes, deep learning significantly outperforms LR with 15.33% relative improvement. A gene-wise comparative analysis shows that deep learning achieves lower error than LR in 99.97% of the target genes. We also tested the performance of our learned model on an independent RNA-Seq-based GTEx dataset, which consists of 2921 expression profiles. Deep learning still outperforms LR with 6.57% relative improvement, and achieves lower error in 81.31% of the target genes. D-GEX is available at https://github.com/uci-cbcl/D-GEX CONTACT: xhx@ics.uci.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.

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    Neutelings Godfrey

    2010-04-01

    Full Text Available Abstract Background Quantitative real-time PCR (qRT-PCR is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs. Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L. Results Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups. qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59. LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both ge

  7. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.).

    Science.gov (United States)

    Huis, Rudy; Hawkins, Simon; Neutelings, Godfrey

    2010-04-19

    Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L). Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and Norm

  8. Endogenous biotin expression in renal and testicular tumours and literature review.

    Science.gov (United States)

    Fahmy, Nader; Woo, Mark; Alameldin, Mona; Lee, Joe King; MacDonald, Kyle; Goneau, Lee W; Cadieux, Peter; Burton, Jeremy; Pautler, Stephen

    2014-07-01

    The aim of this study was to examine endogenous biotin levels in tumour specimens collected from patients with renal and testicular tumours and compare them to the surrounding non-neoplastic surgical margin. Frozen samples were obtained from the Ontario Tumour Bank. Renal and testicular tumour tissue were included in this study. Normal tissue from the negative surgical margins of each tumour served as a control. Biotin detection in tissue specimens was determined using immunohistochemistry (IHC). Specimens collected from 56 patients (36 men and 20 women) were included in this study. Histopathology of the 52 renal tumours included 31 (60%) conventional type RCC, 5 (10%) chromophobe RCC, 5 (10%) papillary RCC, 1 (2%) oncocytoma and 10 (19%) upper tract urothelial carcinoma (UC). The 4 testicular tumours included 1 seminomatous (25%) germ cell tumour and 3 (75%) non seminomatous germ cell tumours. No biotin signal was perceived in all tested tumour samples. Endogenous biotin expression was detected in the matching non-neoplastic surgical margin of tested renal tissues. This lack of staining may prove to be a valuable tool in future studies.

  9. Determinants of human adipose tissue gene expression

    DEFF Research Database (Denmark)

    Viguerie, Nathalie; Montastier, Emilie; Maoret, Jean-José

    2012-01-01

    weight maintenance diets. For 175 genes, opposite regulation was observed during calorie restriction and weight maintenance phases, independently of variations in body weight. Metabolism and immunity genes showed inverse profiles. During the dietary intervention, network-based analyses revealed strong...... interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index...... on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently...

  10. Deriving Trading Rules Using Gene Expression Programming

    Directory of Open Access Journals (Sweden)

    Adrian VISOIU

    2011-01-01

    Full Text Available This paper presents how buy and sell trading rules are generated using gene expression programming with special setup. Market concepts are presented and market analysis is discussed with emphasis on technical analysis and quantitative methods. The use of genetic algorithms in deriving trading rules is presented. Gene expression programming is applied in a form where multiple types of operators and operands are used. This gives birth to multiple gene contexts and references between genes in order to keep the linear structure of the gene expression programming chromosome. The setup of multiple gene contexts is presented. The case study shows how to use the proposed gene setup to derive trading rules encoded by Boolean expressions, using a dataset with the reference exchange rates between the Euro and the Romanian leu. The conclusions highlight the positive results obtained in deriving useful trading rules.

  11. Analysis of gene expression of myo1c and inpp5k genes involved in endometrial adenocarcinoma

    International Nuclear Information System (INIS)

    Koul, A.M.; Nadeem, A.; Baryalai, P.

    2012-01-01

    Abstract: Inpp5k gene encodes a protein which plays a very vital role in a number of metabolic pathways. It is very significant in the glucose metabolism where it regulates the signalling of the insulin pathway. But the full molecular details of the pathways regulated by Inpp5k encoded protein are not known. It is speculated that Inpp5k gene expression is altered in case of endometrial adenocarcinoma. Myolc gene encodes for a protein called Myosin-lc which acts an actin-based molecular motor in the cells. II has been studied that this gene down-regulates during endometrial adenocarcinoma and colorectal cancers. In this study the expression analysis of these two was carried out using multiplex PCR. An endogenous control was used for this PCR. ACTS gene served as the endogenous control because of it being a house keeping gene. It thus shows a universal expression in all cells. Thus in this study the gene expression of Inpp5k and Myulc genes was comparatively analysed with ACTS gene. The results that came out of this study showed an over-expression of Inpp5k gene and down-regulation of myolc gene with respect to ACTS gene in cancer cell lines as was indicated by the previous studies with these genes. Expression of both genes i.e. Inpp5k and Myolc was statistically compared between normal and cancerous cell lines and was found statistically significant at a value of P< O.O I in most of the cases. (author)

  12. A dominant control region from the human β-globin locus conferring integration site-independent gene expression.

    OpenAIRE

    Talbot, D.; Collis, P.; Antoniou, Michael; Vidal, M.; Grosveld, Frank; Greaves, David

    1989-01-01

    textabstractThe regulatory elements that determine the expression pattern of a number of eukaryotic genes expressed specifically in certain tissues have been defined and studied in detail. In general, however, the expression conferred by these elements on genes reintroduced into the genomes of cell lines and transgenic animals has turned out to be at a low level relative to that of endogenous genes, and influenced by the chromosomal site of insertion of the exogenous construct. We have previo...

  13. Polycistronic gene expression in Aspergillus niger.

    Science.gov (United States)

    Schuetze, Tabea; Meyer, Vera

    2017-09-25

    Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at

  14. Profiling Gene Expression in Germinating Brassica Roots.

    Science.gov (United States)

    Park, Myoung Ryoul; Wang, Yi-Hong; Hasenstein, Karl H

    2014-01-01

    Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

  15. Expression and regulation of the endogenous retrovirus 3 (ERV3 in Hodgkin’s lymphoma cells

    Directory of Open Access Journals (Sweden)

    Stefanie eKewitz

    2013-07-01

    Full Text Available Human endogenous retroviruses (ERV are an integral part of our genome. Expression of ERV is usually switched off but reactivation of ERV has been observed in varying human diseases including cancer. Recently, reactivation of ERV associated promoters in Hodgkin’s lymphoma (HL cells has been described. Despite relatively good prognosis, not all patients with HL can be cured with the established therapy and this therapy is associated with severe late side effects. Therefore, new targets are required for the development of future treatment strategies. Reactivated ERV might represent such target structures. Therefore, we asked which ERV loci are expressed in HL cells. Using DNA microarray analysis, we found no evidence for a general activation of ERV transcription in HL cells. In contrast, we observed down-regulation of ERV3, an ERV with potential tumor suppressor function, in HL cells in comparison to normal blood cells. Interestingly, ERV3 was also differentially expressed in published DNA microarray data from resting versus cycling B cells. Treatment of HL cells with the histone deacetylase inhibitor vorinostat strongly up-regulated ERV3 expression. In addition, we observed up-regulation in HL cells after treatment with hypoxia-mimetic cobalt(II chloride. Like vorinostat, cobalt(II chloride inhibited cell growth of HL cells. Our results suggest that cell cycle inhibition of HL cells is accompanied by up-regulation of ERV3.

  16. Endogenous angiotensin II modulates nNOS expression in renovascular hypertension

    Directory of Open Access Journals (Sweden)

    T.M.C. Pereira

    2009-07-01

    Full Text Available Nitric oxide (NO influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS. Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight divided into 2K1C (N = 19 and sham-operated (N = 19 groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9 was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan (10 mg·kg-1·day-1; N = 5 or the superoxide scavenger tempol (0.2 mmol·kg-1·day-1; N = 5, which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

  17. Variable RNA expression from recently acquired, endogenous viral elements (EVE) of white spot syndrome virus (WSSV) in shrimp.

    Science.gov (United States)

    Utari, Heny Budi; Soowannayan, Chumporn; Flegel, Timothy W; Whityachumnarnkul, Boonsirm; Kruatrachue, Maleeya

    2017-11-01

    The viral accommodation hypothesis proposes that endogenous viral elements (EVE) from both RNA and DNA viruses are being continually integrated into the shrimp genome by natural host processes and that they can result in tolerance to viral infection by fortuitous production of antisense, immunospecific RNA (imRNA). Thus, we hypothesized that previously reported microarray results for the presence of white spot syndrome virus (WSSV) open reading frames (ORFs) formerly called 151, 366 and 427 in a domesticated giant tiger shrimp (Penaeus monodon) breeding stock might have represented expression from EVE, since the stock had shown uninterrupted freedom from white spot disease (WSD) for many generations. To test this hypothesis, 128 specimens from a current stock generation were confirmed for freedom from WSSV infection using two nested PCR detection methods. Subsequent nested-PCR testing revealed 33/128 specimens (26%) positive for at least one of the ORF at very high sequence identity (95-99%) to extant WSSV. Positive results for ORF 366 (now known to be a fragment of the WSSV capsid protein gene) dominated (28/33 = 84.8%), so 9 arbitrarily selected 366-positive specimens were tested by strand-specific, nested RT-PCR using DNase-treated RNA templates. This revealed variable RNA expression in individual shrimp including no RNA transcripts (n = 1), sense transcripts only (n = 1), antisense transcripts only (n = 2) or transcripts of both sense (n = 5). The latter 7 expression products indicated specimens producing putative imRNA. The variable types and numbers of the EVE and the variable RNA expression (including potential imRNA) support predictions of the viral accommodation hypothesis that EVE are randomly produced and expressed. Positive nested PCR test results for EVE of ORF 366 using DNA templates derived from shrimp sperm (germ cells), indicated that they were heritable. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Ethanol extract of Portulaca oleracea L. protects against hypoxia-induced neuro damage through modulating endogenous erythropoietin expression.

    Science.gov (United States)

    Wanyin, Wang; Liwei, Dong; Lin, Jia; Hailiang, Xin; Changquan, Ling; Min, Li

    2012-04-01

    In addition to its role in erythropoiesis, erythropoietin is also appreciated for its neuroprotective effects, and it has been suggested for treatment of some ischemic-hypoxic neurovascular diseases. The protective effects of endogenous erythropoietin in the brain give rise to the hypothesis that modulating erythropoietin expression might be a better way for treatment of ischemia-hypoxia neurovascular diseases. We have found that ethanol extract of Portulaca oleracea L. (EEPO) could increase erythropoietin expression in hypoxic mouse brain in our previous study. The present study is to investigate whether EEPO exerts its neuroprotective effects against hypoxia injury through regulating endogenous erythropoietin expression. The results demonstrated that EEPO decreased the serum neuron specific enolase level in hypoxia mice and the activity of caspase-3 in neuron, increased the neuron viability and attenuated the pathological damages caused by the hypoxia condition. Importantly, we also found that EEPO stimulated the endogenous erythropoietin expression at both mRNA and protein levels. Using the conditioned medium containing soluble erythropoietin receptor, we found that the neuroprotective effects of EEPO were dependent, at least partly, on erythropoietin expression. Although EEPO did not affect transcription of hypoxia inducible factor-1α (HIF-1α), it did stabilize expression of HIF-1α. It is concluded that EEPO has neuroprotective effects against hypoxia injury, which is at least partly through stimulating endogenous erythropoietin expression by stabilizing HIF-1α. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Chromatin loops, gene positioning, and gene expression

    NARCIS (Netherlands)

    Holwerda, S.; de Laat, W.

    2012-01-01

    Technological developments and intense research over the last years have led to a better understanding of the 3D structure of the genome and its influence on genome function inside the cell nucleus. We will summarize topological studies performed on four model gene loci: the alpha- and beta-globin

  20. Using riboswitches to regulate gene expression and define gene function in mycobacteria.

    Science.gov (United States)

    Van Vlack, Erik R; Seeliger, Jessica C

    2015-01-01

    Mycobacteria include both environmental species and many pathogenic species such as Mycobacterium tuberculosis, an intracellular pathogen that is the causative agent of tuberculosis in humans. Inducible gene expression is a powerful tool for examining gene function and essentiality, both in in vitro culture and in host cell infections. The theophylline-inducible artificial riboswitch has recently emerged as an alternative to protein repressor-based systems. The riboswitch is translationally regulated and is combined with a mycobacterial promoter that provides transcriptional control. We here provide methods used by our laboratory to characterize the riboswitch response to theophylline in reporter strains, recombinant organisms containing riboswitch-regulated endogenous genes, and in host cell infections. These protocols should facilitate the application of both existing and novel artificial riboswitches to the exploration of gene function in mycobacteria. © 2015 Elsevier Inc. All rights reserved.

  1. Serial analysis of gene expression (SAGE)

    NARCIS (Netherlands)

    van Ruissen, Fred; Baas, Frank

    2007-01-01

    In 1995, serial analysis of gene expression (SAGE) was developed as a versatile tool for gene expression studies. SAGE technology does not require pre-existing knowledge of the genome that is being examined and therefore SAGE can be applied to many different model systems. In this chapter, the SAGE

  2. Evolution of endogenous non-retroviral genes integrated into plant genomes

    Directory of Open Access Journals (Sweden)

    Hyosub Chu

    2014-08-01

    Full Text Available Numerous comparative genome analyses have revealed the wide extent of horizontal gene transfer (HGT in living organisms, which contributes to their evolution and genetic diversity. Viruses play important roles in HGT. Endogenous viral elements (EVEs are defined as viral DNA sequences present within the genomes of non-viral organisms. In eukaryotic cells, the majority of EVEs are derived from RNA viruses using reverse transcription. In contrast, endogenous non-retroviral elements (ENREs are poorly studied. However, the increasing availability of genomic data and the rapid development of bioinformatics tools have enabled the identification of several ENREs in various eukaryotic organisms. To date, a small number of ENREs integrated into plant genomes have been identified. Of the known non-retroviruses, most identified ENREs are derived from double-strand (ds RNA viruses, followed by single-strand (ss DNA and ssRNA viruses. At least eight virus families have been identified. Of these, viruses in the family Partitiviridae are dominant, followed by viruses of the families Chrysoviridae and Geminiviridae. The identified ENREs have been primarily identified in eudicots, followed by monocots. In this review, we briefly discuss the current view on non-retroviral sequences integrated into plant genomes that are associated with plant-virus evolution and their possible roles in antiviral resistance.

  3. HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells

    DEFF Research Database (Denmark)

    Jørgensen, Steffen; Coshun, Mehmet; Mikkelsen Homburg, Keld

    2016-01-01

    analysis and expression data from Caco2 cells also suggests a role for CDX2 in the regulation of HOXB4 gene expression in the intestinal epithelium. Thus, the aim of this study was to investigate whether HOXB4 gene expression is regulated by CDX2 in the intestinal epithelium. We demonstrated binding of CDX......The mammalian Caudal-related homeobox transcription factor 2 (CDX2) plays a key role in the homeobox regulatory network and is essential in regulating the expression of several homeobox (HOX) genes during embryonic development, particularly in the gut. Genome-wide CDX2 chromatin immunoprecipitation......2 to four different CDX2 binding sites in an enhancer region located upstream of the HOXB4 transcription start site. Mutations in the CDX2 binding sites reduced HOXB4 gene activity, and knock down of endogenous CDX2 expression by shRNA reduced HOXB4 gene expression. This is the first report...

  4. Expression of Sox genes in tooth development.

    Science.gov (United States)

    Kawasaki, Katsushige; Kawasaki, Maiko; Watanabe, Momoko; Idrus, Erik; Nagai, Takahiro; Oommen, Shelly; Maeda, Takeyasu; Hagiwara, Nobuko; Que, Jianwen; Sharpe, Paul T; Ohazama, Atsushi

    2015-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

  5. Positron emission tomography imaging of gene expression

    International Nuclear Information System (INIS)

    Tang Ganghua

    2001-01-01

    The merging of molecular biology and nuclear medicine is developed into molecular nuclear medicine. Positron emission tomography (PET) of gene expression in molecular nuclear medicine has become an attractive area. Positron emission tomography imaging gene expression includes the antisense PET imaging and the reporter gene PET imaging. It is likely that the antisense PET imaging will lag behind the reporter gene PET imaging because of the numerous issues that have not yet to be resolved with this approach. The reporter gene PET imaging has wide application into animal experimental research and human applications of this approach will likely be reported soon

  6. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

    DEFF Research Database (Denmark)

    Sørensen, Brita Singers; Alsner, Jan; Overgaard, Jens

    2007-01-01

    BACKGROUND: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression...

  7. Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

    Science.gov (United States)

    Bunker, T W; Koetje, D S; Stephenson, L C; Creelman, R A; Mullet, J E; Grimes, H D

    1995-08-01

    The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves.

  8. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    Science.gov (United States)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  9. The endogenous retroviral locus ERVWE1 is a bona fide gene involved in hominoid placental physiology

    Science.gov (United States)

    Mallet, François; Bouton, Olivier; Prudhomme, Sarah; Cheynet, Valérie; Oriol, Guy; Bonnaud, Bertrand; Lucotte, Gérard; Duret, Laurent; Mandrand, Bernard

    2004-01-01

    The definitive demonstration of a role for a recently acquired gene is a difficult task, requiring exhaustive genetic investigations and functional analysis. The situation is indeed much more complicated when facing multicopy gene families, because most or portions of the gene are conserved among the hundred copies of the family. This is the case for the ERVWE1 locus of the human endogenous retrovirus W family (HERV-W), which encodes an envelope glycoprotein (syncytin) likely involved in trophoblast differentiation. Here we describe, in 155 individuals, the positional conservation of this locus and the preservation of the envelope ORF. Sequencing of the critical elements of the ERVWE1 provirus showed a striking conservation among the 48 alleles of 24 individuals, including the LTR elements involved in the transcriptional machinery, the splice sites involved in the maturation of subgenomic Env mRNA, and the Env ORF. The functionality and tissue specificity of the 5′ LTR were demonstrated, as well as the fusogenic activity of the envelope polymorphic variants. Such functions were also shown to be preserved in the orthologous loci isolated from chimpanzee, gorilla, orangutan, and gibbon. This functional preservation among humans and during evolution strongly argued for the involvement of this recently acquired retroviral envelope glycoprotein in hominoid placental physiology. PMID:14757826

  10. The functional landscape of mouse gene expression

    Directory of Open Access Journals (Sweden)

    Zhang Wen

    2004-12-01

    Full Text Available Abstract Background Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function. Results We generated microarray expression data for nearly 40,000 known and predicted mRNAs in 55 mouse tissues, using custom-built oligonucleotide arrays. We show that quantitative transcriptional co-expression is a powerful predictor of gene function. Hundreds of functional categories, as defined by Gene Ontology 'Biological Processes', are associated with characteristic expression patterns across all tissues, including categories that bear no overt relationship to the tissue of origin. In contrast, simple tissue-specific restriction of expression is a poor predictor of which genes are in which functional categories. As an example, the highly conserved mouse gene PWP1 is widely expressed across different tissues but is co-expressed with many RNA-processing genes; we show that the uncharacterized yeast homolog of PWP1 is required for rRNA biogenesis. Conclusions We conclude that 'functional genomics' strategies based on quantitative transcriptional co-expression will be as fruitful in mammals as they have been in simpler organisms, and that transcriptional control of mammalian physiology is more modular than is generally appreciated. Our data and analyses provide a public resource for mammalian functional genomics.

  11. Overexpression of Arachis hypogaea AREB1 Gene Enhances Drought Tolerance by Modulating ROS Scavenging and Maintaining Endogenous ABA Content

    Directory of Open Access Journals (Sweden)

    Ling Li

    2013-06-01

    Full Text Available AhAREB1 (Arachis hypogaea Abscisic-acid Response Element Binding Protein 1 is a member of the basic domain leucine zipper (bZIP-type transcription factor in peanut. Previously, we found that expression of AhAREB1 was specifically induced by abscisic acid (ABA, dehydration and drought. To understand the drought defense mechanism regulated by AhAREB1, transgenic Arabidopsis overexpressing AhAREB1 was conducted in wild-type (WT, and a complementation experiment was employed to ABA non-sensitivity mutant abi5 (abscisic acid-insensitive 5. Constitutive expression of AhAREB1 confers water stress tolerance and is highly sensitive to exogenous ABA. Microarray and further real-time PCR analysis revealed that drought stress, reactive oxygen species (ROS scavenging, ABA synthesis/metabolism-related genes and others were regulated in transgenic Arabidopsis overexpressing AhAREB1. Accordingly, low level of ROS, but higher ABA content was detected in the transgenic Arabidopsis plants’ overexpression of AhAREB1. Taken together, it was concluded that AhAREB1 modulates ROS accumulation and endogenous ABA level to improve drought tolerance in transgenic Arabidopsis.

  12. Identification and detection of a novel human endogenous retrovirus-related gene, and structural characterization of its related elements

    Directory of Open Access Journals (Sweden)

    Qiaoyi Liang

    2009-01-01

    Full Text Available Up-regulation of human endogenous retroviruses (HERVs is associated with many diseases, including cancer. In this study, an H family HERV (HERV-H-related gene was identified and characterized. Its spliced transcript lacks protein-coding capacity and may belong to the emerging class of noncoding RNAs (ncRNAs. The 1.3-kb RNA consisting of four exons is transcribed from an Alu element upstream of a 5.0-kb structurally incomplete HERV-H element. RT-PCR and quantitative RT-PCR results indicated that expression of this HERV-related transcript was negatively associated with colon, stomach, and kidney cancers. Its expression was induced upon treatment with DNA methylation and histone deacetylation inhibitors. A BLAT search using long terminal repeats (LTRs identified 50 other LTR homogenous HERV-H elements. Further analysis of these elements revealed that all are structurally incomplete and only five exert transcriptional activity. The results presented here recommend further investigation into a potentially functional HERV-H-related ncRNA.

  13. A comparative gene expression database for invertebrates

    Directory of Open Access Journals (Sweden)

    Ormestad Mattias

    2011-08-01

    Full Text Available Abstract Background As whole genome and transcriptome sequencing gets cheaper and faster, a great number of 'exotic' animal models are emerging, rapidly adding valuable data to the ever-expanding Evo-Devo field. All these new organisms serve as a fantastic resource for the research community, but the sheer amount of data, some published, some not, makes detailed comparison of gene expression patterns very difficult to summarize - a problem sometimes even noticeable within a single lab. The need to merge existing data with new information in an organized manner that is publicly available to the research community is now more necessary than ever. Description In order to offer a homogenous way of storing and handling gene expression patterns from a variety of organisms, we have developed the first web-based comparative gene expression database for invertebrates that allows species-specific as well as cross-species gene expression comparisons. The database can be queried by gene name, developmental stage and/or expression domains. Conclusions This database provides a unique tool for the Evo-Devo research community that allows the retrieval, analysis and comparison of gene expression patterns within or among species. In addition, this database enables a quick identification of putative syn-expression groups that can be used to initiate, among other things, gene regulatory network (GRN projects.

  14. Adaptive Evolution of Gene Expression in Drosophila.

    Science.gov (United States)

    Nourmohammad, Armita; Rambeau, Joachim; Held, Torsten; Kovacova, Viera; Berg, Johannes; Lässig, Michael

    2017-08-08

    Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Differential gene expression during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Marco Aurelio Krieger

    1999-09-01

    Full Text Available The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE. The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells, while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

  16. Gene-mutation assays in lambda-lacZ transgenic mice : comparison of lacZ with endogenous genes in splenocytes and small intestinal epithelium

    NARCIS (Netherlands)

    Delft, J.H.M. van; Bergmans, A.; Dam, F.J. van; Tates, A.D.; Howard, L.; Winton, D.J.; Baan, R.A.

    1998-01-01

    Comparison of results derived from transgenic animal gene-mutation assays with those from mutation analyses in endogenous genes is an important step in the validation of the former. We have used λlacZ transgenic mice to study alkylation-induced mutagenesis in vivo in (a) lacZ and hprt in spleen

  17. Learning gene regulatory networks from gene expression data using weighted consensus

    KAUST Repository

    Fujii, Chisato; Kuwahara, Hiroyuki; Yu, Ge; Guo, Lili; Gao, Xin

    2016-01-01

    An accurate determination of the network structure of gene regulatory systems from high-throughput gene expression data is an essential yet challenging step in studying how the expression of endogenous genes is controlled through a complex interaction of gene products and DNA. While numerous methods have been proposed to infer the structure of gene regulatory networks, none of them seem to work consistently over different data sets with high accuracy. A recent study to compare gene network inference methods showed that an average-ranking-based consensus method consistently performs well under various settings. Here, we propose a linear programming-based consensus method for the inference of gene regulatory networks. Unlike the average-ranking-based one, which treats the contribution of each individual method equally, our new consensus method assigns a weight to each method based on its credibility. As a case study, we applied the proposed consensus method on synthetic and real microarray data sets, and compared its performance to that of the average-ranking-based consensus and individual inference methods. Our results show that our weighted consensus method achieves superior performance over the unweighted one, suggesting that assigning weights to different individual methods rather than giving them equal weights improves the accuracy. © 2016 Elsevier B.V.

  18. Learning gene regulatory networks from gene expression data using weighted consensus

    KAUST Repository

    Fujii, Chisato

    2016-08-25

    An accurate determination of the network structure of gene regulatory systems from high-throughput gene expression data is an essential yet challenging step in studying how the expression of endogenous genes is controlled through a complex interaction of gene products and DNA. While numerous methods have been proposed to infer the structure of gene regulatory networks, none of them seem to work consistently over different data sets with high accuracy. A recent study to compare gene network inference methods showed that an average-ranking-based consensus method consistently performs well under various settings. Here, we propose a linear programming-based consensus method for the inference of gene regulatory networks. Unlike the average-ranking-based one, which treats the contribution of each individual method equally, our new consensus method assigns a weight to each method based on its credibility. As a case study, we applied the proposed consensus method on synthetic and real microarray data sets, and compared its performance to that of the average-ranking-based consensus and individual inference methods. Our results show that our weighted consensus method achieves superior performance over the unweighted one, suggesting that assigning weights to different individual methods rather than giving them equal weights improves the accuracy. © 2016 Elsevier B.V.

  19. Promotion of growth by Coenzyme Q10 is linked to gene expression in C. elegans.

    Science.gov (United States)

    Fischer, Alexandra; Niklowitz, Petra; Menke, Thomas; Döring, Frank

    2014-10-03

    Coenzyme Q (CoQ, ubiquinone) is an essential component of the respiratory chain, a cofactor of pyrimidine biosynthesis and acts as an antioxidant in extra mitochondrial membranes. More recently CoQ has been identified as a modulator of apoptosis, inflammation and gene expression. CoQ deficient Caenorhabditis elegans clk-1 mutants show several phenotypes including a delayed postembryonic growth. Using wild type and two clk-1 mutants, here we established an experimental set-up to study the consequences of endogenous CoQ deficiency or exogenous CoQ supply on gene expression and growth. We found that a deficiency of endogenous CoQ synthesis down-regulates a cluster of genes that are important for growth (i.e., RNA polymerase II, eukaryotic initiation factor) and up-regulates oxidation reactions (i.e., cytochrome P450, superoxide dismutase) and protein interactions (i.e., F-Box proteins). Exogenous CoQ supply partially restores the expression of these genes as well as the growth retardation of CoQ deficient clk-1 mutants. On the other hand exogenous CoQ supply does not alter the expression of a further sub-set of genes. These genes are involved in metabolism (i.e., succinate dehydrogenase complex), cell signalling or synthesis of lectins. Thus, our work provides a comprehensive overview of genes which can be modulated in their expression by endogenous or exogenous CoQ. As growth retardation in CoQ deficiency is linked to the gene expression profile we suggest that CoQ promotes growth via gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Dietary exposure of largemouth bass to OCPs changes expression of genes important for reproduction.

    Science.gov (United States)

    Garcia-Reyero, Natàlia; Barber, David S; Gross, Timothy S; Johnson, Kevin G; Sepúlveda, María S; Szabo, Nancy J; Denslow, Nancy D

    2006-07-20

    Dieldrin and p,p'-DDE are ubiquitous contaminants known to act as endocrine disruptors, causing impaired development and reproduction in fish and wildlife. In order to elucidate the mechanisms by which dieldrin and p,p'-DDE cause endocrine disruption in largemouth bass (Micropterus salmoides), fish were exposed subchronically through the diet to both contaminants. Following 120 days of exposure, p,p'-DDE decreased estradiol in females, but increased 11-ketotestosterone in both sexes. Dieldrin on the other hand, decreased estradiol and 11-ketotestosterone in both sexes. Both pesticides also altered steady state mRNA expression levels of a set of genes chosen to represent three possible mechanisms of endocrine disruption: (1) direct interaction with soluble sex steroid receptors, (2) biosynthesis of endogenous sex hormones, and (3) metabolism of endogenous hormones. p,p'-DDE acted as a weak estrogen, increasing the expression of vitellogenin and estrogen receptor alpha in the liver. p,p'-DDE also altered the expression of genes involved in the synthesis of endogenous hormones as well as their metabolism. Dieldrin, on the other hand, only altered expression of vitellogenin and not estrogen receptor alpha. Dieldrin also altered the expression of genes involved in hormone synthesis and metabolism, and it dramatically lowered plasma hormone levels. Both pesticides targeted expression of genes involved in all three modes of action, suggesting that they each have multiple modes of action.

  1. Dietary exposure of largemouth bass to OCPs changes expression of genes important for reproduction

    Science.gov (United States)

    Garcia-Reyero, Natalia; Barber, D.S.; Gross, T.S.; Johnson, K.G.; Sepulveda, M.S.; Szabo, N.J.; Denslow, N.D.

    2006-01-01

    Dieldrin and p,p???-DDE are ubiquitous contaminants known to act as endocrine disruptors, causing impaired development and reproduction in fish and wildlife. In order to elucidate the mechanisms by which dieldrin and p,p???-DDE cause endocrine disruption in largemouth bass (Micropterus salmoides), fish were exposed subchronically through the diet to both contaminants. Following 120 days of exposure, p,p???-DDE decreased estradiol in females, but increased 11-ketotestosterone in both sexes. Dieldrin on the other hand, decreased estradiol and 11-ketotestosterone in both sexes. Both pesticides also altered steady state mRNA expression levels of a set of genes chosen to represent three possible mechanisms of endocrine disruption: (1) direct interaction with soluble sex steroid receptors, (2) biosynthesis of endogenous sex hormones, and (3) metabolism of endogenous hormones. p,p???-DDE acted as a weak estrogen, increasing the expression of vitellogenin and estrogen receptor ?? in the liver. p,p???-DDE also altered the expression of genes involved in the synthesis of endogenous hormones as well as their metabolism. Dieldrin, on the other hand, only altered expression of vitellogenin and not estrogen receptor ??. Dieldrin also altered the expression of genes involved in hormone synthesis and metabolism, and it dramatically lowered plasma hormone levels. Both pesticides targeted expression of genes involved in all three modes of action, suggesting that they each have multiple modes of action. ?? 2006 Elsevier B.V. All rights reserved.

  2. Effects of Argonaute on Gene Expression in Thermus thermophilus.

    Directory of Open Access Journals (Sweden)

    Daan C Swarts

    Full Text Available Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo.To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago, and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels.In the absence of exogenous DNA (plasmid, TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid-encoded RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation.

  3. Stochastic gene expression in Arabidopsis thaliana.

    Science.gov (United States)

    Araújo, Ilka Schultheiß; Pietsch, Jessica Magdalena; Keizer, Emma Mathilde; Greese, Bettina; Balkunde, Rachappa; Fleck, Christian; Hülskamp, Martin

    2017-12-14

    Although plant development is highly reproducible, some stochasticity exists. This developmental stochasticity may be caused by noisy gene expression. Here we analyze the fluctuation of protein expression in Arabidopsis thaliana. Using the photoconvertible KikGR marker, we show that the protein expressions of individual cells fluctuate over time. A dual reporter system was used to study extrinsic and intrinsic noise of marker gene expression. We report that extrinsic noise is higher than intrinsic noise and that extrinsic noise in stomata is clearly lower in comparison to several other tissues/cell types. Finally, we show that cells are coupled with respect to stochastic protein expression in young leaves, hypocotyls and roots but not in mature leaves. Our data indicate that stochasticity of gene expression can vary between tissues/cell types and that it can be coupled in a non-cell-autonomous manner.

  4. Interactions between Bmp-4 and Msx-1 act to restrict gene expression to odontogenic mesenchyme.

    Science.gov (United States)

    Tucker, A S; Al Khamis, A; Sharpe, P T

    1998-08-01

    Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. Bmp4 has been identified as a candidate signalling molecule in these interactions, initially as an epithelial signal and then later at the bud stage as a mesenchymal signal (Vainio et al. [1993] Cell 75:45-58). A target gene for Bmp4 signalling is the homeobox gene Msx-1, identified by the ability of recombinant Bmp4 protein to induce expression in mesenchyme. There is, however, no evidence that Bmp4 is the endogenous inducer of Msx-1 expression. Msx-1 and Bmp-4 show dynamic, interactive patterns of expression in oral epithelium and ectomesenchyme during the early stages of tooth development. In this study, we compare the temporal and spatial expression of these two genes to determine whether the changing expression patterns of these genes are consistent with interactions between the two molecules. We show that changes in Bmp-4 expression precede changes in Msx-1 expression. At embryonic day (E)10.5-E11.0, expression patterns are consistent with BMP4 from the epithelium, inducing or maintaining Msx-1 in underlying mesenchyme. At E11.5, Bmp-4 expression shifts from epithelium to mesenchyme and is rapidly followed by localised up-regulation of Msx-1 expression at the sites of Bmp-4 expression. Using cultured explants of developing mandibles, we confirm that exogenous BMP4 is capable of replacing the endogenous source in epithelium and inducing Msx-1 gene expression in mesenchyme. By using noggin, a BMP inhibitor, we show that endogenous Msx-1 expression can be inhibited at E10.5 and E11.5, providing the first evidence that endogenous Bmp-4 from the epithelium is responsible for regulating the early spatial expression of Msx-1. We also show that the mesenchymal shift in Bmp-4 is responsible for up-regulating Msx-1 specifically at the sites of future tooth formation. Thus, we establish that a reciprocal series of interactions act to restrict expression of both genes to future

  5. Gene expression in periodontal tissues following treatment

    Directory of Open Access Journals (Sweden)

    Eisenacher Martin

    2008-07-01

    Full Text Available Abstract Background In periodontitis, treatment aimed at controlling the periodontal biofilm infection results in a resolution of the clinical and histological signs of inflammation. Although the cell types found in periodontal tissues following treatment have been well described, information on gene expression is limited to few candidate genes. Therefore, the aim of the study was to determine the expression profiles of immune and inflammatory genes in periodontal tissues from sites with severe chronic periodontitis following periodontal therapy in order to identify genes involved in tissue homeostasis. Gingival biopsies from 12 patients with severe chronic periodontitis were taken six to eight weeks following non-surgical periodontal therapy, and from 11 healthy controls. As internal standard, RNA of an immortalized human keratinocyte line (HaCaT was used. Total RNA was subjected to gene expression profiling using a commercially available microarray system focusing on inflammation-related genes. Post-hoc confirmation of selected genes was done by Realtime-PCR. Results Out of the 136 genes analyzed, the 5% most strongly expressed genes compared to healthy controls were Interleukin-12A (IL-12A, Versican (CSPG-2, Matrixmetalloproteinase-1 (MMP-1, Down syndrome critical region protein-1 (DSCR-1, Macrophage inflammatory protein-2β (Cxcl-3, Inhibitor of apoptosis protein-1 (BIRC-1, Cluster of differentiation antigen 38 (CD38, Regulator of G-protein signalling-1 (RGS-1, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene (C-FOS; the 5% least strongly expressed genes were Receptor-interacting Serine/Threonine Kinase-2 (RIP-2, Complement component 3 (C3, Prostaglandin-endoperoxide synthase-2 (COX-2, Interleukin-8 (IL-8, Endothelin-1 (EDN-1, Plasminogen activator inhibitor type-2 (PAI-2, Matrix-metalloproteinase-14 (MMP-14, and Interferon regulating factor-7 (IRF-7. Conclusion Gene expression profiles found in periodontal tissues following

  6. Finding the joker among the maize endogenous reference genes for genetically modified organism (GMO) detection.

    Science.gov (United States)

    Paternò, Annalisa; Marchesi, Ugo; Gatto, Francesco; Verginelli, Daniela; Quarchioni, Cinzia; Fusco, Cristiana; Zepparoni, Alessia; Amaddeo, Demetrio; Ciabatti, Ilaria

    2009-12-09

    The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed.

  7. Redox-Active Antibiotics Control Gene Expression and Community Behavior in Divergent Bacteria

    OpenAIRE

    Dietrich, Lars E. P.; Teal, Tracy K.; Price-Whelan, Alexa; Newman, Dianne K.

    2008-01-01

    It is thought that bacteria excrete redox-active pigments as antibiotics to inhibit competitors. In Pseudomonas aeruginosa, the endogenous antibiotic pyocyanin activates SoxR, a transcription factor conserved in Proteo- and Actinobacteria. In Escherichia coli, SoxR regulates the superoxide stress response. Bioinformatic analysis coupled with gene expression studies in P. aeruginosa and Streptomyces coelicolor revealed that the majority of SoxR regulons in bacteria lack the genes required for ...

  8. Daily rhythms of catalase and glutathione peroxidase expression and activity are endogenously driven in the hippocampus and are modified by a vitamin A-free diet.

    Science.gov (United States)

    Navigatore-Fonzo, Lorena S; Delgado, Silvia M; Gimenez, Maria Sofia; Anzulovich, Ana C

    2014-01-01

    Alterations in enzymatic antioxidant defense systems lead to a deficit of cognitive functions and altered hippocampal synaptic plasticity. The objectives of this study were to investigate endogenous rhythms of catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as CREB1 mRNA, in the rat hippocampus, and to evaluate to which extent the vitamin A deficiency could affect those temporal patterns. Rats from control and vitamin A-deficient (VAD) groups received a diet containing 4000 IU of vitamin A/kg diet, or the same diet devoid of vitamin A, respectively, during 3 months. Rats were maintained under 12-hour-dark conditions, during 10 days before the sacrifice. Circadian rhythms of CAT, GPx, RXRγ, and CREB1 mRNA levels were determined by reverse transcriptrase polymerase chain reaction in hippocampus samples isolated every 4 hours during a 24-hour period. CAT and GPx enzymatic activities were also determined by kinetic assays. Regulatory regions of clock and antioxidant enzymes genes were scanned for E-box, RXRE, and CRE sites. E-box, RXRE, and CRE sites were found on regulatory regions of GPx and CAT genes, which display a circadian expression in the rat hippocampus. VAD phase shifted CAT, GPx, and RXRγ endogenous rhythms without affecting circadian expression of CREB1. CAT and GPx expression and enzymatic activity are circadian in the rat hippocampus. The VAD affected the temporal patterns antioxidant genes expression, probably by altering circadian rhythms of its RXR receptors and clock factors; thus, it would impair the temporal orchestration of hippocampal daily cognitive performance.

  9. Widespread ectopic expression of olfactory receptor genes

    Directory of Open Access Journals (Sweden)

    Yanai Itai

    2006-05-01

    Full Text Available Abstract Background Olfactory receptors (ORs are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information.

  10. Regulation of Gene Expression in Protozoa Parasites

    Directory of Open Access Journals (Sweden)

    Consuelo Gomez

    2010-01-01

    Full Text Available Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

  11. Regulation of gene expression in protozoa parasites.

    Science.gov (United States)

    Gomez, Consuelo; Esther Ramirez, M; Calixto-Galvez, Mercedes; Medel, Olivia; Rodríguez, Mario A

    2010-01-01

    Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

  12. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.; Mallick, B. K.

    2013-01-01

    graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which

  13. Gene Expression and Microarray Investigation of Dendrobium ...

    African Journals Online (AJOL)

    blood glucose > 16.7 mmol/L were used as the model group and treated with Dendrobium mixture. (DEN ... Keywords: Diabetes, Gene expression, Dendrobium mixture, Microarray testing ..... homeostasis in airway smooth muscle. Am J.

  14. Identification of genes showing differential expression profile ...

    Indian Academy of Sciences (India)

    3Department of Natural Sciences, International Christian University, Mitaka, Tokyo 181-8585, Japan ... the changes of expression predicted from gene function suggested association ... ate School of Science and Technology, Niigata University.

  15. Drosophila melanogaster gene expression changes after spaceflight.

    Data.gov (United States)

    National Aeronautics and Space Administration — Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms...

  16. Exertional Heat Illness and Human Gene Expression

    National Research Council Canada - National Science Library

    Sonna, L.A; Sawka, M. N; Lilly, C. M

    2007-01-01

    Microarray analysis of gene expression at the level of RNA has generated new insights into the relationship between cellular responses to acute heat shock in vitro, exercise, and exertional heat illness...

  17. Expression Profiling of Tyrosine Kinase Genes

    National Research Council Canada - National Science Library

    Weier, Heinz

    2000-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  18. Regulation of meiotic gene expression in plants

    Directory of Open Access Journals (Sweden)

    Adele eZhou

    2014-08-01

    Full Text Available With the recent advances in genomics and sequencing technologies, databases of transcriptomes representing many cellular processes have been built. Meiotic transcriptomes in plants have been studied in Arabidopsis thaliana, rice (Oryza sativa, wheat (Triticum aestivum, petunia (Petunia hybrida, sunflower (Helianthus annuus, and maize (Zea mays. Studies in all organisms, but particularly in plants, indicate that a very large number of genes are expressed during meiosis, though relatively few of them seem to be required for the completion of meiosis. In this review, we focus on gene expression at the RNA level and analyze the meiotic transcriptome datasets and explore expression patterns of known meiotic genes to elucidate how gene expression could be regulated during meiosis. We also discuss mechanisms, such as chromatin organization and non-coding RNAs, that might be involved in the regulation of meiotic transcription patterns.

  19. Identification of genes preferentially expressed during

    African Journals Online (AJOL)

    雨林木风

    2012-08-16

    Aug 16, 2012 ... The suppression subtractive hybridization (SSH) method conducted to generate ... which showed the lack of genomic information currently available for lily. ..... characterization of genes expressed during somatic embryo.

  20. Expression profiles and functional associations of endogenous androgen receptor and caveolin-1 in prostate cancer cell lines.

    Science.gov (United States)

    Bennett, Nigel C; Hooper, John D; Johnson, David W; Gobe, Glenda C

    2014-05-01

    In prostate cancer (PCa) patients, the protein target for androgen deprivation and blockade therapies is androgen receptor (AR). AR interacts with many proteins that function to either co-activate or co-repress its activity. Caveolin-1 (Cav-1) is not found in normal prostatic epithelium, but is found in PCa, and may be an AR co-regulator protein. We investigated cell line-specific signatures and associations of endogenous AR and Cav-1 in six PCa cell lines of known androgen sensitivity: LNCaP (androgen sensitive); 22Rv1 (androgen responsive); PC3, DU145, and ALVA41 (androgen non-reliant); and RWPE1 (non-malignant). Protein and mRNA expression profiles were compared and electron microscopy used to identify cells with caveolar structures. For cell lines expressing both AR and Cav-1, knockdown techniques using small interfering RNA against AR or Cav-1 were used to test whether diminished expression of one affected the other. Co-sedimentation of AR and Cav-1 was used to test their association. A reporter assay for AR genomic activity was utilized following Cav-1 knockdown. AR-expressing LNCaP and 22Rv1 cells had low endogenous Cav-1 mRNA and protein. Cell lines that expressed little or no AR (DU145, PC3, ALVA41, and RWPE1) expressed high endogenous levels of Cav-1. AR knockdown in LNCaP cells had little effect on Cav-1, but Cav-1 knockdown inhibited AR expression and genomic activity. These data show endogenous AR and Cav-1 mRNA and protein expression is inversely related in PCa cells, with Cav-1 acting on the androgen/AR signaling axis possibly as an AR co-activator, demonstrated by diminished AR genomic activity following Cav-1 knockdown. © 2013 Wiley Periodicals, Inc.

  1. Mining gene expression data of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Pi Guo

    Full Text Available Microarray produces a large amount of gene expression data, containing various biological implications. The challenge is to detect a panel of discriminative genes associated with disease. This study proposed a robust classification model for gene selection using gene expression data, and performed an analysis to identify disease-related genes using multiple sclerosis as an example.Gene expression profiles based on the transcriptome of peripheral blood mononuclear cells from a total of 44 samples from 26 multiple sclerosis patients and 18 individuals with other neurological diseases (control were analyzed. Feature selection algorithms including Support Vector Machine based on Recursive Feature Elimination, Receiver Operating Characteristic Curve, and Boruta algorithms were jointly performed to select candidate genes associating with multiple sclerosis. Multiple classification models categorized samples into two different groups based on the identified genes. Models' performance was evaluated using cross-validation methods, and an optimal classifier for gene selection was determined.An overlapping feature set was identified consisting of 8 genes that were differentially expressed between the two phenotype groups. The genes were significantly associated with the pathways of apoptosis and cytokine-cytokine receptor interaction. TNFSF10 was significantly associated with multiple sclerosis. A Support Vector Machine model was established based on the featured genes and gave a practical accuracy of ∼86%. This binary classification model also outperformed the other models in terms of Sensitivity, Specificity and F1 score.The combined analytical framework integrating feature ranking algorithms and Support Vector Machine model could be used for selecting genes for other diseases.

  2. Construction of a novel, stable, food-grade expression system by engineering the endogenous toxin-antitoxin system in Bacillus subtilis.

    Science.gov (United States)

    Yang, Sen; Kang, Zhen; Cao, Wenlong; Du, Guocheng; Chen, Jian

    2016-02-10

    Bacillus subtilis as an important workhorse that has been widely used to produce enzymes and metabolites. To broaden its applications, especially in the food and feed industry, we constructed a novel, stable, food-grade expression system by engineering its type II toxin-antitoxin system. The expression of the toxin EndoA, encoded by the chromosomal ydcE gene, was regulated by an endogenous, xylose-inducible promoter, while the ydcD gene, which encodes the unstable antitoxin EndoB, was inserted into a food-grade vector backbone, where its expression was driven by the native, constitutive promoter PylxM. By maintaining the xylose concentration above 2.0 g L(-1), this auto-regulated expression system was absolutely stable after 100 generations. Compared with traditional antibiotic-dependent expression systems, this novel expression system resulted in greater biomass and higher titers of desired products (enzymes or metabolites). Our results demonstrate that this stable, food-grade expression system is suitable for enzyme production and pathway engineering, especially for the production of food-grade enzymes and metabolites. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis

    Science.gov (United States)

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928

  4. Evaluation of suitable reference genes for gene expression studies ...

    Indian Academy of Sciences (India)

    2011-12-14

    Dec 14, 2011 ... MADS family of TFs control floral organ identity within each whorl of the flower by activating downstream genes. Measuring gene expression in different tissue types and developmental stages is of fundamental importance in TFs functional research. In last few years, quantitative real-time. PCR (qRT-PCR) ...

  5. PRAME gene expression profile in medulloblastoma

    Directory of Open Access Journals (Sweden)

    Tânia Maria Vulcani-Freitas

    2011-02-01

    Full Text Available Medulloblastoma is the most common malignant tumors of central nervous system in the childhood. The treatment is severe, harmful and, thus, has a dismal prognosis. As PRAME is present in various cancers, including meduloblastoma, and has limited expression in normal tissues, this antigen can be an ideal vaccine target for tumor immunotherapy. In order to find a potential molecular target, we investigated PRAME expression in medulloblastoma fragments and we compare the results with the clinical features of each patient. Analysis of gene expression was performed by real-time quantitative PCR from 37 tumor samples. The Mann-Whitney test was used to analysis the relationship between gene expression and clinical characteristics. Kaplan-Meier curves were used to evaluate survival. PRAME was overexpressed in 84% samples. But no statistical association was found between clinical features and PRAME overexpression. Despite that PRAME gene could be a strong candidate for immunotherapy since it is highly expressed in medulloblastomas.

  6. Comparative gene expression between two yeast species

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    Guan Yuanfang

    2013-01-01

    Full Text Available Abstract Background Comparative genomics brings insight into sequence evolution, but even more may be learned by coupling sequence analyses with experimental tests of gene function and regulation. However, the reliability of such comparisons is often limited by biased sampling of expression conditions and incomplete knowledge of gene functions across species. To address these challenges, we previously systematically generated expression profiles in Saccharomyces bayanus to maximize functional coverage as compared to an existing Saccharomyces cerevisiae data repository. Results In this paper, we take advantage of these two data repositories to compare patterns of ortholog expression in a wide variety of conditions. First, we developed a scalable metric for expression divergence that enabled us to detect a significant correlation between sequence and expression conservation on the global level, which previous smaller-scale expression studies failed to detect. Despite this global conservation trend, between-species gene expression neighborhoods were less well-conserved than within-species comparisons across different environmental perturbations, and approximately 4% of orthologs exhibited a significant change in co-expression partners. Furthermore, our analysis of matched perturbations collected in both species (such as diauxic shift and cell cycle synchrony demonstrated that approximately a quarter of orthologs exhibit condition-specific expression pattern differences. Conclusions Taken together, these analyses provide a global view of gene expression patterns between two species, both in terms of the conditions and timing of a gene's expression as well as co-expression partners. Our results provide testable hypotheses that will direct future experiments to determine how these changes may be specified in the genome.

  7. Competing endogenous RNA network analysis identifies critical genes among the different breast cancer subtypes.

    Science.gov (United States)

    Chen, Juan; Xu, Juan; Li, Yongsheng; Zhang, Jinwen; Chen, Hong; Lu, Jianping; Wang, Zishan; Zhao, Xueying; Xu, Kang; Li, Yixue; Li, Xia; Zhang, Yan

    2017-02-07

    Although competing endogenous RNAs (ceRNAs) have been implicated in many solid tumors, their roles in breast cancer subtypes are not well understood. We therefore generated a ceRNA network for each subtype based on the significance of both, positive co-expression and the shared miRNAs, in the corresponding subtype miRNA dys-regulatory network, which was constructed based on negative regulations between differentially expressed miRNAs and targets. All four subtype ceRNA networks exhibited scale-free architecture and showed that the common ceRNAs were at the core of the networks. Furthermore, the common ceRNA hubs had greater connectivity than the subtype-specific hubs. Functional analysis of the common subtype ceRNA hubs highlighted factors involved in proliferation, MAPK signaling pathways and tube morphogenesis. Subtype-specific ceRNA hubs highlighted unique subtype-specific pathways, like the estrogen response and inflammatory pathways in the luminal subtypes or the factors involved in the coagulation process that participates in the basal-like subtype. Ultimately, we identified 29 critical subtype-specific ceRNA hubs that characterized the different breast cancer subtypes. Our study thus provides new insight into the common and specific subtype ceRNA interactions that define the different categories of breast cancer and enhances our understanding of the pathology underlying the different breast cancer subtypes, which can have prognostic and therapeutic implications in the future.

  8. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.

    2013-07-18

    The modeling of gene networks from transcriptional expression data is an important tool in biomedical research to reveal signaling pathways and to identify treatment targets. Current gene network modeling is primarily based on the use of Gaussian graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which generate counts of mRNAtranscripts in cell samples.We propose a generalized linear model to fit the discrete gene expression data and assume that the log ratios of the mean expression levels follow a Gaussian distribution.We restrict the gene network structures to decomposable graphs and derive the graphs by selecting the covariance matrix of the Gaussian distribution with the hyper-inverse Wishart priors. Furthermore, we incorporate prior network models based on gene ontology information, which avails existing biological information on the genes of interest. We conduct simulation studies to examine the performance of our discrete graphical model and apply the method to two real datasets for gene network inference. © The Author 2013. Published by Oxford University Press. All rights reserved.

  9. Gene expression profiling in autoimmune diseases

    DEFF Research Database (Denmark)

    Bovin, Lone Frier; Brynskov, Jørn; Hegedüs, Laszlo

    2007-01-01

    A central issue in autoimmune disease is whether the underlying inflammation is a repeated stereotypical process or whether disease specific gene expression is involved. To shed light on this, we analysed whether genes previously found to be differentially regulated in rheumatoid arthritis (RA...

  10. Bayesian assignment of gene ontology terms to gene expression experiments.

    Science.gov (United States)

    Sykacek, P

    2012-09-15

    Gene expression assays allow for genome scale analyses of molecular biological mechanisms. State-of-the-art data analysis provides lists of involved genes, either by calculating significance levels of mRNA abundance or by Bayesian assessments of gene activity. A common problem of such approaches is the difficulty of interpreting the biological implication of the resulting gene lists. This lead to an increased interest in methods for inferring high-level biological information. A common approach for representing high level information is by inferring gene ontology (GO) terms which may be attributed to the expression data experiment. This article proposes a probabilistic model for GO term inference. Modelling assumes that gene annotations to GO terms are available and gene involvement in an experiment is represented by a posterior probabilities over gene-specific indicator variables. Such probability measures result from many Bayesian approaches for expression data analysis. The proposed model combines these indicator probabilities in a probabilistic fashion and provides a probabilistic GO term assignment as a result. Experiments on synthetic and microarray data suggest that advantages of the proposed probabilistic GO term inference over statistical test-based approaches are in particular evident for sparsely annotated GO terms and in situations of large uncertainty about gene activity. Provided that appropriate annotations exist, the proposed approach is easily applied to inferring other high level assignments like pathways. Source code under GPL license is available from the author. peter.sykacek@boku.ac.at.

  11. Bayesian assignment of gene ontology terms to gene expression experiments

    Science.gov (United States)

    Sykacek, P.

    2012-01-01

    Motivation: Gene expression assays allow for genome scale analyses of molecular biological mechanisms. State-of-the-art data analysis provides lists of involved genes, either by calculating significance levels of mRNA abundance or by Bayesian assessments of gene activity. A common problem of such approaches is the difficulty of interpreting the biological implication of the resulting gene lists. This lead to an increased interest in methods for inferring high-level biological information. A common approach for representing high level information is by inferring gene ontology (GO) terms which may be attributed to the expression data experiment. Results: This article proposes a probabilistic model for GO term inference. Modelling assumes that gene annotations to GO terms are available and gene involvement in an experiment is represented by a posterior probabilities over gene-specific indicator variables. Such probability measures result from many Bayesian approaches for expression data analysis. The proposed model combines these indicator probabilities in a probabilistic fashion and provides a probabilistic GO term assignment as a result. Experiments on synthetic and microarray data suggest that advantages of the proposed probabilistic GO term inference over statistical test-based approaches are in particular evident for sparsely annotated GO terms and in situations of large uncertainty about gene activity. Provided that appropriate annotations exist, the proposed approach is easily applied to inferring other high level assignments like pathways. Availability: Source code under GPL license is available from the author. Contact: peter.sykacek@boku.ac.at PMID:22962488

  12. Quantification of endogenous and exogenous protein expressions of Na,K-ATPase with super-resolution PALM/STORM imaging.

    Science.gov (United States)

    Bernhem, Kristoffer; Blom, Hans; Brismar, Hjalmar

    2018-01-01

    Transient transfection of fluorescent fusion proteins is a key enabling technology in fluorescent microscopy to spatio-temporally map cellular protein distributions. Transient transfection of proteins may however bypass normal regulation of expression, leading to overexpression artefacts like misallocations and excess amounts. In this study we investigate the use of STORM and PALM microscopy to quantitatively monitor endogenous and exogenous protein expression. Through incorporation of an N-terminal hemagglutinin epitope to a mMaple3 fused Na,K-ATPase (α1 isoform), we analyze the spatial and quantitative changes of plasma membrane Na,K-ATPase localization during competitive transient expression. Quantification of plasma membrane protein density revealed a time dependent increase of Na,K-ATPase, but no increase in size of protein clusters. Results show that after 41h transfection, the total plasma membrane density of Na,K-ATPase increased by 63% while the endogenous contribution was reduced by 16%.

  13. Preimplantation Factor (PIF Promotes HLA-G, -E, -F, -C Expression in JEG-3 Choriocarcinoma Cells and Endogenous Progesterone Activity

    Directory of Open Access Journals (Sweden)

    Miya Soukaina Hakam

    2017-10-01

    Full Text Available Background/Aims: Pregnancy success requires mandatory maternal tolerance of the semi/ allogeneic embryo involving embryo-derived signals. Expression levels of PreImplantation Factor (PIF, a novel peptide secreted by viable embryos, correlate with embryo development, and its early detection in circulation correlates with a favourable pregnancy outcome. PIF enhances endometrial receptivity to promote embryo implantation. Via the p53 pathway, it increases trophoblast invasion, improving cell survival / immune privilege. PIF also reduces spontaneous and LPS-induced foetal death in immune naïve murine model. We examined PIF effect on gene expression of human leukocyte antigen (HLA-G, -E -F and –C and the influence of PIF on local progesterone activity in JEG-3 choriocarcinoma cells. Methods: PIF and progesterone (P4 effects on JEG-3 cells surface and intracellular HLA molecules was tested using monoclonal antibodies, flow cytometry, and Western blotting. PIF and IL17 effects on P4 and cytokines secretion was determined by ELISA. PIF and P4 effects on JEG-3 cells proteome was examined using 2D gel staining followed by spot analysis, mass spectrometry and bioinformatic analysis. Results: In cytotrophoblastic JEG-3 cells PIF increased intracellular expression of HLA-G, HLA-F, HLA-E and HLA-C and surface expression of HLA-G, HLA-E and HLA-C in dose and time dependent manner. In case of HLA-E, -F results were confirmed also by Western blot. Proteome analysis confirmed an increase in HLA-G, pro-tolerance FOXP3+ regulatory T cells (Tregs, coagulation factors and complement regulator. In contrast, PIF reduced PRDX2 and HSP70s to negate oxidative stress and protein misfolding. PIF enhanced local progesterone activity, increasing steroid secretion and the receptor protein. It also promoted the secretion of the Th1/Th2 cytokines (IL-10, IL-1β, IL-8, GM-CSF and TGF-β1, resulting in improved maternal signalling. Conclusion: PIF can generate a pro

  14. Reference Gene Screening for Analyzing Gene Expression Across Goat Tissue

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    2013-12-01

    Full Text Available Real-time quantitative PCR (qRT-PCR is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2 in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

  15. Dissecting Daily and Circadian Expression Rhythms of Clock-Controlled Genes in Human Blood.

    Science.gov (United States)

    Lech, Karolina; Ackermann, Katrin; Revell, Victoria L; Lao, Oscar; Skene, Debra J; Kayser, Manfred

    2016-02-01

    The identification and investigation of novel clock-controlled genes (CCGs) has been conducted thus far mainly in model organisms such as nocturnal rodents, with limited information in humans. Here, we aimed to characterize daily and circadian expression rhythms of CCGs in human peripheral blood during a sleep/sleep deprivation (S/SD) study and a constant routine (CR) study. Blood expression levels of 9 candidate CCGs (SREBF1, TRIB1, USF1, THRA1, SIRT1, STAT3, CAPRIN1, MKNK2, and ROCK2), were measured across 48 h in 12 participants in the S/SD study and across 33 h in 12 participants in the CR study. Statistically significant rhythms in expression were observed for STAT3, SREBF1, TRIB1, and THRA1 in samples from both the S/SD and the CR studies, indicating that their rhythmicity is driven by the endogenous clock. The MKNK2 gene was significantly rhythmic in the S/SD but not the CR study, which implies its exogenously driven rhythmic expression. In addition, we confirmed the circadian expression of PER1, PER3, and REV-ERBα in the CR study samples, while BMAL1 and HSPA1B were not significantly rhythmic in the CR samples; all 5 genes previously showed significant expression in the S/SD study samples. Overall, our results demonstrate that rhythmic expression patterns of clock and selected clock-controlled genes in human blood cells are in part determined by exogenous factors (sleep and fasting state) and in part by the endogenous circadian timing system. Knowledge of the exogenous and endogenous regulation of gene expression rhythms is needed prior to the selection of potential candidate marker genes for future applications in medical and forensic settings. © 2015 The Author(s).

  16. Novel Strategy to Control Transgene Expression Mediated by a Sendai Virus-Based Vector Using a Nonstructural C Protein and Endogenous MicroRNAs.

    Directory of Open Access Journals (Sweden)

    Masayuki Sano

    Full Text Available Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However, existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study, we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp. Because of the capacity of Sendai virus (SeV nonstructural C proteins to specifically inhibit viral RNA synthesis, overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression, target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore, the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications.

  17. Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase.

    Science.gov (United States)

    Yu, Kyung Ok; Jung, Ju; Kim, Seung Wook; Park, Chul Hwan; Han, Sung Ok

    2012-01-01

    The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol. Copyright © 2011 Wiley Periodicals, Inc.

  18. Noise minimization in eukaryotic gene expression.

    Directory of Open Access Journals (Sweden)

    Hunter B Fraser

    2004-06-01

    Full Text Available All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or "noise." Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  19. Noise minimization in eukaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-15

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  20. Noise minimization in eukaryotic gene expression

    International Nuclear Information System (INIS)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-01

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection

  1. KAP1 regulates type I interferon/STAT1-mediated IRF-1 gene expression

    International Nuclear Information System (INIS)

    Kamitani, Shinya; Ohbayashi, Norihiko; Ikeda, Osamu; Togi, Sumihito; Muromoto, Ryuta; Sekine, Yuichi; Ohta, Kazuhide; Ishiyama, Hironobu; Matsuda, Tadashi

    2008-01-01

    Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation, and survival in immune responses, hematopoiesis, neurogenesis, and other biological processes. Recently, we showed that KAP1 is a novel STAT-binding partner that regulates STAT3-mediated transactivation. KAP1 is a universal co-repressor protein for the KRAB zinc finger protein superfamily of transcriptional repressors. In this study, we found KAP1-dependent repression of interferon (IFN)/STAT1-mediated signaling. We also demonstrated that endogenous KAP1 associates with endogenous STAT1 in vivo. Importantly, a small-interfering RNA-mediated reduction in KAP1 expression enhanced IFN-induced STAT1-dependent IRF-1 gene expression. These results indicate that KAP1 may act as an endogenous regulator of the IFN/STAT1 signaling pathway

  2. Circadian cycles of gene expression in the coral, Acropora millepora.

    Directory of Open Access Journals (Sweden)

    Aisling K Brady

    Full Text Available Circadian rhythms regulate many physiological, behavioral and reproductive processes. These rhythms are often controlled by light, and daily cycles of solar illumination entrain many clock regulated processes. In scleractinian corals a number of different processes and behaviors are associated with specific periods of solar illumination or non-illumination--for example, skeletal deposition, feeding and both brooding and broadcast spawning.We have undertaken an analysis of diurnal expression of the whole transcriptome and more focused studies on a number of candidate circadian genes in the coral Acropora millepora using deep RNA sequencing and quantitative PCR. Many examples of diurnal cycles of RNA abundance were identified, some of which are light responsive and damped quickly under constant darkness, for example, cryptochrome 1 and timeless, but others that continue to cycle in a robust manner when kept in constant darkness, for example, clock, cryptochrome 2, cycle and eyes absent, indicating that their transcription is regulated by an endogenous clock entrained to the light-dark cycle. Many other biological processes that varied between day and night were also identified by a clustering analysis of gene ontology annotations.Corals exhibit diurnal patterns of gene expression that may participate in the regulation of circadian biological processes. Rhythmic cycles of gene expression occur under constant darkness in both populations of coral larvae that lack zooxanthellae and in individual adult tissue containing zooxanthellae, indicating that transcription is under the control of a biological clock. In addition to genes potentially involved in regulating circadian processes, many other pathways were found to display diel cycles of transcription.

  3. Flexible tools for gene expression and silencing in tomato.

    Science.gov (United States)

    Fernandez, Ana I; Viron, Nicolas; Alhagdow, Moftah; Karimi, Mansour; Jones, Matthew; Amsellem, Ziva; Sicard, Adrien; Czerednik, Anna; Angenent, Gerco; Grierson, Donald; May, Sean; Seymour, Graham; Eshed, Yuval; Lemaire-Chamley, Martine; Rothan, Christophe; Hilson, Pierre

    2009-12-01

    As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collections and ease of cultivation and transformation that enable the analysis of biological processes impossible to investigate in other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato and CRC from Arabidopsis (Arabidopsis thaliana). Corresponding transcriptional fusions were made with the GUS gene, a nuclear-localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues was confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR Clonase reactions. Efficient silencing of the endogenous phytoene desaturase gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the cauliflower mosaic virus 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well-characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new Nottingham Arabidopsis Stock Centre service unit dedicated to Solanaceae resources.

  4. TAF6delta controls apoptosis and gene expression in the absence of p53.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Wilhelm

    Full Text Available BACKGROUND: Life and death decisions of metazoan cells hinge on the balance between the expression of pro- versus anti-apoptotic gene products. The general RNA polymerase II transcription factor, TFIID, plays a central role in the regulation of gene expression through its core promoter recognition and co-activator functions. The core TFIID subunit TAF6 acts in vitro as an essential co-activator of transcription for the p53 tumor suppressor protein. We previously identified a splice variant of TAF6, termed TAF6delta that can be induced during apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the impact of TAF6delta on cell death and gene expression, we have employed modified antisense oligonucleotides to enforce expression of endogenous TAF6delta. The induction of endogenous TAF6delta triggered apoptosis in tumor cell lines, including cells devoid of p53. Microarray experiments revealed that TAF6delta activates gene expression independently of cellular p53 status. CONCLUSIONS: Our data define TAF6delta as a pivotal node in a signaling pathway that controls gene expression programs and apoptosis in the absence of p53.

  5. Sex hormones and gene expression signatures in peripheral blood from postmenopausal women - the NOWAC postgenome study

    Directory of Open Access Journals (Sweden)

    Rylander Charlotta

    2011-03-01

    Full Text Available Abstract Background Postmenopausal hormone therapy (HT influences endogenous hormone concentrations and increases the risk of breast cancer. Gene expression profiling may reveal the mechanisms behind this relationship. Our objective was to explore potential associations between sex hormones and gene expression in whole blood from a population-based, random sample of postmenopausal women Methods Gene expression, as measured by the Applied Biosystems microarray platform, was compared between hormone therapy (HT users and non-users and between high and low hormone plasma concentrations using both gene-wise analysis and gene set analysis. Gene sets found to be associated with HT use were further analysed for enrichment in functional clusters and network predictions. The gene expression matrix included 285 samples and 16185 probes and was adjusted for significant technical variables. Results Gene-wise analysis revealed several genes significantly associated with different types of HT use. The functional cluster analyses provided limited information on these genes. Gene set analysis revealed 22 gene sets that were enriched between high and low estradiol concentration (HT-users excluded. Among these were seven oestrogen related gene sets, including our gene list associated with systemic estradiol use, which thereby represents a novel oestrogen signature. Seven gene sets were related to immune response. Among the 15 gene sets enriched for progesterone, 11 overlapped with estradiol. No significant gene expression patterns were found for testosterone, follicle stimulating hormone (FSH or sex hormone binding globulin (SHBG. Conclusions Distinct gene expression patterns associated with sex hormones are detectable in a random group of postmenopausal women, as demonstrated by the finding of a novel oestrogen signature.

  6. Expression Study of Banana Pathogenic Resistance Genes

    Directory of Open Access Journals (Sweden)

    Fenny M. Dwivany

    2016-10-01

    Full Text Available Banana is one of the world's most important trade commodities. However, infection of banana pathogenic fungi (Fusarium oxysporum race 4 is one of the major causes of decreasing production in Indonesia. Genetic engineering has become an alternative way to control this problem by isolating genes that involved in plant defense mechanism against pathogens. Two of the important genes are API5 and ChiI1, each gene encodes apoptosis inhibitory protein and chitinase enzymes. The purpose of this study was to study the expression of API5 and ChiI1 genes as candidate pathogenic resistance genes. The amplified fragments were then cloned, sequenced, and confirmed with in silico studies. Based on sequence analysis, it is showed that partial API5 gene has putative transactivation domain and ChiI1 has 9 chitinase family GH19 protein motifs. Data obtained from this study will contribute in banana genetic improvement.

  7. Dlx homeobox gene family expression in osteoclasts.

    Science.gov (United States)

    Lézot, F; Thomas, B L; Blin-Wakkach, C; Castaneda, B; Bolanos, A; Hotton, D; Sharpe, P T; Heymann, D; Carles, G F; Grigoriadis, A E; Berdal, A

    2010-06-01

    Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time- and site-specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts. (c) 2010 Wiley-Liss, Inc.

  8. Hepatocyte specific expression of human cloned genes

    Energy Technology Data Exchange (ETDEWEB)

    Cortese, R

    1986-01-01

    A large number of proteins are specifically synthesized in the hepatocyte. Only the adult liver expresses the complete repertoire of functions which are required at various stages during development. There is therefore a complex series of regulatory mechanisms responsible for the maintenance of the differentiated state and for the developmental and physiological variations in the pattern of gene expression. Human hepatoma cell lines HepG2 and Hep3B display a pattern of gene expression similar to adult and fetal liver, respectively; in contrast, cultured fibroblasts or HeLa cells do not express most of the liver specific genes. They have used these cell lines for transfection experiments with cloned human liver specific genes. DNA segments coding for alpha1-antitrypsin and retinol binding protein (two proteins synthesized both in fetal and adult liver) are expressed in the hepatoma cell lines HepG2 and Hep3B, but not in HeLa cells or fibroblasts. A DNA segment coding for haptoglobin (a protein synthesized only after birth) is only expressed in the hepatoma cell line HepG2 but not in Hep3B nor in non hepatic cell lines. The information for tissue specific expression is located in the 5' flanking region of all three genes. In vivo competition experiments show that these DNA segments bind to a common, apparently limiting, transacting factor. Conventional techniques (Bal deletions, site directed mutagenesis, etc.) have been used to precisely identify the DNA sequences responsible for these effects. The emerging picture is complex: they have identified multiple, separate transcriptional signals, essential for maximal promoter activation and tissue specific expression. Some of these signals show a negative effect on transcription in fibroblast cell lines.

  9. Gene expression profiles in skeletal muscle after gene electrotransfer

    DEFF Research Database (Denmark)

    Hojman, Pernille; Zibert, John R; Gissel, Hanne

    2007-01-01

    BACKGROUND: Gene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have...... caused down-regulation of structural proteins e.g. sarcospan and catalytic enzymes. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related......) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment. RESULTS: Differentially expressed genes were...

  10. Gene expression analysis of flax seed development

    Science.gov (United States)

    2011-01-01

    Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise

  11. Gene expression analysis of flax seed development

    Directory of Open Access Journals (Sweden)

    Sharpe Andrew

    2011-04-01

    Full Text Available Abstract Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages seed coats (globular and torpedo stages and endosperm (pooled globular to torpedo stages and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST (GenBank accessions LIBEST_026995 to LIBEST_027011 were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152 had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid

  12. Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum

    NARCIS (Netherlands)

    Peters, Dorien J.M.; Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van; Spek, Wouter; Schaap, Pauline

    1989-01-01

    We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl.

  13. Scaling of gene expression data allowing the comparison of different gene expression platforms

    NARCIS (Netherlands)

    van Ruissen, Fred; Schaaf, Gerben J.; Kool, Marcel; Baas, Frank; Ruijter, Jan M.

    2008-01-01

    Serial analysis of gene expression (SAGE) and microarrays have found a widespread application, but much ambiguity exists regarding the amalgamation of the data resulting from these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce

  14. Renal Gene Expression Database (RGED): a relational database of gene expression profiles in kidney disease.

    Science.gov (United States)

    Zhang, Qingzhou; Yang, Bo; Chen, Xujiao; Xu, Jing; Mei, Changlin; Mao, Zhiguo

    2014-01-01

    We present a bioinformatics database named Renal Gene Expression Database (RGED), which contains comprehensive gene expression data sets from renal disease research. The web-based interface of RGED allows users to query the gene expression profiles in various kidney-related samples, including renal cell lines, human kidney tissues and murine model kidneys. Researchers can explore certain gene profiles, the relationships between genes of interests and identify biomarkers or even drug targets in kidney diseases. The aim of this work is to provide a user-friendly utility for the renal disease research community to query expression profiles of genes of their own interest without the requirement of advanced computational skills. Website is implemented in PHP, R, MySQL and Nginx and freely available from http://rged.wall-eva.net. http://rged.wall-eva.net. © The Author(s) 2014. Published by Oxford University Press.

  15. Renal Gene Expression Database (RGED): a relational database of gene expression profiles in kidney disease

    Science.gov (United States)

    Zhang, Qingzhou; Yang, Bo; Chen, Xujiao; Xu, Jing; Mei, Changlin; Mao, Zhiguo

    2014-01-01

    We present a bioinformatics database named Renal Gene Expression Database (RGED), which contains comprehensive gene expression data sets from renal disease research. The web-based interface of RGED allows users to query the gene expression profiles in various kidney-related samples, including renal cell lines, human kidney tissues and murine model kidneys. Researchers can explore certain gene profiles, the relationships between genes of interests and identify biomarkers or even drug targets in kidney diseases. The aim of this work is to provide a user-friendly utility for the renal disease research community to query expression profiles of genes of their own interest without the requirement of advanced computational skills. Availability and implementation: Website is implemented in PHP, R, MySQL and Nginx and freely available from http://rged.wall-eva.net. Database URL: http://rged.wall-eva.net PMID:25252782

  16. Pseudogenes regulate parental gene expression via ceRNA network.

    Science.gov (United States)

    An, Yang; Furber, Kendra L; Ji, Shaoping

    2017-01-01

    The concept of competitive endogenous RNA (ceRNA) was first proposed by Salmena and colleagues. Evidence suggests that pseudogene RNAs can act as a 'sponge' through competitive binding of common miRNA, releasing or attenuating repression through sequestering miRNAs away from parental mRNA. In theory, ceRNAs refer to all transcripts such as mRNA, tRNA, rRNA, long non-coding RNA, pseudogene RNA and circular RNA, because all of them may become the targets of miRNA depending on spatiotemporal situation. As binding of miRNA to the target RNA is not 100% complementary, it is possible that one miRNA can bind to multiple target RNAs and vice versa. All RNAs crosstalk through competitively binding to miRNAvia miRNA response elements (MREs) contained within the RNA sequences, thus forming a complex regulatory network. The ratio of a subset of miRNAs to the corresponding number of MREs determines repression strength on a given mRNA translation or stability. An increase in pseudogene RNA level can sequester miRNA and release repression on the parental gene, leading to an increase in parental gene expression. A massive number of transcripts constitute a complicated network that regulates each other through this proposed mechanism, though some regulatory significance may be mild or even undetectable. It is possible that the regulation of gene and pseudogene expression occurring in this manor involves all RNAs bearing common MREs. In this review, we will primarily discuss how pseudogene transcripts regulate expression of parental genes via ceRNA network and biological significance of regulation. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  17. Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

    Science.gov (United States)

    de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

    2013-05-01

    Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

  18. Collaborative ring trial of the papaya endogenous reference gene and its polymerase chain reaction assays for genetically modified organism analysis.

    Science.gov (United States)

    Wei, Jiaojun; Li, Feiwu; Guo, Jinchao; Li, Xiang; Xu, Junfeng; Wu, Gang; Zhang, Dabing; Yang, Litao

    2013-11-27

    The papaya (Carica papaya L.) Chymopapain (CHY) gene has been reported as a suitable endogenous reference gene for genetically modified (GM) papaya detection in previous studies. Herein, we further validated the use of the CHY gene and its qualitative and quantitative polymerase chain reaction (PCR) assays through an interlaboratory collaborative ring trial. A total of 12 laboratories working on detection of genetically modified organisms participated in the ring trial and returned test results. Statistical analysis of the returned results confirmed the species specificity, low heterogeneity, and single-copy number of the CHY gene among different papaya varieties. The limit of detection of the CHY qualitative PCR assay was 0.1%, while the limit of quantification of the quantitative PCR assay was ∼25 copies of haploid papaya genome with acceptable PCR efficiency and linearity. The differences between the tested and true values of papaya content in 10 blind samples ranged from 0.84 to 6.58%. These results indicated that the CHY gene was suitable as an endogenous reference gene for the identification and quantification of GM papaya.

  19. Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins.

    Science.gov (United States)

    Sakai, Miho; Sakamoto, Tomoaki; Saito, Tamio; Matsuoka, Makoto; Tanaka, Hiroshi; Kobayashi, Masatomo

    2003-04-01

    We have cloned two genes for gibberellin (GA) 2-oxidase from rice ( Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA(1) to GA(8) and GA(20) to GA(29)-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice.

  20. Expression of Versican 3′-Untranslated Region Modulates Endogenous MicroRNA Functions

    Science.gov (United States)

    Lee, Daniel Y.; Jeyapalan, Zina; Fang, Ling; Yang, Jennifer; Zhang, Yaou; Yee, Albert Y.; Li, Minhui; Du, William W.; Shatseva, Tatiana; Yang, Burton B.

    2010-01-01

    Background Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3′UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. Methods and Findings Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3′UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3′UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3′UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3′UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3′UTR formed smaller tumors compared with cells transfected with a control vector. Conclusion Our results demonstrated that a 3′UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3′UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities. PMID:21049042

  1. Expression of versican 3'-untranslated region modulates endogenous microRNA functions.

    Science.gov (United States)

    Lee, Daniel Y; Jeyapalan, Zina; Fang, Ling; Yang, Jennifer; Zhang, Yaou; Yee, Albert Y; Li, Minhui; Du, William W; Shatseva, Tatiana; Yang, Burton B

    2010-10-25

    Mature microRNAs (miRNAs) are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

  2. Expression of versican 3'-untranslated region modulates endogenous microRNA functions.

    Directory of Open Access Journals (Sweden)

    Daniel Y Lee

    Full Text Available BACKGROUND: Mature microRNAs (miRNAs are single-stranded RNAs that regulate post-transcriptional gene expression. In our previous study, we have shown that versican 3'UTR, a fragment of non-coding transcript, has the ability to antagonize miR-199a-3p function thereby regulating expression of the matrix proteins versican and fibronectin, and thus resulting in enhanced cell-cell adhesion and organ adhesion. However, the impact of this non-coding fragment on tumorigenesis is yet to be determined. METHODS AND FINDINGS: Using computational prediction confirmed with in vitro and in vivo experiments, we report that the expression of versican 3'UTR not only antagonizes miR-199a-3p but can also lower its steady state expression. We found that expression of versican 3'UTR in a mouse breast carcinoma cell line, 4T1, decreased miR-199a-3p levels. The decrease in miRNA activity consequently translated into differences in tumor growth. Computational analysis indicated that both miR-199a-3p and miR-144 targeted a cell cycle regulator, Rb1. In addition, miR-144 and miR-136, which have also been shown to interact with versican 3'UTR, was found to target PTEN. Expression of Rb1 and PTEN were up-regulated synergistically in vitro and in vivo, suggesting that the 3'UTR binds and modulates miRNA activities, freeing Rb1 and PTEN mRNAs for translation. In tumor formation assays, cells transfected with the 3'UTR formed smaller tumors compared with cells transfected with a control vector. CONCLUSION: Our results demonstrated that a 3'UTR fragment can be used to modulate miRNA functions. Our study also suggests that miRNAs in the cancer cells are more susceptible to degradation, due to its interaction with a non-coding 3'UTR. This non-coding component of mRNA may be used retrospectively to modulate miRNA activities.

  3. Gene expression analysis identifies global gene dosage sensitivity in cancer

    DEFF Research Database (Denmark)

    Fehrmann, Rudolf S. N.; Karjalainen, Juha M.; Krajewska, Malgorzata

    2015-01-01

    Many cancer-associated somatic copy number alterations (SCNAs) are known. Currently, one of the challenges is to identify the molecular downstream effects of these variants. Although several SCNAs are known to change gene expression levels, it is not clear whether each individual SCNA affects gen...

  4. Metallothionein gene expression in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Deeksha Pal

    2014-01-01

    Full Text Available Introduction: Metallothioneins (MTs are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1 the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2 apoptosis and (3 the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC. No information is available on the gene expression of MT2A isoform in different types and grades of RCC. Materials and Methods: In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchyma was taken as control. Real-time polymerase chain reaction (RT PCR analysis was done for the MT2A gene expression using b-actin as an internal control. All statistical calculations were performed using SPSS software. Results: The MT2A gene expression was found to be significantly increased (P < 0.01 in clear cell RCC in comparison with the adjacent normal renal parenchyma. The expression of MT2A was two to three-fold higher in sarcomatoid RCC, whereas there was no change in papillary and collecting duct RCC. MT2A gene expression was significantly higher in lower grade (grades I and II, P < 0.05, while no change was observed in high-grade tumor (grade III and IV in comparison to adjacent normal renal tissue. Conclusion: The first report of the expression of MT2A in different types and grades of RCC and also these data further support the role of MT2A in tumorigenesis.

  5. Analysis of baseline gene expression levels from ...

    Science.gov (United States)

    The use of gene expression profiling to predict chemical mode of action would be enhanced by better characterization of variance due to individual, environmental, and technical factors. Meta-analysis of microarray data from untreated or vehicle-treated animals within the control arm of toxicogenomics studies has yielded useful information on baseline fluctuations in gene expression. A dataset of control animal microarray expression data was assembled by a working group of the Health and Environmental Sciences Institute's Technical Committee on the Application of Genomics in Mechanism Based Risk Assessment in order to provide a public resource for assessments of variability in baseline gene expression. Data from over 500 Affymetrix microarrays from control rat liver and kidney were collected from 16 different institutions. Thirty-five biological and technical factors were obtained for each animal, describing a wide range of study characteristics, and a subset were evaluated in detail for their contribution to total variability using multivariate statistical and graphical techniques. The study factors that emerged as key sources of variability included gender, organ section, strain, and fasting state. These and other study factors were identified as key descriptors that should be included in the minimal information about a toxicogenomics study needed for interpretation of results by an independent source. Genes that are the most and least variable, gender-selectiv

  6. Gene expression of the endolymphatic sac

    DEFF Research Database (Denmark)

    Friis, Morten; Martin-Bertelsen, Tomas; Friis-Hansen, Lennart

    2011-01-01

    that the endolymphatic sac has multiple and diverse functions in the inner ear. Objectives:The objective of this study was to provide a comprehensive review of the genes expressed in the endolymphatic sac in the rat and perform a functional characterization based on measured mRNA abundance. Methods:Microarray technology...

  7. Gene expression in early stage cervical cancer

    NARCIS (Netherlands)

    Biewenga, Petra; Buist, Marrije R.; Moerland, Perry D.; van Thernaat, Emiel Ver Loren; van Kampen, Antoine H. C.; ten Kate, Fiebo J. W.; Baas, Frank

    2008-01-01

    Objective. Pelvic lymph node metastases are the main prognostic factor for survival in early stage cervical cancer, yet accurate detection methods before surgery are lacking. In this study, we examined whether gene expression profiling can predict the presence of lymph node metastasis in early stage

  8. Shrinkage Approach for Gene Expression Data Analysis

    Czech Academy of Sciences Publication Activity Database

    Haman, Jiří; Valenta, Zdeněk; Kalina, Jan

    2013-01-01

    Roč. 1, č. 1 (2013), s. 65-65 ISSN 1805-8698. [EFMI 2013 Special Topic Conference. 17.04.2013-19.04.2013, Prague] Institutional support: RVO:67985807 Keywords : shrinkage estimation * covariance matrix * high dimensional data * gene expression Subject RIV: IN - Informatics, Computer Science

  9. Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

    Directory of Open Access Journals (Sweden)

    Zanatta Daniela B

    2010-06-01

    Full Text Available Abstract Background Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods B16 (mouse melanoma and C6 (rat glioma cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet

  10. Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

    International Nuclear Information System (INIS)

    Merkel, Christian A; Silva Soares, Rafael B da; Carvalho, Anna Carolina V de; Zanatta, Daniela B; Bajgelman, Marcio C; Fratini, Paula; Costanzi-Strauss, Eugenia; Strauss, Bryan E

    2010-01-01

    Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19

  11. Temporal expression of genes involved in the biosynthesis of ...

    African Journals Online (AJOL)

    Gibberellins (GAs) are a large family of endogenous plant growth regulators. Bioactive GAs influence nearly all processes during plant growth and development. In the present study, we cloned and identified 10 unique genes that are potentially involved in the biosynthesis of GAs, including one BpGGDP gene, two BpCPS ...

  12. Regulation of methane genes and genome expression

    Energy Technology Data Exchange (ETDEWEB)

    John N. Reeve

    2009-09-09

    At the start of this project, it was known that methanogens were Archaeabacteria (now Archaea) and were therefore predicted to have gene expression and regulatory systems different from Bacteria, but few of the molecular biology details were established. The goals were then to establish the structures and organizations of genes in methanogens, and to develop the genetic technologies needed to investigate and dissect methanogen gene expression and regulation in vivo. By cloning and sequencing, we established the gene and operon structures of all of the “methane” genes that encode the enzymes that catalyze methane biosynthesis from carbon dioxide and hydrogen. This work identified unique sequences in the methane gene that we designated mcrA, that encodes the largest subunit of methyl-coenzyme M reductase, that could be used to identify methanogen DNA and establish methanogen phylogenetic relationships. McrA sequences are now the accepted standard and used extensively as hybridization probes to identify and quantify methanogens in environmental research. With the methane genes in hand, we used northern blot and then later whole-genome microarray hybridization analyses to establish how growth phase and substrate availability regulated methane gene expression in Methanobacterium thermautotrophicus ΔH (now Methanothermobacter thermautotrophicus). Isoenzymes or pairs of functionally equivalent enzymes catalyze several steps in the hydrogen-dependent reduction of carbon dioxide to methane. We established that hydrogen availability determine which of these pairs of methane genes is expressed and therefore which of the alternative enzymes is employed to catalyze methane biosynthesis under different environmental conditions. As were unable to establish a reliable genetic system for M. thermautotrophicus, we developed in vitro transcription as an alternative system to investigate methanogen gene expression and regulation. This led to the discovery that an archaeal protein

  13. Fluid Mechanics, Arterial Disease, and Gene Expression.

    Science.gov (United States)

    Tarbell, John M; Shi, Zhong-Dong; Dunn, Jessilyn; Jo, Hanjoong

    2014-01-01

    This review places modern research developments in vascular mechanobiology in the context of hemodynamic phenomena in the cardiovascular system and the discrete localization of vascular disease. The modern origins of this field are traced, beginning in the 1960s when associations between flow characteristics, particularly blood flow-induced wall shear stress, and the localization of atherosclerotic plaques were uncovered, and continuing to fluid shear stress effects on the vascular lining endothelial) cells (ECs), including their effects on EC morphology, biochemical production, and gene expression. The earliest single-gene studies and genome-wide analyses are considered. The final section moves from the ECs lining the vessel wall to the smooth muscle cells and fibroblasts within the wall that are fluid me chanically activated by interstitial flow that imposes shear stresses on their surfaces comparable with those of flowing blood on EC surfaces. Interstitial flow stimulates biochemical production and gene expression, much like blood flow on ECs.

  14. Gene Expression Commons: an open platform for absolute gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Jun Seita

    Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  15. Comparative gene expression of intestinal metabolizing enzymes.

    Science.gov (United States)

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs.

  16. Endogene CGRP

    OpenAIRE

    Höfer, Martina

    2010-01-01

    Hintergrund und Ziele Die vorliegende tierexperimentelle Arbeit beschäftigt sich mit der Frage, welche Rolle endogenes Calcitonin-gene related peptide (CGRP) in der Niere spielt. Hierbei untersuchten wir die renale CGRP Freisetzung aus renalen Afferenzen in vitro anhand von gesunden Tieren und einem pathologischen Modell der Glomerulonephritis. Man weiß bereits, dass sowohl sympathische als auch primär sensorische Neuronen die Entzündung und die Immunantwort in der Peripherie regulieren (68)....

  17. Modeling insertional mutagenesis using gene length and expression in murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Alex S Nord

    2007-07-01

    Full Text Available High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of gene-trapping events have not been tested on a genome-wide scale.In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identified hotspots of gene-trap activity, which represent loci where the high likelihood of vector insertion is controlled by factors other than length and expression. These formalized statistical models describe a high proportion of the variance in the likelihood of a gene being trapped by expression-dependent vectors and a lower, but still significant, proportion of the variance for vectors that are predicted to be independent of endogenous gene expression.The findings of significant expression and length effects reported here further the understanding of the determinants of vector insertion. Results from this analysis can be applied to help identify other important determinants of this important biological phenomenon and could assist planning of large-scale mutagenesis efforts.

  18. Regulation of laminin beta2 chain gene expression in human cancer cell lines

    DEFF Research Database (Denmark)

    Durkin, M E; Nielsen, F C; Loechel, F

    2001-01-01

    of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain m......RNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates....

  19. Structure and expression of thyroglobulin gene

    Energy Technology Data Exchange (ETDEWEB)

    Vassart, G; Brocas, H; Christophe, D; de Martynoff, G; Leriche, A; Mercken, L; Pohl, V; van Heuverswyn, B [Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucleaire (IRIBHN), Faculte de Medecine, Universite libre de Bruxelles, Campus Hopital Erasme, Brussels (Belgium)

    1982-01-01

    Thyroglobulin is composed of two 300000 dalton polypeptide chains, translated from an 8000 base mRNA. Preparation of a full length cDNA and its cloning in E. coli have lead to the demonstration that the polypeptides of thyroglobulin protomers were identical. Used as molecular probes, the cloned cDNA allowed the isolation of a fragment of thyroglobulin gene. Electron microscopic studies have demonstrated that this gene contains more than 90 % intronic material separating small size exons (<200 bp). Sequencing of bovine thyroglobulin structural gene is in progress. Preliminary results show evidence for the existence of repetitive segments. Availability of cloned DNA complementary to bovine and human thyroglobulin mRNA allows the study of genetic defects of thyroglobulin gene expression in the human and in various animal models.

  20. Cerebrovascular gene expression in spontaneously hypertensive rats

    DEFF Research Database (Denmark)

    Grell, Anne-Sofie; Frederiksen, Simona Denise; Edvinsson, Lars

    2017-01-01

    Hypertension is a hemodynamic disorder and one of the most important and well-established risk factors for vascular diseases such as stroke. Blood vessels exposed to chronic shear stress develop structural changes and remodeling of the vascular wall through many complex mechanisms. However......, the molecular mechanisms involved are not fully understood. Hypertension-susceptible genes may provide a novel insight into potential molecular mechanisms of hypertension and secondary complications associated with hypertension. The aim of this exploratory study was to identify gene expression differences......, the identified genes in the middle cerebral arteries from spontaneously hypertensive rats could be possible mediators of the vascular changes and secondary complications associated with hypertension. This study supports the selection of key genes to investigate in the future research of hypertension-induced end...

  1. miR-370 suppresses HBV gene expression and replication by targeting nuclear factor IA.

    Science.gov (United States)

    Fan, Hongxia; Lv, Ping; Lv, Jing; Zhao, Xiaopei; Liu, Min; Zhang, Guangling; Tang, Hua

    2017-05-01

    Hepatitis B virus (HBV) infection is a major health problem worldwide. The roles of microRNAs in the regulation of HBV expression are being increasingly recognized. In this study, we found that overexpression of miR-370 suppressed HBV gene expression and replication in Huh7 cells, whereas antisense knockdown of endogenous miR-370 enhanced HBV gene expression and replication in Huh7 cells and HepG2.2.15 cells. Further, we identified the transcription factor nuclear factor IA (NFIA) as a new host target of miR-370. Overexpression and knockdown studies showed that NFIA stimulated HBV gene expression and replication. Importantly, overexpression of NFIA counteracted the effect of miR-370 on HBV gene expression and replication. Further mechanistic studies showed that miR-370 suppressed HBV replication and gene expression by repressing HBV Enhancer I activity, and one of the NFIA binding site in the Enhancer I element was responsible for the repressive effect of miR-370 on HBV Enhancer I activity. Altogether, our results demonstrated that miR-370 suppressed HBV gene expression and replication through repressing NFIA expression, which stimulates HBV replication via direct regulation on HBV Enhancer I activities. Our findings may provide a new antiviral strategy for HBV infection. J. Med. Virol. 89:834-844, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Digital gene expression analysis of gene expression differences within Brassica diploids and allopolyploids.

    Science.gov (United States)

    Jiang, Jinjin; Wang, Yue; Zhu, Bao; Fang, Tingting; Fang, Yujie; Wang, Youping

    2015-01-27

    Brassica includes many successfully cultivated crop species of polyploid origin, either by ancestral genome triplication or by hybridization between two diploid progenitors, displaying complex repetitive sequences and transposons. The U's triangle, which consists of three diploids and three amphidiploids, is optimal for the analysis of complicated genomes after polyploidization. Next-generation sequencing enables the transcriptome profiling of polyploids on a global scale. We examined the gene expression patterns of three diploids (Brassica rapa, B. nigra, and B. oleracea) and three amphidiploids (B. napus, B. juncea, and B. carinata) via digital gene expression analysis. In total, the libraries generated between 5.7 and 6.1 million raw reads, and the clean tags of each library were mapped to 18547-21995 genes of B. rapa genome. The unambiguous tag-mapped genes in the libraries were compared. Moreover, the majority of differentially expressed genes (DEGs) were explored among diploids as well as between diploids and amphidiploids. Gene ontological analysis was performed to functionally categorize these DEGs into different classes. The Kyoto Encyclopedia of Genes and Genomes analysis was performed to assign these DEGs into approximately 120 pathways, among which the metabolic pathway, biosynthesis of secondary metabolites, and peroxisomal pathway were enriched. The non-additive genes in Brassica amphidiploids were analyzed, and the results indicated that orthologous genes in polyploids are frequently expressed in a non-additive pattern. Methyltransferase genes showed differential expression pattern in Brassica species. Our results provided an understanding of the transcriptome complexity of natural Brassica species. The gene expression changes in diploids and allopolyploids may help elucidate the morphological and physiological differences among Brassica species.

  3. Global gene expression in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, K.; Klemm, Per

    2003-01-01

    It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance to antimicr......It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance...... the transition to biofilm growth, and these included genes expressed under oxygen-limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered...... in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces....

  4. Gene Therapy with Endogenous Inhibitors of Angiogenesis for Neovascular Age-Related Macular Degeneration: Beyond Anti-VEGF Therapy

    Directory of Open Access Journals (Sweden)

    Selwyn M. Prea

    2015-01-01

    Full Text Available Age-related macular degeneration (AMD is the leading cause of substantial and irreversible vision loss amongst elderly populations in industrialized countries. The advanced neovascular (or “wet” form of the disease is responsible for severe and aggressive loss of central vision. Current treatments aim to seal off leaky blood vessels via laser therapy or to suppress vessel leakage and neovascular growth through intraocular injections of antibodies that target vascular endothelial growth factor (VEGF. However, the long-term success of anti-VEGF therapy can be hampered by limitations such as low or variable efficacy, high frequency of administration (usually monthly, potentially serious side effects, and, most importantly, loss of efficacy with prolonged treatment. Gene transfer of endogenous antiangiogenic proteins is an alternative approach that has the potential to provide long-term suppression of neovascularization and/or excessive vascular leakage in the eye. Preclinical studies of gene transfer in a large animal model have provided impressive preliminary results with a number of transgenes. In addition, a clinical trial in patients suffering from advanced neovascular AMD has provided proof-of-concept for successful gene transfer. In this mini review, we summarize current theories pertaining to the application of gene therapy for neovascular AMD and the potential benefits when used in conjunction with endogenous antiangiogenic proteins.

  5. Aberrant Gene Expression in Acute Myeloid Leukaemia

    DEFF Research Database (Denmark)

    Bagger, Frederik Otzen

    model to investigate the role of telomerase in AML, we were able to translate the observed effect into human AML patients and identify specific genes involved, which also predict survival patterns in AML patients. During these studies we have applied methods for investigating differentially expressed......-based gene-lookup webservices, called HemaExplorer and BloodSpot. These web-services support the aim of making data and analysis of haematopoietic cells from mouse and human accessible for researchers without bioinformatics expertise. Finally, in order to aid the analysis of the very limited number...

  6. A Single Argonaute Gene Participates in Exogenous and Endogenous RNAi and Controls Cellular Functions in the Basal Fungus Mucor circinelloides

    Science.gov (United States)

    Nicolás, Francisco E.; Moxon, Simon; de Haro, Juan P.; Dalmay, Tamas; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M

    2013-01-01

    The mechanism of RNAi is well described in metazoans where it plays a role in diverse cellular functions. However, although different classes of endogenous small RNAs (esRNAs) have been identified in fungi, their biological roles are poorly described due, in part, to the lack of phenotype of mutants affected in the biogenesis of these esRNAs. Argonaute proteins are one of the key components of the RNAi pathways, in which different members of this protein family participate in the biogenesis of a wide repertoire of esRNAs molecules. Here we identified three argonaute genes of the fungus Mucor circinelloides and investigated their participation in exogenous and endogenous RNAi. We found that only one of the ago genes, ago-1, is involved in RNAi during vegetative growth and is required for both transgene-induced RNA silencing and the accumulation of distinct classes of esRNAs derived from exons (ex-siRNAs). Classes I and II ex-siRNAs bind to Ago-1 to control mRNA accumulation of the target protein coding genes. Class III ex-siRNAs do not specifically bind to Ago-1, but requires this protein for their production, revealing the complexity of the biogenesis pathways of ex-siRNAs. We also show that ago-1 is involved in the response to environmental signals, since vegetative development and autolysis induced by nutritional stress are affected in ago-1 − M. circinelloides mutants. Our results demonstrate that a single Ago protein participates in the production of different classes of esRNAs that are generated through different pathways. They also highlight the role of ex-siRNAs in the regulation of endogenous genes in fungi and expand the range of biological functions modulated by RNAi. PMID:23935973

  7. A subset of osteoblasts expressing high endogenous levels of PPARgamma switches fate to adipocytes in the rat calvaria cell culture model.

    Directory of Open Access Journals (Sweden)

    Yuji Yoshiko

    2010-07-01

    Full Text Available Understanding fate choice and fate switching between the osteoblast lineage (ObL and adipocyte lineage (AdL is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPARgamma.Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL, a synthetic ligand for PPARgamma. About 30% of the total single cell-derived colonies expressed adipogenic potential (defined cytochemically when BRL was present. Profiling of ObL and AdL markers by qRT-PCR on amplified cRNA from over 160 colonies revealed that BRL-dependent adipogenic potential correlated with endogenous PPARgamma mRNA levels. Unexpectedly, a significant subset of relatively mature ObL cells exhibited osteo-adipogenic bipotentiality. Western blotting and immunocytochemistry confirmed that ObL cells co-expressed multiple mesenchymal lineage determinants (runt-related transcription factor 2 (Runx2, PPARgamma, Sox9 and MyoD which localized in the cytoplasm initially, and only Runx2 translocated to the nucleus during ObL progression. Notably, however, some cells exhibited both PPARgamma and Runx2 nuclear labeling with concomitant upregulation of expression of their target genes with BRL treatment.We conclude that not only immature but a subset of relatively mature ObL cells characterized by relatively high levels of endogenous PPARgamma expression can be switched to the AdL. The fact that some ObL cells maintain capacity for adipogenic fate selection even at relatively

  8. DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas

    Czech Academy of Sciences Publication Activity Database

    Benešová, Martina; Trejbalová, Kateřina; Kovářová, Denisa; Vernerová, Z.; Hron, Tomáš; Kučerová, Dana; Hejnar, Jiří

    2017-01-01

    Roč. 14, č. 1 (2017), č. článku 20. ISSN 1742-4690 R&D Projects: GA ČR GA13-37600S; GA MZd NT14601 Institutional support: RVO:68378050 Keywords : Human endogenous retrovirus * ERVWE1 * Germ cell tumor * Seminoma * Promoter DNA methylation * 5-Hydroxymethylcytosine * Transcription * RNA splicing Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Virology Impact factor: 3.867, year: 2016

  9. Methylated DNA Immunoprecipitation Analysis of Mammalian Endogenous Retroviruses.

    Science.gov (United States)

    Rebollo, Rita; Mager, Dixie L

    2016-01-01

    Endogenous retroviruses are repetitive sequences found abundantly in mammalian genomes which are capable of modulating host gene expression. Nevertheless, most endogenous retrovirus copies are under tight epigenetic control via histone-repressive modifications and DNA methylation. Here we describe a common method used in our laboratory to detect, quantify, and compare mammalian endogenous retrovirus DNA methylation. More specifically we describe methylated DNA immunoprecipitation (MeDIP) followed by quantitative PCR.

  10. Selection of Housekeeping Genes for Transgene Expression Analysis in Eucommia ulmoides Oliver Using Real-Time RT-PCR

    Directory of Open Access Journals (Sweden)

    Ren Chen

    2010-01-01

    Full Text Available In order to select appropriate housekeeping genes for accurate calibration of experimental variations in real-time (RT- PCR results in transgene expression analysis, particularly with respect to the influence of transgene on stability of endogenous housekeeping gene expression in transgenic plants, we outline a reliable strategy to identify the optimal housekeeping genes from a set of candidates by combining statistical analyses of their (RT- PCR amplification efficiency, gene expression stability, and transgene influences. We used the strategy to select two genes, ACTα and EF1α, from 10 candidate housekeeping genes, as the optimal housekeeping genes to evaluate transgenic Eucommia ulmoides Oliver root lines overexpressing IPPI or FPPS1 genes, which are involved in isoprenoid biosynthesis.

  11. Gene expression in Pseudomonas aeruginosa swarming motility

    Directory of Open Access Journals (Sweden)

    Déziel Eric

    2010-10-01

    Full Text Available Abstract Background The bacterium Pseudomonas aeruginosa is capable of three types of motilities: swimming, twitching and swarming. The latter is characterized by a fast and coordinated group movement over a semi-solid surface resulting from intercellular interactions and morphological differentiation. A striking feature of swarming motility is the complex fractal-like patterns displayed by migrating bacteria while they move away from their inoculation point. This type of group behaviour is still poorly understood and its characterization provides important information on bacterial structured communities such as biofilms. Using GeneChip® Affymetrix microarrays, we obtained the transcriptomic profiles of both bacterial populations located at the tip of migrating tendrils and swarm center of swarming colonies and compared these profiles to that of a bacterial control population grown on the same media but solidified to not allow swarming motility. Results Microarray raw data were corrected for background noise with the RMA algorithm and quantile normalized. Differentially expressed genes between the three conditions were selected using a threshold of 1.5 log2-fold, which gave a total of 378 selected genes (6.3% of the predicted open reading frames of strain PA14. Major shifts in gene expression patterns are observed in each growth conditions, highlighting the presence of distinct bacterial subpopulations within a swarming colony (tendril tips vs. swarm center. Unexpectedly, microarrays expression data reveal that a minority of genes are up-regulated in tendril tip populations. Among them, we found energy metabolism, ribosomal protein and transport of small molecules related genes. On the other hand, many well-known virulence factors genes were globally repressed in tendril tip cells. Swarm center cells are distinct and appear to be under oxidative and copper stress responses. Conclusions Results reported in this study show that, as opposed to

  12. Decomposition of gene expression state space trajectories.

    Directory of Open Access Journals (Sweden)

    Jessica C Mar

    2009-12-01

    Full Text Available Representing and analyzing complex networks remains a roadblock to creating dynamic network models of biological processes and pathways. The study of cell fate transitions can reveal much about the transcriptional regulatory programs that underlie these phenotypic changes and give rise to the coordinated patterns in expression changes that we observe. The application of gene expression state space trajectories to capture cell fate transitions at the genome-wide level is one approach currently used in the literature. In this paper, we analyze the gene expression dataset of Huang et al. (2005 which follows the differentiation of promyelocytes into neutrophil-like cells in the presence of inducers dimethyl sulfoxide and all-trans retinoic acid. Huang et al. (2005 build on the work of Kauffman (2004 who raised the attractor hypothesis, stating that cells exist in an expression landscape and their expression trajectories converge towards attractive sites in this landscape. We propose an alternative interpretation that explains this convergent behavior by recognizing that there are two types of processes participating in these cell fate transitions-core processes that include the specific differentiation pathways of promyelocytes to neutrophils, and transient processes that capture those pathways and responses specific to the inducer. Using functional enrichment analyses, specific biological examples and an analysis of the trajectories and their core and transient components we provide a validation of our hypothesis using the Huang et al. (2005 dataset.

  13. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

    Science.gov (United States)

    Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

    2001-11-01

    Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

  14. Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

    Directory of Open Access Journals (Sweden)

    Ming-Wai Poon

    Full Text Available A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs. Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2 reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%. Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs. This cell type may also have advantages in retinal pigmented epithelial differentiation.

  15. Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

    Science.gov (United States)

    Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

    2015-01-01

    A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

  16. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko; Harushima, Yoshiaki; Fujisawa, Hironori; Mochizuki, Takako; Fujita, Masahiro; Ohyanagi, Hajime; Kurata, Nori

    2015-01-01

    Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue

  17. A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron

    Directory of Open Access Journals (Sweden)

    Regis Stentz

    2016-07-01

    Full Text Available There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterised Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter–region of an α-1,2-mannosidase gene (BT_3784, a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products.

  18. Blood Gene Expression Predicts Bronchiolitis Obliterans Syndrome

    Directory of Open Access Journals (Sweden)

    Richard Danger

    2018-01-01

    Full Text Available Bronchiolitis obliterans syndrome (BOS, the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group, and 26 samples at or after BOS diagnosis (diagnosis group. An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group. We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1, T-cell leukemia/lymphoma protein 1A (TCL1A, and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p < 0.01 and are not associated with time posttransplantation. This is the first published large-scale gene expression analysis of blood after lung transplantation. The three-gene blood signature could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS.

  19. Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

    Science.gov (United States)

    Pasion, S G; Brown, G W; Brown, L M; Ray, D S

    1994-12-01

    In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

  20. Wheat-specific gene, ribosomal protein l21, used as the endogenous reference gene for qualitative and real-time quantitative polymerase chain reaction detection of transgenes.

    Science.gov (United States)

    Liu, Yi-Ke; Li, He-Ping; Huang, Tao; Cheng, Wei; Gao, Chun-Sheng; Zuo, Dong-Yun; Zhao, Zheng-Xi; Liao, Yu-Cai

    2014-10-29

    Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5-4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified.

  1. Gene expression pattern at different time points following ALA-PDT

    International Nuclear Information System (INIS)

    Verwanger, T.; Sanovic, R.; Ruhdorfer, S.; Aberger, F.; Frischauf, A.; Krammer, B.

    2003-01-01

    Full text: The photo sensitizer protoporphyrin IX, endogenously accumulated from the precursor aminolevulinic acid (ALA), is a successful agent in photodynamic tumor therapy. In spite of encouraging clinical results, the basic mechanisms leading to cell death are not fully understood. We therefore set out to analyze the alteration of the gene expression pattern in the squamous cell carcinoma cell line A-431 at different time points after photodynamic treatment with endogenous protoporphyrin IX by cDNA-array technique. Cells were incubated for 16 hours with 100 μg/ml ALA and irradiated with a fluence of 3.5 J/cm 2 resulting in 50 % survival until 8 hours post treatment. RNA was isolated at 1.5, 3, 5 and 8 hours post treatment as well as of 3 controls (untreated, light only and dark), radioactively labelled by reverse transcription with 33P-dCTP and hybridized onto macroarray PCR filters containing PCR products of 2135 genes, which were selected for relevance in tumors, stress response and signal transduction. Verification of observed expression changes was carried out by real time PCR. We found a strong induction of expression of immediate early genes like c-fos as well as decreased expression of genes involved in proliferation like myc and the proliferating cell nuclear antigen (PCNA). (author)

  2. Gene expression in the lignin biosynthesis pathway during soybean seed development.

    Science.gov (United States)

    Baldoni, A; Von Pinho, E V R; Fernandes, J S; Abreu, V M; Carvalho, M L M

    2013-02-28

    The study of gene expression in plants is fundamental, and understanding the molecular mechanisms involved in important biological processes, such as biochemical pathways or signaling that are used or manipulated in improvement programs, are key for the production of high-quality soybean seeds. Reports related to gene expression of lignin in seeds are scarce in the literature. We studied the expression of the phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase, 4-hydroxycinnamate 3-hydroxylase, and cinnamyl alcohol dehydrogenase genes involved in lignin biosynthesis during the development of soybean (Glycine max L. Merrill) seeds. As the endogenous control, the eukaryotic elongation factor 1-beta gene was used in two biological replicates performed in triplicate. Relative quantitative expression of these genes during the R4, R5, R6, and R7 development stages was analyzed. Real-time polymerase chain reaction was used for the gene expression study. The analyses were carried out in an ABI PRISM 7500 thermocycler using the comparative Ct method and SYBR Green to detect amplification. The seed samples at the R4 stage were chosen as calibrators. Increased expression of the cinnamate-4-hydroxylase and PAL genes occurred in soybean seeds at the R5 and R6 development stages. The cinnamyl alcohol dehydrogenase gene was expressed during the final development phases of soybean seeds. In low-lignin soybean cultivars, the higher expression of the PAL gene occurs at development stages R6 and R7. Activation of the genes involved in the lignin biosynthesis pathway occurs at the beginning of soybean seed development.

  3. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    International Nuclear Information System (INIS)

    Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

    2013-01-01

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  4. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  5. Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

    2014-05-01

    Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  6. Selective Expression of an Endogenous Inhibitor of FAK Regulates Proliferation and Migration of Vascular Smooth Muscle Cells

    Science.gov (United States)

    Taylor, Joan M.; Mack, Christopher P.; Nolan, Kate; Regan, Christopher P.; Owens, Gary K.; Parsons, J. Thomas

    2001-01-01

    Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538–540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [3H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals. PMID:11238893

  7. Endogenous Reference Genes and Their Quantitative Real-Time PCR Assays for Genetically Modified Bread Wheat (Triticum aestivum L.) Detection.

    Science.gov (United States)

    Yang, Litao; Quan, Sheng; Zhang, Dabing

    2017-01-01

    Endogenous reference genes (ERG) and their derivate analytical methods are standard requirements for analysis of genetically modified organisms (GMOs). Development and validation of suitable ERGs is the primary step for establishing assays that monitoring the genetically modified (GM) contents in food/feed samples. Herein, we give a review of the ERGs currently used for GM wheat analysis, such as ACC1, PKABA1, ALMT1, and Waxy-D1, as well as their performances in GM wheat analysis. Also, we discussed one model for developing and validating one ideal RG for one plant species based on our previous research work.

  8. Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression

    DEFF Research Database (Denmark)

    Pinto, Rita; Hansen, Lars; Hintze, John

    2017-01-01

    to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii......Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven......) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide...

  9. Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically

    Directory of Open Access Journals (Sweden)

    Chan Anthony WS

    2008-02-01

    Full Text Available Abstract Background Alzheimer's disease (AD is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD. Some instances of FAD have been linked to mutations in the β-amyloid protein precursor (APP. Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated. Results Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Aβ40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Aβ42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP. Conclusion This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue

  10. Availability of endogenous peptides limits expression of an M3a-Ld major histocompatibility complex class I chimera

    Science.gov (United States)

    1994-01-01

    Taking advantage of our understanding of the peptide specificity of the major histocompatibility complex class I-b molecule M3a, we sought to determine why these molecules are poorly represented on the cell surface. To this end we constructed a chimeric molecule with the alpha 1 and alpha 2 domains of M3a and alpha 3 of Ld thereby allowing use of available monoclonal antibodies to quantify surface expression. Transfected, but not control, B10.CAS2 (H-2M3b) cells were lysed readily by M3a-restricted monoclonal cytotoxic T lymphocytes. Thus, the chimera bound, trafficked, and presented endogenous mitochondrial peptides. However, despite high levels of M3a-Ld mRNA, transfectants were negative by surface staining. This finding was consistent with inefficient trafficking to the cell surface. Incubation at 26 degrees C, thought to permit trafficking of unoccupied heavy (H) chains, resulted in detectable cell surface expression of chimeric molecules. Incubation with exogenous peptide at 26 degrees C (but not at 37 degrees C) greatly enhanced expression of M3a-Ld molecules in a dose- dependent manner, suggesting stabilization of unoccupied molecules. Stable association of beta 2-microglobulin with the chimeric H chain was observed in labeled cell lysates only in the presence of exogenous specific peptide, indicating that peptide is required for the formation of a ternary complex. These results indicate that surface expression of M3a-Ld is limited largely by the steady-state availability of endogenous peptides. Since most known M3a-binding peptides are N- formylated, native M3a may normally be expressed at high levels only during infection by intracellular bacteria. PMID:8270862

  11. Gene Expression Profiling of Xeroderma Pigmentosum

    Directory of Open Access Journals (Sweden)

    Bowden Nikola A

    2006-05-01

    Full Text Available Abstract Xeroderma pigmentosum (XP is a rare recessive disorder that is characterized by extreme sensitivity to UV light. UV light exposure results in the formation of DNA damage such as cyclobutane dimers and (6-4 photoproducts. Nucleotide excision repair (NER orchestrates the removal of cyclobutane dimers and (6-4 photoproducts as well as some forms of bulky chemical DNA adducts. The disease XP is comprised of 7 complementation groups (XP-A to XP-G, which represent functional deficiencies in seven different genes, all of which are believed to be involved in NER. The main clinical feature of XP is various forms of skin cancers; however, neurological degeneration is present in XPA, XPB, XPD and XPG complementation groups. The relationship between NER and other types of DNA repair processes is now becoming evident but the exact relationships between the different complementation groups remains to be precisely determined. Using gene expression analysis we have identified similarities and differences after UV light exposure between the complementation groups XP-A, XP-C, XP-D, XP-E, XP-F, XP-G and an unaffected control. The results reveal that there is a graded change in gene expression patterns between the mildest, most similar to the control response (XP-E and the severest form (XP-A of the disease, with the exception of XP-D. Distinct differences between the complementation groups with neurological symptoms (XP-A, XP-D and XP-G and without (XP-C, XP-E and XP-F were also identified. Therefore, this analysis has revealed distinct gene expression profiles for the XP complementation groups and the first step towards understanding the neurological symptoms of XP.

  12. Changes in gene expression following androgen receptor blockade ...

    Indian Academy of Sciences (India)

    Madhu urs

    of gene expression in the ventral prostate, it is not clear whether all the gene expression ... These include clusterin, methionine adenosyl transferase IIα, and prostate-specific ..... MAGEE1 melanoma antigen and no similarity was found with the ...

  13. Rubisco activity and gene expression of tropical tree species under ...

    African Journals Online (AJOL)

    Young

    2013-05-15

    May 15, 2013 ... Proteomics analysis associated with gene expression of plants reveal .... Consequently, Rubisco enzyme plays a role in assi- milating into ... technique for examining gene expression encoded at the. mRNA level .... Ammonia.

  14. Gene structure, phylogeny and expression profile of the sucrose ...

    Indian Academy of Sciences (India)

    Gene structure, phylogeny and expression profile of the sucrose synthase gene family in .... 24, 701–713. Bate N. and Twell D. 1998 Functional architecture of a late pollen .... Manzara T. and Gruissem W. 1988 Organization and expression.

  15. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  16. Gene Regulation, Modulation, and Their Applications in Gene Expression Data Analysis

    Directory of Open Access Journals (Sweden)

    Mario Flores

    2013-01-01

    Full Text Available Common microarray and next-generation sequencing data analysis concentrate on tumor subtype classification, marker detection, and transcriptional regulation discovery during biological processes by exploring the correlated gene expression patterns and their shared functions. Genetic regulatory network (GRN based approaches have been employed in many large studies in order to scrutinize for dysregulation and potential treatment controls. In addition to gene regulation and network construction, the concept of the network modulator that has significant systemic impact has been proposed, and detection algorithms have been developed in past years. Here we provide a unified mathematic description of these methods, followed with a brief survey of these modulator identification algorithms. As an early attempt to extend the concept to new RNA regulation mechanism, competitive endogenous RNA (ceRNA, into a modulator framework, we provide two applications to illustrate the network construction, modulation effect, and the preliminary finding from these networks. Those methods we surveyed and developed are used to dissect the regulated network under different modulators. Not limit to these, the concept of “modulation” can adapt to various biological mechanisms to discover the novel gene regulation mechanisms.

  17. Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

    Directory of Open Access Journals (Sweden)

    Yao Han

    Full Text Available MIGS (miRNA-induced gene silencing is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs. MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase and PDS (phytoene desaturase gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant

  18. Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

    Science.gov (United States)

    Han, Yao; Zhang, Bin; Qin, Xiaoting; Li, Mingyang; Guo, Yulong

    2015-01-01

    MIGS (miRNA-induced gene silencing) is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs). MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase) and PDS (phytoene desaturase) gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5'- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant functions.

  19. Over-expression of KdSOC1 gene affected plantlet morphogenesis in Kalanchoe daigremontiana.

    Science.gov (United States)

    Zhu, Chen; Wang, Li; Chen, Jinhua; Liu, Chenglan; Zeng, Huiming; Wang, Huafang

    2017-07-17

    Kalanchoe daigremontiana reproduces asexually by producing plantlets along the leaf margin. The aim of this study was to identify the function of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 gene in Kalanchoe daigremontiana (KdSOC1) during plantlet morphogenesis. In this study, KdSOC1 gene expression was detected at stem cell niche during in vitro somatic embryogenesis and plantlet morphogenesis. Disrupting endogenous auxin transportation suppressed the KdSOC1 gene response. Knockdown of the KdSOC1 gene caused a defect in cotyledon formation during the early heart stage of somatic embryogenesis. Over-expression (OE) of the KdSOC1 gene resulted in asymmetric plantlet distribution, a reduced number of plantlets, thicker leaves, and thicker vascular fibers. Higher KdPIN1 gene expression and auxin content were found in OE plant compared to those of wild-type plant leaves, which indicated possible KdSOC1 gene role in affecting auxin distribution and accumulation. KdSOC1 gene OE in DR5-GUS Arabidopsis reporting lines resulted in an abnormal auxin response pattern during different stages of somatic embryogenesis. In summary, the KdSOC1 gene OE might alter auxin distribution and accumulation along leaf margin to initiate plantlet formation and distribution, which is crucial for plasticity during plantlet formation under various environmental conditions.

  20. Endogenous MCM7 microRNA cluster as a novel platform to multiplex small interfering and nucleolar RNAs for combinational HIV-1 gene therapy.

    Science.gov (United States)

    Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L; DiGiusto, David L; Rossi, John J

    2012-11-01

    Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications.

  1. Nuclear AXIN2 represses MYC gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S., E-mail: gsy3@psu.edu

    2014-01-03

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling.

  2. Nuclear AXIN2 represses MYC gene expression

    International Nuclear Information System (INIS)

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S.

    2014-01-01

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling

  3. Molecular mechanisms of curcumin action: gene expression.

    Science.gov (United States)

    Shishodia, Shishir

    2013-01-01

    Curcumin derived from the tropical plant Curcuma longa has a long history of use as a dietary agent, food preservative, and in traditional Asian medicine. It has been used for centuries to treat biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism, and sinusitis. The preventive and therapeutic properties of curcumin are associated with its antioxidant, anti-inflammatory, and anticancer properties. Extensive research over several decades has attempted to identify the molecular mechanisms of curcumin action. Curcumin modulates numerous molecular targets by altering their gene expression, signaling pathways, or through direct interaction. Curcumin regulates the expression of inflammatory cytokines (e.g., TNF, IL-1), growth factors (e.g., VEGF, EGF, FGF), growth factor receptors (e.g., EGFR, HER-2, AR), enzymes (e.g., COX-2, LOX, MMP9, MAPK, mTOR, Akt), adhesion molecules (e.g., ELAM-1, ICAM-1, VCAM-1), apoptosis related proteins (e.g., Bcl-2, caspases, DR, Fas), and cell cycle proteins (e.g., cyclin D1). Curcumin modulates the activity of several transcription factors (e.g., NF-κB, AP-1, STAT) and their signaling pathways. Based on its ability to affect multiple targets, curcumin has the potential for the prevention and treatment of various diseases including cancers, arthritis, allergies, atherosclerosis, aging, neurodegenerative disease, hepatic disorders, obesity, diabetes, psoriasis, and autoimmune diseases. This review summarizes the molecular mechanisms of modulation of gene expression by curcumin. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  4. Studying the Complex Expression Dependences between Sets of Coexpressed Genes

    Directory of Open Access Journals (Sweden)

    Mario Huerta

    2014-01-01

    Full Text Available Organisms simplify the orchestration of gene expression by coregulating genes whose products function together in the cell. The use of clustering methods to obtain sets of coexpressed genes from expression arrays is very common; nevertheless there are no appropriate tools to study the expression networks among these sets of coexpressed genes. The aim of the developed tools is to allow studying the complex expression dependences that exist between sets of coexpressed genes. For this purpose, we start detecting the nonlinear expression relationships between pairs of genes, plus the coexpressed genes. Next, we form networks among sets of coexpressed genes that maintain nonlinear expression dependences between all of them. The expression relationship between the sets of coexpressed genes is defined by the expression relationship between the skeletons of these sets, where this skeleton represents the coexpressed genes with a well-defined nonlinear expression relationship with the skeleton of the other sets. As a result, we can study the nonlinear expression relationships between a target gene and other sets of coexpressed genes, or start the study from the skeleton of the sets, to study the complex relationships of activation and deactivation between the sets of coexpressed genes that carry out the different cellular processes present in the expression experiments.

  5. The relationship among gene expression, the evolution of gene dosage, and the rate of protein evolution.

    Directory of Open Access Journals (Sweden)

    Jean-François Gout

    2010-05-01

    Full Text Available The understanding of selective constraints affecting genes is a major issue in biology. It is well established that gene expression level is a major determinant of the rate of protein evolution, but the reasons for this relationship remain highly debated. Here we demonstrate that gene expression is also a major determinant of the evolution of gene dosage: the rate of gene losses after whole genome duplications in the Paramecium lineage is negatively correlated to the level of gene expression, and this relationship is not a byproduct of other factors known to affect the fate of gene duplicates. This indicates that changes in gene dosage are generally more deleterious for highly expressed genes. This rule also holds for other taxa: in yeast, we find a clear relationship between gene expression level and the fitness impact of reduction in gene dosage. To explain these observations, we propose a model based on the fact that the optimal expression level of a gene corresponds to a trade-off between the benefit and cost of its expression. This COSTEX model predicts that selective pressure against mutations changing gene expression level or affecting the encoded protein should on average be stronger in highly expressed genes and hence that both the frequency of gene loss and the rate of protein evolution should correlate negatively with gene expression. Thus, the COSTEX model provides a simple and common explanation for the general relationship observed between the level of gene expression and the different facets of gene evolution.

  6. Retrotransposons as regulators of gene expression.

    Science.gov (United States)

    Elbarbary, Reyad A; Lucas, Bronwyn A; Maquat, Lynne E

    2016-02-12

    Transposable elements (TEs) are both a boon and a bane to eukaryotic organisms, depending on where they integrate into the genome and how their sequences function once integrated. We focus on two types of TEs: long interspersed elements (LINEs) and short interspersed elements (SINEs). LINEs and SINEs are retrotransposons; that is, they transpose via an RNA intermediate. We discuss how LINEs and SINEs have expanded in eukaryotic genomes and contribute to genome evolution. An emerging body of evidence indicates that LINEs and SINEs function to regulate gene expression by affecting chromatin structure, gene transcription, pre-mRNA processing, or aspects of mRNA metabolism. We also describe how adenosine-to-inosine editing influences SINE function and how ongoing retrotransposition is countered by the body's defense mechanisms. Copyright © 2016, American Association for the Advancement of Science.

  7. Gene expression profiling of cutaneous wound healing

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    Wang Ena

    2007-02-01

    Full Text Available Abstract Background Although the sequence of events leading to wound repair has been described at the cellular and, to a limited extent, at the protein level this process has yet to be fully elucidated. Genome wide transcriptional analysis tools promise to further define the global picture of this complex progression of events. Study Design This study was part of a placebo-controlled double-blind clinical trial in which basal cell carcinomas were treated topically with an immunomodifier – toll-like receptor 7 agonist: imiquimod. The fourteen patients with basal cell carcinoma in the placebo arm of the trial received placebo treatment consisting solely of vehicle cream. A skin punch biopsy was obtained immediately before treatment and at the end of the placebo treatment (after 2, 4 or 8 days. 17.5K cDNA microarrays were utilized to profile the biopsy material. Results Four gene signatures whose expression changed relative to baseline (before wound induction by the pre-treatment biopsy were identified. The largest group was comprised predominantly of inflammatory genes whose expression was increased throughout the study. Two additional signatures were observed which included preferentially pro-inflammatory genes in the early post-treatment biopsies (2 days after pre-treatment biopsies and repair and angiogenesis genes in the later (4 to 8 days biopsies. The fourth and smallest set of genes was down-regulated throughout the study. Early in wound healing the expression of markers of both M1 and M2 macrophages were increased, but later M2 markers predominated. Conclusion The initial response to a cutaneous wound induces powerful transcriptional activation of pro-inflammatory stimuli which may alert the host defense. Subsequently and in the absence of infection, inflammation subsides and it is replaced by angiogenesis and remodeling. Understanding this transition which may be driven by a change from a mixed macrophage population to predominately M2

  8. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... in cell adhesion and the cytoskeleton. If the proteins involved in tethering cells to the extracellular matrix are important in conferring drug resistance, it may be possible to improve chemotherapy by designing drugs that target these proteins....

  9. Network Completion for Static Gene Expression Data

    Directory of Open Access Journals (Sweden)

    Natsu Nakajima

    2014-01-01

    Full Text Available We tackle the problem of completing and inferring genetic networks under stationary conditions from static data, where network completion is to make the minimum amount of modifications to an initial network so that the completed network is most consistent with the expression data in which addition of edges and deletion of edges are basic modification operations. For this problem, we present a new method for network completion using dynamic programming and least-squares fitting. This method can find an optimal solution in polynomial time if the maximum indegree of the network is bounded by a constant. We evaluate the effectiveness of our method through computational experiments using synthetic data. Furthermore, we demonstrate that our proposed method can distinguish the differences between two types of genetic networks under stationary conditions from lung cancer and normal gene expression data.

  10. Tropism, Cytotoxicity, and Inflammatory Properties of Two Envelope Genes of Murine Leukemia Virus Type-Endogenous Retroviruses of C57BL/6J Mice

    Directory of Open Access Journals (Sweden)

    Young-Kwan Lee

    2011-01-01

    Full Text Available Envelope (env proteins of certain endogenous retroviruses (ERVs participate in various pathophysiological processes. In this study, we characterized pathophysiologic properties of two murine leukemia virus-type ERV (MuLV-ERV env genes cloned from the ovary of C57BL/6J mice. The two env genes (named ENVOV1 and ENVOV2, with 1,926\\,bp coding region, originated from two MuLV-ERV loci on chromosomes 8 and 18, respectively. ENVOV1 and ENVOV2 were ~75 kDa and predominantly expressed on the cell membrane. They were capable of producing pseudotype murine leukemia virus virions. Tropism trait and infectivity of ENVOV2 were similar to the polytropic env; however, ENVOV1 had very low level of infectivity. Overexpression of ENVOV2, but not ENVOV1, exerted cytotoxic effects and induced expression of COX-2, IL-1β, IL-6, and iNOS. These findings suggest that the ENVOV1 and ENVOV2 are capable of serving as an env protein for virion assembly, and they exert differential cytotoxicity and modulation of inflammatory mediators.

  11. Inferring gene expression dynamics via functional regression analysis

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    Leng Xiaoyan

    2008-01-01

    Full Text Available Abstract Background Temporal gene expression profiles characterize the time-dynamics of expression of specific genes and are increasingly collected in current gene expression experiments. In the analysis of experiments where gene expression is obtained over the life cycle, it is of interest to relate temporal patterns of gene expression associated with different developmental stages to each other to study patterns of long-term developmental gene regulation. We use tools from functional data analysis to study dynamic changes by relating temporal gene expression profiles of different developmental stages to each other. Results We demonstrate that functional regression methodology can pinpoint relationships that exist between temporary gene expression profiles for different life cycle phases and incorporates dimension reduction as needed for these high-dimensional data. By applying these tools, gene expression profiles for pupa and adult phases are found to be strongly related to the profiles of the same genes obtained during the embryo phase. Moreover, one can distinguish between gene groups that exhibit relationships with positive and others with negative associations between later life and embryonal expression profiles. Specifically, we find a positive relationship in expression for muscle development related genes, and a negative relationship for strictly maternal genes for Drosophila, using temporal gene expression profiles. Conclusion Our findings point to specific reactivation patterns of gene expression during the Drosophila life cycle which differ in characteristic ways between various gene groups. Functional regression emerges as a useful tool for relating gene expression patterns from different developmental stages, and avoids the problems with large numbers of parameters and multiple testing that affect alternative approaches.

  12. Analysis of swine leukocyte antigen class I gene profiles and porcine endogenous retrovirus viremia level in a transgenic porcine herd inbred for xenotransplantation research

    Science.gov (United States)

    Sypniewski, Daniel; Gałka, Sabina; Sołtysik, Dagna; Loch, Tomasz; Nowak, Ewa; Smorąg, Zdzisław; Bednarek, Ilona

    2018-01-01

    Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety. PMID:29366300

  13. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

  14. Individual Differences in the Expression of Conditioned Fear Are Associated with Endogenous Fibroblast Growth Factor 2

    Science.gov (United States)

    Graham, Bronwyn M.; Richardson, Rick

    2016-01-01

    These experiments examined the relationship between the neurotrophic factor fibroblast growth factor 2 (FGF2) and individual differences in the expression of conditioned fear. Experiments 1 and 2 demonstrated that rats naturally expressing low levels of contextual or cued fear have higher levels of hippocampal FGF2 relative to rats that express…

  15. Interactive visualization of gene regulatory networks with associated gene expression time series data

    NARCIS (Netherlands)

    Westenberg, M.A.; Hijum, van S.A.F.T.; Lulko, A.T.; Kuipers, O.P.; Roerdink, J.B.T.M.; Linsen, L.; Hagen, H.; Hamann, B.

    2008-01-01

    We present GENeVis, an application to visualize gene expression time series data in a gene regulatory network context. This is a network of regulator proteins that regulate the expression of their respective target genes. The networks are represented as graphs, in which the nodes represent genes,

  16. A recombinant lactobacillus strain expressing genes coding for ...

    African Journals Online (AJOL)

    Using genetically engineered endogenous lactobacillus strains colonizing the vagina mucosa to express heterogenous proteins has of late joined the novel strategies aimed at developing a microbicides against HIV. Using the lactobacillus metabolic genome pathway, we found that these bacteria do not naturally produce ...

  17. Positive selection on gene expression in the human brain

    DEFF Research Database (Denmark)

    Khaitovich, Philipp; Tang, Kun; Franz, Henriette

    2006-01-01

    Recent work has shown that the expression levels of genes transcribed in the brains of humans and chimpanzees have changed less than those of genes transcribed in other tissues [1] . However, when gene expression changes are mapped onto the evolutionary lineage in which they occurred, the brain...... shows more changes than other tissues in the human lineage compared to the chimpanzee lineage [1] , [2] and [3] . There are two possible explanations for this: either positive selection drove more gene expression changes to fixation in the human brain than in the chimpanzee brain, or genes expressed...... in the brain experienced less purifying selection in humans than in chimpanzees, i.e. gene expression in the human brain is functionally less constrained. The first scenario would be supported if genes that changed their expression in the brain in the human lineage showed more selective sweeps than other genes...

  18. GENE EXPRESSION DYNAMICS IN PATIENTS WITH SEVERE THERAPY-RESISTANT ASTHMA DURING TREATMENT PERIOD

    Directory of Open Access Journals (Sweden)

    Ye. S. Kulikov

    2014-01-01

    Full Text Available Introduction: The leading mechanisms and causes of severe therapy resistant asthma are poorly understood. The aim of this study was to define global patterns of gene expression in adults with severe therapy-resistant asthma in dynamic during treatment period.Methods: Performed 24-week prospective interventional study in parallel groups. Severe asthma patients was aposterior divided at therapy sensitive and resistant patients according to ATS criteria. Global transcriptome profile was characterized using the Affymetrix HuGene ST1.0 chip. Cluster analysis was performed.Results and conclusion: According to our data several mechanisms of therapy resistance may be considered: increased levels of nitric oxide and beta2-agonists nitration, dysregulation of endogenous steroids secretion and involvement in the pathogenesis of Staphylococcus aureus. Absence of suppression of gene expression KEGG-pathway “asthma" may reflect the low efficiency or long period of anti-inflammatory therapy effect realization.

  19. Redox-active antibiotics control gene expression and community behavior in divergent bacteria.

    Science.gov (United States)

    Dietrich, Lars E P; Teal, Tracy K; Price-Whelan, Alexa; Newman, Dianne K

    2008-08-29

    It is thought that bacteria excrete redox-active pigments as antibiotics to inhibit competitors. In Pseudomonas aeruginosa, the endogenous antibiotic pyocyanin activates SoxR, a transcription factor conserved in Proteo- and Actinobacteria. In Escherichia coli, SoxR regulates the superoxide stress response. Bioinformatic analysis coupled with gene expression studies in P. aeruginosa and Streptomyces coelicolor revealed that the majority of SoxR regulons in bacteria lack the genes required for stress responses, despite the fact that many of these organisms still produce redox-active small molecules, which indicates that redox-active pigments play a role independent of oxidative stress. These compounds had profound effects on the structural organization of colony biofilms in both P. aeruginosa and S. coelicolor, which shows that "secondary metabolites" play important conserved roles in gene expression and development.

  20. Identification of Human HK Genes and Gene Expression Regulation Study in Cancer from Transcriptomics Data Analysis

    Science.gov (United States)

    Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun

    2013-01-01

    The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867

  1. Extensive Differences in Gene Expression Between Symbiotic and Aposymbiotic Cnidarians

    Science.gov (United States)

    Lehnert, Erik M.; Mouchka, Morgan E.; Burriesci, Matthew S.; Gallo, Natalya D.; Schwarz, Jodi A.; Pringle, John R.

    2013-01-01

    Coral reefs provide habitats for a disproportionate number of marine species relative to the small area of the oceans that they occupy. The mutualism between the cnidarian animal hosts and their intracellular dinoflagellate symbionts provides the nutritional foundation for coral growth and formation of reef structures, because algal photosynthesis can provide >90% of the total energy of the host. Disruption of this symbiosis (“coral bleaching”) is occurring on a large scale due primarily to anthropogenic factors and poses a major threat to the future of coral reefs. Despite the importance of this symbiosis, the cellular mechanisms involved in its establishment, maintenance, and breakdown remain largely unknown. We report our continued development of genomic tools to study these mechanisms in Aiptasia, a small sea anemone with great promise as a model system for studies of cnidarian–dinoflagellate symbiosis. Specifically, we have generated de novo assemblies of the transcriptomes of both a clonal line of symbiotic anemones and their endogenous dinoflagellate symbionts. We then compared transcript abundances in animals with and without dinoflagellates. This analysis identified >900 differentially expressed genes and allowed us to generate testable hypotheses about the cellular functions affected by symbiosis establishment. The differentially regulated transcripts include >60 encoding proteins that may play roles in transporting various nutrients between the symbiotic partners; many more encoding proteins functioning in several metabolic pathways, providing clues regarding how the transported nutrients may be used by the partners; and several encoding proteins that may be involved in host recognition and tolerance of the dinoflagellate. PMID:24368779

  2. Predicting cellular growth from gene expression signatures.

    Directory of Open Access Journals (Sweden)

    Edoardo M Airoldi

    2009-01-01

    Full Text Available Maintaining balanced growth in a changing environment is a fundamental systems-level challenge for cellular physiology, particularly in microorganisms. While the complete set of regulatory and functional pathways supporting growth and cellular proliferation are not yet known, portions of them are well understood. In particular, cellular proliferation is governed by mechanisms that are highly conserved from unicellular to multicellular organisms, and the disruption of these processes in metazoans is a major factor in the development of cancer. In this paper, we develop statistical methodology to identify quantitative aspects of the regulatory mechanisms underlying cellular proliferation in Saccharomyces cerevisiae. We find that the expression levels of a small set of genes can be exploited to predict the instantaneous growth rate of any cellular culture with high accuracy. The predictions obtained in this fashion are robust to changing biological conditions, experimental methods, and technological platforms. The proposed model is also effective in predicting growth rates for the related yeast Saccharomyces bayanus and the highly diverged yeast Schizosaccharomyces pombe, suggesting that the underlying regulatory signature is conserved across a wide range of unicellular evolution. We investigate the biological significance of the gene expression signature that the predictions are based upon from multiple perspectives: by perturbing the regulatory network through the Ras/PKA pathway, observing strong upregulation of growth rate even in the absence of appropriate nutrients, and discovering putative transcription factor binding sites, observing enrichment in growth-correlated genes. More broadly, the proposed methodology enables biological insights about growth at an instantaneous time scale, inaccessible by direct experimental methods. Data and tools enabling others to apply our methods are available at http://function.princeton.edu/growthrate.

  3. Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells.

    Science.gov (United States)

    Gunthorpe, M J; Smith, G D; Davis, J B; Randall, A D

    2001-08-01

    Acid-sensing ion channels (ASICs) are a new and expanding family of proton-gated cation (Na+/Ca2+) channels that are widely expressed in sensory neurons and the central nervous system. Their distribution suggests that they may play a critical role in the sensation of the pain that accompanies tissue acidosis and may also be important in detecting the subtle pH variations that occur during neuronal signalling. Here, using whole-cell patch-clamp electrophysiology and reverse transcriptase-polymerase chain reaction (RT-PCR), we show that HEK293 cells, a commonly used cell line for the expression and characterisation of many ion channels, functionally express an endogenous proton-gated conductance attributable to the activity of human ASIC1a. These data therefore represent the first functional characterisation of hASIC1 and have many important implications for the use of HEK293 cells as a host cell system for the study of ASICs, vanilloid receptor-1 and any other proton-gated channel. With this latter point in mind we have devised a simple desensitisation strategy to selectively remove the contribution of hASIC1a from proton-gated currents recorded from HEK293 cells expressing vanilloid receptor-1.

  4. Is really endogenous ghrelin a hunger signal in chickens? Association of GHSR SNPs with increase appetite, growth traits, expression and serum level of GHRL, and GH.

    Science.gov (United States)

    El-Magd, Mohammed Abu; Saleh, Ayman A; Abdel-Hamid, Tamer M; Saleh, Rasha M; Afifi, Mohammed A

    2016-10-01

    Chicken growth hormone secretagogue receptor (GHSR) is a receptor for ghrelin (GHRL), a peptide hormone produced by chicken proventriculus, which stimulates growth hormone (GH) release and food intake. The purpose of this study was to search for single nucleotide polymorphisms (SNPs) in exon 2 of GHSR gene and to analyze their effect on the appetite, growth traits and expression levels of GHSR, GHRL, and GH genes as well as serum levels of GH and GHRL in Mandara chicken. Two adjacent SNPs, A239G and G244A, were detected in exon 2 of GHSR gene. G244A SNP was non-synonymous mutation and led to replacement of lysine amino acid (aa) by arginine aa, while A239G SNP was synonymous mutation. The combined genotypes of A239G and G244A SNPs produced three haplotypes; GG/GG, GG/AG, AG/AG, which associated significantly (P4 to 16w. Chickens with the homozygous GG/GG haplotype showed higher growth performance than other chickens. The two SNPs were also correlated with mRNA levels of GHSR and GH (in pituitary gland), and GHRL (in proventriculus and hypothalamus) as well as with serum level of GH and GHRL. Also, chickens with GG/GG haplotype showed higher mRNA and serum levels. This is the first study to demonstrate that SNPs in GHSR can increase appetite, growth traits, expression and level of GHRL, suggesting a hunger signal role for endogenous GHRL. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Computational design and application of endogenous promoters for transcriptionally targeted gene therapy for rheumatoid arthritis.

    NARCIS (Netherlands)

    Geurts, J.; Joosten, L.A.B.; Takahashi, N.; Arntz, O.J.; Gluck, A.; Bennink, M.B.; Berg, W.B. van den; Loo, F.A.J. van de

    2009-01-01

    The promoter regions of genes that are differentially regulated in the synovial membrane during the course of rheumatoid arthritis (RA) represent attractive candidates for application in transcriptionally targeted gene therapy. In this study, we applied an unbiased computational approach to define

  6. Analysis of multiplex gene expression maps obtained by voxelation.

    Science.gov (United States)

    An, Li; Xie, Hongbo; Chin, Mark H; Obradovic, Zoran; Smith, Desmond J; Megalooikonomou, Vasileios

    2009-04-29

    Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in cortex and corpus callosum. The experimental

  7. Analysis of multiplex gene expression maps obtained by voxelation

    Directory of Open Access Journals (Sweden)

    Smith Desmond J

    2009-04-01

    Full Text Available Abstract Background Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. Results To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in

  8. In situ PCR detection and significance of IL-3 gene expression in irradiated hematopoietic cells of mouse bone marrow

    International Nuclear Information System (INIS)

    Peng Ruiyun; Wang Dewen; Xiong Chengqi; Gao Yabing; Li Yanping; Yang Hong; Cui Yufang

    2000-01-01

    Objective: To study the significance of endogenous interleukin 3(IL-3) gene expression in repair of irradiated mouse bone marrow. Methods: Seventy-eight LACA mice were subjected to total body irradiation with 60 Co γ-rays and were sacrificed within 4 weeks after irradiation. The bone marrow histopathological sections were stained with HE, and the expression of endogenous IL-3 gene was detected by means of immunocytochemistry,in situ hybridization(ISH) and in situ reverse transcription PCR(IS RT-PCR). Results: Obvious injury of bone marrow occurred after irradiation and then recovered within 4 weeks. IL-3 protein was obviously increased in the cytoplasm of recovering hematopoietic cells(HCs), especially on day 21 after irradiation, while its mRNA was poorly positive by ISH on days 10-21, especially day 15.IS RT-PCR showed that IL-3 mRNA was strongly positive in recovering HCs cytoplasm, especially on days 10 to 15. Conclusion: In situ RT-PCR can objectively reflect the regulation of IL-3 gene expression in bone marrow after irradiation, and the expression of endogenous IL-3 gene may play an important role in hematopoietic reconstruction of irradiated bone marrow

  9. Over-expression of Gene FaASR Promotes Strawberry Fruit Coloring

    Directory of Open Access Journals (Sweden)

    Liu Zhongjie

    2015-11-01

    Full Text Available Fruit development and ripening is a complicate process. Although much progress has been made on the ripenig process, the molecular mechamism of fruit development is not yet clear. In this study, we used ‘Sweet Charlie’ strawberry as test materials, based on cloning the strawberries ASR homologous gene, we carried out the bioinformatics and temporal expression analysis of FaASR, by manipulating ASR gene expression level in strawberry fruit, we tested the changes of physiological indicators, including sugar, ABA, pigments content, and fruit firmness, as well as phenotypic changes. In addition, we measured the expression changes of some anthocyanin-related gene, such as CHS and UFGT, by which we revealed the regulation mechanisms of ASR gene over strawberry fruit ripening. Strawberry ASR contained a typical domain of ABA/WDS that was related to fruit ripening and stress-resistance, and ASR gene over-expression could promote strawberry fruit coloring, endogenous ABA and sucrose accumulation, fruit softening, and induced the transcription levels of anthocyanin-related genes CHS and UFGT. The present study will further reveal the molecular mechanisms of information transmission in fruit development, and will also play an important foundation for future molecular improvement of strawberries breeding.

  10. Regulation of root hair initiation and expansin gene expression in Arabidopsis

    Science.gov (United States)

    Cho, Hyung-Taeg; Cosgrove, Daniel J.

    2002-01-01

    The expression of two Arabidopsis expansin genes (AtEXP7 and AtEXP18) is tightly linked to root hair initiation; thus, the regulation of these genes was studied to elucidate how developmental, hormonal, and environmental factors orchestrate root hair formation. Exogenous ethylene and auxin, as well as separation of the root from the medium, stimulated root hair formation and the expression of these expansin genes. The effects of exogenous auxin and root separation on root hair formation required the ethylene signaling pathway. By contrast, blocking the endogenous ethylene pathway, either by genetic mutations or by a chemical inhibitor, did not affect normal root hair formation and expansin gene expression. These results indicate that the normal developmental pathway for root hair formation (i.e., not induced by external stimuli) is independent of the ethylene pathway. Promoter analyses of the expansin genes show that the same promoter elements that determine cell specificity also determine inducibility by ethylene, auxin, and root separation. Our study suggests that two distinctive signaling pathways, one developmental and the other environmental/hormonal, converge to modulate the initiation of the root hair and the expression of its specific expansin gene set.

  11. Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

    Science.gov (United States)

    Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

    2015-03-10

    Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification.

  12. Natural selection in avian protein-coding genes expressed in brain.

    Science.gov (United States)

    Axelsson, Erik; Hultin-Rosenberg, Lina; Brandström, Mikael; Zwahlén, Martin; Clayton, David F; Ellegren, Hans

    2008-06-01

    The evolution of birds from theropod dinosaurs took place approximately 150 million years ago, and was associated with a number of specific adaptations that are still evident among extant birds, including feathers, song and extravagant secondary sexual characteristics. Knowledge about the molecular evolutionary background to such adaptations is lacking. Here, we analyse the evolution of > 5000 protein-coding gene sequences expressed in zebra finch brain by comparison to orthologous sequences in chicken. Mean d(N)/d(S) is 0.085 and genes with their maximal expression in the eye and central nervous system have the lowest mean d(N)/d(S) value, while those expressed in digestive and reproductive tissues exhibit the highest. We find that fast-evolving genes (those which have higher than expected rate of nonsynonymous substitution, indicative of adaptive evolution) are enriched for biological functions such as fertilization, muscle contraction, defence response, response to stress, wounding and endogenous stimulus, and cell death. After alignment to mammalian orthologues, we identify a catalogue of 228 genes that show a significantly higher rate of protein evolution in the two bird lineages than in mammals. These accelerated bird genes, representing candidates for avian-specific adaptations, include genes implicated in vocal learning and other cognitive processes. Moreover, colouration genes evolve faster in birds than in mammals, which may have been driven by sexual selection for extravagant plumage characteristics.

  13. Classification across gene expression microarray studies

    Directory of Open Access Journals (Sweden)

    Kuner Ruprecht

    2009-12-01

    Full Text Available Abstract Background The increasing number of gene expression microarray studies represents an important resource in biomedical research. As a result, gene expression based diagnosis has entered clinical practice for patient stratification in breast cancer. However, the integration and combined analysis of microarray studies remains still a challenge. We assessed the potential benefit of data integration on the classification accuracy and systematically evaluated the generalization performance of selected methods on four breast cancer studies comprising almost 1000 independent samples. To this end, we introduced an evaluation framework which aims to establish good statistical practice and a graphical way to monitor differences. The classification goal was to correctly predict estrogen receptor status (negative/positive and histological grade (low/high of each tumor sample in an independent study which was not used for the training. For the classification we chose support vector machines (SVM, predictive analysis of microarrays (PAM, random forest (RF and k-top scoring pairs (kTSP. Guided by considerations relevant for classification across studies we developed a generalization of kTSP which we evaluated in addition. Our derived version (DV aims to improve the robustness of the intrinsic invariance of kTSP with respect to technologies and preprocessing. Results For each individual study the generalization error was benchmarked via complete cross-validation and was found to be similar for all classification methods. The misclassification rates were substantially higher in classification across studies, when each single study was used as an independent test set while all remaining studies were combined for the training of the classifier. However, with increasing number of independent microarray studies used in the training, the overall classification performance improved. DV performed better than the average and showed slightly less variance. In

  14. Identification of Suitable Endogenous Normalizers for qRT- PCR Analysis of Plasma microRNA Expression in Essential Hypertension

    Science.gov (United States)

    Solayman, Mohamed Hassan M.; Langaee, Taimour; Patel, Archanakumari; El-Wakeel, Lamia; El-Hamamsy, Manal; Badary, Osama; Johnson, Julie A.

    2016-01-01

    Circulating microRNAs (miRNAs) are promising biomarkers for many diseases. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a gold standard for miRNA expression profiling that requires proper data normalization. Since there is no universal normalizer, it is recommended to evaluate normalizers under every experimental condition. This study describes the identification of suitable endogenous normalizer(s) (ENs) for plasma miRNA expression in essential hypertension. Expression levels of 5 candidate ENs and 2 plasma quality markers were determined by qRT-PCR in plasma samples from 18 hypertensive patients and 10 healthy controls. NormFinder, GeNorm, and DataAssist software programs were used to select the best EN(s). Expression levels of the 5 candidate ENs were also analyzed in urine samples from hypertensive patients and compared to the plasma samples of the hypertensive patients. Among the analyzed candidates, hsa-miR-92a-3p was identified as the best EN, and hsa-miR-21-5p and hsa-miR-16-5p as next best. Moreover, hsa-miR-92a-3p showed the most consistent expression between plasma and urine In conclusion, this study showed that hsa-miR-92a-3p, hsa-miR-21-5p, and hsa-miR-16-5p may be used as normalizers for plasma miRNA expression data in essential hypertension studies. PMID:26798072

  15. [Expression analysis of a transformer gene in Daphnia pulex after RNAi].

    Science.gov (United States)

    Guo, C Y; Chen, P; Zhang, M M; Ning, J J; Wang, С L; Wang, D L; Zhao, Y L

    2016-01-01

    In order to explore the importance of the transformer (tra) gene in reproductive mode switching in Daphnia pulex, we studied the effect of silencing of this gene using RNA interference (RNAi). We obtained Dptra dsRNA by constructing and using a dsRNA expression vector and transcription method in vitro. D. pulex individuals in different reproductive modes were treated by soaking in a solution of Dptra dsRNA. We then assayed the expression of the endogenous Dptra mRNA after RNAi treatment using RT-PCR and obtained the suppression ratio. Expression of the tra gene in the RNAi groups was down-regulated compared with the controls after 16 h (p < 0.05). We also analyzed the effect of RNAi on the expression of the TRA protein using Western blot, which showed that the expression level of the TRA protein was reduced after RNAi treatment. Our experimental results showed that soaking of D. pulex adults in tra-specific dsRNA transcribed in vitro can specifically reduce the level of tra mRNA and also reduce the expression of the TRA protein, demonstrating effective in vivo silencing of the tra gene.

  16. Codon usage and amino acid usage influence genes expression level.

    Science.gov (United States)

    Paul, Prosenjit; Malakar, Arup Kumar; Chakraborty, Supriyo

    2018-02-01

    Highly expressed genes in any species differ in the usage frequency of synonymous codons. The relative recurrence of an event of the favored codon pair (amino acid pairs) varies between gene and genomes due to varying gene expression and different base composition. Here we propose a new measure for predicting the gene expression level, i.e., codon plus amino bias index (CABI). Our approach is based on the relative bias of the favored codon pair inclination among the genes, illustrated by analyzing the CABI score of the Medicago truncatula genes. CABI showed strong correlation with all other widely used measures (CAI, RCBS, SCUO) for gene expression analysis. Surprisingly, CABI outperforms all other measures by showing better correlation with the wet-lab data. This emphasizes the importance of the neighboring codons of the favored codon in a synonymous group while estimating the expression level of a gene.

  17. An Approximation to the Temporal Order in Endogenous Circadian Rhythms of Genes Implicated in Human Adipose Tissue Metabolism

    Science.gov (United States)

    GARAULET, MARTA; ORDOVÁS, JOSÉ M.; GÓMEZ-ABELLÁN, PURIFICACIÓN; MARTÍNEZ, JOSE A.; MADRID, JUAN A.

    2015-01-01

    Although it is well established that human adipose tissue (AT) shows circadian rhythmicity, published studies have been discussed as if tissues or systems showed only one or few circadian rhythms at a time. To provide an overall view of the internal temporal order of circadian rhythms in human AT including genes implicated in metabolic processes such as energy intake and expenditure, insulin resistance, adipocyte differentiation, dyslipidemia, and body fat distribution. Visceral and subcutaneous abdominal AT biopsies (n = 6) were obtained from morbid obese women (BMI ≥ 40 kg/m2). To investigate rhythmic expression pattern, AT explants were cultured during 24-h and gene expression was analyzed at the following times: 08:00, 14:00, 20:00, 02:00 h using quantitative real-time PCR. Clock genes, glucocorticoid metabolism-related genes, leptin, adiponectin and their receptors were studied. Significant differences were found both in achrophases and relative-amplitude among genes (P 30%). When interpreting the phase map of gene expression in both depots, data indicated that circadian rhythmicity of the genes studied followed a predictable physiological pattern, particularly for subcutaneous AT. Interesting are the relationships between adiponectin, leptin, and glucocorticoid metabolism-related genes circadian profiles. Their metabolic significance is discussed. Visceral AT behaved in a different way than subcutaneous for most of the genes studied. For every gene, protein mRNA levels fluctuated during the day in synchrony with its receptors. We have provided an overall view of the internal temporal order of circadian rhythms in human adipose tissue. PMID:21520059

  18. Understanding gene expression in coronary artery disease through ...

    Indian Academy of Sciences (India)

    Understanding gene expression in coronary artery disease through global profiling, network analysis and independent validation of key candidate genes. Prathima ... Table 2. Differentially expressed genes in CAD compared to age and gender matched controls. .... Regulation of nuclear pre-mRNA domain containing 1A.

  19. Improved gene expression signature of testicular carcinoma in situ

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Leffers, Henrik; Lothe, Ragnhild A

    2007-01-01

    on global gene expression in testicular CIS have been previously published. We have merged the two data sets on CIS samples (n = 6) and identified the shared gene expression signature in relation to expression in normal testis. Among the top-20 highest expressed genes, one-third was transcription factors...... development' were significantly altered and could collectively affect cellular pathways like the WNT signalling cascade, which thus may be disrupted in testicular CIS. The merged CIS data from two different microarray platforms, to our knowledge, provide the most precise CIS gene expression signature to date....

  20. Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn’s disease

    Science.gov (United States)

    Robertson, Jack; Haas, Carolin T.; Pele, Laetitia C.; Monie, Tom P.; Charalambos, Charles; Parkes, Miles; Hewitt, Rachel E.; Powell, Jonathan J.

    2016-01-01

    Crohn’s disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn’s disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn’s disease show selective failure in PD-L1 expression. Therefore, in Crohn’s disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease. PMID:27226337

  1. Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn's disease.

    Science.gov (United States)

    Robertson, Jack; Haas, Carolin T; Pele, Laetitia C; Monie, Tom P; Charalambos, Charles; Parkes, Miles; Hewitt, Rachel E; Powell, Jonathan J

    2016-05-26

    Crohn's disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn's disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn's disease show selective failure in PD-L1 expression. Therefore, in Crohn's disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease.

  2. Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

    Science.gov (United States)

    Stec, Wojciech J; Rosiak, Kamila; Siejka, Paulina; Peciak, Joanna; Popeda, Marta; Banaszczyk, Mateusz; Pawlowska, Roza; Treda, Cezary; Hulas-Bigoszewska, Krystyna; Piaskowski, Sylwester; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

    2016-05-31

    Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII.The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

  3. Peak flood estimation using gene expression programming

    Science.gov (United States)

    Zorn, Conrad R.; Shamseldin, Asaad Y.

    2015-12-01

    As a case study for the Auckland Region of New Zealand, this paper investigates the potential use of gene-expression programming (GEP) in predicting specific return period events in comparison to the established and widely used Regional Flood Estimation (RFE) method. Initially calibrated to 14 gauged sites, the GEP derived model was further validated to 10 and 100 year flood events with a relative errors of 29% and 18%, respectively. This is compared to the RFE method providing 48% and 44% errors for the same flood events. While the effectiveness of GEP in predicting specific return period events is made apparent, it is argued that the derived equations should be used in conjunction with those existing methodologies rather than as a replacement.

  4. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    DEFF Research Database (Denmark)

    Møller-Larsen, A; Brudek, T; Petersen, T

    2013-01-01

    on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used...

  5. Genetic Variants Contribute to Gene Expression Variability in Humans

    Science.gov (United States)

    Hulse, Amanda M.; Cai, James J.

    2013-01-01

    Expression quantitative trait loci (eQTL) studies have established convincing relationships between genetic variants and gene expression. Most of these studies focused on the mean of gene expression level, but not the variance of gene expression level (i.e., gene expression variability). In the present study, we systematically explore genome-wide association between genetic variants and gene expression variability in humans. We adapt the double generalized linear model (dglm) to simultaneously fit the means and the variances of gene expression among the three possible genotypes of a biallelic SNP. The genomic loci showing significant association between the variances of gene expression and the genotypes are termed expression variability QTL (evQTL). Using a data set of gene expression in lymphoblastoid cell lines (LCLs) derived from 210 HapMap individuals, we identify cis-acting evQTL involving 218 distinct genes, among which 8 genes, ADCY1, CTNNA2, DAAM2, FERMT2, IL6, PLOD2, SNX7, and TNFRSF11B, are cross-validated using an extra expression data set of the same LCLs. We also identify ∼300 trans-acting evQTL between >13,000 common SNPs and 500 randomly selected representative genes. We employ two distinct scenarios, emphasizing single-SNP and multiple-SNP effects on expression variability, to explain the formation of evQTL. We argue that detecting evQTL may represent a novel method for effectively screening for genetic interactions, especially when the multiple-SNP influence on expression variability is implied. The implication of our results for revealing genetic mechanisms of gene expression variability is discussed. PMID:23150607

  6. Expression regulation of design process gene in product design

    DEFF Research Database (Denmark)

    Li, Bo; Fang, Lusheng; Li, Bo

    2011-01-01

    To improve the design process efficiency, this paper proposes the principle and methodology that design process gene controls the characteristics of design process under the framework of design process reuse and optimization based on design process gene. First, the concept of design process gene...... is proposed and analyzed, as well as its three categories i.e., the operator gene, the structural gene and the regulator gene. Second, the trigger mechanism that design objectives and constraints trigger the operator gene is constructed. Third, the expression principle of structural gene is analyzed...... with the example of design management gene. Last, the regulation mode that the regulator gene regulates the expression of the structural gene is established and it is illustrated by taking the design process management gene as an example. © (2011) Trans Tech Publications....

  7. Dissecting specific and global transcriptional regulation of bacterial gene expression

    NARCIS (Netherlands)

    Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe

    Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an

  8. Retroviral-mediated transfer and expression of human β-globin genes in cultured murine and human erythroid cells

    International Nuclear Information System (INIS)

    Weber-Benarous, A.; Cone, R.D.; London, I.M.; Mulligan, R.C.

    1988-01-01

    The authors cloned human β-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 β-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the β-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human β-globin gene transferred into mouse erythroleukemia cells in the same way. They have also introduced the normal human β-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal β-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, they suggest that the lack of expression of the endogenous β-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for β-lobin gene transcription. Retroviral-mediated transfer of the human β-globin gene may, however, uniquely influence expression of the gene K562 cells

  9. Gene expression of the mismatch repair gene MSH2 in primary colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Kuramochi, Hidekazu; Crüger, Dorthe Gylling

    2011-01-01

    promoter was only detected in 14 samples and only at a low level with no correlation to gene expression. MSH2 gene expression was not a prognostic factor for overall survival in univariate or multivariate analysis. The gene expression of MSH2 is a potential quantitative marker ready for further clinical...

  10. Using RNA-Seq data to select refence genes for normalizing gene expression in apple roots

    Science.gov (United States)

    Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for t...

  11. CDX2 gene expression in acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Arnaoaut, H.H.; Mokhtar, D.A.; Samy, R.M.; Omar, Sh.A.; Khames, S.A.

    2014-01-01

    CDX genes are classically known as regulators of axial elongation during early embryogenesis. An unsuspected role for CDX genes has been revealed during hematopoietic development. The CDX gene family member CDX2 belongs to the most frequent aberrantly expressed proto-oncogenes in human acute leukemias and is highly leukemogenic in experimental models. We used reversed transcriptase polymerase chain reaction (RT-PCR) to determine the expression level of CDX2 gene in 30 pediatric patients with acute lymphoblastic leukemia (ALL) at diagnosis and 30 healthy volunteers. ALL patients were followed up to detect minimal residual disease (MRD) on days 15 and 42 of induction. We found that CDX2 gene was expressed in 50% of patients and not expressed in controls. Associations between gene expression and different clinical and laboratory data of patients revealed no impact on different findings. With follow up, we could not confirm that CDX2 expression had a prognostic significance.

  12. Cytokinins and Expression of SWEET, SUT, CWINV and AAP Genes Increase as Pea Seeds Germinate

    Directory of Open Access Journals (Sweden)

    Paula E. Jameson

    2016-12-01

    Full Text Available Transporter genes and cytokinins are key targets for crop improvement. These genes are active during the development of the seed and its establishment as a strong sink. However, during germination, the seed transitions to being a source for the developing root and shoot. To determine if the sucrose transporter (SUT, amino acid permease (AAP, Sugar Will Eventually be Exported Transporter (SWEET, cell wall invertase (CWINV, cytokinin biosynthesis (IPT, activation (LOG and degradation (CKX gene family members are involved in both the sink and source activities of seeds, we used RT-qPCR to determine the expression of multiple gene family members, and LC-MS/MS to ascertain endogenous cytokinin levels in germinating Pisum sativum L. We show that genes that are actively expressed when the seed is a strong sink during its development, are also expressed when the seed is in the reverse role of being an active source during germination and early seedling growth. Cytokinins were detected in the imbibing seeds and were actively biosynthesised during germination. We conclude that, when the above gene family members are targeted for seed yield improvement, a downstream effect on subsequent seed germination or seedling vigour must be taken into consideration.

  13. Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression.

    Science.gov (United States)

    Kabadi, Ami M; Gersbach, Charles A

    2014-09-01

    Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Developmentally regulated expression of reporter gene in adult ...

    Indian Academy of Sciences (India)

    pression of reporter gene in adult brain specific GAL4 enhancer traps of. Drosophila ... genes based on their expression pattern, thus enabling us to overcome the ... order association and storage centres of olfactory learning and memory, and ...

  15. Expression profiling identifies genes involved in emphysema severity

    Directory of Open Access Journals (Sweden)

    Bowman Rayleen V

    2009-09-01

    Full Text Available Abstract Chronic obstructive pulmonary disease (COPD is a major public health problem. The aim of this study was to identify genes involved in emphysema severity in COPD patients. Gene expression profiling was performed on total RNA extracted from non-tumor lung tissue from 30 smokers with emphysema. Class comparison analysis based on gas transfer measurement was performed to identify differentially expressed genes. Genes were then selected for technical validation by quantitative reverse transcriptase-PCR (qRT-PCR if also represented on microarray platforms used in previously published emphysema studies. Genes technically validated advanced to tests of biological replication by qRT-PCR using an independent test set of 62 lung samples. Class comparison identified 98 differentially expressed genes (p p Gene expression profiling of lung from emphysema patients identified seven candidate genes associated with emphysema severity including COL6A3, SERPINF1, ZNHIT6, NEDD4, CDKN2A, NRN1 and GSTM3.

  16. GSEH: A Novel Approach to Select Prostate Cancer-Associated Genes Using Gene Expression Heterogeneity.

    Science.gov (United States)

    Kim, Hyunjin; Choi, Sang-Min; Park, Sanghyun

    2018-01-01

    When a gene shows varying levels of expression among normal people but similar levels in disease patients or shows similar levels of expression among normal people but different levels in disease patients, we can assume that the gene is associated with the disease. By utilizing this gene expression heterogeneity, we can obtain additional information that abets discovery of disease-associated genes. In this study, we used collaborative filtering to calculate the degree of gene expression heterogeneity between classes and then scored the genes on the basis of the degree of gene expression heterogeneity to find "differentially predicted" genes. Through the proposed method, we discovered more prostate cancer-associated genes than 10 comparable methods. The genes prioritized by the proposed method are potentially significant to biological processes of a disease and can provide insight into them.

  17. Automated discovery of functional generality of human gene expression programs.

    Directory of Open Access Journals (Sweden)

    Georg K Gerber

    2007-08-01

    Full Text Available An important research problem in computational biology is the identification of expression programs, sets of co-expressed genes orchestrating normal or pathological processes, and the characterization of the functional breadth of these programs. The use of human expression data compendia for discovery of such programs presents several challenges including cellular inhomogeneity within samples, genetic and environmental variation across samples, uncertainty in the numbers of programs and sample populations, and temporal behavior. We developed GeneProgram, a new unsupervised computational framework based on Hierarchical Dirichlet Processes that addresses each of the above challenges. GeneProgram uses expression data to simultaneously organize tissues into groups and genes into overlapping programs with consistent temporal behavior, to produce maps of expression programs, which are sorted by generality scores that exploit the automatically learned groupings. Using synthetic and real gene expression data, we showed that GeneProgram outperformed several popular expression analysis methods. We applied GeneProgram to a compendium of 62 short time-series gene expression datasets exploring the responses of human cells to infectious agents and immune-modulating molecules. GeneProgram produced a map of 104 expression programs, a substantial number of which were significantly enriched for genes involved in key signaling pathways and/or bound by NF-kappaB transcription factors in genome-wide experiments. Further, GeneProgram discovered expression programs that appear to implicate surprising signaling pathways or receptor types in the response to infection, including Wnt signaling and neurotransmitter receptors. We believe the discovered map of expression programs involved in the response to infection will be useful for guiding future biological experiments; genes from programs with low generality scores might serve as new drug targets that exhibit minimal

  18. Analysis of multiplex gene expression maps obtained by voxelation

    OpenAIRE

    An, L; Xie, H; Chin, MH; Obradovic, Z; Smith, DJ; Megalooikonomou, V

    2009-01-01

    Abstract Background Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we presen...

  19. Human endogenous retrovirus type W (HERV-W) in schizophrenia: a new avenue of research at the gene-environment interface.

    Science.gov (United States)

    Leboyer, Marion; Tamouza, Ryad; Charron, Dominique; Faucard, Raphaél; Perron, Hervé

    2013-03-01

    Provide a synthetic review of recent studies evidencing an association between human endogenous retrovirus-W (HERV-W) and schizophrenia. Bibliography analysis and contextual synthesis. Epidemiological studies suggest that the aetiology of schizophrenia is complex and involves a complex interplay of genetic and environmental factors such as infections. Eight percentof the human genome consists of human endogenous retroviruses (HERV), and this part of the genome was previously thought to be without importance, but new research has refuted this. HERVs share similarities with viruses and it is assumed that HERVs are present in the genome as a result of retroviruses infecting germ line cells many million years ago. A specific type of HERVs, called HERV-W, has through several recent studies been associated with schizophrenia. Elevated transcription of HERV-W elements has been documented, and antigens of HERV-W envelope and capsid proteins have been found in blood samples from patients. Viruses that have been implicated in pathology of schizophrenia, such as herpes and influenza, have been shown to activate HERV-W elements, and such activation has been associated with elevated biomarkers of systemic inflammation. New research indicates that HERV-W may be an important genetic factor interplaying with the environmental risk factor of infections and that, through this, HERV-W may be important for disease pathogenesis. A lifelong scenario of a detrimental interaction between infectious agents and HERV-W genes may decipher the actual development and course of schizophrenia. Further research is needed to find out if specific treatment strategies could reduce the expression of HERV-W and if this will be associated with remission.

  20. Host Gene Expression Analysis in Sri Lankan Melioidosis Patients

    Science.gov (United States)

    2017-06-19

    CCL5 Chemokine (C-C motif) ligand 5 /RANTES. IFNγ Interferon gamma TNFα Tumor necrosis factor alpha HMGB1 High mobility group box 1 protein /high...aim of this study was to analyze gene expression levels of human host factors in melioidosis patients and establish useful correlation with disease...PBMC’s) of study subjects. Gene expression profiles of 25 gene targets including 19 immune response genes and 6 epigenetic factors were analyzed by

  1. Expression of human ARGONAUTE 2 inhibits endogenous microRNA activity in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Ira eDeveson

    2013-04-01

    Full Text Available Plant and animal microRNA (miRNA pathways share many analogous components, the ARGONAUTE (AGO proteins being foremost among them. We sought to ascertain the degree of functional conservation shared by Homo sapiens ARGONAUTE 2 (HsAGO2 and Arabidopsis thaliana ARGONAUTE 1 (AtAGO1, which are the predominant AGO family members involved with miRNA activity in their respective species. Transgenic Arabidopsis plants expressing HsAGO2 were indistinguishable from counterparts over-expressing AtAGO1, each group exhibiting the morphological and molecular hallmarks of miRNA-pathway loss-of-function alleles. However, unlike AtAGO1, HsAGO2 was unable to rescue the ago1-27 allele. We conclude that, despite the evolutionary gulf between them, HsAGO2 is likely capable of interacting with some component/s of the Arabidopsis miRNA pathway, thereby perturbing its operation, although differences have arisen such that HsAGO2 alone is insufficient to confer efficient silencing of miRNA targets in planta.

  2. Effects of 2G on Gene Expression of Stress-Related Hormones in Rat Placenta

    Science.gov (United States)

    Benson, S.; Talyansky, Y.; Moyer, E. L.; Lowe, M.; Baer, L. A.; Ronca, A. E.

    2017-01-01

    Understanding the effects of spaceflight on mammalian reproductive and developmental physiology is important to future human space exploration and permanent settlement beyond Earth orbit. Fetal developmental programming, including modulation of the HPA axis, is thought to originate at the placental-uterine interface, where both transfer of maternal hormones to the fetus and synthesis of endogenous hormones occurs. In healthy rats, fetal corticosterone levels are kept significantly lower by 11BetaHSD-2, which inactivates corticosterone by conversion into cortisone. Placental tissues express endogenous HPA axis-associated hormones including corticotropin-releasing hormone (CRH), pre-opiomelanocortin (POMC), and vasopressin, which may contribute to fetal programming alongside maternal hormones. DNA methylase 3A, 11BetaHSD-2, and 11BetaHSD-1, which are involved in the regulation of maternal cortisol transfer and modulation of the HPA axis, are also expressed in placental tissues along with glucocorticoid receptor and may be affected by differential gravity exposure during pregnancy. Fetuses may respond differently to maternal glucocorticoid exposure during gestation through sexually dimorphic expression of corticosterone-modulating hormones. To elucidate effects of altered gravity on placental gene expression, here we present a ground-based analogue study involving continuous centrifugation to produce 2g hypergravity. We hypothesized that exposure to 2g would induce a decrease in 11BetaHSD-2 expression through the downregulation of DNA methylase 3a and GC receptor, along with concurrent upregulation in endogenous CRH, POMC, and vasopressin expression. Timed pregnant female rats were exposed to 2G from Gestational day 6 to Gestational day 20, and comparisons made with Stationary Control (SC) and Vivarium Control (VC) dams at 1G. Dams were euthanized and placentas harvested on G20. We homogenized placental tissues, extracted and purified RNA, synthesized cDNA, and

  3. Endogenous expression pattern of resolvin D1 in a rat model of self-resolution of lipopolysaccharide-induced acute respiratory distress syndrome and inflammation.

    Science.gov (United States)

    Sun, Wei; Wang, Zai-ping; Gui, Ping; Xia, Weiyi; Xia, Zhengyuan; Zhang, Xing-cai; Deng, Qing-zhu; Xuan, Wei; Marie, Christelle; Wang, Lin-lin; Wu, Qing-ping; Wang, Tingting; Lin, Yun

    2014-11-01

    Resolvin D1 (RvD1), an endogenous lipid mediator derived from docosahexaenoic acid, has been reported to promote a biphasic activity in anti-inflammatory response and regulate inflammatory resolution. The present study aimed to determine the endogenous expression pattern of RvD1 in a rat model of self-resolution of lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) and inflammation. The ARDS model was induced by administrating LPS (2mg/kg) via tracheotomy in 138 male Sprague-Dawley rats. At specified time points, lung injury and inflammation were respectively assessed by lung histology and analysis of bronchoalveolar lavage fluid and cytokine levels. The expression of endogenous RvD1 was detected by high performance liquid chromatography and tandem mass spectrometry. The results showed that histological lung injury peaked between 6h (LPS6h) and day 3, followed by recovery over 4-10 days after LPS administration. Lung tissue polymorph nuclear cell (PMN) was significantly increased at LPS6h, and peaked between 6h to day 2. The levels of interleukin (IL)-6 and IL-10 were significantly increased at LPS6h and remained higher over day 10 as compared to baseline. Intriguingly, the endogenous RvD1 expression was decreased gradually during the first 3 days, followed by almost completely recovery over days 9-10. The finding indicated that endogenous RvD1 underwent a decrease in expression followed by gradual increase that was basically coincident with the lung injury recovery in a rat model of self-resolution LPS-induced ARDS and inflammation. Our results may help define the optimal therapeutic window for endogenous RvD1 to prevent or treat LPS-induced ARDS and inflammation. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae using reverse-transcription quantitative PCR.

    Directory of Open Access Journals (Sweden)

    Miao Yuan

    Full Text Available The brown planthopper (BPH, Nilaparvata lugens (Hemiptera, Delphacidae, is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR. Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT, muscle actin (MACT, ribosomal protein S11 (RPS11, ribosomal protein S15e (RPS15, alpha 2-tubulin (TUB, elongation factor 1 delta (EF, 18S ribosomal RNA (18S, and arginine kinase (AK and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for

  5. Heterologous gene expression in filamentous fungi.

    Science.gov (United States)

    Su, Xiaoyun; Schmitz, George; Zhang, Meiling; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Rhythmic diel pattern of gene expression in juvenile maize leaf.

    Directory of Open Access Journals (Sweden)

    Maciej Jończyk

    Full Text Available BACKGROUND: Numerous biochemical and physiological parameters of living organisms follow a circadian rhythm. Although such rhythmic behavior is particularly pronounced in plants, which are strictly dependent on the daily photoperiod, data on the molecular aspects of the diurnal cycle in plants is scarce and mostly concerns the model species Arabidopsis thaliana. Here we studied the leaf transcriptome in seedlings of maize, an important C4 crop only distantly related to A. thaliana, throughout a cycle of 10 h darkness and 14 h light to look for rhythmic patterns of gene expression. RESULTS: Using DNA microarrays comprising ca. 43,000 maize-specific probes we found that ca. 12% of all genes showed clear-cut diel rhythms of expression. Cluster analysis identified 35 groups containing from four to ca. 1,000 genes, each comprising genes of similar expression patterns. Perhaps unexpectedly, the most pronounced and most common (concerning the highest number of genes expression maxima were observed towards and during the dark phase. Using Gene Ontology classification several meaningful functional associations were found among genes showing similar diel expression patterns, including massive induction of expression of genes related to gene expression, translation, protein modification and folding at dusk and night. Additionally, we found a clear-cut tendency among genes belonging to individual clusters to share defined transcription factor-binding sequences. CONCLUSIONS: Co-expressed genes belonging to individual clusters are likely to be regulated by common mechanisms. The nocturnal phase of the diurnal cycle involves gross induction of fundamental biochemical processes and should be studied more thoroughly than was appreciated in most earlier physiological studies. Although some general mechanisms responsible for the diel regulation of gene expression might be shared among plants, details of the diurnal regulation of gene expression seem to differ

  7. FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data

    DEFF Research Database (Denmark)

    Manijak, Mieszko P.; Nielsen, Henrik Bjørn

    2011-01-01

    circumvented by instead matching gene expression signatures to signatures of other experiments. FINDINGS: To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700...... Arabidopsis microarray experiments. CONCLUSIONS: Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/....

  8. International collaborative study of the endogenous reference gene, sucrose phosphate synthase (SPS), used for qualitative and quantitative analysis of genetically modified rice.

    Science.gov (United States)

    Jiang, Lingxi; Yang, Litao; Zhang, Haibo; Guo, Jinchao; Mazzara, Marco; Van den Eede, Guy; Zhang, Dabing

    2009-05-13

    One rice ( Oryza sativa ) gene, sucrose phosphate synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results. The validated results confirmed the species specificity of the method through testing 10 plant genomic DNAs, low heterogeneity, and a stable single-copy number of the rice SPS gene among 7 indica varieties and 5 japonica varieties. The SPS qualitative PCR assay was validated with a limit of detection (LOD) of 0.1%, which corresponded to about 230 copies of haploid rice genomic DNA, while the limit of quantification (LOQ) for the quantitative PCR system was about 23 copies of haploid rice genomic DNA, with acceptable PCR efficiency and linearity. Furthermore, the bias between the test and true values of eight blind samples ranged from 5.22 to 26.53%. Thus, we believe that the SPS gene is suitable for use as an endogenous reference gene for the identification and quantification of GM rice and its derivates.

  9. PRAME Gene Expression in Acute Leukemia and Its Clinical Significance

    International Nuclear Information System (INIS)

    Ding, Kai; Wang, Xiao-ming; Fu, Rong; Ruan, Er-bao; Liu, Hui; Shao, Zong-hong

    2012-01-01

    To investigate the expression of the preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia and its clinical significance. The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia (AL) and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR. Correlation analyses between PRAME gene expression and the clinical characteristics (gender, age, white blood count, immunophenotype of leukemia, percentage of blast cells, and karyotype) of the patients were performed. The PRAME gene was expressed in 38.2% of all 34 patients, in 40.7% of the patients with acute myelogenous leukemia (AML, n=27), and in 28.6% of the patients with acute lymphoblastic leukemia (ALL, n=7), but was not expressed in the healthy volunteers. The difference in the expression levels between AML and ALL patients was statistically significant. The rate of gene expression was 80% in M 3 , 33.3% in M 2 , and 28.6% in M 5 . Gene expression was also found to be correlated with CD15 and CD33 expression and abnormal karyotype, but not with age, gender, white blood count or percentage of blast cells. The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute leukemia

  10. Demonstration of the dynamic mass redistribution label-free technology as a useful cell-based pharmacological assay for endogenously expressed GABAA receptors

    DEFF Research Database (Denmark)

    Klein, Anders Bue; Nittegaard-Nielsen, Mia; Christensen, Julie T.

    2015-01-01

    the immortalized IMR-32 neuroblastoma cell line, which expresses relatively high levels of several endogenous GABAA receptor subunits, we show that GABA produces concentration-dependent cellular responses that can be measured and quantified in real-time. With the aid of the GABAA receptor-specific agonist muscimol...

  11. Global gene expression analysis for evaluation and design of biomaterials

    Directory of Open Access Journals (Sweden)

    Nobutaka Hanagata, Taro Takemura and Takashi Minowa

    2010-01-01

    Full Text Available Comprehensive gene expression analysis using DNA microarrays has become a widespread technique in molecular biological research. In the biomaterials field, it is used to evaluate the biocompatibility or cellular toxicity of metals, polymers and ceramics. Studies in this field have extracted differentially expressed genes in the context of differences in cellular responses among multiple materials. Based on these genes, the effects of materials on cells at the molecular level have been examined. Expression data ranging from several to tens of thousands of genes can be obtained from DNA microarrays. For this reason, several tens or hundreds of differentially expressed genes are often present in different materials. In this review, we outline the principles of DNA microarrays, and provide an introduction to methods of extracting information which is useful for evaluating and designing biomaterials from comprehensive gene expression data.

  12. Global gene expression analysis for evaluation and design of biomaterials

    International Nuclear Information System (INIS)

    Hanagata, Nobutaka; Takemura, Taro; Minowa, Takashi

    2010-01-01

    Comprehensive gene expression analysis using DNA microarrays has become a widespread technique in molecular biological research. In the biomaterials field, it is used to evaluate the biocompatibility or cellular toxicity of metals, polymers and ceramics. Studies in this field have extracted differentially expressed genes in the context of differences in cellular responses among multiple materials. Based on these genes, the effects of materials on cells at the molecular level have been examined. Expression data ranging from several to tens of thousands of genes can be obtained from DNA microarrays. For this reason, several tens or hundreds of differentially expressed genes are often present in different materials. In this review, we outline the principles of DNA microarrays, and provide an introduction to methods of extracting information which is useful for evaluating and designing biomaterials from comprehensive gene expression data. (topical review)

  13. Targeting the human lysozyme gene on bovine αs1- casein gene ...

    African Journals Online (AJOL)

    ajl yemi

    2011-11-28

    Nov 28, 2011 ... Targeting an exogenous gene into a favorable gene locus and for expression under endogenous regulators is ... case, the expression of human lysozyme could be regulated by the endogenous cis-element of αs1- casein gene in .... Mouse mammary epithelial C127 cells (Cell Bank, Chinese. Academy of ...

  14. Redox regulation of photosynthetic gene expression.

    Science.gov (United States)

    Queval, Guillaume; Foyer, Christine H

    2012-12-19

    Redox chemistry and redox regulation are central to the operation of photosynthesis and respiration. However, the roles of different oxidants and antioxidants in the regulation of photosynthetic or respiratory gene expression remain poorly understood. Leaf transcriptome profiles of a range of Arabidopsis thaliana genotypes that are deficient in either hydrogen peroxide processing enzymes or in low molecular weight antioxidant were therefore compared to determine how different antioxidant systems that process hydrogen peroxide influence transcripts encoding proteins targeted to the chloroplasts or mitochondria. Less than 10 per cent overlap was observed in the transcriptome patterns of leaves that are deficient in either photorespiratory (catalase (cat)2) or chloroplastic (thylakoid ascorbate peroxidase (tapx)) hydrogen peroxide processing. Transcripts encoding photosystem II (PSII) repair cycle components were lower in glutathione-deficient leaves, as were the thylakoid NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) dehydrogenases (NDH) mRNAs. Some thylakoid NDH mRNAs were also less abundant in tAPX-deficient and ascorbate-deficient leaves. Transcripts encoding the external and internal respiratory NDHs were increased by low glutathione and low ascorbate. Regulation of transcripts encoding specific components of the photosynthetic and respiratory electron transport chains by hydrogen peroxide, ascorbate and glutathione may serve to balance non-cyclic and cyclic electron flow pathways in relation to oxidant production and reductant availability.

  15. Social Regulation of Gene Expression in Threespine Sticklebacks.

    Directory of Open Access Journals (Sweden)

    Anna K Greenwood

    Full Text Available Identifying genes that are differentially expressed in response to social interactions is informative for understanding the molecular basis of social behavior. To address this question, we described changes in gene expression as a result of differences in the extent of social interactions. We housed threespine stickleback (Gasterosteus aculeatus females in either group conditions or individually for one week, then measured levels of gene expression in three brain regions using RNA-sequencing. We found that numerous genes in the hindbrain/cerebellum had altered expression in response to group or individual housing. However, relatively few genes were differentially expressed in either the diencephalon or telencephalon. The list of genes upregulated in fish from social groups included many genes related to neural development and cell adhesion as well as genes with functions in sensory signaling, stress, and social and reproductive behavior. The list of genes expressed at higher levels in individually-housed fish included several genes previously identified as regulated by social interactions in other animals. The identified genes are interesting targets for future research on the molecular mechanisms of normal social interactions.

  16. Large scale gene expression meta-analysis reveals tissue-specific, sex-biased gene expression in humans

    Directory of Open Access Journals (Sweden)

    Benjamin Mayne

    2016-10-01

    Full Text Available The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analysed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes, followed by the heart (375 genes, kidney (224 genes, colon (218 genes and thyroid (163 genes. More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases.

  17. Microarray gene expression profiling and analysis in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sadhukhan Provash

    2004-06-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  18. A stochastic approach to multi-gene expression dynamics

    International Nuclear Information System (INIS)

    Ochiai, T.; Nacher, J.C.; Akutsu, T.

    2005-01-01

    In the last years, tens of thousands gene expression profiles for cells of several organisms have been monitored. Gene expression is a complex transcriptional process where mRNA molecules are translated into proteins, which control most of the cell functions. In this process, the correlation among genes is crucial to determine the specific functions of genes. Here, we propose a novel multi-dimensional stochastic approach to deal with the gene correlation phenomena. Interestingly, our stochastic framework suggests that the study of the gene correlation requires only one theoretical assumption-Markov property-and the experimental transition probability, which characterizes the gene correlation system. Finally, a gene expression experiment is proposed for future applications of the model

  19. Clinicopathologic and gene expression parameters predict liver cancer prognosis

    International Nuclear Information System (INIS)

    Hao, Ke; Zhong, Hua; Greenawalt, Danielle; Ferguson, Mark D; Ng, Irene O; Sham, Pak C; Poon, Ronnie T; Molony, Cliona; Schadt, Eric E; Dai, Hongyue; Luk, John M; Lamb, John; Zhang, Chunsheng; Xie, Tao; Wang, Kai; Zhang, Bin; Chudin, Eugene; Lee, Nikki P; Mao, Mao

    2011-01-01

    The prognosis of hepatocellular carcinoma (HCC) varies following surgical resection and the large variation remains largely unexplained. Studies have revealed the ability of clinicopathologic parameters and gene expression to predict HCC prognosis. However, there has been little systematic effort to compare the performance of these two types of predictors or combine them in a comprehensive model. Tumor and adjacent non-tumor liver tissues were collected from 272 ethnic Chinese HCC patients who received curative surgery. We combined clinicopathologic parameters and gene expression data (from both tissue types) in predicting HCC prognosis. Cross-validation and independent studies were employed to assess prediction. HCC prognosis was significantly associated with six clinicopathologic parameters, which can partition the patients into good- and poor-prognosis groups. Within each group, gene expression data further divide patients into distinct prognostic subgroups. Our predictive genes significantly overlap with previously published gene sets predictive of prognosis. Moreover, the predictive genes were enriched for genes that underwent normal-to-tumor gene network transformation. Previously documented liver eSNPs underlying the HCC predictive gene signatures were enriched for SNPs that associated with HCC prognosis, providing support that these genes are involved in key processes of tumorigenesis. When applied individually, clinicopathologic parameters and gene expression offered similar predictive power for HCC prognosis. In contrast, a combination of the two types of data dramatically improved the power to predict HCC prognosis. Our results also provided a framework for understanding the impact of gene expression on the processes of tumorigenesis and clinical outcome

  20. Exogenous And Endogenous Factors Connected With P16 Gene Alteration In Egyptian Patients With Oesophageal Cancer

    International Nuclear Information System (INIS)

    EL-KASHEF, H.S.; KAYED, A.; ELMAGHRABY, T.K.; EL-GANZURI, M.A.; SELIEM, A.H.

    2010-01-01

    Certain areas of Egypt have a high incidence of oesophageal cancer which is one of the most common causes of cancer related deaths in the world. Comparisons of the dietary and cultural habits of people from geographically distinct high-incidence areas in the world have revealed very few similarities to suggest a common induction mechanism. The present study aimed to investigate the effects of sex, age and smoking on some biochemical parameters, p16 gene mutations, methylation and incidence of oesophageal cancer. The study included 50 Egyptian patients with oesophageal cancer with average age 55.6 years (aged between 23-79 years). The results showed significant decrease in superoxide dismutase (SOD), increase in glutathione reductase (GR), increase in lipid peroxidation end product (malonaldehyde) and incidence of oesophageal cancer. Moreover, two mutations were detected in exon 2 of gene p16 and significant increase in p16 methylation in tissues and plasma of oesophageal cancer patients, as compared to healthy control, were observed.

  1. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    Directory of Open Access Journals (Sweden)

    Zongli eHu

    2015-01-01

    Full Text Available Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi technology to partially silence three different genes (FOW2, FRP1 and OPR in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

  2. Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

    Science.gov (United States)

    Hu, Zongli; Parekh, Urvi; Maruta, Natsumi; Trusov, Yuri; Botella, Jimmy

    2015-01-01

    Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Derived RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1 and OPR) in the hemi-biotrophic fungus Fusarium oxysporum f. sp. conglutinans. Expression of double stranded RNA molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75%, 83% and 72% reduction for FOW2, FRP1 and OPR respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and FOW2 between 45-70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

  3. Characterization of differentially expressed genes using high-dimensional co-expression networks

    DEFF Research Database (Denmark)

    Coelho Goncalves de Abreu, Gabriel; Labouriau, Rodrigo S.

    2010-01-01

    We present a technique to characterize differentially expressed genes in terms of their position in a high-dimensional co-expression network. The set-up of Gaussian graphical models is used to construct representations of the co-expression network in such a way that redundancy and the propagation...... that allow to make effective inference in problems with high degree of complexity (e.g. several thousands of genes) and small number of observations (e.g. 10-100) as typically occurs in high throughput gene expression studies. Taking advantage of the internal structure of decomposable graphical models, we...... construct a compact representation of the co-expression network that allows to identify the regions with high concentration of differentially expressed genes. It is argued that differentially expressed genes located in highly interconnected regions of the co-expression network are less informative than...

  4. Gene Expression Measurement Module (GEMM) - a fully automated, miniaturized instrument for measuring gene expression in space

    Science.gov (United States)

    Karouia, Fathi; Ricco, Antonio; Pohorille, Andrew; Peyvan, Kianoosh

    2012-07-01

    The capability to measure gene expression on board spacecrafts opens the doors to a large number of experiments on the influence of space environment on biological systems that will profoundly impact our ability to conduct safe and effective space travel, and might also shed light on terrestrial physiology or biological function and human disease and aging processes. Measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, determine metabolic basis of microbial pathogenicity and drug resistance, test our ability to sustain and grow in space organisms that can be used for life support and in situ resource utilization during long-duration space exploration, and monitor both the spacecraft environment and crew health. These and other applications hold significant potential for discoveries in space biology, biotechnology and medicine. Accordingly, supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measuring microbial expression of thousands of genes from multiple samples. The instrument will be capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing it on a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. The prototype under development is suitable for deployment on nanosatellite platforms developed by the NASA Small Spacecraft Office. The first target application is to cultivate and measure gene expression of the photosynthetic bacterium Synechococcus elongatus, i.e. a cyanobacterium known to exhibit remarkable metabolic diversity and resilience to adverse conditions

  5. Differentially expressed genes in iron-induced prion protein conversion

    International Nuclear Information System (INIS)

    Kim, Minsun; Kim, Eun-hee; Choi, Bo-Ran; Woo, Hee-Jong

    2016-01-01

    The conversion of the cellular prion protein (PrP C ) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrP res ) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases. - Highlights: • Differential genes correlated with prion degeneration by Fe(III) were identified. • Genes were identified in cell proliferation and intra- and extracellular transport. • In PrP degeneration, redox related genes were suggested. • Cbr2, Rsad2, Slc40a1, Amph and Mvd were expressed significantly.

  6. Gene expression profile data for mouse facial development

    Directory of Open Access Journals (Sweden)

    Sonia M. Leach

    2017-08-01

    Full Text Available This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009 [1] and “Systems Biology of facial development: contributions of ectoderm and mesenchyme” (Hooper et al., 2017 In press [2]. Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press [1,2]. Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.

  7. Protection of germline gene expression by the C. elegans Argonaute CSR-1.

    Science.gov (United States)

    Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

    2013-12-23

    In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.

  8. ANALYSES ON DIFFERENTIALLY EXPRESSED GENES ASSOCIATED WITH HUMAN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    MENG Xu-li; DING Xiao-wen; XU Xiao-hong

    2006-01-01

    Objective: To investigate the molecular etiology of breast cancer by way of studying the differential expression and initial function of the related genes in the occurrence and development of breast cancer. Methods: Two hundred and eighty-eight human tumor related genes were chosen for preparation of the oligochips probe. mRNA was extracted from 16 breast cancer tissues and the corresponding normal breast tissues, and cDNA probe was prepared through reverse-transcription and hybridized with the gene chip. A laser focused fluorescent scanner was used to scan the chip. The different gene expressions were thereafter automatically compared and analyzed between the two sample groups. Cy3/Cy5>3.5 meant significant up-regulation. Cy3/Cy5<0.25 meant significant down-regulation. Results: The comparison between the breast cancer tissues and their corresponding normal tissues showed that 84 genes had differential expression in the Chip. Among the differently expressed genes, there were 4 genes with significant down-regulation and 6 with significant up-regulation. Compared with normal breast tissues, differentially expressed genes did partially exist in the breast cancer tissues. Conclusion: Changes in multi-gene expression regulations take place during the occurrence and development of breast cancer; and the research on related genes can help understanding the mechanism of tumor occurrence.

  9. Regulation of mitochondrial gene expression, the epigenetic enigma

    NARCIS (Netherlands)

    Mposhi, Archibold; van der Wijst, Monique G. P.; Faber, Klaas Nico; Rots, Marianne G.

    2017-01-01

    Epigenetics provides an important layer of information on top of the DNA sequence and is essential for establishing gene expression profiles. Extensive studies have shown that nuclear DNA methylation and histone modifications influence nuclear gene expression. However, it remains unclear whether

  10. Expression of KLK2 gene in prostate cancer

    Directory of Open Access Journals (Sweden)

    Sajad Shafai

    2018-01-01

    Conclusion: The expression of KLK2 gene in people with prostate cancer is the higher than the healthy person; finally, according to the results, it could be mentioned that the KLK2 gene considered as a useful factor in prostate cancer, whose expression is associated with progression and development of the prostate cancer.

  11. Comparative genomics of the relationship between gene structure and expression

    NARCIS (Netherlands)

    Ren, X.

    2006-01-01

    The relationship between the structure of genes and their expression is a relatively new aspect of genome organization and regulation. With more genome sequences and expression data becoming available, bioinformatics approaches can help the further elucidation of the relationships between gene

  12. The gene expressions of DNA methylation/demethylation enzymes ...

    African Journals Online (AJOL)

    user

    2011-01-31

    Jan 31, 2011 ... A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases.

  13. Genome organization and expression of the rat ACBP gene family

    DEFF Research Database (Denmark)

    Mandrup, S; Andreasen, P H; Knudsen, J

    1993-01-01

    pool former. We have molecularly cloned and characterized the rat ACBP gene family which comprises one expressed and four processed pseudogenes. One of these was shown to exist in two allelic forms. A comprehensive computer-aided analysis of the promoter region of the expressed ACBP gene revealed...

  14. Effects of heat stress on gene expression in eggplant ( Solanum ...

    African Journals Online (AJOL)

    In order to identify differentially expressed genes involved in heat shock response, cDNA amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time polymerase chain reaction (QPCR) were used to study gene expression of eggplant seedlings subjected to 0, 6 and 12 h at 43°C. A total of 53 of over ...

  15. RNA preparation and characterization for gene expression studies

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2009-01-01

    Much information can be obtained from knowledge of the relative expression level of each gene in the transcriptome. With the current advances in technology as little as a single cell is required as starting material for gene expression experiments. The mRNA from a single cell may be linearly...

  16. The gene expressions of DNA methylation/demethylation enzymes ...

    African Journals Online (AJOL)

    A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases (Dnmts), DNA ...

  17. Microarray analysis of the gene expression profile in triethylene ...

    African Journals Online (AJOL)

    Microarray analysis of the gene expression profile in triethylene glycol dimethacrylate-treated human dental pulp cells. ... Conclusions: Our results suggest that TEGDMA can change the many functions of hDPCs through large changes in gene expression levels and complex interactions with different signaling pathways.

  18. Identification of pyrG Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Pleurotus ostreatus.

    Science.gov (United States)

    Zheng, Shi; Shan, Luying; Zhuang, Yongliang; Shang, Ying

    2018-03-01

    As a well-known edible fungus rich in nutrients, Pleurotus ostreatus has been used as an alternative to expensive wild edible fungi. Specifically, the fact that using P. ostreatus instead of other expensive wild edible fungi has damaged the rights and interests of consumers. Among the existing methods for detection of food adulteration, the amplification of endogenous reference gene is the most accurate method. However, an ideal endogenous reference gene for P. ostreatus has yet to be developed. In this study, a DNA extraction method for P. ostreatus was optimized, and pyrG was selected as a species-specific gene through sequence alignment. This gene was subsequently subjected to qualitative and quantitative Polymerase Chain Reaction (PCR) assays with 3 different P. ostreatus varieties and 7 other species. A low detection limit of 5 pg/μL was obtained by TaqMan quantitative PCR, and no pyrG amplification product was observed in the 7 other species. No allelic variation was detected in P. ostreatus varieties. These experiments confirmed that pyrG was an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus. This method was also suitable for the examination of processed P. ostreatus samples and determination of adulteration in wild mushrooms. The pyrG gene was chosen as an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus, and the detection limit was 5 pg/μL for the quantification. This method is used not only for raw materials but also for processed P. ostreatus products and other processed mushroom foods. © 2018 Institute of Food Technologists®.

  19. DNA alkylating agents alleviate silencing of class II transactivator gene expression in L1210 lymphoma cells.

    Science.gov (United States)

    Murphy, Shawn P; Holtz, Renae; Lewandowski, Nicole; Tomasi, Thomas B; Fuji, Hiroshi

    2002-09-15

    MHC class II (Ia) Ag expression is inversely correlated with tumorigenicity and directly correlated with immunogenicity in clones of the mouse L1210 lymphoma (1 ). Understanding the mechanisms by which class II Ag expression is regulated in L1210 lymphoma may facilitate the development of immunotherapeutic approaches for the treatment of some types of lymphoma and leukemia. This study demonstrates that the variation in MHC class II Ag expression among clones of L1210 lymphoma is due to differences in the expression of the class II transactivator (CIITA). Analysis of stable hybrids suggests that CIITA expression is repressed by a dominant mechanism in class II-negative L1210 clones. DNA-alkylating agents such as ethyl methanesulfonate and the chemotherapeutic drug melphalan activate CIITA and class II expression in class II negative L1210 cells, and this effect appears to be restricted to transformed cell lines derived from the early stages of B cell ontogeny. Transient transfection assays demonstrated that the CIITA type III promoter is active in class II(-) L1210 cells, despite the fact that the endogenous gene is not expressed, which suggests that these cells have all of the transacting factors necessary for CIITA transcription. An inverse correlation between methylation of the CIITA transcriptional regulatory region and CIITA expression was observed among L1210 clones. Furthermore, 5-azacytidine treatment activated CIITA expression in class II-negative L1210 cells. Collectively, our results suggest that 1) CIITA gene expression is repressed in class II(-) L1210 cells by methylation of the CIITA upstream regulatory region, and 2) treatment with DNA-alkylating agents overcomes methylation-based silencing of the CIITA gene in L1210 cells.

  20. Fungal and plant gene expression in arbuscular mycorrhizal symbiosis.

    Science.gov (United States)

    Balestrini, Raffaella; Lanfranco, Luisa

    2006-11-01

    Arbuscular mycorrhizas (AMs) are a unique example of symbiosis between two eukaryotes, soil fungi and plants. This association induces important physiological changes in each partner that lead to reciprocal benefits, mainly in nutrient supply. The symbiosis results from modifications in plant and fungal cell organization caused by specific changes in gene expression. Recently, much effort has gone into studying these gene expression patterns to identify a wider spectrum of genes involved. We aim in this review to describe AM symbiosis in terms of current knowledge on plant and fungal gene expression profiles.

  1. Expression and clinical significance of Pax6 gene in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Hai-Dong Huang

    2013-07-01

    Full Text Available AIM: To discuss the expression and clinical significance of Pax6 gene in retinoblastoma(Rb. METHODS: Totally 15 cases of fresh Rb organizations were selected as observation group and 15 normal retinal organizations as control group. Western-Blot and reverse transcriptase polymerase chain reaction(RT-PCRmethods were used to detect Pax6 protein and Pax6 mRNA expressions of the normal retina organizations and Rb organizations. At the same time, Western Blot method was used to detect the Pax6 gene downstream MATH5 and BRN3b differentiation gene protein level expression. After the comparison between two groups, the expression and clinical significance of Pax6 gene in Rb were discussed. RESULTS: In the observation group, average value of mRNA expression of Pax6 gene was 0.99±0.03; average value of Pax6 gene protein expression was 2.07±0.15; average value of BRN3b protein expression was 0.195±0.016; average value of MATH5 protein expression was 0.190±0.031. They were significantly higher than the control group, and the differences were statistically significant(PCONCLUSION: Abnormal expression of Pax6 gene is likely to accelerate the occurrence of Rb.

  2. Gene expression in cerebral ischemia: a new approach for neuroprotection.

    Science.gov (United States)

    Millán, Mónica; Arenillas, Juan

    2006-01-01

    Cerebral ischemia is one of the strongest stimuli for gene induction in the brain. Hundreds of genes have been found to be induced by brain ischemia. Many genes are involved in neurodestructive functions such as excitotoxicity, inflammatory response and neuronal apoptosis. However, cerebral ischemia is also a powerful reformatting and reprogramming stimulus for the brain through neuroprotective gene expression. Several genes may participate in both cellular responses. Thus, isolation of candidate genes for neuroprotection strategies and interpretation of expression changes have been proven difficult. Nevertheless, many studies are being carried out to improve the knowledge of the gene activation and protein expression following ischemic stroke, as well as in the development of new therapies that modify biochemical, molecular and genetic changes underlying cerebral ischemia. Owing to the complexity of the process involving numerous critical genes expressed differentially in time, space and concentration, ongoing therapeutic efforts should be based on multiple interventions at different levels. By modification of the acute gene expression induced by ischemia or the apoptotic gene program, gene therapy is a promising treatment but is still in a very experimental phase. Some hurdles will have to be overcome before these therapies can be introduced into human clinical stroke trials. Copyright 2006 S. Karger AG, Basel.

  3. Genetic architecture of gene expression in the chicken

    Directory of Open Access Journals (Sweden)

    Stanley Dragana

    2013-01-01

    Full Text Available Abstract Background The annotation of many genomes is limited, with a large proportion of identified genes lacking functional assignments. The construction of gene co-expression networks is a powerful approach that presents a way of integrating information from diverse gene expression datasets into a unified analysis which allows inferences to be drawn about the role of previously uncharacterised genes. Using this approach, we generated a condition-free gene co-expression network for the chicken using data from 1,043 publically available Affymetrix GeneChip Chicken Genome Arrays. This data was generated from a diverse range of experiments, including different tissues and experimental conditions. Our aim was to identify gene co-expression modules and generate a tool to facilitate exploration of the functional chicken genome. Results Fifteen modules, containing between 24 and 473 genes, were identified in the condition-free network. Most of the modules showed strong functional enrichment for particular Gene Ontology categories. However, a few showed no enrichment. Transcription factor binding site enrichment was also noted. Conclusions We have demonstrated that this chicken gene co-expression network is a useful tool in gene function prediction and the identification of putative novel transcription factors and binding sites. This work highlights the relevance of this methodology for functional prediction in poorly annotated genomes such as the chicken.

  4. Decoupling Linear and Nonlinear Associations of Gene Expression

    KAUST Repository

    Itakura, Alan

    2013-01-01

    The FANTOM consortium has generated a large gene expression dataset of different cell lines and tissue cultures using the single-molecule sequencing technology of HeliscopeCAGE. This provides a unique opportunity to investigate novel associations between gene expression over time and different cell types. Here, we create a MatLab wrapper for a powerful and computationally intensive set of statistics known as Maximal Information Coefficient, and then calculate this statistic for a large, comprehensive dataset containing gene expression of a variety of differentiating tissues. We then distinguish between linear and nonlinear associations, and then create gene association networks. Following this analysis, we are then able to identify clusters of linear gene associations that then associate nonlinearly with other clusters of linearity, providing insight to much more complex connections between gene expression patterns than previously anticipated.

  5. Decoupling Linear and Nonlinear Associations of Gene Expression

    KAUST Repository

    Itakura, Alan

    2013-05-01

    The FANTOM consortium has generated a large gene expression dataset of different cell lines and tissue cultures using the single-molecule sequencing technology of HeliscopeCAGE. This provides a unique opportunity to investigate novel associations between gene expression over time and different cell types. Here, we create a MatLab wrapper for a powerful and computationally intensive set of statistics known as Maximal Information Coefficient, and then calculate this statistic for a large, comprehensive dataset containing gene expression of a variety of differentiating tissues. We then distinguish between linear and nonlinear associations, and then create gene association networks. Following this analysis, we are then able to identify clusters of linear gene associations that then associate nonlinearly with other clusters of linearity, providing insight to much more complex connections between gene expression patterns than previously anticipated.

  6. Gene expression profiling of placentas affected by pre-eclampsia

    DEFF Research Database (Denmark)

    Hoegh, Anne Mette; Borup, Rehannah; Nielsen, Finn Cilius

    2010-01-01

    Several studies point to the placenta as the primary cause of pre-eclampsia. Our objective was to identify placental genes that may contribute to the development of pre-eclampsia. RNA was purified from tissue biopsies from eleven pre-eclamptic placentas and eighteen normal controls. Messenger RNA...... expression from pooled samples was analysed by microarrays. Verification of the expression of selected genes was performed using real-time PCR. A surprisingly low number of genes (21 out of 15,000) were identified as differentially expressed. Among these were genes not previously associated with pre-eclampsia...... as bradykinin B1 receptor and a 14-3-3 protein, but also genes that have already been connected with pre-eclampsia, for example, inhibin beta A subunit and leptin. A low number of genes were repeatedly identified as differentially expressed, because they may represent the endpoint of a cascade of events...

  7. Expression Profile of Genes Related to Drug Metabolism in Human Brain Tumors.

    Directory of Open Access Journals (Sweden)

    Pantelis Stavrinou

    Full Text Available Endogenous and exogenous compounds as well as carcinogens are metabolized and detoxified by phase I and II enzymes, the activity of which could be crucial to the inactivation and hence susceptibility to carcinogenic factors. The expression of these enzymes in human brain tumor tissue has not been investigated sufficiently. We studied the association between tumor pathology and the expression profile of seven phase I and II drug metabolizing genes (CYP1A1, CYP1B1, ALDH3A1, AOX1, GSTP1, GSTT1 and GSTM3 and some of their proteins.Using qRT-PCR and western blotting analysis the gene and protein expression in a cohort of 77 tumors were investigated. The major tumor subtypes were meningioma, astrocytoma and brain metastases, -the later all adenocarcinomas from a lung primary.Meningeal tumors showed higher expression levels for AOX1, CYP1B1, GSTM3 and GSTP1. For AOX1, GSTM and GSTP1 this could be verified on a protein level as well. A negative correlation between the WHO degree of malignancy and the strength of expression was identified on both transcriptional and translational level for AOX1, GSTM3 and GSTP1, although the results could have been biased by the prevalence of meningiomas and glioblastomas in the inevitably bipolar distribution of the WHO grades. A correlation between the gene expression and the protein product was observed for AOX1, GSTP1 and GSTM3 in astrocytomas.The various CNS tumors show different patterns of drug metabolizing gene expression. Our results suggest that the most important factor governing the expression of these enzymes is the histological subtype and to a far lesser extent the degree of malignancy itself.

  8. Isolation and characterization of LHY homolog gene expressed in ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-02

    May 2, 2008 ... responsible in negative feedback loop reaction of central oscillator in plant circadian clock system. The level of gene expression was found to be high four hours after dawn in flowering shoots and flower. This paper reported the isolation and characterization of the gene. Key words: LHY gene, circadian ...

  9. Gene mining a marama bean expressed sequence tags (ESTs ...

    African Journals Online (AJOL)

    The authors reported the identification of genes associated with embryonic development and microsatellite sequences. The future direction will entail characterization of these genes using gene over-expression and mutant assays. Key words: Namibia, simple sequence repeats (SSR), data mining, homology searches, ...

  10. Expression profiles of genes involved in tanshinone biosynthesis of ...

    Indian Academy of Sciences (India)

    Expression profiles of genes involved in tanshinone biosynthesis of two. Salvia miltiorrhiza genotypes with different tanshinone contents. Zhenqiao Song, Jianhua Wang and Xingfeng Li. J. Genet. 95, 433–439. Table 1. S. miltiorrhiza genes and primer pairs used for qRT-PCR. Gene. GenBank accession. Primer name.

  11. Differentially expressed genes in the midgut of Silkworm infected ...

    African Journals Online (AJOL)

    In this report, we employed suppression subtractive hybridization to compare differentially expressed genes in the midguts of CPV-infected and normal silkworm larvae. 36 genes and 20 novel ESTs were obtained from 2 reciprocal subtractive libraries. Three up-regulated genes (ferritin, rpL11 and alkaline nuclease) and 3 ...

  12. Adaptive differences in gene expression in European flounder ( Platichthys flesus )

    DEFF Research Database (Denmark)

    Larsen, Peter Foged; Eg Nielsen, Einar; Williams, T.D.

    2007-01-01

    levels of neutral genetic divergence, a high number of genes were significantly differentially expressed between North Sea and Baltic Sea flounders maintained in a long-term reciprocal transplantation experiment mimicking natural salinities. Several of the differentially regulated genes could be directly...... linked to fitness traits. These findings demonstrate that flounders, despite little neutral genetic divergence between populations, are differently adapted to local environmental conditions and imply that adaptation in gene expression could be common in other marine organisms with similar low levels...

  13. Gene Expression and the Diversity of Identified Neurons

    OpenAIRE

    Buck, L.; Stein, R.; Palazzolo, M.; Anderson, D. J.; Axel, R.

    1983-01-01

    Nervous systems consist of diverse populations of neurons that are anatomically and functionally distinct. The diversity of neurons and the precision with which they are interconnected suggest that specific genes or sets of genes are activated in some neurons but not expressed in others. Experimentally, this problem may be considered at two levels. First, what is the total number of genes expressed in the brain, and how are they distributed among the different populations of neurons? Second, ...

  14. Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data

    Directory of Open Access Journals (Sweden)

    Tintle Nathan L

    2012-08-01

    Full Text Available Abstract Background Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. Results We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Conclusions Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

  15. Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

    Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

  16. Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco.

    Science.gov (United States)

    Baek, Eseul; Yoon, Ju-Yeon; Palukaitis, Peter

    2017-10-01

    To facilitate quantification of gene expression changes in virus-infected tobacco plants, eight housekeeping genes were evaluated for their stability of expression during infection by one of three systemically-infecting viruses (cucumber mosaic virus, potato virus X, potato virus Y) or a hypersensitive-response-inducing virus (tobacco mosaic virus; TMV) limited to the inoculated leaf. Five reference-gene validation programs were used to establish the order of the most stable genes for the systemically-infecting viruses as ribosomal protein L25 > β-Tubulin > Actin, and the least stable genes Ubiquitin-conjugating enzyme (UCE) genes were EF1α > Cysteine protease > Actin, and the least stable genes were GAPDH genes, three defense responsive genes were examined to compare their relative changes in gene expression caused by each virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. A Gene Expression Classifier of Node-Positive Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Paul F. Meeh

    2009-10-01

    Full Text Available We used digital long serial analysis of gene expression to discover gene expression differences between node-negative and node-positive colorectal tumors and developed a multigene classifier able to discriminate between these two tumor types. We prepared and sequenced long serial analysis of gene expression libraries from one node-negative and one node-positive colorectal tumor, sequenced to a depth of 26,060 unique tags, and identified 262 tags significantly differentially expressed between these two tumors (P < 2 x 10-6. We confirmed the tag-to-gene assignments and differential expression of 31 genes by quantitative real-time polymerase chain reaction, 12 of which were elevated in the node-positive tumor. We analyzed the expression levels of these 12 upregulated genes in a validation panel of 23 additional tumors and developed an optimized seven-gene logistic regression classifier. The classifier discriminated between node-negative and node-positive tumors with 86% sensitivity and 80% specificity. Receiver operating characteristic analysis of the classifier revealed an area under the curve of 0.86. Experimental manipulation of the function of one classification gene, Fibronectin, caused profound effects on invasion and migration of colorectal cancer cells in vitro. These results suggest that the development of node-positive colorectal cancer occurs in part through elevated epithelial FN1 expression and suggest novel strategies for the diagnosis and treatment of advanced disease.

  18. Gene expression profiling of resting and activated vascular smooth muscle cells by serial analysis of gene expression and clustering analysis

    NARCIS (Netherlands)

    Beauchamp, Nicholas J.; van Achterberg, Tanja A. E.; Engelse, Marten A.; Pannekoek, Hans; de Vries, Carlie J. M.

    2003-01-01

    Migration and proliferation of vascular smooth muscle cells (SMCs) are key events in atherosclerosis. However, little is known about alterations in gene expression upon transition of the quiescent, contractile SMC to the proliferative SMC. We performed serial analysis of gene expression (SAGE) of

  19. Using PCR to Target Misconceptions about Gene Expression

    Directory of Open Access Journals (Sweden)

    Leslie K. Wright

    2013-02-01

    Full Text Available We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA and gene expression (mRNA/protein and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression.

  20. Overexpression of transcription factor Sp1 leads to gene expression perturbations and cell cycle inhibition.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Deniaud

    Full Text Available BACKGROUND: The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression. METHODOLOGY AND PRINCIPAL FINDINGS: We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis. CONCLUSION: This study shows that the binding to DNA

  1. Dynamic association rules for gene expression data analysis.

    Science.gov (United States)

    Chen, Shu-Chuan; Tsai, Tsung-Hsien; Chung, Cheng-Han; Li, Wen-Hsiung

    2015-10-14

    The purpose of gene expression analysis is to look for the association between regulation of gene expression levels and phenotypic variations. This association based on gene expression profile has been used to determine whether the induction/repression of genes correspond to phenotypic variations including cell regulations, clinical diagnoses and drug development. Statistical analyses on microarray data have been developed to resolve gene selection issue. However, these methods do not inform us of causality between genes and phenotypes. In this paper, we propose the dynamic association rule algorithm (DAR algorithm) which helps ones to efficiently select a subset of significant genes for subsequent analysis. The DAR algorithm is based on association rules from market basket analysis in marketing. We first propose a statistical way, based on constructing a one-sided confidence interval and hypothesis testing, to determine if an association rule is meaningful. Based on the proposed statistical method, we then developed the DAR algorithm for gene expression data analysis. The method was applied to analyze four microarray datasets and one Next Generation Sequencing (NGS) dataset: the Mice Apo A1 dataset, the whole genome expression dataset of mouse embryonic stem cells, expression profiling of the bone marrow of Leukemia patients, Microarray Quality Control (MAQC) data set and the RNA-seq dataset of a mouse genomic imprinting study. A comparison of the proposed method with the t-test on the expression profiling of the bone marrow of Leukemia patients was conducted. We developed a statistical way, based on the concept of confidence interval, to determine the minimum support and minimum confidence for mining association relationships among items. With the minimum support and minimum confidence, one can find significant rules in one single step. The DAR algorithm was then developed for gene expression data analysis. Four gene expression datasets showed that the proposed

  2. Dietary Berries and Ellagic Acid Prevent Oxidative DNA Damage and Modulate Expression of DNA Repair Genes

    Directory of Open Access Journals (Sweden)

    Ramesh C. Gupta

    2008-03-01

    Full Text Available DNA damage is a pre-requisite for the initiation of cancer and agents that reduce this damage are useful in cancer prevention. In this study, we evaluated the ability of whole berries and berry phytochemical, ellagic acid to reduce endogenous oxidative DNA damage. Ellagic acid was selected based on > 95% inhibition of 8-oxodeoxyguosine (8-oxodG and other unidentified oxidative DNA adducts induced by 4-hydroxy-17B;-estradiol and CuCl2 in vitro. Inhibition of the latter occurred at lower concentrations (10 u(microM than that for 8-oxodG (100 u(microM. In the in vivo study, female CD-1 mice (n=6 were fed either a control diet or diet supplemented with ellagic acid (400 ppm and dehydrated berries (5% w/w with varying ellagic acid contents -- blueberry (low, strawberry (medium and red raspberry (high, for 3 weeks. Blueberry and strawberry diets showed moderate reductions in endogenous DNA adducts (25%. However, both red raspberry and ellagic acid diets showed a significant reduction of 59% (p < 0.001 and 48% (p < 0.01, respectively. Both diets also resulted in a 3-8 fold over-expression of genes involved in DNA repair such as xeroderma pigmentosum group A complementing protein (XPA, DNA excision repair protein (ERCC5 and DNA ligase III (DNL3. These results suggest that red raspberry and ellagic acid reduce endogenous oxidative DNA damage by mechanisms which may involve increase in DNA repair.

  3. Epigenetic regulation on the gene expression signature in esophagus adenocarcinoma.

    Science.gov (United States)

    Xi, Ting; Zhang, Guizhi

    2017-02-01

    Understanding the molecular mechanisms represents an important step in the development of diagnostic and therapeutic measures of esophagus adenocarcinoma (NOS). The objective of this study is to identify the epigenetic regulation on gene expression in NOS, shedding light on the molecular mechanisms of NOS. In this study, 78 patients with NOS were included and the data of mRNA, miRNA and DNA methylation of were downloaded from The Cancer Genome Atlas (TCGA). Differential analysis between NOS and controls was performed in terms of gene expression, miRNA expression, and DNA methylation. Bioinformatic analysis was followed to explore the regulation mechanisms of miRNA and DNA methylationon gene expression. Totally, up to 1320 differentially expressed genes (DEGs) and 32 differentially expressed miRNAs were identified. 240 DEGs that were not only the target genes but also negatively correlated with the screened differentially expressed miRNAs. 101 DEGs were found to be highlymethylated in CpG islands. Then, 8 differentially methylated genes (DMGs) were selected, which showed down-regulated expression in NOS. Among of these genes, 6 genes including ADHFE1, DPP6, GRIA4, CNKSR2, RPS6KA6 and ZNF135 were target genes of differentially expressed miRNAs (hsa-mir-335, hsa-mir-18a, hsa-mir-93, hsa-mir-106b and hsa-mir-21). The identified altered miRNA, genes and DNA methylation site may be applied as biomarkers for diagnosis and prognosis of NOS. Copyright © 2016 Elsevier GmbH. All rights reserved.

  4. Dietary TiO2 particles modulate expression of hormone-related genes in Bombyx mori.

    Science.gov (United States)

    Shi, Guofang; Zhan, Pengfei; Jin, Weiming; Fei, JianMing; Zhao, Lihua

    2017-08-01

    Silkworm (Bombyx mori) is an economically beneficial insect. Its growth and development are regulated by endogenous hormones. In the present study, we found that feeding titanium dioxide nanoparticles (TiO 2 NP) caused a significant increase of body size. TiO 2 NP stimulated the transcription of several genes, including the insulin-related hormone bombyxin, PI3K/Akt/TOR (where PI3K is phosphatidylinositol 3-kinase and TOR is target of rapamycin), and the adenosine 5'-monophosphateactivated protein kinase (AMPK)/target of rapamycin (TOR) pathways. Differentially expressed gene (DEG) analysis documented 26 developmental hormone signaling related genes that were differentially expressed following dietary TiO 2 NP treatment. qPCR analysis confirmed the upregulation of insulin/ecdysteroid signaling genes, such as bombyxin B-1, bombyxin B-4, bombyxin B-7, MAPK, P70S6K, PI3k, eIF4E, E75, ecdysteroid receptor (EcR), and insulin-related peptide binding protein precursor 2 (IBP2). We infer from the upregulated expression of bombyxins and the signaling network that they act in bombyxin-stimulated ecdysteroidogenesis. © 2017 Wiley Periodicals, Inc.

  5. Identifying potential maternal genes of Bombyx mori using digital gene expression profiling

    Science.gov (United States)

    Xu, Pingzhen

    2018-01-01

    Maternal genes present in mature oocytes play a crucial role in the early development of silkworm. Although maternal genes have been widely studied in many other species, there has been limited research in Bombyx mori. High-throughput next generation sequencing provides a practical method for gene discovery on a genome-wide level. Herein, a transcriptome study was used to identify maternal-related genes from silkworm eggs. Unfertilized eggs from five different stages of early development were used to detect the changing situation of gene expression. The expressed genes showed different patterns over time. Seventy-six maternal genes were annotated according to homology analysis with Drosophila melanogaster. More than half of the differentially expressed maternal genes fell into four expression patterns, while the expression patterns showed a downward trend over time. The functional annotation of these material genes was mainly related to transcription factor activity, growth factor activity, nucleic acid binding, RNA binding, ATP binding, and ion binding. Additionally, twenty-two gene clusters including maternal genes were identified from 18 scaffolds. Altogether, we plotted a profile for the maternal genes of Bombyx mori using a digital gene expression profiling method. This will provide the basis for maternal-specific signature research and improve the understanding of the early development of silkworm. PMID:29462160

  6. Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis.

    Science.gov (United States)

    Sagor, G H M; Berberich, Thomas; Kojima, Seiji; Niitsu, Masaru; Kusano, Tomonobu

    2016-06-01

    Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine. Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

  7. Temperature and Development Impacts on Housekeeping Gene Expression in Cowpea Aphid, Aphis craccivora (Hemiptera: Aphidiae.

    Directory of Open Access Journals (Sweden)

    Chunxiao Yang

    Full Text Available Quantitative real-time PCR (qRT-PCR is a powerful technique to quantify gene expression. To standardize gene expression studies and obtain more accurate qRT-PCR analysis, normalization relative to consistently expressed housekeeping genes (HKGs is required. In this study, ten candidate HKGs including elongation factor 1 α (EF1A, ribosomal protein L11 (RPL11, ribosomal protein L14 (RPL14, ribosomal protein S8 (RPS8, ribosomal protein S23 (RPS23, NADH-ubiquinone oxidoreductase (NADH, vacuolar-type H+-ATPase (ATPase, heat shock protein 70 (HSP70, 18S ribosomal RNA (18S, and 12S ribosomal RNA (12S from the cowpea aphid, Aphis craccivora Koch were selected. Four algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method were employed to evaluate the expression profiles of these HKGs as endogenous controls across different developmental stages and temperature regimes. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidate HKGs, RPS8, RPL14, and RPL11 were the three most stable HKGs across different developmental stages and temperature conditions. This study is the first step to establish a standardized qRT-PCR analysis in A. craccivora following the MIQE guideline. Results from this study lay a foundation for the genomics and functional genomics research in this sap-sucking insect pest with substantial economic impact.

  8. The tobacco smoke component acrolein induces glucocorticoid resistant gene expression via inhibition of histone deacetylase.

    Science.gov (United States)

    Randall, Matthew J; Haenen, Guido R M M; Bouwman, Freek G; van der Vliet, Albert; Bast, Aalt

    2016-01-05

    Chronic obstructive pulmonary disease (COPD) is the leading cause of cigarette smoke-related death worldwide. Acrolein, a crucial reactive electrophile found in cigarette smoke mimics many of the toxic effects of cigarette smoke-exposure in the lung. In macrophages, cigarette smoke is known to hinder histone deacetylases (HDACs), glucocorticoid-regulated enzymes that play an important role in the pathogenesis of glucocorticoid resistant inflammation, a common feature of COPD. Thus, we hypothesize that acrolein plays a role in COPD-associated glucocorticoid resistance. To examine the role of acrolein on glucocorticoid resistance, U937 monocytes, differentiated with PMA to macrophage-like cells were treated with acrolein for 0.5h followed by stimulation with hydrocortisone for 8h, or treated simultaneously with LPS and hydrocortisone for 8h without acrolein. GSH and nuclear HDAC activity were measured, or gene expression was analyzed by qPCR. Acrolein-mediated TNFα gene expression was not suppressed by hydrocortisone whereas LPS-induced TNFα expression was suppressed. Acrolein also significantly inhibited nuclear HDAC activity in macrophage-like cells. Incubation of recombinant HDAC2 with acrolein led to the formation of an HDAC2-acrolein adduct identified by mass spectrometry. Therefore, these results suggest that acrolein-induced inflammatory gene expression is resistant to suppression by the endogenous glucocorticoid, hydrocortisone. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Evidence for an indirect transcriptional regulation of glucose-6-phosphatase gene expression by liver X receptors

    International Nuclear Information System (INIS)

    Grempler, Rolf; Guenther, Susanne; Steffensen, Knut R.; Nilsson, Maria; Barthel, Andreas; Schmoll, Dieter; Walther, Reinhard

    2005-01-01

    Liver X receptor (LXR) paralogues α and β (LXRα and LXRβ) are members of the nuclear hormone receptor family and have oxysterols as endogenous ligands. LXR activation reduces hepatic glucose production in vivo through the inhibition of transcription of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase). In the present study, we investigated the molecular mechanisms involved in the regulation of G6Pase gene expression by LXR. Both T0901317, a synthetic LXR agonist, and the adenoviral overexpression of either LXRα or LXRβ suppressed G6Pase gene expression in H4IIE hepatoma cells. However, compared to the suppression of G6Pase expression seen by insulin, the decrease of G6Pase mRNA by LXR activation was delayed and was blocked by cycloheximide, an inhibitor of protein synthesis. These observations, together with the absence of a conserved LXR-binding element within the G6Pase promoter, suggest an indirect inhibition of G6Pase gene expression by liver X receptors

  10. Extracting gene expression patterns and identifying co-expressed genes from microarray data reveals biologically responsive processes

    Directory of Open Access Journals (Sweden)

    Paules Richard S

    2007-11-01

    Full Text Available Abstract Background A common observation in the analysis of gene expression data is that many genes display similarity in their expression patterns and therefore appear to be co-regulated. However, the variation associated with microarray data and the complexity of the experimental designs make the acquisition of co-expressed genes a challenge. We developed a novel method for Extracting microarray gene expression Patterns and Identifying co-expressed Genes, designated as EPIG. The approach utilizes the underlying structure of gene expression data to extract patterns and identify co-expressed genes that are responsive to experimental conditions. Results Through evaluation of the correlations among profiles, the magnitude of variation in gene expression profiles, and profile signal-to-noise ratio's, EPIG extracts a set of patterns representing co-expressed genes. The method is shown to work well with a simulated data set and microarray data obtained from time-series studies of dauer recovery and L1 starvation in C. elegans and after ultraviolet (UV or ionizing radiation (IR-induced DNA damage in diploid human fibroblasts. With the simulated data set, EPIG extracted the appropriate number of patterns which were more stable and homogeneous than the set of patterns that were determined using the CLICK or CAST clustering algorithms. However, CLICK performed better than EPIG and CAST with respect to the average correlation between clusters/patterns of the simulated data. With real biological data, EPIG extracted more dauer-specific patterns than CLICK. Furthermore, analysis of the IR/UV data revealed 18 unique patterns and 2661 genes out of approximately 17,000 that were identified as significantly expressed and categorized to the patterns by EPIG. The time-dependent patterns displayed similar and dissimilar responses between IR and UV treatments. Gene Ontology analysis applied to each pattern-related subset of co-expressed genes revealed underlying

  11. Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes

    Directory of Open Access Journals (Sweden)

    Øvstebø Reidun

    2010-05-01

    Full Text Available Abstract Background Gene expression in lipopolysaccharide (LPS-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase or ACTB (beta-actin as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system. Results Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B, B2M (beta-2-microglobulin and PPIA (cyclophilin A as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-α (tumor necrosis factor-alpha and IL10 (interleukin 10 expression. Moreover, a significant difference in TNF-α expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH. Conclusions Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

  12. Expression profiles for six zebrafish genes during gonadal sex differentiation

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Morthorst, Jane Ebsen; Andersen, Ole

    2008-01-01

    BACKGROUND: The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine...... the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. RESULTS......: In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high...

  13. Interplay of bistable kinetics of gene expression during cellular growth

    International Nuclear Information System (INIS)

    Zhdanov, Vladimir P

    2009-01-01

    In cells, the bistable kinetics of gene expression can be observed on the level of (i) one gene with positive feedback between protein and mRNA production, (ii) two genes with negative mutual feedback between protein and mRNA production, or (iii) in more complex cases. We analyse the interplay of two genes of type (ii) governed by a gene of type (i) during cellular growth. In particular, using kinetic Monte Carlo simulations, we show that in the case where gene 1, operating in the bistable regime, regulates mutually inhibiting genes 2 and 3, also operating in the bistable regime, the latter genes may eventually be trapped either to the state with high transcriptional activity of gene 2 and low activity of gene 3 or to the state with high transcriptional activity of gene 3 and low activity of gene 2. The probability to get to one of these states depends on the values of the model parameters. If genes 2 and 3 are kinetically equivalent, the probability is equal to 0.5. Thus, our model illustrates how different intracellular states can be chosen at random with predetermined probabilities. This type of kinetics of gene expression may be behind complex processes occurring in cells, e.g., behind the choice of the fate by stem cells

  14. Detecting microRNA activity from gene expression data

    LENUS (Irish Health Repository)

    Madden, Stephen F

    2010-05-18

    Abstract Background MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions. Results Here we combine correspondence analysis, between group analysis and co-inertia analysis (CIA) to determine which miRNAs are associated with differences in gene expression levels in microarray data sets. Using a da