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Sample records for gene oct4b mrna

  1. EWS-Oct-4B, an alternative EWS-Oct-4 fusion gene, is a potent oncogene linked to human epithelial tumours

    PubMed Central

    Kim, S; Lim, B; Kim, J

    2010-01-01

    Background: Characterisation of EWS-Oct-4 translocation fusion product in bone and soft-tissue tumours revealed a chimeric gene resulting from an in-frame fusion between EWS (Ewing's sarcoma gene) exons 1–6 and Oct-4 exons 1–4. Recently, an alternative form of the fusion protein between the EWS and Oct-4 genes, named EWS-Oct-4B, was reported in two types of epithelial tumours, a hidradenoma of the skin and a mucoepidermoid carcinoma of the salivary glands. As the N-terminal and POU domains of the EWS-Oct-4 and EWS-Oct-4B proteins are not structurally identical, we decided to investigate the functional consequences of the EWS-Oct-4B fusion. Methods: In this report, we have characterised the EWS-Oct-4B fusion protein. To investigate how the EWS-Oct-4B protein contributes to tumourigenesis in human cancers, we analysed its DNA-binding activity, subcellular localisation, transcriptional activation behaviour, and oncogenic properties. Results: We found that this new chimeric gene encodes a nuclear protein that binds DNA with the same sequence specificity as the parental Oct-4 protein or the fusion EWS-Oct-4 protein. We show that the nuclear localisation signal of EWS-Oct-4B is dependent on the POU DNA-binding domain, and we identified a cluster of basic amino acids, 269RKRKR273, in the POU domain that specifically mediates the nuclear localisation of EWS-Oct-4B. Comparison of the properties of EWS-Oct-4B and EWS-Oct-4 indicated that EWS-Oct-4B is a less-potent transcriptional activator of a reporter construct carrying the Oct-4-binding sites. Deletion analysis of the functional domains of EWS-Oct-4B revealed that the EWS N-terminal domain (NTD)B, POU, and C-terminal domain (CTD) are necessary for its full transactivation potential. Despite its reduced activity as a transcriptional activator, EWS-Oct-4B regulated the expression of fgf-4 (fibroblast growth factor-4) and nanog, which are potent mitogens, as well as of Oct-4 downstream target genes, the promoters of

  2. Linking gene regulation to mRNA production and export.

    PubMed

    Rodríguez-Navarro, Susana; Hurt, Ed

    2011-06-01

    Regulation of gene expression can occur at many different levels. One important step in the gene expression process is the transport of mRNA from the nucleus to the cytoplasm. In recent years, studies have described how nuclear mRNA export depends on the steps preceding and following transport through nuclear pore complexes. These include gene activation, transcription, mRNA processing and mRNP assembly and disassembly. In this review, we summarise recent insights into the links between these steps in the gene expression cascade.

  3. Post-transcriptional gene regulation by mRNA modifications

    PubMed Central

    Zhao, Boxuan Simen; Roundtree, Ian A.; He, Chuan

    2016-01-01

    The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis. PMID:27808276

  4. Mechanisms of mRNA translation of interferon stimulated genes.

    PubMed

    Joshi, Sonali; Kaur, Surinder; Kroczynska, Barbara; Platanias, Leonidas C

    2010-01-01

    Over the last two decades, a lot of research work has been focused on the interferon (IFN)-regulated JAK-STAT pathway and understanding the mechanisms governing the transcription of interferon stimulated genes (ISGs). Evidence suggests that the JAK-STAT pathway alone does not account in its entirety for mediating cellular responses to IFNs. There is emerging evidence that non-Stat pathways play important roles in mediating signals for the generation of IFN-responses. Various studies have underscored the importance of mitogen activated protein kinases (MAPKs), especially p38 and ERK1/2, as well as the PI 3'K/AKT pathway in transmitting signals that are of critical importance for the biological effects of IFNs. Besides regulating the transcription of ISGs in some cases, engagement of these signaling pathways by the IFN-receptor (IFNR) associated complexes also plays an important role in mediating the translation of ISGs. The mechanisms regulating mRNA translation of ISGs is an area of ongoing active research and a lot more efforts will be required to complete our understanding of the various cellular elements involved in this process. In this review we highlight the mechanisms regulating translation of ISGs. We focus on the proteins regulated by the PI 3'K/AKT pathway, their role in mediating mRNA translation of ISGs and the functional consequences of this regulation. In addition, MAPKs are known to regulate the phosphorylation of various eukaryotic initiation factors and we summarize the roles of eIF4B and eIF4E phosphorylations on the translation of ISGs. The emerging roles of microRNAs in mRNA translation of ISGs are also discussed.

  5. GeneTack database: genes with frameshifts in prokaryotic genomes and eukaryotic mRNA sequences.

    PubMed

    Antonov, Ivan; Baranov, Pavel; Borodovsky, Mark

    2013-01-01

    Database annotations of prokaryotic genomes and eukaryotic mRNA sequences pay relatively low attention to frame transitions that disrupt protein-coding genes. Frame transitions (frameshifts) could be caused by sequencing errors or indel mutations inside protein-coding regions. Other observed frameshifts are related to recoding events (that evolved to control expression of some genes). Earlier, we have developed an algorithm and software program GeneTack for ab initio frameshift finding in intronless genes. Here, we describe a database (freely available at http://topaz.gatech.edu/GeneTack/db.html) containing genes with frameshifts (fs-genes) predicted by GeneTack. The database includes 206 991 fs-genes from 1106 complete prokaryotic genomes and 45 295 frameshifts predicted in mRNA sequences from 100 eukaryotic genomes. The whole set of fs-genes was grouped into clusters based on sequence similarity between fs-proteins (conceptually translated fs-genes), conservation of the frameshift position and frameshift direction (-1, +1). The fs-genes can be retrieved by similarity search to a given query sequence via a web interface, by fs-gene cluster browsing, etc. Clusters of fs-genes are characterized with respect to their likely origin, such as pseudogenization, phase variation, etc. The largest clusters contain fs-genes with programed frameshifts (related to recoding events).

  6. Molecular structure of the human argininosuccinate synthetase gene: Occurrence of alternative mRNA splicing

    SciTech Connect

    Freytag, S.O.; Beaudet, A.L.; Bock, H.G.O.; O'Brien, W.E.

    1984-10-01

    The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.

  7. MRNA expression of genes regulating lipid metabolism in ringed seals (Pusa hispida) from differently polluted areas.

    PubMed

    Castelli, Martina Galatea; Rusten, Marte; Goksøyr, Anders; Routti, Heli

    2014-01-01

    There is a growing concern about the ability of persistent organic pollutants (POPs) to influence lipid metabolism. Although POPs are found at high concentrations in some populations of marine mammals, for example in the ringed seal (Pusa hispida) from the Baltic Sea, little is known about the effects of POPs on their lipid metabolism. An optimal regulation of lipid metabolism is crucial for ringed seals during the fasting/molting season. This is a physiologically stressful period, during which they rely on the energy stored in their fat reserves. The mRNA expression levels for seven genes involved in lipid metabolism were analyzed in liver and/or blubber tissue from molting ringed seals from the polluted Baltic Sea and a less polluted reference location, Svalbard (Norway). mRNA expression of genes encoding peroxisome proliferator-activated receptors (PPAR) α and γ and their target genes acyl-coenzyme A oxidase 1 (ACOX1) and cluster of differentiation 36 (CD36) were analyzed in liver. mRNA expression level of genes encoding PPARβ, PPARγ and their target genes encoding fatty acid binding protein 4 (FABP4) and adiponectin (ADIPOQ) were measured in inner and middle blubber layers. In addition, we evaluated the influence of molting status on hepatic mRNA expression of genes encoding PPARs and their target genes in ringed seals from Svalbard. Our results show higher mRNA expression of genes encoding hepatic PPARγ and adipose PPARβ, FABP4, and ADIPOQ in the Baltic seals compared to the Svalbard seals. A positive relationship between mRNA expressions of genes encoding hepatic PPARγ, adipose FABP4, adipose ADIPOQ and ΣPOP concentrations was observed. These findings suggest that lipid metabolism may be affected by contaminant exposure in the Baltic population. mRNA expression of genes encoding PPARβ, PPARγ, FABP4 and ADIPOQ were similar between the mid and inner adipose layer. Hepatic mRNA expression of genes encoding PPARα and PPARγ was higher in the pre

  8. Propionate induces mRNA expression of gluconeogenic genes in bovine calf hepatocytes.

    PubMed

    Zhang, Qian; Koser, Stephanie L; Donkin, Shawn S

    2016-05-01

    Hepatocytes monolayers from neonatal calves were used to determine the responses of the cytosolic phosphoenolpyruvate carboxykinase (PCK1) mRNA expression to propionate and direct hormonal cues including cyclic AMP (cAMP), dexamethasone, and insulin. The responses of other key gluconeogenic genes, including mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphotase (G6PC), were also measured. Expression of PCK1 was linearly induced with increasing propionate concentrations in media and 2.5 mM propionate increased PCK1 mRNA at 3 and 6h of incubation; however, the induction disappeared at 12 and 24 h. The induction of PCK1 mRNA by propionate was mimicked by 1 mM cAMP, or in combination with 5 µM dexamethasone, but not by dexamethasone alone. The induction of PCK1 mRNA by propionate or cAMP was eliminated by addition of 100 nM insulin. Additionally, expression of PCK2 and PC mRNA was also induced by propionate in a concentration-dependent manner. Consistent with PCK1, propionate-stimulated PCK2 and PC mRNA expression was inhibited by insulin. Expression of G6PC mRNA was neither affected by propionate nor cAMP, dexamethasone, insulin, or their combinations. These findings demonstrate that propionate can directly regulate its own metabolism in bovine calf hepatocytes through upregulation of PCK1, PCK2, and PC mRNA expression.

  9. Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

    PubMed

    Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

    2015-12-01

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon.

  10. Modulation of metabolic and clock gene mRNA rhythms by pineal and retinal circadian oscillators

    PubMed Central

    Karaganis, Stephen P.; Bartell, Paul A.; Shende, Vikram R.; Moore, Ashli F.; Cassone, Vincent M.

    2009-01-01

    Avian circadian organization involves interactions between three neural pacemakers: the suprachiasmatic nuclei (SCN), pineal, and retina. Each of these structures is linked within a neuroendocrine loop to influence downstream processes and peripheral oscillations. However, the contribution of each structure to drive or synchronize peripheral oscillators or circadian outputs in avian species is largely unknown. To explore these interactions in the chick, we measured 2-deoxy[14C]-glucose (2DG) uptake and mRNA expression of the chick clock genes bmal1, cry1, and per3 in three brain areas and in two peripheral organs in chicks that underwent pinealectomy, enucleation, or sham surgery. We found that 2DG uptake rhythms damp under constant darkness in intact animals, while clock gene mRNA levels continue to cycle, demonstrating that metabolic rhythms are not directly driven by clock gene transcription. Moreover, 2DG rhythms are not phase-locked to rhythms of clock gene mRNA. However, pinealectomy and enucleation had similar disruptive effects on both metabolic and clock gene rhythms, suggesting that both of these oscillators act similarly to reinforce molecular and physiological rhythms in the chicken. Finally, we show that the relative phasing of at least one clock gene, cry1, varies between central and peripheral oscillators in a tissue specific manner. These data point to a complex, differential orchestration of central and peripheral oscillators in the chick, and, importantly, indicate a disconnect between canonical clock gene regulation and circadian control of metabolism. PMID:19136000

  11. Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

    PubMed

    Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W

    2011-09-01

    Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.

  12. Nonsense-mediated mRNA decay among coagulation factor genes

    PubMed Central

    Shahbazi, Shirin

    2016-01-01

    Objective(s): Haemostasis prevents blood loss following vascular injury. It depends on the unique concert of events involving platelets and specific blood proteins, known as coagulation factors. The clotting system requires precise regulation and coordinated reactions to maintain the integrity of the vasculature. Clotting insufficiency mostly occurs due to genetically inherited coagulation factor deficiencies such as hemophilia. Materials and Methods: A relevant literature search of PubMed was performed using the keywords coagulation factors, Nonsense-mediated mRNA decay and premature translation termination codons. Search limitations included English language and human-based studies. Results: Mutations that cause premature translation termination codons probably account for one-third of genetically inherited diseases. Transcripts bearing aberrant termination codons are selectively identified and eliminated by an evolutionarily conserved posttranscriptional pathway known as nonsense-mediated mRNA decay (NMD). There are many pieces of evidence of decay among coagulation factor genes. However, the hemophilia gene (F8) does not seem to be subjected to NMD. Since the F8 gene is located on the X-chromosome, a connection between X-linked traits and mRNA decay could be assumed. Conclusion: Considering that not all genes go through decay, this review focuses on the basics of the mechanism in coagulation genes. It is interesting to determine whether this translation-coupled surveillance system represents a general rule for the genes encoding components of the same physiological cascade. PMID:27279976

  13. Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

    PubMed

    Dandoy-Dron, F; Benboudjema, L; Guillo, F; Jaegly, A; Jasmin, C; Dormont, D; Tovey, M G; Dron, M

    2000-03-10

    The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

  14. Low-level lasers and mRNA levels of reference genes used in Escherichia coli

    NASA Astrophysics Data System (ADS)

    Teixeira, A. F.; Machado, Y. L. R. C.; Fonseca, A. S.; Mencalha, A. L.

    2016-11-01

    Low-level lasers are widely used for the treatment of diseases and antimicrobial photodynamic therapy. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is widely used to evaluate mRNA levels and output data from a target gene are commonly relative to a reference mRNA that cannot vary according to treatment. In this study, the level of reference genes from Escherichia coli exposed to red or infrared lasers at different fluences was evaluated. E. coli AB1157 cultures were exposed to red (660 nm) and infrared (808 nm) lasers, incubated (20 min, 37 °C), the total RNA was extracted, and cDNA synthesis was performed to evaluate mRNA levels from arcA, gyrA and rpoA genes by RT-qPCR. Melting curves and agarose gel electrophoresis were carried out to evaluate specific amplification. Data were analyzed by geNorm, NormFinder and BestKeeper. The melting curve and agarose gel electrophoresis showed specific amplification. Although mRNA levels from arcA, gyrA or rpoA genes presented no significant variations trough a traditional statistical analysis, Excel-based tools revealed that these reference genes are not suitable for E. coli cultures exposed to lasers. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from arcA, gyrA and rpoA in E. coli cells.

  15. Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells.

    PubMed

    Spivack, Simon D; Hurteau, Gregory J; Jain, Ritu; Kumar, Shalini V; Aldous, Kenneth M; Gierthy, John F; Kaminsky, Laurence S

    2004-09-15

    Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogen- and oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals (Chi2 = 52.91, P < 0.001). Finally, quantitative real-time reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYP1B1, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.

  16. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

    PubMed Central

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

  17. Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

    PubMed Central

    Staudacher, Jonas J.; Naarmann-de Vries, Isabel S.; Ujvari, Stefanie J.; Klinger, Bertram; Kasim, Mumtaz; Benko, Edgar; Ostareck-Lederer, Antje; Ostareck, Dirk H.; Bondke Persson, Anja; Lorenzen, Stephan; Meier, Jochen C.; Blüthgen, Nils; Persson, Pontus B.; Henrion-Caude, Alexandra; Mrowka, Ralf; Fähling, Michael

    2015-01-01

    Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5′- and 3′-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5′- as well as 3′-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. PMID:25753659

  18. Characterization of the ptr5+ gene involved in nuclear mRNA export in fission yeast.

    PubMed

    Watanabe, Nobuyoshi; Ikeda, Terumasa; Mizuki, Fumitaka; Tani, Tokio

    2012-02-03

    To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A)(+) RNA transport] 1 to 11, which accumulate poly(A)(+) RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5-1 mutant shows dots- or a ring-like accumulation of poly(A)(+) RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5(+) gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5-1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5-1 mutation. In addition, we found that the ptr5-1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5-1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

  19. Secondary structure of bacteriophage T4 gene 60 mRNA: implications for translational bypassing.

    PubMed

    Todd, Gabrielle C; Walter, Nils G

    2013-05-01

    Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb³⁺ ions and the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb³⁺ as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem-loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.

  20. On a stochastic gene expression with pre-mRNA, mRNA and protein contribution.

    PubMed

    Rudnicki, Ryszard; Tomski, Andrzej

    2015-12-21

    In this paper we develop a model of stochastic gene expression, which is an extension of the model investigated in the paper [T. Lipniacki, P. Paszek, A. Marciniak-Czochra, A.R. Brasier, M. Kimmel, Transcriptional stochasticity in gene expression, J. Theor. Biol. 238 (2006) 348-367]. In our model, stochastic effects still originate from random fluctuations in gene activity status, but we precede mRNA production by the formation of pre-mRNA, which enriches classical transcription phase. We obtain a stochastically regulated system of ordinary differential equations (ODEs) describing evolution of pre-mRNA, mRNA and protein levels. We perform mathematical analysis of a long-time behavior of this stochastic process, identified as a piece-wise deterministic Markov process (PDMP). We check exact results using numerical simulations for the distributions of all three types of particles. Moreover, we investigate the deterministic (adiabatic) limit state of the process, when depending on parameters it can exhibit two specific types of behavior: bistability and the existence of the limit cycle. The latter one is not present when only two kinds of gene expression products are considered.

  1. Genomic Analysis and mRNA Expression of Equine Type I Interferon Genes

    PubMed Central

    Detournay, Olivier; Morrison, David A.; Wagner, Bettina; Zarnegar, Behdad

    2013-01-01

    This study aimed at identifying all of the type I interferon (IFN) genes of the horse and at monitoring their expression in equine cells on in vitro induction. We identified 32 putative type I IFN loci on horse chromosome 23 and an unplaced genomic scaffold. A phylogentic analysis characterized these into 8 different type I IFN classes, that is, putative functional genes for 6 IFN-α, 4 IFN-β, 8 IFN-ω (plus 4 pseudogenes), 3 IFN-δ (plus 1 pseudogene), 1 IFN-κ and 1 IFN-ɛ, plus 1 IFN-ν pseudogene, and 3 loci belonging to what has previously been called IFN-αω. Our analyses indicate that the IFN-αω genes are quite distinct from both IFN-α and IFN-ω, and we refer to this type I IFN as IFN-μ. Results from cell cultures showed that leukocytes readily expressed IFN-α, IFN-β, IFN-δ, IFN-μ, and IFN-ω mRNA on induction with, for example, live virus; while fibroblasts only expressed IFN-β mRNA on stimulation. IFN-κ or IFN-ɛ expression was not consistently induced in these cell cultures. Thus, the equine type I IFN family comprised 8 classes, 7 of which had putative functional genes, and mRNA expression of 5 was induced in vitro. Moreover, a relatively low number of IFN-α subtypes was found in the horse compared with other eutherian mammals. PMID:23772953

  2. Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

    PubMed

    Kitessa, Soressa M; Nattrass, Gregory S; Forder, Rebecca E A; McGrice, Hayley A; Wu, Shu-Biao; Hughes, Robert J

    2014-09-01

    The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis

  3. Sugar regulates mRNA abundance of H(+)-ATPase gene family members in tomato.

    PubMed Central

    Mito, N; Wimmers, L E; Bennett, A B

    1996-01-01

    The plant plasma membrane H(+)-ATPase energizes the secondary uptake of nutrients and may facilitate cell expansion by acidifying the cell wall. In yeast, Glc stimulates the accumulation of H(+)-ATPase mRNA, and the growth rate supported by various sugars is correlated with H(+)-ATPase protein abundance. Expression of three H(+)-ATPase genes, LHA1, LHA2, and LHA4, was previously detected in tomato (Lycopersicon esculentum). We have characterized the sequence of the LHA4 gene and examined the expression of these three tomato H(+)-ATPase genes in growing tissues and in response to exogenous sugars. LHA4 is a member of the H(+)-ATPase subfamily, including the Arabidopsis thaliana genes AHA1, AHA2, and AHA3. The 5' untranslated region of the deduced LHA4 cDNA contains a short, open reading frame very similar to that in the Nicotiana plumbaginifolia gene PMA1. LHA4 transcript abundance in seedlings is correlated with cell growth, being 2.5 times greater in hypocotyls of dark- versus light-grown plants. The accumulation of both LHA4 and LHA2 mRNAs is induced by the addition of exogenous sugars and this induction appears to be dependent on sugar uptake and metabolism, because mannitol and 3-O-methylglucose do not stimulate mRNA accumulation. These results suggest that the induction of expression of H(+)-ATPase genes by metabolizable sugars may be part of a generalized cellular response to increased cell growth and metabolism promoted by the availability of an abundant carbon source. PMID:8938420

  4. Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

    SciTech Connect

    Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S.

    2015-12-15

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Rift Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.

  5. Regulation of interferon-dependent mRNA translation of target genes.

    PubMed

    Kroczynska, Barbara; Mehrotra, Swarna; Arslan, Ahmet Dirim; Kaur, Surinder; Platanias, Leonidas C

    2014-04-01

    Interferons (IFNs) are released by cells on exposure to various stimuli, including viruses, double-stranded RNA, and other cytokines and various polypeptides. These IFNs play important physiological and pathophysiological roles in humans. Many clinical studies have established activity for these cytokines in the treatment of several malignancies, viral syndromes, and autoimmune disorders. In this review, the regulatory effects of type I and II IFN receptors on the translation-initiation process mediated by mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways and the known mechanisms of control of mRNA translation of IFN-stimulated genes are summarized and discussed.

  6. Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display

    PubMed Central

    Li, Xiang; Bi, Zhenghong; Di, Rong; Liang, Peng; He, Qiguang; Liu, Wenbo; Miao, Weiguo; Zheng, Fucong

    2016-01-01

    Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant–pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew. PMID:26840302

  7. Identification of Powdery Mildew Responsive Genes in Hevea brasiliensis through mRNA Differential Display.

    PubMed

    Li, Xiang; Bi, Zhenghong; Di, Rong; Liang, Peng; He, Qiguang; Liu, Wenbo; Miao, Weiguo; Zheng, Fucong

    2016-01-29

    Powdery mildew is an important disease of rubber trees caused by Oidium heveae B. A. Steinmann. As far as we know, none of the resistance genes related to powdery mildew have been isolated from the rubber tree. There is little information available at the molecular level regarding how a rubber tree develops defense mechanisms against this pathogen. We have studied rubber tree mRNA transcripts from the resistant RRIC52 cultivar by differential display analysis. Leaves inoculated with the spores of O. heveae were collected from 0 to 120 hpi in order to identify pathogen-regulated genes at different infection stages. We identified 78 rubber tree genes that were differentially expressed during the plant-pathogen interaction. BLAST analysis for these 78 ESTs classified them into seven functional groups: cell wall and membrane pathways, transcription factor and regulatory proteins, transporters, signal transduction, phytoalexin biosynthesis, other metabolism functions, and unknown functions. The gene expression for eight of these genes was validated by qRT-PCR in both RRIC52 and the partially susceptible Reyan 7-33-97 cultivars, revealing the similar or differential changes of gene expressions between these two cultivars. This study has improved our overall understanding of the molecular mechanisms of rubber tree resistance to powdery mildew.

  8. Streamlining gene expression analysis: integration of co-culture and mRNA purification.

    PubMed

    Berry, Scott M; Singh, Chandresh; Lang, Jessica D; Strotman, Lindsay N; Alarid, Elaine T; Beebe, David J

    2014-02-01

    Co-culture of multiple cell types within a single device enables the study of paracrine signaling events. However, extracting gene expression endpoints from co-culture experiments is laborious, due in part to pre-PCR processing of the sample (i.e., post-culture cell sorting and nucleic acid purification). Also, a significant loss of nucleic acid may occur during these steps, especially with microfluidic cell culture where lysate volumes are small and difficult to access. Here, we describe an integrated platform for performing microfluidic cell culture and extraction of mRNA for gene expression analysis. This platform was able to recover 30-fold more mRNA than a similar, non-integrated system. Additionally, using a breast cancer/bone marrow stroma co-culture, we recapitulated stromal-dependent, estrogen-independent growth of the breast cancer cells, coincident with transcriptional changes. We anticipate that this platform will be used for streamlined analysis of paracrine signaling events as well as for screening potential drugs and/or patient samples.

  9. The mRNA expression profile of metabolic genes relative to MHC isoform pattern in human skeletal muscles.

    PubMed

    Plomgaard, Peter; Penkowa, Milena; Leick, Lotte; Pedersen, Bente K; Saltin, Bengt; Pilegaard, Henriette

    2006-09-01

    The metabolic profile of rodent muscle is generally reflected in the myosin heavy chain (MHC) fiber-type composition. The present study was conducted to test the hypothesis that metabolic gene expression is not tightly coupled with MHC fiber-type composition for all genes in human skeletal muscle. Triceps brachii, vastus lateralis quadriceps, and soleus muscle biopsies were obtained from normally physically active, healthy, young male volunteers, because these muscles are characterized by different fiber-type compositions. As expected, citrate synthase and 3-hydroxyacyl dehydrogenase activity was more than twofold higher in soleus and vastus than in triceps. Contrary, phosphofructokinase and total lactate dehydrogenase (LDH) activity was approximately three- and twofold higher in triceps than in both soleus and vastus. Expression of metabolic genes was assessed by determining the mRNA content of a broad range of metabolic genes. The triceps muscle had two- to fivefold higher MHC IIa, phosphofructokinase, and LDH A mRNA content and two- to fourfold lower MHC I, lipoprotein lipase, CD36, hormone-sensitive lipase, and LDH B and hexokinase II mRNA than vastus lateralis or soleus. Interestingly, such mRNA differences were not evident for any of the genes encoding mitochondrial oxidative proteins, 3-hydroxyacyl dehydrogenase, carnitine palmitoyl transferase I, citrate synthase, alpha-ketogluterate dehydrogenase, and cytochrome c, nor for the transcriptional regulators peroxisome proliferator activator receptor gamma coactivator-1alpha, forkhead box O1, or peroxisome proliferator activator receptor-alpha. Thus the mRNA expression of genes encoding mitochondrial proteins and transcriptional regulators does not seem to be fiber type specific as the genes encoding glycolytic and lipid metabolism genes, which suggests that basal mRNA regulation of genes encoding mitochondrial proteins does not match the wide differences in mitochondrial content of these muscles.

  10. Predicting gene targets of perturbations via network-based filtering of mRNA expression compendia

    PubMed Central

    Cosgrove, Elissa J.; Zhou, Yingchun; Gardner, Timothy S.; Kolaczyk, Eric D.

    2008-01-01

    Motivation: DNA microarrays are routinely applied to study diseased or drug-treated cell populations. A critical challenge is distinguishing the genes directly affected by these perturbations from the hundreds of genes that are indirectly affected. Here, we developed a sparse simultaneous equation model (SSEM) of mRNA expression data and applied Lasso regression to estimate the model parameters, thus constructing a network model of gene interaction effects. This inferred network model was then used to filter data from a given experimental condition of interest and predict the genes directly targeted by that perturbation. Results: Our proposed SSEM–Lasso method demonstrated substantial improvement in sensitivity compared with other tested methods for predicting the targets of perturbations in both simulated datasets and microarray compendia. In simulated data, for two different network types, and over a wide range of signal-to-noise ratios, our algorithm demonstrated a 167% increase in sensitivity on average for the top 100 ranked genes, compared with the next best method. Our method also performed well in identifying targets of genetic perturbations in microarray compendia, with up to a 24% improvement in sensitivity on average for the top 100 ranked genes. The overall performance of our network-filtering method shows promise for identifying the direct targets of genetic dysregulation in cancer and disease from expression profiles. Availability: Microarray data are available at the Many Microbe Microarrays Database (M3D, http://m3d.bu.edu). Algorithm scripts are available at the Gardner Lab website (http://gardnerlab.bu.edu/SSEMLasso). Contact: kolaczyk@math.bu.edu Supplementary information: Supplementary Data are available at Bioinformatics on line. PMID:18779235

  11. The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons

    PubMed Central

    García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A.; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E.

    2016-01-01

    We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay. The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. PMID:26717982

  12. The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

    PubMed

    García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

    2016-05-05

    We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth.

  13. Identification of an additional gene required for eukaryotic nonsense mRNA turnover.

    PubMed Central

    Lee, B S; Culbertson, M R

    1995-01-01

    Loss of function of any one of three UPF genes prevents the accelerated decay of nonsense mRNAs in Saccharomyces cerevisiae. We report the identification and DNA sequence of UPF3, which is present in one nonessential copy on chromosome VII. Upf3 contains three putative nuclear localization signal sequences, suggesting that it may be located in a different compartment than the cytoplasmic Upf1 protein. Epitope-tagged Upf3 (FLAG-Upf3) does not cofractionate with polyribosomes or 80S ribosomal particles. Double disruptions of UPF1 and UPF3 affect nonsense mRNA decay in a manner indistinguishable from single disruptions. These results suggest that the Upf proteins perform related functions in a common pathway. Images Fig. 1 Fig. 3 PMID:7479783

  14. Aly/ REF, a factor for mRNA transport, activates RH gene promoter function.

    PubMed

    Suganuma, Hiroshi; Kumada, Maki; Omi, Toshinori; Gotoh, Takaya; Lkhagvasuren, Munkhtulga; Okuda, Hiroshi; Kamesaki, Toyomi; Kajii, Eiji; Iwamoto, Sadahiko

    2005-06-01

    The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity. We identified a major DNaseI hypersensitive site at the 5' flanking regions of both RHD and RHCE exon 1. A 34 bp fragment located at -191 to -158 from a translation start position, and containing the TCCCCTCCC sequence, was involved in enhancing promoter activity, which was assessed by luciferase reporter gene assay. A biotin-labelled 34 bp probe isolated an mRNA transporter protein, Aly/REF. The specific binding of Aly/REF to RH promoter in erythroid was confirmed by chromatin immunoprecipitation assay. The silencing of Aly/REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome. These facts provide second proof of Aly/REF as a transcription coactivator, initially identified as a coactivator for the TCRalpha enhancer function. Aly/REF might be a novel transcription cofactor for erythroid-specific genes.

  15. Comprehensive expression analysis of FSHD candidate genes at the mRNA and protein level.

    PubMed

    Klooster, Rinse; Straasheijm, Kirsten; Shah, Bharati; Sowden, Janet; Frants, Rune; Thornton, Charles; Tawil, Rabi; van der Maarel, Silvère

    2009-12-01

    In facioscapulohumeral muscular dystrophy (FSHD) the majority of patients carry a D4Z4 macrosatellite repeat contraction in the subtelomere of chromosome 4q. Several disease mechanisms have been proposed to explain how repeat contraction causes muscular dystrophy. All proposed mechanisms foresee a change from a closed to a more open chromatin structure followed by loss of control over expression of genes in or proximal to D4Z4. Initially, a distance and residual repeat size-dependent upregulation of the candidate genes FRG2, FRG1 and ANT1 was observed, but most successive expression studies failed to support transcriptional upregulation of 4qter genes. Moreover, chromatin studies do not provide evidence for a cis-spreading mechanism operating at 4qter in FSHD. In part, this inconsistency may be explained by differences in the techniques used, and the use of RNA samples obtained from different muscle groups. The aim of this study is to comprehensively and uniformly study the expression of the FSHD candidate genes FRG1, FRG2, CRYM, ANT1, ALP, PITX1 and LRP2BP at the RNA and protein level in identically processed primary myoblasts, myotubes and quadriceps muscle. Expression was compared between samples obtained from FSHD patients and normal controls with samples from myotonic dystrophy type 1 patients as disease controls. No consistent changes in RNA or protein expression levels were observed between the samples. The one exception was a selective increase in FRG2 mRNA expression in FSHD myotubes. This study provides further evidence that there is no demonstrable consistent, large magnitude, overexpression of any of the FSHD candidate genes.

  16. Comparative mRNA Expression Profiles of Riboflavin Biosynthesis Genes in Lactobacilli Isolated from Human Feces and Fermented Bamboo Shoots

    PubMed Central

    Thakur, Kiran; Tomar, Sudhir K.; Wei, Zhao-Jun

    2017-01-01

    With the aim to bioprospect potent riboflavin producing lactobacilli, the present study was carried out to evaluate the relative mRNA expression of riboflavin biosynthesis genes namely Rib 1, Rib 2, Rib 3, and Rib 4 from potent riboflavin producers obtained from our previous studies. All the four genes were successfully cloned and sequenced for further analysis by in silico procedures. As studied by non-denaturing Polyacrylamide gel electrophoresis, no difference in size of all the four genes among those of various lactobacilli was observed. The relative fold increase in mRNA expression in Rib 1, Rib 2, Rib 3, and Rib 4 genes has been observed to be 10-, 1-, 0.7-, and 8.5-fold, respectively. Due to increase in relative mRNA expression for all the Rib genes as well as phenotypic production attribute, KTLF1 strain was used further for expression studies in milk and whey. The fold increase in mRNA expression for all the four Rib genes was higher at 12 and 18 h in milk and whey respectively. After exposure to roseoflavin, resistant variant of KTLF1 showed considerable increase in expression of all the targets genes. This is the first ever study to compare the mRNA expression of riboflavin biosynthesis pathway genes in lactobacilli and it also under lines the effect of media and harvesting time which significantly affect the expression of rib genes. The use of roseoflavin-resistant strains capable of synthesizing riboflavin in milk and whey paves a way for an exciting and economically viable biotechnological approach to develop novel riboflavin bio-enriched functional foods. PMID:28367143

  17. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    SciTech Connect

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  18. Global SUMO Proteome Responses Guide Gene Regulation, mRNA Biogenesis, and Plant Stress Responses.

    PubMed

    Mazur, Magdalena J; van den Burg, Harrold A

    2012-01-01

    Small Ubiquitin-like MOdifier (SUMO) is a key regulator of abiotic stress, disease resistance, and development in plants. The identification of >350 plant SUMO targets has revealed many processes modulated by SUMO and potential consequences of SUMO on its targets. Importantly, highly related proteins are SUMO-modified in plants, yeast, and metazoans. Overlapping SUMO targets include heat-shock proteins (HSPs), transcription regulators, histones, histone-modifying enzymes, proteins involved in DNA damage repair, but also proteins involved in mRNA biogenesis and nucleo-cytoplasmic transport. Proteomics studies indicate key roles for SUMO in gene repression by controlling histone (de)acetylation activity at genomic loci. The responsible heavily sumoylated transcriptional repressor complexes are recruited by plant transcription factors (TFs) containing an (ERF)-associated Amphiphilic Repression (EAR) motif. These TFs are not necessarily themselves a SUMO target. Conversely, SUMO acetylation (Ac) prevents binding of downstream partners by blocking binding of their SUMO-interaction peptide motifs to Ac-SUMO. In addition, SUMO acetylation has emerged as a mechanism to recruit specifically bromodomains. Bromodomains are generally linked with gene activation. These findings strengthen the idea of a bi-directional sumo-acetylation switch in gene regulation. Quantitative proteomics has highlighted that global sumoylation provides a dynamic response to protein damage involving SUMO chain-mediated protein degradation, but also SUMO E3 ligase-dependent transcription of HSP genes. With these insights in SUMO function and novel technical advancements, we can now study SUMO dynamics in responses to (a)biotic stress in plants.

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

    PubMed

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

    2014-06-15

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

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

    PubMed Central

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

    2014-01-01

    In situ hybridization is an important technique for measuring the spatial expression patterns of mRNA in cells, tissues, and whole animals. However, mRNA levels cannot be compared across experiments using typical protocols. Here we present a semi-quantitative method to compare mRNA levels of a gene across multiple samples. This method yields an estimate of the error in the measurement to allow statistical comparison. Our method uses a typical in situ hybridization protocol to stain for a target gene and an internal standard, which we refer to as a co-stain. As a proof of concept, we apply this method to multiple lines of transgenic Drosophila embryos, harboring constructs that express reporter genes to different levels. We generated this test set by mutating enhancer sequences to contain different numbers of binding sites for Zelda, a transcriptional activator. We demonstrate that using a co-stain with in situ hybridization is an effective method to compare mRNA levels across samples. This method requires only minor modifications to existing in situ hybridization protocols and uses straightforward analysis techniques. This strategy can be broadly applied to detect quantitative, spatially resolved changes in mRNA levels. PMID:24434507

  1. The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI.

    PubMed

    Scholthof, H B; Gowda, S; Wu, F C; Shepherd, R J

    1992-05-01

    Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes.

  2. mRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus.

    PubMed

    Legendre, Matthieu; Audic, Stéphane; Poirot, Olivier; Hingamp, Pascal; Seltzer, Virginie; Byrne, Deborah; Lartigue, Audrey; Lescot, Magali; Bernadac, Alain; Poulain, Julie; Abergel, Chantal; Claverie, Jean-Michel

    2010-05-01

    Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.

  3. A Candida albicans gene expressed in Saccharomyces cerevisiae results in a distinct pattern of mRNA processing.

    PubMed

    Iborra, A; Sentandreu, R; Gozalbo, D

    1996-09-01

    Two plasmids (derived from YCplac22 and YEplac112) carrying a Candida albicans gene (including the 5' non-coding promoter sequences) coding for a 30 kDa membrane-bound protein, were used to transform Saccharomyces cerevisiae cells. A 30 kDa protein was immunodetected by Western blot in the membrane fraction of transformants. Northern analysis showed the presence of three mRNA species (of about 1.1, 0.7 and 0.5 kb) hybridizing with the C. albicans gene as a probe. The same result was obtained using the 5' and 3' regions of the gene as probes, whereas only a 1.1 kb mRNA was found in C. albicans and none was detected in S. cerevisiae control transformants. Thus, heterologous expression of this gene in S. cerevisiae results in a distinct pattern of mRNA processing, either due to the location on plasmid vectors and/or to differences in the mRNA processing systems in the two microorganisms.

  4. Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase.

    PubMed

    Kimura, Tominori; Hashimoto, Iwao; Nishikawa, Masao; Yamada, Hisao

    2009-06-01

    Human immunodeficiency virus type 1 Rev (regulator of the expression of the virion) protein was shown to reduce the expression level of the co-transfected luciferase reporter gene (luc+) introduced to monitor transfection efficiency. We studied the mechanism of the inhibitory Rev effect. The effect, caused by nuclear retention of luc+ mRNA, was reversed if rev had a point mutation that makes its nuclear export signal (NES) unable to associate with cellular transport factors. The Rev NES receptor CRM1 (chromosome region maintenance 1)-specific inhibitor, leptomycin B, blocked luc+ mRNA export. This finding was also supported by the overexpression of delta CAN, another specific CRM1 inhibitor that caused inhibition of luciferase gene expression. Experiments involving tsBN2 cells, which have a temperature-sensitive RCC1 (regulator of chromosome condensation 1) allele, demonstrated that luc+ expression required generation of the GTP-bound form of RanGTPase (RanGTP) by RCC1. The constitutive transport element (CTE)-mediated nuclear export of luc+ mRNA was found to also depend upon RanGTP. Nuclear export of luc+ mRNA is thus suggested to involve CRM1 and RanGTP, which Rev employs to transport viral mRNA. The Rev effect is therefore considered to involve competition between two molecules for common transport factors.

  5. Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

    PubMed

    Dandoy-Dron, F; Guillo, F; Benboudjema, L; Deslys, J P; Lasmézas, C; Dormont, D; Tovey, M G; Dron, M

    1998-03-27

    To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

  6. Exome sequencing coupled with mRNA analysis identifies NDUFAF6 as a Leigh gene.

    PubMed

    Bianciardi, Laura; Imperatore, Valentina; Fernandez-Vizarra, Erika; Lopomo, Angela; Falabella, Micol; Furini, Simone; Galluzzi, Paolo; Grosso, Salvatore; Zeviani, Massimo; Renieri, Alessandra; Mari, Francesca; Frullanti, Elisa

    2016-11-01

    We report here the case of a young male who started to show verbal fluency disturbance, clumsiness and gait anomalies at the age of 3.5years and presented bilateral striatal necrosis. Clinically, the diagnosis was compatible with Leigh syndrome but the underlying molecular defect remained elusive even after exome analysis using autosomal/X-linked recessive or de novo models. Dosage of respiratory chain activity on fibroblasts, but not in muscle, underlined a deficit in complex I. Re-analysis of heterozygous probably pathogenic variants, inherited from one healthy parent, identified the p.Ala178Pro in NDUFAF6, a complex I assembly factor. RNA analysis showed an almost mono-allelic expression of the mutated allele in blood and fibroblasts and puromycin treatment on cultured fibroblasts did not lead to the rescue of the maternal allele expression, not supporting the involvement of nonsense-mediated RNA decay mechanism. Complementation assay underlined a recovery of complex I activity after transduction of the wild-type gene. Since the second mutation was not detected and promoter methylation analysis resulted normal, we hypothesized a non-exonic event in the maternal allele affecting a regulatory element that, in conjunction with the paternal mutation, leads to the autosomal recessive disorder and the different allele expression in various tissues. This paper confirms NDUFAF6 as a genuine morbid gene and proposes the coupling of exome sequencing with mRNA analysis as a method useful for enhancing the exome sequencing detection rate when the simple application of classical inheritance models fails.

  7. Characterization of the ptr5{sup +} gene involved in nuclear mRNA export in fission yeast

    SciTech Connect

    Watanabe, Nobuyoshi; Ikeda, Terumasa; Mizuki, Fumitaka; Tani, Tokio

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer We cloned the ptr5{sup +} gene involved in nuclear mRNA export in fission yeast. Black-Right-Pointing-Pointer The ptr5{sup +} gene was found to encode nucleoporin 85 (Nup85). Black-Right-Pointing-Pointer Seh1p and Mlo3p are multi-copy suppressors for the ptr5 mutation. Black-Right-Pointing-Pointer Ptr5p/Nup85p functions in nuclear mRNA export through the mRNA export factor Rae1p. Black-Right-Pointing-Pointer Ptr5p/Nup85p interacts genetically with pre-mRNA splicing factors. -- Abstract: To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A){sup +} RNA transport] 1 to 11, which accumulate poly(A){sup +} RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5-1 mutant shows dots- or a ring-like accumulation of poly(A){sup +} RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5{sup +} gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5-1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5-1 mutation. In addition, we found that the ptr5-1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5-1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

  8. Pyruvate dehydrogenase complex: mRNA and protein expression patterns of E1α subunit genes in human spermatogenesis.

    PubMed

    Pinheiro, Ana; Silva, Maria João; Graça, Inês; Silva, Joaquina; Sá, Rosália; Sousa, Mário; Barros, Alberto; Tavares de Almeida, Isabel; Rivera, Isabel

    2012-09-10

    During spermatogenesis, germ cells undergo a complex process of cell differentiation and morphological restructuring, which depends on the coordinated expression of different genes. Some vital examples are those involved in cell energy metabolism, namely the genes encoding the E1α subunit of pyruvate dehydrogenase complex: the somatic PDHA1 (X-linked) and the testis-specific PDHA2 (autosomal). There are no data related to the study at the RNA and protein levels of PDHA genes during human spermatogenesis. The present study aimed to describe the mRNA and protein expression patterns of the human PDHA genes during spermatogenesis. Expression profiles of the PDHA1 and PDHA2 genes were characterized using different human tissues and cells. Diploid and haploid germ cells fractions were obtained from testis tissues. The mRNA profiles were analyzed by quantitative RT-PCR, whereas the protein profiles were evaluated by immunohistochemistry, western blotting and two-dimensional electrophoresis. Expression of the PDHA1 gene was found in all somatic cells, whereas expression of PDHA2 gene was restricted to germ cells. The switch from X-linked to autosomic gene expression occurred in spermatocytes. Data suggest the activation of PDHA2 gene expression is most probably a mechanism to ensure the continued expression of the protein, thus allowing germ cell viability and functionality.

  9. Spaceflight has compartment- and gene-specific effects on mRNA levels for bone matrix proteins in rat femur

    NASA Technical Reports Server (NTRS)

    Evans, G. L.; Morey-Holton, E.; Turner, R. T.

    1998-01-01

    In the present study, we evaluated the possibility that the abnormal bone matrix produced during spaceflight may be associated with reduced expression of bone matrix protein genes. To test this possibility, we investigated the effects of a 14-day spaceflight (SLS-2 experiment) on steady-state mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), osteocalcin, osteonectin, and prepro-alpha(1) subunit of type I collagen in the major bone compartments of rat femur. There were pronounced site-specific differences in the steady-state levels of expression of the mRNAs for the three bone matrix proteins and GAPDH in normal weight-bearing rats, and these relationships were altered after spaceflight. Specifically, spaceflight resulted in decreases in mRNA levels for GAPDH (decreased in proximal metaphysis), osteocalcin (decreased in proximal metaphysis), osteonectin (decreased in proximal and distal metaphysis), and collagen (decreased in proximal and distal metaphysis) compared with ground controls. There were no changes in mRNA levels for matrix proteins or GAPDH in the shaft and distal epiphysis. These results demonstrate that spaceflight leads to site- and gene-specific decreases in mRNA levels for bone matrix proteins. These findings are consistent with the hypothesis that spaceflight-induced decreases in bone formation are caused by concomitant decreases in expression of genes for bone matrix proteins.

  10. [Expression of the Drosophila melanogaster limk1 gene 3'-UTRs mRNA in Yeast Saccharomyces cerevisiae].

    PubMed

    Rumyantsev, A M; Zakharov, G A; Zhuravlev, A V; Padkina, M V; Savvateeva-Popova, E V; Sambuk, E V

    2014-06-01

    The stability of mRNA and its translation efficacy in higher eukaryotes are influenced by the interaction of 3'-untranscribed regions (3'-UTRs) with microRNAs and RNA-binding proteins. Since Saccharomyces cerevisiae lack microRNAs, it is possible to evaluate the contribution of only 3'-UTRs' and RNA-binding proteins' interaction in post-transcriptional regulation. For this, the post-transcriptional regulation of Drosophila limk1 gene encoding for the key enzyme of actin remodeling was studied in yeast. Analysis of limkl mRNA 3'-UTRs revealed the potential sites of yeast transcriptional termination. Computer remodeling demonstrated the possibility of secondary structure formation in limkl mRNA 3'-UTRs. For an evaluation of the functional activity of Drosophila 3'-UTRs in yeast, the reporter gene PHO5 encoding for yeast acid phosphatase (AP) fused to different variants of Drosophila limk1 mRNA 3'-UTRs (513, 1075, 1554 bp) was used. Assessments of AP activity and RT-PCR demonstrated that Drosophila limkl gene 3'-UTRs were functionally active and recognized in yeast. Therefore, yeast might be used as an appropriate model system for studies of 3'-UTR's role in post-transcriptional regulation.

  11. Sequence analysis of choriogenin H gene of medaka (Oryzias latipes) and mRNA expression.

    PubMed

    Lee, Chulwoo; Jeon, Seong Hwan; Na, Jin Gyun; Park, Kwangsik

    2002-08-01

    Zona radiata proteins of medaka (Oryzias latipes) consist of two major subunit groups, ZI-1,2 and ZI-3. The precursor of ZI-1,2, which is a glycoprotein with a molecular weight of 74,000 to 76,000 Da, is called choriogenin H, and the precursor of ZI-3, with a molecular weight of 49,000 Da, is called choriogenin L. The precursor proteins are synthesized in the liver in response to estrogen in sexually mature female medaka. However, they are also induced in the male medaka when fish are exposed to estrogenic chemicals. Therefore, choriogenin is known as a possible sensitive biomarker for endocrine disruption in fish. In this study, the choriogenin H cDNA sequence was reanalyzed and the genomic DNA sequence was newly analyzed. This was done for the selection of proper reverse transcription-polymerase chain reaction (RT-PCR) primers to measure the choriogenin mRNA induction by estrogenic chemicals. In the results, the full cDNA sequence was found to be 2,109 bp long and the size of the open reading frame (ORF) was found to be a total of 1,998 bp encoding 666 predicted amino acids, which was found to be different from previously reported cDNA sequence of medaka choriogenin. In the choriogenin H cDNA, a repetitive domain and a nonrepetitive domain were shown. Regarding the repetitive domain, seven complete repeats of the 45 bp of 5'-ccc cag tac cca tca aag cct cag ccc cct cag aat cct cag gtc-3' encoding PQYPSKPQPPQNPQV were found. Medaka choriogenin H gene was found to possess seven exons and six introns, and the total length was 2,643 bp long. The seven repetitive nucleotide residues described above existed in exon 1, which was found to be 1,033 bp long. Based on the full sequence information, proper primers were synthesized for RT-PCR to detect choriogenin H mRNA induction in male and juvenile medaka by 17alpha-ethinylestradiol, and this type of measurement system was found to be effective as a simple tool for the screening of endocrine-disrupting chemicals.

  12. mRNA Noise Reveals that Activators Induce a Biphasic Response in the Promoter Kinetics of Highly Regulated Genes

    NASA Astrophysics Data System (ADS)

    Quinn, Katie; To, Tsz-Leung; Maheshri, Narendra

    2012-02-01

    A dominant source of fluctuations in gene expression is thought to be the process of transcription. The statistics of these fluctuations arise from the kinetics of transcription. Multiple studies suggest the bulk of fluctuations can be understood by a simple process where genes are inactive for exponentially distributed times punctuated by geometric bursts of mRNA. Yet it's largely unknown how cis and trans factors affect the two lumped kinetic parameters, burst size and burst frequency, that describe this process. Importantly, how these parameters are regulated in a single gene can qualitatively affect the dynamical behavior of the network it is embedded within. Here, we ask whether transcriptional activators increase gene expression by increasing the burst size or burst frequency. We do so by deducing these parameters from steady-state mRNA distributions measured in individual yeast cells using single molecule mRNA FISH. We find that for both a synthetic and natural promoter, activators appear to first increase burst size, then burst frequency. We suggest this biphasic response may be common to all highly regulated genes and was previously unappreciated because of measurement techniques. Furthermore, its origins appear to relate to cis events at the promoter, and may arise from combinations of basal and activator-dependent bursts. Our measurements shed new light on transcriptional mechanisms and should assist in building synthetic promoters with tunable statistics.

  13. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

    PubMed

    Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2016-04-26

    Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

  14. mRNA export protein THOC5 as a tool for identification of target genes for cancer therapy.

    PubMed

    Tran, Doan Duy Hai; Saran, Shashank; Koch, Alexandra; Tamura, Teruko

    2016-04-10

    Recent evidence indicates that mRNA export is selective, giving priority to a subset of mRNAs that control diverse biological processes including cell proliferation, differentiation, stress response, and cell survival as well as tumor development. The depletion of a member of the mRNA export complex, the THO complex, impairs the expression of only a subset of genes, but causes dramatic changes in phenotype, such as cell cycle inhibition, abnormal differentiation, and importantly apoptosis of stem cells and cancer cells but not normal epithelial cells, hepatocytes, or fibroblasts. Recent exosome sequence data revealed that over 100 driver gene mutations with a number of signaling pathways are involved in human cancer formation, indicating that multiple signaling pathways will need to be inhibited for cancer therapy. In this review we firstly describe a basic feature and function of the mRNA export complex, THO, secondly, the biological alteration upon depletion of a member of the THO complex in normal and cancer cells, and thirdly, identification of its target genes. Finally we describe our recent data on selection of targeting candidates from THOC5 dependent genes for application in cancer therapy.

  15. Database for mRNA Half-Life of 19 977 Genes Obtained by DNA Microarray Analysis of Pluripotent and Differentiating Mouse Embryonic Stem Cells

    PubMed Central

    Sharova, Lioudmila V.; Sharov, Alexei A.; Nedorezov, Timur; Piao, Yulan; Shaik, Nabeebi; Ko, Minoru S.H.

    2009-01-01

    Degradation of mRNA is one of the key processes that control the steady-state level of gene expression. However, the rate of mRNA decay for the majority of genes is not known. We successfully obtained the rate of mRNA decay for 19 977 non-redundant genes by microarray analysis of RNA samples obtained from mouse embryonic stem (ES) cells. Median estimated half-life was 7.1 h and only <100 genes, including Prdm1, Myc, Gadd45 g, Foxa2, Hes5 and Trib1, showed half-life less than 1 h. In general, mRNA species with short half-life were enriched among genes with regulatory functions (transcription factors), whereas mRNA species with long half-life were enriched among genes related to metabolism and structure (extracellular matrix, cytoskeleton). The stability of mRNAs correlated more significantly with the structural features of genes than the function of genes: mRNA stability showed the most significant positive correlation with the number of exon junctions per open reading frame length, and negative correlation with the presence of PUF-binding motifs and AU-rich elements in 3′-untranslated region (UTR) and CpG di-nucleotides in the 5′-UTR. The mRNA decay rates presented in this report are the largest data set for mammals and the first for ES cells. PMID:19001483

  16. Structural and functional characterization of a Dictyostelium gene encoding a DIF inducible, prestalk-enriched mRNA sequence.

    PubMed Central

    Ceccarelli, A; McRobbie, S J; Jermyn, K A; Duffy, K; Early, A; Williams, J G

    1987-01-01

    The pDd56 mRNA sequence is highly enriched in prestalk over prespore cells and is inducible by DIF, the putative Dictyostelium stalk-specific morphogen. We show that the pDd56 gene is composed of forty one copies of a twenty four amino acid, cysteine rich repeat. This is highly homologus to a repeat which we have previously shown to compose the major fraction of the pDd63 mRNA, another DIF inducible, prestalk-enriched sequence. The predicted pDd56 protein contains a putative signal peptide but does not appear to contain a transmembrane segment. In combination these features suggest it to be an extrinsic protein and we confirm this elsewhere by showing that the pDd56 gene encodes a known, extracellular protein of the stalk. The pDd56 mRNA is dependent upon exogenous DIF for its accumulation. We show that this control is exerted at the transcriptional level and that a restriction fragment containing 1.7Kb of upstream sequence directs temporally-regulated expression of the gene. Images PMID:3658700

  17. Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique.

    PubMed

    Nestorova, Gergana G; Hasenstein, Karl; Nguyen, Nam; DeCoster, Mark A; Crews, Niel D

    2017-03-14

    Extraction and purification of high quality RNA is a crucial initial step required for a variety of genomic assays. We report a solid phase gene extraction (SPGE) method for automated extraction, purification and reverse transcription of mRNA in a microfluidic device. This is performed using a 130 μm diameter stainless steel needle that is amino-linked to dT(15) oligonucleotides for selective hybridization of mRNA. By inserting this probe into the biological sample for only 30 seconds, mRNA is captured with high selectivity and a yield greater than 10 pg per mm of probe length. The probe is then inserted into a lab-on-a-chip device, where the bound poly-adenylated RNA is thermally released and immediately reverse transcribed for subsequent PCR amplification. The insertion of the probe into the microfluidic device is straightforward: the microchannel is formed with an elastomer (PDMS) that, when punctured, will seal around the probe. The specificity and RNA loading capacity of the probes were evaluated using conventional qPCR. This procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes. Analysis of the product confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps. Integrating this elegant sample preparation method into a complete lab-on-a-chip system will substantially enhance the speed and automation of mRNA assays for research and clinical diagnostics.

  18. Selenium Deficiency Affects the mRNA Expression of Inflammatory Factors and Selenoprotein Genes in the Kidneys of Broiler Chicks.

    PubMed

    Zhang, Jiu-Li; Xu, Bo; Huang, Xiao-Dan; Gao, Yu-Hong; Chen, Yu; Shan, An-Shan

    2016-05-01

    The aim of this study was to investigate the influence of Se deficiency on the transcription of inflammatory factors and selenoprotein genes in the kidneys of broiler chicks. One hundred fifty 1-day-old broiler chicks were randomly assigned to two groups fed with either a low-Se diet (L group, 0.033 mg/kg Se) or an adequate Se diet (C group, 0.2 mg/kg Se). The levels of uric acid (UA) and creatinine (Cr) in the serum and the mRNA levels of 6 inflammatory factors and 25 selenoprotein genes in the kidneys were measured as the clinical signs of Se deficiency occurred at 20 days old. The results indicated that the contents of UA and Cr in the serum increased in L group (p < 0.05), and the mRNA levels of the inflammatory factors (NF-κB, iNOS, COX-2, and TNF-α) increased in L group (p < 0.05). Meanwhile, the mRNA levels of PTGEs and HO-1 were not changed. In addition, 25 selenoprotein transcripts displayed ubiquitous expression in the kidneys of the chicks. The mRNA levels of 14 selenoprotein genes (Dio1, Dio2, GPx3, Sepp1, SelH, SelI, SelK, Sepn1, SelO, SelW, Sep15, SelT, SelU, and SelS) decreased, and 9 selenoprotein genes (GPx1, GPx2, GPx4, SelPb, Txnrd1, Txnrd2, Txnrd3, SPS2, and SelM) increased in L group (p < 0.05), but the Dio3 and Sepx1 mRNA levels did not change. The results indicated that Se deficiency resulted in kidney dysfunction, activation of the NF-κB pathway, and a change in selenoprotein gene expression. The changes of inflammatory factor and selenoprotein gene expression levels were directly related to the abnormal renal functions induced by Se deficiency.

  19. Quantification and assessment of viability of Cryptococcus neoformans by LightCycler amplification of capsule gene mRNA.

    PubMed

    Amjad, Muhammad; Kfoury, Najla; Cha, Raymond; Mobarak, Reem

    2004-12-01

    Cryptococcus neoformans is an opportunistic fungal pathogen. It infects the central nervous system causing meningitis, which is fatal if untreated, especially in AIDS and immunosuppressed patients. In this study a method of quantification and assessment of viability of C. neoformans by LightCycler RT-PCR amplification of the capsule gene mRNA is established. The sequence of primers and probes were derived from C. neoformans capsular CAP10 gene mRNA (GenBank accession number AF144574), and were species specific. Agarose gel electrophoresis analysis of LightCycler RT-PCR product showed a single band of 223 bp in length. In order to develop an internal control a 223 bp exon fragment of capsule mRNA was cloned in the pCR2.1 plasmid vector and RNA was generated by in vitro transcription. To determine the sensitivity of the assay, serial dilutions of in vitro-transcribed RNA with known concentrations and copy numbers, and serially diluted cultures of viable and nonviable C. neoformans were used. Under optimal conditions as little as 0.472 fg of capsule mRNA could be detected, corresponding to 1-10 c.f.u. ml(-1) of the sample. No amplification was observed from up to 10(5) heat/UV radiation-killed yeast cells and RNA of other bacterial and fungal pathogens and human genomic DNA or RNA. The amplification of capsule mRNA represents a sensitive, specific and quantitative means of detection of viable C. neoformans in clinical specimens and can be useful in the evaluation of the therapeutic efficacy of antifungal drugs in the treatment of C. neoformans meningitis.

  20. Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels.

    PubMed Central

    Simpson, S B; Stoltzfus, C M

    1994-01-01

    A portion of the avian sarcoma virus (ASV) primary RNA transcripts is alternatively spliced in chicken embryo fibroblast cells to two different messages, the src and env mRNAs. Frameshift mutations of the viral genome causing premature translation termination within the src gene result in a decreased steady-state level of the src mRNA. In marked contrast, frameshift mutations at various positions of the env gene do not decrease the level of the env mRNA. We show that the src gene product is not required in trans for splicing and accumulation of src mRNA. Conversely, the truncated Src proteins do not act negatively in trans to decrease specifically the levels of src mRNA. Taken together, these results indicate that the frameshift mutations act in cis to reduce src mRNA levels. A double mutant with a lesion in the src initiator AUG and a frameshift within the src gene demonstrated wild-type RNA levels, indicating that the src mRNA must be recognized as a translatable mRNA for the effect on src mRNA levels to occur. Our results indicate that the reduced levels do not result from decreased cytoplasmic stability of the mature src mRNA. We also show that the src gene frameshift mutations affect src mRNA levels when expressed from intronless src cDNA clones. We conclude that the reduction of src mRNA levels triggered by the presence of frameshift mutations within the src gene occurs while it is associated with the nucleus. Our data also strongly suggest that this occurs at a step of RNA processing or transport independent of RNA splicing. Images PMID:8114716

  1. Identification of reference housekeeping-genes for mRNA expression studies in patients with type 1 diabetes.

    PubMed

    Kar, Parmita; Chawla, Himika; Saha, Soma; Tandon, Nikhil; Goswami, Ravinder

    2016-06-01

    Selection of appropriate housekeeping-genes as reference is important in mRNA expression-related experiments. It is more important in diabetes since hyperglycemia per se can influence expression of housekeeping-genes. RNA expression of Glyceraldehyde-3-phosphate-dehydrogenase, β-actin and 18S-ribosomal-RNA, Hypoxanthine-phosphoribosyl-transferase (HPRT), Tyrosine-3-monooxygenase/tryptophan (YHWAZ), β2-microglobin (β2M), TATA-binding-protein (TBP), and Ubiquitin C and cytochrome1 (CYC1) assessed in circulating-lymphocytes-(PBMC) of patients with type-1-diabetes and healthy controls. The stability ('M' value <1.02) and number of housekeeping-genes required for normalization in qRT-PCR were determined by 'ge-norm software.' Vitamin-D-receptor (VDR) was used as a target gene. All the nine genes tested had sufficient 'M' value in diabetes and healthy controls. However, housekeeping-genes indicated a relatively higher stability of expression in healthy controls in comparison to diabetes. Use of single housekeeping-genes brought gross variation in the calculation of VDR-mRNA copies. The ge-norm software suggested geometric mean of five housekeeping-genes for ideal normalization in diabetes (CYC1, β-actin, YHWAZ, HPRT, and β2M) and only three in controls (CYC1, β-actin, and TBP). HbA1c did not correlate with expression of any of the nine housekeeping-genes. Thus, geometric mean of CYC1, β-actin, YHWAZ, HPRT, and β2M needs to be used for ideal normalization of mRNA in type-1-diabetes. Similar studies are required in other population.

  2. Effect of gene location, mRNA secondary structures, and RNase sites on expression of two genes in an engineered operon.

    PubMed

    Smolke, Christina D; Keasling, Jay D

    2002-12-30

    The effects of endoribonuclease sites, secondary structures in mRNA, and gene placement on protein production and mRNA stability and steady-state levels were tested in a dual-gene operon containing the genes encoding beta-galactosidase (lacZ) from Escherichia coli and green fluorescent protein (gfp) from Aequorea victoria. Two previously identified RNase E sites were placed separately between the coding regions to direct cleavage in this area and produce two secondary transcripts, each containing a single-gene coding region. Novel secondary structures were engineered into the 3' and 5' ends of each of the coding regions to protect the transcript from inactivation by endoribonucleases (5' hairpins) and degradation by exoribonucleases (3' hairpins). In addition, the effects of relative gene placement were examined by switching the locations of the two coding regions. Depending on the particular secondary structures and RNase E sites placed between the genes the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 2.5-fold and 4-fold, respectively. By changing gene location and incorporating secondary structures and RNase E sites the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 100-fold and 750-fold, respectively.

  3. A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR

    PubMed Central

    Maier, Helena J.; Van Borm, Steven; Young, John R.; Fife, Mark

    2016-01-01

    Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. PMID:27537060

  4. Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout

    USGS Publications Warehouse

    Yada, T.; Hyodo, S.; Schreck, C.B.

    2008-01-01

    Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems. ?? 2008 Elsevier Inc. All rights reserved.

  5. Accumulation of catechins in tea in relation to accumulation of mRNA from genes involved in catechin biosynthesis.

    PubMed

    Eungwanichayapant, P D; Popluechai, S

    2009-02-01

    Catechins are a group of polyphenols found in tea (Camellia sinensis var. sinensis) at high levels. They are beneficial for health. From the study on accumulation of catechins in shoots and mature leaves of a tea cultivar, Oolong No. 17, using high-performance liquid chromatography (HPLC), it was found that the amounts of most catechins in the shoots were higher than those in the mature leaves, with an exception of catechins gallate (CG) that was found in trace amounts in both the shoots and mature leaves. mRNA accumulation of genes involved in catechin synthesis was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the mRNA accumulation of the genes were higher in the shoots than in the mature leaves. These genes included genes of phenylalanine ammonia-lyase 1 (PAL1; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), leucoanthocyanidin reductase (LCR; EC 1.17.1.3), and flavanone 3-hydroxylase (F3H; EC 1.14.11.9).

  6. Human intronless genes: Functional groups, associated diseases, evolution, and mRNA processing in absence of splicing

    SciTech Connect

    Grzybowska, Ewa A.

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Functional characteristics of intronless genes (IGs). Black-Right-Pointing-Pointer Diseases associated with IGs. Black-Right-Pointing-Pointer Origin and evolution of IGs. Black-Right-Pointing-Pointer mRNA processing without splicing. -- Abstract: Intronless genes (IGs) constitute approximately 3% of the human genome. Human IGs are essentially different in evolution and functionality from the IGs of unicellular eukaryotes, which represent the majority in their genomes. Functional analysis of IGs has revealed a massive over-representation of signal transduction genes and genes encoding regulatory proteins important for growth, proliferation, and development. IGs also often display tissue-specific expression, usually in the nervous system and testis. These characteristics translate into IG-associated diseases, mainly neuropathies, developmental disorders, and cancer. IGs represent recent additions to the genome, created mostly by retroposition of processed mRNAs with retained functionality. Processing, nuclear export, and translation of these mRNAs should be hampered dramatically by the lack of splice factors, which normally tightly cover mature transcripts and govern their fate. However, natural IGs manage to maintain satisfactory expression levels. Different mechanisms by which IGs solve the problem of mRNA processing and nuclear export are discussed here, along with their possible impact on reporter studies.

  7. Target and specificity of a nuclear gene product that participates in mRNA 3'-end formation in Chlamydomonas chloroplasts.

    PubMed

    Levy, H; Kindle, K L; Stern, D B

    1999-12-10

    Chloroplast mRNA maturation is catalyzed by nucleus-encoded processing enzymes. We previously described a recessive nuclear mutation (crp3) that affects 3'-end formation of several chloroplast mRNAs in Chlamydomonas reinhardtii (Levy, H., Kindle, K. L., and Stern, D. B. (1997) Plant Cell 9, 825-836). In the crp3 background, atpB mRNA lacking a 3'-inverted repeat normally required for stability accumulates as a discrete transcript. The mutation also affects the atpA gene cluster; polycistronic mRNAs with psbI or cemA 3'-ends accumulate to a lower level in the crp3 background. Here, we demonstrate that the crp3 mutation also alters 3'-end formation of psbI mRNA and cemA-containing mRNAs. A novel 3'-end is formed in monocistronic psbI transcripts, and this is the only terminus observed when the psbI 3'-untranslated region is fused to an aadA reporter gene. Accumulation of mRNAs with 3'-ends between cemA and atpH, which is immediately downstream, was reduced. However, this sequence was not recognized as a 3'-end formation element in chimeric genes. The crp3 mutation was able to confer stability to three different atpB 3'-stem-loop-disrupting mutations that lack sequence similarity, but are located at a similar distance from the translation termination codon. We propose that the wild-type CRP3 gene product is part of the general 3' --> 5' processing machinery.

  8. Gene expression profiling by mRNA array reveals different pattern in Chinese glioblastoma patients between Uygur and Han populations

    PubMed Central

    Liu, Liang; Xia, Haichen; Luan, Xinping; Dun, Zhiping; Zhu, Zhengquan; Dushan, Bieke; Li, Wenting

    2015-01-01

    Objective: To identify differentially expressed genes in Chinese glioblastoma patients of Uygur and Han populations, and investigate their potential clinical value for pathogenesis determination and progress prediction. Methods: Gene expression profiling was obtained from three patients of each Uygur and Han nationalities, respectively, by mRNA expression array. Data were processed by the GenomeStudio software and language R of the Lumi package, followed by GO (Gene Ontology) term and KEGG pathway annotation analysis by the Web Gestalt software. Results: The comparative analysis of genome-scale gene expression in glioblastomas revealed 1,475 differentially expressed genes, with 669 and 807 genes up-regulated and down-regulated, respectively. These included the STRC gene, which has two transcripts, one up-regulated and one down-regulated. GO term analysis suggested that 1,175 out of 1,475 key genes were involved in small GTPase mediated signal transduction, Ras protein signal transduction, bioprocess of neuronal response regulation, and central nervous system myelination. The KEGG pathway enrichment analysis showed that the differentially expressed genes were covered by 28 signaling pathways associated with tumorigenesis, including metabolic pathways, tumor suppressor pathways, MAP kinase signaling pathways, TGF-β signaling pathway, neurotrophin signaling pathways, and mTOR signaling pathway. Conclusion: The comparative study of gene expression profiling in glioblastomas between Uygur and Han nationalities revealed differentially expressed genes, whose functions and expression localization were analyzed by GO term analysis and KEGG pathway enrichment analysis. Different pathogenesis mechanisms were proposed for glioblastomas in Chinese patients of Uygur and Han nationalities from a molecular biology perspective. PMID:26309555

  9. Final report: FASEB Summer Research Conference on ''Post-transcriptional control of gene expression: Effectors of mRNA decay'' [agenda and attendees list

    SciTech Connect

    Maquat, Lynne

    2002-12-01

    The goal of this meeting was to provide an interactive forum for scientists working on prokaryotic and eukaryotic mRNA decay. A special seminar presented by a leader in the field of mRNA decay in S. cerevisiae focused on what is known and what needs to be determined, not only for yeast but for other organisms. The large attendance (110 participants) reflects the awareness that mRNA decay is a key player in gene regulation in a way that is affected by the many steps that precede mRNA formation. Sessions were held on the following topics: mRNA transport and mRNP; multicomponent eukaryotic nucleases; nonsense-mediated mRNA decay and nonsense-associated altered splicing; Cis-acting sequences/Trans-acting factors of mRNA decay; translational accuracy; multicomponent bacterial nucleases; interplay between mRNA polyadenylation, translation and decay in prokaryotes and prokaryotic organelles; and RNA interference and other RNA mediators of gene expression. In addition to the talks and two poster sessions, there were three round tables: (1) Does translation occur in the nucleus? (2) Differences and similarities in the mechanisms of mRNA decay in different eukaryotes, and (3) RNA surveillance in bacteria?

  10. Detection of Mutant NPM1 mRNA in Acute Myeloid Leukemia Using Custom Gene Expression Arrays

    PubMed Central

    van Vliet, Martin H.; Dumee, Belinda; Simons, Erik; Bullinger, Lars; Döhner, Konstanze; Döhner, Hartmut; Viëtor, Henk; Löwenberg, Bob; Valk, Peter J.M.

    2013-01-01

    Mutations in the gene encoding nucleophosmin (NPM1) carry a prognostic value for patients with acute myeloid leukemia (AML). Various techniques are currently being used to detect these mutations in routine molecular diagnostics. Incorporation of accurate NPM1 mutation detection on a gene expression platform would enable simultaneous detection with various other expression biomarkers. Here we present an array-based mutation detection using custom probes for NPM1 WT mRNA and NPM1 type A, B, and D mutant mRNA. This method was 100% accurate on a training cohort of 505 newly diagnosed unselected AML cases. Validation on an independent cohort of 143 normal-karyotype AML cases revealed no false-negative results, and one false positive (sensitivity 100.0% and specificity 98.7%). Based on this, we conclude that this method provides a reliable method for NPM1 mutation detection. The method can be applied to other genes/mutations as long as the mutant alleles are sufficiently highly expressed. PMID:23530539

  11. [Measurement of gene expression of CYP mRNA in liver of rats exposed to toluene and 1-butanol vapors].

    PubMed

    Ishidao, Toru; Ishimatsu, Sumiyo; Hirohashi, Masami; Morimoto, Yasuo; Hori, Hajime

    2006-12-01

    A reduction of the biological half life of toluene in blood in the simultaneous exposure to toluene and alcohol vapors has been reported. To clarify the cause of this reduction, gene expression of CYP mRNA in liver of rats exposed to bi-component organic vapors was investigated. Wistar male rats were repeatedly exposed to 500 ppm of toluene and 300 ppm of 1-butanol vapors individually and simultaneously by inhalation 6 hours a day, five days a week for 4 weeks. After the exposure, the rats were sacrificed and the livers were collected and homogenized. RNA was extracted from the livers, and gene expression of CYP mRNA was observed by reverse transcription-polymerase chain reaction (RT-PCR). The gene expression of CYP3A2 in the simultaneous exposure group was significantly higher than that in the toluene exposure group. However, there was no significant difference in that of CYP1A2, CYP2B1, CYP2C11, CYP2E1 and CYP4A1.

  12. Effects of AFP gene silencing on Survivin mRNA expression inhibition in HepG2 cells.

    PubMed

    Fang, Z L; Fang, N; Han, X N; Huang, G; Fu, X J; Xie, G S; Wang, N R; Xiong, J P

    2015-04-10

    We investigated the effects of alpha-fetoprotein (AFP) gene silencing on Survivin expression in HepG2 cells. Small interfering RNA technology was used to downregulate AFP expression in HepG2 cells. An enzyme-linked immunosorbent assay was used to measure AFP concentration in the supernatant before and after transfection. An MTT assay was used to detect cell proliferation activity before and after transfection. We performed flow cytometric analysis to detect the cell apoptosis rate, and reverse transcription-polymerase chain reaction to detect Survivin mRNA levels before and after transfection. Forty-eight hours after transfection, AFP concentration in the supernatant of the experimental group significantly decreased, hepatocellular carcinoma cell growth was inhibited by 43.1%, and the apoptosis rate increased by 24.3%. Survivin mRNA expression was reduced by 78.0% in HepG2 cells. These indicators in the control group and in the blank group did not change significantly. Silencing of AFP expression in HepG2 cells can effectively inhibit the growth of hepatoma cells and promote apoptosis, which may be useful for reducing intracellular Survivin mRNA levels.

  13. Quantification of diatom gene expression in the sea by selecting uniformly transcribed mRNA as the basis for normalization.

    PubMed

    Kang, Lee-Kuo; Tsui, Feng-Hsiu; Chang, Jeng

    2012-09-01

    To quantify gene expressions by quantitative reverse transcription-PCR (Q-RT-PCR) in natural diatom assemblages, it is necessary to seek a biomass reference specific to the target species. Two housekeeping genes, TBP (encoding the TATA box-binding protein) and EFL (encoding the translation elongation factor-like protein), were evaluated as candidates for reference genes in Q-RT-PCR assays. Transcript levels of TBP and EFL were relatively stable under various test conditions including growth stages, light-dark cycle phases, and nutrient stresses in Skeletonema costatum and Chaetoceros affinis, and TBP expression was more stable than that of EFL. Next, the sequence diversity of diatom assemblages was evaluated by obtaining 32 EFL and 29 TBP homologous gene fragments from the East China Sea (ECS). Based on sequence alignments, EFL and TBP primer sets were designed for Chaetoceros and Skeletonema groups in the ECS. An evaluation of primer specificity and PCR efficiency indicated that the EFL primer sets performed better. To demonstrate the applicability of EFL primer sets in the ECS, they were employed to measure mRNA levels of the FcpB (fucoxanthin-chlorophyll protein) gene in diatoms. The results correctly revealed prominent diel variations in FcpB expression and confirmed EFL as a good reference gene.

  14. Control of gluconeogenic genes during intense/prolonged exercise: hormone-independent effect of muscle-derived IL-6 on hepatic tissue and PEPCK mRNA.

    PubMed

    Banzet, Sébastien; Koulmann, Nathalie; Simler, Nadine; Sanchez, Hervé; Chapot, Rachel; Serrurier, Bernard; Peinnequin, André; Bigard, Xavier

    2009-12-01

    Prolonged intense exercise is challenging for the liver to maintain plasma glucose levels. Hormonal changes cannot fully account for exercise-induced hepatic glucose production (HGP). Contracting skeletal muscles release interleukin-6 (IL-6), a cytokine able to increase endogenous glucose production during exercise. However, whether this is attributable to a direct effect of IL-6 on liver remains unknown. Here, we studied hepatic glycogen, gluconeogenic genes, and IL-6 signaling in response to one bout of exhaustive running exercise in rats. To determine whether IL-6 can modulate gluconeogenic gene mRNA independently of exercise, we injected resting rats with recombinant IL-6. Exhaustive exercise resulted in a profound decrease in liver glycogen and an increase in gluconeogenic gene mRNA levels, phosphoenolpyruvate-carboxykinase (PEPCK), glucose-6-phosphatase (G6P), and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), suggesting a key role for gluconeogenesis in hepatic glucose production. This was associated to an active IL-6 signaling in liver tissue, as shown by signal transducer and activator of transcription and CAAT/enhancer binding protein-beta phosphorylation and IL-6-responsive gene mRNA levels at the end of exercise. Recombinant IL-6 injection resulted in an increase in IL-6-responsive gene mRNA levels in the liver. We found a dose-dependent increase in PEPCK gene mRNA strongly correlated with IL-6-induced gene mRNA levels. No changes in G6P and PGC-1alpha mRNA levels were found. Taken together, our results suggest that, during very demanding exercise, muscle-derived IL-6 could help increase HGP by directly upregulating PEPCK mRNA abundance.

  15. Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA

    PubMed Central

    1993-01-01

    We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation- independent mRNA stabilization mechanism. PMID:8436593

  16. Impact of estrogen receptor gene polymorphisms and mRNA levels on obesity and lipolysis – a cohort study

    PubMed Central

    Nilsson, Maria; Dahlman, Ingrid; Jiao, Hong; Gustafsson, Jan-Åke; Arner, Peter; Dahlman-Wright, Karin

    2007-01-01

    Background The estrogen receptors α and β (ESR1, ESR2) have been implicated in adiposity, lipid metabolism and feeding behaviour. In this report we analyse ESR1 and ESR2 gene single nucleotide polymorphisms (SNPs) for association with obesity. We also relate adipose tissue ESR1 mRNA levels and ESR1 SNPs to adipocyte lipolysis and lipogenesis phenotypes. Methods 23 ESR1 and 11 ESR2 tag-SNPs, covering most of the common haplotype variation in each gene according to HAPMAP data, were analysed by Chi2 for association with obesity in a cohort comprising 705 adults with severe obesity and 402 lean individuals. Results were replicated in a cohort comprising 837 obese and 613 lean subjects. About 80% of both cohorts comprised women and 20% men. Adipose tissue ESR1 mRNA was quantified in 122 women and related to lipolysis and lipogenesis by multiple regression. ESR1 SNPs were analysed for association with adipocyte lipolysis and lipogenesis phenotypes in 204 obese women by simple regression. Results No ESR1 SNP was associated with obesity. Five ESR2 SNPs displayed nominal significant allelic association with obesity in women and one in men. The two ESR2 SNPs associated with obesity with nominal P value < 0.01 were genotyped in a second cohort where no association with obesity was observed. There was an inverse correlation between ESR1 mRNA levels in abdominal subcutaneous (sc) adipose tissue and basal lipolysis, as well as responsiveness to adrenoceptor agonists independent of age and BMI (P value 0.009–0.045). ESR1 rs532010 was associated with lipolytic sensitivity to noradrenaline (nominal P value 0.012), and ESR1 rs1884051 with responsiveness to the non-selective beta-adrenoceptor agonist isoprenaline (nominal P value 0.05). These associations became non-significant after Bonferroni correction. Conclusion ESR1 gene alleles are unlikely to be a major cause of obesity in women. A minor importance of ESR2 on severe obesity cannot be excluded. The inverse correlation

  17. Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

    PubMed

    Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

    2016-04-15

    Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy.

  18. Effect of copy number and mRNA processing and stabilization on transcript and protein levels from an engineered dual-gene operon.

    PubMed

    Smolke, Christina D; Keasling, Jay D

    2002-05-20

    To study the effect of mRNA stability and DNA copy number on protein production from a dual-gene operon, a synthetic operon containing the reporter genes gfp and lacZ under the control of the araBAD promoter was placed in pMB1-based (approximately 100 copies/cell) and F plasmid-based (approximately 1 copy/cell) vectors. DNA cassettes encoding secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the two genes. Although the copy number of the pMB1-based vectors was approximately 100-fold greater than the copy number of the F plasmid-based vectors, transcript and protein levels from the pMB1-based vector were not 100-fold greater than from the F plasmid-based vectors. In identical plasmid backbones, different combinations of mRNA control elements were used to alter steady-state levels of transcripts. Control elements that amplified the stability of one coding region relative to another amplified the ratio of protein produced from those transcripts. The effects of mRNA stability control elements were greater at low inducer concentrations, where mRNA levels limit protein production, than at high inducer concentrations. Although we can alter mRNA and protein levels through copy number, induction level, and mRNA stability control elements, some aspect of gene expression remains dependent on inherent characteristics of the coding region.

  19. Quantification of mRNA stability of stress-responsive yeast genes following conditional excision of open reading frames.

    PubMed

    Talarek, Nicolas; Bontron, Séverine; De Virgilio, Claudio

    2013-08-01

    Eukaryotic cells rapidly adjust the levels of mRNAs in response to environmental stress primarily by controlling transcription and mRNA turnover. How different stress conditions influence the fate of stress-responsive mRNAs, however, is relatively poorly understood. This is largely due to the fact that mRNA half-life assays are traditionally based on interventions (e.g., temperature-shifts using temperature-sensitive RNA polymerase II alleles or treatment with general transcription inhibitory drugs), which, rather than blocking, specifically induce transcription of stress-responsive genes. To study the half-lives of the latter suite of mRNAs, we developed and describe here a minimally perturbing alternative method, coined CEO, which is based on discontinuance of transcription following the conditional excision of open reading frames. Using CEO, we confirm that the target of rapamycin complex I (TORC1), a nutrient-activated, central stimulator of eukaryotic cell growth, favors the decay of mRNAs that depend on the stress- and/or nutrient-regulated transcription factors Msn2/4 and Gis1 for their transcription. We further demonstrate that TORC1 controls the stability of these mRNAs via the Rim15-Igo1/2-PP2A(Cdc55) effector branch, which reportedly also controls Gis1 promoter recruitment. These data pinpoint PP2A(Cdc55) as a central node in homo-directional coordination of transcription and post-transcriptional mRNA stabilization of a specific array of nutrient-regulated genes.

  20. Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.

    PubMed

    Hu, Yan; Liu, Hongxiang; Song, Chi; Xu, Wenjuan; Ji, Gaige; Zhu, Chunhong; Shu, Jingting; Li, Huifang

    2015-03-15

    In this study, the ontogeny of body and liver weight and the pattern of related gene mRNA expression in the hypothalamus-pituitary growth axis (HPGA) of two different duck breeds (Anas platyrhynchos domestica) were compared during embryonic and post-hatch development. Duck hypothalamic growth hormone release hormone (GHRH), somatostatin (SS), pituitary growth hormone (GH), liver growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-1) mRNA were first detected on the 13th embryonic day. During early duck development, SS maintained a lower expression status, whereas the other four genes exhibited highly significant variations in an age-specific manner. Highly significant breed specificity was observed with respect to hepatic IGF-1 mRNA expression, which showed a significant breed-age interaction effect. Compared with previous studies on chickens, significant species differences were observed regarding the mRNA expression of bird embryonic HPGA-related genes. During early development, highly significant breed and age specificity were observed with respect to developmental changes in body and liver weight, and varying degrees of significant linear correlation were found between these performances and the mRNA expression of HPGA-related genes in the duck HPGA. These results suggest that different genetic backgrounds may lead to differences in duck growth and HPGA-related gene mRNA expression, and the differential mRNA expression of related genes in the duck HPGA may be particularly important in the early growth of ducks. Furthermore, hepatic IGF-1 mRNA expression presented highly significant breed specificity, and evidence suggests the involvement of hepatic IGF-1 in mediating genetic effects on embryo and offspring growth in ducks.

  1. Structure of the coding region and mRNA variants of the apyrase gene from pea (Pisum sativum)

    NASA Technical Reports Server (NTRS)

    Shibata, K.; Abe, S.; Davies, E.

    2001-01-01

    Partial amino acid sequences of a 49 kDa apyrase (ATP diphosphohydrolase, EC 3.6.1.5) from the cytoskeletal fraction of etiolated pea stems were used to derive oligonucleotide DNA primers to generate a cDNA fragment of pea apyrase mRNA by RT-PCR and these primers were used to screen a pea stem cDNA library. Two almost identical cDNAs differing in just 6 nucleotides within the coding regions were found, and these cDNA sequences were used to clone genomic fragments by PCR. Two nearly identical gene fragments containing 8 exons and 7 introns were obtained. One of them (H-type) encoded the mRNA sequence described by Hsieh et al. (1996) (DDBJ/EMBL/GenBank Z32743), while the other (S-type) differed by the same 6 nucleotides as the mRNAs, suggesting that these genes may be alleles. The six nucleotide differences between these two alleles were found solely in the first exon, and these mutation sites had two types of consensus sequences. These mRNAs were found with varying lengths of 3' untranslated regions (3'-UTR). There are some similarities between the 3'-UTR of these mRNAs and those of actin and actin binding proteins in plants. The putative roles of the 3'-UTR and alternative polyadenylation sites are discussed in relation to their possible role in targeting the mRNAs to different subcellular compartments.

  2. Acute physiological stress down-regulates mRNA expressions of growth-related genes in coho salmon.

    PubMed

    Nakano, Toshiki; Afonso, Luis O B; Beckman, Brian R; Iwama, George K; Devlin, Robert H

    2013-01-01

    Growth and development in fish are regulated to a major extent by growth-related factors, such as liver-derived insulin-like growth factor (IGF) -1 in response to pituitary-secreted growth hormone (GH) binding to the GH receptor (GHR). Here, we report on the changes in the expressions of gh, ghr, and igf1 genes and the circulating levels of GH and IGF-1 proteins in juvenile coho salmon (Oncorhynchus kisutch) in response to handling as an acute physiological stressor. Plasma GH levels were not significantly different between stressed fish and prestressed control. Plasma IGF-1 concentrations in stressed fish 1.5 h post-stress were the same as in control fish, but levels in stressed fish decreased significantly 16 h post-stress. Real-time quantitative PCR (qPCR) analysis showed that ghr mRNA levels in pituitary, liver, and muscle decreased gradually in response to the stressor. After exposure to stress, hepatic igf1 expression transiently increased, whereas levels decreased 16 h post-stress. On the other hand, the pituitary gh mRNA level did not change in response to the stressor. These observations indicate that expression of gh, ghr, and igf1 responded differently to stress. Our results show that acute physiological stress can mainly down-regulate the expressions of growth-related genes in coho salmon in vivo. This study also suggests that a relationship between the neuroendocrine stress response and growth-related factors exists in fish.

  3. Acute Physiological Stress Down-Regulates mRNA Expressions of Growth-Related Genes in Coho Salmon

    PubMed Central

    Nakano, Toshiki; Afonso, Luis O. B.; Beckman, Brian R.; Iwama, George K.; Devlin, Robert H.

    2013-01-01

    Growth and development in fish are regulated to a major extent by growth-related factors, such as liver-derived insulin-like growth factor (IGF) -1 in response to pituitary-secreted growth hormone (GH) binding to the GH receptor (GHR). Here, we report on the changes in the expressions of gh, ghr, and igf1 genes and the circulating levels of GH and IGF-1 proteins in juvenile coho salmon (Oncorhynchus kisutch) in response to handling as an acute physiological stressor. Plasma GH levels were not significantly different between stressed fish and prestressed control. Plasma IGF-1 concentrations in stressed fish 1.5 h post-stress were the same as in control fish, but levels in stressed fish decreased significantly 16 h post-stress. Real-time quantitative PCR (qPCR) analysis showed that ghr mRNA levels in pituitary, liver, and muscle decreased gradually in response to the stressor. After exposure to stress, hepatic igf1 expression transiently increased, whereas levels decreased 16 h post-stress. On the other hand, the pituitary gh mRNA level did not change in response to the stressor. These observations indicate that expression of gh, ghr, and igf1 responded differently to stress. Our results show that acute physiological stress can mainly down-regulate the expressions of growth-related genes in coho salmon in vivo. This study also suggests that a relationship between the neuroendocrine stress response and growth-related factors exists in fish. PMID:23990952

  4. Untranslated regions of mRNA and their role in regulation of gene expression in protozoan parasites.

    PubMed

    Rao, Shilpa J; Chatterjee, Sangeeta; Pal, Jayantapal K

    2017-03-01

    Protozoan parasites are one of the oldest living entities in this world that throughout their existence have shown excellent resilience to the odds of survival and have adapted beautifully to ever changing rigors of the environment. In view of the dynamic environment encountered by them throughout their life cycle, and in establishing pathogenesis, it is unsurprising that modulation of gene expression plays a fundamental role in their survival. In higher eukaryotes, untranslated regions (UTRs) of transcripts are one of the crucial regulators of gene expression (influencing mRNA stability and translation efficiency). Parasitic protozoan genome studies have led to the characterization (in silico, in vitro and in vivo) of a large number of their genes. Comparison of higher eukaryotic UTRs with parasitic protozoan UTRs reveals the existence of several similar and dissimilar facets of the UTRs. This review focuses on the elements of UTRs of medically important protozoan parasites and their regulatory role in gene expression. Such information may be useful to researchers in designing gene targeting strategies linked with perturbation of host-parasite relationships leading to control of specific parasites.

  5. Integrated Analysis of DNA Methylation and mRNA Expression Profiles Data to Identify Key Genes in Lung Adenocarcinoma

    PubMed Central

    Jin, Xiang; Li, Xiaodan; Guan, Yinghui

    2016-01-01

    Introduction. Lung adenocarcinoma (LAC) is the most frequent type of lung cancer and has a high metastatic rate at an early stage. This study is aimed at identifying LAC-associated genes. Materials and Methods. GSE62950 downloaded from Gene Expression Omnibus included a DNA methylation dataset and an mRNA expression profiles dataset, both of which included 28 LAC tissue samples and 28 adjacent normal tissue samples. The differentially expressed genes (DEGs) were screened by Limma package in R, and their functions were predicted by enrichment analysis using TargetMine online tool. Then, protein-protein interaction (PPI) network was constructed using STRING and Cytoscape. Finally, LAC-associated methylation sites were identified by CpGassoc package in R and mapped to the DEGs to obtain LAC-associated DEGs. Results. Total 913 DEGs were identified in LAC tissues. In the PPI networks, MAD2L1, AURKB, CCNB2, CDC20, and WNT3A had higher degrees, and the first four genes might be involved in LAC through interaction. Total 8856 LAC-associated methylation sites were identified and mapped to the DEGs. And there were 29 LAC-associated methylation sites located in 27 DEGs (e.g., SH3GL2, BAI3, CDH13, JAM2, MT1A, LHX6, and IGFBP3). Conclusions. These key genes might play a role in pathogenesis of LAC. PMID:27610375

  6. Product of Saccharomyces cerevisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA.

    PubMed Central

    Costanzo, M C; Fox, T D

    1986-01-01

    The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII). Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-beta-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria. To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion. These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII. The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs. These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA. Images PMID:3099165

  7. Effects of mRNA amplification on gene expression ratios in cDNA experiments estimated by analysis of variance

    PubMed Central

    Nygaard, Vigdis; Løland, Anders; Holden, Marit; Langaas, Mette; Rue, Håvard; Liu, Fang; Myklebost, Ola; Fodstad, Øystein; Hovig, Eivind; Smith-Sørensen, Birgitte

    2003-01-01

    Background A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20–200 micro-grams total RNA or 0.5–2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material. Results Using analysis of variance (ANOVA) and multiple hypothesis testing, we estimated the impact of amplification on the preservation of gene expression ratios. Both methods showed that the gene expression ratios were not completely preserved between amplified and non-amplified material. We also compared the expression ratios between the two cell lines for the amplified material with expression ratios between the two cell lines for the non-amplified material for each gene. With the aid of multiple t-testing with a false discovery rate of 5%, we found that 10% of the genes investigated showed significantly different expression ratios. Conclusion Although the ratios were not fully preserved, amplification may prove to be extremely useful with respect to characterizing low expressing genes. PMID:12659661

  8. Screening of genes involved in isooctane tolerance in Saccharomyces cerevisiae by using mRNA differential display.

    PubMed

    Miura, S; Zou, W; Ueda, M; Tanaka, A

    2000-11-01

    A Saccharomyces cerevisiae strain, KK-211, isolated by the long-term bioprocess of stereoselective reduction in isooctane, showed extremely high tolerance to the solvent, which is toxic to yeast cells, but, in comparison with its wild-type parent, DY-1, showed low tolerance to hydrophilic organic solvents, such as dimethyl sulfoxide and ethanol. In order to detect the isooctane tolerance-associated genes, mRNA differential display (DD) was employed using mRNAs isolated from strains DY-1 and KK-211 cultivated without isooctane, and from strain KK-211 cultivated with isooctane. Thirty genes were identified as being differentially expressed in these three types of cells and were classified into three groups according to their expression patterns. These patterns were further confirmed and quantified by Northern blot analysis. On the DD fingerprints, the expression of 14 genes, including MUQ1, PRY2, HAC1, AGT1, GAC1, and ICT1 (YLR099c) was induced, while the expression of the remaining 16 genes, including JEN1, PRY1, PRY3, and KRE1, was decreased, in strain KK-211 cultivated with isooctane. The genes represented by HAC1, PRY1, and ICT1 have been reported to be associated with cell stress, and AGT1 and GAC1 have been reported to be involved in the uptake of trehalose and the production of glycogen, respectively. MUQ1 and KRE1, encoding proteins associated with cell surface maintenance, were also detected. Based on these results, we concluded that alteration of expression levels of multiple genes, not of a single gene, might be the critical determinant for isooctane tolerance in strain KK-211.

  9. Phase I Metabolic Genes and Risk of Lung Cancer: Multiple Polymorphisms and mRNA Expression

    PubMed Central

    Rotunno, Melissa; Yu, Kai; Lubin, Jay H.; Consonni, Dario; Pesatori, Angela C.; Goldstein, Alisa M.; Goldin, Lynn R.; Wacholder, Sholom; Burdette, Laurie; Chanock, Stephen J.; Bertazzi, Pier Alberto; Tucker, Margaret A.; Caporaso, Neil E.; Chatterjee, Nilanjan; Bergen, Andrew W.; Landi, Maria Teresa

    2009-01-01

    Polymorphisms in genes coding for enzymes that activate tobacco lung carcinogens may generate inter-individual differences in lung cancer risk. Previous studies had limited sample sizes, poor exposure characterization, and a few single nucleotide polymorphisms (SNPs) tested in candidate genes. We analyzed 25 SNPs (some previously untested) in 2101 primary lung cancer cases and 2120 population controls from the Environment And Genetics in Lung cancer Etiology (EAGLE) study from six phase I metabolic genes, including cytochrome P450s, microsomal epoxide hydrolase, and myeloperoxidase. We evaluated the main genotype effects and genotype-smoking interactions in lung cancer risk overall and in the major histology subtypes. We tested the combined effect of multiple SNPs on lung cancer risk and on gene expression. Findings were prioritized based on significance thresholds and consistency across different analyses, and accounted for multiple testing and prior knowledge. Two haplotypes in EPHX1 were significantly associated with lung cancer risk in the overall population. In addition, CYP1B1 and CYP2A6 polymorphisms were inversely associated with adenocarcinoma and squamous cell carcinoma risk, respectively. Moreover, the association between CYP1A1 rs2606345 genotype and lung cancer was significantly modified by intensity of cigarette smoking, suggesting an underling dose-response mechanism. Finally, increasing number of variants at CYP1A1/A2 genes revealed significant protection in never smokers and risk in ever smokers. Results were supported by differential gene expression in non-tumor lung tissue samples with down-regulation of CYP1A1 in never smokers and up-regulation in smokers from CYP1A1/A2 SNPs. The significant haplotype associations emphasize that the effect of multiple SNPs may be important despite null single SNP-associations, and warrants consideration in genome-wide association studies (GWAS). Our findings emphasize the necessity of post-GWAS fine mapping and

  10. Temperature-sensitive mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 genes result in defects in mRNA turnover.

    PubMed Central

    Zuk, D; Belk, J P; Jacobson, A

    1999-01-01

    In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37 degrees. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p. PMID:10471698

  11. A selection of reference genes and early-warning mRNA biomarkers for environmental monitoring using Mytilus spp. as sentinel species.

    PubMed

    Lacroix, C; Coquillé, V; Guyomarch, J; Auffret, M; Moraga, D

    2014-09-15

    mRNA biomarkers are promising tools for environmental health assessment and reference genes are needed to perform relevant qPCR analyses in tissue samples of sentinel species. In the present study, potential reference genes and mRNA biomarkers were tested in the gills and digestive glands of native and caged mussels (Mytilus spp.) exposed to harbor pollution. Results highlighted the difficulty to find stable reference genes in wild, non-model species and suggested the use of normalization indices instead of single genes as they exhibit a higher stability. Several target genes were found differentially expressed between mussel groups, especially in gills where cyp32, π-gst and CuZn-sod mRNA levels could be biomarker candidates. Multivariate analyses confirmed the ability of mRNA levels to highlight site-effects and suggested the use of several combined markers instead of individual ones. These findings support the use of qPCR technology and mRNA levels as early-warning biomarkers in marine monitoring programs.

  12. mRNA stabilization controls the expression of a class of developmentally regulated genes in Dictyostelium discoideum

    PubMed Central

    Mangiarotti, Giorgio; Giorda, Roberto; Ceccarelli, Adriano; Perlo, Carla

    1985-01-01

    During the development of Dictyostelium discoideum, several thousand new mRNA species appear in the cytoplasm after the cells have formed stable aggregates. Here we show that six of these late mRNAs, corresponding to six clones randomly chosen from a genomic library, are synthesized from the very beginning of development at a rate comparable to that observed late in development but that transcripts do not accumulate until after aggregation. The early- and late-synthesized mRNAs are identical in size and compete with each other for hybridization to the genomic clones. The early-synthesized mRNAs do not accumulate in the cytoplasm in the preaggregation stage because they are very unstable. Their stability, estimated from the kinetics of incorporation during continuous labeling with 32P, increases by perhaps an order of magnitude in the postaggregation stage. We conclude that mRNA stabilization is the major controlling factor of the expression of these genes. Images PMID:16593597

  13. Activin Acts with Nerve Growth Factor to Regulate Calcitonin Gene-Related Peptide mRNA in Sensory Neurons

    PubMed Central

    Xu, Pin; Hall, Alison K.

    2009-01-01

    Calcitonin Gene-Related Peptide (CGRP) increases in sensory neurons after inflammation and plays an important role in abnormal pain responses, but how this neuropeptide is regulated is not well understood. Both activin A and Nerve Growth Factor (NGF) increase in skin after inflammation and induce CGRP in neurons in vivo and in vitro. This study was designed to understand how neurons integrate these two signals to regulate the neuropeptide important for inflammatory pain. In adult dorsal root ganglion neurons, NGF but not activin alone produced a dose-dependent increase in CGRP mRNA. When added together with NGF, activin synergistically increased CGRP mRNA, indicating that sensory neurons combine these signals. Studies were then designed to learn if that combination occurred at a common receptor or shared intracellular signals. Studies with Activin IB receptor or trkA inhibitors suggested that each ligand required its cognate receptor to stimulate the neuropeptide. Further, activin did not augment NGF-initiated intracellular MAPK signals but instead stimulated Smad phosphorylation, suggesting these ligands initiated parallel signals in the cytoplasm. Activin synergy required several NGF intracellular signals to be present. Because activin did not further stimulate, but did require NGF intracellular signals, it appears that activin and NGF converge not in receptor or cytoplasmic signals, but in transcriptional mechanisms to regulate CGRP in sensory neurons after inflammation. PMID:17964731

  14. Early gene expression in bacteriophage T7. I. In vivo synthesis, inactivation, and translational utilization of early mRNA's.

    PubMed Central

    Hercules, K; Jovanovich, S; Sauerbrier, W

    1976-01-01

    In vivo decay rates for the individual T7 early mRNA species were determined. The physical half-lives, measured at 37 C, range from 1.1 min for gene 0.7 RNA to 4.5 min for gene 0.3 RNA. Physical half-lives, as observed after rifampin inhibition of RNA synthesis and polyacylamide electrophoresis of RNAs, are approximately 30% longer than functional half-lives, as observed by 14C-labeled amino acid uptake into individual T7 early proteins. The different RNA species are synthesized at grossly different rates, 0.3 RNA at four times the rate of 1.0 RNA, 0.7 RNA at twice the rate, and 1.1 and 1.3 RNAs at about the same or a slightly lower rate than 1.0 RNA. Rho-factor-mediated termination of transcription behind genes 0.3, 0.7, and perhaps behind 1.0 is inferred from these data. The in vivo translational utilization of the individual T7 early-message species was found to vary by not more than a factor of 2. Images PMID:1255850

  15. Analysis of mutations and alternative splicing patterns in the CFTR gene using mRNA derived from nasal epithelial cells.

    PubMed

    Hull, J; Shackleton, S; Harris, A

    1994-07-01

    Ten to fifteen percent of CF chromosomes carry mutations which are not detected by routine screening of the CFTR gene for known mutations. Many techniques have been used to screen the CFTR gene for these remaining mutations. Most of the methods use genomic DNA, and since the CFTR gene contains 27 exons, are necessarily labour intensive. We have screened the entire coding region of CFTR, by chemical cleavage of 7 overlapping segments of amplified cDNA. Using this method we have identified 4 sequence changes which had not been detected by screening genomic DNA, and successfully detected 10 out of 13 known mutations. In addition, we have identified 8 alternatively spliced forms of CFTR mRNA, 4 of which have not been described previously. These include transcripts lacking a) exon 3, b) exons 2 + 3, c) exons 9 + 12, and d) the final 357 bp of exon 15 as a result of use of the cryptic splice donor site CA2863/GTTCGT).

  16. Differential gene expression in liver and small intestine from lactating rats compared to age-matched virgin controls detects increased mRNA of cholesterol biosynthetic genes

    PubMed Central

    2011-01-01

    Background Lactation increases energy demands four- to five-fold, leading to a two- to three-fold increase in food consumption, requiring a proportional adjustment in the ability of the lactating dam to absorb nutrients and to synthesize critical biomolecules, such as cholesterol, to meet the dietary needs of both the offspring and the dam. The size and hydrophobicity of the bile acid pool increases during lactation, implying an increased absorption and disposition of lipids, sterols, nutrients, and xenobiotics. In order to investigate changes at the transcriptomics level, we utilized an exon array and calculated expression levels to investigate changes in gene expression in the liver, duodenum, jejunum, and ileum of lactating dams when compared against age-matched virgin controls. Results A two-way mixed models ANOVA was applied to detect differentially expressed genes. Significance calls were defined as a p < 0.05 for the overall physiologic state effect (lactation vs. control), and a within tissue pairwise comparison of p < 0.01. The proportion of false positives, an estimate of the ratio of false positives in the list of differentially expressed genes, was calculated for each tissue. The number of differentially expressed genes was 420 in the liver, 337 in the duodenum, 402 in the jejunum, and 523 in the ileum. The list of differentially expressed genes was in turn analyzed by Ingenuity Pathways Analysis (IPA) to detect biological pathways that were overrepresented. In all tissues, sterol regulatory element binding protein (Srebp)-regulated genes involved in cholesterol synthesis showed increased mRNA expression, with the fewest changes detected in the jejunum. We detected increased Scap mRNA in the liver only, suggesting an explanation for the difference in response to lactation between the liver and small intestine. Expression of Cyp7a1, which catalyzes the rate limiting step in the bile acid biosynthetic pathway, was also significantly increased in liver. In

  17. An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1.

    PubMed

    Devonshire, Alison S; Sanders, Rebecca; Whale, Alexandra S; Nixon, Gavin J; Cowen, Simon; Ellison, Stephen L R; Parkes, Helen; Pine, P Scott; Salit, Marc; McDaniel, Jennifer; Munro, Sarah; Lund, Steve; Matsukura, Satoko; Sekiguchi, Yuji; Kawaharasaki, Mamoru; Granjeiro, José Mauro; Falagan-Lotsch, Priscila; Saraiva, Antonio Marcos; Couto, Paulo; Yang, Inchul; Kwon, Hyerim; Park, Sang-Ryoul; Demšar, Tina; Žel, Jana; Blejec, Andrej; Milavec, Mojca; Dong, Lianhua; Zhang, Ling; Sui, Zhiwei; Wang, Jing; Viroonudomphol, Duangkamol; Prawettongsopon, Chaiwat; Partis, Lina; Baoutina, Anna; Emslie, Kerry; Takatsu, Akiko; Akyurek, Sema; Akgoz, Muslum; Vonsky, Maxim; Konopelko, L A; Cundapi, Edna Matus; Urquiza, Melina Pérez; Huggett, Jim F; Foy, Carole A

    2016-06-01

    Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals ('measurement uncertainties') were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the

  18. Structure and expression of the guinea pig preproenkephalin gene: site-specific cleavage in the 3' untranslated region yields truncated mRNA transcripts in specific brain regions.

    PubMed Central

    LaForge, K S; Unterwald, E M; Kreek, M J

    1995-01-01

    We isolated the guinea pig preproenkephalin gene from a genomic library by hybridization to a rat cDNA probe. The entire nucleotide sequence of the gene was determined. Genomic Southern blot hybridization demonstrated that the gene exists in a single copy within the genome. On the basis of RNase protection transcript mapping and homology comparisons with known preproenkephalin sequences from other species and assuming a poly(A) tail length of 100 residues, we predicted an mRNA transcript of approximately 1,400 nucleotides encoded by three exons. Northern (RNA) blot analysis of total RNA from several brain regions showed high levels of preproenkephalin mRNA in the caudate putamen, nucleus accumbens, and hypothalamus, with detectable levels in the amygdala, ventral tegmental area, and central gray and also in the pituitary. Unexpectedly, in several brain regions, the mRNA appeared not only in the 1,400-nucleotide length but also in a shorter length of approximately 1,130 bases. Significant amounts of the shorter mRNA were found in the caudate putamen, nucleus accumbens, and amygdala. The longer, but not the shorter, transcripts from the caudate putamen were found to be polyadenylated, but the difference in size was not due solely to the presence of poly(A) tails. Northern gel analysis of total RNA from the caudate putamen with probes from each exon, together with RNase protection mapping of the 3' end of the mRNA demonstrated that the 1,400-base preproenkephalin mRNA transcripts are cleaved in a site-specific manner in some brain regions, yielding a 1,130-base transcript and a 165-base polyadenylated fragment derived from the terminal end of the 3' untranslated region of the mRNA. This cleavage may serve as a preliminary step in RNA degradation and provide a mechanism for control of preproenkephalin mRNA abundance through selective degradation. PMID:7891703

  19. Associations between genetic variants in mRNA splicing-related genes and risk of lung cancer: a pathway-based analysis from published GWASs

    PubMed Central

    Pan, Yongchu; Liu, Hongliang; Wang, Yanru; Kang, Xiaozheng; Liu, Zhensheng; Owzar, Kouros; Han, Younghun; Su, Li; Wei, Yongyue; Hung, Rayjean J.; Brhane, Yonathan; McLaughlin, John; Brennan, Paul; Bickeböller, Heike; Rosenberger, Albert; Houlston, Richard S.; Caporaso, Neil; Teresa Landi, Maria; Heinrich, Joachim; Risch, Angela; Wu, Xifeng; Ye, Yuanqing; Christiani, David C.; Amos, Christopher I.; Wei, Qingyi

    2017-01-01

    mRNA splicing is an important mechanism to regulate mRNA expression. Abnormal regulation of this process may lead to lung cancer. Here, we investigated the associations of 11,966 single-nucleotide polymorphisms (SNPs) in 206 mRNA splicing-related genes with lung cancer risk by using the summary data from six published genome-wide association studies (GWASs) of Transdisciplinary Research in Cancer of the Lung (TRICL) (12,160 cases and 16,838 controls) and another two lung cancer GWASs of Harvard University (984 cases and 970 controls) and deCODE (1,319 cases and 26,380 controls). We found that a total of 12 significant SNPs with false discovery rate (FDR) ≤0.05 were mapped to one novel gene PRPF6 and two previously reported genes (DHX16 and LSM2) that were also confirmed in this study. The six novel SNPs in PRPF6 were in high linkage disequilibrium and associated with PRPF6 mRNA expression in lymphoblastoid cells from 373 Europeans in the 1000 Genomes Project. Taken together, our studies shed new light on the role of mRNA splicing genes in the development of lung cancer. PMID:28304396

  20. The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells.

    PubMed Central

    Bond, U; Schlesinger, M J

    1986-01-01

    A chicken genomic library was screened to obtain genomic clones for ubiquitin genes. Two genes that differ in their genomic location and organization were identified. One gene, designated Ub I, contains four copies of the protein-coding sequence arranged in tandem, while the second gene, Ub II, contains three. The origin of the two major mRNAs that are induced after heat shock in chicken embryo fibroblasts was determined by generating DNA probes from the 5'-and 3'-noncoding regions of the two genes. Both mRNAs are transcribed from Ub I, the larger being the unspliced precursor of the smaller. A 674-base-pair intron was located within the 5'-noncoding region of Ub I. The second gene, Ub II, does not appear to code for an RNA species in normal or heat-shocked chicken embryo fibroblasts. The expression of ubiquitin mRNA during heat shock and recovery was examined. Addition of actinomycin D before heat shock completely abolished the response of ubiquitin mRNA to the stress. Analysis of the stability of the mRNA during recovery revealed that the mRNA accumulated during the heat shock is rapidly degraded with a half-life of approximately 1.5 h, suggesting a specialized but transient role for ubiquitin during heat shock. Images PMID:3025663

  1. Comparative analyses of gene copy number and mRNA expression in GBM tumors and GBM xenografts

    SciTech Connect

    Hodgson, J. Graeme; Yeh, Ru-Fang; Ray, Amrita; Wang, Nicholas J.; Smirnov, Ivan; Yu, Mamie; Hariono, Sujatmi; Silber, Joachim; Feiler, Heidi S.; Gray, Joe W.; Spellman, Paul T.; Vandenberg, Scott R.; Berger, Mitchel S.; James, C. David

    2009-04-03

    Development of model systems that recapitulate the molecular heterogeneity observed among glioblastoma multiforme (GBM) tumors will expedite the testing of targeted molecular therapeutic strategies for GBM treatment. In this study, we profiled DNA copy number and mRNA expression in 21 independent GBM tumor lines maintained as subcutaneous xenografts (GBMX), and compared GBMX molecular signatures to those observed in GBM clinical specimens derived from the Cancer Genome Atlas (TCGA). The predominant copy number signature in both tumor groups was defined by chromosome-7 gain/chromosome-10 loss, a poor-prognosis genetic signature. We also observed, at frequencies similar to that detected in TCGA GBM tumors, genomic amplification and overexpression of known GBM oncogenes, such as EGFR, MDM2, CDK6, and MYCN, and novel genes, including NUP107, SLC35E3, MMP1, MMP13, and DDX1. The transcriptional signature of GBMX tumors, which was stable over multiple subcutaneous passages, was defined by overexpression of genes involved in M phase, DNA replication, and chromosome organization (MRC) and was highly similar to the poor-prognosis mitosis and cell-cycle module (MCM) in GBM. Assessment of gene expression in TCGA-derived GBMs revealed overexpression of MRC cancer genes AURKB, BIRC5, CCNB1, CCNB2, CDC2, CDK2, and FOXM1, which form a transcriptional network important for G2/M progression and/or checkpoint activation. Our study supports propagation of GBM tumors as subcutaneous xenografts as a useful approach for sustaining key molecular characteristics of patient tumors, and highlights therapeutic opportunities conferred by this GBMX tumor panel for testing targeted therapeutic strategies for GBM treatment.

  2. The use of R-looping for structural gene identification and mRNA purification.

    PubMed Central

    Woolford, J L; Rosbash, M

    1979-01-01

    A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules. RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences. These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt. The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code. We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means. Images PMID:379820

  3. mRNA leader length and initiation codon context determine alternative AUG selection for the yeast gene MOD5.

    PubMed Central

    Slusher, L B; Gillman, E C; Martin, N C; Hopper, A K

    1991-01-01

    MOD5, a nuclear gene of Saccharomyces cerevisiae, encodes two isozymic forms of a tRNA-modification enzyme. These enzymes modify both cytoplasmic and mitochondrial tRNAs. Two inframe ATGs of the MOD5 gene are used for initiation of translation, and the form of the protein translated from the first AUG is imported into mitochondria. Protein translated from the second AUG functions in the cytoplasm. Since all transcripts contain both of these translational start sites and two proteins are made, the question arises as to the factors that influence the translation start-site choice. Extending the 5' ends of the MOD5 mRNA to include leader sequences of the ADH1 (alcohol dehydrogenase defective) transcript produces significant changes in the choice of AUGs. This suggests that for wild-type MOD5 transcripts, the length or structure of the leader sequence plays a role in AUG choice. The nucleotides surrounding the first ATG of MOD5 also have an effect on translation initiation. Altering these nucleotides changes initiation choice and suggests that ribosomal bypass of a suboptimal AUG is another mechanism controlling the alternate use of two initiation codons. Our data support the model that at least one MOD5 transcript is able to produce two proteins with different N-terminal sequences. Images PMID:1946403

  4. Neuronal function of the mRNA decapping complex determines survival of Caenorhabditis elegans at high temperature through temporal regulation of heterochronic gene expression

    PubMed Central

    Borbolis, Fivos; Flessa, Christina-Maria; Roumelioti, Fani; Diallinas, George; Stravopodis, Dimitrios J.

    2017-01-01

    In response to adverse environmental cues, Caenorhabditis elegans larvae can temporarily arrest development at the second moult and form dauers, a diapause stage that allows for long-term survival. This process is largely regulated by certain evolutionarily conserved signal transduction pathways, but it is also affected by miRNA-mediated post-transcriptional control of gene expression. The 5′–3′ mRNA decay mechanism contributes to miRNA-mediated silencing of target mRNAs in many organisms but how it affects developmental decisions during normal or stress conditions is largely unknown. Here, we show that loss of the mRNA decapping complex activity acting in the 5′–3′ mRNA decay pathway inhibits dauer formation at the stressful high temperature of 27.5°C, and instead promotes early developmental arrest. Our genetic data suggest that this arrest phenotype correlates with dysregulation of heterochronic gene expression and an aberrant stabilization of lin-14 mRNA at early larval stages. Restoration of neuronal dcap-1 activity was sufficient to rescue growth phenotypes of dcap-1 mutants at both high and normal temperatures, implying the involvement of common developmental timing mechanisms. Our work unveils the crucial role of 5′–3′ mRNA degradation in proper regulation of heterochronic gene expression programmes, which proved to be essential for survival under stressful conditions. PMID:28250105

  5. Determination of the henipavirus phosphoprotein gene mRNA editing frequencies and detection of the C, V and W proteins of Nipah virus in virus-infected cells.

    PubMed

    Lo, Michael K; Harcourt, Brian H; Mungall, Bruce A; Tamin, Azaibi; Peeples, Mark E; Bellini, William J; Rota, Paul A

    2009-02-01

    The henipaviruses, Nipah virus (NiV) and Hendra virus (HeV), are highly pathogenic zoonotic paramyxoviruses. Like many other paramyxoviruses, henipaviruses employ a process of co-transcriptional mRNA editing during transcription of the phosphoprotein (P) gene to generate additional mRNAs encoding the V and W proteins. The C protein is translated from the P mRNA, but in an alternate reading frame. Sequence analysis of multiple, cloned mRNAs showed that the mRNA editing frequencies of the P genes of the henipaviruses are higher than those reported for other paramyxoviruses. Antisera to synthetic peptides from the P, V, W and C proteins of NiV were generated to study their expression in infected cells. All proteins were detected in both infected cells and purified virions. In infected cells, the W protein was detected in the nucleus while P, V and C were found in the cytoplasm.

  6. Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3′ ends

    PubMed Central

    Liu, Bo; Kearns, Daniel B.; Bechhofer, David H.

    2016-01-01

    Timely turnover of RNA is an important element in the control of bacterial gene expression, but relatively few specific targets of RNA turnover regulation are known. Deletion of the Bacillus subtilis pnpA gene, encoding the major 3′ exonuclease turnover enzyme, polynucleotide phosphorylase (PNPase), was shown previously to cause a motility defect correlated with a reduced level of the 32-gene fla/che flagellar biosynthesis operon transcript. fla/che operon transcript abundance has been shown to be inhibited by an excess of the small regulatory protein, SlrA, and here we find that slrA mRNA accumulated in the pnpA-deletion mutant. Mutation of slrA was epistatic to mutation of pnpA for the motility-related phenotype. Further, Rho-dependent termination was required for PNPase turnover of slrA mRNA. When the slrA gene was provided with a Rho-independent transcription terminator, gene regulation was no longer PNPase-dependent. Thus we show that the slrA transcript is a direct target of PNPase and that regulation of RNA turnover is a major determinant of motility gene expression. The interplay of specific transcription termination and mRNA decay mechanisms suggests selection for fine-tuning of gene expression. PMID:26857544

  7. The BAX gene as a candidate for negative autophagy-related genes regulator on mRNA levels in colorectal cancer.

    PubMed

    Gil, Justyna; Ramsey, David; Szmida, Elzbieta; Leszczynski, Przemyslaw; Pawlowski, Pawel; Bebenek, Marek; Sasiadek, Maria M

    2017-02-01

    Autophagy is a catabolic process, which is involved in the maintenance of intracellular homeostasis by degrading redundant molecules and organelles. Autophagy begins with the formation of a double-membrane phagophore, followed by its enclosure, thus leading to the appearance of an autophagosome which fuses with lysosome. This process is highly conserved, precisely orchestrated and regulated by autophagy-related genes. Recently, autophagy has been widely studied in different types of cancers, including colorectal cancer. As it has been revealed, autophagy plays two opposite roles in tumorigenesis, as a tumor suppressor and a tumor enhancer/activator, and therefore is called a double-edge sword. Recently, interaction between autophagy and apoptosis has been found. Therefore, we aimed to study the mRNA levels of genes engaged in autophagy and apoptosis in colorectal cancer tissues. Colorectal cancer and adjacent healthy tissues were obtained from 73 patients diagnosed with primary colorectal cancer. Real-time PCR analysis employing Universal Probe Library was used to assess the expression of the seven following selected genes: BECN1, UVRAG, ULK1, ATG13, Bif-1, BCL2 and BAX. For all but one of the tested genes, a decrease in expression was observed. An increase in expression was observed for BAX. BAX expression decreases consistently from early to more advanced stages. High expression of BAX was strongly associated with negative UVRAG expression. The high expression of the BAX gene seems to be a negative regulator of autophagy in colorectal cancer cells. The relative downregulation of autophagy-related genes was observed in colorectal cancer samples.

  8. Leading the way: finding genes for neurologic disease in dogs using genome-wide mRNA sequencing.

    PubMed

    Ostrander, Elaine A; Beale, Holly C

    2012-07-10

    Because of dogs' unique population structure, human-like disease biology, and advantageous genomic features, the canine system has risen dramatically in popularity as a tool for discovering disease alleles that have been difficult to find by studying human families or populations. To date, disease studies in dogs have primarily employed either linkage analysis, leveraging the typically large family size, or genome-wide association, which requires only modest-sized case and control groups in dogs. Both have been successful but, like most techniques, each requires a specific combination of time and money, and there are inherent problems associated with each. Here we review the first report of mRNA-Seq in the dog, a study that provides insights into the potential value of applying high-throughput sequencing to the study of genetic diseases in dogs. Forman and colleagues apply high-throughput sequencing to a single case of canine neonatal cerebellar cortical degeneration. This implementation of whole genome mRNA sequencing, the first reported in dog, is additionally unusual due to the analysis: the data was used not to examine transcript levels or annotate genes, but as a form of target capture that revealed the sequence of transcripts of genes associated with ataxia in humans. This approach entails risks. It would fail if, for example, the relevant transcripts were not sufficiently expressed for genotyping or were not associated with ataxia in humans. But here it pays off handsomely, identifying a single frameshift mutation that segregates with the disease. This work sets the stage for similar studies that take advantage of recent advances in genomics while exploiting the historical background of dog breeds to identify disease-causing mutations.

  9. Cholesterol Side-Chain Cleavage Gene Expression in Theca Cells: Augmented Transcriptional Regulation and mRNA Stability in Polycystic Ovary Syndrome

    PubMed Central

    Nelson-DeGrave, Velen L.; Legro, Richard S.; Strauss, Jerome F.; McAllister, Jan M.

    2012-01-01

    Hyperandrogenism is characteristic of women with polycystic ovary syndrome (PCOS). Ovarian theca cells isolated from PCOS follicles and maintained in long-term culture produce elevated levels of progestins and androgens compared to normal theca cells. Augmented steroid production in PCOS theca cells is associated with changes in the expression of genes for several steroidogenic enzymes, including CYP11A1, which encodes cytochrome P450 cholesterol side-chain cleavage. Here, we further examined CYP11A1 gene expression, at both the transcriptional and post-transcriptional level in normal and PCOS theca cells propagated in long-term culture utilizing quantitative RT-PCR, functional promoter analyses, and mRNA degradation studies. The minimal element(s) that conferred increased basal and cAMP-dependent CYP11A1 promoter function were determined. CYP11A1 mRNA half-life in normal and PCOS theca cells was compared. Results of these cumulative studies showed that basal and forskolin stimulated steady state CYP11A1 mRNA abundance and CYP11A1 promoter activity were increased in PCOS theca cells. Deletion analysis of the CYP11A1 promoter demonstrated that augmented promoter function in PCOS theca cells results from increased basal regulation conferred by a minimal sequence between −160 and −90 bp of the transcriptional start site. The transcription factor, nuclear factor 1C2, was observed to regulate basal activity of this minimal CYP11A1 element. Examination of mRNA stability in normal and PCOS theca cells demonstrated that CYP11A1 mRNA half-life increased >2-fold, from approximately 9.22+/−1.62 h in normal cells, to 22.38+/−0.92 h in PCOS cells. Forskolin treatment did not prolong CYP11A1 mRNA stability in either normal or PCOS theca cells. The 5′-UTR of CYP11A1 mRNA confers increased basal mRNA stability in PCOS cells. In conclusion, these studies show that elevated steady state CYP11A1 mRNA abundance in PCOS cells results from increased transactivation of the CYP

  10. Activation of gene expression by herpes simplex virus type 1 ICP0 occurs at the level of mRNA synthesis.

    PubMed Central

    Jordan, R; Schaffer, P A

    1997-01-01

    ICP0 is a nuclear phosphoprotein involved in the activation of herpes simplex virus type 1 (HSV-1) gene expression during lytic infection and reactivation from viral latency. Although available evidence suggests that ICP0 acts at the level of transcription, definitive studies specifically addressing this issue have not been reported. In the present study we measured the ability of ICP0 to activate gene expression (i) from promoters representing the major kinetic classes of viral genes in transient expression assays and (ii) from the same promoters during viral infection at multiplicities of infection ranging from 0.1 to 5.0 PFU/cell. The levels of synthesis and steady-state accumulation of mRNA, mRNA stability, and levels of protein synthesis were compared in cells transfected with a reporter plasmid in the presence and absence of ICP0 and in cells infected with wild-type HSV-1 or an ICP0 null mutant, n212. In transient expression assays and during viral infection at all multiplicities tested, the levels of steady-state mRNA and protein were significantly lower in the absence of ICP0, indicating that ICP0 activates gene expression at the level of mRNA accumulation. In transient expression assays and during infection at low multiplicities (< 1 PFU/cell) in the presence or absence of ICP0, marked increases in the levels of viral mRNAs accompanied by proportional increases in the levels of protein synthesis were observed with increasing multiplicity. At a high multiplicity (5 PFU/cell) in the presence or absence of ICP0, mRNA levels did not increase as a function of multiplicity and changes in the levels of protein were no longer related to changes in the levels of mRNA. Collectively, these tests indicate that transcription of viral genes is rate limiting at low multiplicities and that translation is rate limiting at high multiplicities, independent of ICP0. Consistent with the lower levels of mRNA detected in the absence of ICP0, the rates of transcription initiation

  11. Expression of Caspase-1 Gene Transcript Variant mRNA in Peripheral Blood Mononuclear Cells of Patients with Primary Gout in Different TCM Syndromes

    PubMed Central

    Dang, Wan-Tai; Xu, Dan; Xie, Wen-Guang; Zhou, Jing-Guo

    2015-01-01

    A large number of studies have shown that cysteinyl aspartate specific protease-1 (CASP1) played an important role in the inflammatory response of primary gout, but the decreased expression of different CASP1 transcript variant could inhibit the activation of IL-1β. Our study mainly analyzed the expression level and function of CASP1 gene transcript variant mRNA in peripheral blood mononuclear cells of patients with gout in different TCM syndromes. The expression of CASP1 gene transcript variant and IL-1β mRNA in PBMCs were detected in patients with PG [acute phase (AP: 44 cases); nonacute phase (NAP: 52 cases)] and healthy controls (HC: 30 cases) by reverse transcription-polymerase chain reaction and/or real-time quantitative polymerase chain reaction. The expressions of plasma IL-1β in patients with PG and HC were detected by enzyme-linked immunosorbent assay. Dysregulated expression of the CASP1 gene and its transcript variant, plasma proinflammatory cytokines in all patients with primary gout in different TCM syndromes, correlation analysis showed that there was negative correlation between the expression of CASP1-gamma gene transcript variant mRNA and IL-1β protein in APPG group. The study suggested that CASP1 gene and its transcript variant may play a critical role in the inflammatory response of patients with PG in different phases and TCM syndromes. PMID:26557856

  12. Effects of malachite green on the mRNA expression of detoxification-related genes in Nile tilapia (Oreochromis niloticus) and other major Chinese freshwater fishes.

    PubMed

    Li, Guangyu; Shen, Dan; Liang, Xu-Fang; He, Yan; He, Shan

    2013-03-01

    The use of malachite green (MG) in fish farming is prohibited in China due to its potentially toxicological and carcinogenic nature, but it is still illegally used in some places. The aim of this study was to investigate the time and concentration-dependent responses of xenobiotic metabolizing and detoxification-related genes in diverse fishes exposed to MG both in vivo and in vitro. Experimental fish were administered to two exposure groups of malachite green (MG) (0.10 and 0.50 mg L⁻¹) for 8 h. The hepatocytes isolated from Nile tilapia were incubated with MG (0.5, 1.0, and 2.0 mg L⁻¹) for 8 and 24 h, respectively. In vivo, exposure to 0.10 and 0.50 mg L⁻¹ MG for 8 h caused significant changes of the detoxification-related genes on the mRNA expression levels. Low-concentration (0.10 mg L⁻¹) level of MG induced significant increase on the mRNA expression level of GSTR gene in Nile tilapia and other fishes. The mRNA expression of grass carp UCP2 was significantly induced when exposed to 0.5 mg L⁻¹ MG. However, the mRNA expression levels of GSTA, CYP1A, and GPX were inhibited significantly by 0.5 mg L⁻¹ MG in Nile tilapia, grass carp, and Taiwan snakehead. In vitro, the significant increase of mRNA expression of these genes was detected after exposure to 0.5 mg L⁻¹ MG (UCP2), and 1.0 mg L⁻¹ MG (CYP1A1, GSTA, GSTR, and UCP2). The induction of hepatic CYP1A1, GSTA, GSTR, and UCP2 in response to MG suggested a potential role of fish CYP1A1, GSTA, GSTR, and UCP2 in MG metabolism.

  13. Effects of decabromodiphenyl ether (BDE-209) on mRNA transcription of thyroid hormone pathway and spermatogenesis associated genes in Chinese rare minnow (Gobiocypris rarus).

    PubMed

    Li, Wei; Zhu, Lifei; Zha, Jinmiao; Wang, Zijian

    2014-01-01

    Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants, which are ubiquitous environmental contaminant found in both abiotic and biotic environmental samples. Deca-BDE (BDE-209) is the principal component, which is currently used worldwide. In this study, the effect of BDE-209 on the mRNA levels of thyroid hormone (TH) related genes and spermatogenesis associated genes were determined from larvae and adult rare minnow (Gobiocypris rarus) exposed to concentrations 0.01, 0.1, 1, and 10 μg/L for 21 days. The results showed that the type II deiodinase (dio2) and sodium iodide symporter (nis) mRNA levels were significantly up-regulated in the larvae at 10 μg/L treatment. In adult, histopathological observations showed that liver of female fish were degenerated at 10 μg/L treatment, and inhibition of spermatogenesis were observed in testis of male fish. In addition, the thyroid hormone receptor α (trα), dio2, and nis mRNA levels in the liver of male and female fish were significantly up-regulated, whereas dio2 and nis mRNA levels were significantly down-regulated in the brain. These results indicate that exposure to BDE-209 could result in tissue-specific alternations of TH-related genes expression in adults. Moreover, the mRNA levels of the testis-specific apoptosis genes, the spermatogenesis-associated 4 (spata4) and spermatogenesis-associated 17 (spata17), were down-regulated at 10 μg/L treatment in testis of male fish. Our results suggest that BDE-209 may pose threat to normal thyroid and reproductive function in fish.

  14. Glucocorticoid receptor 1B and 1C mRNA transcript alterations in schizophrenia and bipolar disorder, and their possible regulation by GR gene variants.

    PubMed

    Sinclair, Duncan; Fullerton, Janice M; Webster, Maree J; Shannon Weickert, Cynthia

    2012-01-01

    Abnormal patterns of HPA axis activation, under basal conditions and in response to stress, are found in individuals with schizophrenia and bipolar disorder. Altered glucocorticoid receptor (GR) mRNA and protein expression in the dorsolateral prefrontal cortex (DLPFC) in psychiatric illness have also been reported, but the cause of these abnormalities is not known. We quantified expression of GR mRNA transcript variants which employ different 5' promoters, in 35 schizophrenia cases, 31 bipolar disorder cases and 34 controls. We also explored whether sequence variation within the NR3C1 (GR) gene is related to GR mRNA variant expression. Total GR mRNA was decreased in the DLPFC in schizophrenia cases relative to controls (15.1%, p<0.0005) and also relative to bipolar disorder cases (8.9%, p<0.05). GR-1B mRNA was decreased in schizophrenia cases relative to controls (20.2%, p<0.05), while GR-1C mRNA was decreased in both schizophrenia and bipolar disorder cases relative to controls (16.1% and 17.2% respectively, both p<0.005). A dose-dependent effect of rs10052957 genotype on GR-1B mRNA expression was observed, where CC homozygotes displayed 18.4% lower expression than TC heterozygotes (p<0.05), and 31.8% lower expression than TT homozygotes (p<0.005). Similarly, a relationship between rs6190 (R23K) genotype and GR-1C expression was seen, with 24.8% lower expression in GG homozygotes than GA heterozygotes (p<0.01). We also observed an effect of rs41423247 (Bcl1) SNP on expression of 67 kDa GRα isoform, the most abundant GRα isoform in the DLPFC. These findings suggest possible roles for the GR-1B and GR-1C promoter regions in mediating GR gene expression changes in psychotic illness, and highlight the potential importance of sequence variation within the NR3C1 gene in modulating GR mRNA expression in the DLPFC.

  15. Intrahepatic mRNA Expression of FAS, FASL, and FOXP3 Genes Is Associated with the Pathophysiology of Chronic HCV Infection

    PubMed Central

    Amoras, Ednelza da Silva Graça; Gomes, Samara Tatielle Monteiro; Freitas, Felipe Bonfim; Santana, Bárbara Brasil; Ishak, Geraldo; Ferreira de Araújo, Marialva Tereza; Demachki, Sâmia; Conde, Simone Regina Souza da Silva; Ishak, Marluísa de Oliveira Guimarães; Ishak, Ricardo; Vallinoto, Antonio Carlos Rosário

    2016-01-01

    This study aimed to evaluate the relative mRNA expression of Fas receptor (FAS), Fas ligand (FASL), and forkhead box protein 3 (FOXP3) in liver biopsy specimens obtained from patients with viral and non-viral chronic hepatitis and correlate their expression with the fibrosis stage. A total of 51 liver biopsy specimens obtained from HBV (n = 6), HCV (n = 28), and non-viral hepatic disease (NVHD) (n = 9) patients and from individuals with normal liver histology (n = 8) (control—CT) were analyzed. Quantifications of the target genes were assessed using qPCR, and liver biopsies according to the METAVIR classification. The mRNA expression levels of FAS and FASL were lower in the CT group compared to the groups of patients. The increase in the mRNA expression of FAS and FASL was correlated with higher levels of inflammation and disease progression, followed by a decline in tissues with cirrhosis, and it was also associated with increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Higher mRNA expression of FOXP3 was observed in the HCV and NVHD groups, with the peak observed among patients with cirrhosis. The increased FOXP3 mRNA expression was positively correlated with increased FAS and FASL mRNA expression and the AST and ALT levels in all patients. Conclusions: These results suggest that regardless of the cause, the course of chronic liver disease may be modulated by the analyzed genes and correlated with an increase in regulatory T cells during the liver damage followed by hepatocyte destruction by Fas/FasL system and subsequent non specific lymphocytic infiltrate accumulation. PMID:27243827

  16. Molecular cloning of the SMAD4 gene and its mRNA expression analysis in ovarian follicles of the Yangzhou goose (Anser cygnoides).

    PubMed

    Huang, Z; Yuan, X; Wang, M; Wu, N; Song, Y; Chen, Y; Zhang, Y; Xu, Q; Chen, G; Zhao, W

    2016-08-01

    Mothers against decapentaplegic homolog 4 (SMAD4) is an important protein in animal reproduction. It plays pivotal roles in cellular pathways, including apoptosis. The expression profile of the SMAD4 gene in goose ovarian follicles has not been reported. In this study, the SMAD4 coding sequence was cloned from the Yangzhou goose. A phylogenetic analysis was performed and mRNA expression was examined in various tissues using quantitative real-time PCR. An alternative splice form of SMAD4, SMAD4-b having 1656 bp, was identified. SMAD4-a mRNA was widely expressed in various healthy tissues, whereas SMAD4-b was very weakly expressed. SMAD4 mRNA in the ovary and oviduct was significantly higher than that in the pituitary and hypothalamus. SMAD4 mRNA expression analysis in hierarchical follicles showed that the level of SMAD4 mRNA was higher in large white follicles and post-ovulatory follicles than in the other follicles. The results indicate that SMAD4 might be involved in the recruitment of hierarchical follicles.

  17. tRNA regulation of gene expression: Interactions of an mRNA 5′-UTR with a regulatory tRNA

    PubMed Central

    Nelson, Audrey R.; Henkin, Tina M.; Agris, Paul F.

    2006-01-01

    Many genes encoding aminoacyl-tRNA synthetases and other amino acid–related products in Gram-positive bacteria, including important pathogens, are regulated through interaction of unacylated tRNA with the 5′-untranslated region (5′-UTR) of the mRNA. Each gene regulated by this mechanism responds specifically to the cognate tRNA, and specificity is determined by pairing of the anticodon of the tRNA with a codon sequence in the “Specifier Loop” of the 5′-UTR. For the 5′-UTR to function in gene regulation, the mRNA folding interactions must be sufficiently stable to present the codon sequence for productive binding to the anticodon of the matching tRNA. A model bimolecular system was developed in which the interaction between two half molecules (“Common” and “Specifier”) would reconstitute the Specifier Loop region of the 5′-UTR of the Bacillus subtilis glyQS gene, encoding GlyRS mRNA. Gel mobility shift analysis and fluorescence spectroscopy yielded experimental K ds of 27.6 ± 1.0 μM and 10.5 ± 0.7 μM, respectively, for complex formation between Common and Specifier half molecules. The reconstituted 5′-UTR of the glyQS mRNA bound the anticodon stem and loop of tRNAGly (ASLGlyGCC) specifically and with a significant affinity (K d = 20.2 ± 1.4 μM). Thus, the bimolecular 5′-UTR and ASLGlyGCC models mimic the RNA–RNA interaction required for T box gene regulation in vivo. PMID:16741230

  18. In Vivo mRNA Profiling of Uropathogenic Escherichia coli from Diverse Phylogroups Reveals Common and Group-Specific Gene Expression Profiles

    PubMed Central

    Bielecki, Piotr; Muthukumarasamy, Uthayakumar; Eckweiler, Denitsa; Bielecka, Agata; Pohl, Sarah; Schanz, Ansgar; Niemeyer, Ute; Oumeraci, Tonio; von Neuhoff, Nils; Ghigo, Jean-Marc

    2014-01-01

    ABSTRACT mRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression of Escherichia coli pathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associated E. coli isolates to different phylogenetic groups. Whereas the in vivo gene expression profiles of the majority of genes were conserved among 21 E. coli strains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribed in vivo relative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease. PMID:25096872

  19. An interaction type of genetic screen reveals a role of the Rab11 gene in oskar mRNA localization in the developing Drosophila melanogaster oocyte.

    PubMed Central

    Jankovics, F; Sinka, R; Erdélyi, M

    2001-01-01

    Abdomen and germ cell development of Drosophila melanogaster embryo requires proper localization of oskar mRNA to the posterior pole of the developing oocyte. oskar mRNA localization depends on complex cell biological events like cell-cell communication, dynamic rearrangement of the microtubule network, and function of the actin cytoskeleton of the oocyte. To investigate the cellular mechanisms involved, we developed a novel interaction type of genetic screen by which we isolated 14 dominant enhancers of a sensitized genetic background composed of mutations in oskar and in TropomyosinII, an actin binding protein. Here we describe the detailed analysis of two allelic modifiers that identify Drosophila Rab11, a gene encoding small monomeric GTPase. We demonstrate that mutation of the Rab11 gene, involved in various vesicle transport processes, results in ectopic localization of oskar mRNA, whereas localization of gurken and bicoid mRNAs and signaling between the oocyte and the somatic follicle cells are unaffected. We show that the ectopic oskar mRNA localization in the Rab11 mutants is a consequence of an abnormally polarized oocyte microtubule cytoskeleton. Our results indicate that the internal membranous structures play an important role in the microtubule organization in the Drosophila oocyte and, thus, in oskar RNA localization. PMID:11454766

  20. Alternative splicing and developmental and hormonal regulation of porcine comparative gene identification-58 (CGI-58) mRNA.

    PubMed

    Li, X; Suh, Y; Kim, E; Moeller, S J; Lee, K

    2012-12-01

    The process of lipolysis is essential for regulating the catabolism of cellular fat stores. Therefore, knowledge of lipolysis contributes to improving porcine production, such as reducing back fat, enhancing lean meat, and controlling marbling. Comparative gene identification-58 (CGI-58) plays an important role in the multi-enzyme-mediated process of lipolysis. It was identified as the co-activator of adipose triglyceride lipase (ATGL), which performs the first step in breaking down triacylglycerol and generating diacylglycerol and NEFA. We cloned and sequenced the CGI-58 cDNA and deduced the AA sequences in 3 breeds of swine (Duroc, Berkshire, and Landrace). Homologies were found with the human, mouse, and chicken for the lipid droplet binding domain, the α/β hydrolase domain, and the lysophosphatidic acid acyltransferase (LPAAT) domain, which demonstrates conservation of CGI-58 across species. An alternatively spliced isoform with an exon 3 deletion was identified. Interestingly, this unique isoform contains the lipid droplet-binding domain but lacks the LPAAT domain due to an open reading frame (ORF) shift that creates a premature stop codon. Furthermore, porcine CGI-58 is expressed in multiple organs and tissues but is most predominant in adipose tissue. Porcine adipose and stromal-vascular (SV) cell fractionation reveals that CGI-58 and ATGL are highly expressed (P < 0.01) in mature adipocytes. The expressions of both CGI-58 and ATGL mRNA were found to increase (P < 0.05) at d 6 of SV cell culture, confirming their upregulation during adipogenesis and differentiation. Also, the results from in vitro cell culture showed that insulin decreased (P < 0.05) the expressions of both CGI-58 and ATGL in a dose-dependent manner. Overall, these results report the cDNA and AA sequences of porcine CGI-58 with identification of its unique alternatively spliced variant. The results of the study also reveal the developmental and hormonal regulation of porcine CGI-58 gene

  1. mRIN for direct assessment of genome-wide and gene-specific mRNA integrity from large-scale RNA-sequencing data

    PubMed Central

    Feng, Huijuan; Zhang, Xuegong; Zhang, Chaolin

    2015-01-01

    The volume of RNA-Seq data sets in public repositories has been expanding exponentially, providing unprecedented opportunities to study gene expression regulation. Because degraded RNA samples, such as those collected from post-mortem tissues, can result in distinct expression profiles with potential biases, a particularly important step in mining these data is quality control. Here we develop a method named mRIN to directly assess mRNA integrity from RNA-Seq data at the sample and individual gene level. We systematically analyse large-scale RNA-Seq data sets of the human brain transcriptome generated by different consortia. Our analysis demonstrates that 3′ bias resulting from partial RNA fragmentation in post-mortem tissues has a marked impact on global expression profiles, and that mRIN effectively identifies samples with different levels of mRNA degradation. Unexpectedly, this process has a reproducible and gene-specific component, and transcripts with different stabilities are associated with distinct functions and structural features reminiscent of mRNA decay in living cells. PMID:26234653

  2. Haematopoietic cell lines capable of colonizing the thymus following in vivo transfer expressed T-cell receptor gamma-gene immature mRNA.

    PubMed Central

    Shimamura, M; Oku, M; Ohta, S; Yamagata, T

    1992-01-01

    To clarify the mechanism by which progenitor T (pro-T) cells recognize and enter the thymus, an attempt was made to produce haematopoietic cell lines by the fusion of BALB/c nude mouse bone marrow or foetal liver cells (gestation 14 and 15 days) with AKR thymoma BW5147, thereby immortalizing cells with potency to colonize the thymus, a characteristic of pro-T cells rarely found in adult bone marrow or foetal liver. The hybridomas thus produced were classified according to the phenotype of surface markers, T-cell receptor (TcR) gene configuration and expression. All hybridomas were negative in the surface expression of T-cell markers such as TcR alpha beta, TcR gamma delta, CD3, CD4 and CD8. They had TcR beta-, gamma- and delta-genes, each with a different status with respect to configuration and transcription. Some possessed partially rearranged TcR genes and others expressed immature TcR mRNA. The cell lines were examined for their capacity to colonize the thymus following intravenous injection into recipient mice. It was found that the cells with capacity of colonizing the thymus expressed immature TcR delta mRNA, while the cell lines lacking TcR delta-genes did not home to the thymus. These findings imply that the potency for migrating to thymus is closely associated with the particular stage of prethymic cell differentiation which could be estimated by the analysis of TcR genes, and that some cell lines with the expression of TcR delta-gene mRNA and the ability to colonize the thymus are derived from pro-T cells. Images Figure 2 Figure 3 PMID:1478683

  3. Translational regulation: identification of the site on bacteriophage T4 rIIB mRNA recognized by the regA gene function.

    PubMed Central

    Karam, J; Gold, L; Singer, B S; Dawson, M

    1981-01-01

    The bacteriophage T4 gene regA encodes a protein that diminishes the expression of many unlinked early T4 genes. Previous work demonstrated that regA-mediated repression occurs after transcription. We report here on the identification of the target site on one regA-sensitive mRNA, the message encoding the phage T4 rIIB protein. The target for regA-mediated action overlaps the translational initiation domain of the rIIB messenger. The regA protein may be a repressor that operates translationally on a significant and interesting set of early phage T4 mRNAs. Images PMID:7029523

  4. Comparison of the Dictyostelium rasD and ecmA genes reveals two distinct mechanisms whereby an mRNA may become enriched in prestalk cells.

    PubMed

    Jermyn, K; Wiliams, J

    1995-04-01

    The Dictyostelium ras gene, rasD, encodes an mRNA that is more abundant in prestalk than prespore cells in the migratory slug. Its expression is inducible by extracellular cAMP but is not inducible by the prestalk and stalk cell morphogen differentiation inducing factor (DIF). We show that a rasD-lacZ fusion gene is first expressed in approximately one half of the cells in the aggregate, including some cells that also express a prespore-specific marker. The amount of rasD-lacZ fusion protein in prespore cells then diminishes as the slug is formed. Analysis of a rasD-lacZ fusion protein with an N terminal substitution that reduces protein stability within the cell provides strong confirmatory evidence that the ras gene product becomes enriched in prestalk cells by selective repression of gene expression in prespore cells. In contrast, the DIF-inducible ecmA gene is expressed only in those cells that will become prestalk cells in the migratory slug. These results show that there are two different ways in which an mRNA may become enriched in prestalk cells and support the view that DIF is the inducer of prestalk cell differentiation.

  5. The Arabidopsis translatome cell-specific mRNA atlas: Mining suberin and cutin lipid monomer biosynthesis genes as an example for data application.

    PubMed

    Mustroph, Angelika; Bailey-Serres, Julia

    2010-03-01

    Plants consist of distinct cell types distinguished by position, morphological features and metabolic activities. We recently developed a method to extract cell-type specific mRNA populations by immunopurification of ribosome-associated mRNAs. Microarray profiles of 21 cell-specific mRNA populations from seedling roots and shoots comprise the Arabidopsis Translatome dataset. This gene expression atlas provides a new tool for the study of cell-specific processes. Here we provide an example of how genes involved in a pathway limited to one or few cell-types can be further characterized and new candidate genes can be predicted. Cells of the root endodermis produce suberin as an inner barrier between the cortex and stele, whereas the shoot epidermal cells form cutin as a barrier to the external environment. Both polymers consist of fatty acid derivates, and share biosynthetic origins. We use the Arabidopsis Translatome dataset to demonstrate the significant cell-specific expression patterns of genes involved in those biosynthetic processes and suggest new candidate genes in the biosynthesis of suberin and cutin.

  6. Importance of cis determinants and nitrogenase activity in regulated stability of the Klebsiella pneumoniae nitrogenase structural gene mRNA.

    PubMed

    Simon, H M; Gosink, M M; Roberts, G P

    1999-06-01

    The Klebsiella pneumoniae nitrogen fixation (nif) mRNAs are unusually stable, with half-lives of 20 to 30 min under conditions favorable to nitrogen fixation (limiting nitrogen, anaerobiosis, temperatures of 30 degrees C). Addition of O2 or fixed nitrogen or temperature increases to 37 degrees C or more result in the dramatic destabilization of the nif mRNAs, decreasing the half-lives by a factor of 3 to 5. A plasmid expression system, independent of nif transcriptional regulation, was used to define cis determinants required for the regulated stability of the 5.2-kb nifHDKTY mRNA and to test the model suggested by earlier work that NifA is required in trans to stabilize nif mRNA under nif-derepressing conditions. O2 regulation of nifHDKTY mRNA stability is impaired in a plasmid containing a deletion of a 499-bp region of nifH, indicating that a site(s) required for the O2-regulated stability of the mRNA is located within this region. The simple model suggested from earlier work that NifA is required for stabilizing nif mRNA under conditions favorable for nitrogen fixation was disproved, and in its place, a more complicated model involving the sensing of nitrogenase activity as a component of the system regulating mRNA stability is proposed. Analysis of nifY mutants and overexpression suggests a possible involvement of the protein in this sensing process.

  7. The mRNA expression of cortisol axis related genes differs in Atlantic cod (Gadus morhua) categorized as high or low responders.

    PubMed

    Hori, Tiago S; Rise, Matthew L; Johnson, Stewart C; Afonso, Luis O B; Gamperl, A Kurt

    2012-01-15

    Cortisol is a major stress hormone in fish and is known, under normal or stressful conditions, to affect several physiological processes including growth and immunity. Thus, efforts have been made for several cultured finfish species, including the Atlantic cod, to determine whether fish with a high or low cortisol response to stress can be identified and selected. However, we have a limited understanding of the mechanisms that determine these two phenotypes. Thus, we measured total and free plasma cortisol levels in high and low responding cod when subjected to a 30 s handling stress, and the mRNA expression of four key genes in the glucocorticoid (i.e. cortisol) stress axis both pre- and post-stress. The cortisol data is consistent with our previous findings for cod, with high responding (HR) fish having ∼3-fold higher total and free plasma cortisol levels when compared to low responding (LR) fish. Three of the transcripts studied encode key proteins involved in steroidogenesis (StAR, P450scc and 3βHSD), and the constitutive mRNA expression of all three genes was significantly higher (∼2-fold) in the head kidney of HR fish when compared to LR cod. The other gene of interest was the glucocorticoid receptor (GR). We partly cloned and characterized a cDNA from Atlantic cod likely to be this fish's ortholog of the teleost GR1, and showed that while there was no difference in hepatic constitutive GR mRNA expression between groups, HR fish had liver GR mRNA levels that were significantly (1.8-fold) higher at 3 h post-stress as compared to LR fish. Our results suggest that the different magnitude of cortisol response between LR and HR fish is at least partially determined by the capacity of the interrenal tissue to produce steroids.

  8. mRNA expression profile of multidrug-resistant genes in acute lymphoblastic leukemia of children, a prognostic value for ABCA3 and ABCA2.

    PubMed

    Rahgozar, Soheila; Moafi, Alireza; Abedi, Marjan; Entezar-E-Ghaem, Mansureh; Moshtaghian, Jamal; Ghaedi, Kamran; Esmaeili, Abolghasem; Montazeri, Fatemeh

    2014-01-01

    Multidrug resistance (MDR) is an important cause of treatment failure in acute lymphoblastic leukemia (ALL). The ABC family of membrane transporters is proposed, albeit with controversy, to be involved in this process. The present study aims to investigate the mRNA expression profile of several genes of this family, including ABCA2, ABCA3, ABCB1/MDR1, MRP1/ABCC1, MRP3/ABCC3, ABCG2/BCRP, and the intracellular transporter MVP/LRP, in childhood ALL, and to evaluate their association with response to therapy. Some genes in the present research are being studied for the first time in Iran. Using quantitative real-time PCR, we evaluated 27 children with ALL at diagnosis and 15 children with normal bone marrow. The status of response to therapy was assessed one year after the onset of therapy through investigating the IgH/TCRγ gene rearrangements. Our findings indicate a considerable and direct relationship between mRNA expression levels of ABCA2, ABCA3, MDR1, and MRP1 genes and positive minimal residual disease (MRD) measured after one year of treatment. Statistical analysis revealed that expression of these genes higher than the cutoff point will raise the risk of MRD by 15-, 6.25-, 12-, and 9-fold, respectively. No relationship was found between of MVP/LRP, MRP3 and ABCG2 genes expression and ALL prognoses. Considering the direct and significant relationship between the increased expression of ABCA2, ABCA3, MDR1, and MRP1 genes and positive risk of MRD in children with ALL, evaluating the expression profile of these genes on diagnosis may identify high risk individuals and help plan a more efficient treatment strategy.

  9. mRNA expression profile of multidrug-resistant genes in acute lymphoblastic leukemia of children, a prognostic value for ABCA3 and ABCA2

    PubMed Central

    Rahgozar, Soheila; Moafi, Alireza; Abedi, Marjan; Entezar-e-ghaem, Mansureh; Moshtaghian, Jamal; Ghaedi, Kamran; Esmaeili, Abolghasem; Montazeri, Fatemeh

    2014-01-01

    Multidrug resistance (MDR) is an important cause of treatment failure in acute lymphoblastic leukemia (ALL). The ABC family of membrane transporters is proposed, albeit with controversy, to be involved in this process. The present study aims to investigate the mRNA expression profile of several genes of this family, including ABCA2, ABCA3, ABCB1/MDR1, MRP1/ABCC1, MRP3/ABCC3, ABCG2/BCRP, and the intracellular transporter MVP/LRP, in childhood ALL, and to evaluate their association with response to therapy. Some genes in the present research are being studied for the first time in Iran. Using quantitative real-time PCR, we evaluated 27 children with ALL at diagnosis and 15 children with normal bone marrow. The status of response to therapy was assessed one year after the onset of therapy through investigating the IgH/TCRγ gene rearrangements. Our findings indicate a considerable and direct relationship between mRNA expression levels of ABCA2, ABCA3, MDR1, and MRP1 genes and positive minimal residual disease (MRD) measured after one year of treatment. Statistical analysis revealed that expression of these genes higher than the cutoff point will raise the risk of MRD by 15-, 6.25-, 12-, and 9-fold, respectively. No relationship was found between of MVP/LRP, MRP3 and ABCG2 genes expression and ALL prognoses. Considering the direct and significant relationship between the increased expression of ABCA2, ABCA3, MDR1, and MRP1 genes and positive risk of MRD in children with ALL, evaluating the expression profile of these genes on diagnosis may identify high risk individuals and help plan a more efficient treatment strategy. PMID:24145140

  10. In vivo mRNA profiling of uropathogenic Escherichia coli from diverse phylogroups reveals common and group-specific gene expression profiles.

    PubMed

    Bielecki, Piotr; Muthukumarasamy, Uthayakumar; Eckweiler, Denitsa; Bielecka, Agata; Pohl, Sarah; Schanz, Ansgar; Niemeyer, Ute; Oumeraci, Tonio; von Neuhoff, Nils; Ghigo, Jean-Marc; Häussler, Susanne

    2014-08-05

    mRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression of Escherichia coli pathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associated E. coli isolates to different phylogenetic groups. Whereas the in vivo gene expression profiles of the majority of genes were conserved among 21 E. coli strains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribed in vivo relative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease. Importance: Urinary tract infections (UTI) are very common; at least half of all women experience UTI, most of which are caused by pathogenic Escherichia coli strains. In this study, we applied massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and accurate insight into the nature and the dimension of the uropathogenic E. coli gene expression profile during an acute UTI within the human host. This work was undertaken to identify key players in physiological adaptation processes and, hence, potential targets for new infection prevention and therapy interventions specifically aimed at sabotaging bacterial adaptation to the human host.

  11. Response of detoxification gene mRNA expression and selection of molecular biomarkers in the clam Ruditapes philippinarum exposed to benzo[a]pyrene.

    PubMed

    Liu, Dong; Pan, Luqing; Cai, Yuefeng; Li, Zhen; Miao, Jingjing

    2014-06-01

    Benzo[a]pyrene (B[a]P) has a high carcinogenic potential. B[a]P concentrations and molecular biomarkers (mRNA expressions of Pgp, AhR, CYP4, CYP414A1, GST-pi, GST-S2, Cu/Zn-SOD and Mn-SOD) were assayed in gills and digestive glands of the clam Ruditapes philippinarum exposed to 0.03, 0.3 and 3 μg/L B[a]P for 21 days and then exposed to natural seawater for 15 days. Results showed that B[a]P was rapidly accumulated in and then eliminated from tissues of the clams. All gene mRNA expressions in the treated groups were induced significantly with the exception of CYP414A1 and Cu/Zn-SOD in the 0.03 μg/L B[a]P group. According to correlation analysis, mRNA expressions of AhR, GST-pi and Mn-SOD in gills and GST-pi in digestive glands had good correlations with B[a]P concentrations and could be used as molecular biomarkers of B[a]P exposure. This study investigated the molecular response of the genes mentioned above and selected useful molecular biomarkers for B[a]P pollution monitoring.

  12. Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain

    PubMed Central

    Chen, Li; Tao, Yu; Song, Fan; Yuan, Xi; Wang, Jian; Saffen, David

    2016-01-01

    RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders. PMID:26743651

  13. Differential regulation by MK801 of immediate-early genes, brain-derived neurotrophic factor and trk receptor mRNA induced by a kindling after-discharge.

    PubMed

    Hughes, P E; Young, D; Preston, K M; Yan, Q; Dragunow, M

    1998-01-01

    Transient changes in immediate-early genes and neurotrophin expression produced by kindling stimulation may mediate secondary downstream events involved in kindling development. Recent experiments have demonstrated conclusively that both kindling progression and mossy fibre sprouting are significantly impaired by administration of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801. To further examine the link between kindling, changes in gene expression and the NMDA receptor, we examined the effects of MK801 on neuronal induction of immediate-early genes, brain-derived neurotrophic factor (BDNF) and trk receptor mRNA expression produced by a single electrically induced hippocampal after-discharge in rats. The after-discharge produced a rapid (after 1 h) increase in Fos, Jun-B, c-Jun, Krox-24 mRNA and protein and Krox-20 protein in dentate granule neurons and a delayed, selective expression of Fos, Jun-D and Krox-24 in hilar interneurons. MK801 pretreatment produced a very strong inhibition of Fos, Jun-D and Krox-20 increases in dentate neurons but had a much smaller effect on Jun-B and c-Jun expression. MK801 did not inhibit Krox-24 expression in granule neurons or the delayed expression of Fos, Jun-D and Krox-24 in hilar interneurons. BDNF protein and trk B and trk C mRNA expression were also strongly induced in dentate granule cells 4 h following an after-discharge. MK801 abolished the increase in BDNF protein and trk B, but not trk C mRNA in granule cells at 4 h. These results demonstrate that MK801 differentially regulates the AD-increased expression of a group of genes previously identified as being likely candidates for an involvement in kindling. Because MK801 significantly retards the development of kindling and mossy fibre sprouting, it can be argued that those genes whose induction is not significantly attenuated by MK801 are unlikely to play an important role in the MK801-sensitive component of kindling and the changes in neural connectivity

  14. Expression of the cercosporin toxin resistance gene ( CRG1) as a dicistronic mRNA in the filamentous fungus Cercospora nicotianae.

    PubMed

    Chung, Kuang-Ren; Daub, Margaret E; Ehrenshaft, Marilyn

    2003-09-01

    The CRG1 gene in Cercospora nicotianae encodes a transcription factor and is required for cercosporin toxin resistance and production. Cloning and sequencing of the downstream region of the CRG1 gene led to the discovery of an adjacent gene ( PUT1) encoding a putative uracil transporter. Expression of CRG1 and PUT1 as assessed by Northern analysis indicated that, in addition to the expected monocistronic mRNAs (2.6 kb and 2.0 kb, respectively), a common 4.5-kb mRNA could be identified, using either a CRG1 or a PUT1 gene probe. The 2.6-kb transcript identified only by the CRG1 probe was expressed constitutively, whereas the 2.0-kb transcript identified only by the PUT1 probe was differentially expressed in various media. Four cDNA clones containing CRG1, PUT1, and the CRG1- PUT1 intergenic region were identified as part of the products from the 4.5-kb transcript. Both the 4.5-kb and 2.6-kb transcripts were not detectable in three crg1-disrupted mutants, using the CRG1 probe. The 2.0-kb transcript, but not the 4.5-kb one was detected using the PUT1 probe in the three crg1-disrupted mutants. Taken together, we conclude that the 4.5-kb transcript is a dicistronic mRNA of both CRG1 and PUT1 in the fungus C. nicotianae. This is the first example of a dicistronic mRNA identified in filamentous fungi.

  15. Dietary sunflower oil modulates milk fatty acid composition without major changes in adipose and mammary tissue fatty acid profile or related gene mRNA abundance in sheep.

    PubMed

    Castro-Carrera, T; Frutos, P; Leroux, C; Chilliard, Y; Hervás, G; Belenguer, A; Bernard, L; Toral, P G

    2015-04-01

    There are very few studies in ruminants characterizing mammary and adipose tissue (AT) expression of genes and gene networks for diets causing variations in milk fatty acid (FA) composition without altering milk fat secretion, and even less complementing this information with data on tissue FA profiles. This work was conducted in sheep in order to investigate the response of the mammary gland and the subcutaneous and perirenal AT, in terms of FA profile and mRNA abundance of genes involved in lipid metabolism, to a diet known to modify milk FA composition. Ten lactating Assaf ewes were randomly assigned to two treatments consisting of a total mixed ration based on alfalfa hay and a concentrate (60 : 40) supplemented with 0 (control diet) or 25 (SO diet) g of sunflower oil/kg of diet dry matter for 7 weeks. Milk composition, including FA profile, was analysed after 48 days on treatments. On day 49, the animals were euthanized and tissue samples were collected to analyse FA and mRNA abundance of 16 candidate genes. Feeding SO did not affect animal performance but modified milk FA composition. Major changes included decreases in the concentration of FA derived from de novo synthesis (e.g. 12:0, 14:0 and 16:0) and increases in that of long-chain FA (e.g. 18:0, c9-18:1, trans-18:1 isomers and c9,t11-CLA); however, they were not accompanied by significant variations in the mRNA abundance of the studied lipogenic genes (i.e. ACACA, FASN, LPL, CD36, FABP3, SCD1 and SCD5) and transcription factors (SREBF1 and PPARG), or in the constituent FA of mammary tissue. Regarding the FA composition of AT, the little influence of SO did not appear to be linked to changes in gene mRNA abundance (decreases of GPAM and SREBF1 in both tissues, and of PPARG in the subcutaneous depot). Similarly, the great variation between AT (higher contents of saturated FA and trans-18:1 isomers in the perirenal, and of cis-18:1, c9,t11-CLA and n-3 PUFA in the subcutaneous AT) could not be related to

  16. TATA boxes in gene transcription and poly (A) tails in mRNA stability: New perspective on the effects of berberine

    PubMed Central

    Yuan, Zhi-Yi; Lu, Xi; Lei, Fan; Chai, Yu-Shuang; Wang, Yu-Gang; Jiang, Jing-Fei; Feng, Tian-Shi; Wang, Xin-Pei; Yu, Xuan; Yan, Xiao-Jin; Xing, Dong-Ming; Du, Li-Jun

    2015-01-01

    Berberine (BBR) is a natural compound with variable pharmacological effects and a broad panel of target genes. We investigated berberine’s pharmacological activities from the perspective of its nucleotide-binding ability and discovered that BBR directly regulates gene expression by targeting TATA boxes in transcriptional regulatory regions as well as the poly adenine (poly (A)) tail at the mRNA terminus. BBR inhibits gene transcription by binding the TATA boxes in the transcriptional regulatory region, but it promotes higher levels of expression by targeting the poly (A) tails of mRNAs. The present study demonstrates that TATA boxes and poly (A) tails are the first and second primary targets by which BBR regulates gene expression. The final outcome of gene regulation by BBR depends on the structure of the individual gene. This is the first study to reveal that TATA boxes and poly (A) tails are direct targets for BBR in its regulation of gene expression. Our findings provide a novel explanation for the complex activities of a small molecule compound in a biological system and a novel horizon for small molecule-compound pharmacological studies. PMID:26671652

  17. Interaction of CCR4–NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis

    PubMed Central

    Yang, Cheng-Yuan; Ramamoorthy, Senthilkumar; Boller, Sören; Rosenbaum, Marc; Rodriguez Gil, Alfonso; Mittler, Gerhard; Imai, Yumiko; Kuba, Keiji; Grosschedl, Rudolf

    2016-01-01

    Transcription factor EBF1 (early B-cell factor 1) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. To identify proteins that regulate the diverse functions of EBF1, we used SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4–NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and we identified histidine 240 in EBF1 as a critical residue for this interaction. Complementation of Ebf1−/− progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but we also detected genes with altered occupancy, suggesting that the CCR4–NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which we found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4–NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation. PMID:27807034

  18. Interaction of CCR4-NOT with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis.

    PubMed

    Yang, Cheng-Yuan; Ramamoorthy, Senthilkumar; Boller, Sören; Rosenbaum, Marc; Rodriguez Gil, Alfonso; Mittler, Gerhard; Imai, Yumiko; Kuba, Keiji; Grosschedl, Rudolf

    2016-10-15

    Transcription factor EBF1 (early B-cell factor 1) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. To identify proteins that regulate the diverse functions of EBF1, we used SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4-NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and we identified histidine 240 in EBF1 as a critical residue for this interaction. Complementation of Ebf1(-/-) progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but we also detected genes with altered occupancy, suggesting that the CCR4-NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which we found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4-NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation.

  19. m6A-Driver: Identifying Context-Specific mRNA m6A Methylation-Driven Gene Interaction Networks

    PubMed Central

    Zhang, Song-Yao; Zhang, Shao-Wu; Liu, Lian; Huang, Yufei

    2016-01-01

    As the most prevalent mammalian mRNA epigenetic modification, N6-methyladenosine (m6A) has been shown to possess important post-transcriptional regulatory functions. However, the regulatory mechanisms and functional circuits of m6A are still largely elusive. To help unveil the regulatory circuitry mediated by mRNA m6A methylation, we develop here m6A-Driver, an algorithm for predicting m6A-driven genes and associated networks, whose functional interactions are likely to be actively modulated by m6A methylation under a specific condition. Specifically, m6A-Driver integrates the PPI network and the predicted differential m6A methylation sites from methylated RNA immunoprecipitation sequencing (MeRIP-Seq) data using a Random Walk with Restart (RWR) algorithm and then builds a consensus m6A-driven network of m6A-driven genes. To evaluate the performance, we applied m6A-Driver to build the context-specific m6A-driven networks for 4 known m6A (de)methylases, i.e., FTO, METTL3, METTL14 and WTAP. Our results suggest that m6A-Driver can robustly and efficiently identify m6A-driven genes that are functionally more enriched and associated with higher degree of differential expression than differential m6A methylated genes. Pathway analysis of the constructed context-specific m6A-driven gene networks further revealed the regulatory circuitry underlying the dynamic interplays between the methyltransferases and demethylase at the epitranscriptomic layer of gene regulation. PMID:28027310

  20. Genomic organisation of the mouse gene encoding endothelin-converting enzyme-1 (ECE-1) and mRNA expression of ECE-1 isoforms in murine tissues.

    PubMed

    Lindenau, Steffi; von Langsdorff, Christian; Saxena, Amit; Paul, Martin; Orzechowski, Hans-Dieter

    2006-05-24

    Mouse knockout-models have previously revealed important biological functions of endothelin-converting enzyme-1 (ECE-1) in normal cardiac and craniofacial development. Since human ECE-1 is expressed in various isoforms, termed a, b, c, and d, expression of which is controlled by alternative promoters, we postulated that corresponding isoforms may also be transcribed from the murine Ece1 gene. By comparative sequence analysis using exon-specific sequences of human and rat ECE-1 we have resolved the complete exon-intron structure of the murine Ece1 locus on chromosome 4. The murine Ece1 gene comprises 23 exons distributed over 100 kb of genomic DNA and was found to be structurally highly conserved when compared to the human ECE1 gene. As with the human gene, the exons containing isoform-specific sequences were localised in the 5' terminal region of the murine Ece1 gene. Using specific sense primers, isoform-specific expression of murine ECE-1 mRNA in various mouse tissues was confirmed by RT-PCR. Using real-time PCR we demonstrated that ECE-1c was the most abundantly expressed isoform in most tissues, except for heart and aorta displaying a more even isoform distribution. We detected an additional isoform-specific exon, designated c2, which was apparently constitutively spliced and expressed only as minor fraction of ECE-1c transcripts. Our results provide evidence of structural conservation of mammalian genes encoding ECE-1 and will facilitate a more refined analysis of ECE-1 mRNA expression in the mouse model organism.

  1. Non-Viral, Lipid-Mediated DNA and mRNA Gene Therapy of the Central Nervous System (CNS): Chemical-Based Transfection.

    PubMed

    Hecker, James G

    2016-01-01

    Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and

  2. Silent exonic mutations in the low-density lipoprotein receptor gene that cause familial hypercholesterolemia by affecting mRNA splicing.

    PubMed

    Defesche, J C; Schuurman, E J M; Klaaijsen, L N; Khoo, K L; Wiegman, A; Stalenhoef, A F H

    2008-06-01

    In a large group of patients with the clinical phenotype of familial hypercholesterolemia, such as elevated low-density lipoprotein (LDL) cholesterol and premature atherosclerosis, but without functional mutations in the genes coding for the LDL receptor and apolipoprotein B, we examined the effect of 128 seemingly neutral exonic and intronic DNA variants, discovered by routine sequencing of these genes. Two variants, G186G and R385R, were found to be associated with altered splicing. The nucleotide change leading to G186G resulted in the generation of new 3'-splice donor site in exon 4 and R385R was associated with a new 5'-splice acceptor site in exon 9 of the LDL receptor gene. Splicing of these alternate splice sites leads to an in-frame 75-base pair deletion in a stable mRNA of exon 4 in case of G186G and R385R resulted in a 31-base pair frame-shift deletion in exon 9 and non-sense-mediated mRNA decay.

  3. [Differential expression of genes related to photoperiod-temperature sensitive genic male sterility in wheat, revealed by mRNA differential display using G-box family primer].

    PubMed

    Cao, Shuang-He; Liu, Dong-Cheng; Liu, Li-Ke; Guo, Xiao-Li; Zhang, Ai-Min

    2003-01-01

    mRNA differential display with G-box family primer was used to analyze the differential expression of genes of the photoperiod-temperature sensitive genic male sterile(PTSGMS) line of wheat, BAU3338, between the sterile and fertile conditions. The result indicated that gene expression was significantly different between the two types of condition during the fertility transformation phase. The twelve qualitatively different DNA bands were identified with reverse Northern blot hybridization and five positive clones, HT1-G10, HT1-G3, HT2-G2, HT1-G4 and HT2-G5 were sequenced. The homology search indicated that HT1-G10 was highly homological (96%) to the partial sequences of Triticum aestivum chloroplast genes, rbcL and atpB, HT1-G3 was also homological (88%) to Triticum aestivum histone H2A gene and the other three gene fragments were new sequences in Gen-Bank. The analysis of the candidate gene fragments supplied some effective evidences to reveal the developmental mechanism of PTSGMS.

  4. Association analysis between SNPs in the 5'-flanking region of the chicken GRP78 gene, thermotolerance parameters, and tissue mRNA expression.

    PubMed

    Kong, L N; Zhang, D X; Ji, C L; Zhang, X Q; Luo, Q B

    2015-06-10

    Glucose-regulated protein 78 (GRP78) is a molecular chaperone in the endoplasmic reticulum and can be induced by different kinds of environmental and physiological stress. Thus far, the role of the GRP78 gene in thermotolerance in chickens has not been investigated. In the present study, we detected sequence variations in the 5ꞌ-flanking region of the GRP78 gene and evaluated several thermotolerance parameters, such as T3, corticosterone, H/L ratio, and levels of CD3(+), CD4(+), and CD8(+) T cells, to further determine its associations at 35° and 15°C. The sequencing results revealed 10 SNPs in the 5'-flanking region of the GRP78 gene, and seven mutations were chosen for further genotyping in a White Recessive Rock (WRR) chicken population. The SNP C.-744C>G in WRR chickens was significantly correlated with heat tolerance parameters under both conditions; it may therefore exert a potential hereditary effect on heat tolerance, and the genotype GG may be advantageous for thermotolerance. The heart, liver, brain, and leg muscle tissues of 8-day-old WRR chickens were sampled from heat stress groups, which were defined by exposure to 1, 2, 3, and 6 h of persistent thermal stress, and a control group, which was not exposed to thermal stress. Quantitative real-time polymerase chain reaction assay indicated that the mRNA expression level of the GRP78 gene increased gradually under heat stress, peaked at 3 h, and then decreased. We conclude that the mRNA expression of the GRP78 gene is time- and tissue-dependent.

  5. Correlation of the A-FABP Gene Polymorphism and mRNA Expression with Intramuscular Fat Content in Three-Yellow Chicken and Hetian-Black Chicken.

    PubMed

    Wang, Yong; Chen, Hongwei; Han, Diangang; Chen, Ying; Muhatai, Gemingguli; Kurban, Tursunjan; Xing, Jinming; He, Jianzhong

    2017-01-02

    The adipocyte-type fatty acid-binding protein (A-FABP) is considered a candidate gene for fat metabolism; thus, it affects fat deposition in chickens. The present study was designed to examine the polymorphism and mRNA abundance of the A-FABP gene with intramuscular fat (IMF) in the pectoralis muscles (PM) and leg muscles (LM) of Three-yellow Chicken (TYC) and Hetian-black Chicken (HTBC). In total, 60 TYCs and 60 HTBCs were sacrificed using exsanguination at market age. The IMF contents of the PM and LM in the HTBC were significantly higher than those in the TYC. Three genotypes of the A-FABP gene first exon, AA, AB, and BB, were examined by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and a C51 T mutational site, which is a silent substitution mutation, was revealed. The IMF contents of the AA genotype in the PM of the HTBC were significantly higher than those in the AB genotype; thus, the C51 T mutable site is a gene marker for selecting a higher IMF content in the PM of the HTBC. The relative expression of the A-FABP mRNA in the LM of the HTBC, which was measured by quantitative real-time PCR, was significantly higher than in the TYC. A significantly positive association was detected between A-FABP expression with the IMF contents of the PM and LM of both the TYC and the HTBC. These results provide basic data that might be helpful to further research the role of the A-FABP gene in fat deposition and fatty acid metabolism in chickens.

  6. The association of SNPs in Hsp90β gene 5' flanking region with thermo tolerance traits and tissue mRNA expression in two chicken breeds.

    PubMed

    Chen, Zhuo-Yu; Gan, Jian-Kang; Xiao, Xiong; Jiang, Li-Yan; Zhang, Xi-Quan; Luo, Qing-Bin

    2013-09-01

    Thermo stress induces heat shock proteins (HSPs) expression and HSP90 family is one of them that has been reported to involve in cellular protection against heat stress. But whether there is any association of genetic variation in the Hsp90β gene in chicken with thermo tolerance is still unknown. Direct sequencing was used to detect possible SNPs in Hsp90β gene 5' flanking region in 3 chicken breeds (n = 663). Six mutations, among which 2 SNPs were chosen and genotypes were analyzed with PCR-RFLP method, were found in Hsp90β gene in these 3 chicken breeds. Association analysis indicated that SNP of C.-141G>A in the 5' flanking region of the Hsp90β gene in chicken had some effect on thermo tolerance traits, which may be a potential molecular marker of thermo tolerance, and the genotype GG was the thermo tolerance genotype. Hsp90β gene mRNA expression in different tissues detected by quantitative real-time PCR assay were demonstrated to be tissue dependent, implying that different tissues have distinct sensibilities to thermo stress. Besides, it was shown time specific and varieties differences. The expression of Hsp90β mRNA in Lingshan chickens in some tissues including heart, liver, brain and spleen were significantly higher or lower than that of White Recessive Rock (WRR). In this study, we presume that these mutations could be used in marker assisted selection for anti-heat stress chickens in our breeding program, and WRR were vulnerable to tropical thermo stress whereas Lingshan chickens were well adapted.

  7. Effect of ration size on fillet fatty acid composition, phospholipid allostasis and mRNA expression patterns of lipid regulatory genes in gilthead sea bream (Sparus aurata).

    PubMed

    Benedito-Palos, Laura; Calduch-Giner, Josep A; Ballester-Lozano, Gabriel F; Pérez-Sánchez, Jaume

    2013-04-14

    The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA.

  8. Effects of Saturated Long-chain Fatty Acid on mRNA Expression of Genes Associated with Milk Fat and Protein Biosynthesis in Bovine Mammary Epithelial Cells.

    PubMed

    Qi, Lizhi; Yan, Sumei; Sheng, Ran; Zhao, Yanli; Guo, Xiaoyu

    2014-03-01

    This study was conducted to determine the effects of saturated long-chain fatty acids (LCFA) on cell proliferation and triacylglycerol (TAG) content, as well as mRNA expression of αs1-casein (CSN1S1) and genes associated with lipid and protein synthesis in bovine mammary epithelial cells (BMECs). Primary cells were isolated from the mammary glands of Holstein dairy cows, and were passaged twice. Then cells were cultured with different levels of palmitate or stearate (0, 200, 300, 400, 500, and 600 μM) for 48 h and fetal bovine serum in the culture solution was replaced with fatty acid-free BSA (1 g/L). The results showed that cell proliferation tended to be increased quadratically with increasing addition of stearate. Treatments with palmitate or stearate induced an increase in TAG contents at 0 to 600 μM in a concentration-dependent manner, and the addition of 600 μM was less effective in improving TAG accumulation. The expression of acetyl-coenzyme A carboxylase alpha, fatty acid synthase and fatty acid-binding protein 3 was inhibited when palmitate or stearate were added in culture medium, whereas cluster of differentiation 36 and CSN1S1 mRNA abundance was increased in a concentration-dependent manner. The mRNA expressions of peroxisome proliferator-activated receptor gamma, mammalian target of rapamycin and signal transducer and activator of transcription 5 with palmitate or stearate had no significant differences relative to the control. These results implied that certain concentrations of saturated LCFA could stimulate cell proliferation and the accumulation of TAG, whereas a reduction may occur with the addition of an overdose of saturated LCFA. Saturated LCFA could up-regulate CSN1S1 mRNA abundance, but further studies are necessary to elucidate the mechanism for regulating milk fat and protein synthesis.

  9. Modulation of DNA repair capacity and mRNA expression levels of XRCC1, hOGG1 and XPC genes in styrene-exposed workers

    SciTech Connect

    Hanova, Monika; Stetina, Rudolf; Vodickova, Ludmila; Vaclavikova, Radka; Hlavac, Pavel; Smerhovsky, Zdenek; Naccarati, Alessio; Polakova, Veronika; Soucek, Pavel; Kuricova, Miroslava; Manini, Paola; Kumar, Rajiv; Hemminki, Kari; Vodicka, Pavel

    2010-11-01

    Decreased levels of single-strand breaks in DNA (SSBs), reflecting DNA damage, have previously been observed with increased styrene exposure in contrast to a dose-dependent increase in the base-excision repair capacity. To clarify further the above aspects, we have investigated the associations between SSBs, micronuclei, DNA repair capacity and mRNA expression in XRCC1, hOGG1 and XPC genes on 71 styrene-exposed and 51 control individuals. Styrene concentrations at workplace and in blood characterized occupational exposure. The workers were divided into low (below 50 mg/m{sup 3}) and high (above 50 mg/m{sup 3}) styrene exposure groups. DNA damage and DNA repair capacity were analyzed in peripheral blood lymphocytes by Comet assay. The mRNA expression levels were determined by qPCR. A significant negative correlation was observed between SSBs and styrene concentration at workplace (R = - 0.38, p = 0.001); SSBs were also significantly higher in men (p = 0.001). The capacity to repair irradiation-induced DNA damage was the highest in the low exposure group (1.34 {+-} 1.00 SSB/10{sup 9} Da), followed by high exposure group (0.72 {+-} 0.81 SSB/10{sup 9} Da) and controls (0.65 {+-} 0.82 SSB/10{sup 9} Da). The mRNA expression levels of XRCC1, hOGG1 and XPC negatively correlated with styrene concentrations in blood and at workplace (p < 0.001) and positively with SSBs (p < 0.001). Micronuclei were not affected by styrene exposure, but were higher in older persons and in women (p < 0.001). In this study, we did not confirm previous findings on an increased DNA repair response to styrene-induced genotoxicity. However, negative correlations of SSBs and mRNA expression levels of XRCC1, hOGG1 and XPC with styrene exposure warrant further highly-targeted study.

  10. Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog

    PubMed Central

    Wolff, Stephanie E.; Veldhoen, Nik; Helbing, Caren C.; Ramirez, Claire A.; Malpas, Janae M.; Propper, Catherine R.

    2015-01-01

    Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring for endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10−9 M) or 4-tert-octylphenol (OP; 10−9 M, 10−8 M, and 10−7 M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10−7 M OP increased testicular transcript levels. Treatment with 10−9 and 10−8 M OP, but not 10−7 M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed

  11. Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog.

    PubMed

    Wolff, Stephanie E; Veldhoen, Nik; Helbing, Caren C; Ramirez, Claire A; Malpas, Janae M; Propper, Catherine R

    2015-07-15

    Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring of endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10(-9)M) or 4-tert-octylphenol (OP; 10(-9)M, 10(-8)M, and 10(-7)M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10(-7)M OP increased testicular transcript levels. Treatment with 10(-9) and 10(-8)M OP, but not 10(-7)M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed within

  12. Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes.

    PubMed

    Wang, Y-K; Li, X; Song, Z-Q; Yang, C-X

    2017-04-13

    To ensure accurate normalization and quantification of target RNA transcripts using reverse transcription quantitative polymerase chain reaction (RT-qPCR), most studies focus on the identification of stably expressed gene(s) as internal reference. However, RNA preparation methods could also be an important factor, especially for test samples of limited quantity (e.g. oocytes). In this study, we aimed to select appropriate reference gene(s), and evaluate the effect of RNA preparation methods on gene expression quantification in porcine oocytes and cumulus cells during in vitro maturation. Expression profiles of seven genes (GAPDH, 18S, YWHAG, BACT, RPL4, HPRT1 and PPIA) were examined, on RNA samples extracted from cumulus cells (RNeasy Kit) and oocytes (RNeasy Kit and Lysis Kit) during in vitro maturation, respectively. Interestingly, different RNA preparation methods were found to potentially affect the quantification of reference gene expression in pig oocytes cultured in vitro. After geNorm analyses, the most suitable genes for normalization were identified, GAPDH/18S for cumulus cells and YWHAG/BACT for oocytes, respectively. Thus, our results provide useful data and information on the selection of better reference genes and RNA preparation method for related functional studies.

  13. Zinc-bearing zeolite clinoptilolite improves tissue zinc accumulation in laying hens by enhancing zinc transporter gene mRNA abundance.

    PubMed

    Li, Linfeng; Li, Ping; Chen, Yueping; Wen, Chao; Zhuang, Su; Zhou, Yanmin

    2015-08-01

    A study was conducted to investigate effects of zinc-bearing zeolite clinoptilolite (ZnCP), as an alternative for zinc sulfate (ZnSO4), on laying performance, tissue Zn accumulation and Zn transporter genes expression in laying hens. Hy-Line Brown laying hens were allocated to three treatments, each of which had six replicates with 15 hens per replicate, receiving basal diet supplemented with ZnSO4 (control, 80 mg Zn/kg diet), 0.23% ZnCP (40.25 mg Zn/kg diet) and 0.46% ZnCP (80.50 mg Zn/kg diet) for 8 weeks, respectively. Compared with control, hens fed diet containing 0.23% ZnCP had similar Zn content in measured tissues (P > 0.05). A higher ZnCP inclusion (0.46%) enhanced Zn accumulation in liver (P < 0.05) and pancreas (P < 0.05). In addition, ZnCP inclusion increased blood iron (Fe) content (P < 0.05). ZnCP supplementation enhanced jejunal metallothionein-4 (MT-4) messenger RNA (mRNA) abundance (P < 0.05). ZnCP inclusion at a higher level (0.46%) increased mRNA expression of MT-4 in pancreas (P < 0.05) and zinc transporter-1 (ZnT-1) in jejunum (P < 0.05). The highest ZnT-2 mRNA abundance in jejunum was found in hens fed 0.23% ZnCP inclusion diet (P < 0.05). The results indicated that ZnCP reached a higher bioavailability as compared with ZnSO4 as evidenced by enhanced tissue Zn accumulation and Zn transporter genes expression.

  14. Familial retinoblastoma due to intronic LINE-1 insertion causes aberrant and noncanonical mRNA splicing of the RB1 gene.

    PubMed

    Rodríguez-Martín, Carlos; Cidre, Florencia; Fernández-Teijeiro, Ana; Gómez-Mariano, Gema; de la Vega, Leticia; Ramos, Patricia; Zaballos, Ángel; Monzón, Sara; Alonso, Javier

    2016-05-01

    Retinoblastoma (RB, MIM 180200) is the paradigm of hereditary cancer. Individuals harboring a constitutional mutation in one allele of the RB1 gene have a high predisposition to develop RB. Here, we present the first case of familial RB caused by a de novo insertion of a full-length long interspersed element-1 (LINE-1) into intron 14 of the RB1 gene that caused a highly heterogeneous splicing pattern of RB1 mRNA. LINE-1 insertion was inferred by mRNA studies and full-length sequenced by massive parallel sequencing. Some of the aberrant mRNAs were produced by noncanonical acceptor splice sites, a new finding that up to date has not been described to occur upon LINE-1 retrotransposition. Our results clearly show that RNA-based strategies have the potential to detect disease-causing transposon insertions. It also confirms that the incorporation of new genetic approaches, such as massive parallel sequencing, contributes to characterize at the sequence level these unique and exceptional genetic alterations.

  15. Effects of Dietary Selenium Against Lead Toxicity on mRNA Levels of 25 Selenoprotein Genes in the Cartilage Tissue of Broiler Chicken.

    PubMed

    Gao, H; Liu, C P; Song, S Q; Fu, J

    2016-07-01

    The interactions between the essential element selenium (Se) and the toxic element lead (Pb) have been reported extensively; however, little is known about the effect of Se on Pb toxicity and the expression pattern of selenoproteins in the cartilage of chicken. To investigate the effects of Se on Pb toxicity and the messenger RNA (mRNA) expressions of selenoproteins in cartilage tissue, an in vitro study was performed on 1-day-old broiler chickens (randomly allocated into four groups) with diet of different concentration of Se and Pb. After 90 days, the meniscus cartilage and sword cartilage tissue were examined for the mRNA levels of 25 selenoprotein genes. The results showed that Se and Pb influenced the expression of selenoprotein genes in the chicken cartilage tissue. In detail, Se could alleviate the downtrend of the expression of Gpx1, Gpx2, Gpx4, Txnrd2, Txnrd3, Dio1, Dio2, Seli, Selu, Sepx1, Selk, Selw, Selo, Selm, Sep15, Sepnn1, Sels, and Selt induced by Pb exposure in the meniscus cartilage. In the sword cartilage, Se alleviated the downtrend of the expression of Gpx2, Gpx3, Gpx4, Txnrd1, Txnrd2, Dio2, Dio3, Seli, Selh, SPS2, Sepx1, Selk, Selw, Selo, Selm, Sep15, Selpb, Sepn1, and Selt induced by Pb exposure. The present study provided some compensated data about the roles of Se against Pb toxicity in the regulation of selenoprotein expression.

  16. Resistance to antidepressant treatment is associated with polymorphisms in the leptin gene, decreased leptin mRNA expression, and decreased leptin serum levels.

    PubMed

    Kloiber, Stefan; Ripke, Stephan; Kohli, Martin A; Reppermund, Simone; Salyakina, Daria; Uher, Rudolf; McGuffin, Peter; Perlis, Roy H; Hamilton, Steven P; Pütz, Benno; Hennings, Johannes; Brückl, Tanja; Klengel, Torsten; Bettecken, Thomas; Ising, Marcus; Uhr, Manfred; Dose, Tatjana; Unschuld, Paul G; Zihl, Josef; Binder, Elisabeth; Müller-Myhsok, Bertram; Holsboer, Florian; Lucae, Susanne

    2013-07-01

    Leptin, a peptide hormone from adipose tissue and key player in weight regulation, has been suggested to be involved in sleep and cognition and to exert antidepressant-like effects, presumably via its action on the HPA-axis and hippocampal function. This led us to investigate whether genetic variants in the leptin gene, the level of leptin mRNA-expression and leptin serum concentrations are associated with response to antidepressant treatment. Our sample consisted of inpatients from the Munich Antidepressant Response Signature (MARS) project with weekly Hamilton Depression ratings, divided into two subsamples. In the exploratory sample (n=251) 17 single nucleotide polymorphisms (SNPs) covering the leptin gene region were genotyped. We found significant associations of several SNPs with impaired antidepressant treatment outcome and impaired cognitive performance after correction for multiple testing. The SNP (rs10487506) showing the highest association with treatment response (p=3.9×10(-5)) was analyzed in the replication sample (n=358) and the association could be verified (p=0.021) with response to tricyclic antidepressants. In an additional meta-analysis combining results from the MARS study with data from the Genome-based Therapeutic Drugs for Depression (GENDEP) and the Sequenced Treatment Alternatives to Relieve Depression (STAR(⁎)D) studies, nominal associations of several polymorphisms in the upstream vicinity of rs10487506 with treatment outcome were detected (p=0.001). In addition, we determined leptin mRNA expression in lymphocytes and leptin serum levels in subsamples of the MARS study. Unfavorable treatment outcome was accompanied with decreased leptin mRNA and leptin serum levels. Our results suggest an involvement of leptin in antidepressant action and cognitive function in depression with genetic polymorphisms in the leptin gene, decreased leptin gene expression and leptin deficiency in serum being risk factors for resistance to antidepressant

  17. Relative abundance of tissue mRNA and association of the single nucleotide polymorphism of the goat NGF gene with prolificacy.

    PubMed

    Naicy, Thomas; Venkatachalapathy, R Thirupathy; Aravindakshan, T V; Raghavan, K C; Mini, M; Shyama, K

    2016-10-01

    Nerve Growth Factor (NGF) promotes the development of pre-antral ovarian follicles through ovarian innervations and regulation of ovarian response to gonadotropins. The present study was conducted to study the tissue gene expression profile, to characterize the genetic variants, find associations of the NGF gene with prolificacy in the prolific Malabari and less prolific Attappady Black goats because NGF has an important role in reproduction by augmenting ovarian folliculogenesis. Relative abundance of NGF mRNA was greatest in reproductive tissues signifying its role in reproduction. The PCR-SSCP analysis of a 251bp fragment of Exon 3 of the NGF gene from the 277 goats revealed four diplotypes (EE, EF, FF and EG) with respective frequencies of 0.76, 0.22, 0.01 and 0.01. Sequencing of the representative samples revealed one synonymous and one novel non synonymous mutations (g.705G>A and g.715C>T). Statistical analysis indicated that the SNP g.705G>A was associated with litter size in Attappady Black goats (P<0.05) and a PCR-RFLP was designed using the restriction enzyme, BpiI, for rapid screening of the SNP. The results of the present study suggest that the NGF gene is a primary candidate gene affecting prolificacy in goats and may be used for Marker Assisted Selection (MAS) in goats, especially in lowly prolific Attappady Black goats.

  18. Effects of anti-IgM suppression on polyclonally activated murine B cells: analysis of immunoglobulin mRNA, gene specific nuclear factors and cell cycle distribution.

    PubMed Central

    Marcuzzi, A; Van Ness, B; Rouse, T; Lafrenz, D

    1989-01-01

    Polyclonal activation of murine B cells with bacterial lipopolysaccharide (LPS) and dextran sulfate (DxS) results in cell proliferation as well as increased immunoglobulin gene transcription and antibody secretion. When added to B cell cultures during mitogen activation, anti-mu antibody suppresses the rate of proliferation and immunoglobulin gene expression. Using this model system we show that co-cultures of B cells with LPS/DxS and anti-mu resulted in a decrease of both mu and kappa chain mRNA. Suppression did not prevent B cell entry into cycle nor a significant alteration in the distribution of cells in phases of cell cycle, although it did prolong the cycle transit time in a dose dependent fashion as determined by bromodeoxyuridine pulse labelling. Analysis of B cell specific nuclear binding factors, which previously have been shown to be important in regulating immunoglobulin gene transcription were examined. Results show that the kappa-specific enhancer binding activity of NF-kappa B was induced in activated as well as suppressed cultures. The lymphoid specific factor NF-A2, which recognizes the octamer sequence motif in the promoters of immunoglobulin genes, was induced by the polyclonal activation but was selectively lost in extracts from suppressed cells. Thus, specific regulation of the nuclear factor which plays a critical role in both heavy and light chain immunoglobulin gene expression may contribute to the transcriptional suppression observed in anti-mu treated B cells. Images PMID:2481271

  19. Gene microarray analysis of lncRNA and mRNA expression profiles in patients with hypopharyngeal squamous cell carcinoma

    PubMed Central

    Zhou, Jieyu; Li, Wenming; Jin, Tong; Xiang, Xuan; Li, Maocai; Wang, Juan; Li, Guojun; Pan, Xinliang; Lei, Dapeng

    2015-01-01

    Background: Studies have shown that long noncoding RNAs (lncRNAs) are involved in the development and progression of many types of cancer. However, the mechanisms by which lncRNAs influence development and progression of hypopharyngeal squamous cell carcinoma (HSCC) are unclear. Method: We investigated differences in lncRNA and mRNA expression profiles between 3 pairs of HSCC tissues and adjacent nontumor tissues by microarray analysis. Results: In HSCC tissues, 1299 lncRNAs were significantly upregulated (n=669) or downregulated (n=630) compared to levels in adjacent nontumor tissues. Moreover, 1432 mRNAs were significantly upregulated (n=684) or downregulated (n=748) in HSCC tissues. We randomly selected 2 differentially expressed lncRNAs (AB209630, AB019562) and 2 differentially expressed mRNAs (SPP1, TJP2) for confirmation of microarray results using qRT-PCR. The qRT-PCR results matched well with the microarray data. The differentially expressed lncRNAs and mRNAs were distributed on each of the chromosomes, including the X and Y chromosomes. Pathway analysis indicated that the biological functions of differentially expressed mRNAs were related to 48 cellular pathways that may be associated with HSCC development. GO analysis revealed that 593 mRNAs involved in biological processes, 50 mRNAs involved in cellular components, and 46 mRNAs involved in molecular functions were upregulated in the carcinomas; 280 mRNAs involved in biological processes, 58 mRNAs involved in cellular components, and 71 mRNAs involved in molecular functions were downregulated in the carcinomas. In addition, 8 enhancer-like lncRNAs and 21 intergenic lncRNAs with their adjacent mRNA pairs were identified as coregulated transcripts. Conclusion: These findings provide insight into the mechanisms underlying HSCC tumorigenesis and will facilitate identification of new therapeutic targets and diagnostic biomarkers for this disease. PMID:26131061

  20. Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene.

    PubMed Central

    Linton, J P; Yen, J Y; Selby, E; Chen, Z; Chinsky, J M; Liu, K; Kellems, R E; Crouse, G F

    1989-01-01

    The mouse dihydrofolate reductase gene (dhfr) is a housekeeping gene expressed under the control of a promoter region embedded in a CpG island--a region rich in unmethylated CpG dinucleotides. A divergent transcription unit exists immediately upstream of the dhfr gene which is coamplified with dhfr in some but not all methotrexate-resistant cell lines. We show that the promoter region for this gene pair consists of two bidirectional promoters, a major and minor promoter, which are situated within a 660-base-pair region upstream of the dhfr ATG translation initiation codon. The major promoter controls over 90% of dhfr transcription, while the minor promoter directs the transcription of the remaining dhfr mRNAs. The major promoter functions bidirectionally, transcribing a divergent 4.0-kilobase poly(A) mRNA (class A) in the direction opposite that of dhfr transcription. The predicted protein product of this mRNA is 105 kilodaltons. The minor promoter also functions bidirectionally, directing the transcription of at least two divergent RNAs (class B). These RNAs, present in quantities approximately 1/10 to 1/50 that of the class A mRNAs, are 4.4- and 1.6-kilobase poly(A) mRNAs. cDNAs representing both class A and class B mRNAs have been cloned from a mouse fibroblast cell line which has amplified the dhfr locus (3T3R500). DNA sequence analysis of these cDNAs reveals that the class A and class B mRNAs share, for the most part, the same exons. On the basis of S1 nuclease protection analysis of RNA preparations from several mouse tissues, both dhfr and divergent genes showed similar levels of expression but did show some specificity in start site utilization. Computer homology searches have revealed sequence similarity of the divergent transcripts with bacterial genes involved in DNA mismatch repair, and we therefore have named the divergently transcribed gene Rep-1. Images PMID:2674679

  1. Quantitative mRNA expression analysis of selected genes in patients with early-stage hypothyroidism induced by treatment with iodine-131.

    PubMed

    Guo, Kun; Gao, Rui; Yu, Yan; Zhang, Weixiao; Yang, Yuxuan; Yang, Aimin

    2015-11-01

    The present study aimed to investigate the molecular markers indicative of early-stage hypothyroidism induced by treatment with iodine-131, in order to assist in further investigations of radio iodine‑induced hypothyroidism. A total of 59 patients diagnosed with hyperthyroidism (male/female, 16/43; median age, 46.4 years) and 27 healthy subjects (male/female, 7/21; median age, 44.6 years) were included in the present study. All patients were treated with appropriate doses of iodine‑131 and, three months following treatment, the patients were subdivided into two groups: A group with early‑stage hypothyroidism symptoms, and a group with non‑early‑stage hypothyroidism, including euthyroid patients and patients remaining with hyperthyroidism. Tissue samples from the patients and healthy subjects were collected by fine needle biopsies, and the mRNA expression levels of B-cell lymphoma 2 (Bcl‑2), nuclear factor (NF)‑κB, Ku70, epidermal growth factor receptor (EGFR), early growth response 1 (Egr‑1), TP53 and ataxia telangiectasia mutated were analyzed using reverse transcription‑quantitative polymerase chain reaction prior to iodine‑131 treatment. The association of the variation of target genes with susceptibility to early‑stage hypothyroidism was analyzed. Compared with normal subjects, the mRNA expression levels of Ku70 (0.768, vs. 3.304, respectively; P<0.001) and EGFR (0.859, vs. 1.752, respectively; P<0.05) were significantly higher, whereas those of NF‑κB (0.884, vs. 0.578, respectively; P<0.05) and Bcl‑2 (1.235, vs. 0.834, respectively; P<0.05) were lower in the hyperthyroid patients. Following treatment with iodine‑131, 30 of the 59 (50.8%) patients with hyperthyroidism were diagnosed with early‑stage hypothyroidism, and in the early‑stage hypothyroidism group, the mRNA expression levels of Bcl‑2 were significantly decreased (P<0.05), whereas those of Egr‑1 (P<0.05) were significantly increased, compared with the non

  2. Cyp1B1 mRNA expression in correlation to cotinine levels with respect to the Cyp1B1 L432V gene polymorphism.

    PubMed

    Helmig, Simone; Seelinger, Jens Udo; Philipp-Gehlhaar, Monika; Döhrel, Juliane; Schneider, Joachim

    2010-12-01

    Cytochrome P450 1B1 (CYP1B1) is involved in the activation of a broad spectrum of procarcinogens. An association of the Cyp1B1 Leu432Val polymorphism with cancer as well as an impact on the enzyme activity has been described. To study gene-environmental interactions we investigated the quantitative Cyp1B1 mRNA expression in smokers (N = 102) and non-smokers (N = 192) with regards to the Cyp1B1 L432V gene polymorphism. Tobacco smoke exposure was assessed by serum cotinine levels. Genotypes were analysed by melting curve analysis and quantification of Cyp1B1 mRNA by real-time PCR. In comparing Cyp1B1 expression, significant differences between the two homozygote genotypes *1/*1 and *3/*3 (0.105 ± 0.019; n = 26 vs. 0.051 ± 0.017; n = 14; P = 0.039) and between the heterozygote genotype *1/*3 and *3/*3 (0.121 ± 0.029; n = 55 vs. 0.051 ± 0.017; n = 14; P = 0.039) of smokers were revealed. According to the serum cotinine levels, three subgroups (low; medium; high) were build. The group "high" (0.248 ± 0.089; n = 32) showed proportionally high Cyp1B1 mRNA expression compared to "medium" (0.101 ± 0.024; n = 33), "low" (0.086 ± 0.015; n = 32) and non-smokers (0.084 ± 0.007; n = 176). This result was reflected in the homozygote *1/*1 and the heterozygote *1/*3 genotypes. In contrast the homozygote *3/*3 genotype was missing the high Cyp1B1 mRNA expression in the cotinine subgroup "high". Our results suggest that genotypes carrying the C-allele (*1/*1 and *1/*3) at Cyp1B1 Leu432Val polymorphism show a higher response to environmental factors, such as tobacco smoke than homozygote *3/*3 genotypes.

  3. Sequence polymorphism of GroEL gene in natural population of Bacillus and Brevibacillus spp. that showed variation in thermal tolerance capacity and mRNA expression.

    PubMed

    Sen, R; Tripathy, S; Padhi, S K; Mohanty, S; Maiti, N K

    2014-10-01

    GroEL, a class I chaperonin, plays an important role in the thermal adaptation of the cell and helps to maintain the viability of the cell under heat shock condition. Function of groEL in vivo depends on the maintenance of proper structure of the protein which in turn depends on the nucleotide and amino acid sequence of the gene. In this study, we investigated the changes in nucleotide and amino acid sequences of the partial groEL gene that may affect the thermotolerance capacity as well as mRNA expression of bacterial isolates. Sequences among the same species having differences in the amino acid level were identified as different alleles. The effect of allelic variation on the groEL gene expression was analyzed by comparison and relative quantification in each allele under thermal shock condition by RT-PCR. Evaluation of K a/K s ratio among the strains of same species showed that the groEL gene of all the species had undergone similar functional constrain during evolution. The strains showing similar thermotolerance capacity was found to carry same allele of groEL gene. The isolates carrying allele having amino acid substitution inside the highly ATP/ADP or Mg(2+)-binding region could not tolerate thermal stress and showed lower expression of the groEL gene. Our results indicate that during evolution of these bacterial species the groEL gene has undergone the process of natural selection, and the isolates have evolved with the groEL allelic sequences that help them to withstand the thermal stress during their interaction with the environment.

  4. Detection of three nonsense mutations and one missense mutation in the interleukin-2 receptor [gamma] chain gene in SCIDX1 that differently affect the mRNA processing

    SciTech Connect

    Markiewicz, S.; Fischer, A.; Saint Basile, G. de ); Subtil, A.; Dautry-Varsat, A. )

    1994-05-01

    The interleukin-2 receptor [gamma] (IL-2R[gamma]) chain gene encodes a 64-kDa protein that not only composes the high-affinity form of the IL-2 binding receptor in association with the 2R [alpha] and [beta] chains, but also participates in at least the IL-4 and IL-7 receptor complexes. Mutations in this gene have recently been shown to cause X-linked severe combined immunodeficiency (SCIDX1). This disease of the immune system results from an early block of T lymphocyte and natural killer (NK) cell differentiation, which leads to a severe cellular and humoral immune defect that is lethal unless treated by bone marrow transplantation. Analysis of the IL-2R[gamma] gene in SCIDX1 patients has revealed the presence of heterogeneous mutations principally located in the extracellular domain of the molecule. We report here three intraexonic mutations and one deletion in the IL-2R[gamma] gene in four SCIDX1 patients. These mutations appear to differentially affect RNA processing, either by decreasing IL-2R[gamma] mRNA level or by the skipping of a constitutive exon. 16 refs., 1 fig.

  5. Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity.

    PubMed Central

    Jares, P.; Campo, E.; Pinyol, M.; Bosch, F.; Miquel, R.; Fernandez, P. L.; Sanchez-Beato, M.; Soler, F.; Perez-Losada, A.; Nayach, I.; Mallofré, C.; Piris, M. A.; Montserrat, E.; Cardesa, A.

    1996-01-01

    Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb. Images

  6. Association between mRNA expression of chemotherapy-related genes and clinicopathological features in colorectal cancer: A large-scale population analysis

    PubMed Central

    SHIMAMOTO, YUJI; NUKATSUKA, MAMORU; TAKECHI, TEIJI; FUKUSHIMA, MASAKAZU

    2016-01-01

    To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right- and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right-sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was

  7. One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator

    PubMed Central

    Di Martino, Maria Letizia; Romilly, Cédric; Colonna, Bianca

    2016-01-01

    ABSTRACT VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF30 (30 kDa), and the shorter VirF21 (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF30 and VirF21 and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF21 is also translated from a leaderless mRNA (llmRNA) whose 5′ end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF21. The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF30 is responsible for activation of the virulence system, VirF21 negatively autoregulates virF expression itself. Since VirF21 modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA. PMID:27834204

  8. Molecular cloning and mRNA expression of geraniol-inducible genes in cultured shoot primordia of Matricaria chamomilla.

    PubMed

    Ashida, Yoshiyuki; Nishimoto, Masaki; Matsushima, Akihito; Watanabe, Junko; Hirata, Toshifumi

    2002-11-01

    Genes for two geraniol-responsive factors, designated McEREBP1 and McWRKY1, from cultured shoot primordia of Matricaria chamomilla were cloned. The deduced amino acid sequences of these genes were highly similar to those of the family of ethylene-responsive element binding proteins and elicitor-induced DNA-binding proteins containing a WRKY domain, respectively. The levels of McEREBP1 and McWRKY1 mRNAs were maximum when measured 1 h after treatment of the cultured cells with geraniol.

  9. Asymptotic behavior of distributions of mRNA and protein levels in a model of stochastic gene expression

    NASA Astrophysics Data System (ADS)

    Bobrowski, Adam; Lipniacki, Tomasz; Pichór, Katarzyna; Rudnicki, Ryszard

    2007-09-01

    The paper is devoted to a stochastic process introduced in the recent paper by Lipniacki et al. [T. Lipniacki, P. Paszek, A. Marciniak-Czochra, A.RE Brasier, M. Kimmel, Transcriptional stochasticity in gene expression, JE Theor. Biol. 238 (2006) 348-367] in modelling gene expression in eukaryotes. Starting from the full generator of the process we show that its distributions satisfy a (Fokker-Planck-type) system of partial differential equations. Then, we construct a c0 Markov semigroup in L1 space corresponding to this system. The main result of the paper is asymptotic stability of the involved semigroup in the set of densities.

  10. Noncoding 3' sequences of the transferrin receptor gene are required for mRNA regulation by iron.

    PubMed Central

    Owen, D; Kühn, L C

    1987-01-01

    The cell-surface receptor for transferrin mediates cellular uptake of iron from serum. Transferrin receptor protein and mRNA levels are increased in cells treated with iron chelating agents, and are decreased by treatment with iron salts or hemin. Here we report that expression of human transferrin receptor cDNA constructions in stably transfected mouse fibroblasts is regulated both by the iron chelator, desferrioxamine, and by hemin. We found that sequences within the 3' noncoding region are required for the iron-dependent feed-back regulation of receptor expression, whereas the presence of the transferrin receptor promoter region is not necessary. Regulation by iron is observed when transcription is initiated at either the SV-40 early promoter or the transferrin receptor promoter, but deletion of a 2.3 kb fragment within the 2.6 kb 3' noncoding region of the cDNA abolishes regulation and increases the constitutive level of receptor expression. Furthermore, the 3' deletion does not affect the decrease in receptors which is observed in response to growth arrest, indicating that transferrin receptor expression is controlled by at least two distinct mechanisms. Images Fig. 3. Fig. 6. Fig. 8. PMID:3608980

  11. Trinucleotide Repeat Expansion in the Transcription Factor 4 (TCF4) Gene Leads to Widespread mRNA Splicing Changes in Fuchs' Endothelial Corneal Dystrophy

    PubMed Central

    Wieben, Eric D.; Aleff, Ross A.; Tang, Xiaojia; Butz, Malinda L.; Kalari, Krishna R.; Highsmith, Edward W.; Jen, Jin; Vasmatzis, George; Patel, Sanjay V.; Maguire, Leo J.; Baratz, Keith H.; Fautsch, Michael P.

    2017-01-01

    Purpose To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD). Methods Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system. Results Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins. Conclusions Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events. PMID:28118661

  12. Gene expression as a circular process: cross-talk between transcription and mRNA degradation in eukaryotes; International University of Andalusia (UNIA) Baeza, Spain.

    PubMed

    Collart, Martine A; Reese, Joseph C

    2014-01-01

    Studies on the regulation of gene expression in eukaryotes in the past 20 years have consistently revealed increasing levels of complexity. Thirty years ago it seemed that we had understood the basic principles of gene regulation in eukaryotes. It was thought that regulation of transcription was the first and most important stage at which gene expression was regulated, and transcriptional regulation was considered to be very simple, with DNA-binding activators and repressors talking to the basic transcription machinery. This simple model was overthrown when it became clear that other stages of gene expression are also highly regulated. More recently, other dogmas have started to collapse. In particular, the idea that a linkage between the different steps in gene expression is restricted to processes ongoing in the same compartment has fallen out of favor. It is now evident that functional and physical linkage occurs in eukaryotes. We know that factors contributing to transcription in the nucleus can be found in the cytoplasm, and that RNA binding proteins that contribute to RNA decay in the cytoplasm are present in the nucleus. However, shuttling of such factors between nucleus and cytoplasm has traditionally been thought to serve a simple regulatory purpose, for instance, to avoid untimely activation of a transcription factor in the nucleus. Alternatively, it was thought to be necessary to recruit RNA binding proteins to the relevant RNAs. The notion that is now emerging is that factors thought to have evolved to specialize in regulating a single step of gene regulation in one cellular compartment may be contributing to the regulation of mRNAs at multiple steps along the lifecycle of an mRNA.

  13. Calmodulin Gene Family in Potato: Developmental and Touch-Induced Expression of the mRNA Encoding a Novel Isoform

    NASA Technical Reports Server (NTRS)

    Takezawa, D.; Liu, Z. H.; An, G.; Poovaiah, B. W.

    1995-01-01

    Eight genomic clones of potato calmodulin (PCM1 to 8) were isolated and characterized. Sequence comparisons of different genes revealed that the deduced amino acid sequence of PCM1 had several unique substitutions, especially in the fourth Ca(2+)-binding area. The expression patterns of different genes were studied by northern analysis using the 3'-untranslated regions as probes. The expression of PCM1, 5, and 8 was highest in the stolon tip and it decreased during tuber development. The expression of PCM6 did not vary much in the tissues tested, except in the leaves, where the expression was lower; whereas, the expression of PCM4 was very low in all the tissues. The expression of PCM2 and PCM3 was not detected in any of the tissues tested. Among these genes, only PCM1 showed increased expression following touch stimulation. To study the regulation of PCM1, transgenic potato plants carrying the PCM1 promoter fused to the beta-glucuronidase (GUS) reporter gene were produced. GUS expression was found to be developmentally regulated and touch-responsive, indicating a positive correlation between the expression of PCM1 and GUS mRNAs. These results suggest that the 5'-flanking region of PCM1 controls developmental and touch-induced expression. X-Gluc staining patterns revealed that GUS localization is high in meristematic tissues such as the stem apex, stolon tip, and vascular regions.

  14. The arabidopsis polyamine transporter LHRI/AtPUT3 modulates heat responsive gene expression by regulating mRNA stability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyamines (PA) involve in the gene regulation by interacting with various anionic macromolecules such as DNA, RNA and proteins and modulating their structure and function. Previous studies have showed that changing in polyamine biosynthesis alters plant response to different abiotic stresses. Here,...

  15. mRNA abundance of genes involved in mammary lipogenesis during fish oil- or trans-10,cis-12 CLA-induced milk fat depression in dairy ewes.

    PubMed

    Toral, P G; Hervás, G; Belenguer, A; Carreño, D; Frutos, P

    2017-04-01

    Milk fat depression (MFD) caused by trans-10,cis-12 18:2 is known to be mediated in cows and ewes by downregulation of mammary lipogenic genes. However, transcriptional mechanisms underlying marine lipid-induced MFD have not been well defined yet and the few available studies in ovine are not consistent. This trial was conducted to directly compare changes in animal performance, milk fatty acid composition, and particularly mammary mRNA abundance of candidate lipogenic genes and transcription factors in response to the inclusion of fish oil or trans-10,cis-12 18:2 in the dairy sheep diet. To meet this objective, 12 lactating Assaf ewes (on average, 64 days in milk, producing 1.72 kg of milk/d with 5.17% of fat) were divided into 3 groups and offered a total mixed ration without supplementation (control) or supplemented with 2.4% dry matter of fish oil (FO treatment) or 1% dry matter of a commercial product rich in trans-10,cis-12 18:2 (CLA treatment) for 39 d. Measurements and samplings were conducted before starting the treatments and at the end of the trial. Milk samples were used for RNA extraction from somatic cells. Feed intake was not affected by lipid supplements, and as designed, reductions in milk fat concentration (-31%) were similar in the 2 treatments, although the unpredicted increase in milk production with FO counteracted the anticipated reduction in milk fat yield. Nevertheless, this did not preclude the detection of FO-induced decreases in the mRNA abundance of candidate lipogenic genes [e.g., acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid synthase (FASN), and lipin 1 (LPIN1)], thus supporting the hypothesis that transcriptional regulation would be a relevant component of this type of MFD in sheep. Expected CLA-induced downregulation of some genes, such as FASN or sterol regulatory element binding transcription factor 1 (SREBF1), could not be detected in our samples, which might be related, at least in part, to high inter

  16. Beyond the known functions of the CCR4-NOT complex in gene expression regulatory mechanisms: New structural insights to unravel CCR4-NOT mRNA processing machinery.

    PubMed

    Ukleja, Marta; Valpuesta, José María; Dziembowski, Andrzej; Cuellar, Jorge

    2016-10-01

    Large protein assemblies are usually the effectors of major cellular processes. The intricate cell homeostasis network is divided into numerous interconnected pathways, each controlled by a set of protein machines. One of these master regulators is the CCR4-NOT complex, which ultimately controls protein expression levels. This multisubunit complex assembles around a scaffold platform, which enables a wide variety of well-studied functions from mRNA synthesis to transcript decay, as well as other tasks still being identified. Solving the structure of the entire CCR4-NOT complex will help to define the distribution of its functions. The recently published three-dimensional reconstruction of the complex, in combination with the known crystal structures of some of the components, has begun to address this. Methodological improvements in structural biology, especially in cryoelectron microscopy, encourage further structural and protein-protein interaction studies, which will advance our comprehension of the gene expression machinery.

  17. Low-level laser irradiation alters mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts

    NASA Astrophysics Data System (ADS)

    Trajano, L. A. S. N.; Sergio, L. P. S.; Silva, C. L.; Carvalho, L.; Mencalha, A. L.; Stumbo, A. C.; Fonseca, A. S.

    2016-07-01

    Low-level lasers are used for the treatment of diseases in soft and bone tissues, but few data are available regarding their effects on genomic stability. In this study, we investigated mRNA expression from genes involved in DNA repair and genomic stabilization in myoblasts exposed to low-level infrared laser. C2C12 myoblast cultures in different fetal bovine serum concentrations were exposed to low-level infrared laser (10, 35 and 70 J cm-2), and collected for the evaluation of DNA repair gene expression. Laser exposure increased gene expression related to base excision repair (8-oxoguanine DNA glycosylase and apurinic/apyrimidinic endonuclease 1), nucleotide excision repair (excision repair cross-complementation group 1 and xeroderma pigmentosum C protein) and genomic stabilization (ATM serine/threonine kinase and tumor protein p53) in normal and low fetal bovine serum concentrations. Results suggest that genomic stability could be part of a biostimulation effect of low-level laser therapy in injured muscles.

  18. Polymorphisms of the Steroid Sulfatase [STS] Gene are Associated With Attention Deficit Hyperactivity Disorder and Influence Brain Tissue mRNA Expression

    PubMed Central

    Brookes, KJ; Hawi, Z; Park, J; Scott, S; Gill, M; Kent, L

    2010-01-01

    Previous studies in animals and humans have implicated the X-chromosome STS gene in the etiology of attentional difficulties and attention deficit hyperactivity disorder (ADHD). This family based association study has fine mapped a region of the STS gene across intron 1 and 2 previously associated with ADHD, in an extended sample of 450 ADHD probands and their parents. Significant association across this region is demonstrated individually with 7 of the 12 genotyped SNPs, as well as an allele specific haplotype of the 12 SNPs. The over transmitted risk allele of rs12861247 was also associated with reduced STS mRNA expression in normal human post-mortem frontal cortex brain tissue compared to the non-risk allele (P = 0.01). These results are consistent with the hypothesis arising from previous literature demonstrating that boys with deletions of the STS gene, and hence no STS protein are at a significantly increased risk of developing ADHD. Furthermore, this study has established the brain tissue transcript of STS, which except from adipose tissue, differs from that seen in all other tissues investigated. © 2010 Wiley-Liss, Inc. PMID:20862695

  19. Effect of insoluble fibre on intestinal morphology and mRNA expression pattern of inflammatory, cell cycle and growth marker genes in a piglet model.

    PubMed

    Schedle, Karl; Pfaffl, Michael W; Plitzner, Christian; Meyer, Heinrich H D; Windisch, Wilhelm

    2008-12-01

    The effects of insoluble dietary fibre differing in lignin content on intestinal morphology and mRNA expression was tested in an animal model of 48 weaned piglets. Engaged fibre sources were wheat bran (rich in cellulose and hemicellulose) and pollen from Chinese Masson pine (Pinus massoniana) (rich in lignin), respectively. The fibre sources were added to a basal diet as follows: no addition (control), 3.0% wheat bran, 1.27% pine pollen, and 2.55% pine pollen. The 12 animals of each feeding group were fed four experimental diets ad libitum for 37 days and were then slaughtered for retrieving tissue samples from stomach, jejunum, ileum, colon and mesenterial lymph nodes. Both fibre sources increased villus height of mucosa in jejunum (+10% on average) and ileum (+16% on average). Results of mRNA expression rates of inflammatory, cell cycle and growth marker genes (NFkappaB, TNFalpha, TGFbeta, Caspase3, CDK4, IGF1) were specific to fibre source and tissue: wheat bran induced an up-regulation of NFkappaB in stomach and jejunum, as well as TNFalpha and TGFbeta, and Caspase3 in jejunum. Pine pollen induced down regulation of NFkappaB, TNFalpha, TGFbeta, Caspase3, CDK4 and IGF1 in the colon as well as up-regulation of NFkappaB and TGFbeta in mesenterial lymph nodes. Finally, an overall data comparison based on a hierarchical cluster analysis showed a close relation between gene regulation in different gut sections and organs, as well as between small intestine morphology and zootechnical performance.

  20. In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity

    PubMed Central

    Mazzari, Andre L. D. A.; Milton, Flora; Frangos, Samantha; Carvalho, Ana C. B.; Silveira, Dâmaris; de Assis Rocha Neves, Francisco; Prieto, Jose M.

    2016-01-01

    Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum. PMID:27594838

  1. Zearalenone activates pregnane X receptor, constitutive androstane receptor and aryl hydrocarbon receptor and corresponding phase I target genes mRNA in primary cultures of human hepatocytes.

    PubMed

    Ayed-Boussema, Imen; Pascussi, Jean Marc; Maurel, Patrick; Bacha, Hassen; Hassen, Wafa

    2011-01-01

    The mycotoxin zearalenone (ZEN) is found worldwide as a contaminant in cereals and grains. ZEN subchronic and chronic toxicities are dominated by reproductive disorders in different mammalian species which have made ZEN established mammalian endocrine disrupter. Over the last 30 years of ZEN biotransformation study, the toxin was thought to undergo reductive metabolism only, with the generation in several species of α- and β-isomers of zearalenol. However, recent investigations have noticed that the mycoestrogen is prone to oxidative metabolism leading to hydroxylation of ZEN though the involvement of different cytochromes P450 (CYPs) isoforms. The aim of the present study was to further explore the effect of ZEN on regulation of some CYPs using primary cultures of human hepatocytes. For this aim, using real time RT-PCR, we monitored in a first time, the effect of ZEN on mRNA levels of pregnane X receptor (PXR), constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR), nuclear receptors known to be involved in the regulation of some CYPs. In a second time, we looked for ZEN effect on expression of PXR, CAR and AhR corresponding phase I target genes (CYP3A4, CYP3A5, CYP2B6, CYP2C9, CYP1A1 and CYP1A2). Finally, we realised the luciferase assay in HepG2 treated with the toxin and transiently transfected with p-CYP3A4-Luc in the presence of a hPXR vector or transfected with p-CYPA1-Luc.Our results clearly showed that ZEN activated human PXR, CAR and AhR mRNA levels in addition to some of their phase I target genes mainly CYP3A4, CYP2B6 and CYP1A1 and at lesser extent CYP3A5 and CYP2C9 at ZEN concentrations as low as 0.1 μM.

  2. Increased readthrough transcription across the simian virus 5 M-F gene junction leads to growth defects and a global inhibition of viral mRNA synthesis.

    PubMed

    Parks, G D; Ward, K R; Rassa, J C

    2001-03-01

    Recombinant simian virus 5 (rSV5) mutants containing substitutions in the M-F intergenic region were generated to determine the effect of increased readthrough transcription on the paramyxovirus growth cycle. We have previously shown, using an SV5 dicistronic minigenome, that replacement of the 22-base M-F intergenic region with a foreign sequence results in a template (Rep22) that directs very high levels of M-F readthrough transcription. An rSV5 containing the Rep22 substitution grew slower and to final titers that were 50- to 80-fold lower than those of wild-type (WT) rSV5. Cells infected with the Rep22 virus produced very low levels of monocistronic M and F mRNA, consistent with the M-F readthrough phenotype. Surprisingly, Rep22 virus-infected cells also displayed a global decrease in the accumulation of viral mRNA from genes located upstream and downstream of the M-F junction, and overall viral protein synthesis was reduced. Second-site revertants of the Rep22 virus that had regained WT transcription and growth properties contained a single base substitution that increased the M gene end U tract from four to eight residues, suggesting that the growth defects originated from higher-than-normal M-F readthrough transcription. Thus, the primary growth defect for the Rep22 virus appears to be in viral RNA synthesis and not in morphogenesis. A second rSV5 virus (G14), which contained a different foreign M-F intergenic sequence, grew to similar or slightly higher titers than WT rSV5 in some cell types and produced ~1.5- to 2-fold more mRNA and viral protein. The data support the hypothesis that inhibition of Rep22 virus growth is due to increased access by the polymerase to the 5' end of the genome and to the resulting overexpression of L protein. We propose that the elevated naturally occurring M-F readthrough which is characteristic of many paramyxoviruses serves as a mechanism to fine-tune the level of polymerase that is optimal for virus growth.

  3. Influence of functional polymorphisms in TNF-α, IL-8, and IL-10 cytokine genes on mRNA expression levels and risk of gastric cancer.

    PubMed

    de Oliveira, Juliana Garcia; Rossi, Ana Flávia Teixeira; Nizato, Daniela Manchini; Cadamuro, Aline Cristina Targa; Jorge, Yvana Cristina; Valsechi, Marina Curado; Venâncio, Larissa Paola Rodrigues; Rahal, Paula; Pavarino, Érika Cristina; Goloni-Bertollo, Eny Maria; Silva, Ana Elizabete

    2015-12-01

    Functional polymorphisms in promoter regions can produce changes in the affinity of transcription factors, thus altering the messenger ribonucleic acid (mRNA) expression levels of inflammatory cytokines associated with the risk of cancer development. The goal of this study was to evaluate the influence that polymorphisms in the cytokine genes known as TNF-α-308 G/A (rs1800629), TNF-α-857 C/T (rs1799724), IL-8-251 T/A (rs4073), IL-8-845 T/C (rs2227532), and IL-10-592 C/A (rs1800872) have on changes to mRNA expression levels and on the risks of chronic gastritis (CG) and gastric cancer (GC). A sample of 723 individuals was genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Relative mRNA expression levels were measured using quantitative real-time PCR (qPCR). Polymorphisms TNF-α-308 G/A and IL-8-251 A/T were not associated with risks of these gastric lesions. However, TNF-α-857 C/T, IL-8-845 T/C, and IL-10-592 C/A were found to be associated with a higher risk of GC, and IL-10-592 C/A was found to be associated with a higher risk of CG. The relative mRNA expression levels (RQ) of TNF-α, IL-8, and IL-10 were markedly downregulated in the CG group (median RQs = 0.128, 0.247, and 0.614, respectively), while the RQ levels of TNF-α in the GC group were upregulated (RQ = 2.749), but were basal for IL-8 (RQ = 1.053) and downregulated for IL-10 (RQ = 0.179). When the groups were stratified according to wild-type and polymorphic alleles, only for IL-8-845 T/C the polymorphic allele was found to influence the expression levels of this cytokine. IL-8-845 C allele carriers were significantly upregulated in both groups (GC and CG; RQ = 3.138 and 2.181, respectively) when compared to TT homozygotes (RQ = -0.407 and 0.165, respectively). In silico analysis in the IL-8 promoter region revealed that the presence of the variant C allele in position -845 is responsible for the presence of the binding

  4. LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

    PubMed

    Singh, Parmit Kumar; Plumb, Matthew R; Ferris, Andrea L; Iben, James R; Wu, Xiaolin; Fadel, Hind J; Luke, Brian T; Esnault, Caroline; Poeschla, Eric M; Hughes, Stephen H; Kvaratskhelia, Mamuka; Levin, Henry L

    2015-11-01

    The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

  5. The imprinted SNRPN gene is associated with a polycistronic mRNA and an imprinting control element

    SciTech Connect

    Saitoh, S.; Nicholls, R.D.; Seip, J.

    1994-09-01

    The small nuclear ribonucleoprotein-associated protein SmN (SNRPN) gene is located in the Prader-Willi syndrome (PWS) critical region in chromosome 15q11-q13. We have previously shown that it is functionally imprinted in humans, being only expressed from the paternal allele and differentially methylated on parental alleles. Therefore, SNRPN may have a role in PWS, although genetic studies suggest that at least two genes may be necessary for the classical PWS phenotype. We have characterized the SNRPN genomic structure, and shown that it comprises ten exons. Surprisingly, we identified an open reading frame (ORF) in the first three exons, 190-bp 5{prime} to the SmN ORF. Notably, the majority of base substitutions bewteen human and rodents in the upstream ORF occurred in the wobble position of codons, suggesting selection for a protein coding function. This ORF, which we name SNURF (SNRPN upstream reading frame) encodes a putative polypeptide of 71 amino acids. By analogy to prokaryotic operons that encode proteins with related functions, it is possible that SNURF may have a role in pre-mRNA splicing.

  6. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications.

    PubMed

    Curran, Kathleen A; Karim, Ashty S; Gupta, Akash; Alper, Hal S

    2013-09-01

    Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering. While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of terminators in yeast. In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and determine mRNA half-life changes to be the major cause of the varied protein and transcript expression level. We demonstrate that the difference in transcript level can be over 6.5-fold even for high strength promoters. The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between an expression-enhancing terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline. This is the first time that terminators have been investigated in the context of multiple promoters spanning orders of magnitude in activity. Finally, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept. Through pairing an expression-enhancing terminator with a low-expression promoter, we were able to achieve the same phenotypic result as with a promoter considerably higher in strength. Moreover, we can further boost the phenotype of the high-strength promoter by pairing it with an expression-enhancing terminator. This work highlights how terminator elements can be used to control metabolic pathways in the same way that promoters are traditionally used in yeast. Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering for yeast in the future.

  7. Deep mRNA Sequencing of the Tritonia diomedea Brain Transcriptome Provides Access to Gene Homologues for Neuronal Excitability, Synaptic Transmission and Peptidergic Signalling

    PubMed Central

    Senatore, Adriano; Edirisinghe, Neranjan; Katz, Paul S.

    2015-01-01

    Background The sea slug Tritonia diomedea (Mollusca, Gastropoda, Nudibranchia), has a simple and highly accessible nervous system, making it useful for studying neuronal and synaptic mechanisms underlying behavior. Although many important contributions have been made using Tritonia, until now, a lack of genetic information has impeded exploration at the molecular level. Results We performed Illumina sequencing of central nervous system mRNAs from Tritonia, generating 133.1 million 100 base pair, paired-end reads. De novo reconstruction of the RNA-Seq data yielded a total of 185,546 contigs, which partitioned into 123,154 non-redundant gene clusters (unigenes). BLAST comparison with RefSeq and Swiss-Prot protein databases, as well as mRNA data from other invertebrates (gastropod molluscs: Aplysia californica, Lymnaea stagnalis and Biomphalaria glabrata; cnidarian: Nematostella vectensis) revealed that up to 76,292 unigenes in the Tritonia transcriptome have putative homologues in other databases, 18,246 of which are below a more stringent E-value cut-off of 1x10-6. In silico prediction of secreted proteins from the Tritonia transcriptome shotgun assembly (TSA) produced a database of 579 unique sequences of secreted proteins, which also exhibited markedly higher expression levels compared to other genes in the TSA. Conclusions Our efforts greatly expand the availability of gene sequences available for Tritonia diomedea. We were able to extract full length protein sequences for most queried genes, including those involved in electrical excitability, synaptic vesicle release and neurotransmission, thus confirming that the transcriptome will serve as a useful tool for probing the molecular correlates of behavior in this species. We also generated a neurosecretome database that will serve as a useful tool for probing peptidergic signalling systems in the Tritonia brain. PMID:25719197

  8. Effect of leptin during in vitro maturation of prepubertal calf oocytes: embryonic development and relative mRNA abundances of genes involved in apoptosis and oocyte competence.

    PubMed

    Córdova, Bladimir; Morató, Roser; de Frutos, Celia; Bermejo-Álvarez, Pablo; Paramio, Teresa; Gutiérrez-Adán, Alfonso; Mogas, Teresa

    2011-12-01

    During the in vitro maturation of adult bovine oocytes, leptin has beneficial effects on blastocyst development, apoptosis and transcription levels of developmentally important genes. The present study analyzes the differential effects of leptin on prepubertal bovine oocytes and cumulus cells. Effects were determined of leptin treatment during oocyte maturation on their developmental capacity after fertilization (Exp. 1), incidence of apoptosis in cumulus oocyte complexes (COCs) (Exp. 2) or on relative mRNA abundances of genes in cumulus cells and oocytes (Exp. 3). COCs were matured in serum-free medium containing 1 mg/mL polyvinyl alcohol and 0, 10, 100, or 1000 ng/mL leptin (L0, L10, L100, and L1000, respectively), or in medium supplemented with 10% fetal calf serum (FCS) as a positive control. Addition of leptin during oocyte maturation had no effect on cleavage rates after fertilization (FCS, 68.6%; L0, 62.9%; L10, 66.9%; L100, 63.4%; L1000, 60.9%). Similarly, no significant differences in blastocyst rates were observed when oocytes were matured in the presence of L0 (8.4%), L10 (9.3%), L100 (6.7%), L1000 (8.2%), compared to control FCS (9.4%). In Experiment 2, maturation in the presence of 1000 ng/mL of leptin increased the proportion of TUNEL-positive cumulus cell (6.9%) with respect to those matured in the presence of FCS (4.96%), but not at the lower leptin doses. When relative mRNA abundances were examined for seven genes by qRT-PCR, five (TP53, BAX, DNMT3A, PGTS2 and LEPR) showed differences among groups. LEPR expression was significantly higher in the oocytes matured with FCS compared with the other groups and in those matured with PVA (L0) without leptin compared with the three groups of oocytes matured in the presence of leptin. In conclusion, the addition of leptin to the in vitro maturation medium used for prepubertal bovine oocytes does not increase the development potential of the oocytes or reduce the percentage of apoptosis in cumulus cells

  9. Expression of homothorax and extradenticle mRNA in the legs of the crustacean Parhyale hawaiensis: evidence for a reversal of gene expression regulation in the pancrustacean lineage.

    PubMed

    Prpic, Nikola-Michael; Telford, Maximilian J

    2008-06-01

    In Drosophila leg development, the extradenticle (exd) gene is expressed ubiquitously and its co-factor homothorax (hth) is restricted to the proximal leg portion. This condition is conserved in other insect species but is reversed in chelicerates and myriapods. As the region of co-expression does not differ in the two groups and transcripts from both are necessary for function, this difference in expression is likely to be functionally neutral. Here, we report the expression patterns of exd and hth in a crustacean, the amphipod shrimp Parhyale hawaiensis. The patterns in P. hawaiensis are similar to the insect patterns, supporting the close relationship between crustaceans and insects in the taxon Tetraconata. However, mRNA expression of exd in P. hawaiensis is weak in the distal leg parts, thus being intermediate between the complete lack of distal exd expression in chelicerates and myriapods and the strong distal exd expression in insects. Our data suggest that the reversal of the gene expression regulation of hth and exd occurred in the pancrustacean lineage.

  10. Generation of Interleukin-2 Receptor Gamma Gene Knockout Pigs from Somatic Cells Genetically Modified by Zinc Finger Nuclease-Encoding mRNA

    PubMed Central

    Watanabe, Masahito; Nakano, Kazuaki; Matsunari, Hitomi; Matsuda, Taisuke; Maehara, Miki; Kanai, Takahiro; Kobayashi, Mirina; Matsumura, Yukina; Sakai, Rieko; Kuramoto, Momoko; Hayashida, Gota; Asano, Yoshinori; Takayanagi, Shuko; Arai, Yoshikazu; Umeyama, Kazuhiro; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

    2013-01-01

    Zinc finger nuclease (ZFN) is a powerful tool for genome editing. ZFN-encoding plasmid DNA expression systems have been recently employed for the generation of gene knockout (KO) pigs, although one major limitation of this technology is the use of potentially harmful genome-integrating plasmid DNAs. Here we describe a simple, non-integrating strategy for generating KO pigs using ZFN-encoding mRNA. The interleukin-2 receptor gamma (IL2RG) gene was knocked out in porcine fetal fibroblasts using ZFN-encoding mRNAs, and IL2RG KO pigs were subsequently generated using these KO cells through somatic cell nuclear transfer (SCNT). The resulting IL2RG KO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patients. Our findings demonstrate that the combination of ZFN-encoding mRNAs and SCNT provides a simple robust method for producing KO pigs without genomic integration. PMID:24130776

  11. Altered mRNA Splicing, Chondrocyte Gene Expression and Abnormal Skeletal Development due to SF3B4 Mutations in Rodriguez Acrofacial Dysostosis

    PubMed Central

    Nevarez, Lisette; Pogue, Robert; Krakow, Deborah; Cohn, Daniel H.

    2016-01-01

    The acrofacial dysostoses (AFD) are a genetically heterogeneous group of inherited disorders with craniofacial and limb abnormalities. Rodriguez syndrome is a severe, usually perinatal lethal AFD, characterized by severe retrognathia, oligodactyly and lower limb abnormalities. Rodriguez syndrome has been proposed to be a severe form of Nager syndrome, a non-lethal AFD that results from mutations in SF3B4, a component of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Furthermore, a case with a phenotype intermediate between Rodriguez and Nager syndromes has been shown to have an SF3B4 mutation. We identified heterozygosity for SF3B4 mutations in Rodriguez syndrome, confirming that the phenotype is a dominant disorder that is allelic with Nager syndrome. The mutations led to reduced SF3B4 synthesis and defects in mRNA splicing, primarily exon skipping. The mutations also led to reduced expression in growth plate chondrocytes of target genes, including the DLX5, DLX6, SOX9, and SOX6 transcription factor genes, which are known to be important for skeletal development. These data provide mechanistic insight toward understanding how SF3B4 mutations lead to the skeletal abnormalities observed in the acrofacial dysostoses. PMID:27622494

  12. Concordant Association of Insulin Degrading Enzyme Gene (IDE) Variants with IDE mRNA, Aß, and Alzheimer's Disease

    PubMed Central

    Ertekin-Taner, Nilufer; Ansari, Morad; Wilcox, Samantha L.; Kashino, Mariah R.; Ma, Li; Younkin, Linda H.; Younkin, Samuel G.; Younkin, Curtis S.; Dincman, Toros A.; Howard, Melissa E.; Howell, Chanley C.; Stanton, Chloe M.; Watson, Christopher M.; Crump, Michael; Vitart, Veronique; Hayward, Caroline; Hastie, Nicholas D.; Rudan, Igor; Campbell, Harry; Polasek, Ozren; Brown, Kristelle; Passmore, Peter; Craig, David; McGuinness, Bernadette; Todd, Stephen; Kehoe, Patrick G.; Mann, David M.; Smith, A. David; Beaumont, Helen; Warden, Donald; Holmes, Clive; Heun, Reinhard; Kölsch, Heike; Kalsheker, Noor; Pankratz, V. Shane; Dickson, Dennis W.; Graff-Radford, Neill R.; Petersen, Ronald C.; Wright, Alan F.; Younkin, Steven G.; Morgan, Kevin

    2010-01-01

    Background The insulin-degrading enzyme gene (IDE) is a strong functional and positional candidate for late onset Alzheimer's disease (LOAD). Methodology/Principal Findings We examined conserved regions of IDE and its 10 kb flanks in 269 AD cases and 252 controls thereby identifying 17 putative functional polymorphisms. These variants formed eleven haplotypes that were tagged with ten variants. Four of these showed significant association with IDE transcript levels in samples from 194 LOAD cerebella. The strongest, rs6583817, which has not previously been reported, showed unequivocal association (p = 1.5×10−8, fold-increase = 2.12,); the eleven haplotypes were also significantly associated with transcript levels (global p = 0.003). Using an in vitro dual luciferase reporter assay, we found that rs6583817 increases reporter gene expression in Be(2)-C (p = 0.006) and HepG2 (p = 0.02) cell lines. Furthermore, using data from a recent genome-wide association study of two Croatian isolated populations (n = 1,879), we identified a proxy for rs6583817 that associated significantly with decreased plasma Aβ40 levels (ß = −0.124, p = 0.011) and total measured plasma Aβ levels (b = −0.130, p = 0.009). Finally, rs6583817 was associated with decreased risk of LOAD in 3,891 AD cases and 3,605 controls. (OR = 0.87, p = 0.03), and the eleven IDE haplotypes (global p = 0.02) also showed significant association. Conclusions Thus, a previously unreported variant unequivocally associated with increased IDE expression was also associated with reduced plasma Aß40 and decreased LOAD susceptibility. Genetic association between LOAD and IDE has been difficult to replicate. Our findings suggest that targeted testing of expression SNPs (eSNPs) strongly associated with altered transcript levels in autopsy brain samples may be a powerful way to identify genetic associations with LOAD that would otherwise be difficult to detect

  13. Case-control study and mRNA expression analysis reveal the MyD88 gene is associated with digestive disorders in rabbit.

    PubMed

    Chen, Shi-Yi; Zhang, Wen-Xiu; Zhang, Gong-Wei; Peng, Jin; Zhao, Xiao-Bing; Lai, Song-Jia

    2013-12-01

    As in humans, significant associations between Toll-like receptor 4 (TLR4) and digestive disorders have been identified in rabbit and dog. However, as an essential adaptor downstream of TLR4, the genetic variation of myeloid differentiating factor 88 (MyD88) and its association with digestive disorders have remained unknown. In this study, we detected 10 single nucleotide polymorphisms (SNPs) in the entire genomic region of rabbit MyD88. The genetic variation in susceptibility to digestive disorders for the only coding SNP (synonymous c.699T>C) was studied in Yaan (183 cases and 142 controls) and Chengdu populations (145 cases and 140 controls). A case-control association study revealed that individuals with the C allele had significant protection against digestive disorders in the Yaan population (OR = 0.71; 95% CI, 0.51-0.99; P < 0.05), the Chengdu population (OR = 0.55; 95% CI, 0.39-0.78; P < 0.01) and for joint analysis (OR = 0.62; 95% CI, 0.49-0.79; P < 0.01). We also experimentally induced digestive disorders by feeding a fiber-deficient diet and found that increased susceptibility was significantly associated with higher MyD88 mRNA expression (P < 0.05). The lowest MyD88 mRNA expression was observed in individuals carrying the protective CC genotype. These results suggest that MyD88 is one of the most plausible candidate genes in relation to digestive disorders in rabbit. Further studies are required to explore the biological implications of MyD88 in the pathogenesis of digestive disorders.

  14. Changes of Antioxidant Function and the mRNA Expression Levels of Apoptosis Genes in Duck Ovaries Caused by Molybdenum or/and Cadmium.

    PubMed

    Cao, Huabin; Xia, Bing; Zhang, Mengmeng; Liao, Yilin; Yang, Zhi; Hu, Guoliang; Zhang, Caiying

    2016-06-01

    To investigate the effects of molybdenum (Mo) combined with cadmium (Cd) on the antioxidant function and the mRNA expression levels of apoptosis-related genes in duck ovaries, 60 healthy 11-old-day female ducks were treated with hexaammonium molybdate ([(NH4)6Mo7O24·4H2O]) or/and cadmium sulfate (3CdSO4·8H2O) at different doses on a daily basis for 120 days. On the 120th day, ten female birds in each group were euthanized, and the ovaries and blood were collected to determine the antioxidant indexes and the mRNA expression levels of Bak-1, Bcl-2, and caspase-3 in ovaries. In addition, ovary tissues were subjected to histopathological analysis with optical microscope. The total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity decreased significantly (P < 0.01) in treated groups comparing with control while the nitric oxide synthase (NOS) activity increased (P < 0.01) both in ovary tissue and serum. The Bak-1 and caspase-3 expressions were upregulated while the Bcl-2 was downgraded by Mo or/and Cd. Biomolecules were affected in all metal-treated groups, whereas combined-treated animals showed greater effects. What is more, pathological damage in Mo and Cd combination treated groups was more severe. The results from the present study indicated that Mo or/and Cd caused oxidative stress and apoptosis in duck ovaries. Combination of Mo and Cd showed additive or synergistic effect leading to apoptosis and oxidative stress, and the pathway might be the mitochondrial pathway.

  15. Silencing of lipid metabolism genes through IRE1α-mediated mRNA decay lowers plasma lipids in mice.

    PubMed

    So, Jae-Seon; Hur, Kyu Yeon; Tarrio, Margarite; Ruda, Vera; Frank-Kamenetsky, Maria; Fitzgerald, Kevin; Koteliansky, Victor; Lichtman, Andrew H; Iwawaki, Takao; Glimcher, Laurie H; Lee, Ann-Hwee

    2012-10-03

    XBP1 is a key regulator of the unfolded protein response (UPR), which is involved in a wide range of physiological and pathological processes. XBP1 ablation in liver causes profound hypolipidemia in mice, highlighting its critical role in lipid metabolism. XBP1 deficiency triggers feedback activation of its upstream enzyme IRE1α, instigating regulated IRE1-dependent decay (RIDD) of cytosolic mRNAs. Here, we identify RIDD as a crucial control mechanism of lipid homeostasis. Suppression of RIDD by RNA interference or genetic ablation of IRE1α reversed hypolipidemia in XBP1-deficient mice. Comprehensive microarray analysis of XBP1 and/or IRE1α-deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1α. Ablation of XBP1 ameliorated hepatosteatosis, liver damage, and hypercholesterolemia in dyslipidemic animal models, suggesting that direct targeting of either IRE1α or XBP1 might be a feasible strategy to treat dyslipidemias.

  16. A Genome-Wide mRNA Screen and Functional Analysis Reveal FOXO3 as a Candidate Gene for Chicken Growth.

    PubMed

    Chen, Biao; Xu, Jiguo; He, Xiaomei; Xu, Haiping; Li, Guihuan; Du, Hongli; Nie, Qinghua; Zhang, Xiquan

    2015-01-01

    Chicken growth performance provides direct economic benefits to the poultry industry. However, the underlying genetic mechanisms are unclear. The objective of this study was to identify candidate genes associated with chicken growth and investigate their potential mechanisms. We used RNA-Seq to study the breast muscle transcriptome in high and low tails of Recessive White Rock (WRRh, WRRl) and Xinghua chickens (XHh, XHl). A total of 60, 23, 153 and 359 differentially expressed genes were detected in WRRh vs. WRRl, XHh vs. XHl, WRRh vs. XHh and WRRl vs. XHl, respectively. GO, KEGG pathway and gene network analyses showed that CEBPB, FBXO32, FOXO3 and MYOD1 played key roles in growth. The functions of FBXO32 and FOXO3 were validated. FBXO32 was predominantly expressed in leg muscle, heart and breast muscle. After decreased FBXO32 expression, growth-related genes such as PDK4, IGF2R and IGF2BP3 were significantly down-regulated (P < 0.05). FBXO32 was significantly (P < 0.05) associated with carcass and meat quality traits, but not growth traits. FOXO3 was predominantly expressed in breast and leg muscle. In both of these tissues, the FOXO3 mRNA level in XH was significantly higher than that in WRR chickens with normal body weight (P < 0.05). In DF-1 cells, siRNA knockdown of FOXO3 significantly (P < 0.01) inhibited the MYOD expression and significantly up-regulated (P < 0.01 or P < 0.05) the expression of growth-related genes including CEBPB, FBXO32, GH, GHR, IGF1R, IGF2R, IGF2BP1, IGF2BP3, INSR, PDK1 and PDK4. Moreover, 18 SNPs were identified in FOXO3. G66716193A was significantly (P < 0.05) associated with growth traits. The sites C66716002T, C66716195T and A66716179G were significantly (P < 0.05) associated with growth or carcass traits. These results demonstrated that FOXO3 is a candidate gene influencing chicken growth. Our observations provide new clues to understand the molecular basis of chicken growth.

  17. A Genome-Wide mRNA Screen and Functional Analysis Reveal FOXO3 as a Candidate Gene for Chicken Growth

    PubMed Central

    Chen, Biao; Xu, Jiguo; He, Xiaomei; Xu, Haiping; Li, Guihuan; Du, Hongli; Nie, Qinghua; Zhang, Xiquan

    2015-01-01

    Chicken growth performance provides direct economic benefits to the poultry industry. However, the underlying genetic mechanisms are unclear. The objective of this study was to identify candidate genes associated with chicken growth and investigate their potential mechanisms. We used RNA-Seq to study the breast muscle transcriptome in high and low tails of Recessive White Rock (WRRh, WRRl) and Xinghua chickens (XHh, XHl). A total of 60, 23, 153 and 359 differentially expressed genes were detected in WRRh vs. WRRl, XHh vs. XHl, WRRh vs. XHh and WRRl vs. XHl, respectively. GO, KEGG pathway and gene network analyses showed that CEBPB, FBXO32, FOXO3 and MYOD1 played key roles in growth. The functions of FBXO32 and FOXO3 were validated. FBXO32 was predominantly expressed in leg muscle, heart and breast muscle. After decreased FBXO32 expression, growth-related genes such as PDK4, IGF2R and IGF2BP3 were significantly down-regulated (P < 0.05). FBXO32 was significantly (P < 0.05) associated with carcass and meat quality traits, but not growth traits. FOXO3 was predominantly expressed in breast and leg muscle. In both of these tissues, the FOXO3 mRNA level in XH was significantly higher than that in WRR chickens with normal body weight (P < 0.05). In DF-1 cells, siRNA knockdown of FOXO3 significantly (P < 0.01) inhibited the MYOD expression and significantly up-regulated (P < 0.01 or P < 0.05) the expression of growth-related genes including CEBPB, FBXO32, GH, GHR, IGF1R, IGF2R, IGF2BP1, IGF2BP3, INSR, PDK1 and PDK4. Moreover, 18 SNPs were identified in FOXO3. G66716193A was significantly (P < 0.05) associated with growth traits. The sites C66716002T, C66716195T and A66716179G were significantly (P < 0.05) associated with growth or carcass traits. These results demonstrated that FOXO3 is a candidate gene influencing chicken growth. Our observations provide new clues to understand the molecular basis of chicken growth. PMID:26366565

  18. Molecular cloning and mRNA expression of a hepcidin gene from the spinyhead croaker, Collichthys lucidus.

    PubMed

    Sang, C; Lin, Y; Jiang, K; Zhang, F; Song, W

    2015-12-07

    Antimicrobial peptides are important components that participate in host innate immune activities and play crucial roles in host defense against microbial invasion. Hepcidin is an antimicrobial peptide and iron-regulatory molecule that primarily functions in the liver. In the present study, we first obtained a full-length cDNA sequence of hepcidin and its corresponding genomic DNA sequence from Collichthys lucidus using RT-PCR and rapid amplification of cDNA ends (RACE), and then analyzed these sequences using bioinformatics software. The results showed that C. lucidus hepcidin (CL-hepc) possesses two introns and three exons in the genomic DNA, with a length of 816 bp. The open reading frame was 264 bp, encoding an 87 amino acid peptide, and with high similarity (88.89%) to 83416593 Larimichthys crocea (ABC18307) and relatively low similarity (47.73%) to 158358729 L. crocea (ABY84845.1). The pre-peptide contained a signal peptide (28 amino acids), a prodomain (34 amino acids), and a mature peptide (25 amino acids). The predicted 25 amino acid hepcidin mature peptide included 8 conserved cysteine residues. Quantitative real-time reverse transcription-PCR analysis revealed specific expression patterns of CL-hepc, with the highest expression observed in the liver, relatively low expression observed in the gill and spleen, and almost no expression detected in other tissues analyzed. In conclusion, we identified a hepcidin from C. lucidus that has common expression patterns with other hepcidins. However, as this hepcidin is inconsistent with two other hepcidins from L. crocea in terms of the phylogenetic tree, the presence of another hepcidin gene warrants further investigation.

  19. Codon and mRNA sequence optimization of microdystrophin transgenes improves expression and physiological outcome in dystrophic mdx mice following AAV2/8 gene transfer.

    PubMed

    Foster, Helen; Sharp, Paul S; Athanasopoulos, Takis; Trollet, Capucine; Graham, Ian R; Foster, Keith; Wells, Dominic J; Dickson, George

    2008-11-01

    Duchenne muscular dystrophy is a fatal muscle-wasting disorder. Lack of dystrophin compromises the integrity of the sarcolemma and results in myofibers that are highly prone to contraction-induced injury. Recombinant adeno-associated virus (rAAV)-mediated dystrophin gene transfer strategies to muscle for the treatment of Duchenne muscular dystrophy (DMD) have been limited by the small cloning capacity of rAAV vectors and high titers necessary to achieve efficient systemic gene transfer. In this study, we assess the impact of codon optimization on microdystrophin (DeltaAB/R3-R18/DeltaCT) expression and function in the mdx mouse and compare the function of two different configurations of codon-optimized microdystrophin genes (DeltaAB/R3-R18/DeltaCT and DeltaR4-R23/DeltaCT) under the control of a muscle-restrictive promoter (Spc5-12). Codon optimization of microdystrophin significantly increases levels of microdystrophin mRNA and protein after intramuscular and systemic administration of plasmid DNA or rAAV2/8. Physiological assessment demonstrates that codon optimization of DeltaAB/R3-R18/DeltaCT results in significant improvement in specific force, but does not improve resistance to eccentric contractions compared with noncodon-optimized DeltaAB/R3-R18/DeltaCT. However, codon-optimized microdystrophin DeltaR4-R23/DeltaCT completely restored specific force generation and provided substantial protection from contraction-induced injury. These results demonstrate that codon optimization of microdystrophin under the control of a muscle-specific promoter can significantly improve expression levels such that reduced titers of rAAV vectors will be required for efficient systemic administration.

  20. Gene expression profiling by mRNA sequencing reveals increased expression of immune/inflammation-related genes in the hippocampus of individuals with schizophrenia.

    PubMed

    Hwang, Y; Kim, J; Shin, J Y; Kim, J Ii; Seo, J S; Webster, M J; Lee, D; Kim, S

    2013-10-29

    Whole-genome expression profiling in postmortem brain tissue has recently provided insight into the pathophysiology of schizophrenia. Previous microarray and RNA-Seq studies identified several biological processes including synaptic function, mitochondrial function and immune/inflammation response as altered in the cortex of subjects with schizophrenia. Now using RNA-Seq data from the hippocampus, we have identified 144 differentially expressed genes in schizophrenia cases as compared with unaffected controls. Immune/inflammation response was the main biological process over-represented in these genes. The upregulation of several of these genes, IFITM1, IFITM2, IFITM3, APOL1 (Apolipoprotein L1), ADORA2A (adenosine receptor 2A), IGFBP4 and CD163 were validated in the schizophrenia subjects using data from the SNCID database and with quantitative RT-PCR. We identified a co-expression module associated with schizophrenia that includes the majority of differentially expressed genes related to immune/inflammation response as well as with the density of parvalbumin-containing neurons in the hippocampus. The results indicate that abnormal immune/inflammation response in the hippocampus may underlie the pathophysiology of schizophrenia and may be associated with abnormalities in the parvalbumin-containing neurons that lead to the cognitive deficits of the disease.

  1. Detection of aberrant transcription of major histocompatibility complex class II antigen presentation genes in chronic lymphocytic leukaemia identifies HLA-DOA mRNA as a prognostic factor for survival.

    PubMed

    Souwer, Yuri; Chamuleau, Martine E D; van de Loosdrecht, Arjan A; Tolosa, Eva; Jorritsma, Tineke; Muris, Jettie J F; Dinnissen-van Poppel, Marion J; Snel, Sander N; van de Corput, Lisette; Ossenkoppele, Gert J; Meijer, Chris J L M; Neefjes, Jacques J; Marieke van Ham, S

    2009-05-01

    In human B cells, effective major histocompatibility complex (MHC) class II-antigen presentation depends not only on MHC class II, but also on the invariant chain (CD74 or Ii), HLA-DM (DM) and HLA-DO (DO), the chaperones regulating the antigen loading process of MHC class II molecules. We analysed immediate ex vivo expression of HLA-DR (DR), CD74, DM and DO in B cell chronic lymphocytic leukaemia (B-CLL). Real-time reverse transcription polymerase chain reaction demonstrated a highly significant upregulation of DRA, CD74, DMB, DOA and DOB mRNA in purified malignant cells compared to B cells from healthy donors. The increased mRNA levels were not translated into enhanced protein levels but could reflect aberrant transcriptional regulation. Indeed, upregulation of DRA, DMB, DOA and DOB mRNA correlated with enhanced expression of class II transactivator (CIITA). In-depth analysis of the various CIITA transcripts demonstrated a significant increased activity of the interferon-gamma-inducible promoter CIITA-PIV in B-CLL. Comparison of the aberrant mRNA levels with clinical outcome identified DOA mRNA as a prognostic indicator for survival. Multivariate analysis revealed that the prognostic value of DOA mRNA was independent of the mutational status of the IGHV genes. Thus, aberrant transcription of DOA forms a novel and additional prognostic indicator for survival in B-CLL.

  2. Expression at mRNA level of cytokines and A238L gene in porcine blood-derived macrophages infected in vitro with African swine fever virus (ASFV) isolates of different virulence.

    PubMed

    Gil, S; Spagnuolo-Weaver, M; Canals, A; Sepúlveda, N; Oliveira, J; Aleixo, A; Allan, G; Leitão, A; Martins, C L V

    2003-11-01

    Porcine macrophage cultures were infected with two ASFV isolates of variable virulence and mRNA levels of several relevant macrophage-derived cytokines were quantified by real time PCR. At six hours post infection, a clear enhancement of mRNA expression of TNFalpha, IL6, IL12 and IL15 was observed in macrophages infected with the low virulent ASFV/NH/P68 (NHV) when compared to those infected with the highly virulent ASFV/L60 (L60). The sequence of the A238L gene homologue to the cellular IkappaB was found identical in both viral isolates and its expression at mRNA level was higher in macrophages infected with NHV when compared to macrophages infected with L60. Furthermore our results suggest a negative correlation between the mRNA expression of A238L gene and the mRNA expression of the above mentioned cytokines (with the exception of IL10) in L60 infected macrophages in opposition to the positive correlation (with exception of the IL1) suggested in NHV infection. Overall, our data strongly emphasize that virulence of ASFV isolates may depend on their capacity to regulate the expression of macrophage-derived cytokines relevant for the development of host protective responses by yet unknown mechanisms triggered by the virus at early stages of the cellular infection.

  3. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  4. Molecular characterization and expression of buffalo (Bubalus bubalis) DEAD-box family VASA gene and mRNA transcript variants isolated from testis tissue.

    PubMed

    Kaushik, Ramakant; Singh, Karn Pratap; Bahuguna, Vivek; Rameshbabu, K; Singh, Manoj Kumar; Manik, Radhey Shyam; Palta, Prabhat; Singla, Suresh Kumar; Chauhan, Manmohan Singh

    2015-11-01

    VASA is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. In the present study, we isolated, sequenced, and characterized VASA gene in buffalo testis. Here, we demonstrated that VASA mRNA is expressed as multiple isoforms and uses four alternative transcriptional start sites (TSSs) and four different polyadenylation sites. The TSSs identified by 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-5'-RACE) were positioned at 48, 53, 85, and 88 nucleotides upstream relative to the translation initiation codon. 3'-RACE experiment revealed the presence of tandem polyadenylation signals, which lead to the expression of at least four different 3'-untranslated regions (209, 233, 239 and 605 nucleotides). The full-length coding region of VASA was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. VASA variants are highly expressed in testis of adult buffalo. We found five variants, one full length VASA (729 aa) and four splice variants VASA 2, 4, 5, 6 (683, 685, 679, 703 aa). The expression level of VASA 1 was significantly higher than rest of all (P < 0.05) except VASA 6. The relative ratio for VASA 1:2:4:5:6 was 100:1.0:1.6:0.9:48.

  5. SAC3B, a central component of the mRNA export complex TREX-2, is required for prevention of epigenetic gene silencing in Arabidopsis

    PubMed Central

    Yang, Yu; La, Honggui; Tang, Kai; Miki, Daisuke; Yang, Lan; Wang, Bangshing; Duan, Cheng-Guo; Nie, Wenfeng; Wang, Xingang; Wang, Siwen; Pan, Yufeng; Tran, Elizabeth J.; An, Lizhe; Zhang, Huiming; Zhu, Jian-Kang

    2017-01-01

    Epigenetic regulation is important for organismal development and response to the environment. Alteration in epigenetic status has been known mostly from the perspective of enzymatic actions of DNA methylation and/or histone modifications. In a genetic screen for cellular factors involved in preventing epigenetic silencing, we isolated an Arabidopsis mutant defective in SAC3B, a component of the conserved TREX-2 complex that couples mRNA transcription with nuleo-cytoplasmic export. Arabidopsis SAC3B dysfunction causes gene silencing at transgenic and endogenous loci, accompanied by elevation in the repressive histone mark H3K9me2 and by reduction in RNA polymerase Pol II occupancy. SAC3B dysfunction does not alter promoter DNA methylation level of the transgene d35S::LUC, although the DNA demethylase ROS1 is also required for d35S::LUC anti-silencing. THP1 and NUA were identified as SAC3B-associated proteins whose mutations also caused d35S::LUC silencing. RNA-DNA hybrid exists at the repressed loci but is unrelated to gene suppression by the sac3b mutation. Genome-wide analyses demonstrated minor but clear involvement of SAC3B in regulating siRNAs and DNA methylation, particularly at a group of TAS and TAS-like loci. Together our results revealed not only a critical role of mRNA-export factors in transcriptional anti-silencing but also the contribution of SAC3B in shaping plant epigenetic landscapes. PMID:27672037

  6. Myeloperoxidase activity and its corresponding mRNA expression as well as gene polymorphism in the population living in the coal-burning endemic fluorosis area in Guizhou of China.

    PubMed

    Zhang, Ting; Shan, Ke-Ren; Tu, Xi; He, Yan; Pei, Jin-Jing; Guan, Zhi-Zhong

    2013-06-01

    The myeloperoxidase (MPO) activity and its corresponding mRNA expression as well as gene polymorphism were investigated in the population who live in the endemic fluorosis area. In the study, 150 people were selected from the coal-burning endemic fluorosis area and 150 normal persons from the non-fluorosis area in Guizhou province of China. The blood samples were collected from these people. The activity of MPO in the plasma was determined by spectrophotometer; the expression of MPO mRNA was measured by employing real-time polymerase chain reaction; DNAs were extracted from the leucocytes in blood and five SNP genotypes of MPO promoter gene detected by a multiplex genotyping method, adapter-ligation-mediated allele-specific amplification. The results showed that the MPO activity and its corresponding mRNA in blood were significantly increased in the population living in the area of fluorosis. The different genotype frequencies of MPO, including -1228G/A, -585T/C, -463G/A, and -163C/T, and the three haplotypes with higher frequencies, including -163C-463G-585T-1228G-1276T, -163C-463G-585T-1228G-1276C, and -163C-463G-585T-1228A-1276T, were significantly associated with fluorosis. The results indicated that the elevated activity of MPO induced by endemic fluorosis may be connected in mechanism to the stimulated expression of MPO mRNA and the changed gene polymorphism.

  7. Molecular characterization, tissue distribution, and mRNA expression profiles of two Kiss genes in the adult male and female chub mackerel (Scomber japonicus) during different gonadal stages.

    PubMed

    Selvaraj, Sethu; Kitano, Hajime; Fujinaga, Yoichiro; Ohga, Hirofumi; Yoneda, Michio; Yamaguchi, Akihiko; Shimizu, Akio; Matsuyama, Michiya

    2010-10-01

    Kisspeptins, encoded by the Kiss1 gene, have emerged as key modulators of reproduction in mammals. In contrast to the placental mammals, some teleosts express two Kiss genes, Kiss1 and Kiss2. In the present study, full-length cDNAs of Kiss1 and Kiss2 in the chub mackerel were cloned and sequenced. Chub mackerel Kiss1 and Kiss2 cDNAs encode 105 and 123 amino acids, respectively. A comparison of the deduced amino acid sequences of chub mackerel Kiss1 and Kiss2 with those of other vertebrate species showed a high degree of conservation only in the kisspeptin-10 region (Kp-10). The Kp-10 of chub mackerel Kiss1 (YNFNSFGLRY) and Kiss2 (FNFNPFGLRF) showed variations at three amino acids. Tissue distribution analysis using quantitative real-time PCR (qRT-PCR) revealed that the Kiss1 and Kiss2 transcripts were expressed in different tissues of adult chub mackerel. In addition, their levels in the adipose tissue exhibited sexually dimorphic expression. Further, to have a basic understanding on the involvement of Kiss1 and Kiss2 in the seasonal gonadal development, their relative mRNA expression profiles in the brain, pituitary, and gonads at different gonadal stages were analyzed using qRT-PCR. Kiss1 and Kiss2 levels in the brain showed a differential expression profile between male and female fish. In males, Kiss1 and Kiss2 levels gradually decreased from the immature stage to spermiation and reached a minimal level during the post-spawning period. In contrast, Kiss1 levels in the brain of females did not vary significantly among the different gonadal stages. However, Kiss2 levels fluctuated as that of males, gradually declining from the immature stage to the post-spawning period. The pituitary Kiss1 levels did not show significant fluctuations. However, Kiss1 levels in the gonads were highly elevated during spermiation and late vitellogenesis compared to the immature and post-spawning period. These results suggest the possible involvement of two Kiss genes in the brain and

  8. Quantitative analysis of mRNA transcripts of Hox, SHH, PTCH, Wnt, and Fzd genes in canine hematopoietic progenitor cells and various in vitro colonies differentiated from the cells.

    PubMed

    Ide, Kaori; Goto-Koshino, Yuko; Momoi, Yasuyuki; Fujino, Yasuhito; Ohno, Koichi; Tsujimoto, Hajime

    2009-01-01

    Homeobox (Hox), Sonic hedgehog (SHH), and Wingless-type MMTV integration site family (Wnt) are known to modulate the self-renewal and expansion of hematopoietic progenitor/stem cells in humans and mice. Frizzled (Fzd) and Patched1 (PTCH1) represent the receptors of Wnt and SHH, respectively. In this study, the amounts of mRNA transcripts of the genes associated with the self-renewal of hematopoietic stem cells, HoxB3, HoxB4, HoxA10, Wnt5a, Wnt2b, Fzd1, Fzd6, SHH, and PTCH1, were measured in canine unfractionated bone marrow cells, CD34-enriched cells, and various colony-forming units in culture (CFU-C). Partial cDNA sequences of these 9 canine genes were determined in this study. Quantitative real-time polymerase chain reaction was employed to indicate their relative amounts of mRNA transcripts. Amounts of mRNA transcripts of HoxB3, HoxA10, PTCH1, and Wnt5a genes in canine CD34-enriched cell fraction were significantly larger than those in the CD34-depleted cell fraction. Amounts of mRNA transcripts of HoxB3, HoxA10, PTCH1, Wnt5a, and Wnt2b genes in various CFU-C cells were significantly smaller than those in the seeded CD34-enriched cell fraction. These results suggested important roles of the products of these genes in self-renewal, expansion, and survival of hematopoietic progenitor cells in dogs as shown in humans and rodents.

  9. Comparison of the QuantiGene 2.0 Assay and Real-Time RT-PCR in the Detection of p53 Isoform mRNA Expression in Formalin-Fixed Paraffin-Embedded Tissues- A Preliminary Study

    PubMed Central

    Morten, Brianna C.; Scott, Rodney J.; Avery-Kiejda, Kelly A.

    2016-01-01

    p53 is expressed as multiple smaller isoforms whose functions in cancer are not well understood. The p53 isoforms demonstrate abnormal expression in different cancers, suggesting they are important in modulating the function of full-length p53 (FLp53). The quantification of relative mRNA expression has routinely been performed using real-time PCR (qPCR). However, there are serious limitations when detecting p53 isoforms using this method, particularly for formalin-fixed paraffin-embedded (FFPE) tissues. The use of FFPE tumours would be advantageous to correlate expression of p53 isoforms with important clinical features of cancer. One alternative method of RNA detection is the hybridization-based QuantiGene 2.0 Assay, which has been shown to be advantageous for the detection of RNA from FFPE tissues. In this pilot study, we compared the QuantiGene 2.0 Assay to qPCR for the detection of FLp53 and its isoform Δ40p53 in matched fresh frozen (FF) and FFPE breast tumours. FLp53 mRNA expression was detected using qPCR in FF and FFPE tissues, but Δ40p53 mRNA was only detectable in FF tissues. Similar results were obtained for the QuantiGene 2.0 Assay. FLp53 relative mRNA expression was shown to be strongly correlated between the two methods (R2 = 0.9927, p = 0.0031) in FF tissues, however Δ40p53 was not (R2 = 0.4429, p = 0.3345). When comparing the different methods for the detection of FLp53 mRNA from FFPE and FF samples, no correlation (R2 = 0.0002, p = 0.9863) was shown using the QuantiGene 2.0 Assay, and in contrast, the level of expression was highly correlated between the two tissues using qPCR (R2 = 0.8753, p = 0.0644). These results suggest that both the QuantiGene 2.0 Assay and qPCR methods are inadequate for the quantification of Δ40p53 mRNA in FFPE tissues. Therefore, alternative methods of RNA detection and quantification are required to study the relative expression of Δ40p53 in FFPE samples. PMID:27832134

  10. The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501.

    PubMed

    Zhan, Yuhua; Yan, Yongliang; Deng, Zhiping; Chen, Ming; Lu, Wei; Lu, Chao; Shang, Liguo; Yang, Zhimin; Zhang, Wei; Wang, Wei; Li, Yun; Ke, Qi; Lu, Jiasi; Xu, Yuquan; Zhang, Liwen; Xie, Zhihong; Cheng, Qi; Elmerich, Claudine; Lin, Min

    2016-07-26

    Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade. The nfiS-deficient mutant displayed reduced nitrogenase activity, as well as increased sensitivity to multiple stresses, such as osmotic and oxidative stresses. Secondary structure prediction and complementation studies confirmed that a stem-loop structure was essential for NfiS to regulate the nitrogenase gene nifK mRNA synthesis and thus nitrogenase activity. Microscale thermophoresis and physiological analysis showed that NfiS directly pairs with nifK mRNA and ultimately enhances nitrogenase activity by increasing the translation efficiency and the half-life of nifK mRNA. Our data also suggest structural and functional divergence of NfiS evolution in diazotrophic and nondiazotrophic backgrounds. It is proposed that NfiS was recruited by nifK mRNA as a novel regulator to integrate the horizontally acquired nif island into host global networks.

  11. Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA.

    PubMed

    Miller, Jason B; Zhang, Shuyuan; Kos, Petra; Xiong, Hu; Zhou, Kejin; Perelman, Sofya S; Zhu, Hao; Siegwart, Daniel J

    2017-01-19

    CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg(-1) ). Intravenous co-delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.

  12. Genes for collagen types I, IV, and V are transcribed in HeLa cells but a postinitiation block prevents the accumulation of type I mRNA

    SciTech Connect

    Furth, J.J.; Wroth, T.H.; Ackerman, S. )

    1991-01-01

    Collagen mRNA synthesis in HeLa cells was evaluated by in vitro transcription of type I collagen DNA, nuclear run-on studies, and steady-state mRNA analysis. Type I collagen mRNA was accurately initiated by HeLa cell RNQA polymerase II in nuclear extracts, and run-on analysis indicated that mRNAs for collagen types {alpha}1(I), {alpha}2(I), {alpha}1(III), {alpha}1(IV), and {alpha}2(V) were synthesized in HeLa cells. However, on assessing the steady-state levels of mRNAs of collagen types {alpha}1(I), {alpha}2(I), {alpha}1(IV), and {alpha}2(V), no type I mRNA was found in HeLa cells while types {alpha}1(IV) and {alpha}2(V) collagen mRNAs were observed. These results suggest that a postinitiation process prevents the accumulation of type I collagen mRNAs in HeLa cells. Persistence of types IV and V collagen mRNAs is consistent with the involvement of types IV and V collagen in adhesion of HeLa cells to glass or plastic.

  13. The ultraspiracle gene of the spruce budworm, Choristoneura fumiferana: cloning of cDNA and developmental expression of mRNA.

    PubMed

    Perera, S C; Palli, S R; Ladd, T R; Krell, P J; Retnakaran, A

    1998-01-01

    Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression.

  14. Numerical solution of the chemical master equation uniqueness and stability of the stationary distribution for chemical networks, and mRNA bursting in a gene network with negative feedback regulation.

    PubMed

    Zeron, E S; Santillán, M

    2011-01-01

    In this work, we introduce a couple of algorithms to compute the stationary probability distribution for the chemical master equation (CME) of arbitrary chemical networks. We further find the conditions guaranteeing the algorithms' convergence and the unity and stability of the stationary distribution. Next, we employ these algorithms to study the mRNA and protein probability distributions in a gene regulatory network subject to negative feedback regulation. In particular, we analyze the influence of the promoter activation/deactivation speed on the shape of such distributions. We find that a reduction of the promoter activation/deactivation speed modifies the shape of those distributions in a way consistent with the phenomenon known as mRNA (or transcription) bursting.

  15. Effects of PCB 126 and PCB 153 on secretion of steroid hormones and mRNA expression of steroidogenic genes (STAR, HSD3B, CYP19A1) and estrogen receptors (ERα, ERβ) in prehierarchical chicken ovarian follicles.

    PubMed

    Sechman, Andrzej; Batoryna, Marta; Antos, Piotr A; Hrabia, Anna

    2016-12-15

    The objective of this study was to assess the in vitro effects of dioxin-like PCB 126 and non-dioxin-like PCB 153 on basal and ovine LH (oLH)-stimulated testosterone (T) and estradiol (E2) secretion and expression of steroidogenic genes (STAR, HSD3B and CYP19A1) and estrogen receptors α (ERα) and β (ERβ) in white (WF) and yellowish (YF) prehierarchical follicles of the hen ovary. Steroid concentrations in a medium and gene expression in follicles following 6h of exposition were determined by RIA and real-time qPCR, respectively. Both PCBs increased basal and oLH-stimulated T secretion by the WF follicles. PCB 126 reduced basal E2 secretion by the WF follicles. PCB 153 elevated but PCB 126 reduced oLH-stimulated E2 secretion by the prehierarchical follicles. PCB 126 increased basal STAR and HSD3B and reduced CYP19A1 mRNA expression in these follicles. PCB 153 increased basal expression of STAR and HSD3B in YF follicles, but diminished HSD3B mRNA levels in the WF. The studied PCBs had an opposite effect on basal and oLH-stimulated CYP19A1 mRNA expression in prehierarchical follicles. Both PCBs modulated basal and inhibited oLH-stimulated ERα and ERβ gene expression in the prehierarchical follicles. In conclusion, data of the current study demonstrate the congener-specific effects of PCBs on sex steroid secretion by prehierarchical follicles of the chicken ovary, which are at least partly related to STAR, HSD3B and CYP19A1 gene expression. It is suggested that PCBs, by influencing follicular steroidogenesis and expression of estrogen receptors, may impair development and selection of yellowish follicles to the preovulatory hierarchy.

  16. Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA-messenger RNA molecular mimicry.

    PubMed Central

    Guillier, Maude; Allemand, Frédéric; Raibaud, Sophie; Dardel, Frédéric; Springer, Mathias; Chiaruttini, Claude

    2002-01-01

    In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor. L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20. L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling. All of the cis-acting sequences required for repression by L20, called the operator, are found on an mRNA segment extending from the middle of the IF3 gene to the start of the L35 gene. L20-mediated repression requires a long-range base-pairing interaction between nucleotide residues within the IF3 gene and residues just upstream of the L35 gene. This interaction results in the formation of a pseudoknot. Here we show that L20 causes protection of nucleotide residues in two regions of the operator in vitro. The first region is the pseudoknot itself and the second lies in an irregular stem located upstream of the L35 gene. By primer extension analysis, we show that L20 specifically induces reverse transcriptase stops in both regions. Therefore, these two regions define two L20-binding sites in the operator. Using mutations and deletions of rpml'-'lacZ fusions, we show that both sites are essential for repression in vivo. However L20 can bind to each site independently in vitro. One site is similar to the L20-binding site on 23S rRNA. Here we propose that L20 recognizes its mRNA and its rRNA in similar way. PMID:12166643

  17. Influence of the Cyp1B1 L432V gene polymorphism and exposure to tobacco smoke on Cyp1B1 mRNA expression in human leukocytes.

    PubMed

    Helmig, Simone; Hadzaad, Bahar; Döhrel, Juliane; Schneider, Joachim

    2009-07-01

    Cytochrome P450 1B1 (CYP1B1), a phase I enzyme, is involved in the activation of a broad spectrum of procarcinogens. An association of the Cyp1B1 L432V polymorphism with diverse types of cancer, as well as an impact on the catalytic activity of the enzyme, has been described. To show the functional impact of the allelic variant Cyp1B1*3, we investigated the quantitative Cyp1B1 mRNA expression in a population of smokers, nonsmokers, and ex-smokers and determined their genotypes. Detection of the L432V polymorphism in exon 3 of the Cyp1B1 gene was performed by rapid capillary polymerase chain reaction (PCR) with melting curve analysis. For quantitative comparison of Cyp1B1 mRNA levels, real-time PCR was performed using SYBR Green fluorescence in a LightCycler system. Calculations of expression were made with the 2(-DeltaDeltaCT) method. In comparing relative Cyp1B1 mRNA expression, highly significant differences between the two homozygote genotypes *1/*1 and *3/*3 (0.185 +/- 0.027, n = 118 versus 0.071 +/- 0.013, n = 56; p = 0.000), as well as between the heterozygote genotype *1/*3 and the homozygote genotype *3/*3 (0.178 +/- 0.025, n = 171 versus 0.071 +/- 0.013, n = 56; p = 0.000), were revealed. Significant differences between the genotypes were also detected within the subgroups of smokers, nonsmokers, and ex-smokers. No significant differences were determined in comparing the relative Cyp1B1 mRNA expression with regard to tobacco smoke exposure. Our results suggest that genotypes carrying the C allele (*1/*1 and *1/*3) at Cyp1B1 L432V polymorphism have a significantly higher Cyp1B1 mRNA expression compared with the genotype without the C allele (*3/*3). Gene expression of Cyp1B1 mRNA cannot be used as a biomarker for exposure of tobacco smoke.

  18. Evolution of the Antisense Overlap between Genes for Thyroid Hormone Receptor and Rev-erbα and Characterization of an Exonic G-Rich Element That Regulates Splicing of TRα2 mRNA

    PubMed Central

    Munroe, Stephen H.; Morales, Christopher H.; Duyck, Tessa H.; Waters, Paul D.

    2015-01-01

    The α-thyroid hormone receptor gene (TRα) codes for two functionally distinct proteins: TRα1, the α-thyroid hormone receptor; and TRα2, a non-hormone-binding variant. The final exon of TRα2 mRNA overlaps the 3’ end of Rev-erbα mRNA, which encodes another nuclear receptor on the opposite strand of DNA. To understand the evolution of this antisense overlap, we sequenced these genes and mRNAs in the platypus Orthorhynchus anatinus. Despite its strong homology with other mammals, the platypus TRα/Rev-erbα locus lacks elements essential for expression of TRα2. Comparative analysis suggests that alternative splicing of TRα2 mRNA expression evolved in a stepwise fashion before the divergence of eutherian and marsupial mammals. A short G-rich element (G30) located downstream of the alternative 3’splice site of TRα2 mRNA and antisense to the 3’UTR of Rev-erbα plays an important role in regulating TRα2 splicing. G30 is tightly conserved in eutherian mammals, but is absent in marsupials and monotremes. Systematic deletions and substitutions within G30 have dramatically different effects on TRα2 splicing, leading to either its inhibition or its enhancement. Mutations that disrupt one or more clusters of G residues enhance splicing two- to three-fold. These results suggest the G30 sequence can adopt a highly structured conformation, possibly a G-quadruplex, and that it is part of a complex splicing regulatory element which exerts both positive and negative effects on TRα2 expression. Since mutations that strongly enhance splicing in vivo have no effect on splicing in vitro, it is likely that the regulatory role of G30 is mediated through linkage of transcription and splicing. PMID:26368571

  19. Analysis of mRNA expression for genes associated with regulatory T lymphocytes (CD25, FoxP3, CTLA4, and IDO) after experimental infection with bovine viral diarrhea virus of low or high virulence in beef calves.

    PubMed

    Palomares, Roberto A; Hurley, David J; Woolums, Amelia R; Parrish, Jacqueline E; Brock, Kenny V

    2014-12-01

    Immunosuppression caused by bovine viral diarrhea virus (BVDV) has been associated with lymphocyte depletion, leukopenia and impairment of leukocyte function; however, no work has been done on the relationship between BVDV and regulatory T lymphocytes (Tregs). The objective of this study was to compare the mRNA expression of genes associated with Tregs (CD25, FoxP3, CTLA4, and IDO), after experimental infection of beef calves with low (LV) or high (HV) virulence BVDV. Thirty BVDV-naïve calves were randomly assigned to three groups. Calves were intra-nasally inoculated with LV (n=10, strain SD-1) or HV (n=10, strain 1373) BVDV or BVDV-free cell culture medium (control, n=10). Quantitative RT-PCR was used to determine the expression of target genes in tracheo-bronchial lymph nodes and spleen on day 5 post-infection. The mRNA expression of CD25 was up-regulated in tracheo-bronchial lymph nodes of LV (P<0.05), but not in HV compared to the control group. The expression of FoxP3 and CTLA4 was not increased in tracheo-bronchial lymph nodes of either of the BVDV-inoculated groups. A dramatic up-regulation of IDO mRNA was observed in tracheo-bronchial lymph nodes of LV (P<0.05), but not HV compared to the control calves. In conclusion, experimental infection with BVDV did not provide evidence of Treg activation based on expression of FoxP3 and CTL4. Differential expression of CD25 and IDO mRNA on day 5 post-infection with HV or LV BVDV might reflect temporal differences in transcription occurring during the immune response elicited by these viral strains, or differences in viral infectivity of the host cells.

  20. The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression

    PubMed Central

    Lamana, Amalia; López-Santalla, Mercedes; Castillo-González, Raquel; Ortiz, Ana María; Martín, Javier; García-Vicuña, Rosario; González-Álvaro, Isidoro

    2015-01-01

    Objective The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression. Methods We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models. Results After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations. Conclusion Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway. PMID:26569609

  1. High throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry

    PubMed Central

    Porichis, Filippos; Hart, Meghan G.; Griesbeck, Morgane; Everett, Holly L.; Hassan, Muska; Baxter, Amy E.; Lindqvist, Madelene; Miller, Sara M.; Soghoian, Damien Z.; Kavanagh, Daniel G.; Reynolds, Susan; Norris, Brett; Mordecai, Scott K.; Nguyen, Quan; Lai, Chunfai; Kaufmann, Daniel E.

    2014-01-01

    Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to addressa variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by Image Stream technology. PMID:25472703

  2. Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene.

    PubMed Central

    Majors, J E; Varmus, H E

    1983-01-01

    To extend our understanding of the organization and expression of the mouse mammary tumor virus genome, we determined the nucleotide sequence of large regions of a cloned mouse mammary tumor virus strain C3H provirus that appears to be a DNA copy of env mRNA. In conjunction with analysis of several additional clones of integrated and unintegrated mouse mammary tumor virus DNAs, we came to the following conclusions: (i) the mRNA for env is generated by splicing mechanisms that recognize conventional eucaryotic signals at donor and acceptor sites with a leader of at least 289 bases in length; (ii) the first of three possible initiation codons for translation of env follows the splice junction by a single nucleotide and produces a signal peptide of 98 amino acids; (iii) the amino terminal sequence of the major virion glycoprotein gp52env is confirmed by nucleotide sequencing and is encoded by a sequence beginning 584 nucleotides from the 5' end of env mRNA; (iv) the final 17 amino acids at the carboxyl terminus of the primary product of env are encoded within the long terminal repeat by the 51 bases at the 5' end of the U3 domain; and (v) bases 2 through 4 at the 5' end of the long terminal repeat constitute an initiation codon that commences an open reading frame capable of directing the synthesis of a 36-kilodalton protein. PMID:6312081

  3. The translocation (6; 9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA

    SciTech Connect

    Von Lindern, M.; Fornerod, M.; Van Baal, S.; Jaegle, M.; De Wit, T.; Buijs, A.; Grosveld, G. )

    1992-04-01

    The translocation (6;9) is associated with a specific subtype of acute myeloid leukemia (AML). Previously, it was found that breakpoints on chromosome 9 are clustered in one of the introns of a large gene named Cain (can). cDNA probes derived from the 3' part of can detect an aberrant, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. cDNA cloning of this mRNA revealed that it is a fusion of sequences encoded on chromosome 6 and 3' can. A novel gene on chromosome 6 which was named dek was isolated. In dek the t(6;9) breakpoints also occur in one intron. As a result the dek-can fusion gene, present in t(6;9) AML, encodes an invariable dek-can transcript. Sequence analysis of the dek-can cDNA showed that dek and can are merged without disruption of the original open reading frames and therefore the fusion mRNA encodes a chimeric DEK-CAN protein of 165 kDa. The predicted DEK and CAN proteins have molecular masses of 43 and 220 kDa, respectively. Sequence comparison with the EMBL data base failed to show consistent homology with any known protein sequences. 50 refs., 8 figs.

  4. Cyclin-Dependent Kinase 7 Controls mRNA Synthesis by Affecting Stability of Preinitiation Complexes, Leading to Altered Gene Expression, Cell Cycle Progression, and Survival of Tumor Cells

    PubMed Central

    Kelso, Timothy W. R.; Baumgart, Karen; Eickhoff, Jan; Albert, Thomas; Antrecht, Claudia; Lemcke, Sarah; Klebl, Bert

    2014-01-01

    Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis. PMID:25047832

  5. The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA.

    PubMed Central

    von Lindern, M; Fornerod, M; van Baal, S; Jaegle, M; de Wit, T; Buijs, A; Grosveld, G

    1992-01-01

    The translocation (6;9) is associated with a specific subtype of acute myeloid leukemia (AML). Previously, it was found that breakpoints on chromosome 9 are clustered in one of the introns of a large gene named Cain (can). cDNA probes derived from the 3' part of can detect an aberrant, leukemia-specific 5.5-kb transcript in bone marrow cells from t(6;9) AML patients. cDNA cloning of this mRNA revealed that it is a fusion of sequences encoded on chromosome 6 and 3' can. A novel gene on chromosome 6 which was named dek was isolated. In dek the t(6;9) breakpoints also occur in one intron. As a result the dek-can fusion gene, present in t(6;9) AML, encodes an invariable dek-can transcript. Sequence analysis of the dek-can cDNA showed that dek and can are merged without disruption of the original open reading frames and therefore the fusion mRNA encodes a chimeric DEK-CAN protein of 165 kDa. The predicted DEK and CAN proteins have molecular masses of 43 and 220 kDa, respectively. Sequence comparison with the EMBL data base failed to show consistent homology with any known protein sequences. Images PMID:1549122

  6. Effects of the PPARα agonist WY-14,643 on plasma lipids, enzymatic activities and mRNA expression of lipid metabolism genes in a marine flatfish, Scophthalmus maximus.

    PubMed

    Urbatzka, R; Galante-Oliveira, S; Rocha, E; Lobo-da-Cunha, A; Castro, L F C; Cunha, I

    2015-07-01

    Fibrates and other lipid regulator drugs are widespread in the aquatic environment including estuaries and coastal zones, but little is known on their chronic effects on non-target organisms as marine fish. In the present study, turbot juveniles were exposed to the PPARα model agonist WY-14,643 for 21 days by repeated injections at the concentrations of 5mg/kg (lo-WY) and 50mg/kg (hi-WY), and samples taken after 7 and 21 days. Enzyme activity and mRNA expression of palmitoyl-CoA oxidase and catalase in the liver were analyzed as first response, which validated the experiment by demonstrating interactions with the peroxisomal fatty acid oxidation and oxidative stress pathways in the hi-WY treatment. In order to get mechanistic insights, alterations of plasma lipids (free cholesterol, FC; HDL associated cholesterol, C-HDL; triglycerides, TG; non-esterified fatty acids, NEFA) and hepatic mRNA expression of 17 genes involved in fatty acid and lipid metabolism were studied. The exposure to hi-WY reduced the quantity of plasma FC, C-HDL, and NEFA. Microsomal triglyceride transfer protein and apolipoprotein E mRNA expression were higher in hi-WY, and indicated an increased formation of VLDL particles and energy mobilization from liver. It is speculated that energy depletion by PPARα agonists may contribute to a higher susceptibility to environmental stressors.

  7. The alteration of mRNA expression of SOD and GPX genes, and proteins in tomato (Lycopersicon esculentum Mill) under stress of NaCl and/or ZnO nanoparticles.

    PubMed

    Alharby, Hesham F; Metwali, Ehab M R; Fuller, Michael P; Aldhebiani, Amal Y

    2016-11-01

    Five cultivars of tomato having different levels of salt stress tolerance were exposed to different treatments of NaCl (0, 3 and 6 g L(-1)) and ZnO-NPs (0, 15 and 30 mg L(-1)). Treatments with NaCl at both 3 and 6 g L(-1) suppressed the mRNA levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) genes in all cultivars while plants treated with ZnO-NPs in the presence of NaCl, showed increments in the mRNA expression levels. This indicated that ZnO-NPs had a positive response on plant metabolism under salt stress. Superior expression levels of mRNA were observed in the salt tolerant cultivars, Sandpoint and Edkawy while the lowest level was detected in the salt sensitive cultivar, Anna Aasa. SDS-PAGE showed clear differences in patterns of protein expression among the cultivars. A negative protein marker for salt sensitivity and ZnO-NPs was detected in cv. Anna Aasa at a molecular weight of 19.162 kDa, while the tolerant cultivar Edkawy had two positive markers at molecular weights of 74.991 and 79.735 kDa.

  8. Analysis of methylation and mRNA expression status ofFADD andFAS genes in patients with oral squamous cell carcinoma

    PubMed Central

    Saberi, Eshaghali; Jamali, Sara; Rigi-Ladez, Mohammad A.; Augend, Arsalan

    2014-01-01

    Background: Apoptosis is an important mechanism that is responsible for the physiological deletion of harmful, damaged, or unwanted cells. Changed expression of apoptosis-related genes may lead to abnormal cell proliferation and finally to tumorigenesis. Our aims were to analyze the promoter methylation and gene expression profiles of FADD and FAS genes in risk of OSCC. Material and Methods: we analyze the promoter methylation status of FADD and FAS genes using Methylation - Specific PCR (MSP) in 86 OSCC tissues were kept in paraffin and 68 normal oral tissues applied as control. Also, FADD and FAS genes expression were analyzed in 19 cases and 20 normal specimens by Real-Time Reverse-Transcripts PCR. Results: Aberrant promoter methylation of FADD and FAS genes were detected in 12.79 % (11 of 86) and 60.46 % (52 of 86) of the OSCC cases, respectively, with a significant difference between cases and healthy controls for both FADD and FAS genes (P<0.001). The gene expression analysis showed statistically significant difference between cases and healthy controls for both FADD (p<0.02) and FAS (p<0.007) genes. Conclusions: To the best our knowledge, the data of this study are the first report regarding, the effect of promoter hypermethylation of the FADD and FAS genes in development of OSCC. To confirm the data, it is recommended doing further study in large sample sizes in various genetic populations. Key words:OSCC, FADD, FAS, DNA methylation, gene expression. PMID:25129245

  9. Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

    PubMed

    Sihto, Henna-Maria; Tasara, Taurai; Stephan, Roger; Johler, Sophia

    2014-07-01

    Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models.

  10. Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.

    PubMed Central

    Minvielle-Sebastia, L; Winsor, B; Bonneaud, N; Lacroute, F

    1991-01-01

    In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome H, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rna14 or the rna15 mutation, suggesting that the encoded proteins could interact with each other. Images PMID:1674817

  11. The novel regulatory ncRNA, NfiS, optimizes nitrogen fixation via base pairing with the nitrogenase gene nifK mRNA in Pseudomonas stutzeri A1501

    PubMed Central

    Zhan, Yuhua; Yan, Yongliang; Deng, Zhiping; Chen, Ming; Lu, Wei; Lu, Chao; Shang, Liguo; Yang, Zhimin; Zhang, Wei; Wang, Wei; Li, Yun; Ke, Qi; Lu, Jiasi; Xu, Yuquan; Zhang, Liwen; Xie, Zhihong; Cheng, Qi; Elmerich, Claudine; Lin, Min

    2016-01-01

    Unlike most Pseudomonas, the root-associated bacterium Pseudomonas stutzeri A1501 fixes nitrogen after the horizontal acquisition of a nitrogen-fixing (nif) island. A genome-wide search for small noncoding RNAs (ncRNAs) in P. stutzeri A1501 identified the novel P. stutzeri-specific ncRNA NfiS in the core genome, whose synthesis was significantly induced under nitrogen fixation or sorbitol stress conditions. The expression of NfiS was RNA chaperone Hfq-dependent and activated by the sigma factor RpoN/global nitrogen activator NtrC/nif-specific activator NifA regulatory cascade. The nfiS-deficient mutant displayed reduced nitrogenase activity, as well as increased sensitivity to multiple stresses, such as osmotic and oxidative stresses. Secondary structure prediction and complementation studies confirmed that a stem-loop structure was essential for NfiS to regulate the nitrogenase gene nifK mRNA synthesis and thus nitrogenase activity. Microscale thermophoresis and physiological analysis showed that NfiS directly pairs with nifK mRNA and ultimately enhances nitrogenase activity by increasing the translation efficiency and the half-life of nifK mRNA. Our data also suggest structural and functional divergence of NfiS evolution in diazotrophic and nondiazotrophic backgrounds. It is proposed that NfiS was recruited by nifK mRNA as a novel regulator to integrate the horizontally acquired nif island into host global networks. PMID:27407147

  12. Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

    PubMed Central

    2010-01-01

    Background The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance. Methods Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05. Results IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p < 0.001), bronchioles (0.294 ± 0.139 vs. 0.646 ± 0.172, p < 0.001) and in the septal interstitium (0.027 ± 0.014 vs. 0.067 ± 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002) and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression (p < 0.0001). Conclusions Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment. PMID:20047687

  13. Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses.

    PubMed

    Alves-Costa, Fernanda A; Wasko, A P

    2010-03-01

    Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues. Therefore, we attempted to identify sexually dimorphic gene expression in the brains of adult males and females of Leporinus macrocephalus, a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase-PCR and real-time PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragments presented no significant identity with previously reported gene sequences. The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein-binding, family A, member 2) and to the Rab 37 gene, a member of the Ras oncogene family. The overexpression of these genes has been associated to a greater production of the beta-amyloid protein which, in turns, is the major factor that leads to Alzheimer's disease, and to the development of brain-tumors, respectively. Quantitative RT-PCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus may represent an interesting animal model to the understanding of the function of several vertebrate genes, including those involved in neurodegenerative and cancer diseases.

  14. Deep sequencing reveals different compositions of mRNA transcribed from the F8 gene in a panel of FVIII-producing CHO cell lines.

    PubMed

    Kaas, Christian S; Bolt, Gert; Hansen, Jens J; Andersen, Mikael R; Kristensen, Claus

    2015-07-01

    Coagulation factor VIII (FVIII) is one of the most complex biopharmaceuticals due to the large size, poor protein stability and extensive post-translational modifications. As a consequence, efficient production of FVIII in mammalian cells poses a major challenge, with typical yields two to three orders of magnitude lower than for antibodies. In the present study we investigated CHO DXB11 cells transfected with a plasmid encoding human coagulation factor VIII. Single cell clones were isolated from the pool of transfectants and a panel of 14 clones representing a dynamic range of FVIII productivities was selected for RNA sequencing analysis. The analysis showed distinct differences in F8 RNA composition between the clones. The exogenous F8-dhfr transcript was found to make up the most abundant transcript in the present clones. No correlation was seen between F8 mRNA levels and the measured FVIII productivity. It was found that three MTX resistant, nonproducing clones had different truncations of the F8 transcripts. We find that by using deep sequencing, in contrast to microarray technology, for determining the transcriptome from CHO transfectants, we are able to accurately deduce the mature mRNA composition of the transgene and identify significant truncations that would probably otherwise have remained undetected.

  15. The role of the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene in cytochrome oxidase assembly: mutation causes lowered levels of COX (cytochrome c oxidase) I and COX III mRNA

    PubMed Central

    2004-01-01

    Leigh syndrome French Canadian (LSFC) is a variant of cytochrome oxidase deficiency found in Québec and caused by mutations in the LRPPRC (leucine-rich pentatricopeptide repeat cassette) gene. Northern blots showed that the LRPPRC mRNA levels seen in skeletal muscle>heart>placenta>kidney>liver>lung=brain were proportionally almost opposite in strength to the severity of the enzymic cytochrome oxidase defect. The levels of COX (cytochrome c oxidase) I and COX III mRNA visible on Northern blots were reduced in LSFC patients due to the common (A354V, Ala354→Val) founder mutation. The amount of LRPPRC protein found in both fibroblast and liver mitochondria from LSFC patients was consistently reduced to <30% of control levels. Import of [35S]methionine LRPPRC into rat liver mitochondria was slower for the mutant (A354V) protein. A titre of LRPPRC protein was also found in nuclear fractions that could not be easily accounted for by mitochondrial contamination. [35S]Methionine labelling of mitochondrial translation products showed that the translation of COX I, and perhaps COX III, was specifically reduced in the presence of the mutation. These results suggest that the gene product of LRPPRC, like PET 309p, has a role in the translation or stability of the mRNA for mitochondrially encoded COX subunits. A more diffuse distribution of LRPPRC in LSFC cells compared with controls was evident when viewed by immunofluorescence microscopy, with less LRPPRC present in peripheral mitochondria. PMID:15139850

  16. Contrasted survival under field or controlled conditions displays associations between mRNA levels of candidate genes and response to OsHV-1 infection in the Pacific oyster Crassostrea gigas.

    PubMed

    Normand, Julien; Li, Ronghua; Quillien, Virgile; Nicolas, Jean-Louis; Boudry, Pierre; Pernet, Fabrice; Huvet, Arnaud

    2014-06-01

    Pacific oyster Crassostrea gigas suffers from chronic or sporadic mortality outbreaks worldwide, resulting from infectious diseases and/or physiological disorders triggered by environmental factors. Since 2008, ostreid herpesvirus OsHV-1 μVar has been identified as the main agent responsible for mass mortality of juvenile oysters in Europe. Previous studies of genome-wide expression profiling have provided candidate genes that potentially contribute to genetically-based resistance to summer mortality. To assess their value in determining resistance to the juvenile mass mortality that has occurred in France since 2008, we analyzed the expression of 17 candidate genes in an experimental infection by OsHV-1 μVar, and in an in vivo field experiment. Individual quantification of mRNA levels of 10 out of the 17 targeted genes revealed significant variation, of which 7 genes were showed differences between conditions that created significant differences in mortality, and 6 depended on the number of OsHV-1 genome copies individually quantified in mantle tissue. Complex SOD metalloenzymes known to be part of the antioxidant defense strategies may at least partly determine susceptibility or resistance to OsHV-1-associated mortality. Furthermore, inhibitor 2 of NF-κB, termed CgIκB2, exhibited highly significant variation of mRNA levels depending on OsHV-1 load in both experiments, suggesting its implication in the antiviral immune response of C. gigas. Our results suggest that CgIκB2 expression would make a good starting point for further functional research and that it could be used in marker-assisted selection.

  17. Promoter region hypermethylation and mRNA expression of MGMT and p16 genes in tissue and blood samples of human premalignant oral lesions and oral squamous cell carcinoma.

    PubMed

    Bhatia, Vikram; Goel, Madhu Mati; Makker, Annu; Tewari, Shikha; Yadu, Alka; Shilpi, Priyanka; Kumar, Sandeep; Agarwal, S P; Goel, Sudhir K

    2014-01-01

    Promoter methylation and relative gene expression of O(6)-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

  18. Promoter Region Hypermethylation and mRNA Expression of MGMT and p16 Genes in Tissue and Blood Samples of Human Premalignant Oral Lesions and Oral Squamous Cell Carcinoma

    PubMed Central

    Bhatia, Vikram; Makker, Annu; Tewari, Shikha; Yadu, Alka; Shilpi, Priyanka; Kumar, Sandeep; Agarwal, S. P.; Goel, Sudhir K.

    2014-01-01

    Promoter methylation and relative gene expression of O6-methyguanine-DNA-methyltransferase (MGMT) and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs) and oral squamous cell carcinoma (OSCC). Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P = 0.0010) and 57% (P = 0.0016) of tissue samples, respectively, and 39% (P = 0.0135) and 33% (P = 0.0074) of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P = 0.0001) and 82% (P = 0.0001) in tissue and 57% (P = 0.0002) and 70% (P = 0.0001) in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC. PMID:24991542

  19. Sequences of the 5' portion of the human c-sis gene: characterization of the transcriptional promoter and regulation of expression of the protein product by 5' untranslated mRNA sequences.

    PubMed Central

    Ratner, L; Thielan, B; Collins, T

    1987-01-01

    The c-sis gene encodes the B polypeptide chain of platelet-derived growth factor (PDGF), and is expressed in a number of normal and pathological conditions. In order to study the control of synthesis of the human c-sis product, we have initiated a study of two regions of this genetic locus which regulate transcription and translation. A clone of the 5' portion of the gene was obtained which included 1361 nucleotides upstream of the RNA initiation site. Transcriptional promoter activity of this region was demonstrated in normal and transformed cells using a plasmid with the sequences upstream of the c-sis RNA initiation site fused to an indicator gene, chloramphenicol acetyl transferase. Experiments were also performed to identify other possible regulatory regions of the c-sis gene. These data demonstrated that a portion of the c-sis first exon encoding the 5' untranslated region of the c-sis mRNA inhibited synthesis of the PDGF B product in vitro. These results define regions of the c-sis gene whose activity may be important in the regulation of transcription and translation under normal conditions and in the pathogenesis several human diseases. Images PMID:3627977

  20. CYP1B1 mRNA inducibility due to benzo(a)pyrene is modified by the CYP1B1 L432V gene polymorphism.

    PubMed

    Helmig, Simone; Wenzel, Sibylle; Maxeiner, Hagen; Schneider, Joachim

    2014-07-01

    Benzo(a)pyrene (BaP), a primary component of tobacco smoke, is activated by cytochrome P450 1B1 (CYP1B1). Smokers homozygous for the C-allele (*1/*1) at the CYP1B1 Leu432Val polymorphism have shown increased CYP1B1 expression, compared to smokers homozygous for the G-allele *3/*3. Since no difference has been shown in CYP1B1 expression between both genotypes in non-smokers, we assumed that the genetic impact is produced in combination with an exogenous induction (e.g. BaP). To confirm this theory and to quantify the effect, we induced human leucocytes with increasing BaP concentrations and determined CYP1B1 mRNA expression with real-time polymerase chain reaction (PCR). We incubated human leucocytes from 27 healthy donors with BaP concentrations ranging from 2.5 to 250 µM. We identified the CYP1B1 genotypes by melting curve analysis and assessed relative CYP1B1 mRNA expression using real-time PCR. Expression was related to β-2-microglobulin with the 2(-ΔΔCT) method. Inducibility of CYP1B1 mRNA by BaP was higher in leucocytes carrying the CYP1B1*1/*1 genotype than in leucocytes carrying the CYP1B1*3/*3 genotype (P = 0.012). We revealed significant differences, with BaP concentrations of 2.5 µM (P = 0.0094), 5 µM (P = 0.027), 10 µM (P = 0.0006), 25 µM (P = 0.0007) and 50 µM (P = 0.017). Homozygous carriers of the C-allele (*1/*1) at the CYP1B1 Leu432Val polymorphism show a higher response to environmental factors, such as carcinogenic BaP, than homozygous carriers of the G-allele *3/*3.

  1. Increased apolipoprotein E and c-fms gene expression without elevated interleukin 1 or 6 mRNA levels indicates selective activation of macrophage functions in advanced human atheroma.

    PubMed Central

    Salomon, R N; Underwood, R; Doyle, M V; Wang, A; Libby, P

    1992-01-01

    Cells found within atherosclerotic lesions can produce in culture protein mediators that may participate in atherogenesis. To test whether human atheromata actually contain transcripts for certain of these genes, we compared levels of mRNAs in carotid or coronary atheromata and in nonatherosclerotic human vessels by polymerase chain reaction (PCR) amplification of cDNAs reverse-transcribed from RNA. We measured PCR products (generated during exponential amplification) by incorporation of 32P-labeled primers. Levels of interleukin 1 alpha, 1 beta, or 6 mRNAs in plaques and controls did not differ. Compared to uninvolved vessels, plaques did contain higher levels of mRNA encoding platelet-derived growth factor A chain (42 +/- 24 vs. 12 +/- 10 fmol of product; mean +/- SD; n = 8 and 8, respectively; P = 0.007) and B chain (41 +/- 36 vs. 4 +/- 3 fmol of product, n = 14 and 6, respectively; P = 0.024). Atherosclerotic lesions consistently had much higher levels of apolipoprotein E (apoE) mRNA than did control vessels (131 +/- 71 vs. 5 +/- 3 fmol of product; n = 12 and 10, respectively; P less than 0.001). Direct RNA blot analyses confirmed elevated levels of apoE mRNA in plaque extracts. To test whether mononuclear phagocytes might be a source of the apoE mRNA, we studied a selective marker for cells of the monocytic lineage, the c-fms protooncogene, which encodes the receptor for macrophage colony-stimulating factor. Plaques also contained elevated levels of c-fms mRNA (30 +/- 17 vs. 5 +/- 3 fmol of product; n = 10 and 7, respectively; P = 0.002). Immunohistochemical colocalization demonstrated apoE protein in association with macrophages in plaques, whereas nonatherosclerotic vessels showed no immunoreactive apoE. ApoE produced locally in atheroma might modulate the functions of lesional T cells or promote "reverse cholesterol transport" by associating with high density lipoprotein particles, thus targeting them for peripheral uptake. Macrophages within the advanced

  2. Cytoplasmic mRNA turnover and ageing

    PubMed Central

    Borbolis, Fivos; Syntichaki, Popi

    2015-01-01

    Messenger RNA (mRNA) turnover that determines the lifetime of cytoplasmic mRNAs is a means to control gene expression under both normal and stress conditions, whereas its impact on ageing and age-related disorders has just become evident. Gene expression control is achieved at the level of the mRNA clearance as well as mRNA stability and accessibility to other molecules. All these processes are regulated by cis-acting motifs and trans-acting factors that determine the rates of translation and degradation of transcripts. Specific messenger RNA granules that harbor the mRNA decay machinery or various factors, involved in translational repression and transient storage of mRNAs, are also part of the mRNA fate regulation. Their assembly and function can be modulated to promote stress resistance to adverse conditions and over time affect the ageing process and the lifespan of the organism. Here, we provide insights into the complex relationships of ageing modulators and mRNA turnover mechanisms. PMID:26432921

  3. Analysis of mRNA With Microsomal Fractionation Using a SAGE-Based DNA Microarray System Facilitates Identification of the Genes Encoding Secretory Proteins

    PubMed Central

    Toyoda , Nobuaki; Nagai, Shigenori; Terashima, Yuya; Motomura, Kazushi; Haino, Makoto; Hashimoto, Shin-ichi; Takizawa, Hajime; Matsushima, Kouji

    2003-01-01

    In the regulation of host defense responses such as inflammation and immunity, the secretory proteins, including membrane proteins, play central roles. Although many secretory proteins have been identified by using methods such as differential display, random screening, or the signal sequence trap method, each method suffers from poor reproducibility, low sensitivity, or time-consuming or laborious work. Therefore, the strategy for facilitating the selection of the genes encoding the secretory proteins is desired. In this paper, we describe a system for isolating the genes encoding secretory proteins by analyzing mRNAs with microsomal fractionation on serial analysis of gene expression (SAGE)–based DNA microarray system. This system succeeded in discriminating the genes encoding secretory proteins from ones encoding nonsecretory proteins with 80% accuracy. We applied this system to human T lymphocytes. As a result, we were able to identify the genes that are not only encoding secretory proteins but also expressing selectively in a specific subset of T lymphocytes. The SAGE-based DNA microarray system is a promising system to identify the genes encoding specific secretory proteins. PMID:12805275

  4. A targeted gene expression platform allows for rapid analysis of chemical-induced antioxidant mRNA expression in zebrafish larvae

    PubMed Central

    Mills, Margaret G.; Gallagher, Evan P.

    2017-01-01

    Chemical-induced oxidative stress and the biochemical pathways that protect against oxidative damage are of particular interest in the field of toxicology. To rapidly identify oxidative stress-responsive gene expression changes in zebrafish, we developed a targeted panel of antioxidant genes using the Affymetrix QuantiGene Plex (QGP) platform. The genes contained in our panel include eight putative Nrf2 (Nfe2l2a)-dependent antioxidant genes (hmox1a, gstp1, gclc, nqo1, prdx1, gpx1a, sod1, sod2), a stress response gene (hsp70), an inducible DNA damage repair gene (gadd45bb), and three reference genes (actb1, gapdh, hprt1). We tested this platform on larval zebrafish exposed to tert-butyl hydroperoxide (tBHP) and cadmium (Cd), two model oxidative stressors with different modes of action, and compared our results with those obtained using the more common quantitative PCR (qPCR) method. Both methods showed that exposure to tBHP and Cd induced expression of prdx1, gstp1, and hmox1a (2- to 12-fold increase via QGP), indicative of an activated Nrf2 response in larval zebrafish. Both compounds also elicited a general stress response as reflected by elevation of hsp70 and gadd45bb, with Cd being the more potent inducer. Transient changes were observed in sod2 and gpx1a expression, whereas nqo1, an Nrf2-responsive gene in mammalian cells, was minimally affected by either tBHP or Cd chemical exposures. Developmental expression analysis of the target genes by QGP revealed marked upregulation of sod2 between 0-96hpf, and to a lesser extent, of sod1 and gstp1. Once optimized, QGP analysis of these experiments was accomplished more rapidly, using far less tissue, and at lower total costs than qPCR analysis. In summary, the QGP platform as applied to higher-throughput zebrafish studies provides a reasonable cost-effective alternative to qPCR or more comprehensive transcriptomics approaches to rapidly assess the potential for chemicals to elicit oxidative stress as a mechanism of

  5. A targeted gene expression platform allows for rapid analysis of chemical-induced antioxidant mRNA expression in zebrafish larvae.

    PubMed

    Mills, Margaret G; Gallagher, Evan P

    2017-01-01

    Chemical-induced oxidative stress and the biochemical pathways that protect against oxidative damage are of particular interest in the field of toxicology. To rapidly identify oxidative stress-responsive gene expression changes in zebrafish, we developed a targeted panel of antioxidant genes using the Affymetrix QuantiGene Plex (QGP) platform. The genes contained in our panel include eight putative Nrf2 (Nfe2l2a)-dependent antioxidant genes (hmox1a, gstp1, gclc, nqo1, prdx1, gpx1a, sod1, sod2), a stress response gene (hsp70), an inducible DNA damage repair gene (gadd45bb), and three reference genes (actb1, gapdh, hprt1). We tested this platform on larval zebrafish exposed to tert-butyl hydroperoxide (tBHP) and cadmium (Cd), two model oxidative stressors with different modes of action, and compared our results with those obtained using the more common quantitative PCR (qPCR) method. Both methods showed that exposure to tBHP and Cd induced expression of prdx1, gstp1, and hmox1a (2- to 12-fold increase via QGP), indicative of an activated Nrf2 response in larval zebrafish. Both compounds also elicited a general stress response as reflected by elevation of hsp70 and gadd45bb, with Cd being the more potent inducer. Transient changes were observed in sod2 and gpx1a expression, whereas nqo1, an Nrf2-responsive gene in mammalian cells, was minimally affected by either tBHP or Cd chemical exposures. Developmental expression analysis of the target genes by QGP revealed marked upregulation of sod2 between 0-96hpf, and to a lesser extent, of sod1 and gstp1. Once optimized, QGP analysis of these experiments was accomplished more rapidly, using far less tissue, and at lower total costs than qPCR analysis. In summary, the QGP platform as applied to higher-throughput zebrafish studies provides a reasonable cost-effective alternative to qPCR or more comprehensive transcriptomics approaches to rapidly assess the potential for chemicals to elicit oxidative stress as a mechanism of

  6. Simulated microgravity reduces mRNA levels of multidrug resistance genes 4 and 5 in non-metastatic human melanoma cells

    NASA Astrophysics Data System (ADS)

    Eiermann, Peter; Tsiockas, Wasiliki; Hauslage, Jens; Hemmersbach, Ruth; Gerzer, Rupert; Ivanova, Krassimira

    Multidrug resistance proteins (MRP) are members of the ATP-binding cassette transporter superfamily that are able to export a large variety of substances into the extracellular space in-cluding nucleoside and nucleotide base analogs used in antiviral and anticancer therapy. MRP4 and 5 (MRP4/5) particularly transport cyclic nucleotides, e.g. guanosine 3',5'-cyclic monophos-phate (cGMP). The second messenger cGMP, which is synthesized by the catalytic activity of the guanylyl cyclase (GC), plays an import role in vasodilatation, smooth muscle relaxation, and nitric oxide (NO)-induced perturbation of melanocyte-extracellular matrix interactions. In previous studies we have reported that different GC isoforms are responsible for cGMP synthe-sis in melanocytic cells. Normal human melanocytes and non-metastatic melanoma cell lines predominantly express the NO-sensitive soluble GC isoform (sGC), a heterodimeric protein consisting of α and β subunits. Metastatic melanoma cells lack the expression of the β sub-unit and show up-regulated activities of the particulate isoforms. We have further found that long-term exposure to hypergravity (5 g for 24 h) induced an increased cGMP export in normal human melanocytes, and non-metastatic, but not in metastatic human melanoma cells as a re-sult of up-regulated MRP4/5 expression. The aim of the present study is to investigate whether simulated microgravity may also alter the expression of MRP4/5 in non-metastatic melanoma cells. Experiments were performed using a fast-rotating clinostat (60 rpm) with one rotation axis. The non-metastatic 1F6 melanoma cells were exposed to simulated microgravity (up to 1.21x10-2 g) for 24 h. The mRNA analyses were performed by a relative calibrator-normalized and efficiency corrected quantitative polymerase chain reaction (Light Cycler R , Roche). Our data show a reduced expression of approximately 35% for MRP4 and of 50% for MRP5 in simulated microgravity in comparison to 1 g controls. Also, the

  7. Two NF-κB inhibitor-alpha (IκBα) genes from rock bream (Oplegnathus fasciatus): molecular characterization, genomic organization and mRNA expression analysis after immune stimulation.

    PubMed

    Lee, Youngdeuk; Umasuthan, Navaneethaiyer; Whang, Ilson; Revathy, Kasthuri Saranya; Lee, Sukkyoung; De Zoysa, Mahanama; Oh, Chulhong; Kang, Do-Hyung; Noh, Jae Koo; Lee, Jehee

    2014-12-01

    IkBa is a member of IkB family, which sequesters NF-kB in an inactivate form in the cytoplasm and blocks the translocation of NF-kB to nucleus. The IkBa paralogs of rock bream (OfIkBa-A and OfIkBa-B) encoded IkBa proteins with typical features including, highly conserved IkB degradation motif, six ankyrin repeats and a PEST sequence. However, their amino acid identity and similarity were only 55.6 and 69.7%, respectively suggesting that these two genes could be the two different isoforms of IkBa. The number and size of the exons of OfIkBa-A and OfIkBa-B were conserved well with all the compared vertebrate species, although they have significantly different genomic sizes. Phylogenetic analysis revealed that OfIkBa-A and OfIkBa-B proteins cluster with IkBa family members; however, they were grouped with different subclades in IkBa family. Tissue specific expression of OfIkBa mRNA was constitutively detected in all the tested tissues, and they showed the higher transcription level in heart, liver, gill and peripheral blood cells, respectively. The injection of flagellin stimulated the mRNA expression of OfIkBa paralogs in head kidney and intestine. Moreover, the OfIkBa mRNA expression in gill and liver was significantly upregulated by LPS, poly I:C and Edwardsiella tarda challenges. The transcription of OfIkBa was up-regulated in early-phase of injection and then rapidly restored. These results suggest that the OfIkBa paralogs might be involved in rapid immune responsive reactions in rock bream against bacterial and viral pathogens.

  8. Identification of adaptation-specific differences in mRNA expression of sessile and pedunculate oak based on osmotic-stress-induced genes.

    PubMed

    Porth, Ilga; Koch, Margit; Berenyi, Maria; Burg, Agnes; Burg, Kornel

    2005-10-01

    Quercus petraea (Matt.) Liebl. and Q. robur L. hybridize frequently and occupy similar, though distinct, ecological niches. So far, genetic discrimination between these species at the molecular level has been based mainly on neutral markers. Because such markers often exhibit low species differentiation because of high genetic compatibility and exchange between Q. robur and Q. petraea at these loci, we used adaptation-related expressed genes as markers. Accordingly, we identified osmotic-stress-induced genes in a Q. petraea cell line grown under moderate osmotic stress conditions. Two subtraction libraries were established from callus cells cultured under hyperosmotic stress for 1 or 48 h. Thirty-three differentially expressed sequence tags (ESTs) (from 70 originally isolated) were classified according to their putative functions. At least five of these gene products may contribute to osmotic-stress tolerance in oak: betaine aldehyde dehydrogenase, two trans-acting transcription factors (one abscsic acid (ABA)-responsive, the other ABA-independent), a glutathione-S- transferase and a heat-shock cognate protein. Seven genes were selected based on their putative function and their expression monitored in vivo. Leaf tissue from Q. petraea and Q. robur plantlets grown hydroponically under hyperosmotic conditions was harvested after 0, 1, 6, 24 or 72 h and analyzed by real-time polymerase chain reaction (PCR). We found indications of osmotic stress adaptation in Q. petraea based on up-regulation of genes related to protective functions, whereas down-regulation of these genes was evident in Q. robur. Thus, genetic markers related to adaptive traits may be useful for differentiating Q. petraea and Q. robur genotypes.

  9. Critical evaluation of KCNJ3 gene product detection in human breast cancer: mRNA in situ hybridisation is superior to immunohistochemistry

    PubMed Central

    Kammerer, Sarah; Jahn, Stephan Wenzel; Winter, Elke; Eidenhammer, Sylvia; Rezania, Simin; Regitnig, Peter; Pichler, Martin; Schreibmayer, Wolfgang; Bauernhofer, Thomas

    2016-01-01

    Increased expression levels of KCNJ3 have been correlated with lymph node metastases and poor prognosis in patients with breast cancer, suggesting a prognostic role of KCNJ3. We aimed to establish protocols for the detection of KCNJ3 in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue. Several antibodies were tested for sensitivity and specificity by western blot, followed by optimisation of the immunohistochemistry (IHC) procedure and establishment of KCNJ3 mRNA in situ hybridisation (ISH). Methods were validated by processing 15 FFPE breast cancer samples for which microarray data were available. Spearman's rank correlation analysis resulted in borderline significant correlation for IHC versus ISH (rS: 0.625; p<0.05) and IHC versus microarray (rS: 0.668; p<0.01), but in significant correlation for ISH versus microarray (rS: 0.861; p<0.001). The ISH method was superior to IHC, regarding robustness, sensitivity and specificity and will aid to further study expression levels of KCNJ3 in both malignant and physiological conditions. PMID:27698251

  10. Critical evaluation of KCNJ3 gene product detection in human breast cancer: mRNA in situ hybridisation is superior to immunohistochemistry.

    PubMed

    Kammerer, Sarah; Jahn, Stephan Wenzel; Winter, Elke; Eidenhammer, Sylvia; Rezania, Simin; Regitnig, Peter; Pichler, Martin; Schreibmayer, Wolfgang; Bauernhofer, Thomas

    2016-12-01

    Increased expression levels of KCNJ3 have been correlated with lymph node metastases and poor prognosis in patients with breast cancer, suggesting a prognostic role of KCNJ3 We aimed to establish protocols for the detection of KCNJ3 in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue. Several antibodies were tested for sensitivity and specificity by western blot, followed by optimisation of the immunohistochemistry (IHC) procedure and establishment of KCNJ3 mRNA in situ hybridisation (ISH). Methods were validated by processing 15 FFPE breast cancer samples for which microarray data were available. Spearman's rank correlation analysis resulted in borderline significant correlation for IHC versus ISH (rS: 0.625; p<0.05) and IHC versus microarray (rS: 0.668; p<0.01), but in significant correlation for ISH versus microarray (rS: 0.861; p<0.001). The ISH method was superior to IHC, regarding robustness, sensitivity and specificity and will aid to further study expression levels of KCNJ3 in both malignant and physiological conditions.

  11. mRNA levels of imprinted genes in bovine in vivo oocytes, embryos and cross species comparisons in humans, mice and pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty-six confirmed imprinted genes in the bovine were quantified in in vivo produced oocytes and embryos. Eighteen were detectable and their transcriptional abundance were categorized into five patterns: largely decreased (MEST and PLAGL1); first decreased and then increased (CDKN1C and IGF2R); p...

  12. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

    PubMed Central

    Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

    2016-01-01

    The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries. PMID:27886048

  13. Molecular Characterization, mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy)

    PubMed Central

    Wang, Ke-Yi; Jiang, Xuan-Zhao; Yuan, Guo-Rui; Shang, Feng; Wang, Jin-Jun

    2015-01-01

    Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action. PMID:26154764

  14. Molecular identification and characterization of a gene associated with the onset of tapping panel dryness (TPD) syndrome in rubber tree (Hevea brasiliensis Muell.) by mRNA differential display.

    PubMed

    Venkatachalam, Perumal; Thulaseedharan, Arjunan; Raghothama, Kashchandra

    2009-01-01

    In rubber tree (Hevea brasiliensis), tapping panel dryness (TPD) syndrome is considered as a complex physiological disorder which affects latex biosynthesis. To identify differentially expressed genes between healthy and TPD-affected trees, mRNA differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) analysis was performed. We isolated 10 differentially expressed cDNA fragments of which one cDNA encoding a putative TOM20 like protein was identified. The cDNA (1,024 bp), corresponding to the HbTOM20 gene (H evea b rasiliensis Translocase of the Outer Mitochondrial Membrane), contained an open reading frame to code for 202 amino acid protein with a theoretical pI value of 9.5 and the calculated protein M (W) was 23.5 kDa. The predicted amino acid sequence contained conserved domains of TOM20 like proteins in the N-terminal. The protein HbTOM20 has 32% and 27% similarity to Populus TOM20 and Solanum TOM20, respectively. Both semi-quantitative RT-PCR and Northern blot results revealed that the HbTOM20 expression was significantly down-regulated in TPD-affected trees compared to healthy one. Accumulation of HbTOM20 mRNA transcripts was significantly higher in the bark tissues collected from healthy region than that of partially affected by TPD (partially dried) while barely detectable in completely TPD-affected area. Differential expression pattern was noticed in three rubber clones representing various degrees of TPD tolerance. These results suggest that down-regulation of HbTOM20 in TPD-affected trees may play an important role in alteration of mitochondrial metabolism resulting in impaired latex biosynthesis.

  15. Guggulipid and nimesulide differentially regulated inflammatory genes mRNA expressions via inhibition of NF-kB and CHOP activation in LPS-stimulated rat astrocytoma cells, C6.

    PubMed

    Niranjan, Rituraj; Nath, Chandishwar; Shukla, Rakesh

    2011-07-01

    Neuroinflammation is an integral part of neurodegenerative diseases. Lipo-polysacharide (LPS) induces reactive astrogliosis, the cellular manifestation of neuroinflammation, in various models of neurological diseases, but its mechanism of action is still not properly known. The effect of guggulipid and nimesulide on LPS-induced neuroinflammatory changes is also not properly understood. This work demonstrated the mechanism of actions of guggulipid and nimesulide on inflammatory genes expressions in LPS-stimulated rat astrocytoma cells, C6. We observed that LPS (10 μg/ml) treatment of rat astrocytoma cells, C6, for 24 h significantly increased intracellular Ca(2+) ion and expression of inducible nitric oxide synthase (iNOS), nuclear factor kappa-B (NF-kB), C/EBP homologous protein 10 (CHOP), c-fos, and c-jun proteins. At transcriptional stage, LPS upregulated mRNA levels of cyclooxygenase-2 and IL-6 with downregulation in IL-1α, IL-1β, and microsomal prostaglandin E synthase-1 (mPGES-1) through activating NF-kB translocation. Treatment with guggulipid reversed these LPS-induced changes in rat astrocytoma cells. Treatment with nimesulide also attenuated LPS-induced Ca(2+) ion, iNOS, NF-kB, and c-fos expressions, but does not significantly influence CHOP, c-jun protein expressions, and mRNA levels of IL-6, IL-1α, IL-1β, and mPGES-1 genes. In conclusion, our findings elucidated the molecular mechanism of neuroinflammation in response to LPS and its modulation by guggulipid and nimesulide in rat astrocytoma cells (C6), which suggest the use of these drugs in the treatment of neuroinflammation-associated disorders.

  16. Molecular cloning and characterization of cDNAs coding for apo-polysialoglycoprotein of rainbow trout eggs. Multiple mRNA species transcribed from multiple genes contain diverged numbers of exact 39-base (13-amino acid) repeats.

    PubMed

    Sorimachi, H; Emori, Y; Kawasaki, H; Kitajima, K; Inoue, S; Suzuki, K; Inoue, Y

    1988-11-25

    Polysialoglycoprotein (PSGP) of unfertilized eggs of rainbow trout (Salmo gairdneri) consists of tandem repeats (about 25) of a glycotridecapeptide, Asp-Asp-Ala-Thr*-Ser*-Glu-Ala-Ala-Thr*-Gly-Pro-Ser-Gly (* denotes the attachment site of a polysialoglycan chain) (Kitajima, K., Inoue, Y., and Inoue, S. (1986) J. Biol. Chem. 261, 5262-5269). By using oligodeoxynucleotide probes based on the above sequence, we isolated a genomic clone for apoPSGP which contains 39-base pair repeats (5'-GACGACGCCACCTCTGAAGCT-GCGACCGGCCCGTCTGGC-3') encoding the tridecapeptide. Using a fragment of this genomic DNA as a probe, we next screened a cDNA library constructed with mRNA from immature ovaries of rainbow trout. Nucleotide sequencing analyses of cDNA clones thus obtained revealed that apoPSGP is encoded by multiple mRNA species consisting of diverged numbers (6-32) of the 39-base repeat encoding the tridecapeptide unit and homologous 5'- and 3'-bordering regions. The encoded protein consists of three distinct regions: the N-region consisting of a putative signal peptide and a pro-peptide, the R-region containing diverged numbers of the tandem repeat of 13-amino acid residues, and the C-region with six amino acid residues. Southern blot analysis showed that multiple mRNAs are transcribed from multiple genes for apoPSGP containing diverged numbers of the 39-base pair repeat. Thus, the genes for apoPSGP constitute a multigene family. Expression of the mRNAs is stage and organ specific, i.e. they are expressed only in immature ovaries and not in mature ovaries or in any other organ.

  17. Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

    PubMed Central

    Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

    2013-01-01

    Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role. PMID:24287918

  18. Up-regulation of mRNA ventricular PRNP prion protein gene expression in air pollution highly exposed young urbanites: endoplasmic reticulum stress, glucose regulated protein 78, and nanosized particles.

    PubMed

    Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

    2013-11-28

    Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.

  19. The effect of two endogenous retinoids on the mRNA expression profile in human primary keratinocytes, focusing on genes causing autosomal recessive congenital ichthyosis.

    PubMed

    Törmä, H; Bergström, A; Ghiasifarahani, G; Berne, B

    2014-10-01

    Retinoids (natural forms and synthetic derivatives of vitamin A) are used as therapeutic agents for numerous skin diseases such as keratinization disorders (e.g. ichthyoses) and psoriasis. Two endogenous ligands for retinoic acid receptors exist, retinoic acid (atRA) and 3,4-didehydroretinoic acid (ddRA). In primary human epidermal keratinocytes many transcriptional targets for atRA are known, whereas the targets for ddRA are unknown. In an attempt to determine the targets, we compared the effect of atRA and ddRA on transcriptional profiles in undifferentiated and differentiating human primary keratinocytes. First, as expected, many genes were induced or suppressed in response to keratinocyte differentiation. Furthermore, the two retinoids affected substantially more genes in differentiated keratinocytes (>350) than in proliferating keratinocytes (≈20). In differentiating keratinocytes markers of cornification were suppressed suggesting a de-differentiating effect by the two retinoids. When comparing the expression profile of atRA to that of ddRA, no differently regulated genes were found. The array analysis also found that a minor number of miRNAs and a large number of non-coding transcripts were changed during differentiation and in response to the two retinoids. Furthermore, the expression of all, except one, genes known to cause autosomal recessive congenital ichthyosis (ARCI) were found to be induced by differentiation. These results comprehensively document that atRA and ddRA exert similar transcriptional changes in keratinocytes and also add new insights into the molecular mechanism influenced by retinoids in the epidermis. Furthermore, it suggests which ARCI patients could benefit from therapy with retinoids.

  20. Understanding of altered N-glycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting.

    PubMed

    Ha, Tae Kwang; Kim, Yeon-Gu; Lee, Gyun Min

    2015-08-01

    To understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10 mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes. Among these queried genes, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5 times in the culture with ammonium addition after 5 days of culture; however, none of the 54 genes were significantly different after 3 days of culture. In particular, the expression level of neu1 (sialidase-1) and neu3 (sialidase-3), which play a role in reduction of sialylation, increased over 2 times. Likewise, the protein expression levels of sialidase-1 and sialidase-3 determined by Western blot analysis were also increased significantly in the culture with ammonium addition. Transient transfection of neu-1 or neu3-targeted siRNAs significantly improved the sialic acid content of the Fc-fusion protein in the culture with ammonium addition, indicating that the decreased sialic acid content was in part due to the increased expression level of sialidase. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of ammonium on N-glycosylation, especially sialylation, in rCHO cells.

  1. Digital mRNA profiling of N-glycosylation gene expression in recombinant Chinese hamster ovary cells treated with sodium butyrate.

    PubMed

    Lee, Sang Min; Kim, Yeon-Gu; Lee, Eun Gyo; Lee, Gyun Min

    2014-02-10

    To understand the effects of sodium butyrate (NaBu) on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing Fc-fusion glycoprotein were subjected to 3mM NaBu. The addition of NaBu to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of the glycoprotein. Fifty-two N-glycosylation-related gene expressions were also assessed by the NanoString nCounter system, which can provide a direct digital readout using custom-designed color-coded probes. Among them, ten genes (ugp, slc35a2, ganc, man1a, man1c, mgat5a, st3gal5, glb1, neu1, and neu3) were up-regulated and three genes (b4galt2, st3gal3, and neu2) were down-regulated significantly. Altered expression patterns in st3gal3, neu1, and neu3, which have roles in the sialic acid biosynthesis pathway, correlated with reduced sialic acid content of the glycoprotein by NaBu. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of NaBu on N-glycosylation in rCHO cells.

  2. Messenger RNA (mRNA) nanoparticle tumour vaccination

    NASA Astrophysics Data System (ADS)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  3. A mutation in the promoter of the chicken β,β-carotene 15,15'-monooxygenase 1 gene alters xanthophyll metabolism through a selective effect on its mRNA abundance in the breast muscle.

    PubMed

    Jlali, M; Graulet, B; Chauveau-Duriot, B; Chabault, M; Godet, E; Leroux, S; Praud, C; Le Bihan-Duval, E; Duclos, M J; Berri, C

    2012-12-01

    A polymorphism in the promoter of the β,β-carotene 15,15'-monooxygenase 1 (BCMO1) gene recently was identified in an experimental cross between 2 chicken lines divergently selected on growth rate and found to be associated with variations in the yellow color of the breast meat. In this study, the effects of the polymorphism on several aspects of carotenoid metabolism were evaluated in chickens sharing the same genetic background except for their genotype at the BCMO1 locus. We confirmed that BCMO1 mRNA abundance varied (P < 0.001) between the 2 homozygous genotypes (GG < AA) and in the pectoralis major muscle. By contrast, BCMO1 mRNA expression was not affected (P > 0.05) by the polymorphism in the duodenum, liver, or sartorius muscle. The breast meat of GG chickens was more (P < 0.001) yellow and richer in lutein (P < 0.01) and zeaxanthin (P < 0.05) compared to that of AA chickens whereas these variables did not differ (P > 0.05) in the other tissues tested. The GG were also characterized by reduced (P < 0.01) plasma lutein and zeaxanthin concentrations than AA without affecting plasma and tissue content of fat-soluble vitamins A and E. As lutein and zeaxanthin are usually not considered as substrates of the BCMO1 enzyme, the impact of BCMO1 polymorphism on the activity of other genes involved in carotenoid transport (SCARB1 and CD36 encoding the scavenger receptor class B type I and the cluster determinant 36, respectively) and metabolism (BCDO2 encoding β,β-carotene 9',10'-dioxygenase 2) was evaluated. The BCMO1 polymorphism did not affect mRNA abundance of BCDO2, SCARB1, or CD36, regardless of tissue considered. Taken together, these results indicated that a genetic variant of BCMO1 specifically changes lutein and zeaxanthin content in the chicken plasma and breast muscle without impairing vitamin A and E metabolism.

  4. Nerve Growth Factor, Brain-Derived Neurotrophic Factor and Osteocalcin Gene Relationship in Energy Regulation, Bone Homeostasis and Reproductive Organs Analyzed by mRNA Quantitative Evaluation and Linear Correlation Analysis

    PubMed Central

    Camerino, Claudia; Conte, Elena; Cannone, Maria; Caloiero, Roberta; Fonzino, Adriano; Tricarico, Domenico

    2016-01-01

    Nerve Growth Factor (NGF)/Brain-derived Neurotrophic Factor (BDNF) and osteocalcin share common effects regulating energy, bone mass, reproduction and neuronal functions. To investigate on the gene-relationship between NGF, BDNF, and Osteocalcin we compared by RT-PCR the transcript levels of Ngf, Bdnf and Osteocalcin as well as of their receptors p75NTR/NTRK1, NTRK2, and Gprc6a in brain, bone, white/brown adipose tissue (WAT/BAT) and reproductive organs of 3 months old female and male mice. Brain and bone were used as positive controls for NGF/BDNF and Osteocalcin respectively. The role of oxitocin(Oxt) and its receptor(Oxtr) was also investigated. Ngf expression shows an opposite trend compared to Bdnf. Ngf /p75NTR expression is 50% higher in BAT than brain, in both genders, but lower in bone. In contrast, Bdnf expression in bone is higher than in brain, but low in BAT/WAT. We found Osteocalcin gene expressed in brain in both genders, but Gprc6a expression is low in brain and BAT/WAT. As expected, Gprc6a gene is expressed in bone. Oxt gene was markedly expressed in brain, Oxtr in the ovaries and in fat and bone in both genders. Ngf is highly expressed in reproductive tissues and p75NTR mRNA levels are respectively 300, 100, and 50% higher in testis/ovaries/uterus than in brain. In contrast, BDNF genes are not expressed in reproductive tissues. As expected, Gprc6a is expressed in testis but not in the ovaries/uterus. A significant correlation was found between the expression levels of the gene ligands and their receptors in brain, BAT and testis suggesting a common pathway of different genes in these tissues in either male and female. Changes in the expression levels of osteocalcin, Ngf, or Bdnf genes may mutually affect the expression levels of the others. Moreover, it may be possible that different ligands may operate through different receptor subtypes. Oxt and Oxtr failed to show significant correlation. The up-regulation of Ngf /p75NTR in BAT is consistent

  5. Nerve Growth Factor, Brain-Derived Neurotrophic Factor and Osteocalcin Gene Relationship in Energy Regulation, Bone Homeostasis and Reproductive Organs Analyzed by mRNA Quantitative Evaluation and Linear Correlation Analysis.

    PubMed

    Camerino, Claudia; Conte, Elena; Cannone, Maria; Caloiero, Roberta; Fonzino, Adriano; Tricarico, Domenico

    2016-01-01

    Nerve Growth Factor (NGF)/Brain-derived Neurotrophic Factor (BDNF) and osteocalcin share common effects regulating energy, bone mass, reproduction and neuronal functions. To investigate on the gene-relationship between NGF, BDNF, and Osteocalcin we compared by RT-PCR the transcript levels of Ngf, Bdnf and Osteocalcin as well as of their receptors p75NTR/NTRK1, NTRK2, and Gprc6a in brain, bone, white/brown adipose tissue (WAT/BAT) and reproductive organs of 3 months old female and male mice. Brain and bone were used as positive controls for NGF/BDNF and Osteocalcin respectively. The role of oxitocin(Oxt) and its receptor(Oxtr) was also investigated. Ngf expression shows an opposite trend compared to Bdnf. Ngf /p75NTR expression is 50% higher in BAT than brain, in both genders, but lower in bone. In contrast, Bdnf expression in bone is higher than in brain, but low in BAT/WAT. We found Osteocalcin gene expressed in brain in both genders, but Gprc6a expression is low in brain and BAT/WAT. As expected, Gprc6a gene is expressed in bone. Oxt gene was markedly expressed in brain, Oxtr in the ovaries and in fat and bone in both genders. Ngf is highly expressed in reproductive tissues and p75NTR mRNA levels are respectively 300, 100, and 50% higher in testis/ovaries/uterus than in brain. In contrast, BDNF genes are not expressed in reproductive tissues. As expected, Gprc6a is expressed in testis but not in the ovaries/uterus. A significant correlation was found between the expression levels of the gene ligands and their receptors in brain, BAT and testis suggesting a common pathway of different genes in these tissues in either male and female. Changes in the expression levels of osteocalcin, Ngf, or Bdnf genes may mutually affect the expression levels of the others. Moreover, it may be possible that different ligands may operate through different receptor subtypes. Oxt and Oxtr failed to show significant correlation. The up-regulation of Ngf /p75NTR in BAT is consistent

  6. A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus.

    PubMed Central

    Dorsch-Häsler, K; Keil, G M; Weber, F; Jasin, M; Schaffner, W; Koszinowski, U H

    1985-01-01

    Using the simian virus 40 "enhancer trap" approach, we have identified a transcription enhancer located just upstream of the major immediate early gene of murine cytomegalovirus. This enhancer has several striking properties. (i) Together with the enhancer of human cytomegalovirus, it is the strongest transcription enhancer found to date. (ii) It is an extremely long enhancer, spanning greater than 700 base pairs. (iii) It consists of a rather complex pattern of sequence repeats, the longest of which is 181 base pairs. Also, several types of short sequence motifs are scattered throughout the enhancer in monomeric, heterodimeric, or homodimeric (palindromic) form. These motifs have been identified to be components of other enhancers and promoters, and they are presumably binding sites for specific nuclear factors. Our analysis suggests that enhancers are composed of a modular arrangement of short conserved sequence motifs and that enhancer strength is correlated with the redundancy of these motifs. Images PMID:3001696

  7. Identification of the mouse and rat orthologs of the gene mutated in Usher syndrome type IIA and the cellular source of USH2A mRNA in retina, a target tissue of the disease.

    PubMed

    Huang, Dali; Eudy, James D; Uzvolgyi, Eva; Davis, Jack R; Talmadge, Catherine B; Pretto, Dalyir; Weston, Michael D; Lehman, Janae E; Zhou, Ming; Seemayer, Thomas A; Ahmad, Iqbal; Kimberling, William J; Sumegi, Janos

    2002-08-01

    Usher syndrome type IIA (MIM: 27601) is an autosomal recessive disorder characterized by moderate to severe congenital deafness and progressive retinitis pigmentosa. We recently identified the human Usher syndrome type IIA gene (USH2A) on chromosome 1q41, which encodes a protein possessing 10 laminin epidermal growth factor and four fibronectin type 3 domains, both commonly observed in extracellular matrix proteins. To gain insight into the pathogenesis of Usher syndrome type IIA, we isolated and characterized the murine (Ush2a) and rat (rat Ush2a) orthologs of human USH2A. We mapped mouse Ush2a by fluorescence in situ hybridization to mouse chromosome 1 in the region syntenic to human chromosome 1q41. Rat Ush2a has been localized by radiation hybrid mapping to rat chromosome 13 between d13rat49 and d13rat76. The mouse and rat genes, similar to human USH2A, are expressed primarily in retina and cochlea. Mouse Ush2a encodes a 161-kDa protein that shows 68% identity and 9% similarity to the human USH2A protein. Rat Ush2a encodes a 167-kDa protein with 64% identity and 10% similarity to the human protein and 81% identity and 5% similarity to the mouse USH2A protein. The predicted amino acid sequence of the mouse and rat proteins, like their human counterpart, contains a leader sequence, an amino-terminal globular domain, 10 laminin epidermal growth factor domains, and four carboxy-terminal fibronectin type III motifs. With in situ hybridization, we compared the cellular expression of the USH2A gene in rat, mouse, and human retinas. USH2A mRNA in the adult rat, mouse, and human is expressed in the cells of the outer nuclear layer of the retina, one of the target tissues of the disease. In the developing rat retina, Ush2a mRNA expression appears in the neuroepithelium at embryonic day 17.

  8. V kappa gene family in (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies that express CGAT (or pGAT) public idiotypic specificities. Protein and mRNA sequencing of eight monoclonal V kappa chains

    PubMed Central

    1983-01-01

    A large proportion of (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies expresses public idiotypic specificities, termed CGAT (or pGAT), that require the presence of both the heavy and the light chains in order to be expressed. We report in this paper the complete sequence of eight V kappa regions pertaining to eight anti-GAT monoclonal antibodies derived from three strains of mice: BALB/c, DBA/2, and C57BL/6. The methodology used a combination of NH2-terminal amino acid and mRNA nucleotide sequencing. All eight sequences analyzed, although highly homologous and all pertaining to the same V kappa 1 subgroup, allowed definition of three germline genes that are likely to be present in all three strains of mice and also in NZB. It seems likely, however, that any given strain may not necessarily use all three genes for making anti-GAT antibodies. The search for structural correlates of idiotypes could not be framed in a simple picture, but our data suggest that similar idiotopes may result from different interacting primary structures, leading to structural homologies that should be visualized at three-dimensional level. PMID:6415205

  9. The effect of iron and fat in a diet containing green tea extract (Camellia sinensis) on the antioxidant capacity of some organs and the mRNA expression of specific genes in mice.

    PubMed

    Koutelidakis, Antonios E; Kizis, Dimosthenis; Argyri, Konstantina; Kyriakou, Alkistis; Komaitis, Michael; Kapsokefalou, Maria

    2014-11-01

    The hypothesis that iron and fat in the diet may affect green tea extract (GTE) bioactivity, in particular antioxidant capacity and gene expression, was proposed and tested in mice. Thirty mice were randomly assigned to have for 37 days free access to standard or high-fat diets with or without GTE and ferrous lactate. Mice were euthanized and specific organs were removed. Total antioxidant capacity (TAC) was measured using the ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity assays. Polymerase chain reaction was performed on liver and heart mRNA extracts. The FRAP assay showed that GTE from the standard diet did not affect plasma TAC but increased TAC of heart, aorta, and duodenum. GTE from diets enriched with iron resulted to lower TAC of liver and heart than diets with GTE alone. GTE from the fatty diet did not have any effect on TAC compared with fatty control diet, but increased TAC in heart and aorta compared with standard control diet. An effect on expression of the mapk-1 and NF-kB genes in heart was observed in the presence of GTE. These results suggest that GTE may exhibit bioactivity in some organs affected by dietary fat and iron. The findings of this study contribute to the elucidation of the role of dietary components on tea bioactivity.

  10. Molecular Cloning, Characterization and mRNA Expression of a Chitin Synthase 2 Gene from the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae)

    PubMed Central

    Chen, Li; Yang, Wen-Jia; Cong, Lin; Xu, Kang-Kang; Wang, Jin-Jun

    2013-01-01

    Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis. PMID:23965972

  11. Molecular cloning, characterization and mRNA expression of a chitin synthase 2 gene from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

    PubMed

    Chen, Li; Yang, Wen-Jia; Cong, Lin; Xu, Kang-Kang; Wang, Jin-Jun

    2013-08-19

    Chitin synthase (CHS), a potential target for eco-friendly insecticides, plays an essential role in chitin formation in insects. In this study, a full-length cDNA encoding chitin synthase 2 (BdCHS2) was cloned and characterized in the oriental fruit fly, Bactrocera dorsalis. The BdCHS2 cDNA had 4417 nucleotides, containing an open reading frame of 4122 nucleotides, which encoded 1373 amino acid residues with a predicted molecular weight of 158.5 kDa. Phylogenetic analysis with other insect CHSs suggested that BdCHS2 belongs to insect CHS2. The BdCHS2 transcript was predominately found in midgut but was detected at low levels in fat body, Malpighian tubules, integument, and trachea. Moreover, BdCHS2 was expressed in all developmental stages, and highly expressed in the feeding stages. There was a positive relationship between BdCHS2 expression and total chitin content during development. Furthermore, both the gene expression and chitin content in midgut decreased when the insect was fed for 24 h, then starved for 24 h, while they increased dramatically and rapidly under the condition of starvation for 24 h then feeding for 24 h. These results suggest that BdCHS2 may play an important role in regulating chitin content of the midgut, and subsequently affect the growth and development of B. dorsalis.

  12. Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: a European multicenter study.

    PubMed

    Willasch, A M; Gruhn, B; Coliva, T; Kalinova, M; Schneider, G; Kreyenberg, H; Steinbach, D; Weber, G; Hollink, I H I M; Zwaan, C M; Biondi, A; van der Velden, V H J; Reinhardt, D; Cazzaniga, G; Bader, P; Trka, J

    2009-08-01

    A standardized, sensitive and universal method for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) is still pending. Although hyperexpression of Wilms' tumor (WT1) gene transcript has been frequently proposed as an MRD marker in AML, wide comparability of the various methods used for evaluating WT1 expression has not been given. We established and standardized a multicenter approach for quantifying WT1 expression by quantitative reverse transcriptase PCR (qRT-PCR), on the basis of a primer/probe set combination at exons 6 and 7. In a series of quality-control rounds, we analyzed 69 childhood AML samples and 47 normal bone marrow (BM) samples from 4 participating centers. Differences in the individual WT1 expressions levels ranged within <0.5 log of the mean in 82% of the cases. In AML samples, the median WT1/1E+04 Abelson (ABL) expression was 3.5E+03 compared with that of 2.3E+01 in healthy BM samples. As 11.5% of childhood AML samples in this cohort harbored WT1 mutations in exon 7, the effect of mutations on WT1 expression has been investigated, showing that mutated cases expressed significantly higher WT1 levels than wild-type cases. Hence, our approach showed high reproducibility and applicability, even in patients with WT1 mutations; therefore, it can be widely used for the quantitation of WT1 expression in future clinical trials.

  13. Molecular cloning, mRNA expression and tissue distribution analysis of Slc7a11 gene in alpaca (Lama paco) skins associated with different coat colors.

    PubMed

    Tian, Xue; Meng, Xiaolin; Wang, Liangyan; Song, Yunfei; Zhang, Danli; Ji, Yuankai; Li, Xuejun; Dong, Changsheng

    2015-01-25

    Slc7a11 encoding solute carrier family 7 member 11 (amionic amino acid transporter light chain, xCT), has been identified to be a critical genetic regulator of pheomelanin synthesis in hair and melanocytes. To better understand the molecular characterization of Slc7a11 and the expression patterns in skin of white versus brown alpaca (lama paco), we cloned the full length coding sequence (CDS) of alpaca Slc7a11 gene and analyzed the expression patterns using Real Time PCR, Western blotting and immunohistochemistry. The full length CDS of 1512bp encodes a 503 amino acid polypeptide. Sequence analysis showed that alpaca xCT contains 12 transmembrane regions consistent with the highly conserved amino acid permease (AA_permease_2) domain similar to other vertebrates. Sequence alignment and phylogenetic analysis revealed that alpaca xCT had the highest identity and shared the same branch with Camelus ferus. Real Time PCR and Western blotting suggested that xCT was expressed at significantly high levels in brown alpaca skin, and transcripts and protein possessed the same expression pattern in white and brown alpaca skins. Additionally, immunohistochemical analysis further demonstrated that xCT staining was robustly increased in the matrix and root sheath of brown alpaca skin compared with that of white. These results suggest that Slc7a11 functions in alpaca coat color regulation and offer essential information for further exploration on the role of Slc7a11 in melanogenesis.

  14. Effects of DNA replication on mRNA noise.

    PubMed

    Peterson, Joseph R; Cole, John A; Fei, Jingyi; Ha, Taekjip; Luthey-Schulten, Zaida A

    2015-12-29

    There are several sources of fluctuations in gene expression. Here we study the effects of time-dependent DNA replication, itself a tightly controlled process, on noise in mRNA levels. Stochastic simulations of constitutive and regulated gene expression are used to analyze the time-averaged mean and variation in each case. The simulations demonstrate that to capture mRNA distributions correctly, chromosome replication must be realistically modeled. Slow relaxation of mRNA from the low copy number steady state before gene replication to the high steady state after replication is set by the transcript's half-life and contributes significantly to the shape of the mRNA distribution. Consequently both the intrinsic kinetics and the gene location play an important role in accounting for the mRNA average and variance. Exact analytic expressions for moments of the mRNA distributions that depend on the DNA copy number, gene location, cell doubling time, and the rates of transcription and degradation are derived for the case of constitutive expression and subsequently extended to provide approximate corrections for regulated expression and RNA polymerase variability. Comparisons of the simulated models and analytical expressions to experimentally measured mRNA distributions show that they better capture the physics of the system than previous theories.

  15. Molecular Characteristics, mRNA Expression, and Alternative Splicing of a Ryanodine Receptor Gene in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

    PubMed Central

    Yuan, Guo-Rui; Shi, Wen-Zhi; Yang, Wen-Jia; Jiang, Xuan-Zhao; Dou, Wei; Wang, Jin-Jun

    2014-01-01

    Ryanodine receptors (RyRs) are a distinct class of ligand-gated channels controlling the release of calcium from intracellular stores. The emergence of diamide insecticides, which selectively target insect RyRs, has promoted the study of insect RyRs. In the present study, the full-length RyR cDNA (BdRyR) was cloned and characterized from the oriental fruit fly, Bactrocera dorsalis (Hendel), a serious pest of fruits and vegetables throughout East Asia and the Pacific Rim. The cDNA of BdRyR contains a 15,420-bp open reading frame encoding 5,140 amino acids with a predicted molecular weight of 582.4 kDa and an isoelectric point of 5.38. BdRyR shows a high level of amino acid sequence identity (78 to 97%) to other insect RyR isoforms. All common structural features of the RyRs are present in the BdRyR, including a well-conserved C-terminal domain containing consensus calcium-binding EF-hands and six transmembrane domains, and a large N-terminal domain. Quantitative real-time PCR analyses revealed that BdRyR was expressed at the lowest and highest levels in egg and adult, respectively, and that the BdRyR expression levels in the third instar larva, pupa and adult were 166.99-, 157.56- and 808.56-fold higher, respectively, than that in the egg. Among different adult body parts, the highest expression level was observed in the thorax compared with the head and abdomen. In addition, four alternative splice sites were identified in the BdRyR gene, with the first, ASI, being located in the central part of the predicted second spore lysis A/RyR domain. Diagnostic PCR analyses revealed that alternative splice variants were generated not only in a tissue-specific manner but also in a developmentally regulated manner. These results lay the foundation for further understanding the structural and functional properties of BdRyR, and the molecular mechanisms for target site resistance in B. dorsalis. PMID:24740254

  16. Sequences controlling histone H4 mRNA abundance.

    PubMed Central

    Capasso, O; Bleecker, G C; Heintz, N

    1987-01-01

    The post-transcriptional regulation of histone mRNA abundance is manifest both by accumulation of histone mRNA during the S phase, and by the rapid degradation of mature histone mRNA following the inhibition of DNA synthesis. We have constructed a comprehensive series of substitution mutants within a human H4 histone gene, introduced them into the mouse L cell genome, and analyzed their effects on the post-transcriptional control of the H4 mRNA. Our results demonstrate that most of the H4 mRNA is dispensable for proper regulation of histone mRNA abundance. However, recognition of the 3' terminus of the mature H4 mRNA is critically important for regulating its cytoplasmic half-life. Thus, this region of the mRNA functions both in the nucleus as a signal for proper processing of the mRNA terminus, and in the cytoplasm as an essential element in the control of mRNA stability. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3608993

  17. Short-term hypertonic exposure enhances in vitro follicle growth and meiotic competence of enclosed oocytes while modestly affecting mRNA expression of aquaporin and steroidogenic genes in the domestic cat model.

    PubMed

    Songsasen, N; Thongkittidilok, C; Yamamizu, K; Wildt, D E; Comizzoli, P

    2017-03-01

    Using the domestic cat as a non-rodent, larger animal model, the objective was to determine the impact of a brief incubation in a hypertonic microenvironment on (1) ovarian follicle and oocyte growth in vitro, (2) developmental capacity of the resident oocyte, and (3) expression of aquaporin (AQP) genes in parallel with genes involved in regulation of folliculogenesis. In Study 1: Secondary or early antral follicles encapsulated in 0.5% alginate were allocated to one of three treatment groups: 1) culture in standard medium at 290 mOsm for 15 d (Control); 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for 15 d (Hypertonic-1h); or 3) incubation in 350 mOsm medium for 24 h followed by incubation in standard medium for additional 14 d (Hypertonic-24h). After measuring follicle and oocyte diameters on Day 15, in vitro-grown oocytes were incubated for 24 h before assessing nuclear status. In Study 2: secondary or early antral follicles were subjected to one of the three treatments: 1) culture in standard medium at 290 mOsm for 48 h; 2) incubation in 350 mOsm medium for 1 h followed by culture in standard medium for additional 47 h; or 3) incubation in 350 mOsm medium for 24 h followed by culture in standard medium for additional 24 h. At the end of the culture period, all follicles were assessed for mRNA level of Cyp17a1, Cyp19a1, Star, Aqp1, 3, 5, 7 and 8 as well as Fshr using qPCR. Freshly collected follicles also were subjected to gene expression analysis and served as the 'Non-cultured control'. Hypertonic-24h follicles grew larger (P < 0.05) than the control, whereas those in Hypertonic-1h group exhibited intermediate growth, especially when the culture started at the early antral stage. Oocytes in the Hypertonic-24h group were larger and resumed meiosis at a higher rate than in the other treatments. In vitro culture affected (P < 0.05) mRNA expression of Cyp19a1, Star, Aqp1, and Aqp7 in both the secondary and early

  18. Two-level inhibition of galK expression by Spot 42: Degradation of mRNA mK2 and enhanced transcription termination before the galK gene

    PubMed Central

    Wang, Xun; Ji, Sang Chun; Jeon, Heung Jin; Lee, Yonho; Lim, Heon M.

    2015-01-01

    The Escherichia coli gal operon has the structure Pgal-galE-galT-galK-galM. During early log growth, a gradient in gene expression, named type 2 polarity, is established, as follows: galE > galT > galK > galM. However, during late-log growth, type 1 polarity is established in which galK is greater than galT, as follows: galE > galK > galT > galM. We found that type 2 polarity occurs as a result of the down-regulation of galK, which is caused by two different molecular mechanisms: Spot 42-mediated degradation of the galK-specific mRNA, mK2, and Spot 42-mediated Rho-dependent transcription termination at the end of galT. Because the concentration of Spot 42 drops during the transition period of the polarity type switch, these results demonstrate that type 1 polarity is the result of alleviation of Spot 42-mediated galK down-regulation. Because the Spot 42-binding site overlaps with a putative Rho-binding site, a molecular mechanism is proposed to explain how Spot 42, possibly with Hfq, enhances Rho-mediated transcription termination at the end of galT. PMID:26045496

  19. A novel approach to understanding the role of polymorphic forms of the NR3C1 and TGF-β1 genes in the modulation of the expression of IL-5 and IL-15 mRNA in asthmatic inflammation.

    PubMed

    Panek, Michał; Jonakowski, Mateusz; Zioło, Jan; Wieteska, Łukasz; Małachowska, Beata; Pietras, Tadeusz; Szemraj, Janusz; Kuna, Piotr

    2016-06-01

    The aim of the present study was to identify polymorphic forms of the nuclear receptor subfamily 3, group C, member 1 (NR3C1) and transforming growth factor β1 (TGF-β1) genes and evaluate their impact on the expression levels of interleukin (IL)-5 and IL‑15 in asthma. The study was conducted on a control group consisting of 91 people (54 women and 37 men). The patient group consisted of 130 participants (86 women and 44 men). Genotyping was performed by polymerase chain reaction‑restriction fragment length polymorphism (PCR‑RFLP) and PCR‑high resolution melting (HRM) methods. Interleukin expression was measured by reverse transcription‑quantitative polymerase chain reaction. The frequency of the polymorphic forms in the analyzed group were observed to be: Tth111I (rs10052957) controls AA 0.0440, AG 0.5714, GG 0.3846, patients AA 0.1538/AG 0.4692, GG 0.3769; ER22/23EK (rs6189 /rs6190) controls AG 0.0556, GG 0.9444, patients AG 0.0385, GG 0.9615; N363S (rs6195) controls AA 0.6444, AG 0.2667, GG 0.0889, patients AA 0.7846, AG 0.1385, GG 0.0769; BclI (rs41423247) controls CC 0.0879, CG 0.5604, GG 0.3516, patients CC 0.1008, CG 0.5736, GG 0.3256; C‑509T (rs1800469) controls TT 0.0805, CT 0.6322, CC 0.2874, patients TT 0.1102, CT 0.5669, CC 0.3228. The results indicated that the C‑509T single nucleotide polymorphism (SNP) of the TGF-β1 gene contributed to an increase in the IL‑5 mRNA expression levels. The GG genotype of the N363S SNP of the NR3C1 gene was observed to result in an increase in the expression levels of IL‑15. The present study indicated that the selected SNPs of the NR3C1 and TGF‑β1 genes demonstrate a regulatory effect on the expression of IL‑5 and IL‑15. Therefore, genetic variation affects inflammation in asthma and the clinical course of the disease.

  20. Silencing of two alternative splicing-derived mRNA variants of chitin synthase 1 gene by RNAi is lethal to the oriental migratory locust, Locusta migratoria manilensis (Meyen).

    PubMed

    Zhang, Jianzhen; Liu, Xiaojian; Zhang, Jianqin; Li, Daqi; Sun, Yi; Guo, Yaping; Ma, Enbo; Zhu, Kun Yan

    2010-11-01

    Chitin synthases are crucial enzymes responsible for chitin biosynthesis in fungi, nematodes and arthropods. We characterized two alternative splicing-derived variants of chitin synthase 1 gene (LmCHS1) from the oriental migratory locust, Locusta migratoria manilensis (Meyen). Each cDNA of the two variants (LmCHS1A and LmCHS1B) consists of 5116 nucleotides that include a 4728-nucleotide open reading frame (ORF) encoding 1576 amino acid residues, and 67- and 321-bp non-coding regions at the 5'- and 3'-ends of the cDNA, respectively. The two variants differ only in one exon consisting of 177 nucleotides that encode 59 amino acid residues. The amino acid sequences within this alternative splicing region are 75% identical between the two variants. Both variants were expressed in all the developmental stages. However, LmCHS1A was predominately expressed in the integument whereas LmCHS1B was mainly expressed in the trachea. Our RNAi-based gene silencing study resulted in a dramatic reduction in the levels of the corresponding mRNA in the locust nymphs injected with dsRNA of LmCHS1, or either of its two variants, LmCHS1A and LmCHS1B. Consequentially, 95, 88 and 51% of mortalities were observed in the locusts injected with the LmCHS1, LmCHS1A and LmCHS1B dsRNA, respectively. The phenotypes resulted from the injection of LmCHS1A dsRNA were similar to those from the injection of LmCHS1 dsRNA, whereas the locusts injected with LmCHS1B dsRNA exhibited crimpled cuticle phenotype. Our results suggest that both variants of chitin synthase 1 are essential for insect growth and development.

  1. Complementation of non-tumorigenicity of HPV18-positive cervical carcinoma cells involves differential mRNA expression of cellular genes including potential tumor suppressor genes on chromosome 11q13.

    PubMed

    Kehrmann, Angela; Truong, Ha; Repenning, Antje; Boger, Regina; Klein-Hitpass, Ludger; Pascheberg, Ulrich; Beckmann, Alf; Opalka, Bertram; Kleine-Lowinski, Kerstin

    2013-01-01

    The fusion between human tumorigenic cells and normal human diploid fibroblasts results in non-tumorigenic hybrid cells, suggesting a dominant role for tumor suppressor genes in the generated hybrid cells. After long-term cultivation in vitro, tumorigenic segregants may arise. The loss of tumor suppressor genes on chromosome 11q13 has been postulated to be involved in the induction of the tumorigenic phenotype of human papillomavirus (HPV)18-positive cervical carcinoma cells and their derived tumorigenic hybrid cells after subcutaneous injection in immunocompromised mice. The aim of this study was the identification of novel cellular genes that may contribute to the suppression of the tumorigenic phenotype of non-tumorigenic hybrid cells in vivo. We used cDNA microarray technology to identify differentially expressed cellular genes in tumorigenic HPV18-positive hybrid and parental HeLa cells compared to non-tumorigenic HPV18-positive hybrid cells. We detected several as yet unknown cellular genes that play a role in cell differentiation, cell cycle progression, cell-cell communication, metastasis formation, angiogenesis, antigen presentation, and immune response. Apart from the known differentially expressed genes on 11q13 (e.g., phosphofurin acidic cluster sorting protein 1 (PACS1) and FOS ligand 1 (FOSL1 or Fra-1)), we detected novel differentially expressed cellular genes located within the tumor suppressor gene region (e.g., EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) and leucine rich repeat containing 32 (LRRC32) (also known as glycoprotein-A repetitions predominant (GARP)) that may have potential tumor suppressor functions in this model system of non-tumorigenic and tumorigenic HeLa x fibroblast hybrid cells.

  2. Sensitivity of mRNA Translation.

    PubMed

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-08-04

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5' end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies.

  3. Sensitivity of mRNA Translation

    PubMed Central

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-01-01

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5′ end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

  4. INDUCTION OF GENE EXPRESSION IN SHEEPSHEAD MINNOWS (CYPRINODON VARIEGATUS) TREATED WITH 17B-ESTRADIOL, DIETHYLSTILBESTROL, OR ETHINYLESTRADIOL: THE USE OF MRNA FINGERPRINTS AS AN INDICATOR OF GENE REGULATION

    EPA Science Inventory

    The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variega...

  5. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  6. The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells.

    PubMed

    Mobarak, C D; Anderson, K D; Morin, M; Beckel-Mitchener, A; Rogers, S L; Furneaux, H; King, P; Perrone-Bizzozero, N I

    2000-09-01

    The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.

  7. A novel cis-acting element from the 3'UTR of DNA damage-binding protein 2 mRNA links transcriptional and post-transcriptional regulation of gene expression.

    PubMed

    Melanson, Brian D; Cabrita, Miguel A; Bose, Reetesh; Hamill, Jeffrey D; Pan, Elysia; Brochu, Christian; Marcellus, Kristen A; Zhao, Tong T; Holcik, Martin; McKay, Bruce C

    2013-06-01

    The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis-acting determinants of mRNA stability in its 3' untranslated region (3'UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3'UTR sequences from DDB2 to identify novel cis-acting elements that regulate mRNA stability. Synthetic 3'UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3'UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3'UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis-acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked.

  8. Signaling Pathways That Control mRNA Turnover

    PubMed Central

    Thapar, Roopa; Denmon, Andria P.

    2013-01-01

    Cells regulate their genomes mainly at the level of transcription and at the level of mRNA decay. While regulation at the level of transcription is clearly important, the regulation of mRNA turnover by signaling networks is essential for a rapid response to external stimuli. Signaling pathways result in posttranslational modification of RNA binding proteins by phosphorylation, ubiquitination, methylation, acetylation etc. These modifications are important for rapid remodeling of dynamic ribonucleoprotein complexes and triggering mRNA decay. Understanding how these posttranslational modifications alter gene expression is therefore a fundamental question in biology. In this review we highlight recent findings on how signaling pathways and cell cycle checkpoints involving phosphorylation, ubiquitination, and arginine methylation affect mRNA turnover. PMID:23602935

  9. Nuclear Retention of mRNA in Mammalian Tissues

    PubMed Central

    Bahar Halpern, Keren; Caspi, Inbal; Lemze, Doron; Levy, Maayan; Landen, Shanie; Elinav, Eran; Ulitsky, Igor; Itzkovitz, Shalev

    2015-01-01

    Summary mRNA is thought to predominantly reside in the cytoplasm, where it is translated and eventually degraded. Although nuclear retention of mRNA has a regulatory potential, it is considered extremely rare in mammals. Here, to explore the extent of mRNA retention in metabolic tissues, we combine deep sequencing of nuclear and cytoplasmic RNA fractions with single-molecule transcript imaging in mouse beta cells, liver, and gut. We identify a wide range of protein-coding genes for which the levels of spliced polyadenylated mRNA are higher in the nucleus than in the cytoplasm. These include genes such as the transcription factor ChREBP, Nlrp6, Glucokinase, and Glucagon receptor. We demonstrate that nuclear retention of mRNA can efficiently buffer cytoplasmic transcript levels from noise that emanates from transcriptional bursts. Our study challenges the view that transcripts predominantly reside in the cytoplasm and reveals a role of the nucleus in dampening gene expression noise. PMID:26711333

  10. Links between mRNA splicing, mRNA quality control, and intellectual disability

    PubMed Central

    Fasken, Milo B.; Corbett, Anita H.

    2016-01-01

    In recent years, the impairment of RNA binding proteins that play key roles in the post-transcriptional regulation of gene expression has been linked to numerous neurological diseases. These RNA binding proteins perform critical mRNA processing steps in the nucleus, including splicing, polyadenylation, and export. In many cases, these RNA binding proteins are ubiquitously expressed raising key questions about why only brain function is impaired. Recently, mutations in the ZC3H14 gene, encoding an evolutionarily conserved, polyadenosine RNA binding protein, have been linked to a nonsyndromic form of autosomal recessive intellectual disability. Thus far, research on ZC3H14 and its Nab2 orthologs in budding yeast and Drosophila reveals that ZC3H14/Nab2 is important for mRNA processing and neuronal patterning. Two recent studies now provide evidence that ZC3H14/Nab2 may function in the quality control of mRNA splicing and export and could help to explain the molecular defects that cause neuronal dysfunction and lead to an inherited form of intellectual disability. These studies on ZC3H14/Nab2 reveal new clues to the puzzle of why loss of the ubiquitously expressed ZC3H14 protein specifically affects neurons. PMID:27868086

  11. mRNA expression profile of the TNF-α system in LH-induced bovine preovulatory follicles and effects of TNF-α on gene expression, ultrastructure and expansion of cumulus-oocyte complexes cultured in vitro.

    PubMed

    Silva, A W B; Bezerra, F T G; Glanzner, W G; Dos Santos, J T; Dau, A M P; Rovani, M T; Ilha, G F; Costa, J J N; Cunha, E V; Donato, M A M; Peixoto, C A; Gonçalves, P B D; Bordignon, V; Silva, J R V

    2017-03-01

    This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.

  12. Nuclear Decay Factors Crack Up mRNA.

    PubMed

    Tudek, Agnieszka; Schmid, Manfred; Jensen, Torben Heick

    2017-03-02

    In this issue of Molecular Cell, Bresson et al. (2017) show that the nuclear RNA decay factors Nab3 and Mtr4 reshape the coding transcriptome during glucose starvation in budding yeast, placing nuclear mRNA metabolism as an important contributor of gene expression regulation.

  13. Sites of amyloid SAA mRNA production

    SciTech Connect

    Meek, R.L.; Benditt, E.P.

    1986-03-01

    To investigate possible extrahepatic sites of SAA production, male BALB/c mice were given a single 0.5 ml injection of either 10% casein or lipopolysaccharide (LPS; 100 mg/ml). Twenty hours after injection, RNA was extracted from liver, kidney, adrenal, testis, brain, spleen, skeletal muscle, heart, lung and small intestine. Northern blots of total RNA were hybridized with nick-translated /sup 32/P-labeled cDNA probes (length approximately 150 base pairs) corresponding to an homologous region of the three known SAA genes. Both casein and LPS elevated the mRNA in liver to about 200-fold above control levels; mRNA was elevated in adrenals from O to approximately 2% of liver. mRNA in some other tissues responded only to LPS injection: levels in kidney reached 15% of liver; pituitary, testis and brain reached 0.02 to 0.5% of liver; no apoSAA mRNA was detected in heart, skeletal muscle, lung, spleen or small intestine. Thus, some organs other than liver appear to have operational genes for apoSAA. The expression of apoSAA genes in different tissues is shared with other apoproteins; it remains to be seen whether all three or only selected genes are transcribed and translated in different tissues.

  14. Identification of a novel P450 gene belonging to the CYP4 family in the clam Ruditapes philippinarum, and analysis of basal- and benzo(a)pyrene-induced mRNA expression levels in selected tissues.

    PubMed

    Pan, Luqing; Liu, Na; Xu, Chaoqun; Miao, Jingjing

    2011-11-01

    A novel full-length cDNA encoding a CYP4 protein was initially cloned from the clam, Ruditapes philippinarum. The nucleotide sequence contained an open reading frame coding for 442 amino acids and the deduced amino acid sequence showed 42.6-49.1% identity with other species CYP4s. The phylogenetic analysis demonstrated that the clam CYP4 was clustered within the CYP4s branch. The clam CYP4 mRNA expression was detected in gill, digestive gland, adductor muscle and mantle, and highest transcription level was observed in digestive gland compared to other tissues. Quantitative real-time RT-PCR analysis revealed that there was no notable change in CYP4 mRNA expression in gill of R. philippinarum exposure to benzo(a)pyrene (BaP), while the mRNA expression was induced significantly in the digestive gland of the clam by 0.2 ppb (μgL(-1)) BaP (p<0.05). The results suggest that CYP4 of the clam may serve as a useful biomarker of marine environmental PAH pollution.

  15. Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

    NASA Technical Reports Server (NTRS)

    Raghavan, V.

    1992-01-01

    Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.

  16. Detection of mRNA for the tax sub 1 /rex sub 1 gene of human T-cell leukemia virus type I in fresh peripheral blood mononuclear cells of adult T-cell leukemia patients and viral carriers by using the polymerase chain reaction

    SciTech Connect

    Kinoshita, Tomohiro; Shimoyama, Masanori; Tobinai, Kensei; Ito, Mizuko; Ito, Shinichiro; Ikeda, Shuichi; Tajima, Kazuo; Shimotohno, Kunitada; Shugimura, Takashi )

    1989-07-01

    Expression of human T-cell leukemia virus type I (HTLV-I) is not detectable by immunofluorescence analysis or RNA blot analysis in most fresh peripheral blood mononuclear cells of patients with adult T-cell leukemia or of asymptomatic HTLV-I carriers. However, in this work, mRNA for the HTLV-I tax{sub 1}/rex{sub 1} genes was detected in fresh peripheral blood mononuclear cells of adult T-cell leukemia patients and asymptomatic HTLV-I carriers by using reverse transcription followed by the polymerase chain reaction. By using fresh peripheral blood mononuclear cells, the expression of tax{sub 1}/rex{sub 1} mRNA was detected in five of the six adult T-cell leukemia patients and four of the eight HTLV-I carriers examined. The amounts of tax{sub 1}/rex{sub 1} mRNA detected corresponded to {approx} 10{sup 5} to 10{sup 6} times less than that in the HTLV-I-infected MT-2 cell line. These results indicate that, in some individuals infected with HTLV-I, the provirus in circulating blood cells is transcribed in vivo. Thus the expression of viral antigens in circulating blood cells in vivo is suggested.

  17. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

    NASA Astrophysics Data System (ADS)

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-10-01

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  18. Transfection efficiency and transgene expression kinetics of mRNA delivered in naked and nanoparticle format.

    PubMed

    Phua, Kyle K L; Leong, Kam W; Nair, Smita K

    2013-03-28

    Transfection efficiencies and transgene expression kinetics of messenger RNA (mRNA), an emerging class of nucleic acid-based therapeutics, have been poorly characterized. In this study, we evaluated transfection efficiencies of mRNA delivered in naked and nanoparticle format in vitro and in vivo using GFP and luciferase as reporters. While mRNA nanoparticles transfect primary human and mouse dendritic cells (DCs) efficiently in vitro, naked mRNA could not produce any detectable gene product. The protein expression of nanoparticle-mediated transfection in vitro peaks rapidly within 5-7h and decays in a biphasic manner. In vivo, naked mRNA is more efficient than mRNA nanoparticles when administered subcutaneously. In contrast, mRNA nanoparticle performs better when administered intranasally and intravenously. Gene expression is most transient when delivered intravenously in nanoparticle format with an apparent half-life of 1.4h and lasts less than 24h, and most sustained when delivered in the naked format subcutaneously at the base of tail with an apparent half-life of 18h and persists for at least 6days. Notably, exponential decreases in protein expression are consistently observed post-delivery of mRNA in vivo regardless of the mode of delivery (naked or nanoparticle) or the site of administration. This study elucidates the performance of mRNA transfection and suggests a niche for mRNA therapeutics when predictable in vivo transgene expression kinetics is imperative.

  19. The ticking tail: daily oscillations in mRNA poly(A) tail length drive circadian cycles in protein synthesis.

    PubMed

    Gotic, Ivana; Schibler, Ueli

    2012-12-15

    In this issue of Genes & Development, Kojima and colleagues (pp. 2724-2736) examined the impact of mRNA poly(A) tail length on circadian gene expression. Their study demonstrates how dynamic changes in transcript poly(A) tail length can lead to rhythmic protein expression, irrespective of whether mRNA accumulation is circadian or constitutive.

  20. Influenza Virus mRNA Trafficking Through Host Nuclear Speckles

    PubMed Central

    Mor, Amir; White, Alexander; Zhang, Ke; Thompson, Matthew; Esparza, Matthew; Muñoz-Moreno, Raquel; Koide, Kazunori; Lynch, Kristen W.; García-Sastre, Adolfo; Fontoura, Beatriz M.A.

    2016-01-01

    Influenza A virus is a human pathogen whose genome is comprised of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. Since nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression. PMID:27347430

  1. Inducible Control of mRNA transport using reprogrammable RNA-binding proteins.

    PubMed

    Abil, Zhanar; Gumy, Laura F; Zhao, Huimin; Hoogenraad, Casper C

    2017-03-06

    Localization of mRNA is important in a number of cellular processes such as embryogenesis, cellular motility, polarity, and a variety of neurological processes. A synthetic device that controls cellular mRNA localization would facilitate investigations on the significance of mRNA localization in cellular function and allow an additional level of controlling gene expression. In this work, we developed the PUF (Puilio and FBF homology domain) -assisted Localization of RNA (PULR) system, which utilizes a eukaryotic cell's cytoskeletal transport machinery to re-position mRNA within a cell. Depending on the cellular motor used, we show ligand-dependent transport of mRNA towards either pole of the microtubular network of cultured cells. In addition, implementation of the re-programmable PUF domain allowed the transport of untagged endogenous mRNA in primary neurons.

  2. Effect of 3,3',5-triiodothyronine and 3,5-diiodothyronine on progesterone production, cAMP synthesis, and mRNA expression of STAR, CYP11A1, and HSD3B genes in granulosa layer of chicken preovulatory follicles.

    PubMed

    Sechman, A; Pawlowska, K; Hrabia, A

    2011-10-01

    In vitro studies were performed to assess whether stimulatory effects of triiodothyronine (T3) on progesterone (P4) production in a granulosa layer (GL) of chicken preovulatory follicles are associated with 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and mRNA expression of STAR protein, CYP11A1, and HSD3B. Effects of 3,5-diiodothyronine (3,5-T2) on steroidogenic function in these follicles were also investigated. The GL of F3 to F1 follicles was incubated in medium supplemented with T3 or 3,5-T2, LH, or forskolin (F), and a combination of each iodothyronine with LH or F. Levels of P4 and cAMP in culture media were determined by RIA. Expression of genes involved in P4 synthesis (ie, STAR protein, CYP11A1, and HSD3B) in the GL of F3 to F1 follicles incubated in medium with T3 or 3,5-T2 and their combination with LH was performed by real-time PCR. Triiodothyronine increased basal and LH- and F-stimulated P4 secretion by preovulatory follicles. The 3,5-T2 elevated P4 synthesis by F3, had no effect on F2 follicles, and diminished P4 production by the GL of F1 follicles. It had no effect on LH-stimulated P4 production; however, it augmented F-stimulated P4 production by F2 and F1 follicles. Although T3 did not affect basal and F-stimulated cAMP synthesis by the GL of preovulatory follicles, it increased LH-stimulated synthesis of this nucleotide. However, 3,5-T2 elevated F-stimulated cAMP synthesis in F3 and F2 follicles; it did not change basal and LH-stimulated cAMP production. Triiodothyronine decreased basal STAR and CYP11A1 mRNAs in F3 follicles, increased them in F1 follicles, and elevated HSD3B mRNA levels in F1 follicles. Triiodothyronine augmented LH-stimulated STAR, CYP11A1, and HSD3B mRNA levels in F2 and CYP11A1 in F1 follicles. However, T3 decreased LH-stimulated STAR and HSD3B mRNA levels in F1 follicles. The 3,5-T2 did not affect basal STAR and CYP11A1 mRNA expression in all investigated follicles; however, it decreased LH-stimulated STAR

  3. Regulation of mRNA decay in plant responses to salt and osmotic stress.

    PubMed

    Kawa, Dorota; Testerink, Christa

    2017-04-01

    Plant acclimation to environmental stresses requires fast signaling to initiate changes in developmental and metabolic responses. Regulation of gene expression by transcription factors and protein kinases acting upstream are important elements of responses to salt and drought. Gene expression can be also controlled at the post-transcriptional level. Recent analyses on mutants in mRNA metabolism factors suggest their contribution to stress signaling. Here we highlight the components of mRNA decay pathways that contribute to responses to osmotic and salt stress. We hypothesize that phosphorylation state of proteins involved in mRNA decapping affect their substrate specificity.

  4. High lib mRNA expression in breast carcinomas.

    PubMed

    Satoh, Kazuki; Hata, Mitsumi; Yokota, Hiroshi

    2004-06-30

    Lib, first identified as a novel beta-amyloid responsive gene in rat astrocytes, has an extracellular domain of 15 leucine-rich repeats (LRRs) followed by a transmembrane domain and a short cytoplasmic region. It is a distinctly inducible gene and is thought to play a key role in inflammatory states via the LRR extracellular motif, an ideal structural framework for protein-protein and protein-matrix interactions. To evaluate potential roles of Lib, we screened various tumors for Lib expression. Lib mRNA expression was high and uniquely expressed in breast tumor tissues, compared to paired normal breast tissues. Lib mRNA was localized in the ductal carcinoma cells and Lib protein displayed a homophilic association on the surface of cultured cells. These data suggest that Lib may play a role in the progression of breast carcinomas and may be a diagnostic marker for breast tumors.

  5. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  6. Quantitative Imaging of Single mRNA Splice Variants in Living Cells

    PubMed Central

    Lee, Kyuwan; Cui, Yi

    2015-01-01

    Alternative mRNA splicing is a fundamental process of gene regulation via the precise control of the post-transcriptional step that occurs before mRNA translation. Errors in RNA splicing have been known to correlate with different diseases; however, a key limitation is the lack of technologies for live cell monitoring and quantification to understand the process of alternative splicing. Here, we report a spectroscopic strategy for quantitative imaging of mRNA splice variants in living cells, using nanoplasmonic dimer antennas. The spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1 were monitored at single copy resolution by measuring the hybridization dynamics of nanoplasmonic antennas targeting complementary mRNA sequences in live cells. Our study provides valuable insights on RNA and its transport in living cells, which has the potential to enhance our understanding of cellular protein complex, pharmacogenomics, genetic diagnosis, and gene therapies. PMID:24747838

  7. Promoter-Bound Trinucleotide Repeat mRNA Drives Epigenetic Silencing in Fragile X Syndrome

    PubMed Central

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S.; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D.; Jaffrey, Samie R.

    2015-01-01

    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide–repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5′ untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA. PMID:24578575

  8. Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome.

    PubMed

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D; Jaffrey, Samie R

    2014-02-28

    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide-repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5' untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA.

  9. The life of an mRNA in space and time.

    PubMed

    Ben-Ari, Ya'ara; Brody, Yehuda; Kinor, Noa; Mor, Amir; Tsukamoto, Toshiro; Spector, David L; Singer, Robert H; Shav-Tal, Yaron

    2010-05-15

    Nuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed beta-actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic beta-actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the beta-actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell.

  10. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function.

    PubMed

    Del Campo, Cristian; Bartholomäus, Alexander; Fedyunin, Ivan; Ignatova, Zoya

    2015-10-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation.

  11. Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function

    PubMed Central

    Fedyunin, Ivan; Ignatova, Zoya

    2015-01-01

    Messenger RNA acts as an informational molecule between DNA and translating ribosomes. Emerging evidence places mRNA in central cellular processes beyond its major function as informational entity. Although individual examples show that specific structural features of mRNA regulate translation and transcript stability, their role and function throughout the bacterial transcriptome remains unknown. Combining three sequencing approaches to provide a high resolution view of global mRNA secondary structure, translation efficiency and mRNA abundance, we unraveled structural features in E. coli mRNA with implications in translation and mRNA degradation. A poorly structured site upstream of the coding sequence serves as an additional unspecific binding site of the ribosomes and the degree of its secondary structure propensity negatively correlates with gene expression. Secondary structures within coding sequences are highly dynamic and influence translation only within a very small subset of positions. A secondary structure upstream of the stop codon is enriched in genes terminated by UAA codon with likely implications in translation termination. The global analysis further substantiates a common recognition signature of RNase E to initiate endonucleolytic cleavage. This work determines for the first time the E. coli RNA structurome, highlighting the contribution of mRNA secondary structure as a direct effector of a variety of processes, including translation and mRNA degradation. PMID:26495981

  12. Application of talcum powder, trichloroacetic acid and silver nitrate in female rats for non-surgical sterilization: evaluation of the apoptotic pathway mRNA and miRNA genes

    PubMed Central

    Yumrutas, Onder; Kara, Murat; Atilgan, Remzi; Kavak, Salih Burcin; Bozgeyik, Ibrahim; Sapmaz, Ekrem

    2015-01-01

    There are several methods used for non-surgical sterilization in birth control including quinacrine, trichloroacetic acid (TCA), erythromycin, tetracycline, silver nitrate and talcum powder. Among these, talcum powder, TCA and silver nitrate are the most commonly used. However, the toxic and carcinogenic activities of these chemicals in ovarian tissue have been poorly elucidated. This study demonstrates the expression levels of antioxidant, apoptotic and anti-apoptotic genes after administration of talc powder, TCA and silver nitrate for non-surgical sterilization in female rat models. The expression changes of some microRNAs (miR-15b, miR-21, miR-34a and miR-98) that play key roles in the apoptosis pathway were also included. All expression analyses were evaluated with real-time PCR. The expression levels of all genes appeared to be upregulated in the talcum powder group, but the results were not statistically significant. Increased expression of Gsr and Sod1 genes was statistically significant in the talcum powder group. In TCA and silver nitrate group, expression of all genes was appeared to be elevated but only the Gsr expression was statistically significant in the TCA-administrated group; there were no statistically significant changes in the silver nitrate group. miRNA expression levels were increased in talcum powder and TCA-administrated groups, but these results were not significant. Expression levels of miR-15b, miR-21 and miR-98 in the silver nitrate group were significantly increased. Consequently, these chemicals appear to be non-carcinogenic agents for rat ovarian tissue which do not induce apoptosis. However, talcum powder and TCA can be considered as agents that are toxic to ovarian tissue. PMID:25885949

  13. Expression of the ctenophore Brain Factor 1 forkhead gene ortholog (ctenoBF-1) mRNA is restricted to the presumptive mouth and feeding apparatus: implications for axial organization in the Metazoa

    NASA Technical Reports Server (NTRS)

    Yamada, Atsuko; Martindale, Mark Q.

    2002-01-01

    Ctenophores are thoroughly modern animals whose ancestors are derived from a separate evolutionary branch than that of other eumetazoans. Their major longitudinal body axis is the oral-aboral axis. An apical sense organ, called the apical organ, is located at the aboral pole and contains a highly innervated statocyst and photodetecting cells. The apical organ integrates sensory information and controls the locomotory apparatus of ctenophores, the eight longitudinal rows of ctene/comb plates. In an effort to understand the developmental and evolutionary organization of axial properties of ctenophores we have isolated a forkhead gene from the Brain Factor 1 (BF-1) family. This gene, ctenoBF-1, is the first full-length nuclear gene reported from ctenophores. This makes ctenophores the most basal metazoan (to date) known to express definitive forkhead class transcription factors. Orthologs of BF-1 in vertebrates, Drosophila, and Caenorhabditis elegans are expressed in anterior neural structures. Surprisingly, in situ hybridizations with ctenoBF-1 antisense riboprobes show that this gene is not expressed in the apical organ of ctenophores. CtenoBF-1 is expressed prior to first cleavage. Transcripts become localized to the aboral pole by the 8-cell stage and are inherited by ectodermal micromeres generated from this region at the 16- and 32-cell stages. Expression in subsets of these cells persists and is seen around the edge of the blastopore (presumptive mouth) and in distinct ectodermal regions along the tentacular poles. Following gastrulation, stomodeal expression begins to fade and intense staining becomes restricted to two distinct domains in each tentacular feeding apparatus. We suggest that the apical organ is not homologous to the brain of bilaterians but that the oral pole of ctenophores corresponds to the anterior pole of bilaterian animals.

  14. Application of talcum powder, trichloroacetic acid and silver nitrate in female rats for non-surgical sterilization: evaluation of the apoptotic pathway mRNA and miRNA genes.

    PubMed

    Yumrutas, Onder; Kara, Murat; Atilgan, Remzi; Kavak, Salih Burcin; Bozgeyik, Ibrahim; Sapmaz, Ekrem

    2015-04-01

    There are several methods used for non-surgical sterilization in birth control including quinacrine, trichloroacetic acid (TCA), erythromycin, tetracycline, silver nitrate and talcum powder. Among these, talcum powder, TCA and silver nitrate are the most commonly used. However, the toxic and carcinogenic activities of these chemicals in ovarian tissue have been poorly elucidated. This study demonstrates the expression levels of antioxidant, apoptotic and anti-apoptotic genes after administration of talc powder, TCA and silver nitrate for non-surgical sterilization in female rat models. The expression changes of some microRNAs (miR-15b, miR-21, miR-34a and miR-98) that play key roles in the apoptosis pathway were also included. All expression analyses were evaluated with real-time PCR. The expression levels of all genes appeared to be upregulated in the talcum powder group, but the results were not statistically significant. Increased expression of Gsr and Sod1 genes was statistically significant in the talcum powder group. In TCA and silver nitrate group, expression of all genes was appeared to be elevated but only the Gsr expression was statistically significant in the TCA-administrated group; there were no statistically significant changes in the silver nitrate group. miRNA expression levels were increased in talcum powder and TCA-administrated groups, but these results were not significant. Expression levels of miR-15b, miR-21 and miR-98 in the silver nitrate group were significantly increased. Consequently, these chemicals appear to be non-carcinogenic agents for rat ovarian tissue which do not induce apoptosis. However, talcum powder and TCA can be considered as agents that are toxic to ovarian tissue.

  15. mRNA degradation machines in eukaryotic cells.

    PubMed

    Tourrière, Hélène; Chebli, Karim; Tazi, Jamal

    2002-08-01

    The steady-state levels of mRNAs depend upon their combined rates of synthesis and processing, transport from the nucleus to cytoplasm, and decay in the cytoplasm. In eukaryotic cells, the degradation of mRNA is an essential determinant in the regulation of gene expression, and it can be modulated in response to developmental, environmental, and metabolic signals. This level of regulation is particularly important for proteins that are active for a brief period, such as growth factors, transcription factors, and proteins that control cell cycle progression. The mechanisms by which mRNAs are degraded and the sequence elements within the mRNAs that affect their stability are the subject of this review. We will summarize the current state of knowledge regarding cis-acting elements in mRNA and trans-acting factors that contribute to mRNA regulation decay. We will then consider the mechanisms by which specific signaling proteins seem to contribute to a dynamic organization of the mRNA degradation machinery in response to physiological stimuli.

  16. Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products.

    PubMed Central

    Lim, S K; Sigmund, C D; Gross, K W; Maquat, L E

    1992-01-01

    Human beta zero-thalassemic beta-globin genes harboring either a frameshift or a nonsense mutation that results in the premature termination of beta-globin mRNA translation have been previously introduced into the germ line of mice (S.-K. Lim, J.J. Mullins, C.-M. Chen, K. Gross, and L.E. Maquat, EMBO J. 8:2613-2619, 1989). Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and II. In this communication, we show by using genetics and S1 nuclease transcript mapping that the premature termination of beta-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human beta-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs. Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of beta-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of

  17. Hypoxia may increase rat insulin mRNA levels by promoting binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich insulin mRNA 3'-untranslated region.

    PubMed Central

    Tillmar, Linda; Welsh, Nils

    2002-01-01

    BACKGROUND: Recent reports identify the 3'-UTR of insulin mRNA as crucial for control of insulin messenger stability. This region contains a pyrimidine-rich sequence, which is similar to the hypoxia-responsive mRNA-stabilizing element of tyrosine hydroxylase. This study aimed to determine whether hypoxia affects insulin mRNA levels. MATERIALS AND METHODS: Rat islets were incubated at normoxic or hypoxic conditions and with or without hydrogen peroxide and a nitric oxide donor. Insulin mRNA was determined by Northern hybridization. Islet homogenates were used for electrophoretic mobility shift assay with an RNA-oligonucleotide, corresponding to the pyrimidine-rich sequence of the 3'-UTR of rat insulin I mRNA. The expression of reporter gene mRNA, in islets transfected with reporter gene constructs containing the wild-type or mutated insulin mRNA pyrimidine-rich sequences, was measured by semiquantitive RT-PCR. RESULTS: Insulin mRNA was increased in response to hypoxia. This was paralleled by increased binding of the polypyrimidine tract-binding protein (PTB) to the pyrimidine-rich sequence of the 3'-UTR of insulin mRNA, which was counteracted by hydrogen peroxide. The reporter gene mRNA level containing the wild-type binding site was not increased in response to hypoxia, but mutation of the site resulted in a destabilization of the mRNA. CONCLUSIONS: The complete understanding of different diabetic conditions requires the elucidation of mechanisms that control insulin gene expression. Our data show that hypoxia may increase insulin mRNA levels by promoting the binding of PTB to the insulin mRNA 3'-UTR. Hydrogen peroxide abolishes the hypoxic effect indicating involvement of reactive oxygen species and/or the redox potential in the oxygen-signaling pathway. PMID:12359957

  18. Modified mRNA Vaccines Protect against Zika Virus Infection.

    PubMed

    Richner, Justin M; Himansu, Sunny; Dowd, Kimberly A; Butler, Scott L; Salazar, Vanessa; Fox, Julie M; Julander, Justin G; Tang, William W; Shresta, Sujan; Pierson, Theodore C; Ciaramella, Giuseppe; Diamond, Michael S

    2017-03-09

    The emergence of ZIKV infection has prompted a global effort to develop safe and effective vaccines. We engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding wild-type or variant ZIKV structural genes and tested immunogenicity and protection in mice. Two doses of modified mRNA LNPs encoding prM-E genes that produced virus-like particles resulted in high neutralizing antibody titers (∼1/100,000) that protected against ZIKV infection and conferred sterilizing immunity. To offset a theoretical concern of ZIKV vaccines inducing antibodies that cross-react with the related dengue virus (DENV), we designed modified prM-E RNA encoding mutations destroying the conserved fusion-loop epitope in the E protein. This variant protected against ZIKV and diminished production of antibodies enhancing DENV infection in cells or mice. A modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of sensitizing individuals to subsequent exposure to DENV, should this become a clinically relevant concern.

  19. Exaptive origins of regulated mRNA decay in eukaryotes

    PubMed Central

    Hamid, Fursham M.

    2016-01-01

    Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense‐mediated decay (NMD) and motif‐specific transcript destabilization by CCCH‐type zinc finger RNA‐binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of “professional” innate and adaptive immunity systems allowed NMD and the motif‐triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post‐transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. PMID:27438915

  20. Aiding and abetting cancer: mRNA export and the nuclear pore.

    PubMed

    Culjkovic-Kraljacic, Biljana; Borden, Katherine L B

    2013-07-01

    mRNA export is a critical step in gene expression. Export of transcripts can be modulated in response to cellular signaling or stress. Consistently, mRNA export is dysregulated in primary human specimens derived from many different forms of cancer. Aberrant expression of export factors can alter the export of specific transcripts encoding proteins involved in proliferation, survival, and oncogenesis. These specific factors, which are not used for bulk mRNA export, are obvious therapeutic targets. Indeed, given the emerging role of mRNA export in cancer, it is not surprising that efforts to target different aspects of this pathway have reached the clinical trial stage. Thus, like transcription and translation, mRNA export may also play a critical role in cancer genesis and maintenance.

  1. Visualization of dynamics of single endogenous mRNA labeled in live mouse.

    PubMed

    Park, Hye Yoon; Lim, Hyungsik; Yoon, Young J; Follenzi, Antonia; Nwokafor, Chiso; Lopez-Jones, Melissa; Meng, Xiuhua; Singer, Robert H

    2014-01-24

    The transcription and transport of messenger RNA (mRNA) are critical steps in regulating the spatial and temporal components of gene expression, but it has not been possible to observe the dynamics of endogenous mRNA in primary mammalian tissues. We have developed a transgenic mouse in which all β-actin mRNA is fluorescently labeled. We found that β-actin mRNA in primary fibroblasts localizes predominantly by diffusion and trapping as single mRNAs. In cultured neurons and acute brain slices, we found that multiple β-actin mRNAs can assemble together, travel by active transport, and disassemble upon depolarization by potassium chloride. Imaging of brain slices revealed immediate early induction of β-actin transcription after depolarization. Studying endogenous mRNA in live mouse tissues provides insight into its dynamic regulation within the context of the cellular and tissue microenvironment.

  2. Molecular cloning of seal myoglobin mRNA.

    PubMed Central

    Wood, D; Blanchetot, A; Jeffreys, A J

    1982-01-01

    Grey seal skeletal muscle containing high levels of myoglobin was used to prepare poly(A)+ RNA. In vitro translation of this RNA produced a range of polypeptides including myoglobin. cDNA was prepared by reverse transcription of muscle poly(A)+ RNA and cloned into the plasmid pAT 153. 4% of cDNA recombinants were shown to contain myoglobin cDNA inserts. DNA sequence analysis of one clone (pSM 178) which contained a relatively large myoglobin cDNA insert showed an incomplete cDNA comprising the terminal 293 nucleotides of 3' non-translated mRNA sequences. Hybridization experiments using this myoglobin cDNA indicated that seal myoglobin is coded by a single gene which is transcribed to give a 1400 nucleotide mRNA considerably longer than related haemoglobin mRNAs. Images PMID:6185919

  3. Control of mRNA Translation in ALS Proteinopathy

    PubMed Central

    Cestra, Gianluca; Rossi, Simona; Di Salvio, Michela; Cozzolino, Mauro

    2017-01-01

    Cells robustly reprogram gene expression during stress generated by protein misfolding and aggregation. In this condition, cells assemble the bulk of mRNAs into translationally silent stress granules (SGs), while they sustain the translation of specific mRNAs coding for proteins that are needed to overcome cellular stress. Alterations of this process are deeply associated to neurodegeneration. This is the case of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by a selective loss of motor neurons. Indeed, impairment of protein homeostasis as well as alterations of RNA metabolism are now recognized as major players in the pathogenesis of ALS. In particular, evidence shows that defective mRNA transport and translation are implicated. Here, we provide a review of what is currently known about altered mRNA translation in ALS and how this impacts on the ability of affected cells to cope with proteotoxic stress. PMID:28386218

  4. Stabilization of a specific nuclear mRNA precursor by thyroid hormone.

    PubMed Central

    Narayan, P; Towle, H C

    1985-01-01

    The regulation of a thyroid hormone-responsive gene in rats, designated spot 14, was explored. The expression of this gene in liver is rapidly (less than 10 min) and markedly (greater than 10-fold) altered by the administration of 3,5,3'-triiodo-L-thyronine (T3) to hypothyroid rats (P. Narayan, C. W. Liaw, and H. C. Towle, Proc. Natl. Acad. Sci. USA 81:4687-4691, 1984). To investigate the cellular site at which T3 acts to induce this hepatic mRNA, we made parallel measurements of the relative levels of spot 14 mRNA and nuclear precursor RNA and of the rate of gene transcription after treatments designed to alter the thyroid status of rats. The relative levels of both the mRNA and nuclear precursor were elevated roughly 5- to 6-fold in euthyroid animals and 9- to 12-fold in hyperthyroid animals over those in hypothyroid controls. However, only a small difference of approximately 1.5-fold was detected in the rate of spot 14 gene transcription. After a single injection of T3 into hypothyroid animals, a small and transient rise in the transcription rate was detected at 30 min. However, the levels of spot 14 mRNA and nuclear precursor RNA increased much more dramatically throughout the first 4 h of treatment. In both cases, changes in the rate of gene transcription were not capable of accounting for the alterations observed in mRNA levels. Thus, the major site of spot 14 gene regulation by T3 is at a posttranscriptional level. The proportional changes observed in the nuclear precursor and mRNA levels suggest that the site of control is at the level of stability of the nuclear precursor RNA for spot 14 mRNA. PMID:3837180

  5. Detection of WIPI1 mRNA as an indicator of autophagosome formation.

    PubMed

    Tsuyuki, Satoshi; Takabayashi, Mei; Kawazu, Manami; Kudo, Kousei; Watanabe, Akari; Nagata, Yoshiki; Kusama, Yusuke; Yoshida, Kenichi

    2014-03-01

    Autophagy is a cellular bulk degradation system for long-lived proteins and organelles that operates during nutrient starvation and is thus a type of recycling system. In recent years, a series of mammalian orthologs of yeast autophagy-related (ATG) genes have been identified; however, the importance of the transcriptional regulation of ATG genes underlying autophagosome formation is poorly understood. In this study, we identified several ATG genes, including the genes ULK1, MAP1LC3B, GABARAPL1, ATG13, WIPI1, and WDR45/WIPI4, with elevated mRNA levels in thapsigargin-, C2-ceramide-, and rapamycin-treated as well as amino acid-depleted HeLa cells except for MAP1LC3B mRNA in rapamycin-treated HeLa cells. Rapamycin had a weaker effect on the expressions of ATG genes. The increase in WIPI1 and MAP1LC3B mRNA was induced prior to the accumulation of the autophagy marker protein MAP1LC3 in the thapsigargin- and C2-ceramide-treated A549 cells. By counting the puncta marked with MAP1LC3B in HeLa cells treated with different autophagy inducers, we revealed that the time-dependent mRNA elevation of a specific set of ATG genes was similar to that of autophagosome accumulation. The transcriptional attenuation of WIPI1 mRNA using RNA interference inhibited the puncta number in thapsigargin-treated HeLa cells. Remarkably, increases in the abundance of WIPI1 mRNA were also manifested in thapsigargin- and C2-ceramide-treated human fibroblasts (WI-38 and TIG-1), human cancer cells (U-2 OS, Saos-2, and MCF7), and rodent fibroblasts (Rat-1). Taken together, these results suggest that the detection of WIPI1 mRNA is likely to be a convenient method of monitoring autophagosome formation in a wide range of cell types.

  6. Definition of global and transcript-specific mRNA export pathways in metazoans.

    PubMed

    Farny, Natalie G; Hurt, Jessica A; Silver, Pamela A

    2008-01-01

    Eukaryotic gene expression requires export of messenger RNAs (mRNAs) from their site of transcription in the nucleus to the cytoplasm where they are translated. While mRNA export has been studied in yeast, the complexity of gene structure and cellular function in metazoan cells has likely led to increased diversification of these organisms' export pathways. Here we report the results of a genome-wide RNAi screen in which we identify 72 factors required for polyadenylated [poly-(A(+))] mRNA export from the nucleus in Drosophila cells. Using structural and functional conservation analysis of yeast and Drosophila mRNA export factors, we expose the evolutionary divergence of eukaryotic mRNA export pathways. Additionally, we demonstrate the differential export requirements of two endogenous heat-inducible transcripts--intronless heat-shock protein 70 (HSP70) and intron-containing HSP83--and identify novel export factors that participate in HSP83 mRNA splicing. We characterize several novel factors and demonstrate their participation in interactions with known components of the Drosophila export machinery. One of these factors, Drosophila melanogaster PCI domain-containing protein 2 (dmPCID2), associates with polysomes and may bridge the transition between exported messenger ribonucleoprotein particles (mRNPs) and polysomes. Our results define the global network of factors involved in Drosophila mRNA export, reveal specificity in the export requirements of different transcripts, and expose new avenues for future work in mRNA export.

  7. Myoglobin expression: early induction and subsequent modulation of myoglobin and myoglobin mRNA during myogenesis.

    PubMed Central

    Weller, P A; Price, M; Isenberg, H; Edwards, Y H; Jeffreys, A J

    1986-01-01

    We showed that myoglobin gene transcription and the appearance of myoglobin occur very early in myogenesis, in both humans and mice. In contrast to the contractile protein genes, there is a subsequent increase of 50- to 100-fold in myoglobin mRNA and protein levels during later muscle development. Myoglobin and myoglobin mRNA are present at elevated levels in fetal heart and are also detectable at low levels in adult smooth muscle. The absolute level of myoglobin mRNA in highly myoglobinized seal muscle is very high [2.8% of the total population of poly(A)+ RNAs]. Levels of myoglobin in seal skeletal muscle and in various human muscle types appear to be determined by the size of the myoglobin mRNA pool. In contrast, low levels of myoglobin in mouse skeletal muscle are not apparently correlated with low levels of myoglobin mRNA. As expected from the early appearance of myoglobin mRNA in embryonic skeletal muscle, both rat and mouse embryonic myoblasts accumulate myoglobin mRNA on fusion and differentiation in vitro. Images PMID:3796609

  8. Dietary oils mediate cortisol kinetics and the hepatic mRNA expression profile of stress-responsive genes in gilthead sea bream (Sparus aurata) exposed to crowding stress. Implications on energy homeostasis and stress susceptibility.

    PubMed

    Pérez-Sánchez, Jaume; Borrel, Míriam; Bermejo-Nogales, Azucena; Benedito-Palos, Laura; Saera-Vila, Alfonso; Calduch-Giner, Josep A; Kaushik, Sadasivam

    2013-06-01

    Juveniles of gilthead sea bream were fed with plant protein-based diets with fish oil (FO diet) or vegetable oils (66VO diet) as dietary lipid sources. No differences in growth performance were found between both groups, and fish with an average body mass of 65-70 g were crowded (90-100 kg/m(3)) to assess the stress response within the 72 h after the onset of stressor. The rise in plasma cortisol and glucose levels was higher in stressed fish of group 66VO (66VO-S) than in FO group (FO-S), but the former stressed group regained more quickly the cortisol resting values of the corresponding non-stressed diet group. The cell-tissue repair response represented by derlin-1, 75 kDa glucose-regulated protein and 170 kDa glucose-regulated protein was triggered at a lower level in 66VO-S than in FO-S fish. This occurred in concert with a long-lasting up-regulation of glucocorticoid receptors, antioxidant enzymes, enzyme subunits of the mitochondrial respiratory chain, and enzymes involved in tissue fatty acid uptake and β-oxidation. This gene expression pattern allows a metabolic phenotype that is prone to "high power" mitochondria, which would support the replacement of fish oil with vegetable oils when theoretical requirements in essential fatty acids for normal growth are met by diet.

  9. Expression of APOBEC3B mRNA in Primary Breast Cancer of Japanese Women

    PubMed Central

    Tokunaga, Eriko; Yamashita, Nami; Tanaka, Kimihiro; Inoue, Yuka; Akiyoshi, Sayuri; Saeki, Hiroshi; Oki, Eiji; Kitao, Hiroyuki; Maehara, Yoshihiko

    2016-01-01

    Recent studies have identified the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3B (APOBEC3B) as a source of mutations in various malignancies. APOBEC3B is overexpressed in several human cancer types, including breast cancer. In this study, we analyzed APOBEC3B mRNA expression in 305 primary breast cancers of Japanese women using quantitative reverse transcription-PCR, and investigated the relationships between the APOBEC3B mRNA expression and clinicopathological characteristics, prognosis, and TP53 mutations. The expression of APOBEC3B mRNA was detected in 277 tumors and not detected in 28 tumors. High APOBEC3B mRNA expression was significantly correlated with ER- and PR-negativity, high grade and high Ki67 index. The APOBEC3B mRNA expression was highest in the triple-negative and lowest in the hormone receptor-positive/HER2-negative subtypes. The TP53 gene was more frequently mutated in the tumors with high APOBEC3B mRNA expression. High APOBEC3B mRNA expression was significantly associated with poor recurrence-free survival in all cases and the ER-positive cases. These findings were almost consistent with the previous reports from the Western countries. In conclusion, high APOBEC3B mRNA expression was related to the aggressive phenotypes of breast cancer, high frequency of TP53 mutation and poor prognosis, especially in ER-positive tumors. PMID:27977754

  10. Analysis of xanthine dehydrogenase mRNA levels in mutants affecting the expression of the rosy locus.

    PubMed Central

    Covington, M; Fleenor, D; Devlin, R B

    1984-01-01

    Xanthine dehydrogenase (XDH) mRNA levels were measured in a number of mutants and natural variants affecting XDH gene expression. Two variants, ry+4 and ry+10, contain cis-acting elements which map to a region flanking the 5' end of the XDH gene. Ry+4, which has 2-3 times more XDH protein than a wild type strain, has 3.2 times more XDH mRNA. Ry+10 has 50% of the wild type XDH level and 54% of the wild type XDH mRNA level. Three rosy mutants which map within the structural gene were also examined. Two of these had little if any XDH mRNA, but the third mutant had 1.3 times more XDH mRNA than wild type flies. Another mutant, ry2 , which contains no XDH protein and has a 9KB transposable element inserted into the XDH gene, has normal levels of XDH mRNA transcripts which are also the same size as those found in the wild type strain. Changes in XDH mRNA levels were measured during Drosophila development and found to parallel changes in the amount of XDH protein. In addition, there were no large changes in the size of XDH mRNA during development. Images PMID:6588363

  11. The mRNA Decay Pathway Regulates the Expression of the Flo11 Adhesin and Biofilm Formation in Saccharomyces cerevisiae

    PubMed Central

    Lo, Tricia L.; Qu, Yue; Uwamahoro, Nathalie; Quenault, Tara; Beilharz, Traude H.; Traven, Ana

    2012-01-01

    Regulation of the FLO11 adhesin is a model for gene expression control by extracellular signals and developmental switches. We establish that the major mRNA decay pathway regulates FLO11 expression. mRNA deadenylation of transcriptional repressors of FLO11 by the exonuclease Ccr4 keeps their levels low, thereby allowing FLO11 transcription. PMID:22595243

  12. Characterization of a type II chlorophyll a/b-binding protein gene (Lhcb2*Pp1) in peach. II. mRNA abundance in developing leaves exposed to sun or shade.

    PubMed

    Bassett, Carole L; Callahan, Ann M

    2003-05-01

    Leaf development of shoots exposed to full sunlight and shoots shaded by the canopy was followed in field-grown, mature peach trees (Prunus persica (L.) Batsch, cv. Loring) during the first half of the 1995 growing season. The architecture and size of shaded shoots and sun-exposed shoots differed significantly. Total number of leaves produced on shaded shoots was significantly less than on sun-exposed shoots throughout the season, and differences in leaf number between light conditions increased as the season progressed. The overall patterns of leaf development along sun-exposed and shaded shoots were qualitatively similar. The expression pattern of the type II chlorophyll a/b-binding protein gene, Lhcb2*Pp1, determined by RNA abundance in leaves at different positions along the shoot, was also similar between the two light conditions. The major difference between sun-exposed and shaded leaves was a lower abundance of Lhcb2*Pp1 RNA in mature, shaded leaves compared with sun-exposed leaves. Although the number of fruit per shoot was significantly lower on shaded shoots than on sun-exposed shoots, the rate of fruit drop was not substantially different during the growing season, indicating that quantitative differences in leaf initiation and growth caused by differences in light exposure did not adversely affect fruit retention. However, based on comparison with a previous study of leaf development in non-fruiting trees, reproductive development slowed the rate of vegetative growth without affecting the overall pattern of leaf development along the shoots.

  13. Single-Molecule mRNA Detection in Live Yeast

    PubMed Central

    Lenstra, Tineke L.

    2016-01-01

    Visualization of single RNA molecules in living cells has enabled the study of synthesis, movement, and localization of mRNAs and has provided insight into gene regulation with sub-second temporal resolution and nanometer spatial resolution. Following transcription in single cells indicates that gene activity is heterogeneous between cells and also exhibits random variability over time even within single cells. Studies of mRNAs in yeast can take advantage of the powerful genetics available in this model organism and allow mechanistic questions to be addressed. In this chapter, we describe an approach for visualizing mRNA and transcription in live yeast cells. The method is based on binding of fluorescently labeled MS2 and PP7 coat proteins to stem loops sequences that are introduced into the gene of interest. We give detailed protocols for the construction of the necessary yeast strains, for image acquisition, and for validation. PMID:27110320

  14. The role of mRNA structure in bacterial translational regulation.

    PubMed

    Meyer, Michelle M

    2017-01-01

    The characteristics of bacterial messenger RNAs (mRNAs) that influence translation efficiency provide many convenient handles for regulation of gene expression, especially when coupled with the processes of transcription termination and mRNA degradation. An mRNA's structure, especially near the site of initiation, has profound consequences for how readily it is translated. This property allows bacterial gene expression to be altered by changes to mRNA structure induced by temperature, or interactions with a wide variety of cellular components including small molecules, other RNAs (such as sRNAs and tRNAs), and RNA-binding proteins. This review discusses the links between mRNA structure and translation efficiency, and how mRNA structure is manipulated by conditions and signals within the cell to regulate gene expression. The range of RNA regulators discussed follows a continuum from very complex tertiary structures such as riboswitch aptamers and ribosomal protein-binding sites to thermosensors and mRNA:sRNA interactions that involve only base-pairing interactions. Furthermore, the high degrees of diversity observed for both mRNA structures and the mechanisms by which inhibition of translation occur have significant consequences for understanding the evolution of bacterial translational regulation. WIREs RNA 2017, 8:e1370. doi: 10.1002/wrna.1370 For further resources related to this article, please visit the WIREs website.

  15. Gravitational loading of a simulated launch alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1996-01-01

    Serum-deprived mouse osteoblastic cells (MC3T3-E1a) were centrifuged under a regime designed to simulate a space shuttle launch (maximum of 3g). Messenger RNA levels for eight genes involved in bone growth and maintenance were determined using RT-PCR. Following 30 min of centrifugation, mRNA level for early response gene c-fos was significantly increased 89% (P < 0.05). The c-fos induction was transient and returned to control levels after 3 h. The mRNA level for the mineralization marker gene osteocalcin was significantly decreased to 44% of control level (P < 0.005) 3 h after centrifugation. No changes in mRNA levels were detected for c-myc, TGFbeta1, TGFbeta2, cyclophilin A, or actin. No basal mRNA level for TGFbeta3 was detected. In addition, no change in the steady-state synthesis of prostaglandin E2 was detected, possibly due to lack of lipid substrates in serum-deprived cells, suggesting that the increase in c-fos mRNA in response to gravitational loading is a result of mechanical stimulation. These results indicate that a small magnitude mechanical loading, such as that experienced during a shuttle launch, can alter mRNA levels in quiescent osteoblastic cells.

  16. Role of mRNA Methylation in Prostate Cancer

    DTIC Science & Technology

    2015-02-01

    TERMS mRNA methylation, FTO, MeRIP-seq, RNA -seq, m6A 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...FTO, MeRIP-seq, RNA -seq, m6A, gene regulation 3. ACCOMPLISHMENTS:  What were the major goals of the project? Task 1. To determine how FTO...knockdown Months 1-2 100% 1/1/2014 Work out MeRIP experiment Months 1-2 100% 8/1/2014 Perform RNA -Seq analysis of control and FTO-knockdown LNCaP

  17. Decreased albumin mRNA in immunodeficient wasted' mice

    SciTech Connect

    Libertin, C.R.; Buczek, N.; Weaver, P.; Mobarhan, S.; Woloschak, G.E. Argonne National Lab., IL )

    1991-03-15

    Mice bearing the autosomal recessive gene wst (wst/wst) develop a wasting syndrome' that leads to death by 28-32 days of age. These mice have faulty repair of damage induced by ionizing radiation, immunodeficiency at secretory sites, and neurologic abnormalities. In addition to a progressively more apparent wasted phenotype, wst/wst mice show other features of failure to thrive and malnutrition. Daily body weights of the animals revealed a loss in weight between 25 and 30 days of age, a time during which normal littermates were progressively and rapidly gaining weight. Albumin mRNA levels were measured by dilution dot blot hybridizations of liver-derived RNA preparations from wasted mice, littermates, and parental controls. In all wasted mice, albumin mRNA levels were reduced 5 to 10 fold compared to controls. Northern blots revealed that the albumin mRNA present in wasted mice was normal in length though reduced in amount. These results suggest there may be a relationship between low albumin synthesis and the wasting syndrome of the wst/wst mouse.

  18. Analysis of specific mRNA destabilization during Dictyostelium development.

    PubMed

    Mangiarotti, G; Bulfone, S; Giorda, R; Morandini, P; Ceccarelli, A; Hames, B D

    1989-07-01

    A number of specific mRNAs are destabilized upon disaggregation of developing Dictyostelium discoideum cells. Analysis of a family of cloned genes indicates that only prespore-enriched mRNAs are affected; constitutive mRNAs that are expressed throughout development and mRNAs that accumulate preferentially in prestalk cells are stable under these conditions. The decay of sensitive prespore mRNAs can be halted by allowing the cells to reaggregate, indicating that destabilization occurs by the progressive selection of individual molecules rather than on all members of an mRNA subpopulation at the time of disaggregation. Individual molecules of the sensitive mRNA species remain engaged in protein synthesis in the disaggregated cells until selected. Destabilization of sensitive mRNAs is induced by cell dissociation even in the presence of concentrations of nogalamycin that inhibit RNA synthesis. The reported prevention of disaggregation-induced mRNA decay by actinomycin D and daunomycin is therefore probably a secondary effect unrelated to the inhibition of transcription.

  19. Maternally inherited npm2 mRNA is crucial for egg developmental competence in zebrafish.

    PubMed

    Bouleau, Aurélien; Desvignes, Thomas; Traverso, Juan Martin; Nguyen, Thaovi; Chesnel, Franck; Fauvel, Christian; Bobe, Julien

    2014-08-01

    The molecular mechanisms underlying and determining egg developmental competence remain poorly understood in vertebrates. Nucleoplasmin (Npm2) is one of the few known maternal effect genes in mammals, but this maternal effect has never been demonstrated in nonmammalian species. A link between developmental competence and the abundance of npm2 maternal mRNA in the egg was previously established using a teleost fish model for egg quality. The importance of maternal npm2 mRNA for egg developmental competence remains unknown in any vertebrate species. In the present study, we aimed to characterize the contribution of npm2 maternal mRNA to early developmental success in zebrafish using a knockdown strategy. We report here the oocyte-specific expression of npm2 and maternal inheritance of npm2 mRNA in zebrafish eggs. The knockdown of the protein translated from this maternal mRNA results in developmental arrest before the onset of epiboly and subsequent embryonic death, a phenotype also observed in embryos lacking zygotic transcription. Npm2 knockdown also results in impaired transcription of the first-wave zygotic genes. Our results show that npm2 is also a maternal effect gene in a nonmammalian vertebrate species and that maternally inherited npm2 mRNA is crucial for egg developmental competence. We also show that de novo protein synthesis from npm2 maternal mRNA is critical for developmental success beyond the blastula stage and required for zygotic genome activation. Finally, our results suggest that npm2 maternal mRNA is an important molecular factor of egg quality in fish and possibly in all vertebrates.

  20. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    SciTech Connect

    Kreamer, G.L.; Squibb, K.; Gioeli, D.; Garte, S.J.; Wirgin, I. )

    1991-06-01

    The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.

  1. Cytokine mRNA expression in postischemic/reperfused myocardium.

    PubMed Central

    Herskowitz, A.; Choi, S.; Ansari, A. A.; Wesselingh, S.

    1995-01-01

    While the role of cytokines in mediating injury during hind limb skeletal muscle ischemia followed by reperfusion has recently been described, the role of cytokines in myocardial infarction and ischemia/reperfusion have remained relatively unexplored. We hypothesize that cytokines play an important role in the regulation of postischemic myocardial inflammation. This study reports the temporal sequence of proinflammatory cytokine gene expression in postischemic/reperfused myocardium and localizes interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha)-protein by immunostaining. Rats were subjected to either permanent left anterior descending (LAD) occlusion or to 35 minutes of LAD occlusion followed by reperfusion and sacrificed up to 7 days later. Rat-specific oligonucleotide probes were used to semiquantitatively assess the relative expression of mRNA for TNF-alpha, IL-1 beta, IL-2, IL-6, interferon-gamma (IFN-gamma), and transforming growth factor-beta 1 (TGF-beta 1) utilizing the reverse transcriptase-polymerase chain reaction amplification technique. Increased cardiac mRNA levels for all cytokines except IL-6 and IFN-gamma were measurable within 15 to 30 minutes of LAD occlusion and increased levels were generally sustained for 3 hours. During early reperfusion, mRNA levels for IL-6 and TGF-beta 1 were significantly reduced compared with permanent LAD occlusion. In both groups, cytokine mRNA levels all returned to baseline levels at 24 hours, while IL-1 beta, TNF-alpha, and TGF-beta 1 mRNA levels again rose significantly at 7 days only in animals with permanent LAD occlusion. Immunostaining for IL-1 beta and TNF-alpha protein revealed two patterns of reactivity: 1) microvascular staining for both IL-1 beta and TNF-alpha protein only in postischemic reperfused myocardium in early post-reperfusion time points; and 2) staining of infiltrating macrophages in healing infarct zones which was most prominent at 7 days after permanent LAD occlusion

  2. Genes

    MedlinePlus

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

  3. A microfluidic multiwell chip for enzyme-free detection of mRNA from few cells.

    PubMed

    Haider, Michaela; Ji, Bozhi; Haselgrübler, Thomas; Sonnleitner, Alois; Aberger, Fritz; Hesse, Jan

    2016-12-15

    Isogenic cell populations possess heterogeneous gene expression patterns. Most methods for mRNA expression analysis start with the reverse transcription of mRNA into cDNA, a process that can introduce strong signal variations not related to the actual mRNA levels. Miniaturized lab-on-a-chip systems offer properties - e.g. low sample dilution, low contamination - that enable new reaction schemes for molecular analyses. To enable transcription-free mRNA expression analysis of few single cells, a one-step cell lysis, target labelling and hybridisation approach as well as a corresponding passive multiwell chip with a volume of 25.5 nL/well were developed. The method enabled the parallel analysis of up to 96 samples and 6 target genes per sample. Preceding light microscopy of the living cells allowed correlating mRNA levels and cell number. As a proof-of-principle, the pancreatic cancer cell line Panc-1 was investigated for expression heterogeneity of a reference gene plus 5 genes reported to be overexpressed in cancer stem cells (CSCs). A good correlation (r(51)=0.739, p<0.001; rs(51)=0.744, p<0.001) between the cell number per well and the number of detected reference gene mRNA confirmed the proper function of the device. Moreover, a heterogeneous expression of the CSC-associated target genes was found which matched well with reports on the presence of CSCs in the Panc-1 cell line.

  4. A new regulatory pathway of mRNA export by an F-box protein, Mdm30.

    PubMed

    Durairaj, Geetha; Lahudkar, Shweta; Bhaumik, Sukesh R

    2014-02-01

    Mdm30, an F-box protein in yeast, has been recently shown to promote mRNA export. However, it remains unknown how Mdm30 facilitates mRNA export. Here, we show that Mdm30 targets the Sub2 component of the TREX (Transcription/Export) complex for ubiquitylation and subsequent proteasomal degradation. Such a targeted degradation of Sub2 enhances the recruitment of the mRNA export adaptor, Yra1, to the active genes to promote mRNA export. Together, these results elucidate that Mdm30 promotes mRNA export by lowering Sub2's stability and consequently enhancing Yra1 recruitment, thus illuminating new regulatory mechanisms of mRNA export by Mdm30.

  5. Synthetically modified mRNA for efficient and fast human iPS cell generation and direct transdifferentiation to myoblasts.

    PubMed

    Preskey, David; Allison, Thomas F; Jones, Mark; Mamchaoui, Kamel; Unger, Christian

    2016-05-06

    Synthetic mRNA transfection enables efficient and controlled gene expression in human cells, without genome integrations. Further, modifications to the mRNA and transfection protocol now allow for repeated transfection and long-term gene expression of an otherwise short-lived mRNA expression. This is mainly achieved through introducing modified nucleosides and interferon suppression. In this study we provide an overview and details of the advanced synthesis and modifications of mRNA originally developed for reprogramming. This mRNA allows for very efficient transfection of fibroblasts enabling the generation of high quality human iPS cells with a six-factor mRNA cocktail in 9 days. Furthermore, we synthesised and transfected modified MYOD1 mRNA to transdifferentiate human fibroblasts into myoblast-like cells without a transgene footprint. This efficient and integration-free mRNA technology opens the door for safe and controlled gene expression to reverse or redirect cell fate.

  6. Antisense oligodeoxynucleotides targeted to MAG mRNA profoundly alter BP and PLP mRNA expression in differentiating oligodendrocytes: a caution.

    PubMed

    Laszkiewicz, I; Wiggins, R C; Konat, G W

    1999-09-01

    The applicability of antisense technology to suppress the expression of myelin associated glycoprotein (MAG) in cultured oligodendrocytes was evaluated. Differentiating oligodendrocyte precursor cells obtained by the shake-off method were exposed to nine unmodified antisense oligodeoxynucleotides (ODNs) targeted to the first seven exons of MAG mRNA. After four days, steady-state levels of MAG, proteolipid protein (PLP) and basic protein (BP) mRNAs were determined by Northern blot analysis. Only ODN annealing to 599-618 nt of the MAG mRNA (the junction of exon 5 and 6) resulted in a significant, 75% decrease in the MAG mRNA level. Unexpectedly, six other anti-MAG ODNs which had no significant effect on the MAG message, greatly increased the level of BP mRNA. The highest upregulation of approximately 12 fold was observed with ODN annealing to 139-168 nt (junction of exon 3 and 4). On the other hand, the 997-1016 ODN decreased the levels of BP and PLP messages by 70-80%. The 599-618 ODN also decreased the PLP mRNA by 85%. The results demonstrate that antisense ODNs targeted to one gene may profoundly alter the expression of other genes, and hence, complicate functional analysis of the targeted protein.

  7. Identify signature regulatory network for glioblastoma prognosis by integrative mRNA and miRNA co-expression analysis.

    PubMed

    Bing, Zhi-Tong; Yang, Guang-Hui; Xiong, Jie; Guo, Ling; Yang, Lei

    2016-12-01

    Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor in adults. Patients with this disease have a poor prognosis. The objective of this study is to identify survival-related individual genes (or miRNAs) and miRNA -mRNA pairs in GBM using a multi-step approach. First, the weighted gene co-expression network analysis and survival analysis are applied to identify survival-related modules from mRNA and miRNA expression profiles, respectively. Subsequently, the role of individual genes (or miRNAs) within these modules in GBM prognosis are highlighted using survival analysis. Finally, the integration analysis of miRNA and mRNA expression as well as miRNA target prediction is used to identify survival-related miRNA -mRNA regulatory network. In this study, five genes and two miRNA modules that significantly correlated to patient's survival. In addition, many individual genes (or miRNAs) assigned to these modules were found to be closely linked with survival. For instance, increased expression of neuropilin-1 gene (a member of module turquoise) indicated poor prognosis for patients and a group of miRNA -mRNA regulatory networks that comprised 38 survival-related miRNA -mRNA pairs. These findings provide a new insight into the underlying molecular regulatory mechanisms of GBM.

  8. Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability.

    PubMed

    Russo, Joseph; Heck, Adam M; Wilusz, Jeffrey; Wilusz, Carol J

    2017-02-20

    Changes in the rate of mRNA decay are closely coordinated with transcriptional changes and together these events have profound effects on gene expression during development and disease. Traditional approaches to assess mRNA decay have relied on inhibition of transcription, which can alter mRNA decay rates and confound interpretation. More recently, metabolic labeling combined with chemical modification and fractionation of labeled RNAs has allowed the isolation of nascent transcripts and the subsequent calculation of mRNA decay rates. This approach has been widely adopted for measuring mRNA half-lives on a global scale, but has proven challenging to use for analysis of single genes. We present a series of normalization and quality assurance steps to be used in combination with 4-thiouridine pulse labeling of cultured eukaryotic cells. Importantly, we demonstrate how the relative amount of 4sU-labeled nascent RNA influences accurate quantification. The approach described facilitates reproducible measurement of individual mRNA half-lives using 4-thiouridine and could be adapted for use with other nucleoside analogs.

  9. Global decay of mRNA is a hallmark of apoptosis in aging Xenopus eggs

    PubMed Central

    Tokmakov, Alexander A.; Iguchi, Sho; Iwasaki, Tetsushi; Fukami, Yasuo; Sato, Ken-Ichi

    2017-01-01

    ABSTRACT Cytoplasmic mRNAs are specifically degraded in somatic cells as a part of early apoptotic response. However, no reports have been presented so far concerning mRNA fate in apoptotic gametes. In the present study, we analyzed the content of various cytoplasmic mRNAs in aging oocytes and eggs of the African clawed frog, Xenopus laevis. To circumvent large gene expression variation among the individual oocytes and eggs, single-cell monitoring of transcript levels has been implemented, using multiple cytoplasmic collections and reverse transcriptase quantitative PCR. It was found that numerous cytoplasmic mRNAs, coding for proteins classified in different functional types, are robustly degraded in apoptotic Xenopus eggs, but not in aging oocytes. mRNA degradation becomes evident in the eggs after meiotic exit at the time of cytochrome c release. A strong correlation between the length of PCR amplicon and specific transcript content was observed, suggesting endonucleolytic cleavage of mRNA. In addition, it was found that mRNA deadenylation also contributes to apoptotic mRNA degradation. Altogether, these findings indicate that the global decay of mRNA represents a hallmark of apoptosis in aging Xenopus eggs. To our knowledge, this is the first description of mRNA degradation in apoptotic gamete cells. PMID:28045588

  10. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    SciTech Connect

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. )

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  11. Dietary glycerol for quail: association between productive performance and COX III mRNA expression.

    PubMed

    Silva, S C C; Gasparino, E; Batista, E; Tanamati, F; Vesco, A P D; Lala, B; de Oliveira, D P

    2016-05-25

    This study was carry out to evaluate mRNA expression of mitochondrial cytochrome c oxidase III in the Pectoralis superficialis muscle of 28-day-old quails fed diets containing 0, 8, and 12% glycerol. Total RNA was extracted (N = 10) and cDNA was amplified using specifics primers for qRT-PCR. Feed efficiency and feed intake were evaluated. COX III mRNA expression in breast muscle was higher in the group fed with 12% glycerol (0.863 AU); no differences were observed in the expression of this gene between the muscle of animals fed diets without glycerol (0.357 AU) and 8% glycerol (0.415 AU). Quails that showed greater COX III mRNA expression also showed the lowest feed efficiency. These results show that there is a difference in COX III mRNA expression in breast muscle of 28-day-old quail fed diets different concentrations of glycerol.

  12. c-fos mRNA in mouse brain after MPTP treatment.

    PubMed

    Duchemin, A M; Gudehithlu, K P; Neff, N H; Hadjiconstantinou, M

    1992-04-01

    The neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces a transient increase of mRNA for the immediate-early gene c-fos in the mouse brain. The c-fos mRNA level is MPTP dose-dependent and is evident in all brain regions tested including striatum, hypothalamus, cortex, hippocampus, cerebellum and midbrain. There are regional differences in the time-course for the rise of c-fos mRNA. Pretreatment with deprenyl, a selective monoamine oxidase B inhibitor, pargyline, a nonselective monoamine oxidase inhibitor, or mazindol, a dopamine uptake transport inhibitor, does not prevent the c-fos mRNA increase, suggesting that the elevation is due to the action of MPTP and not its neurotoxic metabolite MPP+.

  13. Dopaminergic control of prolactin mRNA accumulation in the pituitary of the male rat.

    PubMed

    Brocas, H; van Coevorden, A; Seo, H; Refetoff, S; Vassart, G

    1981-04-01

    Dopaminergic control of the expression of the prolactin gene was investigated by administration of bromoergocryptine (CB154) to male rats. The effects of the drug on the following parameters were measured: (i) circulating levels of GH and PRL; (ii) synthesis of GH and PRl measured by pulse labeling pituitary fragments in vitro; (iii) GH and PRL mRNA activities; and (iv) content of PRL and mRNA. After 1 day of CB154 administration, serum PRL fell to undetectable levels whereas it took 3 days to observe a 50% reduction in PRL synthesis. This effect was accounted for by a parallel decrease in PRL mRNA activity and content. GH synthesis and GH mRNA were not affected by the treatment. Our results show that the dopaminergic inhibition of PRL production involves regulation at a pre-translational level.

  14. Modified mRNA as an alternative to plasmid DNA (pDNA) for transcript replacement and vaccination therapy

    PubMed Central

    Youn, Hyewon; Chung, June-Key

    2015-01-01

    Introduction: Current gene therapy involves replacement of defective gene by delivery of healthy genetic material to precede normal function. Virus-mediated gene delivery is the most successful and efficient method for gene therapy, but it has been challenged due to serious safety concerns. Conversely, gene delivery using plasmid DNA (pDNA) is considered safer, but its transfection efficiency is much lower than virus-mediated gene transfer. Recently, mRNA has been suggested as an alternative option to avoid undesired insertion of delivered DNA sequences with higher transfection efficiency and stability. Area covered: In this review, we summarize the currently available strategies of mRNA modification to increase the therapeutic efficacy; we also highlight the recent improvements of mRNA delivery for in vivo applications of gene therapy. Expert opinion: The use of mRNA-based gene transfer could indeed be a promising new strategy for gene therapy. Notable advantages include no risk of integration into the genomic DNA, adjustable gene expression and easier modulation of the immune system. By reducing or utilizing the immunogenic properties, mRNA offers a promising tool for gene/or transcript replacement. PMID:26125492

  15. Prolyl carboxypeptidase mRNA expression in the mouse brain.

    PubMed

    Jeong, Jin Kwon; Diano, Sabrina

    2014-01-13

    Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and α-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic α-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain.

  16. Biomarkers of endocrine disruption at the mRNA level

    SciTech Connect

    Denslow, N.D.; Bowman, C.J.; Robinson, G.; Lee, H.S.; Ferguson, R.J.; Hemmmer, M.J.; Folmar, L.C.

    1999-07-01

    A large number of estrogen-mimicking, anthropogenic chemicals capable of disrupting normal reproductive function have been identified. The ubiquitous distribution of these compounds, many as components of complex industrial or municipal waste, has spurred an effort to develop methods to screen for chemicals which disrupt normal endocrine regulation of reproduction. The authors have developed assays that both allow exposure of animals in vivo and measure the response at the level of gene activation. The authors have developed a probe for measuring the induction of vitellogenin mRNA by Northern Blot in livers of sheepshead minnows treated with 17-{beta}-estradiol. The authors have also developed a strategy for using Differential Display Polymerase Chain Reaction for determining gene induction profiles following exposure to estradiol. These methods should be adaptable to a variety of structurally diverse estrogen mimics.

  17. Analysis of an mRNA exhibiting anomalous translational specificity.

    PubMed Central

    Vellanoweth, R L; Rabinowitz, J C

    1991-01-01

    Gene 6 mRNA of Bacillus subtilis phage phi 29 is inefficiently translated under standard in vitro conditions by Escherichia coli, while it is efficiently translated by the in vitro system derived from B. subtilis. This is a rare example of the inability of E. coli to translate mRNA translated by B. subtilis. The ionic condition in the translation systems was the key component in the differential recognition of the gene 6 message by E. coli and B. subtilis ribosomes. Its translation by E. coli ribosomes was preferentially inhibited by moderate levels of KCl, while its translation by B. subtilis ribosomes was unaffected by these concentrations of salt. This preferential inhibition with E. coli ribosomes was observed in vitro as well as in vivo. While not influencing the general phenomenon of preferential inhibition, anion-specific effects were observed in overall protein synthesis. Glutamate and acetate promoted efficient synthesis over a broad range of concentrations, whereas chloride was inhibitory at all concentrations tested. Images PMID:1898927

  18. Regulation of mRNA abundance in activated T lymphocytes: identification of mRNA species affected by the inhibition of protein synthesis.

    PubMed Central

    Coleclough, C; Kuhn, L; Lefkovits, I

    1990-01-01

    Inhibition of protein synthesis has often been observed to increase the concentration of mRNAs that encode proteins associated with the regulation of cell division. As two-dimensional gel electrophoresis permits the simultaneous monitoring of individual elements in large populations of gene products, we have used this technique to assess the effect of cycloheximide treatment on the mRNA complement of activated mouse T cells in an objective fashion. Two-dimensional gels of proteins generated by cell-free translation of mRNA from T-cell blasts display about 400 spots; only 5 of these are reproducibly enhanced by cycloheximide treatment and about 4 are diminished. The cDNA cloning vector lambda jac allows analysis of large arrays of molecular clones by cell-free expression, and we have used it in a sibling selection scheme to isolate a clone of one of the prominently induced mRNA species, which we refer to as chx1. chx1 mRNA concentration is increased by cycloheximide treatment of activated B cells, as well as T cells, and it is rapidly and transiently induced, in a cycloheximide-enhanced manner, upon serum stimulation of resting 3T3 fibroblastoid cells. The chx1 protein is hydrophilic, is slightly basic, and has patches of homology with the Jun-D gene product. The chx1 gene is remarkable in its lack of detectable introns and of strong bias against CpG dinucleotides. Images PMID:2308934

  19. Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli.

    PubMed Central

    Wu, C J; Janssen, G R

    1997-01-01

    The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli. PMID:9352935

  20. Self-amplifying mRNA vaccines.

    PubMed

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization.

  1. mRNA transport in yeast: time to reinvestigate the functions of the nucleolus.

    PubMed Central

    Schneiter, R; Kadowaki, T; Tartakoff, A M

    1995-01-01

    Nucleocytoplasmic transport of mRNA is vital to gene expression and may prove to be key to its regulation. Genetic approaches in Saccharomyces cerevisiae have led to the identification of conditional mutants defective in mRNA transport. Mutations in approximately two dozen genes result in accumulation of transcripts, trapped at various sites in the nucleus, as detected by in situ hybridization. Phenotypic and molecular analyses of many of these mRNA transport mutants suggest that, in yeast, the function of the nucleus is not limited to the biogenesis of pre-ribosomes but may also be important for transport of poly(A)+ RNA. A similar function of the animal cell nucleolus is suggested by several observations. Images PMID:7626803

  2. RNA Polymerase II cluster dynamics predict mRNA output in living cells

    PubMed Central

    Cho, Won-Ki; Jayanth, Namrata; English, Brian P; Inoue, Takuma; Andrews, J Owen; Conway, William; Grimm, Jonathan B; Spille, Jan-Hendrik; Lavis, Luke D; Lionnet, Timothée; Cisse, Ibrahim I

    2016-01-01

    Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output. DOI: http://dx.doi.org/10.7554/eLife.13617.001 PMID:27138339

  3. Functional mapping of the translation-dependent instability element of yeast MATalpha1 mRNA.

    PubMed Central

    Hennigan, A N; Jacobson, A

    1996-01-01

    The determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions. We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product. This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure. Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region. Termination of translation 5' to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts. Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling. Detailed mutagenesis of the element's rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element's rare-codon interval is translated. This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA. Deletion of three nucleotides 3' to this sequence shifted the stability boundary one codon 5' to its wild-type location. Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance. Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction

  4. Dendritic transport element of human arc mRNA confers RNA degradation activity in a translation-dependent manner.

    PubMed

    Ninomiya, Kensuke; Ohno, Mutsuhito; Kataoka, Naoyuki

    2016-11-01

    Localization of mRNA in neuronal cells is a critical process for spatiotemporal regulation of gene expression. Cytoplasmic localization of mRNA is often conferred by transport elements in 3' untranslated region (UTR). Activity-regulated cytoskeleton-associated protein (arc) mRNA is one of the localizing mRNAs in neuronal cells, and its localization is mediated by dendritic targeting element (DTE). As arc mRNA has introns in its 3' UTR, it was thought that arc mRNA is a natural target of nonsense-mediated mRNA decay (NMD). Here, we show that DTE in human arc 3' UTR has destabilizing activity of RNA independent of NMD pathway. DTE alone was able to cause instability of the reporter mRNA and this degradation was dependent on translation. Our results indicate that DTE has dual activity in mRNA transport and degradation, which suggests the novel spatiotemporal regulation mechanism of activity-dependent degradation of the mRNA.

  5. FKBP5, SERT and COMT mRNA expressions in the peripheral leukocytes during menstruation cycle in healthy reproductive females.

    PubMed

    Kinouchi, Sawako; Iga, Jun-Ichi; Ueno, Shu-Ichi; Yamauchi, Ken; Numata, Shusuke; Song, Hongwei; Sumitani, Satsuki; Shibuya-Tayoshi, Sumiko; Haku, Mari; Yasui, Toshiyuki; Irahara, Minoru; Morita, Kyoko; Rokutan, Kazuhito; Ohmori, Tetsuro

    2008-03-21

    There have been several evidences that the mRNA expressions in the peripheral leukocytes may indicate not only physical but also psychological states. The purpose of this study is whether the mRNA expressional changes in the leukocytes are related to the mental states across the menstrual cycle in reproductive healthy female subjects. Thirty-eight female subjects (22.4+/-1.4 year-old) were participated in this study at three menstruation cycle periods (menstrual, follicular and luteal phase). The FKBP5 (FK506-binding protein gene), SERT (serotonin transporter gene) and COMT (catechol-o-methyltransferase gene) mRNA expressions in the leukocytes were determined with hormonal data. The psychological changes were assessed with self-rating hospital anxiety and depression scale (HADS). Only one thirds of subjects (n=12) had regular menstrual cycles during the experiment. So we analyzed the data from these 12 subjects. The anxiety score of each subject was changed across the menstrual cycle (Friedman test: P<0.05). The FKBP5 mRNA expression was significantly lower in the follicular phase than in the other phases but no changes were seen in either SERT or COMT mRNA expressions among the phases. In conclusion, there are differences of HADS anxiety score and FKBP5 mRNA expression in the leukocytes across the menstrual cycle but there is no correlation between anxiety scores and FKBP5 mRNA.

  6. Polyamines cause elevation of steroid 5α-reductase mRNA levels by suppressing mRNA degradation in C6 glioma cells.

    PubMed

    Morita, Kyoji; Lee, Mi-Sook; Her, Song; Nishibori, Naoyoshi

    2014-10-01

    Polyamines are widely distributed in living organisms, and considered to play a potential role in various cellular processes. The effects of polyamines on gene expression as well as cell proliferation have been suggested to be closely associated with the physiological and pathological functions. However, it seems necessary to investigate their potential roles in the regulation of cellular metabolism and functions. Previously, glial cells have been suggested to be involved in the protection and preservation of neuronal functions, probably through the production of neurotrophic factors in the brain. On the other hand, neuroactive 5α-reduced steroids promote glial cell differentiation, resulting in enhancement of their ability to produce brain-derived neurotrophic factor (BDNF). Based on these findings, polyamines are assumed to stimulate the expression of the gene encoding steroid 5α-reductase (5α-R), which can induce the production of neuroactive 5α-reduced steroids in glial cells. The effects of polyamines on 5α-R mRNA levels in C6 glioma cells were examined as a model experiment. In consequence, spermine (SPM) and spermidine (SPD), but not putrescine (PUT), have been shown to elevate 5α-R mRNA levels without activating the 5α-R promoter. Furthermore, SPM increased 5α-R mRNA levels under the conditions in which the mRNA biosynthesis was inhibited. Therefore, it can be speculated that polyamines increase 5α-R mRNA levels as a consequence of suppressing the degradation of mRNA.

  7. Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

    PubMed

    Kasprzak, Aldona; Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Małgorzata; Przybyszewska, Wiesława; Kaczmarek, Elżbieta; Koczorowska, Maria; Kościński, Tomasz; Zabel, Maciej; Drews, Michał

    2013-01-01

    The insulin-like growth factor (IGF)-1 gene consists of 6 exons resulting in the expression of 6 variant forms of mRNA (IA, IB, IC, IIA, IIB and IIC) due to an alternative splicing. The mechanisms of IGF-1 gene splicing and the role of local expression manifested by IGF-1 mRNA variants in colorectal carcinoma (CRC) have not been extensively investigated. Therefore, the aim of our study was to analyse the expression of IGF-1 mRNA isoforms [A, B, C, P1 (class I) and P2 (class II)], as well as the protein expression in CRC and control samples isolated from 28 patients. The expression of Ki-67 was also analysed and clinical data were obtained. For this purpose, we used quantitative real-time PCR (qPCR) and immunocytochemistry. The expression of mRNAs coding for all splicing isoforms of IGF-1 was observed in every tissue sample studied, with a significantly lower expression noted in the CRC as compared to the control samples. The cytoplasmic expression of IGF-1 protein was found in 50% of the CRC and in ~40% of the non-tumor tissues; however, no significant quantitative inter-group differences were observed. The expression of the IGF-1 gene in the 2 groups of tissues was controlled by the P1 and P2 promoters in a similar manner. No significant differences were detected in the expression of the IGF-1 A and B isoforms; however, their expression was significantly higher compared to that of isoform C. No significant differences were observed between the expression of Ki-67 mRNA in the CRC and control tissue even though the expression of the Ki-67 protein was higher in the CRC compared to the control samples. Ki-67 protein expression was associated with the macroscopic and microscopic aspects of CRC. A significant positive correlation was found between the local production of total mRNA and isoform A and the expression of Ki-67 mRNA, although only in the non-tumor tissues. In CRC samples, the local expression of the total IGF-1 mRNA and all splicing isoforms of IGF-1 mRNA

  8. Genetic analysis of glucose regulation in saccharomyces cerevisiae: control of transcription versus mRNA turnover.

    PubMed Central

    Cereghino, G P; Scheffler, I E

    1996-01-01

    A major determinant of the steady-state level of the mRNA encoding the iron protein (Ip) subunit of succinate dehydrogenase of yeast is its rate of turnover. This mRNA is significantly more stable in glycerol than in glucose media. Many other genes, for example, SUC2, that are repressed in the presence of glucose are believed to be controlled at the level of transcription. The present study elucidates differences in the regulatory mechanisms by which glucose controls the transcription and turnover of the SUC2 and Ip mRNAs. The signaling pathway for glucose repression at the transcriptional level has been associated with a number of gene products linking glucose uptake with nuclear events. We have investigated whether the same genes are involved in the control of Ip mRNA stability. Phosphorylation of glucose or fructose is critical in triggering the transcript's degradation, but any hexokinase will do. Of the other known genes examined, most, with the exception of REG1, are not involved in determining the differential stability of the Ip transcript. Finally, our results indicate that differential stability on different carbon sources also plays a role in determining the steady-state level of the SUC2 mRNA. Thus, glucose repression includes both transcriptional and post-transcriptional mechanisms. Images PMID:8617211

  9. Evidence That Base-pairing Interaction between Intron and mRNA Leader Sequences Inhibits Initiation of HAC1 mRNA Translation in Yeast*

    PubMed Central

    Sathe, Leena; Bolinger, Cheryl; Mannan, M. Amin-ul; Dever, Thomas E.; Dey, Madhusudan

    2015-01-01

    The Hac1 transcription factor in yeast up-regulates a collection of genes that control protein homeostasis. Base-pairing interactions between sequences in the intron and the 5′-untranslated region (5′ UTR) of the HAC1 mRNA represses Hac1 protein production under basal conditions, whereas cytoplasmic splicing of the intron by the Ire1 kinase-endonuclease, activated under endoplasmic reticulum stress conditions, relieves the inhibition and enables Hac1 synthesis. Using a random mutational screen as well as site-directed mutagenesis, we identify point mutations within the 5′ UTR-intron interaction site that derepress translation of the unspliced HAC1 mRNA. We also show that insertion of an in-frame AUG start codon upstream of the interaction site releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhances production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. These results suggest that the major block of translation occurs at the initiation stage. Supporting this interpretation, the point mutations that enhanced Hac1 production resulted in an increased percentage of the HAC1 mRNA associating with polysomes versus free ribosomal subunits. Thus, our results provide evidence that the 5′ UTR-intron interaction represses translation initiation on the unspliced HAC1 mRNA. PMID:26175153

  10. Cistanches Herba aqueous extract affecting serum BGP and TRAP and bone marrow Smad1 mRNA, Smad5 mRNA, TGF-β1 mRNA and TIEG1 mRNA expression levels in osteoporosis disease.

    PubMed

    Liang, Hai-Dong; Yu, Fang; Tong, Zhi-Hong; Zhang, Hong-Quan; Liang, Wu

    2013-02-01

    We studied molecular mechanism of Cistanches Herba aqueous extract (CHAE) in ovariectomized (OVX) rats, as an experimental model of postmenopausal osteoporosis. Female rats were either sham-operated or bilaterally OVX; and at 60 days postoperatively. The OVX group (n = 8) received an ovariectomy and treatment with normal saline for 90 days commencing from 20th post ovariectomy day. The ovariectomized +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy and were treated with Cistanches Herba aqueous extract of 100 mg/kg body weight daily for 90 days commencing from 22nd post ovariectomy day. The ovariectomy +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy, and were treated with the of 200 mg/kg body weight daily for 90 days commencing from 20th post ovariectomy day. Serum BGP and TRAP, E2, FSH and LH level, bone marrow Smad1, Smad5, TGF-β1 and TIEG1 mRNA expression levels were examined. Results showed that serum BGP and TRAP, FSH and LH levels were significantly increased, whereas E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels were significantly decreased in OVX rats compared to sham rats. 90 days of CHAE treatment could significantly decrease serum BGP and TRAP, FSH and LH levels, and increase E2, Smad1, Smad5, TGF-β1 and TIEG1 mRNA and proteins expression levels in OVX rats. It can be concluded that CHAE play its protective effect against OVX-induced bone degeneration partly by regulating some bone metabolism related genes, e.g. Smad1, Smad5, TGF-β1 and TIEG1.

  11. Tissue-specific mRNA expression profiling in grape berry tissues

    PubMed Central

    Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

    2007-01-01

    Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and

  12. Reduced secreted mu mRNA synthesis in selective IgM deficiency of Bloom's syndrome.

    PubMed Central

    Kondo, N; Ozawa, T; Kato, Y; Motoyoshi, F; Kasahara, K; Kameyama, T; Orii, T

    1992-01-01

    Serum IgM concentrations were low although serum IgG and IgA concentrations were normal in both our patients with Bloom's syndrome. Although the percentages of surface IgM-bearing cells were not reduced, the numbers of IgM-secreting cells were markedly reduced. The membrane-bound mu (microns) and secreted mu (microseconds) mRNAs are produced from transcripts of a single immunoglobulin mu gene by alternative RNA processing pathways. The control of microseconds mRNA synthesis depends on the addition of poly(A) to microseconds C-terminal segment. In both patients, mu mRNA was well detected but microseconds C-terminal mRNA was scarcely detected, suggesting that microns mRNA was well transcribed but microseconds mRNA was not. There was, at least, no mutation or deletion in the microseconds C-terminal coding sequence, the RNA splice site (GG/TAAAC) at the 5' end of microseconds C-terminal segment and the AATAAA poly(A) signal sequence in both patients. Our results suggest that selective IgM deficiency in Bloom's syndrome is due to an abnormality in the maturation of surface IgM-bearing B cells into IgM-secreting cells and a failure of microseconds mRNA synthesis. Moreover, reduced microseconds mRNA synthesis may be due to the defect on developmental regulation of the site at which poly(A) is added to transcripts of the mu gene. Images Fig. 2 PMID:1563106

  13. Deregulated expression of VHL mRNA variants in papillary thyroid cancer.

    PubMed

    Baldini, Enke; Tuccilli, Chiara; Arlot-Bonnemains, Yannick; Chesnel, Frank; Sorrenti, Salvatore; De Vito, Corrado; Catania, Antonio; D'Armiento, Eleonora; Antonelli, Alessandro; Fallahi, Poupak; Watutantrige-Fernando, Sara; Tartaglia, Francesco; Barollo, Susi; Mian, Caterina; Bononi, Marco; Arceri, Stefano; Mascagni, Domenico; Vergine, Massimo; Pironi, Daniele; Monti, Massimo; Filippini, Angelo; Ulisse, Salvatore

    2017-03-05

    Recent findings demonstrated that a subset of papillary thyroid cancers (PTCs) is characterized by reduced expression of the von Hippel-Lindau (VHL) tumor suppressor gene, and that lowest levels associated with more aggressive PTCs. In the present study, the levels of the two VHL mRNA splicing variants, VHL-213 (V1) and VHL-172 (V2), were measured in a series of 96 PTC and corresponding normal matched tissues by means of quantitative RT-PCR. Variations in the mRNA levels were correlated with patients' clinicopathological parameters and disease-free interval (DFI). The analysis of VHL mRNA in tumor tissues, compared to normal matched tissues, revealed that its expression was either up- or down-regulated in the majority of PTC. In particular, V1 and V2 mRNA levels were altered, respectively, in 78 (81.3%) and 65 (67.7%) out of the 96 PTCs analyzed. A significant positive correlation between the two mRNA variants was observed (p < 0.001). Univariate analysis documented the lack of association between each variant and clinicopathological parameters such as age, tumor size, histology, TNM stage, lymph node metastases, and BRAF mutational status. However, a strong correlation was found between altered V1 or V2 mRNA levels and DFI. Multivariate regression analysis indicated higher V1 mRNA values, along with lymph node metastases at diagnosis, as independent prognostic factors predicting DFI. In conclusion, the data reported demonstrate that VHL gene expression is deregulated in the majority of PTC tissues. Of particular interest is the apparent protective role exerted by VHL transcripts against PTC recurrences.

  14. Gallium nitrate regulates rat osteoblast expression of osteocalcin protein and mRNA levels.

    PubMed

    Guidon, P T; Salvatori, R; Bockman, R S

    1993-01-01

    Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

  15. Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

    PubMed

    Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

    2014-12-16

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

  16. Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity

    PubMed Central

    Ling, Jun; Lopez-Dee, Zenaida P.; Cottell, Colby; Wolfe, Laura; Nye, Derek

    2016-01-01

    Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP) was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T), BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl) phthalate (MEHP) as a major metabolite of another important phthalate di (2-ethylhexyl) phthalate (DEHP) inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein) and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29) growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities. PMID:27992464

  17. Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity.

    PubMed

    Ling, Jun; Lopez-Dee, Zenaida P; Cottell, Colby; Wolfe, Laura; Nye, Derek

    2016-01-01

    Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP) was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T), BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl) phthalate (MEHP) as a major metabolite of another important phthalate di (2-ethylhexyl) phthalate (DEHP) inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein) and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29) growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities.

  18. Imaging mRNA and protein interactions within neurons

    PubMed Central

    Eliscovich, Carolina; Shenoy, Shailesh M.

    2017-01-01

    RNA–protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions. PMID:28223507

  19. The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae

    PubMed Central

    Huch, Susanne; Müller, Maren; Muppavarapu, Mridula; Gommlich, Jessie; Balagopal, Vidya; Nissan, Tracy

    2016-01-01

    ABSTRACT The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization. PMID:27543059

  20. Staufen-mediated mRNA decay

    PubMed Central

    Park, Eonyoung; Maquat, Lynne E.

    2013-01-01

    Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

  1. Genomic gems: SINE RNAs regulate mRNA production.

    PubMed

    Ponicsan, Steven L; Kugel, Jennifer F; Goodrich, James A

    2010-04-01

    Mammalian short interspersed elements (SINEs) are abundant retrotransposons that have long been considered junk DNA; however, RNAs transcribed from mouse B2 and human Alu SINEs have recently been found to control mRNA production at multiple levels. Upon cell stress B2 and Alu RNAs bind RNA polymerase II (Pol II) and repress transcription of some protein-encoding genes. Bi-directional transcription of a B2 SINE establishes a boundary that places the growth hormone locus in a permissive chromatin state during mouse development. Alu RNAs embedded in Pol II transcripts can promote evolution and proteome diversity through exonization via alternative splicing. Given the diverse means by which SINE encoded RNAs impact production of mRNAs, this genomic junk is proving to contain hidden gems.

  2. Denervation of rat adrenal glands markedly increases preproenkephalin mRNA.

    PubMed Central

    Kilpatrick, D L; Howells, R D; Fleminger, G; Udenfriend, S

    1984-01-01

    The effect of denervation on the expression of rat adrenal proenkephalin has been examined. Following splanchnicectomy there was a several-fold increase in the steady-state levels of preproenkephalin mRNA, which became maximal after 24-48 hr (greater than 10-fold). These results indicate that the previously observed increase in rat adrenal enkephalin-containing peptides following denervation occurs entirely by a pretranslational mechanism. The increase in preproenkephalin mRNA was accompanied by a 50-75% decrease in rat adrenal poly(A)+ RNA. Neural input thus exerts a profound trophic influence on proenkephalin gene expression and RNA metabolism in rat adrenals. Images PMID:6594691

  3. Denervation of rat adrenal glands markedly increases preproenkephalin mRNA.

    PubMed

    Kilpatrick, D L; Howells, R D; Fleminger, G; Udenfriend, S

    1984-11-01

    The effect of denervation on the expression of rat adrenal proenkephalin has been examined. Following splanchnicectomy there was a several-fold increase in the steady-state levels of preproenkephalin mRNA, which became maximal after 24-48 hr (greater than 10-fold). These results indicate that the previously observed increase in rat adrenal enkephalin-containing peptides following denervation occurs entirely by a pretranslational mechanism. The increase in preproenkephalin mRNA was accompanied by a 50-75% decrease in rat adrenal poly(A)+ RNA. Neural input thus exerts a profound trophic influence on proenkephalin gene expression and RNA metabolism in rat adrenals.

  4. Expressional characterization of mRNA (guanine-7) methyltransferase (rnmt) during early development of Xenopus laevis.

    PubMed

    Lokapally, Ashwin; Metikala, Sanjeeva; Hollemann, Thomas

    2016-01-01

    Methylation of the guanosine cap structure at the 5' end of mRNA is essential for efficient translation of all eukaryotic cellular mRNAs, gene expression and cell viability and promotes transcription, splicing, polyadenylation and nuclear export of mRNA. In the current study, we present the spatial expression pattern of the Xenopus laevis rnmt homologue. A high percentage of protein sequence similarity, especially within the methyltransferase domain, as well as an increased expression in the cells of the transcriptionally active stages, suggests a conserved RNA cap methylation function. Spatial expression analysis identified expression domains in the brain, the retina, the lens, the otic vesicles and the branchial arches.

  5. Quantitative imaging of single mRNA splice variants in living cells

    NASA Astrophysics Data System (ADS)

    Lee, Kyuwan; Cui, Yi; Lee, Luke P.; Irudayaraj, Joseph

    2014-06-01

    Alternative messenger RNA (mRNA) splicing is a fundamental process of gene regulation, and errors in RNA splicing are known to be associated with a variety of different diseases. However, there is currently a lack of quantitative technologies for monitoring mRNA splice variants in cells. Here, we show that a combination of plasmonic dimer probes and hyperspectral imaging can be used to detect and quantify mRNA splice variants in living cells. The probes are made from gold nanoparticles functionalized with oligonucleotides and can hybridize to specific mRNA sequences, forming nanoparticle dimers that exhibit distinct spectral shifts due to plasmonic coupling. With this approach, we show that the spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1, can be monitored at single-copy resolution by measuring the hybridization dynamics of the nanoplasmonic dimers. Our study provides insights into RNA and its transport in living cells, which could improve our understanding of cellular protein complexes, pharmacogenomics, genetic diagnosis and gene therapies.

  6. Internal Initiation of Translation of mRNA in the Methylotrophic Yeast Hansenula polymorpha.

    PubMed

    Mardanova, E S; Beletsky, A V; Ravin, N V

    2016-05-01

    Besides regular cap-dependent translation of mRNA, eukaryotes exploit internal initiation of translation driven by internal ribosome entry sites (IRESs). It is supposed that internal initiation provides translation of cellular mRNAs under stress conditions where the cap-dependent initiation is reduced. A number of IRESs have been characterized in mammalian mRNAs, but only a few examples are known in lower eukaryotes, particularly in yeasts. Here we identified two IRESs in the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1. These sites are located in 5'-untranslated regions of genes HPODL_02249 and HPODL_04025 encoding a hypothetical membrane protein and actin-binding protein, respectively. In Saccharomyces cerevisiae cells, both IRESs drive expression of a second gene of a bicistronic mRNA, as well as translation of hairpin-containing monocistronic mRNA. The possibility of spurious splicing or presence of a cryptic promoter in the IRES sequences was ruled out, indicating that expression of a second gene of a bicistronic mRNA was IRES-dependent. We evaluated IRES activity of both elements and found that under normal physiological conditions its contribution to the overall translation of the respective mRNAs in yeast cells is about 0.3-0.4%. Therefore, these results suggest that the IRES-dependent translation initiation mechanism exists in Hansenula polymorpha.

  7. Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

    SciTech Connect

    Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.; Costa, E.

    1986-03-01

    In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 ..mu..M) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 ..mu..M veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 ..mu..M dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 ..mu..M) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated.

  8. mRNA for low density lipoprotein receptor in brain and spinal cord of immature and mature rabbits

    SciTech Connect

    Hofmann, S.L.; Russell, D.W.; Goldstein, J.L.; Brown, M.S.

    1987-09-01

    Hybridization studies with (/sup 32/P)cDNA probes revealed detectable amounts of mRNA for the low density lipoprotein (LDL) receptor in the central nervous system (CNS) of rabbits. mRNA levels were highest in the medulla/pons and spinal cord, which were the most heavily myelinated regions that were studied. Lower, but detectable levels were present in cerebral cortex, hypothalamus, thalamus, midbrain, and cerebellum. In the medulla/pons and spinal cord, the levels of receptor mRNA were in a range comparable to that detected in the liver. The levels of receptor mRNA in whole brain were constant from 3 days of age to adulthood and, thus, did not vary in proportion to the rate of myelin synthesis. LDL receptor mRNA in the CNS was produced by the same gene that produced the liver and adrenal mRNA as revealed by the demonstration of a deletion in the neural mRNA of Watanabe-heritable hyperlipidemic (WHHL) rabbits identical to the deletion in the LDL receptor gene of these mutant animals. Using antibodies directed against the bovine LDL receptor, the authors showed that LDL receptor protein is present in the medulla/pons of adult cows. The cell types that express LDL receptors in the CNS and the functions of these receptors are unknown.

  9. Regulation of mRNA translation during mitosis.

    PubMed

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

    2015-08-25

    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  10. Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates.

    PubMed

    Rodgers, Buel D; Weber, Gregory M; Kelley, Kevin M; Levine, Michael A

    2003-05-01

    Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indexes in adults, skeletal muscle myostatin mRNA levels were unaffected. By contrast, larval myostatin mRNA levels were sometimes elevated after a short-term fast and were consistently reduced with prolonged fasting. These effects were specific for myostatin, as mRNA levels of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase were unchanged. Cortisol levels were elevated in fasted larvae with reduced myostatin mRNA, whereas in addition immersion of larvae in 1 ppm (2.8 microM) cortisol reduced myostatin mRNA in a time-dependent fashion. These results suggest that larval myostatin mRNA levels may initially rise but ultimately fall during a prolonged fast. The reduction is likely mediated by fasting-induced hypercortisolemia, indicating divergent evolutionary mechanisms of glucocorticoid regulation of myostatin mRNA, since these steroids upregulate myostatin gene expression in mammals.

  11. hnRNP L binds to CA repeats in the 3'UTR of bcl-2 mRNA

    SciTech Connect

    Lee, Dong-Hyoung; Lim, Mi-Hyun; Youn, Dong-Ye; Jung, Seung Eun; Ahn, Young Soo; Tsujimoto, Yoshihide; Lee, Jeong-Hwa

    2009-05-08

    We previously reported that the CA-repeat sequence in the 3'-untranslated region (3'UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3'UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). A super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.

  12. Regulation of histone mRNA in the unperturbed cell cycle: evidence suggesting control at two posttranscriptional steps.

    PubMed Central

    Harris, M E; Böhni, R; Schneiderman, M H; Ramamurthy, L; Schümperli, D; Marzluff, W F

    1991-01-01

    The levels of histone mRNA increase 35-fold as selectively detached mitotic CHO cells progress from mitosis through G1 and into S phase. Using an exogenous gene with a histone 3' end which is not sensitive to transcriptional or half-life regulation, we show that 3' processing is regulated as cells progress from G1 to S phase. The half-life of histone mRNA is similar in G1- and S-phase cells, as measured after inhibition of transcription by actinomycin D (dactinomycin) or indirectly after stabilization by the protein synthesis inhibitor cycloheximide. Taken together, these results suggest that the change in histone mRNA levels between G1- and S-phase cells must be due to an increase in the rate of biosynthesis, a combination of changes in transcription rate and processing efficiency. In G2 phase, there is a rapid 35-fold decrease in the histone mRNA concentration which our results suggest is due primarily to an altered stability of histone mRNA. These results are consistent with a model for cell cycle regulation of histone mRNA levels in which the effects on both RNA 3' processing and transcription, rather than alterations in mRNA stability, are the major mechanisms by which low histone mRNA levels are maintained during G1. Images PMID:2017161

  13. Selecting Reliable mRNA Expression Measurements Across Platforms Improves Downstream Analysis

    PubMed Central

    Tong, Pan; Diao, Lixia; Shen, Li; Li, Lerong; Heymach, John Victor; Girard, Luc; Minna, John D.; Coombes, Kevin R.; Byers, Lauren Averett; Wang, Jing

    2016-01-01

    With increasing use of publicly available gene expression data sets, the quality of the expression data is a critical issue for downstream analysis, gene signature development, and cross-validation of data sets. Thus, identifying reliable expression measurements by leveraging multiple mRNA expression platforms is an important analytical task. In this study, we propose a statistical framework for selecting reliable measurements between platforms by modeling the correlations of mRNA expression levels using a beta-mixture model. The model-based selection provides an effective and objective way to separate good probes from probes with low quality, thereby improving the efficiency and accuracy of the analysis. The proposed method can be used to compare two microarray technologies or microarray and RNA sequencing measurements. We tested the approach in two matched profiling data sets, using microarray gene expression measurements from the same samples profiled on both Affymetrix and Illumina platforms. We also applied the algorithm to mRNA expression data to compare Affymetrix microarray data with RNA sequencing measurements. The algorithm successfully identified probes/genes with reliable measurements. Removing the unreliable measurements resulted in significant improvements for gene signature development and functional annotations. PMID:27199546

  14. Global changes in processing of mRNA 3' untranslated regions characterize clinically distinct cancer subtypes.

    PubMed

    Singh, Priyam; Alley, Travis L; Wright, Sarah M; Kamdar, Sonya; Schott, William; Wilpan, Robert Y; Mills, Kevin D; Graber, Joel H

    2009-12-15

    Molecular cancer diagnostics are an important clinical advance in cancer management, but new methods are still needed. In this context, gene expression signatures obtained by microarray represent a useful molecular diagnostic. Here, we describe novel probe-level microarray analyses that reveal connections between mRNA processing and neoplasia in multiple tumor types, with diagnostic potential. We now show that characteristic differences in mRNA processing, primarily in the 3'-untranslated region, define molecular signatures that can distinguish similar tumor subtypes with different survival characteristics, with at least 74% accuracy. Using a mouse model of B-cell leukemia/lymphoma, we find that differences in transcript isoform abundance are likely due to both alternative polyadenylation (APA) and differential degradation. While truncation of the 3'-UTR is the most common observed pattern, genes with elongated transcripts were also observed, and distinct groups of affected genes are found in related but distinct tumor types. Genes with elongated transcripts are overrepresented in ontology categories related to cell-cell adhesion and morphology. Analysis of microarray data from human primary tumor samples revealed similar phenomena. Western blot analysis of selected proteins confirms that changes in the 3'-UTR can correlate with changes in protein expression. Our work suggests that alternative mRNA processing, particularly APA, can be a powerful molecular biomarker with prognostic potential. Finally, these findings provide insights into the molecular mechanisms of gene deregulation in tumorigenesis.

  15. Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna).

    PubMed

    Hannas, Bethany R; Wang, Ying H; Thomson, Susanne; Kwon, Gwijun; Li, Hong; Leblanc, Gerald A

    2011-01-25

    The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.

  16. Role of miRNAs and alternative mRNA 3'-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy.

    PubMed

    Soetanto, R; Hynes, C J; Patel, H R; Humphreys, D T; Evers, M; Duan, G; Parker, B J; Archer, S K; Clancy, J L; Graham, R M; Beilharz, T H; Smith, N J; Preiss, T

    2016-05-01

    miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3'-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2 days post-TAC), and hypertrophic (7 days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3'-ends, identifying novel 3' untranslated regions (3'UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3'-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3'UTR proportion change and showed that alternative 3'UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart.

  17. mRNA Decay of Most Arabidopsis miRNA Targets Requires Slicer Activity of AGO11[OPEN

    PubMed Central

    2016-01-01

    MicroRNAs (miRNAs) are key posttranscriptional regulators of gene expression in animals and plants. They guide RNA-induced silencing complexes to complementary target mRNA, thereby mediating mRNA degradation or translational repression. ARGONAUTE (AGO) proteins bind directly to miRNAs and may catalyze cleavage (slicing) of target mRNAs. In animals, miRNA target degradation via slicing occurs only exceptionally, and target mRNA decay is induced via AGO-dependent recruitment of deadenylase complexes. Conversely, plant miRNAs generally direct slicing of their targets, but it is unclear whether slicer-independent mechanisms of target mRNA decay also exist, and, if so, how much they contribute to miRNA-induced mRNA decay. Here, we compare phenotypes and transcript profiles of ago1 null and slicer-deficient mutants in Arabidopsis (Arabidopsis thaliana). We also construct conditional loss-of-function mutants of AGO1 to allow transcript profiling in true leaves. Although phenotypic differences between ago1 null and slicer-deficient mutants can be discerned, the results of both transcript profiling approaches indicate that slicer activity is required for mRNA repression of the vast majority of miRNA targets. A set of genes exhibiting up-regulation specifically in ago1 null, but not in ago1 slicer-deficient mutants was also identified, leaving open the possibility that AGO1 may have functions in gene regulation independent of small RNAs. PMID:27208258

  18. Evaluation of diagnostic relevance of mRNA levels in peripheral blood: predictive value for mortality in hemodialysis patients.

    PubMed

    Füth, Reiner; Herder, Christian; Förster, Stefan; Müller-Scholze, Sylvia; Kruse, Niels; Rieckmann, Peter; Heinig, Antonia; Koenig, Wolfgang; Scherbaum, Werner A; Kolb, Hubert; Martin, Stephan

    2004-09-21

    In clinical practice, diagnosis and risk prediction are usually based on the analysis of serum or plasma proteins whereas gene expression analysis is not used on a routine basis. In order to compare the diagnostic and predictive relevance of serum protein and peripheral blood mRNA levels, we determined cytokine levels of end-stage renal failure patients undergoing hemodialysis. These patients face a high mortality mainly due to acceleration of atherosclerosis and subsequent severe vascular events. mRNA expression of the pro-inflammatory cytokine TNF alpha was significantly elevated in hemodialysis patients and further increased after 2 h of dialysis treatment. In contrast, gene expression of the anti-inflammatory cytokine TGF beta was significantly decreased. Patients who died during the observation period of 36 months had significantly increased mRNA levels of TNF alpha and decreased TGF beta mRNA expression at baseline. Survival analysis indicated that increased TNF alpha mRNA levels (P < 0.02) and TNF alpha/TGF beta mRNA ratios (P < 0.001) predict mortality. The corresponding cytokines in serum showed some association with disease, but serum concentrations neither changed during hemodialysis nor predicted mortality. This study shows that gene expression patterns of circulating leukocytes may present an important new diagnostic tool to predict clinical outcome in patients with inflammatory vascular diseases.

  19. Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

    PubMed Central

    Atger, Florian; Gobet, Cédric; Marquis, Julien; Martin, Eva; Wang, Jingkui; Weger, Benjamin; Lefebvre, Grégory; Descombes, Patrick; Naef, Felix; Gachon, Frédéric

    2015-01-01

    Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light–dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5′-Terminal Oligo Pyrimidine tract (5′-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5′-UTR (TISU) motif. The increased translation efficiency of 5′-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation. PMID:26554015

  20. Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver.

    PubMed

    Atger, Florian; Gobet, Cédric; Marquis, Julien; Martin, Eva; Wang, Jingkui; Weger, Benjamin; Lefebvre, Grégory; Descombes, Patrick; Naef, Felix; Gachon, Frédéric

    2015-11-24

    Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5'-UTR (TISU) motif. The increased translation efficiency of 5'-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation.

  1. Degradation of Gadd45 mRNA by nonsense-mediated decay is essential for viability

    PubMed Central

    Nelson, Jonathan O; Moore, Kristin A; Chapin, Alex; Hollien, Julie; Metzstein, Mark M

    2016-01-01

    The nonsense-mediated mRNA decay (NMD) pathway functions to degrade both abnormal and wild-type mRNAs. NMD is essential for viability in most organisms, but the molecular basis for this requirement is unknown. Here we show that a single, conserved NMD target, the mRNA coding for the stress response factor growth arrest and DNA-damage inducible 45 (GADD45) can account for lethality in Drosophila lacking core NMD genes. Moreover, depletion of Gadd45 in mammalian cells rescues the cell survival defects associated with NMD knockdown. Our findings demonstrate that degradation of Gadd45 mRNA is the essential NMD function and, surprisingly, that the surveillance of abnormal mRNAs by this pathway is not necessarily required for viability. DOI: http://dx.doi.org/10.7554/eLife.12876.001 PMID:26952209

  2. CYP1A mRNA expression in redeye mullets (Liza haematocheila) from Bohai Bay, China.

    PubMed

    An, Lihui; Hu, Jianying; Yang, Min; Zheng, Binghui; Wei, An; Shang, Jingjing; Zhao, Xingru

    2011-04-01

    Induction of cytochrome P4501A (CYP1A) has been used as a biomarker in fish for monitoring aromatic and organic contaminants. In this study, a partial of CYP1A gene in redeye mullet (Liza haematocheila) was isolated and sequenced, and then a real-time quantitative reverse-transcription polymerase chain reaction assay was developed for quantification of CYP1A mRNA normalized to β-actin. The developed method was applied to detect CYP1A mRNA expression in redeye mullets collected from Nandaihe (reference site) and Dashentang (impacted site) in Bohai Bay, China. CYP1A mRNA expression values were significantly elevated in redeye mullets from Dashentang compared to a reference site--Nandaihe, which was correlated with the contents of different environmentally relevant pollutants in tissues, particularly with PCBs and PBDEs.

  3. Expression of SART-1 mRNA in canine squamous cell carcinomas.

    PubMed

    Takaishi, Yumi; Yoshida, Yukari; Nakagaki, Kazuhide; Fujita, Michio; Taniguchi, Akiko; Orima, Hiromitsu

    2008-12-01

    SART-1, a squamous cell carcinoma (SCC) antigen recognized by cytotoxic T lymphocytes, has been useful in human cancer therapy. The SART-1(259) peptide is a potential candidate for vaccine. The present study examined an orthologue of the mRNA coding this peptide in canine SCCs. Specimens were obtained from seven canine patients with SCC, and the mRNA was isolated from the samples. The SART-1 and beta-actin genes were amplified by reverse-transcription polymerase chain reaction, using the isolated mRNA as a template. Canine SART-1 was amplified in six of the seven specimens, while beta-actin was detected in all the samples. In dogs, carcinomas expressing SART-1 could be a target for cytotoxic T lymphocyte mediated immunotherapy.

  4. mRNA expression profiling of neonatal rats after 16-day spaceflight

    NASA Astrophysics Data System (ADS)

    Miyake, M.; Ijiri, K.

    Some studies pointed out that postnatal development is the key to realize generation change of mammalians in space. For example, functional changes and hypoplasia in some organs after spaceflight during postnatal development were reported. Though profiling mRNA expression is useful to evaluate what happened in animals, these studies after spaceflight are limited to specific organs for understanding the relationship between phenotype and gene. The organ-wide analysis of mRNA expression is important to evaluate the condition of each animal, and it can find new phenomenon and help precise understanding for effect induced by spaceflight. In this experiment, we analyzed mRNA expression of liver, spleen and intestine in neonatal rats after 16-day spaceflight by Space Shuttle Columbia (STS-90).

  5. Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

    PubMed Central

    Zhang, Hui; Korenková, Vlasta; Sjöback, Robert; Švec, David; Björkman, Jens; Kruhøffer, Mogens; Verderio, Paolo; Pizzamiglio, Sara; Ciniselli, Chiara Maura; Wyrich, Ralf; Oelmueller, Uwe; Kubista, Mikael; Lindahl, Torbjørn; Lönneborg, Anders; Rian, Edith

    2014-01-01

    There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples. PMID:25369468

  6. OPIATE EXPOSURE AND WITHDRAWAL DYNAMICALLY REGULATE mRNA EXPRESSION IN THE SEROTONERGIC DORSAL RAPHE NUCLEUS

    PubMed Central

    Lunden, Jason; Kirby, Lynn G.

    2013-01-01

    Previous results from our lab suggest that hypofunctioning of the serotonergic (5-HT) dorsal raphe nucleus (DRN) is involved in stress-induced opiate reinstatement. To further investigate the effects of morphine dependence and withdrawal on the 5-HT DRN system, we measured gene expression at the level of mRNA in the DRN during a model of morphine dependence, withdrawal and post withdrawal stress exposure in rats. Morphine pellets were implanted for 72h and then either removed or animals were injected with naloxone to produce spontaneous or precipitated withdrawal, respectively. Animals exposed to these conditions exhibited withdrawal symptoms including weight loss, wet dog shakes and jumping behavior. Gene expression for brain-derived neurotrophic factor (BDNF), TrkB, corticotrophin releasing-factor (CRF)-R1, CRF-R2, GABAA-α1, μ-opioid receptor (MOR), 5-HT1A, tryptophan hydroxylase2 and the 5-HT transporter was then measured using quantitative real-time PCR at multiple time-points across the model of morphine exposure, withdrawal and post withdrawal stress. Expression levels of BDNF, TrkB and CRF-R1 mRNA were decreased during both morphine exposure and following seven days of withdrawal. CRF-R2 mRNA expression was elevated after seven days of withdrawal. 5-HT1A receptor mRNA expression was decreased following 3 hours of morphine exposure, while TPH2 mRNA expression was decreased after seven days of withdrawal with swim stress. There were no changes in the expression of GABAA-α1, MOR or 5-HT transporter mRNA. Collectively these results suggest that alterations in neurotrophin support, CRF-dependent stress signaling, 5-HT synthesis and release may underlie 5-HT DRN hypofunction that can potentially lead to stress-induced opiate relapse. PMID:24055683

  7. Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats.

    PubMed

    Alway, Stephen E; Degens, Hans; Lowe, Dawn A; Krishnamurthy, Gururaj

    2002-02-01

    The objective of this study was to determine if levels of repressors to myogenic regulatory factors (MRFs) differ between muscles from young adult and aged animals. Total RNA from plantaris, gastrocnemius, and soleus muscles of Fischer 344 x Brown Norway rats aged 9 mo (young adult, n = 10) and 37 mo (aged, n = 10) was reverse transcribed and then amplified by PCR. To obtain a semiquantitative measure of the mRNA levels, PCR signals were normalized to cyclophilin or 18S signals from the corresponding reverse transcription product. Normalization to cyclophilin and 18S gave similar results. The mRNA levels of MyoD and myogenin were approximately 275-650% (P < 0.001) and approximately 500-1,100% (P < 0.001) greater, respectively, in muscles from aged compared with young adults. In contrast, the protein levels were lower in plantaris and gastrocnemius muscles and similar in the soleus muscle of aged vs. young adult rats. Id repressor mRNA levels were approximately 300-900% greater in fast and slow muscles of aged animals (P < or = 0.02), and Mist 1 mRNA was approximately 50% greater in the plantaris and gastrocnemius muscles (P < 0.01). The mRNA level of Twist mRNA was not significantly affected by aging. Id-1, Id-2, and Id-3 protein levels were approximately 17-740% greater (P < 0.05) in hindlimb muscles of aged rats compared with young adult rats. The elevated levels of Id mRNA and protein suggest that MRF repressors may play a role in gene regulation of fast and slow muscles in aged rats.

  8. Arc mRNA induction in striatal efferent neurons associated with response learning.

    PubMed

    Daberkow, D P; Riedy, M D; Kesner, R P; Keefe, K A

    2007-07-01

    The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate-early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague-Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE(+) and PPE(-) neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE(-) neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motor-response learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons.

  9. Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system

    PubMed Central

    DeRosa, F; Guild, B; Karve, S; Smith, L; Love, K; Dorkin, J R; Kauffman, K J; Zhang, J; Yahalom, B; Anderson, D G; Heartlein, M W

    2016-01-01

    DNA-based gene therapy has considerable therapeutic potential, but the challenges associated with delivery continue to limit progress. Messenger RNA (mRNA) has the potential to provide for transient production of therapeutic proteins, without the need for nuclear delivery and without the risk of insertional mutagenesis. Here we describe the sustained delivery of therapeutic proteins in vivo in both rodents and non-human primates via nanoparticle-formulated mRNA. Nanoparticles formulated with lipids and lipid-like materials were developed for delivery of two separate mRNA transcripts encoding either human erythropoietin (hEPO) or factor IX (hFIX) protein. Dose-dependent protein production was observed for each mRNA construct. Upon delivery of hEPO mRNA in mice, serum EPO protein levels reached several orders of magnitude (>125 000-fold) over normal physiological values. Further, an increase in hematocrit (Hct) was established, demonstrating that the exogenous mRNA-derived protein maintained normal activity. The capacity of producing EPO in non-human primates via delivery of formulated mRNA was also demonstrated as elevated EPO protein levels were observed over a 72-h time course. Exemplifying the possible broad utility of mRNA drugs, therapeutically relevant amounts of human FIX (hFIX) protein were achieved upon a single intravenous dose of hFIX mRNA-loaded lipid nanoparticles in mice. In addition, therapeutic value was established within a hemophilia B (FIX knockout (KO)) mouse model by demonstrating a marked reduction in Hct loss following injury (incision) to FIX KO mice. PMID:27356951

  10. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells.

    PubMed Central

    Stefanovic, B; Hellerbrand, C; Holcik, M; Briendl, M; Aliebhaber, S; Brenner, D A

    1997-01-01

    The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR. PMID:9271398

  11. Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

    PubMed

    Preece, Paul; Virley, David J; Costandi, Moheb; Coombes, Robert; Moss, Stephen J; Mudge, Anne W; Jazin, Elena; Cairns, Nigel J

    2004-03-17

    Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

  12. Detection of MDR1 mRNA expression with optimized gold nanoparticle beacon

    NASA Astrophysics Data System (ADS)

    Zhou, Qiumei; Qian, Zhiyu; Gu, Yueqing

    2016-03-01

    MDR1 (multidrug resistance gene) mRNA expression is a promising biomarker for the prediction of doxorubicin resistance in clinic. However, the traditional technical process in clinic is complicated and cannot perform the real-time detection mRNA in living single cells. In this study, the expression of MDR1 mRNA was analyzed based on optimized gold nanoparticle beacon in tumor cells. Firstly, gold nanoparticle (AuNP) was modified by thiol-PEG, and the MDR1 beacon sequence was screened and optimized using a BLAST bioinformatics strategy. Then, optimized MDR1 molecular beacons were characterized by transmission electron microscope, UV-vis and fluorescence spectroscopies. The cytotoxicity of MDR1 molecular beacon on L-02, K562 and K562/Adr cells were investigated by MTT assay, suggesting that MDR1 molecular beacon was low inherent cytotoxicity. Dark field microscope was used to investigate the cellular uptake of hDAuNP beacon assisted with ultrasound. Finally, laser scanning confocal microscope images showed that there was a significant difference in MDR1 mRNA expression in K562 and K562/Adr cells, which was consistent with the results of q-PCR measurement. In summary, optimized MDR1 molecular beacon designed in this study is a reliable strategy for detection MDR1 mRNA expression in living tumor cells, and will be a promising strategy for in guiding patient treatment and management in individualized medication.

  13. The use of Molecular Beacons to Directly Measure Bacterial mRNA Abundances and Transcript Degradation

    PubMed Central

    Kuechenmeister, Lisa J.; Anderson, Kelsi L.; Morrison, John M.; Dunman, Paul M.

    2009-01-01

    The regulation of mRNA turnover is a dynamic means by which bacteria regulate gene expression. Although current methodologies allow characterization of the stability of individual transcripts, procedures designed to measure alterations in transcript abundance/turnover on a high throughput scale are lacking. In the current report, we describe the development of a rapid and simplified molecular beacon-based procedure to directly measure the mRNA abundances and mRNA degradation properties of well-characterized Staphylococcus aureus pathogenicity factors. This method does not require any PCR-based amplification, can monitor the abundances of multiple transcripts within a single RNA sample, and was successfully implemented into a high throughput screen of transposon mutant library members to detect isolates with altered mRNA turnover properties. It is expected that the described methodology will provide great utility in characterizing components of bacterial RNA degradation processes and can be used to directly measure the mRNA levels of virtually any bacterial transcript. PMID:18992285

  14. Effect of taurine on mRNA expression of thioredoxin interacting protein in Caco-2 cells.

    PubMed

    Gondo, Yusuke; Satsu, Hideo; Ishimoto, Yoko; Iwamoto, Taku; Shimizu, Makoto

    2012-09-28

    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, plays an important role in several essential biological processes; although, the underlying mechanisms for these regulatory functions remain to be elucidated, especially at the genetic level. We investigated the effects of taurine on the gene expression profile in Caco-2 cells using DNA microarray. Taurine increased the mRNA expression of thioredoxin interacting protein (TXNIP), which is involved in various metabolisms and diseases. β-Alanine or γ-aminobutyric acid (GABA), which are structurally or functionally related to taurine, did not increase TXNIP mRNA expression. These suggest the expression of TXNIP mRNA is induced specifically by taurine. β-Alanine is also known to be a substrate of taurine transporter (TAUT) and competitively inhibits taurine uptake. Inhibition of taurine uptake by β-alanine eliminated the up-regulation of TXNIP, which suggests TAUT is involved in inducing TXNIP mRNA expression. The up-regulation of TXNIP mRNA expression by taurine was also observed at the protein level. Furthermore, taurine significantly increased TXNIP promoter activity. Our present study demonstrated the taurine-specific phenomenon of TXNIP up-regulation, which sheds light on the physiological function of taurine.

  15. P-bodies and their functions during mRNA cell cycle: mini-review.

    PubMed

    Olszewska, Marta; Bujarski, Józef J; Kurpisz, Maciej

    2012-04-01

    P-bodies (processing bodies) are observed in different organisms such as yeast, Caenorhabditis elegans and mammals. A typical eukaryotic cell contains several types of spatially formed granules, such as P-bodies, stress granules and a variety of ribonucleoprotein bodies. These microdomains play important role in mRNA processing, including RNA interference, repression of translation and mRNA decay. The P-bodies components as well as stress granules may play an important role in host defense against viral infection. The complete set of P-bodies protein elements is still poor known. They contain conserved protein core limited to different organisms or to stress status of the cell. P-bodies are related also to some neuronal mRNA granules as well as to maternal RNA granules or male germ cell granules. In this mini-review, we focus on the structure of P-bodies and their function in the mRNA utilization and processing because of the high mRNA's dynamics between different cellular compartments and its key role in modulation of gene expression.

  16. Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity.

    PubMed

    Donnelly, Christopher J; Willis, Dianna E; Xu, Mei; Tep, Chhavy; Jiang, Chunsu; Yoo, Soonmoon; Schanen, N Carolyn; Kirn-Safran, Catherine B; van Minnen, Jan; English, Arthur; Yoon, Sung Ok; Bassell, Gary J; Twiss, Jeffery L

    2011-09-30

    Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of β-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of β-actin mRNA, and introducing GFP with the 3'UTR of β-actin mRNA depletes axons of endogenous β-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of β-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.

  17. Changes in the stability of a human H3 histone mRNA during the HeLa cell cycle.

    PubMed Central

    Morris, T D; Weber, L A; Hickey, E; Stein, G S; Stein, J L

    1991-01-01

    A major component of the regulation of histone protein synthesis during the cell cycle is the modulation of the half-life of histone mRNA. We have uncoupled transcriptional and posttranscriptional regulation by using a Drosophila hsp70-human H3 histone fusion gene that produces a marked human H3 histone mRNA upon heat induction. Transcription of this gene can be switched on and off by raising and lowering cell culture temperatures, respectively. HeLa cell lines containing stably integrated copies of the fusion gene were synchronized by double thymidine block. Distinct populations of H3 histone mRNA were produced by heat induction in early S-phase, late S-phase, or G2-phase cells, and the stability of the induced H3 histone mRNA was measured. The H3 histone mRNA induced during early S phase decayed with a half-life of 110 min, whereas the same transcript induced during late S phase had a half-life of 10 to 15 min. The H3 histone mRNA induced in non-S-phase cells is more stable than that induced in late S phase, with a half-life of 40 min. Thus, the stability of histone mRNA is actively regulated throughout the cell cycle. Our results are consistent with an autoregulatory model in which the stability of histone mRNA is determined by the level of free histone protein in the cytoplasm. Images PMID:1986245

  18. A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

    PubMed

    Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

    2016-11-09

    Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication.

  19. Moisturizers change the mRNA expression of enzymes synthesizing skin barrier lipids.

    PubMed

    Buraczewska, Izabela; Berne, Berit; Lindberg, Magnus; Lodén, Marie; Törmä, Hans

    2009-09-01

    In a previous study, 7-week treatment of normal human skin with two test moisturizers, Complex cream and Hydrocarbon cream, was shown to affect mRNA expression of certain genes involved in keratinocyte differentiation. Moreover, the treatment altered transepidermal water loss (TEWL) in opposite directions. In the present study, the mRNA expression of genes important for formation of barrier lipids, i.e., cholesterol, free fatty acids and ceramides, was examined. Treatment with Hydrocarbon cream, which increased TEWL, also elevated the gene expression of GBA, SPTLC2, SMPD1, ALOX12B, ALOXE3, and HMGCS1. In addition, the expression of PPARG was decreased. On the other hand, Complex cream, which decreased TEWL, induced only the expression of PPARG, although not confirmed at the protein level. Furthermore, in the untreated skin, a correlation between the mRNA expression of PPARG and ACACB, and TEWL was found, suggesting that these genes are important for the skin barrier homeostasis. The observed changes further demonstrate that long-term treatment with certain moisturizers may induce dysfunctional skin barrier, and as a consequence several signaling pathways are altered.

  20. An integrated analysis of differential miRNA and mRNA expressions in human gallstones.

    PubMed

    Yang, Bin; Liu, Bin; Bi, Pinduan; Wu, Tao; Wang, Qiang; Zhang, Jie

    2015-04-01

    Gallstone disease, including cholesterol precipitation in bile, increased bile salt hydrophobicity and gallbladder inflammation. Here, we investigated miRNA and mRNA involved in the formation of gallstones, and explored the molecular mechanisms in the development of gallstones. Differentially expressed 17 miRNAs and 525 mRNA were identified based on Illumina sequencing from gallbladder mucosa of patients with or without gallstones, and were validated by randomly selected 6 miRNAs and 8 genes using quantitative RT-PCR. 114 miRNA target genes were identified, whose functions and regulating pathways were related to gallstones. The differentially expressed genes were enriched upon lipoprotein binding and some metabolic pathways, and differentially expressed miRNAs enriched upon ABC transportation and cancer related pathways. A molecular regulatory network consisting of 17 differentially expressed miRNAs, inclusive of their target genes, was constructed. miR-210 and its potential target gene ATP11A were found to be differentially expressed in both miRNA and mRNA profiles. ATP11A was a direct target of miR-210, which was predicted to regulate the ABC-transporters pathway. The expression levels of ATP11A in the gallstone showed inverse correlation with miR-210 expression, and up-regulation of miR-210 could reduce ATP11A expression in HGBEC. This is the first report that indicates the existence of differences in miRNA and mRNA expression in patients with or without gallstones. Our data shed light on further investigating the mechanisms of gallstone formation.

  1. Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life.

    PubMed Central

    Belgrader, P; Cheng, J; Zhou, X; Stephenson, L S; Maquat, L E

    1994-01-01

    Frameshift and nonsense mutations within the gene for human triosephosphate isomerase (TPI) that generate a nonsense codon within the first three-fourths of the protein coding region have been found to reduce the abundance of the product mRNA that copurifies with nuclei. The cellular process and location of the nonsense codon-mediated reduction have proven difficult to elucidate for technical reasons. We show here, using electron microscopy to judge the purity of isolated nuclei, that the previously established reduction to 25% of the normal mRNA level is evident for nuclei that are free of detectable cytoplasmic contamination. Therefore, the reduction is likely to be characteristic of bona fide nuclear RNA. Fully spliced nuclear mRNA is identified by Northern (RNA) blot hybridization and a reverse transcription-PCR assay as the species that undergoes decay in experiments that used the human c-fos promoter to elicit a burst and subsequent shutoff of TPI gene transcription upon the addition of serum to serum-deprived cells. Finally, the finding that deletion of a 5' splice site of the TPI gene results predominantly but not exclusively in the removal by splicing (i.e., skipping) of the upstream exon as a part of the flanking introns has been used to demonstrate that decay is specific to those mRNA products that maintain the nonsense codon. This result, together with our previous results that implicate translation by ribosomes and charged tRNAs in the decay mechanism, indicate that nonsense codon recognition takes place after splicing and triggers decay solely in cis. The possibility that decay takes place during the process of mRNA export from the nucleus to the cytoplasm is discussed. Images PMID:7969159

  2. The influence of eccentric exercise on mRNA expression of skeletal muscle regulators.

    PubMed

    Jensky, Nicole E; Sims, Jennifer K; Rice, Judd C; Dreyer, Hans C; Schroeder, E Todd

    2007-11-01

    To evaluate change in myostatin, follistatin, MyoD and SGT mRNA gene expression using eccentric exercise to study mechanisms of skeletal muscle hypertrophy. Young (28+/-5 years) and older (68+/-6 years) men participated in a bout of maximal single-leg eccentric knee extension on an isokinetic dynamometer at 60 degrees /s: six sets, 12-16 maximal eccentric repetitions. Muscle biopsies of the vastus lateralis were obtained from the dominant leg before exercise and 24 h after exercise. Paired t tests were used to compare change (pre versus post-exercise) for normalized gene expression in all variables. Independent t tests were performed to test group differences (young vs. older). A probability level of PmRNA expression in young subjects 24 h after eccentric exercise. Similarly, we did not observe significant change in myostatin (-3.83+/-8.8; P=0.23), follistatin (-2.66+/-5.2; P=0.17), MyoD (-0.13+/-3.1; P=0.90), or SGT (-1.6+/-3.5; P=0.19) mRNA expression in older subjects. Furthermore, the non-significant changes in mRNA expression were not different between young and older subjects, P>0.23 for all variables. Our data suggests that a single bout of maximal eccentric exercise does not alter myostatin, follistatin, MyoD or SGT mRNA gene expression in young or older subjects.

  3. Global analysis of mRNA decay intermediates in Saccharomyces cerevisiae.

    PubMed

    Harigaya, Yuriko; Parker, Roy

    2012-07-17

    The general pathways of eukaryotic mRNA decay occur via deadenylation followed by 3' to 5' degradation or decapping, although some endonuclease sites have been identified in metazoan mRNAs. To determine the role of endonucleases in mRNA degradation in Saccharomyces cerevisiae, we mapped 5' monophosphate ends on mRNAs in wild-type and dcp2 xrn1 yeast cells, wherein mRNA endonuclease cleavage products are stabilized. This led to three important observations. First, only few mRNAs that undergo low-level endonucleolytic cleavage were observed, suggesting that endonucleases are not a major contributor to yeast mRNA decay. Second, independent of known decapping enzymes, we observed low levels of 5' monophosphates on some mRNAs, suggesting that an unknown mechanism can generate 5' exposed ends, although for all substrates tested, Dcp2 was the primary decapping enzyme. Finally, we identified debranched lariat intermediates from intron-containing genes, demonstrating a significant discard pathway for mRNAs during the second step of pre-mRNA splicing, which is a potential step to regulate gene expression.

  4. CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer

    PubMed Central

    Barrón, Eira Valeria; Roman-Bassaure, Edgar; Sánchez-Sandoval, Ana Laura; Espinosa, Ana María; Guardado-Estrada, Mariano; Medina, Ingrid; Juárez, Eligia; Alfaro, Ana; Bermúdez, Miriam; Zamora, Rubén; García-Ruiz, Carlos; Gomora, Juan Carlos; Kofman, Susana; Pérez-Armendariz, E. Martha; Berumen, Jaime

    2015-01-01

    The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10−6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5–10, p = 3.3 x 10−6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC. PMID:26372210

  5. Tristetraprolin (TTP): Interactions with mRNA and proteins, and current thoughts on mechanisms of action

    PubMed Central

    Brooks, Seth A.; Blackshear, Perry J.

    2013-01-01

    Changes in mRNA stability and translation are critical control points in the regulation of gene expression, particularly genes encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenosine and uridine (AU)-rich elements (ARE), often located in the 3′ untranslated regions (3′UTR) of mRNAs, are known to target transcripts for rapid decay. They are also involved in the regulation of mRNA stability and translation in response to extracellular cues. This review focuses on one of the best characterized ARE binding proteins, tristetraprolin (TTP), the founding member of a small family of CCCH tandem zinc finger proteins. In this survey, we have reviewed the current status of TTP interactions with mRNA and proteins, and discussed current thinking about TTP's mechanism of action to promote mRNA decay. We also review the proposed regulation of TTP's functions by phosphorylation. Finally, we have discussed emerging evidence for TTP operating as a translational regulator. PMID:23428348

  6. DDR2 polymorphisms and mRNA expression in lung cancers of Japanese patients.

    PubMed

    Sasaki, Hidefumi; Shitara, Masayuki; Yokota, Keisuke; Okuda, Katsuhiro; Hikosaka, Yu; Moriyama, Satoru; Yano, Motoki; Fujii, Yoshitaka

    2012-07-01

    Discoidin domain receptor 2, DDR2, is a tyrosine kinase receptor for fibrillar collagen that is involved in postnatal development, tissue repair and primary and metastatic cancer progression. Recently, mutations in the DDR2 kinase gene were identified in squamous cell lung cancer from large-scale Sanger sequencing. The present study investigated the DDR2 gene mutations and mRNA expression in surgically treated non-small cell lung cancer (NSCLC) of squamous histology cases. The presence or absence of DDR2 mutations at the kinase and discoidin domain was analyzed by direct sequencing. In this cohort, DDR2 mutations were not observed in the 166 patients with lung cancer, although DDR2 polymorphisms were observed (H136H, n=14) at the discoidin domain. mRNA levels of DDR2 in lung tumor samples and the adjacent normal lung samples were simultaneously analyzed. DDR2 mRNA levels were significantly decreased in tumor samples compared with normal lung samples. However, the DDR2 mRNA levels were elevated in the DDR2 polymorphism cases.

  7. CDKN3 mRNA as a Biomarker for Survival and Therapeutic Target in Cervical Cancer.

    PubMed

    Barrón, Eira Valeria; Roman-Bassaure, Edgar; Sánchez-Sandoval, Ana Laura; Espinosa, Ana María; Guardado-Estrada, Mariano; Medina, Ingrid; Juárez, Eligia; Alfaro, Ana; Bermúdez, Miriam; Zamora, Rubén; García-Ruiz, Carlos; Gomora, Juan Carlos; Kofman, Susana; Pérez-Armendariz, E Martha; Berumen, Jaime

    2015-01-01

    The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, involved in mitosis, is upregulated in cervical cancer (CC). We investigated CDKN3 mRNA as a survival biomarker and potential therapeutic target for CC. CDKN3 mRNA was measured in 134 CC and 25 controls by quantitative PCR. A 5-year survival study was conducted in 121 of these CC patients. Furthermore, CDKN3-specific siRNAs were used to investigate whether CDKN3 is involved in proliferation, migration, and invasion in CC-derived cell lines (SiHa, CaSki, HeLa). CDKN3 mRNA was on average 6.4-fold higher in tumors than in controls (p = 8 x 10-6, Mann-Whitney). A total of 68.2% of CC patients over expressing CDKN3 gene (fold change ≥ 17) died within two years of diagnosis, independent of the clinical stage and HPV type (Hazard Ratio = 5.0, 95% CI: 2.5-10, p = 3.3 x 10-6, Cox proportional-hazards regression). In contrast, only 19.2% of the patients with lower CDKN3 expression died in the same period. In vitro inactivation of CDKN3 decreased cell proliferation on average 67%, although it had no effect on cell migration and invasion. CDKN3 mRNA may be a good survival biomarker and potential therapeutic target in CC.

  8. Salt stress affects mRNA editing in soybean chloroplasts.

    PubMed

    Rodrigues, Nureyev F; Fonseca, Guilherme C da; Kulcheski, Franceli R; Margis, Rogério

    2017-03-02

    Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress.

  9. Metabolite sensing in eukaryotic mRNA biology

    PubMed Central

    Clingman, Carina C

    2016-01-01

    All living creatures change their gene expression program in response to nutrient availability and metabolic demands. Nutrients and metabolites can directly control transcription and activate second-messenger systems. More recent studies reveal that metabolites also affect post-transcriptional regulatory mechanisms. Here, we review the increasing number of connections between metabolism and post-transcriptional regulation in eukaryotic organisms. First, we present evidence that riboswitches, a common mechanism of metabolite sensing in bacteria, also function in eukaryotes. Next, we review an example of a double stranded RNA modifying enzyme that directly interacts with a metabolite, suggesting a link between RNA editing and metabolic state. Finally, we discuss work that shows some metabolic enzymes bind directly to RNA to affect mRNA stability or translation efficiency. These examples were discovered through gene-specific genetic, biochemical, and structural studies. A directed systems level approach will be necessary to determine whether they are anomalies of evolution or pioneer discoveries in what may be a broadly connected network of metabolism and post-transcriptional regulation. PMID:23653333

  10. PABPN1-Dependent mRNA Processing Induces Muscle Wasting

    PubMed Central

    Raz, Yotam; van Putten, Maaike; Paniagua-Soriano, Guillem; Krom, Yvonne D.; Florea, Bogdan I.; Raz, Vered

    2016-01-01

    Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3’-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting. PMID:27152426

  11. TP53 and ATM mRNA expression in skin and skeletal muscle after low-level laser exposure.

    PubMed

    Guedes de Almeida, Luciana; Silva Sergio, Luiz Philippe da; de Paoli, Flavia; Mencalha, Andre Luiz; da Fonseca, Adenilson de Souza

    2017-02-16

    Low-level lasers are widespread in regenerative medicine, but the molecular mechanisms involved in their biological effects are not fully understood, particularly those on DNA stability. Therefore, this study aimed to investigate mRNA expression of genes related to DNA genomic stability in skin and skeletal muscle tissue from Wistar rats exposed to low-level red and infrared lasers. For this, TP53 (Tumor Protein 53) and ATM (Ataxia Telangiectasia Mutated gene) mRNA expressions were evaluated by real-time quantitative PCR (RT-qPCR) technique 24 hours after low-level red and infrared laser exposure. Our data showed that relative TP53 mRNA expression was not significantly altered in both tissues exposed to lasers. For ATM, relative mRNA expression in skin tissue was not significantly altered, but in muscle tissue, laser exposure increased relative ATM mRNA expression. Low-level red and infrared laser radiations alter ATM mRNA expression related to DNA stability in skeletal muscle tissue.

  12. UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii.

    PubMed

    Serpeloni, Mariana; Jiménez-Ruiz, Elena; Vidal, Newton Medeiros; Kroeber, Constanze; Andenmatten, Nicole; Lemgruber, Leandro; Mörking, Patricia; Pall, Gurman S; Meissner, Markus; Ávila, Andréa R

    2016-11-01

    Nucleo-cytoplasmic RNA export is an essential post-transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans.

  13. The Rtr1p CTD phosphatase autoregulates its mRNA through a degradation pathway involving the REX exonucleases

    PubMed Central

    Hodko, Domagoj; Ward, Taylor; Chanfreau, Guillaume

    2016-01-01

    Rtr1p is a phosphatase that impacts gene expression by modulating the phosphorylation status of the C-terminal domain of the large subunit of RNA polymerase II. Here, we show that Rtr1p is a component of a novel mRNA degradation pathway that promotes its autoregulation through turnover of its own mRNA. We show that the 3′UTR of the RTR1 mRNA contains a cis element that destabilizes this mRNA. RTR1 mRNA turnover is achieved through binding of Rtr1p to the RTR1 mRNP in a manner that is dependent on this cis element. Genetic evidence shows that Rtr1p-mediated decay of the RTR1 mRNA involves the 5′-3′ DExD/H-box RNA helicase Dhh1p and the 3′-5′ exonucleases Rex2p and Rex3p. Rtr1p and Rex3p are found associated with Dhh1p, suggesting a model for recruiting the REX exonucleases to the RTR1 mRNA for degradation. Rtr1p-mediated decay potentially impacts additional transcripts, including the unspliced BMH2 pre-mRNA. We propose that Rtr1p may imprint its RNA targets cotranscriptionally and determine their downstream degradation mechanism by directing these transcripts to a novel turnover pathway that involves Rtr1p, Dhh1p, and the REX family of exonucleases. PMID:26843527

  14. Imaging single mRNAs to study dynamics of mRNA export in the yeast Saccharomyces cerevisiae.

    PubMed

    Bensidoun, Pierre; Raymond, Pascal; Oeffinger, Marlene; Zenklusen, Daniel

    2016-04-01

    Regulation of mRNA and protein expression occurs at many levels, initiated at transcription and followed by mRNA processing, export, localization, translation and mRNA degradation. The ability to study mRNAs in living cells has become a critical tool to study and analyze how the various steps of the gene expression pathway are carried out. Here we describe a detailed protocol for real time fluorescent RNA imaging using the PP7 bacteriophage coat protein, which allows mRNA detection with high spatial and temporal resolution in the yeast Saccharomyces cerevisiae, and can be applied to study various stages of mRNA metabolism. We describe the different parameters required for quantitative single molecule imaging in yeast, including strategies for genomic integration, expression of a PP7 coat protein GFP fusion protein, microscope setup and analysis strategies. We illustrate the method's use by analyzing the behavior of nuclear mRNA in yeast and the role of the nuclear basket in mRNA export.

  15. Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

    PubMed Central

    Nishimura, Yoshiki; Kikis, Elise A.; Zimmer, Sara L.; Komine, Yutaka; Stern, David B.

    2004-01-01

    In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Δ26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Δ26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3′ poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS−, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Δ26, in which atpB mRNA is unstable because of the lack of a 3′ stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3′→5

  16. Frequent up-regulation of WNT5A mRNA in primary gastric cancer.

    PubMed

    Saitoh, Tetsuroh; Mine, Tetsuya; Katoh, Masaru

    2002-05-01

    WNT signal is transduced to the beta-catenin - TCF pathway, the JNK pathway, or the Ca2+-releasing pathway through seven-transmembrane-type WNT receptors encoded by Frizzled genes (FZD1-FZD10). We have previously cloned and characterized human WNT2B/WNT13, WNT3, WNT3A, WNT5B, WNT6, WNT7B, WNT8A, WNT8B, WNT10A, WNT10B, WNT11, WNT14, and WNT14B/WNT15 by using bioinformatics, cDNA-library screening, and cDNA-PCR. Here, we investigated expression of human WNT5A mRNA in various normal tissues, 66 primary tumors derived from various tissues, and 15 human cancer cell lines. WNT5A mRNA was relatively highly expressed in salivary gland, bladder, uterus, placenta, and fetal kidney. Up-regulation of WNT5A mRNA was detected in 5 out of 8 cases of primary gastric cancer, 5 out of 18 cases of primary colorectal tumors, and in 2 out of 7 cases of primary uterus tumors by using matched tumor/normal expression array analysis. Up-regulation of WNT5A mRNA was also detected in 7 out of 10 other cases of primary gastric cancer by using cDNA-PCR. Although low-level expression of WNT5A mRNA was detected in gastric cancer cell line MKN45, WNT5A mRNA was almost undetectable in gastric cancer cell lines OKAJIMA, TMK1, MKN7, MKN28, MKN74, and KATO-III. Compared with frequent up-regulation of WNT5A mRNA in primary gastric cancer, expression levels of WNT5A mRNA in 7 gastric cancer cell lines were significantly lower than that in normal stomach. Frequent up-regulation of WNT5A mRNA in human primary gastric cancer might be due to cancer-stromal interaction.

  17. Regulation of bovine pyruvate carboxylase mRNA and promoter expression by thermal stress.

    PubMed

    White, H M; Koser, S L; Donkin, S S

    2012-09-01

    Pyruvate carboxylase (PC) catalyzes the rate-limiting step in gluconeogenesis from lactate and is a determinant of tricarboxylic acid cycle carbon flux. Bovine PC 5' untranslated region (UTR) mRNA variants are the products of a single PC gene containing 3 promoter regions (P3, P2, and P1, 5' to 3') that are responsive to physiological and nutritional stressors. The objective of this study was to determine the direct effects of thermal stress on PC mRNA and gene expression in bovine hepatocyte monolayer cultures, rat hepatoma (H4IIE) cells, and Madin-Darby bovine kidney epithelial (MDBK) cells. Hepatocytes were isolated from 3 Holstein bull calves and used to prepare monolayer cultures. Rat hepatoma cells and MDBK cells were obtained from American Type Culture Collection, Manassas, VA. Beginning 24 h after initial seeding, cells were subjected to either 37°C (control) or 42°C (thermal stress) for 24 h. Treatments were applied in triplicate in a minimum of 3 independent cell preparations. For bovine primary hepatocytes, endogenous expression of bovine PC mRNA increased (P < 0.1) with 24 h of thermal stress (1.31 vs. 2.79 ± 0.49, arbitrary units, control vs. thermal stress, respectively), but there was no change (P ≥ 0.1) in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) mRNA expression. Similarly, exposure of MDBK cells to thermal stress increased (P < 0.1) expression of bovine PC mRNA without altering (P ≥ 0.1) PEPCK-C mRNA expression. Conversely, there was no effect (P ≥ 0.1) of thermal stress on endogenous rat PC (0.47 vs. 0.30 ± 0.08, control vs. thermal stress) or PEPCK-C (1.61 vs. 1.20 ± 0.48, arbitrary units, control vs. thermal stress, respectively) mRNA expressions in H4IIE cells. To further investigate the regulation of PC, H4IIE cells were transiently transfected with bovine promoter-luciferase constructs containing either P1, P2, or P3, and exposed to thermal stress for 23 h. Activity of P1 was suppressed (P < 0.1) 5-fold, activity of P2

  18. Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA.

    PubMed Central

    Yamashita, M; Krystal, M; Palese, P

    1988-01-01

    In contrast to influenza A and B viruses, which encode their matrix (M) proteins via an unspliced mRNA, the influenza C virus M protein appears to be coded for by a spliced mRNA from RNA segment 6. Although an open reading frame in RNA segment 6 of influenza C/JJ/50 virus could potentially code for a protein of 374 amino acids, a splicing event results in an mRNA coding for a 242-amino-acid M protein. The message for this protein represents the major M gene-specific mRNA species in C virus-infected cells. Despite the difference in coding strategies, there are sequence homologies among the M proteins of influenza A, B, and C viruses which confirm the evolutionary relationship of the three influenza virus types. Images PMID:3404579

  19. Differential responses of intestinal glucose transporter mRNA transcripts to levels of dietary sugars.

    PubMed

    Miyamoto, K; Hase, K; Takagi, T; Fujii, T; Taketani, Y; Minami, H; Oka, T; Nakabou, Y

    1993-10-01

    Dietary sugars are known to stimulate intestinal glucose transport activity, but the specific signals involved are unknown. The Na(+)-dependent glucose co-transporter (SGLT1), the liver-type facilitative glucose transporter (GLUT2) and the intestinal-type facilitative glucose transporter (GLUT5) are all expressed in rat jejunum [Miyamoto, Hase, Taketani, Minami, Oka, Nakabou and Hagihira (1991) Biochem. Biophys. Res. Commun. 181, 1110-1117]. In the present study we have investigated the effects of dietary sugars on these glucose transporter genes. A high-glucose diet stimulated glucose transport activity and increased the levels of SGLT1 and GLUT2 mRNAs in rat jejunum. 3-O-Methylglucose, D-galactose, D-fructose, D-mannose and D-xylose can mimic the regulatory effect of glucose on the SGLT1 mRNA level in rat jejunum. However, only D-galactose and D-fructose increased the levels of GLUT2 mRNA. The GLUT5 mRNA level was increased significantly only by D-fructose. Our results suggest that the increase in intestinal transport activity in rats caused by dietary glucose is due to an increase in the levels of SGLT1 and GLUT2 mRNAs, and that these increases in mRNA may be caused by an enhancement of the transcriptional rate. Furthermore, for expression of the SGLT1 gene, the signal need not be a metabolizable or transportable substrate whereas, for expression of the GLUT2 gene, metabolism of the substrate in the liver may be necessary for signalling. Only D-fructose is an effective signal for expression of the GLUT5 gene.

  20. COMBINING PROTEIN AND mRNA QUANTIFICATION TO DECIPHER TRANSCRIPTIONAL REGULATION

    PubMed Central

    Xu, Heng; Sepúlveda, Leonardo A.; Figard, Lauren; Sokac, Anna Marie; Golding, Ido

    2015-01-01

    We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription-factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo. PMID:26098021

  1. The nonlinear dynamics and fluctuations of mRNA levels in cross-talking pathway activated transcription.

    PubMed

    Yu, Jianshe; Sun, Qiwen; Tang, Moxun

    2014-12-21

    Gene transcription is a stochastic process, and is often activated by multiple signal transduction pathways. In this work, we study gene transcription activated randomly by two cross-talking pathways, with the messenger RNA (mRNA) molecules being produced in a simple birth and death process. We derive the analytical formulas for the mean and the second moment of mRNA copy numbers and characterize the nature of transcription noise. We find that the stationary noise strength Φ is close to its baseline limit 1 when the mRNA level is high due to strong activation or stable transcription, or the mRNA level is low due to unstable transcription or ineffective mRNA production. If Φ stays well above 1, then the gene is infrequently active but mRNAs are accumulated rapidly once it is active. In this case, the system generates a transcriptional bursting, and the mean mRNA level peaks at a finite time. By examining the nonlinear dependance of Φ on transcriptional efficiency, we show that the maximum noise strength is attained only when the gene is silent in the majority of cells as observed in recent experiments. By comparing the current findings with our previous results in sequential pathway model, we come up with a profound conclusion that parallel, cross-talking pathways tend to increase transcription noise, whereas sequential pathways tend to reduce transcription noise. A further study on gene transcription activated by entangling pathways may help us reveal the subtle connection between the characteristics of transcription noise and the topology of genetic network.

  2. Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation

    NASA Technical Reports Server (NTRS)

    Reichler, S. A.; Balk, J.; Brown, M. E.; Woodruff, K.; Clark, G. B.; Roux, S. J.

    2001-01-01

    The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

  3. hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation

    PubMed Central

    Williams, Kathryn R.; McAninch, Damian S.; Stefanovic, Snezana; Xing, Lei; Allen, Megan; Li, Wenqi; Feng, Yue; Mihailescu, Mihaela Rita; Bassell, Gary J.

    2016-01-01

    Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5′ untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1–mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development. PMID:26658614

  4. Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

    PubMed Central

    Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

    2013-01-01

    Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

  5. Evaluation of mRNA marker specificity for the identification of five human body fluids by capillary electrophoresis.

    PubMed

    Richard, Mara L Lennard; Harper, Kathryn A; Craig, Rhonda L; Onorato, Anthony J; Robertson, James M; Donfack, Joseph

    2012-07-01

    The identification of forensically relevant human body fluids through messenger RNA (mRNA) profiling is of interest to the forensic community. Previous studies have proposed several tissue-specific mRNA markers to achieve this goal. Seven markers for the following genes were selected for evaluation in this study: histatin 3 (HTN3) and statherin (STATH) for saliva, mucin 4 (MUC4) for vaginal secretions, matrix metalloproteinase 7 (MMP7) for menstrual blood, delta-aminolevulinate synthase 2 (ALAS2) for peripheral blood, and protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen. The expression of these markers was examined in each body fluid. All mRNA markers were present in their target body fluids. Peripheral blood and saliva showed little cross-reactivity with the selected markers. However, a high level of cross-reactivity was observed between the vaginal secretion marker MUC4 and saliva stains. Semen showed a high level of cross-reactivity with the selected markers. Co-expression of the predicted body fluid markers was detected in menstrual blood and vaginal secretion stains. The expression pattern of these mRNA markers varied through the menstrual cycle time points tested. Differences in gene expression levels and marker cross-reactivity were observed in the donors tested. Despite the presence of cross-reactivity and co-expression, each of the body fluids examined have distinct gene expression profiles, allowing for body fluid identification based on mRNA profiling.

  6. Dexamethasone increases growth hormone (GH)-releasing hormone (GRH) receptor mRNA levels in cultured rat anterior pituitary cells.

    PubMed

    Tamaki, M; Sato, M; Matsubara, S; Wada, Y; Takahara, J

    1996-06-01

    To examine the effects of glucocorticoid (GC) on growth hormone (GH)-releasing hormone (GRH) receptor gene expression, a highly-sensitive and quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method was used in this study. Rat anterior pituitary cells were isolated and cultured for 4 days. The cultured cells were treated with dexamethasone for 2, 6, and 24 h. GRH receptor mRNA levels were determined by competitive RT-PCR using a recombinant RNA as the competitor. Dexamethasone significantly increased GRH receptor mRNA levels at 5 nM after 6- and 24 h-incubations, and the maximal effect was found at 25 nM. The GC receptor-specific antagonist, RU 38486 completely eliminated the dexamethasone-induced enhancement of GRH receptor mRNA levels. Dexamethasone did not alter the mRNA levels of beta-actin and prolactin at 5 nM for 24 h, whereas GH mRNA levels were significantly increased by the same treatment. The GH response to GRH was significantly enhanced by the 24-h incubation with 5 nM dexamethasone. These findings suggest that GC stimulates GRH receptor gene expression through the ligand-activated GC receptors in the rat somatotrophs. The direct effects of GC on the GRH receptor gene could explain the enhancement of GRH-induced GH secretion.

  7. Growth hormone mRNA in mammary gland tumors of dogs and cats.

    PubMed Central

    Mol, J A; van Garderen, E; Selman, P J; Wolfswinkel, J; Rijinberk, A; Rutteman, G R

    1995-01-01

    We have shown recently that in the dog progestin administration results in mammary production of immunoreactive growth hormone (GH). At present we demonstrate the expression of the gene encoding GH in the mammary gland of dogs and cats using reverse-transcriptase PCR. GH mRNA was found in the great majority of normal mammary tissues as well as benign and malignant mammary tumors of the dog and was associated with the presence of immunoreactive GH in cryostat sections. The mammary PCR product proved to be identical to that of the pituitary. The highest expression levels were found after prolonged treatment with progestins. In carcinomas GH mRNA was also found in progesterone receptor-negative tissue samples, indicating that after malignant transformation GH gene expression may become progestin independent. GH mRNA was also present in mammary tissues of cats with progestin-induced fibroadenomatous changes. It is concluded that GH gene expression occurs in normal, hyperplastic, and neoplastic mammary tissue of the dog. The expression in normal tissue is stimulated by progestins and might mediate the progestin-stimulated development of canine mammary tumors. The demonstration of progestin-stimulated GH expression in mammary tissue of cats indicates that the phenomenon is more generalized among mammals. Images PMID:7738169

  8. Ammonium Chloride Ingestion Attenuates Exercise-Induced mRNA Levels in Human Muscle

    PubMed Central

    Mündel, Toby; Pilegaard, Henriette; Hawke, Emma; Leikis, Murray; Lopez-Villalobos, Nicolas; Oliveira, Rodrigo S. F.; Bishop, David J.

    2015-01-01

    Minimizing the decrease in intracellular pH during high-intensity exercise training promotes greater improvements in mitochondrial respiration. This raises the intriguing hypothesis that pH may affect the exercise-induced transcription of genes that regulate mitochondrial biogenesis. Eight males performed 10x2-min cycle intervals at 80% V˙O2peak intensity on two occasions separated by ~2 weeks. Participants ingested either ammonium chloride (ACID) or calcium carbonate (PLA) the day before and on the day of the exercise trial in a randomized, counterbalanced order, using a crossover design. Biopsies were taken from the vastus lateralis muscle before and after exercise. The mRNA level of peroxisome proliferator-activated receptor co-activator 1α (PGC-1α), citrate synthase, cytochome c and FOXO1 was elevated at rest following ACID (P<0.05). During the PLA condition, the mRNA content of mitochondrial- and glucose-regulating proteins was elevated immediately following exercise (P<0.05). In the early phase (0–2 h) of post-exercise recovery during ACID, PGC-1α, citrate synthase, cytochome C, FOXO1, GLUT4, and HKII mRNA levels were not different from resting levels (P>0.05); the difference in PGC-1α mRNA content 2 h post-exercise between ACID and PLA was not significant (P = 0.08). Thus, metabolic acidosis abolished the early post-exercise increase of PGC-1α mRNA and the mRNA of downstream mitochondrial and glucose-regulating proteins. These findings indicate that metabolic acidosis may affect mitochondrial biogenesis, with divergent responses in resting and post-exercise skeletal muscle. PMID:26656911

  9. Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae.

    PubMed Central

    Kshirsagar, Meenakshi; Parker, Roy

    2004-01-01

    The major pathway of mRNA decay in yeast initiates with deadenylation, followed by mRNA decapping and 5'-3' exonuclease digestion. An in silico approach was used to identify new proteins involved in the mRNA decay pathway. One such protein, Edc3p, was identified as a conserved protein of unknown function having extensive two-hybrid interactions with several proteins involved in mRNA decapping and 5'-3' degradation including Dcp1p, Dcp2p, Dhh1p, Lsm1p, and the 5'-3' exonuclease, Xrn1p. We show that Edc3p can stimulate mRNA decapping of both unstable and stable mRNAs in yeast when the decapping enzyme is compromised by temperature-sensitive alleles of either the DCP1 or the DCP2 genes. In these cases, deletion of EDC3 caused a synergistic mRNA-decapping defect at the permissive temperatures. The edc3Delta had no effect when combined with the lsm1Delta, dhh1Delta, or pat1Delta mutations, which appear to affect an early step in the d