Academic literature on the topic 'RNase-L expression'
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Journal articles on the topic "RNase-L expression"
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Orlandi, Elisa, Elisa De Tomi, Rachele Campagnari, Francesca Belpinati, Monica Rodolfo, Elisabetta Vergani, Giovanni Malerba, Macarena Gomez-Lira, Marta Menegazzi, and Maria Romanelli. "Human Melanoma Cells Differentially Express RNASEL/RNase-L and miR-146a-5p under Sex Hormonal Stimulation." Current Issues in Molecular Biology 44, no. 10 (October 11, 2022): 4790–802. http://dx.doi.org/10.3390/cimb44100326.
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Polymorphisms in the ribonuclease L (RNASEL) coding gene and hsa-miR-146a-5p (miR-146a) have been associated with melanoma in a sex-specific manner. We hypothesized that RNASEL and miR-146a expression could be influenced by sex hormones playing a role in the female advantages observed in melanoma incidence and survival. Thus, we explored the effects of testosterone and 17β-estradiol on RNASEL and miR-146a expression in LM-20 and A375 melanoma cell lines. Direct targeting of miR-146a to the 3’ untranslated region (3′UTR) of RNASEL was examined using a luciferase reporter system. Our results indicate that RNASEL is a direct target of miR-146a in both melanoma cell lines. Trough qPCR and western blot analyses, we explored the effect of miR-146a mimic transfection in the presence of each hormone either on RNASEL mRNA level or on protein expression of RNase-L, the enzyme codified by RNASEL gene. In the presence of testosterone or 17β-estradiol, miR-146a overexpression did not influence RNASEL transcript level in LM-20 cell line, but it slightly induced RNASEL mRNA level in A375 cells. Remarkably, miR-146a overexpression was able to repress the protein level of RNase-L in both LM-20 and A375 cells in the presence of each hormone, as well as to elicit high expression levels of the activated form of the extracellular signal-regulated kinases (ERK)1/2, hence confirming the pro-tumorigenic role of miR-146a overexpression in melanoma. Thereafter, we assessed if the administration of each hormone could affect the endogenous expression of RNASEL and miR-146a genes in LM-20 and A375 cell lines. Testosterone exerted no significant effect on RNASEL gene expression in both cell lines, while 17β-estradiol enhanced RNASEL transcript level at least in LM-20 melanoma cells. Conversely, miR-146a transcript augmented only in the presence of testosterone in either melanoma cell line. Importantly, each hormone acted quite the opposite regarding the RNase-L protein expression, i.e., testosterone significantly decreased RNase-L expression, whereas 17β-estradiol increased it. Overall, the data show that, in melanoma cells treated with 17β-estradiol, RNase-L expression increased likely by transcriptional induction of its gene. Testosterone, instead, decreased RNase-L expression in melanoma cell lines with a post-transcriptional mechanism in which miR-146a could play a role. In conclusion, the pro-tumor activity of androgen hormone in melanoma cells could be exacerbated by both miR-146a increase and RNase-L downregulation. These events may contribute to the worse outcome in male melanoma patients.
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Li, Xiao-Ling, John A. Blackford, and Bret A. Hassel. "RNase L Mediates the Antiviral Effect of Interferon through a Selective Reduction in Viral RNA during Encephalomyocarditis Virus Infection." Journal of Virology 72, no. 4 (April 1, 1998): 2752–59. http://dx.doi.org/10.1128/jvi.72.4.2752-2759.1998.
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ABSTRACT The 2′,5′-oligoadenylate (2-5A) system is an RNA degradation pathway which plays an important role in the antipicornavirus effects of interferon (IFN). RNase L, the terminal component of the 2-5A system, is thought to mediate this antiviral activity through the degradation of viral RNA; however, the capacity of RNase L to selectively target viral RNA has not been carefully examined in intact cells. Therefore, the mechanism of RNase L-mediated antiviral activity was investigated following encephalomyocarditis virus (EMCV) infection of cell lines in which expression of transfected RNase L was induced or endogenous RNase L activity was inhibited. RNase L induction markedly enhanced the anti-EMCV activity of IFN via a reduction in EMCV RNA. Inhibition of endogenous RNase L activity inhibited this reduction in viral RNA. RNase L had no effect on IFN-mediated protection from vesicular stomatitis virus. RNase L induction reduced the rate of EMCV RNA synthesis, suggesting that RNase L may target viral RNAs involved in replication early in the virus life cycle. The RNase L-mediated reduction in viral RNA occurred in the absence of detectable effects on specific cellular mRNAs and without any global alteration in the cellular RNA profile. Extensive rRNA cleavage, indicative of high levels of 2-5A, was not observed in RNase L-induced, EMCV-infected cells; however, transfection of 2-5A into cells resulted in widespread degradation of cellular RNAs. These findings provide the first demonstration of the selective capacity of RNase L in intact cells and link this selective activity to cellular levels of 2-5A.
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Birdwell, L. Dillon, Zachary B. Zalinger, Yize Li, Patrick W. Wright, Ruth Elliott, Kristine M. Rose, Robert H. Silverman, and Susan R. Weiss. "Activation of RNase L by Murine Coronavirus in Myeloid Cells Is Dependent on BasalOasGene Expression and Independent of Virus-Induced Interferon." Journal of Virology 90, no. 6 (January 6, 2016): 3160–72. http://dx.doi.org/10.1128/jvi.03036-15.
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ABSTRACTThe oligoadenylate synthetase (OAS)-RNase L pathway is a potent interferon (IFN)-induced antiviral activity. Upon sensing double-stranded RNA, OAS produces 2′,5′-oligoadenylates (2-5A), which activate RNase L. Murine coronavirus (mouse hepatitis virus [MHV]) nonstructural protein 2 (ns2) is a 2′,5′-phosphodiesterase (PDE) that cleaves 2-5A, thereby antagonizing RNase L activation. PDE activity is required for robust replication in myeloid cells, as a mutant of MHV (ns2H126R) encoding an inactive PDE fails to antagonize RNase L activation and replicates poorly in bone marrow-derived macrophages (BMM), while ns2H126Rreplicates to high titer in several types of nonmyeloid cells, as well as in IFN receptor-deficient (Ifnar1−/−) BMM. We reported previously that myeloid cells express significantly higher basal levels of OAS transcripts than nonmyeloid cells. Here, we investigated the contributions ofOasgene expression, basal IFN signaling, and virus-induced IFN to RNase L activation. Infection with ns2H126Ractivated RNase L inIfih1−/−BMM to a similar extent as in wild-type (WT) BMM, despite the lack of IFN induction in the absence of MDA5 expression. However, ns2H126Rfailed to induce RNase L activation in BMM treated with IFNAR1-blocking antibody, as well as inIfnar1−/−BMM, both expressing low basal levels ofOasgenes. Thus, activation of RNase L does not require virus-induced IFN but rather correlates with adequate levels of basalOasgene expression, maintained by basal IFN signaling. Finally, overexpression of RNase L is not sufficient to compensate for inadequate basal OAS levels.IMPORTANCEThe oligoadenylate synthetase (OAS)-RNase L pathway is a potent antiviral activity. Activation of RNase L during murine coronavirus (mouse hepatitis virus [MHV]) infection of myeloid cells correlates with high basalOasgene expression and is independent of virus-induced interferon secretion. Thus, our data suggest that cells with high basalOasgene expression levels can activate RNase L and thereby inhibit virus replication early in infection upon exposure to viral double-stranded RNA (dsRNA) before the induction of interferon and prior to transcription of interferon-stimulated antiviral genes. These findings challenge the notion that activation of the OAS-RNase L pathway requires virus to induce type I IFN, which in turn upregulates OAS gene expression, as well as to provide dsRNA to activate OAS. Our data further suggest that myeloid cells may serve as sentinels to restrict viral replication, thus protecting other cell types from infection.
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Scherbik, Svetlana V., Jayashree M. Paranjape, Bronislava M. Stockman, Robert H. Silverman, and Margo A. Brinton. "RNase L Plays a Role in the Antiviral Response to West Nile Virus." Journal of Virology 80, no. 6 (March 15, 2006): 2987–99. http://dx.doi.org/10.1128/jvi.80.6.2987-2999.2006.
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ABSTRACT Alleles at the Flv locus determine disease outcome after a flavivirus infection in mice. Although comparable numbers of congenic resistant and susceptible mouse embryo fibroblasts (MEFs) are infected by the flavivirus West Nile virus (WNV), resistant MEFs produce ∼100- to 150-fold lower titers than susceptible ones and flavivirus titers in the brains of resistant and susceptible animals can differ by >10,000-fold. The Flv locus was previously identified as the 2′-5′ oligoadenylate synthetase 1b (Oas1b) gene. Oas gene expression is up-regulated by interferon (IFN), and after activation by double-stranded RNA, some mouse synthetases produce 2-5A, which activates latent RNase L to degrade viral and cellular RNAs. To determine whether the lower levels of intracellular flavivirus genomic RNA from resistant mice detected in cells at all times after infection were mediated by RNase L, RNase L activity levels in congenic resistant and susceptible cells were compared. Similar moderate levels of RNase L activation by transfected 2-5A were observed in both types of uninfected cells. After WNV infection, the mRNAs of IFN-β and three Oas genes were up-regulated to similar levels in both types of cells. However, significant levels of RNase L activity were not detected until 72 h after WNV infection and the patterns of viral RNA cleavage products generated were similar in both types of cells. When RNase L activity was down-regulated in resistant cells via stable expression of a dominant negative RNase L mutant, ∼5- to 10-times-higher yields of WNV were produced. Similarly, about ∼5- to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L−/− cells compared to RNase L+/+ cells that were either left untreated or pretreated with IFN and/or poly(I) · poly(C). The data indicate that WNV genomic RNA is susceptible to RNase L cleavage and that RNase L plays a role in the cellular antiviral response to flaviviruses. The results suggest that RNase L activation is not a major component of the Oas1b-mediated flavivirus resistance phenotype.
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Maitra, Ratan K., and Robert H. Silverman. "Regulation of Human Immunodeficiency Virus Replication by 2′,5′-Oligoadenylate-Dependent RNase L." Journal of Virology 72, no. 2 (February 1, 1998): 1146–52. http://dx.doi.org/10.1128/jvi.72.2.1146-1152.1998.
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ABSTRACT Activation of RNase L by 2′,5′-linked oligoadenylates (2-5A) is one of the antiviral pathways of interferon action. To determine the involvement of the 2-5A system in the control of human immunodeficiency virus type 1 (HIV-1) replication, a segment of the HIV-1nef gene was replaced with human RNase L cDNA. HIV-1 provirus containing sense orientation RNase L cDNA caused increased expression of RNase L and 500- to 1,000-fold inhibition of virus replication in Jurkat cells for a period of about 2 weeks. Subsequently, a partial deletion of the RNase L cDNA which coincided with increases in virus production occurred. The anti-HIV activity of RNase L correlated with decreases in HIV-1 RNA and with an acceleration in cell death accompanied by DNA fragmentation. Replication of HIV-1 encoding RNase L was also transiently suppressed in peripheral blood lymphocytes (PBL). In contrast, recombinant HIV containing reverse orientation RNase L cDNA caused decreased levels of RNase L, increases in HIV yields, and reductions in the anti-HIV effect of alpha interferon in PBL and in Jurkat cells. To obtain constitutive and continuous expression of RNase L cDNA, Jurkat cells were cotransfected with HIV-1 proviral DNA and with plasmid containing a cytomegalovirus promoter driving expression of RNase L cDNA. The RNase L plasmid suppressed HIV-1 replication by eightfold, while an antisense RNase L construct enhanced virus production by twofold. These findings demonstrate that RNase L can severely impair HIV replication and suggest involvement of the 2-5A system in the anti-HIV effect of alpha interferon.
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Rath, Sneha, Jesse Donovan, Gena Whitney, Alisha Chitrakar, Wei Wang, and Alexei Korennykh. "Human RNase L tunes gene expression by selectively destabilizing the microRNA-regulated transcriptome." Proceedings of the National Academy of Sciences 112, no. 52 (December 14, 2015): 15916–21. http://dx.doi.org/10.1073/pnas.1513034112.
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Double-stranded RNA (dsRNA) activates the innate immune system of mammalian cells and triggers intracellular RNA decay by the pseudokinase and endoribonuclease RNase L. RNase L protects from pathogens and regulates cell growth and differentiation by destabilizing largely unknown mammalian RNA targets. We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and nontargets. We show that this RNase L-dependent decay selectively affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 and other miRs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these miRs and acts as a suppressor of proliferation and adhesion in mammalian cells. Our data suggest that RNase L-dependent decay serves to establish an antiproliferative state via destabilization of the miR-regulated transcriptome.
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Wei, Ruhan, Guanmin Chen, Naseh Algehainy, Chun Zeng, Chunfang Liu, Hongli Liu, Wendy Liu, Dennis Stacey, and Aimin Zhou. "RNase L Is Involved in Liposaccharide-Induced Lung Inflammation." Viruses 12, no. 1 (January 7, 2020): 73. http://dx.doi.org/10.3390/v12010073.
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RNase L mediates interferon (IFN) function during viral infection and cell proliferation. Furthermore, the role of RNase L in the regulation of gene expression, cell apoptosis, autophagy, and innate immunity has been well established in the last decade. Tissue distribution reveals that RNase L is highly expressed in the lung and other organs. However, the physiological roles of RNase L in the lung are largely unknown. In this study, we found that polysaccharide (LPS)-induced acute lung injury (ALI) was remarkably intensified in mice deficient in RNase L compared to wild type mice under the same condition. Furthermore, we found that RNase L mediated the TLR4 signaling pathway, and regulated the expression of various pro- and anti-inflammatory genes in the lung tissue and blood. Most importantly, RNase L function in macrophages during LPS stimulation may be independent of the 2-5A system. These findings demonstrate a novel role of RNase L in the immune response via an atypical molecular mechanism.
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Li, Yize, Shuvojit Banerjee, Yuyan Wang, Stephen A. Goldstein, Beihua Dong, Christina Gaughan, Robert H. Silverman, and Susan R. Weiss. "Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses." Proceedings of the National Academy of Sciences 113, no. 8 (February 8, 2016): 2241–46. http://dx.doi.org/10.1073/pnas.1519657113.
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The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans—OAS1, OAS2, and OAS3—synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI):poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.
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Zhang, Ao, Beihua Dong, Aurélien J. Doucet, John B. Moldovan, John V. Moran, and Robert H. Silverman. "RNase L restricts the mobility of engineered retrotransposons in cultured human cells." Nucleic Acids Research 42, no. 6 (December 25, 2013): 3803–20. http://dx.doi.org/10.1093/nar/gkt1308.
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Abstract Retrotransposons are mobile genetic elements, and their mobility can lead to genomic instability. Retrotransposon insertions are associated with a diverse range of sporadic diseases, including cancer. Thus, it is not a surprise that multiple host defense mechanisms suppress retrotransposition. The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is a mechanism for restricting viral infections during the interferon antiviral response. Here, we investigated a potential role for the OAS-RNase L system in the restriction of retrotransposons. Expression of wild type (WT) and a constitutively active form of RNase L (NΔ385), but not a catalytically inactive RNase L mutant (R667A), impaired the mobility of engineered human LINE-1 (L1) and mouse intracisternal A-type particle retrotransposons in cultured human cells. Furthermore, WT RNase L, but not an inactive RNase L mutant (R667A), reduced L1 RNA levels and subsequent expression of the L1-encoded proteins (ORF1p and ORF2p). Consistently, confocal immunofluorescent microscopy demonstrated that WT RNase L, but not RNase L R667A, prevented formation of L1 cytoplasmic foci. Finally, siRNA-mediated depletion of endogenous RNase L in a human ovarian cancer cell line (Hey1b) increased the levels of L1 retrotransposition by ∼2-fold. Together, these data suggest that RNase L might function as a suppressor of structurally distinct retrotransposons.
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Martinand, Camille, Céline Montavon, Tamim Salehzada, Michelle Silhol, Bernard Lebleu, and Catherine Bisbal. "RNase L Inhibitor Is Induced during Human Immunodeficiency Virus Type 1 Infection and Down Regulates the 2-5A/RNase L Pathway in Human T Cells." Journal of Virology 73, no. 1 (January 1, 1999): 290–96. http://dx.doi.org/10.1128/jvi.73.1.290-296.1999.
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ABSTRACT The interferon-regulated 2-5A/RNase L pathway plays a major role in the antiviral and antiproliferative activities of these cytokines. Several viruses, however, have evolved strategies to escape the antiviral activity of the 2-5A/RNase L pathway. In this context, we have cloned a cDNA coding for the RNase L inhibitor (RLI), a protein that specifically inhibits RNase L and whose regulated expression in picornavirus-infected cells down regulates the activity of the 2-5A/RNase L pathway. We show here that RLI increases during the course of human immunodeficiency virus type 1 (HIV-1) infection, which may be related to the downregulation of RNase L activity that has been described to occur in HIV-infected cells. In order to establish a possible causal relationship between these observations, we have stably transfected H9 cells with RLI sense or antisense cDNA-expressing vectors. The overexpression of RLI causes a decrease in RNase L activity and a twofold enhancement of HIV production. This increase in HIV replication correlates with an increase in HIV RNA and proteins. In contrast, reduction of RLI levels in RLI antisense cDNA-expressing clones reverses the inhibition of RNase L activity associated with HIV multiplication and leads to a threefold decrease in the viral load. This anti-HIV activity correlated with a decrease in HIV RNA and proteins. These findings demonstrate that the level of RLI, via its modulation of RNase L activity, can severely impair HIV replication and suggest the involvement of RLI in the inhibition of the 2-5A/RNase L system observed during HIV infection.
Dissertations / Theses on the topic "RNase-L expression"
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Souza, Edmárcia Elisa de [UNESP]. "Clonagem, expressão e purificação de domínios da proteína AtRLI2 (RNase L Inhibitor), um supressor endógeno de silenciamento por RNA de Arabidopis thaliana, visando estudos estruturais." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/92439.
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A RNase L inhibitor (RLI) é uma proteína altamente conservada em Eukatyota e Archaea e foi primeiramente identificada em humanos, onde se mostrou reguladora da via 2'-5' - oligoadenilato sintetase/ribonuclease L (OAS/RNase L), principal via induzida por interferon. Novas funções têm sido descritas para RLI em diferentes organismos, dentre elas o controle do silenciamento por RNA e resistência a vírus. Visando futuros estudos estruturais foi possível subclonar a sequência que codifica para o domínio NBDs (Nucleotide Binding Domain) de AtRLI2 de Arabidopsis thaliana em vetor de expressão pET-28a(+) capaz de expressar a proteína NBDs ligada a um His6 tag. A proteína NBDs foi expressa e purificada por cromatografia de afinidade por níquel em condição nativa utilizando detergente N-lauril-sarcosil, com um rendimento da ordem de 8 mg/L e em condição desnaturante utilizando uréia, com um rendimento da ordem de 10 mg/L. Para renaturação da proteína utilizou-se dATP e cloreto de magnésio com posterior diálise obtendo-se uma diminuição significativa dos corpúsculos de inclusão e o aumento da solubilidade da proteína produzida em condição desnaturante. Para confirmar a presença dos resíduos His6 tag em fusão com a proteína NBDs, testes de Western blot foram realizados utilizando extrato total das células induzi das, a proteína purificada e a proteína originada da diálise. Foi possível concluir que houve o reconhecimento do anticorpo monoclonal anti-HiS6 tag à proteína confirmando o sucesso da obtenção e purificação da proteína fusionada à uma sequência de 6 histidinas. Etapas posteriores de purificação, a partir da proteína NBDs obtida, necessitam ser realizadas a fim de obter a proteína com grau de pureza significativo e em quantidades suficientes para a realização dos ensaios biofisicos e cristalográficos
The RNase L inhibitor (RLI) is a protein highly conserved in Eukaryota and Archaea and it was first identified in humans acting as a regulator of Oligoadenylate SynthetaselRNase L system (OASlRNase L), the main pathway induced by interferon. New functions have been described for RLI in different organisms, among them the control of RNA silencing and virus resistance. In order to further structural studies could subcIoned the coding sequence for the domain NBDs (nucIeotide-binding domain) AtRLI2 of Arabidopsis thaliana in expression vector pET-28a (+) capable of expressing the protein NBDs linked to a His6 tag. NBDs protein was expressed and purified by affinity chromatography on nickel in native condition using detergent Nlaurylsarcosine with a yield of about 8 mgIL and denaturing conditions using urea with a yield of about 10 mgIL. For renaturation of the protein was used dATP and magnesium chloride with subsequent dialysis resulting in a significant reduction in inclusion bodies and increasing the solubility of the protein produced in denaturant condition. To confirm the presence of residues HiS(; tag fusion protein with the NBDs, Western hlot tests were conducted using total extract of induced cells, the purified protein and protein originated from dialysis. It was concIuded that there was recognition of the monocIonal anti-His6 tag protein confirming the success of obtaining and purification of protein fused to a sequence of 6 histidines. Later stages of purification from the protein obtained NBDs are being conducted to obtain the pure protein 10 sufficient quantities for the testing biophysical and crystallographic
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Souza, Edmárcia Elisa de. "Clonagem, expressão e purificação de domínios da proteína AtRLI2 (RNase L Inhibitor), um supressor endógeno de silenciamento por RNA de Arabidopis thaliana, visando estudos estruturais /." Botucatu : [s.n.], 2010. http://hdl.handle.net/11449/92439.
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Resumo: A "RNase L inhibitor" (RLI) é uma proteína altamente conservada em Eukatyota e Archaea e foi primeiramente identificada em humanos, onde se mostrou reguladora da via 2'-5' - oligoadenilato sintetase/ribonuclease L (OAS/RNase L), principal via induzida por interferon. Novas funções têm sido descritas para RLI em diferentes organismos, dentre elas o controle do silenciamento por RNA e resistência a vírus. Visando futuros estudos estruturais foi possível subclonar a sequência que codifica para o domínio NBDs (Nucleotide Binding Domain) de AtRLI2 de Arabidopsis thaliana em vetor de expressão pET-28a(+) capaz de expressar a proteína NBDs ligada a um His6 tag. A proteína NBDs foi expressa e purificada por cromatografia de afinidade por níquel em condição nativa utilizando detergente N-lauril-sarcosil, com um rendimento da ordem de 8 mg/L e em condição desnaturante utilizando uréia, com um rendimento da ordem de 10 mg/L. Para renaturação da proteína utilizou-se dATP e cloreto de magnésio com posterior diálise obtendo-se uma diminuição significativa dos corpúsculos de inclusão e o aumento da solubilidade da proteína produzida em condição desnaturante. Para confirmar a presença dos resíduos His6 tag em fusão com a proteína NBDs, testes de Western blot foram realizados utilizando extrato total das células induzi das, a proteína purificada e a proteína originada da diálise. Foi possível concluir que houve o reconhecimento do anticorpo monoclonal anti-HiS6 tag à proteína confirmando o sucesso da obtenção e purificação da proteína fusionada à uma sequência de 6 histidinas. Etapas posteriores de purificação, a partir da proteína NBDs obtida, necessitam ser realizadas a fim de obter a proteína com grau de pureza significativo e em quantidades suficientes para a realização dos ensaios biofisicos e cristalográficosAbstract: The RNase L inhibitor (RLI) is a protein highly conserved in Eukaryota and Archaea and it was first identified in humans acting as a regulator of Oligoadenylate SynthetaselRNase L system (OASlRNase L), the main pathway induced by interferon. New functions have been described for RLI in different organisms, among them the control of RNA silencing and virus resistance. In order to further structural studies could subcIoned the coding sequence for the domain NBDs (nucIeotide-binding domain) AtRLI2 of Arabidopsis thaliana in expression vector pET-28a (+) capable of expressing the protein NBDs linked to a His6 tag. NBDs protein was expressed and purified by affinity chromatography on nickel in native condition using detergent Nlaurylsarcosine with a yield of about 8 mgIL and denaturing conditions using urea with a yield of about 10 mgIL. For renaturation of the protein was used dATP and magnesium chloride with subsequent dialysis resulting in a significant reduction in inclusion bodies and increasing the solubility of the protein produced in denaturant condition. To confirm the presence of residues HiS(; tag fusion protein with the NBDs, Western hlot tests were conducted using total extract of induced cells, the purified protein and protein originated from dialysis. It was concIuded that there was recognition of the monocIonal anti-His6 tag protein confirming the success of obtaining and purification of protein fused to a sequence of 6 histidines. Later stages of purification from the protein obtained NBDs are being conducted to obtain the pure protein 10 sufficient quantities for the testing biophysical and crystallographic
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Castillo-Pérez, Karina. "Étude de l'expression différentielle du génome en relation avec la détermination du sexe chez le palmier dattier (Phoenix dactylifera L.)." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS054.
Full textAbstract:
La compréhension des mécanismes moléculaires impliqués dans la détermination du sexe chez les plantes à fleurs est primordiale d’un point de vue fondamental et appliqué. Des processus liés à la biosynthèse des hormones, tel que l’éthylène, ou la régulation de l’expression génique via des petits ARN et des facteurs de transcription ont été associés à l’unisexualisation des fleurs chez des espèces dioïques. Cependant, les déterminants contrôlant le sexe chez les plantes sont encore largement méconnus. Le palmier dattier, Phoenix dactylifera L, est une espèce dioïque dont le dimorphisme sexuel est observé très tôt au cours du développement des fleurs. Des gènes différentiellement exprimés (DEGs) ont été identifiés pendant les stades précoces du développement floral mâle et femelle. Pour cela, un transcriptome de référence rassemblant des données d’expression relatives aux deux sexes a été généré. L’analyse d'enrichissement GO des DEGs, a révélé des processus biologiques communs aux mâles et aux femelles, associés au développement reproducteur et à la réponse aux stimuli. Ce résultat indique que des mêmes processus peuvent solliciter des gènes différents au cours du développement floral précoce en fonction du sexe. Cette analyse a également mis en évidence que le développement des fleurs mâles requiert des processus biologiques spécifiques impliqués dans la régulation cellulaire et l'expression des gènes. En outre, deux DEGs femelles, une S-adenosylmethionine synthase et une Flap endonuclease et un DEG mâle, un élément transposable, ont été identifiés dans les régions non-recombinantes du génome du palmier dattier.Cette étude est la première analyse globale des processus biologiques associés à l’acquisition du dimorphisme sexuel. Elle contribue également à la compréhension de la détermination du sexe chez le palmier dattier, et plus largement à la connaissance de ces processus chez les espèces dioïquesUnraveling molecular mechanisms involved in sex determination in flowering plants is of outstanding basic and applied interest. Several studies on dioecious species have highlighted the molecular basis of sex determination, such as cell death and ethylene biosynthesis pathway. Sex determination mechanisms in plants are, however, still largely unknown. The date palm, Phoenix dactylifera L, is a dioecious species where sexual dimorphism is observed very early in development of flowers. Differentially expressed genes (DEGs) were identified during the early stages of the male and female flower development. A reference transcriptome including male and female data was constructed to gain insight into this process in the dioecious palm Phoenix dactylifera L. Differentially expressed genes (DEG) were subsequently identified between males and females in the early flower development stages in which the first morphological gender difference occurs in date palms.Gene ontology enrichment analysis of DEG revealed biological processes shared between males and females involved in reproductive development and response to stimulus, indicating that same processes could require different genes during early flower development in date palm. This analysis also suggested that date palm triggers biological processes specifically involved in cellular regulation and gene expression to develop male flowers. Furthermore, two female DEGs related to DNA methylation S-adenosylmethionine synthase and DNA metabolism Flap endonuclease, and one male DEGs, a transposable element were found in non-recombinant date palm regions. This study provided the first insight into biological processes involved in sex determination in date palms and more widely to knowledge of this process in dioecious species