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1
Hao, Shengli, and David Baltimore. "RNA splicing regulates the temporal order of TNF-induced gene expression (167.5)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 167.5. http://dx.doi.org/10.4049/jimmunol.188.supp.167.5.
Повний текст джерелаАнотація:
Abstract When cells are induced to express inflammatory genes by treatment with TNF the mRNAs for the induced genes appear in three distinct waves, defining gene Groups I, II and III or early, intermediate and late genes. To examine the basis for these different kinetic classes, we developed a PCR-based procedure to distinguish pre-mRNA synthesis from mRNA synthesis. It showed that the three Groups initiate transcription virtually simultaneously but that delays in splicing characterize Groups II and III. We examined the elongation times and polyadenylation timing, concluding that pre-mRNA synthesis is coordinate but splicing differences directly regulate the timing of mRNA production. Coupled to previous measurements of the half-life of mRNAs, the kinetic parameters can be combined in a general mathematical model of pre-mRNA conversion to mRNA that reproduces the experimental kinetics without recourse to differences in transcription.This model could be a generally relevant description of sequential transcriptional events in response to inducers.
2
Bette, M., M. K. Schäfer, N. van Rooijen, E. Weihe, and B. Fleischer. "Distribution and kinetics of superantigen-induced cytokine gene expression in mouse spleen." Journal of Experimental Medicine 178, no. 5 (November 1, 1993): 1531–39. http://dx.doi.org/10.1084/jem.178.5.1531.
Повний текст джерелаАнотація:
The polyclonal stimulation of T cells by bacterial superantigens is involved in the pathogenesis of the toxic shock syndrome in certain staphylococcal and streptococcal infections. Here we describe the onset and kinetics of superantigen-induced cytokine production in situ in spleens of normal BALB/c mice monitored at the level of cytokine mRNA expression by in situ hybridization. Messenger RNAs for interleukin 2 (IL-2), interferon gamma, and tumor necrosis factors (TNF) alpha and beta were not expressed at detectable levels in spleens of unstimulated animals but became visible already 30 min after intraperitoneal application of 50 micrograms staphylococcal enterotoxin B. All mRNA levels showed peak expression approximately 3 h after injection and a slow decrease up to 24 h after injection. Expression of the mRNAs was restricted to the T cell-dependent area of the periarteriolar lymphatic sheets of the spleen. Interestingly, TNF-alpha mRNA showed a biphasic response, the early appearing mRNA had the same localization as the other mRNAs, whereas after 3 h TNF-alpha mRNA showed a broader distribution indicating a second cell population producing TNF-alpha. The expression of IL-2 and TNF proteins in the serum increased in parallel to the observed mRNA changes with a slight delay. The presence of macrophages was not required for the expression of the cytokine mRNAs in the spleen as the expression was unchanged in macrophage-depleted mice. Only the second phase of TNF-alpha mRNA expression was abrogated in such animals. The expression of all mRNAs was completely suppressed by prior administration of cyclosporin A. These data show that nonphagocytic cells are the essential superantigen-presenting cells in vivo and indicate that at least part of the pathogenetic TNF-alpha is T cell derived.
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Granelli-Piperno, A., L. Andrus, and R. M. Steinman. "Lymphokine and nonlymphokine mRNA levels in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A." Journal of Experimental Medicine 163, no. 4 (April 1, 1986): 922–37. http://dx.doi.org/10.1084/jem.163.4.922.
Повний текст джерелаАнотація:
Northern and dot blotting with a panel of DNA probes were used to monitor the levels of specific mRNAs in mitogen-stimulated human T cells. The induction of IL-2 and IFN mRNAs required the synergistic action of PMA and either PHA or OKT3 mAb. In contrast, several nonlymphokine genes, the protooncogenes c-fos and c-myc, and the IL-2-R gene, were induced by either PHA or PMA alone. PHA increased the background levels of a 70 kD heat shock protein mRNA, but did not affect the observed background of c-myb mRNA. For all mRNAs that were induced, isolated CD4 and CD8 T cell subsets behaved similarly. Exogenous IL-2 had little (IFN) or no (IL-2) effect on lymphokine mRNAs, but significantly increased c-myc, IL-2-R and heat shock protein mRNAs. Therefore, the stimuli for lymphokine mRNAs differed from those required for several inducible nonlymphokine genes. IL-2 and IFN mRNAs exhibited some important similarities with c-myc, however. The levels of IL-2, IFN, and c-myc mRNA followed similar kinetics, peaking at 3 h in restimulated blasts and at 12 h in unstimulated T cells. The subsequent downregulation of lymphokine and c-myc mRNAs was retarded by cycloheximide. The induction of IL-2, IFN, and c-myc mRNAs was blocked by the immunosuppressive drug CsA, but not by the inactive analog CsH, and this block occurred at the level of nuclear transcription. Since the exogenous stimuli for lymphokine and c-myc gene expression differ, we suggest that intracellular controls must be shared to account for the similarities in their kinetics of expression and CsA sensitivity.
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Liu, C. C., S. Rafii, A. Granelli-Piperno, J. A. Trapani, and J. D. Young. "Perforin and serine esterase gene expression in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A." Journal of Experimental Medicine 170, no. 6 (December 1, 1989): 2105–18. http://dx.doi.org/10.1084/jem.170.6.2105.
Повний текст джерелаАнотація:
A pore-forming protein (PFP; perforin) and various serine esterases (SE) have been identified in the cytoplasmic granules of CTL and NK cells. Perforin and several SE have recently been cloned. Northern blotting analysis was performed here using cDNA probes specific for human perforin and two SE (SE 1/HS and SE 2/GB) to monitor the levels of specific mRNAs in mitogen-stimulated primary human T cells. These mRNAs were rapidly induced by IL-2 with optimal responses at 300 U/ml. After IL-2 treatment, mRNAs for perforin, SE 1, and SE 2 peaked at 12-24 h and decreased after 48 h. The three mRNAs were also induced in T cells treated with a combination of PMA plus lectin, OKT3 mAb, or plastic-adherent accessory cells. However, the induction induced by PMA/mitogen followed a slower kinetics, peaking at 48 h. In general, we found that SE 1 mRNA was more readily induced by IL-2, while SE 2 responded better to PMA/mitogen. Similar patterns of mRNA expression were observed for both unprimed T cells and PHA-primed T blasts. After stimulation with IL-2 and PMA/mitogen, the T8+ subset was shown to be the main producer of perforin, SE 1, and SE 2. Low levels of all three mRNAs, however, were also detected in the T4+ subset. The induction of all three mRNAs by either IL-2 or PMA/mitogen was partially blocked by the immunosuppressive drug cyclosporin A (CsA), but not by the biologically inactive analogue cyclosporin H. Together, these results point to some similarities and differences with upregulation of granule mediator mRNAs relative to lymphokine mRNAs. Both sets of genes require two signals for their induction by mitogens. In contrast to lymphokines, there is a strong response of granule mRNAs to IL-2, and the induction of these transcripts is only partially blocked by CsA.
5
Barton, P. J., A. J. Harris, and M. E. Buckingham. "Myosin light chain gene expression in developing and denervated fetal muscle in the mouse." Development 107, no. 4 (December 1, 1989): 819–24. http://dx.doi.org/10.1242/dev.107.4.819.
Повний текст джерелаАнотація:
We have investigated the accumulation of mRNA transcripts of the atrial (or embryonic) myosin light chain MLC1A (MLC1emb), and the two adult fast muscle myosin light chains (MLC1F and MLC3F) during fetal skeletal muscle development in the mouse. In 15-day fetal muscle, MLC1A is the predominant mRNA detectable, by 18 days MLC1F has become the major transcript and MLC3F mRNA is detectable for the first time. By 12 days after birth, MLC1A transcripts are undetectable and MLC1F and MLC3F are similar in abundance. In fetuses treated with beta-bungarotoxin and which therefore develop in the absence of functional nerve, MLC1A and MLC1F undergo normal transitions but MLC3F mRNA accumulation is significantly retarded. This demonstrates that these myosin light chain mRNAs accumulate with differing kinetics, and that MLC3F mRNA accumulation is nerve-dependent during fetal development. The results are discussed in terms of secondary muscle fibre formation, and in relation to the independent regulation of MLC1F and MLC3F mRNAs which are transcribed from the same gene.
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Rende, Francesca, Ilaria Cavallari, Alberto Corradin, Micol Silic-Benussi, Frederic Toulza, Gianna M. Toffolo, Yuetsu Tanaka, et al. "Kinetics and intracellular compartmentalization of HTLV-1 gene expression: nuclear retention of HBZ mRNAs." Blood 117, no. 18 (May 5, 2011): 4855–59. http://dx.doi.org/10.1182/blood-2010-11-316463.
Повний текст джерелаАнотація:
AbstractHuman T-cell leukemia virus type 1 (HTLV-1) codes for 9 alternatively spliced transcripts and 2 major regulatory proteins named Tax and Rex that function at the transcriptional and posttranscriptional levels, respectively. We investigated the temporal sequence of HTLV-1 gene expression in primary cells from infected patients using splice site-specific quantitative RT-PCR. The results indicated a two-phase kinetics with the tax/rex mRNA preceding expression of other viral transcripts. Analysis of mRNA compartmentalization in cells transfected with HTLV-1 molecular clones demonstrated the strict Rex-dependency of the two-phase kinetics and revealed strong nuclear retention of HBZ mRNAs, supporting their function as noncoding transcripts. Mathematical modeling underscored the importance of a delay between the functions of Tax and Rex, which was supported by experimental evidence of the longer half-life of Rex. These data provide evidence for a temporal pattern of HTLV-1 expression and reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts.
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Snaar, Sabine P., Pauline Verdijk, Hans J. Tanke, and Roeland W. Dirks. "Kinetics of HCMV immediate early mRNA expression in stably transfected fibroblasts." Journal of Cell Science 115, no. 2 (January 15, 2002): 321–28. http://dx.doi.org/10.1242/jcs.115.2.321.
Повний текст джерелаАнотація:
Compelling evidence supports an intimate link in time and space between eukaryotic pre-mRNA synthesis and processing and nucleocytoplasmic transport of mature mRNA. In this study, we analyzed the kinetic behavior of these processes in a quantitative manner. We used FISH and confocal scanning laser microscopy to detect transcripts produced by an inducible human cytomegalovirus immediate early (HCMV-IE) expression system. Upon induction, a large amount of pre-mRNA accumulated in nuclear foci at or near their transcription sites and, at later time, throughout the nucleoplasm. Inhibition of RNA polymerase II activity resulted in a rapid decrease in the number of transcripts in the nuclear RNA foci (half time ∼two minutes), indicating that accumulated transcripts were rapidly spliced and then released. The dispersed nucleoplasmic transcripts exited the nucleus with a half time of ∼10 minutes. Both processes were temperature dependent, suggesting that mRNA export is an active process. RNA polymerase II activation revealed that production of mature HCMV IE mRNAs required less than five minutes. Transcripts radiated from the gene at an average speed of ∼0.13 μm2/sec from this time on. Thus, it appears that these processes are tightly linked in time and space, with the splicing reaction as a rate-limiting factor.
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Feng, Pinghui, David N. Everly, and G. Sullivan Read. "mRNA Decay during Herpesvirus Infections: Interaction between a Putative Viral Nuclease and a Cellular Translation Factor." Journal of Virology 75, no. 21 (November 1, 2001): 10272–80. http://dx.doi.org/10.1128/jvi.75.21.10272-10280.2001.
Повний текст джерелаАнотація:
ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5′ end of mRNAs prior to the 3′ end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using theSaccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathioneS-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.
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Zhao, Renbin, Kurt Gish, Maureen Murphy, Yuxin Yin, Daniel Notterman, William H. Hoffman, Edward Tom, David H. Mack, and Arnold J. Levine. "Analysis of p53-regulated gene expression patterns using oligonucleotide arrays." Genes & Development 14, no. 8 (April 15, 2000): 981–93. http://dx.doi.org/10.1101/gad.14.8.981.
Повний текст джерелаАнотація:
Oligonucleotide microarrays were employed to quantitate mRNA levels from a large number of genes regulated by the p53 transcription factor. Responses to DNA damage and to zinc-inducible p53 were compared for their transcription patterns in cell culture. A cluster analysis of these data demonstrates that genes induced by γ radiation, UV radiation, and the zinc-induced p53 form distinct sets and subsets with a few genes in common to all these treatments. Cell type- or cell line-specific p53 responses were detected. When p53 proteins were induced with zinc, the kinetics of induction or repression of mRNAs from p53-responsive genes fell into eight distinct classes, five different kinetics of induction, and three different kinetics of repression. In addition, low levels of p53 in a cell induced or repressed only a subset of genes observed at higher p53 levels. The results of this study demonstrate that the nature of the p53 response in diverse mRNA species depends on the levels of p53 protein in a cell, the type of inducing agent or event, and the cell type employed. Of 6000 genes examined for p53 regulatory responses, 107 induced and 54 repressed genes fell into categories of apoptosis and growth arrest, cytoskeletal functions, growth factors and their inhibitors, extracellular matrix, and adhesion genes.
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Wu, X., G. J. Dolecki, and J. B. Lefkowith. "GRO chemokines: a transduction, integration, and amplification mechanism in acute renal inflammation." American Journal of Physiology-Renal Physiology 269, no. 2 (August 1, 1995): F248—F256. http://dx.doi.org/10.1152/ajprenal.1995.269.2.f248.
Повний текст джерелаАнотація:
We recently observed that cytokine-induced neutrophil chemoattractant (CINC), a GRO chemokine, contributes to neutrophil migration into the inflamed glomerulus in rat. Therefore, we sought to clarify how expression of the GRO chemokines, CINC and macrophage inflammatory protein-2 (MIP-2), is regulated in mesangial cells in vitro and the kidney in vivo. Mesangial cells expressed both GRO chemokine mRNAs in response to mediators of acute renal inflammation [interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharides (LPS)], but not chronic renal inflammation (transforming growth factor-beta 1), with CINC mRNA expression predominating over MIP-2. The kinetics of GRO chemokine mRNA expression in response to both IL-1 beta and TNF-alpha (but not LPS) paralleled those defined for polymorphonuclear leukocyte (PMN) migration during nephritis in vivo. IL-1 beta and TNF-alpha displayed nonparallel concentration-response relationships for GRO chemokine mRNA expression, and together were synergistic together rather than additive. Expression of GRO chemokine mRNAs in response to both cytokine agonists, however, was inhibited by genistein, a tyrosine kinase inhibitor. GRO chemokine mRNAs were rapidly expressed in inflamed glomeruli during immune complex glomerulonephritis with MIP-2 predominating over CINC. Expression of both chemokines was substantially inhibited by complement, leukocyte, and PMN depletion. In sum, GRO chemokines are expressed coordinately by mesangial cells and inflamed glomeruli and appear both to transduce the response to mediators of acute inflammation into a chemotactic signal and to amplify this response both temporally and quantitatively.(ABSTRACT TRUNCATED AT 250 WORDS)
11
Blattner, Christine, Patricia Kannouche, Margarethe Litfin, Klaus Bender, Hans J. Rahmsdorf, Jaime F. Angulo, and Peter Herrlich. "UV-Induced Stabilization of c-fos and Other Short-Lived mRNAs." Molecular and Cellular Biology 20, no. 10 (May 15, 2000): 3616–25. http://dx.doi.org/10.1128/mcb.20.10.3616-3625.2000.
Повний текст джерелаАнотація:
ABSTRACT Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than 15 min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine fibroblasts irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IκB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced growth factor receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.
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Sonza, Secondo, Helen P. Mutimer, Kate O'Brien, Philip Ellery, Jane L. Howard, Jonathan H. Axelrod, Nicholas J. Deacon, Suzanne M. Crowe, and Damian F. J. Purcell. "Selectively Reduced tat mRNA Heralds the Decline in Productive Human Immunodeficiency Virus Type 1 Infection in Monocyte-Derived Macrophages." Journal of Virology 76, no. 24 (December 15, 2002): 12611–21. http://dx.doi.org/10.1128/jvi.76.24.12611-12621.2002.
Повний текст джерелаАнотація:
ABSTRACT The transcription and splicing of human immunodeficiency virus type 1 (HIV-1) mRNA in primary blood monocyte-derived macrophages (MDM) and CD4+ peripheral blood lymphocytes (PBL) were compared to determine whether any differences might account for the slower noncytopathic infection of cells of the macrophage lineage. The expression of regulatory mRNAs during acute infection of MDM was delayed by about 12 h compared to that of PBL. In each cell type, an increase in spliced viral mRNAs slightly preceded virus production from the culture. Following the peak of productive infection, there was a proportional decrease in the expression of all regulatory mRNAs detected in PBL. In MDM, a dramatic additional decrease specifically in the tat mRNA species heralded a reduction in virus production. This decline in tat mRNA was reflected by a concomitant decrease in Tat activity in the cells and occurred with the same kinetics irrespective of the age of the cells when infected. Addition of exogenous Tat protein elicited a burst of virus production from persistently infected MDM, suggesting that the decrease in virus production from the cultures is a consequence of the reduction in tat mRNA levels. Our results show that modulation of HIV-1 mRNAs in macrophages during long-term infection, which is dependent on the period of infection rather than cell differentiation or maturation, results in a selective reduction of Tat protein levels at the commencement of a persistent, less productive phase of infection. Determination of the mechanism of this mRNA modulation may lead to novel targets for control of replication in these important viral reservoirs.
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Kato, S., H. Mano, T. Kumazawa, Y. Yoshizawa, R. Kojima та S. Masushige. "Effect of retinoid status on α, β and γ retinoic acid receptor mRNA levels in various rat tissues". Biochemical Journal 286, № 3 (15 вересня 1992): 755–60. http://dx.doi.org/10.1042/bj2860755.
Повний текст джерелаАнотація:
We have investigated the effects of retinoids, vitamin D and thyroid hormone on the levels of retinoic acid receptor (RAR)alpha, RAR beta and RAR gamma mRNAs in intact animals. Although vitamin A deficiency caused no significant changes in the levels of RAR alpha and RAR gamma mRNAs, the level of RAR beta transcripts was greatly decreased in various tissues of vitamin A-deficient rats, but was restored rapidly to a normal level after administration of retinoic acid. Retinol also restored the RAR beta mRNA level, but the magnitude and kinetics of the induction differed from those by retinoic acid. The use of specific inhibitors demonstrated that this autoregulation of RAR beta gene expression in vivo occurred at the transcriptional level. In addition, from these results it was postulated that the maintenance of the normal RAR beta mRNA levels seemed to require a threshold serum retinol concentration (about 25 micrograms/dl). Moreover, we found that administration of retinol and retinoic acid to normal rats caused the overexpression of RAR beta transcripts (2-15-fold) when compared with the control levels of RAR beta mRNA, although the levels of RAR alpha and RAR gamma mRNAs were not affected. Vitamin D and thyroid hormone did not modulate the levels of RAR transcripts. These findings clearly indicate the specific ligand regulation of RAR beta gene expression in intact animals. The altered levels of RAR beta according to retinoid status may affect retinoid-inducible gene expression.
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Maderazo, Alan B., Jonathan P. Belk, Feng He, and Allan Jacobson. "Nonsense-Containing mRNAs That Accumulate in the Absence of a Functional Nonsense-Mediated mRNA Decay Pathway Are Destabilized Rapidly upon Its Restitution." Molecular and Cellular Biology 23, no. 3 (February 1, 2003): 842–51. http://dx.doi.org/10.1128/mcb.23.3.842-851.2003.
Повний текст джерелаАнотація:
ABSTRACT Nonsense-mediated mRNA decay (NMD) is a conserved proofreading mechanism that protects eukaryotic cells from the potentially deleterious effects of truncated proteins. Studies of Saccharomyces cerevisiae imply that NMD is a predominantly cytoplasmic decay pathway, while studies of mammalian systems suggest that decay of most substrate mRNAs may occur while they are still associated with the nucleus, possibly during a round of translation that occurs during their export to the cytoplasm. Complete entry of the latter mRNAs into the cytoplasm appears to render them immune to further NMD; i.e., they escape further susceptibility to this decay pathway. To determine if yeast cytoplasmic nonsense-containing mRNAs that evade decay are subsequently immune to NMD, we examined the consequences of placing each of the three UPF/NMD genes under the control of a galactose-inducible promoter. The decay kinetics of ADE2 and PGK1 nonsense-containing mRNAs were then analyzed when expression of UPF1, NMD2, or UPF3 was either repressed or subsequently induced. Results from these experiments demonstrated that activation of NMD caused rapid and immediate degradation of both substrate transcripts, with half-lives of both stable mRNA populations shortened to approximately 7 min. These findings make it unlikely that yeast nonsense-containing mRNAs can escape degradation by NMD and indicate that such mRNAs are available to this decay pathway at each round of translation.
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Guilfoyle, TJ, G. Hagen, Y. Li, T. Ulmasov, ZB Liu, T. Strabala, and M. Gee. "Auxin-Regulated Transcription." Functional Plant Biology 20, no. 5 (1993): 489. http://dx.doi.org/10.1071/pp9930489.
Повний текст джерелаАнотація:
We have cloned and sequenced a number of auxin-responsive cDNAs and their corresponding genes from soybean and Arabidopsis. Each of these genes, with the exception of GH2/4, is transcriptionally regulated specifically by auxins within minutes after hormone application. The auxin-responsive mRNAs are induced some 3-60-fold depending on the type of mRNA analysed, the tissue examined, the dose and duration of auxin application, and the manipulation of the organ tested. Some of the mRNAs show rapid turnover kinetics. The mRNAs show distinct patterns of organ-specific, tissue-specific, and developmental-specific expression. The promoters of the auxin-responsive genes have been fused to the E. coli uidA gene which encodes β-glucuronidase (GUS) and transferred into tobacco and/or Arabidopsis via Agrobacterium T-DNA. These promoters and parts of these promoters have been used to follow the expression patterns and auxin-inducibility of the reporter genes in transgenic plants. We are attempting to identify minimal auxin-responsive elements and gravity-responsive elements within these promoters. We have also fused the auxin-inducible promoters to bacterial genes that encode cytokinin and auxin biosynthetic or conjugating enzymes to study the effects of organ, tissue, and developmental-specific expression of cytokinins and auxins on plant growth, development, and physiology.
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Benard, Lionel, Kathleen Carroll, Rosaura C. P. Valle, and Reed B. Wickner. "Ski6p Is a Homolog of RNA-Processing Enzymes That Affects Translation of Non-Poly(A) mRNAs and 60S Ribosomal Subunit Biogenesis." Molecular and Cellular Biology 18, no. 5 (May 1, 1998): 2688–96. http://dx.doi.org/10.1128/mcb.18.5.2688.
Повний текст джерелаАнотація:
ABSTRACT We mapped and cloned SKI6 of Saccharomyces cerevisiae, a gene that represses the copy number of the L-A double-stranded RNA virus, and found that it encodes an essential 246-residue protein with homology to a tRNA-processing enzyme, RNase PH. The ski6-2 mutant expressed electroporated non-poly(A) luciferase mRNAs 8- to 10-fold better than did the isogenic wild type. No effect of ski6-2 on expression of uncapped or normal mRNAs was found. Kinetics of luciferase synthesis and direct measurement of radiolabeled electroporated mRNA indicate that the primary effect of Ski6p was on efficiency of translation rather than on mRNA stability. Both ski6 and ski2 mutants show hypersensitivity to hygromycin, suggesting functional alteration of the translation apparatus. The ski6-2 mutant has normal amounts of 40S and 60S ribosomal subunits but accumulates a 38S particle containing 5′-truncated 25S rRNA but no 5.8S rRNA, apparently an incomplete or degraded 60S subunit. This suggests an abnormality in 60S subunit assembly. The ski6-2 mutation suppresses the poor expression of the poly(A)− viral mRNA in a strain deficient in the 60S ribosomal protein L4. Thus, a ski6mutation bypasses the requirement of the poly(A) tail for translation, allowing better translation of non-poly(A) mRNA, including the L-A virus mRNA which lacks poly(A). We speculate that the derepressed translation of non-poly(A) mRNAs is due to abnormal (but full-size) 60S subunits.
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Kim, Hyung-Yong, та Yasuko Rikihisa. "Expression of Interleukin-1β, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44". Infection and Immunity 68, № 6 (1 червня 2000): 3394–402. http://dx.doi.org/10.1128/iai.68.6.3394-3402.2000.
Повний текст джерелаАнотація:
ABSTRACT Human granulocytic ehrlichiosis (HGE) is an emerging febrile systemic disease caused by the HGE agent, an obligatory intracellular bacterium of granulocytes. The pathogenicity- and immunity-related mechanisms of HGE are unknown. In this study, several cytokines generated in human peripheral blood leukocytes (PBLs) incubated with the HGE agent or a recombinant 44-kDa major surface protein (rP44) of the HGE agent were examined by reverse transcription-PCR and a capture enzyme-linked immunosorbent assay. The HGE agent induced expression of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 mRNAs and proteins in PBLs in a dose-dependent manner to levels as high as those resulting from Escherichia colilipopolysaccharide stimulation. The kinetics of induction of these three cytokines in PBLs by rP44 and by the HGE agent were similar. Proteinase K treatment of the HGE agent or rP44 eliminated the ability to induce these three cytokines. Induction of these cytokine mRNAs was not dependent on superoxide generation. These results suggest that P44 proteins have a major role in inducing the production of proinflammatory cytokines by PBLs. Expression of IL-8, IL-10, gamma interferon, transforming growth factor β, and IL-2 mRNAs in response to the HGE agent was not remarkable. Among PBLs, neutrophils and lymphocytes expressed IL-1β mRNA but not TNF-α or IL-6 mRNA in response to the HGE agent, whereas monocytes expressed all three of these cytokine mRNAs. These observations suggest that induction of proinflammatory-cytokine gene expression by the major outer membrane protein of the HGE agent in monocytes, which are not the primary host cells of the HGE agent, contributes to HGE pathogenesis and immunomodulation.
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Matus Nicodemos, Rodrigo A., Daniel Douek, and Richard Koup. "Stochastic HIV Gene Expression is a Novel Mechanism for HIV Persistence." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 197.3. http://dx.doi.org/10.4049/jimmunol.202.supp.197.3.
Повний текст джерелаАнотація:
Abstract In HIV-infected people, the virus persists in resting memory CD4 T cells as a latent provirus integrated into transcribed genes. HIV has 9 gene products that are generated from 7 alternatively spliced mRNAs whose expression is regulated by the cell’s transcriptional state. Furthermore, transcription of genes accessible by RNA pol II occurs in bursts, giving rise to the stochastic expression of mRNAs in cells. The HIV protein Nef allows infected cells to evade CD8 T cell recognition through the downregulating of peptide-MHC complexes (pMHCs). The precise kinetics of pMHC downregulation by Nef upon reactivation of a latent provirus remains unclear. We explored this question by direct infection and longitudinal analysis of primary resting CD4 T cells with a CCR5-tropic intact replication-competent virus in which GFP reports the expression of Nef. We detected GFP+ cells 3 to 4 days after infection. These GFP+ cells were resting memory CD4 T cells and had downregulated CD4 and pMHCs. We then performed single-cell RNA-seq on sorted GFP+ cells to examine both cellular and virus gene expression, as well as HIV integration sites. Our dataset identified various transcribed host genes that had an integrated provirus. Strikingly we found GFP+ cells either (1) had no HIV mRNAs or (2) had HIV mRNAs encoding Nef, Vpr, Vif or Vpu-Env, but never HIV mRNAs encoding Gag-Pol, Tat or Rev. We conclude that in resting memory CD4 T cells, an integrated HIV genome is transcribed and alternatively spliced for mRNAs encoding Nef, Vif, Vpr or Vpu-Env. HIV expression is stochastic because infected cells didn’t always express HIV mRNAs. Therefore we propose stochastic HIV gene expression is a novel mechanism for HIV persistence by hiding the infected cells from the immune system.
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Audibert, A., D. Weil, and F. Dautry. "In Vivo Kinetics of mRNA Splicing and Transport in Mammalian Cells." Molecular and Cellular Biology 22, no. 19 (October 1, 2002): 6706–18. http://dx.doi.org/10.1128/mcb.22.19.6706-6718.2002.
Повний текст джерелаАнотація:
ABSTRACT The kinetics of pre-mRNA processing in living cells is poorly known, preventing a detailed analysis of the regulation of these reactions. Using tetracycline-regulated promoters we performed, during a transcriptional induction, a complete analysis of the maturation of two cellular mRNAs, those for LT-α and β-globin. In both cases, splicing was appropriately described by first-order reactions with corresponding half-lives ranging between 0.4 and 7.5 min, depending on the intron. Transport also behaved as a first-order reaction during the early phase of β-globin expression, with a nuclear dwelling time of 4 min. At a later time, analysis was prevented by the progressive accumulation within the nucleus of mature mRNA not directly involved in export. Our results further establish for these genes that (i) splicing components are never limiting, even when expression is induced in naive cells, (ii) there is no significant RNA degradation during splicing and transport, and (iii) precursor-to-product ratios at steady state can be used for the determination of splicing rates. Finally, the comparison between the kinetics of splicing during transcriptional induction and during transcriptional shutoff reveals a novel coupling between transcription and splicing.
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Xu, N., C. Y. Chen, and A. B. Shyu. "Modulation of the fate of cytoplasmic mRNA by AU-rich elements: key sequence features controlling mRNA deadenylation and decay." Molecular and Cellular Biology 17, no. 8 (August 1997): 4611–21. http://dx.doi.org/10.1128/mcb.17.8.4611.
Повний текст джерелаАнотація:
Regulation of cytoplasmic deadenylation has a direct impact on the fate of mRNA and, consequently, its expression in the cytoplasm. AU-rich elements (AREs) found in the 3' untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. AREs direct accelerated deadenylation as the first step in mRNA turnover. Recently we have proposed that AREs can be divided into three different classes. mRNAs bearing either the class I AUUUA-containing ARE or the class III non-AUUUA ARE display synchronous poly(A) shortening, whereas class II ARE-containing mRNAs are deadenylated asynchronously, with the formation of poly(A)- intermediates. In this study, we have systematically characterized the deadenylation kinetics displayed by various AREs and their mutant derivatives. We find that a cluster of five or six copies of AUUUA motifs in close proximity forming various degrees of reiteration is the key feature that dictates the choice between processive versus distributive deadenylation. An AU-rich region 20 to 30 nucleotides long immediately 5' to this cluster of AUUUA motifs can greatly enhance the destabilizing ability of the AUUUA cluster and is, therefore, an integral part of the class I and class II AREs. These two features are the defining characteristics of class II AREs. Our results are consistent with the interpretation that the pentanucleotide AUUUA, rather than the nonamer UUAUUUA(U/A)(U/A), is both an essential and the minimal sequence motif of AREs. Our study provides the groundwork for future characterization of ARE-binding proteins identified by in vitro gel shift assays in order to stringently define their potential role in the ARE-mediated decay pathway. Moreover, transformation of deadenylation kinetics from one type to the other by mutations of AREs implies the existence of cross talk between the ARE and 3' poly(A) tail, which dictates the decay kinetics.
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Wang, Xinkang, Tian-Li Yue, Peter R. Young, Frank C. Barone, and Giora Z. Feuerstein. "Expression of Interleukin-6, c-Fos, and zif268 mRNAs in Rat Ischemic Cortex." Journal of Cerebral Blood Flow & Metabolism 15, no. 1 (January 1995): 166–71. http://dx.doi.org/10.1038/jcbfm.1995.18.
Повний текст джерелаАнотація:
The expression of interleukin-6 (IL-6) mRNA in the focal ischemic rat cortex was studied by means of Northern hybridization. IL-6 mRNA was induced after permanent occlusion of the middle cerebral artery, reached a significant level at 3 h, and peaked at 12 h, i.e., ∼ 10-fold increase in the ischemic zone compared with the nonischemic cortex or sham-operated controls. The increased IL-6 mRNA was elevated for at least 24 h. Low levels of IL-6 mRNA were detected in sham-operated rats or in the contralateral nonischemic cortex. The expression of c- fos and zif268 mRNAs, two early response genes, was rapid (increased by 1 h postischemia) and transient (returned to basal levels by 24 and 12 h, respectively), clearly having different kinetic patterns from that of IL-6 mRNA. The early response kinetic pattern of c- fos and zif268 mRNAs in focal ischemia suggests their transcriptional regulatory roles in response to ischemic insult, while the delayed induction pattern of IL-6 mRNA suggests a role for this pleiotropic cytokine in the inflammatory response to the focal ischemic damage of the brain.
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Lai, Ruanne Y. J., Vladimir Ljubicic, Donna D'souza, and David A. Hood. "Effect of chronic contractile activity on mRNA stability in skeletal muscle." American Journal of Physiology-Cell Physiology 299, no. 1 (July 2010): C155—C163. http://dx.doi.org/10.1152/ajpcell.00523.2009.
Повний текст джерелаАнотація:
Repeated bouts of exercise promote the biogenesis of mitochondria by multiple steps in the gene expression patterning. The role of mRNA stability in controlling the expression of mitochondrial proteins is relatively unexplored. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 or 6 h/day) rat muscle for 7 days. Chronic contractile activity (CCA) increased the protein expression of PGC-1α, c-myc, and mitochondrial transcription factor A (Tfam) by 1.6-, 1.7- and 2.0-fold, respectively. To determine mRNA stability, we incubated total RNA with cytosolic extracts using an in vitro cell-free system. We found that the intrinsic mRNA half-lives ( t1/2) were variable within control muscle. Peroxisome proliferator-activated receptor-γ, coactivator-1α (PGC-1α) and Tfam mRNAs decayed more rapidly ( t1/2 = 22.7 and 31.4 min) than c-myc mRNA ( t1/2 = 99.7 min). Furthermore, CCA resulted in a differential response in degradation kinetics. After CCA, PGC-1α and Tfam mRNA half-lives decreased by 48% and 44%, respectively, whereas c-myc mRNA half-life was unchanged. CCA induced an elevation of both the cytosolic RNA-stabilizing human antigen R (HuR) and destabilizing AUF1 (total) by 2.4- and 1.8-fold, respectively. Increases in the p37AUF1, p40AUF1, and p45AUF1 isoforms were most evident. Thus these data indicate that CCA results in accelerated turnover rates of mRNAs encoding important mitochondrial biogenesis regulators in skeletal muscle. This adaptation is likely beneficial in permitting more rapid phenotypic plasticity in response to subsequent contractile activity.
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Escalante, Ricardo, Alberto García-Sáez, Maria-Asunción Ortega, and Leandro Sastre. "Gene expression after resumption of development of Artemia franciscana cryptobiotic embryos." Biochemistry and Cell Biology 72, no. 3-4 (March 1, 1994): 78–83. http://dx.doi.org/10.1139/o94-014.
Повний текст джерелаАнотація:
The steady-state levels of six different mRNAs have been studied during Artemia franciscana development. Some of these mRNAs are present in the cryptobiotic cyst, like those coding for cytoplasmic actins, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, and the Na+,K+-ATPase α-subunit isoform coded by the clone pArATNa136. The expression of these mRNAs is markedly induced during cyst development. A small increase in mRNA levels can be observed for some genes at very early stages of development (2 h). The main increase is observed between 4 and 16 h of development for all these genes, although the time course of mRNA accumulation is different for each one of the genes studied. Some other genes, like those coding for muscle actin (actin 3) or the Na+,K+-ATPase α-subunit isoform coded by the cDNA clone α2850, are not expressed in the cyst before resumption of development and their expression is induced after 10 or 6 h of development, respectively. These data on the kinetic of mRNA accumulation provide the information required to determine transcriptionally active developmental stages, necessary to study in more detail the mechanisms of transcriptional regulation during activation of cryptobiotic cysts and resumption of embryonic development.Key words: Artemia, gene expression, actin, Na,K-ATPase, Ca2+-ATPase.
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Lin, Shankung, Wengong Wang, Gerald M. Wilson, Xiaoling Yang, Gary Brewer, Nikki J. Holbrook, and Myriam Gorospe. "Down-Regulation of Cyclin D1 Expression by Prostaglandin A2 Is Mediated by Enhanced Cyclin D1 mRNA Turnover." Molecular and Cellular Biology 20, no. 21 (November 1, 2000): 7903–13. http://dx.doi.org/10.1128/mcb.20.21.7903-7913.2000.
Повний текст джерелаАнотація:
ABSTRACT Prostaglandin A2 (PGA2), an experimental chemotherapeutic agent, causes growth arrest associated with decreased cyclin D1 expression in several cancer cell lines. Here, using human non-small-cell lung carcinoma H1299 cells, we investigated the mechanisms whereby PGA2 down-regulates cyclin D1 expression. Transcription rates of the cyclin D1 gene, studied using a cyclin D1 promoter-luciferase construct and nuclear run-on assays, were not affected by PGA2 treatment. Instead, the cyclin D1 mRNA was rendered unstable after exposure to PGA2. Since the stability of labile mRNA is modulated through binding of proteins to specific mRNA sequences, we sought to identify protein(s) recognizing the cyclin D1 mRNA. In electrophoretic mobility-shift assays using radiolabeled RNA probes derived from different regions of cyclin D1 mRNA, we observed that (i) lysates prepared from PGA2-treated cells exhibited enhanced protein-cyclin D1 RNA complex formation; (ii) the kinetics of complex formation correlated closely with that of cyclin D1 mRNA loss; and (iii) binding occurred within a 390-base cyclin D1 3′ untranslated region (UTR) (K12). This binding activity could be cross-linked, revealing proteins ranging from 30 to 47 kDa. The RNA-binding protein AUF1, previously associated with the degradation of target mRNAs, bound cyclin D1 mRNA, because anti-AUF1 antibodies were capable of supershifting or immunoprecipitating cyclin D1 mRNA-protein complexes. Finally, insertion of K12 in the 3′UTR of reporter genes markedly reduced the expression and half-life of the resulting chimeric mRNAs in transfected, PGA2-treated cells. Our data demonstrate that PGA2 down-regulates cyclin D1 expression by decreasing cyclin D1 mRNA stability and implicates a 390-base element in the 3′UTR in this regulation.
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Narayanan, Krishna, Cheng Huang, Kumari Lokugamage, Wataru Kamitani, Tetsuro Ikegami, Chien-Te K. Tseng, and Shinji Makino. "Severe Acute Respiratory Syndrome Coronavirus nsp1 Suppresses Host Gene Expression, Including That of Type I Interferon, in Infected Cells." Journal of Virology 82, no. 9 (February 27, 2008): 4471–79. http://dx.doi.org/10.1128/jvi.02472-07.
Повний текст джерелаАнотація:
ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.
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Lambolez, Florence, Orly Azogui, Anne-Marie Joret, Corinne Garcia, Harald von Boehmer, James Di Santo, Sophie Ezine, and Benedita Rocha. "Characterization of T Cell Differentiation in the Murine Gut." Journal of Experimental Medicine 195, no. 4 (February 11, 2002): 437–49. http://dx.doi.org/10.1084/jem.20010798.
Повний текст джерелаАнотація:
Gut intraepithelial CD8 T lymphocytes (T-IEL) are distinct from thymus-derived cells and are thought to derive locally from cryptopatch (CP) precursors. The intermediate stages of differentiation between CP and mature T-IEL were not identified, and the local differentiation process was not characterized. We identified and characterized six phenotypically distinct lineage-negative populations in the CP and the gut epithelium: (a) we determined the kinetics of their generation from bone marrow precursors; (b) we quantified CD3-ϵ, recombination activating gene (Rag)-1, and pre-Tα mRNAs expression at single cell level; (c) we characterized TCR-β, -γ, and -α locus rearrangements; and (d) we studied the impact of different mutations on the local differentiation. These data allowed us to establish a sequence of T cell precursor differentiation in the gut. We also observed that the gut differentiation varied from that of the thymus by a very low frequency of pre-Tα chain mRNA expression, a different kinetics of Rag-1 mRNA expression, and a much higher impact of CD3 ϵ/δ and pre-Tα deficiencies. Finally, only 3% of CP cells were clearly involved in T cell differentiation, suggesting that these structures may have additional physiological roles in the gut.
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Hao, Shengli, and David Baltimore. "mRNA stability influences the temporal order of inflammatory gene induction (136.1)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 136.1. http://dx.doi.org/10.4049/jimmunol.182.supp.136.1.
Повний текст джерелаАнотація:
Abstract The inflammatory response is regulated by a great number of genes. It is still unknown whether and how these genes are organized to function as groups. We have performed a global analysis of about 200 genes induced in cultured cells by TNF, a proinflammatory cytokine. We found that genes can be divided roughly into three groups with different induction kinetics. Group I genes are activated quickly but then quickly repressed. Group II genes are also activated rapidly but reach maximal activity later and remain at the induced level longer. The Group III genes are activated in a delayed, slow but steady manner. The activation pattern of a gene remains largely the same in different cell types and species and is not specified by the extracellular signals. In addition, some genes within the same group have similar related functions. This suggests that these genes are temporally organized to function as groups. While transcriptional regulation is important, we found that a key parameter characterizing the grouping of genes is the stability of their mRNAs. The early mRNAs are very unstable, while the late mRNAs are highly stable. The instability of mRNAs correlates with the number of AU-rich elements in their 3'-untranslated regions (3'UTR). Changing mRNA stability either through extracellular LPS treatment or by switching the 3'UTRs but not the promoter/enhancer regions that control expression of GFP transgenes resulted in corresponding changes of the gene activation kinetics. These show that the inflammatory response is pre-set at the genome level and that the 3'UTR of a gene is a key element for organizing genes into functional groups.
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Smith, Kelly P., Phillip T. Moen, Karen L. Wydner, John R. Coleman, and Jeanne B. Lawrence. "Processing of Endogenous Pre-mRNAs in Association with SC-35 Domains Is Gene Specific." Journal of Cell Biology 144, no. 4 (February 22, 1999): 617–29. http://dx.doi.org/10.1083/jcb.144.4.617.
Повний текст джерелаАнотація:
Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript “trees” of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional “tracks” of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.
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Vittet, D., MH Prandini, R. Berthier, A. Schweitzer, H. Martin-Sisteron, G. Uzan, and E. Dejana. "Embryonic stem cells differentiate in vitro to endothelial cells through successive maturation steps." Blood 88, no. 9 (November 1, 1996): 3424–31. http://dx.doi.org/10.1182/blood.v88.9.3424.bloodjournal8893424.
Повний текст джерелаАнотація:
The mechanisms involved in the regulation of vasculogenesis still remain unclear in mammals. Totipotent embryonic stem (ES) cells may represent a suitable in vitro model to study molecular events involved in vascular development. In this study, we followed the expression kinetics of a relatively large set of endothelial-specific markers in ES-derived embryoid bodies (EBs). Results of both reverse transcription-polymerase chain reaction and/or immunofluorescence analysis show that a spontaneous endothelial differentiation occurs during EBs development. ES-derived endothelial cells express a full range of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial (VE) cadherin, MECA-32, and MEC-14.7. Analysis of the kinetics of endothelial marker expression allows the distinction of successive maturation steps. Flk-1 was the first to be detected; its mRNA is apparent from day 3 of differentiation. PECAM and tie-2 mRNAs were found to be expressed only from day 4, whereas VE-cadherin and tie-1 mRNAs cannot be detected before day 5. Immunofluorescence stainings of EBs with antibodies directed against Flk-1, PECAM, VE-cadherin, MECA-32, and MEC-14.7 confirmed that the expression of these antigens occurs at different steps of endothelial cell differentiation. The addition of an angiogenic growth factor mixture including erythropoietin, interleukin-6, fibroblast growth factor 2, and vascular endothelial growth factor in the EB culture medium significantly increased the development of primitive vascular-like structures within EBs. These results indicate that this in vitro system contains a large part of the endothelial cell differentiation program and constitutes a suitable model to study the molecular mechanisms involved in vasculogenesis.
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Liu, Xiaomei, Qing Zhang, Weixiao Wang, Dongjiao Zuo, Jing Wang, Feng Zhou, Liping Niu, et al. "Analysis of Long Noncoding RNA and mRNA Expression Profiles in IL-9-Activated Astrocytes and EAE Mice." Cellular Physiology and Biochemistry 45, no. 5 (2018): 1986–98. http://dx.doi.org/10.1159/000487975.
Повний текст джерелаАнотація:
Background/Aims: Multiple sclerosis (MS) is an autoimmune disease in the central nervous system associated with demyelination and axonal injury. Astrocyte activation is involved in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. This study was designed to find potential lncRNAs in EAE mice and activated astrocytes. Methods: we performed microarray analysis of lncRNAs from the brain tissues of EAE mice and primary mouse astrocytes treated with IL-9(50 ng/ml). 12 lncRNAs were validated through real-time PCR. Gene ontology and KEGG pathway analysis were applied to explore the potential functions of lncRNAs. Results: Differentially expressed 3300 lncRNAs and 3250 mRNAs were in the brain tissues of EAE mice, and 3748 lncRNAs and 3332 mRNAs were in activated astrocytes. Notably, there were 2 co-up-regulated lncRNAs and 3 co-down-regulated lncRNAs both in the brain tissues of EAE mice and in activated astrocytes, including Gm14005, Gm12478, mouselincRNA1117, AK080435, and mouselincRNA0681, which regulate the ER calcium flux kinetics, zinc finger protein and cell apoptosis. Similarly, there were 7 mRNAs co-up-regulated and 2 mRNAs co-down-regulated both in vivo and in vitro. Gene ontology and KEGG pathway analysis showed that the biological functions of differentially expressed mRNAs were associated with metabolism, development and inflammation. The results of realtime PCR validation were consistent with the data from the microarrays. Conclusions: Our data uncovered the expression profiles of lncRNAs and mRNAs in vivo and in vitro, which may help delineate the mechanisms of astrocyte activation during MS/EAE process.
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Duval, C., P. Bouvet, F. Omilli, C. Roghi, C. Dorel, R. LeGuellec, J. Paris, and H. B. Osborne. "Stability of maternal mRNA in Xenopus embryos: role of transcription and translation." Molecular and Cellular Biology 10, no. 8 (August 1990): 4123–29. http://dx.doi.org/10.1128/mcb.10.8.4123-4129.1990.
Повний текст джерелаАнотація:
The first 12 cell divisions of Xenopus laevis embryos do not require gene transcription. This means that the regulation of gene expression during this period is controlled at post transcriptional levels and makes Xenopus early development a potentially interesting biological system with which to study the mechanisms involved. We describe here the stability characteristics of several maternal Xenopus mRNAs which are deadenylated soon after fertilisation (J. Paris and M. Philippe, Dev. Biol., in press). We show that these mRNAs were only degraded in the embryo after the midblastula transition (MBT), when gene transcription was initiated. The kinetics with which the deadenylated maternal mRNAs decreased in the post-MBT embryos showed sequence specificity. The degradation of these mRNAs after the MBT was inhibited by cycloheximide but was not affected by dactinomycin. Therefore, the destabilization of these mRNAs does not appear to be initiated by new embryonic gene transcripts. Sequence comparisons of the 3' untranslated region of these mRNAs identified several motifs which may be involved in the posttranscriptional control of these gene products.
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Duval, C., P. Bouvet, F. Omilli, C. Roghi, C. Dorel, R. LeGuellec, J. Paris, and H. B. Osborne. "Stability of maternal mRNA in Xenopus embryos: role of transcription and translation." Molecular and Cellular Biology 10, no. 8 (August 1990): 4123–29. http://dx.doi.org/10.1128/mcb.10.8.4123.
Повний текст джерелаАнотація:
The first 12 cell divisions of Xenopus laevis embryos do not require gene transcription. This means that the regulation of gene expression during this period is controlled at post transcriptional levels and makes Xenopus early development a potentially interesting biological system with which to study the mechanisms involved. We describe here the stability characteristics of several maternal Xenopus mRNAs which are deadenylated soon after fertilisation (J. Paris and M. Philippe, Dev. Biol., in press). We show that these mRNAs were only degraded in the embryo after the midblastula transition (MBT), when gene transcription was initiated. The kinetics with which the deadenylated maternal mRNAs decreased in the post-MBT embryos showed sequence specificity. The degradation of these mRNAs after the MBT was inhibited by cycloheximide but was not affected by dactinomycin. Therefore, the destabilization of these mRNAs does not appear to be initiated by new embryonic gene transcripts. Sequence comparisons of the 3' untranslated region of these mRNAs identified several motifs which may be involved in the posttranscriptional control of these gene products.
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Ye, Jianjiang, Lyndle Gradoville, Derek Daigle, and George Miller. "De Novo Protein Synthesis Is Required for Lytic Cycle Reactivation of Epstein-Barr Virus, but Not Kaposi's Sarcoma-Associated Herpesvirus, in Response to Histone Deacetylase Inhibitors and Protein Kinase C Agonists." Journal of Virology 81, no. 17 (June 27, 2007): 9279–91. http://dx.doi.org/10.1128/jvi.00982-07.
Повний текст джерелаАнотація:
ABSTRACT The oncogenic human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are latent in cultured lymphoma cells. We asked whether reactivation from latency of either virus requires de novo protein synthesis. Using Northern blotting and quantitative reverse transcriptase PCR, we measured the kinetics of expression of the lytic cycle activator genes and determined whether abundance of mRNAs encoding these genes from either virus was reduced by treatment with cycloheximide (CHX), an inhibitor of protein synthesis. CHX blocked expression of mRNAs of EBV BZLF1 and BRLF1, the two EBV lytic cycle activator genes, when HH514-16 Burkitt lymphoma cells were treated with histone deacetylase (HDAC) inhibitors, sodium butyrate or trichostatin A, or a DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine. CHX also inhibited EBV lytic cycle activation in B95-8 marmoset lymphoblastoid cells by phorbol ester phorbol-12-myristate-13-acetate (TPA). EBV lytic cycle induction became resistant to CHX between 4 and 6 h after application of the inducing stimulus. KSHV lytic cycle activation, as assessed by ORF50 mRNA expression, was rapidly induced by the HDAC inhibitors, sodium butyrate and trichostatin A, in HH-B2 primary effusion lymphoma cells. In HH-B2 cells, CHX did not inhibit, but enhanced, expression of the KSHV lytic cycle activator gene, ORF50. In BC-1, a primary effusion lymphoma cell line that is dually infected with EBV and KSHV, CHX blocked EBV BRLF1 lytic gene expression induced by TPA and sodium butyrate; KSHV ORF50 mRNA induced simultaneously in the same cells by the same inducing stimuli was resistant to CHX. The experiments show, for the cell lines and inducing agents studied, that the EBV BZLF1 and BRLF1 genes do not behave with “immediate-early” kinetics upon reactivation from latency. KSHV ORF50 is a true “immediate-early” gene. Our results indicate that the mechanism by which HDAC inhibitors and TPA induce lytic cycle gene expression of the two viruses differs and suggest that EBV but not KSHV requires one or more proteins to be newly synthesized between 4 and 6 h after application of an inducing stimulus.
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Matsushime, H., MF Roussel, K. Matsushima, A. Hishinuma, and CJ Sherr. "Cloning and expression of murine interleukin-1 receptor antagonist in macrophages stimulated by colony-stimulating factor 1." Blood 78, no. 3 (August 1, 1991): 616–23. http://dx.doi.org/10.1182/blood.v78.3.616.616.
Повний текст джерелаАнотація:
Abstract Colony-stimulating factor 1 (CSF-1) can act on mature macrophages to modulate their production of inflammatory cytokines. A cDNA encoding the interleukin-1 receptor antagonist (IL-1Ra) was cloned by subtractive hybridization from a CSF-1-stimulated murine macrophage cell line, sequenced, and expressed in mammalian and bacterial cells. Mouse IL-1Ra is a 22-Kd glycoprotein that is 76% identical to its human counterpart, shows considerably less similarity to IL-1 alpha and IL-1 beta, and competes with IL-1 alpha for binding to the type I IL-1 receptor normally expressed on T cells and fibroblasts. CSF-1 treatment of mouse bone marrow-derived macrophages led to a rapid and sustained increase in IL-1Ra mRNA during the G1 phase of the cell cycle as well as to increases in mRNAs encoding IL-1 alpha and IL-1 beta. Cycloheximide inhibited CSF-1-induced IL-1 alpha mRNA synthesis, but augmented IL-1 beta mRNA production and did not affect induction of IL- 1Ra mRNA. No IL-1Ra mRNA was observed in CSF-1-stimulated mouse fibroblasts engineered to express CSF-1 receptors, demonstrating that its regulation depends on cell context and can be dissociated from the proliferative response. In agreement, bacterial lipopolysaccharide, a nonmitogenic activator, also induced IL-1Ra and IL-1 mRNAs in macrophages. Unlike IL-1 alpha and beta, IL-1Ra contains a signal peptide. The kinetics of its induction and its ability to gain access to the secretory compartment imply that IL-1Ra may be secreted more efficiently than IL-1, and suggest that macrophages both positively and negatively regulate the IL-1 response.
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Matsushime, H., MF Roussel, K. Matsushima, A. Hishinuma, and CJ Sherr. "Cloning and expression of murine interleukin-1 receptor antagonist in macrophages stimulated by colony-stimulating factor 1." Blood 78, no. 3 (August 1, 1991): 616–23. http://dx.doi.org/10.1182/blood.v78.3.616.bloodjournal783616.
Повний текст джерелаАнотація:
Colony-stimulating factor 1 (CSF-1) can act on mature macrophages to modulate their production of inflammatory cytokines. A cDNA encoding the interleukin-1 receptor antagonist (IL-1Ra) was cloned by subtractive hybridization from a CSF-1-stimulated murine macrophage cell line, sequenced, and expressed in mammalian and bacterial cells. Mouse IL-1Ra is a 22-Kd glycoprotein that is 76% identical to its human counterpart, shows considerably less similarity to IL-1 alpha and IL-1 beta, and competes with IL-1 alpha for binding to the type I IL-1 receptor normally expressed on T cells and fibroblasts. CSF-1 treatment of mouse bone marrow-derived macrophages led to a rapid and sustained increase in IL-1Ra mRNA during the G1 phase of the cell cycle as well as to increases in mRNAs encoding IL-1 alpha and IL-1 beta. Cycloheximide inhibited CSF-1-induced IL-1 alpha mRNA synthesis, but augmented IL-1 beta mRNA production and did not affect induction of IL- 1Ra mRNA. No IL-1Ra mRNA was observed in CSF-1-stimulated mouse fibroblasts engineered to express CSF-1 receptors, demonstrating that its regulation depends on cell context and can be dissociated from the proliferative response. In agreement, bacterial lipopolysaccharide, a nonmitogenic activator, also induced IL-1Ra and IL-1 mRNAs in macrophages. Unlike IL-1 alpha and beta, IL-1Ra contains a signal peptide. The kinetics of its induction and its ability to gain access to the secretory compartment imply that IL-1Ra may be secreted more efficiently than IL-1, and suggest that macrophages both positively and negatively regulate the IL-1 response.
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Plata-Guzmán, Laura Y., Rossana Arroyo, Nidia León-Sicairos, Adrián Canizález-Román, Héctor S. López-Moreno, Jeanett Chávez-Ontiveros, José A. Garzón-Tiznado, and Claudia León-Sicairos. "Stem–Loop Structures in Iron-Regulated mRNAs of Giardia duodenalis." International Journal of Environmental Research and Public Health 20, no. 4 (February 17, 2023): 3556. http://dx.doi.org/10.3390/ijerph20043556.
Повний текст джерелаАнотація:
Giardia duodenalis is a significant cause of waterborne and foodborne infections, day-care center outbreaks, and traveler’s diarrhea worldwide. In protozoa such as Trichomonas vaginalis and Entamoeba histolytica, iron affects the growth, pathogenicity mechanisms, and expression of virulence genes. One of the proposed iron regulatory mechanisms is at the post-transcriptional level through an IRE/IRP-like (iron responsive element/iron regulatory protein) system. Recently, the expression of many putative giardial virulence factors in the free-iron levels has been reported in subsequent RNAseq experiments; however, the iron regulatory mechanism remains unknown. Thus, this work aimed to determine the effects of iron on the growth, gene expression, and presence of IRE-like structures in G. duodenalis. First, the parasite’s growth kinetics at different iron concentrations were studied, and the cell viability was determined. It was observed that the parasite can adapt to an iron range from 7.7 to 500 µM; however, in conditions without iron, it is unable to survive in the culture medium. Additionally, the iron modulation of three genes was determined by RT-PCR assays. The results suggested that Actin, glucosamine-6-phosphate deaminase, and cytochrome b5 mRNA were down-regulated by iron. To investigate the presence of IRE-like structures, in silico analyses were performed for different mRNAs from the Giardia genome database. The Zuker mfold v2.4 web server and theoretical analysis were used to predict the secondary structures of the 91 mRNAs analyzed. Interestingly, the iron-induced downregulation of the genes analyzed corresponds to the location of the stem–loop structures found in their UTR regions. In conclusion, iron modulates the growth and expression of specific genes, likely due to the presence of IRE-like structures in G. duodenalis mRNAs.
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AUDIGÉ, A., Z. R. YU, B. M. FREY, D. E. UEHLINGER, F. J. FREY, and B. VOGT. "Epithelial sodium channel (ENaC) subunit mRNA and protein expression in rats with puromycin aminonucleoside-induced nephrotic syndrome." Clinical Science 104, no. 4 (March 13, 2003): 389–95. http://dx.doi.org/10.1042/cs1040389.
Повний текст джерелаАнотація:
In experimental nephrotic syndrome, urinary sodium excretion is decreased during the early phase of the disease. The molecular mechanism(s) leading to salt retention has not been completely elucidated. The rate-limiting constituent of collecting duct sodium transport is the epithelial sodium channel (ENaC). We examined the abundance of ENaC subunit mRNAs and proteins in puromycin aminonucleoside (PAN)-induced nephrotic syndrome. The time courses of urinary sodium excretion, plasma aldosterone concentration and proteinuria were studied in male Sprague–Dawley rats treated with a single dose of either PAN or vehicle. The relative amounts of αENaC, βENaC and γENaC mRNAs were determined in kidneys from these rats by real-time quantitative TaqMan PCR, and the amounts of proteins by Western blot. The kinetics of urinary sodium excretion and the appearance of proteinuria were comparable with those reported previously. Sodium retention occurred on days 2, 3 and 6 after PAN injection. A significant up-regulation of αENaC and βENaC mRNA abundance on days 1 and 2 preceded sodium retention on days 2 and 3. Conversely, down-regulation of αENaC, βENaC and γENaC mRNA expression on day 3 occurred in the presence of high aldosterone concentrations, and was followed by a return of sodium excretion to control values. The amounts of αENaC, βENaC and γENaC proteins were not increased during PAN-induced sodium retention. In conclusion, ENaC mRNA expression, especially αENaC, is increased in the very early phase of the experimental model of PAN-induced nephrotic syndrome in rats, but appears to escape from the regulation by aldosterone after day 3.
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Matthes, T., C. Werner-Favre, H. Tang, X. Zhang, V. Kindler, and R. H. Zubler. "Cytokine mRNA expression during an in vitro response of human B lymphocytes: kinetics of B cell tumor necrosis factor alpha, interleukin (IL)6, IL-10, and transforming growth factor beta 1 mRNAs." Journal of Experimental Medicine 178, no. 2 (August 1, 1993): 521–28. http://dx.doi.org/10.1084/jem.178.2.521.
Повний текст джерелаАнотація:
Expression of mRNA for eight cytokines was analyzed in an in vitro response-proliferation and Ig-secretion--of normal human B lymphocytes. This was made possible by the use of murine thymoma cells as helper cells in conjunction with human T cell supernatant, and the design of human DNA sequence-specific primers for RT-polymerase chain reaction. mRNAs for interleukin (IL)2 and IL-4, but also for IL-1 alpha and IL-1 beta remained undetectable during the whole culture period in highly purified B cells prepared by a three-step purification protocol. However, tumor necrosis factor alpha and IL-6 mRNAs peaked during days 1-3 after culture start and became undetectable after 5-6 d, shortly before bulk B cell proliferation started to decline. In contrast, transforming growth factor beta 1 mRNA, after a progressive increase during the first few days, and IL-10 mRNA, after a peak on days 1-3, remained detectable in immunoglobulin (Ig)-secreting cultures throughout the observation period of 22 d. Clonal analysis on 8-d cultures that had been seeded with single B cells by autocloning with the cell sorter, revealed that 85% of 77 B cell clones studied, expressed TGF-beta 1 mRNA, and only 19% IL-10 mRNA. These findings show a differentiation stage-related cytokine program during a B cell response, whereby (a) B cells can become activated without IL-1 alpha or IL-1 beta expression; (b) mRNA for positive (IL-10) and negative (TGF-beta 1) autoregulatory factors coexists in cell populations during the later phase of the response, although not necessarily in all B cell clones; and (c) normal Ig-secreting cells cease IL-6 expression in contrast to their malignant counterparts, myeloma cells.
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Bascom, C. C., J. R. Wolfshohl, R. J. Coffey, L. Madisen, N. R. Webb, A. R. Purchio, R. Derynck, and H. L. Moses. "Complex regulation of transforming growth factor beta 1, beta 2, and beta 3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors beta 1 and beta 2." Molecular and Cellular Biology 9, no. 12 (December 1989): 5508–15. http://dx.doi.org/10.1128/mcb.9.12.5508-5515.1989.
Повний текст джерелаАнотація:
Regulation of transforming growth factor beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 mRNAs in murine fibroblasts and keratinocytes by TGF beta 1 and TGF beta 2 was studied. In quiescent AKR-2B fibroblasts, in which TGF beta induces delayed stimulation of DNA synthesis, TGF beta 1 autoregulation of TGF beta 1 expression was observed as early as 1 h, with maximal induction (25-fold) after 6 to 12 h. Increased expression of TGF beta 1 mRNA was accompanied by increased TGF beta protein production into conditioned medium of AKR-2B cells. Neither TGF beta 2 nor TGF beta 3 mRNA, however, was significantly induced, but both were apparently down regulated at later times by TGF beta 1. Protein synthesis was not required for autoinduction of TGF beta 1 mRNA in AKR-2B cells. Nuclear run-on analyses and dactinomycin experiments indicated that autoregulation of TGF beta 1 expression is complex, involving both increased transcription and message stabilization. In contrast to TGF beta 1, TGF beta 2 treatment of quiescent AKR-2B cells increased expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs, but with different kinetics. Autoinduction of TGF beta 2 mRNA occurred rapidly with maximal induction at 1 to 3 h, enhanced TGF beta 3 mRNA levels were observed after 3 h, and increased expression of TGF beta 1 occurred later, with maximal mRNA levels obtained after 12 to 24 h. Nuclear run-on analyses indicated that TGF beta 2 regulation of TGF beta 2 and TGF beta 3 mRNA levels is transcriptional, while TGF beta 2 induction of TGF beta 1 expression most likely involves both transcriptional and posttranscriptional controls. In BALB/MK mouse keratinocytes, minimal autoinduction of TGF beta 1 occurred at only the 12- and 24-h time points and protein synthesis was required for this autoinduction. The results of this study provide an example in which TGF beta 1 and TGF beta 2 elicit different responses and demonstrate that expression of TGF beta 1, and TGF beta 3 are regulated differently. The physiological relevance of TGF beta 1 autoinduction in the context of wound healing is discussed.
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Bascom, C. C., J. R. Wolfshohl, R. J. Coffey, L. Madisen, N. R. Webb, A. R. Purchio, R. Derynck, and H. L. Moses. "Complex regulation of transforming growth factor beta 1, beta 2, and beta 3 mRNA expression in mouse fibroblasts and keratinocytes by transforming growth factors beta 1 and beta 2." Molecular and Cellular Biology 9, no. 12 (December 1989): 5508–15. http://dx.doi.org/10.1128/mcb.9.12.5508.
Повний текст джерелаАнотація:
Regulation of transforming growth factor beta 1 (TGF beta 1), TGF beta 2, and TGF beta 3 mRNAs in murine fibroblasts and keratinocytes by TGF beta 1 and TGF beta 2 was studied. In quiescent AKR-2B fibroblasts, in which TGF beta induces delayed stimulation of DNA synthesis, TGF beta 1 autoregulation of TGF beta 1 expression was observed as early as 1 h, with maximal induction (25-fold) after 6 to 12 h. Increased expression of TGF beta 1 mRNA was accompanied by increased TGF beta protein production into conditioned medium of AKR-2B cells. Neither TGF beta 2 nor TGF beta 3 mRNA, however, was significantly induced, but both were apparently down regulated at later times by TGF beta 1. Protein synthesis was not required for autoinduction of TGF beta 1 mRNA in AKR-2B cells. Nuclear run-on analyses and dactinomycin experiments indicated that autoregulation of TGF beta 1 expression is complex, involving both increased transcription and message stabilization. In contrast to TGF beta 1, TGF beta 2 treatment of quiescent AKR-2B cells increased expression of TGF beta 1, TGF beta 2, and TGF beta 3 mRNAs, but with different kinetics. Autoinduction of TGF beta 2 mRNA occurred rapidly with maximal induction at 1 to 3 h, enhanced TGF beta 3 mRNA levels were observed after 3 h, and increased expression of TGF beta 1 occurred later, with maximal mRNA levels obtained after 12 to 24 h. Nuclear run-on analyses indicated that TGF beta 2 regulation of TGF beta 2 and TGF beta 3 mRNA levels is transcriptional, while TGF beta 2 induction of TGF beta 1 expression most likely involves both transcriptional and posttranscriptional controls. In BALB/MK mouse keratinocytes, minimal autoinduction of TGF beta 1 occurred at only the 12- and 24-h time points and protein synthesis was required for this autoinduction. The results of this study provide an example in which TGF beta 1 and TGF beta 2 elicit different responses and demonstrate that expression of TGF beta 1, and TGF beta 3 are regulated differently. The physiological relevance of TGF beta 1 autoinduction in the context of wound healing is discussed.
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Teng, Michael N., and Peter L. Collins. "Altered Growth Characteristics of Recombinant Respiratory Syncytial Viruses Which Do Not Produce NS2 Protein." Journal of Virology 73, no. 1 (January 1, 1999): 466–73. http://dx.doi.org/10.1128/jvi.73.1.466-473.1999.
Повний текст джерелаАнотація:
ABSTRACT The second gene in the 3′-to-5′ gene order in respiratory syncytial virus (RSV) encodes the nonstructural protein NS2, for which there is no assigned function. To study the function of NS2, we have used a recently developed reverse genetics system to ablate expression of NS2 in recombinant RSV. A full-length cDNA copy of the antigenome of RSV A2 strain under the control of a T7 promoter was modified by introduction of tandem termination codons within the NS2 open reading frame (NS2stop) or by deletion of the entire NS2 gene (ΔNS2). The NS2 knockout antigenomic cDNAs were cotransfected with plasmids encoding the N, P, L, and M2-1 proteins of RSV, each controlled by the T7 promoter, into cells infected with a vaccinia virus recombinant expressing T7 RNA polymerase. Recombinant NS2stop and ΔNS2 RSVs were recovered and characterized. Both types of NS2 knockout virus displayed pinpoint plaque morphology and grew more slowly than wild-type RSV. The expression of monocistronic mRNAs for the five genes examined (NS1, NS2, N, F, and L) was unchanged in cells infected with either type of NS2 knockout virus, except that no NS2 mRNA was detected with the ΔNS2 virus. Synthesis of readthrough mRNAs was affected only for the ΔNS2 virus, where the NS1-NS2, NS2-N, and NS1-NS2-N mRNAs were replaced with the predicted novel NS1-N mRNA. Upon passage, the NS2stop virus stock rapidly developed revertants which expressed NS2 protein and grew with similar plaque morphology and kinetics wild-type RSV. Sequence analysis confirmed that the termination codons had reverted to sense, albeit not the wild-type assignments, and provided evidence consistent with biased hypermutation. No revertants were recovered from recombinant ΔNS2 RSV. These results show that the NS2 protein is not essential for RSV replication, although its presence greatly improves virus growth in cell culture. The attenuated phenotype of these mutant viruses, coupled with the expected genetic stability associated with gene deletions, suggests that the ΔNS2 RSV is a candidate for vaccine development.
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Silverstein, Peter S., Vicky L. van Santen, Kenneth E. Nusbaum, and R. Curtis Bird. "Expression Kinetics and Mapping of the Thymidine Kinase Transcript and an Immediate-Early Transcript from Channel Catfish Virus." Journal of Virology 72, no. 5 (May 1, 1998): 3900–3906. http://dx.doi.org/10.1128/jvi.72.5.3900-3906.1998.
Повний текст джерелаАнотація:
ABSTRACT Three transcripts from the terminal repeat of the channel catfish virus (CCV; also known as ictalurid herpesvirus 1) genome were mapped by S1 nuclease and primer extension analyses as well as by cDNA sequencing. These transcripts, TR3, TR5/6, andTR6, are encoded by open reading frame (ORF) 3, ORFs 5 and 6, and ORF 6, respectively, and correspond to those previously identified by sequence analysis (A. J. Davison, Virology 186:9–14, 1992). ORF 5 has previously been determined to encode thymidine kinase, but ORF 3 and ORF 6 encode proteins of unknown function. Although all three transcripts accumulate to high levels in cells infected in the presence of cycloheximide, kinetic analysis demonstrates that TR5/6 and TR6 are either early or late transcripts that leak through the cycloheximide block. In addition, two transcripts from the terminal repeat of the CCV genome that were mapped previously and were thought to be immediate-early in character, TR8a/9 and TR9, exhibit kinetics characteristic of early or late transcripts. TR3 is an immediate-early transcript that appears to have a very short half-life. In the 3′ untranslated region of TR3, there are three copies of an AU-rich element which has previously been shown to be involved in destabilization of the oncogene c-fos and granulocyte/macrophage colony-stimulating factor mRNAs. mRNA destabilization may represent another mechanism by which herpesviruses regulate the rapid switch in expression from immediate-early genes to early genes during the transition to the early phase of infection.
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Guenette, R. S., H. B. Corbeil, J. Léger, K. Wong, V. Mézl, M. Mooibroek, and M. Tenniswood. "Induction of gene expression during involution of the lactating mammary gland of the rat." Journal of Molecular Endocrinology 12, no. 1 (February 1994): 47–60. http://dx.doi.org/10.1677/jme.0.0120047.
Повний текст джерелаАнотація:
ABSTRACT After weaning, the mammary gland ceases lactation and involutes. The wet weight of the gland decreases by 70% within 4 days of weaning. This involves significant tissue remodelling as the ducts regress and return to the resting state. The presence of apoptotic bodies in the luminal epithelial compartment 2 to 3 days after weaning provides clear evidence that a substantial proportion of the regression is attributable to the induction of active cell death (ACD) of the epithelial cells. These changes in the architecture of the gland were found to be mirrored by changes in gene expression. The steady-state level of β-casein mRNA decreased rapidly after weaning from the high levels seen during lactation to undetectable levels by 8 days after weaning. The steady-state levels of expression of a number of genes associated with ACD, including TRPM-2, tissue transglutaminase (TGase) and poly(ADP-ribose) polymerase (PARP), increased transiently during this time-frame. The steady-state level of TRPM-2 mRNA increased 2 days after weaning, reaching a peak on day 4, and decreasing to undetectable levels by day 8 after weaning. The steady-state levels of two other mRNAs, TGase and PARP, showed very similar kinetics. In contrast, the mRNA for Hsp 27, which has been shown to be induced during prostate regression, was not significantly induced in the regressing mammary gland. In-situ hybridization demonstrated that the TRPM-2, TGase and PARP genes were expressed predominantly in the luminal epithelial cells of the ducts. These cells expressed β-casein mRNA during lactation, and underwent ACD after weaning. While the ultrastructural changes in the mammary gland after weaning, and the induction of TRPM-2, TGase and PARP mRNAs, are reminiscent of apoptosis in the prostate, several features of the process are different. Most notably, the disruption of the secretory processes and the lack of increased expression of Hsp 27 in the regressing mammary gland suggest that there may be a number of important events in ACD that are not common to all cells.
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Plumet, Sébastien, W. Paul Duprex, and Denis Gerlier. "Dynamics of Viral RNA Synthesis during Measles Virus Infection." Journal of Virology 79, no. 11 (June 1, 2005): 6900–6908. http://dx.doi.org/10.1128/jvi.79.11.6900-6908.2005.
Повний текст джерелаАнотація:
ABSTRACT We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until ∼24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is ∼3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5′-end abortive replication fragment RNAs.
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Epperson, L. Elaine, and Sandra L. Martin. "Quantitative assessment of ground squirrel mRNA levels in multiple stages of hibernation." Physiological Genomics 10, no. 2 (August 14, 2002): 93–102. http://dx.doi.org/10.1152/physiolgenomics.00004.2002.
Повний текст джерелаАнотація:
Hibernators in torpor dramatically reduce their metabolic, respiratory, and heart rates and core body temperature. These extreme physiological conditions are frequently and rapidly reversed during the winter hibernation season via endogenous mechanisms. This phenotype must derive from regulated expression of the hibernator’s genome; to identify its molecular components, a cDNA subtraction was used to enrich for seasonally upregulated mRNAs in liver of golden-mantled ground squirrels. The relative steady-state levels for seven mRNAs identified by this screen, plus five others, were measured and analyzed for seasonal and stage-specific differences using kinetic RT-PCR. Four mRNAs show seasonal upregulation in which all five winter stages differ significantly from and are higher than summer (α2-macroglobulin, apolipoprotein A1, cathepsin H, and thyroxine-binding globulin). One of these mRNAs, α2-macroglobulin, varies during the winter stages with significantly lower levels at late torpor. None of the 12 mRNAs increased during torpor. The implications for these newly recognized upregulated mRNAs for hibernation as well as more global issues of maintaining steady-state levels of mRNA during torpor are discussed.
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Cohen-Solal, K., JL Villeval, M. Titeux, S. Lok, W. Vainchenker, and F. Wendling. "Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis." Blood 88, no. 7 (October 1, 1996): 2578–84. http://dx.doi.org/10.1182/blood.v88.7.2578.bloodjournal8872578.
Повний текст джерелаАнотація:
Mpl ligand (thrombopoietin [TPO]) is the physiological regulator of platelet production. In mice, mRNA encoding the Mpl ligand (Mpl-L) is predominantly found by Northern blot analysis in the liver and kidney. To investigate the mode of regulation of the Mpl-L gene, we have developed several experimental models of severe thrombocytopenia differing in their kinetics and an opposite model of chronic thrombocytosis. Northern analysis performed at various times after induction of a thrombocytopenic state demonstrates that, whatever the number of circulating platelets, no change in Mpl-L mRNA level occurs in liver and kidney. By ribonuclease protection assays, we analyzed the ratios between mRNAs coding for the wild-type Mpl-L form and various splice variants encoding inactive or nonsecreted Mpl-L proteins. No modification in levels of these various isoforms was detected confirming the data of a previous report. Because the highest level of Mpl-L bioactivity in sera was observed only in mice with drastically reduced numbers of both platelets and megakaryocytes, these results further suggest that not only platelets, but also megakaryocytes, must be involved in the regulation of the level of circulating Mpl-L. In addition, we show that no downregulation of wild-type Mpl-L mRNA and no change in the ratio of Mpl-L mRNA isoforms were detected in mice in which a chronic thrombocytosis was induced. Together, these different models extend and further confirm that the regulation of Mpl-L does not occur at a transcriptional level or by a modulation in the ratios of Mpl-L mRNA isoforms.
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Nishiyama, Atsushi, Douglas N. Ishii, Peter H. Backx, Bruce E. Pulford, Barbara R. Birks, and Michael M. Tamkun. "Altered K+ channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4." American Journal of Physiology-Heart and Circulatory Physiology 281, no. 4 (October 1, 2001): H1800—H1807. http://dx.doi.org/10.1152/ajpheart.2001.281.4.h1800.
Повний текст джерелаАнотація:
Expression of voltage-gated K+ channels encoding the K+independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K+ current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in α-MHC mRNA and a 259% increase in β-MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K+ channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.
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Lasa, Marina, Sonya M. Abraham, Christine Boucheron, Jeremy Saklatvala, and Andrew R. Clark. "Dexamethasone Causes Sustained Expression of Mitogen-Activated Protein Kinase (MAPK) Phosphatase 1 and Phosphatase-Mediated Inhibition of MAPK p38." Molecular and Cellular Biology 22, no. 22 (November 15, 2002): 7802–11. http://dx.doi.org/10.1128/mcb.22.22.7802-7811.2002.
Повний текст джерелаАнотація:
ABSTRACT The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibition of p38 and the induction of MKP-1 by dexamethasone occurred with similar dose dependence and kinetics. No other known p38 phosphatases were induced by dexamethasone, and other cell types which failed to express MKP-1 also failed to inhibit p38 in response to dexamethasone. The proinflammatory cytokine interleukin 1 (IL-1) induced MKP-1 expression in a p38-dependent manner and acted synergistically with dexamethasone to induce MKP-1 expression. In HeLa cells treated with IL-1 or IL-1 and dexamethasone, the dynamics of p38 activation mirrored the expression of MKP-1. These observations suggest that MKP-1 participates in a negative-feedback loop which regulates p38 function and that dexamethasone may inhibit proinflammatory gene expression in part by inducing MKP-1 expression.
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Hallet, MM, V. Praloran, H. Vie, MA Peyrat, G. Wong, J. Witek-Giannotti, JP Soulillou, and JF Moreau. "Macrophage colony-stimulating factor (CSF-1) gene expression in human T- lymphocyte clones." Blood 77, no. 4 (February 15, 1991): 780–86. http://dx.doi.org/10.1182/blood.v77.4.780.780.
Повний текст джерелаАнотація:
Abstract Macrophage colony stimulating factor (CSF-1) is one of several cytokines that control the differentiation, survival, and proliferation of monocytes and macrophages. A set of 11 human T-cell clones, chosen for their phenotypic diversity, were tested for their ability to express CSF-1 mRNA. After 5 hours of stimulation with phorbol myristate acetate (PMA) + calcium ionophore (Cal), all T-cell clones expressed a major 4-kb transcript, a less abundant 2-kb transcript, and several other minor species. This pattern of expression is typical for CSF-1 mRNAs. Furthermore, of the two alloreactive T-cell clones analyzed, only one showed a definitive message for CSF-1 on specific antigenic stimulation, but with delayed kinetics and less efficiency. Both conditions of stimulation induced the release of CSF-1 protein by T cells in the culture medium. Together, these findings demonstrate for the first time that normal T cells are able to produce CSF-1, previous reports being limited to two cases of tumoral cells of the T-cell lineage.
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Hallet, MM, V. Praloran, H. Vie, MA Peyrat, G. Wong, J. Witek-Giannotti, JP Soulillou, and JF Moreau. "Macrophage colony-stimulating factor (CSF-1) gene expression in human T- lymphocyte clones." Blood 77, no. 4 (February 15, 1991): 780–86. http://dx.doi.org/10.1182/blood.v77.4.780.bloodjournal774780.
Повний текст джерелаАнотація:
Macrophage colony stimulating factor (CSF-1) is one of several cytokines that control the differentiation, survival, and proliferation of monocytes and macrophages. A set of 11 human T-cell clones, chosen for their phenotypic diversity, were tested for their ability to express CSF-1 mRNA. After 5 hours of stimulation with phorbol myristate acetate (PMA) + calcium ionophore (Cal), all T-cell clones expressed a major 4-kb transcript, a less abundant 2-kb transcript, and several other minor species. This pattern of expression is typical for CSF-1 mRNAs. Furthermore, of the two alloreactive T-cell clones analyzed, only one showed a definitive message for CSF-1 on specific antigenic stimulation, but with delayed kinetics and less efficiency. Both conditions of stimulation induced the release of CSF-1 protein by T cells in the culture medium. Together, these findings demonstrate for the first time that normal T cells are able to produce CSF-1, previous reports being limited to two cases of tumoral cells of the T-cell lineage.