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Fibroblast growth factor-2 (FGF-2) plays a fundamental role in brain functions. This role may be partly achieved through the control of its expression at the translational level via an internal ribosome entry site (IRES)-dependent mechanism. Transgenic mice expressing a bicistronic mRNA allowed us to study in vivo and ex vivo where this translational mechanism operates. Along brain development, we identified a stringent spatiotemporal regulation of FGF-2 IRES activity showing a peak at post-natal day 7 in most brain regions, which is concomitant with neuronal maturation. At adult age, this activity remained relatively high in forebrain regions. By the enrichment of this activity in forebrain synaptoneurosomes and by the use of primary cultures of cortical neurons or cocultures with astrocytes, we showed that this activity is indeed localized in neurons, is dependent on their maturation, and correlates with endogenous FGF-2 protein expression. In addition, this activity was regulated by astrocyte factors, including FGF-2, and spontaneous electrical activity. Thus, neuronal IRES-driven translation of the FGF-2 mRNA may be involved in synapse formation and maturation.  相似文献   

3.
Fibroblast growth factor 1 (FGF-1) is a powerful angiogenic factor whose gene structure contains four promoters, giving rise to a process of alternative splicing resulting in four mRNAs with alternative 5' untranslated regions (5' UTRs). Here we have identified, by using double luciferase bicistronic vectors, the presence of internal ribosome entry sites (IRESs) in the human FGF-1 5' UTRs, particularly in leaders A and C, with distinct activities in mammalian cells. DNA electrotransfer in mouse muscle revealed that the IRES present in the FGF-1 leader A has a high activity in vivo. We have developed a new regulatable TET OFF bicistronic system, which allowed us to rule out the possibility of any cryptic promoter in the FGF-1 leaders. FGF-1 IRESs A and C, which were mapped in fragments of 118 and 103 nucleotides, respectively, are flexible in regard to the position of the initiation codon, making them interesting from a biotechnological point of view. Furthermore, we show that FGF-1 IRESs A of murine and human origins show similar IRES activity profiles. Enzymatic and chemical probing of the FGF-1 IRES A RNA revealed a structural domain conserved among mammals at both the nucleotide sequence and RNA structure levels. The functional role of this structural motif has been demonstrated by point mutagenesis, including compensatory mutations. These data favor an important role of IRESs in the control of FGF-1 expression and provide a new IRES structural motif that could help IRES prediction in 5' UTR databases.  相似文献   

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Manipulation of angiogenesis in vivo is an example of successful gene therapy strategies. Overexpression of angiogenic genes like VEGF, FGF or PDGF causes new vessel formation and improves the clinical state of patients. Gene therapy is a very promising procedure but requires large amounts of pharmaceutical-grade plasmid DNA. In this regard we have constructed a bicistronic plasmid DNA vector encoding two proangiogenic factors, VEGF165 and FGF-2. The construct (pVIF) contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) which permits both genes to be translated from a single bicistronic mRNA. The IRES sequence allows for a high efficiency of gene expression in vivo. The pVIF vector was characterized in vitro and in vivo. In vivo angiogenesis studies showed that the bicistronic vector encoding two proangiogenic factors induces the formation of new vessels significantly more than pVEGF165 or pFGF-2 alone. In our opinion the combined proangiogenic approach with VEGF165 and FGF-2 is more powerful and efficient than single gene therapy. We also postulate that IRES sequence can serve as a useful device improving efficiency of gene therapy.  相似文献   

6.
将HCVIRES插入双报告基因海肾荧光素酶 (Rluc)基因和萤火虫荧光素酶 (Fluc)基因之间 ,建立了“依赖帽子的扫描机制”翻译表达Rluc ,HCVIRES调控Fluc翻译的双顺反子表达载体pCI Rluc HCVIRES Fluc ,通过酶切反应及转染HepG2细胞鉴定双荧光素酶瞬间表达活性等试验 ,证实获得了表达双荧光素酶的双顺反子载体 .并应用水压转染法将双顺反子表达质粒导入小鼠体内 ,在小鼠肝脏检测到高水平表达的Rluc和Fluc .该研究成功构建一种HCVIRES介导萤火虫荧光素酶基因表达的双顺反子载体 ,并在HepG2细胞及小鼠体内进行了瞬时表达 ,为进一步建立稳定评价靶向HCVIRES药物作用的细胞及小动物模型研究奠定了基础  相似文献   

7.
Bochkov YA  Palmenberg AC 《BioTechniques》2006,41(3):283-4, 286, 288 passim
The internal ribosomal entry site (IRES)from encephalomyocarditis virus (EMCV) is a popular RNA element used widely in experimental and pharmaceutical applications to express proteins in eukaryotic cells or cell-free extracts. Inclusion of the wild-type element in monocistronic or bicistronic messenger RNAs (mRNAs) confers a high level of cap-independent translation activity to appropriately configured cistrons. The history of this element and the experimental consequences of sequence derivations inherent to commercial IRES vectors are less well known. Compared head-to-head with dual-luciferase reporter constructs, a native EMCV IRES in a bicistronic configuration directed 8- to 10-fold more protein than a similarly configured pIRES vector. It also produced nearly twice as much protein as pCITE-1, an early monocistronic iteration, harboring a suboptimal A7 sequence in a crucial structural motif The results indicate that investigators should be aware of and carefully report the sequence of their IRES in any comparative study. The preferred IRES (viral bases 273-845) and the minimum IRES (viral bases 400-836) for optimum activity are illustrated.  相似文献   

8.
Previous studies have described poliovirus genomes in which the internal ribosome entry (IRES) for encephalomyocarditis virus (EMCV) is positioned between the P1 and P2-P3 open reading frames of the poliovirus genome. Although these dicistronic poliovirus genomes were replication competent, most exhibited evidence of genetic instability, and the EMCV IRES was deleted upon serial passage. One possible reason for instability of the genome is that the dicistronic genome was at least 108% larger than the wild-type poliovirus genome, which could reduce the efficiency of encapsidation. To address this possibility, we have constructed dicistronic poliovirus replicons by substituting the EMCV IRES and the gene encoding luciferase in place of the poliovirus P1 region; the resulting dicistronic replicons are smaller than the wild-type poliovirus genome. One dicistronic genome was constructed in which the poliovirus 5' nontranslated region was fused to the gene encoding luciferase, followed by the complete EMCV IRES fused to the P2-P3 region of the poliovirus genome (PV-Luc-EMCV). A second dicistronic genome, EMCV-Luc-PV, was constructed with the first 108 nucleotides of the poliovirus genome fused to the EMCV IRES, followed by the gene encoding luciferase and the poliovirus IRES fused to the remaining P2-P3 region of the poliovirus genome. Both dicistronic replicons expressed abundant luciferase following transfection of in vitro-transcribed RNA into HeLa cells at 30, 33, or 37 degrees C. The luciferase activity detected from PV-Luc-EMCV increased rapidly during the first 4 h following transfection and then plateaued, peaking after approximately 24 h. In contrast, the luciferase activity detected from EMCV-Luc-PV increased for approximately 12 h following transfection; by 24 h posttransfection, the overall levels of luciferase activity were similar to that of PV-Luc-EMCV. To analyze encapsidation of the dicistronic replicons, we used a system in which the capsid protein (P1) is provided in trans from a recombinant vaccinia virus (VV-P1). The PV-Luc-EMCV replicon was unstable upon serial passage in the presence of VV-P1, with deletions of the EMCV IRES region detected even during the initial transfection at 37 degrees C. Following serial passage in the presence of VV-P1 at 33 or 30 degrees C, we detected deleted genomes in which the luciferase gene was fused with the P2-P3 genes of the poliovirus genome so as to maintain the translational reading frame. In contrast, the EMCV-Luc-PV replicon was genetically stable during passage with VV-P1 at 33 or 30 degrees C. The encapsidation of EMCV-Luc-PV was compared to that of monocistronic replicons encoding luciferase with either a poliovirus or EMCV IRES. Analysis of the encapsidated replicons after four serial passages with VV-P1 revealed that the dicistronic replicon was encapsidated more efficiently than the monocistronic replicon with the EMCV IRES but less efficiently than the monicistronic replicon with the poliovirus IRES. The results of this study suggest a genetic predisposition for picornavirus genomes to contain a single IRES region and are discussed with respect to a role of the IRES in encapsidation.  相似文献   

9.
利用脑炎心肌炎病毒的内核糖体进入位点连接人TNF-αcDNA和选择基因NeoR基因,使TNF-α及NeoR基因均受控于病毒LTR启动子,将两基因同时转录至同一mRNA,从而构建成人TNF-α双顺反子逆转录病毒载体pGCEN/TNF-α.在LipofectAMINE介导下将其导入包装细胞PA317,G418筛选得单克隆,病毒滴度为106CFU/ml重组病毒分泌的细胞株.经PCR证明外源基因已整合至细胞基因组,Northern印迹显示出单一LRT转录本.持续G418筛选能明显促进目的基因TNF-α的表达.用重组病毒上清感染小鼠成纤维细胞NIH3T3,G418筛选获得的混合抗性克隆持续高表达TNF-α,40Gyγ线照射后能维持高效表达至7d.实验结果表明,含IRES的双顺反子逆转录病毒载体将是一个很好的基因转移载体.  相似文献   

10.
Mouse cells are not permissive for the replication of human rhinovirus type 2 (HRV2). To determine the role of the HRV2 internal ribosome entry site (IRES) in determining species specificity, a recombinant poliovirus (P1/HRV2) was constructed by substituting the poliovirus IRES with the IRES from HRV2. This recombinant virus replicated in all human and murine cell lines examined, demonstrating that the HRV2 IRES does not limit viral replication in transformed murine cells. P1/HRV2 replicated in the brain and spinal cord in neonatal but not adult mice transgenic for the poliovirus receptor, CD155. Passage of P1/HRV2 in mice led to selection of a virus that caused paralysis in neonatal mice. To determine the relationship between HRV2 IRES-mediated translation and replication of P1/HRV2 in mice, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The results demonstrate that the HRV2 IRES mediates translation in organs of neonatal but not adult mice. These findings show that HRV2 IRES-mediated translation is a determinant of virus replication in the murine brain and spinal cord and suggest that the IRES determines the species specificity of HRV2 infection.  相似文献   

11.
A Pacheco  JL Twiss 《PloS one》2012,7(7):e40788
Transport of neuronal mRNAs into distal nerve terminals and growth cones allows axonal processes to generate proteins autonomous from the cell body. While the mechanisms for targeting mRNAs for transport into axons has received much attention, how specificity is provided to the localized translational apparatus remains largely unknown. In other cellular systems, protein synthesis can be regulated by both cap-dependent and cap-independent mechanisms. The possibility that these mechanisms are used by axons has not been tested. Here, we have used expression constructs encoding axonally targeted bicistronic reporter mRNAs to determine if sensory axons can translate mRNAs through cap-independent mechanisms. Our data show that the well-defined IRES element of encephalomyocarditis virus (EMCV) can drive internal translational initiation of a bicistronic reporter mRNA in distal DRG axons. To test the potential for cap-independent translation of cellular mRNAs, we asked if calreticulin or grp78/BiP mRNA 5'UTRs might have IRES activity in axons. Only grp78/BiP mRNA 5'UTR showed clear IRES activity in axons when placed between the open reading frames of diffusion limited fluorescent reporters. Indeed, calreticulin's 5'UTR provided an excellent control for potential read through by ribosomes, since there was no evidence of internal initiation when this UTR was placed between reporter ORFs in a bicistronic mRNA. This study shows that axons have the capacity to translate through internal ribosome entry sites, but a simple binary choice between cap-dependent and cap-independent translation cannot explain the specificity for translation of individual mRNAs in distal axons.  相似文献   

12.
We established a bicistronic expression system using an encephalomyocarditis virus (EMCV)-derived internal ribosomal entry site (IRES) element to generate stably transformed Drosophila melanogaster Schneider 2 (S2) cells expressing human rotavirus Wa capsid proteins, VP2 and VP6, for the synthesis of VP2/6 double-layered virus-like particle (DVLP). The EMCV-derived IRES permitted bicistronic translation of recombinant VP6. Recombinant VP2 and VP6 were detected in extracellular fractions of stably transformed S2 cells. A wheel-like DVLP (diam ~ 50–55 nm) with short spikes was produced from the extracellular fraction of stably transformed S2 cells. A bicistronic expression system using an EMCV-derived IRES element can thus be used to express two proteins of interest in stably transformed S2 cells. The bi-or tri-cistronic expression of recombinant VP2/6/7 using stably transformed S2 cells can also be used to produce rotavirus VLPs.  相似文献   

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14.
Flaviviruses have a positive-stranded RNA genome, which simultaneously serves as an mRNA for translation of the viral proteins. All of the structural and nonstructural proteins are translated from a cap-dependent cistron as a single polyprotein precursor. In an earlier study (K. K. Orlinger, V. M. Hoenninger, R. M. Kofler, and C. W. Mandl, J. Virol. 80:12197-12208, 2006), it was demonstrated that an artificial bicistronic flavivirus genome, TBEV-bc, in which the region coding for the viral surface glycoproteins prM and E from tick-borne encephalitis virus (TBEV) had been removed from its natural context and inserted into the 3' noncoding region under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) produces viable, infectious virus when cells are transfected with this RNA. The rates of RNA replication and infectious particle formation were significantly lower with TBEV-bc, however, than with wild-type TBEV. In this study, we have identified two types of mutations, selected by passage in BHK-21 cells, that enhance the growth properties of TBEV-bc. The first type occurred in the E protein, and these most likely increase the affinity of the virus for heparan sulfate on the cell surface. The second type occurred in the inserted EMCV IRES, in the oligo(A) loop of the J-K stem-loop structure, a binding site for the eukaryotic translation initiation factor 4G. These included single-nucleotide substitutions as well as insertions of additional adenines in this loop. An A-to-C substitution in the oligo(A) loop decreased the efficiency of the IRES itself but nevertheless resulted in improved rates of virus particle formation and overall replication efficiency. These results demonstrate the need for proper balance in the competition for free template RNA between the viral RNA replication machinery and the cellular translation machinery at the two different start sites and also identify specific target sites for the improvement of bicistronic flavivirus expression vectors.  相似文献   

15.
Cap-independent translation of encephalomyocarditis virus (EMCV) RNA is controlled by a segment of the 5' untranslated region termed the internal ribosomal entry site, or IRES. The IRES contains a series of stem-loop structural elements. The J and K stems (EMCV bases 682 to 795), near the center of the IRES, are well conserved among all cardio-, aphtho-, and hepatoviruses. We have examined the biological roles of these elements by constructing mutations within the J-K sequences of EMCV and testing the mutations for activity in translation, translation competition, UV cross-linking, and viral infectivity assays. Mutations near the helical junction of J and K proved severely detrimental to both cellular translation and cell-free translation of downstream cistrons. The same mutations reduced the ability of the IRES to compete for cellular factors in competition assays and reduced the infectivity of viral genomes carrying these lesions. A mutation in the terminal loop of J gave similar results. In contrast, mutations within the terminal loop of K had minimal impact on in vitro translation activity and IRES competitive ability. However, in vivo analysis of the K-loop mutations revealed deficiencies during cellular translation and further showed markedly reduced infectivity in HeLa cells. UV cross-linking experiments identified a 49-kDa protein which interacts strongly with the J-K region, but the identity of this protein and its contribution to IRES activity are unclear.  相似文献   

16.
Internal ribosome entry sites (IRESs) allow ribosomal access to messenger RNA without a requirement for cap recognition and subsequent scanning to an initiator AUG. Hence, IRESs have been adapted into dicistronic vectors for the expression of more than one gene from a single mRNA. Dicistronic vectors have been used for many applications in mammalian tissue culture and transgenesis. However, whether the IRESs from mammalian viruses function without temporal or spatial restrictions in nonmammalian organisms like zebra fish (Danio rerio) is unknown. Therefore, we have examined the expression capabilities of the encephalomyocarditis virus (EMCV) IRES during zebrafish embryogenesis. We determined that the EMCV IRES was sufficient to permit detectable expression of several second cistron reporters during zebrafish embryogenesis, including luciferase and green fluorescent protein. This suggests that our dicistronic vectors are suitable for general use in any vertebrate system, from fish to humans. Received March 26, 1999; accepted June 14, 1999.  相似文献   

17.
The IRES from poliovirus and from encephalomyocarditis virus (EMCV) added between the cap and the AUG initiator codon were strong inhibitors of chloramphenicol acetyltransferase gene expression in three different cell types. The poliovirus IRES also inhibited bGH (bovine growth hormone) cDNA expression in the HC11 mammary cell line when added between the rabbit whey acidic gene promoter and the cDNA whereas the HTLV-1 IRES showed a stimulatory effect in the same situation. RNA stem loops were added before HTLV-1 (SUR) and the BiP (Immunoglobulin heavy-chain Binding Protein) IRESs followed by the firefly luciferase gene under the control of Rous sarcoma virus (RSV) promoter. The RNA loops abolished the expression of the reporter gene almost completely. These data suggest that the different IRESs may favour or inhibit translation of monocistronic mRNA.  相似文献   

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Internal ribosome entry site (IRES)-mediated translation is an essential replication step for certain viruses. As IRES-mediated translation is regulated differently from cap-dependent translation under various cellular conditions, we sought to investigate whether temperature influences efficiency of viral IRES-mediated translation initiation by using bicistronic reporter constructs containing an IRES element of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), human rhinovirus (HRV) or poliovirus (PV). Under mild hypothermic conditions (30 and 35°C), we observed increases in the efficiency of translation initiation by HCV and HRV IRES elements compared to translation initiation at 37°C. The promotion of HRV IRES activity was observed as early as 2 hours after exposure to mild hypothermia. We also confirmed the promotion of translation initiation by HRV IRES under mild hypothermia in multiple cell lines. The expression levels and locations of polypyrimidine tract-binding protein (PTB) and upstream of N-Ras (unr), the IRES trans-acting factors (ITAFs) of HCV and HRV IRES elements, were not modulated by the temperature shift from 37°C to 30°C. Taken together, this study demonstrates that efficiency of translation initiation by some viral IRES elements is temperature dependent.  相似文献   

20.
Translation initiation on poliovirus and encephalomyocarditis virus (EMCV) mRNAs occurs by a cap-independent mechanism utilizing an internal ribosomal entry site (IRES). However, no unifying mechanism for AUG initiation site selection has been proposed. Analysis of initiation of mRNAs translated in vitro has suggested that initiation of poliovirus mRNA translation likely involves both internal binding of ribosomes and scanning to the first AUG which is in a favorable context for initiation. In contrast, internal initiation on EMCV mRNA may not utilize scanning, since ribosomes bind directly or very close to the initiation codon AUG-11. We have studied in vivo the sequence requirements for internal initiation around the EMCV initiation codon, both in monocistronic and in dicistronic mRNAs. Our studies show that the upstream AUG-10 is normally not used and that there is no specific sequence requirement for nucleotides between AUG-10 and AUG-11. However, the sequence context of AUG-11 does influence the efficiency of initiation at AUG-11. Efficient IRES-mediated internal initiation at AUG-11 exhibits a requirement for an adenine in the -3 position, similar to cap-dependent initiation. These results support a model for internal initiation on EMCV mRNA in which scanning starts at or near AUG-11. Although initiation primarily occurs at AUG-11, initiation at multiple downstream AUG codons can be detected. In addition, a poor sequence context around AUG-11 results in increased initiation at one or more downstream AUG codons, indicative of leaky scanning or jumping by the ribosome from AUG-11 mediated by the EMCV IRES.  相似文献   

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