Synthesis of functional mRNA in mammalian cells by bacteriophage T3 RNA polymerase.

Zhou, Y; Giordano, Thomas J.; Durbin, Russell K.; McAllister, William T.

doi:10.1128/mcb.10.9.4529

Cited by 26 publications

(9 citation statements)

References 16 publications

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“…Transcripts synthesized by these polymerases are not capped and may contain structural elements (hairpins) inhibitory to capping or scanning, but inclusion of an IRES element downstream of the promoter yields RNA that is translated efficiently in variety of cell types by avoiding the limitations of 5' end-dependent initiation. The first application of the EMCV IRES to enhance expression at the level of translation was reported by ELROy-STEIN et al (1989), who incorporated the element into the vaccinia virus expression system, and several related publications followed (DENG et al 1991;ELROy-STEIN et al (1989;ELROy-STEIN and Moss 1990;MiROCHNITCHENKO et al 1994;VENNEMA et al 1991;ZHOU et al 1990). The EMCV IRES was identified by translation of bicistronic mRNAs (JANG et al 1988, and the same principle has been exploited in constructing bicistronic vectors for high level expression of foreign genes (ADAM et al 1991;GHATIAS et al 1991;MORGAN et al 1992;KAUFMAN et al 1991).…”

Section: Applications Of the Encephalomyocarditis Virus Internal Ribomentioning

confidence: 95%

Translation of Encephalomyocarditis Virus RNA by Internal Ribosomal Entry

Hellen

Wimmer

1995

Current Topics in Microbiology and Immunology

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Section: Applications Of the Encephalomyocarditis Virus Internal Ribomentioning

confidence: 95%

Translation of Encephalomyocarditis Virus RNA by Internal Ribosomal Entry

Hellen

Wimmer

1995

Current Topics in Microbiology and Immunology

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“…The FBP cDNA in this pUC 18 derivative had been previously modified to add the Kozak consensus sequence, which provided the Nco I restriction site into which the processed PCR-generated fragment was cloned. This complete cDNA coding region was then excised at flanking Sma I and Tha I sites and cloned between the Stu I and Sma I sites of pYZ28 (17,18). The resulting plasmid, pCAR 17, contains the FBP cDNA sequence between an SV40 early promoter and polyadenylation signals (Fig.…”

Section: Methodsmentioning

confidence: 99%

Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.

Luhrs¹,

Raskin²,

Durbin³

et al. 1992

J. Clin. Invest.

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KB cells express a folate-binding protein that is anchored to the plasma membrane by a glycosylated phosphatidylinositol (GPI) tail and these cells can grow in medium containing a very low folate concentration (1 nM). In contrast, mouse 3T3 cells do not express a membrane-associated folate-binding protein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-binding protein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-binding protein similar to KB cells, 1 and 1.4 ng/,ug protein, respectively. The capacity for binding 13HIfolate to the surface of transfected 3T3 cells cultured in folatedeficient medium is 7.7 pmol/ 106 cells, and this is -50% of the surface binding capacity of KB cells under similar culture conditions. Moreover, after treatment of the transfected 3T3 cells with phospholipase C specific for phosphatidylinositol, the binding of 13HI folate to the surface of these cells is reduced by 90%, indicating that, like the KB cells, the folate-binding protein is anchored to the plasma membrane by a GPI tail. Although the doubling time of wild-type 3T3 cells markedly increases after 13 d of culture in folate-deficient medium, the doubling time of both the transfected 3T3 cells and KB cells do not change. The results of these experiments indicate that the GPI-anchored folate-binding protein provides a mechanism to maintain a level of folate that permits the folate-dependent metabolic functions necessary for cell survival under low folate conditions. (J. Clin. Invest. 1992. 90:840-847.)

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“…Although the liter ature dealing with this subject is as yet sparse, Elroy-Stein et al [53] have used the EMCV 5'NTR under the control of the phage T7 promoter for high expression of heterologous proteins in their vaccinia virus system, which provides phage T7 RNA poly merase in trans. Similarly, plasmids contain ing the EMCV 5'NTR under the control of phage T3 promoter directed good expression of reporter genes in cells supplying T3 RNA polymerase [54], The 1RES of EMCV is a suitable leader for the development of a eukaryotic pro moter selection cassette by combining the EMCV 1RES with a selectable marker gene. As long as insertion occurred in exons, the selectable marker gene will be expressed re gardless of position or of reading frame within the target gene.…”

Section: Application Of 1res Elements In Molecular Biologymentioning

confidence: 99%

Cap-Independent Translation of Picornavirus RN As: Structure and Function of the Internal Ribosomal Entry Site

Jang

Pestova

Hellen

et al. 1990

Enzyme

191

126

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Picornaviruses are mammalian plus-strand RNA viruses whose genomes serve as mRNA. A study of the structure and function of these viral mRNAs has revealed differences among them in events leading to the initiation of protein synthesis. A large segment of the 5' nontranslated region, approximately 400 nucleotides in length, promotes ‘internal’ entry of ribosomes independent of the non-capped 5' end of the mRNA. This segment, which we have called the internal ribosome entry site (IRES), maps approximately 200 nt downstream from the 5' end and is highly structured. IRES elements of different picomaviruses, although functionally similar in vitro and in vivo, are not identical in sequence or structure. However, IRES elements of the genera entero- and rhinoviruses, on the one hand, and cardio- and aphthoviruses, on the other hand, reveal similarities corresponding to phylogenetic kinship. All IRES elements contain a conserved Yn-Xm-AUG unit (Y, pyrimidine; X, nucleotide) which appears essential for IRES function. The IRES elements of cardio-, enteroand aphthoviruses bind a cellular protein, p57. In the case of cardioviruses, the interaction between a specific stem-loop of the IRES is essential for translation in vitro. The IRES elements of entero- and cardioviruses also bind the cellular protein, p52, but the significance of this interaction remains to be shown. The function of p57 or p52 in cellular metabolism is unknown. Since picornaviral IRES elements function in vivo in the absence of any viral gene products, we speculate that IRES-like elements may also occur in specific cellular mRNAs releasing them from cap-dependent translation. 1RES elements are useful tools in the construction of high yield expression vectors, or for tagging cellular genetic elements.

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Synthesis of functional mRNA in mammalian cells by bacteriophage T3 RNA polymerase.

Cited by 26 publications

References 16 publications

Translation of Encephalomyocarditis Virus RNA by Internal Ribosomal Entry

Translation of Encephalomyocarditis Virus RNA by Internal Ribosomal Entry

Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium.

Cap-Independent Translation of Picornavirus RN As: Structure and Function of the Internal Ribosomal Entry Site

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