A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay.

Akusjärvi, Göran; Svensson, Christina; Nygård, O

doi:10.1128/mcb.7.1.549

Cited by 76 publications

(54 citation statements)

References 14 publications

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“…Monolayers of 293 cells were seeded (3 × 105 cells per 60 mm dish) in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. After incubation for 24 h, the cells were transfected with 3 gg of either an expression vector coding for the wild-type or the mutant enzyme, or the control vector pCMV-7 plus 0.3 lag of pVA-1 (a plasmid encoding adenovirus-associated RNA1) [13] using LipofectAMINE (Gibco BRL). After 36 h, cells washed once with ice-cold phosphate-buffered saline (PBS) were harvested with PBS and stored at -80°C [14].…”

Section: Expression Vector and Mutagenesismentioning

confidence: 99%

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

et al. 1996

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The possible active site Cys 441 in the Cys-pocket and Glu 347 and Arg ~s° of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441Ala, Cys441Ser, Cys441His, Glu347Ala and Arg3S°Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1Jmin per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys 441 in the Cyspocket, and Glu 347 and Arg 35° of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

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Section: Expression Vector and Mutagenesismentioning

confidence: 99%

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

et al. 1996

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“…This reflects the ability of EBER RNA to bind and inhibit the double-stranded RNAactivated protein kinase (PKR), a key mediator of the interferon response (Nanbo et al, 2002). PKR phosphorylates translation initiation factor eIF2-a in order to inhibit protein synthesis (Akusjarvi et al, 1987;Davies et al, 1989;Thimmappaya et al, 1982). This can be prevented by EBERs, which bind PKR tightly and block its function (Clarke et al, 1991;Elia et al, 1996;Nanbo et al, 2002;Sharp et al, 1993).…”

Section: Transformation By Pol III Transcripts?mentioning

confidence: 99%

RNA polymerase III transcription and cancer

2004

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RNA polymerase (pol) III synthesizes a range of essential products, including tRNA, 5S rRNA and 7SL RNA, which are required for protein synthesis and trafficking. High rates of pol III transcription are necessary for cells to sustain growth. A wide range of transformed and tumour cell types have been shown to express elevated levels of pol III products. This review will summarize what is known about the mechanisms responsible for this deregulation. Some transforming agents have been shown to stimulate expression of the pol III-specific transcription factors TFIIIB or TFIIIC2. In addition, TFIIIB is bound and activated by several oncogenic proteins, including cMyc. Conversely, TFIIIB interacts in healthy cells with the tumour suppressors RB and p53. Indeed, the ability to limit pol III transcription through TFIIIB may contribute to their growth-suppression capacities. The function of p53 and/or RB is compromised in most if not all transformed cells; the resultant derepression of TFIIIB may provide an almost universal route to deregulate pol III transcription in cancers. In addition to effects on protein synthesis and growth, there is a precedent for a pol III product having oncogenic activity.

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“…On days 1 and 2, the cells were transfected with expression plasmids using 6 l of FuGENE 6 according to the manufacturer's instructions. Transfection studies with KAR contained 0.6 g of pCMV-HA-LCE, 0.6 g of pCMV-HA-KAR, 0.8 g of pVAI (15), and 0.02 g of pCMV ␤-galactosidase plasmids/dish. Transfection studies with TER contained 1.0 g of pCMV-HA-LCE, 0.2 g of pCMV-HA-TER, 0.8 g of pVAI (15), and 0.02 g of pCMV ␤-galactosidase plasmids/dish.…”

Section: Materials-[2-mentioning

confidence: 99%

“…Transfection studies with KAR contained 0.6 g of pCMV-HA-LCE, 0.6 g of pCMV-HA-KAR, 0.8 g of pVAI (15), and 0.02 g of pCMV ␤-galactosidase plasmids/dish. Transfection studies with TER contained 1.0 g of pCMV-HA-LCE, 0.2 g of pCMV-HA-TER, 0.8 g of pVAI (15), and 0.02 g of pCMV ␤-galactosidase plasmids/dish. The total amount of plasmid DNA in each transfection was adjusted to 2 g/dish by adding pCMV-Script.…”

Section: Materials-[2-mentioning

confidence: 99%

Identification of Two Mammalian Reductases Involved in the Two-carbon Fatty Acyl Elongation Cascade

Moon¹,

Horton²

2003

Journal of Biological Chemistry

188

150

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The de novo synthesis of fatty acids occurs in two distinct cellular compartments. Palmitate (16:0) is synthesized from acetyl-CoA and malonyl-CoA in the cytoplasm by the enzymes acetyl-CoA carboxylase 1 and fatty acid synthase. The synthesis of fatty acids longer than 16 carbons takes place in microsomes and utilizes malonyl-CoA as the carbon source. Each two-carbon addition requires four sequential reactions: condensation, reduction, dehydration, and a final reduction to form the elongated fatty acyl-CoA. The initial condensation reaction is the regulated and rate-controlling step in microsomal fatty acyl elongation. We previously reported the cDNA cloning and characterization of a murine long chain fatty acyl elongase (LCE) (1). Overexpression of LCE in cells resulted in the enhanced addition of two-carbon units to C12-C16 fatty acids, and evidence was provided that LCE catalyzed the initial condensation reaction of long chain fatty acid elongation. The remaining three enzymes in the elongation reaction have not been identified in mammals. Here, we report the identification and characterization of two mammalian enzymes that catalyze the 3-ketoacyl-CoA and trans-2,3-enoyl-CoA reduction reactions in long and very long chain fatty acid elongation, respectively.

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A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay.

Cited by 76 publications

References 14 publications

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

RNA polymerase III transcription and cancer

Identification of Two Mammalian Reductases Involved in the Two-carbon Fatty Acyl Elongation Cascade

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A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay.

Cited by 76 publications

References 14 publications

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys‐pocket, and Glu347 and Arg350 of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys‐pocket, and Glu347 and Arg350 of the EXXR motif

RNA polymerase III transcription and cancer

Identification of Two Mammalian Reductases Involved in the Two-carbon Fatty Acyl Elongation Cascade

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Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif

Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys⁴⁴¹ of the Cys‐pocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif