In vitro Transcription and Translation

The cDNA encoding the fusion protein is cloned into a plasmid downstream of a SP6, T7 or T3 RNA polymerase promoter. The plasmid is linearized downstream of the inserts by digesting with an appropriate restriction enzyme, and transcribed in vitro with the appropriate RNA polymerase (75). 

The mRNA is then precipitated with 2.5 volumes ethanol and dissolved in 10 pi H2O containing 10 mM dithiothreitol and 0.1 U/ pi RNasin 

The transcripts is translated in a rabbit reticulocyte lysate system (Promega, Wl, USA), as follows  

1 pi 1 mM amino acide mixture (19 amino acids without methionine) 

To obtain unlabeled fusion protein, the incubation is carried out in the presence of 25 pM unlabelled methionine instead of pS]methionine to obtain maximal yield of fusion protein. 

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Gambacorti-Passerini C., Mologni L, Bertazzoli C., le Coutre P., Marchesi E., Grignani F., Nielsen P.E. In vitro transcription and translation inhibition by anti-promyelocytic leukemia (PML)/re-tinoic acid receptor-alpha and anti-PML peptide nucleic acid. Blood 1996 88 1411-1417. 

Extending the emulsion to a water-in-oil-in-water mixture allowed further refinement of the IVC concept. Compartmentalization of E. coli containing semm paraoxonase variants allowed the accumulation of fluorescent product to a point where it could be detected by FACS [57]. This approach was also used with in vitro transcription and translation to evolve /3-galactosidase activity from the Ebg gene [58]. 

PTT is based on a combination of reverse-transcription polymerase chain reaction (RT-PCR) and linked in vitro transcription and translation (Fig. 3C). This combination of procedures can selectively detect translation-terminating or nonsense mutations. Unfortunately, it does not find missense mutations, which may be etiologic, depending upon location. 

Dihydroflavonol 4-reductase (DFR) catalyzes the stereospecific conversion of 2R,3R)-trans-DHFs to the respective (2R,35, 45)-flavan-2,3-traKi-3,4-cA-diols (leucoanthocyanidins) through a NADPH-dependent reduction at the 4-carbonyl. DNA sequences for DFR were first identified from A. majus and Z. mays, and the identity of the Z. mays sequence confirmed by in vitro transcription and translation of the cDNA and assay of the resultant protein. DNA sequences have now been cloned from many species, with the size of the predicted protein averaging about 38kDa. Stereospecificity to (2R,3R)-dihydroquercetin (DHQ) has been shown for some recombinant DFR proteins. ... 

Fig. 6. Selection for enzymatic activity (DNA methylation) by compartmentalization. DNA encoding HaeIII methylase is diluted with unrelated DNA (encoding dihydrofolate-reductase). This mixture is dispersed together with a reaction mixture for in vitro transcription and translation to form water in oil compartments. The dilution is chosen such that each compartment contains one DNA molecule on average. In the aqueous compartments, the genes are transcribed and translated. In compartments in which an active methylase is translated, the DNA can be methylated and is subsequently recovered from the emulsion and digested by a restriction enzyme. The methylated DNA (encoding Hae III methylase) is protected against the digestion, remains intact and is subsequently amplified by PCR.

Thus far a series of deletion mutations have been introduced into glucocorticoid receptor cDNAs in the region coding for the DNA binding domain [83,84,90,104], These were tested either by in vitro transcription and translation followed by a spe-... 

The mRNA required for in vitro translation can itself be produced by in vitro synthesis. Commercially available kits allow DNA cloned downstream of T7-, T3 or SP6-promoters to be transcribed effectively in vitro by the relevant RNA polymerases. In coupled transcription-translation, it should be remembered that translation of eukaryotic mRNA requires a 5 cap upstream of the initiation codon, and similarly, for prokaryotic translation there should be an appropriately positioned ribosome binding site. Commercial kits are also available for combined in vitro transcription and translation. 

Tymms, M.J. (Ed.) (1995) In vitro Transcription and Translation Protocols, in Methods in Molecular Biology Vol. 37. Humana Press, Totowa, NJ. 

The E. coli harboring a plasmid, pDS-b2 (Fig. 16), designed for in vitro transcription and translation, were noticeably pink in color 147). This results from constitutive expression of flavocytochrome 62 at up to 5% of the total soluble protein in these cells. Expressed enzyme contains full stoichiometric amounts of FMN and heme 147). Expression of flavocytochrome 62 in vivo was not expected with this vector because there is no E. coli ribosome-binding site (rbs). It appears that a fortuitous rbs exists within the region of DNA encoding the mitochondrial targeting sequence and this leads to initiation of translation at Met 6 of the mature flavocytochrome 62 147). The absence of residues 1-5... 

Howe, A.Y. M., Elliott J. F., and Tyrrell, D. L. J. (1992) Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities. Biochem. Biopyhs. Res. Commun. 189, 1170-1176. 

Unique disadvantages to ribosome display relate to the inherent instability of RNA. This is a disadvantage shared by mRNA display but an advantage of ribosome display over mRNA display is that the in vitro transcription and translation can be combined into one step. This renders the mRNA less vulnerable to degradation. 

In order to verify the activity of the chimeric product, in vitro transcription and translation experiments were performed. Active photoprotein formation was monitored by measuring luminescence from the translation mixture in the presence of coelenterazine after the addition of calcium ions. As shown in Fig. 1, Photina produces an intense luminescence signal in response to calcium stimulation (25 mM CaCU) which is generally higher than that observed with natural photoproteins. 

R. Tachibana, H. Harashima, T. Ishida, Y. Shinohara, M. Hino, H. Terada, Y. Baba, H. Kiwada, Effect of cationic liposomes in an in vitro transcription and translation system, Biol. Pharm. Bull, 2002, 25, 529-531. 

Another distinct advantage of microdroplets is their abihty to compartmentalize a single copy of a molecule in one microdroplet [4]. This technique allows the analysis of a single protein or DNA molecule from a library [3, 5]. One particular application is the screening of a DNA library for protein expression. In this respect, previous work has demonstrated enzyme synthesis by in vitro transcription and translation (IVTT) in microdroplets, with the ultimate aim to develop a platform to screen mutant DNA constructs in a library [6, 7]. 

This research has demonstrated an integrated platform for the encapsulatimi of plasmid DNA in individual microdroplets, the synthesis of an enzyme within the microdroplet through in vitro transcription and translation, and activity assays and sorting of the microdroplets based on the enzyme activity. The use of a dilute solution of a homogeneous plasmid DNA population in the work confirms that it could be possible to compartmentalize a single copy of the DNA in one... 

KORANT, B. In In Vitro Transcription and Translation of Viral Genomes. Haenni, A. and Beaud, G. eds.,(l975), p.273 INSERM, Paris. 

Cryopreservation and Freeze-Drying Protocols, edited by John G. Day and Mark R. McLellan, 1995 37. In Vitro Transcription and Translation Protocols, edited by Martin J. Tymms, 1995 36. Peptide Analysis Protocols, edited by Ben M. Dunn and Michael W. Pennington. 1994... 

FIGURE 5 Immunoprecipitation of the autoantigen fibrillarin using autoantibodies. cDNA encoding mouse fibrillarin was radiolabeled with [35S]methionine by in vitro transcription and translation (TnT mFIB). This protein was then used in a protein A-Sepharose bead immunoprecipitation assay to examine human sera (A-L) for antifibrillarin antibodies. Positive sera are identified by an asterisk. POS. CONT, immunoprecipitate from an antifibrillarin-positive serum NEG. CONT, immunoprecipitate from an antifibrillarinnegative serum. 

Porter, A. G., Frisby, D. P., Carey, N. H., and Fellner, P., 1975, Nucleotide sequence studies on picornavirus RNAs, in In Vitro Transcription and Translation of Viral Genomes (A. L. Haenni and G. Beaud, eds.), pp. 169-176, INSERM, Paris. 

Breindl, M., and Holland, J. J., 1975, Coupled in vitro transcription and translation of vesicular stomatitis virus messenger RNA, Proc. Natl. Acad. Sci. USA 72 2545. 

Because of the ease of its elimination by phenol/chloroform extraction, PRI is often preferred to VRC for protecting RNA. In experiments where RNA is either a substrate or a product, e.g., in the isolation of RNA, cDNA synthesis, in vitro transcription and translation, PRI is used at concentrations of 1 U//al. 

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