Reticulocyte lysate system

Inhibitory constants for inhibition of natural globin mRNA translation in a rabbit reticulocyte lysate system. For the tri-, tetra-, and pentaphosphate series, each value for Kx was normalized by dividing with the value for Kj for the cap analog standard for the series m7Gp3G, m7Gp4G, and m7GpsG, respectively. 

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

Dasso, M. C., and Jackson, R. J. (1989). On the fidelity of mRNA translation in the nuclease-treated rabbit reticulocyte lysate system. Nucleic Acids Res. 17, 3129—3144. 

Both proteins have been shown to be translated vitro In a reticulocyte lysate system as preinhibitors, 2000-3000 daltons larger than those synthesized and accumulated vivo (11). The preinhibitors may be Important In the compartmentall-zatlon of the Inhibitors as they are stored In the central vacuole, or plant lysosome, of the plant cells (12). We have now studied the time course of the Increase In translatable mRNA In leaves of wounded plants utilizing poly(A) mRNA Isolated at various times following wounding. 

Additional information <1-7, 11, 14, 15, 17-19, 21, 30> (<7> cell-free synthesis in mRNA-dependent rabbit reticulocyte lysate system [40] <2,4,5> high activities in tissues where turnover of energy from adenine nucleotides is great, e. g. muscle [3] <1-6,11,14,15> tissue distribution [3,46] <2,5> rabbit and human carry a minimum of 2 sets of isozymes within an individual one set in muscle, erythrocytes, brain and another in liver, kidney and spleen [3]) [3, 40, 46]... 

Table 6.8. The incorporation of [3SS] methionine into protein in a rabbit reticulocyte lysate system as directed by eight different RNA preparations from E. granulosus (horse strain). (Data from McManus et al., 1985)...

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

Other Methods SDS-PAGE was performed according to Laemmli (17). The immunoblot assay was performed according to the protocol from Bio-Rad Laboratories using anti-C. reinhardtii periplasmic CA antibody as the primary antibody. In vitro translations were performed using the rabbit reticulocyte lysate system from Stratagene. Protein and chlorophyll concentrations were determined spectrophotometrically. 

In Vitro Translation. TnT Coupled Reticulocyte Lysate System (Promega, Madison, WI) was used according to the manufacturer s instructions. 25 rabbit reticulocyte lysate was added to 2 p of TNT Reaction Buffer, 1 of RNasin ribonuclease inhibitor (40 u///l), 1 of amino acid mixture minus methionine (1 mM), 2 /il [ S] methionine (>1000 Ci/mmol at 10 mCi/ml), 2 of DNA template(s) (0.5 pgJpV), 1 /.tl of T7 polymerase, and 16 /il of nuclease-free water to a final volume of 50 /il. The reaction was incubated at 30° for 1 h. 

On the basis of our previous results discussed above, early viral transcripts were primary candidates in the inhibition of host protein synthesis. Since in vitro transcription produces only the early species of viral transcripts (Kates and Beeson, 1970), we acquired these early species of viral RNA by means of in vitro transcription by viral cores and tested their effect on the translation of various exogenous mRNAs in in vitro cell-free systems rendered messenger dependent. The results from such experiments (Coppola and Bablanian, 1983) revealed that transcripts prepared in vitro of either 8-10 S or 4-7 S size classes inhibit globin, HeLa, and hamster cell mRNA translation in a reticulocyte cell-free protein-synthesizing system. Inhibition was observed not only under conditions where in vitro viral transcripts by themselves were capable of producing viral polypeptides, but also when low concentrations of in v/7ro-synthesized transcripts were used which were incapable of synthesizing polypeptides as determined by polyacrylamide-gel electrophoresis. In contrast, the transcripts synthesized in vitro by vaccinia virus cores had no inhibitory effect on the translation of cytoplasmic RNA obtained from vaccinia virus-infected cells at early times after infection. Therefore, this inhibitory effect, like that seen in vaccinia virus-infected cells, was selective. The vaccinia virus transcripts, synthesized in vitro, also inhibited encephalomyocarditis virus mRNA translation in the cell-free reticulocyte lysate system indicating that this inhibition does not require... 

Total RNAs have been extracted from shoots of 3-day-old maize seedlings and spinach leaves. After ollgo (dT)cellulose chromatography, poly(A) RNAs were translated In a reticulocyte lysate system. The In vitro products were analyzed by SDS-electrophoresls. As shown In Flg.l, several proteins were synthesized, among which high-molecular mass polypeptides were observed. This result Indicates that the preparation of poly(A) RNAs had a good quality. 

Each of the steps involved in RDM has been used separately in many organisms and for the study of various mRNAs. Thus, although the protocol presented herein is for the analysis of yeast cells grown at optimal conditions, it could be adapted easily to other experimental systems. It might also be applied for in vitro systems or extracts (e.g., reticulocytes lysate), yet we have not performed such experiments. 

Preparation of Krebs-2 translation extracts Krebs-2 extracts are an ideal system to screen for compounds that inhibit translation because they faithfully recapitulate the cap dependency and the cap-poly(A) synergism associated with eukaryotic mRNA translation (Svitkin and Sonenberg, 2004), unlike standard rabbit reticulocyte lysates (RRL) (Borman et al., 2000). Furthermore, the translation of many types of IRESes is supported in Krebs-2 extracts. The use of commercially available translation competent extracts prepared from RRL, wheat germ, and E. coli is extremely useful in assessing selectivity of inhibitors identified in primary screens. 

Dittmar KD, Hutchison KA, Owens-Grillo JK, Pratt WB. (1996) Reconstitution of the steroid receptor.hsp90 heterocomplex assembly system of rabbit reticulocyte lysate. J Biol Chem. 271, 12833-12839. 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

In a further experiment we assayed for the presence of a cap structure on the mRNAs for both Inhibitors I and II by competitive inhibition by 7-methyl-guanosine 5 -monophosphate (m G p) of the in vitro translation of these messengers. Concentrations of 40 pM m G p inhibited by 50% the in vitro translation of total tomato leaf poly(A)" " mRNA (Fig. 7A). This level is 40-fold lower than that required to similarly inhibit rabbit globin mRNA translated in a rabbit reticulocyte lysate (17) and 4-fold lower than that required to inhibit the same mRNA in a wheat germ system (18). It was of interest that the translation of Inhibitor I is inhibited to 50% by 20 pM m G p while 50% inhibition of Inhibitor II requires less than 10 pM (Fig. 7B). The basis of this difference is not understood but... 

Pelham, H. R. and Jackson, R. J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67,247-256. 

Different in vitro translation systems are used depending on whether the mRNA is of prokaryotic or eukaryotic origin. For eukaryotic mRNA, translation kits from reticulocyte lysates or wheat germ extract are recommended, and for prokaryotic mRNA, the E. coli 30 S supernatant, all of which are commercially available. These translation kits can be supplemented with [3H]-leudne or... 

Scheele, G. (1983) Methods for the study of protein translocation across the RER membrane using the reticulocyte lysate translation system and canine pancreatic microsomal membranes. Methods Enzymol. 96, 94-111. 

Because CCT is assembled from eight different polypeptides, the prospect of engineering a host/vector system for the expression of recombinant chaperonin is a daunting one hence, all studies of CCT have thus far depended on material purified from a eukaryotic source such as mouse or bovine testis or rabbit reticulocyte lysate. Even if sufficient material could be purified from these tissues, the heterooligomeric nature of the particle might make crystallization extremely challenging. Structural analyses of CCT have therefore been confined to studies by cryo-electron microscopy. Nonetheless, such analyses have proved very useful in identifying some of the unique characteristics of this chaperonin. 

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