Rabbit reticulocyte lysates

Jackson, R. J., and Hunt, T. (1983). Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 96, 50-74. 

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. 

Translation extracts Rabbit reticulocyte lysates (RRL) (Promega), wheat germ (WG) extract (Promega), bacterial S30 extract (Promega). Extracts from Krebs-2 cells were prepared as described (Svitkin and Sonenberg, 2004). 

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. 

Borman, A. M., Michel, Y. M., and Kean, K. M. (2000). Biochemical characterization of cap-poly(A) synergy in rabbit reticulocyte lysates The eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5r-end. Nucleic Acids Res. 28, 4068-4075. 

R. Hough, G. Pratt, M. Reichsteiner, Ubiquitin-Lysosome Conjugates. Identification and Characterization of an ATP-Dependent Protease from Rabbit Reticulocyte Lysates , J. Biol. Chem. 1986, 261, 2400-2408. 

Hoeeman, L., Phatt, G., and Rechsteiner, M. Multiple forms of the 20 S multicatalytic and the 26S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate. J. Biol. Chem. 1992, 267, 22362-22368. 

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. 

The suppressor tRNA developed by the Chamberlin lab for use in a rabbit reticulocyte lysate is based on an E. coli glycyl tRNA, which was initially chosen because glycyl-tRNA synthetases do not rely on a double-sieve editing mechanism for enzymatic hydrolysis of misacylated tRNAs [26]. Two base pair changes were made to the acceptor stem to allow incorporation of the optimal T7 RNA polymerase promoter into the DNA template for tRNA y-Con [27,28],... 

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... 

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]... 

Fig. 12. Dose-response and binding properties of mERa and mERp. (A) Cos-1 cells were transfected with 500 ng mERp (open circles) or mERa (closed circles) expression vectors and 1 pg vitA2-ERE-TKLuc and then incubated for 12 hours with increasing concentrations of Eg as indicated. (B) Specific binding of [2,4,6,7-3H] 17 f-estradiol ([3H]E2) to mERP was determined by using receptors generated from rabbit reticulocyte lysates. Binding was determined over a concentration range of 0.01-3 nM [3H]E2 in the absence or presence of a 200-fold excess of unlabeled E2. The saturation plot is shown in the insert. The results were plotted by Scatchard s method. Each point was determined in triplicate in each experiment, and the above results are representative of at least two separate experiments. (C) Specific binding to mERa using the conditions described in panel B (Tremblay et al., 1997).

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)...

RNAP, DNA-dependent RNA polymerase RNAS, RNA synthesis ROS, reactive oxygen species rRIP, recombinant RIP RRL, rabbit reticulocyte lysate (for in vitro PSI measurement) rRNA, ribosomal RNA RT, reverse transcriptase RTK, receptor tyrosine kinase RY-R, ryanodine receptor,... 

Fig. 10. Cell-free synthesis of t-PA glycoforms. The niRNA coding for t-PA was translated in a rabbit reticulocyte lysate in the presence of dog pancreas microsomes. Microsonies were isolated posttranslationally and the translocated, glycosylated products were separated by SDS-PAGE. Translation was carried out under conditions that either prevented (lane 2) or allowed (lane 3) proper folding of the t-PA molecule, yielding enzymatically active protein that was sensitive to natural inhibitors and stimulators.

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

Some members of the human RNase A superfamily of proteins are known to have host defense activities (reviewed in ref. 9). These include, for example, two of the eosinophil cytotoxic granular proteins, eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) (10). Angiogenin, a protein 65 % homologous to pancreatic RNase (11,12) that was originally isolated on the basis of its angiogenic activity (13), is a potent inhibitor of protein synthesis in the rabbit reticulocyte lysate (14) and when injected into Xenopus oocytes (15). We have therefore sought to fuse RNases to MAbs to evaluate their usefulness as immunotoxins (16,17). 

Fig. 1. Appearance of CCT in the electron microscope. CCT purified from rabbit reticulocyte lysate was examined in the absence (a) or presence (b) of ATP. Reprinted from Gao et al. (1992), with permission.

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. 

Fig. 10. Kinetic analysis of /3-actin translation in rabbit reticulocyte lysate. Triangles /3-actin bound to prefoldin closed circles /bactin bound to CCT squares native monomeric /3-actin open circles /3-actin bound to an unknown protein. Reprinted from Hansen et al. (1999), with permission.

In a recent study, the use of rabbit reticulocyte lysate for the translation of preproteins was discarded, and purified preproteins were instead employed for import studies (Lim et al., 2001). Since cytosolic chaperones could not be acting, the involvement of mitochondria in promoting unfolding of preproteins could be more closely scrutinized than before. 

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