Miscoding in a cell-free system from spleen.

Infidelity of translation of the genetic code has often been widely reported'-'2 in cell-free, protein-synthesizing systems derived from bacteria and, much less extensively,'3-'7 in cell-free systems. However, the degree by which experimental conditions per se contribute to the observed level of infidelity remains undetermined. Nevertheless, some studies on the influence of streptomycin on protein biosynthesis show that this drug causes miscoding only in cell-free systems which contain ribosomes derived from streptomycin-sensitive cells.9' 11 18-21 These findings suggest that the phenomenon observed in cell-free systems reflects the in vivo process.12, 21 Thus, it seems likely that the streptomycin case exemplifies a means whereby a specific, externally administered factor can influence the fidelity of in vivo protein biosynthesis. This paper examines the miscoding properties of cell-free systems derived from rabbit spleen, a tissue capable of antibody production. Investigation of such a system is of special interest since the small differences in amino acid composition among antibodies which have been observed,22-26 and which may be the basis of antibody specificity, could result from miscoding. The experiments described here were conducted with a ribosomal preparation which had been preincubated in order to eliminate endogenous incorporation (incorporation due to residual native messages), thus avoiding possible ambiguities in interpreting the role of the added synthetic polynucleotide. This treatment is important for studying miscoding; however, no mammalian system previously described has been completely freed of endogenous incorporation without the removal of most of its activity. Materials and Methods.-Preparation of ribosomes: Rabbit spleens were washed in a solution containing 0.07 M KCl, 0.0055 M MgC12, 0.05 M tris(hydroxymethyl)aminomethane (Tris) buffer pH 7.6, 0.35 M sucrose, 0.006 M 0-mercaptoethanol (medium A), then weighed, sliced into small pieces, and suspended in medium A 1:2.5 (w/v). The suspension was homogenized carefully in a Potter-Elvehjem homogenizer with a Teflon pestle. The homogenate was centrifuged at 18,000 X g for 10 min. To the upper two thirds of the supernatant fraction Na-DOC (sodium deoxycholate) was added to give a concentration of 1%, then diluted 1:1 (v/v) in a solution equivalent to medium A but containing 0.9M sucrose instead of the usual level (medium B) and centrifuged at 150,000 X g for 90 min. The ribosomal pellet was suspended in medium A and stored at -80'C. Preparation of pH 5 fraction: Rat livers or rabbit spleens were subjected to the procedure described above, except that no Na-DOC was added. The upper two thirds of the last supernatant was collected and the pH brought to 5.1 by addition of 1 N acetic acid. After 30 min of continuous stirring, the precipitate was centrifuged out (10 min at 18,000 X g), dissolved in medium A, and stored at -80'C. (The use of rat liver pH 5 fraction is just a matter of convenience; preliminary experiments showed that pH 5 fraction prepared from rat liver is interchangeable with the same fraction from spleen.) All the operations were carried out at 40C or below. Incubation conditions: The reaction mixture in a final volume of 0.1 ml contained in millimoles: KCl, 8; Tris buffer, 5 (pH 7.6); sucrose, 7; 03-mercaptoethanol, 0.1; adenosine 5'-triphosphate, 0.16; guanosine 5'-triphosphate, 0.04; and phosphoenolpyruvate,