Globin mRNA Translation on Artemia salina Ribosomes with Components from Friend Leukemia Cells

Abstract

Globin mRNA can be translated with relatively high efficiency in a fractionated cell-free system containing ribosomes prepared from cysts of Artemia salina. These ribosomes have unusually low endogenous activity for peptide synthesis in the absence of added mRNA. The system requires components from the postribosomal supernatant and from the 0.5 M KCI ribosomal wash fraction. Both these fractions were derived from either rabbit reticulocytes or unstimulated Friend leukemia cells that produce little or no hemoglobin.The activity of mRNA and enzyme fractions from rabbit reticulocytes and Friend leukemia cells were tested in this system in vitro for their ability to direct the synthesis of the a and p chains of globin. The a : P chain ratio synthesized from mRNA in the rabbit reticulocyte salt wash fraction was 4 : 1. The corresponding value for the 9-S mRNA fraction from the salt-washed reticulocyte ribosomes was 1 : 4, thus these two fractions appear to provide sources enriched in either a or p globin mRNA. Under all conditions tested, the ratio and amounts of peptides formed in vitro appear to reflect mRNA composition. Globin mRNA from dimethylsulfoxidestimulated Friend leukemia cells when translated in vitro produced a and p chains in a ratio of 1 : 1.These peptides are formed in the same ratio in the intact cells.The mechanism involved in the regulation of hemoglobin synthesis in reticulocytes is unclear. Hunt and co-workers [l] observed that fi globin chains were synthesized on larger polysomes than a chains and determined [2] that the rate of a chain elongation was faster than that for p chains. These data indicate that the two different classes of mRNA are translated at different rates within an intact cell. Lodish [3] concluded that there is no difference in the translation time for a and p chain mRNA by using antibiotics that reduced the rate of elongation but seemed not to affect peptide initiation. He observed that under these conditions the ratio of a

Structure and Activity of an Active Site Substitution of Ricin A Chain,

“…Enzyme activity was monitored as dose response curves, and relative activities were deduced by comparing 50% inhibitory concentrations (IC 50 ) under standard conditions. RTA was incubated for 5 min at 25 °C with 350 nM Artemia salinas ribosomes (purified as described by Kramer et al, 1975), the reaction was stopped by addition of anti-RTA antibodies, and residual ribosome activity was measured as previously described (Ready et al, 1983).…”

Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes

“…Yeast tRNAPhc, either unlabeled or labeled at the S'-phosphate, was aminoacylated as previously described (Hardesty et al, 1971) except that a 0.5 M KC1 salt wash of ribosomes from rabbit reticulocytes was used as the source of synthetases. The reaction mixture, in a final volume of 1 mL, contained 50 mM Tris-HCl (pH 7.5), 20 mM Mg(OAc)2,120 mM KC1, 2.5 mM dithioerythritol, 3.75 mM ATP, 60 µ [14C]Phe (100 Ci/ mol), 10 A 260 units of tRNAPhe, and 40 µg of the 0.5 M KC1 ribosomal salt wash fraction prepared as described previously (Kramer et al, 1975). After incubation for 20 min at 37 °C, the tRNA was extracted twice with phenol and collected by ethanol precipitation.…”

Hemin-independent control of globin synthesis in Friend erythroleukemia cells induced to differentiate.

“…Evidence presented here suggests that initiation factors from uninduced FL cells have different properties in recognizing mRNA than those from differentiated normal erythroid cells. This possibility is supported by the findings of Kramer et al (28). They have shown that crude initiation factor preparations from uninduced FL cells are able to support accurate translation of rabbit globin mRNA, using Artemia salina ribosomes but at a much lower efficiency than when the rabbit reticulocyte fractions are used.…”