1. Undegraded and active poly(A)+RNAs were obtained from tissues (rat liver or calf thymus) and cell lines (K562, NS 1 or HMy2) using sucrose gradient ultracentrifugation, guanidinium thiocyanate and rapid phenol-chloroform deproteinization. The above methods yielded 2-3% of total RNA which was as mRNA, found to be an undegraded product by analysis on either PAG or agarose gel electrophoresis. Single cycle oligo(dT) cellulose chromatography was used to purify poly(A)+RNA and the percentage yield was 0.6-4% of total RNA. These poly(A)+RNAs were in fact active templates and gave more than 3 fold increase in the incorporation of radioactivity over the control (without exogenous mRNA) in the rabbit reticulocyte lysate cell-free system. The fidelity of protein synthesis could be shown by gel electrophoresis. The incorporation of radioactivity was directly dependent on a concentration of added poly(A)+RNA. 2. Two types of cell-free systems; wheat germ lysate and rabbit reticulocyte lysate, were studied. Both cell-free systems were shown to have high efficiency in translation of various types of RNA (TMV RNA, globin mRNA, polu U and poly(A)+RNA from rat liver, K562 and NS 1 cells). The rabbit reticulocyte lysate was more efficient than wheat germ lysate in the synthesis of high M.W. protein (TMV protein at M.W. of 176k) but less sensitive to salt concentration. TMV RNA was the most active template since its rate of incorporation of radioactivity was approximately 100 fold above the control (without exogenous mRNA). 3. In studies of the effects of ANAs on protein synthesis in cell-free systems, either rabbit reticulocyte lysate or wheat germ lysate was used to synthesize protein directed by TMV RNA, globin mRNA, poly U or poly(A)+RNA from K562 and NS 1 cells. IgG fractions (36 samples from 8 subgroups of ANAs) prepared from the sera of SLE patients were examined and their effects detected by comparing the incorporation of radioactivity with normal IgG controls. The translation product was further characterized using a single-dimensional gel electrophoresis technique. It was demonstrated that some of these ANA samples had an inhibitory effect on protein synthesis and that the inhibition was not specific to any particular group of ANA. The ability to inhibit protein synthesis by different ANAs was different, since the percentage inhibition varied from 3% to 96%. However, the inhibition was dose-dependent in each case (IgG concentration). These ANA samples inhibited mRNA-directed protein synthesis in either rabbit reticulocyte lysate or wheat germ lysate cell-free systems. ANAs did not appear to inhibit translation preferentially on any particular type of messenger. Messengers from various sources were affected by these ANAs with only slight difference in the percentage inhibition. The results of gel analysis demonstrated that the amount of protein synthesized was dependent on percentage inhibition and the inhibition was not related to any specific type of protein. At high percentage inhibition, there was no protein synthesis and synthesis of low M.W. protein was seen in the samples that exhibited low percentage inhibition. The mechanism of protein synthesis inhibition by different ANAs appeared to be different. AntiRo (Frayne) and antiDNA (Jonas), which showed high percentage inhibition (more than 90%), exerted the effect of inhibition at the level of elongation, whereas, other ANAs with inhibition between 20% and 40% blocked protein synthesis at the initiation step. The above results clearly showed that some ANA samples had inhibitory effects, on protein synthesis in cell-free systems. The significance of the inhibition in these systems by ANAs was unclear. 4. The effects of ANAs on protein synthesis were further examined in intact cells. Several techniques were employed to transfer IgG molecules into cells, including fusion with liposomes or red cell ghosts, transfer via Fc receptors on cells, and permeabilization of cells in the presence of ANA. This study indicated that there was a limitation in transfer of macromolecules into cells. The protein synthesis pattern of cell lines was more complicated than protein synthesized in cell-free systems. Owing to the limitation involved, the ANAs did not show any effects on protein synthesis in the intact cells.
|Date of Award||1984|