Polysomes from expanded tobacco leaves

Abstract

The isolation of intact polysomes from leaves of tobacco (Nicotiana tabacum L.) is dependent on the age and state of development of leaves. Undegraded polysomes from young leaves in the early stages of expansion can be isolated easily by extracting the leaves in ice-cold extraction buffer (200 mM tris(hydroxymethyl)aminomethylmethane(Tris)-HCl, pH 9.0; 400 mM KCl; 200 mM sucrose; 35 mM MgCl₂). Medium-size leaves give best yields of undegraded polysomes when extractions are carried out in the above buffer and in the presence of ethyleneglycol-bis-(β-amino-ethyl ether)-N,N′-tetracetic acid (EGTA) and mercaptoethanol. Isolation of polysomes from large, nearly fully expanded (mature) leaves requires all of the above plus diethyldithiocarbamate (DIECA) in the extraction medium. An extraction medium consisting of 25 mM EGTA, 0.01 M mercaptoethanol, 25 mM DIECA and 0.5% of the nonionic detergent, Nonidet-P40 (NP 40) was found to be very suitable for extraction of polysomes from all developmental stages of leaves. The polysomes extracted in the above medium showed active translation of protein in the wheat-germ in-vitro protein-synthesizing system. The translational products were similar when translations were carried out directly with polysomes or polysomal RNA, or polysomal poly(A)⁺ RNA from tobacco leaves. Poly(A)⁻ polysomal RNA was a poor template in the in-vitro wheat-germ system.