We have developed techniques for isolating free, membrane bound, and total polyribosomes from developing maize endosperm.
Field grown W64A was harvested at 18 days after pollination and frozen at -20 C. Freezing in liquid nitrogen did not substantially increase total yield or the proportion of higher order polyribosomes. Kernels were homogenized in the following grinding buffer: 0.25 M sucrose, 0.2 M Tris-HCl (pH 8.5), 0.06 M KCl and 0.03 M MgCl2 plus 0.1 mg/ml heparin, 4 mg/ml bentonite and 1 mg/ml dithiothreitol. A 3 tissue:1 buffer volume ratio was found to work well while using a Waring blender for homogenization. For homogenization with a mortar and pestle a 9 tissue:4 buffer volume ratio was used. Homogenates were filtered through cheesecloth to remove cellular debris. The filtered homogenate was centrifuged at 16,000xg, for 15 minutes to obtain a supernatant containing polysomes. Supernatants were then layered on a discontinuous gradient consisting of 10 ml 2.5 M sucrose in gradient buffer and 8 ml 1 M sucrose in gradient buffer. The gradient buffer was 0.04 M Tris-HCl (pH 8.5), 0.02 M KCl and 0.01 M MgCl2 (Larkins and Hurkman, 1978). Centrifugation of samples at 90,000xg for 4 hours using an SW27 rotor at 26 Krpm resulted in the banding of polysomes at the sucrose interface. Polyribosomes were removed by aspiration.
Total, free cytoplasmic and membrane-bound polysomes were all isolated using this basic technique. For isolation of total polysomes 10 mg/ml sodium deoxycholate and 13% Triton X-100 were added to the grinding buffer in the above procedure. Separation of free and bound polysomes was accomplished by using the procedure as described without detergents in the grinding buffer to yield free polysomes in the 16,000xg supernatant. The pellet from this was then resuspended in the detergent buffer (that for total polysomes) and recentrifuged at 16,000xg for 15 minutes; the supernatant in this step contained bound polysomes. All other procedures were the same for isolating these three polysome populations.
Polysome profiles for total, free and bound polysomes were obtained by sedimenting polysomes suspended in gradient buffer on 12.5 ml 150-550 mg/ml sucrose gradients. Samples were centrifuged at 190,000xg for 135 minutes in a Beckman SW-41 rotor. Gradients were then monitored at 254 nm while being pumped through a flow cell using a dense chase solution of sucrose.
Polysomes obtained in these experiments will be used for isolation of mRNA to be used in in vitro translation assays in an attempt to determine those polysome fraction(s) responsible for synthesis of the Cat1 isozyme found in maize immature endosperm.
Teresa Kelley and John Sorenso
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