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Cytoplasmic cylindrical inclusions are one of the main characteristics of potyvirus group1,2) and are controlled by portions of the virus genomes1-5). The study of cylindrical inclusions is very important to gain some understanding of their functions in host cells6,7). It also has been pointed out that antisera to inclusion proteins are potentially useful in diagnosing virus infections5) and studying the relationships among potyviruses6). The purification of many cylindrical inclu sions has been reported already6-11). Most experiments conducted so far on their purification have employed sucrose density gradient centrifugation as the final step in this process. How ever, it is difficult to separate cylindrical inclusions as a single band by this method since they are not uniform in size12). This paper describes an improved method fox purification of cylindrical inclusions, induced by turnip mosaic virus (TuMV) obtained from Statice sp.13), bean yellow mosaic virus (BYMV, P 25M strain)14) and potato virus Y (PVY) from potato15), reducing the amount of organic sol vents needed for clarification by treating inclusion preparations with them at a later step in the process of purification, and using isopycnic centrifugation in a sucrose-potassium tartrate density gradient. TuMV13) was propagated in Brassica rapa var. komatsuna. Infected plants, 2-5 weeks after inoculation, were placed in the dark for 48 hr to reduce starch contamination prior to harvesting of leaves. Systemically infected leaf tissue was homogenized in a blender with a cold solution containing 3ml of 500mM potassium phosphate buffer (PB) (pH 7.5) and 5mg sodium sulfite per gram of fresh tissue and then filtered through two-layers of gauze. Triton X-100 was added to the homogenate to a final concentration of 5% and after stirring for 1 hr at 4 C, the mixture

Isopycnic separation of potyviral cylindrical inclusions by sucrose-potassium tartrate density gradient centrifugation.