112 R. MARKHAM 



XIV. The Southern Bean Mosaic Virus 



This virus received its name from its distribution in the southern states 

 of the United States. Under natural conditions it causes a disease in Phaseolus 

 vulgaris (Zaumeyer and Harter, 1942), and it infects several leguminous 

 plants, but its host range is fairly restricted, so that preparations are usually 

 made on bean. 



A. Purification (W. C. Price, 1946) 



Bean plants are harvested 3 weeks after inoculation, frozen, and ground. 

 Clarification is carried out using Na2HP04, which may be used in solution to 

 extract a second quantity of virus from the pulp. The virus is then precipit- 

 ated by the addition of 300 gm. ammonium sulfate to each liter of sap, or 

 it is centrifuged out [the sedimentation coefficient is 115 >S: (Lauffer et al., 

 1952)]. Further purification is carried out by the addition of ethanol to 

 30 % by volume, which causes extraneous material to precipitate. Removal 

 of pigment from the preparations can be carried out by electrophoresis at 

 pH 7. A Tiselius apparatus was used, but no doubt the more recent stabihzed 

 column electrophoresis apparatuses would be better for this purpose (Lauffer 

 and Price, 1947). 



Alternatively the virus can be adsorbed on to Amberlite XE-67, a basic 

 resin, and eluted by NaCl + phosphate at pH 6.85, in which case the pigment 

 is retained on the column. Trouble with pigment would also no doubt be 

 bypassed by suitable treatment of the sap when preparing it. 



The yield of virus is of the order of half a gram per liter of sap. 



B. Crystallization 



Crystals of the virus have been obtained in a number of ways. A 1 % 

 solution of the virus in 20 % saturated (NH4)2S04 is centrifuged, and the 

 peUet free from the supernatant is moistened with water. The peUets become 

 opaque on standing and contain masses of plate-shaped crystals, which 

 are thought to be orthorhombic, but are not birefringent. The crystals are 

 stable if stored in half-saturated (NH4)2S04. The virus also crystallizes out 

 if dialyzed against tap water and pH 5.5 acetate buffer or against distilled 

 water (MiUer and Price, 1946a,b). 



Crystals of this virus are of some interest because they were the first 

 crystals to be examined by the electron microscope. One interesting point 

 about this is that Wyckoff (1949) has published pictures of one of these 

 crystals in which the packing distances in two planes roughly at right angles 

 to each other are 1.5 : 1, suggesting that the virus particles themselves may 

 have an axial ratio of this order (but see Labaw and Wyckoff, 1957). The 



