GILLCHRIEST AND BOCK 3 



wax "4000" to maintain the protoplasts. The turbid solutions were brought to 

 13 ug/ml in crystalline egg white lysozyme, and the turbidity was observed until 

 its rapid decrease ceased. The protoplasts were then collected by low-speed 

 centrifugation, washed once in osmotically adjusted RNP buffer, collected 

 again, and ruptured by osmotic shock upon dilution with 10 times the packed 

 cell volume of RNP buffer to yield a crude extract. During the development 

 of the protoplast procedure, both the formation of the protoplasts and their 

 osmotic rupture upon dilution were followed in the visible and phase contrast 

 microscope. 



PURIFICATION OF RIBONUCLEOPROTEIN PARTICLES (fig. 1) 



The crude extract derived from any of the above three procedures is centri- 

 fuged at 4900g for 30 minutes. The pellet that accumulates consists of cell debris 



PARTICLE 



PREPARATION 



IT 



nr 



CRUDE EXTRACT 



(4,900g x 30min) 



DISCARD SUPERNATE 



(I05,400g x 60min) 



PELLET 



DISCARD 



(8,700 g x 15 min) 



1 

 DISCARD PREPARATION 



Fig. 1. Flow sheet for differential centrifugation of ribonucleoprotein from a crude 

 extract of A. vinelandii prepared by I grinding, II osmotic shock, or III protoplastic osmotic 

 shock. 



