McCLURE AND ROBERTS 153 



pH 7.6 (TS). Suspend with 10 ml TS, and break the cells by forcing the sus- 

 pension through an orifice at 10,000 psi with a flow rate of roughly 2 ml/min. 

 Add TS to bring the total volume to 50 ml, and centrifuge 15 minutes at 

 40,000^. Discard the precipitate, which contains unbroken cells and large frag- 

 ments of cell walls. Centrifuge again at 105,000^ for 15 minutes, and discard 

 the precipitate, which contains smaller fragments of cell walls and a small pro- 

 portion of the ribosomes. Dilute the supernatant fluid with TS to 100 ml, and 

 add MgCL and M11CI2 to make it 0.005 M in each. In 5 to 48 hours the solu- 

 tion will show turbidity because of protomorph formation. The entire proce- 

 dure is carried out at 0° to 5 J C. 



The yield of this procedure is variable, as is the time required for formation 

 of the protomorphs. Some of the sources of variability have been identified; 

 others remain unknown. 



The addition of manganese is essential. No protomorphs were formed when 

 Mn ++ was omitted even though adequate Mg ++ was present. Higher concen- 

 trations of Mn" (0.01 M to 0.5 M) caused the formation of a precipitate of 

 particles of irregular shape and widely variable size. No attempt was made to 

 find other cations that might substitute for the Mn + \ The Mg ++ was not essen- 

 tial but seemed to increase the yield. No difference was noted whether the 

 magnesium was added before or after the cells were broken; thus there would 

 appear to be no difference to protomorph formation whether the ribosomes 

 were in the 80 S form or not (see paper 3 of this volume). 



No protomorphs were formed when the £>H of the solution was outside the 

 limits 7 to 8; a pH of 7.5 seemed to be optimum. 



The concentration of orthophosphate affects the yield. When the cells are 

 carefully washed in TS before breaking, the phosphate of the growth medium 

 is removed and phosphate must be added to give a concentration of 10" 3 M. 

 If higher quantities of phosphate are added the particles become less refractile 

 in appearance and are dark, rough, and "hairy." At still higher concentra- 

 tions of phosphate, precipitates are formed in manganese solutions lacking cel- 

 lular material. Although these inorganic precipitates have only a slight re- 

 semblance to the protomorphs, it is possible that the inorganic material provides 

 a framework on which the organic material deposits. 



The concentration of the cellular material is important. When the usual pro- 

 cedure was followed a twofold dilution of the cell extract prevented the forma- 

 tion of protomorphs. The presence of cell-wall material was not important to 

 the yield; the yield was the same whether or not the centrifugation steps to re- 

 move cell walls and unbroken cells were omitted. When the wall material was 

 present the protomorphs appeared less smooth, as if irregular fragments of wall 

 had been incorporated. 



The constituent responsible for the sensitivity to the concentration is prob- 

 ably deoxyribonucleic acid (DNA). The addition of DNAase invariably pre- 

 vents the formation of protomorphs. The pressure cell routinely used to dis- 

 rupt the cells also degrades DNA, as preparations of DNA lose their viscosity 



