Protein Synthesis in Protoplasts 189 



physical dissolution of the protoplasts. After incubation with 

 either lipase or trypsin at the levels indicated, few protoplasts 

 can be recovered. It is thus important in any given case to 

 demonstrate that an inhibition of enzyme synthesis which is 

 observed to follow a particular treatment is not the result 

 of a generalized destruction. This caution is also relevant to 

 experiments involving ribonuclease (RNAse) and deoxy- 

 ribonuclease (DNAse). Lysis of protoplasts by RNAse has 

 been observed by Brenner (1955) and in our own laboratory. 

 To be interpretable, experiments of this nature must be 

 accompanied by evidence that the enzyme treatment has 

 resulted in the selective removal of the homologous compound. 



An extensive examination has been made (Spiegelman and 

 Li, unpublished) of the effects of both RNAse and DNAse on 

 the synthesis of p-galactosidase in both intact cells and 

 protoplasts. No inhibitions were observed with intact cells 

 under any conditions of test. Striking effects were, however, 

 obtained with protoplasts. A few words may be interposed 

 here on the conditions necessary for consistent results. Cells 

 for an experiment were customarily prepared by inoculation 

 with 2 per cent peptone and incubation with shaking overnight 

 at 30°C. By morning, the cultures were in stationary phase 

 and were put through a "rejuvenation" prior to use. This 

 consisted in diluting the cultures fivefold with fresh medium 

 and reincubating until they had entered logarithmic growth 

 as determined by periodic examination of the optical density. 

 It was noted that extraordinary care had to be exercised in 

 controlling the extent of this rejuvenation if protoplast pre- 

 parations were to be obtained exhibiting uniform behaviour 

 with respect to enzyme-forming ability and response to 

 enzymatic resolution. 



Investigation of the cells during the course of the rejuvena- 

 tion revealed that our procedure had inadvertently led to 

 extensive phasing of the culture. This finding made under- 

 standable the extreme precision with which the timing of the 

 rejuvenation had to be carried out. It also made possible the 

 preparation of protoplasts which responded homogeneously 



