COHEN, HALVORSON, AND SPIEGELMAN 101 



from radioactive glucose and incorporated in the presence of the analog during 

 the linear growth. In addition, /7-fluorophenylalanine was incorporated (Munier 

 and Cohen [2], [3]) to a great extent (up to 200 m|jmoles/g dry weight). In- 

 duced (3-galactosidase was synthesized at the same differential rate as in a con- 

 trol culture without analog. Because of the contradictory results of these two 

 studies, it was decided to re-examine the effects of /7-fluorophenylalanine on 

 yeast. 



MATERIALS AND METHODS 



Saccharomyces italicus Y1225 was used in these experiments. The cultures 

 were grown in synthetic medium (Halvorson and Spiegelman [1]) in Erlen- 

 meyer flasks which were shaken at 30° C. During the exponential phase of 

 growth, the protein content per cell was found to be proportional to the opti- 

 cal density. Therefore, for reasons of convenience, growth was followed by 

 measurements of the optical density in a Beckman DU Spectrophotometer at 

 600 mu. Under these conditions 1.00 O D = 772 ug dry wt./ml. 



Viable counts were determined by plating appropriate dilutions on dextrose 

 broth agar medium. 



The cells were centrifuged, washed, and fractionated for isotope distribution 

 as previously described (Halvorson [4]). The components of the protein hy- 

 drolysates were identified by radioautography. For radioactivity measurements, 

 aliquots were evaporated to dryness on stainless-steel planchets and counted in 

 a gas flow counter, and the radioactivities were corrected to infinite thinness. 



The photomicrographs were taken on a 35-mm microfile film with a 100 

 apochromatic objective, N. A. = 1.30. Magnification on the film was 1250 X. 

 3-C 14 -DL-phenylalanine (Phe) (2.1 mc/mmole), 4-4'-C 14 -DL-valine (1.33 mc/ 

 mmole), and 3-C 14 -DL-p-fluorophenylalanine (/>-FPhe) (2.35mc/mmole) were 

 obtained from the Commissariat a l'Energie Atomique, France. Uniformly 

 labeled glucose (2.4 mc/mmole) was obtained from the Fisher Scientific Com- 

 pany, and carrier-free S 35 sulfate from the Oak Ridge National Laboratory. 



RESULTS 



Effects of p-Fluorophenylalanine and $-2 Thienylalanine on the Growth of 

 Yeast. Figure 1 shows that, upon addition of 10 2 M /7-FPhe or $-2 thienyl- 

 alanine (Thiala) to an exponentially growing culture of yeast, the mass in- 

 creases at a rate which is linear with time, as previously observed with E. coli. 

 In this particular experiment, from the time of addition to the termination of 

 growth, the mass increased 4 times in the presence of /7-FPhe and 2.7 times in 

 the presence of Thiala. Under these conditions, although no component of the 

 medium was limiting for growth, there was no increase in viable count in 

 the presence of the antagonists. The increase in optical density in the presence 

 of /7-FPhe (4 times) can be attributed to an increase in cell size (fig. 2). As- 

 suming that yeast cells are ellipsoids of revolution, the average cell volume in- 

 creased approximately 3.6 times in the presence of p-FPhe. 



