148 MUTATION AND PLANT BREEDING 



plated on appropriate medium for determination of the number of 

 organisms subjected to the radiation. Other aliquots were irradiated 

 with UV or X-ray depending on the experiment. 



In preirradiation supplementation experiments, immediately 

 following irradiation, appropriate dilutions were plated on agar plat 

 ing media by the glass rod spreader technique. 



For postirradiation experiments aliquots of the irradiated culture 

 were diluted 1:4 into appropriately supplemented growth medium 

 and incubated at 37° C on a reciprocal shaker for time intervals 

 indicated in the various experiments prior to plating on agar media. 

 All plates were incubated at 37° C for 2 days in the case of color 

 mutants and for 3 days in the cases of prototrophic reversions before 

 scoring; for survivors and mutants. 



Biochemical determinations 



Culture samples were taken at indicated intervals during pre- 

 irradiation and postirradiation incubation for analysis of RNA, 

 DNA, and protein. The samples were precipitated and washed with 

 0.5N perchloric acid, and the nucleic acids then hydrolyzed by incu- 

 bation in perchloric acid for 50 minutes at 70° C (20). They were 

 then analyzed for DNA content by the Burton (1 ) method. For RNA 

 determination, the UV absorption at 260 mu and 290 mu were deter- 

 mined (25), and the amount of DNA, as determined by the Burton 

 analysis, subtracted with correction for extinction coefficients. Protein 

 was determined by the Folin (15) method. 



Experimental Results and Discussion 



I. Effect of Preirradiation Growth Factor Supplementation on 

 Radiation-Induced Mutations 



Early experiments were carried out using E. coli strain B, and 

 consisted of attempts to identify possible extragenic factors which 

 might be affected by UV so as to produce mutations. These experi- 

 ments have been previously reported (7). The induced mutation- 

 radiation dose curves previously obtained by many investigators 

 suggested that radiation-sensitive material present in the cell was 

 activated or altered by radiation as a prerequisite to mutation indue 

 tion. The material also appeared to be limited in amount since the 

 induced mutation frequency leveled off at high doses of radiation 

 (18, 19, 29). 



