thymidine and hypoxanthine) not required exogenously by the cells for 

 optimal growth. Mutants are detected by screening populations of cells 

 exposed to test compounds for nutritionally deficient forms (auxotrophs) 

 requiring one or more of the nine nutrilites omitted from F12D medium. 



n 

 Operationally, 10 cells are exposed in four parallel cultures to single or 



multiple doses of the test agent, following inoculation and cell attachment 



(point A, Figure 7-3). Because even induced mutation is a rare event, there will 



generally be, following expression, a small number of mutant cells growning 



among millions of nonmutants in enriched medium. To identify the mutants, it 



is necessary to introduce a procedure which will eliminate the prototrophic 



(wild-type) cells, while allowing auxotrophs to survive. This is accomplished by 



taking advantage of the fact that mutants auxotrophic for one or more of the 



nine nutrilites omitted from F12D medium will be unable to grow in this 



medium, whereas wild-types will. Thus, at point B, Figure 7-3, F12 medium is 



replaced with F12D medium. This initiates the selective process by effectively 



terminating protein and nucleic acid synthesis. The thymidine analog, BrdU, is 



then added to the F12D medium from which it is incorporated into the DNA 



of wild-type cells (point C, Figure 7-3). Subsequent illumination of the cell 



population with wliite light is lethal to those cells having incorporated 



sufficient BrdU. Mutant cells do not incorporate BrdU, and survive the 



selective process (point D, Figure 7-3). Wild-type cells survive selection to an 



extent approaching 0.02 percent. In the presence of F12 medium, mutant cells, 



along with some wild-types, grow into macroscopic colonies (point E, Figure 



7-3). These are picked and tested for mutant identification. It is important to 



note that the use of a known mutagenic agent (BrdU) in the selective process is 



of no consequence, as the only cells incorporating BrdU are wild-types destined 



for death. Mutants to be isolated are existent in the population at the end of 



the expression period prior to the application of selective conditions. 



The procedure illustrated in Figure 7-3 and described above was applied in 

 collecting the mutagenesis data presented below. Figure 7-4 shows the ligliting 

 apparatus used to illuminate cell populations following exposure to BrdU. 

 Figure 7-5 shows cell survival in four randomly selected dishes several days 

 after illumination. Cells not killed in selection have grown into macroscopic 

 colonies. Because mutant and wild-type cells differ only in their requirements 

 for exogenous nutrilites, they may be distinguished only by analysis of their 

 growth properties in enriched and deficient media or by biochemical analysis. 



Statistical Analysis of Data 



When testing compounds for mutagenic activity, we usually want to know if 

 the number of mutants per unit number of viable cells screened is sufficiently 

 larger in experimental, versus control situations, to support a conclusion of 



84 



