equation [3] is at present a proposed one and may not be the final model of 

 choice. 



(y) = (x/n) [(A)/(B)] [(C)/(D)(E)] [2] 



(V) = (2X + 3/4)1 /2.(2Y+ 3/4)1/2 [3] 



RESULTS 



Control Investigations 



With the exception of the spontaneous proline auxotroph isolated as the Kl 

 subclone of the CHO cell (44), other spontaneous auxotrophs with 

 requirements for one or more of the nutrilites omitted from F12D medium had 

 not been previously reported for the CHO Cell/BrdU-VL system. Such 

 auxotrophs could easily be suppressed in stock cell populations by maintaining 

 cells in minimal rather than enriched medium. This was not done in order to 

 determine if spontaneously arising auxotrophs could indeed be identified in 

 control or stock populations. Table 7-1 summarizes data from 16 different 

 control experiments carried out over a period of several months. Two glycine 

 mutants were identified among 989 clones picked and tested. The observed 

 frequency of spontaneous auxotrophy is thus two mutants per 1.2 x 10 viable 

 cells. These data, in combination with data for the other parameters of 

 equation [2] , were utilized to obtain an estimate of 0.331 mutants per 10 

 viable control cells. This value was substituted for (Y) in equation [3] . 



Table 7-1. Summary Mutagenesis Data From Several 

 Control Experiments 



The scaling of (x) by equation [2] does not consider the fact that mutant 

 cells may be lost to the effects of starvation during selection. In fact, 

 reconstruction experiments employing known numbers of mutants have 

 demonstrated that this type of loss does occur for the three types of 

 auxotrophs observed (27, 33). Consequently, equation [2] underestimates 

 actual mutant frequencies. 



87 



