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 K1 
subclone of the CHO cell (44), other spontaneous auxotrophs with 
requirements for one or more of the nutrilites omitted from FI 2D 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 
(n) Viable [(A)/(B)] [(C)/(D)(E)] Auxotrophys isolated (y) 
cells Gly Hyp (x) 
16 1.2 x 10 7 1888/989 (800)/(770) (.750) 2 0 2 0.331 
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 
