Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5'-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5'-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5'-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.

The factors that affect reliable estimations of mutation rates (mu) in cultured mammalian somatic cell populations by fluctuation analysis are studied experimentally and statistically. We analyze the differential effect of the final cell population size in each culture (Nt) and the number of parallel cultures (C) on the variation in the rate estimates (mu) inferred from the P0 method. The analysis can be made after the derivation of the variance of mu, which is a measure of variation of mu for a given combination of Nt and C in a number of repeat experiments. The variance of mu is inversely proportional to C and to the square of Nt . Nt determines the probability of occurrence of mutations in a cell culture. By influencing the size of P0, Nt also determines whether a rate estimate is obtainable from the experiment. Since Po is estimated from the fraction of cultures containing no mutation in a set of C cultures, C becomes a determining factor for the accuracy of mu. The rate estimated from P0 is biased, but the bias is in general 2 orders of magnitude smaller than mu. By the selection of an appropriate combination of Nt and C for the experiment, this bias can be reduced even further. Based on the notion of comparing two proportions, we propose a test statistic and have applied it to experimental results for a test of equality of mutation rates in different cell lines. This development places the comparison of mutation rates on a statistical basis.

A systematic comparison of 5 different statistical methods for the estimation of mutation rate (mu) in cultured Chinese hamster V79 cells is presented. Fluctuation tests were performed with several large batches of parallel cell cultures each allowed to grow for a different length of time in order to reach different population size (Nt). Based on Lea and Coulson's theoretical distribution, a comparison has been made between the experimental data and the expected distribution of the number of ouabain-resistant mutants per culture in these hamster cell populations. The sum of squared deviation between the observed and expected values, or SSD, was used as a means of the adequacy of the estimation method; the method which gives the smallest SSD is regarded as the best one for the estimation of mu. Our results show that when Nt is small, the occurrence of mutation is infrequent, and SSDs from different methods are similar. However, when Nt is large, there is a great discrepancy of the SSD values, suggesting a preference of using the maximum likelihood method, the Po method, the median method, the upper quartile method and the mean method, in that order, for the estimation of mu. The order of preference is correlated with estimation efficiencies. Depending on the size of Nt and the method used, the estimated mu may vary up to more than 3-fold. At a large Nt, the mu obtained from the maximum likelihood method is very precise. This suggests the importance of choosing an appropriate Nt as well as method for the estimation of mu.