Mutants of chinese hamster cells deficient in thymidylate synthetase

Stable mutants of Chinese hamster V79 cells deficient in thymidylate synthetase (TS; E.C. 2.1.1.45) have been selected from cultures grown in medium supplemented with folinic acid, aminopterin, and thymidine (FAT). After chemical mutagenesis, the frequency of colonies resistant to the “FAT” medium increased more than 100-fold over the spontaneous frequency. The optimal expression time of the mutant phenotype was 5–7 days after mutagen treatment. The recovery of FAT-resistant colonies in the selective medium was not affected by the presence of wild-type cells at a density below 9,000 cells per cm 2 . All 21 mutants tested exhibited thymidine auxotrophy; neither folinic acid nor deoxyuridine could support mutant cell growth. There was no detectable TS activity in all 11 mutants so far examined and only about 50% of wild-type activity in three prototrophic revertants, as measured by whole-cell and cell-free enzyme assays. The apparent Michaelis-Menten constant (K m ) for deoxyuridine-5′-monophosphate and inhibition constant (K i ) for 5-fluorodeoxyuridine-5′-monophosphate, measured by whole-cell enzyme assay, appear to be similar for the wild-type and revertant cell lines. Using 5-fluoro-[6 3 H]-2′-deoxyuridine 5′-monophosphate as active site titrant, the relative amounts of TS in crude cell extract from the parental, revertant, and mutant cells were shown to exist in a 1:0.5:0 ratio. Furthermore, the enzymes from two revertants were more heat labile than that of V79 cells. These properties, taken together, suggest that the FAT-resistant, thymidine auxotrophic phenotype may be the result of a structural gene mutation at the TS locus. The availability of such a mutant facilitates studies on thymidylate stress in relation to DNA metabolism, cell growth, and mutagenesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49874/1/1041200202_ftp.pd

Evaluation of methods for the estimation of mutation rates in cultured mammalian cell populations

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 P0 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].Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26754/1/0000306.pd

Single-step selection of mammalian cell mutants deficient in CTP synthetase

A single-step selection of Chinese hamster V79 cells deficient in CTP synthetase (CTPS − ) is presented. The underlying principle of the direct selection is the differential and efficient killing of synchronized wild-type cells through incorporation of [ 3 H] uridine and [ 3 H]thymidine. The CTPS − mutant cells were recovered by virtue of their not engaging in DNA synthesis, because (1) CTPS − cells are deficient in CTP synthetase and thus are unable to convert [ 3 H]UTP into [ 3 H]CTP, which eventually is converted into [ 3 H]dCTP and incorporated into DNA; (2) the growth of CTPS − mutant cells was arrested as a result of cytidine deprivation, thus escaping the killing by the incorporation of [ 3 H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 × 10 −7 . The mutant cells have no or very low CTP synthetase activity when tested by in vitro CTP synthetase assay or by whole-cell [ 3 H]uridine labeling assay. This modified “tritium suicide” method combined with the S-phase cell synchronization could provide a powerful means for the recovery from the cell population of nondividing mutant cells that are auxotrophic for some special nutrient requirement .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45536/1/11188_2005_Article_BF01534673.pd

Estimation of mutation rates in cultured mammalian cells

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 () inferred from the P0 method. The analysis can be made after the derivation of the variance of , which is a measure of variation of for a given combination of Nt and C in a number of repeat experiments. The variance of is inversely proportional to C and to the square of Nt. Nt determines the probability of occurrence of mutation 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 . The rate estimated from is biased, but the bias is in general 2 orders of magnitude smaller than . 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25103/1/0000535.pd

A deterministic approach for the estimation of mutation rates in cultured mammalian cells

Unequal growth rates between mutant and wild-type cells in a large population constitute a problem for the estimation of mutation rate. Over a period of cell growth, a selective advantage of one cell type over the other might lead to considerable error in the estimation of mutation rate if equal growth rates are assumed. In this study, we propose a formula and apply it to the estimation of spontaneous mutation rate in a growing population of Chinese hamster V79 cells in which ouabain-resistant mutant cells exhibit a slower growth rate than the wild-type cells. The formula is a generalization of that previously presented by Armitage (1953), and this is the first attempt to apply the deterministic approach for mutation rate estimation to cultured mammalian cells. The value of the estimated rate is compred with that derived from a parallel experiment using the fluctuation test of Luria and Delbruck (1943). The limitations and advantages of taking the deterministic approach to mutation rate estimation in mammalian cell systems are discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25744/1/0000304.pd

Modification of cell membrane in variants of chinese hamster cells resistant to abrin

Stable and heritable variants of Chinese hamster ovary (CHO) cells which are resistant to different levels (0.1, 1.0 and 10 [mu]g/ml) of the toxin abrin have been isolated and characterized. The frequency of resistant colonies to abrin was increased with the concentration of a chemical mutagen. There was no effect of cell density or cross-feeding on the recovery of variants. In experiments using fluorescein-labeled abrin and ricin which bind to terminal (non-sialylated) galactose residues of cell-surface oligosaccharides, parental cells exhibited strong binding toward both toxins, whereas no fluorescence was observed in the resistant clones. A fluorescein-conjugated lectin, BS II, which is specific for terminal N-acetyl--glucosaminyl residues, did not interact with the parental cells, but did with the resistant clones. This suggests that on the surface of resistant cells the number of terminal galactosyl residues of oligosaccharide chains in glycoproteins was reduced, exposing the penultimate N-acetyl--glucosaminyl residues. The number of available endogenous acceptor sites for galactosyl transferase in the abrin-resistant clones was directly proportional to the degree of resistance. In the presence of great excess of exogenous acceptor, the rates of galactosyl transfer were similar in all the abrin-resistant cell types tested, with levels ranging from 1.4 to 1.7 times parental cell values. Studies with tetraploid cell hybrids reveal that resistance was a recessive trait. Fluctuation analysis showed that abrin resistance occurred in CHO cell populations at a rate of 4-7 x 10-8/cell/generation. The system may serve as a new marker for quantitative mutagenesis studies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23138/1/0000062.pd

Comparative maturation of cynomolgus monkey oocytes in vivo and in vitro

BACKGROUND: In vitro maturation (IVM) of oocytes followed by fertilization in vitro (IVF) and embryo transfer offers an alternative to conventional IVF treatment that minimises drug administration and avoids ovarian hyperstimulation. However, the technique is less efficient than maturation in vivo. In the present study, a non-human primate model was used to address the hypothesis that the number of oocytes is increased and their nuclear and cytoplasmic maturity after IVM are improved when maturation is initiated in vivo by priming with hCG. METHODS: Young, adult cynomolgus monkeys were given recombinant human (rh) gonadotropins to stimulate the development of multiple follicles, and oocytes were aspirated 0, 12, 24, or 36 h after injection of an ovulatory dose of rhCG. The nuclear status of oocytes was determined at the time of recovery and after culture for a total elapsed time of 40–44 hours after hCG. RESULTS: Priming with hCG significantly increased the number of oocytes harvested, especially after delaying aspiration for 24 h or longer. Nuclear maturation after the full period in culture was also enhanced by priming: 71.5, 83.6, and 94.6% of oocytes collected at 0, 12, and 24 h hCG had progressed to MII by the end of the culture period, compared to 87.8% of oocytes that were retrieved at 36 h. A large proportion of oocytes reaching the MII stage had either or both abnormal spindles (>40%) and misaligned chromosomes (>60%), judging by immunofluorescence microscopy, but these abnormalities were independent of culture time. The mitochondria were evenly distributed throughout the cytoplasm at all stages of maturation. Importantly, there was no microscopic evidence that the duration of culture had any injurious effects on the cells. CONCLUSION: In conclusion, the evidence supports this non-human primate as a model for human IVM and the practice of priming with hCG to promote developmental potential

Overview of biologically digested leachate treatment using adsorption

Biological process is effective in treating most biodegradable organic matter present in leachate; however, a significant amount of ammonia, metals and refractory organic compounds may still remain in this biologically digested leachate. This effluent cannot be released to receiving bodies until the discharge limit is met. Several physical/chemical processes have been practiced as post-treatment to remove the remaining pollutants including coagulation–flocculation, oxidation and adsorption. Adsorption is often applied in leachate treatment as it enhances removal of refractory organic compounds. This chapter will focus on works related to adsorption as one of the commonly used methods to treat biologically digested leachate further down to acceptable discharge limit

Overview of biologically digested leachate treatment using adsorption

Biological process is effective in treating most biodegradable organic matter present in leachate; however, a significant amount of ammonia, metals and refractory organic compounds may still remain in this biologically digested leachate. This effluent cannot be released to receiving bodies until the discharge limit is met. Several physical/chemical processes have been practiced as post-treatment to remove the remaining pollutants including coagulation–flocculation, oxidation and adsorption. Adsorption is often applied in leachate treatment as it enhances removal of refractory organic compounds. This chapter will focus on works related to adsorption as one of the commonly used methods to treat biologically digested leachate further down to acceptable discharge limit