MUTATION SYSTEMS IN CULTURED MAMMALIAN CELLS *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73274/1/j.1749-6632.1983.tb47827.x.pd

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

Effects of DNA damaging agents on cultured fibroblasts derived from patients with Cockayne syndrome

The cytotoxic action of physical and chemical agents on 10 skin fibroblast strains in culture derived from individuals with Cockayne's syndrome was measured in terms of colony-forming ability. As compared to fibroblasts from normal donors, all Cockayne cell strains tested exhibited a significantly increased sensitivity to UV light and a normal sensitivity to X-rays. Cells from two sets of parents of unrelated Cockayne children showed an intermediate level of UV sensitivity. There was no effect of 0.5 mM caffeine on UV survival in normal and two Cockayne strains tested, indicating that postreplicational repair in Cockayne cells as measured by caffeine sensitivity was probably normal.Sensitivity of normal and Cockayne cells to the chemical carcinogens and mutagens 4NQO, N-AcO-AAF, ICR-170 and EMS was also compared. An increased sensitivity of Cockayne cells to 4NQO of N-AcO-AAF, but not to ICR-170 or EMS, was observed. However, unlike the intermediate UV sensitivity, the cell strains from two parents of Cockayne patients showed the same sensitivity to N-AcO-AAF or 4NQO as fibroblasts from normal individulas.Quantitation of damage to the DNA after 20 J [middle dot] m-2 UV irradiation indicates normal levels of [3H]thymidine incorporation in the Cockayne cells, in contrast to UV-irradiated xeroderma pigmentosum cells (XP 12BE) in which there was a very low level of repair synthesis. Moreover, we have shown previoulsy that excision of UV-induced pyrimidine dimers in 2 of the 10 Cockayne cell strains was normal.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23664/1/0000632.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

The importance of early immunotherapy in patients with faciobrachial dystonic seizures.

Faciobrachial dystonic seizures and limbic encephalitis closely associate with antibodies to leucine-rich glioma-inactivated 1 (LGI1). Here, we describe 103 consecutive patients with faciobrachial dystonic seizures and LGI1 antibodies to understand clinical, therapeutic and serological differences between those with and without cognitive impairment, and to determine whether cessation of faciobrachial dystonic seizures can prevent cognitive impairment. The 22/103 patients without cognitive impairment typically had normal brain MRI, EEGs and serum sodium levels (P < 0.0001). Overall, cessation of faciobrachial dystonic seizures with antiepileptic drugs alone occurred in only 9/89 (10%) patients. By contrast, 51% showed cessation of faciobrachial dystonic seizures 30 days after addition of immunotherapy (P < 0.0001), with earlier cessation in cognitively normal patients (P = 0.038). Indeed, expedited immunotherapy (P = 0.031) and normal cognition (P = 0.0014) also predicted reduced disability at 24 months. Furthermore, of 80 patients with faciobrachial dystonic seizures as their initial feature, 56% developed cognitive impairment after 90 days of active faciobrachial dystonic seizures. Whereas only one patient developed cognitive impairment after cessation of faciobrachial dystonic seizures (P < 0.0001). All patients had IgG4-LGI1 antibodies, but those with cognitive impairment had higher proportions of complement-fixing IgG1 antibodies (P = 0.03). Both subclasses caused LGI1-ADAM22 complex internalization, a potential non-inflammatory epileptogenic mechanism. In summary, faciobrachial dystonic seizures show striking time-sensitive responses to immunotherapy, and their cessation can prevent the development of cognitive impairment.awx323media15681705685001

Apparent synergism between amino donors for CTP synthesis in Chinese hamster fibroblasts

The synergistic effects of potential amino donors were studied in the assay of CTP synthetase in extracts of Chinese hamster fibroblasts. We found that L-glutamine was not effective as the sole amino donor, but combinations of L-glutamine with NH 4 HCO 3 , L-arginine or potassium phosphate did result in the conversion of UTP to CTP. L-arginine or potassium phosphate were also not effective when used alone, and NH 4 HCO 3 was only slightly effective. Our studies demonstrate that the individual synergistic combinations were not additive; multiple combinations of components decreased rather than increased the formation of CTP.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45347/1/11010_2004_Article_BF00223531.pd

Public healthcare financing and provision in Hong Kong : a public-private partnership approach

published_or_final_versionPolitics and Public AdministrationMasterMaster of Public Administratio

Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer

Cytidine-triphosphate synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2.) has been purified over 31 000-fold to homogeneity with 17% recovery from rat liver cytosol, using high-performance liquid chromatography (HPLC) techniques. The presence of CTP synthetase monomer, dimer and tetramer has been demonstrated in the ammonium sulfate fraction of rat liver cytosol. By gel-permeation HPLC, the molecular weights of the three molecular forms of the enzyme have been estimated as 240 000 (tetramer), 120 000 (dimer) and 60 000 (monomer). By gel-permeation chromatography on Bio-Gel A-1.5m column, the molecular weights of dimer and monomer were estimated as 100 000 and 50 000, respectively. The molecular weight of the monomeric subunit is determined to be 66 000 by SDS-polyacrylamide gel electrophoresis. Monomers isolated fresh from 0-30 (NH4)2SO4 fraction of rat liver cytosol are enzymatically active. Purified rat liver CTP synthetase exhibited sigmoidal kinetic plots as a function of the substrate UTP in the presence of the end-product, CTP. Partially purified CTP synthetase usually forms an inactive coagulum on freezing and subsequent thawing. Incubation of CTP synthetase dimer at 25[deg]C for 1 h in the presence of UTP, ATP and Mg2+ resulted in optimum conversion to tetramer with least inactivation. The purified tetramer dissociates to dimers when UTP, ATP and Mg2+ are removed by dialysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27539/1/0000583.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

Inverse relationship between galactokinase activity and 2-deoxygalactose resistance in Chinese hamster ovary cells

Galactokinase activity is reduced in 12 independent clones of Chinese hamster ovary cells resistant to 2-deoxygalactose. The frequency of resistant colonies is increased with chemical mutagens. The resistant phenotype is stable in the absence of selection. There is an inverse correlation between the levels of galactokinase activity and the cloning efficiency in deoxygalactose. Cells with high resistance have 1%or less of the enzyme activity observed in the parental cells; while cells with low resistance have 10–30% galactokinase activity. Studies with tetraploid hybrid cells reveal that resistance to deoxygalactose is a recessive trait and that cells with high resistance do not complement those with low resistance. In cell lines with low resistance, the K m for galactose , K i for deoxygalactose , K m for ATP, and thermolability were not significantly altered compared to sensitive parental cells. Although the possibility of mutation at the structural gene locus has not been ruled out, the reduced enzyme activity may also be due to mutation at a regulatory site which affects the number of galactokinase molecules per cell .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45552/1/11188_2005_Article_BF01543159.pd