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E H Chu

Publications and source records attributed to E H Chu.

At least 37 records · Page 2Linked to original sources
PubMed1984

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 cm2. 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 (Km) for deoxyuridine-5'-monophosphate and inhibition constant (Ki) for 5-fluoro-deoxyuridine-5'-monophosphate, measured by whole-cell enzyme assay, appear to be similar for the wild-type and revertant cell lines. Using 5-fluoro-[6-3H]-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.

Aminopterin↗
PubMed1984

Pleiotropic mutants of Chinese hamster cells with altered cytidine 5'-triphosphate synthetase.

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.

3-Deazauridine↗
PubMed1984

The kinetics and feedback inhibition of cytidine 5'-triphosphate synthetase in wild-type and mutant Chinese hamster cells.

The kinetics and cytidine 5'-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5'-triphosphate (UTP). Three of the mutants had Kmapp and S50 values distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. All four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.

Animals↗
PubMed1983

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 NH4HCO3, 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 NH4HCO3 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. The synergistic combinations of L-glutamine with either NH4HCO3 or L-arginine had an absolute requirement for ATP; when ATP and PEP were absent no conversion of UTP to CTP occurred. The presence of GTP in a reaction mixture slightly increased the formation of CTP when L-glutamine and NH4HCO3 were used and substantially increased CTP formation when L-glutamine and L-arginine were used. De novo CTP synthesis was greatly reduced when nonradioactive CTP was added to an assay mixture, suggesting feedback inhibition. A TLC procedure has been developed that allows for the direct separation of UTP and CTP without requiring prior conversion to the mononucleotide or nucleoside level.

Animals↗
PubMed1983

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 (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.

Animals↗
PubMed1983

Mutation systems in cultured mammalian cells.

Experimental studies on the complex process of mutagenesis are conducted in the belief that they will increase our understanding of the effects of environmental agents on human populations. Cultured mammalian cells offer distinct advantages over other biological systems for studying this problem. Mutagenesis in cultured mammalian cells is unquestionably more relevant to man than are bacterial, fungal, or insect systems. At the same time, compared to whole-animal studies, cultured cells offer the advantage of ease of handling, low cost, and rapidity of assay. Mutation in cultured mammalian cells can be identified and measured at the phenotypic, protein, and DNA levels. Test systems have been developed to detect both gene and chromosome mutations. Direct evidence is available for true mutations occurring in mammalian cells, although some of the heritable variations could be epigenetic. It is now possible to identify the specific molecular alternations in mammalian cell mutants arising in vitro. The development of methods for the quantitation of mutation in cultured mammalian cells has been briefly considered in this presentation, particularly with reference to the choice of cell material, development of selective markers, and the various factors that affect quantitative measurement of mutations. Although notable progress has been made, much still remains to be learned to make these measurements more reliable. In applying the mammalian cell mutation assays for the screening of environmental mutagens, the choice of a system or systems should be based on their technical advantages and relevance to man. The basic parameters of the mutagen screening system must be well defined and controlled. The sensitivity for detection may be increased by the use of hypermutable cell strains. Appropriate statistical models to account for various sources of variability and the choice of statistical methods for the analysis of data are important considerations in screening environmental mutagens and carcinogens.

Animals↗
PubMed1981

BS I-B4 isolectin as a probe for an investigation of membrane alterations and transformation phenotypes of mouse L cells.

BS I-B4, an alpha-D-galactopyranosyl-binding isolectin from Bandeiraea simplicifolia seeds, was found to interact differently with transformed mouse L cells and non-transformed mouse 3T3 cells. The lectin induces detachment of 3T3 cells but increases adhesiveness and clustering of L cells. However, the induced cell aggregation does not lead to cell fusion. A variant clone of L cells, resistant to BS I-B4, which had lost the capacity for agglutination in the presence of the lectin, was isolated. Fluorescence binding studies of this variant suggest a lesion involving alpha and beta-D-galactopyranosyl units on its cell-surface structures. Although the variant cells form colonies in a methylcellulose medium, they do not produce tumours, as do the parental cells, when transplanted in athymic nude mice. The results demonstrate that alterations in cell membrane glyco-conjugates play an important role in tumourigenesis of animal cells, but anchorage-independent growth in vitro, as one of the transformation phenotypes, cannot be correlated absolutely with tumourigenicity in vivo.

Animals↗
PubMed1980

Duplication 8p syndrome: studies in a family with a reciprocal translocation between chromosomes 8 and 12.

We report a family in which six individuals were carriers of a translocation between chromosomes 8 and 12. The balanced carriers had a chromosomes constitution: 46,XX or 46,XY,t(8;12)(021;p13). Six individuals in five generations were mentally retarded. Three of them were examined; their chromosome constitution was 46,XX or 46,XYder(12),t(8;12)(p21;p13); thus they had a duplication of 8pter leads to 8p21 and possible deficiency of 12pter leads to 12p13. The activities of the enzymes that are coded by genes on 8p (glutathione reductase, GSR, E.C. 1.6.4.2.) and 12p (triosephosphate isomerase, TPI, E.C. 5.3.1.1.; lactate dehydrogenase-B, LDH-B, E.C. 1.1.1.27.; and glyceraldehyde-3-phosphate dehydrogenase, G3PD, E.C. 1.2.1.12.) were normal in these individuals. These findings helped in interpreting the position of the break points in the respective chromosomes. The phenotypic findings in our patients are discussed. Segregation analysis indicates no significant variation from a 25% recurrence risk for each of the possible genotypes in the offspring of balanced carriers.

Abnormalities, Multiple↗
PubMed1979

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 or N-AcO-AAF, but not the 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 individuals. Quantiation of damage to the DNA after 20 J . 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 repari synthesis. Moreover, we have shown previously that excision of UV-induced pyrimidine dimers in 2 of the 10 Cockayne cell strains was normal.

Carcinogens↗
PubMed1979

Interaction of wild-type and variant mouse 3T3 cells with lectins from Bandeiraea simplicifolia seeds.

An isolectin (BS I-B4) derived from Bandeiraea simplicifolia seeds and specific for terminal alpha-D-galactopyranosyl groups was found to be cytotoxic to Swiss 3T3 mouse cells. After mutagenesis and selection with BS I-B4, a variant clonal cell line resistant to both this isolectin and the alpha-D- and beta-D-galactose-binding lectin abrin was isolated. The parental cell line showed homogeneous and noninteracting binding sites for BS I-B4, whereas the variant cells exhibited a curved plot with a reduced number of binding regions. Another lectin, BS II, which is derived from the same seeds by specific for terminal N-acetyl-D-glucosaminyl groups, was cytotoxic to the variant but not the parental cells. These results suggest a possible lesion in the biosynthesis of cell surface structures resulting in the exposure of subterminal N-acetyl-D-glucosaminyl moieties in the variant line.

Acetylglucosamine↗
PubMed1978

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 Km for galactose, Ki for deoxygalactose, Km 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.

Animals↗
PubMed1977

Regional chromosomal localization of the human gene for galactose-1-phosphate uridyltransferase.

In the progeny of somatic cell hybrids formed by fusion of human lymphocytes and Chinese hamster mutant cells, a single human chromosome A2 was selectively retained when grown in appropriate medium. Spontaneous breakage of this chromosome in different hybrid subclones led to the assignment of the gene for galactose-1-phosphate uridyltransferase to the centromeric region of this chromosome (2q11 leads to 2q14). This gene is shown to be syntenic to the previously mapped genes for acid phosphatase 1 and malate dehydrogenase 1.

Acid Phosphatase↗
PubMed1977

Partial trisomy of the long arm of human chromosome 1 as demostrated by in situ hybridization with 5S ribosomal RNA.

In a newborn boy with multiple malformations, a tandem duplication was detected at the distal end of the long arm of one human chromosome 1. The Giemsa bands, 1q31 to 1q43--44, were repeated serially. Since 5S rRNA genes are located at 1q42--43, in situ hybridization of 125I 5S rRNA with fixed chromosome preparations was used to confirm the chromosomal duplication. The infant exhibited numerous developmental and clinical abnormalities as might be expected with an abnormality of chromosome structure relating to a ribosome component.

Chromosomes, Human, 1-3↗
PubMed1977

Chromosomes of lemuriformes. II. Chromosome polymorphism in Lemur fulvus collaris (E. Geoffroy 1812).

Two previously unreported diploid numbers, 2N = 50 and 2N = 51, from five individual Lemur fulvus collaris are described. In both chromosome complements, the nombre fondamental is 64. The 2N = 50 complement is composed of 7 pairs of bi-armed chromosomes, 17 pairs of acrocentric chromosomes with small short arms, and acrocentric sex chromosomes. The 2N = 51 complement is identical with these exceptions: Only one member equivalent to bi-armed pair 6 of L.f. collaris (2N = 50) is present, and two extra acrocentrics are found in the 2N = 51 complement. G-banding analyses suggest that these chromosomes are a heteromorphic pair of the Robertsonian type. This conculsion is supported by evidence from studies of meiotic pairing relationships of the three chromosomes and the complements of hybrids resulting from interspecific matings. Comparison of the 2N = 50 and 2N = 51 complements with a published 2N = 48 complement suggests that these new karyotypes do not provide a lineal link between the 2N = 52 and 2N = 48 karyotypes (Rumpler, Y., and R. Albignac 1969 C.R. Soc. Biol., 163: 1989-1992).

Animals↗