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Biomedical subjects

T Seno

Publications and source records attributed to T Seno.

At least 127 records · Page 7Linked to original sources
PubMed1982

Increased level of ribonucleotide reductase and associated resistance to aphidicolin in mouse FM3A cell mutants selected for simultaneous resistance to 9-beta-D-arabinofuranosyladenine and 1-beta-D-arabinofuranosylcytosine.

Mutants simultaneously resistant to 9-beta-D-arabinofuranosyladenine and 1-beta-D-arabinofuranosylcytosine were isolated by single-step selection from mutagenized mouse FM3A cells with a frequency of about 10(-5). Most of the mutants showed cross-resistance to aphidicolin and excess thymidine. In some of these mutants, the level of ribonucleotide reductase was 2 approximately 5 times that of the parent with concomitantly greater resistance to hydroxyurea than that of the parent, and their reductase showed normal sensitivity in vitro to the allosteric negative effector dATP. Consequently, the intracellular pools of dATP and dCTP were expanded in the mutants. These mutants thus differ from the aphidicolin-resistant mutants isolated previously, which have an altered reductase desensitized to dATP with a concomitant increase in the dATP pool. Therefore, either increase in the level of ribonucleotide reductase or its structural change leading to desensitization to dATP seems to confer resistance to the nucleoside analogs and to aphidicolin.

Animals↗
PubMed1981

Conditional thymidine auxotrophic mutants of mouse FM3A cells due to thermosensitive thymidylate synthase and their prototrophic revertants.

A number of temperature-sensitive conditional thymidine auxotrophs were isolated from mutagenized mouse FM3A cells. Upon temperature shift from 33.5 degrees C to 39.5 degrees C, the mutant cells rapidly lost thymidylate synthase activity with concomitant decrease in intracellular dTTP and changes in other dNTP pools. Thymidylate synthase obtained from these mutants was inactivated in a manner following first order kinetics by heat treatment, which did not affect the parental enzyme. When bound covalently to 5-fluoro-2'-deoxyuridine 5'-[32P]monophosphate and 5,10-methylenetetrahydrofolate, the enzyme of one mutant migrated slower than the parental enzyme on nondenaturing polyacrylamide gel electrophoresis, whereas it did not do so on sodium dodecyl sulfate gel electrophoresis. Spontaneous prototrophic revertants were isolated from most of the mutant lines. In some revertants, the enzyme regained the heat resistance of the wild type completely or partially. In other revertants, the enzyme was overproduced, but its heat-sensitive nature was unaltered. The results demonstrate unequivocally that the conditional thymidine auxotrophy in the mutants was caused by thermosensitive thymidylate synthase due, at least in one particular line, to a missense mutation in its structural gene.

Animals↗
PubMed1981

Alteration of ribonucleotide reductase in aphidicolin-resistant mutants of mouse FM3A cells with associated resistance to arabinosyladenine and arabinosylcytosine.

Aphidicolin-resistant mutants of mouse FM3A cells were isolated and characterized. Most of the mutants were of a type showing associated resistance to arabinosyladenine, arabinosylcytosine, deoxyadenosine, and excess thymidine. This phenotype could also be observed in a variant line selected by resistance to a low level of arabinosylcytosine. In cell-cell hybrids, aphidicolin resistance as well as this cross-resistance behaved a codominant traits. The mutants had an increased dATP pool and decreased ability to incorporate labeled deoxycytidine into macromolecules. Genetic and biochemical evidence suggested that the mutation conferring the pleiotropic phenotype resulted from a change in ribonucleotide reductase activity such that the enzyme was desensitized to the allosteric negative effector dATP. This alteration of the enzyme could account for the marked change in deoxynucleotide pools and for the aphidicolin resistance of the mutants.

Adenosine Triphosphate↗
PubMed1981

Selection of mammalian thymidine auxotrophic cell mutants defective in thymidylate synthase by their reduced sensitivity to methotrexate.

Thymidine auxotrophic mutants were selectively isolated from mutagenized mouse FM3A cells by resistance to methotrexate in the presence of thymidine and 5-methyl-tetrahydrofolate with a frequency of 10(-5)-10(-6). In most of the thymidine auxotrophs the activity of thymidylate synthase was very low or undetectable, but dihydrofolate reductase activity was normal. Upon starvation of thymidine, the mutant cells immediately stopped growing and started to lyse within one day. In the presence of thymidine, the mutant cells grew quite normally. This phenotype behaved recessively in cell-cell hybrids, and the segregation profile of its marker indicated that the lesions in the mutants are not linked to the X chromosome. Prototrophic revertants could be isolated from these mutants, and they showed almost the normal level of thymidylate synthase activity. The selection method described here should be useful for isolating large numbers of thymidylate synthase-negative mutants from various mammalian cell lines.

Animals↗
PubMed1981

Heterogeneous forms of poly(rA) . oligo(dT)-directed DNA polymerase activity from rat spleen.

Three forms of DNA polymerase, named enzymes A, B, and C, that preferred (rA)n x (dT)12-18 as a template-primer, were partially purified from an extract of rat spleen. Enzymes B and C, both sedimenting at 9S, appeared to correspond to DNA polymerase gamma. However, they differed in their behavior on phosphocellulose and DNA-cellulose column chromatographies, and in their optimum KCl and divalent cation requirements for activity. Enzyme A showed a unique property. Like DNA polymerase beta, it sedimented at 3.8S, was resistant to reagents blocking sulfhydryl groups, and was inhibited by phosphate, but it differed from DNA polymerase beta with respect to elution positions from DEAE-cellulose, phosphocellulose and DNA-cellulose columns, Km value (lower by one order of magnitude for dTTP), and template-primer preference. Enzyme A was found in the mitochondrial fraction, in which DNA polymerase beta was not detectable. Enzymes A and C were isolated from the nuclear fraction, but this fraction did not contain enzyme B. The cytosol contained only enzyme A. The mitochondrial fraction contained enzyme A and enzyme C-like polymerase. Enzyme B was obtained with enzymes A and C only by extraction of the whole cell homogenate. Enzyme B may be labile or may be an artificial form of DNA polymerase gamma formed during the purification procedures.

Animals↗
PubMed1981

Inhibition of DNA polymerases alpha and gamma by rifamycin derivative AF/013.

The nature of the inhibitory effects of rifamycin derivative AF/013 (O-n-octyloxime of rifamycin SV) on DNA polymerases alpha and gamma were studied. Lineweaver-Burk analysis of the inhibition of DNA polymerases with respect to a substrate and template-primer showed a different mode of inhibition by AF/013 for each: the inhibition of DNA polymerase gamma was competitive with both dTTP and poly(rA)-oligo(dT), while that of DNA polymerase alpha was competitive with activated calf thymus DNA and non-competitive with dTTP. Further analysis of the competitive mode of the inhibition of DNA polymerase alpha, using poly(dT)-oligo(rA) as a template-primer, demonstrated that the primer molecule competed with AF/013. A change of effective divalent metal ion (Mn2+ in place of Mg2+) in the reaction mixture did not alter this competitive mode of inhibition with respect to the template-primer. The results of experiments to obtain further insight into the mechanism of drug-enzyme interaction suggest that AF/013 binds tightly to DNA polymerase alpha, and inhibits the process of chain elongation with DNA polymerases alpha and gamma.

Animals↗
PubMed1981

Resistance to methotrexate in thymidylate synthetase-deficient mutants of cultured mouse mammary tumor FM3A cells.

Growth inhibition by methotrexate (MTX) of a cultured mouse mammary carcinoma FM3A line and its mutants deficient in thymidine kinase and thymidylate synthetase was studied under a variety of conditions. In medium containing 10 microM thymidine, the thymidylate synthetase-deficient mutants were slightly more resistant to MTX than the wild-type line and the thymidine kinase-deficient mutant. The addition of both 10 microM thymidine and 50 microM hypoxanthine to the medium completely eliminated the inhibition by MTX of the wild-type and thymidylate synthetase-deficient mutants but had no effect on inhibition of the thymidine kinase-deficient mutant. Thus, addition of either thymidine or purine alone was not sufficient to protect FM3A cells against the growth inhibitory effect of MTX. In contrast, addition of a small amount of 5-methyltetrahydrofolate to medium containing 10 microM thymidine caused an increase of several orders of magnitude in the resistance of thymidylate synthetase-deficient mutants to MTX but did not affect that of the wild-type line. Wild-type cells became almost as resistant to MTX as did the mutant cells by addition of 1 microM 5-fluorodeoxyuridine as well as a small amount of 5-methyltetrahydrofolate with thymidine. These results show directly that thymidylate synthetase is essential in determining the cytotoxicity of MTX by modulating the intracellular tetrahydrofolate pool.

Animals↗
PubMed1981

Species difference in liver microsomal and cytosolic enzymes involved in mutagenic activation of N-hydroxy-N-2-fluorenylacetamide.

The mutagenic activations of N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) by subcellular fractions of the livers of the Sprague-Dawley rat, C57BL mouse, Hartley guinea pig, Syrian golden hamster, and Macaca fuscata fuscata monkey were examined for sensitivity to paraoxon, which inhibits a deacetylase but not an arylhydroxamic acid acyltransferase. The mutagenic activation by liver microsomes was almost entirely mediated by a paraoxon-sensitive enzyme in all the animals tested. In contrast, the mutagenic activation by liver cytosol was mediated mostly by a paraoxon-sensitive enzyme in mice and guinea pigs, mostly by a paraoxon-resistant enzyme in rats and hamsters, and by both enzyme types in the monkey. In rats and guinea pigs, attempts were made to identify the enzymes causing mutagenic activation in the liver cytosol by a comparison of the elution positions of these enzymes in gel filtration and DEAE-cellulose column chromatography with those of known enzymes. In the rat liver cytosol, the mutagenic activation was mediated not only by acyltransferase but also by an unknown enzyme, which was resistant to paraoxon and differed from acyltransferase in chromatographic behavior. In the guinea pig liver cytosol, the activation was due to deacetylase activities, which could be separated into four fractions by gel filtration. These data indicate that species differ in the kinds of liver cytosolic enzymes involved in mutagenic activation of N-OH-2-FAA but not in the kind of liver microsomal enzyme involved.

2-Acetylaminofluorene↗
PubMed1981

Effect of tunicamycin on production by mouse fibroblast L929 cells of the factor-stimulating differentiation of mouse myeloid leukemic cells and the colony-stimulating factor.

Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor), which is suggested to be a glycoprotein. On the other hand, growth and differentiation of normal precursor cells of macrophages and granulocytes can be stimulated by a glycoprotein termed colony-stimulating factor (CSF). Mouse fibroblast L929 cells were found to produce both the D-factor and CSF. The properties of the D-factor and CSF and the roles of carbohydrates in the molecules of these factors were examined using tunicamycin, a specific inhibitor of asparaginase-linked glycosylation. Although both the D-factor and CSF were produced by L-cells in usual medium containing fetal calf serum, production of D-factor, but not CSF, was reduced by omission of serum from the medium. The activity of the D-factor was slightly decreased by treating the L-cells with tunicamycin (0.5 microgram/ml) in the presence of 2% fetal calf serum, without any decrease in CSF activity. Conditioned medium of L-cells incubated with or without tunicamycin was fractionated by gel filtration on a Sephadex G-200 column. Normal D-factor appeared as a single peak with an apparent molecular weight of 67,000. D-factor produced in the presence of tunicamycin had an apparent molecular weight of 25,000. On the other hand, most of the CSF was eluted in the void volume, even when it was produced in the presence of tunicamycin. The D-factor produced in the presence of tunicamycin was more sensitive than normal D-factor was to trypsin or heat treatment at 70 degrees. The CSF produced in the presence of tunicamycin was resistant to these treatments. These results indicate that the D-factor is distinct from CSF. Furthermore, the results suggest that the D-factor produced by L-cells is also a glycoprotein and that, although carbohydrate is not essential for production or activity of the D-factor, it contributes to stabilizing the protein portion of D-factor.

Animals↗
PubMed1980

DNA synthesis in isolated chromatin. Nature of activities, and relationship to kinetics of DNA polymerase release from chromatin DNA.

Chromatin isolated from Ehrlich ascites tumor cells showed two DNA synthetic activities differing in sensitivity to N-ethylmaleimide. For studies on the nature of activities and relationship to kinetics of DNA polymerase, a new method was developed for detecting the activity of DNA polymerase released from chromatin DNA during DNA synthesis in vitro. The activity of DNA polymerase released was measured in a reaction mixture for DNA synthesis using exogenously added poly(dA-dT) as a template-primer in the presence of actinomycin D. Evidence that the DNA polymerase released was actually involved in DNA synthesis of chromatin was obtained in experiments using chromatin isolated from cells treated with various concentrations of 1-beta-D-arabinofuranosylcytosine and chromatin from adult mouse liver. The experiments showed that chromatin isolated from cells in which only small amount of DNA polymerase was engaged in DNA synthesis released a negligible amount of DNA polymerase, especially N-ethylmaleimide-sensitive polymerase. Kinetic analysis of DNA polymerase during chromatin DNA synthesis by the new method suggested that KCl at the optimal concentration (10-20 mM) for the N-ethylmaleimide-sensitive chromatin activity enhanced the binding of the N-ethylmaleimide-sensitive DNA polymerase to chromatin DNA. From the findings that addition of actinomycin D or omission of dNTPs from the preincubation mixture prevents this binding, it is suggested that the binding of DNA polymerase is followed by the DNA chain synthesis and that the DNA polymerase involved in this reaction is N-ethylmaleimide sensitive. Data on the effect of KCl on the rate of chromatin DNA synthesis and on the size of the DNA chain favor this assumption.

Animals↗
PubMed1980

Structural changes in the glutamine-chargeable Escherichia coli transfer RNA-Trp produced by chemical modification with sodium bisulfite.

Glutamine-mischargeable tRNA produced by sodium bisulfite-treated Escherichia coli tRNA-Trp was isolated by dihydroxyboryl-cellulose affinity column chromatography. This tRNA was shown to have dual specificity tryptophan and glutamine, and, when charged with either amino acid, bound to ribosomes in response to the non-sense codon UAG but not in response to the tryptophan codon UGG. The results were consistent with the reported properties of Su+7 tRNA. The bisulfite-treated tRNA-Trp migrated as two bands during polyacrylamide gel electrophoresis. The faster moving band (band I) coincided with that of untreated tRNA-Trp. The slower moving band (band II) coincided with the glutamine-chargeable tRNA-Trp. Su+7 tRNA behaved like band II tRNA upon gel electrophoresis. Nucleotide sequence analysis showed that a cytidine-uridine transition occurred at the 1st or the 2n position of the anitcodon of band II tRNA. Band I and band II tRNAs differed from each other in their thermal melting profiles. It is suggested that the single base change in the anticodon is responsible for the altered conformation of band II tRNA.

Autoradiography↗
PubMed1980

Single-step selection of mouse FM3A cell mutants defective in thymidylate synthetase.

A tritium-suicide method for isolating thymidine auxotrophic mutants is described. Mutagenized mouse FM3A cells were cultured in medium containing [3H] deoxyuridine. Most of the surviving clones examined showed a phenotype of absolute thymidine auxotrophy. This phenotype is very stable and was found to be genetically recessive in cell-cell hybridization experiments. The growth of these variant clones was not supported by various pyrimidine nucleosides other than thymidine. The activity of thymidylate synthetase in crude extracts of these clones was less than 1% of that of FM3A cells. These results strongly indicate that the thymidine auxotrophic phenotype resulted from a genetic defect in thymidylate synthetase.

Animals↗