Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.

A system of DNA replication in HeLa nuclei treated with inhibitors of protein synthesis.

An in vitro DNA synthesizing system consisting os isolated nuclei from HeLa cells which had been treated with inhibitors of protein synthesis was investigated. Treatment with both 30 microgram/ml cycloheximide and 10 microgram/ml puromycin of S-phase cells reduced the rate of DNA synthesis immediately; however, the overall DNA synthesis continued for up to 4 h with a diminished rate and then ceased. In the nuclei which were isolated from the cells which had been incubated with these drugs for 6 h, little incorporation of [3H]TTP into acid-insoluble materials was observed. Addition of cytosol prepared from cells actively synthesizing DNA induced the incorporation of [3H]TTP in these nuclei, while little induction was observed by the addition of cytosol prepared from drug-treated cells in spite of the fact that the latter cytosol stimulated DNA synthesis in isolated nuclei from non-treated cells. The induced DNA synthesis was shown to require Mg2+, all four deoxyribonucleoside triphosphates and ATP, and to proceed discontinuously. The activity inducing DNA synthesis in drug-treated nuclei fluctuated with the phases in a cell cycle and it was not ascribed solely to DNA polymerase alpha nor to DNA ligase.

DNA repair synthesis in HeLa cell lysate.

In order to study the mechanism of DNA repair, we established an in vitro system for repair (unscheduled) DNA synthesis. HeLa cells synchronized at G2-G1 phase were irradiated with ultraviolet light in the presence of two DNA replication inhibitors, hydroxyurea and 1-beta-D-arabinofuranosyl cytosine (araCyt), to reduce the replicative DNA synthesis as much as possible. Hypotonic treatment of the cells was followed by gentle homogenization, and the resulting cell lysate was incubated with [3H]dTTP. The lysate system required all four dXTPs and Mg2+, but required no ATP. The incorporation of [3H]dTTP was dependent on the dose of ultraviolet light, was linear for 2 min, and reached the maximum at 5 min. The presence of hydroxyurea and araCyt during in vivo incubation was necessary for in vitro DNA synthesis. Accumulation of single-strand breaks was observed under these conditions, and this could explain the very high incorporation of [3H]dTTP in this system.

ATP requirement for the processes of DNA replication in isolated HeLa cell nuclei.

The ATP requirement for two steps of DNA replication, the synthesis and subsequent joining of Okazaki fragments, was investigated by using isolated HeLa cell nuclei. Among adenine nucleotides tested, high levels of dATP and ADP stimulated DNA synthesis. In the presence of high levels of ATP, the addition of high levels of dATP or ADP resulted in about 70% inhibition of DNa synthesis. The optimal concentration of ATP for the stimulation of DNA synthesis varied depending on the magnesium ion concentration. When the molar ratio of magnesium ion to ATP was approximately 1, maximal stimulation was attained. Product analysis by sedimentation in an alkaline sucrose gradient revealed that both Okazaki fragments and high molecular weight DNA were synthesized in the presence of high levels of ATP, whereas in the case of dATP and ADP, little high molecular weight DNA was synthesized. The ability to synthesize high molecular weight DNA was restored to nuclei by adding low levels of ATP in the presence of high levels of ADP but not of dATP.

Metabolism of benzo[A]pyrene and the related enzyme activities in hamster embryo cells.

The rate of metabolism of benzo[a]pyrene (BP) and changes in related enzyme activities in cultured hamster embryo cells during successive subculture were studied. The activity of aryl hydrocarbon hydroxylase (AHH) was the highest when embryo cells were first dispersed in tissue culture flasks and decreased during subsequent passages. On the other hand, UDP-glucuronyl transferase activity increased gradually during successive subculture. Treatment of the cells with 13 nmol/ml of benz[a]anthracene (BA) for 24 h increased the activity of AHH but not that of UDP-glucuronyl transferase. The metabolism of BP was measured in cells of the passages 1, 3 and 7; metabolism of BP was most efficient in cells in passage 3 and their formation of glucuronic acid conjugates of BP, one of the major metabolites found in the medium, was 3- and 10-fold more than those of cells in passages 1 and 7, respectively. Analysis of BP-metabolites extracted from the medium with ethylacetate showed that the main metabolites were 9,10-diol and 7,8-diol. Phenols and quinones were released by treatment of the medium with beta- glucuronidase and their amounts were larger than those of diols at all passages. These results show that in hamster embryo cells in early passage, BP is metabolized to conjugates of phenols with glucuronic acid.

Inhibitory effects of antipromoters and radical scavengers on the promotion process in two-stage cell transformation.

To find the mechanism of promotion process, we have investigated the antipromoting effects of radical scavengers and specific inhibitors for phospholipid metabolism and for protein kinase C using a two-stage transformation assay system in BALB/3T3 cells. All radical scavengers and inhibitors tested showed the antipromoting effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted transformation. Diacylglycerols, activators of protein kinase C, showed promoting effects in vitro and the promoted-transformation by them was suppressed by radical scavengers employed. By an electron spin resonance (ESR) spin-trapping method, inhibitors, which suppressed promoted-transformation by TPA markedly, had .OH scavenging action. It was found using a ESR spin-trapping method that treatment of TPA on BALB/3T3 cells generates .OH in a dose-dependent manner. These results suggest that generation of oxygen radicals, especially .OH, which occurs in the processes of phospholipid metabolism as well as activation of protein kinase C, is essential to the promotion process.

A mammalian cell mutant with temperature-sensitive thymidine kinase.

We have isolated a mutant clone from mouse FM3A cells with temperature-sensitive defects both in cytokinesis and in thymidine kinase enzyme activity. The clone, designated tsCl.B59, was isolated after mutagenesis at 33 degrees C followed by exposure to cytosine arabinoside at 39 degrees C. It was derived from a thymidine kinase deficient, 5-bromodeoxyuridine-resistant clone (S-BUCl.42) which was originally derived from wild-type clone H-5 of FM3A cells. The temperature-sensitive mutant clone grows normally at 33 degrees C, but not at 39 degrees C, where it exhibits an increased frequency of multinucleate cells due to defective cytokinesis. Unlike the parental S-BUCl.42 cells, which have negligible thymidine kinase activity and are unable to incorporate 3H-thymidine, the mutant in corporates substantial amounts of 3H-thymidine at 33 degrees C, although its thymidine kinase activity remains lower than that of wild-type H-5 cells. When cultures of tsCl.B59 cells are transferred to 39 degrees C, incorporation of 3H-thymidine decreases markedly. The decrease has been shown to be due to thermolability of the thymidine kinase in tsCl.B59 cells.

X-linked hypoxanthine-guanine phosphoribosyltransferase deficiency without neurological disorders. a report of a family.

A patient is reported with X-linked hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency. He had gout but no neurological symptoms. The patient had negligible HGPRT activity as determined by thin layer chromatography and liquid scintillation counting. Autoradiography of fibroblast cultures revealed no uptake of -3H-hypoxanthine. His mother and two sisters were shown to be heterozygotes.

Exposure of DNA bases induced by the interaction of DNA and calf thymus DNA helix-destabilizing protein.

The reaction of chloroacetaldehyde with adenine bases in DNA to give a fluorescent product was used to study the availability to intermolecular reaction of positions 1 and 6 of adenine in DNA complexes with calf thymus DNA helix-destabilizing protein. No inhibition of this reaction was observed when heat-denatured DNA was complexed with the protein at a protein/DNA weight ratio of 10:1, compared to free DNA. On the contrary, the same reaction was inhibited markedly for denatured DNA in the presence of calf thymus histone HI at protein/DNA weight ratio of 2:1. Furthermore, the exchange rate for hydrogens of amino and imide groups of DNA bases in DNA strands with deuterium in the solvent was totally unaffected upon complexing of DNA with the DNA helix-destabilizing protein as examined by stopped-flow ultraviolet spectroscopy. These results indicate that the DNA helix-destabilizing protein forms a complex with single-stranded DNA, leaving DNA bases uncovered by the protein. The fluorescence intensity of DNA pretreated with chloroacetaldehyde was amplified by nearly 3-fold upon addition of the DNA helix-destabilizing protein. The possibility of "unstacking" of DNA bases induced by the protein is discussed.

Association of virus specific replicative ribonucleic acid with nuclear membrane in chick embryo cells infected with japanese encephalitis virus.

Using high resolution electron microscopic autoradiography and velocity sedimentation, RNA synthesis was examined in chick embryo cells infected with Japanese encephalitis virus (JEV). RNA was labelled with 3H-uridine for 5 min or 10 min at 15 h after infection in the presence of actinomycin D and D-glucosamine. Microautoradiography showed significantly numbers of silver grains on the nuclear membranes of 5 min pulse-labelled thin cell sections. The RNA species in membrane fractions obtained from the nucleus and cytoplasm of the infected cells were analysed by sucrose density gradient sedimentation. Radioactive 23S replicative form RNA and 8-12S RNA were obtained from the outer membrane fractions of the nuclear envelope. Labelled 42S virus RNA was obtained from the fractions containing large vesicle membranes and plasma membranes. These results suggest that JEV-RNA synthesis is initiated in the perinuclear region in close association with the outer membranes of the nuclear envelope.