Tight association of DNA primase with a subspecies of mouse DNA polymerase alpha.

Evidence was obtained for tight association of DNA primase activity with a subspecies of mouse DNA polymerase alpha by study with immunoadsorption assay using two monoclonal antibodies specific for human DNA polymerase alpha that have been shown to react with mouse murine myeloma DNA polymerase alpha (Tanaka, S., Hu, S.-Z.

Unusual sensitivity to bleomycin and joint resistance to 9-beta-D-arabinofuranosyladenine and 1-beta-D-arabinofuranosylcytosine of mouse FM3A cell mutants with altered ribonucleotide reductase and thymidylate synthase.

This paper describes unusual sensitivity to bleomycin; resistance to aphidicolin, 9-beta-D-arabinofuranosyladenine, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea; and a mutator phenotype as common phenotypes of two classes of previously reported mouse FM3A cell mutants with distinct biochemical defects. One class of mutants, selected for aphidicolin resistance, contains altered ribonucleotide reductase desensitized to deoxyadenosine triphosphate, a negative allosteric effector, and the other class contains temperature-sensitive thymidylate synthase. Abnormal levels of deoxyribonucleoside triphosphate pools, especially those of deoxyadenosine triphosphate, were the common biochemical trait for both classes of mutants.

Unusual aspects of human thymidylate synthase in mouse cells introduced by DNA-mediated gene transfer.

Thymidylate synthase-negative mutants of mouse FM3A cells were transformed to thymidine prototrophs by human DNA. The stable transformants had only human thymidylate synthase and segments of human DNA. They grew normally but had unusually high levels of the human enzyme.

OSa, a 'new' low-frequency red cell antigen.

A new low-frequency red cell antigen, OSa, in a Japanese blood donor is inherited as a Mendelian dominant. A family study showed that OSa is not controlled by ABO, P1, Rh, Kidd, or Diego loci, nor is the antigen X- or Y-borne.

Mouse DNA replicase. DNA polymerase associated with a novel RNA polymerase activity to synthesize initiator RNA of strict size.

De novo DNA synthesis on poly(dT) by a novel mouse DNA polymerase, here named "DNA replicase," was examined for the synthesis of RNA which functions as a primer in the subsequent synthesis of DNA. As has been reported previously (Yagura, T., Kozu, T.

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.

Arrest of chain growth of replicon-sized intermediates by aphidicolin during rat fibroblast cell chromosome replication.

The effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, on size maturation of nascent DNA intermediates was studied in cultured rat fibroblast cells. Results provided the first evidence of DNA synthesis associated with merging of intermediates of larger than replicon size. Aphidicolin at a concentration (1.

Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization.

A mouse DNA polymerase accompanied by a novel RNA polymerase activity and its specific protein factor (stimulating factor) were purified from Ehrlich ascites tumor cells and partially characterized. The DNA polymerase was thought to be a subspecies of DNA polymerase alpha, and to be accompanied by or copurified with RNA polymerase activity capable of synthesizing RNA, which was probably utilized as a primer for subsequent DNA polymerization on a template of poly(dT) or poly(dC). This coupled reaction by RNA and DNA polymerase activities required the stimulating factor in addition to ribo- and deoxyribonucleotide substrates, although the degree of requirement depended on the kind of template and ribonucleotide substrate: the activity to incorporate dATP with poly(dT) plus ATP depended greatly on the stimulating factor, while the activity to incorporate dGTP with poly(dC) did not when GTP was added at high concentrations.

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.

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.

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.

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.

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.

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.

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.

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.

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.