Effect of cyclic nucleotides and hormones of homologous methylation in nuclear homogenates of rat bone marrow

In vitro and in vivo studies showed that methylation of homologous DNA in nuclear homogenates of rat bone marrow is controlled by cyclic nucleotides and some hormones. The cyclic nucleotides and their dibutyryl analogs inhibited homologous methylation of DNA in the presence and absence of phosphodiesterase inhibitors. In the presence of Ca2+ the inhibiting effect of the nucleotides was more pronounced. Prostaglandins inhibited DNA methylation in a weaker degree while insulin and erythropoietin had a stronger effect in comparison with the nucleotides. Histamine stimulated DNA methylation, whereas acute hypoxic hypoxia caused a reduction in the rate of DNA methylation.     

Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase β

In the present study, we investigated mammalian polymerases that consecutively incorporate various fluorophore-labeled nucleotides. We found that rat DNA polymerase β (pol β) consecutively incorporated fluorophore-labeled nucleotides to a greater extent than four bacterial polymerases, Sequenase Version 2.0, Vent_R (exo-), DNA polymerase IIIα and the Klenow fragment, and the mammalian polymerases DNA polymerase α and human DNA polymerase δ, under mesophilic conditions. Furthermore, we investigated the kinetics of correct or mismatched incorporation with labeled nucleotides during synthesis by rat pol β. The kinetic parameters K_m and k_cat were measured and used for evaluating: (i) the discrimination against correct pair incorporation of labeled nucleotides relative to unlabeled nucleotides; and (ii) the fidelity for all nucleotide combinations of mismatched pairs in the presence of labeled or unlabeled nucleotides. We also investigated the effect of fluorophore-labeled nucleotides on terminal deoxynucleotidyl transferase activity of rat pol β. We have demonstrated for the first time that mammalian pol β can consecutively incorporate various fluorophore-labeled dNTPs. These findings suggest that pol β is useful for high-density labeling of DNA probes and single-molecule sequencing for high-speed genome analysis.     

Nuclear matrix-bound DNA primase. Elucidation of an RNA priming system in nuclear matrix isolated from regenerating rat liver

Recent findings in purified systems demonstrate the universality of DNA polymerase-primase complexes which may function in the priming and continuation of eucaryotic DNA replication. In this report we characterize an in vitro, nuclear matrix-associated, priming and continuation system that can utilize either endogenous matrix-bound DNA or exogenous single-stranded DNA as template. 30-40% of total nuclear DNA primase activity was recovered in association with the isolated nuclear matrix fraction from regenerating rat liver. Matrix-bound primase catalyzed the alpha-amanitin, actinomycin D-resistant synthesis of oligonucleotide chains of 8-50 nucleotides on the endogenous template. At least a portion of the RNA primers were continued by DNA polymerase alpha with deoxynucleoside triphosphate incorporation up to 300-600 nucleotides. Nearest neighbor analysis revealed ribodeoxynucleotide covalent linkages in these RNA-DNA chains. The matrix-bound primase preferred single-stranded fd DNA as exogenous template over synthetic homopolymers and was strictly dependent on the presence of ribonucleoside triphosphates. Appropriate subfractionation revealed that the matrix-bound primase activity is exclusively localized in the nuclear matrix interior. The ability of primase and DNA polymerase to synthesize covalently linked RNA-DNA products demonstrates the potentially useful role of the nuclear matrix in vitro system for elucidating the organizational and functional properties of the eucaryotic replication apparatus in the cell nucleus.     

Autonomous 3'-->5' exonucleases can proofread for DNA polymerase beta from rat liver

Autonomous 3'-->5'exonucleases are not bound covalently to DNA polymerases but are often involved in replicative complexes. Such exonucleases from rat liver, calf thymus and Escherichia coli (molecular masses of 28+/-2 kDa) are shown to increase more than 10-fold the accuracy of DNA polymerase beta (the most inaccurate mammalian polymerase) from rat liver in the course of reduplication of the primed DNA of bacteriophage phiX174 amber 3 in vitro. The extent of correction increases together with the rise in 3'-->5' exonuclease concentration. Extrapolation of the in vitro DNA replication fidelity to the cellular levels of rat exonuclease and beta-polymerase suggests that exonucleolytic proofreading could augment the accuracy of DNA synthesis by two orders of magnitude. These results are not explained by exonucleolytic degradation of the primers ("no synthesis-no errors"), since similar data are obtained with the use of the primers 15 or 150 nucleotides long in the course of a fidelity assay of DNA polymerases, both alpha and beta, in the presence of various concentrations of 3'-->5' exonuclease.     

Application of an in vitro system in the study of chemotherapeutic drug effects on DNA replication

DNA replication machinery is an important target for chemotherapeutic drugs. We have used an in vitro system to study the effect of drugs on mammalian DNA replication, either by direct interaction with the DNA structure or with replication proteins and machinery. The anthracycline doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA replication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibition of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside analog, had no significant effect on DNA synthesis. In contrast, araCTP was able to inhibit DNA replication in vitro. Since metabolism is diminished in this in vitro system, the degree of phosphorylation of araC was apparently low. Progesterone showed an increase in nucleotide incorporation (sensitive to BuPdGTP inhibition of replication-specific polymerases α and δ) after preincubation with HeLa cell extracts, although progesterone receptors were not detectable in the HeLa cell extracts. In addition, we observed an inhibition in DNA replication when progesterone was preincubated with DNA and nucleotides. These results suggest that progesterone may have a mechanism of action that is different from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro replication system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication. © 1996 Wiley-Liss, Inc.     

Effects of colchicine on nucleic acid metabolism during metamorphosis of Tenebrio molitor L. and in some mammalian tissues

1. Administration of 10mug. of colchicine/pupa of the beetle Tenebrio molitor L. arrests its differentiation, the pupa remaining alive for 2-3 weeks. 2. The same concentration of colchicine inhibits DNA synthesis and stimulates RNA synthesis (as shown by incorporation into the nucleic acids of labelled adenine, labelled uridine and labelled thymidine). The effects of colchicine on nucleic acid metabolism are first detected 3 days after its administration to first-day pupae. 3. No effects of colchicine are seen on [1-(14)C]glycine incorporation into protein in vivo. 4. Relatively high concentrations of colchicine (e.g. 10mm) suppress incorporation of [8-(14)C]adenine into RNA in dorsal abdominal wall in vitro. Such concentrations have no effect on its incorporation into acid-soluble nucleotides. 5. Colchicine (1mm) suppresses incorporation of [8-(14)C]adenine into DNA to a greater extent than into RNA in various mammalian tissues in vitro (e.g. rat spleen, regenerating rat liver, rat embryo, guinea-pig intestinal mucosa, Ehrlich ascites cells). Colchicine (1mm) has no effect on the rate of respiration of, or on incorporation of radioactivity into acid-soluble nucleotides in, the mammalian tissues tested. 6. Further evidence indicates complex-formation between colchicine and DNA, and it is suggested that the effect of colchicine in suppressing DNA synthesis is due to its combination with the DNA primer (template).     

The four human muscle regulatory helix-loop-helix proteins Myf3-Myf6 exhibit similar hetero-dimerization and DNA binding properties.

The muscle regulatory proteins Myf3, Myf4, Myf5, and Myf6 share a highly conserved DNA binding and dimerization domain consisting of a cluster of basic amino acids and a potential helix-loop-helix structure. Here we demonstrate that the four human muscle-specific HLH proteins have similar DNA binding and dimerization properties. The members of this family form protein complexes of comparable stability with the ubiquitously expressed HLH proteins E12, E2-2, and E2-5 and bind to the conserved DNA sequence CANNTG designated as E-box with similar efficiency in vitro. The binding affinities of the various complexes are greatly influenced by the variable internal and flanking nucleotides of the consensus motif. Combinations of Myf proteins with one another and with lyl-1, and HLH protein from human T cells, do not bind to DNA in vitro. Our results suggest that combinatorial associations of the various tissue-specific and more widely expressed HLH factors do not result in differential recognition of DNA sequences by Myf proteins.     

Studies on ethionine-induced fatty liver

1. The effects of the administration of dl-ethionine on some aspects of lipid and nucleotide metabolism in rat liver were studied. 2. In ethionine-treated animals neutral fat was increased, whereas phospholipids and cholesterol were unchanged. Lipogenesis in vitro was inhibited. 3. The concentration of nicotinamide nucleotides, purine nucleotides and pyrimidine nucleotides was decreased. The decrease was due to free adenine nucleotides, inosine nucleotides, uridine nucleotides and cytidine nucleotides. Also, the protein-bound biotin content was lower. 4. In biotin-deficient rats the development of ethionine-induced fatty liver was inhibited. 5. The possibility was considered that ethionine might produce an inhibition of the synthesis of biotin-dependent acetyl-CoA carboxylase.     

Stimulation of rat placental cell DNA synthesis by transferrin

The purpose of the present investigation was to evaluate the in vitro requirements for rat placental cell DNA synthesis. A cell line established from the labyrinth region of midgestation rat chorioallantoic placentas was used to examine the actions of various agents. Transferrin was found to stimulate rat placental cell DNA synthesis and cell proliferation. The effects of transferrin on rat placental cell growth paralleled those observed with fetal bovine serum. Rat placental cells were responsive to both rat and human transferrin. Iron-saturated (holo-) transferrin was a more potent stimulator of rat placental cell DNA synthesis than was iron-free (apo-) transferrin. Addition of insulin, epidermal growth factor, or insulin-like growth factor-II to serum-free medium supplemented with rat transferrin did not significantly enhance rat placental cell DNA synthesis beyond that observed with only transferrin. The results demonstrate that a population of cells exists within the rat chorioallantoic placenta that are highly responsive to transferrin.     

与TMV-RNA装配起始位点互补的cDNA片段对TMV体外装配的抑制

合成了与TMV-RNA病毒装配起始位点互补的、长度为二十个核苷酸的DNA片段。该片段用~(32)P标记后,代替反义RNA(antisense RNA)与TMV-RNA进行硝基纤维素膜点杂交和溶液杂交。结果表明,该cDNA片段在两种条件下均能与TMV-RNA进行杂交。将溶液杂交的RNA-cDNA复合体经酒精沉淀,再与TMV衣壳蛋白的20S聚合体制剂进行体外装配,用测定310nm吸收光谱变化和电子显微镜观察的方法鉴定装配结果。实验证明,该cDNA片段与TMV-RNA杂交后抑制了装配起始位点的活力,从而使TMV病毒颗粒的装配不能完成。这一结果提示,TMV基因装配起始位点顺宁的cDNA和反义RNA能够在体外抑制TMV病毒颗粒的装配。     

The inhibition of nucleic acid synthesis by mycophenolic acid

1. Mycophenolic acid, an antibiotic of some antiquity that more recently has been found to have marked activity against a range of tumours in mice and rats, strongly inhibits DNA synthesis in the L strain of fibroblasts in vitro. 2. The extent of the inhibition of DNA synthesis is markedly increased by preincubation of the cells with mycophenolic acid before the addition of [(14)C]thymidine. 3. The inhibition of DNA synthesis by mycophenolic acid in L cells in vitro is reversed by guanine in a non-competitive manner, but not by hypoxanthine, xanthine or adenine. 4. The reversal of inhibition by guanine can be suppressed by hypoxanthine, 6-mercaptopurine and adenine. 5. Mycophenolic acid does not inhibit the incorporation of [(14)C]thymidine into DNA in suspensions of Landschütz and Yoshida ascites cells in vitro. 6. Mycophenolic acid inhibits the conversion of [(14)C]hypoxanthine into cold-acid-soluble and -insoluble guanine nucleotides in Landschütz and Yoshida ascites cells and also in L cells in vitro. There is some increase in the radioactivity of the adenine fraction in the presence of the antibiotic. 7. Mycophenolic acid inhibits the conversion of [(14)C]hypoxanthine into xanthine and guanine fractions in a cell-free system from Landschütz cells capable of converting hypoxanthine into IMP, XMP and GMP. 8. Preparations of IMP dehydrogenase from Landschütz ascites cells, calf thymus and LS cells are strongly inhibited by mycophenolic acid. The inhibition showed mixed type kinetics with K(i) values of between 3.03x10(-8) and 4.5x10(-8)m. 9. Evidence was also obtained for a partial, possibly indirect, inhibition by mycophenolic acid of an early stage of biosynthesis of purine nucleotides as indicated by a decrease in the accumulation of formylglycine amide ribonucleotide induced by the antibiotic azaserine in suspensions of Landschütz and Yoshida ascites cells and L cells in vitro.     

Enhanced processivity of nuclear matrix bound DNA polymerase alpha from regenerating rat liver

Translocation of DNA during in vitro DNA synthesis on nuclear matrix bound replicational assemblies from regenerating rat liver was determined by measuring the processivity (average number of nucleotides added following one productive binding event of the polymerase to the DNA template) of nuclear matrix bound DNA polymerase alpha with poly(dT).oligo(A)10 as template primer. The matrix-bound polymerase had an average processivity (28.4 nucleotides) that was severalfold higher than the bulk nuclear DNA polymerase alpha activity extracted during nuclear matrix preparation (8.9 nucleotides). ATP at 1 mM markedly enhanced the activity and processivity of the matrix-bound polymerase but not the corresponding salt-soluble enzyme. The majority of the ATP-dependent activity and processivity enhancement was completed by 100 microM ATP and included products ranging up to full template length (1000-1200 nucleotides). Average processivity of the net ATP-stimulated polymerase activity exceeded 80 nucleotides with virtually all the DNA products greater than 50 nucleotides. Release of nuclear matrix bound DNA polymerase alpha by sonication resulted in a loss of ATP stimulation of activity and a corresponding decrease in processivity to a level similar to that of the salt-soluble polymerase (6.8 nucleotides). All nucleoside di- and triphosphates were as effective as ATP. Stimulation of both activity and processivity by the nonhydrolyzable ATP analogues adenosine 5'-O-(3-thiotriphosphate), 5'-adenylyl imidodiphosphate, and adenosine 5'-O-(1-thiotriphosphate) further suggested that the hydrolysis of ATP is not required for enhancement to occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication blocks and sequence interaction specificities in the codon 12 region of the c-Ha-ras proto-oncogene induced by four carcinogens in vitro

We have examined the region surrounding codon 12 in the human c-Ha-ras proto-oncogene in vitro to determine the reaction intensities at the guanine nucleotides after exposure to the mycotoxin aflatoxin B1, to one of its human metabolites aflatoxin M1, to dimethyl sulfate, and to the major ultimate carcinogen of benzo(a)pyrene, benzo(a)pyrene diol-epoxide. Among the adducts produced, those at N-7-guanyl sites are alkali-labile and can be identified using a variation of the Maxam-Gilbert sequencing procedure. Data indicate that the guanine nucleotides of codon 12 have above average potential for adduct formation by the genotoxins when compared to other guanine sites, but were not the strongest sites. This codon 12 region has been inserted into single-stranded M13 phage, exposed to several of the genotoxins, and used as a template for DNA synthesis in vitro. There is a sequence-specific variation in polymerase inhibition at various adducted nucleotide sites, but replication blocks are not preferentially seen at the carcinogen-adducted guanines of codon 12. These results indicate that the predominance of point mutations which are detected in vivo at codon 12 do not reflect sequence-mediated preferential susceptibility of these sites to initial DNA adduction or to replication errors. The mechanisms controlling these sequence effects are not currently understood, and attempts to predict relative alkylation or termination frequencies based solely on local DNA sequence are not reliable.     

Structure of the rat prolactin gene

The organization and sequence of the rat preprolactin gene has been investigated. Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon. Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments. The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments. The sequence of more than 1800 nucleotides of the cloned DNA has been determined. The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin. These coding regions are separated by an intervening sequence of 597 nucleotides. At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin. Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome.     

An in vitro system for the editing of apolipoprotein B mRNA

A novel form of RNA editing generates two forms of apolipoprotein B (apo-B) mRNA by converting C at nucleotide 6666 to U or a U-like base. We have established an in vitro system for the editing of apo-B mRNA using synthetic RNAs and S100 extracts from rat hepatoma cells. Editing was detected by a sensitive primer extension assay and confirmed by DNA sequencing. The in vitro editing activity is specific and sensitive to proteinase K. Apo-B100 RNAs were synthesized in vitro from deletion mutants spanning nucleotide 6666. Synthetic RNAs containing 2383, 483, and 55 nucleotides of apo-B mRNA sequence were edited in vitro with similar efficiency, but an RNA containing 26 nucleotides was not edited.     

Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase beta and DNA-ligase

Synthetic deoxy-oligo duplexes containing short gaps of 1 and 4 nucleotides were used as model substrates to assess the DNA gap repair ability of the neuronal extracts prepared from cerebral cortex of rats of different ages. Our results demonstrate that gap repair activity in neurons decreases markedly with age. The decreased activity could be restored by supplementing the neuronal extracts with pure recombinant rat liver DNA polymerase beta. High levels of DNA polymerase beta supplementation resulted in gap-filling activity that proceeded essentially through addition of nucleotides through a slow distributive strand displacement mode to achieve full template length (32-mer). However, at lower concentrations of DNA polymerase beta, the gap repair takes place quickly through gap filling followed by ligation to downstream primer, in an energy efficient manner. For this to happen, the conditions required are the presence of 5'-PO4 on the downstream primer and supplementation of aging neuronal extracts with DNA-ligase in addition to recombinant DNA polymerase beta. These results demonstrate that aging neurons are unable to affect base excision repair (BER) due to deficiency of DNA polymerase beta and DNA-ligase and fortifying aged neuronal extracts with these two factors can restore the lost BER activity.     

Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase

We have determined the fidelity of in vitro DNA synthesis catalyzed at high temperature by the DNA polymerase from the thermophilic bacterium Thermus aquaticus. Using a DNA substrate that contains a 3'-OH terminal mismatch, we demonstrate that the purified polymerase lacks detectable exonucleolytic proofreading activity. The fidelity of the Taq polymerase was measured by two assays which score errors produced during in vitro DNA synthesis of the lacZ alpha complementation gene in M13mp2 DNA. In both assays, the Taq polymerase produces single-base substitution errors at a rate of 1 for each 9000 nucleotides polymerized. Frameshift errors are also produced, at a frequency of 1/41,000. These results are discussed in relation to the effects of high temperature on fidelity and the use of the Taq DNA polymerase as a reagent for the in vitro amplification of DNA by the polymerase chain reaction.     

Characterization of the genome of the free-living nematode Panagrellus silusiae: absence of short period interspersion.

The complexity of the DNA of the free-living nematode Panagrellus silusiae has been examined. Reassociation kinetics of pressure-sheared fragments (approximately 290 nucleotides) in 0.18 M Na+ at 60 degrees C showed the presence of foldback, repetitive, and unique DNA sequence elements. The three classes comprise 9.3%, 26.1%, and 61.3% of the total DNA, respectively. The mean length of the foldback duplex DNA after digestion with S1 nuclease is about 185 nucleotides. There are about 1.8 x10(4) inverted repeats per genome. Sequence arrangement was deduced from (1) renaturation kinetic profiles of long and short fragments on hydroxylapatite; (2) the pattern of renaturation of tracer DNA, labeled in vitro with 125I, of various sizes after incubation with excess short fragments; and (3) thermal denaturation behavior of DNA that had been reassociated to various C0t values. It was found that DNA fragments of the repetitive fraction that are, at least, 2000 nucleotides in length are virtually free of unique sequences. Moreover, it is estimated that the repeated segments in this species could extend for 10,000 nucleotide pairs. Thus, Panagrellus DNA lacks the pattern of extensive short period interspersion that is typified by the DNA of Xenopus.