In Vitro DNA Synthesis by Chloroplasts Isolated from Marchantia polymorpha L. Cell Suspension Cultures

Lysate of chloroplasts prepared from liverwort Marchantia polymorpha L. cell suspension cultures incorporated [³H]-dTTP into acid insoluble materials when DNA was added exogenously as a template. The incorporation was highly dependent on the addition of template DNA, four deoxynucleoside triphosphates and magnesium ions (maximum incorporation at 5mM). Magnesium ions could be replaced by manganese ions. DNA synthesis inhibitors, N-ethylmaleimide (NEM) and ethidium bromide (EtBr), strongly inhibited the incorporation. Dideoxythymidine triphosphate (ddTTP), an inhibitor of DNA polymerases β and γ, inhibited the incorporation at the concentration of 50 μM (molar ratio of ddTTP/dTTP = 17). On the other hand, the incorporation by the chloroplast lysate was resistant to arabinofuranosyl cytosine triphosphate (araCTP) and aphidicolin as well as the RNA polymerase inhibitors, rifampicin and α-amanitin. The chloroplast lysate highly utilized denatured calf thymus DNA and bacteriophage ?X174 single-stranded DNA as templates when added exogenously, while a synthetic homopolymer, poly(rA)-oligo(dT)₁₂ ~ ₁₈, did not stimulate the incorporation at all. Autoradiographic analysis of DNA synthesized in isolated chloroplasts showed that the chloroplast DNA synthesis took place at several specific sites on the chloroplast DNA from cells of the liverwort, Marchantia polymorpha.     

Common fragile sites

Aphidicolin-induced common fragile sites are site-specific gaps or breaks seen on metaphase chromosomes after partial inhibition of DNA synthesis. These fragile sites were first recognized during the early studies of the fragile X syndrome and are induced by the same conditions of folate or thymidylate stress used to induce the fragile X site. Common fragile sites are normally stable in cultured human cells. However, following induction with replication inhibitors, they display a number of characteristics of unstable and highly recombinogenic DNA. From the many studies that have cloned and characterized fragile sites, it is now known that these sites extend over large regions, are associated with genes, exhibit late or delayed replication, and contain regions of high flexibility but are otherwise unremarkable in sequence. Studies showing that fragile sites and their associated genes are frequently deleted or rearranged in cancer cells have clearly demonstrated their importance in genome instability in tumorigenesis. Yet until recently, very little was known about the molecular mechanisms involved in their stability. Recent findings showing that the key checkpoint genes ATR and BRCA1 are critical for genome stability at fragile sites have shed new light on these mechanisms and on the biological significance of common fragile sites.     

Thymidylate synthetase inhibitors and fragile site expression in lymphocytes.

The ability of three thymidylate synthetase inhibitors, fluorodeoxyuridine, fluorodeoxycytidine, and trifluorothymidine, to induce the expression of eight different folate-sensitive fragile sites has been investigated in 22 patients and compared with the efficacy of simple folate deprivation for inducing fragile site expression. Fluorodeoxyuridine and fluorodeoxycytidine were equal in their ability to elicit fragile site expression but fluorodeoxycytidine proved less cytotoxic under comparable culture conditions. Both fluorodeoxyuridine and fluorodeoxycytidine were found to be more efficient than trifluorothymidine at comparable concentrations but less efficient than simple folate deprivation in eliciting fragile site expression in lymphocytes. Since the three inhibitors induced expression of eight different folate-sensitive fragile sites, it is likely that all folate-sensitive fragile sites have a common underlying mechanism of expression. The practical application of thymidylate synthetase inhibitors in the routine detection of heritable fragile sites is discussed.     

DNA instability at chromosomal fragile sites in cancer

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.     

Reduced levels of DNA polymerase delta induce chromosome fragile site instability in yeast

Specific regions of genomes (fragile sites) are hot spots for the chromosome rearrangements that are associated with many types of cancer cells. Understanding the molecular mechanisms regulating the stability of chromosome fragile sites, therefore, has important implications in cancer biology. We previously identified two chromosome fragile sites in Saccharomyces cerevisiae that were induced in response to the reduced expression of Pol1p, the catalytic subunit of DNA polymerase α. In the study presented here, we show that reduced levels of Pol3p, the catalytic subunit of DNA polymerase δ, induce instability at these same sites and lead to the generation of a variety of chromosomal aberrations. These findings demonstrate that a change in the stoichiometry of replicative DNA polymerases results in recombinogenic DNA lesions, presumably double-strand DNA breaks.     

ATR regulates fragile site stability

Conditions that partially inhibit DNA replication induce expression of common fragile sites. These sites form gaps and breaks on metaphase chromosomes and are deleted and rearranged in many tumors. Yet, the mechanism of fragile site expression has been elusive. We demonstrate that the replication checkpoint kinase ATR, but not ATM, is critical for maintenance of fragile site stability. ATR deficiency results in fragile site expression with and without addition of replication inhibitors. Thus, we propose that fragile sites are unreplicated chromosomal regions resulting from stalled forks that escape the ATR replication checkpoint. These findings have important implications for understanding both the mechanism of fragile site instability and the consequences of stalled replication in mammalian cells.     

The neurobeachin gene spans the common fragile site FRA13A

Common fragile sites are normal constituents of chromosomal structure prone to chromosomal breakage. In humans, the cytogenetic locations of more than 80 common fragile sites are known. The DNA at 11 of them has been defined and characterized at the molecular level. According to the Genome Database, the common fragile site FRA13A maps to chromosome band 13q13.2. Here, we identify the precise genomic position of FRA13A, and characterize the genetic complexity of the fragile DNA sequence. We show that FRA13A breaks are limited to a 650 kb region within the neurobeachin (NBEA) gene, which genomically spans approximately 730 kb. NBEA encodes a neuron-specific multidomain protein implicated in membrane trafficking that is predominantly expressed in the brain and during development.     

Fragile sites induced by FUdR,caffeine, and aphidicolin

Summary The frequencies of common fragile sites (c-fra) induced in peripheral blood lymphocytes by fluorodeoxyuridine (FUdR), aphidicolin, or caffeine, in eight healthy controls were studied. There was a significantly higher frequency of breaks (P<0.05) in the latter two treatments than the former. Also, significant variation in total number of breaks was observed among the eight individuals within the three treatments. The relative frequency of a fragile site in relation to the total number of fragile sites in an individual rather than its expression in total cells was considered important. Use of a frequency of 4% or more of total fragile sites was proposed to eliminate apparent random breaks that were observed. Using these criteria, a total of 31 c-fra were observed in the three treatments. The distribution of the fragile sites was different in FUdR-treated cells as opposed to caffeine- and aphidicolin-treated cells. Sites 3p14 and 16q23 and Xp22 were the three most frequently observed c-fra. The higher frequency of expression of some fragile sites in normal controls, as observed here, suggests that any relationship between fragile sites and neoplastic transformation has to be carefully evaluated. A classification based on frequency in the population, rather than mode of induction, is suggested.     

Expression of fragile sites in childhood acute lymphoblastic leukemia patients and normal controls

Summary A high concordance has been reported between fragile sites and breakpoints involved in chromosomal rearrangements in cancer. A prospective study on the role of fragile sites in the etiology of childhood acute lymphocytic leukemia (ALL), with appropriate comparisons to results obtained from normal controls, analyzed fluorodeoxyuridine-, aphidicolin-, and caffeine-induced fragile sites in the peripheral blood of seven ALL patients (three with cytogenetically normal karyotype and four with pseudodiploid karyotype) and eight normal controls. While extensive variations in the number and distribution of fragile sites was observed within each group, there was no significant difference in the mean total fragile sites and mean fragile sites per cell between the two groups (P>0.05) in all three treatments. Similarly, within the ALL patients, the two karyotypic groups did not exhibit any significant difference in fragility (P>0.05).     

Synergistic effect of aphidicolin and ethanol on the induction of common fragile sites

Summary The effect of ethanol on the frequency of aphidicolin-induced common fragile sites was studied using lymphocyte cultures from two normal women. Aphidicolin was added to the cultures at a final concentration of 0.2 M and ethanol at 0.02%, 0.1%, 0.2%, 0.5%, and 1%, both during the last 26 h of culture. The frequency of common fragile sites increased from 296% in subject 1 and 201% in subject 2 with aphidicolin plus 0.02% ethanol, to 765% and 823%, respectively, with aphidicolin plus 1% ethanol. Ethanol alone added to cultures did not induce common fragile sites. The gaps and breaks induced by aphidicolin plus ethanol were highly nonrandom. Altogether, 35 common fragile sites were identified. The addition of 1% ethanol to aphidicolin increased both random and nonrandom gaps and breaks as compared with that of 0.02% ethanol. Dimethyl sulfoxide added to culture at final concentrations of 0.02% to 1% did not change the frequency of aphidicolin-induced fragile sites. The frequency of fluorodeoxyuridine-induced fragile sites was not affected by the addition of 0.02% to 1% ethanol. It was thus concluded that ethanol enhances the aphidicolin-induced fragile sites, possibly inhibiting the repair mechanism of gaps and breaks induced by aphidicolin.     

Hedonic Functions of Monosodium Glutamate and Four Basic Taste Substances Used at Various Concentration Levels in Single and Complex Systems

An in vitro DNA-directed protein-synthesizing system of Bacillus subtilis was developed using vegetative and sporulating cell extracts. Protease activity was inhibited by the addition of three kinds of protease inhibitors and removed from the extracts by hemoglobin-Sepharose treatment. Endogenous RNA synthesis was very low because of elimination of endogenous DNA by polyethylene glycol 6000 treatment of the supernatant. Protein synthesis was dependent on the DNA template, ribosomes and supernatant. When pUB110 DNA was used as a template, three proteins (Kl, K2 and K5) which have the same molecular weights as those synthesized in vivo were synthesized in vitro with vegetative, T₂ (2 hr after the end of logarithmic growth) and T₄ cell extracts.     

Postreplication Repair of Ultraviolet Damage to DNA, DNA-Chain Elongation, and Effects of Metabolic Inhibitors in Mouse L Cells

Alkaline sucrose sedimentation studies of DNA from mouse L cells have demonstrated the following effects of several inhibitors of nucleic acid and protein synthesis on postreplication repair of ultraviolet (UV) damage to their DNA. The DNA newly synthesized by a 2 h [³H]thymidine (dThd) label following 254 nm UV irradiation of 20 J/m² is made in smaller segments of the number average mol wt (Mn) of ~10 × 10⁶ than the control of ~40 × 10⁶. The presence of caffeine at a concentration of 2 mM during the labeling of the irradiated cells reduces the Mn value to 5.8 × 10⁶, which is nearly comparable to, but somewhat larger than the expected distance between dimers in parental DNA. Afterwards, such an interrupted DNA made in the irradiated cells is completely repaired to the present maximum Mn value of 40 × 10⁶ in the consecutive 4 h chase in unlabeled dThd. The presence of the nucleic acid inhibitor, either 2 mM hydroxyurea, 50 μM arabinofuranosyl cytosine, 2 mM excess dThd or 5 μg/ml of actinomycin D (AMD) during 2- to 24-h chase periods after a 2 h postirradiation label prevents the repair to various extents, while 2 mM caffeine completely inhibits it. In the unirradiated cells, these agents except excess dThd and caffeine also interfere severely with normal elongation of nascent DNA made by a 3 min pulse label, but do not appreciably induce single chain breaks of either newly synthesized or parental DNA. The inhibition of the repair by AMD suggests that de novo elongation of DNA to close the gaps in new DNA made in the irradiated cells requires at least a template-dependent DNA polymerase. In contrast, 100 μg/ml of cycloheximide allows to complete the gap-filling repair, while it simply reduces the rates of chain growth for the repair and normal replication. Therefore, the similar sensitivity of gap-filling repair and normal replication towards the above inhibitors indicates that a preexisting DNA polymerizing system appears to be responsible and to play a common role without new protein synthesis, as far as the repair at early time after UV is concerned.     

Cyclic Purine Mononucleotides: Induction of Gibberellin Biosynthesis in Barley Endosperm

The de novo synthesis of α-amylase in barley endosperm and isolated aleurone layers is induced by 3′,5′-cyclic purine mononucleotides and gibberellic acid. The induction of α-amylase by cyclic purine mononucleotides is prevented by 2,4-DNP, inhibitors of RNA and protein syntheses, CCC, AMO-1618 and phosfon. The induction of α-amylase formation by 3′,5′-cyclic purine mononucleotides, but not by gibberellic acid, is also blocked by inhibitors of DNA synthesis. Extracts from cyclic AMP-treated endosperm halves exhibit a characteristic gibberellin-like activity which is detectable within 12 hours from the addition of the cyclic AMP. On paper chromatograms this gibberellin-like activity is located at the Rf typical for GA₃. Its formation is prevented by inhibitors of DNA synthesis, CCC and AMO-1618. Glucose inhibits the formation of α-amylase induced by gibberellic acid. Glucose has no effect on the cAMP-induced gibberellin biosynthesis. The evidence shows that the cyclic purine mononucleotides induce DNA synthesis, which results in gibberellin biosynthesis, which in turn activates the synthesis of α-amylase.     

Deoxyribonucleic acid synthesis in mammalian nuclei. Incorporation of deoxyribonucleotides and chain-terminating nucleotide analogues

The properties of a nuclear preparation from rat liver and thymus are described. (1) Nearest-neighbour analysis after incorporation of (32)P-labelled nucleotide residues from dATP, dCTP, dGTP, dTTP and arabinofuranosyl analogues of CTP and ATP shows template-dependent DNA synthesis. (2) Where primer termini are limiting, incorporation of arabinofuranosyl analogues of AMP and CMP residues proceeds to a limit indicating that both of these analogues are DNA chain terminators. (3) No large differences have been found between the priming potentialities or the intrinsic DNA polymerase activities of nuclei from resting or regenerating liver and the relationship of this DNA synthesis in vitro to DNA replication or repair in vivo is briefly discussed.     

Effects of selected inhibitors on growth and cell division inAgmenellum

Summary Concentrations of chloramphenicol and penicillin G which permit growth induce the formation of temporary filaments, morphologically and ultrastructurally identical to stable, chemically-induced filamentous mutants ofAgmenellum quadruplicatum strainBG-1. These induced filaments were propagated by serial transfers in the presence of inhibitor and underwent an immediate, synchronous reversion upon its removal. The reversion of penicillin-induced filaments was insensitive to inhibitors of DNA synthesis but sensitive to inhibitors of protein synthesis until the completion of the mucopolymer septum. Penicillin G blocked the early stages or initiation of cell division. Chloramphenicol blocked the terminal stages of cell division, the cleavage of the mucopolymer septum by the outer wall layers.     

An in vitro polypeptide synthesizing system from methanogenic bacteria: Sensitivity to antibiotics

Summary An in vitro polypeptide synthesis system was set up for three methanogenic bacteria, Methanococcus vannielii, Methanobacterium formicicum and Methanosarcina barkeri, and the effect of classical 70S and 80S protein synthesis inhibitors studied. The following results were obtained: (i) The activity of ribosomes from all three methanogens was unaffected by a number of 70S inhibitors such as tetracycline, chloramphenicol, streptomycin, tiamulin and, probably, erythromycin as well; (ii) However, the ribosomes were sensitive to thiostrepton, virginiamycin and, to varying degrees, to those aminoglycosides containing a 2-deoxystreptamine moiety. Among the aminoglycosides examined, streptomycin induced no translational misreading. The compounds containing 2-deoxystreptamine stimulated misreading, albeit only at high concentrations (neomycin being an exception); (iii) Ribosomes from all three organisms were insensitive to the 80S inhibitors cycloheximide and ricin, but those from Methanobacterium formicicum were highly sensitive to anisomycin and moderately sensitive to verrucarin. The results support those of in vivo studies and provide conclusive evidence that archaebacterial ribosomes despite being 70S ribosomes lack binding sites for many classical eubacterial ribosome inhibitors. At the same time they possess sites for others, as well as for some inhibitors of 80S ribosomes.     

Restriction in vivo

Summary Degradation products of restricted T4 DNA induced filamentation, mutagenesis, and to a lesser extent, synthesis of recA protein in wild type cells but not in recA, lexA or recBC mutants of Escherichia coli. We conclude that the structural damage to the DNA caused by restriction cleavage and exonuclease V degradation can induce SOS functions. Degradation of restricted nonglucosylated T4 DNA by exonuclease V delayed cell division and induced filament formation and mutagenesis in lexA ⁺ but not in lexA ^- cells. Delay of cell division was also dependent upon recA and recBC funtions. Such degradation of DNA also dramatically increased mutagenesis in tif ^- Sfi^- cells at 42°C. The synthesis of recA protein continued in the restricting host after infection by the nonglucosylated T4 phage, but enhanced synthesis is not induced to the extent seen in SOS induced tif ^- cells grown at 42°. We also found that restriction of nonglucosylated T4 was alleviated in UV irradiated cells. The UV induced alleviation of rgl and r _K restriction depended upon post irradiation protein synthesis and was not observed in recA, lexA or recBC mutants.     

High-resolution chromosome banding studies in Cebus apella,Cebus albifrons,and Lagothrix lagothricha: Comparison with the human karyotype

Karyotypes of three species of South American primates (Cebus apella, Cebus albifrons, and Lagothrix lagothricha) were studied using high-resolution banding techniques, and were compared to the human karyotype. The number of homologies was very high for the three species. Some of the breakpoints implicated in chromosome rearrangements corresponded with human fragile sites. The fragile sites in human chromosomes often correspond with the localization of latent centromeres in the platyrrhines or with large heterochromatic regions that may have been lost or newly added during evolution.     

Fluorescent in-situ hybridization of cattle and sheep chromosomes with cloned human fragile-X DNA

An extensive study on spontaneous and 5-Fluorodeoxyuridine induced fragile sites identified Xq31 in cattle (Bos taurus) and (Xq24, Xq26) in sheep (Ovis aries) in addition to several autosomal fragile sites (under publication). A ZOO-FISH study using three cloned human fragile-X probes with CCG/CGG_n trinucleotide repeat sequence was carried out to determine homology between human and bovine fragile-X. The hybridisation results showed only a weak signal on a human chromosome that was not an X with all three fragile site probes. No signals were detected in sheep chromosomes. The signal of all three human fragile-X probes on cattle chromosomes was however, medium-prominent sub-centromeric signal on two homologues. BrdU administration in 12 h before harvesting identified these homologues to be chromosome number 5. In addition retrospective slides of cattle and sheep chromosomes used for fragile site studies showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X.     

Cycloheximide induction of xenotropic type C virus from synchronized mouse cells: metabolic requirements for virus activation.

The information for type C RNA viruses is genetically transmitted within the cellular DNA of the normal mouse cell. These viruses can be induced after exposure of cells to two classes of chemicals, inhibitors of protein synthesis and halogenated pyrimidines. The metabolic requirements for activation of one endogenous virus of BALB/c mouse cells by representatives of each class of drugs were studies. Cycloheximide and iododeoxyuridine each induce virus efficiently from cultures in exponential growth but are inactive on cells in stationary phase. However, cells are maximally sensitive to the actions of each drug at different times within the cell cycle. Further, virus induction in response to each is differentially inhibited under conditions of simultaneous cell exposure to inhibitors of DNA or RNA synthesis. The results provide support for the concept that inhibitors of protein synthesis and halogenated pyrimidines act by different mechanisms to induce type C virus release.