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PubMed2003

Prenatal detection of maternal UPD15 in a new case with i(15p) by Timing Replication Test (TRT) and methylation analysis.

DNA replication kinetics of the Prader-Willi/Angelman Critical Region (PWACR) was studied with and without synchronisation in human amniotic cell cultures obtained from 20 cases with normal karyotype and 4 cases with a marker of chromosome 15, respectively. A Timing Replication Test (TRT) was performed by synchronisation of amniotic cell cultures and followed by interphase FISH to analyse and compare the early/late replication patterns in SNRPN and UBE3A genes between the homologues of chromosome 15. Asynchronous replication patterns of the analysed genes were observed in both amniotic cell cultures but the percentage of interphase nuclei presenting with asynchronous replication was significantly increased in the cultures with synchronisation (40-51%), as compared to those without synchronisation (20-23%). The evaluations, performed by means of TRT, showed asynchronous replication patterns on control values: between 39% and 46% of cells in all the cases with inv dup(15). In contrast, the percentage of cells with asynchronous replication in the case with i(15p) was significantly decreased (3-6%), as compared to the control value, and it may be indicated by uniparental disomy of chromosome 15 (UPD15). In addition, those results have been confirmed by molecular evaluation, using the methylation diagnostic test for diagnosis of the Prader-Willi Syndrome.

Amniotic Fluid↗
PubMed2004

The mammalian beta globin origin of DNA replication.

Initiation of DNA replication is a tightly regulated process aimed to insure that the entire genome is replicated at the appropriate time during each cell cycle. In the human beta globin locus, replication initiates from a region between the two genes that encode the adult subunit of hemoglobin (the beta globin initiation region, or IR). Mammalian beta globin loci replicate early during the S phase of the cell cycle in pre erythroid cells, in which the beta-globin locus is present in a euchromatin form. However, in cells that do not express globin and in which the locus is heterochromatic, these same loci replicate during the later stages of S phase. Both early and late replication patterns utilize similar replication initiation regions. These features make the beta globin locus an attractive model for studying the determinants of replication sites and replication timing, as well as the correlation between gene expression and DNA replication. Two genomic domains are essential for initiation of DNA replication within the locus: the initiation region (IR), and a 40 kb region upstream of the globin gene cluster known as the locus control region (LCR). The IR meets the genetic requirements for a chromosomal replicator, since it can initiate DNA replication at ectopic sites. The LCR regulates transcriptional activity and chromatin structure, and may act as a determinant of replication timing. This review will summarize recent findings characterizing the sequence requirements for initiation of DNA replication in mammalian beta globin loci and will discuss the specific influence of the location and the chromosomal environment in regulating DNA replication at the beta globin IR.

Animals↗
PubMed1993

Replication cycle and associated biosynthetic reactions for a non-oncogenic avian adenovirus.

The replication cycle of the non-oncogenic fowl adenovirus serotype 10 (FAV-10) has been examined. The onset of viral DNA synthesis was shown to commence at about 10 h postinfection (hpi) defining the early period of viral replication as prior to this time and the late phase as that time following the initiation of DNA replication. Virus titre rapidly increased between 18 and 24 hpi with maximum virus yield between 28 and 30 hpi. The late phase transcription profiles of the FAV-10 genome from 10 hpi to 24 hpi were determined. Late translation of virus protein began about 14 hpi increasing rapidly between 18 and 30 hpi.

Aviadenovirus↗
PubMed1981

Initiation of nuclear DNA replication: evidence for formation of committed prereplicative cellular state.

This paper explores the transitional states that bridge the gap between nuclear quiescence and mitogenesis. It presents evidence for the formation of a committed but prereplicative state. Quiescent murine Swiss 3T3 cells were exposed to an external mitogenic stimulant (epidermal growth factor or excess serum) and simultaneously to a synchronizer which inhibits entry into the S phase. Thus, the cells were stimulated to synthesize DNA, but the normal replicative response to this stimulus was blocked. The block to DNA replication was removed at varying times after removal of the stimulant. Experiments were done to monitor the decay of commitment to DNA synthesis after removal of the external stimulant. This decay turned out to be a first-order process. The half-life (time required for loss of the commitment to DNA synthesis in half of the initially sensitized cells) was found to be approximately 5 hr. The same result was found whether total DNA synthesis or individually replicating cells were measured and was independent of the type of external growth stimulant or blocker used. These results point to the existence, on the mitogenic pathway, of a committed but prereplicative state. The committed state appears to represent a unit or global property of the whole cell rather than, for example, a critical concentration of some active inducer molecule because the latter would display multi-hit decay kinetics rather than the single-step lability actually observed.

Animals↗
PubMed1982

Temporal patterns of human cytomegalovirus transcription: mapping the viral RNAs synthesized at immediate early, early, and late times after infection.

The transcription of the human cytomegalovirus genome was investigated at immediate early, early, and late times after infection. Viral RNAs associated with either the whole cell, the nucleus, the cytoplasm, or the polyribosomes were analyzed. At immediate early times, i.e., in the absence of de novo viral protein synthesis, the viral RNA in high abundance originated from a region of the long unique section of the prototype arrangement of the viral genome (0.660 to 0.770 map units). The viral RNA in low abundance originated from the long repeat sequences (0.010 to 0.035 and 0.795 to 0.825 map units) and a region in the long unique section (0.201 to 0.260 map units). Viral RNAs associated with the polyribosomes as polyadenylated RNA were mapped to these restricted regions of the viral genome and characterized according to size class in kilobases. At 24 h after infection in the presence of an inhibitor of viral DNA replication, i.e., at early times, the stable viral RNAs in highest abundance mapped in the long repeat sequences. Viral RNAs at intermediate abundance under these conditions mapped in two regions of the long unique section of the viral genome (0.325 to 0.460 and 0.685 to 0.770 map units). Stable viral RNAs that were associated with the polyribosomes in high abundance as polyadenylated RNA orginated from the long repeat sequences, but not from the long unique section of the viral genome. An analysis of whole-cell RNA at late times (72 h) indicated that the abundant transcription was in the regions of the long unique sequences (0.325 to 0.460 and 0.660 to 0.685 map units), and transcription of intermediate abundance was from the long repeat sequences. However, stable viral mRNA's derived from the long repeat sequences were associated with the polyribosomes at late times after infection. In addition, mRNA's originating from the long and short unique sequences were found associated with the polyribosomes at higher relative concentration than at early times after infection. It is proposed that expression of the immediate early viral genes is required to transcribe the early viral genes in the long repeat and adjacent sequences. These sequences are also transcribed at late times after infection while viral DNA synthesis continues. The expression of viral genes in most of the long and short unique sequences appears to require viral DNA replication.

Cell Nucleus↗
PubMed1966

Replication of chloroplast DNA of tobacco.

An experimental method has been designed for determining the relative rates of replication of the chloroplast and nuclear DNA's of Nicotiana tabacum. By this method chloroplast DNA in week-old seedlings is being replicated several times faster than nuclear DNA.

Cell Division↗
PubMed1979

Conservative segregation of parental histones during replication in the presence of cycloheximide.

Long stretches of protein-free, nonbeaded DNA were observed electron microscopically in nuclear spreads prepared from cells that had replicated their DNA in the absence of protein synthesis. The amount of this DNA increased with increasing time of replication in the presence of cycloheximide and was greatly decreased when replication was inhibited with 1-beta-D-arabinofuranosylcytosine (cytosine arabinoside). This DNA is considered to be "free" DNA because it has the same diameter as marker PM2 DNA and it is preferentially sensitive to DNase I digestion. Reversal of the cycloheximide block resulted in a burst of histone synthesis and repair of the depleted chromatin within 5 min. In addition, 26 presumptive replication forks were observed with beaded chromatin on two arms and free DNA on the third. These results suggest that new histones are usually deposited onto new DNA, that the cellular histone pool is very small, that histone migration is minimal in vivo for at least 18 hr, that for most fibers nuclesome assembly and segregation is conservative for stretches of DNA as long as 100 kbases, and that some part of the octameric histone core may remain bound to DNA during the replication process. The regularity we have observed for the assembly and segregation of nucleosomes is likely to be important for our understanding of how chromosomal information is segregated during development.

Cell Line↗
PubMed1988

Mechanism of cis-diamminedichloroplatinum(II)-induced cytotoxicity: role of G2 arrest and DNA double-strand breaks.

DNA has been implicated as the critical target for cis-diamminedichloroplatinum(II) (cis-DDP)-induced cytotoxicity. In vitro, DNA-platinum adducts inhibit DNA synthesis. An assessment of the inhibition of DNA synthesis in murine leukemia L1210 cells demonstrated that, although cell division was halted, DNA replication continued for a period of time. The DNA underwent almost a complete doubling even in cells that did not divide. Flow cytometric analysis demonstrated a slowed synthetic phase which progressed to a block in the G2 phase of the cell cycle. The duration of the G2 block was proportional to the concentration of cis-DDP. Low concentrations of cis-DDP caused the cells to be transiently blocked in the G2 phase for 24 to 48 h. Higher concentrations of cis-DDP resulted in a G2 arrest that was not reversed by 96 h. After this time, the arrested cells appeared to disintegrate, rather than recover. Cell survival and trypan blue exclusion studies indicated that, at low drug concentrations, cells which had transiently arrested in the G2 phase survived, while at higher concentrations only a limited number of survivors were responsible for the observed recovery of growth. Analysis of DNA double-strand breaks showed that significant numbers of breaks only occurred at concentrations of cis-DDP that subsequently led to debris detectable on the flow cytometer and to loss of trypan blue exclusion. The formation of these breaks appeared to be the first detectable change that was indicative of cell death. It is proposed that cells arrest in the G2 phase because they are unable to transcribe damaged DNA and make mRNA essential for passage into mitosis. DNA repair probably overcomes this arrest. Cell death may therefore be a consequence of the inability to adequately recover transcription.

Cell Survival↗
PubMed1995

Correlation of GC content with replication timing and repair mechanisms in weakly expressed E.coli genes.

Regional variations of DNA GC content are observed in species as different as S.cerevisiae and humans. In vertebrates and yeast they are correlated with replication timing; late replicating chromosomal regions are more AT-rich than early replicating regions. We show here that gene composition in E.coli also has long range variations which are similarly correlated with replication timing. We suggest that the enrichment in AT base pairs in late replicating DNA reflects differences in DNA repair modes. These sequences, which are in single copy for a greater part of the cell cycle than origin-linked genes, have less opportunity to engage in repair via homologous recombination and therefore may resort more often to translesion synthesis involving the misincorporation of adenine opposite modified nucleotides.

Base Composition↗
PubMed1993

Replication timing and Xenopus 5S RNA gene transcription in vitro.

The influence of DNA replication in a chromosomal environment on the transcription of Xenopus laevis 5S RNA genes has been examined using low-speed extracts of Xenopus eggs and Xenopus sperm nuclei. The major oocyte- and somatic-type 5S RNA multigene families of X. laevis are differentially expressed in these extracts dependent on limiting transcription factors and replicate in different compartments of S-phase. Under these particular experimental conditions the timing of replication of the somatic 5S RNA genes appears to be independent of their transcriptional activity. Moreover, differential transcription of the oocyte- and somatic-type 5S RNA genes is not influenced by the replication process.

Animals↗
PubMed2001

Molecular mechanism of sequence-specific termination of lentiviral replication.

The central termination sequence (CTS) terminates (+) strand DNA synthesis in certain lentiviruses. The molecular mechanism underlying this event, catalyzed by equine infectious anemia virus reverse transcriptase (EIAV RT), was evaluated by pre-steady-state kinetic techniques. Time courses in nucleotide incorporation using several DNA substrates were biphasic, consistent with release of enzyme from extended DNA being the rate-limiting step for turnover. While the burst amplitude reflecting the amount of functional RT-DNA complex was sequence-dependent, rate constants for initial product formation were not. Filter binding assays indicate the K(d) for CTS-containing substrate is only 2-fold higher than a random DNA and cannot account entirely for the large diminution in burst amplitudes. Measurements of processive DNA replication on a millisecond time scale indicate that the rate of polymerization is unaffected by the T(6)-tract within the CTS. However, termination products accumulate due to a substantial increase in the rate of nonproductive enzyme-nucleic acid complex formation after incorporation of four to five adenosines of a T(6)-tract within the CTS. During strand displacement synthesis through the CTS, products accumulate after incorporation of three to four adenosines. The rate of polymerization during strand displacement synthesis decreases 2-fold while the rate of nonproductive enzyme-nucleic acid complex formation is identical in the absence or presence of the displacement strand. These results have allowed us to develop a model for CTS-induced termination of (+) strand synthesis.

Base Sequence↗
PubMed1983

On the nature of the RecBC and RecF pathways of conjugal recombination in Escherichia coli.

The molecular mechanisms of the RecBC and RecF pathways for genetic recombination in E. coli were investigated by studying the kinetics of RecA protein function during conjugation. RecF recombination in recBC sbcB mutants is shown to be a much slower process than RecBC recombination in recBC+ sbcB+ strains, and is blocked by a mutation in lexA that prevents induction of RecA protein. Progress of the RecF pathway is greatly accelerated by a recAoc mutation which increases synthesis of RecA protein, but this does not restore recombination proficiency to a recBC sbcB lexA mutant. These results are interpreted to suggest that the RecF pathway directs integration of single-stranded Hfr DNA into the recipient chromosome whereas the RecBC pathway catalyses the exchange of largely double stranded DNA. This is consistent with the known stoichiometry of RecA protein catalysed heteroduplex DNA formation in vitro and with the delayed replication of RecF pathway recombinants which approximates to the time required for one round of DNA replication to generate homoduplex DNA. The regulation of the RecF pathway by lexA repressor is discussed in relation to the factors that govern the relative utilization of the two recombination pathways in wild-type cells.

Bacterial Proteins↗
PubMed1975

Interactions of Polyoma and Mouse DNAs III. Mechanism of Polyoma Pseudovirion Formation.

In primary mouse kidney cell cultures infected with polyoma virus, the processes leading to virion and pseudovirion formation were studied. By photometric DNA quantitation, we followed the kinetics of mouse and polyoma DNA synthesis and the formation of low-molecular-weight fragmented mouse DNA (mouse f-DNA). Virus was harvested at different times and analyzed for its proportion of pseudovirions. The following correlations between the intracellular events and the production of virions and pseudovirions were found. (i) Syntheses of cellular and viral DNA were closely linked, both in time and in rates of synthesis. (ii) An increase of mouse f-DNA could only be detected several hours after the onset of mouse and polyoma DNA replication; its formation coincided in time with the appearance of progeny virus. (iii) The proportion of pseudovirions was not dependent on the amount of mouse f-DNA formed, but seemed to be inversely related to the amount of viral DNA synthesized. This was borne out by experiments in which DNA synthesis was partially inhibited by mitomycin C or after a synchronized onset of DNA replication. Under these conditions, virus preparations with a two- to threefold increased proportion of pseudovirions were obtained as compared with those from uninhibited cultures. Virus isolated from the remaining monolayer always had a higher proportion of pseudovirions than virus isolated at the same time from the supernatant medium only; also, the proportion of pseudovirions increased slightly with time after infection. Thus, according to the experimental conditions used, polyoma virus preparations with a low (10 to 20%) or a high (60 to 80%) proportion of pseudovirions can be obtained.

Journal Article↗
PubMed1994

Dissociation between radioresistant DNA replication and chromosomal radiosensitivity in ataxia telangiectasia cells.

Ataxia telangiectasia (AT) skin fibroblasts in G1 phase and peripheral blood lymphocytes in G0 and G1 phase were studied for their DNA replication response to X-rays. The irradiation of normal cells in G1 but not in G0 phase caused a delay of onset of DNA replication, which was less pronounced in AT cells. However, such radioresistant DNA replication itself cannot be the sole mechanism of the increased sensitivity of AT cells to chromosome aberration formation by X-rays for the following two reasons: (1) due to the intrinsically slow cell cycle progression of AT fibroblasts, the time of traverse to DNA replication of AT cells was comparable with that of normal cells after exposure to 1 Gy while AT cells gave rise to a greatly increased number of chromatid aberrations; (2) in peripheral blood lymphocytes irradiated in G0 phase, the traversal to the DNA replication phase was the same for normal and AT cells in spite of the well documented chromosomal radiosensitivity of G0-irradiated AT cells. The AT factor may be better explained as a key element directly involved in DNA damage processing, which in turn provides messages to suppress replication if recombination and replication are mutually exclusive.

Adolescent↗
PubMed1975

Chromosomes of a cell line of Dipodomys panamintinus (kangaroo rat). A banding and autoradiographic study.

The chromosomes of an established cell line of Dipodomys panamintinus have been characterised in terms of their C, G and Q banding patterns, and the distributions of silver grains in autoradiographs of chromosomes labelled in early or late S phase. No relationship could be established between C, G or Q banding regions of chromosomes and a particular S phase time of replication of the DNA in these banded regions. The implication of this result to the concept of heterochromatin is discussed.

Animals↗
PubMed1999

Eukaryotic DNA replication.

One of the fundamental characteristics of life is the ability of an entity to reproduce itself, which stems from the ability of the DNA molecule to replicate itself. The initiation step of DNA replication, where control over the timing and frequency of replication is exerted, is poorly understood in eukaryotes in general, and in mammalian cells in particular. The cis-acting DNA element defining the position and providing control over initiation is the replication origin. The activation of replication origins seems to be dependent on the presence of both a particular sequence and of structural determinants. In the past few years, the development of new methods for identification and mapping of origins of DNA replication has allowed some understanding of the fundamental elements that control the replication process. This review summarizes some of the major findings of this century, regarding the mechanism of DNA replication, emphasizing what is known about the replication of mammalian DNA. J. Cell. Biochem. Suppls. 32/33:1-14, 1999.

Animals↗
PubMed1997

Analysis of replication timing of ribosomal RNA genes by fluorescence in situ hybridization.

Fluorescence in situ hybridization has been used to study the replication timing of various repeat DNA families in the short arms of human acrocentric chromosomes. In interphase nuclei, unreplicated DNA segments show singlet hybridization signals whereas replicated loci have doublet signals. The distribution of these two patterns in unsynchronized cell cultures revealed that the rRNA gene clusters replicate earlier than the closely juxtaposed alpha- and beta-satellite DNA sequences. Within the rDNA repeat unit, replication of the intergenic spacer appears to precede that of the transcribed rDNA.

DNA Replication↗
PubMed1999

Nuclear organization of mammalian genomes. Polar chromosome territories build up functionally distinct higher order compartments.

We investigated the nuclear higher order compartmentalization of chromatin according to its replication timing (Ferreira et al. 1997) and the relations of this compartmentalization to chromosome structure and the spatial organization of transcription. Our aim was to provide a comprehensive and integrated view on the relations between chromosome structure and functional nuclear architecture. Using different mammalian cell types, we show that distinct higher order compartments whose DNA displays a specific replication timing are stably maintained during all interphase stages. The organizational principle is clonally inherited. We directly demonstrate the presence of polar chromosome territories that align to build up higher order compartments, as previously suggested (Ferreira et al. 1997). Polar chromosome territories display a specific orientation of early and late replicating subregions that correspond to R- or G/C-bands of mitotic chromosomes. Higher order compartments containing G/C-bands replicating during the second half of the S phase display no transcriptional activity detectable by BrUTP pulse labeling and show no evidence of transcriptional competence. Transcriptionally competent and active chromatin is confined to a coherent compartment within the nuclear interior that comprises early replicating R-band sequences. As a whole, the data provide an integrated view on chromosome structure, nuclear higher order compartmentalization, and their relation to the spatial organization of functional nuclear processes.

Acetylation↗