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PubMed2000

A detailed analysis of cyclin A accumulation at the G(1)/S border in normal and transformed cells.

The temporal relationship between cyclin A accumulation and the onset of DNA replication was analyzed in detail. Five untransformed and nine transformed asynchronously growing cell cultures were investigated using a triple immunofluorescence staining protocol combined with computerized evaluation of staining intensities in individual cells. The simultaneous staining of BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position of each cell investigated. Cells at the G(1)/S border were identified on the basis of cyclin E content and were further analyzed with respect to cyclin A and BrdU content. A method was developed to calculate objective thresholds defining the highest staining intensity found in the negative cells in the population. Using the thresholds we could distinguish cells with minute amounts of cyclin A and BrdU from truly negative cells. We show that the onset of cyclin A accumulation and the start of DNA replication occurs at the same time, or deviating by a few minutes at the most. We also show that cyclin A accumulates continuously during S. This study clearly demonstrates that nuclear cyclin A can be used as a reliable marker for the S and G(2) phases in both normal and transformed interphase cells.

Antibodies↗
PubMed2004

SV40 DNA replication inhibition by the monofunctional DNA alkylator Et743.

Ecteinascidin 743 (Et743) is a highly cytotoxic anticancer agent isolated from the squirt Ecteinascidia turbinate, which alkylates DNA in the minor groove at GC-rich sequences resulting in an unusual bending toward the major groove. The ability of Et743 to block DNA replication was studied using the well-established simian virus (SV40) model for mammalian DNA replication in cells and cell-free extracts. Intracellular SV40 DNA isolated from Et743-treated BSC-1 cells was analyzed by native, two-dimensional agarose gel electrophoresis. A low frequency of Et743 adducts detected at 30-100 nM drug concentrations inhibited SV40 origin activity and induced formation of unusual DNA replication intermediates. Under cell-free conditions, only a high Et743 adduct frequency reduced SV40 DNA synthesis. Comparative studies involving related DNA alkylators, tomamycin and saframycin A, revealed inhibition of SV40 DNA replication in cells at concentrations approximately 10 times higher than Et743. Under cell-free conditions tomamycin- or saframycin-A-adducted DNA templates inhibited DNA synthesis similarly to Et743. Et743 appears to be unusual among other alkylators, because its adducts strongly inhibit intracellular SV40 DNA replication but are relatively weak as cis inhibitors as measured under cell-free conditions.

Animals↗
PubMed1982

DNA Synthesis and Gene Expression in Bacillus subtilis Infected with Wild-Type and Hypermodification-Defective Bacteriophage SP10.

A hypermodified base (Y-Thy) replaces 20% of the thymine (Thy) in mature DNA of Bacillus subtilis phage SP10. Two noncomplementing hypermodification-defective (hmd) mutants are described. At 30 degrees C, hmd phage carried out a normal program, but at temperatures of >/=37 degrees C, the infection process was nonproductive. When cells were infected at 37 degrees C with hmd phage, DNA synthesis started at its usual time (12 min), proceeded at about half the normal rate for 6 to 8 min, and then stopped or declined manyfold. All, or nearly all, of the DNA made under hmd conditions consisted of fully hypermodified parental DNA strands H-bonded to unhypermodified nascent strands. The reduced levels of DNA synthesis observed under hmd conditions were accompanied by weak expression of late genes. A sucrose gradient analysis of SP10 hmd(+) replicating DNA intermediates was made. Two intermediates, called VG and F, were identified. VF consisted of condensed DNA complexed to protein; VF also contained negatively supercoiled domains covalently joined to relaxed regions. F was composed of linear concatenates from which mature DNA was cleaved. None of those intermediates was evident in cells infected at 37 degrees C with hmd phage. Shiftup experiments were performed wherein cells infected with hmd phage at 30 degrees C were shifted to 37 degrees C at a time when replication was well under way. DNA synthesis stopped or declined manyfold 10 min after shiftup. The hmd DNA made after shiftup was conserved as a form sedimentationally equivalent to the F intermediate, but little mature DNA was evident. It is proposed that Y-Thy is required for replication and DNA maturation because certain key proteins involved with these processes interact preferentially with hypermodified DNA.

Journal Article↗
PubMed1989

Temporal order of gene replication in Chinese hamster ovary cells.

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.

Animals↗
PubMed1986

Yeast DNA replication.

We have explored various strategies for exploiting the yeast genetic and biochemical system for understanding DNA replication. Because of the long time that has intervened between the isolation of random replication mutants of yeast and the identification of the gene products affected, an alternative approach to elucidating the molecular basis of replication has been needed. One such alternative involves purifying replication proteins, isolating the genes encoding them, and constructing the appropriate mutant from the cloned gene. We have applied this "reverse genetics" strategy to three different replication activities: DNA polymerases, single-stranded DNA binding proteins, and proteins that bind to autonomously replicating sequence (ARS) elements.

DNA Polymerase I↗
PubMed1975

Uridine uptake in a unicellular eukaryote during the interdivision period and after growth arrest.

The rate of uridine uptake was measured in Tetrahymena after shiftdown to non-nutrient physiological salt solution. Uptake follows Michaelis-Menten kinetics and an apparent Km of transport of 2 X 10-minus 6 M has been estimated. This value is in good agreement with those reported for tissue-derived cells in culture. Incorporation of uridine into RNA follows similar kinetics suggesting that uptake is rate limiting for incorporation. Within three hours after shiftdown the rate of uptake is decreased by an order of magnitude. Also at three hours after shiftdown pairing occurs between cells of complementary mating types. It seems likely that the change in uptake is a reflection of a surface change associated with differentiation. The rate of uptake was also measured during the interdivision period using cells synchronized by a physical selection procedure. A change in rate occurs at the time the cells begin replication of DNA and is essentially stable thereafter. These results indicate that there exists in Tetrahymena a relationship between surface properties as assayed by uridine uptake and properties of growth and differentiation.

Animals↗
PubMed1995

Plus-strand DNA synthesis of the yeast retrotransposon Ty1 is initiated at two sites, PPT1 next to the 3' LTR and PPT2 within the pol gene. PPT1 is sufficient for Ty1 transposition.

Long terminal repeat elements and retroviruses require primers for initiation of minus and plus-strand DNA synthesis by reverse transcriptase. Here we demonstrate genetically that plus-strand DNA synthesis of the yeast Ty1 element is initiated at two sites located at the 5' boundary of the 3' long terminal repeat (PPT1) and near the middle of the pol gene in the integrase coding sequence (PPT2). A consequence of the presence of two PPTs is that Ty1 plus-strand DNA exists as segments at some time during replication. Three fragments have been identified: the plus-strand strong-stop DNA initiated at PPT1, a downstream fragment initiated at PPT2 and an upstream fragment spanning the 5'-terminal part of Ty1 and a portion of the TyB gene. Characterization of the 3' ends of the plus-strand DNA fragments reveals (1) that the upstream fragment is elongated beyond PPT2 creating a plus-strand overlap and (2) that the majority of plus-strand strong-stop DNA fragments bear a copy of the minus-strand primer binding site in agreement with the accepted model of retroviral genomic RNA reverse transcription. The two polypurine tracts, PPT1 and PPT2, have an identical sequence GGGTGGTA. Mutations replacing purines by pyrimidines in this sequence significantly diminish or abolish initiation of plus-strand synthesis. Ty1 elements bearing a mutated PPT2 sequence are not defective for transposition whereas mutations in PPT1 abolish transposition.

Base Sequence↗
PubMed1991

Enteric adenovirus type 40:E1B transcription map and identification of novel E1A-E1B cotranscripts in lytically infected cells.

Adenovirus 40 (Ad40) is defective for growth in tissue culture but is complemented when the Ad2/5 or Ad12 E1B 55K protein is supplied in trans. Ad40 E1B mRNA has not been detected in E1-transformed cells, or at early times in lytically infected cells. In cells constitutively expressing the E1B region of Ad2, Ad40 E1B mRNAs are detected at late times in infection, after the onset of DNA replication. We have determined the Ad40 E1B transcription map from RNA produced at late times in infected KB16 cells, using S1 nuclease, primer extension, PCR-cDNA analysis, and Northern blotting. E1B transcripts corresponding to Ad2 14 S, 22 S, and 9 S mRNAs were identified but no 13 S mRNA equivalent was detected, a pattern similar to that seen in the Ad12 transcription map. The coding potential for E1B 19K, 55K, and 15K proteins and for ppIX is retained in the Ad40 transcripts. In addition we find novel E1A-E1B cotranscript counterparts of the 14 S and 22 S mRNAs. These contain the first 40 codons of the E1A first exon linked to a site 4-5 nt downstream of the E1B cap site, retaining all the coding potential of the E1B mRNAs. No new open reading frames are created by the junction, and the E1A ORF terminates with one codon added after the junction. Each E1A-E1B cotranscript is present in abundance comparable to that of its authentic E1B counterpart. The E1A-E1B junction is unusual in that it does not conform to splice consensus sequences and thus may not be generated by a conventional splicing mechanism.

Adenovirus Early Proteins↗
PubMed1994

Herpes simplex virus type 1 DNA replication and gene expression during explant-induced reactivation of latently infected murine sensory ganglia.

Infectious virus assays and PCR amplification of DNA and RNA were used to investigate herpes simplex virus DNA replication and gene expression in two murine in vitro models for virus reactivation. We examined latent infections with wild-type (wt), precisely defined latency-associated transcript-negative (LAT-) mutants, and LAT+ rescuants of these mutants of the 17syn+ strain of virus in both murine trigeminal and lumbosacral ganglia and of the KOS(M) strain in the latter. In explants of ganglia latently infected with the LAT- mutant of strain 17syn+ virus, a reduction in number of cultures exhibiting cytopathic effects due to virus reactivation and measurable delays in virus recovery were observed compared with wt or the LAT+ rescuant. This LAT-specific effect was not seen in explants of lumbosacral ganglia latently infected with mutants derived from the KOS(M) strain of virus. Although there was appreciable variation between individual animals, no significant difference between LAT+ and LAT- virus in time of onset of viral DNA replication in explanted ganglia was seen with use of either virus strain. There was a slight decrease in the relative amount of viral DNA recovered compared with internal cellular controls in latently infected ganglia harboring the LAT- mutant of 17syn+ compared with the wt virus or the LAT+ rescuant. This reduced relative amount ranged from 0 to as much as 50% but averaged 20%. Such differences were not seen in infections with KOS(M)-derived mutants. In contrast, although expression of productive-cycle transcripts could be detected within 4 h following explant cultivation of latently infected ganglia, no differences between LAT+ and LAT- viruses could be seen. As discussed, these data place specific constraints on possible models for the role of LAT expression in in vitro reactivation systems.

Animals↗
PubMed2005

The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle.

We describe a new method for synchronizing bacterial cells. Cells that have transiently expressed an inducible mutant 'sticky' flagellin are adhered to a volume of glass beads suspended in a chromatography column though which growth medium is pumped. Following repression of flagellin synthesis, newborn cells are eluted from the column in large quantities exceeding that of current baby machine techniques by approximately 10-fold. Eluted cultures of 'baby cells' are highly synchronous as determined by analysis of DNA replication, cell division and other events, over time after elution from the column. We also show that use of 'minutes after elution' as a time metric permits much greater temporal resolution among sequential chromosomal events than the commonly used metric of cell size (length). The former approach reveals the existence of transient intermediate stages that are missed by the latter approach. This finding has two important implications. First, at a practical level, the baby cell column is a particularly powerful method for temporal analysis. Second, at a conceptual level, replication-related events are more tightly linked to cell birth (i.e. cell division) than to absolute cell mass.

Adhesins, Bacterial↗
PubMed1990

An abnormal profile of DNA replication intermediates in Bloom's syndrome.

Bloom's syndrome (BS) cells display a characteristic genomic instability, notably an elevated frequency of sister-chromatid exchange. Replicating DNA in cultured BS cells was labeled with [3H]thymidine using several time schedules. Separation of DNA in agarose gels showed high molecular weight DNA and three classes of DNA replication intermediates: 20-kilobase DNA, 10-kilobase DNA, and Okazaki fragments. In contrast newly replicated DNA from normal cells showed no 20-kilobase DNA replication intermediates. Certain BS cells, exceptional in that their characteristic genomic instability has for unknown reasons been corrected, also differed from normal cells in having the 20-kilobase intermediate, but they differed from both normal cells and the other (the uncorrected) BS cells in lacking the 10-kilobase DNA replication intermediates.

Aphidicolin↗
PubMed1984

Reversible inhibition of bovine parvovirus DNA replication by aphidicolin and L-canavanine.

The replication of the autonomous parvovirus, bovine parvovirus (BPV), has been studied in virus-infected cells. Gel electrophoresis was used to determine the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, and L-canavanine, an inhibitor of protein synthesis, on viral DNA replication. Synchronized cell cultures were infected with 32P-labelled or unlabelled BPV in the presence or absence of aphidicolin and L-canavanine. Cells were harvested at various times post-infection, and DNA was electrophoresed and blotted. When aphidicolin was added to cells at the time of infection, then removed 8 h later, BPV replicative form DNA (RF) synthesis began within 2 h after its removal. This preceded the peak of cellular DNA synthesis by 2 h, unlike an uninhibited infection, when viral RF synthesis follows the peak of S phase by 2 to 4 h. Furthermore, if aphidicolin was added at any point during the replication cycle, BPV DNA synthesis stopped. This effect was shown to be completely reversible and indicated that aphidicolin did not disrupt the replication apparatus required for viral DNA synthesis. L-Canavanine inhibited synthesis of the virus-specific proteins NP-1 and VP3 and synthesis of BPV DNA. Upon removal of L-canavanine, viral protein synthesis was detected by 30 min followed by viral DNA synthesis. These results indicate that a specific S phase function other than cellular DNA synthesis is required for initiation of BPV DNA synthesis, that DNA polymerase alpha plays a major role in BPV DNA replication in vivo, and that these inhibitors can be used to inhibit reversibly various stages of BPV DNA replication.

Animals↗
PubMed1999

The karyotype of the Iberian imperial eagle (Aquila adalberti) analyzed by classical and DNA replication banding.

We report here for the first time the karyotype of the Iberian imperial eagle (Aquila adalberti). All eagles examined had a diploid number of 82 chromosomes and a greater number of microchromosomes (12 pairs) than has been found in all other species of the Accipitridae family. This karyotypic evidence corroborates the recent separation of A. adalberti from A. heliaca on the basis of molecular data. RB-FPG banding induced a specific banding pattern that allowed us to identify homologous chromosome pairs and revealed features about late and early replicating regions. Several chromosome banding techniques (C-, CMA3-, and restriction endonuclease banding and silver staining) were used to characterize the karyotype more accurately. Two GC-rich, late-replicating heterochromatin regions were found in the W chromosome. These regions are AluI resistant and can be used for sex determination in this species. All microchromosomes were heterochromatic, GC rich, and late replicating. Silver staining revealed active nucleolus organizing regions on a pair of microchromosomes that were entirely heterochromatic and stained intensely after CMA3-banding. Different chromosome rearrangements are discussed in order to establish the phylogenetic relationship between A. adalberti and its most closely related species, A. heliaca.

Animals↗
PubMed1972

Repair DNA synthesis in differentiated embryonic muscle cells.

The differentiation of embryonic skeletal muscle cells is closely coupled with the cessation of normal DNA replication. Once these cells begin to differentiate, they normally never undergo semiconservative replication of DNA during the entire life time of the muscle cell. Cessation of DNA synthesis has been shown to be accompanied by a loss of 80-90% of the replicative DNA polymerase activity of these cells. Despite this loss the studies reported here demonstrate that muscle cells retain the ability to synthesize DNA of a repair type after UV irradiation. These results suggest that the control exercised over semiconservative DNA synthesis during differentiation of these cells does not extend to repair synthesis after UV irradiation.

Animals↗
PubMed2018

Chromatin architecture changes and DNA replication fork collapse are critical features in cryopreserved cells that are differentially controlled by cryoprotectants.

In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. Moreover, for the first time, we describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. We simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Interestingly, cells with condensed chromatin, which was strongly stimulated by dimethyl sulfoxide (DMSO) prior to freezing had the highest rate of survival after thawing. Our results will facilitate the design of compounds and procedures to decrease injury to cryopreserved cells.

Cell Survival↗
PubMed1993

The effect on chromosome stability of deleting replication origins.

The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase. To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured. In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20% of the time, deletion of either highly active origin increased its rate of loss two- to fourfold. Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20% of cell divisions. This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used. Deletion of the same two origins from the full-length chromosome III, which contains more than six replication origins, had no effect on its rate of loss. These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.

Chromosome Mapping↗
PubMed1997

Transcription and DNA replication of sperm nuclei introduced into blastomeres of 2-cell mouse embryos.

The aim of this study was to investigate the behaviour of sperm nuclei in the cytoplasm of the 2-cell mouse embryo. To this end, we produced hybrids between anucleate fertilised oocyte fragments and blastomeres of the 2-cell embryos. When sperm nuclei at the stage of decondensation or recondensation were introduced into blastomeres the development of male pronuclei was usually retarded and they never reached the size of the blastomere nuclei. These abortive male pronuclei were unable to initiate transcription but they were capable of synthesising DNA. The majority of sperm nuclei introduced into blastomeres as early male pronuclei developed normally and reached the size of the blastomere nuclei. They synthesised DNA simultaneously with blastomere nuclei and were transcriptionally active. In addition they participated in the cleavage division of hybrid cells. This shows that the very early male pronucleus when transmitted from the oocyte cytoplasm to the blastomere cytoplasm can respond positively to the new cytoplasmic factors, i.e. it undertakes both DNA replication and transcription according to the time schedule characteristic of the second cell cycle.

Animals↗
PubMed1984

The coupling of DNA repair-recombination functions with DNA replication in bacteriophage T4: a new DNA repair mutant.

The requirement of DNA repair-recombination functions for T4 phage DNA replication has been known for some time but the underlying basis for this relationship has been unclear. This report is concerned with a new uv-sensitive gene [uvsU], whose function appears to bridge these two major activities of DNA. The [uvsU] mutant fails to complement [uvsX] mutants but uvsU maps in a region distinct from uvsX. Furthermore, the uvsU mutation specifically suppressed the DNA replication defect but not the uv sensitivity of the uvsX mutation. The previously discovered uvsW gene, whose mutations suppress the DNA replication defects of gene 59, 46, and 47 mutations, seems to have an analogous role. As a possible explanation for these observations, it is suggested that the uvsW and uvsU gene products (gps) couple the DNA repair-recombination and replication functions by controlling the entry of DNA intermediates from the replication pool into the DNA repair-recombination pathway. Furthermore the suppression data are interpreted to suggest that the gps uvsW, 59, 46, and 47 function together. Similarly the gps uvsU and uvsX may form a functional unit.

Base Sequence↗