Synchronous Replication Research Articles

Synchronous replication of SV 40 DNA in virus infected TC 7 cells induced by transient hypoxia

We transiently exposed SV 40 infected TC 7 cell cultures to a reduced O 2 tension (4–8 h, about 200 ppm relative to 10 5 Pa total pressure). Under the hypoxic conditions, ‘working’ viral replication forks were greatly retarded or stopped, and initiation of daughter strand synthesis in further SV 40 DNA molecules was suppressed. Reoxygenation released an immediate burst of SV 40 replication which mainly consisted of a synchronous viral replication round. This synchronous in vivo replication began at the known origin of replication and proceeded at normal rates to the known termination region. Viral replicons seemed to accumulate under hypoxia in a state fully prepared to begin replication immediately after recovery of a normal pO 2. The shut-down and sudden reactivation of DNA synthesis under hypoxia and reoxygenation, respectively, were not accompanied by changes of the phosphorylation state of large T antigen. The described synchronization procedure can be applied to optionally large SV 40 infected cell cultures.

Hepatic gene therapy: Efficient retroviral-mediated gene transfer into rat hepatocytes in vivo

The rat is an excellent model for gene therapy because there are many rat models for human diseases. We have developed a simple and efficient method to deliver genes to the rat liver using recombinant retroviral vectors. A 70% partial hepatectomy followed by retroviral infusion into the portal vein results in 10-15% hepatocyte transduction in vivo. This is 10 times more efficient than in the mouse due partially to the observation that the rat livers have much more synchronous hepatocyte replication after partial hepatectomy. Using a recombinant retroviral vector containing the human alpha 1-antitrypsin cDNA, persistent expression of the human protein in recipient rat plasma was observed for at least six months and at a level that is 10 times greater than the mouse. Thus, rats can serve as an excellent model for gene therapy of metabolic disorders secondary to hepatic deficiencies.

Two major replicating simian virus 40 chromosome classes. Synchronous replication fork movement is associated with bound large T antigen during elongation.

We have analyzed the asynchronous progression of replication forks through the early (E) and late (L) gene sides in bidirectionally replicating SV40 chromosomes during lytic infection. By cutting purified replicating DNA with an appropriate single-site restriction endonuclease and measuring the contour lengths of replicated and unreplicated segments by electron microscopy, the positions of the two replication forks in each elongating intermediate were determined. Our results indicate that there are at least two major classes of replicating SV40 chromosomes which differ in their relative rate of E and L fork movement, the presence or absence of bound SV40 large T antigen during elongation, and the termination region utilized. These two classes also have altered apparent start sites for initiating bidirectional replication, flanking either side of core ori. The largest group (67%) replicated synchronously was associated with T antigen during elongation, appeared to initiate bidirectional elongation at nucleotide 5203 or 41 base pairs (bp) toward the E side of 0/5243, at the junction of T binding site I and ori, and terminated at the typical region centered at 0.5 map units. A second group (24%) replicated asynchronously with the L fork moving 3 times faster than the E fork, was not associated with T antigen during elongation, and terminated at a broad region centered at 0.73 map units. This group appeared to initiate at nucleotide 29 at the junction of the AT-rich region of ori, T binding site I, and the start of the 21-bp repeated transcriptional control sequences. A third group (9%) appeared to initiate at nucleotide 5148 or 95 bp to the E side of 0/5243 and replicated asynchronously preferentially on the E side at early times. However, this group is related to the synchronous class in that it contains bound T antigen and both forks move synchronously past 30% elongation, terminating at the same region. The association of T antigen with synchronous but not asynchronous DNA molecules indicates that T functions in regulating fork movement during elongation. A synchronization role implies that both forks are closely associated with one another in replicating molecules with bound T. Replicating molecules lacking T not only elongated highly asynchronously but preferential fork progression occurred almost exclusively on the L side. The ori region in asynchronous compared to synchronous intermediates was differentially sensitive to BglI digestion, indicating that nuclease digestion can distinguish between different populations of replicating molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Processive replication of single-stranded DNA templates by the herpes simplex virus-induced DNA polymerase.

The DNA polymerase encoded by herpes simplex virus 1 consists of a single polypeptide of Mr 136,000 that has both DNA polymerase and 3'----5' exonuclease activities; it lacks a 5'----3' exonuclease. The herpes polymerase is exceptionally slow in extending a synthetic DNA primer annealed to circular single-stranded DNA (turnover number approximately 0.25 nucleotide). Nevertheless, it is highly processive because of its extremely tight binding to a primer terminus (Kd less than 1 nM). The single-stranded DNA-binding protein from Escherichia coli greatly stimulates the rate (turnover number approximately 4.5 nucleotides) by facilitating the efficient binding to and extension of the DNA primers. Synchronous replication by the polymerase of primed single-stranded DNA circles coated with the single-stranded DNA-binding protein proceeds to the last nucleotide of available 5.4-kilobase template without dissociation, despite the 20-30 min required to replicate the circle. Upon completion of synthesis, the polymerase is slow in cycling to other primed single-stranded DNA circles. ATP (or dATP) is not required to initiate or sustain highly processive synthesis. The 3'----5' exonuclease associated with the herpes DNA polymerase binds a 3' terminus tightly (Km less than 50 nM) and is as sensitive as the polymerase activity to inhibition by phosphonoacetic acid (Ki approximately 4 microM), suggesting close communication between the polymerase and exonuclease sites.

An RNA-DNA copolymer whose synthesis is correlated with the transcriptional requirement for chromosomal initiation in Bacillus subtilis contains ribosomal RNA sequences

During synchronous replication induced in a temperature-sensitive initiation mutant of Bacillus subtilis, we previously isolated an RNA covalently linked to DNA. This molecule was synthesized at specific times, correlated with the period of transcription that is required to initiate a new round of replication. In this paper, we show that both RNA and DNA components of the RNA-DNA molecule hybridized with the coding strand for ribosomal RNA. Competition hybridization experiments also demonstrated that ribosomal RNA sequences represent the great majority of the RNA that is linked to DNA. Both RNA and DNA components of the RNA-DNA molecule also hybridized with a region close to the origin of replication, in particular with E19 and E22, two restriction fragments that are replicated early. These fragments in fact form part of the ribosomal operon rrnO. The role of the RNA-linked DNA molecule in initiation in B. subtilis and the possibility that this molecule emanates directly from rrnO rather than from other ribosomal RNA genes are discussed.

Mitotic cell cycle control in Physarum: Unprecedented insights via flow-cytometry

High resolution flow-cytometric studies of isolated macroplasmodial nuclei of the myxomycete, Physarum polycephalum provide definite evidence for the persistence of natural synchrony at mitosis throughout the entire cell cycle, i.e. completely synchronous DNA replication and traverse of G2. Even if nuclei within a given macroplasmodium belong to two distinct genome size classes (mixoploidy), they cycle and traverse mitosis in strict synchrony. This cannot be explained by current models of regulation of division based solely upon nuclear size and/or nuclear/cytoplasm ratios. Constitutional DNA content variation was apparent among all tested strains, and loss of late-replicating, presumably AT-rich DNA accounts for this variation. A constant duration of the S phase is maintained, irrespective of DNA content, via differential slowdown of replication rates during the 2nd and 3rd hours of replication. A frequently described extension of nuclear replication into G2 could not be substantiated. Interference with DNA and protein synthesis provides the first evidence for a brief “G1 phase” equivalent of 3–4 min duration in asynchronous microplasmodial cultures, and temporally assigns a protein synthesis-dependent “transition point” for completion of mitosis and initiation of DNA synthesis at 5 min prior to actual division, nuclei which have passed this point at the time of addition of cycloheximide replicate 5% of their DNA before they become arrested. These findings provide strong experimental support for the transition point concept of cell cycle control, and additionally are commensurate with some form of the replicon-set hypothesis in Physarum.

Periodic enzyme synthesis and oscillatory repression: Why is the period of oscillation close to the cell cycle time?

During exponential growth of a cell culture, some enzymes are synthesized periodically. In a synchronous culture, in which all cells undergo DNA synthesis and division more-or-less synchronously, the burst of enzyme synthesis also occurs synchronously in each cell once per division cycle. However, there are a number of interesting cases in which periodic enzyme synthesis continues in the absence of synchronous DNA replication or cell division. In all cases of periodic enzyme synthesis in asynchronous cultures, the time between bursts of enzyme synthesis, though no longer identical to the cell cycle time, is still close to the interdivision time of the growing, replicating cells. The theory of oscillatory repression looks for an explanation of this phenomenon in the periodic repression of gene transcription caused by periodic fluctuations in the concentration of the endproduct of the metabolic pathway of which the enzyme is a part. A major difficulty with this theory is that there is no obvious relationship between the periodicity of the negative feedback loop, which is determined by the kinetics of synthesis and degradation of the individual components of the feedback loop, and the periodicity of the cell cycle, which is determined by overall net synthetic rates of cellular macromolecules. Why should the period of oscillation of a repressible gene transcription system be close to the interdivision time of a population of growing cells? In this paper, I show that the relationship may be coincidental: the two fundamental periods are close to each other because they are both close to the mass-doubling time of the cell culture. That the mean interdivision time must be close to the mass-doubling time is a consequence of “balanced” growth: there is a stable size distribution of cells in a growing culture. That the period of oscillation of the negative feedback loop is also close to the mass-doubling time is shown to be a consequence of the large, nearly constant demand for endproduct and the assumed stability of the enzyme. The period of oscillation is largely attributable to the slow dilution of the stable enzyme by cell growth. For reasonable values of the parameters describing the gene-control system, I show that the enzyme must be diluted by a factor of two (approximately), that is, by the growth accomplished by one mass-doubling (nearly).

Sequential mutagenesis of drug resistance in Streptococcus mutans during synchronous replication.

The frequency of nitrosoguanidine-induced mutation for streptomycin-, bacitracin- and rifampicin-resistance was measured in Streptococcus mutans during synchronous replication after release from chloramphenicol inhibition. A clear peak of mutagenesis for each marker was observed at certain times during synchronous replication. These times were different for individual markers; the times of peaks for streptomycin-, bacitracin- and rifampicin-resistance were 13, 22 and 12 min, respectively. At a definite time after the first peak, there was a second one. The distances between the first and second peaks during the synchronous replication were identical for all markers and approximately 50 min which represents the doubling time of the organisms. These results indicate that nitrosoguanidine causes sequential mutagenesis for these three markers in Streptococcus mutans when the growth is resumed after chromosome alignment, so that the methods may be useful in determining the sequence of gene replication for various markers in Streptococcus mutans.

The periodic synthesis of tubulin in the Physarum cell cycle. Characterization of Physarum tubulins by affinity for monoclonal antibodies and by peptide mapping.

Polypeptides preferentially labeled in the G2 phase of the synchronous nuclear replication cycle of Physarum macroplasmodia were compared in electrophoretic mobility and peptide map with the tubulins enriched from Physarum myxamoebae. One major and one minor fluorographic species match the myxamoebal alpha and beta chains, respectively. Thus, tubulins are among the proteins of Physarum selectively synthesized before nuclear division. A third species P, prominently labeled in premitotic plasmodia, is distinct from the two myxamoebal tubulins even though it co-polymerizes with microtubules. The nature of P remains unknown. Two rat monoclonal antibodies directed against yeast tubulin were found to bind selectively to the alpha tubulin of porcine brain. These served to confirm the assignment of the 50,000-dalton Physarum myxamoebal tubulin as an alpha-like polypeptide.

Replication terminus of the Bacillus subtilis chromosome.

Bidirectional replication of the Bacillus subtilis chromosome terminates at a point on the circular chromosome which is symmetrically opposite to the replication origin. Since replication rates are similar in both "halves" of the chromosome, termination presumably occurs at the meeting point of the two replication forks. To investigate whether the DNA sequence of this region of the chromosome contributes to the termination event, we have determined the latest replicating region of a chromosome in which this DNA sequence is no longer symmetrically opposite to the origin. The merodiploid strain GSY1127 has a very large nontandem duplication (approximately 25% of the total chromosome length) in the left-hand half of the chromosome, so that size and symmetry of this chromosome are grossly different from those of normal strains. We have examined the replication order of genetic markers in this strain by measuring subtilis terminal marker for replication remains a terminal marker in the merodiploid, i.e., replicates later than a marker situated symmetrically opposite to the replication origin. These results were supported by replication orders determined by pulse-density transfer experiments during synchronous replication. The data obtained indicate that there is a preferred site for the termination of replication in the B. subtilis chromosome.

Homologous early replication patterns of the distal short arms of prometaphasic X and Y chromosomes.

Replication studies on prometaphasic human sex chromosomes reveal a distinct early replicating segment on both distal Xp ad Yp. These segments correspond to high resolution bands Xp22.3 and Yp11.3. The findings demonstrate synchronous replication of these parts of the sex chromosomes and correspond to the comparatively long stretches of Xp and Yp that participate in a synaptonemal complex. Furthermore these observations are compatible with Polani's view that suggests homologous segments with similar genetic information on both sex chromosomes (Polani 1982).

Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication.

After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with high-molecular-weight DNA. This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradient. It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K. Properties of the complex were not altered by prior treatment with RNase H. A control experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex. These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage. In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles. A possible involvement of this RNA in the initiation of chromosome replication is discussed.

Periodic synthesis of microtubular proteins in the cell cycle of Physarum.

Periodic polypeptide labeling over the naturally synchronous nuclear replication cycle of Physarum polycephalum was analyzed by fluorography of two-dimensional electropherograms. Two sets of polypeptides, denoted as P and Q, showed strong periodicity; they were maximally labeled just prior to mitosis. This periodicity was shown to reflect synthesis rather than turnover or recovery. Both P and Q copolymerized with porcine microtubular proteins and displayed electrophoretic properties similar to those of porcine tubulins. The significance of the periodic synthesis of these microtubular proteins is discussed as a possible component within the chain of events that establishes the high mitotic synchrony of Physarum syncytia.

Relationship between tracheary element differentiation and the cell cycle in single cells isolated from the mesophyll of Zinnia elegans

A relationship between tracheary element differentiation and the cell cycle was studied in single cells isolated from the mesophyll of Zinnia elegans L. cv. Canary bird. Almost all nuclei of isolated mesophyll cells were at the 2 C level of DNA, indicating that almost all cells were initially in the G1 phase and that somatic polyploidy was absent. Cultured cells underwent partially synchronous DNA replication at 42 h and mitosis at 54 h of culture, and the first cell cycle time was approximately 58 h.The occurrence and timing of DNA replication and mitosis during cytodifferentiation to tracheary elements were investigated using microspectrophotometry, microfluorometry, tritiated thymidine autoradiography, and serial observation. More than 55% of the nuclei of the immature tracheary elements were at the 2 C level of DNA and were not labeled by continuous feeding with tritiated thymidine, providing clear evidence that these cells differentiated without interventing DNA replication. Some tracheary elements (approximately 30%) were formed after one round of the cell cycle, and others (less than 5%) were formed after passing through the S phase, but without intervening mitosis. All types of tracheary elements appeared simultaneously after 58 h of culture, and their patterns of increase in number were similar. From the results, we propose a hypothesis concerning the relationship between cytodifferentiation and the cell cycle.

Evidence for the synchronous replication of mitochondrial DNA during the yeast cell cycle

The nature of mitochondrial DNA replication during the synchronous cell cycle in the yeast, Saccharomyces cerevisiae has been investigated by examining the rate of labeled DNA precursor incorporation into specific segments of the mitochondrial genome at defined points during synchronous growth. The movement of label uptake from one area of the DNA to another at different times during the synchronous cell cycle indicates mitochondrial DNA replication to be a synchronous process during this time with most or all molecules at the same point in replication at any given time during the cell cycle.

An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles.

Infection of Protoplasts from Soybean Cell Culture with Southern Bean Mosaic and Cowpea Mosaic Viruses

SUMMARY Soybean protoplasts, isolated from liquid suspension culture, were successfully infected with cowpea mosaic virus (CPMV) and with southern bean mosaic virus (SBMV). Poly-l-ornithine (PLO) was required for infection with either virus. As detected by fluorescent antibody, 70 to 90% of the protoplasts were infected by CPMV when the inoculation medium contained 0.4 m-sorbitol, 0.5 µg virus/ml, 1.5 µg PLO/ml, 10 mm-potassium phosphate buffer, pH 6.3, and 0.5 mm-CaCl2, and inoculation was performed at 23 °C. This maximum level of infection was decreased sixfold when CaCl2 was omitted. Inoculation at temperatures below 10 °C also decreased infection substantially. With SBMV a maximum of 30 to 35% of the protoplasts were infected when 0.4 m-sorbitol, 2 to 2.5 µg virus/ml, 2 µg PLO/ml, 10 mm-tris-HCl buffer, pH 8.0, 1 mm-MgSO4 and 0.5 mm-CaCl2 were present in the inoculum. Less than 5% of the protoplasts were infected when magnesium and calcium salts were omitted. The major advantage of the use of soybean cell suspension culture for plant virus studies is that it can provide a reliable and continuous source of cells for protoplast isolation. These soybean protoplasts allow synchronous infection and replication under sterile conditions without antibiotics and also a high degree of reproducibility.

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography.

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with 3H-thymidine and chromosome samples were prepared for the next 32 h at 1--2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.

Genetic analysis of Streptococcus mutans by nitrosoguanidine-induced mutation in synchronous replication.

The frequency of nitrosoguanidine-induced mutation for streptomycin (Str)-, rifampicin (Rif)-and bacitracin (Bac)-resistance, and that of colonial mutation accompanied by elevated production of cell-associated insoluble glucan, were measured in Streptococcus mutans strains 102 and GS 5 during synchronous replication after release from chloramphenicol inhibition. A clear peak of mutagenesis for each drug-resistance was observed at certain times during synchronous replication. Peaks appeared in the order of Rif-r, Str-r and Bac-r during synchronous replication. At a definite time after the first peak, there was a second one. The distances between first and second peaks were identical for all markers and approximately 50 min which represents the doubling time of the organisms. The frequency of colonial mutation showed two peaks (P1sA and PlsB) after the peak for Bac-r, each of which was followed by the second peak 50 min after incubation. These results indicate that the method may be a valuable tool for gene analysis of S. mutans, and that the markers for Rif-r, Str-r, Bac-r, P1sA or PlsB in that order duplicate on the chromosomes of S. mutans.

Cytogenetic analysis of great horned owls (Bubo virginianus).

A chromosome analysis of seven great honored owls (Bubo virginianus, Gmelin) has been performed on peripheral lymphocytes. The modal chromosome number was found to be 2n = 82. Measurements of relative lengths and arm ratios of the macrochromosomes were made. All chromosomes contain a region of constitutive heterochromatin in a centromeric location, with the W chromosome being composed almost entirely of this material. Chromosome replication, as demonstrated by the BudR-acridine orange method, showed synchronous replication of the two Z chromosomes of the male and late replication of the W chromosome of the female. In this species, functional regions of nucleolar organization appear to be localized to the microchromosomes.