Evidence that F'' lac replicates asynchronously during the cell cycle of Escherichia coli B/r.

Summary The replication of F lac was studied in exponentially growing cultures of E. coli B/r. The cells were pulse induced for the synthesis of -galactosidase and their DNA pulse labelled with ³H thymidine. The cells were then separated into age classes by centrifugation through a sucrose gradient in a zonal rotor. Plasmid replication was measured in each age fraction by three methods: the rate at which -galactosidase could be induced, the amount of label incorporated into CCC plasmid DNA which had been separated from chromosomal DNA on agarose gels, and the amount of label incorporated into plasmid DNA which had been separated from chromosomal DNA by ultracentrifugation through CsCl-EtBr gradients. All these methods gave the same result, that replication of F lac occurs in cells of all ages and is not confined to a part of the cell cycle.     

Replication of kinetoplast DNA of Crithidia acanthocephali. I. Density shift experiments using deuterium oxide

The protozoan Crithidia acanthocephali contains, within a modified region of a mitochondrion, a mass of DNA known as kinetoplast DNA (kDNA). This DNA consists mainly of an association of approximately 27,000 covalently closed 0.8-mum circular molecules which are apparently held together in a definite ordered manner by topological interlocking. After culturing of C. acanthocephali cells for 25 generations in medium containing 75% deuterium oxide, both nuclear DNA (rhonative, nondeuterated=1.717 g/cm3) and kDNA (rhonative, nondeuterated=1.702 g/cm3) increased in buoyant density by 0.012 g/cm3. The replication of the two DNAs was studied by cesium chloride buoyant density analysis of DNAs from exponentially growing cells taken at 1.0, 1.4, 2.0, 3.0, and 4.0 cell doublings after transfer of cells from D2O- containing medium into medium containing only normal water. The results obtained from analysis of both native and denatured nuclear DNAs indicate that this DNA replicates semiconservatively. From an analysis of intact associations of kDNA, it appears that this DNA doubles once per generation and that the newly synthesized DNA does not segregate from parental DNA. Fractions of covalently closed single circular molecules and of open circular and unit length linear molecules were obtained from associations of kDNA by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium gradient centrifugation. Buoyant density profiles obtained from these fractions indicate that: (a) doubling of the kDNA results from the replication of each circular molecule rather than from repeated replication of a small fraction of the circular molecules; (b) replication of kDNA is semiconservative rather than conservative, but there is recombination between the circles at an undefined time during the cell cycle.     

Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli.

Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient. In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region. In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA. In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously. In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated. Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions. The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.     

Sequence organization and expression of a yeast plasmid DNA.

Saccharomyces cerevisiae strain A364A D5 contains circular double-stranded DNA molecules of 6230 +/- 30 base pairs (2mu DNA) which are present in 68 copies per cell and make up 2.4% of the haploid genome. About 0.4% of non-poly A containing yeast RNA hybridizes to the yeast DNA circles. When denatured and then self-annealed, the DNA molecules assume a characteristic "dumbbell" shape in the electron microscope indicating that each circle possesses a non-tandem inverted repeat sequence of 630 +/- 10 base pairs. Eco-RI digestion of purified 2mu DNA yields 4 fragments on an agarose gel whose combined molecular mass is twice that of the monomer circle, suggesting that there are 2 populations of circles, each of the same molecular weight. Representatives of each population have been separated by cloning in Escherichia coli via the bacterial plasmid pSC101. Heteroduplex analysis of the cloned circles show that the 2 different populations arise because of intramolecular recombination between the inverted repeat sequences. Acrylamide gel patterns of polypeptides synthesized in bacterial mini-cells containing the hybrid plasmids between 2mu DNA and pSC101 are significantly different than the pattern obtained from mini-cells containing pSC101 alone.     

Repair of lesions induced by bruneomycin in DNA of isolated mitochondria from the mature eggs of the teleost fish Misgurnus fossilis

Addition of a radiomimetic antibiotic bruneomycin (Streptonigrin) to isolated mitochondria from mature quiescent oocytes of the teleost fish loach Misgurnus fossilis leads to the induction of unscheduled synthesis of mitochondrial DNA. Most of the newly synthesized DNA has the sedimentation properties of open circles and up to 15% of the label is present in the fraction of the covalently closed-circular molecules. The size of the newly synthesized DNA stretches determined from the bouyant shift of DNA labeled with 5-bromouracil and [3H]dAMP and sonicated to fragments of different molecular weight, was found to be equal to about 1000 nucleotides for the labeled covalently closed circles and to about 2000 nucleotides for the labeled open-circular DNA. Experiments with the centrifugation of non-sheared and sonicated 5-bromouracil and [3H]dAMP-labeled mitochondrial DNA (mtDNA) in alkaline CsCl density gradients provided evidence of a covalent linkage between newly-synthesized stretches and the parental DNA strands. It is concluded from these data that the unscheduled mtDNA synthesis induced by bruneomycin does at least in part represent mtDNA repair synthesis.     

Characterization of the plasmid DNAs of Rhodopseudomonas capsulata

Presence of extrachromosomal DNa in Rhodopseudomonas capsulata strain BH9 was shown by the appearance of a satellite band in a dye-buoyant density gradient. Radioactively labelled DNA was prepared from this satellite band and examined on a 5–20% sucrose gradient. Three radioactive peaks with sedimentation coefficients of 100 S, 94 S, and 58–64 S, respectively, were consistently observed. Analysis of these sedimentation coefficients suggested that there are two species of plasmid DNA with molecular sizes of 94×10⁶ daltons (named pBH91) and 74×10⁶ daltons (named pBH92). The 58–64 S peak is attributed to open circular molecules. DNAs from each peak of the sucrose gradient were examined by electronmicroscopy, and the results agree closely with those of the sucrose gradient analysis. Reassociation kinetics of the plasmid DNA was also followed. Addition of total DNA of strain BH9 increased the renaturation rate of the plasmid DNA. It was calculated from the magnitude of the increase that approximately 10% of the BH9 total DNA may hybridize with the plasmid sequences. DNA prepared from the gene transfer agent (GTA) produced by R. capsulata increases the renaturation rate of the plasmid to the same extent as total DNA isolated from the GTA producing strain, Y262.     

Characterization of the Polydisperse Closed Circular Deoxyribonucleic Acid Molecules of Bacillus megaterium

The polydisperse circular deoxyribonucleic acid (DNA) molecules which comprise up to 30% of the total extractable DNA of Bacillus megaterium strain 216 have been purified and partially characterized. Banding in cesium chlorideethidium bromide by "gradient relaxation" in a fixed-angle rotor provided good resolution of circular and chromosomal DNAs for preparative separations. Renaturation studies on purified circular DNA failed to reveal a rapidly renaturing fraction, and DNA-DNA hybridization studies indicated that the majority of the chromosomal nucleotide sequences are represented in the heterogeneous-size population of circular molecules. It is concluded that the circular DNA of B. megaterium does not represent typical bacterial plasmid DNA. The possibility that the circular DNA molecules are the result of the expression of a defective bacteriophage is discussed.     

A critical examination of possible fractionations of human DNA according to base composition.

Human DNA has been fractionated according to base composition by sedimentation equilibrium in an HgCl2/Cs2SO4 density gradient, followed by sedimentation equilibrium in an actinomycin/cesium formate density gradient. The fractions of different base composition resulting from this procedure were subsequently analyzed by sedimentation equilibrium in CsCl, DNA renaturation kinetics, and electron microscopy. All fractions contain similar kinetic classes of repeated DNA sequences as judged by renaturation studies. Short (300 nucleotides) interspersed repeated sequences are found in all fractions with no noticeable enrichment for these sequences in any fraction. Repeated sequences from fractions of different base composition are partially able to cross-hybridize, demonstrating that nearly identical repeated sequences occur in molecules of different base composition. These findings are critically compared to reports of successful density gradient fractionations of different human DNA sequence classes.     

Repetitious and unique sequences in the heterogeneous nuclear and cytoplasmic messenger RNA of mammalian and insect cells

Initiation of DNA synthesis has been followed in mouse myeloma cells grown in suspension culture. In cells labeled with ³H-thymidine for short times, label first appears in short fragments of DNA which can be chased into bulk DNA (>50 S) upon further incubation in unlabeled thymidine. In a 15 min pulse, DNA fragments with a sedimentation coefficient of 30 S tend to accumulate. Our results support the contention that DNA synthesis is discontinuous in myeloma cells.However, a search for RNA associated with nascent DNA in the myeloma system was unsuccessful. Newly synthesized DNA was isolated on a benzoylated naphthoylated DEAE cellulose column. After heat denaturation, this fraction was centrifuged to equilibrium in a Cs₂SO₄ density gradient. The nascent DNA displays no shift in density greater than the density of the bulk DNA. When cells were pulse labeled with ³H-uridine and the nascent DNA fraction analyzed on Cs₂SO₄ density gradients, no ³H-labeled RNA was found associated with the DNA peak or at intermediate densities that would be indicative of a RNA-DNA molecule, covalently linked. Unless scission of the RNA primers occurs immediately after the initiation of DNA synthesis, our results indicate that DNA synthesis commences without RNA primers in myeloma cells.     

The origin of nascent single-stranded DNA extracted from mammalian cells.

In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained besides small amounts of fragmented bulk DNA, single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.     

Double-Length, Circular, Single-Stranded DNA from Filamentous Phage

Wild-type and gene 3 mutant filamentous phage stocks, containing different relative amounts of multiple-length particles, were treated exhaustively with DNase and then were highly purified. The phage DNA was extracted and examined by electron microscopy. In all cases, about 0.03% of the molecules were circular dimers. (3)H-labeled phage DNA was separated as to size by sedimentation in a preformed CsCl density gradient. Individual fractions were then examined in the electron microscope, and the percentage of linear and circular monomer and dimer DNAs was determined. A peak of double-length, circular molecules (with the expected sedimentation constant of 38S) was found ahead of the 24S monomer peak. The double-length molecules had been purified 65-fold. As previously found for single-stranded DNA, the contour length of these molecules was strongly dependent upon ionic strength. Possible artifacts were ruled out, and it was shown that the double-length molecules arose from phage particles.     

Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid I. Tertiary Structure of Early Replicative and Recombining Deoxyribonucleic Acid

A replicative hybrid resulting from the infection of heavy (substituted with 5-bromodeoxyuridine) bacteria with light (not substituted with 5-bromodeoxyuridine) radioactive bacteriophage was isolated from a CsCl density gradient. Sedimentation studies indicate that 60% of the deoxyribonucleic acid (DNA) behaves as if it were in units more than four times as large as an intact reference molecule. Under the electron microscope, hybrid molecules appeared tangled, showed puffs and loops, occupied a small area, and often had a total length twice that of mature phage. This indicates that sucrose gradient sedimentation is not applicable as a method for estimating the relative molecular size of replicative forms of DNA. After denaturation, the separated strands of hybrid were of the same size as those of reference DNA. CsCl density gradient analysis revealed no terminal covalent addition of new material to the old parental strand. The possibility of a continuous growth of the DNA molecule, either on a single-stranded level or as a double helical structure, is disproved. When chloramphenicol (CM) was added at critical times after infection, DNA synthesis continued at a constant rate. The parental label soon assumed and retained a hybrid density, despite concomitant synthesis of DNA, throughout the rest of the period of incubation in CM. The hybrid moiety, however, actively participated in replication and exchanged its partner strand for a new one; this was demonstrated by changing the density label during incubation in CM. A new enzyme synthesized shortly after infection introduced single-stranded "nicks" into the parental DNA. Since nicking can be inhibited by chloramphenicol, the responsible enzyme is not of host origin. The time of the appearance of this enzyme coincided with the onset of molecular recombination. Another enzyme, which mediates the repair of the continuity of the polynucleotide chain after recombination, appeared after recombination. If selectively inhibited by chloramphenicol, recombinant molecules remained unrepaired, and, upon denaturation, the parental fragment was liberated in pure form.     

Structure of Replicating Simian Virus 40 Deoxyribonucleic Acid Molecules

Properties of replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) have been examined by sedimentation analysis and by direct observation during a lytic cycle of infection of African green monkey kidney cells. Two types of replicating DNA molecules were observed in the electron microscope. One was an open structure containing two branch points, three branches, and no free ends whose length measurements were consistent with those expected for replicating SV40 DNA molecules. A second species had the same features as the open structure, but in addition it contained a superhelix in the unreplicated portion of the molecule. Eighty to ninety per cent of the replicative intermediates (RI) were in this latter configuration, and length measurements of these molecules also were consistent with replicating SV40 DNA. Replicating DNA molecules with this configuration have not been described previously. RI, when examined in ethidium bromide-cesium chloride (EB-CsCl) isopycnic gradients, banded in a heterogeneous manner. A fraction of the RI banded at the same density as circular SV40 DNA containing one or more single-strand nicks (component II). The remaining radioactive RI banded at densities higher than that of component II, and material was present at all densities between that of supercoiled double-stranded DNA (component I) and component II. When RI that banded at different densities in EB-CsCl were examined in alkaline gradients, cosedimentation of parental DNA and newly replicated DNA did not occur. All newly replicated DNA sedimented more slowly than did intact single-stranded SV40 DNA, a finding that is inconsistent with the rolling circle model of DNA replication. An inverse correlation exists between the extent of replication of the SV40 DNA and the banding density in EB-CsCl. Under alkaline conditions, the parental DNA strands that were contained in the RI sedimented as covalently closed structures. The sedimentation rates in alkali of the covalently closed parental DNA decreased as replication progressed. Based on these observations, some possible models for replication of SV40 DNA are proposed.     

Anatomy of Herpes Simplex Virus DNA. VIII. Properties of the Replicating DNA

This paper concerns the properties of herpes simplex virus 1 DNA replicating in HEp-2 and human embryonic lung cells. The results were as follows. (i) Only a small fraction of input viral DNA entered the replicative pool. The bulk of the input viral DNA cosedimented with marker viral DNA and did not appear to be degraded or dissociated into L and S components. (ii) Nascent DNA sedimented faster and banded at a higher density than that of mature viral DNA extracted from virions. Pulse-chase experiments indicated that nascent DNA acquires the sedimentation rate and buoyant density of viral DNA within 30 to 40 min after its synthesis. (iii) Electron microscopic studies indicated that the DNA extracted from cells replicating viral DNA and banding at the density of viral DNA contained: (a) linear, full-size molecules with internal gaps and single-stranded regions at termini; (b) molecules with lariats, consisting of a linear segment up to 2x the size of mature DNA and a ring ranging from 0.5 x 10(6) to 100 x 10(6) in molecular weight, showing continuous and discontinuous forks; (c) circular, double-stranded molecules, both full-size and multiples of 18 x 10(6) in molecular weight, but without forks or loops; (d) molecules showing "eye" and "D" loops at or near one end of the DNA; (e) large, tangled masses of DNA, similar to those observed for T4 and pseudorabies virus replicating DNAs, containing loops and continuous and discontinuous forks. The electron micrographs are consistent with the hypothesis that the single-stranded ends on the DNA anneal to form a hairpin, that the DNA synthesis is initiated at or near that end and proceeds bidirectionally to form a lariat, and that resulting progeny derived by semiconservative replication are "head-to-head" and "tail-to-tail" dimers.     

Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli.

It has been possible to locate a submembrane domain representing less than 10% of the total membrane that appears to be responsible for sequestering some essential components required for plasmid RK2 DNA replication. This subfraction, whose cellular location in the membrane prior to extraction is still unknown, is derived from the inner membrane fraction, since it possesses enzyme marker activity (NADH oxidase) exclusively associated with the inner membrane. The subfraction was detected by a modification of the methods of Ishidate et al. (K. Ishidate, E. S. Kreeger, J. Zrike, S. Deb, B. Glauner, T. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) in which low pressure in a French pressure cell and lysozyme were used to preserve the supercoil plasmid DNA template during cell disruption. This was followed by successive cycles of sucrose gradient sedimentation and flotation density gradient centrifugation to reveal a number of subfractions, including the one of interest. The characteristics of plasmid interaction with the subfraction include the presence of supercoil DNA after extraction, the binding of the origin of plasmid replication (oriV) in vitro, and the association of the two plasmid-encoded initiation (TrfA) proteins (encoded by overlapping genes). However, another peak, the outer membrane fraction, also binds oriV in vitro, contains plasmid DNA in vivo, and associates with the TrfA initiation proteins. Nevertheless, it contains much less of the initiation proteins, and the specific activity of binding oriV is also much reduced compared with the other subfraction. There is a strong correlation between the association of the TrfA initiation proteins with a particular membrane fraction and the binding of oriV in vitro or plasmid DNA in vivo. Since the proteins are known to bind to repeated sequences in oriV (S. Perri, D. R. Helinski, and A. Toukdarian, J. Biol. Chem. 266:12536-1254, 1991; M. Pinkney, R. Diaz, E. Lanka, and C. M. Thomas, J. Mol. Biol. 203: 927-938, 1988), it appears that the initiation proteins themselves could be responsible, at least in part, for the association of plasmid DNA to the membrane.     

Process of attachment of phi X174 parental DNA to the host cell membrane

The phi X174-DNA membrane complex was isolated from Escherichia coli infected with phi X174 am3 by isopycnic sucrose gradient centrifugation followed by zone electrophoresis. The phi X174 DNA-membrane complex banded at two positions, intermediate density membrane fraction and cytoplasmic membrane fraction, having bouyant densities of 1.195 and 1.150 g/ml, respectively. Immediately after infection with phi X147, replicating DNA was pulse-labeled and then the incorporated label was chased. The radioactivity initially recovered in the intermediate density membrane fraction migrated to the cytoplasmic membrane fraction. The DNAs from both complexes sedimented mainly at the position of parental replicative form I (RFI). The phi X174 DNA-membrane complex contained a speficic membrane-bound protein having a molecular weigth of 80,000 which is accumulated in the host DNA-membrane complex. These results suggest that when phi X174 DNA penetrated into cells in the early phase of infection, single-stranded circular DNA was converted to parental RFI at a wall/membrane adhesion region and migrated to the cytoplasmic membrane fraction, where the parental RF could serve as a template in the replication of progeny RF.     

In vitro system that synthesizes circular viral DNA of bacteriophage phi X 174.

An extract prepared from Escherichia coli cells infected with phi chi 174 bacteriophage was capable of incorporating dTTP into phage-specific DNAs in vitro. The synthesized DNAs were associated with proteins and sedimented with S values of 20, 50, and 90 in a sucrose gradient sedimentation. DNA isolated from 20S material was open circular replicative form (RF), DNA in 50S material was replicative-form DNA with an extended single-stranded viral DNA that ranged up to one genome in length, and DNA in 90S material consisted of circular and linear single-stranded viral DNA of full genome length and single-stranded viral DNA shorter than full genome length. Pulse and pulse-chase experiments indicated that 90S material derived from 50S material.     

Multiple origins and circular structures in replicating T5 bacteriophage DNA.

Replicating T5 phage DNA was gently isolated using NaI density gradient centrifugation and examined by electron microscopy. At the beginning of phage DNA synthesis, linear unit-length T5 DNA molecules containing from one to four replicating "eye-loops" were consistently observed. Replication in these molecules was found to proceed bidirectionally from multiple, internal origins. A primary origin of replication is located near the center of the T5 genome, which does not coincide with the location of any of the nicks (single-strand breaks) found in mature T5 DNA. The initiation of replication at the various origins within an individual molecule does not appear to follow any definite temporal sequence. At later times in the infection, we have observed a significant number of circular T5 DNA molecules-both replicating and nonreplicating-whose average circumference is approximately the length of mature T5 DNA minus the terminal redundancy. The replicating circular molecules appear to be either in a theta configuration, a sigma configuration with the tails all being less than the length of the circle, or a combination of theta and sigma forms.     

High-resolution separation and accurate size determination in pulsed-field gel electrophoresis of DNA. 4. Influence of DNA topology

Pulsed-field gel electrophoresis is a powerful technique for the fractionation of linear DNA molecules with sizes above 50 kilobase pairs (kb). Here it is demonstrated that this technique is also effective for separating smaller DNAs including linear, circular, and supercoiled species. The mobilities of linear DNAs larger than 8 kb can be modulated by pulse times between 0.1 and 100 s. The mobility of supercoiled DNA molecules up to 16 kb is generally unaffected by these pulse times except that 10-s pulse times cause a small but distinct increase in the mobility. The general insensitivity of small supercoiled DNAs to pulse time presumably occurs because these species reorient so rapidly that they spend most of their time undergoing conventional electrophoresis. However, the mobilities of larger supercoiled DNAs are affected by pulse times of less than 1 s, and at 0.1 s the molecules are better resolved by pulsed electrophoresis than by ordinary electrophoresis. The mobility of 3-19 kb nicked and relaxed circular DNA molecules is also affected by pulse time but in a complex way.