Alkaline CsCl Gradients Research Articles

Physico-chemical characterization of mitochondrial DNA from potato tubers

1. A procedure was described for the mass isolation of mitochondria from potato tubers. The deoxyribonuclease-treated crude mitochondrial pellet was fractionated into several layers by sucrose gradient centrifugation. The light layer ( d = 1.18) contained highly purified mitochondria, as seen by electron microscopy. 2. The DNA extracted from this fraction gave only a sharp and symmetrical band at 1.706 g/ml in analytical CsCl gradient ultracentrifugation. Analysis of the very heavy and very light fractions of the preparative CsCl DNA band, revealed only molecules banding at 1.706 g/ml, which is evidence of the intermolecular homogeneity of this plant mtDNA. The buoyant density was shifted by 0.016 and 0.058 g/ml when mtDNA was respectively thermally denatured and run in an alkaline CsCl gradient. No band splitting was observed in an alkaline CsCl gradient. The melting curve was characterized by a sharp transition at T m = 88.8°C in 1 × SSC . 3. The intramolecular homogeneity has been demonstrated by CsCl gradients and thermal denaturations achieved with mtDNA of different molecular weights. No heterogeneity was detectable on the CsCl banding pattern and on derivatives of the melting curves. 4. The mtDNA renatured rapidly as seen by the buoyant density shift and hypochromicity, like other organelle DNAs. Except for the initial minutes the optical reassociation curves appeared homogeneous and the kinetic complexity was close to 100 · 10 6 daltons. 5. The size of the mtDNA molecules was studied by sedimentation velocity and by length measurement on the electron microscope. The molecular weight calculated from the s 2 0, w and from the molecular length was close to 60 · 10 6. Electron microscopy revealed that mtDNA molecules are linear and heterogeneous in size.

Novel properties of satellite DNA from muskmelon.

The purified dense satellite of muskmelon (Cucumis melo L.) represents 30% of the total DNA and forms a sharp, unimodal peak in both neutral and alkaline CsCl gradients. Spectrophotometric melting and DNA reassociation analyses revealed that one-third of the satellite is high melting and has a complexity of about 2.5 x 10(5) daltons, while the remaining two-thirds of the satellite melts 8 degrees lower and has a complexity of about 5 x 10(8) daltons. The thermal stability of reassociated satellite DNA indicates that the multiple copies of the two melting fractions are essentially identical. The sharp, unimodal peak in Cs(2)SO(4) becomes two distinct peaks when either silver or mercuric ions are included in the Cs(2)SO(4) gradient

Hepatic DNA repair synthesis in rats fed 3′-methyl-4-dimethylaminoazobenzene

Rats, whose liver DNA was prelabeled with BdUrd- 14 C, were placed on a diet containing 0.05% of the hepatic carcinogen 3′-methyl-4-dimethylaminoazobenzene. At various times during the second day on the carcinogenic diet animals received an injection of dThd- 3 H and were sacrificed 1 hr later, hepatic DNA was extracted and analyzed by density equilibrium centrifugation in alkaline CsCl gradients. The results of these studies indicate that non-semiconservative DNA synthesis (DNA repair synthesis is an early consequence of carcinogen exposure and that the amount of DNA repair synthesis occurring at a given time depends, in part, on the animals feeding schedule.

The Anatomy of the Mitochondrial DNA

The distribution of the mitochondrial DNA among the allomorphic forms, supercoiled circle, open circle and linear rod is a characteristic feature of each cell line. After pulse‐chase labeling with [Me‐3H]thymidine the bulk of the radioactivity can be located either in the supercoiled circular mitochondrial DNA (monkey Vero, ATCC; CCL 81) or in the open circular mitochondrial DNA (mouse NCTC 929, ATCC; CCL 1).The open circular mitochondrial DNA from Vero cells contains one single nick, which is located in the heavy strand (alkaline CsCl gradient). In contrast, the open circular mitochondrial DNA from mouse cells contains one nick in one strand and at least two nicks in the opposite strand.The bulk of the supercoiled Vero mitochondrial DNA has a heat‐labile site in the heavy strand and one site in both strands at which the phosphodiester backbone can be hydrolyzed by pancreatic RNAase.

Synthesis of mitochondrial DNA in vitro: Two classes of nascent DNAs

1. In vitro synthesis of mitochondrial DNA was studied with isolated newborn rat liver mitochondria. The over-all synthesis of nascent DNA was about one-order higher than that of other systems previously reported. 2. The majority of the nascent DNA was initially observed in the closed circles and subsequently in both open and closed circles. 3. Two classes of the nascent DNA were obtained during the incubation: one class was the heat-dissociable fragments, and the other was alkali-dissociable higher-molecular DNAs. 4. The nascent DNA was found in both light and heavy bands in the alkaline CsCl gradient. 5. A plausible scheme is discussed in which replication of the circular mtDNA in vitro may be modulated by at least two steps: one related to discontinuity; the other operative for the semiconservative mode.

Interaction of polyribonucleotides with plant mitochondrial DNA

DNA is extracted from purified mitochondria of potato tubers. This DNA cannot be resolved into two strands by alkaline CsCl gradients in our experiments. Poly (G), poly (U) and poly (I,G) interact with the plant mitochondrial DNA as shown by the marked shift in buoyant density that they produce on denatured DNA. Poly (A) and poly (C) do not lead to detectable interactions in standard conditions whereas a slight fixation of poly (C) occurs at acidic pH. These results suggest that the plant mitochondrial DNA contains d-A and d-C rich clusters and, in a lesser extent, d-G rich clusters.

Physical properties of cytoplasmic membrane-associated DNA

Some of the physical properties of a cytoplasmic membrane-associated DNA isolated from a diploid human lymphocyte cell line have been examined. Cytoplasmic membrane-associated DNA extracted from lymphocytes labeled with either [ 3H]or [ 14C]thymidine had a specific activity lower than nuclear DNA extracted from the same cells. Analysis of cytoplasmic membrane-associated DNA in the electron microscope shows that the molecules are linear and have a mean length of 1·75 μm; the average sedimentation coefficient of this DNA is 16·6 S, which corresponds to a molecular weight of 4·2×10 6. Cytoplasmic membrane-associated and nuclear DNA band at identical positions in both neutral and alkaline CsCl gradients with buoyant densities of 1·699 g/ml and 1·752 g/ml, respectively. Native cytoplasmic membrane-associated DNA is double-stranded and has a mole fraction of guanine plus cytosine of 40± l %. Sheared, denatured cytoplasmic membrane-associated DNA reassociates as two distinct fractions whose rates of reassociation differ by about four decades: the complexity of the reassociation of this DNA tends to rule out the possibility that it arises from either mycoplasmal or viral contamination of our cell cultures. The slowly reassociating fraction of cytoplasmic membrane-associated DNA reassociates about ten times faster than the unique sequences of nuclear DNA. This could represent potential genetic information for about 100,000 diverse genes of 1000 nucleotide pairs each. At present the function of cytoplasmic membrane-associated DNA in these cells is unknown.

Related satellite DNA's in the genus Mus

Several Thailand species from the genus Mus have been shown to contain satellite DNA's able to cross-reassociate with the Mus musculus satellite. One species, Mus caroli, contains at least three discrete but related light satellite DNA's. All the related Mus satellites band on the light side of the major band in neutral CsCl gradients, separate into complementary strands in alkaline CsCl gradients, and have a relatively low affinity for Ag +. Three of the Mus satellite DNA's have been purified: taken separately, they show very sharp thermal transitions and reassociate at similar rates to give well-matched duplexes. Separated, 32P-labelled strands of M. musculus satellite DNA cross-reassociate rapidly with the purified Thailand mice satellites. Very poorly-matched hybrid duplexes are formed, yet these melt off hydroxylapatite columns over a narrow temperature range. It is concluded that the related Mus satellite DNA's were produced by independent events of massive replication involving a single unstable sequence element inherited from a common ancestor species. The evidence from intersatellite reassociation and from pyrimidine tract analysis of the M. musctdus satellite suggests that the unstable satellite ancestor was itself a diverged repeating sequence, with a basic complexity of only 8 to 20 nucleotide pairs.

Azotobacter phages: Properties of phages A12, A21, A31, A41 and their constituent DNAs

The properties of several Azotobacter vinelandii phages and of the DNA isolated from each have been investigated. Phages A12, A21, and A41 have short noncontractile tails. A12 and A21 have virion weights of 80 × 10 6 daltons and contain 58% DNA. A31 has a long noncontractile tail, a virion weight of 175 × 10 6 daltons and contains 26% DNA. A31 and A41 are unstable in salt solutions of moderate ionic strength. All phages contain linear, double-stranded DNA that sediments as a single component in neutral and alkaline solutions. The molecular weights of A12, A21, and A31 DNAs obtained by equilibrium density gradient sedimentation are (46 ± 7) × 10 6, (42 ± 5) × 10 6 and (47 ± 5) × 10 6, respectively. Electron microscope length measurements using the replicative form of ∅X174 DNA as an internal contour length standard yielded molecular weights of (44.3 ± 2) × 10 6, (45.6 ± 1.2) × 10 6 and (46.5 ± 2.6) × 10 6, respectively. The buoyant densities of A12 and A21 DNA in CsCl and Cs 2SO 4 indicate a G + C content of 58% consistent with the T m . For A31 DNA the buoyant densities suggest a G + C content of 52% whereas the T m corresponds to 69% G + C. A12 and A21 DNAs show strand separation in an alkaline CsCl gradient (Δ ρ⋍0.006 g ml −1).

Heterogeneity of patch size in repair replicated DNA in Escherichia coli

Repair replication in ultraviolet-irradiated Escherichia coli has been detected by the use of [ 3H]bromouracil as a density label and then equilibrium sedimentation in isopycnic CsCl gradients to separate the repaired parental DNA from normally replicated DNA. In the use of this assay it has been generally assumed that the patches of repair synthesis are short enough that the incorporated [ 3H]bromouracil will cause little detectable shift in density of the repaired DNA fragments. However, the repair label is consistently found to be skewed to the denser side of the parental DNA density position, possibly indicating size heterogeneity of the patches. We have now demonstrated such a heterogeneity. Repaired DNA isolated from the parental density regions of neutral CsCl gradients was extensively sheared by sonication to a single-strand molecular weight of 0.5 to 0.8 × 10 6 daltons. When the resulting fragments were sedimented in an alkaline CsCl gradient, a large fraction of the repair label was found in fragments which banded at density position sintermediate between those of fully bromouracil-labeled and unlabeled strands. Evidence is presented that this density-shift of some sonic fragments is due to the fact that repair replication sometimes produces very large patches. Base-composition analysis of the repaired regions indicates that these large patches must be at least as long as the sonic fragments (i.e. 1500 nucleotides). The possibility that the large patches arise from normal replication is eliminated. A model involving two alternative enzymic repair systems in the excision-repair process is presented to account for the existence of heterogeneity of patch size in repair replication.

Studies on the deoxyribonucleic acid from Spirillum

The two strands of DNA from Spirillum SP5 have been separated in alkaline CsCl gradients and on a polynucleotide kieselguhr column. One of the strands is probably richer in guanine, thymine and pyrimidines than the other. The DNA contains an average of 61 % guanine+cytosine and no unusual bases.

Nature of the Complementary Strands Synthesized in Vitro upon the Single-Stranded Circular DNA of Bacteriophage øX174 after Ultraviolet Irradiation

This paper describes experiments intended to decide whether UV lesions in DNA act as absolute blocks to chain elongation by the Escherichia coli DNA polymerase or only slow down the polymerization process. Ultraviolet (UV)-irradiated, single-stranded (SS) circular DNA of bacteriophage øX174 was used as template for the polymerase in a reaction mixture in vitro, under conditions allowing synthesis of not more than one complementary strand per template molecule. The mean length of the newly synthesized complementary strands (as determined by velocity sedimentation in alkaline CsCl gradients), as well as the over-all template activity (as measured by deoxyadenosine monophosphate [dAMP] incorporation) was found to decrease with the number of biologically lethal hits sustained by the irradiated templates. With the increase of time or temperature of reaction, the net synthesis of complementary strands increased (as a consequence of increased initiation), but their mean length remained constant. The mean length of synthesized strands was greater than would be expected if all biologically lethal hits were to block the polymerization process. The lethal hits which serve as blocking lesions are inferred to be pyrimidine dimers because it is possible to obtain synthesis of full-length complementary strands if, when heat-denatured, UV-irradiated, double-stranded replicative form (RF II) DNA of bacteriophage øX174 is used as a template, it is pretreated with yeast photoreactivating enzyme (YPRE) in presence of visible light.

Asymmetric Distribution of Guanine Plus Thymine Between Complementary Strands of Deoxyribonucleic Acid of Members of the Enterobacteriaceae

Analysis of the deoxyribonucleic acid prepared from Proteus mirabilis, Escherichia coli, and Serratia marcescens in an alkaline CsCl gradient has shown that there is an asymmetric distribution of guanine plus thymine residues between the complementary strands of the deoxyribonucleic acid.

Membrane attachment of replicating parental DNA molecules of bacteriophage M13

Early in the infection with bacteriophage M13 the infecting parental DNA strand becomes attached to the host cell membrane. Using a gentle lysis procedure followed by sucrose gradient centrifugation, up to 80% of the parental DNA co-sediment with the bacterial membranes. The membrane fraction was deproteinized by phenol extraction and the solubilized DNA was further analysed by band sedimentation in neutral and alkaline CsCl gradients. Between 5′ and 15′ after infection at least half of the membrane bound parental DNA was found to be incorporated into replicative intermediates with viral strands of more than unit length.

Expression of the mitochondrial genome in HeLa cells: II. Evidence for complete transcription of mitochondrial DNA

The fraction of mitochondrial DNA which is complementary to mitochondria-associated RNA in exponentially growing HeLa cells has been investigated by RNA-DNA hybridization experiments using mitochondrial DNA strands separated by alkaline CsCl density-gradient centrifugation and RNA from cells uniformly labeled with [5- 3H]uridine. It has been found that mitochondrial RNA hybridizes almost exclusively with the heavy strand, in agreement with the complementarity of their base compositions. Less than 2% of the light strand, after purification by two cycles of alkaline CsCl gradient centrifugation, was found to form hybrids with mitochondrial RNA. In hybridization saturation experiments, the RNA sedimenting slower than 22 s or faster than 30 s gave about 100% saturation level, whereas the components in the intermediate range of sedimentation coefficients gave about 85% saturation level. An analysis in Cs 2SO 4 gradients of the density of the RNA-DNA hybrids formed at saturation showed that the hybrids formed with the RNA of less than 22 s and greater than 30 s banded at a density of 1.491 g/cm 3, i.e. the density expected for fully base-paired hybrids; whereas the hybrids formed with the components of intermediate sedimentation coefficients banded at a slightly lower density (1.485 g/cm 3), in agreement with the lower saturation level obtained with these components. An analysis by electron microscopy of the hybrids separated in a Cs 2SO 4 density-gradient showed that essentially all the material from the 1.491 g/cm 3 band consists of nucleic acid duplexes. In view of the available evidence for the absence of major internal repetitions in mammalian mitochondrial DNA, the results obtained by the three approaches described here indicate that the whole or almost whole mitochondrial genome is transcribed in HeLa cells. The possibility that this transcription occurs in the form of a continuous RNA chain is discussed.

Structural alteration of intracellular φX174 DNA following infection of host cells in which protein synthesis is inhibited

The structure of the intracellular form of φX174 DNA was studied in cells treated to inhibit protein synthesis before infection. Inhibition of protein synthesis was accomplished in Escherichia coli 15 THU by 90 minutes incubation in medium lacking histidine and uracil, and in E. coli THU or HF4704 by addition of 135 μg chloramphenicol/ml. five minutes before infection. φXDNA containing parental label was isolated from treated cells. Its sedimentation rate in a neutral high salt gradient was 5% faster than RFI † † Abbreviations used: RF, replicating form; BNC, benzoyl naphthoyl DEAE-cellulose. , and the buoyant density in neutral cesium chloride was more broadly distributed and somewhat denser than RFI. When the intracellular parental labeled DNA was fractionated by chromatography on benzoyl naphthoyl DEAE-cellulose 10 to 20% chromatographed as RFI or II, while 80 to 90% was tightly bound and could only be eluted by 1.0 m-NaCl plus 2% caffeine. The tightly bound DNA was infective in the spheroplast assay, but infectivity was lost after denaturation. In alkaline CsCl equilibrium gradients the parental label was found in the position of viral DNA but no infectivity was found in the region of viral or complementary strand. Hybrid ( 13C, 15N parental: 12C, 14N post-infection) DNA had a lower buoyant density than normal hybrid RF. Parental strands sedimented slower than viral DNA on alkaline CsCl gradients with a peak around ~ 8 s. Post-infection DNA had a heterogeneous sedimentation distribution in alkali, but a majority of the counts sedimented faster than viral DNA. Most of the parental label was membrane associated, and all infected cells were able to contribute to initial progeny phage production. The structural and biological significance of these findings is discussed.

Characterization of the Bacillus stearothermophilus phage φμ-4 and Its DNA

Bacteriophage φμ-4 was purified from lysates by isoelectric precipitation and CsCl gradient centrifugation. This phage displays an Escherichia coli phage lambda-like appearance in electron microscopy with polyhedrical heads and long tails, but without terminal plates and tail fibers. Bacteriophage φμ-4 is a DNA virus. The DNA has a molecular weight 26 × 10 6 daltons, a buoyant density of 1.719 g/cm 3, a G + C-content of 58% and appears to contain no unusual base residues. The DNA strands can be separated by alkaline CsCl gradient centrifugation or annealing to poly U,G, and subsequent CsCl gradient centrifugation. DNA of phage φμ-4 is an excellent template for in vitro transcription by both E. coli RNA-polymerase and host RNA-polymerase ( Bacillus stearothermophilus). There are 15 polymerase binding sites on μμ-4 DNA for both enzymes as determined bv the heparin technique. φμ-4 DNA is transcribed completely in vitro. In vitro synthesized RNA is, however, not as well translated as other templates in a cell-free E. coli system.

Further studies on high molecular weight fowlpox virus DNA and its hydrodynamic properties

The intrinsic viscosity of fowlpox virus DNA measured at low shear stress in a rotating cylinder viscometer averages 447 dl/g. Calculation of the molecular weight from intrinsic viscosity data gives a value of approximately 200 × 10 6 daltons and agrees well with the molecular weight previously determined by other methods. Fowlpox DNA shows an anomalous behavior after heat denaturation with as much as 50% of the DNA exhibiting a buoyant density in CsCl similar to that of the native DNA. Denaturation in the presence of 1% formaldehyde virtually eliminates this nativelike material. Sedimentation velocity and buoyant density studies on alkaline-denatured fowlpox DNA give no evidence for covalent cross-links in the molecule. Centrifugation of denatured fowlpox DNA in an alkaline CsCl gradient revealed only one band, indicating that there is no significant difference in the guanine + thymine content of the complementary strands.

Fate of donor DNA in pneumococcal transformation

The molecular fate of homologous (pneumococcal) and heterologous ( Escherichia coli) [ 32P]DNA was examined after introduction into pneumococcal cells. In both cases the composition of 32P-containing products at the earliest time of observation was the same. About 50% of the incorporated donor material was in the form of single strands, identified by their density in alkaline CsCl gradients. Of the incorporated 32P, 20 to 30% was in the form of native DNA of pneumococcal composition. Since this was true for donor DNA from E. coli as well as for donor DNA from pneumococcus, it appears that this label entered native DNA by way of de novo synthesis of DNA, presumably from nucleotides. The remaining 32P in the cell was in the form of dialyzable fragments composed of large amounts of inorganic phosphate and l-α-glycerophosphate and smaller amounts of the four 5′-deoxynucleotides. A plausible origin of labeled inorganic phosphate is the dephosphorylation of nucleotides. The labeled glycerophosphate, in turn, may be derived from inorganic phosphate, since artificially introduced [ 32P]phosphate rapidly equilibrated with a large pool of intracellular glycerophosphate. Thus, the initial products of entry are envisioned to be equal amounts of single strands and 5′-deoxynucleotides. This would be compatible with their origin in the degradation by a pneumococcal exonuclease of one strand of an incoming DNA duplex. On subsequent incubation, single strands of heterologous origin were slowly degraded; their breakdown products were used for de novo DNA synthesis. The disappearance of homologous single strands was much faster and corresponded to an increase of 32P in native DNA as well as to the recovery from eclipse of donor marker-transforming activity. It is concluded that single strands are precursors of genetically integrated DNA and that recombination involves the interaction of homologous single strands with host DNA. The ratio of genetically integrated DNA to total DNA incorporated suggests that only half, or somewhat more, of the single-stranded DNA was inserted as intact sequences. Inhibition of DNA synthesis by aminopterin gave results which were compatible with these conclusions.