The secondary structure of bacteriophage DNA in situ. 3. Reaction of Sd phage with O-methylhydroxylamine

Cytosine nuclei in denatured DNA and in DNA inside the S_d phage readily react with O-methylhydroxylamine, whereas no reaction with native DNA is observed under the same experimental conditions. In the intraphage DNA, about 20% of cytosine participates in the reaction, the rate of modification of cytosine being close to that of modification of cytosine nuclei in monomers. “Reactive” cytosine of the intraphage DNA is likely to be localised in the sites having no base stacking.     

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10⁶ daltons (native) DNA.In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA^Val, tRNA₁^Met and tRNA₂^Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm^?3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.     

Structure and physico-chemical properties of bacteriophage G: III. A homogeneous DNA of molecular weight 5 × 108

The physico-chemical properties of the DNA released from bacteriophage G (active on Bacillus megatherium) are described. Phage G, an unusually large bacteriophage, has a nucleic acid content of 4 to 6 × 10⁸ daltons.Sedimentation velocity analysis at low angular speed and examination by electron microscopy, indicate that a single DNA molecule, sedimenting with s_{20, w}⁰ = 125 ± 1.5 S and at least 200 ± 20 μm long, is released upon thermal or osmotic shock. Melting temperature data and chromatographic analysis indicate a mean base composition of 70% A + T. CsCl and Cs₂SO₄ buoyant density data, circular dichroism spectra and sensitivity to specific nucleases indicate that phage G DNA is similar to the DNAs from T-even phages and is more glucosylated than phage T6 DNA. Direct glucose determination indicates a 185% molar ratio of glucose to cytosine. Linear density extrapolated from literature data and contour length measurement yield a lower limit for the molecular weight of phage G DNA of 4.9 × 10⁸. Comparison of this value with the s_20,w⁰ measured with the analytical ultracentrifuge seems to confirm the validity of the empirical relationship proposed by Freifelder (1970), between s_{20, w}⁰ and molecular weight, over a larger range than that previously known. A possible systematic error in defect in length determination, however, prevents a discrimination between this and other empirical formulae proposed by various authors, which predict a molecular weight that is 20 to 25% higher.     

Unique Properties of Nucleic Acid from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Phage SP-15

BACTERIOPHAGE SP-15 is a large, generalized transducing phage of Bacillus subtilis and B. licheniformis. The DNA extracted from the purified phage has unusual physical properties: its melting temperature (T_m) in 0.15 M NaCl, 0.015 M sodium citrate (SSC) is very low, 61.5° C and its buoyant density in neutral CsCl is very high, 1.761 g/ml.¹. We describe here additional unique features of SP-15 DNA: the presence of (1) a new modified pyrimidine which partially replaces the thymine; (2) a compound which reacts with orcinol as a pentose; (3) alkali-sensitive phosphodiester bonds; and (4) glucose.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.     

Isolation and structure of phage lambda head-mutant DNA

High molecular weight DNA accumulates in bacteria in which λ is multiplying but cannot complete the formation of new phage particles due to a defect in head assembly. Accumulated λ DNA has been isolated from induced mitomycin C-treated lysogens by means of a shift in buoyant density labels from heavy to light and fractionation by density-gradient sedimentation for completely light DNA. Head formation was blocked in these lysogens by amber mutations in genes D or E, which specify the two major head proteins. The purified DNA is at least 80% λ by DNA-DNA hybridization and some preparations are close to 100% λ by this test.     

Reciprocal induction of cell division by cells of complementary mating types in Tetrahymena

Chick embryo fibroblasts in monolayer culture were synchronized by contact inhibition and serum starvation. Nuclear DNA isolated from the [³H] thymidine pulse-labelled cells throughout the period of DNA synthesis (S phase) was analysed by hydroxylapatite chromatography after renaturation at different C₀t values. It is shown that repeated sequences having different frequencies of reassociation, replicate differently throughout the S period. In order to study the distribution of the repeated sequences, DNA isolated during the S period was fractionated according to its buoyant density. It is shown that only some of the highly reiterated sequences which are included in the high buoyant density DNA fractions, replicate equally well during the early and the late S periods. By contrast, reiterated sequences of the low buoyant density DNA fractions replicate mainly during the late S period.     

ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Mitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm³ has been isolated from unfertilized eggs of Drosophila melanogaster. This DNA is a circular molecule with an average length of 5.3 µm; it reassociates with a low C₀t_1/2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm³. MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm³, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function.     

Biochemical and biophysical properties of hyphomicrobium bacteriophage hyphi30

Hyphomicrobium bacteriophage Hy30 and its nucleic acid were studied to determine some of their biochemical and biophysical properties. The molecular weight of the phage is 55.4 × 10⁶, and its buoyant density is 1.508 g/ml. The nucleic acid of Hy30 is linear, double-stranded DNA with a molecular weight of 29.7 × 10⁶. The guanine-plus-cytosine content of the DNA was 62% as determined from its melting temperature and buoyant density.     

Plant Disease and the Regulation of Enzymes Involved in Lignification: Increased Rate of De Novo Synthesis of the Three Tobacco O-Methyltransferases during the Hypersensitive Response to Infection by Tobacco Mosaic Virus

The mechanism underlying the increase of activity of the three O-methyltransferases of tobacco (Nicotiana tabacum) after infection by tobacco mosaic virus (TMV) has been investigated with a density-labeling method. The three O-methyltransferases from healthy or TMV-infected leaves fed with H₂O or ²H₂O were purified by ion-exchange chromatography and their mean buoyant densities were calculated from their respective distribution profiles after centrifugation to equilibrium on RbCl gradients. Densities were corrected with respect to the mean buoyant density of a radioactive density marker prepared from tobacco leaves floated on a solution containing l-[³H]leucine and selected on a preparative gradient for its density close to those of the O-methyltransferases. The introduction of ²H into the pool of amino acids from which the enzymic proteins were synthesized was monitored. By measurement of the labeling of β-galactosidase synthesized by bacteria from the plant amino acids, it was shown that infection did not alter the rate of labeling of the pool of amino acids. The buoyant-density values of the three O-methyltransferases were determined, and density-labeled enzymes from healthy and infected materials were compared. The largest density shifts from the native enzyme were measured for O-methyltransferases from infected leaves. These results show that the increase in activity of the three enzymes after infection is due to the stimulation of the rate of de novo synthesis of enzyme proteins.     

Satellite DNA associated with heterochromatin in Rhynchosciara

The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm³. The larger of the two minor bands has a buoyant density of 1.680 g/cm³ while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm³. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA.     

Effect of nickel(II) on DNA-protein interactions

Alterations in DNA-protein interactions (DPI) may play an important role in carcinogenesis. Although the mechanism of nickel carcinogenesis is unknown, nickel reportedly affects DPI. A microfiltration, nitrocellulose filter assay was utilized to study DPI in intact Chinese hamster ovary (CHO) cells and in isolated nuclei. Prior to exposure of CHO cells or isolated CHO cell nuclei, DNA and proteins were radiolabeled using³H-thymidine and³⁵S-methionine, respectively. Nuclei were exposed to NiCl₂ in 10 mM HEPES buffer (pH 6.8). CHO cells were exposed in either complete or a salts-glucose medium. Following exposure, nuclei or cells were incubated at 37°C for 20 min in a high salt lysis solution; aliquots were loaded onto nitrocellulose filters and washed with a low salt solution. DNA (³H) retained on each filter was normalized to protein (³⁵S) bound on the filter. Exposure of either whole cells or isolated nuclei to increasing, noncytotoxic concentrations of NiCl₂ resulted in a dose dependent decrease in DPI. The effect of nickel on specific DNA-protein interactions was examined using a band shift assay and a cloned satellite DNA sequence. Nickel inhibited specific protein binding to the satellite DNA probe. The results of these two independent assays, which were conducted at physiological pH, indicate that NiCl₂ inhibits specific DNA-protein interactions.     

Characterization of Bacteriophage S13 suN15 Single-Strand and Replicative-Form Deoxyribonucleic Acid

The deoxyribonucleic acid (DNA) of bacteriophage S13 was shown to be single-stranded by the criteria of reactivity with formaldehyde, dependence of optical density on ionic strength, broad temperature-absorbance profile, and lack of molar equivalence of the purine and pyrimidine bases. The DNA has a molecular weight of 1.8 × 10⁶ daltons, an S°₂₀ of 24.6 in SSC (0.15 m NaCl plus 0.015 m sodium citrate), and a buoyant density of 1.726 g/cc in CsCl. Electron microscopy showed the molecule to be circular. S13 replicative-form DNA was shown to be a double-stranded, circular molecule with a molecular weight of 3.5 × 10⁶ daltons, an S_[ill] of 20.7 in SSC, and a buoyant density in CsCl of 1.710 g/cc. The finding that S13 DNA is slightly more pyrimidine-rich than X174 DNA but is indistinguishable by all other parameters supports the close genetic relationship between the two bacteriophages.     

Extent and location of DNA synthesis associated with a class of Rec-mediated recombinants of bacteriophage lambda

The extent and location of DNA synthesis associated with Rec recombination of a lambda phage mutant has been determined approximately for recombinants arising under conditions that restrict DNA duplication. The mutant bio1 contains a substitution in its DNA, and nearly all phage maturing under these conditions have undergone a recombination event within a short region in or near the inserted DNA. Density labeled phage bio1 were used to prepare a lysate under these conditions and the extent of new DNA synthesis was determined by analyzing the density of the progeny phage. On the average, about 6% of the phage chromosome was resynthesized in such a cross.DNA was extracted from bio1 phage crossed under similar conditions in the presence of ³²PO₄. The position of incorporated ³²PO₄ was determined by cleaving the DNA with EcoRI restriction endonuclease and resolving the resulting fragments by electrophoresis on agarose gels. The fragment found to have the most newly synthesized DNA and the highest average amount of synthesis per nucleotide contains the bio1 insertion near its left end and the “hot spot” for Rec-mediated recombination near its center. It appears that in these crosses recombination-associated DNA synthesis is localized about the region of the Rec-mediated recombination event.     

Physical properties of the DNA of bacteriophage SP50

Summary The following properties of the DNA of B. subtilis phage SP50 were established: Molecular weight (in Daltons) 102×10⁶ (sedimentation velocity) 97×10⁶ (viscosity) 97×10⁶ (contour lengths of electron micrographs) Base Composition (in % GC) 41.7 (chemical analysis) 44 (melting point) 44 (buoyant density) No unusual bases were observed. The complementary strands of the DNA can be separated. The phage DNA has genuine single strand breaks. The number and distribution of such breaks appears to be determined by the host on which phages were grown.This investigation was supported in part by a Public Health Service research grant GM 13,666 from the National Institutes of General Medical Sciences, AI 01267 from the National Institutes of Allergy and Infectious Diseases, AM 04763 from the National Institutes of Arthritis and Metabolic Diseases; cancer research funds from the University of California; and a grant from the Hartford Foundation.     

Sequence of the cryptic satellite DNA from the plant Sinapis alba

A satellite DNA with a buoyant density equal to that of main band DNA in neutral cesium chloride (‘cryptic satellite’) can be isolated from the DNA of mustard (Sinapis alba) nuclei by Ag⁺/Cs₂SO₄ density gradient centrifugation. This satellite is cleaved into 172 bp repeat units by HinfI, AluI or HaeIII. The HinfI fragments have been further cleaved by AluI, and seven AluI subfragments have been sequenced. As a result two versions of a basic 172 HinfI repeat have been found, one (A + B) with an additional HinfI site. These two sequences (A + B and C) are the most frequent versions of the basic repeat of mustard satellite DNA. The basic 172 bp unit does not contain subrepeats or palindromic sequences. It is not similar (at a criterion of 15 common bases) with any known satellite sequence. It is not unusually highly methylated in the native state.     

Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1

O⁶-Methylguanine (O⁶-MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O⁶-MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O⁶-MeG by gp90 exo⁻. O⁶-MeG partially inhibited full-length extension by gp90 exo⁻. O⁶-MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo⁻ extends beyond T:O⁶-MeG 2-fold more efficiently than C:O⁶-MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O⁶-MeG show fast burst phases. The pre-steady-state incorporation efficiency (k_pol/K_d,dNTP) is decreased in the order of dCTP:G > dTTP:O⁶-MeG > dCTP:O⁶-MeG. The presence of O⁶-MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg²⁺. Misincorporation of dTTP opposite O⁶-MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O⁶-MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O⁶-MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1.     

The degree of unwinding of the DNA helix by ethidium. I. Titration of twisted PM2 DNA molecules in alkaline cesium chloride density gradients

A series of covalently closed bacteriophage PM2 DNA samples with varying degrees of superhelicity were prepared in vitro. The amount of bound ethidium per DNA nueleotide needed for the removal of all superhelical turns, v_c⁰, was determined for each sample by a number of methods. In order to evaluate the unwinding angle for the binding of one ethidium molecule to a DNA double helix, the pH dependence of the buoyant densities in CsCI of these samples was examined. A new calibration relating the change in buoyant density of a DNA to the fraction of bases titrated has been obtained, by measuring the buoyant densities of a number of catenanes (interlocked rings) containing both single-stranded and double-stranded λ DNA rings, at a pH such that the single-stranded DNA is fully titrated while the double-stranded DNA is not titrated. This calibration was used to obtain the pH dependence of the fraction of DNA bases titrated for the phage PM2 DNAs with differing extents of supercoiling. A simple theoretical analysis shows that in a restricted pH range close to pH_m, the melting pH of the DNA in the absence of the topological constraint associated with covalently closed double-stranded DNAs, the difference in the fraction of bases titrated at a certain pH between two covalently closed DNAs with different degrees of superhelicity is directly proportional to the difference in the v_c⁰ values of the DNAs. The unwinding angle per bound ethidium molecule can be obtained from the proportionality constant. In this way, it is not necessary to know precisely the actual pH value for either DNA, pH_e, at which the DNA is titrated to the extent that it contains no superhelical turns. The conclusion of the theoretical analysis and the experimental results is that the binding of an ethidium molecule to a double-stranded DNA unwinds the DNA helix by an angle φ_e = 26 °. The uncertainty in this value is estimated to be less than 10%. The new value for φ_e is approximately a factor of two larger than the value 12 °, which has been in use in the past decade. In the earlier alkaline titration results for polyoma DNA (Vinograd et al., 1968), which had been interpreted as supporting the 12 ° value, the calculation of φ_e was critically dependent on knowing pH_e. It is believed that pH_e was underestimated in the earlier work, resulting in a low φ_e value. Since the previous value φ_e = 12 ° has been widely used in the determination of the number of superhelical turns for many DNAs, and in measurements on the angular alterations of the DNA helix by the binding of a variety of small and large molecules and by solvent and temperature changes, the new value φ_e = 26 ° requires proportional adjustments of many previous results.