Single-stranded fraction of deoxyribonucleic acid from Bacillus subtilis.

About 13% of the deoxyribonucleic acid (DNA) of various strains of Bacillus subtilis, independent of the stage of growth or competence for transformation, was rendered acid soluble by endonuclease S1. In a pH 11.2 CsCl gradient, 4% of the untreated DNA banded at the density typical for single-stranded molecules, whereas 9% of the remaining DNA (main band) was sensitive to endonuclease S1. Selective inhibition of DNA polymerase III, or of DNA-dependent ribonucleic acid polymerase, did not increase or abolish single-strandedness. The DNA purification procedure did affect the level of single-stranded DNA, indicating its binding to cell constituents containing ribonucleic acid, protein, and membranous material. The molecular weight of the single-stranded fraction resembled that of total denatured DNA, and its buoyant density in an alkaline CsCl gradient was centered partially at a density of 1.772 g/cm3 and partially at a density of 7.759 g/cm3. Incubation of DNA under conditions leading to renaturation of its single-stranded fraction led to an increase in transforming activity for the purA16+ marker (close to the origin of replication) relative to leu-8+ and metC3+ markers (located in the middle of the chromosome), indicating this region is the main source of the single-stranded fraction.     

The gene transfer agent of Rhodopseudomonas capsulata,: Purification and characterization of its nucleic acid

Photosynthetic bacteria of the species Rhodopseudomonas capsulata are capable of exchanging genetic information via a recently discovered gene transfer agent (GTA). The 70S particle mediating the genetic exchange was purified and its nucleic acid was analyzed. Cell-free filtrates containing GTA were prepared by filtration of stationary cultures of R. capsulata on an Amicon thin-channel filtration apparatus. Purification of this filtrate was achieved by successive membrane filtration, diafiltration, agarose-gel filtration, ion-exchange chromatography on diethylaminoethyl cellulose, and sucrose gradient sedimentation, resulting in an overall purification of 4000-fold with a yield of 2–4%. [³H]thymidine-labeled nucleic acid isolated from this material was identified as deoxyribonucleic acid on the basis of its resistance to alkaline hydrolysis and its buoyant density of 1.718 g/ml in CsCl. The double-stranded nature of the deoxyribonucleic was demonstrated by its resistance to degradation by the single-strand-specific S₁-nuclease and the density shift in CsCl of +0.016 g/ml upon denaturation. Its molecular weight was estimated to be 3.6 × 10⁶ from sucrose gradient sedimentation in the presence of markers, and the linear, unnicked nature of the molecule was evident from sedimentation in an alkaline sucrose gradient.     

Changing proportions of DNA components in Euglena gracilis

The ratios of satellite deoxyribonucleic acid components to chromosomal deoxyribonucleic acid in Euglena gracilis Z were measured by analytical density gradient ultracentrifugation. Chloroplast deoxyribonucleic acid with a buoyant density of 1.685 g/cm³ exhibited a constant ratio to chromosomal deoxyribonucleic acid during exponential growth and increased twofold as the culture reached the end of the exponential growth phase. The quantity of a satellite deoxyribonucleic acid with a buoyant density of 1.691 g/cm³ was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but increased to approximately equal the quantity of chloroplast deoxyribonucleic acid as the culture approached the end of the exponential growth phase. The quantity of a deoxyribonucleic acid component with a buoyant density of 1.700 g/cm³ was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but represented approximately one-third of the total deoxyribonucleic acid as the culture entered the stationary phase of growth.     

Characterization of the deoxyribonucleic acid of various strains of halophilic bacteria

Bacteria classified as extreme halophiles, in the genera Halobacterium and Halococcus, contain deoxyribonucleic acid (DNA) which displays two components in a CsCl equilibrium density gradient. The base composition of the major DNA component ranges from 66 to 68% guanine plus cytosine (GC), whereas that of the satellite DNA comprising some 11 to 36% of the total, is between 57 and 60% GC. Purification of the bacterial cells in a CsCl density gradient and other more conventional strain purification procedures both indicated that the presence of the satellite DNA component is not a result of mixed cultures.     

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.     

Analysis of Euglena gracilis chloroplast deoxyribonucleic acid with a restriction endonuclease, EcoRI.

Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoRI) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight of the circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosomal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.     

Replication of Coliphage M-13 II. Intracellular Deoxyribonucleic Acid Forms Associated with M-13 Infection of Mitomycin C-treated Cells

Intracellular deoxyribonucleic acid (DNA) forms associated with bacteriophage M-13 infection have been isolated and characterized. Escherichia coli HF4704 (F⁺, hcr⁻, thy⁻) cells were treated with mitomycin C to inhibit host-cell DNA synthesis and were then infected with phage M-13. This treatment permitted radioactive labeling of phage-specific DNA forms with ³H-thymine. These labeled DNA components were characterized by sucrose density sedimentation and equilibrium density gradient centrifugation in neutral and ethidium bromide CsCl gradient. Two double-stranded circular forms were found with properties analogous to the replicative form I and replicative form II of X174. A third component, identified as single-stranded DNA, was isolated in some samples removed 45 min after phage synthesis was initiated.     

A rapid, simple method for nuclei isolation from plant protoplasts

A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.     

Ribosomal RNA cistrons in Euglena gracilis

Euglena gracilis chloroplasts contain about 12 fg DNA of average density 1.686 g cm^?3 and 1.7 pg RNA. The large (1.1 × 10⁶ mol. wt) and small (0.56 × 10⁶ mol. wt) ribosomal RNA components are coded for by separate cistrons, both of which band at a density of 1.696 g cm^?3 in a CsCl gradient. About 6% of the chloroplast DNA codes for rRNA indicating that there are 240 cistrons for rRNA in each chloroplast or about three to six cistrons per chloroplast genome. Similar studies with rRNA from cytoplasmic ribosomes indicate that the cistrons for cytoplasmic rRNA band at a density of 1.716 g cm^?3, denser than that of the main-band DNA, and that there are 1000 cistrons for cytoplasmic rRNA per cell. Fractionation of E. gracilis DNA on CsCl gradients and subsequent hybridization experiments, as well as melting curves of DNA-RNA hybrids, show that chloroplast rRNA does not anneal specifically with either the cistrons for cytoplasmic rRNA or any DNA in the dark-grown cell, in contrast to those results found in some higher plants.     

Liverwort genomes display extensive structural variations

PIKE, L. M., HU, A., RENZAGLIA, K. S. & MUSICH, P. R., 1992. Liverwort genomes display extensive structural variations. Analyses of the total genomic DNA of eight species of liverworts and two species of green algae by thermal denaturation and CsCl buoyant density gradient centrifugation reveal a high degree of structural complexity and interspecific heterogeneity. The hepatic taxa exhibit two or more DNA components of varying base composition. Average G4-C contents of total cellular DNA calculated from melting profiles are similarly variable, ranging from 38% to 53% G + C. The green alga Chara , a member of the ancestral line to land plants, shows similarities with liverworts in possessing multiple DNA components of comparable complexity, whereas Hydrodiciyon DNA displays a single component. Detailed hybridization analyses of individual density gradient fractions using α-tubulin, rRNA and ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene probes were performed to locate the low-copy number and moderately repetitive nuclear genes, and the chloroplast chromosome, respectively. The location of each gene within the density gradient is highly variable among the organisms examined; a-tubulin occurs in fractions ranging from 44–64% G + C, rDNA in 50–64% G + C fractions, and the RbcL gene is located in fractions from 30–59% G + C. For a given species, the two nuclear genes normally overlap in their distributions within the gradient. In most instances, neither gene occurs in the major DNA components, indicating that these components may contain repetitive DNAs. The observed variation in the density of the rbcL gene implies substantial reorganization of the chloroplast genome. The overall differences in the genomic components within and between taxa provide insight into the dynamics of DNA structure that have occurred during the extended evolutionary history of these organisms.     

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.     

Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids

Birnboim, H. C. (Albert Einstein College of Medicine, New York, N.Y.). Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids. J. Bacteriol. 91:1004-1011. 1966.-A nuclease, identified by a marked preference for single-stranded nucleic acids, has been demonstrated in extracts of Bacillus subtilis. The enzyme was associated with the cell wall-membrane fraction of mechanically disrupted cells and was released from cells which had been converted to protoplasts by lysozyme. The nuclease activity prepared by the latter procedure was found to be activated and solubilized by treatment with trypsin. The enzyme had about 2% activity on native deoxyribonucleic acid (DNA) as compared with denatured DNA. By use of CsCl analytical density gradient ultracentrifugation, this preparation was shown to degrade denatured DNA selectively in mixtures of native and denatured DNA.     

Isolation of twelve satellite DNAs from Drosophila hydei

The genome of a Drosophila hydei genotype with a reduced amount of heterochromatin was fractionated by three cycles of preparative gradients: firstly in Ag⁺/Cs₂SO₄, secondly in actimomycin D/CsCl, and finally in neutral CsCl. Using this method, twelve highly repetitive simple-sequence satellites were isolated. Ten of them comprised only a minor amount of the genome in contrast to the two major satellites found earlier¹ (p = 1.696 and 1,714 g/cm³). These minor satellites were characterized by their banding in the gradient systems used, by their density in neutral CsCl, and by their melting point. Using these characteristics, it was found that the fractions of the Ag^?/Cs₂SO₄ gradient do not contain purified single components, because up to five different satellites band in the same position of the Ag^?/Cs₂SO₄ gradient. It was possible to isolate a high number of satellites even from a genome with a reduced amount of heterochromatin. Thus, the D hydei heterochromatin does not domain one unique highly repetitive sequence DNA, but is comprised of many different satellite sequences.     

The physical state of herpes simplex virus DNA in infected human cells.

Treatment of herpes simplex virus type 1 (HSV-1)-infected human embryo lung (HEL) cells with phosphonoacetic acid (PAA) resulted in complete inhibition of HSV DNA replication. DNA was extracted from PAA-treated HEL cells infected with HSV-1 and centrifuged in a neutral CsCl density gradient. The HSV DNA sequences in the nuclei of PAA treated cells at 24 hr post infection banded at the same density as free HSV DNA (1.725 g/cm3), but a significant amount of viral DNA sequences were detected in the regions of cell DNA (1.700 g/cm3) as well as in the intermediate fractions as determined by hybridization with 3H HSV complementary RNA. The viral DNA sequences of lower deisntiy did not change in density by recentrifugation in a CsCl density gradient, but did change to the density of free viral DNA after treatment with EcoR1 restriction endonuclease. When the DNA from the nuclei of PAA treated cells was analyzed in an alkaline glycerol gradient, more than 95% of the viral DNA sequences were found in the free viral DNA fractions. Since the viral and cellular hybrid DNA represented approximately 33% of the total viral DNA sequences, it is concluded that some of the HSV DNA sequences in PAA treated, infected cells are associated with cell DNA by alkali-labile bonds.     

Isolation of the Ochromanas danica plasma membrane and identification of several membrane enzymes

Ochromonas danica cell homogenate can be fractionated by differential centrifugation into chloroplast, mitochondrial, ribosome, lysosomal, plasma membrane and soluble fractions. The plasma membrane fraction was further purified by discontinuous sucrose density gradient centrifugation and was found to be enriched 4–16-fold in the following enzymes: β-galactosidase, acid phosphatase, alkaline phosphatase, 5′-nucleotidase, and (Na⁺, K⁺)-ATPase. The role of plasma membrane phosphatase in the phosphate metabolism of plants is discussed.     

Molecular Fate of Heterologous Bacterial DNA in Competent BACILLUS SUBTILIS. I. Processing of B. PUMILUS and B. LICHENIFORMIS DNA in B. SUBTILIS

Competent Bacillus subtilis cells were exposed to radioactive and density labeled donor DNA extracted from B. pumilus and B. licheniformis. The DNA from these strains hybridized with B. subtilis DNA in vitro at a rate of 24% and 11%, respectively. After entry the vast majority of heterologous DNA was found at the single-strand DNA position in CsCl gradients, and was gradually degraded during incubation. Much less donor DNA than expected from the hybridization values participated in the formation of the donorrecipient complex (DRC). By subjecting the heterologous DRC to sonication and alkaline CsCl gradient centrifugation, it was established that the DRC consisted of three components: (1) recipient DNA in which breakdown products of donor DNA were incorporated through DNA synthesis, (2) recipient DNA in which donor DNA was covalently integrated and (3) recipient DNA in which the donor moiety was not covalently integrated.     

Effect of Iododeoxyuridine upon Conjugation and the Fate of Transferred Deoxyribonucleic Acid in Escherichia coli K-12

The incorporation of 5-iododeoxyuridine (IUdR) into Escherichia coli K-12 deoxyribonucleic acid (DNA) has been found to decrease significantly the viability of female strains A288 and JC411(r) but to have only minor effect upon their ability to act as conjugational recipients and to perform recombination after conjugation. In contrast, IUdR incorporation into male strain HfrC appears to interfere with both chromosome transfer and genetic recombination. By using IUdR to densitylabel female DNA, and carrying out large-scale matings with (3)H-thymidine-labeled male cells, we examined the fate of transferred DNA. After a 30-min mating, the T6-sensitive male cells were lysed, and the DNA of the merozygotes and remaining female cells was isolated. Initial centrifugation of this DNA in a CsCl gradient showed that the male and female DNA species were associated. The nature of this association of the parental DNA species was determined by formaldehyde denaturation followed by CsCl centrifugation. Denaturation of DNA isolated immediately after T6 lysis gave a peak of radioactivity banding at the density of light single-stranded DNA. However, denaturation of DNA isolated after T6 lysis and dilution of the cells into fresh medium, exhibited peaks of radioactivity banding at positions corresponding to single-stranded, density-labeled DNA. The results indicate that recombination after conjugation in E. coli takes place by a breakage-and-reunion mechanism. The process of recombination can be separated into two stages. In the first stage, the donor and recipient DNA molecules become associated. The second stage consists of the formation of phosphodiester bonds between the donor and recipient segments comprising the recombinant molecule.     

DNA Products in In Vitro DNA Synthesis Using Bacteriophage ϕX174 Single-stranded DNA

We described product analysis of DNA synthesized in chloroplast lysate from liverwort Marchantia polymorpha L. cell suspension cultures. Characteristics of in vitro DNA synthesis by chloroplast lysate using bacteriophage ?X174 single-stranded DNA were very similar to those in the case of double-stranded calf thymus DNA reported previously. Autoradiographic analysis clearly showed the incorporation of radioactive [α-³²P]-dCTP into DNA molecules associated with bacteriophage ?X174 single-stranded template DNA, indicating conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III, double-stranded linear molecule). Experiments on the fate of [³²P]-labeled single-stranded DNA also showed a clear conversion of the single-stranded DNA to double-stranded DNA. Furthermore, patterns of sucrose density gradient centrifugations (neutral and alkaline) showed the production of two major components in in vitro DNA synthesis by chloroplast lysate. This also indicated conversion of bacteriophage ?X174 single-stranded DNA to double-stranded DNA (RF III form). Our results suggest that the mechanism of chloroplast DNA replication could be the mode of strand-displacement DNA synthesis as seen in animal mitochondrial DNA synthesis.     

Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza

Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size.