Isolation and characterization of satellite DNA from mustard seedlings

The DNA from mustard (Sinapis alba L.) seedlings was examined by neutral CsCl and Ag⁺/Cs₂SO₄ density gradient centrifugation. Different satellite fractions were revealed by these two methods. The satellite fractions obtained from the Ag⁺/Cs₂SO₄ density gradient could not be generally correlated with satellite DNA fractions observed in CsCl. In CsCl density gradient centrifugation, a main band at density 1,695 g/cm³ and a heavy shoulder at density 1,703 g/cm³ are found. By preparative CsCl gradient centrifugation the heavy shoulder can be enriched but not completely separated from the main band DNA.—Gradient centrifugation by complexing the DNA with Ag⁺ rf. 0.25 to DNA phosphate reveals three distinct fractions which are further characterized: The heavy satelite DNA fraction revealed by Ag⁺/Cs₂SO₄ gradient centrifugation has the same density in a CsCl gradient and the same Tm value as the main band, but differs from main band DNA in the details of its melting profile and in its renaturation kinetics. The light Ag⁺/Cs₂SO₄ satellite DNA fraction had a higher melting temperature corresponding to a GC-rich base composition. Differences between these 3 fractions are observed in thermal denaturation and renaturation profiles, hybridization in situ with ribosomal RNA, and their response to restriction endonuclease digestion. The light satellite fraction from the Ag⁺/Cs₂SO₄ gradient, rich in ribosomal cistrons corresponds to the heavy shoulder DNA of neutral CsCl gradients which also is rich in ribosomal cistrons. The heavy satellite fraction from Ag⁺/Cs₂SO₄ gradient which contains highly repetitive short nucleotide sequences could not be revealed by the classical CsCl gradient centrifugation technique.     

Occurrence of a kinetoplast DNA-protein complex in Trypanosoma cruzi

The kinetoplast DNA has been purified in high yield from $\text{Trypanosoma cruzi}$ by centrifugation on 3 M CsCl after lysis with a detergent. In exponentially growing trypanosomes, a light component causing a density shift of the kinetoplast DNA in CsCl density gradients is observed. This component is presumably a protein since it is removed by digestion with proteolytic enzymes. The DNA-protein complex is resistant to phenol extraction and 4 M guanidinium chloride, which indicates the possibility of a covalent bond between the protein and the DNA. The Kinetoplast DNA isolated from trypanosomes at the stationary phase by centrifugation on 3 M CsCl is free of proteins.     

Extraction, separation and labeling with 14C-glucose of HeLa cell polyglucose, RNA and DNA and comparison of the molecular weights and buoyant densities of polyglucose from poliovirus-infected and noninfected cultures

The aqueous phase of phenol extracts of HeLa cells contains polyglucose (CHO)_n, RNA, and DNA. These macromolecules were precipitated together and removed from 50% (v/v) ethanol solutions with a stirring rod. The viscous precipitate had the classical white appearance of DNA, but contained an average of 439, 670, and 220 μg (from 3 × 10⁷ cells) of (CHO)_n, RNA, and DNA, respectively. The (CHO)_n was separated from the RNA, either by CsCl density gradient centrifugation or by precipitating the RNA with trichloroacetic acid (TCA). Both methods of separation resulted in preparations of (CHO)_n with similar specific activities (radioactive counts/μg min). However, electron micrographs showed that the (CHO)_n separated by using TCA had a greater variation in particle size when compared with (CHO)_n separated by CsCl centrifugation. With the CsCl methods, the number-average molecular weights, as determined by electron microscope particle-counting, and the buoyant densities of (CHO)_n whose synthesis was stimulated by poliovirus infection and (CHO)_n from noninfected cultures, were found to be similar. When the (CHO)_n was extracted from HeLa cells with TCA, rather than phenol, the yield was 1.68-fold greater and its specific activity was an average of twice that of the (CHO)_n extracted with phenol. The time at which cells were pulse-labeled with ¹⁴C-glucose, after reducing the glucose in the culture medium to 0.01 of normal, was found to be important, in that the specific activity of the (CHO)_n increased 23.4-fold over a 4-hr period and the amount extracted decreased 8.2-fold. The increase in the specific activities of RNA and DNA was not as large as that of the (CHO)_n and the amounts extracted were not significantly changed. The sedimentation coefficients of RNA and (CHO)_n which were separated from each other with TCA were 6.4 and 116 S, respectively, whereas, without separation, two peaks were seen, with values of 25.4 and 31.4 S. Chloride ions reduce the sensitivity of the Burton test for DNA. However, the Burton reagent will detect (CHO)_n even in the presence of DNA if the assay mixture is heated. Chloride ions increase the sensitivity of the Burton reagent to detect melizitose and, at concentrations above l.5M, synthetic- polyglucose by increasing the absorption of the colored (CHO)_n reaction product(s).     

Analysis by isopycnic centrifugation of isolated nucleoids of Escherichia coli.

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 +/- 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 +/- 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.     

Deoxyribonucleic Acid Polymerase Associated with Avian Tumor Viruses: Secondary Structure of the Deoxyribonucleic Acid Product

The products of the deoxyribonucleic acid (DNA) polymerase associated with Rous sarcoma virus and avian myeloblastosis virus were characterized by correlative analyses with equilibrium centrifugation and stepwise elution from hydroxyapatite. The initial enzymatic product consists of nascent DNA chains which are hydrogen-bonded to 70S viral ribonucleic acid (RNA), whereas the final enzymatic product is double-stranded DNA. Appreciable amounts of free single-stranded DNA were not detected at any point during the course of the enzymatic reaction, but the data in this regard are not decisive. The time course of synthesis of DNA:RNA hybrids and double-stranded DNA has been analyzed. It is concluded that the synthesis of double-stranded DNA is a sequel to and is probably dependent upon the synthesis of DNA:RNA hybrid.     

ISOLATION AND PARTIAL CHARACTERIZATION OF MACRO- AND MICRONUCLEI FROM PARAMECIUM AURELIA

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs₂SO₄. These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 10⁶ and 2.8 x 10⁶ daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.     

The maize mitochondrial plasmid RNA b is associated with protein during synthesis but is not encapsidated

RNA b is the most abundant member of a family of autonomously replicating single- and double-stranded RNA plasmids found in maize mitochondria. The extent to which this molecule is associated with proteins was investigated by rate zonal and CsCl equilibrium density gradient centrifugation of clarified lysates of S cytoplasm maize mitochondria. A soluble complex of RNA b, responsible for synthesis of the more abundant (+) RNA b strand in mitochondrial lysates, was identified. The complex had a buoyant density of 1.49 g/cm³, indicating a substantial non-nucleic acids content. The sedimentation coefficient of the complex, however, was only slightly larger than that of deproteinized RNA b. Synthesis of RNA b as well as the larger RNA plasmid, RNA a, was resistant to heparin, suggesting that, for both RNAs, preformed complexes between an RNA template and an RNA-dependent RNA polymerase capable of elongating in vivo preinitiated RNA plasmid strands, were present in the lysate. Only a small fraction of RNA b molecules were bound in the complex; the bulk of RNA b sedimented at the same rate as the deproteinized RNA. Thus, after replication, maize mitochondrial plasmids are not associated with nucleoprotein capsids although their synthesis takes place through ribonucleoprotein replication complexes.     

Structure of DNA in Spores of Bacillus cereus

The structure of DNA extracted from dormant and germinating spores of B. cereus T was investigated using circular dichroism and other methods. No significant differences between DNAs extracted from vegetative cells and from spores of various stages could be found by analyses of CD spectra, CsCl density gradient centrifugation, melting profiles and template activity. All the DNA preparations were in B conformation and had the same density (1.695), Tm (83°C) and template activity in the reaction of DNA-dependent RNA polymerase. An abnormal DNA fraction of high density which was formerly found in B. cereus spores or a stable DNA complex with protein and/or RNA was not detected in the present extracts of spores. Preliminary X-ray analyses of intact spores indicate that the structure of DNA in spores is not so different from B form.     

Isolation of viral RNA using cesium chloride-ethidium bromide equilibrium sedimentation

The effect of ethidium bromide (EB) on the buoyant density of reovirus RNA during equilibrium sedimentation has been investigated. The addition of the dye ethidium bromide was found to reduce the buoyant density of reovirus RNA in a Cs₂SO₄ gradient by a value of 0.13 to 0.15 g/cc, and provided a separation limit of 0.10 g/cc relative to the ? of marker DNA. Ethidium bromide was found also to reduce the ? of reovirus RNA to allow this RNA to band on a CsCl gradient. The separation factor between DNA and RNA on a CsCl-EB gradient was found to be 0.23 g/cc, indicating this type of gradient to be highly effective for separating the two types of polynucleotides.     

Isolation of nucleoli from Tetrahymena pyriformis.

A procedure is described to isolate nucleoli from Tetrahymena pyriformis which contain extrachromosomal ribosomal DNA. Macronuclei isolated by the Nonidet procedure were sonicated at a reduced magnesium concentration, and the sonicate was fractionated by isopycnic centrifugation in a metrizamide density gradient. The heaviest band, designated Band IIb, contains exclusively ribosomal DNA, thus constituting the nucleolar fraction. The purity of the nucleolar fraction on a DNA basis, which is defined as the percentage of ribosomal DNA and determined by equilibrium centrifugation in a CsCl density gradient, was around 70%. Electron microscopic examination revealed that the isolated nucleoli retained fairly well the ultrastructure of the in situ nucleoli. Some of the biochemical properties of the isolated nucleoli are also presented.     

The genes for cytoplasmic ribosomal ribonucleic Acid in higher plants

The genes for cytoplasmic ribosomal RNA are partially resolved from the bulk of the DNA by CsCl equilibrium centrifugation. Although in some plants the buoyant density of the ribosomal RNA genes is as expected from the base composition of ribosomal RNA, others show a large discrepancy which cannot be due to the presence of low G-C spacer-DNA. The cross-hybridization observed with 1.3 and 0.7 × 10⁶ molecular weight ribosomal RNAs and DNA, which varies greatly with different plant species, is not due to contamination of the ribosomal RNAs, and is specific for the ribosomal DNA of each species, probably largely restricted to those sequences coding for the two stable ribosomal RNAs. The double reciprocal plot may be used for the extrapolation of saturation values only with caution, because in these cases such plots are not linear over the whole of the hybridization reaction.     

Uptake of exogenous DNA by pea seedlings and tobacco cells

1.

(1) The uptake of Pseudomonas aeruginosa DNA by pea seedlings, and uptake of tobacco DNA or P. aeruginosa DNA by tobacco cells in shake cultures has been investigated. The fate of the DNA has been followed by CsCl density gradient equilibrium centrifugation, using radiolabeled donor DNA of high density.     

Analysis of plant genomes. V. Comparative study of molecular properties of DNAs of seven Allium species

The genomes of seven plant species belonging to the genus Allium and exhibiting a threefold variation in their nuclear DNA content were analyzed by studying their reassociation kinetics, equilibrium centrifugation behavior in neutral CsCl gradients, and melting properties. The reassociation kinetics experiments revealed the presence of 44–65% repeated DNA sequences. A comparison between DNA contents and the proportion of repeated DNA sequences indicated that, in Allium, increase in the genome size is not exclusively due to variations in the proportions of repetitive DNA. The total DNA as well as the various repetitive DNA fractions in all the Allium species examined exhibited, in spite of a few differences, a gross similarity in their behavior in neutral CsCl gradients and in their melting properties.     

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.     

Inhibition of thymidine phosphorylase in vivo provides a rapid method for switching DNA labeling

Summary The interaction between nuclear ploidy and chloroplast DNA content was investigated in the unicellular green alga Chlamydomonas reinhardtii. DNA was extracted from both exponentially growing and synchronized haploid and diploid strains and analysed by CsCl equilibrium density centrifugation in an analytical ultracentrifuge. It was found that the doubling of the nuclear genome in diploids was linked to a doubling of the chloroplast DNA content per cell.     

Initiation of recombination during transformation of Bacillus subtilis requires no extensive homologous sequences

Summary Lysates obtained shortly after entry of transforming DNA to Bacillus subtilis contain donor-recipient DNA complexes, in which the donor moiety is associated with the recipient DNA in an unstable way. The complexes could be artificially stabilized by crosslinking with 4,5,8-trimethylpsoralen. The unstable complexes dissociated upon helix-destabilizing treatments, such as heating at 70°C, and CsCl gradient centrifugation at pH 11.2, but remained stable during CsCl gradient centrifugation at pH 10. Donor-recipient DNA complexes were not formed after entry of heterologous pUB110 DNA. These observations suggest that base-pairing is involved in the unstable association. The donor moiety of the unstable complexes was completely, or almost completely, digestible by nuclease S₁, indicating that the donor and recipient base-sequences are only paired over very short distances.The unstable donor-recipient DNA complexes are true recombination intermediates because (i) strain 7G224 (recE4) was impaired in the formation of the unstable complexes, and (ii) the unstable complexes were rapidly converted to stable complexes in recombination proficient strains, whereas their conversion was delayed in the recombination deficient strain 7G84.Unstable complexes were also formed with Escherichia coli donor DNA, but to a lesser extent. Apparently a limited degree of base-sequence homology is sufficient to initiate recombination.     

A particulate DNA polymerase activity in adult rat brain

A particulate fraction of adult rat brain (sucrose buoyant density 1.24 gm/ml) catalyzed the incorporation of [³H]dTTP into an acid-insoluble product in an endogenously templated reaction sensitive to ribonuclease pretreatment. Upon fractionation, this activity was identified in the cerebellum, pons, frontal lobes and base. The DNA polymerase present in these brain fractions exhibited a strong preference for the synthetic template dT_12–18·poly rA rather than dT_12–18·poly dA; dT₁₀ was completely inactive. Purification and equilibrium Cs₂SO₄ gradient centrifugation of the [³H]DNA product-endogenous template complex suggested that RNA was serving as primer for endogenous DNA synthesis.     

Separation and characterization of some ribonucleic acids from imaginal discs of Drosophila melanogaster

Summary The RNA obtained from mass isolated imaginal discs of Drosophila melanogaster cultured in vitro in ³H-5-uridine has been studied. A separation procedure employing benzoylated, naphthoylated DEAE cellulose column chromatography was used to obtain several fractions of RNA, including one consisting primarily of ribosomal RNA, and three others relatively free of rRNA and transfer RNA. Other than its behavior in BND-cellulose chromatography, the RNA fractions have been examined with the following procedures: (1) sucrose gradient centrifugation, (2) heat labilities with apparent shifts in S-values, (3) changes in apparent secondary structure due to monovalent cation concentration, (4) Cs₂SO₄–CsCl equilibrium gradient centrifugation, (5) dimethyl sulfoxide gradient centrifugation, (6) melting out studies in different salt (Na⁺) concentrations, (7) limited digestion with different kinds of RNase, (8) kinetics of labeling, and (9) DNA-RNA hybridization. Marked differences were found among the RNA fractions using these techniques. The addition of -ecdysone to the cultured imaginal discs induces the production of quantitatively and perhaps qualitatively different RNA when compared with RNA obtained from discs cultured without the hormone. The possible secondary structures of the RNA fractions are also discussed.