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.     

Nuclear DNA in normal and refed Amoeba proteus

Amoeba proteus synthesizes DNA in G2 phase of the cell cycle upon feeding after starvation. The characteristics of the DNA synthesized in G2 have been studied by microscope photometry of individual Feulgen-stained nuclei and by buoyant density centrifugation of nuclear DNA in CsCl. Amoeba nuclei were found to contain 42.8 pg of DNA. This DNA bands in CsCl at a density of 1.693 g/cm³ with a satellite at 1.714 g/cm³ which makes up 24% of nuclear DNA. DNA from whole cells has an additional non-nuclear satellite at 1.726 g/cm³. When cells are starved and re-fed with food labeled with [³H]thymidine, the DNA synthesized is predominantly the 1.714 satellite. The amount of DNA synthesized in G2 is small since there is no measurable difference in Feulgen dye binding to nuclei of starved vs starved and re-fed cells. The data suggest that refeeding induces a resumption of late S phase DNA synthesis, or the preferential synthesis of specific DNA sequences such as rRNA genes.     

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.     

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.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Physicochemical characterization of cyanophage SM-2

Cyanophage SM-2 which infects two unicellular cyanobacteria, Synechococcus elongatus UTEX 563 and Microcystis aeruginosa NRC-1 (Synechococcus sp. NRC-1) UTEX 1937 has a buoyant density of 1.483 g/cm³, a DNA buoyant density of 1.729 g/cm³ and a guanine + cytosine (G+C) content of 69–70%. The protein patterns of cyanophage SM-2 particles showed 11 bands, as determined by polyacrylamide gel electrophoresis, with the bulk of the protein mass concentrated at the 39,000 Mr band. There appear to be no cross-reacting anibodies to whole virus particles of cyanophages SM-1, SM-2 and AS-1. Cyanophage SM-2 requires the presence of cations for viral stability.     

The plastome of a brown alga,Dictyota dichotoma

Summary Plastids of the brown algaDictyota dichotoma contain a single homogeneous DNA species which bands at a buoyant density of 1.693 g/cm³ in neutral CsCl equilibrium density gradients. The corresponding nuclear DNA has a density of 1.715 g/cm³. The molecular size of the plastid DNA is 123 kbp as calculated by both electron microscopy of spread intact circular molecules and gel electrophoresis following single and double digestions with various restriction enzymes. A restriction map has been constructed using the endonucleases Sal I, Bam HI, and Bgl II which cleave theDictyota plastome into 6, 12, and 17 fragments, respectively. No large repeated regions, as found in chlorophycean andEuglena plastid DNAs, were detected.Dictyota dichotoma is the first member from the chlorophyll c-line of the algal pedigree for which a physical map of plastid DNA has been established. Dedicated to Professor Dr. W. Stubbe on the occasion of his 65th birthday.     

Circular DNA from the water mold Saprolegnia

Summary Circular DNA has been investigated in the water mold Saprolegnia. This organism was shown to contain two satellite DNA components at 1.685 and 1.707 g/cm³ in addition to main band DNA at a buoyant density of 1.717 g/cm³. The component banding at a density of 1.685 g/cm³ was found to occur as closed circles of length 14 m in the relaxed form. On the basis of sucrose gradient studies this DNA is thought to be mitochondrial in origin. No other class of circular molecules was observed.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Location of Satellite and Homogeneous DNA Sequences on Human Chromosomes

HUMAN DNA^1,2 contains at least two satellite fractions—satellite I (0.5% of the genome) which bands at a density of 1.687 g/cm³ in neutral CsCl and satellite II (2% of the genome) which bands at 1.693 g/cm³. The main band DNA has an average buoyant density between 1.670 and 1.720 g/cm³ and a light shoulder (constituting 15% of the genome) with a buoyant density of 1.696 g/cm³, referred to as homogeneous mainband. Satellite DNA has been observed in many higher organisms³, usually with an unknown function, notable exceptions being cistrons coding for ribosomal RNA⁴ and the DNA coding for histone messenger RNA⁵. To investigate possible functions of human repetitive DNA we have studied the annealing of complementary RNA fractions to chromosomes using in situ hybridization^6,7. We describe here preliminary observations using human satellite II and homogeneous mainband fractions.     

High-affinity microtubule protein-higher organism DNA complexes. Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs

We have examined aspects of the interaction of cycled microtubule protein preparations with ³⁵S-labeled mouse DNA tracer in a competition system with unlabelled competitor E. coli or mouse DNA. The nitrocellulose filter binding assay was used to measure interaction by scintillation counting. DNA molecular weight affected the levels of filter retained ³⁵S-labelled mouse tracer DNA. Filter retention levels increased if ³⁵S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased. There was a sizeable, reproducible difference in the ³⁵S-labelled mouse DNA tracer binding level of about 1% when E. coli or mouse DNA competitors were compared. Mouse DNA more effectively competed with ³⁵S-labelled mouse DNA for microtubule protein binding than did E. coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein. From other studies we know this to be the MAP fraction (Marx, K.A. and Denial, T. (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R. Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R. and Avila, J. (1981) Nucleic Acids Res. 9, 895–908). We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor [DNA]) we developed for examining high-affinity complexes. Under high-stringency conditions we isolated 1.2% and 0.6% of ³⁵S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes. At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S₁ nuclease solution reassociation kinetics. The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes. The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm³) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm³) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A. (1977) Biochem. Biophys. Res. Commun. 78, 777–784). That the high-affinity microtubule-bound DNA was some 3–5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern     

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.     

Isolation and partial characterization of a mitochondrial deoxyribonucleic acid-protein complex from sea urchin embryos

Summary Lysis of mitochondria from sea urchin embryos with Triton X-100 led to a complete conversion of DNA-containing mitochondrial residues into protein-DNA complex with a density higher than 1.22 g/cm³ in sucrose solutions. This complex banded isopycnically in metrizamide gradients at a density of 1..26 g/cm³. Exposure to mixtures of Triton X-100 with Tween 80 resulted in progressively less delipitated and disorganized mitochondria over Tween/Triton weight ratios from 1 to 2, with the retention of the starting buoyant density in sucrose of approximately 1.16 g/cm³ at Tween/Triton ratios above 2.5. The DNA-internal protein complex sedimented with the bulk of the surviving mitochondrial structure under all conditions studied. No free DNA could be detected under any conditions of membrane removal.     

Polypyrimidine segments in drosophila melanogaster DNA: I. Detection of a cryptic satellite containing polypyrimidine/polypurine DNA

Very long runs of pyrimidine nucleotides (polypyrimidines), previously detected in DNA from Drosophila melanogaster, have now been localized to a “cryptic” satellite. These polypyrimidines have an average length of 750 nucleotides and account for about 3% of the thymine residues in total DNA. The buoyant density of the DNA component which contains the polypyrimidines was detected by centrifuging native DNA to equilibrium in a CsCI gradient, and then assaying each fraction for its content of polypyrimidines. A peak was detected at a density of about 1.707 gm/cm³, distinctly heavier than the main band of DNA (1.702 gm/cm³). The buoyant density of polypyrimidine-containing molecules was little affected by differences in the molecular weight of the starting DNA in the range 10⁵-10⁷ daltons (single-stranded). Thus polypyrimidines (and their complementary polypurines) appear to form all or part of a “cryptic” satellite.Polypyrimidines have been isolated and characterized with respect to composition and buoyant density. Direct nucleoside analysis of unlabeled material indicated 34.5% deoxycytidine, 65.5% thymidine. Their banding position in neutral and alkaline CsCI gradients was consistent with a single-stranded DNA polymer of this composition.     

Buoyant densities of natural and synthetic DNA's in CsCl and Cs2SO4 considered as sequence-dependent properties

The buoyant densities of natural and synthetic DNA's can be accurately interrelated if second-neighbor influences are taken into account. We derive the following expressions, based partly on the buoyant densities of six synthetic DNA's, for the buoyant densities ρ (g/cm³) of DNA's having random sequences. In CsCl, and in Cs₂SO₄, . In these equations, H_G is the mole fraction of G : C base pairs in the DNA and the buoyant densities are calculated relative to densities for E. coli DNA of 1.703 and 1.426 (g/cm³) in CsCl and Cs₂SO₄, respectively.     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.     

Polytene chromosomes of Oxytricha: Biochemical and morphological changes during macronuclear development in a ciliated protozoan

After conjugation in the ciliated protozoan, Oxytricha, polytene chromosomes are formed during the development of a macronucleus from a micronucleus. Here we report a microscopic study of these chromosomes and an analysis of their DNA. The polytene chromosomes of Oxytricha bear a strong morphological resemblance to the polytene chromosomes of the Dipteran salivary gland. The nucleus of a developing macronuclear anlage contains 120±2 polytene chromosomes and each chromosome has an average of 81 bands; a total of about 10,000 bands per nucleus. At a later stage in development, the number of bands per chromosome is reduced by a factor of four, presumably due to fusion of adjacent bands. The polytene chromosomes then break up into their constituent bands, each of which is encased in a vesicle. There are about 2,700 vesicles per nucleus. — During the growth of polytene chromosomes, there is a change in the relative proportion of sequences in the DNA. The DNA from polytene nuclei has a buoyant density of 1.695 g/cc, significantly lighter than the density of the original micronuclear DNA (1.698 g/cc to 1.702 g/cc). We interpret this buoyant density change to be the result of differential replication of DNA sequences during polytene chromosome growth. A second change in DNA composition occurs after the polytene stage of development, shown by a shift in buoyant density to 1.701 g/cc in the DNA of the mature macronucleus. During this second process, the molecular weight of the DNA is reduced from greater than 50×10⁶ daltons to about 2×10⁶ daltons.This paper is No. VI in the series, DNA of Ciliated Protozoa.     

Structural dynamics of bacterial ribosomes. III. Quantitative conversion of vacant ribosome couples into an initiation complex with R17 RNA as messenger

The arrangement of the DNA sequences coding for the ribosomal 5.8 S RNA in the genome of Xenopus laevis has been studied. In Xenopus the 5.8 S cistrons, like the ribosomal 28 S and 18 S cistrons, are reiterated some 600-fold (Clarkson et al., 1973a). When banded in caesium chloride, the 5.8 S cistrons separate from somatic DNA of high molecular weight and band as a distinct satellite, indicating a clustered arrangement in the genome. The buoyant density of this satellite (1.723 g cm^?3) corresponds to that of the ribosomal DNA satellite.It has previously been shown that the ribosomal DNA sequences have been deleted from the genome of the anucleotide Xenopus mutant. Our findings, first that the anucleolate mutant does not synthesize 5.8 S RNA and second that somatic DNA from this mutant does not detectably hybridize with 5.8 S RNA, demonstrate that the 5.8 S cistronic complement has been similarly deleted. This finding supports our contention that 5.8 S sequences are clustered on chromosomal DNA and further suggests that they are located close to or within the rDNA complements in the nucleolus organizer region.Pre-hybridization to saturation with unlabelled 5.8 S RNA results in only a slight increase in the buoyant density of denatured 5.8 S coding sequences from low molecular weight DNA. Since a contiguous arrangement of the 5.8 S sequences would give rise to a much larger increase in density, it follows that, although clustered, the sequences must be intercalated within stretches of other DNA. By contrast, pre-hybridization of the somatic DNA with unlabelled 28 S or 18 S ribosomal RNAs results in large shifts in the buoyant density of the 5.8 S sequences. These shifts indicate that the 5.8 S sequences are closely linked to both 28 S and 18 S coding sequences.It is concluded that the 5.8 S cistrons are interspersed along the ribosomal DNA sense strand and that each is located together with a 28 S and an 18 S cistron in a ribosomal repeat unit. Estimates, obtained from the pre-hybridization experiments, of the separations between the 5.8 S and the 28 S and 18 S sequences, are combined in a model of the ribosomal repeat unit. In this model the 5.8 S cistron is located within the transcribed spacer which links the 28 S and 18 S coding sequences.     

Buoyant Densities and Dry-Matter Contents of Microorganisms: Conversion of a Measured Biovolume into Biomass

Several isolates of bacteria and fungi from soil, together with cells released directly from soil, were studied with respect to buoyant density and dry weight. The specific volume (cubic centimeters per gram) of wet cells as measured in density gradients of colloidal silica was correlated with the percent dry weight of the cells and found to be in general agreement with calculations based on the partial specific volume of major cell components. The buoyant density of pure bacterial cultures ranged from 1.035 to 1.093 g/cm³, and their dry-matter content ranged from 12 to 33% (wt/wt). Average values proposed for the conversion of bacterial biovolume into biomass dry weight are 1.09 g/cm³ and 30% dry matter. Fungal hyphae had buoyant densities ranging from 1.08 to 1.11 g/cm³, and their dry-matter content ranged from 18 to 25% (wt/wt). Average values proposed for the conversion of hyphal biovolume into biomass dry weight are 1.09 g/cm³ and 21% dry matter. Three of the bacterial isolates were found to have cell capsules. The calculated buoyant density and percent dry weight of these capsules varied from 1.029 g/cm³ and 7% dry weight to 1.084 g/cm³ and 44% dry weight. The majority of the fungi were found to produce large amounts of extracellular material when grown in liquid cultures. This material was not produced when the fungi were grown on either sterile spruce needles or membrane filters on an agar surface. Fungal hyphae in litter were shown to be free from extracellular materials.