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.     

Characterization and localization of cryptic satellite DNAs in barley (Hordeum vulgare)

Summary Three satellites on the heavy side of the main band and two satellites on the light side were isolated in a pure from by preparative ultracentrifugation of H. vulgare DNA in Ag⁺/Cs₂SO₄ density gradients. The satellites were characterised in terms of their buoyant densities in CsCl and their thermal dissocation temperature in both native and reassociated forms to Cot 4. In CsCl gradients, heavy satellites formed a single peak whereas light satellites resolved into more than one component. Thermal transitions of some satellites indicated the presence of more than one molecular species. The multicomponent nature of thermal denaturation profiles was evident on differential analysis. Radioactive RNAs complementary to the three heavy satellites of H. vulgare were localised by in situ hybridization onto its nuclei and chromosomes. One heavy satellite (H₃) was found to be distributed on all chromosomes, although one pair showed less hybridization compared to the others. The other satellite (H₁) appeared to be present in a much lower amount on the chromosomes.     

Fractionation and characterization of satellite DNAs of the kangaroo rat (Dipodomys ordii)

Nuclear DNA from liver cells of the kangaroo rat species Dipodomysordii was fractionated and characterized with the aid of buoyant density gradients in neutral and alkaline CsCl and in Ag⁺-Cs₂SO₄. More than one-half of the DNA was present in three density satellites, a greater proportion than in any other species yet reported; the purified satellite DNAs were denser than principal DNA. All satellite fractions revealed sharp isopycnic bands and narrow denaturation profiles. Two had identical buoyant densities but differed substantially in T_m, base composition, and reassociation kinetics. In alkaline CsCl all three satellites, as well as a shoulder of intermediate repetitive DNA on the heavy side of the principal band, revealed unique strand densities. The most highly repetitive satellite was unusually rich in (G + C) and contained 6.7% of 5-methylcytosine. A survey of internal organs and spermatozoa of an adult male revealed no significant differences in distribution of the satellites among tissues.     

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.     

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.     

RNase-sensitive DNA polymerase activity in cell fractions and mutants of Neurospora crassa

RNase-sensitive DNA polymerase activity (RSDP) was tested in different cell fractions of Neurospora crassa cell types and its morphological mutants. This RSDP was found localized in the microsomal pellet fraction and absent in the purified nuclear pellets isolated from different N. crassa cell types: conidia, germinated conidia, and mycelia. This enzyme is capable of synthesizing a DNA product only in the presence of all four deoxyribonucleoside-5-triphosphates and Mg²⁺. Removal of RNA from the pellet fraction by RNase strongly inhibited the DNA synthesis. The endogenous synthesis of DNA in the microsomal pellet fraction was associated with the formation of an RNA:DNA hybrid as analyzed by Cs₂SO₄ equilibrium density gradient centrifugation. The DNA product after alkali hydrolysis hybridizes with the RNA isolated from the same pellet fraction, as analyzed by elution from hydroxylapatite column at 60 C. This DNA product did not hybridize with poly(A). A few mutants tested showed this RNase-sensitive DNA polymerase activity.This work was supported in part by a contract with the U.S. Department of Energy and a grant from the U.S. Naval Research.     

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.     

Repetitive sequences in nuclear deoxyribonucleic acid ofSaccharomyces cerevisiae

Nuclear DNA isolated from aSaccharomyces cerevisiae ρ mutant was studied for the presence of repetitive sequences. A main-band DNA preparation free of rRNA genes and 2-μm plasmid DNA was prepared by density gradient centrifugation in Cs₂SO₄−Ag⁺. A fast renaturing fraction was obtained from this mainband DNA by 3 cycles of reassociation at a low C₀t value (0.2). This fraction reassociated 10 times faster than the bulk of the main-band DNA. Its sequences comprised about 3% of the genome and showed a considerable heterogeneity in respect to repetitiveness. The relationship of this fraction to the repetitive transposable elements recently found in yeast cells is discussed.     

Two highly repetitive DNA satellites of Drosophila hydei localized within the α-heterochromatin of specific chromosomes

Two major highly repetitive satellites have been isolated from nuclear DNA of Drosophila hydei by sequential centrifugations in Ag⁺/Cs₂SO₄, actinomycin D/CsCl and CsCl. Their CsCl-densities are 1.696 and 1.714 g/cm³. In diploid larval brains they comprise about 13% and 4% respectively of the DNA. Both satellites are localized and chromosome specific. The 1.696 component was shown to be derived from the X-heterochromatin by comparison of different stocks containing different amounts of X-heterochromatin and by in situ hybridization of the ¹²⁵I-labelled light single strand of this satellite. Since the amount of X-heterochromatin equals the amount of this satellite it was concluded that the 1.696 satellite is the only major DNA component of the X-heterochromatin besides minor DNA fractions (e.g. rDNA). The other highly repetitive satellite (1.714 g/cm³) hybridized in situ to all four acrocentric autosome pairs of D. hydei, but neither to the X nor to the small dot-like sixth chromosome, and not to the Y.     

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.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag⁺-Cs₂SO₄ density gradient centrifugation. — According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2×10⁴ copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).— Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides. —Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides. — The organization of mouse genome analyzed by Ag⁺-Cs₂SO₄ density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

Characterisation of DNA from condensed and dispersed human chromatin

Condensed and dispersed chromatin fractions were isolated from human placental nuclei. The DNA of each fraction was purified and characterised by isopycnic centrifugation, thermal fractionation on hydroxylapatite (HAP) and sequence complexity studies. The DNAs had identical buoyant densities in neutral CsCl (1.698 g/cm³) and similar melting profiles on HAP. Analytical ultracentrifugation in Ag⁺-Cs₂SO₄, however, showed that satellite DNAs were present in the condensed fraction DNA (DNA_C) but were not visible in the dispersed fraction DNA (DNA_D). In addition, DNA_C was found to be enriched in highly reiterated sequences (20% reassociated by C₀t 10^?3) which can be correlated with the presence of satellite DNAs, whereas DNA_D contained only 3% of these fast reassociating sequences. In contrast DNA_D contained 30% intermediate sequences (reassociating between C₀t 10^?3 and C₀t 100) which represent only 10% of DNA_C. The reassociated highly repeated sequences of DNA_C showed the presence of two components in both CsCl density gradients and HAP thermal elution studies. This suggests that either there are sequence relationships resulting in partial mismatching between the different highly repeated DNA sequences in this fraction, or that highly repeated sequences are associated with less repetitious DNA. The results are discussed in terms of possible differences in genetic activity between the chromatin fractions.     

Organization,replication and modification of the human genome: Differential methylation of two classes of HeLa nuclear DNA separated on Ag+−Cs2SO4 gradients

HeLa nuclear DNA sediments as a single peak, in neutral CsCl, while it is separated in a heavier and a lighter components, in alkaline Ag⁺–Cs₂SO₄. The heavy fraction, on the average, represents about 20% of the total DNA. CsCl analytical ultracentrifugation shows that heavy DNA bands at 1.715 g/cm³ and contains 53% GC (10% of the total GC), whereas light DNA bands at 1.703 g/cm³ and contains 40% GC (32% of the total GC). Coherently, Tm values in 0.1 x SSC are 82.5°C, for heavy DNA, and 72.5°C, for light DNA. After treatment with [³H-methyl-S-adenosyl-L-methionine in isolated nuclei, the concentration of labelled 5-methylcytosine was found to be highest in the more dense regions of the heavy peak and in the less dense regions of the light peak. Exposure to ultrasound modifies the quantitative relationship of the two peaks and improves the separation of supermethylated AT- and GC-rich DNAs. Four possible triplets as sites for DNA-methylase recognition are discussed.     

Complex organization of a cryptic satellite DNA in the genome of the marine invertebrate Rapana thomasiana Grosse (Gastropoda)

Isopicnic centrifugation in Cs₂SO₄-Ag⁺ gradients at pH 7.0 reveals that the genome of the marine snail Rapana thomasiana Grosse (Gastropoda) contains an AT-rich satellite fraction comprising 5% of the DNA. Restriction enzyme analysis shows that the satellite DNA is composed of a number of related subsets arranged in tandem arrays. They have evolved from the segmental amplification of an 1460 bp long monomer unit with a complex inner organization. Most probably, the present basic repeat originates from an ancestral 400–500 bp long sequence in which some insertions and/or deletions have occurred.     

A method for fractionating rat liver cell nuclei on equilibrium density gradients of CS2SO4.

Sonically disrupted nuclei from proliferating liver cells were fractionated in Cs₂SO₄ equilibrium density gradients. Nuclear constituents were concentrated in three bands designated as light band (LB, 1.21 g/cm³), middle band (MB, 1.26 g/cm³), and heavy band (HB, 1.32 g/cm³). Analysis of protein and nucleic acid distribution in gradients suggests preservation of some macromolecular interactions. Studies comparing distributions of radioactively labeled DNA after 1- or 120-min intervals following tritiated thymidine injection indicate enrichment of nascent DNA in LB and MB. This enrichment is sensitive to time and pressure of sonication. Furthermore, DNA-polymerase activity was demonstrated in the gradient fractions following removal of Cs₂SO₄, with most activity once again in the LB and MB. These results suggest this procedure as an initial step in the isolation of an enzymatically active DNA replication complex.     

Organization,replication and modification of the human genome: Temporal order of synthesis and methylation of two classes of HeLa nDNA separated in Ag+−Cs2SO4 gradients

During theHeLa S-phase, DNA was methylated, at 1-hr intervals in isolated nuclei and fractionated in Ag⁺–Cs₂SO₄ gradients providing a heavy GC-rich peak and a main light AT-rich peak. Both size and specific methylation of these peaks changed during the nDNA duplicative phase. Replication of the heavy GC-rich nDNA fraction, which contains genes for ribosomal RNA, occurred inearly S; in contrast, replication of the main AT-rich nDNA fraction was maximal inlate S. Concomitantly, specific methylation of the GC-rich nDNA was maximal in the first part of S, while that of the AT-rich nDNA was maximal in the second part of S. This suggested that genes are replicated and methylated with order during the S-phase.     

Variation in satellite DNA from some higher plants

Pure satellite DNAs were prepared as minor components after centrifugation to equilibrium on CsCl gradients. A single satellite DNA band was isolated from flax (Linum usitatissimum) DNA and two bands were resolved in cucumber (Cucumis sativus) DNA. These apparently homogeneous components of the plant genomes were further analyzed by thermal denaturation and renaturation. The flax satellite DNA appeared homogeneous on thermal denaturation but was shown to contain several components of renaturation. The two cucumber satellite DNAs were different from each other, but both showed at least two components in denaturation and renaturation analyses. Renaturation in the three satellites, particularly in flax, was inaccurate, indicating a considerable degree of sequence divergence. Although each satellite contained quite large amounts of simple repetitious sequences, a residual heterogeneous DNA fraction was always present. It is considered that this was too large a portion of the satellite DNA to be due to organelle or ribosomal DNA in cucumber. The latter possibility is precluded in flax, where the satellite is completely resolved in buoyant density from both organelle and ribosomal 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.     

Chromosomes of antelope squirrels (genus Ammospermophilus): A systematic banding analysis of four species with unusual constitutive heterochromatin

The G- and C-banding patterns of mitotic chromosomes from four species of antelope squirrels (Ammospermophilus harrisi, interpres, leucurus and nelsoni) are discussed with special attention payed to the unusual quantities and position of constitutive heterochromatin. Heterochromatin appears to be responsible for the observation that cells from antelope squirrels contain over 70% more DNA than cells from another ground squirrel. A substantial fraction of this excess DNA consists of sequences that band as satellites in neutral CsCl or Cs₂SO₄-Ag⁺ density gradients. Interspecies similarities in the distribution of heterochromatin suggest that it has a function of some importance to these species and has therefore been conserved.     

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.