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.     

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.     

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.     

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.     

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.     

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.     

Organization of a repetitive human 1.8 kb KpnI sequence localized in the heterochromatin of chromosome 15

We have isolated a repetitive 1.8 kb Kpnl DNA sequence which is amplified in the homogeneously staining regions of a human melanoma cell line. Under low stringency conditions this sequence (D15Z1) hybridized in situ to the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and distal Yq as well as to the the short arms of the other acrocentric chromosomes. Under conditions of high stringency, labelling was predominantly on the short arm of chromosome 15. D15Z1 was shown to be present at approximately 3,000 copies per haploid genome and organized in long tandem arrays showing restriction site heterogeneity. Sequences homologous to D15Z1 were highly enriched in the less dense shoulder region of a Ag⁺—Cs₂SO₄ gradient. Analysis of D15Z1 indicated that this sequence is composed of tandemly arranged imperfect repeats of the consensus 5 AATGG 3 similar to previously identified satellite III sequences. Digestion of D15Z1 with HinfI resulted in a series of restriction fragments making up a subset of the HinfI ladder components of satellites III and IV. These data suggest that D15Z1 represents a chromosome 15 specific domain of human satellites III or IV and that it makes up the major fraction of the heterochromatin of this chromosome. Possible relationships between this sequence and the cytochemical staining properties of human chromosomes with distamycin A/DAPI, D280/170, and antiserum to 5-methylcytosine are discussed.     

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.     

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.     

Absence of satellite DNA synthesis during meiotic prophase in mouse and human spermatocytes

Mouse spermatocytes were labelled in situ with ³H-thymidine at successive stages of meiosis. Isolated mouse as well as human spermatocytes were similarly labelled under in vitro conditions. DNA synthesis was followed either by tracking radioactivities in Cs₂SO₄ gradients or by measuring reassociation kinetics. Mouse satellite DNA and the 3 satellites of human DNA are labelled during S-phase but not during pachytene. In the mouse genome, there is a preferential labelling of regions containing foldbacks (human spermatocytes were not analyzed in this respect). The absence of detectable pachytene synthesis in satellite DNA is consistent with genetic evidence on the absence of crossing-over in constitutive heterochromatin.     

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.     

The replication of mouse skin epidermal DNA to which is bound 7,12-dimethylbenz(a)anthracene

Experiments were carried out to determine in the intact mouse whether or not mouse skin epidermal DNA to which the polycyclic hydrocarbon DMBA was bound could serve as a template for further DNA replication. Mice which were treated topically with [³H]7,12-dimethylbenz(a)anthracene ([³H]DMBA) received 5-bromodeoxyuridine (BUdR) and 5-fluorouracil (5-FU) in order to incorporate BUdR into replicating DNA which was stimulated to undergo synthesis one or two days later. Epidermal DNA was put on a neutral CsCl gradient and binding of [³H]DMBA was found to both replicated and non-replicated DNA. Separation of the BUdR substituted and non-substituted parental strands of newly replicated DNA an on alkaline CsCl, Cs₂SO₄ gradient showed that the great majority of DMBA was bound to parental strand DNA. The possibility that [³H]DMBA binding was taking place at the same time that labeling with BUdR occurred was eliminated. Thus, these experiments showed that in the intact mouse, skin epidermal DNA to which DMBA is bound can serve as a template for further DNA synthesis.     

Related satellite DNA's in the genus Mus

Several Thailand species from the genus Mus have been shown to contain satellite DNA's able to cross-reassociate with the Mus musculus satellite. One species, Mus caroli, contains at least three discrete but related light satellite DNA's. All the related Mus satellites band on the light side of the major band in neutral CsCl gradients, separate into complementary strands in alkaline CsCl gradients, and have a relatively low affinity for Ag⁺. Three of the Mus satellite DNA's have been purified: taken separately, they show very sharp thermal transitions and reassociate at similar rates to give well-matched duplexes.     

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.     

The chromosomes and DNA of Allium cepa

Giemsa C-banded idiograms that allow the identification of all chromosomes have been prepared for Allium cepa, Ornithogalum virens, and Secale cereale. An analysis of A. cepa DNA has determined that: (1) It has the lowest GC content so far reported for an angiosperm (32%). (2) It appears to have no satellite DNA detectable by CsCl or Cs₂SO₄-Ag⁺ density gradient centrifugation. (3) Aside from fold back DNA and unreactable fragments, a C₀t curve indicates that most of the DNA can be adequately described as two major middle repetitive components (Fractions I and II) and a single copy component (Fraction III). And (4) most of the repeated DNA sequences are involved in a short period interspersion pattern with single copy and other repetitive sequences. In situ hybridization of tritiated cRNAs to fold back, long repeated, and Fraction I DNA from A. cepa to squash preparations of chromosomes and nuclei from A. cepa, O. virens, and S. cereale root tips indicates: (1) Sequences complementary to fold back DNA are scattered throughout the genome of A. cepa except for telomeric heterochromatin and nucleolus organizers while they are not detectable in the genomes of O. virens or S. cereale. (2) Although long repeated sequences are scattered throughout the genome of A. cepa, they are concentrated to some extent in telomeric heterochromatin and nucleolus organizers (NOs). Sequences complementary to long repeats of A. cepa occur primarily in chromosome three of O. virens while these sequences are more common in the genome of more distantly related S. cereale. (3) Fraction I DNA is scattered throughout the genome of A. cepa while it is hardly detectable in the genomes of O. virens and S. cereale. These results are discussed in regard to the evolutionary conservation and function of repeated DNA sequences.     

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.     

Density gradient proportionality constants,density distributions,and pressure effects for three salt solutions

Earlier determinations of density gradient proportionality constants β⁰, density distributions ρ(r), and the effect of pressure on density gradients in the analytical ultracentrifuge have been of limited precision and usefulness in the study of proteins and polypeptides. Reasons for these difficulties are that numerous intermediate relationships were required in the calculations, and the density ranges studied were generally above 1.2 g/ml. Relations are derived in the present paper to directly compute β⁰(ρ) values and β⁰′(ρ) values from the original data without any intermediate expansions or approximations. Data are presented for CsCl, CsBr, and Cs₂SO₄ and compared with literature values. Density distributions are computed for all three salts under a wide variety of experimental conditions of density, column length, and angular velocity. These values of ρ(r) and r_e are obtained by a numerical iterative technique. Values obtained by this new method are compared with values obtained using closed-form expressions. The effects of pressure on the composition density gradient for the three salts given above are calculated and found to be significant for Cs₂SO₄ solutions.     

Observations on centromeric heterochromatin and satellite DNA in salamanders of the genus Plethodon

DNA from Plethodon cinereus cinereus separates into two fractions on centrifugation to equilibrium in neutral CsCl. The smaller of these fractions has been described as a high-density satellite. It represents about 2% of nuclear DNA from this species, and it has a density of 1.728 g/cm³. It is cytologically localized near the centromeres of all 14 chromosomes of the haploid set. In P. c. cinereus the heavy satellite DNA constitutes about 1/4 of the DNA in centromeric heterochromatin. The nature of the rest of the DNA in centromeric heterochromatin is unknown. The number of heavy satellite sequences clustered around the centromeres in a chromosome from P. c. cinereus is roughly proportional to the size of the chromosome, as determined by in situ hybridization with satellite-complementary RNA, and autoradiography. Likewise the amount of contromeric heterochromatin, as identified by its differential stainability with Giemsa, shows a clear relationship to chromosome size. — The heavy satellite sequences identified in DNA from P. c. cinereus are also present in smaller amounts in other closely related forms of Plethodon. Plethodon cinereus polycentratus and P. richmondi have approximately half as many of these sequences per haploid genome as P. c. cinereus. P. hoffmani and P. nettingi shenandoah have about 1/3 as many of these sequences as P. c. cinereus. P. c. cinereus, P. c. polycentratus, and P. richmondii all have detectable heavy satellites with densities of 1.728 g/cm³. Among these forms, satellite size as determined by optical density measurements, and number of satellite sequences as determined from hybridization studies, vary co-ordinately. P. c. cinereus heavy satellite sequences are not detectable in P. nettingi, P. n. hubrichti, or P. dorsalis. The latter species has a heavy satellite with a density of 1.718 g/cm³, representing about 8% of the genomic DNA, and two light satellites whose properties have not been investigated. The heavy satellite of P. dorsalis is cytologically localized in the centromeric heterochromatin of this species. — These observations are discussed in relation to the function and evolution of highly repetitive DNA sequences in the centromeric heterochromatin of salamanders and other organisms.