Satellite DNA in differentiating chick tissues

Embryonic chick DNA from different tissues was examined for differences which might indicate specific DNA amplification in somatic cells. The problem was approached by determining the DNA compositional heterogeneity and searching for possible variation in different tissues of the 12-day chick. Neural retina, muscle, and whole decapitated (general) chick DNA were analyzed in CsCl and Cs2SO4 density gradients. While overloaded CsCl gradients showed a main band (rho = 1.701 g/cm3) and a heavy shoulder (rho = 1.716 g/cm3), overloaded Cs2SO4 gradients displayed a main band (rho = 1.426 g/cm3) and a discrete heavy satellite (rho = 1.447 g/cm3). This satellite, comprising approximately 1% of the whole cell DNA, appeared to be of nuclear origin and not related to mitochondrial DNA, which was found to have a density of 1.426 g/cm3 in Cs2SO4. No differences were found in the densities of the main band or the satellite DNA in the DNA samples isolated from the different tissues. However, the method of DNA isolation was found to be of crucial importance when comparing satellite DNA's among different tissues.     

Ribosomal DNA in spores of Physarum polycephalum

DNA was isolated from plasmodia, spores and newly hatched amoebae of the slime mould Physarum polycephalum. The DNA preparations were fractionated in CsCl gradients and each fraction hybridised to combined 19 S + 26 S rRNA. In all three DNA preparations hybridisation was found to be limited to satellite DNA (rho = 1.714 g/cm3) and at saturation was found to reach a level of 0.16--0.18 % of total DNA. The main band of nuclear DNA (rho = 1.702 g/cm3) did not hybridise appreciably. Further experiments using analytical CsCl gradients revealed that the ratio of satellite to main band DNA was similar in all three preparations. It is concluded that the genes for ribosomal RNA are equally reiterated in spores, hatching amoebae and in plasmodia. They appear to be similarly organised in all stages of the life cycle examined so far.     

Satellite Dnas in the Embryos of Various Species of the Genus Drosophila

A tentative evolutionary pattern has been found for two classes of the multiple satellite DNA's found in the genus Drosophila. The satellite DNA's from five Drosophila species (D. melanogaster, D. simulans, D. nasuta, D. virilis and D. hydei) were analyzed and found to fall into three arbitrary CsCl buoyant density classes: Class I, rho = 1.661-1.669 g cm(-3), DNA molecules composed of primarily dA and dT moieties; Class II, rho = 1.685 and rho = 1.692, DNA molecules of low GC content; and Class III, rho = 1.711, a DNA of high GC composition. The dAT satellite DNA's appear in all the species studied except D. hydei, the species of most recent evolutionary divergence, whereas the heavy satellite appears only in the two species of most recent divergence, D. virilis and D. hydei.     

Sequence analysis of bovine satellite I DNA (1.715 gm/cm3).

The 1402 bp Eco RI repeating unit of bovine satellite I DNA (rho CsCl = 1.715 gm/cm3) has been cloned in pBR322. The sequence of this cloned repeat has been determined and is greater than 97% homologous to the sequence reported for another clone of satellite I (48) and for uncloned satellite I DNA (49). The internal sequence structure of the Eco RI repeat contains imperfect direct and inverted repeats of a variety of lengths and frequencies. The most outstanding repeat structures center on the hexanucleotide CTCGAG which, at a stringency of greater than 80% sequence homology, occurs at 26 locations within the RI repeat. Two of these 6 bp units are found within the 31 bp consensus sequence of a repeating structure which spans the entire length of the 1402 bp repeat (49). The 31 bp consensus sequence contains an internal dodecanucleotide repeat, as do the consensus sequences of the repeat units determined for 3 other bovine satellite DNAs (rho CsCl = 1.706, 1.711a, 1.720 gm/cm3). Based on this evidence, we present a model for the evolutionary relationship between satellite I and the other bovine satellites.     

Structure of 1.71 lb gm/cm(3) bovine satellite DNA: evolutionary relationship to satellite I

The Eco RI fragments from the 2600 bp repeating unit of 1.711b gm/Cm(3) bovine satellite DNA were cloned in pBR322. The structure of the repeat unit was determined and compared to bovine satellite I DNA (rho CsCl = 1.715 gm/cm(3)). All of the DNA in the 1402 bp repeat of satellite I is represented in the sequence of the 2600 bp 1.711b gm/cm(3) repeat. The difference between the two repeats is due to a 1200 bp piece of DNA (INS) residing in the middle of the 1.711b gm/cm(3) repeat. The INS is AT-rich and has some repetitive components; it bears only limited similarity to the structure of eukaryotic transposable elements. We propose that the 1.711b gm/cm(3) satellite DNA arose via the amplification of a 1.715 gm/cm(3) satellite repeat altered by a 1200 bp insertion of 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.     

On the question of integration of Agrobacterium tumefaciens deoxyribonucleic acid by tomato plants.

Treatment of tomato plants with Agrobacterium tumefaciens causes subsequently administered [3H]thymidine to be preferentially incorporated into a satellite deoxyribonucleic acid (DNA) whose buoyant density is between that of bacterial DNA (rho = 1.718 g/cm3) and plant main band DNA (rho = 1.692 g/cm3). Satellite DNA upon shearing or sonic treatment releases fragments of higher and lower buoyant density, as reported by earlier investigators. The satellite has no significant base sequence homology with A. tumefaciens DNA, for its rate of reassociation is not accelerated by the addition of high concentrations of the latter. Tomato DNA isolated from shoots or from leaf nuclei accelerates renaturation of labeled satellite DNA. We conclude that the intermediate density labeled DNA is a plant satellite and not the product of covalent joining of bacterial and plant DNA as suggested by earlier investigators.     

DNA nuclear satellites of the genus Brassica: variation between species.

The distribution of nuclear DNAs of nine species of the genus Brassica in CsCl density gradients was investigated. The amount of satellite DNA with buoyant density of 1.704 g - cm-minus3 varies widely between the species. The satellite component is completely absent in B. oleracea; in B. nigra its amount reaches 37%, and in the other species it occupies an intermediate position. The absence of satellite DNA in B. oleracea was demonstrated by equilibrium centrifugation using a Cs2SO4 density gradient, containing Hg2+.     

Characterization of guinea pig cytomegalovirus DNA.

The genome of guinea pig cytomegalovirus (GPCMV) was analyzed and compared with that of human cytomegalovirus (HCMV). GPCMV and HCMV DNAs were isolated from virions and further purified by CsCl centrifugation. Purified GPCMV DNA sedimented as a single peak in a neutral sucrose gradient and was infectious when transfected into guinea pig embryo fibroblast cells. The cytopathology was characteristic of that seen after infection with GPCMV. Virus DNA purified from virions isolated from infected GPEF or 104C1 cells had a CsCl buoyant density of 1.713 g/cm3, which corresponds to a guanine plus cytosine content of 54.1%. The CsCl buoyant density of GPCMV DNA was slightly less than that of HCMV DNA (1.716 g/cm3), but sufficiently different so that the two virus DNA peaks did not coincide. GPCMV DNA cosedimented with T4 DNA in a neutral sucrose gradient. Restriction endonuclease cleavage of GPCMV or HCMV DNAs with HindIII, XbaI, or EcoRI yielded fragments easily separable by agarose gel electrophoresis and ranging from 1.0 X 10(6) to 25.8 X 10(6) daltons. The number, size, and molarity of GPCMV DNA fragments generated by restriction enzymes were determined. Hybridization of restriction endonuclease-cleaved GPCMV DNA with radioactively labeled HCMV DNA and, conversely, hybridization of restriction endonuclease-cleaved HCMV DNA with radioactively labeled GPCMV DNA indicated sequence homology between the two virus DNAs.     

Analysis of eucaryotic DNAs with a restriction endonuclease from H.influenzae: isolation of “hidden” satellite DNAs

The restriction enzymes from H.influenzae have been used to study various eucaryotic DNAs. Fractions resistant to the action of these enzymes have been isolated from rat, mouse and calf DNAs. The method revealed the existence in rat of several components having the properties of satellite DNAs. Mouse DNA was shown to contain a new satellite of buoyant density 1. 704. Therefore, this appears to be a powerfull method for the isolation of "hidden" satellite DNAs.Calf satellite DNAs rho = 1.705 and rho = 1.723 were those resistant to the action of the restriction enzymes whilst satellite DNAs rho = 1.714 and rho = 1.710 gave fragments of discrete lengths, suggesting internal repeat units of 1 500 and 5 000 base pairs respectively.     

Analysis of the genome of plants. II. Characterization of repetitive DNA in barley (Hordeum vulgare) and wheat (Triticum aestivum).

Barley and wheat DNAs have been characterized by studying their kinetics of reassociation, melting properties and sedimentation behaviour in neutral CsCl gradients as well as in Cs2SO4 gradients containing Ag+ or Hg2+. In both species, reassociation kinetics have revealed the presence of approx. 76% redundant nucleotide sequences which have been grouped into very rapidly reassociating (Cot 0-0.01), rapidly reassociating (Cot 0.01-1.0) and slowly reassociating (Cot 1-100) fractions. The barley Cot 0-0.01 and Cot 0.01-1.0 fractions as well as the wheat Cot 0.01-1.0 fraction form narrow bands upon centrifugation in CsCl gradients. Under similar experimental conditions both Cot 0.01 and Cot 1.0-100 wheat fractions and the barley Cot 1.0-100 fraction form broad bands each having several shoulders. Thermal denaturation studies of most of the above reassociated fractions have shown a considerable degree of order in their duplexes with an average hyperchromicity of 21.5%. When native, high molecular weight barley DNA is centrifuged in Ag+/CS2SO4 density gradients (RF = 0.2), two satellites appear on the heavier side of the main band, as against one in the case of wheat. The two minor peaks, designated as satellites I and II, have buoyant densities of 1.702 and 1.698 g/cm3, respectively, in neutral CsCl gradients and together represent about 8-9% of total barley DNA. Upon centrifugation in Hg2+/CS2SO4 density gradients, one satellite is observed in both barley and wheat and it accounts for 1-2% of their genomes.     

Compositional patterns in the nuclear genome of cold-blooded vertebrates

Summary DNA preparations obtained from 122 species of fishes, 5 species of amphibians, and 13 species of reptiles were investigated in their compositional properties by analytical equilibrium centrifugation in CsCl density gradients. These species represented 21 orders of Osteichthyes, 3 orders of Chondrichthyes, 2 orders of amphibians, and 3 orders of reptiles. Modal buoyant densities of fish DNAs ranged from 1.696 to 1.707 g/cm³, the vast majority of values falling, however, between 1.699 and 1.704 g/cm³, which is the range covered by the DNAs of amphibians and reptiles. In all cases, DNA bands in CsCl were only weakly asymmetrical and only very rarely were accompanied by separate satellite bands (mostly on the GC-rich side). Intermolecular compositional heterogeneities were low in the vast majority of cases, and, like CsCl band asymmetries, at least partially due to cryptic or poorly resolved satellites. The present findings indicate, therefore, that DNAs from cold-blooded vertebrates are characterized by a number of common properties, namely a very wide spectrum of modal buoyant densities, low intermolecular compositional heterogeneities, low CsCl band asymmetries, and, in most cases, small amounts of satellite DNAs. In the case of fish DNAs a negative correlation was found between the GC level and the haploid size (c value) of the genome. If polyploidization is neglected, this phenomenon appears to be mainly due to the fact that increases and decreases in GC are associated with contraction and expansion phenomena, respectively, of intergenic noncoding sequences, which are GC poor relative to coding sequences.     

Localization of repeated DNA sequences in CsCl gradients by hybridization with complementary RNA

Single-stranded ends of mouse satellite DNA sequences exposed during the extraction of nuclear DNA, or by mechanical shear, can hybridize with highly labelled RNA, complementary to satellite DNA. In neutral CsCl gradients, the hybrid sediments as a sharp peak at the density of mouse satellite DNA (1.691 g/cm³). The possibility of exploiting this observation in locating and isolating cryptic repetitive DNAs from nuclear DNA or the DNA of heterochromatin is discussed.     

High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families

We have examined high affinity interactions of chick brain microtubule proteins with ³⁵S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1–4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65–75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. ³⁵S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM^–1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three ³⁵S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720–1.740 g/cm³; 1.750–1.765 g/cm³). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.     

Mitochondrial DNA of Yoshida ascites tumour cells. Physicochemical properties and biological function.

Mitochondrial DNA from Yoshida A.H. 130 cells, has been characterized by determination of the buoyant density by CsCl equilibrium density gradient centrifugation and the thermal denaturation and renaturation behaviour. These studies have been carried out parallelly on nuclear DNA from the same cells in order to search for possible differences between both DNAs. Mitochondrial DNA of Yoshida cells presents an equilbrium in CsCl of 1.7154 g/cm3 and a sharp melting with a Tm of 92 degrees C. Nuclear DNA presents an equilibrium of 1.7030 g/cm3 and a Tm of 88 degrees C. The guanine plus cytosine content in both DNAs has been calculated from tumour results and compared with the content in normal rat liver cells M-DNA of tumour cells presents a higher guanine plus cytosine content than N-DNA, whereas in normal liver cells is higher in N-DNA. N-DNAs of both normal and tumour cells have the same guanine plus cytosine content, whereas M-DNA from tumour cells presents a significant increase (about 35%) with regard to this from normal liver cells.     

Buoyant densities of DNA of mammals

One characteristic of DNA, CsCl buoyant density peak values, was determined for DNA samples isolated from 93 species belonging to 11 orders of mammals. The CsCl buoyant density values varied over a very narrow range, 1.696–1.701 g/cm³. Satellite DNAs were found in a number of species. The function and origin of these satellite DNAs are not known.This work was supported by grant DRG-269 from the Damon Runyon Memorial Fund for Cancer Research, Inc., and GB-6657 from the National Science Foundation.     

Molecular structure of adeno-associated virus variant DNA

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.     

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.     

Properties of satellite DNA of Phaseolus vulgaris]

Nuclear DNA components of Ph. vulgaris were preparatively separated by equilibrium ultracentrifugation in Hg++-Cs2SO4 density gradient (buoyant density of the major component in CsCl density gradient 1.694 g/cm3., satellite component--1.703 g/cm3). The properties of individual DNA fractions were investigated. The melting curve of satellite DNA of Ph. vulgaris has biphasic character. The observed heterogeneity of satellite DNA component is of intermolecular nature. This is illustrated by the splitting of unsheared satellite DNA into two components during renaturation, as well as by its behaviour in Hg++-Cs2SO4 density gradient at high rf value. The width of satellite DNA reassociation curve covers three decades of Cot. The length of the major repeating sequences of the satellite component is close to the length of phage T2 DNA. During chromatography on MAK column satellite DNA elutes earlier than the major component due to its higher GC-content. It is suggested that one of the satellite DNA fractions of Ph. vulgaris contains rRNA genes.     

The fractionation of calf thymus DNA by multiple formation of sedimentable complexes with homologous lysine-rich histone KAP. Derivative melting curves and ultracentrifugation in CsCl concentration gradients

The process of fractionation of total calf thymus DNA using a step precipitation of DNA by means of increasing concentrations of the homologous histone KAP was investigated. In addition to the known fractions three so far undescribed ones/in thymus/,characterized by buoyant densities in CsCl equal 1.692, 1.706 and 1.728 g/ccm, were identified. Considerable amounts of preparations seriously enriched in individual satellite fractions were obtained. The ability of GC-rich satellite DNAs to form more soluble complexes with histone KAP is suggested as reason for the observed fractionation.