Denaturation of mouse satellite and ribosomal DNA during hydroxyapatite thermal chromatography of chromatin

Mouse DNA and chromatin were melted on hydroxyapatite and the denaturation profiles of ribosomal and satellite DNAs were followed by hybridization with their complementary RNAs. Neither ribosomal nor bulk DNA had significantly different melting profiles in chromatin as compared to DNA. However, most of satellite DNA eluted at higher temperature from chromatin than from purified DNA. One explanation for the higher melting temperature of mouse satellite DNA in chromatin suggests that the complex between this particular DNA component and at least some proteins in chromatin is more stable than the average DNA-protein interaction.     

Localisation of foldback DNA sequences in nuclei chromosomes of Scilla, Secale, and of mouse.

Foldback DNA, prepared from mouse and Scilla sibirica main band DNA, and from rye (Secale cereale) total DNA, was characterised by denaturation, renaturation, and electron microscopy. 3H-cRNA of this DNA was hybridised in situ to nuclei and chromosomes of the respective species. There is no universal labelling pattern among the three species. In mouse, highly repetitive foldback DNA is present in the whole chromatin including the satellite DNA-containing regions. In Scilla sibirica, on the contrary, the highly repetitive foldback sequences are excluded form the satellite DNA loci and are arranged in clusters in the remaining chromatin. In rye, there is a clear preferential labelling of the chromocenters in the interphase nuclei as well as metaphase chromosomes, indicating that highly repetitive foldback DNA is preferentially located among other highly repetitive sequences.     

An analysis of repeated sequence heterogeneity

High-resolution thermal denaturation was used to measure the heterogeneity within repeated DNA sequences. An analysis of combined denaturation/redenaturation experiments on mouse satellite DNA suggests the existence of two minor components, one of which does not appear in the prepared EcoRII monomer. The resolving power of the denaturation/redenaturation experiment is estimated and contrasted with that of the reassociation experiment, often used to estimate repeated sequence heterogeneity. A mathematical model of the redenaturation experiment was developed and applied to mouse satellite data; the results suggest that only one-fourth of the mismatched base pairs are energetically significant in the reduction of heteroduplex stability.     

Effect of non-histone proteins on thermal transition of chromatin and of DNA.

The effect of chromatin non-histone protein on DNA and chromatin stability is investigated by differential thermal denaturation method. 1) Chromatin (rat liver) yields a multiphasic melting profile. The major part of the melting curve of this chromatin is situated at temperatures higher than pure DNA, with a distinct contribution due to nucleosomes melting. A minor part melts at temperatures lower than DNA which may be assigned to chromatin non-histone protein-DNA complex which destabilized DNA structure. 2) Heparin which extracts histones lowers the melting profile of chromatin and one observes also a contribution with a Tm lower that of pure DNA. In contrast, extraction on non-histone proteins by urea supresses the low Tm peak. 3) Reconstitution of chromatin non-histone protein-DNA complexes confirms the existence of a fraction of chromatin non-histone protein which lowers the melting temperature when compared to pure DNA. It is concluded that chromatin non-histone proteins contain different fractions of proteins which are causing stabilizing and destabilizing effect on DNA structure.     

Interspersion of mouse satellite deoxyribonucleic acid sequences

DNA sequences with homology to the major (A + T)-rich mouse satellite component were localized in CsCl gradients by hybridization with a labeled satellite cRNA probe. Although, as expected, most of the hybridization was to DNA in the satellite-rich shoulder, substantial radioactive cRNA hybridized with DNA from denser regions of the gradient. Further examination revealed that hybridization to main-band DNA was not due to physical trapping of satellite DNA in the gradient, and melting experiments argue that the associated radioactivity was due to true RNA/DNA hybridization. Nearest-neighbor analysis of hybridized [alpha-32P]CTP-labeled l-strand cRNA indicates that hybridization to main-band DNA is by the satellite cRNA and not a contaminant. Together, these data argue that mouse satellite-like sequences are interspersed within the main-band fraction of DNA. For the support of this contention, total mouse DNA, purified main-band DNA, and purified satellite DNA were digested with EcoRI, sedimented in a sucrose gradient, and hybridized with labeled satellite cRNA. Mouse satellite DNA is not cleaved with EcoRI, so that purified EcoRI-digested satellite DNA sediments as a high molecular weight component. When total mouse DNA is digested with EcoRI, the majority of satellite-like sequences remain as high molecular weight DNA; however, significant amounts of satellite-like sequences sediment with the bulk of the lower molecular weight digested DNA, lending further credence to the argument that satellite-like sequences are interspersed with main-band DNA.     

Determination of Melting Sequences in DNA and DNA-Protein Complexes by Difference Spectra

A graphical formula is presented for determining the base ratio of melted DNA. By use of this formula, the composition of sequences which melt in different portions of the melting curves of Clostridium DNA, Escherichia coli DNA, and mouse DNA were determined. As the DNA melts, the per cent of adenine and thymine (AT) in the melted sequences decreases linearly with temperature. The average composition of sequences which melt in a given part of the melting curve is proportional to the base ratio of the DNA. The concentration and average composition of sequences were determined for three parts of the melting curves of the DNA samples, and a frequency distribution curve was constructed. The curve is symmetrical and has a maximum at about 56% AT. The distribution of GC-rich sequences on the E. coli chromosome was estimated by shearing, partially melting, and fractionating the DNA on hydroxylapatite. GC-rich sequences appear to occur every thousand base pairs, and have a maximum length of about 180 base pairs. The graphical formula was applied to the determination of the composition of sequences which melt in different parts of the melting curve of chromatin. Throughout the melting curve, the composition of the melting sequences is about 60% AT, which appears to suggest that relatively long sequences are melting simultaneously. Their melting temperature may be a function of the composition of the protein on different parts of the DNA. The problem of light scattering in DNA-protein and DNA was also investigated. A formula is presented which corrects for light scattering by relating the intensity of the scattered light to the rate of change of absorbance of DNA with wavelength.     

A high amount of satellite DNA in the genome of Lupinus angustifolius L.

Embryo DNA, isolated from ungerminated seeds of Lupinus angustifolius L., contains an exceptionally high amount of guanine-cytosine-rich satellite DNA. The thermal denaturation curve of total embryo DNA is biphasic with an inflexion point at 62% denaturation, indicating the presence of satellite DNA. The satellite fraction could be separated from the mainband DNA by three successive preparative CsCl-gradient centrifugations. The densities of the DNA fractions are 1.7045 g cm^-3 and 1.6925 g cm^-3, respectively. The percentages of guanine-cytosine calculated from these densities are comparable to the percentages of GC calculated from the melting temperatures. Finally, ressociation studies prove that foldback DNA and highly repeated sequences are much more frequent in the satellite DNA fraction than in the mainband DNA.Abbreviation C _o t the product of the DNA concentration (mol nucleotides l^-1) and the time (s) of incubation in a DNA reassociation reaction - GC guanine-cytosine - np nucleotide parirs - T temperature interval between 16 and 84% denaturation     

Attempted separation of transcriptively active and inactive chromatin by hydroxylapatite thermal chromatography

Chromatin and purified DNA were fractionated by hydroxylapatite thermal chromatography. Fractions of varying thermal stability were tested for the proportions of transcribed sequences and repetitive sequences relative to the unfractionated genome. The first 80--85% of either total chromatin or purified DNA eluted from hydroxylapatite contained the same proportion of hybridizable sequences as total DNA. The remaining 15--20% of chromatin eluting at the highest temperatures was depleted of transcribed sequences. Analysis of the 20% highest melting fraction of purified DNA showed that, while the first two-thirds of this fraction contained the same proportion of transcribed sequences as unfractionated DNA, the last third, comprising about 6% of total DNA, was depleted of active sequences. Although no major differences were detected in nonrepetitive sequence complexity of chromatin fractions, there was a correlation between relative thermal stability and repetitive sequence content in fractions of both chromatin and DNA separated by thermal chromatography. Fragments eluting at higher temperatures contained a greater proportion of repetitive sequences, as indicated by a rapidly renaturing component. Most likely, the latest eluting fractions from both chromatin and purified DNA were enriched for a nontranscribed, highly reiterated, G+C rich satellite component of the chicken genome.     

Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

Characterization of the most rapidly renaturing sequences in mouse main-band DNA

The most rapidly renaturing sequences in the main-band DNA of Mus musculus, isolated on hydroxyapatite, are found to consist of two discrete families: a presumed “foldback” DNA fraction and a fraction renaturing bimolecularly. The latter family, which we call “main-band hydroxyapatite-isolated rapidly renaturing DNA”, has a kinetic complexity about an order of magnitude greater than that of mouse satellite DNA. It shows about twice as much mismatching as renatured mouse satellite, as judged by its thermal denaturation curve. In situ hybridization localizes the sequences to all chromosomes in the mouse karyotype, and to at least several regions of each chromosome. The in situ result and solution hybridization studies eliminate the possibility that the main-band rapidly renaturing DNA is composed of mouse satellite sequences attached to sequences of higher buoyant density. Nuelease S₁ digestion experiments disclose that even at low molecular weight there are unrenatured “tails” attached to the rapidly renaturing sequences. When the main-band DNA fragment size is increased the amount of rapidly renaturing sequences remains constant, but the amount of attached tails of unrenatured DNA increases as judged by S₁ nuclease digestibility, hyperchromicity and buoyant density. It is concluded that at least 5% of the mouse genome is composed of segments of the rapidly renaturing sequences averaging about 1500 base pairs, alternating with segments of more complex DNA averaging about 2200 base pairs. This interspersion of sequences is compared to that found in several other organisms. The properties of the foldback DNA are similarly investigated as a function of DNA fragment size.     

Nucleosomes are positioned on mouse satellite DNA in multiple highly specific frames that are correlated with a diverged subrepeat of nine base-pairs

Nucleosome phasing on highly repetitive DNA was investigated using a novel strategy. Nucleosome cores were prepared from mouse liver nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population that contains satellite sequences was then purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value and hydroxyapatite chromatography to separate the renatured satellite fraction. After end-labeling, the termini of the satellite core DNA fragments were mapped with an accuracy of +/- 1 base-pair relative to known restriction sites on the satellite DNA. Sixteen dominant nucleosome positions were detected. There is a striking correlation between these nucleosome frames and an internal highly diverged 9 base-pair subrepeat of the satellite DNA. The results are consistent with a sequence-dependent association of histone octamers with the satellite DNA. Our finding that histone octamers can interact with a given DNA in a number of different defined frames has important implications for the possible biological significance of nucleosome phasing.     

Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH

Human blood leukocytes within an agarose matrix were deproteinized and exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.     

Effects of shearing on chromatin structure

The effects of mechanical shearing on chromatin structure were investigated by using thermal denaturation and circular dichroism (CD) spectroscopy. Under ordinary conditions of mechanical shearing used for preparation of soluble chromatin, we observed only minor changes (less than 10%) of chromatin properties with respect to (a) absorption melting curves, (b) CD spectra, (c) CD melting curves and (d) histone transfer from chromatin to exogenous DNA. Such small pertubation of structural properties could be due to the generation of free ends when a large chromatin was cut into smaller fragments and by weakening the binding of histones to DNA near these free ends. In addition to mechanical shearing, sonication was used to shear some samples of chromatin. The effect of sonication on chromatin structure was investigated by the same physical methods used for mechanically sheared chromatin. The results indicate that sonication only slightly changes the chromatin properties with respect to CD spectra, similar to the results obtained by mechanical shearing, but sonication at high settings has a greater effect on the thermal denaturation property of chromatin as contrasted to our results from mechanically sheared chromatin.     

Variations in the satellite DNA content of Cucumis melo in relation to dedifferentiation and hormone concentration.

Total DNA from Cucumis melo contains a 1.706 satellite DNA which can be resolved into two components; one of these components has a higher temperature of melting (component I) then the other component II). In this study, we have further investigated these components by thermal denaturation and by Eco R1 digestion. Component I reveals a homogeneous melting profile and is only partially cleaved by Eco R1, whereas component II reveals a heterogeneous melting profile and is entirely digested by Eco R1. A possible mitochondrial origin for component II is discussed. When an in vitro culture of root tissues from Cucumis melo was initiated or when the phytohormone (NAA) concentration of established cultures was modified, a new satellite DNA (1.719) appears transitorily and the satellite DNAs already present in seedlings undergo quantitative and possibly qualitative variations. Satellite DNAs therefore seem to be involved in the response to some physiological variations.