Preparation, properties and fractionation of the nuclear deoxyribonucleic acid from guinea pig brain

L ittle work has been done on the chemical and physical properties of brain nuclear DNA. Recently D u B uy , M attern and R iley (1966) described the heat denaturation of mouse brain nuclear DNA, which apparently differs from the related mitochondrial DNA and the various protozoal DNA's tested. For such studies, it is essential to prepare the nuclear DNA from brain tissues in a native and highly polymerized state. In this brief communication preparation procedures of the native DNA from isolated brain nuclei are described, and some physical and chemical properties are given.     

Preparative isolation and study of the reassociation kinetics of a T2 phage DNA fraction containing readily melting regions]

A DNA fraction comprising 6% of total DNA and containing readily-melting regions is isolated from phage T2 DNA using preparative chromatography on MAK columns at T congruent to T m--3 degrees C. Two denaturation regions, differing in the stability for 7 degrees, were observed on this DNA melting curve. A sharp increase of the reassociation rate at initial moments under reassociation temperatures T r approximately less than m --25 degrees C was observed. Thermodynamic characteristics obtained under the repeated melting of DNA fragments after reassociation confirm the fact, that under these reassociation temperatures the incorporation of readily-melting regions into spiral duplexes takes place.     

DNA changes during sequential leaf senescence of tobacco (Nicotiana tabacum)

Age related DNA changes in tobacco (Nicotiana tabacum) leaf nuclei were investigated by Feulgen cytophotometry, thermal denaturation, renaturation, and DNA-DNA hybridization studies during sequential leaf senescence. Cytophotometric Feulgen-DNA comparison measurements between young and senescing nuclei displayed 18% reduction in Feulgen-DNA values, with a corresponding decrease in nuclear area in senescing nuclei. Hydrolysis kinetics indicated that the loss was not due to compactness of the DNA as the curves for older nuclei were consistently lower than curves generated from younger nuclei. DNA loss in senescing nuclei was associated with a decrease in euchromatin or shift from euchromatin to facultative heterochromatin. Purified DNA from young and senescing leaf nuclei did not display different thermal profiles nor did hydroxylapatite chromatography reassociation curves. DNA-DNA hybridization in free solution from young and senescing leaf DNA performed by a Gilford thermo-programmer system indicated that DNA of senescing tobacco nuclei reassociated more slowly than DNA from young nuclei and the mixture of young and senescing leaf DNA displayed intermediate reassociation values. The study indicates that the DNA changes during senescence involve a complex phenomenon which includes the possibility of small single strand nicks undetectable by thermal denaturation, and a loss of small double strand fragments which were detectable only by precise DNA-DNA free solution reassociation and not by hydroxylapatite chromatography reassociation.     

Molecular characterization of an insect genome: Chironomus thummi.

DNA extracted from Chironomus thummi larvae was studied by isopycnic centrifugation in CsCl, thermal denaturation and DNA-DNA reassociation techniques. The mean G+C content of the C. thummi DNA is 28-29% as indicated both by centrifugation in CsCl and thermal denaturation. According to optical reassociation analysis of total DNA and of isolated DNA fractions the C. thummi genome is composed of at least four components. About 80% of the DNA is classified as unique with a kinetic complexity of nearly 7 X 10(10) daltons. 6-8% intermediate DNA exhibits a kinetic complexity slightly above 10(8) daltons with a mean repetition frequency of 35. 11-13% fast-reassociating DNA has a kinetic complexity slightly above 10(6) daltons with a mean repetition frequency of 6000. 3-5% of the DNA cannot be properly studied by the optical reassociation technique and probably contains inverted repeats. The thermal denaturation behaviour of isolated DNA fractions indicated that most of the repetitive sequences in the C. thummi genome are tightly interspersed.     

Genetic relatedness of the genomes of equine herpesvirus types 1, 2, and 3.

Genomic DNAs of equine herpesvirus type 1 (EHV-1), EHV-2 (equine cytomegalovirus), and EHV-3 were examined by reassociation kinetic and thermal denaturation analyses to determine the extent and degree of homology among the three viral DNAs. Results of reassociation analyses indicated a limited homology among the three EHV genomes. Homologous DNA sequences equivalent to 1.8 to 3.7 megadaltons between EHV-1 and equine cytomegalovirus, 7.6 to 8.2 megadaltons between EHV-1 and EHV-3, and 1.3 to 1.9 megadaltons between equine cytomegalovirus and EHV-3 were detected. Examination by thermal denaturation of the DNA homoduplexes and heteroduplexes formed during reassociation revealed a high degree of base pairing within the duplexes, suggesting that closely related sequences may be conserved among the genomes of EHV.     

Chromosomal DNA denaturation and reassociation in situ.

The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10^?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization.     

Acridine-orange differential fluorescence of fast- and slow-reassociating chromosomal DNA after in situ DNA denaturation and reassociation

A cytological technique based on heat denaturation of in situ chromosomal DNA followed by differential reassociation and staining with acridine orange was developed. Mouse nuclei and chromosomes in fixed cytological preparations show a red-orange fluorescence after thermal DNA denaturation (2–4 minutes at 100° C), and fluoresce green if denaturation is followed by a total DNA reassociation (two minutes or more at 65–66°C). — A reassociation time between a few and 60–90 seconds demonstrates the centromeric heterochromatin of chromosomes (which sometimes aggregate in the form of clusters) and the interphase chromocenters in green, the chromosomal arms fluorescing red-orange. Under the same conditions, the Y chromosome presents a pale green or yellow-green fluorescence along its chromatids, but its centromeric region fluoresces weakly. — The interpretation is suggested that the fast-reassociating chromosomal DNA (as detected by AO in centromeric heterochromatin and interphase chromocenters), represents repetitive DNA.     

Selective staining of X chromosome segments in Schistocerca gregaria after denaturation and reassociation procedures

The DNA of fixed mitotic and meiotic chromosomes and of spermatides of Schistocerca gregaria males was heat denaturated and then differentially reassociated in a Giemsa buffer or in acridine orange buffer solution. After this procedure, two to three large, selectively stained regions are seen in the X chromosome of spermatocytes and spermatides. Denaturation and reassociation experiments have shown that after differential reassociation such a selective stainability of chromosome regions is characteristic for the presence of fast-reassociating, i.e., repetitive DNA (Stockert and Lisanti, 1972). The possible presence of repetitive DNA in the X chromosome regions concerned can not be the only reason for the occurrence of the heavily stained segments after reassociation because (1) these segments are obtained in positively heteropycnotic X chromosomes, but not in negatively heteropycnotic Xs and (2) they do not occur in positively heteropycnotic X chromosomes when the histones have been extracted before the denaturation and reassociation processes. Contrary to the latter statement, the heavily stained X chromosomal regions are preserved when the histones are removed after the denaturation and reassociation steps. — It is assumed that the heavily stained X chromosome segments represent DNA reassociation complexes which are only formed if histones are present. It is discussed whether the formation of the X chromosome complexes depends on a specific chromatin configuration within positively heteropycnotic X chromosomes.     

A method of equalizing the level of DNA sequences of varying amount in a heterogeneous DNA mixture]

A method for the equalization of double-stranded DNA concentrations in the mixture which may be used for equalizing double-stranded cDNA concentrations involves thermal denaturation of the double-stranded DNA mixture followed by reassociation. The initial reassociation rate is Vi = Ki.(single-stranded DNA)2, and by the end of the process the concentrations of the unreassociated molecules for different DNAs should be approximately equal. Using hydroxylapatite chromatography one can separate single-stranded DNAs from double-stranded DNAs and carry out complete single-stranded DNAs reassociation. The new ratio of different double-stranded DNA concentrations would be almost 1.     

Quantitation of bovine papilloma viral DNA in viral-induced tumors.

Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching.     

Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization.

The fine structure of the melting curve for the linear colE1 DNA has been obtained. To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/. Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range. Thereby the whole process of colE1 DNA melting was reconstructed in detail. Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule. The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease. The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde.     

Reassociation Kinetics and Cytophotometric Characterization of Peanut (Arachis hypogaea L.) DNA

The base composition of peanut (var. NC-17) DNA determined from thermal denaturation profiles showed an average guanine plus cystosine content of 34% which was in close approximation to 36% guanine plus cytosine calculated from the buoyant density. Buoyant density also indicated the absence of satellite DNA. The genome size, 2.0 × 10⁹ base pairs, as determined by reassociation kinetics of the single copy DNA was close to the genome size determined by cytophotometry, 2.1 × 10⁹ base pairs. Peanut DNA averaging 450 to 600 base pairs long, reassociated in phosphate buffer and fractionated by hydroxylapatite, indicated a DNA genome composition of 36% nonrepetitive or single copy DNA; reassociation in formamide and followed by optical methods indicated the repetitive DNA possesses highly repeated, intermediately repeated and rarely repeated components of DNA with DNA sequences repeated on the average about 38,000, 6,700, and 200 times each. Different criteria of reassociation in formamide revealed further subdivisions of these four separate components of DNA. The DNA of above mentioned NC-17 variety compared to Florigiant variety showed no differences in thermal denaturation profiles, buoyant density, or in genome size.     

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.     

Isolation of intracellular symbiotes by immune lysis of flagellate protozoa and characterization of their DNA

A new method dependent on immune lysis is described for the isolation of intracellular symbiotes from two species of flagellate protozoa Blastocrithidia culicis and Crithidia oncopelti. The symbiote- containing flagellates are exposed to complement and antisera prepared in rabbits against symbiote-free organisms. The immune lysis seems to weaken the plasma membranes of the flagellates so that subsequent application of gentle shearing force liberates the intracellular entities in an undamaged condition. The symbiotes are then separated from other cellular components by DNAse digestion and differential centrifugation. The average recovery of symbiotes isolated by this method is 20%. Light and electron microscopy establishes the structural integrity and numerical abundance of isolated symbiotes in the final fractions. Integrity of symbiotes is further indicated by the high activity of a marker enzyme, uroporphyrinogen I synthetase. The DNA's of symbiote-containing and symbiote-free flagellates, and of isolated symbiotes were purified and compared after isopycnic centrifugation. The comparison establishes the presence of DNA's in symbiotes of both species. The guanine-cytosine (G-C) content of symbiote DNA differs from that of host DNA's in C. oncopelti, but resembles that of kinetoplast DNA in B. culicis. The latter observation was further shown by heat denaturation study. Renaturation kinetics indicate that the genome complexity of symbiote DNA in B. culicis is similar to that of bacteria.     

In situ localization and characterization of different classes of chromosomal DNA: acridine orange and quinacrine mustard fluorescence

In situ denaturation of metaphase chromosomes with alkali results in a shift from green to yellow, orange, brown and red fluorescence with acridine orange, indicating increasing denaturation of chromosomal DNA. The kinetics and characteristics of denaturation are described. Mouse and Microtus agrestis chromosomes denature uniformly but human cells show sequential denaturation. With increasing concentrations of alkali, the secondary constrictions in chromosomes 1, 9 and 16 are the first, and the distal half of the Y chromosome the last, to become denatured. — Reassociation of chromosomal DNA occurs within seconds after the start of incubation in salt solution. Areas containing repetitious DNA, e.g. mouse centromeres, fluoresce much more strongly than other regions with acridine orange after prolonged reassociation. Since human and Microtus centromeric regions behave similarly, it is proposed that they, too, contain repetitious DNA. — Reassociation treatment leads to enhancement of bright quinacrine mustard fluorescence in regions already bright before treatment. Furthermore, regions containing repetitious DNA, e.g. the secondary constrictions in human chromosomes 1, 9 and 16, whose fluorescence is dull before treatment, turn bright after reassociation. — The methods of fluorescence analysis of mammalian chromosomes with acridine orange and quinacrine mustard permit the localization and study of different classes of chromosomal DNA.     

Influence of formamide on the thermal stability of DNA

The utility of formamide in the denaturation and renaturation of DNA has been examined. The melting temperature of duplex DNA is lowered by 0·6°C per per cent formamide. The depression of melting temperature is independent of the GC content. Formamide also increases the width of the thermal transition. Upto 30%, it does not affect the rate of DNA reassociation     

Methyl green staining and highly repetitive DNA in polytene chromosomes

Fixed polytene chromosomes of Rhynchosciara angelae were stained with methyl green-pyronin after different pretreatments. Methyl green affinity for chromatin in the polytene chromosomes is lost after acid, alkali and heatformaldehyde treatments as a consequence of DNA denaturation, but reappears if conditions for DNA strand reassociation are provided. When such conditions are absent, only some chromosomal regions recover their affinity for methyl green. The resulting differential staining pattern is described and its possible relation to highly repetitive DNA sequences is discussed.     

Mouse satellite DNA isolated by Ag+ -- CsSO4 density gradients contains G+C rich, slow reassociating sequences

Mouse satellite DNA sequences isolated by centrifugation in CS2SO4--Ag+ gradients are analyzed for buoyant density by CSCl density gradients and for their content of fast reassociating sequences by denaturation and partial reassociation. Our data suggest that in CS2SO4 gradients silver ions separate a satellite band which contains both fast reassociating G+C rich sequences and slow reassociating, A+T rich DNA sequences.     

The distribution of satellite DNA in the chromosome complements of Vicia species (Leguminosae)

When centrifuged to equilibrium in neutral CsCl approximately 5% of the total nuclear DNA of V. melanops separated into a light satellite fraction. Buoyant density gradient analysis, thermal denaturation analysis and Cot reassociation experiments were used to find out the base sequence organisation of the satellite DNA. Using the method of in situ hybridisation its distribution in the chromosome complements of V. melanops and three other Vicia species were compared.     

Eight different highly specific nucleosome phases on alpha-satellite DNA in the African green monkey.

The question of nucleosome phasing on African Green Monkey (AGM) alpha-satellite DNA has been addressed by employing a new approach. Nucleosome cores were prepared from AGM nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population derived from alpha-satellite DNA containing chromatin was 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 alpha-satellite containing core DNA fragments were mapped by high resolution gel electrophoresis relative to known restriction sites along the 172 bp repeat unit of the satellite DNA. The results show that nucleosomes occupy eight strictly defined positions on the alpha-satellite DNA which could be determined with an accuracy of +/- 1 base pair. Approximately 35% of all nucleosomes are organized in one of these frames while the other seven registers contribute about 10% each.