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.     

Characterization of foldback sequences in hamster DNA using electron microsocpy.

Foldback sequences in nuclear DNA from cultured Hamster fibroblasts (BHK-21/C13 cells) have been characterized by electron microscopy. One half of the structures observed when denatured hamster DNA is allowed to anneal in the range O less than Cot1 less than 1 x 10(-4) M sec result from the annealing of inverted sequences forming foldback DNA. The remainder have a probable bimolecular origin. arising from rapidly-annealing sequences of satellite-like complexity. The average length of the inverted sequences in the foldback molecules is about 0.9 kilobases. There is estimated to be about 42,000 such sequences (21,000 pairs) in the hamster genome, approximately 45% of which form looped structures with a mean loop length of 1.74 kilobases. Contrary to previous reports, binding of the renatured duplex molecules to hydroxyapatite results in a poor recovery of structures containing identifiable foldback sequences, due to preferential enrichment of the bound fraction with duplexes formed by intermolecular annealing.     

Inverted repeated sequences in yeast nuclear DNA.

The inverted repeated sequences (foldback DNA) of yeast nuclear DNA have been examined by electron microscopy and hydroxyapatite chromatography. Of the inverted repeat structures seen in the electron microscope, 34% were hairpins and 66% had a single stranded loop at the end of a duplex stem. The number average length of the repeat was 0.3 kb and the single stranded loop was 1.6 kb. It is estimated that there are approximately 250 inverted repeats per haploid genome. A statistical analysis of the frequency of molecules containing multiple inverted repeats showed that these sequences are non-randomly distributed. The distribution of inverted repeats was also examined by measuring the fraction of total DNA in the foldback fraction that bound to hydroxyapatite as a function of single strand fragment size. This analysis also indicated that the inverted repeats are clustered. Renaturation kinetic analysis of isolated foldback and inverted repeat stem sequence DNA showed that these sequences are enriched for repetitive DNA.     

Characterization of inverted repeated sequences in Ascaris nuclear DNA

The inverted repeated sequences of the chromatin-eliminating nematode Ascaris lumbricoides var. suum have been examined by electron microscopy and by hydroxyapatite chromatography, both in the germ-line and in the somatic DNA. 38% of the inverted repeats of the germ-line DNA analysed in the electron microscope have a single-stranded loop, in comparison to about 50% of looped structures in the somatic DNA. The loops are on average 2.3 X 10(3) base pairs (bp) long. The rest of the foldback DNA consists of simple hairpins. The average length of looped and unlooped inverted repeats is of the order of 300-400 bp in the germ-line and in the somatic DNA. The content of S1-resistant foldback duplexes isolated by hydroxyapatite chromatography amounts to 1.3% in spermatids, with an average length of 350 bp, and to 1.1% in intestinal or larval cell nuclei, with a length of about 320 bp. We estimate by two different methods that there exist approximately 12500 inverted repeats per haploid germ-line genome and approximately 8000 in the haploid somatic genome. A statistical analysis of the data indicates that the great majority of the foldback sequences are randomly distributed in the Ascaris genome, with a spacing of about (40-80) X 10(3) bp, both in the germ-line and in the somatic DNA.     

Sequence organization in nuclear deoxyribonucleic acid from Physarum polycephalum. Physical properties of foldback sequences.

An investigation was performed with the use of physical techniques, to determine the nature and organization of inverted repeat sequences in nuclear DNA fragments from Physarum polycephalum. From the average size of foldback duplexes (550 nucleotide pairs), and the foldback duplex yield as determined by treatment of DNA with S1 deoxyribonuclease followed by hydroxyapatite chromatography, it is estimated that there are at least 25000 foldback sequences in the Physarum genome. Foldback DNA molecules exhibit properties intermediate between single-stranded DNA and native duplexes on elution from hydroxyapatite with a salt gradient. In addition, thermal-elution chromatography of foldback DNA from hydroxyapatite crystals shows that foldback duplexes are less stable than native DNA. These properties can be explained on the basis that inverted repeat sequences are mismatched when in the foldback configuration. The results of experiments in which the binding of foldback DNA molecules to hydroxyapatite was determined, by using fragments of different single-chain size, agree with previous studies indicating that inverted repeat sequences are located, on average, every 7000 residues throughout the Physarum genome. The inverted repeats are derived from both the repetitive and single-copy components in Physarum nuclear DNA.     

Characterization of inverted repeated sequences in wheat nuclear DNA.

The properties of inverted repeated sequences in wheat nuclear DNA have been studied by HAP(1) chromatography, nuclease S1 digestion and electron microscopy. Inverted repeated sequences comprise 1.7% of wheat genome. The HAP studies show that the amount of "foldback HAP bound DNA" depends on DNA length. Inverted repeats appear to be clustered with an average intercluster distance of 25 kb. It is estimated that there are approximately 3 x 10(6) inverted repeats per haploid wheat genome. The sequences around inverted repeats involve all families of repetition frequencies. Inverted repeats are observed as hairpins in electron microscopy. 20% of hairpins are terminated by a single-stranded spacer ranging from 0.3 to 1.5 kb in length. Duplex regions of the inverted repeats range from 0.1 to 0.45 kb with number average values of 0.24 kb and 0.18 kb for unlooped and looped hairpin respectively. Thermal denaturations and nuclease S1 digestions have revealed a length of about 100 bases for duplex regions. The methods used to study inverted repeated sequences are compared and discussed.     

Periodic organisation of foldback sequences in Physarum polycephalum nuclear DNA.

Nuclear DNA from the slime mould Physarum polycephalum is shown to contain interspersed inverted repeat sequences, such that denatured fragments of DNA containing pairs of these sequences form intra-chain duplexes under appropriate conditions. The organisation and distribution of the nucleotide sequences responsible for the formation of foldback structures in Physarum DNA have been investigated using the electron microscope. The majority of foldback duplexes have sizes ranging up to 800 base pairs, and about 60-80% of DNA molecules 2.2 X 10(4) bases in length contain interspersed foldback elements. The size of individual foldback duplexes, and also the length of the intervening sequences which separate them, are non-random. The results can best be explained by a model in which separate foldback foci in Physarum DNA are spaced periodically at regular intervals. The regions containing foldback foci are thought to contain smaller, tandemly-arranged sequences of discrete sizes, in some cases related to other nucleotide sequences of a similar nature in the same locality in Physarum DNA.     

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.     

Interspersion and transcription of repeated sequences of rat DNA.

Repetitive rat DNA reassociated to Cot=0.1 and deprived of "foldback" sequences showed close interspersion with unique sequences. As measured by electron microscopy, the average length of repetitive segments was about 600 +/- 400, and of unique segments 1800-3600 base pairs. Pyrimidine tracts over 80 nucleotides in length were found mainly in foldback and repetitive fractions. Oligo(dT) tracts, 20-30 bases in length prevailed in the DNA fraction reassociated to Cot=0.1. Repetitive and unique DNA fractions were annealed to Millipore filters and hybridized with hnRNA. Up to 1.6% of repetitive DNA reassociated to Cot=0.05 showed base complementarity with hnRNA, whereas the comparative figures for DNA reassociated to Cot=10 and for the unique fraction were 0.8% and 0.3% respectively. When hybridization of hnRNA was carried out in solution in vast DNA excess, no hybrid formation with repetitive sequences reassociated to Cot=0.1 was observed, although hybridization with DNA reassociated to Cot=10 was noticeable.     

Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.     

Structure of nuclear pre-mRNA. VI. "Reversed repeats" in animal DNA and their hybridization with double-stranded regions of pre-mRNA.

About 2% of mouse DNA reassociates at extremely low Cot values (10-7-10-6 mole-liter-1-sec). This DNA fraction was isolated with the aid of nuclease S1 and purified by chromatography on hydroxyapatite. The study of this fraction suggests that it has a structure of hairpin type. The DNA of the hairpins can hybridize with sequences forming the double-stranded regions in pre-mRNA. Probably at least some of the DNA of the hairpins, described as "reversed repeating sequences" of DNA, is transcribed with the formation of structures of hairpin type in pre-mRNA.     

Characterization of foldback sequences in Physarum polycephalum nuclear DNA using the electron microscope

An examination of the foldback fraction of nuclear DNA from Physarum polycephalum has been carried out using the electron microscope. Results show that the inverted repeat sequences responsible for the formation of foldback DNA range from 150-3000 bases in length, with a number-average size of 340 bases. About one-half of the inverted sequences form looped structures with loop sizes averaging 1200 bases in length. The distance between adjacent foldback sequences is estimated to be in the range 100-1500 bases.     

Melting studies of short DNA hairpins containing the universal base 5-nitroindole.

Effects of the universal base 5-nitroindole on the thermodynamic stability of DNA hairpins having a 6 bp stem and four base loops were investigated by optical absorbance and differential scanning calorimetry techniques. Melting studies were conducted in buffer containing 115 mM Na(+). Five different modified versions of DNA hairpins containing a 5-nitroindole base or bases substituted at different positions in the stem and loop regions were examined. Thermo-dynamic parameters of the melting transitions estimated from a two-state analysis of optical melting curves and measured directly by calorimetry revealed that the presence of 5-nitroindole bases in the duplex stem or loop regions of short DNA hairpins significantly affects both their enthalpic and entropic melting components in a compensating manner, while the transition free energy varies linearly with the transition temperature. The calorimetrically determined enthalpy and entropy values of the modified hairpins were considerably smaller (43-53%) than the two-state optical parameters, suggesting that solvent effects may be significant in the melting processes of these hairpins. Results of circular dichroism measurements also revealed slight differences between the modified hairpins and the control in both the duplex and melted states, suggesting subtle structural differences between the control and DNA hairpins containing a 5-nitroindole base or bases.     

Organisation of inverted repeat sequences in hamster cell nuclear DNA.

Hamster cell nuclear DNA is shown to contain inverted repeat (foldback) sequences, in some respects similar to the foldback fraction in DNA from other animal cell types. Using electron microscopy the majority of foldback duplexes are shown to be located in simple hairpin-like DNA structures, formed from individual pairs of complementary inverted repeated sequences 50--1000 nucleotides in length, in some cases arranged in tandem, and in other cases separated by intervening sequences, up to 16000 nucleotide residues long. In addition, a novel class of foldback structure, referred to as 'bubbled hairpins' is reported, which appear to be formed from clusters of inverted repeat sequences that are separated from adjacent clusters of complementary inverted repeats by large intervening sequences which vary in length from 5000 to over 20000 nucleotide residues. Due to the special pattern of distribution of these latter inverted repeat sequences, 'bubbled hairpins' are observed only in long foldback DNA. Evidence is presented that the distribution of foldback sequences in hamster cell DNA is highly ordered. The lengths of the intervening single chains in foldback structures appear to vary non-randomly. This gives rise to a localised periodic pattern of organisation that is believed to be a consequence of regular alternating arrangements of foldback and non-foldback sequences in the segments of DNA from which foldback structures are derived.     

Distribution of inverted repeat sequences in nuclear DNA from Physarum polycephalum.

Inverted repeat sequences, capable of forming stable intra-chain foldback duplexes, are shown using electron microscopy to be located in over 90% of fragments of nuclear DNA from Physarum polycephalum. A statistical treatment of the data indicates that, on average, foldback sequence foci are spaced every 7,000 nucleotides and that they are distributed uniformly amongst the DNA chains. The majority of inverted repeat sequences give rise to the simple types of foldback structure observed in DNA from other eukaryotic species, but a significant proportion of the DNA fragments also contain novel foldback structures with a more complex appearance, referred to as 'bubbled' hairpins. The latter structures appear to be formed by the annealing of several distinct segments of homologous inverted repeat sequence, each separated by interspersed non-foldback sequences of variable sizes up to 15,000 nucleotides in length. The size, both of the foldback duplexes and of the intervening single-chain segments of DNA, are not random. Instead, they appear to form a regular, arithmetic series of lengths. These observations suggest that the different segments of Physarum DNA from which foldback structures are derived contain nucleotide sequences that share a highly ordered and unform pattern of structural organisation. These regular units of organisation in Physarum DNA in some cases extend over distances up to 50,000 nucleotides in length.     

Isolation of foldback DNA utilizing nuclease S1 digestion in aqueous dioxane.

An improved method for the isolation of the inverted repetitive (foldback) sequences present in mammalian DNAs is described. It makes use of the new observation that nuclease S1 digestion of denatured DNA occurred at a faster rate and was more extensive in medium containing dioxane. The temperature-absorbance characteristics of nuclease S1-resistant DNA were systematically studied as a function of the temperature employed during the step of enzymic hydrolysis. Specimens of human placental and calf thymus DNA which had been denatured and renatured to C₀t ≤ 10^?3 mol s liter^?1 were used as substrates. Foldback DNA was isolated from the enzymic digests by means of hydroxylapatite chromatography. Temperature-absorbance studies showed the enzyme-resistant DNA had a high degree of thermal stability; the hyperchromic rises equaled those obtained in the native speciments. The amount of foldback DNA which could be obtained was not influenced by the fragment size of the starting material, above a certain molecular weight range. Foldback DNA represented about 4% of the human genome and at least 5% of the bovine genome. The size distributions of these strands were studied by means of polyacrylamide gel electrophoresis.     

Sequence organisation in nuclear DNA from Physarum polycephalum. Arrangement of highly-repeated sequences

Recombinant plasmids containing highly repetitive Physarum DNA segments were identified by colony hybridisation using a radioactively-labelled total Physarum DNA probe. A large number of these clones also hybridised to a foldback DNA probe purified from Physarum nuclear DNA. The foldback DNA probe was characterised by reassociation kinetic analysis. About one-half of this component was shown to consist of highly repeated sequences with a kinetic complexity of 1100 bp and an average repetition frequency of 5200. Direct screening of 67 recombinant plasmids for foldback sequences using the electron microscope revealed that about one-half were located in segments of DNA containing highly repetitive sequences; the remainder were present in clones containing low-copy number repeated elements. Analysis of two DNA clones showed that they contained repetitive elements located in over half of all DNA segments containing highly repetitive DNA and that the foci containing these highly repetitive sequences had different sequence arrangements. The results are consistent with the hypothesis that the most highly repeated DNA sequence families in the Physarum genome are few in number and are clustered together in different arrangements in about one-sixth of the genome. Over one-half of the foldback DNA complement in the Physarum genome is derived from these segments of DNA.     

Sequence organization of the rat genome by electron microscopy.

The size and arrangement of repetitive and inverted repeat (foldback) sequences in rat DNA were studied by visualization of hybrid and heteroduplex structures in the electron microscope. The self-reassociation of repetitive sequence-bearing DNA strands often results in the formation of four-ended "H" structures, whose duplex regions equal the repetitive sequence length and can be measured in the electron microscope. In this way, it was determined that the average size of the class of numerous short repetitive sequences is 0.40 +/- 0.15 kbp. Heteroduplex structures were prepared between long whole DNA single strands and short repeat-sequence-bearing strands. The analysis of these structures confirms that the size of the repetitive sequences in 0.4 kbp on average. Length measurements between adjacent duplexes show that the average spacing between two interspersed repeats is at least 1.5-1.8 kbp. By examining 29.4-kbp single strands after brief renaturation, the size and distribution of foldback sequences were determined. There are 1.9 X 10(5) foldback apirs per rat genome, spaced an average of 9.7 kbp apart according to our measurement. Repetitive, inverted repeat and unique sequences are interspersed with each other in at least half the genome.     

Characterisation of ribosomal satellite in total nuclear DNA from Physarum polycephalum.

The distinctive properties of satellite DNA molecules containing the genes for ribosomal RNA in Physarum polycephalum permits their identification in total, unfractionated nuclear DNA in the foldback form, after denaturation and fast annealing. Using the electron microscope the location and properties of three characteristic regions containing tandemly-repeated, inverted sequences have been investigated. At least two additional regions, also containing tandem repeats, are shown to be present and located towards each end of the rDNA molecule, at a site adjacent to the segment coding for the 26 S rRNA. All the regions which contain tandem repeats are composed of sequences which, within experimental error, appear to share a common unit repeat length of about 90 nucleotides.