Repetitive DNA in differentiating chick tissues

Embryonic chick DNA from different tissues was examined for differences in relative content of highly repetitive DNA which might indicate specific DNA amplification in somatic cells. The content of repetitive sequences in DNA isolated from cerebrum, muscle, and neural retina tissues, at the same and at different embryonic stages, was determined by hydroxyapatite fractionation of partially reassociated DNA samples. An unrenatured marker DNA (C14-labeled E. coli DNA) was added to each chick DNA sample in order to monitor the nonspecific single-stranded DNA retention by each hydroxyapatite column. When chick DNA samples were sheared to a double-stranded length of 1,300 nucleotide pairs, an average of 20.2% +/- 2.2% of the DNA was found to reassociate at a Cot value of 10. The quantity of the fast reassociating sequences was found to constitute the same fraction of the DNA in all the tissues studied. In addition, all the reassociated DNA samples exhibited the same CsCl density classes. The studies also indicated that most chick DNA repetitive sequences are interspersed with nonrepetitive sequences.     

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.     

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.     

Exploration of long and short repetitive sequence relationships in the sea urchin genome.

Long and short repetitive sequences of sea urchin DNA were prepared by reassociation of 2000 nucleotide long fragments to Cot 4 and digestion with the single strand specific nuclease S1. The S1 resistant duplexes were separated into long repetitive and short repetitive fractions on Agarose A50. The extent of shared sequences was studied by reassociating a labeled preparation of short repetitive DNA with an excess of unlabeled long repetitive DNA. Less than 10% of the long repetitive DNA preparation was able to reassociate with the short repetitive DNA. Thus the long and short repetitive elements appear to be principally independent sequence classes in sea urchin DNA. Precisely reassociating repetitive DNA was prepared by four successive steps of reassociation and thermal chromatography on hydroxyapatite. This fraction (3% of the genome) was reassociated by itself or with a great excess of total sea urchin DNA. The thermal stability of the products was identical in both cases (Tm=81 degrees C), indicating that precisely repeated sequences do not have many imprecise copies in sea urchin DNA.     

Evolutionary divergence and length of repetitive sequences in sea urchin DNA

Summary The organization of repetitive and single copy DNA sequences in sea urchin DNA has been examined with the single strand specific nuclease Sl fromAspergillus. Conditions and levels of enzyme were established so that single strand DNA was effectively digested while reassociated divergent repetitive duplexes remained enzyme resistant. About 25% of sea urchin DNA reassociates with repetitive kinetics to form Sl resistant duplexes of two distinct size classes derived from long and short repetitive sequences in the sea urchin genome. Fragments 2,000 nucleotides long were reassociated to Cot 20 and subjected to controlled digestion with Sl nuclease. About half of the resistant duplexes (13% of the DNA) are short, with a mode size of about 300 nucleotide pairs. This class exhibits significant sequence divergence, and principally consists of repetitive sequences which were interspersed with single copy sequences. About one-third of the long duplexes (4% of the DNA) are reduced in size after extensive Sl nuclease digestion to about 300 nucleotide pairs. About two-thirds of the long resistant duplexes (8% of the DNA) remains long after extensive SI nuclease digestion. These long reassociated duplexes are precisely base paired. The short duplexes are imprecisely paired with a melting temperature about 9°C below that of precisely paired duplexes of the same length. The relationship between length of repetitive duplex and precision of repetition is confirmed by an independent method and has been observed in the DNA of a number of species over a wide phylogenetic area.Also Staff Member, Carnegie Institution of Washington     

Clustered and interspersed repetitive DNA sequences in four amphibian species with different genome size.

We have compared the amount of clustered and interspersed repetitive sequences in the genome of four Amphibia with different DNA contents per haploid nucleus: two Anura (Xenopus laevis, 3 pg and Bufo bufo, 7 pg) and two Urodela (Triturus cristatus, 23 pg and Necturus maculosus, 52 pg). High molecular weight DNA of the four species was denatured and reassociated to the same Cot in order to obtain duplex sequences with a similar reiteration frequency. Single-stranded DNA was digested off with the Aspergillus S1 nuclease. DNA was then fractionated according to the molecular weight through an agarose A-50 column. We found that the amount of long repetitive sequences is roughly proportional to the genome size in the four species, while the number of short (about 300 base pairs) repetitive sequences is increased many-fold in the species with the larger DNA content, both in Anura and in Urodela.     

Evolutionary change in the repetition frequency of sea urchin DNA sequences

The frequency of occurrence of particular repetitive sequence families has been estimated in the DNA of the three sea urchin species Strongylocentrotus purpuratus, Strongylocentrotus franciscanus and Lytechinus pictus using individual cloned S. purpuratus repetitive sequence elements. Cloned repetitive sequence elements as described by Scheller et al. (1977a) were prepared by reassociation of S. purpuratus DNA fragments to repetitive Cot, digestion with single-strand-specific nuclease S1 and ligation of synthetic restriction sites to their ends. The sequences were cloned by insertion at the Eco RI site of plasmid RSF2124, labeled, strand-separated and reassociated with 800–900 nucleotide long unlabeled DNA. Both kinetic (genomic DNA excess) and saturation (cloned DNA excess) estimates of frequencies were made. For nine cloned fragments, the ratio of the repetition frequency in S. purpuratus DNA to that in S. franciscanus DNA ranges from about 20 to about 1. In the four cases examined, only a few copies were detected in the DNA of L. pictus. Estimates have also been made of frequency changes in many repetitive families by measuring the reassociation of labeled repetitive DNA fractions of each species with total DNA from other species. In each reciprocal comparison, the labeled repetitive sequences reassociate more slowly with DNA of other species than with DNA of the species from which they were prepared. Thus it appears that the dominant repetitive sequence families in the DNA of each species are present at lower frequencies in the DNA of closely related species. Measurements of thermal stability have been made of S. purpuratus cloned repetitive sequences reassociated with S. franciscanus DNA or S. purpuratus DNA. Most families have changed both in frequency and sequence, although some have changed little in sequence but show great changes in frequency.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

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.     

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.     

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.     

Absence of short period interspersion of repetitive and non-repetitive sequences in the DNA of Drosophila melanogaster

A sensitive search has been made in Drosophila melanogaster DNA for short repetitive sequences interspersed with single copy sequences. Five kinds of measurements all yield the conclusion that there are few short repetitive sequences in this genome: 1) Comparison of the kinetics of reassociation of short (360 nucleotide) and long (1,830 nucleotide) fragments of DNA; 2) reassociation kinetics of long fragments (2,200 nucleotide) with an excess of short (390 short nucleotide) fragments; 3) measurement of the size of S1 nuclease resistant reassociated repeated sequences; 4) measurement of the hyperchromicity of reassociated repetitive fragments as a function of length; 5) direct assay by kinetics of reassociation of the amount of single copy sequence present on 1,200 nucleotide long fragments which also contain repetitive sequences.     

Analysis of plant genomes. V. Comparative study of molecular properties of DNAs of seven Allium species

The genomes of seven plant species belonging to the genus Allium and exhibiting a threefold variation in their nuclear DNA content were analyzed by studying their reassociation kinetics, equilibrium centrifugation behavior in neutral CsCl gradients, and melting properties. The reassociation kinetics experiments revealed the presence of 44–65% repeated DNA sequences. A comparison between DNA contents and the proportion of repeated DNA sequences indicated that, in Allium, increase in the genome size is not exclusively due to variations in the proportions of repetitive DNA. The total DNA as well as the various repetitive DNA fractions in all the Allium species examined exhibited, in spite of a few differences, a gross similarity in their behavior in neutral CsCl gradients and in their melting properties.     

Cytoplasmic DNA synthesis in rhinovirus type 14-inoculated KB cells.

Rhinovirus type 14 (RV14) incuced a transient statistically significant stimulation in synthesis of DNA which appeared between 0 and 3 h post-inoculation in the cytoplasm of high density monolayer cultures of KB cells. Newly synthesized DNA was measured by incorporation of [3H] thymidine into acid-insoluble DNAase-sensitive material and the cytoplasmic location established by cell fractionation and electron microscope radioautographic methods. A minimum of 10 plaque-forming units per cell of RV14 was required to stimulate DNA synthesis which did not occur above 34.5 degrees C, a temperature optimal for virus replication. Cytoplasmic DNA taken from RV14-infected or control cells could be differentiated from the bulk of cell (nuclear) DNA by several criteria, including: (1) RV14 induction of synthesis; (2) lower buoyant density and greater heterogeneity in CsCl and ethidium bromide/CsCl gradients; and (3) a different kinetic complexity upon reannealing. The Cot 1/2 value of cytoplasmic DNA, calclated as 50--100 from reassociation profiles, was about 10-fold less complex than the Cot 1/2 value of nuclear DNA (800-1000). These data rule out the possibility that cytoplasmic DNA arises by random breakage of nuclear DNA during cell disruption and extraction and are compatible with the hypothesis that inoculation of KB cells with RV14 results in stimulation of synthesis of a specific class of cell DNA which is detected in the cytoplasm.     

Deoxyribonucleic acid sequence organization in the genome of the dinoflagellate Crypthecodinium cohnii

Details of the general DNA sequence organization in the dinoflagellate Crypthecodinium cohnii have been obtained by using hydroxylapatite binding experiments, S1 nuclease digestion .and electron microscopy of reassociated DNA. It has been found that roughly half of the genome is made up of unique sequences interspersed with repeated sequence elements with a period of approximately 600 nucleotides. This class represents roughly 95% of the total number of interspersed unique elements in the genome. The remaining 5% are uninterrupted by repeated sequences for at least 4000 nucleotide pairs. The interspersed repeated elements are narrowly distributed in length with 80% under 300 nucleotide pairs in length. About half of the repeated DNA (20-30% of the genome) is not interspersed among unique sequences. The close spacing of the short repeats interspersed throughout much of the genome is consistent with the occurrence of the huge network structures observed in the electron microscope for low Cot reassociation of moderately long fragments. An unusual class of heteroduplexes was detected in the electron microscope which is believed to derive from the reassociation of repeated sequences from different families which are frequently found adjacent to one another in different locations in the genome. The occurrence of this novel arrangement of repeated sequences may reflect the unusual organization of the dinoflagellate nucleus. However, in most respects the sequence arrangement in this unicellular alga is very typical of higher plants and animals.     

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.     

Identification and separation of components of calf thymus DNA using a CsCl-netropsin density gradient

Calf thymus DNA containing satellite components of various densities was used as a model to study the effect of netropsin on the density of DNA in a CsCl gradient. The binding of netropsin resulted in a decrease in density which depended upon the quantity of netropsin added and on the average composition of the DNA. Differences in density of DNA components were higher in CsCl - netropsin gradients than in simple CsCl gradients. By use of netropsin a main band and four satellite bands could be differentiated in calf thymus DNA. Satellite DNA's were isolated using preparative CsCl - netropsin gradient centrifugation and were characterised by density and homogeneity in native and in reassociated state. Two of the satellite components, with densities of 1.722 and 1.714 g/cm³, are probably of homogenous sequence, the other two components of densities 1.709 and 1.705 g/cm³ appear to be heterogeneous.     

The evolution of repetitive DNA sequences in sea urchins.

Molecular hybridization of nuclear DNAs has been employed to study the evolution of the repetitive DNA sequences in four species of sea urchin. The data show that relative to S. purpuratus there has been approximately 0.1% sequence divergence per million years in the repetitive DNA sequences of S. droebachiensis, S. franciscanus, and L. pictus. These results confirm that repetitive DNA sequences are strongly conserved during evolution. However, comparison of the extent of base pair mismatch in the repetitive DNA heteroduplexes formed at Cot 20 with those formed at Cot 200 during the hybridization of S. purpuratus and L. pictus DNAs reveals that highly repetitive sequences of sea urchins may diverge more rapidly than do the more moderately repetitive sequences.     

An alternative view of mammalian DNA sequence organization: II. Short repetitive sequences are organized into scrambled tandem clusters in Syrian hamster DNA

Hyperchromicity, S₁ nuclease digestion, and reassociation studies of Syrian hamster repetitive DNA have led to novel conclusions about repetitive sequence organization. Re-evaluation of the hyperchromicity techniques commonly used to determine the average length of genomic repetitive DNA regions indicates that both the extent of reassociation, and the possibility of non-random elution of hyperpolymers from hydroxyapatite can radically affect the observed hyperchromicity. An alternative interpretation of hyperchromicity experiments, presented here, suggests that the average length of repetitive regions in Syrian hamster DNA must be greater than 4000 nucleotides.S₁ nuclease digestion of reassociated 3200 nucleotide Syrian hamster repetitive DNA, on the other hand, yields both long (>2000 nucleotides) and short (300 nucleotides) resistant DNA duplexes. Calculations indicate that the observed mass of short nuclease-resistant duplexes (>60%) is too large to have arisen only from independent short repetitive DNA sequences alternating with non-repetitive regions. Reassociation experiments using long and short S₁ nuclease-resistant duplexes as driver DNA indicate that all repetitive sequences are present in both fractions at approximately the same concentration. Isolated long S₁ nuclease-resistant duplexes, after denaturation, renaturation, and a second S₁ nuclease digestion, again produce both long and short DNA duplexes. Reassociation experiments indicate that all repetitive DNA sequences are still present in the “recycled” long S₁ nuclease-resistant duplexes. These experiments imply that many of the short S₁ nuclease-resistant repetitive DNA duplex regions present in reassociated Syrian hamster DNA were initially present in the genome as part of longer repetitive sequence blocks. This conclusion suggests that the majority of “short” repetitive regions in Syrian hamster DNA are organized into scrambled tandem clusters rather than being individually interspersed with non-repetitive regions.     

Construction of a small Mus musculus repetitive DNA library: identification of a new satellite sequence in Mus musculus.

We report the construction of a small library of recombinant plasmids containing Mus musculus repetitive DNA inserts. The repetitive cloned fraction was derived from denatured genomic DNA by reassociation to a Cot value at which repetitive, but not unique, sequences have reannealed followed by exhaustive S1 nuclease treatment to degrade single stranded DNA. Initial characterizations of this library by colony filter hybridizations have led to the identification of a previously undetected M. musculus minor satellite as well as to clones containing M. musculus major satellite sequences. This new satellite is repeated 10-20 times less than the major satellite in the M. musculus genome. It has a repeat length of 130 nucleotides compared with the M. musculus major satellite with a repeat length of 234 nucleotides. Sequence analysis of the minor satellite has shown that it has a 29 base pair region with extensive homology to one of the major satellite repeating subunits. We also show by in situ hybridization that this minor satellite sequence is located at the centromeres and possibly the arms of at least half the M musculus chromosomes. Sequences related to the minor satellite have been found in the DNA of a related Mus species, Mus spretus, and may represent the major satellite of that species.