Ring theory

In what follows we demonstrate that the minimum requirement for the formation of a DNA ring is a pair of ordinary (ABC … ABC) or inverted (ABC … C′B′A′) repetitions. DNA fragments that are partly degraded from their ends by a 3′ (or 5′) specific exonuclease such as exonuclease III (or λ exonuelease) produce resected fragments that can only form rings by virtue of ordinary repetitions.Next we analyze how random fragments cut from DNA molecules containing ordinary repetitions would be expected to form rings. Since longer fragments (>5 to 10 μm) cyclize less efficiently than do shorter ones (2 μm), we are led to the view that the chromatid is composed of thousands of distinctive regions, called g-regions, within which characteristic repetitious sequences are clustered in an intermittent or tandem fashion. Mathematical expressions are derived that allow one to measure the length and number of these g-regions from the ring frequency, R, and its dependence on the length of the fragment.The interior organization of the g-regions is considered in terms of two models and their variants: intermittent repetition and tandem repetition. These are depicted in Figure 2. The objective of this effort is to calculate the frequency of rings that can be generated from these two models, and to explain the “shortside fall-off”, that is, the decrease in ring frequency as the fragment length becomes shorter. This could not be due to the stiffness of the DNA double helix and must reflect a distribution of spacing of the repetitious sequences within the g-regions. Mathematical expressions are obtained that allow one to estimate the average values of the repetitive or partly repetitive unit. These estimates may be obtained from the dependence of ring frequency on the extent of resection, and from the dependence of ring frequency on the length of shorter fragments.The mathematical expressions derived here are employed in the previous papers of this group, and lead to the conclusion that the g-regions are composed of tandemly repeating sequences.     

On travelling wave solutions in a model for the Belousov-Zhabotinskii reaction.

DNA fragments partially digested by a 3′- or 5′-specific nuclease to produce single chain ends of opposite polarity will form a ring if the ends contain complementary sequences and are allowed to anneal. The frequency of rings can then be used as an assay to determine where and how identical repetitious sequences are arranged in the DNA. Thomas et al. (1973b) showed that all eucaryote chromosomes studied contain similar if not identical repetitious sequences clustered into regions called g-regions. To account for the observed ring frequency under different experimental conditions Thomas, Zimm &; Dancis (1973c) derived equations for two possible models of g-region organization. In the pure tandem model, the repetitious sequences are contiguous and occupy the entire g-region. In the intermittent repetition model, the repetitious sequences are simple copolymers and are irregularly arranged among non-repetitious sequences which are heterogenous in length. In the present paper, the results of Thomas et al. (1973c) are extended to cover the fractional tandem model. In this model, adjacent repetitious sequences are separated by non-repetitious sequences of uniform length. In addition, the equations for both the pure tandem and intermittent repetition models are shown to be special cases of the fractional tandem model but not vice versa.The capabilities and limitations of an analysis of ring formation are demonstrated using data from Drosophila. Although it is not possible to rule out any of the three models, the analysis can limit the ranges of the parameters describing each of the models that are consistent with the data. Previous conclusions that the data could only be explained by a pure tandem model which lacks any intervening unique sequences (Bick, Huang &; Thomas, 1973; Thomas et al., 1973b), are shown to be incorrect, in part because the equations for the fractional tandem model had not then been derived. Thus ring theory equations can be used to show the presence of clusters of similar if not identical sequences from ring-forming experiments, but they may not be able to determine the exact spacing and arrangement of these sequences within the clusters.     

The cyclization of Xenopus laevis 5 S DNA

DNA from Xenopus laevis containing the sequences complementary to 5 S RNA has been studied by the formation of folded rings. Maximal cyclization for fragments 1 to 2 μm in length is 45 to 55%. Thus the efficiency of folded ring formation from this tandemly-repeating DNA is about 50%, assuming that all fragments are 5 S DNA. From the ring frequency as a function of the number of nucleotides removed from the 3′ terminals of the shear-broken fragments, one may calculate that the repeating sequence is approximately 750 nucleotides long, a number that agrees with earlier partial denaturation mapping. The circumference of the folded rings confirms this repeating length since most rings correspond to modular size classes of 0.25-μm increments. Fragments 12 μm long cyclize almost as readily as 1 to 2-μm fragments do. Therefore, the length of the regions (g-regions) containing the tandemly-repeating 5 S DNA is more than 12 μm. The folded rings are about as stable to linearization by increasing concentrations of formamide as the duplex DNA is to denaturation. This indicates that the local, non-transcribed, spacer portions which represent the majority (83%) of the nucleotides in the tandemly-repeating unit, are probably homogeneous in sequence. The exonuclease-treated 5 S DNA fragments cyclize more rapidly than phage T7 DNA, and the kinetics are in accord with theoretical expectation.     

Tandemly Repeated Sequence Families in Micronuclear DNA of the Ciliate Stylonychia pustulata1

One third of a collection of cloned Stylonychia pustulata micronuclear DNA PstI fragments were found to be of a similar size, consistent with their being members of a repetitious sequence family with a repeat size of about 160 base pairs. Cross-hybridization experiments confirmed that these small cloned fragments are related by sequence homology. Hybridization of the cloned repetitious sequences to PstI digested micronuclear DNA revealed a “ladder” of bands (step size = 160 base pairs), indicating that the repeats are found in tandem arrays. This is the first demonstration of highly repetitious, tandemly repeated sequences in a ciliated protozoan.     

Stability and fine structure of eukaryotic DNA rings in formamide

Folded rings formed from Drosophila and Necturus DNA fragments were examined by electron microscopy in increasing concentrations of formamide, in an effort to identify regions of non-homology within the closure region. Unusual closure regions of this type were not found, in spite of an extensive search. If such regions exist, they must be too short to be detectable (<50 nucleotides), or longer than 1000 nucleotides. In this latter case, they could not be contained within the overlap region of the ring. A study of the thermal (formamide) stability of these rings in relation to the observed closure lengths suggests that extensive (>2 to 3%) mismatching is not possible. At higher formamide concentrations, some rings will partly denature, yet remain circular because the closure region remains intact.     

Electron microscopy and restriction enzyme mapping reveal additional intervening sequences in the chicken ovalbumin split gene

The Eco RI fragment “b” of chicken DNA (Breathnach, Mandel and Chambon, 1977), which contains the sequences coding for the 5′ quarter of ovalbumin mRNA (ov mRNA), has been isolated by molecular cloning using a “shotgun” approach. Electron microscopy and restriction enzyme analysis have revealed that the sequences coding for the 5′ quarter (～500 nucleotides) of ov mRNA are split into four regions separated by three intervening sequences. The cloning procedure seems to be reliable, since the restriction enzyme pattern of the cloned Eco RI fragment “b” is similar to that of the corresponding chromosomal DNA fragment. There is no evidence supporting the existence of a 150–200 nucleotide long sequence at the 5′ end of the ov mRNA similar to the “leader” sequences found at the 5′ end of some adenovirus and SV40 mRNAs.     

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.     

Regions of the terminal repetitions of the herpes simplex virus type 1 genome. Relationship to immunoglobulin switch-like DNA sequences

Several recombinant clones isolated from a mouse genomic library were previously shown to hybridize with a SmaI fragment located in the terminal repetition of the S component of herpes simplex virus DNA. We report here the nucleotide sequence of the related regions in two mouse clones, TGL19 and TGL35, as well as that of the SmaI fragment of HSV-1. The mouse DNA clones have a core of repetitive sequences 80% homologous to a tandem repeat (reiteration II) in the viral fragment. The regions of homology are in turn related to immunoglobulin class-switch sequences, due mostly to the presence of the pentamer TGGG(G), involved in class-switch recombination. These results suggest that the HSV genome has recombination sequences identical to those of the host cell and provide a possible explanation for the high frequency of recombination events observed in this region of the viral genome.     

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.     

Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA.

The inverted terminal repetition in adeno-associated virus type 2 DNA has been sequenced. The terminal repetition contain 145 nucleotides of which the first 125 nucleotides can self-base pair to form a T-shaped hairpin structure. Both restriction endonuclease analysis with SmaI and BglI and direct sequence analysis of the SmaI fragments provide evidence for two sequences in the region of the terminal repetition between nucleotides 44 and 81. The two sequences represent an inversion of the first 125 nucleotides of the terminal repetition. Based on these data a model for adeno-associated virus DNA replication is presented which agrees in detail with a general model for eucaryotic DNA replication originally proposed by Cavalier-Smith (T. Cavalier-Smith, Nature [London] 18:672--684, 1976).     

Sequence and secondary structure of mouse 28S rRNA 5''terminal domain. Organisation of the 5.8S-28S rRNA complex.

We present the sequence of the 5' terminal 585 nucleotides of mouse 28S rRNA as inferred from the DNA sequence of a cloned gene fragment. The comparison of mouse 28S rRNA sequence with its yeast homolog, the only known complete sequence of eukaryotic nucleus-encoded large rRNA (see ref. 1, 2) reveals the strong conservation of two large stretches which are interspersed with completely divergent sequences. These two blocks of homology span the two segments which have been recently proposed to participate directly in the 5.8S-large rRNA complex in yeast (see ref. 1) through base-pairing with both termini of 5.8S rRNA. The validity of the proposed structural model for 5.8S-28S rRNA complex in eukaryotes is strongly supported by comparative analysis of mouse and yeast sequences: despite a number of mutations in 28S and 5.8S rRNA sequences in interacting regions, the secondary structure that can be proposed for mouse complex is perfectly identical with yeast's, with all the 41 base-pairings between the two molecules maintained through 11 pairs of compensatory base changes. The other regions of the mouse 28S rRNA 5'terminal domain, which have extensively diverged in primary sequence, can nevertheless be folded in a secondary structure pattern highly reminiscent of their yeast' homolog. A minor revision is proposed for mouse 5.8S rRNA sequence.     

Gene amplification in methotrexate-resistant mouse cells. II. Rearrangement and amplification of non-dihydrofolate reductase gene sequences accompany chromosomal changes

Total amplified DNA in methotrexate-resistant mouse lymphoma EL4 cells and mouse melanoma PG19 cells has been characterized in two ways. Metaphase spreads show the presence of additional chromosome forms that are either “homogeneously staining” chromosomes or “double minute” and ring chromosomes. Gel electrophoresis of restriction enzyme-digested nuclear DNA shows the presence of amplified sequences, the pattern of which is unique in each of five cell lines. We conclude that extensive DNA rearrangement has taken place during amplification.     

Occurrence of reiterated sequences in an untranslated region of Simian virus 40 DNA determined by nucleotide sequence analysis.

An earlier report (Subramanian, Dhar, and Weissman, 1977c) presented the nucleotide sequence of Eco RII-G fragment of SV40 DNA, which contains the origin of DNA replication. The nucleotide sequence of Eco RII-N fragment located next to Eco RII-G on the physical map of SV40 DNA is presented in this report. Eco RII-N is found to be a tandem duplication of the last 55 nucleotides of Eco RII-G. This tandem repeat is immediately preceded by two other reiterated sequences occurring within Eco RII-G, one of them being a tandem repeat of 21 nucleotides and the other a nontandem repeat of 10 nucleotides. These repetitive sequences occur in close proximity to the origin of DNA replication which is known to contain other specialized sequences such as a few palindromes (one of which is 27 long and possesses a perfect 2-fold axis of symmetry), one "true" palindrome, and a long A/T-rich cluster. The repeats (and the replication origin) occur within an untranslated region of SV40 DNA flanked by (the few) structural genes coding for the "late" proteins on the one side and that (those) coding for the "early" protein(s) on the other side. The reiterated sequences are comparable in some respects to repetitive sequences occurring in eucaryotic DNAs. Possible biological functions of the repeats are discussed.     

VNTRs in review

In the last decade the study of human genetic variation has made a quantum leap from the analysis of protein and antigen intermediaries to the investigation of DNA itself.¹ The DNA double helix codes genetic information as a sequence of four different nucleotides: adenine (A), guanine (G), cytosine (C), and thymine (T). Nucleotides are nitrogenous bases that bind the complementary strands of the double helix, giving rise to the use of base pairs (bp) as a unit of DNA length. So far so good. Within the human genome there are DNA sequences that do not code for proteins and that consist of short runs of nucleotides, say GTGGACAGG, repeated in tandem hundreds, or even thousands of times. This particular sequence, known as MS1 for minisatellite 1, is found on human chromosome 1 at a locus called D1S7. Although it is old news that there are a lot of repetitive DNAs in the human genome, it is new and very interesting to find that many repetitive DNA loci have arrays of different numbers of repeats in different individuals. These loci are referred to as VNTRs, shorthand for “variable numbers of tandem repeats” or, more flippantly for “very nasty types of repeat.” The finding of hundreds of VNTR loci distributed across all chromosomes exposes a richness of genetic diversity for anthropologists studying human evolutionary history.     

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.     

Mechanism of DNA chain growth. XI. Structure of RNA-linked DNA fragments of Escherichia coli

The size of RNA attached to nascent DNA fragments of Escherichia coli with a chain length of 400 to 2000 nucleotides is estimated to be about 50 to 100 nucleotides from: (a) the density of the molecules of known sizes; (b) the decrease of the molecular size produced by hydrolysis with RNases or alkali; and (c) the size of RNA released by DNase treatment. Only a small decrease in molecular size is produced by RNase or alkali treatment, excluding the possibility that the RNA is located in the middle of the fragment or that ribonucleotide sequences are scattered in the molecule. The RNA is not located at the 3′ end of the molecule either, since the DNA is degraded by 3′ → 5′ exonuclease action of bacteriophage T4 DNA polymerase which has neither RNase nor DNA endonuclease activity. Positive evidence for the covalent attachment of the RNA to the 5′ end of the DNA is provided by the finding that one 5′-OH terminus of DNA is created from each RNA-linked DNA fragment by alkaline hydrolysis. The quantitative production of the 5′-OH group at the 5′ end of DNA is also found upon hydrolysis with pancreatic RNase, indicating that the 3′-terminal base of the RNA segment of the fragments is a pyrimidine. On the other hand, when the RNA-linked DNA fragments hydrolysed with alkali or pancreatic RNase are incubated with [γ-³²P]ATP and polynucleotide kinase and the DNA thus labelled is degraded to constituent 5′-mononucleotides, the ³²P is found only in dCMP. Therefore, C is the specific 5′-terminal base of the DNA segment of the RNA-linked DNA fragments, and the RNA-DNA junction has the structure … p(rPy)p(dC)p …     

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses

Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.