Analysis of nucleosome arrangement on satellite DNA of rat liver chromatin

The arrangement of nucleosomes on the nucleotide sequence of satellite DNA of Oceanian rat (Rattus rattus) has been studied. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonucleaseIII and nuclease Sl. From the total population of core DNA fragments, the satellite-containing fragments were selected by molecular cloning and the complete nucleotide sequence of these clones was determined. The data show that nucleosomes occupy a number of preferred positions on satellite DNA. These positions are strictly defined. Thus location of nucleosomes along the satellite sequence is non-random. Such finding may have important biological significance.     

Analysis of rat repetitive DNA sequences.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.     

A non-curved chicken lysozyme 5'' matrix attachment site is 3'' followed by a strongly curved DNA sequence.

Matrix attachment regions (MARs) partition the genome into functional and structural loop-domains. Here, we determined the relative matrix affinity of cloned fragments of the chicken lysozyme 5' MAR. We show that this region contains a non-curved high-affinity binding site, which is 3' followed by a strongly curved DNA sequence that exhibits weak matrix binding. DNA curvature is not a physical property required for strong matrix binding. Possible biological functions of this sequence arrangement, particularly of the strongly curved DNA, are discussed.     

Satellite DNA from Xenopus laevis: comparative analysis of 745 and 1037 base pair Hind III tandem repeats.

Highly repetitive Hind III restriction fragments of 0.72-0.76 KBP from total Xenopus laevis genomic DNA are organized in a tandem like arrangement. Cloning of these fragments in pBR 322 with subsequent restriction site mapping and nucleotide sequence analysis of some selected clones showed two different types of sequences. 25-30% of material represent the oocyte specific 5 S DNA repeat units, 70-75% are similar to the recently described repeat elements of satellite 1 DNA. Hybridization of a genomic DNA library to such a 745 BP monomeric repeat unit and investigation of some clones with positive autoradiographic signals revealed structural heterogeneities of repeat elements, in that the 745 BP sequence cross-hybridized with 1037 BP Hind III repeat units. Nucleotide sequence analysis demonstrated that the two types of sequences show a homology of 84.3% and that the 1037 BP sequence additionally contains duplicated elements of the 745 BP sequence as well as apparently unrelated DNA sequences.     

Herpesvirus papio DNA is similar in organization to Epstein-Barr virus DNA.

EcoRI, HindII, SalI, nd XbaI restriction endonuclease maps of herpesvirus papio (HVPapio) DNA were derived by determining the fragment sizes and the linkage relationships between fragments generated by the different enzymes. The data indicate that HVPapio DNA has a single molecular arrangement which is similar to that of Epstein-Barr virus DNA. The size of the DNA was 110 X 10(6) to 114 X 10(6) daltons. Restriction fragments from both ends varied in the number of repeats of a 4 X 10(5)-dalton sequence, TR, and hybridized to each other. This suggests that there is an identical repeating unit, TR, at both ends of the DNA. There were usually six tandem repetitions (range, 1 to 11) of a 2 X 10(6)-dalton sequence, IR, within the DNA. IR separated the DNA into two domains of largely unique sequence complexity, a 9 X 10(6)-dalton segment, Us, and an 88 X 10(6)-dalton segment, UL. There was homology between DNA fragments which mapped at 25 X 10(6) to 29 X 10(6) to 91 X 10(6) to 95 X 10(6) daltons in UL.     

Sequence of 1019 nucleotides encompassing one of the inverted repeats from the yeast 2 micrometer plasmid.

A sequence of 1019 nucleotides encompassing one of the 600 base inverted repeats and non-repeated flanking regions has been determined in the type A yeast 2 micrometers plasmid cloned in pMB9. Methods are described for applying the Maxam-Gilbert sequencing procedure to DNA fragments labelled at the 3'-end using a T4-polymerase exchange/repair reaction and for sequencing 5'-end labelled fragments using dideoxy-nucleotides as chain terminators in the presence of E. coli DNA polymerase (nach Klenow). A notable feature of the sequence is its unusual content of symmetry elements. In one region of 140 nucleotides, 137 are involved in a complex arrangement of direct and inverted repeats linked by palindromic sequences.     

Mode of DNA binding by SopA protein of Escherichia coli F plasmid

The binding of SopA to the promoter region of its own gene, in which four copies of SopA's recognition sequence, 5'-CTTTGC-3', are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5'-CTTTGC-3' was changed to 5'-CTTACG-3'. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA's DNA binding is based on its intrinsic protein-protein interaction modified by DNA interaction.     

Specific protection of DNA by distamycin A, netropsin and bis-netropsins against the action of DNAse I

Interaction of netropsin, distamycin A and a number of bis-netropsins with DNA fragments of definite nucleotide sequence was studied by footprinting technique. The nuclease protection experiments were made at fixed DNA concentration and varying ligand concentrations. The affinity of ligand for a DNA site was estimated from measurements of ligand concentration that causes 50% protection of the DNA site. Distribution pattern of the protected and unprotected regions along the DNA fragment was compared with the theoretically expected arrangement of the ligand along the same DNA. The comparison led us to the following conclusions: 1. Footprinting experiments show that at high levels of binding the arrangement of netropsin molecules along the DNA corresponds closely to the distribution pattern expected from theoretical calculations based on the known geometry of netropsin--DNA complex. However, the observed differences in the affinity of netropsin for various DNA sequences is markedly greater than that expected from theoretical calculations. 2. Netropsin exhibits a greater selectivity of binding than that expected for a ligand with three specific reaction centers associated with the antibiotic amide groups. It binds preferentially to DNA regions containing four or more successive AT pairs. Among 13 putative binding sites for netropsin with four or more successive AT pairs there are 11 strong binding sites and two weaker sites which are occupied at 2 D/P less than or equal to 1/9 and 2 D/P = 1/4, respectively. 3. The extent of specificity manifested by distamycin A is comparable to that shown by netropsin although the molecule of distamycin A contains four rather than three amide groups. At high levels of binding distamycin A occupies the same binding sites on DNA as netropsin does. 4. The binding specificity of bis-netropsins is greater than that of netropsin. Bis-netropsins can bind to DNA in such a way that the two netropsin-like fragments are implicated in specific interaction with DNA base pairs. However, the apparent affinity of bis-netropsins estimated from footprinting experiments is comparable with that of netropsin for the same DNA region. 5. At high levels of binding bis-netropsins and distamycin A (but not netropsin) can occupy any potential site on DNA irrespectively of the DNA sequence. 6. Complex formation with netropsin increases sensitivity to DNase I at certain DNA sites along with the protection effect observed at neighboring sites.     

Anatomy of Herpes Simplex Virus DNA XII. Accumulation of Head-to-Tail Concatemers in Nuclei of Infected Cells and Their Role in the Generation of the Four Isomeric Arrangements of Viral DNA

Previous reports (H. Delius and J. B. Clements, J. Gen. Virol. 33:125-134, 1976; G. S. Hayward, R. J. Jacob, S. C. Wadsworth, and B. Roizman, Proc. Natl. Acad. Sci. U.S.A. 72:4243-4247, 1975; B. Roizman, G. S. Hayward, R. Jacob, S. W. Wadsworth, and R. W. Honess, Excerpta Med. Int. Congr. Ser. 2:188-198, 1974) have shown that herpes simplex virus DNA extracted from virions accumulating in the cytoplasm of infected cells consists of four populations of linear molecules differing in the orientation of the covalently linked large (L) and small (S) components relative to each other. Together, these four isomeric arrangements of viral DNA display four different termini and four different L-S component junctions. In the studies reported in this paper, we analyzed with restriction endonucleases the newly replicated viral DNA shortly after the onset of viral DNA synthesis, the progeny DNA accumulating in the nuclei late in infection, and rapidly sedimenting DNA present in nuclei of infected cells at 8 h after infection. In each instance the nuclear viral DNA contained a decreased concentration of all four terminal fragments and an increase in the concentration of fragments spanning the junction of L and S components relative to the concentration of other DNA fragments. The results are consistent with the hypothesis that the viral DNA accumulating in the nuclei consists of head-to-tail concatemers arising from the replication of DNA by a rolling-circle mechanism. A model is presented for generation of all four isomeric arrangements of herpes simplex virus DNA from one arrangement based on excision and repair of unit length DNA from head-to-tail concatemers and known features of the sequence arrangement of viral DNA.     

Herpesvirus saimiri DNA in tumor cells--deleted sequences and sequence rearrangements.

Herpesvirus saimiri DNA in continuous lymphoblastoid cell lines obtained from viral induced tumors in marmosets has been analyzed by gel electrophoresis of restricted DNA. Southern transfer to nitrocellulose filters, and hybridization to 32P-labeled viral DNA or DNA fragments. The viral DNA fragments EcoRI-G, -H, -D, and -I, KpnI-A, and BamHI-D and -E were not detected in Southern transfers of DNA from the nonproducing 1670 cell line. For each restriction endonuclease, a new fragment appeared, consistent with a 13.0-megadalton deletion of viral DNA sequences. This deletion encompassed 35 to 48 +/- 0.6 megadaltons from the left end of the unique DNA region. A sequence arrangement map is presented for the major population of H. saimiri DNA sequences in the 1670 cell line. Although H. saimiri DNA in the nonproducing 70N2 cell line can be distinguished from viral DNA in the 1670 cell line by several criteria, the same sequences were found to be deleted in the major population of viral DNA molecules. Unlike 1670 and 70N2 cells, restricted DNA from the virus-producing cell lines 77/5 and 1926 contained all of the DNA fragments present in the parental virion DNA. DNA from 1670, 70N2, and 77/5 cells contained additional viral DNA fragments that did not comigrate with any virion DNA fragments. Most of these unexplained fragments were confined to or highly enriched in partially purified circular or linear DNA fractions. DNA from tumor cells taken directly from a tumor-bearing animal contained viral DNA indistinguishable from the parental virion DNA by the assay conditions used. These results indicate that viral DNA sequence rearrangements can occur upon cultivation of tumor cells in vitro and that excision of DNA sequences from the viral genome may play a role in establishing the nonproducing state of some tumor cell lines.     

AFLP: a new technique for DNA fingerprinting.

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.     

A highly bent fragment of Crithidia fasciculata kinetoplast DNA

Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two-dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation.     

Characterization of the genome of the free-living nematode Panagrellus silusiae: absence of short period interspersion.

The complexity of the DNA of the free-living nematode Panagrellus silusiae has been examined. Reassociation kinetics of pressure-sheared fragments (approximately 290 nucleotides) in 0.18 M Na+ at 60 degrees C showed the presence of foldback, repetitive, and unique DNA sequence elements. The three classes comprise 9.3%, 26.1%, and 61.3% of the total DNA, respectively. The mean length of the foldback duplex DNA after digestion with S1 nuclease is about 185 nucleotides. There are about 1.8 x10(4) inverted repeats per genome. Sequence arrangement was deduced from (1) renaturation kinetic profiles of long and short fragments on hydroxylapatite; (2) the pattern of renaturation of tracer DNA, labeled in vitro with 125I, of various sizes after incubation with excess short fragments; and (3) thermal denaturation behavior of DNA that had been reassociated to various C0t values. It was found that DNA fragments of the repetitive fraction that are, at least, 2000 nucleotides in length are virtually free of unique sequences. Moreover, it is estimated that the repeated segments in this species could extend for 10,000 nucleotide pairs. Thus, Panagrellus DNA lacks the pattern of extensive short period interspersion that is typified by the DNA of Xenopus.     

Kinetoplast DNA of Crithidia oncopelti. Recognition of the principles of structural organization of minicircular DNA molecules

The heterogeneity of the kinetoplast minicircular DNAs from Crithidia oncopelti was studied by electron microscopy and restriction analysis. The associate of kinetoplast DNA comprises five main classes of minicircles sizing 0.42, 0.51, 0.62, 0.78 and 0.83 microns. Examination of cleavage patterns revealed that each class of the minicircles is heterogeneous in base sequence, but there are restriction fragments of the same size common for all these classes. A model of structural arrangement of minicircles of Crithidia oncopelti was proposed. This model is based on the block structure of minicircular molecules. Each minicircle is composed of one variable block (V-block), 810 base pairs, and from 1 to 4 more conservative blocks (C-blocks) each containing 445 base pairs.     

Map of restriction sites on bacteriophage T4 cytosine-containing DNA for endonucleases bamHI, BglII, KpnI, PvuI, SalI, and XbaI.

A complete map of the cleavage sites of restriction endonucleases BamHI, BglII, KpnI, PvuI, SalI, and XbaI was determined for the cytosine-containing DNA of a bacteriophage T4 alc mutant. The 56 sequence-specific sites were assigned map coordinates based on a least-squares analysis of measured fragment lengths. Altogether, the lengths of 118 fragments from single and double enzyme digestions were measured by electrophoresis of the fragments in agarose gels. DNA fragments of known sequence or DNA fragments calibrated with fragments of known sequence were used as standards. The greatest deviation between an experimentally measured fragment length and its computed map coordinates was 3.0%; the average deviation was 0.8%. The total length of the wild-type T4 genome was calculated to be 166,200 base pairs.     

Structure and function of Y chromosomal DNA

The sequence organization of four different families of Y chromosomal repetitive DNA is characterized at three levels of spatial extension along the Y chromosome of Drosophila hydei. At the lowest level of resolution, DNA blot analysis of Y chromosomal fragments of different lengths and in situ hybridization experiments on metaphase chromosomes demonstrate the clustering of each particular sequence family within one defined region of the chromosome. At a higher level of resolution, family specific repeats can be detected within these clusters by crosshybridization within 10–20 kb long continuous stretches of cloned DNA in EMBL3 phages. At the highest level of resolution, detailed sequence analysis of representative subclones about 1 kb in length reveals a satellite-like head to tail arrangement of family specific degenerated subrepeats as the building scheme common to all four families. Our results provide the first comparative sequence analysis of three novel families of repetitive DNA on the long arm of the F chromosome of D. hydei. Additional data are presented which support the existence of two related subfamilies of repetitive DNA on the short arm of the Y chromosome.     

Sequence replication and banding organization in the polytene chromosomes of Drosophila melanogaster

The relative proportions of cloned DNA fragments from all known hierarchies of sequence organization in polytene and diploid chromosomes were compared. It was found that unique sequences of varying sizes and chromosomal locations are equally replicated in salivary gland chromosomes. Sequences of euchromatic polydisperse gene families are also replicated proportionately in polytene and diploid tissues. Perhaps the most significant finding is that the histone gene repeats, despite their normal banding organization, are under-replicated in the polytene chromosome of Drosophila melanogaster. However, the clustered and well-banded 5S genes are most likely equally replicated. It is therefore concluded that differential sequence replication plays no apparent role in either the assembly or morphology of a band; and likewise, the assembly of polytenic DNA into band units is not affected by either the local abundancy or arrangement of middle repetitive sequences. The likelihood that the clustered arrangement is an important factor in the selection of sequences for under-replication is discussed.     

DNA of Epstein-Barr virus. IV. Linkage map of restriction enzyme fragments of the B95-8 and W91 strains of Epstein-Barr Virus.

The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).     

DNA sequence organization in the genomes of five marine invertebrates

The arrangement of repetitive and non-repetitive sequence was studied in the genomic DNA of the oyster (Crassostrea virginica), the surf clam (Spisula solidissima), the horseshoe crab (Limulus polyphemus), a nemertean worm (Cerebratulus lacteus) and a jelly-fish (Aurelia aurita). Except for the jellyfish these animals belong to the protostomial branch of animal evolution, for which little information regarding DNA sequence organization has previously been available. The reassociation kinetics of short (250-300 nucleotide) and long (2,000-3,000 nucleotide) DNA fragments was studied by the hydroxyapatite method. It was shown that in each case a major fraction of the DNA consists of single copy sequences less than about 3,000 nucleotides in length, interspersed with short repetitive sequences. The lengths of the repetitive sequences were estimated by optical hyperchromicity and S1 nuclease measurements made on renaturation products. All the genomes studied include a prominent fraction of interspersed repetitive sequences about 300 nucleotides in length, as well as longer repetitive sequence regions.