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At least 19 recordsLinked to original sources

Cell-cycle-associated rearrangement of inverted repeat DNA sequences.

Inverted repeat DNA sequences of Caulobacter crescentus have been isolated, characterized, and cloned in a bacteriophage lambda vector. Both whole populations and individual clones of these sequences were hybridized to restriction endonuclease-generated fragments of chromosomal DNA isolated from cells that were in different stages of the cell cycle. Some inverted repeat DNA sequences were observed to hybridize to different regions of the chromosomal DNA isolated from the morphologically and biochemically distinct swarmer cell and stalked cell populations. These results suggest that the inverted repeat sequences have the capacity to rearrange and thus be located at different sites on the genomes of the different cell types.

Bacteria

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.

Animals

Mapping of inverted repeated DNA sequences within the genome of simian virus 40.

Single-stranded, linear DNA of simian virus 40 (SV40) created by denaturing the endonuclease EcoRI- or Hpa II-generated, linear, double-stranded products from form I DNA of SV40 was analyzed for regions of inverted repeated sequences by visualization with the electron microscope. Six hairpin loops were found at positions 0.11-0.30 (two loops forming a "rabbit ears" structure), 0.47-0.52, 0.63-0.68, 0.70-0.76, and 0.90-0.96. The nucleotide sequences within all of these inverted repeats may be related since the looped regions can crosshybridize with one another and, thus, the SV40 genome may contain regions of interspersed repeated and unique sequences. The map positions of the 3' and 5' ends of the early and late messenger RNAs, as determined by others, lie within regions of inverted repeated sequences. Previously recorded recombination events that occurred either within the SV40 genome or between SV40 DNA and other genomes have apparently occurred frequently at positions of inverted repeated sequences within the SV40 DNA.

Base Sequence

Arrangement of a highly repeated DNA sequence in the genome and chromatin of the African green monkey.

The DNA of the African green monkey contains three components that are distinguishable by the kinetics of reassociation. The rapidly reassociating component represents about 20% of the total DNA and is composed almost entirely of a sequence (AGMr(HindIII)-1) which is repeated 6.8 x 10(6) times. The majority of the AGMr(HindIII)-1 sequences are organized in long tandem repeats of a segment of 172 base pairs in length. However, a fraction of the AGMr (HindIII)-1 sequences is interspersed with another 37% of the genome. The structure of the chromatin containing the AGMr-(HindIII)-1 sequence is indistinguishable from that containing total DNA. Furthermore, there is nothing inherent in the nucleotide sequence of AGMr(HindIII)-1 which specifies a unique location for nucleosomes.

Animals

Conservation of repeated DNA sequences in aneuploid human tumor cells.

A series of human neuroectodermal tumors, all containing more than the normal diploid DNA, and each with its own distinct chromosome mode, were studied using restriction enzyme cleavage and specific DNA sequence hybridization. Methods described were quite sensitive and quantitative and as few as 40 molecules with a given restriction site were reproducibly detected in total nuclear DNA. Analysis of several fluorescent gel bands associated with different chromosomal domains revealed no changes between any of the tumor and normal cells. Specific probe hybridization, using purified complex repeating sequences, indicated fidelity of base sequence, as well as preservation of the relative amounts of each of a number of minor related multimers in both the tumor and normal cells. Centromeric regions containing arrays of such sequences may be maintained in these tumor cells and furthermore it is possible that some of these cells are polyploid with respect to DNA sequences, rather than aneuploid as their chromosome profiles suggest.

Aneuploidy

Hybridization of mouse leukemia virus c-DNA to mouse repeated DNA sequences.

Experiments of hybridization between mouse leukemia virus synthetic 3H-DNA probe and mouse main band and satellite DNAs indicate that there is not a higher concentration of viral sequences in the satellite DNA. On the contrary, viral sequences appear to be enriched in the fast renaturing intermediate main band DNA.

Animals

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.

Animals

Evidence that populations of Dictyostelium single-copy mRNA transcripts carry common repeat sequences.

Two recombinant plasmids, M4 and KH10, carrying Dictyostelium DNA inserted into the Eco RI restriction endonuclease site of pMB9 by poly(dA)-poly(dT) tailing, were selected for study because they are complementary to abundant mRNA populations from Dictyostelium. Both plasmids have been shown to hybridize a heterogeneous size class of mRNAs which, in the case of KH10, comprise 5-10% of the pulse-labeled poly(A)+ RNA from vegetative cells. Analysis of the sequence organization of the two pieces of Dictyostelium DNA shows that they consist mostly of single-copy sequences with a short DNA sequence which is repeated in the genome and interspersed with single-copy DNA. These and other results suggest that the majority of the hybridization of pulse-labeled mRNA to M4 and KH10 is to the short "repeated" DNA sequences. In the genome, members of these repeat families appear to be transcribed onto a population of different single-copy mRNAs. Additional results show that M4 DNA contains a sequence which is entirely complementary to a discrete mRNA.

Base Sequence

Transcription of a defective polyoma virus genome.

The circular genome of the cloned defective polyoma virus D-50 consists of tandemly repeated copies of the DNA sequence between 67 and 84 units on the wild-type polyoma virus DNA map. Each repeated copy thus contains the origin of viral DNA replication, which is located at about 71 map units. Viral RNA was synthesized in vitro using viral transcription complexes extracted late (30 hr) after infection from mouse cells co-infected with D-50 and helper wild-type virus. Both wild-type and D-50 DNA molecules were active as templates for in vitro transcription. Approximately 84% of the RNA transcribed in vitro from wild-type DNA was complementary to the L DNA strand. This is normal for wild-type transcription late after infection. By contrast, at least 90% of the RNA transcribed from D-50 DNA molecules was complementary to the E DNA strand. After normalization of the data to account for the observed molar ratio of D-50 DNA repeated sequences to unit length wild-type DNA, we estimate that transcription of the E DNA strand of each D-50 repeated unit is about 1.4 times as efficient as transcription of the wild-type E DNA strand. Transcription of the D-50 L DNA strand, however, is only 0.03 times as efficient as transcription of the wild-type L DNA strand. The implications of these results concerning the nature and location of promoter sequences in polyoma DNA are discussed.

Binding Sites

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.

Base Sequence

Studies on DNA sequences in the Osmundaceae.

Phylogenetic relationships of Osmunda cinnamomea, O. claytoniana, and O. regalis were explored by means of DNA sequence comparisons. Hydroxyapatite thermal elution profiles of self-reassociated repetitive DNA fragments were very similar, indicating the absence of gross differences in the amount of recent amplification or addition of repetitive DNA in any of these three genomes. Interspecific DNA sequence comparisons showed, in contrast to our earlier interpretation, that repeated DNA sequences of O. claytoniana are nearly equally diverged from those of O. cinnamomea and O. regalis. Differences between repetitive sequences of the three species can be interpreted as reflecting amplification events which occurred subsequent to speciation. The data obtained suggest that the three Osmunda species most likely arose more or less simultaneously from a common ancestor. These findings were verified in experiments with tracer DNA preparations enriched for single copy sequences. On the basis of the hydridization data presented here and of the fossil record, the rate of single copy sequence divergence in the ferns is comparable to that in the primates, although slower than that observed in other animal taxa. From this first evaluation of rates of DNA evolution in plants it would seem that the rates for plants and animals are roughly comparable. The evidence suggests that species divergence is accompanied by further reiteration of preexisting repeat sequences. The rate of addition of repetitive sequences probably is slower in ferns than in angiosperms. This difference might be attributable to the much larger effective generation time in ferns.

Base Sequence

And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break.

Heterochromatin is mostly composed of repeated DNA sequences prone to aberrant recombination. How cells maintain the stability of these sequences during double-strand break (DSB) repair has been a long-standing mystery. Studies in Drosophila cells revealed that faithful homologous recombination repair of heterochromatic DSBs relies on the striking relocalization of repair sites to the nuclear periphery before Rad51 recruitment and repair progression. Here, we summarize our current understanding of this response, including the molecular mechanisms involved, and conserved pathways in mammalian cells. We will highlight important similarities with pathways identified in budding yeast for repair of other types of repeated sequences, including rDNA and short telomeres. We will also discuss the emerging role of chromatin composition and regulation in heterochromatin repair progression. Together, these discoveries challenged previous assumptions that repair sites are substantially static in multicellular eukaryotes, that heterochromatin is largely inert in the presence of DSBs, and that silencing and compaction in this domain are obstacles to repair.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

Animals

A critical examination of possible fractionations of human DNA according to base composition.

Human DNA has been fractionated according to base composition by sedimentation equilibrium in an HgCl2/Cs2SO4 density gradient, followed by sedimentation equilibrium in an actinomycin/cesium formate density gradient. The fractions of different base composition resulting from this procedure were subsequently analyzed by sedimentation equilibrium in CsCl, DNA renaturation kinetics, and electron microscopy. All fractions contain similar kinetic classes of repeated DNA sequences as judged by renaturation studies. Short (300 nucleotides) interspersed repeated sequences are found in all fractions with no noticeable enrichment for these sequences in any fraction. Repeated sequences from fractions of different base composition are partially able to cross-hybridize, demonstrating that nearly identical repeated sequences occur in molecules of different base composition. These findings are critically compared to reports of successful density gradient fractionations of different human DNA sequence classes.

Centrifugation, Density Gradient