Structural organization of a hypermethylated nuclear DNA component in Physarum polycephalum

Digestion of Physarum polycephalum nuclear DNA using the restriction endonuclease HpaII generates two components, distinguishable on the basis of their molecular size. The high-molecular-weight, HpaII-resistant component, which accounts for 20% of the DNA, contains a fivefold greater concentration of 5-methylcytosine residues than the low-molecular-weight HpaII-digested fraction. Segments of hypermethylated (M+) DNA are largely composed of a single, long, highly repeated sequence, and this major element is sometimes associated with other less highly repetitive sequences in the M+ DNA fraction. Restriction mapping of cloned Physarum M+ DNA segments, and Southern blot analysis of genomic DNA using subcloned segments of M+ DNA as a probe, provide evidence for sequence variation within different copies of the dominant highly repeated element, and possibly the other associated repeats in M+ DNA, and additionally that almost complete tandemly repeated copies of the major repeat are found in some M+ DNA segments.     

Transposon-like properties of the major, long repetitive sequence family in the genome of Physarum polycephalum

A family of long, highly-repetitive sequences, referred to previously as `HpaII-repeats', dominates the genome of the eukaryotic slime mould Physarum polycephalum. These sequences are found exclusively in scrambled clusters. They account for about one-half of the total complement of repetitive DNA in Physarum, and represent the major sequence component found in hypermethylated, 20-50 kb segments of Physarum genomic DNA that fail to be cleaved using the restriction endonuclease HpaII. The structure of this abundant repetitive element was investigated by analysing cloned segments derived from the hypermethylated genomic DNA compartment. We show that the `HpaII-repeat' forms part of a larger repetitive DNA structure, ~8.6 kb in length, with several structural features in common with recognised eukaryotic transposable genetic elements. Scrambled clusters of the sequence probably arise as a result of transposition-like events, during which the element preferentially recombines in either orientation with target sites located in other copies of the same repeated sequence. The target sites for transposition/recombination are not related in sequence but in all cases studied they are potentially capable of promoting the formation of small `cruciforms' or `Z-DNA' structures which might be recognised during the recombination process.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Genomic organisation of DNA segments containing foldback sequences

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hydridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.     

Methylation of nuclear DNA in Physarum polycephalum.

The restriction endonucleases HpaII and HhaI, whose action is inhibited by the presence of methylated base analogues at the recognition sequences in the DNA substrate, were used to investigate the distribution of 5-methylcytosine in nuclear DNA from Physarum polycephalum. Physarum DNA is digested into two fractions by these enzymes: a low-molecular-weight (M--) compartment comprising 80% of the DNA, and a high-molecular-weight (M+) compartment containing 20% of the DNA. The DNA fraction showing resistance to digestion by restriction endonuclease HpaII is cleaved by its isoschizomer MspI, indicating that methylated endonuclease-HpaII-specific sites are present in M + DNA. Additional properties of sequences in the M+ compartment were investigated.     

Clusters of repeated sequences of chicken DNA are extensively methylated but contain specific undermethylated regions

In the chicken genome there are middle repetitive DNA sequences with a clustered organization. Each cluster is composed of members of different families of repeated DNA sequences and usually contains only one member of each family. Many clusters have the same assortment of repeated sequences but they are in scrambled order from cluster to cluster. These clusters usually exceed 20 × 10³ bases in length and comprise at least 10% of the repeated DNA of the chicken. The repeated sequences that are cluster components are extensively methylated. Methylation was detected by comparing HpaII and MspI digests of total DNA, where the occurrence of the sequence C-m⁵C-G-G is indicated when HpaII (cleaves C-C-G-G) fragments are larger than those generated by MspI (cleaves C-m⁵C-G-G or C-C-G-G). In hybridization experiments with Southern (1975) blots of total DNA digested with either HpaII or MspI, the cloned probes representing clustered repeated sequences showed a dramatic difference in the lengths of restriction fragments detected in the two digests. Many of the sequences that comprise these clusters are methylated in most of their genomic occurrences. There are patterns of methylation that are reproduced faithfully from copy to copy. The overall distribution of methylation within clusters seems to be regional, with long methylated DNA segments interrupted by specific undermethylated regions.     

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 organisation in nuclear DNA from Physarum polycephalum: methylation of repetitive sequences.

Nuclear DNA from the slime mould Physarum polycephalum is digested by the restriction endonuclease HpaII to generate a high molecular weight and a low molecular weight component. These are referred to as the M+ and the M- compartment, respectively. Sequences that are present in the M+ compartment are cleaved by MspI, the restriction enzyme isoschizomer of HpaII, thus showing that the recognition sequences for these enzymes in M+ DNA contain methylated CpG doublets. The distribution of repetitive sequences in the M+ and M- DNA compartments was investigated by comparison of the 'fingerprint' patterns of total Physarum DNA and isolated M+ DNA after digestion using different restriction endonucleases, and by probing for the presence of specific repetitive sequences in Southern blots of M+ and M- DNA by the use of cloned DNA segments. Both types of experiment indicate that many repetitive sequences are shared by both compartments, though some repetitive sequences appear to be considerably enriched, or are present exclusively, either in M+ DNA or in M- DNA.     

The analysis of some new Drosophila repetitive DNA sequences isolated and cloned from two-dimensional agarose gels

Drosophila melanogaster DNA (Dm) was sequentially cleaved by BamHI and EcoRI and separated by two-dimensional gel electrophoresis. Six different prominent bands, which are derived primarily from the cleavage of long sequences that are repeated 20-100 times per genome, were recovered from the gel and cloned in pBR322. Hybridization and restriction analysis of the cloned Dm segments showed that three of these bands are mainly derived from the ribosomal and histone gene repeating units. Segments cloned from the other three bands are not homologous to any known repeating elements that we have tested. They represent long repetitive sequences of moderate multiplicity that appear not to have been hitherto described. These segments have been restriction-mapped and hybridized to cDNA prepared from poly(A)RNA from adult flies. While two minority segments did hybridize to this probe, the majority failed to hybridize. The arrangement of genomic sequences homologous to each plasmid was tested by restriction analysis and Southern hybridization. The results indicate that the repetitive element is largely conserved intact although occupying numerous different positions in the genome. The DNAs from four different strains of D. melanogaster and two of D. simulans produced restriction patterns having some segment lengths in common and some showing clear differences, a fact that indicates that these sequences can move about to occupy different genomic locations in different strains.     

Genomic representation of the Hind II 1.9 kb repeated DNA.

The genomic representation and organization of sequences homologous to a cloned Hind III 1.9 kb repeated DNA fragment were studied. Approximately 80% of homologous repeated DNA was contained in a genomic Hind III cleavage band of 1.9 kb. Double digestion studies indicated that the genomic family, in the majority, followed the arrangement of the sequenced clone, with minor restriction cleavage variations compatible with a few base changes. Common restriction sites external to the 1.9 kb sequence were mapped, and hybridization of segments of the cloned sequence indicated the 1.9 kb DNA was itself not tandemly repeated. Kpn I bands which were homologous to the sequence contained specific regions of the repeat, and the molecular weight of these larger fragments could be simply explained. Mapping of common external restriction sites indicated that in some but not all cases the repeat could be organized in larger defined blocks of greater than or equal to 5.5 kb. In some instances, flanking regions adjacent to the repeat may contain common DNA elements such as other repeated DNA sequences, or possibly rearranged segments of the 1.9 kb sequence. It is suggested that although the 1.9 kb sequence is not strictly contiguous, at least some of these repeated sequences in the human genome are arranged in clustered or intercalary arrays. A region of the 1.9 kb sequence hybridized to a mouse repeated DNA, indicating homology beyond the primates.     

Centromeric tandem repeat from the chaffinch genome: isolation and molecular characterization.

A new family of avian centromeric satellites is described. The highly repeated sequence, designated FCP (Fringilla coelebs PstI element), was cloned from the 500-bp PstI digest fraction of the chaffinch (Fringilla coelebs L.) genomic DNA, sequenced, and characterized. The FCP repeat was found to have 505-506 bp length of monomer, 57% content of GC, to compose about 0.9% of the chaffinch genome, and to be highly methylated. Results of Southern-blot hybridization of cloned FCP element onto genomic DNA digested with different restriction enzymes, and sequencing directly from total genomic DNA using FCP-specific primers and ThermoFidelase enzyme (Fidelity Systems Inc.) were in agreement with a tandem arrangement of this repeat in the chaffinch genome. Five positions of single-nucleotide polymorphism (SNP) were found in the FCP monomers using direct genomic sequencing. Fluorescence in situ hybridization (FISH) with FCP probe and primed in situ labelling (PRINS) with FCP specific primers showed that the FCP elements occupy pericentric regions of all chaffinch chromosomes. On chromosome spreads, the fluorescent signals were also observed in the intercentromeric connectives between nonhomologous chromosomes. The results suggest that the centromeric FCP repeat is responsible for chromosome ordering during mitosis in chaffinch.     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

Analysis of foldback sequences and repetitive sequences in cloned segments of nuclear DNA from Physarum polycephalum

The properties of DNA segments containing foldback elements were studied after their selection from a ‘random’ recombinant library of Physarum polycephalum nuclear DNA sequences, cloned using the plasmid vector pBR322. Hybridisation of in vitro labelled recombinant plasmids to Southern blots of genomic restriction fragments demonstrated that each cloned segment contained repetitive elements located at several hundred sites in the genome. Two of the DNA clones generated hybridisation patterns which suggested that they contain repetitive elements with internal cleavage sites for the restriction endonuclease HaeIII. Cross-hybridisation of all combinations of the cloned sequences showed that most contain different arrangements of repetitive elements derived from different sequence families. The results are consistent with a model proposed previously on the basis of studies on total nuclear DNA, for the organisation of sequences closely associated with foldback elements in the Physarum genome     

Rapid identification of hybridization probes for chromosomal walking

The presence of repeated elements in restriction fragments used as hybridization probes for chromosomal walking poses a major obstacle to the success of this gene-cloning strategy. This report describes a simple and rapid means of identifying restriction fragments devoid of repeated sequences and therefore useful as hybridization probes for chromosomal walking. Restriction fragments derived from a genomic DNA clone are Southern blotted and hybridized to nick-translated total genomic [32P]DNA. Fragments of the genomic clone that contain high abundance sequences (i.e., repeated elements) hybridize strongly to their nick-translated counterparts, which, due to their high copy number, comprise a significant proportion of the total genomic DNA probe. Conversely, fragments containing single-copy or low-abundance sequences do not hybridize, as their nick-translated counterparts are poorly represented in the total genomic DNA probe. These latter fragments, by virtue of their low-abundance sequences, are well suited as probes for chromosomal walking. Ensuring the absence of repeated elements in restriction fragments prior to their purification and utilization as chromosomal walking probes results in marked savings of time, effort and materials.     

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.     

A family of moderately repetitive sequences in mouse DNA.

When mouse DNA is digested to completion with restriction endonuclease Eco R1, a distinct band of 1.3 kb segments comprising about 0.5-3% of the genome is observed upon agarose gel electrophoresis. This DNA is not tandemly repeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kb band and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 10(4) times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco R1 band.     

Abundance and polymorphism of di-, tri-and tetra-nucleotide tandem repeats in chickpea (Cicer arietinum L.)

The abundance and polymorphism of 38 different simple-sequence repeat motifs was studied in four accessions of cultivated chickpea (Cicer arietinum L.) by in-gel hybridization of synthetic oligonucleotides to genomic DNA digested with 14 different restriction enzymes. Among 38 probes tested, 35 yielded detectable hybridization signals. The abundance and level of polymorphism of the target sequences varied considerably. The probes fell into three broad categories: (1) probes yielding distinct, polymorphic banding patterns; (2) probes yielding distinct, monomorphic banding patterns, and (3) probes yielding blurred patterns, or diffused bands superimposed on a high in lane background. No obvious correlation existed between abundance, fingerprint quality, and the sequence characteristics of a particular motif. Digestion with methyl-sensitive enzymes revealed that simple-sequence motifs are enriched in highly methylated genomic regions. The high level of intraspecific polymorphism detected by oligonucleotide fingerprinting suggests the suitability of simple-sequence repeat probes as molecular markers for genome mapping.     

DNA methylation in the fungi

A systematic study on the incidence and patterns of cytosine methylation in the fungi has been carried out by restriction and nearest-neighbor analysis of DNAs isolated from undifferentiated cells of several fungal species. With respect to DNA modification, the fungi appear to be a heterogeneous group, with a 5-methylcytosine content ranging from undetectable levels (less than or equal to 0.1% of cytosine residues methylated in 18 out of 20 species tested) to low but detectable levels (e.g. congruent to 0.2 and congruent to 0.5% of the total cytosines methylated in Sporotrichum dimorphosporum and Phycomyces blakesleeanus, respectively). In the species where it has been detected, 5-methylcytosine is located mostly at CpG doublets, and the methylated sites are clustered in long tracts (10-30 kilobase pairs) separated from essentially unmethylated regions. This methylated compartment, which comprises a small fraction (1-11%) of the total DNA, contains at least a specific set of repetitive sequences. These results contrast with the higher 5-methylcytosine content found in the fungus Physarum polycephalum and in vertebrates and higher plants.     

Methylated and unmethylated DNA compartments in the sea urchin genome.

Sea urchin (Echinus esculentus) DNA has been separated into high and low molecular weight fractions by digestion with the mCpG-sensitive restriction endonucleases Hpa II, Hha I and Ava I. The separation was due to differences in methylation at the recognition sequences for these enzymes because an mCpG-insensitive isoschizomer of Hpa II (Msp I) digested Hpa II-resistant DNA to low molecular weight, showing that many Hpa II sites were in fact present in this fraction; and because 3H-methyl methionine administered to embryos was incorporated into the high molecular weight Hpa II-, Hha I- and Ava I-resistant fraction, but not significantly into the low molecular weight fraction. The fraction resistant to Hpa II, Hha I and Ava I amounted to about 40% of the total DNA. It consisted of long sequence tracts between 15 and well over 50 kg in length, in which many sites for each of these enzymes were methylated consecutively. The remaining 60% of the genome, (m-), was not significantly methylated. Methylated and unmethylated fractions were considered to be subfractions of the genome because enriched unique sequences from one fraction cross-reassociated poorly with the other fraction and specific sequences were found in either (m+) or (m-) but not in both (see below). Similar (m+) and (m-) compartments were found in embryos, germ cells and adult somatic tissues. Furthermor, we found no evidence for changes in the sequence composition of (m+) or (m-) between sperm, embryo or intestine DNAs, although low levels of exchange would not have been detected. Using cloned Echinus histone DNA, heterologous 5S DNA and ribosomal DNA probes, we have found that each of these gene families belongs to the unmethylated DNA compartment in all the tissues examined. In particular, there was no detectable methylation of histone DNA either in early embryos, which are thought to be actively transcribing the bulk of histone genes, or in sperm and gastrulae, in which most histone genes are not being transcribed. In contrast to these gene families, sequences complementary to an internally repetitious Echinus DNA clone were found primarily in the methylated DNA compartment.