Mapping and positioning DNA-binding proteins along genomic DNA. Structure of D. melanogaster ribosomal 'Alu-repeats' and 1.688 satellite chromatin.

Chromatin structure of so-called 'Alu-repeat' in D. melanogaster ribosomal non-transcribed spacer that contains sequences homologous to the promoter of ribosomal genes has been studied. Using the 'protein image' hybridization assay based on UV-light-induced DNA-protein crosslinking and 2-D gel retardation electrophoresis, two proteins of the molecular mass of 50 kD (rABP50) and 70 kD (rABP70), associated with 'Alu-repeat' DNA have been found. Exo III mapping of crosslinking sites and DNase I footprinting have provided a detailed map of H1, rABP50 and rABP70 contacts within the 'Alu-repeat' and H1 and a non-histone protein contacts on satellite DNA. These data indicate precise positioning of non-histone proteins, histone H1 and nucleosomes within genomic regions studied and account for the presence of unusual 240 bp long nucleosomal particles in 'Alu-repeats'. The same approach can be adapted for successive mapping and positioning proteins on genomic DNA.     

Different repeat lengths in rat satellite I DNA containing chromatin and bulk chromatin.

The nucleosome repeat structure of a rat liver chromatin component containing the satellite I DNA (repeat length 370 bp) was investigated. Digestion experiments with micrococcal nuclease, DNAase II, and the Ca2+/Mg2+-dependent endogenous nuclease of rat liver nuclei revealed a repeat unit of 185 nucleotide pairs which is shorter by approximately 10 bp than the repeat unit of the bulk chromatin of this cell type. The difference seems not to be related to the histone composition which was found to be similar in the two types of chromatin.     

Preparation of chromatin containing ribosomal deoxyribonucleic acid from the macronucleus of Tetrahymena pyriformis.

A method is described that enables a chromatin fraction containing ribosomal DNA (DNA containing sequences coding for rRNA) to be prepared from the macronuclei of growing or stationary cultures of Tetrahymena pyriformis. This material is obtained in yields of between 25 and 75% of the theoretical maximum. The DNA in this fraction was identified as ribosomal DNA on the basis of its density and molecular weight, and it appears not to be appreciably contaminated by other DNA. The method relies on the approximate assumption that ribosomal DNA is the smallest species of DNA in chromatin in the nucleus, and avoids the use of mechanical force, or enzyme action, to fractionate chromatin.     

A general method for preparing chromatin containing intact DNA.

A simple and general method is described for preparing chromatin from eukaryotic cells using isotonic conditions. First, cells are encapsulated in agarose microbeads and then lysed using Triton X-100 in the presence of a chelating agent and a physiological concentration of salt. Most cytoplasmic proteins and RNA diffuse rapidly out through pores in the beads to leave encapsulated chromatin which is nevertheless completely accessible to enzymes and other probes. This chromatin can be manipulated freely without aggregation in a variety of different salt and detergent concentrations. It also contains intact DNA since removal of the histones releases superhelical DNA. Conditions are described for incubating this chromatin at 37 degrees C in the presence of Mg2+ ions without any nicking of the DNA. We illustrate the usefulness of this chromatin in investigations on the attachment of nascent RNA to the nucleoskeleton, the accessibility of the ribosomal locus to EcoRI and the properties of the endogenous RNA polymerase II. This type of chromatin preparation should prove useful for both structural and functional studies.     

Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum.

We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.     

Structure of the extrachromosomal ribosomal RNA chromatin of Physarum polycephalum

Isolated nucleoli from exponentially growing microplasmodia of Physarum polycephalum were digested with micrococcal nuclease or DNAase I, or were photoreacted with trimethyl psoralen. In the coding region for the precursor of the ribosomal RNA, micrococcal nuclease and DNAase I digestions show predominantly a smear, and treatment with psoralen leads to a fairly continuous crosslinking of the DNA. All three assays are compatible with the absence of a typical nucleosomal array in most of the gene copies. In contrast, in the central non-transcribed spacer, except in the immediate 5'-flanking region, micrococcal nuclease and DNAase I digestions yield fragments that are multiples of a basic repeat, compatible with a nucleosomal packing of this region. The crosslinking pattern with psoralen confirms this conclusion. In addition, there are three sites over 400 base-pairs long that are inaccessible for psoralen crosslinking. Two of these sites have been mapped to the putative origins of replication. In the terminal non-transcribed spacer, except in the immediate 3'-flanking region, digestions with micrococcal nuclease and DNAase I give a smeared repeat. The crosslinking pattern after treatment with psoralen suggests that this region is packed in nucleosomes, except for about 900 base-pairs constituting the telomere regions of the linear extrachromosomal palindromic rDNA. Micrococcal nuclease digestion of the immediate 5'-flanking region shows a complete absence of any nucleosomal repeat, but digestion with DNAase I leads to a faint ten base-pair repeat. In contrast, in the 3'-flanking regions both nuclease assays indicate a chromatin structure similar to the coding region. Both flanking regions are unusual with respect to psoralen crosslinking, in that crosslinking is reduced both in chromatin and deproteinized DNA. On the basis of the known sequence-dependent psoralen crosslinking and the established sequences in these regions, crosslinking should be expected to occur. However, it does not and we therefore propose the presence of an unusual DNA conformation in these regions.     

The ribosomal DNA of Calliphora erythrocephala; an analysis of hybrid plasmids containing ribosomal DNA

We have analysed the ribosomal DNA of Calliphora erythrocephala, a Dipteran fly of the same sub-order as Drosophila melanogaster, through a series of rDNA² fragments cloned in a plasmid vector. We have mapped the sites for eight restriction enzymes within these plasmids, and positioned the regions coding for the 18 S and 28 S rRNAs within the maps of selected plasmids using the S₁ endonuclease mapping procedure of Berk & Sharp (1977). This analysis establishes that some rDNA cistrons of C. erythrocephala contain an “intron” (Gilbert, 1978) which interrupts the 28 S coding region at the same position as that of D. melanogaster rDNA. Two introns of 2.85 kilobases in length and part of a longer, sequence-related variant were isolated in these cloned fragments. Restriction enzyme site analysis and preliminary hybridization data indicate that the 2.85 kb intron of C. erythrocephala is largely unrelated in sequence to the two classes of D. melanogaster rDNA introns.     

Analysis of highly purified satellite DNA containing chromatin from the mouse.

A purification scheme for satellite DNA containing chromatin from mouse liver has been developed. It is based on the highly condensed state of the satellite chromatin and also takes advantage of its resistance to digestion by certain restriction nucleases. Nuclei are first treated with micrococcal nuclease and the satellite chromatin enriched 3-5 fold by extraction of the digested nuclei under appropriate conditions. Further purification is achieved by digestion of the chromatin with a restriction nuclease that leaves satellite DNA largely intact but degrades non-satellite DNA extensively. In subsequent sucrose gradient centrifugation the rapidly sedimenting chromatin contains more than 70% satellite DNA. This material has the same histone composition as bulk chromatin. No significant differences were detected in an analysis of minor histone variants. Nonhistone proteins are present only in very low amounts in the satellite chromatin fraction, notably the HMG proteins are strongly depleted.     

Structure of cloned ribosomal DNA cistrons from Bacillus thuringiensis.

A library of B. thuringiensis DNA has been prepared by using the plasmid pBR322 as a cloning vehicle and E. coli as a host cell. By screening this collection with specific probes, 17 clones were identified whose hybrid plasmids contain rRNA genes of B. thuringiensis. Several of these plasmids have been mapped with restriction endonucleases and by DNA-RNA hybridization. By using maps of overlapping fragments, we have been able to establish an overall map of the ribosomal gene cluster.     

DNA damage alters nuclear mechanics through chromatin reorganization

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.     

Characterisation of ribosomal satellite in total nuclear DNA from Physarum polycephalum.

The distinctive properties of satellite DNA molecules containing the genes for ribosomal RNA in Physarum polycephalum permits their identification in total, unfractionated nuclear DNA in the foldback form, after denaturation and fast annealing. Using the electron microscope the location and properties of three characteristic regions containing tandemly-repeated, inverted sequences have been investigated. At least two additional regions, also containing tandem repeats, are shown to be present and located towards each end of the rDNA molecule, at a site adjacent to the segment coding for the 26 S rRNA. All the regions which contain tandem repeats are composed of sequences which, within experimental error, appear to share a common unit repeat length of about 90 nucleotides.     

Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardii

Summary Hybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×10⁶ d and two SalI ribosomal fragments of 3.8 and 1.5×10⁶ d. The ribosomal DNA (rDNA) units, 5.3×10⁶ d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.Abbreviations used rRNA ribosomal RNA - rDNA ribosomal DNA d, dalton     

Genetic analysis of nuclear ribosomal DNA of Lentinula edodes

Genetic analysis of nuclear ribosomal DNA (rDNA) of Lentinula edodes was carried out using rDNA restriction fragment length polymorphisms (RFLPs) as genetic markers. Two compatible monokaryotic strains that differed in the endonuclease digestion patterns of their rDNA were used. The dikaryotic strain established by crossing them produced mixed RFLP patterns. Single-spore isolates derived from the dikaryotic strain showed three types of rDNA RFLP patterns: either one of the two parental types or a mixed type. From the frequency of the mixed type, the recombination value of rDNA tandem repeats was calculated to be 31.4%. Linkage analysis between rDNA and two incompatibility factors (A and B) revealed that rDNA was not linked to either factor. The rDNA genotypes did not affect mycelial growth among the single-spore isolates.     

Characterization of the nuclear ribosomal DNA of Euglena gracilis

A phage lambda recombinant library containing Euglena gracilis genomic DNA was screened for nuclear rDNA sequences. A recombinant phage was isolated that contained an 11.5-kb nuclear rDNA sequence. The 11.5-kb insert was mapped with restriction endonucleases and was shown to represent a complete rDNA repeat unit that carried the genes for the 19S, 25S, 5.8 S and 5 S cytoplasmic rRNAs. The 2000 rDNA repeat units per haploid genome are organized in the form of identical tandem repeats.