Tightly bound matrix DNA probably plays an important role in organization of chromosome centromeres

A large variety of DNA sequences have been described in matrix attachment regions (MARs), This is a result of the different methods used for their isolation. Here we used the conventional procedure for preparing nuclear matrix from Friend-S cells and found that the fibrogranular network of the matrix is formed by uniform-looking tightly packed granules. The residual DNA extracted from matrix is enriched in highly repetitive sequences. Our previous investigations indicated that these MARs-DNA are probably constitutive in the cell cycle. The DNA was partially cloned to obtain additional information about the nature of these sequences, In sits hybridization with cloned DNAs showed that the formation of clusters in interphase nuclei is a result of close disposition of the centromeric regions of a few chromosomes, Computer analysis of the sequences of two MARs did not reveal any significant homologies with the known sequences in the NCBI database. The comparison of other two clones showed a high degree of similarity to mouse repetitive families, In one of the clones we found four TGGAA motifs which could nucleate stem-loop structures. This unusual structure could explain the unique morphology and function of the mouse centromere in mitosis

The induction of DNA strand breaks at specific sites by N-nitroso-N-ethylurea depends on the phases of the cell cycle

Synchronized root meristems of Pisum sativum were treated at each phase of the cell cycle with 6.25 mM N-nitroso-N-ethylurea. DNA extracted from treated cells and run in agarose gel electrophoresis exhibits a series of discrete fragments with length below 2500 bp and a significant number of unspecific single-stranded breaks (or alkali-labile sites). Experiments with micrococcal nuclease indicated that the nucleosomal organization of the chromatin is not responsible for the generation of the discrete fragments: it seems that their appearance is associated with a preferable attack of the mutagen at specific sites, characteristic for the plant genome. Moreover, a cell cycle dependent release of the discrete fragments was found with maximum at G1-S and minimum at mitosis. The model experiments designed to clarify this observation suggest that it might be determined from the cell cycle dependent fluctuation in the accessibility of the chromatin DNA and/or the process of excision-repair

Localisation of DNA topoisomerase II alpha in mouse erythroleukemia cells

The presence of DNA topoisomerase II alpha was investigated in interphase and metaphase mouse erythroleukemia (MEL) Friend-S cells, and in extracted with 25 mM lithium diiodosalicylate buffer (Lis) nuclei using indirect immunofluorescence. The results showed that DNA topoisomerase II alpha is localised in the nuclei. In the metaphase cells, we found high concentrations of this enzyme in the mitotic chromosomes. Our results support the idea of the accumulation of DNA topoisomerase II alpha at the end of the cell cycle. The extractions of nuclei with 25 mM Lis led to the complete depletion of DNA topoisomerase II alpha from the residual nuclear matrix. Using a high dilution of the first antibody, we established that the high level of heterochromatin compactisation in the interphase nuclei is caused by the high concentration of DNA topoisomerase II alpha

Interphase Chromosomes of Friend-S Cells Are Attached to the Matrix Structures Through the Centromeric/Telomeric Regions

DNA of the attachment sites of Friend erythroleukemia cells, isolated according to the conventional procedure, represents short, nuclease-resistant fragments with sizes below 400 bp, belonging to the class of mouse satellite. A number of experiments have indicated that their unusual resistance is due to complexing with RNA. By various approaches, it was confirmed that similar fragments might be recovered from total DNA following extensive digestion with DNase I. In situ hybridizations revealed further that at mitosis the sequences of the attachment sites are located at the centromeric/telomeric regions of the chromosomes, while at interphase they are redistributed into 9-13 well-defined clusters spread throughout the entire nuclear area. Parallel biochemical and electronomicroscopic studies have clarified, moreover, that the all three compartments of the matrix harbor such sequences. Thus, it appears that the attachment sites described function only at interphase, anchoring the both ends of each interphase chromosome to the matrix structures

Spatial and structural segregation of the transcribed and nontranscribed alleles of c-myc in Namalva-S cells

By using various approaches we received evidence that, in Namalva-S cells carrying a t(8;14) translocation and highly expressing c-myc, the two alleles of the gene are spatially and structurally segregated. Spatial segregation of the alleles was observed in all nuclei analyzed by in situ hybridization technique. Their structural segregation, i.e., association with different intranuclear structures, was confirmed in a number of experiments. When high-salt extracted nuclei were digested with EcoRI, which is known to produce fragments containing the entire c-myc locus, the sequences of the gene were found separated between the pellet, containing sequences firmly associated with the heavier matrix structures, and the supernatant, containing sequences from the free length of the DNA loops. Southern hybridization performed with a probe representative for the constant region of the human IgH locus revealed that this fractionation in fact segregates the reorganized from the normal allele of c-myc. Run-on experiments carried out with two fractions, topologically equivalent to the above P and S but isolated as intact chromatin structures, indicated that the allele associated with nuclear matrix is actively transcribed, while that located in the free length of the chromatin loops is practically nontranscribed. Studies on the chromatin organization of transcribed and nontranscribed alleles revealed the existence in them of two alternative chromatin structures. Control experiments with beta-globin gene, performed with cells constitutively nontranscribing or actively transcribing this gene, confirmed our conclusions about the spatial segregation of the two alleles and clarified that their structural segregation occurs when the gene is activated for transcription