DNA topology in heterochromatin (a hypothesis).

A hypothesis explaining the known heterochromatin features--a compact DNA packaging, transcriptional inactivity, propensity to aggregate (stickiness) and position effect variegation-is described. The hypothesis is based on the assumption that DNA molecules in heterochromatin are topologically open and contain single-strand breaks in the regions with identical or similar primary sequences.

[Maintenance of torsional tension in DNA of transactivated genes during the cell cycle]. / Sokhranenie v kletochnom tsikle torsionnogo napriazheniia v DNK transkriptsionno-aktivnykh genov.

Changes in DNA topology during the cell cycle of murine fibroblasts in vitro were studied by microfluorometric method. At interphase stage, 21% of DNA were found to be torsionally stressed and hypersensitive to the relaxing activity of DNAase I. This DNA is presumably transcriptionally active. At metaphase stage, about 75% of DNA are under torsional stress with low sensitivity to DNAase I. After cytokinesis, one half of the stressed DNA relaxes, the other half of DNA remains under the stress. Later, one hour after cytokinesis, the stressed DNA attains hypersensitivity to DNAase I and remains hypersensitive during interphase. Taking into account that the cells in vitro steady maintain some domains of torsionally stressed DNA during the whole cell cycle, we suppose that these DNA domains contain genes, activated to transcription after mitosis. This may mean that the torsional stress preserved after mitosis is necessary for activation of the genes, which were active at the previous interphase stage.

[Torsional tension in metaphase chromosome DNA]. / Torsionnoe napriazhenie v DNK metafaznykh khromosom.

The torsional tension in DNA of isolated metaphase chromosomes from murine fibroblasts was measured by the microfluorescent method. The method is based on the ability of a fluorescent dye ethidium bromide to compensate for the negative torsional tension in topologically closed DNA by intercalation between DNA base pairs. The value of the relative twist difference delta Tw/Tw = -0.1 was found in a bulk (about 3/4) of unconstrained chromosomal DNA. In interphase nuclei, the torsionally stressed DNA comprises about 15%, with value of delta Tw/Tw = -0.075. We suppose that the tension in chromosomal DNA was created in the prophase stage of mitosis by condensines, the drivers of chromosomal condensation.

[Heterochromatin and single-strand DNA breaks (a hypothesis)]. / Geterokhromatin i odnonitevye razryvy DNK (gipoteza).

A hypothesis is put forward which explains the known heterochromatin properties: dense DNA packing, transcriptional inactivity, a tendency for aggregation (adhesiveness) and ectopic contacts. The basic assumption of the hypothesis is that the DNA molecules in heterochromatin are topologically open and contain single-strand breaks in the regions with the same or similar base sequence.

[Preparation of Drosophila mitotic chromosomes for electron-microscopic studies]. / Prigotovlenie preparatov mitoticheskikh khromosom drozofily dlia élektronno-mikroskopicheskogo issledovaniia.

A method of chromosome spreading on microscopic slides was modified for electron microscopy of metaphase chromosomes in Drosophila tissues. The slides covered with an electron transparent film were plasmochemically modified to make them hydrophilic. A piece of fixed tissue was macerated in 60% propionic acid before spreading chromosomes over the slide. The parts of preparation selected under light microscope for electron microscopic examination were cut and peeled of the slide to the top of a water drop. It was shown that the resolution of chromosomal structures was significantly higher than seen under optical microscope, but lower than in serial sections.

Torsional state of DNA in a transcriptionally hyperactive Balbiani ring of polytene chromosomes.

The torsional tension of unconstrained double-helical DNA was determined in transcriptionally hyperactive Balbiani ring 2 (BR2) and in inactive polytene chromosome bands of Chironomus tentans. The method used is based on the dual ability of small intercalating ligands to (a) sense, by differential binding, twists that deviate from that of regular B-form DNA and (b) create positive torsional tension in closed double-stranded DNA, thereby compensating for any negative torsional tension that existed before intercalation. Isolated nuclei of salivary glands were stained with the intercalating fluorescent dye ethidium bromide (EtBr) at various concentrations, and the temporal fluorescence intensity changes (deltaI/I per min) occurring in BR2 and in inactive bands were monitored under a confocal laser scanning microscope during the process of DNA nicking by laser irradiation or DNAase I. From the EtBr concentration at which deltaI/I per min was neither positive nor negative after nicking (i.e. at the equivalence point), the relative twist difference (RTD) was calculated. In bands, it was found to be very small, suggesting that their unconstrained DNA is under low torsional stress. In contrast, the RTD of DNA in highly expanded areas of BR2 was estimated to be negative and of a significant magnitude in absolute terms. This indicates that transcriptionally hyperactive DNA is under considerable negative torsional tension.

Topological state of DNA in polytene chromosomes.

A new microfluorometric method was developed for measuring two topological characteristics of DNA in isolated nuclei, chromosomes and other DNA containing structures: (1) the relative amount of the topologically non-closed DNA (tncDNA) and (2) the supercoiling density of the topologically closed unconstrained DNA (tcDNA). The method was applied to isolated polytene nuclei and chromosomes of Chironomus thummi. The relative amount of tncDNA was found to be 0.21. Evidence in favour of the tncDNA localization in transcriptionally active loci (puffs) of the polytene chromosomes is presented. The supercoiling density of tcDNA localized, presumably, in inactive loci (bands) of the polytene chromosomes is about -0.001.

[Structure of virions of the M13 phage containing chimeric B-protein molecules]. / Izuchenie struktury virionov faga M13, soderzhashchikh molekuly khimernykh B-belkov.

We have studied the virion structure of M13 strains (M13B1, M13BOM1, M13BOM2, M13BOL1) with chimeric variants of B-protein. Data concerning the spatial structure of chimeric B-protein molecules and their interaction with intraphage DNA were obtained. The phage contour lengths were measured under electron microscope and the DNA/protein ratios were obtained by spectrophotometry. These data testified that the insertion of foreign peptide affected neither DNA packaging nor the compactness of molecular arrangement of proteins in the virion. By linear dichroism and fluorescence spectra of phages it was determined, that the insert can influence the polarity of amino acid environment and the orientation of amino acids in the B-protein central part. It was shown by quenching of phage fluorescence by KI that the inward or outward amino acids location in the capsid is invariable. The carboxyl residues have been titrated in the phage strains by Auramine O. It was shown that there is no correlation between the number of the titrated carboxyl groups and the number of the carboxyl groups as a whole.

Functional and structural units in the chromomere.

Electron microscopic observations demonstrate the existence of several DNA packing levels in the chromomere. A linear DNA molecule forms a big (chromomere) loop anchored to the chromosomal scaffold. The loop forms a set of smaller loops in the rosette pattern. Packing of the DNA by the histone octamer particles results in nucleosomes and nucleomeres. To establish the possible correspondence between the structural units of a chromomere and the genetical units (genes, exons, introns) in it, we compared the lengths of the units. Statistical analysis of the 315 sequenced genes indicate that the average gene size corresponds to the average length of a rosette loop. It means that a chromomere contains one or more genes. Assuming that exon-intron boundaries cannot bind nucleosomes we constructed DNA-packing models of the 88 genes. They demonstrate that the first (in 77.8 per cent of the genes) and the last (in 52.7 per cent) exons of the genes are too short to bind nucleosomes. Many genes contain long (nucleosome binding) pieces of DNA. Long packed pieces are introns in vertebrates; they are exons in invertebrates and plants. The average size gene contains two nucleomeres.

[Changes in the cellular structure of the salivary glands in Chironomus larvae resulting from mechanical cell damage]. / Izmenenie struktury kletok sliunnykh zhelez lichinok khironomusa v rezul'tate mekhanicheskogo povrezhdeniia kletok.

Rapid morphological changes were observed in some cells of hand-isolated salivary glands of Ch. thummi larvae. The nuclear envelope, routinely closely fitting the tightly packaged polytene chromosomes, was seen to lose its contact with the chromosomes and to attain a smooth round shape. Then unfolding of the chromosomes occurred, their banding patterns becoming clearly evident, probably through widening the interband regions; the chromosome length increased by about 20%. We argue that the changes observed were induced during gland isolation by lesions of the cell basal envelope in the sites of the fat body connections to the salivary gland.

[Effect of the length of internucleosome regions of DNA on the conformation of chromatid fibrils]. / Vliianie dliny mezhnukleosomnykh uchastkov DNK na konformatsiiu khromatidnykh fibrill.

Models of chromatin fibers structures with linear regions of linker DNA were analysed. Limitations put by end dimensions of linker DNA and nucleosomes are considered. Good agreement between the structural properties of model and real chromatin fibers was obtained. It has been shown that the models with three and more configurations of closely located nucleosomes have linker DNA of 19-53 base pairs length, which is characteristic of real chromatin of the majority of somatic cells.

Fluorescence polarization study of tertiary structure of DNA within bacteriophage lambda.

The fluorescence polarization of acridine orange-stained, oriented lambda phages was measured. The parameters of DNA packing within the phage head cos2 theta and cos4 theta were calculated (theta, angle between the direction of a small segment of DNA and the phage axis). It is shown that simple models of lambda phage DNA tertiary structure are not consistent with calculated values. A new model is proposed.

[Models of chromatin fibril organization]. / Modeli organizatsii fibrill khromatina.

Idealized models of chromatin fibers are presented. They are based on the data of nucleosome structure and on the postulate about lengths congruence and rectilinearity of internucleosomal DNA fragments. It has been shown that the majority of models are in the form of spirals with the diameter 30--40 nm consisting of chains of successively located nucleosomes. With some lengths of internucleosomal stretches the spirals are transformed to linear columns with two, three of four rows of nucleosomes. The DNA packing coefficient strongly depends on the shape of the model. The structure of real chromatin fibers is believed to be close to that of compact models. Corrections of the models due to supercoiling of internucleosomal DNA stretches are considered qualitatively.

[Site-specific scission of DNA of polytene chromosomes of Chironomus thummi in situ by the oligoadenylate alkylating derivatives]. / Induktsiia napravlennykh razryvov v DNK politennykh khromosom Chironomus thummi in situ pri deistvii alkiliruiushchikh proizvodnykh oligoadenilatov.

The possibility of site-specific scission of DNA in polytene chromosomes in situ by means of the method of complementarily addressed fragmentation is demonstrated. The fragmentation of polytene chromosomes of Chironomus thummi resulting from the alkylation of the denatured chromosomes with oligoadenylate derivatives, followed by scission of the DNA in specific sites and enlargement of nicks with exonucleases was investigated. Single-stranded regions were registered by means of luminescence microscopy after staining the chromosomes with acridine orange. The pattern of degradation of the chromosomes depends on the length of the oligoadenylate part of the reagents and is different from that obtained with uridine-5'-methylphosphate alkylating derivative which does not form any complexes with DNA. Oligoadenylates inhibit the action of the alkylating derivatives on the chromosomes.

[Evidence of single-strandedness in eukaryotic chromosomes]. / Dokazatel'stvo odnonitchatosti khromosom éukariot.

Polytene salivary gland chromosomes of Chironomus thummi were stretched in pronase solution by 176 +/- 26 times up to their break (fig. 1). The DNA packed arrangement coefficient, determined as a ratio of DNA length, equal to 85 +/- 5 mm in a haploid set, to the length of 520 +/- 40 microns of a set of polytene chromosomes was found to be 164 +/- 22. The coincidence of these two values is a very strong evidence in favour of the uninemity of chromatids of the Chironomus chromosomes. The effect of ethidium bromide on elastic properties of chromosomes, preliminary stretched in pronase (fig. 2), and the lengthening of these chromosomes after ethidium staining prove that DNA molecules are double-stranded and supercoiled until chromosomes are broken. This enables us to conclude that each chromatid of Chironomus consists of a single DNA molecule or, more probably, of a single chain of linked DNA molecules whose both ends are located in telomeres of chromosomes.

[Model of chromonema packing in the mitotic chromosome]. / Model' ukladki khromonemy v mitoticheskoi khromosome.

Based on available data, a model of packing of a chromonemata in mitotic chromosomes is presented (fig. 1). The model reflects three well established facts: 1) chromosomes are uninemic, i.e. each chromonema consists of a single DNA molecule (or a single chain of linked DNA molecules), whose ends are located in telomeres; 2) a proteinaceous fibre is a structural basis of a chromatid; the fibre is folded, its ends are located near a centromere; 3) a chromonema makes a spiral in the chromatid. The model reveals half-chromatid structure of mitiotic chromosomes and may describe morphological changes of chromosomes induced by substances affecting the states of chromonemata or fibres.