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.

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.

[Relative positions of stainable portions of DNA in deoxyribonucleoprotein fibrils]. / Vzaimnoe raspolozhenie okrashivaemykh uchastkov DNK v fibrillakh dezoksiribonukleoproteidov.

The effect of acridine orange (AO) concentration on fluorescence polarization of DNA, DNP100 and DNP250 solutions (100 A and 249 A thick DNP fibers, respectively) was studied. It was shown that fluorescence depolarization by an excitation energy migration mechanisms is 3.5 times more efficient for DNP250 than for DNP100. Higher efficiency of energy migration may be explained by a tighter packing of stained DNA regions inside the 250 A fiber because both DNP100 and DNP250 have equal numbers of AO binding sites. The calculated average number of packed DNA strands in DNP250 is 3, the average angle between them is 26.5 degrees. A model of the 250 A DNP fiber is proposed. In this model nucleosomes form a tight 250 A coil with a 430 A pitch, internucleosomal DNA strands are oriented approximately along the fiber axis.

[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.

[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.

[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.

[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.

[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.

[Nonhomogeneity of the distribution of the absorbing substance in cytophotometry]. / Neravnomernost' respredeleniia pogloshchaiushchego veshchestva v tsitofotometrii.

Results of two-wavelengths cytophotometry may be used to calculate m--the quantity of light absorbing substance, and gamma--the degree of non-homogeneity of distribution of the substance on a preparation. Formula (1) is almost free from any distributional error. Optical density limits of applicability of formula (3) for gamma are shown on fig. 2. An algorithm for calculation of gamma at higher optical densities is proposed.

[Preparation and properties of stretched polytene chromosomes. 2. Mechanism of stretching]. / Poluchenie i svoitsva rasianutykh politennykh khromosom. 2. Mechanizm rastiazheniia.

Isolated polytene chromosomes were stretched in a 0.125 M NaCl solution with constant speed, by constant force and by cyclically changing force. For each regime, the dependence of chromosome length on the time and force magnitude were recorded. From this it may be concluded that three processes are involved in chromosome stretching: viscoelastic deformation, viscous flow of DNP segments, and cristallization, i.e. intermolecular cross-linking of neighbour segments. At a high rate stretching (V greater than Vo) chromosome may be torn like at small deformation; when rate is V greater than Vo chromosome deformation is mostly viscoelastic; at rates V approximately Vo viscous flow of DNP segments if predominant. We estimate Vo approximately less than 3--6 mum/s. Electron microscopy shows that during chromosome stretching its DNP fibers are oriented along chromosome axis without detectable breaks.

[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.

[Stretched polytene chromosomes--model for studying the functional organization of eukaryotic chromosomes]. / Rastianutye politennye khromosomy--model' dlia izucheniia funktsional'noi organizatsii khromosom éukariot

To localize functional loci on cytological maps of polytene chromosomes we propose to use 10-100 times stretched chromosomes. Three different ways of stretchening are briefly considered: the squash tissue preparation, when chromosomes are stretched by hydrodynamical forces; the treatment of isolated polytene chromosomes in 10-minus 4M EDTA OR 0.8M NaCL with subsequent change of these solution for saline when abrupt structural changes occur in chromosomes and they become morphologically homogeneous threads (Gruzdev and Belaya, 1973); and, finally, the use of microneedles of the micromanipulator. After an intense (ca. 100 times) stretchening, the autoradiography is sufficient to localize the loci within one micron length of double helical DNA molecule.

[Preparation and properties of elongated polytene chromosomes. I. The method]. / Poluchenie i svoistva rastianutykh politennykh khromosom. I. Metod.

Compactness of eukaryotic, particularly polytene chromosomes pose difficulties for investigation of their functional organization. For example, 0.1 --1.0 micrometer thick bands of polytene chromosomes contain some dozens microns pieces of DNA molecules. Therefore the useful resolving power of autoradigraphical methods is reduced by 10--100 times of its upper limit. To overcome the mentioned difficulty, a new method has been developed which permits to attain the upper limit of resolution. An isolated polytene chromosome from salivary gland nucleus of Chironomus thummi larva is stretched by microneedles to obtain a bundle of oriented DNP-fibers. A previously chosen small region of the chromosome (band or puff) is stretched simultaneously in a transverse direction by a stick frame made of another chromosome. Electron microscopy of the preparation reveals a meshwork of DNP fibers as presented on fig. 6.

[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.

[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.

[Cytogenetic effect of ultraviolet rays in mammalian cells at the DNA synthesis stage]. / Tsitogeneticheskii éffekt ul'trafioletovykh luchei v kletkakh mlekopitaiushchikh na stadii sinteza DNK

The frequency of chromosome aberrations induced by UV light at various wavelengths in the primary culture of mouse embryonic fibroblasts during the S-phase was studied. The aberration frequency is wavelength-dependent and reaches a maximum at 265 nm. The action spectrum for the chromosome aberrations determined at 254, 265, 280 and 302 nm closely conforms to the absorption spectra of thymidine. The value of caffeine potentiation was the same for 265 and 280 nm UV-induced aberrations. This indicates that primary chromosome damages and their transformation in cells are similar at these two wavelengths. The data obtained suggest that the formation of DNA cross-links following thymine dimerization is the first step in the formation of UV-induced chromosome aberrations in mammalian cells at the S phase.

[DNA packing in the DNP fibrils of polytene chromosomes]. / Ukladka DNK v fibrillakh DNP politennykh khromosom.

Isolated polytene chromosomes stained with acridine orange were stretched with micromanipulator needles. Changes of chromosome fluorescence polarization led to the conclusions: 1) almost complete orientation of DNP fibers is attained at very low levels of chromosome stretching (3--8 times) without irreversible disruption of chromomeres; 2) internucleosomal DNA in native DNP fibers is supercoiled and makes 30 degrees angle to the fiber axis.

[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.

[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.

[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.