Morphological Criteria of Cell Differentiation Stages in Experimental Hepatocarcinoma and Evaluation of Antitumor Drug Efficiency.

Structural polymorphism of 5 cell differentiation stages of hepatocarcinoma-29 from ascitic fluid is detected and the morphological criteria for identification of these stages are defined on the base of optic and electron microscopy findings, cytofluorometry, and DNA cytometry. The percentage of cells at differentiation stages 4 and 5 in the tumor structure increases after hepatocarcinoma cell inoculation into the hip. Injection of a cell cycle-modulating substance to animals with tumor growth shifts the proportion of cells with various differentiation stages. The morphological criteria of 5 stages of hepatocarcinoma-29 cell differentiation can be used for prospective drug testing.

Raman spectroscopy for DNA quantification in cell nucleus.

Here we demonstrate the feasibility of a novel approach to quantify DNA in cell nuclei. This approach is based on spectroscopy analysis of Raman light scattering, and avoids the problem of nonstoichiometric binding of dyes to DNA, as it directly measures the signal from DNA. Quantitative analysis of nuclear DNA contribution to Raman spectrum could be reliably performed using intensity of a phosphate mode at 1096 cm(-1) . When compared to the known DNA standards from cells of different animals, our results matched those values at error of 10%. We therefore suggest that this approach will be useful to expand the list of DNA standards, to properly adjust the duration of hydrolysis in Feulgen staining, to assay the applicability of fuchsines for DNA quantification, as well as to measure DNA content in cells with complex hydrolysis patterns, when Feulgen densitometry is inappropriate.

[Chromomeric organization of interphase chromosomes in Drosophila melanogaster].

As a result of treatment of bioinformatic data on the genome localization of structural proteins, histone modifications, DNase-hypersensitive regions, replication origins (taken from modENCODE) and their cytological localization to polytene chromosome structures, it is shown here that two types of interphase chromosomes -polytene chromosomes from salivary glands and from mitotically dividing cells cultures - demonstrate identical pictures of interband/band, i. e. the same localization and length on physical map and the same sets of proteins. In the interbands of both chromosome types we find the proteins that control initiation of transcription (RNA-polymerase II, transcription factors), replication (ORC2) as well as proteins modifying nucleosome structure (WDS, NURF) and proteins of insulators (BEAF). The nucleosome density and H1 histone concentration in the interbands are depleted; localization of DNase-hypersensitive regions corresponds strictly to the interbands. So, we conclude that both polytene and cell line interphase chromosomes are arranged according to general principle and polytene chromosomes represent precise model of interphase chromosomes. The interbands play a critical role in the initiation of transcription and replication. The interbands of interphase chromosomes are the sites of 5' parts of genes, while the 3' gene ends are located in the adjacent bands. The constancy of interbands decondensation results in the conclusion that the "interbands" genes are constantly active, i. e. they contain "house-keeping" genes. The large late replicating bands contain genes that do not have direct contact to the adjoining interbands are usually polygenic and contain tissue-specific genes.

[Identification and molecular genetic characterization of the polytene chromosome interbands in Drosophila melanogaster].

Being inserted into the polytene chromosome interbands, P transposable elements integrated in the genome of Drosophila produce new bands, enabling their use as markers of interband positions on the physical map. Molecular genetic analysis of 13 interbands marked as described showed that in most cases these regions were represented by intergenic spacers and by 5' noncoding regions of the genes. The interband regions consist of unique chromatin type whose decondensation is not obviously associated with transcription. In addition, interbands are enriched with the specific CHRIZ protein. Comparison of chromosomal protein sets and histone modifications in the polytene chromosome interband regions and in the corresponding sequences of the diploid cell chromosomes demonstrated their complete similarity relative these characteristics. In both cell types, interband regions contained open chromatin markers, including RNA polymerase II, ORC, GAF, TRX, and acetylated histones. At the same time, these regions appeared to be depleted of the repressed chromatin proteins, PC, E(Z), H3K9Me3, H3K27Me3, and some others. The similarity between interband chromosomal regions from different cell types is also manifested in the sets of DNAse I hypersensitive sites, which proved to be hot spots for transposon insertions. Our results suggest that band-interband structure is a fundamental principle of the interphase chromosome organization.

[DNA contents in nuclei of Cyclops kolensis and C. insignis (Crustacea, Copepoda)].

Chromatin diminution (CD) in two Cyclopoida species, Cyclops kolensis and C. insignis, was studied by static digital Feulgen cytophotometry. DNA content (pg/cell) was evaluated by standard curves builded up using blood cells of five organisms with known DNA content, which ranged from 1.25 to 14.70 pg. According to data obtained, diploid genome of C. kolensis has about 40 pg DNA before CD and 1.8-2.0 pg DNA after CD. These values are similar for both Moscow and Baikal populations of C. kolensis and 6-10 times exceed estimates made earlier (Grishanin, 2008), Our data confirm that CD in C. kolensis is 94-96% of DNA. In mitotic dividing cells of C. insignis, DNA content was about 7.5 pg both in early and late embryos, and CD was not revealed for this species. The data obtained show that, among Cyclopoida studied, the genome of C. kolensis before CD has a maximum content of DNA.

[An intergral method for quantitative cell DNA content measurements by digital microphotography].

Quantitative nuclei DNA content measurement based on Feulgen reaction and the analysis of CCD images was tested. The measurements were performed in monochorome CCD option (650 X 514 pixels) with the wavelength 551 nm. The linear dependence of photomatrix elements signals on the falling light was shown with the use of multigraded light absorption filter. The optimal microscope and camera setting and an approach for elimination of the optic blur were found. We have shown that the proper Feulgen fluorescence does not affect our measurements. Densitometric DNA content measurements of blood cells of four vertebrate species (Gallus domesticus, Danio rerio, Homo sapiens and Rana arvalis) showed good consistence to the literature data (http://www.genomesize.com). The precision of our approach is comparable to the other known methods. Current improvement of CCD technical parameters and wide usage of CCD cameras in biological applications gives perspectives for the suggested approach.

[Intercalary heterochromatin in the genome of Drosophila].

The modern concept of intercalary heterochromatin as polytene chromosome regions exhibiting a number of specific characteristics is formulated. DNA constituting these regions is replicated late in the S period; therefore, some strands of polytene chromosomes are underrepresented; i.e., they are underreplicated. Late-replicating regions account for about 7% of the genome; genes are located there in clusters of as many as 40. In general, the gene density in the clusters is substantially lower than in the main part of the genome. Late-replicating regions have an inactivating capacity: genes incorporated into these regions as parts of transposons are inactivated with a higher probability. These regions contain a specific protein SUUR affecting the rate of replication completion.

[Functional organization of interbands in Drosophila polytene chromosomes].

The functional organization of particular chromosome regions is tightly associated with their function in eukaryotic cells. Details of this association are among the most topical problems of modem genetics. The paper characterizes the results of recent research of the specifics of the genetic organization and chromatin decondensation in interbands of Drosophila polytene chromosomes. Data on functional heterogeneity of interbands are considered. Experimental findings point to a lack of correlation between the decondensed chromatin state and the observed transcription level in particular interbands. The DNA sequences responsible for the interband formation are principally identifiable via site-specific homologous FRT/FLP recombination between two P transposons contained in chromosomes. The results allow a search for particular protein factors that are involved in the decondensed state of interbands and structural and functional differentiation of polytene chromosomes.

The Drosophila dosage compensation complex binds to polytene chromosomes independently of developmental changes in transcription.

In Drosophila, the dosage compensation complex (DCC) mediates upregulation of transcription from the single male X chromosome. Despite coating the polytene male X, the DCC pattern looks discontinuous and probably reflects DCC dynamic associations with genes active at a given moment of development in a salivary gland. To test this hypothesis, we compared binding patterns of the DCC and of the elongating form of RNA polymerase II (PolIIo). We found that, unlike PolIIo, the DCC demonstrates a stable banded pattern throughout larval development and escapes binding to a subset of transcriptionally active areas, including developmental puffs. Moreover, these proteins are not completely colocalized at the electron microscopy level. These data combined imply that simple recognition of PolII machinery or of general features of active chromatin is either insufficient or not involved in DCC recruitment to its targets. We propose that DCC-mediated site-specific upregulation of transcription is not the fate of all active X-linked genes in males. Additionally, we found that DCC subunit MLE associates dynamically with developmental and heat-shock-induced puffs and, surprisingly, with those developing within DCC-devoid regions of the male X, thus resembling the PolIIo pattern. These data imply that, independently of other MSL proteins, the RNA-helicase MLE might participate in general transcriptional regulation or RNA processing.

Polytene chromosomes: 70 years of genetic research.

Polytene chromosomes were described in 1881 and since 1934 they have served as an outstanding model for a variety of genetic experiments. Using the polytene chromosomes, numerous biological phenomena were discovered. First the polytene chromosomes served as a model of the interphase chromosomes in general. In polytene chromosomes, condensed (bands), decondensed (interbands), genetically active (puffs), and silent (pericentric and intercalary heterochromatin as well as regions subject to position effect variegation) regions were found and their features were described in detail. Analysis of the general organization of replication and transcription at the cytological level has become possible using polytene chromosomes. In studies of sequential puff formation it was found for the first time that the steroid hormone (ecdysone) exerts its action through gene activation, and that the process of gene activation upon ecdysone proceeds as a cascade. Namely on the polytene chromosomes a new phenomenon of cellular stress response (heat shock) was discovered. Subsequently chromatin boundaries (insulators) were discovered to flank the heat shock puffs. Major progress in solving the problems of dosage compensation and position effect variegation phenomena was mainly related to studies on polytene chromosomes. This review summarizes the current status of studies of polytene chromosomes and of various phenomena described using this successful model.

[Use of immunogold labelling technique for immunoelectron microscope localization of proteins in Drosophila polytene chromosomes]. / Immunoélektronno-mikroskopicheskaia lokalizatsiia belkov na politennykh khromosomakh drozofily s pomoshch'iu immunnoi tekhniki mecheniia zolotom.

Using gold labeled antibodies, we developed and tested an immunoelectron microscope (IEM) method for detection of protein localization in Drosophila melanogaster polytene chromosomes. This method is based on procedures widely used for indirect immunofluorescent (IF) staining of salivary gland polytene chromosome squashes. The application of IEM was evaluated by using specific antibodies against proteins earlier localized in both decondensed (interbands and puffs) and compact (bands) regions of polytene chromosomes. In all the experiments, IEM and IF images for homologous chromosome regions were compared. When applied to regions of loose structures, IEM enabled us to localize, with high precision, signals in fine bands, interbands and puffs. There was a good correspondence between immunogold EM and IF data. However, there was no correspondence for dense bands: gold particles were distributed at their boundaries, while the entire bands showed bright fluorescence. This discrepancy probably resulted from a poor penetration of antibodies conjugated to gold particles in the tightly packaged structures. From the results obtained it may by concluded that the IEM method is advantageous for studying the fine protein topography of loose decompacted regions of polytene chromosomes. And this must be taken into consideration when protein localization in polytene chromosomes is performed.

Formation and morphology of dark puffs in Drosophila melanogaster polytene chromosomes.

The formation of unusual dark puffs in Drosophila melanogaster polytene chromosomes has been studied by electron microscopic (EM) analysis. Fly stocks transformed by the P[ry; Prat:bw] and P[hs-BRC-z1] constructs were used. In the former the bw gene is under the promoter of a housekeeping gene, Prat; in the latter the Br-C locus, mapping to the dark puff 2B, is under the promoter of a heat-shock gene, hsp70. Inserted into region 65A of the 3L chromosome, the Prat:bw copies give rise to structures which are morphologically reminiscent of the so-called "dark" puffs. In contrast, insertion of P[hs-BRC-z1] into region 99B of the 3R chromosome causes a regular "light" puff of form. Comparative analysis of the dark puffs--both transgenic and natural--suggests that there might be at least two mechanisms underlying their formation. One is a local incomplete decondensation of activated bands, characteristic of the so-called small puffs. The other is the formation of ectopic-looking contacts between the bands adjacent to the puffing zone. Transposition of the DNA, from which such a puff develops, causes a regular light puff to form at the new location. Heterochromatic regions do not appear to be directly involved in puffing.

[Modeling dark puffs using P-transposons in Drosophila melanogaster polytene chromosomes]. / Modelirovanie temnykh pufov s pomoshch'iu P-transpozonov v politennykh khromosomakh Drosophila melanogaster.

Modeling of morphologically unusual "dark" puffs was conducted using Drosophila melanogaster strains transformed by construct P[ry; Prat:bw], in which gene brown is controlled by the promoter of the housekeeping gene Prat. In polytene chromosomes, insertions of this type were shown to form structures that are morphologically similar to small puffs. By contrast, the Broad-Complex (Br-C) locus, which normally produce a dark puff in the 2B region of the X chromosome, forms a typical light-colored puffs when transferred to the 99B region of chromosome 3R using P[hs-BRC-z1]. A comparison of transposon-induced puffs with those appearing during normal development indicates that these puff types are formed via two different mechanisms. One mechanism involves decompaction of weakly transcribed bands and is characteristic of small puffs. The other mechanism is associated with contacts between bands adjacent to the puffing zone, which leads to mixing of inactive condensed and actively transcribed decondensed material and forming of large dark puffs.

The bithorax complex of Drosophila melanogaster: Underreplication and morphology in polytene chromosomes.

The level of polyteny of the Drosophila salivary gland chromosomes was determined throughout the chromosome region 89E1-4, the locus of the Bithorax Complex. A zone of underreplication spans the 300 kb of DNA from the Ubx to Abd-B loci. From the centromere proximal end of the complex, a 70-kb-long gradual decrease of polytenization starts with the Ubx transcription unit and, after a floor corresponding to the abd-A locus, raises gradually back to the maximum over 70 kb in the region of the Abd-B transcription unit. In flies carrying the mutation Suppressor of DNA Underreplication [Su(UR)ES], the underreplication of the Bithorax Complex is fully suppressed. In the wild type, the Bithorax Complex forms a weak point featuring thinner bands separated by clefts or constrictions. In Su(UR)ES strain in contrast, the 89E1-4 band looks like a single solid band consisting of homogenous dense material. We speculate that the wild-type Su(UR)ES protein hampers DNA replication of silenced domains and leads to their underreplication in salivary gland polytene chromosomes.

[Analysis of DNA interband regions 3A5/A6, 3C5-6/c7 and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes]. / Analiz DNK mezhdiskovykh raionov 3A5/A6, 3C5-6/C7 i 60E8-9/E10 politennykh khromosom.

Using electron microscopic (EM) data on the formation of a novel band from the P-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequences removed by deletion Df(1)faswb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophila genome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19 transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5' and 3' nontranslated gene regions are located. These results suggest that Drosophila interbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.

[Interline differences in morphology of the precentromeric region of polytene X-chromosome in Drosophila melanogaster salivary glands]. / Mezhlineinye razlichiia v morfologii pritsentromernogo raiona politennoi X-khromosomy v sliunnykh zhelezakh Drosophila melanogaster.

Morphology of the Drosophila melanogaster polytene X chromosome section 20 in normal flies, in strains carrying inversions that break pericentric heterochromatin at different points, and at the background of the Su(UR)ES mutation has been examined. In all of the strains carrying the Su(UR)ES mutation section 20 displayed a distinct banding pattern till to the section 20F, while in the wild-type strains this region was represented by beta-heterochromatin. The strains carrying different inversions substantially differed in the number and morphology of bands forming section 20. In the Su(UR)ES mutants the most proximal X chromosome euchromatin gene, su(f), is mapped to the boundary between sections 20E and F, while rDNA forming the middle part of the X chromosome mitotic heterochromatin is located in the proximal part of section 20F. All large bands observed in section 20 of the w; Su(UR)ES strain were also present in In(1)sc4; Su(UR)ES, which breaks heterochromatin in the distal part. Hence, the bands of polytene chromosome section 20 are virtually devoid of mitotic heterochromatin.

[Electron microscopic in situ hybridization of dUTP-digoxigenin labeled probes with polytene chromosomes of Drosophila melanogaster]. / Elektronno-mikroskopicheskaia gibridizatsiia i situ prob, mechennykh dUTP-digoksigenninom, s politennymi khromosomami Drosophila melanogaster.

Using DNA probes labeled with digoxygenin-11-dUTP, a simplified method of electron microscopic (EM) in situ hybridization was developed for standard squashes of Drosophila melanogaster polytene chromosomes. The developed method is efficient and reproducible: its high resolution and specificity was shown for the transformed strain 148, in which the insertion was localized by EM as a new thin band. The method was applied for fine mapping of the developmentally regulated complex gene, muscleblind (mbl), which was shown to cover the 54B1-2 large band and the adjacent interbands in 2R polytene chromosome.

Electron microscopic in situ hybridization of digoxigenin-dUTP-labelled DNA probes with Drosophila melanogaster polytene chromosomes.

We report a simplified method of electron microscopic (EM) in situ hybridization for standard squashes of Drosophila melanogaster polytene chromosomes using digoxigenin-11-dUTP labelled DNA probes. The method is efficient and reproducible: its high resolution and specificity were demonstrated for the transformed strain 148, in which the insertion was localized precisely as a new thin band both by conventional EM and according to our method. In addition, the method was applied to the fine mapping of the developmentally regulated gene muscle-blind (mbl). On the one hand, mbl was shown to cover the 54B1-2 large band and the adjacent interbands in the 2R polytene chromosome. On the other hand, the use of distantly located DNA probes in the mbl gene allowed us to orientate the transcription unit in the chromosome.