[COMPARATIVE CYTOGENETIC ANALYSIS OF MONOLAYER AND SUSPENSION CHINESE HAMSTER OVARY CELL LINES CHO(dhfr-)].

The karyotypes of CHO(dhfr-) and CHO(dhfr-)/susp Chinese hamster ovary cell lines were investigated with the use of GTG-staining. Modal chromosome set consists of 20 and 18 chromosomes respectively. The karyotypes of both cell lines were stable with constant ratio of normal chromosomes and chromosomes with structural rearrangements. Monosomy for chromosomes 1, 2, 4, 5, 8 was observed in both cell lines and for chromosome 9 in CHO(dhfr-)/susp cell line. The differences between CHO(dhfr-) cell lines studied by us consists of inclusion of part of chromosome 7 in der(6)t(1;6), rearrangement of del(5) and monosomy of chromosome 9. It was shown that in karyotypes of all CHO cell lines studied up today there are 5 common structurally chromosome rearrangements: del(2), inv(3), add(6), del(9) and mar1. In both CHO(dhfr-) cell lines investigated by us three unique chromosome rearrangements: del(1), der(6)t(1,6) and mar3 were revealed. Necessity of simultaneous GTG and FISH analysis of chromosomes rearrangements in the CHO cell lines under study is discussed.

[Classifier of domestic pig (Sus scrofa domesticus) automatic chromosome identification].

Automated chromosome classification is an essential task in cytogenetics of animals and plants. Until now the automatic karyotyping systems were obtained only for human chromosomes. The main aim of this study was to develop the automatic pig chromosome classifier using image processing software "VideoTest-Karyo 3.1". To solve this problem 1578 chromosomes from 47 metaphases were used. The constructed classifier was checked with the use of additional sample of metaphases classified in fully automatic regime: error rate was 8.2%, this corresponds to 3.12 ± 0.26 errors per metaphase plate (these values are within acceptable limits for such kind of studies). In further studies the extra sample of pig acrocentric chromosomes was added to classifier up to 1807 chromosomes. This addition reduced the error rate up to 6.1%, which correspondes to 2.78 ± 0.18 errors per metaphase plate. It should be underlined that the revealed errors can immediately be corrected by an operator on every stage of analysis. The classifier was also verified using the chromosomes of boar with rcp(1p-; 11p+) in fully automatic regime and routine stained metaphases of Siberian minipigs with rob(16;17) in semi automatic regime. In both cases the chromosomes were identified correctly. The area of application of obtained pig automatic chromosome classifier is discussed.

[Differentiation of interphase nucleolus organizers in embryonic pig kidney cells (PK cell line)].

The prometaphase karyotype of cell line PK contains two heteromorphous pairs of nucleolus organizers that belong to chromosomes 8a and 8, and to 10L and 10s. It was proposed that such heteromorphism may promote chromosome differentiating of interphase nucleolus organizers (INOs) with linear configuration. To test this assumption, we used two-dimensional (2D) preparations of methanol fixed PK cells surface stretched without hypotonic treatment. It was shown that in these preparations the large bulk of interphase PK cells contained 3-4 necklace-like linear structures arranged in nucleolar domains. The observed structures were positive in phase contrast and after DAPI-staining. Complimentary rDNA-FISH revealed that these structures were INOs, the largest iNO in individual cells containing prominent terminal rDNA FISH/DAPI signal. In accordance with the data on prometaphase analysis, the latter INOs belong to chromosomes 8a. As reported by Smetana and coworkers (1999), proteins of the nucleolar fibrillar center reacted preferentially with silver in methanol fixed unwashed smears of human peripheral lymphocytes. It was established that the same specific silver reaction is characteristic most probably of 2D preparations of methanol fixed PK cells. Both silver stained and rDNA-FISH linearized INOs had necklace-like or banded structure with different degrees of resolution. Banded INOs consisted of transverse argyrophilic structures: dense bands and loose interbands. High resolved banded INOs revealed a longitudinal splitting (binemic structure) of interband zone. Necklace-like INOs consisted of argyrophilic beads nearly two-fold more narrow than argyrophilic bands, and uninemic or silver-negative interbead zones. Our findings evidence that necklace-like INOs are typical for G1 and S phase cells, whereas banded INOs are characteristic of G2 cells. Among high resolved linear INOs, we found four reproducible patterns of silver staining, which could be combined it two homologous groups. Because each given pattern is unique for individual PK cells, we concluded that the patterns under study were chromosome specific. Using prometaphase analysis data, we determined chromosome affiliation for each of the four tested patterns of INO silver staining. High resolved INOs, belonging to different chromosomes, were further compared with regard to their average length and the mean of argyrophilic bead number per individual INO, in addition to the length and argyrophilic bead number ratios calculated for different INO pairs of individual cells. Surprisingly, we found that both the ratios, detected for most heteromorphous pair of homologous chromosomes 8a and 8, made only 1.26 +/- 0.02. In comparison, the similar length ratio for nucleolus organizers in chromosomes 8a and 8, calculated for individual prometaphase cells, reached 2.92 +/- 0.30.

[Transmission of ribosomal chromatin during mitosis in embryonal pig kidney cells (PK cell line)].

Mammalian nucleolar organizers (NORs) contain ribosomal (rRNA) genes associated with argyrophilic proteins and can be specifically stained by silver. These genes are clustered in four loci of PK meta- and submetacentric chromosomes 8 and 10. According to our data reported elsewhere, one of PK NORs contains a large amplification of rRNA that is manifested on silver stained chromosomes as abnormally large Ag+-NOR 8a. It is proposed that such a redundancy of rRNA genes induced their dose compensation in the form of transcriptional silencing, triggering the rise of Ag-negative ribosomal chromatin. To test this assumption, we carried out a morphometrical analysis of Ag+-NOR (bearing) chromosomes from prometaphase sets with modal chromosome number. We found that in the individual chromosome set a longer homologue 10L of chromosome 10 had always a larger Ag(+)-NOR area than did a shorter homologue 10S. Thus, PK karyotype consists of two pairs of heteromorphous Ag(+)-NORs: 8a and 8, 10L and 10S. One half of tested chromosome sets revealed four Ag(+)-NOR chromosomes, the other half had one Ag-negative NOR nearly equally belonging to chromosome either 8 or 10S. The majority of prometaphase Ag+-NORs showed partial or full chromatid splitting that allows for Ag(+)-NOR area measurements of (sister) chromatid. The area ratio of larger to smaller chromatid Ag(+)-NOR strongly varied for all chromosomes except chromosomes 8a. The maximum value of this ratio reached 5.1 for chromosome 8 and 3.4, and 2.3--for 10L and 10S (vs. 1.6 for 8a). Proportions of chromosomes with the ratio 1.25 and more, were: near two thirds--among chromosomes 8, near half--among 10L and 10S, but less than a quarter--among chromosomes 8a. These findings show that progressive differentiation of sister chromatid NORs in regard of the content of Ag-negative ribosomal chromatin may lead to an unequal Ag(+)-NOR distribution between daughter cells. To test this prediction, we developed a new technique for obtaining two dimensional (2D) preparations of stretched PK cells, which makes it possible to avoid the stage of hypotonic treatment of living cells, since this treatment levels the silver staining of NORs and prenucleolar bodies of fixed telophase cells. We found that some daughter nuclei from early telophase cells revealed the value of Ag(+)-NOR separation equal to 4:3 instead of the common value equal to 4:4. Complimentary 2D FISH with 28S + 18S mink rDNA probe showed that early telophase rRNA loci, detected as four bright large spots, are close by area and shape to Ag(+)-NORs of the corresponding cells. Sometimes, however, one of the daughter nuclei showed three such domains, in addition to one slight linear FISH signal that most probably represented Ag-negative NOR. A delayed separation of sister chromatids is the main structural characteristic of inactive chromatin (Azuara et al., 2003). It was established that the largest PK Ag(+)-NOR (chromosome 8a) showed a high level of cohesion and (or) twisting of sister chromatids that is characteristic of prophase rather than prometaphase PK chromosomes. These findings, together with the above cited literature data, give evidence for enrichment of Ag(+)-NOR by either inactive or (and) low active late-replicating chromatin.

[Differentiation of interphase domains of the homologous human X-chromosomes].

In accordance with molecular biology data reported elsewhere, homologous interphase X-chromosome territories differ greatly in the abundance of inactive condensed chromatin. On the other hand, a three dimensional FISH (3D FISH) method has revealed that domains of both inactive and active X-chromosome have similar volumes and similar maximum section areas (Smax). To solve this contradiction, we examined differences between homologous human interphase X-chromosome territories using two dimensional FISH (2D FISH) preparations of clustered PHA-stimulated lymphocytes. For obtaining such preparations, we developed a new technique to avoid a stage of hypotonic treatment of living cells, since this treatment levels the chromatin compactness degree. According to our 2D FISH data, the mean ratios of Smax for larger and smaller homologous X-chromosomes, calculated for individual flattened nuclei, were 1.83 +/- 0.08 and 2.02 +/- 0.09, respectively, for clumped cells and groups of loosely associated and separated lymphocytes. In comparison, the same ratio calculated for individual 3D nuclei of PHA-stimulated lymphocytes was 1.38 +/- 0.05 (Falk et al., 2002). Our findings give evidence for enrichment of inactive X-chromosomes by low stretchable condensed chromatin. In addition, these findings show that an active X is enriched by a high stretchable form of chromatin, whose content may distinctly vary from cell to cell.

[Differential decondensation of mitotic chromosomes in SPEV tissue cultured cells induced by repeated hypotonic shock ]. / Differentsial'naia dekondensatsiia mitoticheskikh khromosom v kletkakh kul'tury tkani SPEV, indutsirovannaia dvukratnym gipotonicheskim shokom.

A new method of differential decondensation of mitotic chromosomes has been proposed by means of repeated treatment of live cells with 15% Hanks' balanced salt solution. The procedure of cell treatment includes three stages: the first hypotonic shock, cultivation in isotonic medium, and the second hypotonic shock. As a result, after a standard methanol-acetic acid fixation and Giemsa staining some discrete Giemsa-positive globules are revealed in mitotic chromosomes. Such globules are symmetrically arranged in axial regions of sister chromatids. The comparative analysis of marker chromosomes has revealed a topological conformity of these globules to G-bands of chromosomes. It has been shown that it is the first hypotonic shock that triggers induction of structural modification of chromatin in interphase nuclei and in mitotic chromosomes. Of interest is the fact that the effect of the first shock is prolonged in time and is realized during at least one cell cycle, with the normal structure of mitotic chromosomes being restored after S-phase of the successive cell cycle.

[The study of chromatin and chromosome structure on preparations of interphase nucleus derivatives resulting from nuclear wall removal.III Structural heterogeneity of chromatin and argyrophilic zone of the nucleolus in stretched membrane-free nuclei and chromatin bodies from human peripheral lymphocytes]. / Issledovanie struktury khromatina i khromosom na preparatakh derivatov interfaznykh iader, poluchennykh putem udaleniia obolochek iader III. Strukturnaia geterogennost' khromatina i argentofil'noizony iadrysh ka v rastianutykh bezmembrannykh iadrakh i khromatinovykh telakh iz perifericheskikh limfotsitov cheloveka.

Viewed by light microscopy, the majority of lymphocytes in smears of human peripheral blood display a deep staining (with any chromatin- or DNA-specific dye) of the nucleus consisting of densely aggregated chromatin in addition to one or several small nucleoli with a dot- or spot-like argyrophilic zone. Amembraneous nuclei and "free chromatin" structures were isolated from intact lymphocytes gently treated with Triton X-100. Surface stretching of both these nuclei and structures, shortly fixed in methanol--glacial acetic acid (3:1), resulted in spatial separation of thin and thick chromatin or argyrophilic fibres, nucleoli, intranuclear bodies, polymorphous aggregations of chromatin or argyrophilic fibres and incidentally observed splitted or beaded thick chromatin fibres and the chromocenter. The light microscopic pattern of chromatin fibres of stretched amembraneous nuclei, isolated from peripheral lymphocytes, well compares with that of deconvolved images of intact lymphocyte nucleus obtained with optical tomography.

The RNA polymerase I transcription factor UBF and rDNA are located at the same major sites in both interphase and mitotic pig embryonic kidney (PK) cells.

Indirect immunolabeling with anti-UBF antibodies, in situ hybridization with an rDNA probe, and confocal scanning laser microscopy were used to study nucleolar organizer regions (NORs) during the cell cycle in pig embryonic kidney (PK) cells. The chromosomal distribution of the polymerase I transcription factor UBF and rDNA was compared with the number of silver-stained NORs (Ag-NORs) present and nucleolar size. It was shown, both at interphase and mitosis, that the majority of UBF and rDNA signals were located at the same foci and that the amounts of UBF and rDNA at any given site were in a striking positive correlation. At mitosis, only the NORs were labeled; at interphase, the signals for both UBF and rDNA were arranged in necklace-like structures around the nucleoli. No chromosomal NORs without Ag-proteins or UBF were present, indicating that all NORs in PK cells are active at interphase. It was concluded that (1) UBF and rDNA co-localize throughout the cell cycle in PK cells; (2) their association with mitotic NORs is determined by the number of rDNA repeats, rather than by any differential ability of NORs to recruit the transcription factor; and (3) the amount of UBF can be correlated with the size and activity of the nucleoli at interphase.

[The ultrastructural characteristics of the metaphase chromosomal regions (telomere, centromere, nucleolar organizer) in the pig]. / Osobennosti ul'trastruktury nekotorykh raionov metafaznykh khromosom svin'i (telomera, tsentromera, iadryshkovyi organizator).

The ultrastructural organization of some chromosome regions (telomere, centromere, nucleolus organizer region), which can be identified undoubtedly under electron microscope after in situ fixation, was studied using serial ultrathin sections of pig embryo kidney cells. The ultrastructure of these regions was compared with patterns of their differential staining revealed by C-method and by fluorochromes, chromomycin A3 and DAPI, specific for GC- and AT-rich base pair regions. It is shown that telomere regions of chromosome are organized primarily by 20 nm fibrils of DNP, which form a clear chromonema--a thread of chromatin with thickness nearly 100 nm. In the centromere regions of metacentric chromosomes, fibrils 20 nm in diameter predominate, while in several acrocentric chromosomes--fibrils 10 nm thick are prevailing. It was suggested that the observed differences may depend on the DNA composition in these regions. The nucleolus organizer regions contain the fibrillar material 5-11 nm in diameter.

[An analysis of the action of nonspecific and restriction nucleases on metaphase chromosomes in situ]. / Analiz deistviia nespetsificheskikh i restriktsionnykh nukleaz na metafaznye khromosomy in situ.

Patterns of differential staining of Drosophila, mouse, rat, cattle and pig chromosomes were examined after the treatment with nucleases (DNAase I, DNAase II) and restriction enzymes (AluI, HpaII, MspI, BpE, EcoRI). The above effects depend on the species used, on the enzymes and substitution of thymine for bromodeoxyuridine in the chromosomal DNA. It is supposed that such a phenomenon may not only result from the irregular distribution of specific restriction sites along chromosomes but also depend on the specificity of supramolecular organization of the chromosomal DNA.

[Quantitative characteristics of the nucleolus organizer region of the chromosomes in the domestic pig]. / Kolichestvennaia kharakteristika iadryshkoobrazuiushchikh raionov khromosom domashnei svin'i.

Absolute and relative dimensions of Ag+-NORs and secondary constrictions (SC) of pig chromosomes were determined. Interchromosomal and interindividual variability of these indices were marked. An attempt was undertaken to standardize the absolute sizes of Ag+-NORs and SC, degrees of chromosome condensation being taken into consideration. A high correlation between the dimensions of Ag+-NOR and of SC was noted.