[Uniparametric analysis of chromosomes from human fibroblasts using flow cytofluorometry]. / Odnoparamtericheskii analiz khromosom iz fibroblastov cheloveka metodom tsitofluorimetrii v potoke.

Metaphase chromosomes from human diploid fibroblasts were isolated by the polyamine method and stained with different fluorochromes: propidium iodide, chromomycine A2, bisbensimide H33258, and DAPI. The fluorescently stained chromosomes were analyzed using a cell sorter ATC-3000 ("Bruker-Spectrospine"), and the flow histograms were investigated to identify the peaks of individual chromosomes and to choose the chromosomes which can be isolated to a high purity. High resolution was obtained with H33258 or DAPI. It was demonstrated that the peaks of chromosomes 13, 17, 20 are well distinguished from the adjacent peaks, so that these chromosomes can be sorted with a high purity. The peaks formed by chromosomes 5, 6, 8, 14, 15 are slightly overlapped by the adjacent peaks, and the sorting on the basis of monovariate analysis cannot provide sufficient purity of the sorted fractions (< 0.5). Chromosomes 1-2, 3-4, 7-X, 9-12, 16-18, 19, 21-22 cannot be discriminated for successful sorting of an individual chromosome type. The data obtained provided an experimental basis for choosing the chromosomes to be sorted with high purity for the subsequent cloning of their DNA.

[Genomic DNA as compact particles: morphology, biochemical analysis and optical properties]. / Genomnaia DNK v forme kompaktnykh chastits: morfologiia, biokhimicheskii analiz i opticheskie svoistva.

Compact DNA particles have been obtained by a new method we developed to isolate high-molecular-weight DNA. The method includes exhaustive proteolysis while the cellular DNA is folded in compact state in polyethylene glycol-containing aqueous-salt media. The DNA particles derived from interphase nuclei have a diameter of 8-10 microns and each of them contains the genome of an individual cell. The DNA particles derived from isolated metaphase chromosomes present tightly packed DNA of individual chromosomes. The compact particles contain no proteins detectable by electrophoretic analysis and so appear to be formed by DNA only. These data have been confirmed by electron microscopy, when the particles investigated with the protein monolayer technique have revealed only spread DNA molecules. Simultaneously the microscopic observations of DNA particles of different origin and of their behaviour in the absence of compactizing agents support the idea that during formation of compact particles the intrinsic features of DNA folding in nuclei and chromosomes have been conserved.

[Electron microscopic study of changes in chromosomal structural organization under the effect of formamide]. / Elektronno-mikroskopicheskoe issledovanie izmenenii strukturnoi organizatsii khromosom pod deistviem formamida.

Isolated human metaphase chromosomes were treated with formamide at different (0-70%) concentrations and examined electronmicroscopically by protein monolayer technique. At increasing formamide concentration chromosomes gradually decondense, the scaffold becomes more clearly visible, the loops of chromatin fibres coming off the central part of chromosomes lose their nucleosomal appearance. Electrophoretic analysis of chromosomal proteins data show that formamide-treated chromosomes have approximately the same histone content as those before treatment, although chromosomes treated with 70% formamide look very similar to histone-depleted ones described elsewhere.

[Patterns in the occurrence and the submicroscopic organization of elongated metaphase chromosomes isolated from the cell]. / Zakonomernosti vozniknoveniia i subkikroskopicheskaia organizatsiia udlinennykh metafaznykh khromosom, izolirovannykh iz kletki.

Chinese hamster metaphase chromosomes were investigated under different conditions of isolation. Light microscopic study demonstrated different forms of stretched chromosomes, from those in which merely a small region is stretched to rope-like structures 20-25 microns long with diameter of about 0.4 micron. The ratio between the number of stretched and of compact chromosomes is dependent on the concentration of bivalent cations, on the pH and temperature of the isolation buffer. A study of the submicroscopic organization of stretched chromosomes revealed lengthwise fibrils that disappeared after the treatment with 0.6 NaCl and staphylococcal nuclease. Distinct aggregates were seen, whose array is maintaining the stretched chromosome structure. It is suggested that stretched chromosomes appear due to the existing in vivo lability of bonds between the main chromosome components involved in organization of chromatin fiber packing. It is proposed that the structure obtains rigidity in the course of isolation with bivalent cations.

[Residual structures revealed after the action of staphylococcal nuclease on decondensed metaphase chromosomes]. / Ostatochnye struktury, obnaruzhivaemye posle deistviia stafilokokkovoi nukleazy na dekondensirovannye metafaznye khromosomy.

The isolated Chinese hamster metaphase chromosomes were decondensed in 0.6 M NaCl solution and digested by staphylococcal nuclease on electron microscope grids. The residual structures as seen in electron microscope as isolated aggregates scattered over the entire area occupied by the chromosome. Prior to digestion, the material in the chromosome did not form presumably an integral protein structure similar to a scaffold.

[Changes in the structure of the chromosomal nucleohistone fibrils on removal of divalent cations and histone H1]. / Izmeneniia v strukture nukleogistonovykh fibrill khromosomy pri udalenii bivalentnykh kationov i gistona H1.

A technique of freeze--drying was applied in studies on submicroscopic organization of metaphase chromosomes from Chinese hamster. In the buffer without bivalent cations the chromosomes increased in size; the removal of bivalent cations with EDTA resulted in a large swelling of chromosomes. Regularly organized nucleosomal filaments of a "beads-on-a-string" type appeared on the periphery of chromosomes. The regular organization of the fibers is damaged as soon as histone H1 is removed. An increase in the ionic strength of solution in the absence of bivalent cations results in chromosome compactization, although the regular fibrillar structure being not restored.

Differences in AT- and GC-base pair ratios in DNA of the paracentromeric heterochromatin of chromosome 1 in two human cell types. I. Autoradiographic analysis of the incorporation of 3H-labeled thymidine and 3H-labeled deoxycytidine.

The distribution of AT- and GC-base pairs in DNA along chromosomes 1 and 2 has been studied in primary cultures of human embryo fibroblasts and peripheral blood leukocytes by an autoradiographic method using 3H-labeled thymidine and 3H-labeled deoxycytidine. The two cell types differed in their relative contents of DNA and in the ratio of AT and GC pairs at the centromere and the adjacent region of heterochromatin in chromosome 1. The DNA content of this section was higher in fibroblasts than in leukocytes, mainly because of AT pairs. In both cell types, the telomere in the short arm of this chromosome had a higher content of GC pairs than AT pairs. No differences were observed in base pair distribution along chromosome 2 in the two types. This phenomenon may be due to incomplete replication, or to loss by some means of part of the genetic material during the development and differentiation of the cellular systems.