Dependence of heterochromatin differential staining on the time of its reduplication and the degree of condensation]

A comparison of the chromosomes banding pattern after G-and C-staining with the time of DNA reduplication and the degree of chromosome condensation, was carried out using Chinese hamster metaphase chromosomes. Chromosome condensation was studied under 5-bromodeoxyuridine and 5-bromodeoxycytidine treatment. All the chromosomal segments stained with C-technique are also stainable with G-technique, while only some G-positive segments are capable to be C-bands. C-bands are heterochromatic segments characterized by extremely late replication and great delay in condensation under the analog action, while G-bands are segments with earlier labelling and irregular decondensation. The data obtained suggest a close correlation between the capability of chromosomal region of G- and C- staining and the degree of its heterochromatinization.     

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light-scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield- or phase-contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding. The condensation of free DNA, single or double stranded, natural or synthetic, the latter of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands. The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light-scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome "counter-staining" can be explored by flow cytometry.     

Chromosome condensation in mitosis and meiosis of rye (Secale cereale L.)

Structural investigation and morphometry of meiotic chromosomes by scanning electron microscopy (in comparison to light microscopy) of all stages of condensation of meiosis I + II show remarkable differences during chromosome condensation in mitosis and meiosis I of rye (Secale cereale) with respect to initiation, mode and degree of condensation. Mitotic chromosomes condense in a linear fashion, shorten in length and increase moderately in diameter. In contrast, in meiosis I, condensation of chromosomes in length and diameter is a sigmoidal process with a retardation in zygotene and pachytene and an acceleration from diplotene to diakinesis. The basic structural components of mitotic chromosomes of rye are "parallel fibers" and "chromomeres" which become highly compacted in metaphase. Although chromosome architecture in early prophase of meiosis seems similar to mitosis in principle, there is no equivalent stage during transition to metaphase I when chromosomes condense to a much higher degree and show a characteristic "smooth" surface. No indication was found for helical winding of chromosomes either in mitosis or in meiosis. Based on measurements, we propose a mechanism for chromosome dynamics in mitosis and meiosis, which involves three individual processes: (i) aggregation of chromatin subdomains into a chromosome filament, (ii) condensation in length, which involves a progressive increase in diameter and (iii) separation of chromatids.     

Reorganization of chromatin in Xenopus egg extracts: electron microscopic studies.

The structural basis of mitotic condensation of chromosomes is one of the problems of cell biology yet to be elucidated. A variety of approaches have been used to study this problem and a large number of hypotheses have been proposed to explain the different levels of compaction of chromatin. Xenopus egg extracts, now widely used to study various aspects of cell biology, provide a valuable tool to study mitotic condensation of chromosomes. No detailed study has however yet been reported on the submicroscopic organization of condensed chromosomes in vitro in egg extracts. We present here the results of our electron microscopic studies on the organization of condensed chromosomes in vitro, using demembranated sperm nuclei and mitotic (CSF-arrested) extracts of Xenopus laevis eggs, clarified by high speed centrifugation. Upon introduction of sperm nuclei in egg extracts, the nuclei swell and the chromatin undergoes a rapid decondensation; at this stage the chromatin is formed of 10 nm fibrils. After longer incubation, the chromatin condenses, and by 2 h chromosomal structures can be visualized by staining with DAPI or Hoechst 33258. Our results on the organization of chromosomes in different stages of condensation are discussed in relation to the different hypotheses proposed to explain the process of mitotic condensation of chromosomes. Finally, this study demonstrates the feasibility of high-resolution analysis of the process of chromosome condensation.     

Somatic chromosome map of rice by imaging methods

Summary Rice somatic chromosomes were completely identified and quantitatively mapped based on an image parameter, condensation pattern (CP), or a chromosomal density profile determined by imaging methods. The CP corresponds to the compactness of the chromatin fibers along the chromatid, which is characteristic in small plant chromosomes such as rice chromosomes at the mitotic pro-metaphase stage. The standard CP for every chromosome was obtained by averaging 60 CPs from 30 chromosome spreads. Each standard CP exhibited a characteristic pattern of the chromosome, which enabled it to be distinguished from the other chromosomes. An ideogram based on the numerical data and the standard CP was established. The chromosomal address was also determined based on the degree of condensation, and the fractional length of each chromosomal address was quantitatively presented.     

Inducing chromosome pairing through premature condensation: analysis of wheat interspecific hybrids

At the onset of meiosis, chromosomes first decondense and then condense as the process of recognition and intimate pairing occurs between homologous chromosomes. We show here that okadaic acid, a drug known to induce chromosome condensation, can be introduced into wheat interspecific hybrids prior to meiosis to induce chromosome pairing. This pairing occurs in the presence of the Ph1 locus, which usually suppresses pairing of related chromosomes and which we show here delays condensation. Thus the timing of chromosome condensation during the onset of meiosis is an important factor in controlling chromosome pairing.     

Heteropycnosis of an underreplicating chromosome

Drosophila nasutoides has an extraordinary genome since 62% of its DNA resides in chromosome4. This element mainly consists of constitutive heterochromatin which does not polytenize. Earlier studies of heterochromatin attributed little attention to the fact that condensed chromosomes often vary in condensation. This paper reports that chromosomes of the same complement display different degrees and kinetics of condensation. InD. nasutoides, even sex specific differences can be observed. The results of a comparative microphotometric study on neuroblast metaphases in both sexes revealed the following picture. The process of chromosome condensation is not restricted to mitotic prophase but continues into the metaphase. The mean condensation is not equal for all chromosomes. In the metaphase of the female, Feulgen density increases from theX chromosome, via3 and2, to chromosome4. In the male, the order isX, 2, 3, Y, and4. During the metaphase of the male, chromosomes condense with similar kinetics. In contrast, chromosomes of the female display asynchrony as monitored by area and length determinations. TheX chromosomes of the female probably have enhanced shortening during prophase. This would explain the metaphase of the female where theX chromosomes shorten less than the autosomes, and why each of theX chromosomes is 15% shorter than theX chromosome in the metaphase of the male. Further differences were observed in the longitudinal and lateral compaction of the chromosomes in males and females. The sex chromosomes and chromosome3 condense by shortening, while chromosomes2 and4 preferentially reduce their diameter. The large amount of DNA engaged in heteropycnosis and the isochromosome nature allow the identification of chromosome4 during interphase. At this stage, a new category of extreme DNA packaging was detected. The interphase density of chromosome4 can exceed that of metaphase by a factor of up to 8. Two events account for this high degree of condensation:(1) the homologues are particularly associated due to somatic pairing and (2) the arms are further tightened as a result of pericentric folding. The features of the isochromosome suggest that the interaction of chromatids during interphase is essentially caused by specific DNA sequences. The data confirm that heteropycnosis not only interferes with gene expression but also strongly inhibits DNA synthesis in endocycles.     

Polyteny and endomitosis in supergiant trophoblast cells of the gray vole Microtus subarvalis

A cytomorphological study was made of peculiarly structured polytene chromosomes in supergiant trophoblast cells of Microtus subarvalis. The polyteny level was extremely high (over 1024C). The polytene chromosomes are characterized by a rather high degree of condensation of single chromosomes, and, as a consequence, close chromosome junctions and the typical disk pattern are lacking. The presence of complex nucleoli in the nuclei of these cells also testifies to a great detachment of chromonemes in polytene chromosomes of the studied supergiant trophoblast cells. Compared to other rodent species, a lower degree of chromoneme junction in the vole polytene chromosomes may cause their easy dissociation into single chromonemata, whose further condensation results in endomitotic chromosome formation. The chromosome depolytenization, earlier suggested from the analysis of interphase nucleus markers, has been traced here in detail. The process of polytene chromosome splitting was most obvious in the nucleolus-organizing chromosomes. A hony-combed nucleolus splits into numerous micronucleoli. The nucleus pattern becomes altered. Once in the polytene nucleus, chromosome bundles were located below the nuclear membrane and the central zone of the karyoplasm was not completely filled up. However, after dissociation of polytene chromosomes the whole karyoplasm was filled up with small nucleoli, and a thin layer of endomitotic chromosomes was seen beneath the nuclear membrane. The correlation between endomitosis and polyteny is discussed in terms of the dissociation of polytene chromosomes and formation of endomitotic chromosomes.     

The replication of human heterochromatin in serial culture

The human C group chromosomes late to start replication in asynchronous and in FUdR synchronized cell lines are X chromosomes. These same chromosomes are also heterochromatic during interphase. During metaphase these allocyclic Xs cannot be identified simply by metaphase position or morphology and show a wide range of measurements for arm ratio, centromere index and total length. Replication starts in the short arm and extends over the entire chromosome during the 2nd and 3rd hr of S until by the 4th hr distinction from other C group chromosomes cannot be made by means of the labeling pattern. When the allocyclic X chromosomes start replication the pattern of H³TdR label over interphase sex chromatin and non-specific heterochromatin shifts from unlabeled to labeled in FUdR synchronized human cell lines. The overall time required for replication of the allocyclic X is less than that for the other chromosomes in both asynchronous and FUdR treated cells. A hypothesis is presented for a direct relation between the delay of onset of replication in heterochromatin and its degree of interphase condensation.The present study was supported by research grants: No. HD-00777 from the National Institutes of Health and No. E-487 from the American Cancer Society, Inc.     

Pachytene variations in Ricinus

The pachytene complement in microsporocytes was compared among ten races of Ricinus communis L., the castor plant (2n=20). Four kinds of pachytene variations were observed: (1) variation in degree of spreading of the chromosomes; (2) variation in degree of attenuation of heterochromatin; (3) variation in morphology, which appeared to be restricted to the two nucleolar organizing bivalents; (4) variation in frequency of association of each of these two bivalents with the nucleolus. It is suggested that variations in the pachytene chromosomes occur ubiquitously in this species.     

A developmental study of constitutive heterochromatin in Microtus agrestis

In the vole, Microtus agrestis, the constitutive heterochromatin is largely restricted to the giant sex chromosomes but varies in its degree of condensation in various cell types. In the cleavage embryos and fibroblasts it formed one or two long and extended heterochromatic fibers, in hepatocytes it formed two large and diffuse masses and in neurons, spermatogonia and oogonia it formed two large and compact masses. The basic patterns of all differentiated cells were essentially unchanged throughout development.—At all stages of development and in cells of all types, mitotic nuclei displayed two large heteropycnotic chromosomes in prophase and persistent condensation in telophase. Apposition and delayed separation of chromatids of the giant chromosomes was also observed in metaphase and anaphase, respectively. During the first meiotic prophase of spermatocytes and oocytes, the giant chromosomes were also heteropycnotic.—The results strongly suggest that constitutive heterochromatin is localized in the same chromosomes throughout development and represents a specific entity.     

The location of 5S RNA genes in lampbrush polytene chromosomes from Drosophila

Drosophila polytene chromosomes were transformed into lampbrush-like structures by exposure to solutions of alkali-urea. In this process, the chromosomes shorten and widen, and the bands (chromomeres) extend laterally into loops leaving a central core between the paired homologues. The expanded polytene chromosomes are very similar in appearance to the true lampbrush chromosomes of amphibian oocytes and to ordinary chromosomes in pachytene. The denaturing effects of alkali-urea were partially counteracted by return of the treated chromosomes to Ringer solution. These observations are interpreted in terms of recent findings on protein backbones in chromosomes, and indicate that chromosomes generally may have very similar basic organization, despite differences due to species, polyteny and degree of condensation. To gain more information on the specific location of a structural gene, ¹²⁵I-labelled low molecular weight (containing 5S RNA) was hybridized in situ to normal and lampbrush-like polytene chromosomes. Autoradiography showed silver grain distribution for 5S RNA consistent with hybridization primarily to the loop regions of the lampbrush chromosomes rather than the core. This provides further indirect evidence that structural genes like 5S RNA may be located on the bands (chromomeres) and not the interbands of normal polytene chromosomes.     

Changes in the organization of chromosomes during the cell cycle: response to ultraviolet light.

Fusion between mitotic and interphase cells results in the premature condensation of the interphase chromosomes into a morphology related to the position in the cell cycle at the time of fusion. These prematurely condensed chromosomes (PCC) have been used in conjunction with u.v. irradiation to examine the interphase chromosome condensation cycle of HeLa cells. The following observations have been made: (I) There is a progressive decondensation of the chromosomes during G1 which is accentuated by u.v. irradiation: (2) The chromosomes become more resistant to u.v.-induced decondensation during G2 and mitosis. (3) There is a close correlation between the degree of chromosome decondensation and the amount of unscheduled DNA synthesis induced by u.v. irradiation during G1 and mitosis: (4) Hydroxyurea enhances the ability of u.v. irradiation to promote the decondensation of chromosomes during G1, G2 and mitosis. Hydroxyurea also potentiates the lethal action of u.v. irradiation during mitosis and G1. These data are discussed in relation to the suggestion that chromosomes undergo a progressive decondensation during G1 and condensation during G2.     

Human cells in suspension 2

Summary When PHA stimulated human lymphoid cells are allowed to proliferate in vitro and chromosomal slides are prepared from the culture after 48,72, 96,120, and 144h of growth, gradual changes in chromosome morphology can be observed after traditional Giemsa staining of the slides. Keeping culture conditions, colcemid exposure, and fixation procedure constant for all samples, it is found that average chromosome length decreases with increasing culture time. A shift from high frequencies of subbanded chromosomes (sample 48 h and sample 72 h) to high frequencies of unbanded and G banded chromosomes (sample 120 h and sample 144 h) takes place simultaneously with the general compaction of the chromosomes. Examination of trypsin-induced G bands as well as examination of untreated G banded chromosomes from all samples clearly indicate that the basic G band pattern is not altered during proliferation and differentiation, although the progressive compaction of the chromosome observed with increasing culture time results in a phenomenon similar to that observed during mitosis, where the compact late metaphase chromosome after trypsin treatment exhibits fewer but more prominent bands than the prophase/prometaphase chromosomes. Thus the progressive compaction of metaphase chromosomes observed during in vitro aging seems to resemble the condensation processes during the G₂ phase and mitosis.It has been suggested that the chromomeres serve as centers for chromosome condensation during mitosis, probably mediated by a sulphydryl-disulphide transition in chromosomal proteins. The data presented here further suggest that the chromomeres may also serve as centers for chromosomal differentiation, presumable by a mechanism similar to that acting during chromosome condensation in mitosis.     

Chromosomal instability in lemon grass,Cymbopogon flexuosus (Steudel) Wats

Somatic mitoses in C. flexuosus exhibit a significant degree of chromosomal instability leading to nearly 33% cells with chromosome elimination. A range of chromosome numbers between 20-8 (most common being 2n=20, the somatic number for this species) was encountered from root tip cells. The course of variation suggests a gradual elimination of somatic chromosomes. The larger chromosomes are less stable and are eliminated earlier. The variation in chromosome number in somatic cells within individual plants is possibly controlled by genetic factors, which result from weaker spindle operation and minute chromosomes.CIMAP Publication No. 571 (1984)     

Elektronenmikroskopische Untersuchungen zum Formwechsel der Kinetochoren während der Spermatocytenteilungen von Pales ferruginea (Nematocera)

Longitudinal thin sections of preselected spermatocytes were studied with the electron microscope. The kinetochores of autosomes and sex chromosomes show a characteristic change of their form during the meiotic divisions. Just after nuclear membrane breakdown the kinetochore profiles have the form of circles, in early prometaphase they have flame shape, and in metaphase appear as straight zones. As early as prometaphase I two sister kinetochores are discernible in each kinetochore region of a dyad. In prometaphase the sister kinetochores of the sex chromosomes are connected with each other through condensation zones which are continuous with both kinetochores. A double line structure is often seen in kinetochores and condensation zones. The morphological change of kinetochores can be asynchronous, as is especially conspicuous in the sex chromosomes. —The mitotic apparatuses of Pales behave in hexylenglycol medium like mitotic apparatuses of marine eggs. Crystalloids (Fuge, 1970) and microfilament bundles (Bajer and Molè-Bajer, 1969) occur in mitotic apparatuses in early and middle prometaphase.     

Karyotype variation in aminoethylcysteine resistant cell and callus cultures and regenerated plants of a dihaploid potato (Solanum tuberosum)

Karyotypic studies on cell suspensions and calli of an S-(2-aminoethyl)cysteine resistant cell line of the interdihaploid potato H²578 (2n=24) revealed a high degree of variation in the number of chromosomes (33–217) and dicentric chromosomes (0–8). The suspensions also exhibited megachromosomes and fused chromosomes. Differential staining showed that in suspensions dicentrics survived mitotic cycles mainly by parallel separation of the chromatids during anaphase and hardly by the breakage-fusion-bridge cycle. Two phenotypes of plantlets regenerated, each with 34 or 35 chromosomes with gross structural mutations and with the triploid amount of DNA. Chromosomal variation was related to the degree of tissue organization.