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.     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.     

UV micro-irradiation of the Chinese hamster cell nucleus and caffeine post-treatment immunocytochemical localization of DNA photolesions in cells with partial and generalized chromosome shattering

UV micro-irradiation of a small part of the Chinese hamster nucleus and caffeine post-incubation often results in shattered chromosomes at the first post-irradiation mitosis. In some of these mitotic cells, chromosome shattering is restricted to a few chromosomes spatially related in a small area of the metaphase spread; in others, shattering includes the whole chromosome complement. These 2 types of damage have been called partial and generalized chromosome shattering (PCS and GCS).Using antisera that specifically react with UV-irradiated DNA, we identified micro-irradiated chromatin in interphase nuclei and in mitotic cells with PCS or GCS by indirect immunofluorescence microscopy. In PCS, immunofluorescence staining was found in the damaged area, while the surrounding intact chromosomes were not stained. In GCS, staining was also restricted to a small region of the shattered chromosome complement. In other experiments, cells synchronized in G1 were micro-irradiated in the nucleus, pulse-labelled with [³H]thymidine and post-incubated with caffeine. Autoradiographs of cells with GCS showed unscheduled DNA synthesis restricted to a small chromatin region.Our data present direct evidence that the distribution of DNA photolesions does not coincide with the sites of chromosomal damage in GCS. As a working hypothesis, we propose that an indirect mechanism is involved in the induction of GCS by which DNA photolesions in a small nuclear segment induce shattering of both micro-irradiated and non-irradiated chromosomes.     

Unscheduled DNA synthesis after partial UV irradiation of the cell nucleus. Distribution in interphase and metaphase.

Cells of an euploid strain of the Chinese hamster synchronized in the G1 phase were microirradiated in the nucleus with a laser UV microbeam (λ = 257 nm) and pulse-labelled with [³H]thymidine. In autoradiographs of cells fixed immediately after the pulse unscheduled DNA synthesis (UDS) was found restricted to the microirradiated part of the nucleus. The rate of UDS varied with the UV energy applied and the post-irradiation incubation time. In other experiments chromosome preparations were established after an additional chase and a subsequent growth period. In 28 mitotic cells autoradiographic label was found concentrated on a few chromosomes which lay adjacent to each other in one part of the metaphase plate. The distribution of label on the chromosomes could clearly be distinguished from patterns which originate from semi-conservative DNA synthesis within S phase. The label on chromosomes of microirradiated cells thus represents UDS. Our findings support the following ideas on the arrangement of interphase chromosomes: (1) Decondensed interphase chromosomes may occupy rather compact territories. (2) Chromosomes do not necessarily exhibit a close and permanent association with their respective homologues.     

AN ANALYSIS OF HETEROCHROMATIN IN MAIZE ROOT TIPS

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15–20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.     

Asynchrony of DNA replication and mitotic spiralization along heterochromatic portions of Chinese hamster chromosomes

Sequence of DNA synthesis and mitotic chromosome spiralization along heterochromatic portions of the sex (X₁X₂) and of some marker chromosomes in cultured Chinese hamster cells were studied, employing two methods: study of segmentation pattern caused in chromosomes with colcemid, and autoradiography with tritiated thymidine. The heterochromatic portions of all chromosomes studied were characterized by striking internal asynchrony of DNA replication. In particular, they had segments that replicated relatively early. The short arm of the X₂ chromosome, heterochromatic in female somatic cells, had at least three such segments. Replication patterns of the long arms of the X₁ and X₂ chromosomes were different. In X₁ this arm contains several segments showing relatively early replication. The long arm of X₂ had no similar segments. The possible significance of the data obtained is discussed with regard to the problem of genetic inertness of heterochromatin. At the terminal stage of the S period, H³-thymidine seems to be incorporated into condensed chromatin of interphase nuclei. On the basis of the data obtained, it is proposed that during replication of heterochromatin consecutive despiralization of parts of it takes place.     

Topological origins of chromosomal territories

Using freely jointed polymer model we compare equilibrium properties of crowded polymer chains whose segments are either permeable or not permeable for other segments to pass through. In particular, we addressed the question whether non-permeability of long chain molecules, in the absence of excluded volume effect, is sufficient to compartmentalize highly crowded polymer chains, similarly to what happens during formation of chromosomal territories in interphase nuclei. Our results indicate that even polymers without excluded volume compartmentalize and show strongly reduced intermingling when they are mutually non-permeable. Judging from the known fact that chromatin fibres originating from different chromosomes show very limited intermingling in interphase nuclei, we propose that regular chromatin fibres during chromosome decondensation can hardly serve as a substrate of cellular type II DNA topoisomerases.     

Cell-cycle-dependent dissociation of histone H1 from chromatin in nuclei of P. polycephalum.

The dissociation curves of histone H1 from chromatin in interphase and metaphase nuclei from Physarum polycephalum have been determined using CaCl2 as dissociating agent. H1 is less strongly bound to metaphase chromosomes than to interphase chromatin. However, no differences could be detected in the binding of Hl to early S, late S or G2 phase chromatin. The number of CaCl2 molecules involved in binding one H1 molecule to chromatin was reduced from 5 in interphase to 4 in metaphase. The non-electrostatic contribution to the free-energy of binding was small in both cases. A comparison of the binding properties of H1 to sheared chromatin, native chromatin and metaphase chromosomes suggests that the electrostatic binding functions of H1 are completely satisfied within the nucleosome and that further electrostatic interactions are not involved in folding the nucleosomal fibre into the 300 A "solenoid" or the more tightly folded metaphase chromosome.     

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.     

Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells

Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis. J. Cell. Biochem. 65:114–130. © 1997 Wiley-Liss, Inc.     

Do individual allocyclic chromosomes in metaphase reflect their interphase domains?

Summary Individual S phase allocyclic chromosomes have been analyzed in Bloom syndrome lymphocytes, in cells with an r(9), and in hypotetraploid Ehrlich mouse ascites cells treated with 1-methyl-2-benzyl hydrazine. On the basis of the following observations, we conclude that such chromosomes more or less reflect their domains in interphase: (1) The S phase allocyclic chromosomes have the same structure as S phase prematurely condensed chromatin (PCC) in fused cells; in other words they form limited areas of chromatin dots; (2) the allocyclic chromosome is the only chromosome in a metaphase plate which synthesizes DNA simultanneously with interphase nuclei; (3) the size of the allocyclic chromosomes is related to the size of the corresponding metaphase chromosome; and (4) the S phase allocyclic chromosomes resemble closely the chromosome domains in interphase made visible with biotinylated human DNA. A variety of evidence shows that most allocyclic chromosomes are simply left behind in their cycle, which presumably is caused by a deletion or inactivation of a hypothetical coiling center situated on each chromosome arm.     

Premature chromosome condensation and cell cycle analysis.

The application of the phenomenon of premature chromosome condensation for cell cycle analysis in HeLa and CHO cells has been examined. Random populations of HeLa and CHO cells pulse labelled with H3-TdR were separately fused with mitotic HeLa cells using U.V. inactivated Sendai virus. The resulting prematurely condensed chromosomes (PCC) were scored and classified into G1, S and G2-PCC on the basis of both morphological and autoradiographic data, The results of this study indicated that the G1, S and G2 phase cells are equally susceptible to virus-induced fusion with mitotic cells and subsequent induction into PCC. Hence the PCC method for cell cycle analysis is both practical and accurate. This study also revealed that the process of chromosome decondensation initiated during the telophase of mitosis continues throughout the G1 period reaching an ultimate state of decondensation by the end of G1, at which point the fusion of such cells with those in mitosis yield PCC with the most diffused morphology instead of the discrete single stranded structures characteristic of early G1-PCC. Thus, the decondensation of chromatin during G1 appears to be a prerequisite for the subsequent initiation of DNA synthesis.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G1 period of the HeLa cell cycle. HeLa cells synchronized in the G1 period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N2O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G1 period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G1 period, when DNA synthesis is initiated.     

Association of BAF53 with mitotic chromosomes

The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis.     

HETEROCHROMATIN IN HUMAN MALE LEUKOCYTES

Tritiated thymidine was added to peripheral blood cultures containing phytohemagglutinin so that DNA synthesis in interphase nuclei of white blood cells in the human male could be studied. After 57 hours in culture, a large heterochromatic body with a central position is seen in unlabeled Feulgen-stained nuclei. In labeled nuclei in which DNA synthesis was taking place in both the eu- and heterochromatin at the time the thymidine became available, the heterochromatin shows a higher number of silver grains per unit area, accompanied by a stronger Feulgen reaction, an indication of its higher DNA content. The time of DNA synthesis in the heterochromatin blocks is different from that in the surrounding euchromatin. The large heterochromatic block is composed of chromosome segments gathered together around the nucleolus but it is not part of this organelle. In preparations stained with azure A and acid fuchsin for demonstrating both the nucleolus and the chromosomes, six distinctly heteropyenotic chromosome segments can be seen associated with the nucleolus. Cells of all size categories were found to incorporate tritiated thymidine. The distinct appearance of autosomal heterochromatin in white blood cells may be the result of the new physiological conditions to which the cells are subjected in the medium containing phytohemagglutinin.     

DNA synthesis and endomitoses in the giant nuclei of the silkgland of Bombyx mori.

At the first symptoms of organisation of the silkgland in the embryo, mitoses stop and nuclei start to grow. Through autoradiographic studies, performed after sequenced labeling with [3H] and [14C] thymidine, the durations of the different phases of DNA synthesis cycles (T = 42 to 48 h, S = 22 to 25 h, G = 20 to 23 h) are established. These durations are in fact identical during the second and the third instar, and the same, whatever region is concerned : posterior, middle or anterior parts. A model has been established to account for the distribution of the S phases during the second and third instars. The DNA synthesis in all nuclei of a given region has been followed during the first four instars by labelling with [3H]thymidine. The activity goes through maxima and minima, depending on the percent of nuclei synthesizing DNA and their synchronism, both being characteristic of each region; long resting periods are observed during the molting stages of the first three instars in the middle and the anterior parts. The coincidence is obvious between the maxima and minima and respectively the S and G phases of the model. DNA assays agree with the distribution of cycles established by autoradiography if one admits that each cycle does lead to a doubling of the amount of DNA. The overall DNA synthesis from the diploid value is estimated to correspond to 18-19 endomitoic cycles in the nucleic of the posterior part, 19-20 cycles of those of the middle part and 13 in those of the anterior part. The analysis of chromosome structures, by squashing the nuclear content, shows that existence of a complete endomitotic cycle: the doubling of chromosomes is associated with condensed structures, alternating with a decondensed state of chromatin, responsible for the DNA synthesis. The female heterochromatin undergoes a restricted morphological cycle delayed with respect to bulk chromatin. It is characterized by a late DNA duplication and by non dispersed daughter chromosomes. Some of these aspects are, to a lesser extent, reproduced in groups of autosomic chromosomes.     

THE FINE STRUCTURE OF THE DNP COMPONENT OF THE NUCLEUS : An Electron Microscopic Study Utilizing Autoradiography to Localize DNA Synthesis

In the present investigation, the sites of deoxyribonucleic acid (DNA) synthesis and the fate of labeled deoxyribonucleoprotein (DNP) were studied in autoradiographs of ultrathin sections viewed with the electron microscope. Tritiated thymidine was employed as a label for DNA in the nuclei of proliferating cells of regenerating salamander limbs. In the autoradiographic method reported here, dilute NaOH was used to remove the gelatin of the emulsion after exposure and development. The exposed silver grains are not displaced by this treatment and the resolution of fine structure in the underlying section is greatly improved. Our observations suggest that the DNP component is a meshwork of interconnected filaments 50 to 75 A in diameter, which may be cross-linked to form what Frey-Wyssling would term a "reticular gel." The filamentous DNP meshwork is dispersed throughout the interphase nucleus during DNA synthesis, whereas in chromosomes, which are relatively inert metabolically, the meshwork is denser and is aggregated into compact masses. Dense chromatin centers in interphase nuclei are similar in fine structure to chromosomes and are also inert with respect to DNA synthesis. In the Discussion, the structure of the filamentous meshwork in chromatin is compared with that in chromosomes, and speculations are made as to the functional significance of the variations in DNP fine structure observed.     

Quinacrine fluorescence of specific chromosome regions

The pattern of intense fluorescence of interphase nuclei and metaphase chromosomes after staining with quinacrine is described in Samoaia leonensis. Autoradiographic analysis of interphase nuclei after pulse labeling with tritiated thymidine indicates that there is little or no overlap in the time of replication of the intensely fluorescing and weakly fluorescing regions. Autoradiographic analysis of metaphase figures after continuous labeling with tritiated thymidine shows that the intensely fluorescing regions are late replicating and establishes their order of replication. Autoradiographic analysis of interphase nuclei after pulse labeling with tritiated deoxycytidine and of metaphase figures after continuous labeling with this tracer show that there is little, if any, incorporation of deoxycytidine into those chromosome regions which fluoresce intensely after staining with quinacrine and quinacrine mustard. These results indicate that such chromosome regions are characterized chemically by an extremely high, if not exclusive, content of adenine and thymine.