Meiosis of trisomy 21 in the human pachytene oocyte

Association modalities of the three 21 chromosomes were studied during pachytene in three trisomy 21 fetuses whose chromosomal constitution was identified following amniocentesis. -- Three classes of images were observed: a trivalent, a trivalent presenting an important asynaptic region of the long arm, and a bivalent accompanied by a univalent. Such behaviour is analagous to that observed in all trisomic organisms. -- We have been able to establish the sequence of chromomeres, whose number varies from 9 to 14 according to the state of contraction in the 21 chromosome. Each band is thus subdivided into several sub-bands: at maximal elongation 2 sub-bands for band p11, 4 for q21 and 3 for q222. In addition, the interchromomeric clear bands q221 and q223 are also subdivided by the presence of a very small chromomere. In this way, the G-bands visible on mitotic metaphase chromosomes result from the compression together of several chromomeres whose individuality disappears as chromosomal condensation increases with progression of prophase.     

Structure of chromatin and chromosomes in preparations of interphase nucleus derivatives, prepared by removal of the nucleuar envelopes. II. Structure of chromatin and associations of chromosomes in stretched amembranous nuclei and mitotic figures]

Preparations of surface stretched amembranous nuclei and mitotic figures were used for revealing the high order nuclear and chromosomal structures. The preparations were obtained by dropping amembraneous nuclei and mitotic figures suspension in methanol-glacial acetic acid mixture (3:1) on wetted superclean slides. Amembraneous nuclei and mitotic figures were isolated from intact murine and human cells (lines L1210, SK-UT-1B, PHA-stimulated lymphocytes) by means of their 1-5 min prefixational capillary pipetting with freshly prepared 0.018-0.06% Triton X-100 solution in the conditional cultural medium. Stretched amembraneous nuclei and mitotic figures had no features of induced chromatin dispersion and compaction. Stretched interphase amembraneous nuclei showed spatially separated individual structures (thin chromatin fibres, nucleoli, intranuclear bodies), polymorphous pattern of perinucleolar chromatin aggregation and episodically expressed beaded thick chromatin fibres and a chromocenter. The chromomeric pattern of the spread chromosomes of mitotic figures was quite similar but hardly identical with that of G-banding. The stretched prometaphase mitotic figures in all tested cell types always contained loose "residual" nucleoli looking like typical prophase nucleoli as concerns their shape and number per cell (mitotic figure). The majority of chromosomes of stretched mitotic figures and of prophase amembraneous nuclei were attached to the nucleolar material. All tested cell lines showed almost the same variation in number of nucleolus-attached chromosomes, per both prophase amembraneous nucleus and prometaphase mitotic figure. Some chromosomes of stretched mitotic figures were colocated with "residual" nucleoli and looked shortened and strongly condensed. Other chromosomes, locally associated with "residual" nucleoli, were straight and oriented radially to these. Mutual chromosomal arrangements in mitotic cells on smears and in stretched mitotic figures were analogous. Equatorial plates from PBS-washed SK-UT-1B cells displayed a better stretching capacity than those from untreated cells. In the former case metaphase chromosomes were seen more uniformly stretched and well identified after GTG-banding procedure. The number of interchromosomal (mainly telomere-telomeric and telomere-centromeric) connections per stretched mitotic figure (or per stretched prophase amembraneous nucleus) was minimum in late prometaphase, maximum in prophase and early prometaphase, and intermediate in metaphase. The obtained data are discussed in terms of topology and longitudinal heterogeneity of mitotic chromosomes.     

The Chromosome Number of Psammosilene tunicoides Endemic to China

In this paper,Psammosilene tunicoides,an endemic species to China,was cytologically studied for the first time.The morphology of the nuclei at resting stage was categorized to be simple chromocentre type.The morphology of mitotic prophase chromosomes was categorized to be the interstitial type.28 chromosomes were observed at the mitotic metaphase,and 14 bivalent chromosomes were observed at diakinesis.So,the basic chromosome number was confirmed to be x=14.Psammosilene tunicoides is different from Silene rubicunda in the basic chromosome number and the morphology of the nuclei at resting stage and mitotic prophase chromosomes,because Silene rubicund has the basic chromosome number of x=10 and 12,and its nuclei at resting stage and mitotic prophase chromosomes is sparsely diffuse type and continuous type respectivrly.     

Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line

The replication of chromosomes in meiosis is an important first step for subsequent chromosomal interactions that promote accurate disjunction in the first of two segregation events to generate haploid gametes. We have developed an assay to monitor DNA replication in vivo in mitotic and meiotic germline nuclei of the nematode Caenorhabditis elegans. Using mutants that affect the mitosis/meiosis switch, we show that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cell nuclei. Furthermore, our assay reveals that different regions of the genome replicate at different times, with the heterochromatic-like X chromosomes replicating at a distinct time from the autosomes. Finally, we have exploited S-phase labeling to monitor the timing of progression through meiotic prophase. Meiotic prophase for oocyte production in hermaphrodites lasts 54-60 h. Further, we find that the duration of the pachytene sub-stage is modulated by the presence of sperm. On the other hand, meiotic prophase for sperm production in males is completed by 20-24 h. Possible sources for the sex-specific differences in meiotic prophase kinetics are discussed.     

Characterization of G-banded chromosomes of the Indian muntjac and progression of banding patterns through different stages of condensation

Muntjac prophase and metaphase chromosomes were G-banded following methotrexate-mediated synchronization of peripheral lymphocytes. Bands and subbands were characterized from prophase through metaphase, and the progression of band patterns from late prophase to mid-metaphase was analyzed. Extended prophase chromosomes exhibited more bands and subbands, a number of which became fused with each other, giving rise to fewer and thicker bands in the condensed metaphase chromosomes. It appeared that the dark bands condensed relatively more than the light bands. Precise delineation of the bands and subbands on extended prophase chromosomes and the usage of a proposed banding pattern nomenclature should aid in better detection and localization of induced chromosomal rearrangements with this extremely useful experimental material.     

Immunocytochemical localization of the DNA-binding protein HCc during the cell cycle of the histone-less dinoflagellate protoctista Crypthecodinium cohnii B

The major basic nuclear protein HCc (previously named Histone-like) of the dinoflagellate Crypthecodinium cohnii B was immunolocalized in light and electron microscopy using an affinity-purified polyclonal antibody. Complementary conventional and cryo-techniques were used to study the distribution of the DNA-binding protein in interphase cells and to follow its behaviour throughout the mitotic cycle. In non-dividing cells, the HCc protein was found to be located on extra-chromosomal loops and chromosomal nucleofilaments dispersed in the nucleoplasm. In mitotic cells, from prophase to early telophase, it was homogeneously distributed in the (whole) dividing chromosomes. HCc protein was also detected in two compartments of all the permanently observable nucleoli: the nucleolar organizing region and the fibrillo-granular region. In this paper we discuss the hypothetical roles, structural and/or functional, of this DNA-binding protein, which is specific to dinoflagellates, the only eukaryotes whose chromatin is devoid of histones and nucleosomes.     

Microtubule disassembly delays the G2-M transition in vertebrates

When cell cultures in growth are treated with drugs that cause microtubules to disassemble, the mitotic index (MI) progressively increases as the cells accumulate in a C-mitosis. For many cell types, however, including rat kangaroo kidney PtK(1) cells, the MI does not increase during the first several hours of treatment [1-3] (Figure 1). This 'lag' implies either that cells are entering mitosis but rapidly escaping the block, or that they are delayed from entering division. To differentiate between these possibilities, we fixed PtK(1) cultures 0, 90 and 270 minutes after treatment with nocodazole, colcemid, lumi-colcemid, taxol or cytochalasin D. After 90 minutes, we found that the numbers of prophase cells in cultures treated with nocodazole or colcemid were reduced by approximately 80% relative to cultures treated with lumi-colcemid, cytochalasin D or taxol. Thus, destroying microtubules delays late G(2 )cells from entering prophase and, as the MI does not increase during this time, existing prophase cells do not enter prometaphase. When mid-prophase cells were treated with nocodazole, the majority (70%) decondensed their chromosomes and returned to G(2) before re-entering and completing prophase 3-10 hours later. Thus, a pathway exists in vertebrates that delays the G(2)-M transition when microtubules are disassembled during the terminal stages of G(2). As this pathway induces mid-prophase cells to transiently decondense their chromosomes, it is likely that it downregulates the cyclin A-cyclin-dependent kinase 2 (CDK2) complex, which is required in vertebrates for the early stages of prophase [4].     

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK1. When the laser power delivered to sample surface was 90-190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360-420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50-70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

Complete pachytene chromomere karyotypes of human spermatocyte bivalents

Summary Well-spread human pachytene spermatocyte bivalents were obtained allowing specific identification of each bivalent within its total complement according to its chromomere sequence combined with further staining of its centromeric heterochromatin. The total number of chromomeres was found to be related to the degree of bivalent contraction: 396 in condensed bivalents and 511 in decondensed bivalents. A striking correspondance between chromomeres and mitotic G-bands was observed; on account of the variability of bivalent contraction, condensed bivalents corresponded to prometaphase somatic chromosomes and decondensed bivalents to mid/late prophase chromosomes.     

Effect of methotrexate and actinomycin D on the frequency and ratio of early mitotic cells

Different methods for obtaining early mitotic chromosomes have been comparatively analyzed. Lymphocyte cultures have been treated with methotrexate, actinomycin-D and with a combination of both agents. It is shown that a high rate of chromosomes in prophase, prometaphase and mid-metaphase can be achieved by the combined methotrexate and actinomycin-D treatment of the lymphocyte culture. Methotrexate treatment of cultures is preferable to obtain the great number of early metaphase chromosomes.     

Karyomorphology of four species in Ancylostemon,Briggsiopsis and Lysionotus (Gesneriaceae)

Reported in the present paper are chromosome numbers and karyotypes of three genera of the Gesneriaceae, i.e. Ancylostemon Craib. , Briggsiopsis (Franch.) K. Y. Pan and Lysionotus D. Don. The former two genera are endemic to China. The karyotype of Ancylostemon aureus (Franch.) Burtt is formulated as 2n = 34 = 20m(1sat) + 14sm, with the same chromosome number as its allied species A. convexus Craib. This species is characterized by the interphase nucleus of complex chromocenter type and the proximal type of chromosomes in the mitotic prophase. The chromosome number of the monospecific genus Briggsiopsis is 2n = 34, the same as the lowest chromosome number reported in Briggsia. The karyotype of Briggsiopsis, which is formulated as 2n = 25m + 6sm + 3st, also seems to be primitive among the species of the two genera. Briggsiopsis is characterized by the interphase nucleus of simple-complex chromocenter type and the interstitial-gradient type of chromosomes in the mitotic prophase. The chromosome number of Lysionotus carnosus Hemsl. is the lowest reported in this genus. Its karyotype is formulated as 2n= 30 = 21m + 5sm + 3st + lt. Lysionotus serratus var. pterocaulis, with the karyotype being formulated as 2n= 32 = 2lm + 10sm + lt, has the same chromosome number as var. serratus. These two species show a remarkable differentiation of karyotypes and are characterized by the interphase nuclei of simple-complex chromocenter type and the gradient type of chromosomes in the mitotic prophase. _ .     

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK₁. When the laser power delivered to sample surface was 90–190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360–420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50–70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

INTRAPOPULATION KINETICS OF THE MITOTIC CYCLE

Data obtained with time lapse cinemicrographic techniques showed that the distribution of generation times for exponentially proliferating human amnion cells in culture is skewed to the right and that reciprocals of generation times appear normally distributed. As shown for bacteria, the true age distribution is much broader than theoretical distributions which fail to take into account the dispersion of generation times. By means of the technique utilizing autoradiographic detection of tritiated thymidine in cells whose mitotic histories were recorded by time lapse cinemicrography, it was shown that the G1 distribution is similar to the generation time distribution but is more variable. In our experiments, the G2 + prophase distribution resembled the generation time and G1 distributions. The data suggested two possibilities for S: either it is relatively constant, or it is inversely related to the lengths of G1 and G2 + prophase. Since G1 is more variable than the total cycle, and G2 + prophase more variable than the computed sum of S + G2 + prophase + metaphase, it was concluded that the relationships between parts of the cycle are non-random and that compensating mechanisms apparently help regulate the lengths of successive parts of the mitotic cycle in individual cells.     

Evidence of activity-specific, radial organization of mitotic chromosomes in Drosophila

The organization and the mechanisms of condensation of mitotic chromosomes remain unsolved despite many decades of efforts. The lack of resolution, tight compaction, and the absence of function-specific chromatin labels have been the key technical obstacles. The correlation between DNA sequence composition and its contribution to the chromosome-scale structure has been suggested before; it is unclear though if all DNA sequences equally participate in intra- or inter-chromatin or DNA-protein interactions that lead to formation of mitotic chromosomes and if their mitotic positions are reproduced radially. Using high-resolution fluorescence microscopy of live or minimally perturbed, fixed chromosomes in Drosophila embryonic cultures or tissues expressing MSL3-GFP fusion protein, we studied positioning of specific MSL3-binding sites. Actively transcribed, dosage compensated Drosophila genes are distributed along the euchromatic arm of the male X chromosome. Several novel features of mitotic chromosomes have been observed. MSL3-GFP is always found at the periphery of mitotic chromosomes, suggesting that active, dosage compensated genes are also found at the periphery of mitotic chromosomes. Furthermore, radial distribution of chromatin loci on mitotic chromosomes was found to be correlated with their functional activity as judged by core histone modifications. Histone modifications specific to active chromatin were found peripheral with respect to silent chromatin. MSL3-GFP-labeled chromatin loci become peripheral starting in late prophase. In early prophase, dosage compensated chromatin regions traverse the entire width of chromosomes. These findings suggest large-scale internal rearrangements within chromosomes during the prophase condensation step, arguing against consecutive coiling models. Our results suggest that the organization of mitotic chromosomes is reproducible not only longitudinally, as demonstrated by chromosome-specific banding patterns, but also radially. Specific MSL3-binding sites, the majority of which have been demonstrated earlier to be dosage compensated DNA sequences, located on the X chromosomes, and actively transcribed in interphase, are positioned at the periphery of mitotic chromosomes. This potentially describes a connection between the DNA/protein content of chromatin loci and their contribution to mitotic chromosome structure. Live high-resolution observations of consecutive condensation states in MSL3-GFP expressing cells could provide additional details regarding the condensation mechanisms.     

Studies on the Mitotic Prophase Chromosomal Fibers in Vicia faba

The observations have been made on the structures of mitotic prophase nuclei and chromosomes in Vicia faba root meristematic cells. Two methods, i.e., the cell squashing and the nucleus isolation methods, were applied in present study to prepare the specimen of chromosomes and nuclei. Chromosomal fibers 0.3—0.5 μm in diameter were observed in the squashed preparations stained with Giemsa, and in the isolated nucleus preparations treated with 0.05% EDTA followed by Giemsa staining. Using Feulgen reaction, it has been demonstrated that these fibers are nuclear origin containing DNA. The results suggest that this order of chromosomal fiber may be one structural level in the chromosomes in Vicia faba. This conclusion is in support of the view which holds that there exists an intermediate level of structure between the 250–300Å chromatin fiber and the chromosome.     

Entamoeba histolytica: ultrastructure of the chromosomes and the mitotic spindle

We have analyzed by transmission electron microscopy the mitotic process of Entamoeba histolytica trophozoites in an asynchronous population of axenically cultured parasites. Our observations showed that nuclear microtubules, initially located at random in the karyosome during prophase, formed in subsequent stages a mitotic spindle closely related to the nuclear membrane at the polar regions of dividing nuclei. In late prophase and in anaphase, chromosomes appeared as dense bodies 0.1-0.5 microm. At least 15 chromosomes appeared in favorable planes of section, arranged as an incomplete elliptical circle, in close contact with microtubules. There was no morphological evidence of structures resembling the kinetochores of higher eukaryotes. When cut in cross-section, the mitotic spindle was made of 28-35 microtubular rosette assemblies. The latter probably correspond to a similar number of chromosomes, as has been shown by others with pulse-field electrophoresis and fluorescence microscopy of trophozoite spreads. In turn, each microtubular rosette was constituted by 7-12 parallel microtubules. In later stages of the metaphase, two sets of chromosomes were disposed forming a pair of elliptical circles. An additional finding in the dividing nuclei of E. histolytica trophozoites was the presence of compact conglomerates of numerous particles 50 nm in diameter, of similar electron density, shape, and size, probably corresponding to RNA episomes.     

The vesicular compartment of the mitotic apparatus in mammalian cells

Intracellular membranes might play an eminent role in regulating several events during mitosis: In this paper the appearance and changing configurations of the vesicular compartment of the mitotic apparatus of HeLa cells was studied from anaphase to telophase. In early prophase electron opaque and transparent membranous vesicles are found in the pericentriolar region outside the nucleus. During prometaphase when the nuclear envelope opens and starts to disappear, an increasing number of these vesicles appears in the mitotic apparatus near the chromosomes. During metaphase vesicles are spread all over the mitotic apparatus, the number of electron opaque vesicles decreases while the total amount of vesicles does not change significantly. Anaphase shows the same pattern of distribution in the half-spindle and in the midbody. In telophase the amount of electron opaque vesicles increases again. They are now found around vacuoles and near the newly appearing Golgi-cisternae. We assume that the electron opaque vesicles are derived from the Golgi- apparatus which disintegrates during prophase and reappears in late telophase. The change in the appearance of the different types of vesicles during metaphase coincides with drastic changes in the ionic milieu in the mitotic apparatus (Wolniak et al., 1983).     

Evidence for a relatively random array of human chromosomes on the mitotic ring

We used fluorescence in situ hybridization (FISH) to study the positions of human chromosomes on the mitotic rings of cultured human lymphocytes, MRC-5 fibroblasts, and CCD-34Lu fibroblasts. The homologous chromosomes of all three cell types had relatively random positions with respect to each other on the mitotic rings of prometaphase rosettes and anaphase cells. Also, the positions of the X and Y chromosomes, colocalized with the somatic homologues in male cells, were highly variable from one mitotic ring to another. Although random chromosomal positions were found in different pairs of CCD-34Lu and MRC-5 late-anaphases, the separations between the same homologous chromosomes in paired late-anaphase and telophase chromosomal masses were highly correlated. Thus, although some loose spatial associations of chromosomes secondary to interphase positioning may exist on the mitotic rings of some cells, a fixed order of human chromosomes and/or a rigorous separation of homologous chromosomes on the mitotic ring are not necessary for normal mitosis. Furthermore, the relative chromosomal positions on each individual metaphase plate are most likely carried through anaphase into telophase.     

Controlled somatic and germline copy number variation in the mouse model

Changes in the number of chromosomes, but also variations in the copy number of chromosomal regions have been described in various pathological conditions, such as cancer and aneuploidy, but also in normal physiological condition. Our classical view of DNA replication and mitotic preservation of the chromosomal integrity is now challenged as new technologies allow us to observe such mosaic somatic changes in copy number affecting regions of chromosomes with various sizes. In order to go further in the understanding of copy number influence in normal condition we could take advantage of the novel strategy called Targeted Asymmetric Sister Chromatin Event of Recombination (TASCER) to induce recombination during the G2 phase so that we can generate deletions and duplications of regions of interest prior to mitosis. Using this approach in the mouse we could address the effects of copy number variation and segmental aneuploidy in daughter cells and allow us to explore somatic mosaics for large region of interest in the mouse.     

Events associated with the initiation of mitosis in fused multinucleate HeLa cells

Large multinucleate (LMN) HeLa cells with more than 10–50 nuclei were produced by random fusion with polyethylene glycol. The number of nuclei in a particular stage of the cell cycle at the time of fusion was proportionate to the duration of the phase relative to the total cell cycle. The fused cells did not gain generation time. Interaction of various nuclei in these cells has been observed. The nuclei initially belonging to the G1-or S-phase required a much longer time to complete DNA synthesis than in mononucleate cells. Some of the cells reached mitosis 15 h after fusion, whereas others required 24 h. The cells dividing early, contained a larger number of initially early G1-phase nuclei than those cells dividing late. The former very often showed prematurely condensed chromosome (PCC) groups. In cells with a large number of advanced nuclei the few less advanced nuclei could enter mitosis prematurely. On the other hand, the cells having a large number of nuclei belonging initially to late S-or G2-phase took longer to reach mitosis. These nuclei have been taken out of the normal sequence and therefore failed to synthesize the mitotic factors and depended on others to supply them. Therefore the cells as a whole required a longer period to enter mitosis. Although the nuclei became synchronized at metaphase, the cells revealed a gradation in prophase progression in the different nuclei. At the ultrastructural level the effect of advanced nuclei on the less advanced ones was evident with respect to chromosome condensation and nuclear envelope breakdown. Less advanced nuclei trapped among advanced nuclei showed PCC and nuclear envelope breakdown prematurely, whereas mitotic nuclei near interphase or early prophase nuclei retained their nuclear envelopes for a much longer time. PCC is closely related to premature breakdown of the nuclear envelope. Our observations clearly indicate that chromosome condensation and nuclear envelope breakdown are two distinct events. Kinetochores with attached microtubules could be observed on prematurely condensed chromosomes. Kinetochores of fully condensed chromosomes often failed to become connected to spindle elements. This indicates that the formation of a functional spindle is distinct from the other events and may depend on different factors.