Genetic control of mitosis. Premature beginning of telophase chromatin reorganization in cells of the Drosophila melanogaster strain with ff3 mutation

The effect of cell cycle mutation ff3 on chromosome segregation was studied on fixed cells of neural ganglia. The cell distributions by diameter of interphase nuclei and by distance between sister chromatid sets were compared at anaphase and telophase. In the control wild-type strain Lausenne, the cell distribution by distance between sister chromatids in anaphase was similar to their distribution by nuclear size. The mean distance between segregating chromatids at anaphase (lcp) coincided with the mean diameter of interphase nuclei (dcp) and was 8.3 microns. Cells passed to telophase when chromatids were at least 10 microns apart. The mutant ff3 strain differed from the control strain Lausenne in cell distribution by interphase nuclear diameter and distance between sister chromatids in anaphase; the mean nuclear diameter and mean distance between segregating chromatids similarly increased to 9.3 microns. A specific feature of mitosis in mutant strain ff3 was a premature beginning of telophase chromatin reorganization. This caused the occurrence of cells with abnormally short (less then the interphase nuclear diameter) distance between sister chromatid sets in telophase but not in anaphase, as if these cells had passed from anaphase to telophase prematurely, during the chromatid movement toward poles in anaphase A.     

Actinomycin D effects on mitosis and chromosomes: sticky chromatids and localized lesions

When Indian muntjac and Chinese hamster cells in culture were treated with Actinomycin D (1 g/ml) for 1–2 hours, the sister chromatids, especially the distal segments, appeared to have difficulty separating in anaphase. The separated proximal segments progressively became stretched. The nucleolus organizer regions seemed to be most susceptible to stretching, and breaks in these regions were frequently observed. Electron microscopic observations showed that the sticky chromatids (and less frequently sticky chromosomes) contain connecting submicroscopic chromosome strands. When the treated cells were allowed to grow in a drug-free medium for several days, a high frequency of endoreduplicated mitotic figures was found. Chromosome and chromatid breaks and other aberrations were common, mainly localized at G band negative areas particularly nucleolus organizer regions.     

Nucleolus-like structures detected in the cytoplasm ofBrodiaea uniflora: Light- and electron-microscopic surveys during mitosis

Summary Both light- and electron-microscopic studies confirmed the presence of cytoplasmic nucleolus-like structures in the root-tip meristems ofBrodiacea uniflora, Liliaceae. This structures were found to be different from all the types of nucleolus-like structures previously detected in the cytoplasm. The nucleolus-like structures in the present material usually appeared as complex structures with two types of elements, although the two elements sometimes lay separately in the cytoplasm. The two elements were differentially stained by the silver staining technique: one reacted more intensely with silver forming black-stained dot while the other was stained brown. The ultrastructural examination revealed that the black-stained dots were spherical, about 1.0 m in diameter, and consisted of highly electron-dense material in which many lacunar areas were always seen, while the brown-stained spherules were consisted of loosely packed RNP- or ribosome-like granules. The nucleolus-like structures were found almost exclusively at telophase and they were never or seldom detected from interphase to anaphase.Their peculiar behavior during mitosis and their ultrastructural organization are discussed with reference to their origin.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation. Received: 3 July 1996 / Accepted: 4 October 1996     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

Genetic Control of Mitosis: Features of Anaphase Separation of Sister Chromatid Sets in Mutant Strain aar V158 in Drosophila melanogaster

The effect of mutation aar ^V158 on anaphase separation of chromatids was studied on fixed cells of neural ganglia of Drosophila melanogaster larvae. It was shown that mutation aar ^V158 causes three types of defective chromosome segregation manifested as (1) monopolar anaphase, (2) separation of chromatids to an abnormally short distance in anaphase, and (3) bridging and lagging of some chromatids or prolonged asynchronous separation of sister chromatid sets to the poles in anaphase. We believe that the former two types of defective segregation are caused by disturbed centrosome separation at the beginning of mitosis and the third type, by defects in chromatid separation during anaphase. During the two-year maintenance of the mutation in a heterozygous state, partial correction (adaptive modification) of the defects of type 1 and type 2 (but not type 3) occurred. The correction of type 1 and type 2 defects during adaptogenesis depended on the genotype: in heterozygotes and homozygotes, respectively type 1 and type 2 were preferentially corrected. The frequency of type 3 defects remained constant during the two-year period of maintenance of the mutation in a heterozygous state. However, in all variants of the experiment, their frequency decreased with increasing distance between the sister chromatid sets. In the cells that completed the previous division with abnormalities, the checkpoint system is supposed to effectively arrest the cell cycle in the subsequent division.     

Shoot organogenesis and plant regeneration in mulberry (Morus bombycis Koidz): Factors influencing morphogenetic potential in callus cultures

Regeneration of multiple shoots via callus induction and organogenesis was achieved in mulberry (Morus bombycis). Pre-soaked internodal explants in 4.4–8.9 M benzyladenine (BA) formed callus on Linsmaier and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (9.05 M), -naphthaleneacetic acid (2.85 M) and BA (2.2 M). Explants soaked for 48 to 72 h in low levels of BA produced loose and nodular callus that showed regeneration ability. Calluses developed adventitious shoot buds within 3–4 weeks on medium containing BA (8.9 M). Fifteen-week-old calluses developed fewer shoot buds than five-week-old calluses, indicating a decrease in morphogenetic potential with increasing duration of callus cultures. Semi-thin section microscopy was used to evaluate incapability of sustained regeneration. Development of normal shoot bud primordia, due to sub-surface reorganisation, was high in young calluses. The decline in the frequency of shoot bud primordia formation with callus ageing is due to reduced cell division activity in epidermal as well as sub-epidermal layers.     

Nuclear reformation following metaphase in HeLa S3 cells: Three-dimensional visualization of chromatid rearrangements

Nuclear reformation from chromatids following metaphase was visualized three-dimensionally for the first time in mammalian cells (HeLa S₃) by scanning electron microscopy (SEM). Anaphase and telophase configurations free of mitotic apparatus, cytoskeletal elements and nuclear envelope were prepared using a slightly modified standard cytological procedure which permitted visualization of chromatid position and orientation. Mid-anaphase alignments were observed to be more complex than previously revealed by light and transmission electron microscopy (TEM). One pole consisted of chromatids joined along their lateral length, the other pole consisted of telomeres, apparently of the longest chromatids, aligned in a double concentric layer. As anaphase progressed, re-association of these chromatids appeared to occur progressively along their lateral length toward their telomeres. Morphological evidence is presented suggesting that this lateral re-association may involve interchromatid fibers. After complete joining, structures resembling a hollow half sphere had formed. Based on different preparative procedures for SEM and published TEM analysis, it is this shell-like configuration upon which the nuclear envelope is reestablished in early telophase. As telophase progressed there was loss in depth of the internal chamber resulting in a disc configuration. Following loss of chromatid outline from the surface of this structure, interphase nuclear shape was assumed. Morphometric determinations revealed relative dimensions of chromatid configurations and supported the conclusion that nuclear reformation proceeded by discrete steps. The complexity of such a process, as revealed by SEM analysis, is discussed.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation.     

Colchicine-resistant assembly of tubulin in plant mitosis

Summary Tubulin distribution in c-mitoses (induced by 1 mM colchicine) has been studied by indirect immunofluorescence with monoclonal antibodies inAllium cepa L. meristems proliferating under steady state kinetics. Two hours after colchicine treatment was initiated tubulin is detected in approximately 25% of the cells as arrowheads on the kinetochores, as if these structures stabilize microtubules against disassembly. Total disassembly of microtubules occurs in 70% of the c-mitoses six hours after the initiation of the colchicine treatment, when restitution nuclei also start appearing. After 2 to 14 hours of colchicine treatment, tubulin is detected in about 30% of the c-mitoses, both in small kinetochores-like dots and in a strand which apparently connects sister kinetochores. Other larger microtubule-like structures, up to 20 m long, apparently unassociated with kinetochores, are assembled in the presence of cholchicine in c-mitoses after 10 hours. Such structures disappear when chromosomes decondense and the nuclear envelope reforms in the restitution nucleus; they do not seem to be related to interphase cortical microtubules which reappear in control telophase.     

Fine structure of abscission zones

Summary Electron micrographs of the zone of separation in flower pedicels of Lycopersicon esculentum and Nicotiana tabacum are presented with particular reference to the indentation of epidermal tissue in the abscission zone, subcellular organelles, and the cell wall. The indentation or groove which delineates the abscission zone extends some distance into the pedicel with branchings off the main groove. These branches are approximately 20 m in width while the main groove averages approximately 200 m in width. Invaginations of the plasmalemma are observed with considerable frequency. within these invaginations one observes a material of about the same density as the cell wall except that it is more fibrillar. Plasmodesmata are also observed, with considerable branching into middle lamellae of cells comprising the abscission zones. Microbodies with crystalloid cores appear with considerable frequency in cells of the abscission zone. The crystalloids appear to be cubical in shape and are composed of parallel sheets of osmiophilic material. The sheets average about 6 m in thickness and are spaced at 4 m intervals. The microbodies with crystalloid cores are observed to be characteristically of two size groupings. In tobacco the microbodies average 900 m and 1,500 m in profile. In tomato they average 300 m and 500 m. Chloroplasts contain a granular component which is membrane-enclosed. The component is large in comparison with the plastid in which it occurs, averaging 1.2–1.4 in diameter in chloroplasts ranging from 1.6 to 2.2 in diameter. The inner membrane of the chloroplast is highly invaginated, and DNA- and phytoferritin-like materials are observed within the plastids. Microtubules with an average diameter of 20 m are observed adjacent and parallel to the plasmalemma, primarily in the corners of the cells. Micrographs of other normally occurring cytoplasmic inclusions are also presented.     

Nuclear envelope projections from pronuclei ofSpirogyra zygotes

Summary Spirogyra zygospores were fixed in buffered 2% osmium tetroxide and embedded in Epon. Serial sectioning established that the pronuclei, in the zygotes studied, had come to lie in very close proximity but had not started to fuse.The pronuclei had interesting projections from their nuclear envelopes. These were regular finger-like structures containing both membranes of the nuclear envelope but lacking nuclear pores. The projections measured 0.22 m in diameter and had an indeterminate length around 2.0 m. They had an inner band on the nucleoplasmic side which appeared to describe a helix along the evagination maintaining a distance of 20 nm from the inner membrane, except for occasional small regions of contact.The possibility that these evaginations were important in nuclear movement or fusion was considered less likely than that they were involved in some kind of nucleocytoplasmic exchange, possibly organellogenesis like that described inPteridium.     

Spindel und kinetochoren in der Mitose und Meiose der Baumwollwanze Dysdercus intermedius (Heteroptera)

In spermatogonial mitoses of Dysdercus intermedius, chromosomal spindle microtubules insert in discrete kinetochores. The latter consist of two electron dense plates of about 0,3 m diameter located at opposite sites at the poleward surfaces of the daughter chromatids. The evidence presented permits the interpretation that a localized kinetochore owes its layered substructure to successive binding sites for kinetochore material at the ends of bundled microtubules. A working hypothesis, based upon different distributions of genetic loci for kinetochore material in species with localized and diffuse kinetochores is proposed. In Dysdercus, holokinetic chromosome behavior in mitotic anaphase movement is not due to truly diffuse kinetochores but to chromosomal interconnections which are established at prometaphase. Such interconnections are absent in the first meiotic division when each bivalent is enclosed by a sheath of endoplasmic reticulum. Spindle microtubules penetrate through terminal openings in this sheath and end singly in dense spots of kinetochore material.     

Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach

Kinetochores and chromatid cores of meiotic chromosomes of the grasshopper species Arcyptera fusca and Eyprepocnemis plorans were differentially silver stained to analyse the possible involvement of both structures in chromatid cohesiveness and meiotic chromosome segregation. Special attention was paid to the behaviour of these structures in the univalent sex chromosome, and in B univalents with different orientations during the first meiotic division. It was observed that while sister chromatid of univalents are associated at metaphase I, chromatid cores are individualised independently of their orientation. We think that cohesive proteins on the inner surface of sister chromatids, and not the chromatid cores, are involved in the chromatid cohesiveness that maintains associated sister chromatids of bivalents and univalents until anaphase I. At anaphase I sister chromatids of amphitelically oriented B univalents or spontaneous autosomal univalents separate but do not reach the poles because they remain connected at the centromere by a long strand which can be visualized by silver staining, that joins stretched sister kinetochores. This strand is normally observed between sister kinetochores of half-bivalents at metaphase II and early anaphase II. We suggest that certain centromere proteins that form the silver-stainable strand assure chromosome integrity until metaphase II. These cohesive centromere proteins would be released or modified during anaphase II to allow normal chromatid segregation. Failure of this process during the first meiotic division could lead to the lagging of amphitelically oriented univalents. Based on our results we propose a model of meiotic chromosome segregation. During mitosis the cohesive proteins located at the centromere and chromosome arms are released during the same cellular division. During meiosis those proteins must be sequentially inactivated, i.e. those situated on the inner surface of the chromatids must be eliminated during the first meiotic division while those located at the centromere must be released during the second meiotic division.by D.P. Bazett-Jones     

Genetic Control of Mitosis: Is Protein MASTν40 an Element of the Checkpoint System?

The effect of the mast ^v40 mutation was studied using neural ganglion cells of third-instar larvae of Drosophila melanogaster. The distributions of the cells by the interphase nucleus diameter and by the distance between the sister chromosome sets in anaphase were analyzed. Three following types of defects induced by the mutation were described: (1) Monopolar mitosis or, in the case of bipolar mitosis, an abnormally short distance between the sister chromosome sets in anaphase and early telophase. We suppose that these abnormalities are caused by damage of the start and (or) motor mechanisms of centrosome separation at the beginning and in the end of mitosis. (2) Lagging and bridging of chromosomes in anaphase and early telophase. These defects seem to be related to the disruption of functioning of mitotic spindle microtubules and (or) their defective attachment to the appropriate kinetochores. (3) Unlimited division of aneuploid and polyploid cells, which may be explained either by inactivation of the checkpoint system controlling the genome ploidy or by checkpoint adaptation. Taken collectively, our results and literature data suggest that the MAST protein is an element of the checkpoint system and that division of aneuploid and polyploid cells results from inactivation of the checkpoints.     

Localization of kinetochore fragments isolated from single chromatids in mitotic CHO cells

Summary Chinese hamster ovary (CHO) cells are treated with hydroxurea followed by a caffeine treatment to form detached kinetochore fragments in the absence of sister chromatids. Detached kinetochores in mitotic CHO cells display a functional association with MTs initiated from one or both centrosomes such that these association(s) have a significant influence on the location and orientation of detached kinetochores and/or their fragments. Kinetochore fragments which are amphitelically oriented are positioned approximately midway between the two centrosomes. Thus, a kinetochore isolated from a single chromatid can capture MTs from both poles. Monotelic orientation of these fragments is more frequently observed with kinetochore fragments located an average distance of 2.5 m from the nearest centrosome, compared to an average distance of 4.4 m in amphitelically oriented fragments. In cells treated with the potent MT poison, nocodazole, kinetochore isolation also occurs and therefore is not dependent on the presence of MTs. CHO cells treated to produce isolated kinetochores or kinetochore fragments then subsequently hyperosmotically shocked show no MTs directly inserted into kinetochore lamina, similar to the response of sucrose-treated metapbase PtK₁ cells. This treatment shows circular kinetochores tangentially associated with bundles of MTs that are located an average of 1.5 m from the centrosome. Our results suggest that a single kinetochore fragment can attach to MTs initiated from one or both centrosomes and that their specific association to MT fibers defines orientation of detached kinetochores within the spindle domain.     

MITOSIS AND CYTOKINESIS IN THE CHLOROMONADOPHYCEAN ALGA GONYOSTOMUM SEMEN1

Mitosis and cytokinesis in Gonyostomum semen (Ehrenberg) Diesing have been investigated with the light microscope. During prophase nucleoli disappear and the chromatid structure of the chromosomes becomes apparent. Separation of chromatids at anaphase is accompanied by progressive fusion of the progeny chromosomes. This process continues into telophase by which stage the progeny nuclei consist of dense masses of chromatin with occasional chromosomes extending from their equatorial surfaces. By the end of telophase, nucleoli are reforming and the interphase nuclear morphology is reestablished. Mitosis is followed by cytokinesis, which is a relatively lengthy phase. In early cytokinesis the 2 interphase nuclei are present, and there is no indication of the forthcoming division of the cytoplasm. Later in cytokinesis a membrane is formed between the 2 nuclei. Final separation of the progeny individuals is accomplished by vigorous movements of swimming cells or, in the case of palmelloid cells, by the deposition of a mucilaginous layer.     

Endomitosis in the mealy bug,Planococcus citri (Homoptera: Coccoidea)

Endomitosis in the Malpighian tubules of the mealy bug Planococcus citri (Risso) is described. The stages are identified on the basis of the length of the chromosomes and the distance between the sister chromatids or chromosomes. The appearance of the chromosomes in the various stages of endomitosis is compared to that in other hemipteran insects. During anaphase and telophase of endomitosis the ends of the sister chromatids and chromosomes tend to stay together longer than the other parts. It is suggested that in holokinetic chromosomes special regions for holding the chromatids together are concentrated near the ends of the chromosomes.Supported by grant GB1585 from the National Science Foundation, Washington, D.C.     

Structural analysis of the mitotic cycle in pre-gastrula Xenopus embryos

The long-known phenomenon of karyomere (chromosome vesicle) formation at early telophase of the nuclear cycle during early embryogenesis of a wide range of organisms including amphibians (Rubaschkin 1905; for review, see Richards 1917) was investigated in the early cleavage cycles of Xenopus laevis embryos before the mid blastula transition. Embryos were fixed and Epon embedded at successive time intervals and consecutive thick (3 m) and ultrathin sections cut. Using conventional light microscopy at low magnification as well as phase and/or interference contrast video microscopy at high magnification, a substantial amount of information could be obtained from the analysis of optical sections in thick-sectioned material. In addition, details of the ultrastructural organization could be analysed from corresponding ultrathin sections by electron microscopy. The light microscopic analysis of serial thick sections allowed precise determination of the arrangement and sizes of telophase karyomere structures during the embryonic nuclear division cycle. It was found that small, widely spaced 1st order karyomeres fuse to larger (2nd order) karyomeres which then progressively exhibit lateral fusion of neighbouring karyomeres. The final coalescence of adjacent karyomeres marks the onset of the reorganization of the typical interphase nuclear structure. The data are discussed with regard to the occurrence of karyomeres during the embryonic nuclear cycle of arthropods, dipteran insects, and echinoderms as well as recent progress in the use of Xenopus egg extracts for in vitro assembly of nuclear structures around protein-free DNA.     

Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine

An in vivo system for the detection of sister chromatid exchange (SCE) in the central mudminnow, Umbra limi, is presented. Sister chromatid differential (SCD) and SCE were demonstrated by fluorescent and Giemsa procedures 5 to 6 days after the fish were injected with 500 g/g of BrdU. The exchange rate was found to be 2.64 SCEs metaphase in the intestines and 2.42 SCEs/metaphase in the gills. SCE analysis in U. limi should be a useful tool for measuring the mutagenicity of water-borne chemicals.