Mitotic force generators and chromosome segregation

The mitotic spindle uses dynamic microtubules and mitotic motors to generate the pico-Newton scale forces that are needed to drive the mitotic movements that underlie chromosome capture, alignment and segregation. Here, we consider the biophysical and molecular basis of force-generation for chromosome movements in the spindle, and, with reference to the Drosophila embryo mitotic spindle, we briefly discuss how mathematical modeling can complement experimental analysis to illuminate the mechanisms of chromosome-to-pole motility during anaphase A and spindle elongation during anaphase B.     

Genetic characterization of the new morphological and UV-sensitive mutants inCoprinus cinereus. I. A UV-sensitive mutationrad1 associated with elevated frequencies of mitotic and meiotic recombination

Summary Studies on the effect of an UV-sensitive mutation,rad1, in meiotic and mitotic recombination inCoprinus indicated that, in homozygous condition,rad1 increased the spontaneous meiotic recombination by 50% and UV-induced mitotic intergenic recombination by about 5-fold. The homozygousrad1 diploid was shwon to be much more sensitive to the recombinogenic effects of polyfunctional than of mono- or non-functional alkylating agents.This research, carried out in partial fulfilment of Ph.D. requirements at the University of London, was supported by a grant from The School of Public Health, University of Tehran, Iran. My thanks are due to Dr J.W. Cowan for his advice and constructive criticism. I am also indebted to Dr A.B. Borkovec of The Institute of Chemosterilants Laboratory, US Ministry of Agriculture for the most generous supply of aziridinyl phosphine compounds.     

Der Einfluss von Giften auf die zur Meiose führenden Stoffwechselvorgänge beiSabellaria spinulosa

Summary The metabolic processes leading to meiosis inSabellaria spinulosa were investigated with cytochemical methods (PAS-reaction for polysaccharides, and basic dyes in combination with ribonuclease applied for the detection of ribonucleic acid) and the use of specific inhibitors. Before the beginning of meiotic prophase, polysaccharides and later ribonucleic acid appear in the nuclear sap and become incorporated into the spindle. 2,4-dinitrophenol and NaN₃, which inhibit the formation of energy-rich phosphates, prevent the accumulation of polysaccharides and RNA in the nucleus and the formation of the meiotic spindle. KCN and mono-iodoacetic acid are without influence on polysaccharide and RNA metabolism of the nucleus. The first and second meiotic division are normal, but cleavage is completely suppressed. From these results the following conclusions may be drawn: (1) the division of the nucleus is dependant on the presence of energy-rich phosphate, (2) the division occurs without respiration, and (3) respiration is important for cell division only in so far as it provides the synthesis of energy-rich phosphates.     

Genetic characterization of the new morphological and UV-sensitive mutants in Coprinus cinereus. I. A UV-sensitive mutation rad 1 associated with elevated frequencies of mitotic and meiotic recombination

Studies on the effect of an UV-sensitive mutation, rad 1, in meiotic and mitotic recombination in Coprinus indicated that, in homozygous condition, rad 1 increased the spontaneous meiotic recombination by 50% and UV-induced mitotic intergenic recombination by about 5-fold. The homozygous rad 1 diploid was shown to be much more sensitive to the recombinogenic effects of polyfunctional than than of mono- or non-functional alkylating agents.     

Der Mitosezyklus im diploiden und triploiden Wurzelmeristem vonScilla sibirica

Summary In meristematic root tip cells ofScilla sibirica (2n=12 and 3n=18) the following results were obtained with the aid of autoradiography: 1. The average duration of the mitotic cycle (2n=12) is 69.5 h. The G₁-phase lasts 36.5 h, the DNA synthetic phase 17.5 h, the G₂-phase 8 h and mitosis 7.5 h. 2. There are no marked differences in the lengths of the cell cycles nor in the duration of the various phases between diploid and triploid plants.     

Ultrastructure of muscle spindles in C57BL/6Jdy 2J/dy 2J dystrophic mice

Summary The sensory organs of skeletal muscles, the muscle spindles, were examined using electron microscopy indy ^2J/dy ^2J dystrophic mice. Despite widespread damage to the extrafusal (skeletomotor) fibres the intrafusal (spindle) fibres appeared normal and seemed resistant to the aetiological factors for murine dystrophy.This work was supported by grants from the Medical Research Council and the Science Research Council of Great Britain.     

Application de méthodes d'analyse biochimique à une étude taxonomique: les corégones du lac de Neuchâtel

Summary Two closely related forms ofCoregonus from Lake Neuchatel were examined cytologically and biochemically, in order to ascertain the chromosome number and the DNA content of haploid and diploid nuclei.Coregonus fera has 2N=78 ± 2 chromosomes, and a DNA content (diploid) of 5.8 × 10⁻⁹ mg;Coregonus macrophthalmus, 2N=78⁺ ± 3, DNA content of 6.1 × 10⁻⁹ mg. The difference between the two DNA constants is statistically significant. These results do not support the hypothesis which postulates that polyploidy may be a determining factor in the speciation of these fishes.      

Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein

S. cerevisiae anaphase spindle elongation is accomplished by the overlapping function of dynein and the kinesin-5 motor proteins, Cin8 and Kip1. Cin8 and dynein are synthetically lethal, yet the arrest phenotypes of cells eliminated for their function had not been identified. We found that at a non-permissive temperature, dyn1Δ cells that carry a temperature-sensitive cin8 – 3 mutation arrest at mid-anaphase with a unique phenotype, which we named TAN (two microtubule asters in one nucleus). These cells enter anaphase, but fail to proceed through the slow phase of anaphase B. At a permissive temperature, dyn1Δ, cin8 – 3 or dyn1Δcin8 – 3 cells exhibit perturbed spindle midzone morphologies, with dyn1Δcin8 – 3 anaphase spindles also being profoundly bent and nonrigid. Sorbitol, which has been suggested to stabilize microtubules, corrects these defects and suppresses the TAN phenotype. We conclude that dynein and Cin8 cooperate in anaphase midzone organization and influence microtubule dynamics, thus enabling progression through the slow phase of anaphase B. Received 10 August 2008; received after revision 22 October 2008; accepted 27 October 2008     

Evolution chromosomique chez genre Sorex: nouvelle information

Summary InSorex unguiculatus, a species closely related to the common shrew, 41 chromosomes were observed in the spermatogonia of two male specimens. The meiotic cells show about 21 formations at diakinesis and first maturation division metaphase. The mode of sex determination still remains unsatisfactorily clarified. However, the species investigated is of interest as a further instance of the very complex chromosomal evolution characterizing the genus Sorex. Within this genus, the observed variation in chromosome numbers now ranges from 21 to 41 at the diploid level.     

The exotoxin ofBacillus thuringiensis: A new C-mitotic agent

Summary Thuringiensin A, an exotoxin fromBacillus thuringiensis, a constituent of the microbial insecticide thuricide has been found to inhibit mitotic spindle, condense and scatter chromosomes. It may therefore be a promizing tool in future cell biological studies.We thank Prof.H. Sharatchandra for facilities. CBSR is a National Associate of the University Grants Commission, New Delhi.     

Homologous recombination in fission yeast: Absence of crossover interference and synaptonemal complex

The study of homologous recombination in the fission yeastSchizosaccharomyces pombe has recently been extended to the cytological analysis of meiotic prophase. Unlike in most eukaryotes no tripartite SC structure is detectable, but linear elements resembling axial cores of other eukaryotes are retained. They may be indispensable for meiotic recombination and proper chromosome segregation in meiosis I. In addition fission yeast shows interesting features of chromosome organization in vegetative and meiotic cells: Centromeres and telomeres cluster and associate with the spindle pole body. The special properties of fission yeast meiosis correlate with the absence of crossover interference in meiotic recombination. These findings are discussed. In addition homologous recombination in fission yeast is reviewed briefly.This article is dedicated to Urs Leupold, the founder of fission yeast genetics.     

Epigenetic mechanisms in mammals

DNA and histone methylation are linked and subjected to mitotic inheritance in mammals. Yet how methylation is propagated and maintained between successive cell divisions is not fully understood. A series of enzyme families that can add methylation marks to cytosine nucleobases, and lysine and arginine amino acid residues has been discovered. Apart from methyltransferases, there are also histone modification enzymes and accessory proteins, which can facilitate and/or target epigenetic marks. Several lysine and arginine demethylases have been discovered recently, and the presence of an active DNA demethylase is speculated in mammalian cells. A mammalian methyl DNA binding protein MBD2 and de novo DNA methyltransferase DNMT3A and DNMT3B are shown experimentally to possess DNA demethylase activity. Thus, complex mammalian epigenetic mechanisms appear to be dynamic yet reversible along with a well-choreographed set of events that take place during mammalian development.     

Über die Beeinträchtigung der Wirkung von Spindelgiften in der Zellkultur durch menschliches Serum sowie durch Seren verschiedener Tierspezies

Summary Chick embryo fibroblasts, cultivatedin vitro, require higher concentrations of podophyllotoxin derivatives for complete mitotic arrest when the medium contains human adult or cord serum than when it contains horse, bovine, guinea pig, rat, mouse, or cock serum. This difference is due to a higher binding capacity of human serum for these compounds. No such difference was found with a colchicine and a peltatin derivative.     

Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase

The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.     

Overexpression of SUMO perturbs the growth and development of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Small ubiquitin-related modifiers (SUMOs) are important regulator proteins. Caenorhabditis elegans contains a single SUMO ortholog, SMO-1, necessary for the reproduction of C. elegans. In this study, we constructed transgenic C. elegans strains expressing human SUMO-1 under the control of pan-neuronal (aex-3) or pan-muscular (myo-4) promoter and SUMO-2 under the control of myo-4 promoter. Interestingly, muscular overexpression of SUMO-1 or -2 resulted in morphological changes of the posterior part of the nematode. Movement, reproduction and aging of C. elegans were perturbed by the overexpression of SUMO-1 or -2. Genome-wide expression analyses revealed that several genes encoding components of SUMOylation pathway and ubiquitin-proteasome system were upregulated in SUMO-overexpressing nematodes. Since muscular overexpression of SMO-1 also brought up reproductive and mobility perturbations, our results imply that the phenotypes were largely due to an excess of SUMO, suggesting that a tight control of SUMO levels is important for the normal development of multicellular organisms.     

Génétique et évolution des Nématodes libres. Perspectives tirées de l'étude deCaenorhabditis elegans

Summary Free-living nematodes are endowed with a set of properties that make them specially attractive for experimental work. Geneticists and evolutionists may be especially interested in their easy cultivation, great fecundity and polymorphism. As an example, some results are brought concerningCaenorhabditis elegans and related species: (1) Sex determination occurs as the expression of a genic balance involving differences in the number of chromosomes. The alternatives gynogenesis, amphimixis, or auto-heterofecundation take a prominent place in the reproductive processes of these species. (2) Polyploidy may be obtained by heat-shock, giving rise to individuals showing characteristic chromosomal unbalance and variability. (3)C. elegans, which cannot normally be raised at a higher temperature than 22°C, has been gradually adapted to temperatures up to 24.5°C. This acclimatization implies an adaptive transformation of the ovarian physiology; this effect is obtained after long training, taking more than 1000 generations. Study of the process of acclimatization shows that its genetical basis may be partly of a non-genic nature, partly of a genic type. Hypotheses are developed for explaining this phenomenon and for the understanding of the evolution from free-living to parasitic nematodes.     

Chromosomal translocations in 2 Italian populations ofReticulitermes lucifugus (Rossi) (Insecta,Isoptera: Rhinotermitidae)

Summary The chromosomes of 2 Italian populations ofReticulitermes lucifugus (Rossi) were studied in both spermatogonial and oogonial divisions. The diploid chromosome complement is 2=42. All the first meiotic metaphases in Sardinian males show a complex translocation arranged in chains of 4 bivalents. The Apulian males show either 21 bivalents or 19 bivalents and a chain of 2.The authors express their thanks to Prof. A. Springhetti for his kind help in supplying the materials and for criticism in preparation of this paper, and to Prof. G. Colombo for valuable advice and criticism.     

Selectable genetic markers for nematode transgenesis

The nematode Caenorhabditis elegans has been used to study genetics and development since the mid-1970s. Over the years, the arsenal of techniques employed in this field has grown steadily in parallel with the number of researchers using this model. Since the introduction of C. elegans transgenesis, nearly 20 years ago, this system has been extensively used in areas such as rescue experiments, gene expression studies, and protein localization. The completion of the C. elegans genome sequence paved the way for genome-wide studies requiring higher throughput and improved scalability than provided by traditional genetic markers. The development of antibiotic selection systems for nematode transgenesis addresses these requirements and opens the possibility to apply transgenesis to investigate biological functions in other nematode species for which no genetic markers had been developed to date.