Division Block Research Articles

On the functional overlap between two Drosophila POU homeo domain genes and the cell fate specification of a CNS neural precursor.

The approximately 200 distinct neurons comprising each hemisegment of the Drosophila embryonic CNS are derived from a stereotypic array of approximately 30 progenitor stem cells, called neuroblasts (NBs). Each NB undergoes repeated asymmetric divisions to produce several smaller ganglion mother cells (GMCs), each of which, in turn, divides to produce two neurons and/or glia cells. To understand the process by which cell type diversity is generated in the CNS, we are focusing on identifying genes that affect cell identity in the NB4-2 lineage from which the RP2 motoneuron is derived. We show here that within the early part of the NB4-2 lineage, two closely linked and structurally related POU homeo domain genes, pdm-2 (dPOU28) and pdm-1 (dPOU19), both encode proteins that accumulate to high levels only in the first GMC (GMC4-2a) and not in its progeny, the RP2 motoneuron. Our results from the genetic and developmental analysis of pdm-1 and pdm-2 demonstrate that these genes are not required for the birth of GMC4-2a; however, they are both involved in specifying the identity of GMC4-2a and, ultimately, in the genesis of RP2 neurons, with pdm-2 being the more dominant player in this process. In mutant animals where both pdm-1 and pdm-2 functions are removed, GMC4-2a fails to express markers consistent with a GMC4-2a identity and no mature (Eve protein expressing) RP2 neurons are produced. We demonstrate that in some mutant combinations in which no mature RP2 neurons are produced, some GMC4-2a cells can nevertheless divide. Hence, the failure of the POU mutants to produce mature RP2 neurons is not attributable to a block in GMC4-2a cell division per se but, rather, because the GMC4-2a cells fail to acquire their correct cellular identity.

Carbon and Energetic Uncoupling Are Associated with Block of Division at Different Stages of the Cell Cycle in Several cdc Mutants of Saccharomyces cerevisiae

Cell proliferation arrest at 37°C (restrictive temperature) of the cell division cycle (cdc) mutants of Saccharomyces cerevisiae cdc28, cdc35, cdc19, cdc21, and cdc17 was correlated with carbon and energy uncoupling. At 37°C, cdc mutants diverted to biomass synthesis only 3 to 4% and 8 to 24% of the fluxes of carbon consumed and ATP obtained by catabolism, respectively, compared with 48 and 34% in the wild-type strain A364A. At the permissive temperature (25°C), the wild type showed similar carbon and energy coupling indexes as at 37°C. However, carbon and energy coupling indexes were two- to sevenfold higher at 25° than at 37°C in cdc mutants; e.g., at 25°C two- to seven-fold higher amounts of carbon and ATP were directed to biomass production than at 37°C. The wild-type strain exhibited a purely oxidative glucose catabolism at 37°C (RQ ∼ 1.0), while the cell proliferation arrest of cdc mutants at the same temperature was characterized by fermentative metabolism. At 37°C, cdc mutants directed 50 to 60% of the carbon to ethanol production; 3 to 12% of the carbon was recovered as glycerol in cdc mutants as well as in the wild type. The proliferation arrest of the cell division cycle mutant cdc28 correlated with a significant decrease in the incorporation of radio-active precursors into DNA, RNA, and proteins. In the presence of 8-hydroxyquinoline, the wild-type strain underwent cell proliferation arrest and also exhibited metabolic uncoupling with bioenergetic and catabolic behavior similar to that of the cdc mutants at 37°C. Experimental evidence obtained with cdc19, whose defective gene product is pyruvate kinase, suggests that the primary defect of cdc mutants correlates with a metabolically, highly uncoupled yeast cell. The results presented point to the existence of strong carbon and energy uncoupling together with cell division arrest exhibited by cdc mutants at the restrictive temperature. The degree of uncoupling appears to be tuned, at least in part, by the increase in flux of sugar catabolism through the ethanol fermentative pathway.

Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli.

The product of the ftsW gene has been identified as a polypeptide that, like the related RodA protein, shows anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FtsW is produced at low levels that can be increased by altering the translation initiation region of the mRNA. Overproduction of FtsW strongly inhibits cell growth. A new mutant allele, ftsW201, causes a temperature-dependent block in the initiation stage of cell division which is similar to the division block in ftsZ mutants. The block in initiation of division in the ftsW201 allele is shown to be independent of FtsZ or the FtsZ inhibitor, SulA. In addition, the ftsW201 mutant is hypersensitive to overproduction of the division initiation protein FtsZ at the permissive temperature. Our results suggest a role for FtsW in an early stage of division which may involve an interaction with FtsZ.

Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality.

Precise chromosome transmission in cell division cycle is maintained by a number of genes. The attempt made in the present study was to isolate temperature-sensitive (ts) fission yeast mutants that display high loss rates of minichromosomes at permissive or semipermissive temperature (designated mis). By colony color assay of 539 ts strains that contain a minichromosome, we have identified 12 genetic loci (mis1-mis12) and determined their phenotypes at restrictive temperature. Seven of them are related to cell cycle block phenotype at restrictive temperature, three of them in mitosis. Unequal distribution of regular chromosomes in the daughters is extensive in mis6 and mis12. Cells become inviable after rounds of cell division due to missegregation. The phenotype of mis5 is DNA replication defect and hypersensitivity to UV ray and hydroxyurea. mis5+ encodes a novel member of the ubiquitous MCM family required for the onset of replication. The mis5+ gene is essential for viability and functionally distinct from other previously identified members in fission yeast, cdc21+, nda1+, and nda4+. The mis11 mutant phenotype was the cell division block with reduced cell size. Progression of the G1 and G2 phases is blocked in mis11. The cloned mis11+ gene is identical to prp2+, which is essential for RNA splicing and similar to a mammalian splicing factor U2AF65.

Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis.

CENP-E is a kinesin-like protein that binds to kinetochores through the early stages of mitosis, but after initiation of anaphase, it relocalizes to the overlapping microtubules in the midzone, ultimately concentration in the developing midbody. By immunoblotting of cells separated at various positions in the cell cycle using centrifugal elutriation, we show that CENP-E levels increase progressively across the cycle peaking at approximately 22,000 molecules/cell early in mitosis, followed by an abrupt (> 10 fold) loss at the end of mitosis. Pulse-labeling with [35S]methionine reveals that beyond a twofold increase in synthesis between G1 and G2, interphase accumulation results primarily from stabilization of CENP-E during S and G2. Despite localizing in the midbody during normal cell division, CENP-E loss at the end of mitosis is independent of cytokinesis, since complete blockage of division with cytochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degradation of as yet unidentified components at the metaphase/anaphase transition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of timed proteolysis.

Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp.

The Escherichia coli strain known as GC2553, FB8, UTH1038, or K12S (Luria), considered an F- lambda- wild-type strain, is shown here to carry a cryptic mutation, ftsR1, causing nonlethal filamentation during exponential growth in Luria-Bertani (LB) broth at 42 degrees C and the inability to grow in salt-free LB broth at 42 degrees C. The ftsR1 mutation is completely suppressed in genetic backgrounds which increase RelA-dependent synthesis of the nucleotide ppGpp, i.e., argS201 (Mecr) and alaS21 (Mecr) mutations, affecting aminoacyl-tRNA synthetases, or the presence of a plac-relA' plasmid. These backgrounds also confer resistance in LB broth to the beta-lactam mecillinam, an antibiotic which specifically inhibits penicillin-binding protein 2 and, in wild-type cells, causes an indirect block in cell division. Furthermore, the ftsR1 mutant (but not an isogenic ftsR+ strain) is sensitive to mecillinam in minimal glucose medium at 37 degrees C. Since the division block caused by mecillinam can be overcome by overproduction of the cell division protein FtsZ, we tested the effect of plasmid pZAQ (carrying the ftsZ, ftsA, and ftsQ genes) on the ftsR1 mutant; it suppressed the filamentation in LB broth and the mecillinam sensitivity on minimal glucose medium at 37 degrees C but not the growth defect in salt-free LB broth at 42 degrees C. Genetic analysis indicated that the full phenotype of the ftsR1 mutant is due to a single mutation in the rpoB gene (90 min), coding for the beta subunit of RNA polymerase; we call this allele rpoB369(Fts). We propose that the rpoB369(Fts) mutation alters the specificity of the polymerase and that the mutant enzyme can recover normal activity in the presence of high salt concentrations or via interaction with the nucleotide ppGpp.

Expression of p60v-src in Saccharomyces cerevisiae results in elevation of p34CDC28 kinase activity and release of the dependence of DNA replication on mitosis.

Expression of the oncogenic protein tyrosine kinase p60v-src in the yeast Saccharomyces cerevisiae has been shown to result in rapid cell death (J. S. Brugge, G. Jarosik, J. Andersen, A. Queral-Lustig, M. Fedor-Chaiken, and J. R. Broach, Mol. Cell. Biol. 7:2180-2187, 1987). Work described here demonstrates that v-Src expression results in accumulation of large-budded cells and a nuclear division block without blocking cytokinesis. Flow-cytometric analysis indicates that the DNA content of these cells is elevated beyond the G2 DNA content, and genetic studies indicate that v-Src expression causes aneuploidy. The activity of Cdc28 kinase, which controls the G1/S and G2/M transitions in S. cerevisiae, increases during galactose induction in a Src+ strain but not in an isogenic Src- strain. These observations indicate that v-Src expression disrupts p34CDC28 kinase regulation, allowing DNA replication to proceed in the absence of a prior mitotic event.

Expression of p60v-src in Saccharomyces cerevisiae results in elevation of p34CDC28 kinase activity and release of the dependence of DNA replication on mitosis.

Expression of the oncogenic protein tyrosine kinase p60v-src in the yeast Saccharomyces cerevisiae has been shown to result in rapid cell death (J. S. Brugge, G. Jarosik, J. Andersen, A. Queral-Lustig, M. Fedor-Chaiken, and J. R. Broach, Mol. Cell. Biol. 7:2180-2187, 1987). Work described here demonstrates that v-Src expression results in accumulation of large-budded cells and a nuclear division block without blocking cytokinesis. Flow-cytometric analysis indicates that the DNA content of these cells is elevated beyond the G2 DNA content, and genetic studies indicate that v-Src expression causes aneuploidy. The activity of Cdc28 kinase, which controls the G1/S and G2/M transitions in S. cerevisiae, increases during galactose induction in a Src+ strain but not in an isogenic Src- strain. These observations indicate that v-Src expression disrupts p34CDC28 kinase regulation, allowing DNA replication to proceed in the absence of a prior mitotic event.

Temporally different poly(adenosine diphosphate-ribosylation) signals are required for DNA replication and cell division in early embryos of sea urchins.

To analyze the temporal relationship of poly(adenosine diphosphate [ADP]-ribosylation) signal with DNA replication and cell divisions, the effect of 3 aminobenzamide (3ABA), an inhibitor of the poly(ADP-ribose)synthetase, was determined in vivo during the first cleavage division of sea urchins. The incorporation of 3H-thymidine into DNA was monitored and cleavage division was examined by light microscopy. The poly(ADP-ribose) neosynthesized on CS histone variants was measured by labeling with 3H-adenosine during the two initial embryonic cell cycles and the inhibitory effect of 3ABA on this poly(ADP-ribosylation) was determined. The results obtained indicate that the CS histone variants are poly(ADP-ribosylated) de novo during the initial cell cycles of embryonic development. The synthesis of poly(ADP-ribose) is decreased but not abolished by 20 mM of 3ABA. The incubation of zygotes in 3ABA at the entrance into S1 phase decreased 3H-thymidine incorporation into DNA in phase S2, while S1 was unaltered. Alternatively, when the same treatment was applied to zygotes at the exit of S1 phase, a block of the first cleavage division and a retardation of S2 phase were observed. The inhibitory effect of 3ABA on both DNA replication and cell division was totally reversible by a release of the zygotes from this inhibition. Taking together these observations it may be concluded that the poly(ADP-ribosylation) signals related to embryonic DNA replication are not contemporaneous with S phase progression but are a requirement before its initiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that requires the coordinate action of the MinC, MinD, and MinE proteins. In the absence of MinE, the coordinate expression of MinC and MinD leads to a general inhibition of cell division. MinE gives topological specificity to the division inhibition process, so that the septation block is restricted to the cell poles. At normal levels of expression, both MinC and MinD are required for the division block. We show here that, when expressed at high levels, MinC acts as a division inhibitor even in the absence of MinD. The division inhibition that results from MinC overexpression in the absence of MinD is insensitive to the MinE topological specificity factor. The results suggest that MinC is the proximate cause of the septation block and that MinD plays two roles in the MinCDE system--it activates the MinC-dependent division inhibition mechanism and is also required for the sensitivity of the division inhibition system to the MinE topological specificity factor.

The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site.

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that is mediated by the MinC, MinD and MinE proteins. During the normal division cycle MinD plays two roles. It activates the MinC-dependent mechanism that is responsible for the inactivation of potential division sites and it also renders the division inhibition system sensitive to the topological specificity factor MinE. MinE suppresses the division block at the normal division site at mid-cell but not all cell poles, thereby ensuring the normal division pattern. In this study the MinD protein was purified to homogeneity and shown to bind ATP and to have ATPase activity. When the putative ATP binding domain of MinD was altered by site-directed mutagenesis, the mutant protein was no longer able to activate the MinC-dependent division inhibition system. Immunoelectron microscopy showed that MinD was located in the inner membrane region of the cell envelope. These results show that MinD is a membrane ATPase and suggest that the ATPase activity plays an essential role in the functions of the MinD protein during the normal division process.

Pertussis Toxin Selectively Interferes With the Responses of the HL-60 Human Promyelocytic Cell Line to Dimethylsulfoxide

AbstractThe effects of pertussis toxin (PT) on the growth and dimethylsulfoxide (Me2SO4)-induced differentiation of the HL-60 human promyelocytic leukemia cell line were tested. Cell growth was quantified by direct cell counts. Cell differentiation was estimated by measuring the expression of myeloid-specific cell-surface antigens (Mo-1 and fMet-Leu-Phe [fMLP] receptors), the ability of the cells to produce superoxide anions on stimulation with fMLP, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA), and by monitoring the level of expression of messenger RNA (mRIMA) for tumor necrosis factor α (TNFα). By itself, PT did not affect the proliferation of HL-60 cells in serum-containing medium. In contrast, PT (but not its B-oligomer) dose-dependently inhibited the Me2SO4-induced expression of Mo-1, fMLP receptors, and the oxidative responses to the chemotactic factor and to A23187, but not to PMA. The addition of Me2SO4 induced a significant increase in the steady-state levels of TNFα mRNA, and this effect was strongly inhibited by PT. Finally, the bacterial toxin did not reverse the block of cell division that follows the addition of Me2SO4. These results provide evidence for the involvement of a PT substrate (presumably a guanine nucleotide-binding protein) in the regulation of the maturation of the excitation-response coupling sequence in human myeloid cell precursors and show that the regulation of cell division and maturation of HL-60 cells are under distinct sets of control mechanisms.

Pertussis toxin selectively interferes with the responses of the HL-60 human promyelocytic cell line to dimethylsulfoxide

The effects of pertussis toxin (PT) on the growth and dimethylsulfoxide (Me2SO4)-induced differentiation of the HL-60 human promyelocytic leukemia cell line were tested. Cell growth was quantified by direct cell counts. Cell differentiation was estimated by measuring the expression of myeloid-specific cell-surface antigens (Mo-1 and fMet-Leu- Phe [fMLP] receptors), the ability of the cells to produce superoxide anions on stimulation with fMLP, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA), and by monitoring the level of expression of messenger RNA (mRNA) for tumor necrosis factor alpha (TNF alpha). By itself, PT did not affect the proliferation of HL-60 cells in serum-containing medium. In contrast, PT (but not its B-oligomer) dose-dependently inhibited the Me2SO4-induced expression of Mo-1, fMLP receptors, and the oxidative responses to the chemotactic factor and to A23187, but not to PMA. The addition of Me2SO4 induced a significant increase in the steady-state levels of TNF alpha mRNA, and this effect was strongly inhibited by PT. Finally, the bacterial toxin did not reverse the block of cell division that follows the addition of Me2SO4. These results provide evidence for the involvement of a PT substrate (presumably a guanine nucleotide-binding protein) in the regulation of the maturation of the excitation-response coupling sequence in human myeloid cell precursors and show that the regulation of cell division and maturation of HL-60 cells are under distinct sets of control mechanisms.

Two alpha-tubulin genes of Aspergillus nidulans encode divergent proteins.

We have isolated and analyzed the tubA and tubB alpha-tubulin genes of Aspergillus nidulans. The nucleotide sequences of these genes predict polypeptides of 447 amino acids for tubA and 450 for tubB. The predicted amino acids sequences exhibit 28% divergence between the two polypeptides. This is the second known case of such high divergence between alpha-tubulins within the same species. The tubB gene is unique in that it codes for an extra glycine residue between what are usually the second and third amino acids. RNA blot analysis demonstrates that the tubA and tubB transcripts are each 1.8 kb long. The level of tubA transcript remains the same throughout the cell cycle. The level of tubB transcript does not change at any particular stage in the cell cycle but increases continuously during spore germination. The tubA gene was previously mapped to linkage group eight, and we have now mapped the tubB gene to linkage group four. Gene disruption in heterokaryons suggests that the phenotypic consequences of disruption are different for the tubA and tubB genes. Molecular disruption of tubA results in a block in nuclear division whereas in tubB it gives rise to abnormal cell and nuclear morphology.

Equiblock-sized efficiency-balanced designs

Methods of construction of proper efficiency-balanced designs from group divisible designs and balanced incomplete block designs are presented together with the tables of these designs in the range of ri≤ 20 or 30 and k ≥ 10.

Central role for the Escherichia coli minC gene product in two different cell division-inhibition systems.

In Escherichia coli, selection of the proper division site at midcell requires the specific inhibition of septation at two other potential division sites, located at each of the cell poles. This site-specific inhibition of septation is mediated by the gene products of the minicell locus (the minB operon) that includes three genes, minC, minD, and minE. In this paper we show that one of the components of this division-inhibition system, the minC gene product, is also an essential component of another division-inhibition system, which is induced by derepression of the dicB gene and leads to inhibition of septation at all potential division sites. The two minC-dependent division-inhibition systems could be functionally distinguished by their different responses to the minE gene product. The results suggest a model in which a common mechanism, mediated by MinC, is responsible for the division block in a class of division-inhibition systems that can be independently activated by different proteins that determine the specific properties of these systems.

Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants

Penicillin-binding protein 2 (PBP2), target of the beta-lactam mecillinam, is required for rod morphology and cell wall elongation in Escherichia coli. A new temperature-sensitive PBP2 allele and an in vitro-constructed insertion deletion allele were shown to be lethal in wild-type strains, establishing that the activity of this protein is essential. Mutations in the lov or cya genes, conferring mecillinam resistance, compensated for the deleterious effect of the absence of PBP2. The resulting double mutants grew as spheres. In a cya mutant lacking PBP2, the restoration of a Cya+ phenotype by addition of cyclic AMP caused lethality and a block in cell division. These results show that in wild-type cells, PBP2 is essential for growth and division.

The transient division block in the D9ts mutant of Tetrahymena is inducible at every cell cycle stage.

The temperature-sensitive mutant D9 of Tetrahymena thermophila doubles its size at restrictive temperature. It does so by complete cessation of cell division for a limited time. After resumption of proliferation, division rate and specific growth rate are the same as at the permissive temperature, thereby maintaining the new cell size. In this study a detailed analysis of the process of controlling the new cell size is presented, by probing the temperature sensitivity of cell cycle phases. It will be shown that high temperature affects the size-controlling system immediately upon shift in temperature. Temperature pulses are effective at every stage in the cycle and are executed at the time of expected division. After return to the permissive temperature, cells gradually recover from the temperature pulse as seen by a decrease in division delay. Preparation for the next division is unaffected by the temperature pulse. It occurs at the same time as in untreated controls. The results allow us to describe some features of the division initiating system.

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and flavin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.

A conditional mutant having paralyzed cilia and a block in cytokinesis is rescued by cytoplasmic exchange in Tetrahymena thermophila.

Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.