Feedback control of mitosis in budding yeast.

We have investigated the feedback control that prevents cells with incompletely assembled spindles from leaving mitosis. We isolated budding yeast mutants sensitive to the anti-microtubule drug benomyl. Mitotic arrest-deficient (mad) mutants are the subclass of benomyl-sensitive mutants in which the completion of mitosis is not delayed in the presence of benomyl and that die as a consequence of their premature exit from mitosis. A number of properties of the mad mutants indicate that they are defective in the feedback control over the exit from mitosis: their killing by benomyl requires passage through mitosis; their benomyl sensitivity can be suppressed by an independent method for delaying the exit from mitosis; they have normal microtubules; and they have increased frequencies of chromosome loss. We cloned MAD2, which encodes a putative calcium-binding protein whose disruption is lethal. We discuss the role of feedback controls in coordinating events in the cell cycle.     

Suppression of microtubule dynamics by benomyl decreases tension across kinetochore pairs and induces apoptosis in cancer cells

We found that benomyl, a benzimidazole fungicide, strongly suppressed the reassembly of cold-depolymerized spindle microtubules in HeLa cells. Benomyl perturbed microtubule-kinetochore attachment and chromosome alignment at the metaphase plate. Benomyl also significantly decreased the distance between the sister kinetochore pairs in metaphase cells and increased the level of the checkpoint protein BubR1 at the kinetochore region, indicating that benomyl caused loss of tension across the kinetochores. In addition, benomyl decreased the intercentrosomal distance in mitotic HeLa cells and blocked the cells at mitosis. Further, we analyzed the effects of benomyl on the signal transduction pathways in relation to mitotic block, bcl2 phosphorylation and induction of apoptosis. The results suggest that benomyl causes loss of tension across the kinetochores, blocks the cell cycle progression at mitosis and subsequently, induces apoptosis through the bcl2-bax pathway in a manner qualitatively similar to the powerful microtubule targeted anticancer drugs like the vinca alkaloids and paclitaxel. Considering the very high toxicity of the potent anticancer drugs and the low toxicity of benomyl in humans, we suggest that benomyl could be useful as an adjuvant in combination with the powerful anticancer drugs in cancer therapy.     

Yeast kinetochore microtubule dynamics analyzed by high-resolution three-dimensional microscopy

We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.     

Morphological and genetic effects of benomyl on polyploid brewing yeasts: isolation of auxotrophic mutants

An enrichment procedure after ethyl methanesulfonate mutagenesis and exposure to the fungicide benomyl yielded mutants auxotrophic for several amino acids from two polyploid Saccharomyces spp. Benomyl treatment was found to have a marked morphological effect on polyploid Saccharomyces cerevisiae, causing cells to adopt a characteristic doublet cell morphology in which buds are nearly as large as the parent cells. Experiments in which nuclear division was monitored in benomyl-induced doublet cells by Giemsa nuclear staining demonstrated an unusual sequence of cytological events which culminated in the formation of binucleate parental and mononucleate bud components. The frequency of formation of doublet and binucleate parent cells was found to depend on the strain employed and the benomyl concentration administered.     

Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans

We have examined the effects of the antimicrotubule agent benomyl and several mutations on nuclear and mitochondrial movement in germlings of the filamentous fungus Aspergillus nidulans. While, as previously reported, benomyl inhibited nuclear division and movement, it did not inhibit mitochondrial movement. To test the effects of benomyl more rigorously, we germinated two benomyl super-sensitive, beta-tubulin mutants at a benomyl concentration 50-100 times greater than that required to inhibit colony formation completely. Again nuclear division and movement were inhibited, but mitochondrial movement was not. We also examined conditionally lethal beta-tubulin mutations that disrupt microtubule function under restrictive conditions. Nuclear division and movement were inhibited but, again, mitochondrial movement was not. Finally we examined the effects of five heat-sensitive mutations that inhibit nuclear movement but not nuclear division at restrictive temperatures. These mutations strongly inhibited nuclear movement at a restrictive temperature but did not inhibit mitochondrial movement. These data demonstrate that the mechanisms of nuclear and mitochondrial movement in Aspergillus nidulans are not identical and suggest that mitochondrial movement does not require functional microtubules.     

Morphological and genetic effects of benomyl on polyploid brewing yeasts: isolation of auxotrophic mutants.

An enrichment procedure after ethyl methanesulfonate mutagenesis and exposure to the fungicide benomyl yielded mutants auxotrophic for several amino acids from two polyploid Saccharomyces spp. Benomyl treatment was found to have a marked morphological effect on polyploid Saccharomyces cerevisiae, causing cells to adopt a characteristic doublet cell morphology in which buds are nearly as large as the parent cells. Experiments in which nuclear division was monitored in benomyl-induced doublet cells by Giemsa nuclear staining demonstrated an unusual sequence of cytological events which culminated in the formation of binucleate parental and mononucleate bud components. The frequency of formation of doublet and binucleate parent cells was found to depend on the strain employed and the benomyl concentration administered.     

Cytogenetic action of the pesticides captan and benomyl in a lymphocyte culture of human peripheral blood in the absence and presence of a system of metabolic activation

Results of the investigation of mutagenic effects of fungicides captan and benomyl in the culture of human peripheral lymphocytes in vitro with and without metabolic activation showed that captan did not manifest cytogenetic activity in any experimental variants, while benomyl induced colchicine-like and weak clastogenic effect in the concentration 10 micrograms/ml in the presence of microsomal activating mixture only.     

Effects of fungicide residues on the survival, fecundity, and predation of the mites Tetranychus urticae (Acari: Tetranychidae) and Galendromus occidentalis (Acari: Phytoseiidae)

Representative fungicides from three or four families used for management of powdery mildew and other diseases in tree fruits were evaluated for their effects on a common spider mite and predator mite species, respectively. A modified Munger cell technique was effective in measuring the response of phytophagous and predaceous mites to fungicide residues on detached leaves in the laboratory. Demethylation-inhibiting (DMI) (imidazole [triflumazole] and triazole [myclobutanil]) and strobilurin (trifloxystrobin) fungicides were not toxic to female Tetranychus urticae Koch and Galendromus occidentalis (Nesbitt), and no sublethal effects were found on fecundity and predation rate after 3-5-d exposure to residues. Benomyl, a benzimidazole fungicide, increased adult mortality and reduced fecundity for both mite species; however, it did not alter the predation rate of G. occidentalis females on T. urticae eggs and larvae. Female G. occidentalis that survived the lethal effects of benomyl and the comparison acaricide pyridaben were unimpaired in predation. Our results for benomyl substantiate those of earlier studies and provide evidence for nontoxic effects of DMI and strobilurin fungicides on mites. We propose that DMI and strobilurin fungicides are a good fit for integrated mite management programs due to conservation of phytoseiid predatory mites.     

Darwinism and human affairs: By R. D. Alexander. Seattle: University of Washington Press. 317 pp. $14.95

We have examined nuclear transport in Aspergillus nidulans to determine whether microtubles function in the movement of this organelle. Nuclear movement was found to be inhibited in germinating conidia (uninucleate asexual spores) by the microtubule inhibitor benomyl. To show that the benomyl inhibition was due to its effect on microtubules, the test was repeated with mutants which have genetic lesions in β-tubulin which produce resistance to benomyl in one case (benA15) and super-sensitivity in another (benA16). Nuclear movement was resistant to benomyl in the strain carrying benA15 and super-sensitive in the strain carrying benAl6. Since altered sensitivity to benomyl in these strains is specifically due to alterations in β-tubulin, these results show that β-tubulin is involved in nuclear movement. To eliminate the possibility that nuclear movement blockage was a secondary consequence of nuclear division blockage, this experiment was repeated with temperature-sensitive nuclear division mutants. At restrictive temperature, nuclear division was blocked in these mutants but nuclear movement was not. In the presence of benomyl, nuclear division and migration were blocked at permissive and restrictive temperatures. Thus nuclear division blockage alone is not sufficient to block nuclear movement. These experiments were corroborated by similar experiments on a temperature-sensitive nuclear movement mutant. Five previously isolated nonallelic temperature-sensitive nuclear movement mutants, nudA-E, were analyzed genetically and found not to be allelic to the benA (β-tubulin) tubA α-tubulin genes.     

The disappearance of benomyl from mushroom casing

The failure of Benlate (50% benomyl) to control Mycogone perniciosa on mushroom farms where isolates of the organism were benomyl sensitive was investigated. A comparison was made of carbendazim levels, the major fungitoxic breakdown product of benomyl, in unsterilised casing, autoclaved casing and autoclaved casing mixed with a small proportion (1.7%) of casing previously treated with benomyl but from which all detectable levels of carbendazim had disappeared. Added benomyl was effective in controlling M. perniciosa in autoclaved casing, less effective in unamended casing and ineffective in the amended casing mixture. The recovery of carbendazim from these treatments was directly related to disease occurrence. Studies on farms where benomyl or other carbendazim generating fungicides had been used indicated that a lack of control of M. perniciosa was associated with the disappearance of fungicide from the casing before cropping began. This was not so on a farm where carbendazim generating fungicides had not been used before these investigations. Thiabendazole, a fungicide closely related to the carbendazim generators showed only a slight decline in concentration in casing capable of degrading benomyl. Bacteria isolated from the casing were examined in vitro for their ability to degrade benomyl. Five groups of bacteria were compared and three bacteria, Pseudomonas putida, Pseudomonas aeruginosa and an unidentified member of the Entero-bacteriaceae were most able to degrade benomyl.     

Influence of benomyl on photosynthetic capacity in soybean leaves

This investigation was performed to study the influence of benomyl on photosynthetic pigments and enzymes in soybean leaves. Chlorophyll and pheophytin levels were reduced by benomyl 45 days after greening. These results indicate that chlorophylla andb, and pheophytin must be controlled by benomyl. SDS-PAGE analysis showed that 50 and 14.5 kD polypeptides represented as the large and small subunits of rubisco. In the both of these subunits, the band intensity of the control was significantly higher than that after benomyl treatment, indicating that these two subunits are affected by benomyl. Benomyl strongly inhibited both the activity and content of rubisco as its concentration was gradually increased. However, it remains unclear whether this reduction of rubisco level was due to a reduced level of rubisco activase. Two major polypeptides of 46 and 42 kD were identified as rubisco activase subunits by SDS-PAGE. The intensity of these two bands was shown to be higher in the control than after benomyl treatment. These results indicate that the rubisco decrease resulting from increased benomyl concentrations was caused by rubisco activase. A significant decrease in both the activity and content of rubisco activase by benomyl was also observed. These results suggest that the decrease in rubisco level caused by benomyl is accompanied by a decrease in both the activity and content of rubisco activase.     

Induced Phytoextraction of Lead Through Chemical Manipulation of Switchgrass and Corn; Role of Iron Supplement

The effects of combined chemical application of benomyl, ethylenedianinetetraacetate (EDTA), and iron (Fe) (foliar and root) on lead (Pb) phytoextraction by switchgrass (Panicum virgatum) and corn (Zea mays) was examined. Switchgrass was grown in Pb-contaminated urban topsoil with the following treatments: (C) Control, (B) benomyl, (E) EDTA, (F) foliar-Fe, (BE) benomyl + EDTA, (BF) benomyl + foliar-Fe, (FE) foliar-Fe + EDTA, (BFE) benomyl + foliar-Fe + EDTA. Corn was grown in sand-culture supplemented with Pb (500 mg kg^?1) with the following treatments: (C) control, (B) benomyl, (E) EDTA, (F) root-Fe, (BE) benomyl + EDTA, (BF) benomyl + root-Fe, (FE) root-iron + EDTA, and, (BFE) benomyl + root-Fe + EDTA. All treatments were replicated three times and pots were arranged in a completely randomized design. Plants were analyzed for element concentration (Fe, Zn, P, and Pb) using either inductively coupled plasma (argon) atomic emission spectroscopy (ICP-AES) or graphite furnace atomic absorption spectrometer. Iron supplementation (foliar and root) affected Pb-translocation in plants. Foliar-Fe treatment increased translocation ratio of Pb (TF-Pb) significantly compared to other treatments with the exception of plants treated with benomyl and BF. Root-Fe treatment in combination with EDTA (FE) increased TF-Pb significantly compared to other treatments. Phytoextraction was improved by the combined chemical application; plants treated with BFE treatment increased Pb-total-phytoextraction by 424% compared to Control plants.     

Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae.

The fungicide benomyl inhibited spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae when applied at doses of 21.25 microg/ml (agronomic dose), 10.62 microg/ml and 10 microg/ml. G. mosseae was able to germinate in the presence of 2.12 microg/ml of benomyl, and the percentage of spore germination was unaffected by dosis of 0.1, 0.01 and 0.001 microg/ml of the fungicide. However, all doses of fungicide tested in this study decreased the hyphal length. When ungerminated G. mosseae spores previously exposed to benomyl were transferred to water-agar medium without benomyl, the maximum germination was 16%. Small spores of G. mosseae were more resistant to benomyl than the larger ones. Our results show some of the factors which can explain the variability of the effect of benomyl on arbuscular mycorrhizal fungi.     

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.     

Effect of some fungicides on the yield of grass swards

When benomyl, drazoxolon, thiabendazole or zineb were applied as soil drenches to established grassland drazoxolon reduced the incidence of rhizoplane fungi. Benomyl and thiabendazole significantly increased yield at one harvest but only benomyl had a significant effect on total yield. When benomyl was applied at monthly intervals from March to July significant increases in total yield were obtained with the June applications which also significantly increased yield at the first harvest in the following year. Repeated applications from March to July also had a significant residual effect on total yield. Yield increases were not significantly affected by increasing the benomyl level from 2.5 to 5.0 g/m².     

The effects of fungicide benomyl (benlate) on growth and mitosis in onion (Allium cepa L.) root apical meristem

In this study, the effects of benomyl, a systemic fungicide were investigated in the mitotic cell division in onion (Allium cepa) root tip cells during germination. For this aim, different concentrations (1, 2, 5, 10 and 20 mM) of benomyl solutions were used. All the concentrations used caused several abnormalities in mitotic cell divisions and the mitotic frequency in the onion root tip cells decreased as the concentration of benomyl solution increased. Based on our findings, it is reported that benomyl has some negative effects on mitotic divisions in onion root tip cells.     

The effect of benomyl on growth and ultrastructure of two isolates of Phytophthora infestans from Egypt

The effect of benomyl as a fungicide on the growth rate and ultrastructure of two isolates (P1319 and P623) of Phytophthora infestans is compared. Benomyl caused a concentration-dependent inhibition of the mycelial growth of both isolates. The isolate P1319 was found to be more sensitive to benomyl than the isolate P623. Ultarstructural studies confirmed these observations. The hyphae of isolate P1319 subjected to 100 and 500 ppm benomyl showed more severe changes in the cytoplasm than those of isolate P623. An increase in lipid bodies and vacuoles in the hyphal cytoplasm was the characteristic phenomenon after treatment with benomyl, particularly at a concentration of 500 ppm.