Isolation and characterization of a rice mutant hypersensitive to Al

Rice (Oryza sativa L.) is a highly Al-resistant species among small grain crops, but the mechanism responsible for the high Al resistance has not been elucidated. In this study, rice mutants sensitive to Al were isolated from M(3) lines derived from an Al-resistant cultivar, Koshihikari, irradiated with gamma-rays. Relative root elongation was used as a parameter for evaluating Al resistance. After initial screening plus two rounds of confirmatory testing, a mutant (als1) was isolated from a total of 560 lines. This mutant showed a phenotype similar to the wild-type plant in the absence of Al. However, in the presence of 10 microM Al, root elongation was inhibited 70% in the mutant, but only 8% in the wild-type plant. The mutant also showed poorer root growth in acid soil. The Al content of root apices (0-1 cm) was much lower in the wild-type plant. The sensitivity to other metals including Cd and La did not differ between the mutant and the wild-type plants. A small amount of citrate was secreted from the roots of the mutant in response to Al stress, but there was no difference from that secreted by the wild-type plant. Genetic analysis of F(2) populations between als1 and wild-type plants showed that the Al-resistant seedlings and Al-sensitive seedlings segregated at a 3 : 1 ratio, indicating that the high sensitivity to Al in als1 is controlled by a single recessive gene. The gene was mapped to the long arm of chromosome 6, flanked by InDel markers MaOs0619 and MaOs0615.     

A unique type II topoisomerase mutant that is hypersensitive to a broad range of cleavage-inducing antitumor agents

Bacteriophage T4 provides a useful model system for dissecting the mechanism of action of antitumor agents that target type II DNA topoisomerases. Many of these inhibitors act by trapping the cleavage complex, a covalent complex of enzyme and broken DNA. Previous analysis showed that a drug-resistant T4 mutant harbored two amino acid substitutions (S79F, G269V) in topoisomerase subunit gp52. Surprisingly, the single amino acid substitution, G269V, was shown to confer hypersensitivity in vivo to m-AMSA and oxolinic acid [Freudenreich, C. H., et al. (1998) Cancer Res. 58, 1260-1267]. We purified this G269V mutant enzyme and found it to be hypersensitive to a number of cleavage-inducing inhibitors including m-AMSA, VP-16, mitoxantrone, ellipticine, and oxolinic acid. While the mutant enzyme did not exhibit altered DNA cleavage site specificity compared to the wild-type enzyme, it did display an apparent 10-fold increase in drug-independent DNA cleavage. This suggests a novel mechanism of altered drug sensitivity in which the enzyme equilibrium has been shifted to favor the cleavage complex, resulting in an increase in the concentration of cleavage intermediates available to inhibitors. Mutations that alter drug sensitivities tend to cluster within two specific regions of all type II topoisomerases. Residue G269 of gp52 lies outside of these regions, and it is therefore not surprising that G269V leads to a unique mechanism of drug hypersensitivity. We believe that this mutant defines a new category of type II topoisomerase mutants, namely, those that are hypersensitive to all inhibitors that stabilize the cleavage complex.     

Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle

Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3'-5' helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.     

Induction of diplochromosomes in mammalian cells by inhibitors of topoisomerase II

Diplochromosomes, consisting of four chromatids lying side-by-side, instead of the normal two, are produced when cells go through two rounds of DNA replication without separation of chromatids. They are thus an indication of the failure of the normal chromosome separation mechanism. In the present experiments, induction of diplochromosomes by inhibitors of topoisomerase II (Topo II) was used to provide further evidence that Topo II is required for separation of daughter chromosomes. Actively growing cultures of CHO cells were treated with Colcemid, and separated into metaphase and interphase fractions, each of which was treated for 2 h with the Topo II inhibitor being tested. The cells were then cultivated in fresh medium without inhibitor for periods of between 18 and 44 h, and metaphase cells once again accumulated by treatment with Colcemid. Chromosome preparations were made in the standard way and stained with Giemsa. Up to 2,000 metaphases were counted from each culture, and the proportion with diplochromosomes calculated. At appropriate concentrations, the Topo II inhibitors etoposide and mitoxantrone induced substantial levels of metaphases with diplochromosomes in cultures that had been treated when the cells were in interphase (up to 30% and 11%, respectively). Amsacrine, however, only produced a smaller proportion (4.7%) of metaphases with diplochromosomes after a much longer culture period following treatment. All the inhibitors caused severe chromosome damage. When used to treat metaphase cells, mitoxantrone and amsacrine only induced diplochromosomes after prolonged culture, although a small number of diplochromosomes were seen after etoposide treatment and a shorter period of culture. Results with cells treated in metaphase might indicate that Topo II is, in fact, not required for anaphase chromosome separation, although it is clearly important for segregation of newly replicated DNA. Received: 8 August 1998 / Accepted: 13 September 1998     

Netropsin and bis-netropsin analogs as inhibitors of the catalytic activity of mammalian DNA topoisomerase II and topoisomerase cleavable complexes.

This study examined the ability of netropsin and related minor groove binders to interfere with the actions of DNA topoisomerases II and I. We evaluated a series of netropsin dimers linked with flexible aliphatic chains of different lengths. These agents are potentially able to occupy longer stretches of DNA than the parental drug as a result of bidentate binding. Both netropsin and its dimers were found: (i) to inhibit the catalytic activity of isolated topoisomerase II and (ii) to interfere with the stabilization of the cleavable complexes of topoisomerase II and I in nuclei. Dimers with linkers consisting of 0-4 and 6-9 methylene groups (n) were far more inhibitory than netropsin against isolated enzyme and in the nuclear system. The compound with n = 5 was less active than netropsin in both assays while the dimer with n = 10 inhibited only the isolated enzyme. The comparison of dimers with fixed linker length (n = 2) but varying number of N-methylpyrrole residues (from 1 to 3) revealed that the inhibitory properties were enhanced with increasing number of N-methylpyrrole units. For dimers with varying linker length, drug ability to inhibit catalytic activity of isolated topoisomerase II was positively correlated with calf thymus DNA association constants. In contrast, no such correlation existed in nuclei. However, the inhibitory effects in the nuclear system were correlated with the association constants for poly(dAdT). The results indicate that bidentate binding can significantly enhance anti-topoisomerase activity of netropsin related dimeric minor groove binders. However, other factors such as the length of the linker, the number of pyrrole moieties and the nature of the target (isolated enzyme/DNA versus chromatin in nuclei) also contribute to these activities.     

Overproduction of topoisomerase II in an ataxia telangiectasia fibroblast cell line: comparison with a topoisomerase II-overproducing hamster cell mutant.

Ataxia telangiectasia (AT) cell lines are characterised by their hypersensitivity to ionizing radiation and bleomycin, and their failure to inhibit DNA synthesis after DNA damage. A recent report [Singh et al. (1988) Nucl. Acids Res. 16, 3919-3929] indicated that a reduction in topoisomerase II (topo II) activity was a feature of AT lymphoblast cell lines. We have studied the possible role of DNA topoisomerases in determining the phenotype of an AT fibroblast cell line. AT5BIVA cells are sensitive to the topo II inhibitors etoposide (VP16) and amsacrine (m-AMSA), compared to normal human fibroblasts (MRC5-V1 and VA13). AT5BIVA cells express a 3-fold higher level of topo II protein than MRC5-V1 cells, and 6-fold higher than VA13. This is reflected in elevated topo II activity in AT5BIVA cells. Untransformed AT5BI cells also show elevated topo II activity compared to untransformed normal cells. The extent of overproduction of topo II in AT5BIVA cells is comparable with that seen in a mutant Chinese hamster cell line, ADR-1, which is similarly hypersensitive to both bleomycin and topo II inhibitors. However, ADR-1 cells show neither hypersensitivity to ionizing radiation nor abnormal inhibition of DNA synthesis following DNA damage. Topo II overproduction per se does not appear sufficient to generate an "AT-like" phenotype. AT5BIVA cells express a reduced level of topoisomerase I (topo I) and are hypersensitive to the topo I inhibitor, camptothecin. ADR-1 cells express a normal level of topo I, indicating that a reduction in the level of topo I is not the inevitable consequence of an elevation in topo II.     

Development and use of a high-throughput bacterial DNA gyrase assay to identify mammalian topoisomerase II inhibitors with whole-cell anticancer activity

A high-throughput screen (HTS) was developed and used to identify inhibitors of bacterial DNA gyrase. Among the validated hits were 53 compounds that also inhibited mammalian topoisomerase II with IC(50) values of <12.5 micro g/mL for 51 of them. Using computational methods, these compounds were subjected to cluster analysis to categorize them according to their chemical and structural properties. Nine compounds from different clusters were tested for their whole-cell inhibitory activity against 3 cancer cell lines-NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS)-at a concentration of 100 micro M. Five compounds inhibited cell growth by >50% for all 3 cell lines tested. These compounds were tested further against a panel of 53 to 57 cell lines representing leukemia, melanoma, colon, CNS, ovarian, renal, prostate, breast, and non-small cell lung cancers. In this assay, PGE-7143417 was found to be the most potent compound, which inhibited the growth of all the cell lines by 50% at a concentration range of 0.31 to 2.58 micro M, with an average of 1.21 micro M. An additional 17 compounds were also tested separately against a panel of 10 cell lines representing melanoma, colon, lung, mammary, ovarian, prostate, and renal cancers. In this assay, 4 compounds-PGE-3782569, PGE-7411516, PGE-2908955, and PGE-3521917-were found to have activity with concentrations for 50% cell growth inhibition in the 0.59 to 3.33, 22.5 to 59.1, 7.1 to >100, and 24.7 to >100 micro M range.     

Molecular characterization of a nuclear topoisomerase II from Nicotiana tabacum that functionally complements a temperature-sensitive topoisomerase II yeast mutant

We have successfully expressed enzymatically active plant topoisomerase II in Escherichia coli for the first time, which has enabled its biochemical characterization. Using a PCR-based strategy, we obtained a full-length cDNA and the corresponding genomic clone of tobacco topoisomerase II. The genomic clone has 18 exons interrupted by 17 introns. Most of the 5 and 3 splice junctions follow the typical canonical consensus dinucleotide sequence GU-AG present in other plant introns. The position of introns and phasing with respect to primary amino acid sequence in tobacco TopII and Arabidopsis TopII are highly conserved, suggesting that the two genes are evolved from the common ancestral type II topoisomerase gene. The cDNA encodes a polypeptide of 1482 amino acids. The primary amino acid sequence shows a striking sequence similarity, preserving all the structural domains that are conserved among eukaryotic type II topoisomerases in an identical spatial order. We have expressed the full-length polypeptide in E. coli and purified the recombinant protein to homogeneity. The full-length polypeptide relaxed supercoiled DNA and decatenated the catenated DNA in a Mg²⁺- and ATP-dependent manner, and this activity was inhibited by 4-(9-acridinylamino)-3-methoxymethanesulfonanilide (m-AMSA). The immunofluorescence and confocal microscopic studies, with antibodies developed against the N-terminal region of tobacco recombinant topoisomerase II, established the nuclear localization of topoisomerase II in tobacco BY2 cells. The regulated expression of tobacco topoisomerase II gene under the GAL1 promoter functionally complemented a temperature-sensitive TopII ^ts yeast mutant.     

Isolation and partial characterization of a non-motile mutant ofClostridium acetobutylicum

Summary A non-motile mutant ofClostridium acetobutylicum, morphologically indistinguishable from the motile parent, was capable of solvent production, suggesting that motility is not a regulatory trigger for solventogenesis. However, solventogenesis was considerably weaker than that of the parent, providing evidence of a positive relationship between solvent production and motility.     

Isolation and partial characterization of a lead-accumulating Brassica juncea mutant

A new screening method for non-destructive, high-sensitivity, high-throughput isolation of plant mutants capable of accumulating large amounts of heavy metals has been developed. This method is based on incubating seedlings in a solution containing radioisotopes of the metals of interest and visualizing the tissue accumulation of these metals with a phosphorimager. We used this technique to isolate mutants of Brassica juncea (L.) Czern with increased accumulation of Cd and Pb for use in phytoremediation, an emerging technology using plants to remediate polluted soil and water. Approximately 50,000 M2 seedlings were screened and 21 mutants were recovered that retained increased accumulation through the third generation. Mutant 7/15–1 is characterized by enhanced Pb accumulation per unit of root fresh weight, stunted root growth, and decreased root cell size. Data indicate that roots of 7/15–1 contain more cell-wall material on a fresh-weight basis than roots of the wild-type, which may at least partially explain its ability to accumulate more Pb. Received: 22 September 1998 / Accepted:19 December 1998     

Cleavage of DNA by mammalian DNA topoisomerase II

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.     

Isolation and partial characterisation of dehalogenase-deficient mutants of a Rhizobium sp.

Abstract The use of a mutant of Streptococcus pneumoniae deleted in the lytA gene coding for the N-acetyl-muramyl- l -alanine amidase, and therefore devoid of any amidase, has allowed the identification of a new murein hydrolase activity in this bacterium. This enzyme (or enzymes) acted as an autolysin when the cultures were grown at 30°C. Our results strongly suggest that the new lytic activity corresponds to one or more glycosidases.     

The identification and characterisation of a functional interaction between arginyl-tRNA-protein transferase and topoisomerase II

Topoisomerase II is required for the viability of all eukaryotic cells. It plays important roles in DNA replication, recombination, chromosome segregation, and the maintenance of the nuclear scaffold. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between arginyl-tRNA-protein transferase (Ate1) and the N-terminal domain of the S. cerevisiae topoisomerase II, including the potential site of interaction. Ate1 is a component of the N-end rule protein degradation pathway which targets proteins for degradation. We also propose a previously unidentified role for Ate1 in modulating the level of topoisomerase II through the cell cycle.     

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G₂. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.     

Isolation and characterisation of a partial peptide synthetase gene from Trichoderma asperellum

Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a lambda FIXII genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1.     

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G₂. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.Key words: topoisomerase II, mitosis, G₂, conditional knockdown, S phase, mitotic catastrophe     

Isolation and characterization of a mammalian cell mutant defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase

A somatic cell mutant of the Chinese hamster ovary (CHO)-K1 cell auxotrophic for mevalonic acid has been isolated by means of the bromodeoxyuridine-visible light technique. This mutant can incorporate labeled mevalonate but not labeled acetate into cholesterol and, thus, is apparently defective in mevalonate biosynthesis. The mutant is recessive with respect to the parental cell phenotype. Assessment of the in vitro activities of the enzymes responsible for mevalonate biosynthesis under varying growth conditions indicates that the mutant, Mev-1, is defective in 3-hydroxy-3-methylglutaryl coenzyme A synthase.