Mitosis in the free-living flagellate Bodo saltans strain PS+ (Kinetoplastidea, Bodonida)

The mitosis in the free-living flagellate Bodo saltans Ps+ with prokaryotic cytobionts in perinuclear space has been studied. The nuclear division in B. saltans Ps+ occurs by closed mitosis type without condensation of chromosomes. Two spatially separated mitotic spindles begin to form consistently at the initial stages of nuclear division. The spindle including about 20 microtubules appears first and later the second spindle with half the number of microtubules comes at the angle of 30-40 degrees. Both spindles rest their ends against the inner nuclear membrane and form 4 distinct poles. The microtubules of the first spindle are associated with 4 pairs of kinetochores, the microtubules of the second one are associated with 2 pairs of kinetochores. The divergence of the kinetochores towards the poles occurs independently in each spindle. The equatorial phase is not revealed in B. saltans Ps+. The poles of both spindles unite in pairs at the elongation phase of mitosis and form the integrated bipolar structure. At this stage of the nuclear division, the kinetochores reach the poles of subspindles and become indistinguishable. Then the nucleus takes the shape of a dumbbell. The inner nuclear membranes of just formed nuclei have layers of condensed chromatin characteristic of the interphase nuclei of kinetoplastidea. The daughter nuclei separate at the phase of reorganization. There are 1-2 prokaryotic endocytobionts in the perinuclear space of the interphase nuclei in B. saltans Ps+. The symbionts multiply during mitosis and their number reaches more than 20 specimens par nucleus.     

Mitosis of the free-living flagellate Bodo saltans strain Ps+ (Kinetoplastidea, Bodonida)

Mitosis of the free-living flagellate Bodo saltans of the Ps+ strain characterized by the presence of prokaryotic cytobionts in the perinuclear space was studied. Division of B. saltans Ps+ nuclei occurs by the closed intranuclear type of mitosis without condensation of chromosomes. At the initial stages of nuclear division, consecutive anlage of two spatially separated microtubular spindles begins. The spindle containing about 20 microtubules appears first, then, at an angle of 30–40° to it, the second spindle containing half as many microtubules is formed. The microtubules of the first spindle are associated with 4 pairs of kinetochores, the microtubules of the second one—with 2 pairs. The kinetochores of B. saltans Ps+ have a pronounced laminar structure. Both spindles rest with their ends directly on the internal membrane of the nuclear envelope and form 4 well-pronounced poles. The equatorial phase of mitosis in B. saltans Ps+ is not revealed. The divergence of sister kinetochores towards the poles occurs independently in each spindle. At the elongation phase of mitosis, the poles of both spindles are united in pairs to form a single bipolar structure composed of two loose bundles of microtubules. At this stage of nuclear division, the kinetochores reach the poles of the subspindles and cease to be visible. At subsequent nuclear division stages the nucleus acquires a dumbbell shape. During the reorganization phase the sister nuclei are separated. In the perinuclear space of the interphase nuclei of B. saltans Ps+, 1–2 prokaryotic cytobionts are present. In the course of mitosis, these organisms divide intensively, such that their number can reach 20 and more per nucleus. During separation of sister nuclei, the “excessive” cytobionts are released into the cytoplasmic vacuoles formed by external membranes of the nuclear envelope.     

Identification of a single nuclear localization signal in the C-terminal domain of an Aspergillus DNA topoisomerase II

DNA topoisomerase II (topo II) is a major nuclear protein that plays an important role in DNA metabolism. We have isolated the gene for topo II ( TOP2) from the filamentous fungus Aspergillus terreus. The deduced amino acid sequence revealed that topo II consists of 1,587 amino acids and has a calculated molecular weight of 180 kDa; the protein expressed in Escherichia coli has an estimated molecular weight of 185 kDa. Expression of topo II polypeptides tagged with yellow fluorescent protein (YFP) in budding yeast suggests that the C-terminal region of the topo II is essential for transport of the fusion protein into the nucleus. The nuclear localization signal (NLS) sequence of topo II is a non-classical bipartite type containing two interdependent, positively charged clusters separated by 15 amino acids. Alanine scanning mutagenesis and deletion analyses showed further that a stretch of 23 amino acid residues (positions 1,234-1,256) is necessary for nuclear import. In addition, we confirmed, using co-immunoprecipitation and two-hybrid analysis, that this non-classical NLS interacts with importin alpha in budding yeast. These results suggest that the fungal topo II NLS is functional in yeast cells.     

Localization of a type II DNA topoisomerase to two sites at the periphery of the kinetoplast DNA of Crithidia fasciculata

A type II DNA topoisomerase (topollmt), purified to near homogeneity from the trypanosomatid C. fasciculata has been shown to be localized to the single mitochondrion of these kinetoplastid protozoa. Immunoblots show at least a 10-fold higher level of topollmt (per milligram of protein) in preparations of partially purified mitochondria as compared with those from whole cells. Analyses of type I and type II topoisomerase activities in both mitochondrial and whole cell extracts show a 4- to 5-fold higher specific activity of topollmt in mitochondrial extracts while a nuclear type I topoisomerase has a 4- to 5-fold lower specific activity in the same extract. Immunolocalizations using anti-topollmt antibodies show the enzyme to be present in close association with the mitochondrial DNA networks (kinetoplast DNA or kDNA). This association appears at two distinct locations on opposite sides of the kDNA network.     

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T. borreli. We identified 1.4-kb circular DNAs as the B. saltans equivalent of minicircles via sequence analysis of 4 complete minicircles, 14 minicircle fragments, and 14 gRNAs. We show that each minicircle harbors two gRNA gene cassettes of opposite polarity residing in variable regions of about 200 nt in otherwise highly conserved molecules. In the conserved region, B. saltans minicircles contain a putative bent helix sequence and a degenerate dodecamer motif (CSB-3). Electron microscopy, sedimentation, and gel electrophoresis analyses showed no evidence for the existence of large minicircle networks in B. saltans, the large majority of the minicircles being present as circular and linear monomers (85-90%) with small amounts of catenated dimers and trimers. Our results provide the first example of a kinetoplastid species with noncatenated, gRNA gene-containing minicircles, which implies that the creation of minicircles and minicircle networks are separate evolutionary events.     

Multiple forms and cellular localization of Drosophila DNA topoisomerase II

Purified type II topoisomerase from Drosophila melanogaster embryos was reported earlier to contain a major polypeptide of 166,000 daltons and several smaller peptides between 132,000 and 145,000 daltons (Shelton, E. R., Osheroff, N. and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9530-9535). Using purified topoisomerase II we have raised antibodies against the 132,000-166,000-dalton cluster of polypeptides. In this paper we demonstrate that at least three of these polypeptides are also present in embryos immediately upon lysis. Using antigen-affinity purified antibody from the cluster of purified topoisomerase II antigens, we have also discovered several smaller polypeptides in the molecular size range of 30,000-40,000 daltons in embryo extracts. These observations suggest the presence of multiple forms of DNA topoisomerases in the cell. In addition, we demonstrate that purified Drosophila topoisomerase II antibody recognizes yeast topoisomerase II antigens expressed by lambda gt 11-yeast topoisomerase II recombinants (Goto, T. and Wang, J. C. (1984) Cell 36, 1073-1080) establishing a structural homology between yeast and Drosophila enzymes. Antibody preparations were also used to localize the distribution of topoisomerase II in polytene nuclei. In contrast with the distribution of topoisomerase I which is located primarily at puffs, the Drosophila topoisomerase II is distributed generally along the chromosomes paralleling the distribution of DNA itself.     

Expression and localization of DNA topoisomerase II during rat spermatogenesis

The potential role(s) of DNA topoiosmerase II (topo II) during chromatin changes that characterize different stages of spermatogenesis was investigated in the rat by an analysis of the expression and localization of topo II mRNA and protein in individual spermatogenic cells. Expression of topo II was restricted to spermatogonia, spermatocytes, and round and early-elongating spermatids. Two protein bands of 177 and 170 kDa were detected in immunoblots of spermatocytes and round spermatids, while bands of 148 and 142 kDa were prominent in preparations of elongating spermatids. Topo II levels and distribution patterns, as observed by immunofluorescent microscopy, exhibited cell type-specific variations. Differences in topo II staining patterns were also apparent when nuclear matrices of spermatogenic cells were prepared with different extraction conditions. In addition to its possible function as a structural component, topo II, associated with nuclear matrix preparations from spermatogenic cells, possessed catalytic activity. These observations indicate that both the 177 and 170 kDa and the 148 and 142 kDa forms of topo II share similar structural and functional properties. Topo IIβ mRNA was transcribed in rat spermatogenic cells at 6.2 kb. Relative levels of topo IIβ mRNA were high in spermatogonia and spermatocytes, and decreased in both round and early-elongating spermatids. Changes in topo II expression levels and localization patterns represent distinct stage-specific markers for the maturation of spermatogenic cells, and are consistent with the involvement of topo II in mediating DNA modifications and chromatin changes during spermatogenesis. © 1996 Wiley-Liss, Inc.     

Drug-promoted cleavage of kinetoplast DNA minicircles. Evidence for type II topoisomerase activity in trypanosome mitochondria

Minicircle DNA, the major component of the mitochondrial DNA of trypanosomes (kinetoplast DNA), is linearized when living Trypanosoma equiperdum cells are treated with inhibitors of mammalian type II topoisomerases and then lysed with sodium dodecyl sulfate. A variety of intercalating and nonintercalating compounds (the epipodophyllotoxins, 4'-(9-acridinylamino)-methanesulfon-m-anisidine, 2-methyl-9-hydroxyellipticine, and acriflavine) are active, but novobiocin and specific gyrase inhibitors (the quinolones) are not. The linearized minicircles are in a DNA-protein complex, as their electrophoretic mobility is increased by Proteinase K treatment. They are digested by exonuclease III but not by lambda exonuclease, indicating that the protein must be linked to both 5' ends. Drug-induced cleavage sites vary with different compounds and are found throughout the minicircle sequence. These results indicate that trypanosome mitochondria contain a type II topoisomerase with some properties similar to those of type II topoisomerases in the nucleus of higher eukaryotes. A maximum of 12% of all minicircles is cleaved in the presence of VP16-213, indicating there are at least 600 molecules of mitochondrial type II topoisomerase/cell or about one enzyme/8 kilobases of minicircle DNA.     

Phylogeny of the Drosophila saltans species group based on combined analysis of nuclear and mitochondrial DNA sequences

Nucleotide sequences from two nuclear loci, alcohol dehydrogenase and internal transcribed spacer-1 of the nuclear ribosomal DNA repeats, and two mitochondrial genes, cytochrome oxidase I and cytochrome oxidase II, were determined from nine species in the Drosophila saltans species group. The partition homogeneity test and partitioned Bremer support were used to measure incongruence between phylogenetic hypotheses generated from individual partitions. Individual loci were generally congruent with each other and consistent with the previously proposed morphological hypothesis, although they differed in level of resolution. Since extreme conflict between partitions did not exist, the data were combined and analyzed simultaneously. The total evidence method gave a more resolved and highly supported phylogeny, as indicated by bootstrap proportions and decay indices, than did any of the individual analyses. The cordata and elliptica subgroups, considered to have diverged early in the history of the D. saltans group, were sister taxa to the remainder of the saltans group. The sturtevanti subgroup, represented by D. milleri and D. sturtevanti, occupies an intermediate position in this phylogeny. The saltans and parasaltans subgroups are sister clades and occupy the most recently derived portion of the phylogeny. As with previous morphological studies, phylogenetic relationships within the saltans subgroup were not satisfactorily resolved by the molecular data.      

Sequence determinants of nuclear localization in the alpha and beta isoforms of human topoisomerase II.

The alpha and beta isoforms of DNA topoisomerase II (topo II) are targets for several widely used chemotherapeutic agents, and resistance to some of these drugs may be associated with reduced nuclear localization of the alpha isoform. Human topo IIalpha contains a strong bipartite nuclear localization signal (NLS) sequence between amino acids 1454 and 1497 (alphaNLS(1454-1497)). In the present study, we show that human topo IIalpha tagged with green fluorescence protein is still detectable in the nucleus when alphaNLS(1454-1497) has been disrupted. Seven additional regions in topo IIalpha containing overlapping potential bipartite NLSs were evaluated for their nuclear targeting abilities using a beta-galactosidase reporter system. A moderately functional NLS was identified between amino acids 1259 and 1296. When human topo IIbeta was examined in a similar fashion, it was found to contain two strongly functional sequences betaNLS(1522-1548) and betaNLS(1538-1573) in the region of topo IIbeta comparable to the region in topo IIalpha that contains the strongly functional alphaNLS(1454-1497). The third, betaNLS(1294-1332), although weaker than the other two beta sequences, is significantly stronger than the analogous alphaNLS(1259-1296). Differences in the NLS sequences of human topo II isoforms may contribute to their differences in subnuclear localization.     

DNA fragments which specifically bind to isolated nuclear matrix in vitro interact with matrix-associated DNA topoisomerase II

A matrix-associated region (MAR)-containing fragment has been selected from the library of cloned chicken nuclear matrix-associated DNA fragments. Factors, which determine the specific binding of DNA fragments have been studied. Using topoisomerase II-specific inhibitor VM 26 we established that nuclear matrix-associated topoisomerase II interacted with the MAR-containing DNA fragment producing specific cleavage sites on DNA of the fragment.     

Mitochondrial and nuclear DNA analysis revealed a cryptic species and genetic introgression in Littorina sitkana (Mollusca, Gastropoda)

We investigated mitochondrial and nuclear DNA genotypes in nominal Littorina sitkana samples from 2 localities in Eastern Hokkaido, northern Japan. Our results indicated the existence of cryptic species. In the analysis of partial mitochondrial Cytchrome b gene sequences, haplotypes of L. sitkana samples were monophyletic in a phylogenetic tree with orthologous sequences from other Littorina species, but were apparently separated in 2 clades. One included typical L. sitkana (CBa clade) samples, which formed a clade with an allopatric species, L. horikawai. The other, CBb, was independent from CBa and L. horikawai. Haplotypes of the mitochondrial 16S rRNA gene also separated into 2 clades. We additionally examined intron sequence of the heat shock cognate 70 (HSC70) nuclear gene and identified 17 haplotypes. These were also separated into 2 clades, HSCa and HSCb. Among the examined Hokkaido samples, 60% of individuals were heterozygotes. However, each heterozygote consisted of haplotypes from the same clade, HSCa or HSCb, and no admixture of HSCa and HSCb haplotypes was observed. These results indicate reproductive isolation between the 2 clades. Among the genotyped Hokkaido samples, 93% of individuals had CBa?+?HSCa or CBb?+?HSCb genotypes, and 7% had CBb?+?HSCa genotypes. The discrepancy between the mtDNA and nuclear DNA haplotypes in a few individuals may have been caused by genetic introgression due to past hybridization.     

Prophage induction by DNA topoisomerase II poisons and reactive-oxygen species: role of DNA breaks.

Various compounds were evaluated for their ability to induce prophage lambda in the Escherichia coli WP2s(lambda) microscreen assay. The inability of a DNA gyrase subunit B inhibitor (novobiocin) to induce prophage indicated that inhibition of the gyrase's ATPase was insufficient to elicit the SOS response. In contrast, poisons of DNA gyrase subunit A (nalidixic acid and oxolinic acid) were the most potent inducers of prophage among the agents examined here. This suggested that inhibition of the ligation function of subunit A, which also has a DNA nicking activity, likely resulted in DNA breaks that were available (as single-stranded DNA) to act as strong SOS-inducing signals, leading to prophage induction. Agents that both intercalated and produced reactive-oxygen species (the mammalian DNA topoisomerase II poisons, adriamycin, ellipticine, and m-AMSA) were the next most potent inducers of prophage. Agents that produced reactive-oxygen species only (hydrogen peroxide and paraquat) were less potent than adriamycin and ellipticine but more potent than m-AMSA. Agents that intercalated but did not generate reactive-oxygen species (actinomycin D) or that did neither (teniposide) were unable to induce prophage, suggesting that intercalation alone may be insufficient to induce prophage. These results illustrate the variety of mechanisms (and the relative effectiveness of these mechanisms) by which agents can induce prophage. Nonetheless, these agents may induce prophage by producing essentially the same type of DNA damage, i.e., DNA strand breaks. The potent genotoxicity of the DNA gyrase subunit A poisons illustrates the genotoxic consequences of perturbing an important DNA-protein complex such as that formed by DNA and DNA topoisomerase.     

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24 h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10 h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.     

Effects of VM26 (teniposide), a specific inhibitor of type II DNA topoisomerase, on SV40 DNA replication in vivo.

Simian Virus 40 (SV40) infected cells were pulse labeled with (3H) thymidine and chased either in the absence or in the presence of the cytotoxic drug VM26 (teniposide). We investigated the structure of labeled SV40 DNA and found that VM26 had no significant effect on replicative chain elongation but strongly inhibited the conversion of late replication intermediates to mature DNA daughter molecules. The late replicative SV40 DNA intermediates which accumulate in VM26 treated cells contained essentially full length labeled DNA strands. These newly synthesized strands were not part of two catenated interlocked SV40 monomers suggesting that the block occurred prior to the final ligation reaction. Since VM26 is known to be a specific inhibitor of DNA topoisomerase II we conclude that this enzyme is dispensable for the chain elongation of replicating SV40 DNA, but that it is essential for the termination of SV40 DNA replication cycles.     

Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform.

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)