A temperature-sensitive DNA topoisomerase II mutant of the yeast Saccharomyces cerevisiae has been identified. Genetic analysis shows that a single recessive nuclear mutation is responsible for both temperature-sensitive growth and enzymatic activity. Thus, topoisomerase II is essential for viability and the mutation is most probably in the structural gene. Experiments with synchronized mutant cells show that at the nonpermissive temperature cells can undergo one, and only one, round of DNA replication. These cells are arrested at medial nuclear division. Analysis of 2-,um plasmid DNA from these cells shows it to be in the form of multiply intertwined catenated dimers. The results suggest that DNA topoisomerase II is necessary for the segregation of chromosomes at the termination of DNA replication.DNA topoisomerases are enzymes that catalyze the concerted breakage and rejoining of DNA backbone bonds (1). Topoisomerases can be divided into several categories, depending on their source and mode of action (reviewed in ref.2). Eukaryotic type 2 topoisomerases, the subject of this paper, can catalyze several different DNA isomerization reactions, including the relaxation, catenation, decatenation, knotting, and unknotting of closed double-stranded DNA circles (3-5). Although the eukaryotic type 2 DNA topoisomerases are fairly well characterized in vitro, nothing is known about their in vivo roles. It has been suggested that these enzymes might be involved in initiation of DNA replication (3) or in the segregation of daughter DNA molecules at the termination of DNA replication (6, 7).The properties of the yeast topoisomerases are quite similar to their counterparts from mammalian cells (8,9 MATa adel ade2 ura3-52, top2-1(ts), a segregant from the last backcross, was used for phenotype studies.Yeast Growth Conditions. Medium for growth of yeast was YPD (11) or YM-5 (12). The in vivo uniform labeling experiments were carried out essentially as described (12), except that [5,6-3H]uracil at 3 tCi/ml (41 Ci/mmol; 1 Ci = 37 GBq) was used. Cells were synchronized with a-factor pheromone as described (13). Nuclear staining was carried out as described (14), except that Hoechst 33258 dye was used instead of DAPI. Progress through the yeast cell cycle was monitored morphologically by phase-contrast microscopy (13).Topoisomerase Assays. Cells were grown in 25 ml of YPD medium at 250C. During exponential growth cultures were shifted to 370C for 20 min, chilled, centrifuged, washed with cold H20, and recentrifuged. The cell pellet was resuspended in 0.5 ml of yeast lysis buffer (20 mM Tris HCl, pH 7.5/1 mM Na2EDTA/1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/500 mM KCl/10% glycerol). Onethird volume of glass beads (Sigma, type IV,tm) was added and the cells were lysed by brief sonication. The lysate was centrifuged for 10 min in a desk top centrifuge.One microliter of the supernatant (undiluted or diluted in yeast lysis buffer plus 100 ,ug of bovine serum albumin per ml) was used for topoisomerase assays. D...

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Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome, near cysB. Strains carrying deletions of the top gene are viable. The top mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerase I have a more rapid rate of induction and a higher level of catabolite-sensitive enzymes including tryptophanase and -galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished. The frequency of transposition of Tn5, a transposon carrying kanamycin resistance, is decreased by a factor of 40 or more in top mutants. A direct or indirect role of the topoisomerase in transposition is discussed. The transposition frequency ofTn3, however, is not dependent on top. Based on the studies of the E. coli top mutants, it appears that the supX gene, which was originally studied in SalmoneUa typhimurium [Dubnau, E. & Margolin, P. (1972) Mol. Gen. Genet. 117, 91-112] is likely to be the structural gene for DNA topoisomerase I.Escherichia coli DNA topoisomerase I, also known as w protein, has been the subject of extensive studies (for a review, see ref.1). The enzyme is a single polypeptide ofabout 110,000 daltons. In vitro it catalyzes the relaxation of negatively supercoiled DNA (2), the knotting and unknotting of single-stranded DNA rings (3), the linking of single-stranded DNA rings of complementary sequences into intertwined duplex rings (4), and the catenation and decatenation of duplex DNA rings when at least one member of a pair of participating duplex rings has a singlechain scission (5). The catalysis of these topological isomerization reactions by the enzyme is believed to involve transient single-stranded breakage of DNA phosphodiester bonds (1).In contrast to the extensive in vitro studies, there has been little work on the functions of the enzyme in vivo. We have therefore undertaken the isolation and characterization of mutants deficient in DNA topoisomerase I activity. In this communication, we report the identification of a number of mutations in the structural gene for the enzyme and the genetic mapping ofthis gene, which we have termed top, on the E. coli chromosome. Roles of the enzyme in the regulation of transcription ofa number ofoperons and in the transposition of several transposons are also implicated based on our initial characterization of the mutants.MATERIALS AND METHODS Bacterial Strains. All strains used were E. coli K-12 derivatives. The strain used to produce the collection oftemperaturesensitive mutants was PA3092 F-thr leu thi argH thyA his trp lacYl mtl xyl malA mel tonA str supE. Two mutants from this collection were found to have top mutations, JE10010 topl0 and JE10250 top250 (6). Other strains used include: P4X8 Hfr met; KV385 met lacYl xyl mtl str topl0; PLK831 F-trpE pyrF gal-25 nirA strA195; JTT1, a trp+ top+...

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Fly ash was used as a model for ambient particulate matter which is under suspicion to cause adverse pulmonary health effects. The fly ash was pre-sized and contained only particles < 20 microm including an ultrafine fraction (< 100 nm) that contributed 31% to the particle number. In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the formation of ROS with regard to the mass of particles applied. Lipopolysaccharide (LPS) added as a co-stimulus did not increase the formation of ROS induced by fly ash. Furthermore, in LPS (0.1 microg/ml) and tumour necrosis factor-alpha (TNF-alpha; 1 ng/ml) pre-treated cells no increase in reactive oxygen species comparable to fly ash alone is observable. In presence of the metal chelator, desferrioxamine (DFO), ROS formation can be significantly reduced. Neither fly ash nor LPS induced a significant NO release in RLE-6TN cells.

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