Hydrogen peroxide-induced oxidative damages in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H₂O₂-treated and non-treated control cells. It was observed that increased H₂O₂ concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.     

A replication-time-controlling sequence element in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Eukaryotic replication origins are highly variable in their activity and replication timing. The nature and role of cis-acting regulatory sequences that control chromosomal replication timing is not well defined. In the fission yeast, Schizosaccharomyces pombe, a 200-bp late-replication-enforcing element (LRE), has been shown to enforce late replication of ARS elements in plasmids. Here, we show that a short (133-bp) fragment of the LRE (shLRE) is required for causing late replication of adjoining origins in its native as well as in an ectopic early-replicating chromosomal location. Active from both sides of an early-replicating origin, the shLRE is a bona fide cis-acting regulatory element that imposes late replication timing in the chromosome.     

Recombinational repair in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>: The role of mediator proteins

Repair of double-strand breaks (DSBs), which arise in DNA spontaneously or under the influence of external factors, is critical for cell survival. The evolutionarily conserved mechanism of error-free recombinational repair plays a major role in maintaining the genome integrity and has a number of similarities between lower eukaryotes and vertebrates. The review considers the currently available data on the mechanism of recombinational DSB repair in the fission yeast Schizosaccharomyces pombe and its differences from the corresponding mechanisms of Saccharomyces cerevisiae and higher eukaryotes.     

Characterization of chromate-sensitive and-tolerant mutants of<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Stable chromium(VI)-sensitive and -tolerant mutants were obtained by induced mutagenesis of Schizosaccharomyces pombe lysine and leucine auxotrophic heterothallic strains 6chr+ and 9chr+. Eleven of them were selected for further studies. Fast transport of 51CrO4(2-) was detected in a representative sensitive mutant, chr-51S, while the tolerant mutant chr1-66T and the parental strain 6chr+ exhibited significantly lower 51CrO4(2-) uptake. The segregation of tetrads of three selected CrVI-tolerant mutants, chr1-66T, chr1-14T and chr2-04T, strongly indicated that tolerance was determined by single mutations. Random spore analysis proved that the mutations of chr1-66T and chr1-14T were allelic and the mutation of mutant chr2-04T was not allelic with the mutation of chr1-66T. Recombinants carrying the ura4D18 selective marker were created for transformation experiments. Two of them (chr1-661T and chr2-046T) can be used to clone and identify the genes responsible for their CrVI tolerance phenotype.     

Expression,purification, and crystallization of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> eIF2B

Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution.     

The <Emphasis Type="Italic">alm1</Emphasis><Superscript><Emphasis Type="Italic">+</Emphasis></Superscript> gene from <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> encodes a coiled-coil protein that associates with the medial region during mitosis

We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4 ⁺ disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4 ⁺ diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1 ⁺ / alm1::ura4 ⁺ diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Identification of thermostable glyoxalase I in the fission yeast <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Glyoxalase I is a ubiquitous enzyme that detoxifies methylglyoxal, which is derived from glycolysis but inhibits the growth of cells from microorganisms to mammals. Here, the structural gene for glyoxalase I (glo1⁺) from the fission yeast Schizosaccharomyces pombe was identified. Disruption of glo1⁺ enhanced susceptibility to methylglyoxal, while expression of glo1⁺ in a glo1 mutant of Saccharomyces cerevisiae restored tolerance to this aldehyde. The glo1⁺ gene product was purified. The glyoxalase I of S. pombe was a monomeric enzyme with a molecular weight of 34,000 and the k_cat/K_m value for methylglyoxal was 4.3×10⁷ M^–1 min^–1. Treatment of purified enzyme with EDTA in imidazole buffer completely abolished enzyme activity, whereas the EDTA-treated enzyme was reactivated by several divalent metal ions, such as Zn²⁺, Co²⁺, Ni²⁺ and Mn²⁺. The glyoxalase I of S. pombe exhibited fairly high thermal stability, and almost 100% activity was retained after incubating the enzyme at 60°C for 4 h.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Hierarchical Chromatin Structure of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> Revealed by Atomic Force Microscopy

Many structural studies on higher eukaryotic chromatin have been carried out, but chromatin structure in fungi remains unclear. Schizosaccharomyces pombe has been used for investigations of chromosome function; however, the structural details of S. pombe chromatin have not been clarified owing to its small nucleus. We used atomic force microscopy for nano-scale imaging of chromatin isolated from S. pombe. Topographic images indicated that nuclear chromatin contained at least three hierarchical structures: large-scale chromatin fibers, spherical domains in the fibers, and nodules in the domains. The average diameters of the domain and the nodule were 363 ± 85.2 nm and 46.2 ± 9.30 nm. Each structure comprising the hierarchy was similar to higher eukaryotic chromatin thus far observed, despite definite differences in chromatin organization at the nucleosomal level. The presence of histone H1 suggested that there might be an alternative to compensate for histone H1 lacking in S. pombe.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.