Production of antithrombotic hirudin in <Emphasis Type="Italic">GAL1</Emphasis>-disrupted <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A gratuitous strain was developed by disrupting the GAL1 gene (galactokinase) of recombinant Saccharomyces cerevisiae harboring the antithrombotic hirudin gene in the chromosome under the control of the GAL10 promoter. A series of glucose-limited fed-batch cultures were carried out to examine the effects of glucose supply on hirudin expression in the gratuitous strain. Controlled feeding of glucose successfully supported both cell growth and hirudin expression in the gratuitous strain. The optimum fed-batch culture done by feeding glucose at a rate of 0.3 g h^–1 produced a maximum hirudin concentration of 62.1 mg l^–1, which corresponded to a 4.5-fold increase when compared with a simple batch culture done with the same strain.     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.     

<Emphasis Type="Italic">Pm37</Emphasis>, a new broadly effective powdery mildew resistance gene from <Emphasis Type="Italic">Triticum </Emphasis><Emphasis Type="Italic">timopheevii</Emphasis>

Powdery mildew is an important foliar disease in wheat, especially in areas with a cool or maritime climate. A dominant powdery mildew resistance gene transferred to the hexaploid germplasm line NC99BGTAG11 from T. timopheevii subsp. armeniacum was mapped distally on the long arm of chromosome 7A. Differential reactions were observed between the resistance gene in NC99BGTAG11 and the alleles of the Pm1 locus that is also located on chromosome arm 7AL. Observed segregation in F_2:3 lines from the cross NC99BGTAG11 × Axminster (Pm1a) demonstrate that germplasm line NC99BGTAG11 carries a novel powdery mildew resistance gene, which is now designated as Pm37. This new gene is highly effective against all powdery mildew isolates tested so far. Analyses of the population with molecular markers indicate that Pm37 is located 16 cM proximal to the Pm1 complex. Simple sequence repeat (SSR) markers Xgwm332 and Xwmc790 were located 0.5 cM proximal and distal, respectively, to Pm37. In order to identify new markers in the region, wheat expressed sequence tags (ESTs) located in the distal 10% of 7AL that were orthologous to sequences from chromosome 6 of rice were targeted. The two new EST-derived STS markers were located distal to Pm37 and one marker was closely linked to the Pm1a region. These new markers can be used in marker-assisted selection schemes to develop wheat cultivars with pyramids of powdery mildew resistance genes, including combinations of Pm37 in coupling linkage with alleles of the Pm1 locus.     

DNA elements modulating the <Emphasis Type="Italic">KARS12</Emphasis> chromosomal replicator in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.     

Functional expression and purification of cytokinin dehydrogenase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (AtCKX2) in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cytokinin dehydrogenase (CKX) is responsible for regulating the endogenous cytokinin content by oxidative removal of the side chain and seven distinct genes, AtCKX1 to AtCKX7, code for the enzyme in Arabidopsis thaliana. The recombinant enzyme AtCKX2 was produced in Saccharomyces cerevisiae after expressing the corresponding gene from a plasmid (pDR197) or following chromosomal integration, under either the constitutive promoter PMA1 or the inducible promoter GAL1. The recombinant protein was purified from yeast culture media using a sequence of chromatographic steps. The purified enzyme had a molecular mass of 61 kDa and a typical flavoprotein spectrum. The specific activity of the enzyme was 87.8 μkat g⁻¹, with isopentenyladenine as a substrate and 2,3-dimethoxy-5-methyl-p-benzoquinone as an electron acceptor. The pH optimum lay between 7.0 and 8.0, depending on the electron acceptor used. AtCKX2 reacts both with isoprenoid and aromatic cytokinins, the activity with isoprenoid cytokinins being two to three orders of magnitude higher. AtCKX2 prefers p-quinones and the synthetic dye 2,6-dichlorophenol indophenol as electron acceptors, although low reactivity with oxygen can also be observed. This study presents the first purification and characterization of the enzyme from Arabidopsis thaliana.     

Genetic instability in the <Emphasis Type="Italic">RAD51</Emphasis> and <Emphasis Type="Italic">BRCA1</Emphasis> regions in breast cancer

Breast cancer is the most prevalent cancer type in women. Accumulating evidence indicates that the fidelity of double-strand break repair in response to DNA damage is an important step in mammary neoplasias. The RAD51 and BRCA1 proteins are involved in the repair of double-strand DNA breaks by homologous recombination. In this study, we evaluated loss of heterozygosity (LOH) in the RAD51 and BRCA1 regions, and their association with breast cancer. The polymorphic markers D15S118, D15S214 and D15S1006 were the focus for RAD51, and D17S855 and D17S1323 for BRCA1. Genomic deletion detected by allelic loss varied according to the regions tested, and ranged from 29 to 46% of informative cases for the RAD51 region and from 38 to 42% of informative cases for the BRCA1 region. 25% of breast cancer cases displayed LOH for at least one studied marker in the RAD51 region exclusively. On the other hand, 31% of breast cancer cases manifested LOH for at least one microsatellite marker concomitantly in the RAD51 and BRCA1 regions. LOH in the RAD51 region, similarly as in the BRCA1 region, appeared to correlate with steroid receptor status. The obtained results indicate that alteration in the RAD51 region may contribute to the disturbances of DNA repair involving RAD51 and BRCA1 and thus enhance the risk of breast cancer development.     

Expression of CoQ10-producing <Emphasis Type="Italic">ddsA</Emphasis> transgene by efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Panicum meyerianum</Emphasis>

Panicum meyerianum Nees is a wild relative of Panicum maximum Jacq. (guinea grass), which is an important warm-season forage grass and biomass crop. We investigated the conditions that maximized the transformation efficiency of P. meyerianum by Agrobacterium infection by monitoring the expression of the β-glucuronidase (GUS) gene. The highest activities of GUS in calli were achieved by the co-cultivation of plants with Agrobacterium at 28°C for 6 days. We transferred the ddsA gene, which encodes decaprenyl diphosphate synthase and is required for coenzyme Q10 (CoQ10) synthesis, into P. meyerianum by using our optimized co-cultivation procedure for transformation. We confirmed by PCR and DNA gel blot hybridization that all hygromycin-resistant plants retained stable insertion of the hpt and ddsA genes. We also demonstrated strong expression of S14:DdsA protein in the leaves of transgenic P. meyerianum. Furthermore, we showed that transgenic P. meyerianum produced CoQ10 at levels 11–20 times higher than that of non-transformants. By comparison, the CoQ9 level in transgenic plants was dramatically reduced. This is the first report of efficient Agrobacterium-mediated transfer of a foreign gene into the warm-season grass P. meyerianum.     

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.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Sarcocystis</Emphasis> sp. in red foxes (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) from northern Croatia

The prevalence of Sarcocystis sp. was assessed in foxes from northern Croatia (city of Varadin). The intestine of 50 (29 male and 21 female) foxes aged 1–5 years, killed during the rabies control campaign, were examined. Sarcocystis sp. was identified in 62% of the fox intestinal samples examined. There was no sex difference in the rate of invasion. However, the rate of invasion differed significantly between the animals aged 1 year (47%), and those aged 2 (71%) and 3–5 years (71%).     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

Atypical non-lipid-dependent strains of <Emphasis Type="Italic">Malassezia furfur</Emphasis>

Members of the yeast genus Malassezia, including atypical strains, are lipophilic except for Malassezia pachydermatis. New physiological features that characterize atypical Malassezia strains are mainly associated with alteration in Tween assimilation pattern — such isolates still require lipids for growth. We isolated three non-lipid-dependent strains of Malassezia from patients with diagnosed atopic dermatitis (AD). These isolates could not be identified to the species level via their physiological properties. Phylogenetic trees, based on the D1/D2 regions of the 26S rDNA gene sequences and nucleotide sequences of the ITS1-5.8S-ITS2 rRNA region, showed the isolates to belong to Malassezia furfur. Three non-lipid dependent isolates from AD skin were conspecific, and sequences analysis proved them to be M. furfur.     

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.     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.