Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Expression and Immunogenicity of Enterotoxigenic <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis> Heat-Labile Toxin B Subunit in Transgenic Rice Callus

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G_M1-ganglioside, a receptor for biologically active LTB, was confirmed by G_M1-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G_M1-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

We have devised a method for cloning an entire mammalian mitochondrial genome (mtDNA) in Escherichia coli using PCR-based amplification and sequential ligation. Here we test this approach by cloning the complete mouse mtDNA. The mtDNA was divided into four to five fragments based on unique restriction enzyme sites and amplified by high-fidelity long-range DNA polymerase. The synthesized fragments were cloned individually to test their toxicity in the E. coli host and then combined sequentially into a vector containing the E. coli R6K origin of DNA replication. The synthetic complete mouse mtDNA clones were replicated stably and faithfully in E. coli when maintained at moderately low copy numbers per cell. The sequence integrity of the synthetic mouse mtDNA clones was confirmed by nucleotide sequencing; no mutations or rearrangements in the genome were found. This approach can facilitate the cloning of entire mammalian mitochondrial genomes in E. coli and assist in the introduction of desired modifications into the mitochondrial genome.     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

Expression and characterization of a cold-active and xylose-stimulated β-glucosidase from <Emphasis Type="Italic">Marinomonas</Emphasis> MWYL1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding a cold-active and xylose-stimulated β-glucosidase of Marinomonas MWYL1 was synthesized and expressed in Escherichia coli. The recombinant enzyme (reBglM1) was purified and characterized. The molecular mass of the purified reBglM1 determined by SDS-PAGE agree with the calculated values (50.6 Da). Optima of temperature and pH for enzyme activity were 40°C and 7.0, respectively. The enzyme exhibited about 20% activity at 5°C and was stable over the range of pH 5.5–10.0. The presence of xylose significantly enhanced enzyme activity even at higher concentrations up to 600 mM, with maximal stimulatory effect (about 1.45-fold) around 300 mM. The enzyme is active with both glucosides and galactosides and showed high catalytic efficiency (k _cat = 500.5 s⁻¹) for oNPGlc. These characterizations enable the enzyme to be a promising candidate for industrial applications.     

Expression profiles of <Emphasis Type="Italic">amh</Emphasis> and <Emphasis Type="Italic">foxl2</Emphasis> in <Emphasis Type="Italic">Schizothorax kozlovi</Emphasis>, and their response to temperature during the early developmental stage

To elucidate the role of amh and foxl2 in sex differentiation of the teleost fish Schizothorax kozlovi, the full-length cDNAs were cloned from the mature testis and ovary by rapid amplification of cDNA ends (RACE), and their relative mRNA expression levels were determined by quantitative real-time polymerase chain reaction among tissues and temperature groups. The complete amh and foxl2 cDNAs of S. kozlovi were 2060 bp and 1750 bp, which encoded 568 and 306 amino acids, respectively. The amh were expressed only in gonads, while foxl2 was expressed in the gills, brain and gonads, both exhibiting relatively high tissue specificity. The amh exhibited sex-specific expression pattern in the gonads. No sex differences in the foxl2 expression were observed in the brain and gonads, but significant sex differences were found in the gills. No significant differences were found in the foxl2 expression, from the larval to the juvenile stage, and also between different temperature groups. However, significant differences were found in the expression levels of amh from the larval (12–63 days posthatching (dph)) to the juvenile stage (190 dph), and also among the \(18{^{\circ }}\hbox {C}\) and \(10{^{\circ }}\hbox {C}\) groups at 31 dph. This result suggested that amh plays an important role in male sex differentiation of S. kozlovi during the early developmental stage, but no similar effect was observed in foxl2.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Fertility and precocity of <Emphasis Type="Italic">Osmunda</Emphasis> × <Emphasis Type="Italic">intermedia</Emphasis> offspring in culture

The feasibility of later-generation hybrid production in ferns has not been previously studied, although it is a significant factor in relation to reproductive isolation. Osmunda × intermedia, a hybrid between O. japonica and O. lancea, is semifertile and has moderate spore germination rates. Under the artificial conditions of this study, F2 and F3 offspring were formed. Some of the F2 offspring showed precocity, and some of the F3 offspring also showed precocity. This fertility suggests that introgressive hybridization might be ongoing in nature. This also indicates a currently unknown genetic control over the timing of fertile frond production in Osmunda.     

Gill cell toxicity of northern boreal scyphomedusae <Emphasis Type="Italic">Cyanea</Emphasis> <Emphasis Type="Italic">capillata</Emphasis> and <Emphasis Type="Italic">Aurelia</Emphasis> <Emphasis Type="Italic">aurita</Emphasis> measured by an in vitro cell assay

Scyphozoan medusae are very successful foragers which occasionally occur in high abundances in boreal waters and may impact many different groups in the marine ecosystem by means of a variety of toxins. A rainbow trout gill cell line, RTgill-W1, was tested for its suitability as quantitative indicator of the cytotoxicity of Cyanea capillata and Aurelia aurita; the major scyphozoan species in the North and Baltic seas. Cultures of rainbow trout gill cells were exposed to whole venoms extracted from fishing tentacles and oral arms at increasing protein concentrations. The venom caused detachment, clumping and lysis of cells, as well as a drop in vitality, in a dose-dependent manner. Morphological changes in the cells were evident within 1 h after venom addition. The damage to gill cells was quantified by measuring the metabolic activity of the cells by means of the fluorescence of resorufin derived from the nonfluorescent substrate, resazurin. In general, a decrease in the metabolic activity of the cells was detected at a venom (protein) concentration above 2.0 μg ml⁻¹ (corresponding to 0.2 μg 10⁴ cells⁻¹), and a total loss of activity was observed above 40.0 μg ml⁻¹ (corresponding to 4.0 μg 10⁴ cells⁻¹). C. capillata venoms had increased cytotoxic activity as compared to A. aurita venoms at the same concentration. Cnidocyst extracts from oral arms of A. aurita induced an 85% loss of gill cell viability at concentrations of 0.2 μg 10⁴ cells⁻¹, whereas crude venoms from fishing tentacles reduced cell viability by 18% at the same concentration. Gel electrophoresis of the venoms indicated that these consist of a large number of proteins in a fairly wide size range, from 6 to 200 kDa, including some that are the same size as those found in cubomedusae. It also appears that larger (i.e., older) medusae have more complex venoms and, in some cases, more potent venoms than smaller animals.     

Involvement of Carbohydrates in Response to Preconditioning Flooding in Two Clones of <Emphasis Type="Italic">Populus deltoides</Emphasis> Marsh. × <Emphasis Type="Italic">P.</Emphasis> <Emphasis Type="Italic">nigra</Emphasis> L.

Poplar, a fast-growing species widely used outside its original area of distribution, was evaluated in the present study to verify its tolerance and hardening to waterlogging in anticipation of its implementation in rehabilitation practices for marginal lands subjected to flooding. Three water regimes were applied to the plants, grown in pots, with two clones (I-488 and D-64) with different sensitivities to flooding. These plants included a lot consisting of control plants (C), which were irrigated with good drainage, a non-preconditioned lot (NPr), and a third lot that was preconditioned to flooding (Pr). Furthermore, flooding was imposed on NPr and Pr plants by submerging pots up to 5 cm above the collar for 60 days followed by a 40-day recovery period. At the end of these two periods, shoot dry mass, foliar concentrations of certain ions, soluble sugars, starch and proline, and the relative electrolyte leakage were evaluated. The preconditioning treatment conferred different degrees of hardness on the treated plants compared with NPr plants. Our results revealed that high carbohydrate availability (soluble sugars and starch) is suggested to participate in sustaining membrane integrity which may affect the recuperative potential of Pr plants, notably those of clone I-488, from flooding damage.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of mint with <Emphasis Type="Italic">E.</Emphasis> <Emphasis Type="Italic">coli</Emphasis> glutathione synthetase gene

Morphologically identical transgenic mint (Mentha arvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of leaf disks with Agrobacterium tumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E. coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented with 25% coconut water, 1.12 mg l⁻¹ BAP, 0.2 mg l⁻¹ NAA, 50 mg l⁻¹ kanamycin and 125 mg l⁻¹ cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2 mg l⁻¹ NAA and 50 mg l⁻¹ kanamycin. The presence and expression of transgenes in transgenics (T₀) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall transformation frequency of 15% was achieved in approximately 3 months of time period. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications. Akhilesh Kumar and Amrita Chakraborty contributed equally.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

High production of bioactive depsides in shoot and callus cultures of <Emphasis Type="Italic">Aronia arbutifolia</Emphasis> and <Emphasis Type="Italic">Aronia</Emphasis> × <Emphasis Type="Italic">prunifolia</Emphasis>

Methanolic extracts from calluses and shoots of Aronia arbutifolia and Aronia × prunifolia cultivated in vitro were quantitatively analysed for phenolic acids by DAD-HPLC. The cultures were grown on ten variants of Murashige–Skoog medium variants enriched with various concentrations of growth regulators (GRs), BA and NAA, in the concentration range 0.1–3.0 mg/L. The analysed extracts were confirmed to contain from four to six compounds (depsides—chlorogenic acid, neochlorogenic acid, and rosmarinic acid, and also protocatechuic acid, p-hydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid). The total amounts of the metabolites varied considerably, depending on the amounts of the GRs in the tested medium variants, and increased in the callus and shoot extracts, respectively, up to 1.7 and 3.2 times (A. arbutifolia), and 2.2 and 2.7 times (A. × prunifolia). Maximum total amounts were confirmed in shoot extracts of both plants (approx. 200 and 600 mg/100 g DW, respectively). The main compounds in A. arbutifolia cultures were the depsides—chlorogenic acid, rosmarinic acid, and neochlorogenic acid (max. 91.94, 77.03, 32.57 mg/100 g DW, respectively). The same depsides dominated quantitatively in the cultures of A. × prunifolia (max. 131.82, 206.62 and 257.39 mg/100 g DW, respectively).     

Butterfly diversity in Mediterranean islands and in Pentadaktylos <Emphasis Type="Italic">Pinus</Emphasis><Emphasis Type="BoldItalic"> </Emphasis><Emphasis Type="Italic">brutia</Emphasis> forests of Cyprus

We analysed the influence of contemporary geography on butterfly diversity for islands in the Mediterranean Basin. We found that island size and distance from the mainland has a significant effect on the number of species. We also used butterflies as an indicator group to identify the importance of forest habitats for biodiversity conservation in the island of Cyprus. To understand the relative importance of local vegetation characteristics of butterflies in the Pentadaktylos mountains transect counts were used to assess the abundance and butterfly diversity in two different forest types. A total of 1,602 butterflies and 23 species were recorded during this research. We observed highly significant effects of forest type on abundance and species richness of butterflies. For example, number of butterflies was significantly higher in old forest than young pine forest. Also, the abundance of endemic butterflies was highest in old forest habitats. Therefore, the survival of the majority of endemic butterflies in Cyprus may depend on conservation of old forests and their understorey plants.     

Karyotype reshufflings of <Emphasis Type="Italic">Festuca pratensis</Emphasis> × <Emphasis Type="Italic">Lolium perenne</Emphasis> hybrids

Many different processes have an impact on the shape of plant karyotype. Recently, cytogenetic examination of Lolium species has revealed the occurrence of spontaneous fragile sites (FSs) associated with 35S rDNA regions. The FSs are defined as the chromosomal regions that are sensitive to forming gaps or breaks on chromosomes. The shape of karyotype can also be determined by interstitial telomeric sequences (ITSs), what was recognized for the first time in this paper in chromosomes of Festuca pratensis × Lolium perenne hybrids. Both FSs and ITSs can contribute to genome instabilities and chromosome rearrangements. To evaluate whether these cytogenetic phenomena have an impact on karyotype reshuffling observed in Festuca × Lolium hybrids, we examined F₁ F. pratensis × L. perenne plants and generated F₂-F₉ progeny by fluorescent in situ hybridization (FISH) using rDNA sequences, telomere and centromere probes, as well as by genomic in situ hybridization (GISH). Analyses using a combination of FISH and GISH revealed that intergenomic rearrangements did not correspond to FSs but overlapped with ITSs for several analyzed genotypes. It suggests that internal telomeric repeats can affect the shape of F. pratensis × L. perenne karyotypes. However, other factors that are involved in rearrangements and have a more crucial impact could exist, but they are still unknown.