Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

A binary vector, designated PROGMO, was constructed to assess the potential of the Zygosaccharomyces rouxii R/Rs recombination system for generating marker- and backbone-free transgenic potato (Solanum tuberosum) plants with high transgene expression and low copy number insertion. The PROGMO vector utilises a constitutively expressed plant-adapted R recombinase and a codA-nptII bi-functional, positive/negative selectable marker gene. It carries only the right border (RB) of T-DNA and consequently the whole plasmid will be inserted as one long T-DNA into the plant genome. The recognition sites (Rs) are located at such positions that recombinase enzyme activity will recombine and delete both the bi-functional marker genes as well as the backbone of the binary vector, leaving only the gene of interest flanked by a copy of Rs␣and RB. Efficiency of PROGMO transformation was tested by introduction of the GUS reporter gene into potato. It was shown that after 21 days of positive selection and using 300 mgl⁻¹ 5-fluorocytosine for negative selection, 29% of regenerated shoots carried only the GUS gene flanked by a copy of Rs and RB. The PROGMO vector approach is simple and might be widely applicable for the production of marker- and backbone-free transgenic plants of many crop species.     

Subsequent effect of subacute T-2 toxicosis on spermatozoa, seminal plasma and testosterone production in rabbits

Pannon White (n=12) male rabbits (weight: 4050 to 4500 g, age: 9 months) received 2 ml of a suspension containing purified T-2 toxin by gavage for 3 days. The daily toxin intake was 4 mg/animal (0.78 to 0.99 mg/kg body weight (BW)). Control animals (n=12) received toxin-free suspension for 3 days. Since a feed-refusal effect was observed on the second day after T-2 administration, a group of bucks (n=10) were kept as controls (no toxin treatment) but on a restricted feeding schedule, that is, the same amount of feed was provided to them as was consumed by the exposed animals. On day 51 of the experiment (i.e. 48 days after the 3-day toxin treatment), semen was collected, and pH, concentration, motility and morphology of the spermatozoa, as well as concentration of citric acid, zinc and fructose in the seminal plasma, were measured. After gonadotropin-releasing hormone (GnRH) analogue treatment, the testosterone level was examined. One day of T-2 toxin treatment dramatically decreased voluntary feed intake (by 27% compared to control, P<0.05) and remained lower (P<0.05) during the first 2 weeks after the withdrawal of the toxin. BW of the contaminated rabbits decreased by 88% on days 17 and 29 compared to controls (P<0.05). No effect of toxin treatment was detected on pH and quantity of the semen or concentration of spermatozoa. The ratio of spermatozoa showing progressive forward motility decreased from 65% to 53% in the semen samples of toxin-treated animals compared to controls (P>0.05). The ratio of spermatozoa with abnormal morphology increased (P<0.05) in the ejaculates collected from the toxin-treated animals. T-2 toxin applied in high doses decreased the concentration of citric acid in seminal plasma (P<0.05). No effect of T-2 toxin on the concentrations of the other seminal plasma parameters (fructose and zinc) was observed. T-2 toxin decreased the basic testosterone level by 45% compared to control (P<0.01) and resulted in lower (P<0.05) GnRH-induced testosterone concentration. Feed restriction, that is, less nutrient intake, resulted in more morphologically abnormal spermatozoa in the semen, but it did not cause significant loss in BW, motility of the spermatozoa, composition of the seminal plasma or testosterone concentration--its effect needs further examination.     

Iron homeostasis in chronic inflammation

Inflammation induced anemia and resistance to erythropoietin are common features in patients with chronic kidney disease (CKD). Elevated levels of cytokines and enhanced oxidative stress, conditions associated with inflammatory states, are implicated in the development of anemia. Accumulating evidence suggests that activation of cytokine cascade and the associated acute-phase response, as it often occurs in patients with CKD, divert iron from erythropoiesis to storage sites within the reticuloendothelial system leading to functional iron deficiency and subsequently to anemia or resistance to erythropoietin. Other processes have also been shown to be involved in the pathogenesis of anemia provoked by the activated immune system including an inhibition of erythroid progenitor proliferation and differentiation, a suppression of erythropoietin production and a blunted response to erythropoietin. The present review concerns the underlying alterations in iron metabolism induced by chronic inflammation that result in anemia.     

Terminal electron transport system (ETS)-activity in the sediment of Lake Balaton,Hungary

Terminal electron transport system (ETS)-activity of the sediment and plankton of Lake Balaton, the largest shallow lake of Central Europe was measured by tetrazolium-reduction biweekly during 1989–1990 and in the spring of 1991. Sediment proved to be enzymatically active to 30-35 cm down in the hypertrophic Keszthely Bay and to 15–20 cm down in the meso-eutrophic Siófok Basin. Sediment ETS-activity exceeded planktonic activity 15 to 24 fold.The total activity m^–2 showed one or two order of magnitude higher respiratory potential in Lake Balaton than needed for complete oxidation of the planktonic primary production; most of this potential was detected in the upper 3–5 cm sediment layer in springs. Incubations of cell-free homogenates of sediment bacteria showed that ETS remains active days after death of organisms at low temperature. Accumulated postmortem ETS-activity derived from the benthic diatoms, bacteria, plankton deposit and dead summer macrophytes seems to be responsible for the high ETS-activity of the sediment in the warming periods in springs. These enzyme fractions may contribute to the rapid oxidation of the alkaline, well-aerated lake.     

Determination of the basicity of Mannich ketones by capillary electrophoresis

Capillary electrophoretic (CE) method to characterise Mannich ketones (MKs) containing morpholine moiety was developed. Basicity (pKa,exp) of the MKs was characterised by measured data derived from the electrophoretic mobility values obtained in the CE separation. The MKs were found to be weaker bases than the parent morpholine molecule itself and the experimentally determined basicity values proved to be dependent on the chemical structure of the MKs. Since the basicity of the MKs has an influence on their reactivity and biological activity it seems to be useful to determine experimentally the pKa,exp values of the newly synthesised compounds to support rational drug design or screening of the molecule libraries.     

Soil Drench Treatment with ?-Aminobutyric Acid Increases Drought Tolerance of Potato

The non-protein amino acid β-aminobutyric acid (BABA) is known to be a priming agent for a more efficient activation of cellular defence responses and a potent inducer of resistance against biotic and abiotic stresses in plants. Nevertheless, most of the studies on priming have been carried out in Arabidopsis. In potato, the effect of BABA was demonstrated only on biotic stress tolerance. We investigated the effect of BABA on the drought tolerance of potato and found that soil drenched with BABA at a final concentration of 0.3 mM improves the drought tolerance of potato. Water loss from the leaves of the primed plants is attenuated and the yield is increased compared to the unprimed drought-stressed plants. The metabolite composition of the tubers of the BABA-treated plants is less affected by drought than the tuber composition of the non-treated plants. Nitric oxide and ROS (reactive oxygen species) production is increased in the BABA-treated roots but not in the leaves. In the leaves of the BABA-treated plants, the expression of the drought-inducible gene StDS2 is delayed, but the expression of ETR1, encoding an ethylene receptor, is maintained for a longer period under the drought conditions than in the leaves of the non-treated, drought-stressed control plants. This result suggests that the ethylene-inducible gene expression remains suppressed in primed plants leading to a longer leaf life and increased tuber yield compared to the non-treated, drought-stressed plants. The priming effect of BABA in potato, however, is transient and reverts to an unprimed state within a few weeks.     

Conservation of the drought-inducible DS2 genes and divergences from their ASR paralogues in solanaceous species.

The drought-inducible DS2 genes of potatoes are members of the ASR (abscisic acid, stress and ripening) gene family. Previously it was shown that expression of DS2 genes is highly dehydration-specific in potato leaves, is not inducible by cold, heat, salt, hypoxia or oxidative stresses, and is independent of abscisic acid (ABA). Now it is shown that StDS2 does not respond either to sucrose or any plant hormones. Conservation of DS2 genes with this unique mode of regulation was studied in the solanaceous species with different relationships to potatoes. DS2 orthologues were identified by DNA sequence alignment in the closely related Lycopersicon and Capsicum species but not in the more distantly related Nicotiana sp. DNA and RNA gel blot analysis revealed the presence of a gene highly homologous to the potato gene StDS2 in tomato (LeDS2) with the same desiccation-specific expression in leaves and organ-specific expression in flowers and green fruits. The LeDS2 promoter was isolated and found to be almost identical in sequence with the promoter of StDS2, except for a 45-bp insertion in tomato. In contrast, no gene highly similar to StDS2 was detected in Nicotiana species on DNA gel blots. Neither StDS2 nor LeDS2 promoter regions were able to confer expression for the beta-glucuronidase (GUS) reporter gene in transgenic tobacco plants indicating that the trans regulatory factors necessary for DS2 expression are not conserved either in Nicotiana tabacum. These data suggest a narrow species-specificity and late evolution of the DS2-type genes within the family Solanaceae.     

Old nests are cues for suitable breeding sites in the Eurasian penduline tit Remiz pendulinus

Nest-sites often have a major influence on avian reproductive success. The use of reliable cues that assist nest-site selection should thus be favoured by natural selection. The old nests have been known to serve as a cue in nest-site selection in several species. To find out whether the old nests act as cue in nest-site selection in the Eurasian penduline tit Remiz pendulinus , we carried out two experiments in southern Hungary, where the penduline tits breed around fishponds and build sophisticated pendulous nests on tree branches that often hang over water. In April 2006, we choose 20 groups of two nearby trees, and hung an old nest on one of the trees in each group. The male penduline tits choose 12 of these groups to build a new nest, and every of the twelve nests were built on trees with an old nest. This suggests that the old nests serve as a cue in the selection of breeding sites for males when they enter a habitat. To find out whether the old nests are cues of plentiful nest building material, or to signal high quality breeding areas, we carried out a second experiment in 2007 by selecting 13 groups of three nearby trees. A "worn-out" old nest was hung on one of the trees, a "re-utilize" type of old nest on another tree, whereas the third tree was left without an old nest in each group. The rationale was that while the worn-out material of the old nests is of no use in building the new nests, material of the "re-utilize" nests is good enough to be used for building new nests. Males built a new nest in 10 of the 13 groups, and eight of the new nests were built on trees with an old nest. Of the eight new nests, five were built on trees with a "worn-out" old nest and three on trees with a "re-utilize" type of old nest. It appears that for the penduline tit males it is the presence of an existing old nest and not the quality of the old nest material that serves as cue during the selection of the suitable breeding sites.