Synthesis of Fluorinated Indoles as RNA Analogues

Nucleoside analogues are chemical means to investigate hydrogen bonds, base stacking, and solvation as the three predominant forces that are responsible for the stability of secondary structure of nucleic acids. To obtain deeper insight into the contributions of these interactions to RNA stability apart from the ones exerted by the predominant nucleosides we decided to synthesize some novel nucleic acid analogues where the nucleobases are replaced by fluoroindoles. Fluorinated indoles can be compared to fluorinated benzimidazoles to determine the role of nitrogen in five membered ring system. The synthesis of fluoroindole ribonucleosides is described here.     

Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Adding value to cocoa (Theobroma cacao L.) germplasm information with domestication history and admixture mapping

A sound understanding of crop history can provide the basis for deriving novel genetic information through admixture mapping. We confirmed this, by using characterization data from an international collection of cocoa, collected 25 years ago, and from a contemporary plantation. We focus on the trees derived from three centuries of admixture between Meso-American Criollo and South American Forastero genomes. In both cacao sets of individuals, linkage disequilibrium extended over long genetic distances along chromosome regions, as expected in populations derived from recent admixture. Based on loose genome scans, genomic regions involved in useful traits were identified. Fifteen genomic regions involved in seed and fruit weight variation were highlighted. They correspond to ten previously identified QTLs and five novel ones. Admixture mapping can help to add value to genetic resources and thus, help to encourage investment in their conservation. Maria Marcano and Tatiana Pugh contributed equally to this work.     

Rapid effects of nitrogen form on leaf morphogenesis in tobacco

Ammonium (NH4+) instead of nitrate (NO3-) as the nitrogen (N) source for tobacco (Nicotiana tabacum L.) cultivated in a pH-buffered nutrient solution resulted in decreased shoot and root biomass. Reduction of shoot fresh weight was mainly related to inhibition of leaf growth, which was already detectable after short-term NH4+ treatments of 24 h, and even at a moderate concentration level of 2 mM. Microscopic analysis of the epidermis of fully expanded leaves revealed a decrease in cell number (50%) and in cell size (30%) indicating that both cell division and cell elongation were affected by NH4+ application. Changes in various physiological parameters known to be associated with NH4(+)-induced growth depression were examined both in long-term and short-term experiments: the concentrations of total N, soluble sugars and starch as well as the osmotic potential, the apparent hydraulic conductivity and the rate of water uptake were not reduced by NH4+ treatments (duration 1-12 d), suggesting that leaf growth was neither limited by the availability of N and carbohydrates, nor by a lack of osmotica or water supply. Although the concentration of K+ in leaf press sap declined in expanding leaves by approximately 15% in response to NH4+ nutrition, limitation of mineral nutrients seems to be unlikely in view of the fast response of leaf growth at 24 h after the start of the NH4+ treatment. No inhibitory effects were observed when NH4+ and NO3- were applied simultaneously (each 1 mM) resulting in a NO3-/NH4+ net uptake ratio of 6:4. These findings suggest that the rapid inhibition of leaf growth was not primarily related to NH4+ toxicity, but to the lack of NO3(-)-supply. Growth inhibition of plants fed solely with NH4+ was associated with a 60% reduction of the zeatine + zeatine riboside (Z + ZR) cytokinin fraction in the xylem sap after 24 h. Furthermore Z + ZR levels declined to almost zero within the next 4 d after start of the NH4+ treatment. In contrast, the concentrations of the putative Z + ZR precursors isopentenyl-adenine and isopentenyl-adenosine (i-Ade + i-Ado) were not affected by NH4+ application. Since cytokinins are involved in the regulation of both cell division and cell elongation, it seems likely that the presence of NO3- is required to maintain biosynthesis and/or root to shoot transfer of cytokinins at a level that is sufficient to mediate normal leaf morphogenesis.     

Occurrence of vitellogenin in drone honeybees (Apis mellifica)

Summary

The occurrence of vitellogenin in adult haploid drones of the honeybee, Apis mellifica, was determined by sensitive immunotechniques, i.e. two-dimensional Immunoelectrophoresis and SDS-PAGE immunoblotting using a monospecific anti-vitellogenin-serum. In drones vitellogenin is one of the minor fractions of the hemolymph proteins. Genetic and regulatory aspects of vitellogenin synthesis in male bees are discussed.     

Phenylalkyl backbone modified oligodeoxynucleotides, their synthesis and the influence of the alkyl chain length

Phenylalkyl modified phosphoramidites (alkyl chain length n = 1, 2, 3, 5; Fig. 1) were synthesised and incorporated into a DNA hexamer (5'-d(GCCp-GCG); p = place of modification). The obtained diastereomeres were separated by RP-HPLC. After hybridisation with the complementary DNA strand Tm-value and thermodynamic data were measured. The stability of duplexes depends on the linker length and the absolute configuration of the backbone modified oligodeoxynucleotides (Rp, Sp).     

Root Morphology and Anchorage of Six Native Tree Species from a Tropical Montane Forest and an Elfin Forest in Ecuador

Root architecture of tree species was investigated at two different altitudes in tropical forests in Ecuador. Increasing altitude was accompanied by higher wind speeds and more shallow soils, while slope angles of both sites were comparable (20–50°). Three tree species typical for the montane forest at 1900 m (Graffenrieda emarginata (Ruiz & Pav.) Triana (Melastomataceae), Clethra revoluta (Ruiz & Pav.) Spreng. (Clethraceae), Vismia tomentosa Ruiz & Pav. (Clusiaceae)) and for the elfin forest at 3000 m (Weinmannia loxensis Harling (Cunoniaceae), Clusia spec. (Clusiacaea) Styrax foveolaria Perkins (Styraceae)) were examined. At 1900 m, 92% of the trees grew upright, in comparison to 52% at 3000 m. At 3000 m, 48% of the trees were inclined, lying or even partly uprooted. At this altitude, all trees with tap roots or with shoots connected by coarse rhizomes, 83% of the trees with stilt roots, and 50% of the trees in which stems or roots were supported by other trees grew upright, suggesting that these characteristics were relevant for tree stability. Root system morphology differed markedly between altitudes. In contrast to 1900 m, where 20% of structural roots originated in the deeper mineral soil, root origin at 3000 m was restricted to the forest floor. The mean ratio of root cross sectional area to tree height decreased significantly from 6.1 × 10⁻³ m² m⁻¹ at 1900 m to 3.2 × 10⁻³ m² m⁻¹ at 3000 m. The extent of root asymmetry increased significantly from 0.29 at 1900 m to 0.62 at 3000 m. This was accompanied by a significantly lower number of dominant roots at 3000 m (2.3 compared to 3.8 at 1900 m). In conclusion, native tree species growing in tropical montane and elfin forests show a variety of root traits that improve tree stability. Root system asymmetry is less important for tree stability where anchorage is provided by a deep and solid root–soil plate. When deep rooting is impeded, root traits improving the horizontal extension of the root–soil plate are more pronounced or occur more frequently. Furthermore, mutual mechanical support of roots and stems of neighboring trees seems to be an appropriate mechanism to provide anchorage in soils with low bulk density and in environments with high wind speeds.     

Role of biotic interactions and physical factors in determining the distribution of marsh species along an estuarine salinity gradient

Understanding the mechanisms that shape plant distribution patterns is a major goal in ecology. We investigated the role of biotic interactions (competition and facilitation) and abiotic factors in creating horizontal plant zonation along salinity gradients in the Elbe estuary. We conducted reciprocal transplant experiments with four dominant species from salt and tidal freshwater marshes at two tidal elevations. Ten individuals of each species were transplanted as sods to the opposing marsh type and within their native marsh (two sites each). Transplants were placed at the centre of 9‐m² plots along a line parallel to the river bank. In order to disentangle abiotic and biotic influences, we set up plots with and without neighbouring vegetation, resulting in five replicates per site. Freshwater species (Bolboschoenus maritimus and Phragmites australis) transplanted to salt marshes performed poorly regardless of whether neighbouring vegetation was present or not, although 50–70% of the transplants did survive. Growth of Phragmites transplants was impaired also by competition in freshwater marshes. Salt marsh species (Spartina anglica and Puccinellia maritima) had extremely low biomass when transplanted to freshwater marshes and 80–100% died in the presence of neighbours. Without neighbours, biomass of salt marsh species in freshwater marshes was similar to or higher than that in salt marshes. Our results indicate that salt marsh species are precluded from freshwater marshes by competition, whereas freshwater species are excluded from salt marshes by physical stress. Thus, our study provides the first experimental evidence from a European estuary for the general theory that species boundaries along environmental gradients are determined by physical factors towards the harsh end and by competitive ability towards the benign end of the gradient. We generally found no significant impact of competition in salt marshes, indicating a shift in the importance of competition along the estuarine gradient.     

Establishing genomic tools and resources for Guizotia abyssinica (L.f.) Cass.-the development of a library of expressed sequence tags, microsatellite loci, and the sequencing of its chloroplast genome

We present an EST library, chloroplast genome sequence, and nuclear microsatellite markers that were developed for the semi-domesticated oilseed crop noug (Guizotia abyssinica) from Ethiopia. The EST library consists of 25 711 Sanger reads, assembled into 17 538 contigs and singletons, of which 4781 were functionally annotated using the Arabidopsis Information Resource (TAIR). The age distribution of duplicated genes in the EST library shows evidence of two paleopolyploidizations—a pattern that noug shares with several other species in the Heliantheae tribe (Compositae family). From the EST library, we selected 43 microsatellites and then designed and tested primers for their amplification. The number of microsatellite alleles varied between 2 and 10 (average 4.67), and the average observed and expected heterozygosities were 0.49 and 0.54, respectively. The chloroplast genome was sequenced de novo using Illumina’s sequencing technology and completed with traditional Sanger sequencing. No large re-arrangements were found between the noug and sunflower chloroplast genomes, but 1.4% of sites have indels and 1.8% show sequence divergence between the two species. We identified 34 tRNAs, 4 rRNA sequences, and 80 coding sequences, including one region (trnH-psbA) with 15% sequence divergence between noug and sunflower that may be particularly useful for phylogeographic studies in noug and its wild relatives.