Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences,short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing

In genetically transformed plants, transgene silencing has been correlated with multiple and complex insertions of foreign DNA, e.g. T-DNA and vector backbone sequences. Occasionally, single-copy transgenes also suffer transgene silencing. We have compared integration patterns and T-DNA/plant DNA junctions in a collection of 37 single-copy T-DNA-transformed Arabidopsis lines, of which 13 displayed silencing. Vector sequences were found integrated in five lines, but only one of these displayed silencing. Truncated T-DNA copies, positioned in inverse orientation to an intact T-DNA copy, were discovered in three lines. The whole nptII gene with pnos promoter was present in the truncated copy of one such line in which heavy silencing has been observed. In the two other lines no silencing has been observed over five generations. Thus, vector sequences and short additional T-DNA sequences are not sufficient or necessary to induce transgene silencing. DNA methylation of selected restriction endonuclease sites could not be correlated with silencing. Our collection of T-DNA/plant DNA junctions has also been used to evaluate current models of T-DNA integration. Data for some of our lines are compatible with T-DNA integration in double-strand breaks, while for others initial invasion of plant DNA by the left or by the right T-DNA end seem important.     

A nuclear export sequence located on a beta-strand in fibroblast growth factor-1

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C delta induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a beta-strand, which has not been reported before, since NESs usually are alpha-helical.     

4-Phenylbutyrate restores the functionality of a misfolded mutant low-density lipoprotein receptor

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low-density lipoprotein receptor. To date, more than 900 different mutations have been described. Transport-defective mutations (class 2) causing partial or complete retention of the receptor in the endoplasmic reticulum are the predominant class of mutations. In a cell culture system (Chinese hamster ovary cells), we show that chemical chaperones are able to mediate rescue of a transport-defective mutant (G544V), and that the ability to obtain rescue is mutation dependent. In particular, the low molecular mass fatty acid derivative 4-phenylbutyrate mediated a marked increase in the transport of G544V-mutant low-density lipoprotein receptor to the plasma membrane. Thirty per cent of the mutant receptor was able to escape from the endoplasmic reticulum and reach the cell surface. The rescued receptor had reduced stability, but was found to be as efficient as the wild-type low-density lipoprotein receptor in binding and internalizing low-density lipoprotein. In addition to 4-phenylbutyrate, we also studied 3-phenylpropionate and 5-phenylvalerate, and compared their effect on rescue of the G544V-mutant low-density lipoprotein receptor with their ability to increase overall gene expression caused by their histone deacetylase inhibitor activity. No correlation was found. Our results indicate that the effect of these agents was not solely mediated by their ability to induce gene expression of proteins involved in intracellular transport, but rather could be due to a direct chemical chaperone activity. These data suggest that rescue of mutant low-density lipoprotein receptor is possible and that it might be feasible to develop pharmacologic chaperones to treat familial hypercholesterolemia patients with class 2 mutations.     

Identity, abundance and ecophysiology of filamentous Chloroflexi species present in activated sludge treatment plants

Filamentous Chloroflexi species are often present in activated sludge wastewater treatment plants in relatively low numbers, although bulking incidences caused by Chloroflexi filaments have been observed. A new species-specific gene probe for FISH was designed and using phylum-, subdivision-, morphotype 1851- and species-specific gene probes, the abundance of Chloroflexi filaments were monitored in samples from 126 industrial wastewater treatment plants from five European countries. Chloroflexi filaments were present in 50% of the samples, although in low quantities. In most treatment plants the filaments could only be identified with phylum or subdivision probes, indicating the presence of great undescribed biodiversity. The ecophysiology of various Chloroflexi filaments was investigated by a suite of in situ methods. The experiments revealed that Chloroflexi constituted a specialized group of filamentous bacteria only active under aerobic conditions consuming primarily carbohydrates. Many exo-enzymes were excreted, e.g. chitinase, glucuronidase and galactosidase, suggesting growth on complex polysaccharides. The surface of Chloroflexi filaments appeared to be hydrophilic compared to other filaments present. These results are generally supported by physiological studies of two new isolates. Based on the results obtained in this study, the potential role of filamentous Chloroflexi species in activated sludge is discussed.     

The cda GenoTox assay: a new and sensitive method for detection of environmental genotoxins, including nitroarenes and aromatic amines

A new bacterial test system for detection of genotoxic compounds was developed, based on two new Salmonella typhimurium tester strains, TGO1 and TGO2. Both strains contain a gene fusion between a strong SOS-promotor, P(cda), and the gfp gene, which allows detection of genotoxic compounds that induce the SOS response. SOS induction was detected by means of flow cytometry. TGO1 showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine compared with a previously developed strain, which had an Escherichia coli strain as host instead of S. typhimurium. S9 mix was introduced into the assay, making the test system suitable for detection of indirect mutagens. Furthermore, the genes for bacterial nitro-reductase (NR) and o-acetyl transferase (o-AT) were inserted into TGO2, making it an NR- and o-AT-over-expressing strain. This resulted in an assay that was able to detect the nitroarene 1-nitropyrene and the aromatic amine 2-aminoanthracene with high sensitivity.     

Organ and cell specificity of base excision repair mutants in mice

Genetically modified mouse models are a powerful approach to study the relation of a single gene-deletion to processes such as mutagenesis and carcinogenesis. The generation of base excision repair (BER) deficient mouse models has resulted in a re-examination of the cellular defence mechanisms that exist to counteract DNA base damage. This review discusses novel insights into the relation between specific gene-deletions and the organ and cell specificity of visible and molecular phenotypes, including accumulation of base lesions in genomic DNA and carcinogenesis. Although promising models exist, there is still a need for new models. These models should comprise combined deficiencies of DNA glycosylases which initiate the BER pathway, to elaborate on the repair redundancy, as well as conditional models of the intermediate steps of BER.     

Quantification of Compositional Changes of Petroleum Hydrocarbons by GC/FID and GC/MS during a Long-Term Bioremediation Experiment

Samples from a long-term bioremediation experiment contaminated with two crude oils, Arabian Heavy and Gullfax, was used to analyze the compositional change of petroleum hydrocarbons. A time course of five different homologous series of petroleum hydrocarbons were analysed by GC/FID and GC/MS. The homologous series were n-alkanes, acyclic isoprenoids, alkylated naphthalenes, alkylated phenanthrenes, and alkylated dibenzothiophenes. Several biomarker compounds were monitored during the experiment to evaluate the possible use as conserved reference compounds for the quantification of other oil compounds, that is, nor-hopanes, hopanes, methyl-hopanes, steranes, mono- og triaromatic steranes. The 17α(H),21β(H)-hopane was found to be stable toward biodegradation and was used as reference compound. The internal standard quantification method was used to quantify changes of the homologous series of oil compounds, and a graphic presentation was used to compare the decrease of the individual compounds. This was found to be an easy way of comparing relative changes in oil. The disappearance of the compounds was extensive and in 6 to 7 months less than 6% remained. The decrease of the n-alkanes (>C15) and acyclic isoprenoids was almost uniform within each homologous series and thus independent of physical-chemical characteristics. Evaporation affected compounds with boiling points lower than n-C15. The alkylated aromatic and sulfur-aromatic compounds decreased according to the degree of alkylation and the decrease showed to be delayed by 10 to 20% by each additional alkyl group. The lack of isomeric-specific degradation of most of the aromatic and sulfur-aromatic compounds, until extensive decrease in concentration had occurred, suggests these compounds have to be dissolved, before any biodegradation occurs.     

Molecular Diversity of the Rumen Microbiome of Norwegian Reindeer on Natural Summer Pasture

The molecular diversity of the rumen microbiome was investigated in five semi-domesticated adult female Norwegian reindeer (Rangifer tarandus tarandus) grazing on natural summer pastures on the coast of northern Norway (71.00° N, 25.30° E). Mean population densities (numbers per gram wet weight) of methanogenic archaea, rumen bacteria and ciliate protozoa, estimated using quantitative real-time polymerase chain reaction (PCR), were 3.17 × 10⁹, 5.17 × 10¹¹ and 4.02 × 10⁷, respectively. Molecular diversity of rumen methanogens was revealed using a 16S rRNA gene library (54 clones) constructed using pooled PCR products from the whole rumen contents of the five individual reindeer. Based upon a similarity criterion of <97%, a total of 19 distinct operational taxonomic units (OTUs) were identified, nine of which are potential new species. The 16S rRNA sequences generated from the reindeer rumen exhibited a high degree of sequence similarity to methanogens affiliated with the families Methanobacteriaceae (14 OTUs) and Methanosarcinaceae (one OTU). Four of the OTUs detected belonged to a group of uncultivated archaea previously found in domestic ruminants and thought to be dominant in the rumen together with Methanobrevibacter spp. Denaturing gradient gel electrophoresis profiling of the rumen bacterial 16S rRNA gene and the protozoal 18S rRNA gene indicated a high degree of animal variation, although some bands were common to all individuals. Automated ribosomal intergenic spacer analysis (ARISA) profiling of the ruminal Neocallimastigales population indicated that the reindeer are likely to contain more than one type of anaerobic fungus. The ARISA profile from one animal was distinct from the other four. This is the first molecular investigation of the ruminal methanogenic archaea in reindeer, revealing higher numbers than expected based on methane emission data available. Also, many of the reindeer archaeal 16S rRNA gene sequences were similar to those reported in domesticated ruminants in Australia, Canada, China, New Zealand and Venezuela, supporting previous findings that there seems to be no host type or geographical effect on the methanogenic archaea community structure in ruminants.