;Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1-or PTS2-mediated peroxisomal import.

Altered microRNA (miRNA) expression is a hallmark of many cancer types. The combined analysis of miRNA and messenger RNA (mRNA) expression profiles is crucial to identifying links between deregulated miRNAs and oncogenic pathways. Therefore, we investigated the small non-coding (snc) transcriptomes of nine clear cell renal cell carcinomas (ccRCCs) and adjacent normal tissues for alterations in miRNA expression using a publicly available small RNA-Sequencing (sRNA-Seq) raw-dataset. We constructed a network of deregulated miRNAs and a set of differentially expressed genes publicly available from an independent study to in silico determine miRNAs that contribute to clear cell renal cell carcinogenesis. From a total of 1,672 sncRNAs, 61 were differentially expressed across all ccRCC tissue samples. Several with known implications in ccRCC development, like the upregulated miR-21-5p, miR-142-5p, as well as the downregulated miR-106a-5p, miR-135a-5p, or miR-206. Additionally, novel promising candidates like miR-3065, which i.a. targets NRP2 and FLT1, were detected in this study. Interaction network analysis revealed pivotal roles for miR-106a-5p, whose loss might contribute to the upregulation of 49 target mRNAs, miR-135a-5p (32 targets), miR-206 (28 targets), miR-363-3p (22 targets), and miR-216b (13 targets). Among these targets are the angiogenesis, metastasis, and motility promoting oncogenes c-MET, VEGFA, NRP2, and FLT1, the latter two coding for VEGFA receptors.

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one-and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.All members in the sulfurtransferase (Str)/rhodanese protein family in archaea, eubacteria, and eukaryota are unified by characteristic well-defined sequence domains (Bordo and Bork, 2002). These domains are found as tandem repeats, with the C-terminal domain containing the active site Cys residue, as singledomain proteins or as members of multidomain proteins (Bordo and Bork, 2002). The 18 proteins identified in Arabidopsis which contain at least one Str signature were classified into six groups on the basis of their sequence homology (Bauer and Papenbrock, 2002; http://arabidopsis.org/info/ genefamily/STR_genefamily.html). Group I consists of two Str proteins with two-domain; the five proteins in group VI contain only the C-terminal Str signature and thus possess similarity to the single-domain Str from bacteria.Strs catalyze the transfer of a sulfur atom from suitable sulfur donors to a nucleophilic acceptor. Specific biological roles for most members of this superfamily have not been established (Spallarossa et al., 2001). Proposed roles include cyanide detoxification (Vennesland et al., 1982), involvement in sulfate assimilation (Donadio et al., 1990), and mobilization of sulfur for iron-sulfur cluster biosynthes...

The jellyfish (Aequorea victoria) green fluorescent protein (GFP) and its variants (CFP [cyan] and YFP [yellow]) were successfully used as a vital marker system for the transformation of hybrid poplar (Populus tremula x P. alba). Our results show that, in this woody plant, fluorescent proteins can be expressed: (i) transiently in protoplasts after PEG-mediated transformation, as well as in leaf cells after particle bombardment, and (ii) stably in callus cells and plants after Agrobacterium-mediated transformation. For these studies, we constructed vectors permitting easy recloning of any promoter fragments of interest. Confocal laser scanning microscopy was used both for visualization and differentiation between the different colours of the GFP variants and autofluorescence of chlorophyll and lignified xylem vessels. Peroxisomes were chosen as target organelles for GFP translocation via the peroxisomal targeting sequence PTS1 because this allowed us to concentrate the fluorochrome in the small volume of a few peroxisomes, giving a bright fluorescence over background noise.

The potential of double-stranded RNA interference (RNAi) technology was studied for down-regulation of gene expression in poplar. A set of vectors was constructed generating RNAs capable of duplex formation of sequences specific for the beta-glucuronidase (GUS) reporter gene system. These gene cassettes are driven by the CaMV-35S promoter. To address the question of gene silencing, we tested the functionality of these vectors, both in transient assays by transforming protoplasts with the RNAi constructs, and in stably transformed GUSexpressing poplar plants. Agrobacterium-mediated transformation of those GUS-expressing plants with a GUS-specific RNAi construct showed a strong down-regulation of the reporter gene. From these results we conclude that RNAi is also functional in poplar.