Biomedical subjects

Rudy Vanderhaeghen

Publications and source records attributed to Rudy Vanderhaeghen.

5 recordsLinked to original sources

PubMed2006

Leader sequence of a plant ribosomal protein gene with complementarity to the 18S rRNA triggers in vitro cap-independent translation.

Cap-independent translation (CIT) occurs at the leader sequences of uncapped plant viral RNAs, but also at a number of normally capped cellular mRNAs and has been correlated with sequence complementarity to 18S rRNA. The ribosomal protein S18 (RPS18) is a component of the small ribosomal subunit and is encoded by three gene copies (A, B, and C) in the Arabidopsis thaliana genome. The RPS18C mRNA was most abundant and contained a short 5' untranslated region of 84 bp that is complementary to a novel putative interaction site at the 3' end of the 18S rRNA. The RPS18C leader mediated CIT as demonstrated by dicistronic constructs consisting of luciferase and chloramphenicol acetyl transferase reporter genes in an in vitro wheat germ extract system. CIT was rapidly inhibited upon addition of an oligonucleotide that competed for the 18S rRNA site complementary to the RPS18C leader and interfered with polysome assembly at the transcript.

3' Untranslated Regions↗

PubMed2006

Simultaneous high-throughput recombinational cloning of open reading frames in closed and open configurations.

Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.

Arabidopsis↗

PubMed2005

Benchmarking the CATMA microarray. A novel tool for Arabidopsis transcriptome analysis.

Transcript profiling is crucial to study biological systems, and various platforms have been implemented to survey mRNAs at the genome scale. We have assessed the performance of the CATMA microarray designed for Arabidopsis (Arabidopsis thaliana) transcriptome analysis and compared it with the Agilent and Affymetrix commercial platforms. The CATMA array consists of gene-specific sequence tags of 150 to 500 bp, the Agilent (Arabidopsis 2) array of 60mer oligonucleotides, and the Affymetrix gene chip (ATH1) of 25mer oligonucleotide sets. We have matched each probe repertoire with the Arabidopsis genome annotation (The Institute for Genomic Research release 5.0) and determined the correspondence between them. Array performance was analyzed by hybridization with labeled targets derived from eight RNA samples made of shoot total RNA spiked with a calibrated series of 14 control transcripts. CATMA arrays showed the largest dynamic range extending over three to four logs. Agilent and Affymetrix arrays displayed a narrower range, presumably because signal saturation occurred for transcripts at concentrations beyond 1,000 copies per cell. Sensitivity was comparable for all three platforms. For Affymetrix GeneChip data, the RMA software package outperformed Microarray Suite 5.0 for all investigated criteria, confirming that the information provided by the mismatch oligonucleotides has no added value. In addition, taking advantage of replicates in our dataset, we conducted a robust statistical analysis of the platform propensity to yield false positive and false negative differentially expressed genes, and all gave satisfactory results. The results establish the CATMA array as a mature alternative to the Affymetrix and Agilent platforms.

Arabidopsis↗

PubMed2005

Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells.

Although sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca2+ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed.

Arabidopsis↗

PubMed2003

DRL1, a homolog of the yeast TOT4/KTI12 protein, has a function in meristem activity and organ growth in plants.

The DEFORMED ROOTS AND LEAVES1 (DRL1) gene is single copy in the Arabidopsis genome, and based on overall amino acid similarity and conservation of functional domains, the DRL1 protein is homologous with yeast TOT4/KTI12. TOT4/KTI12 associates with Elongator, a multisubunit complex that binds the RNA polymerase II transcription elongation complex. Recessive mutations at the DRL1 locus caused defective organ formation indicative of disorganized shoot, inflorescence, flower, and root meristems. DRL1 is a putative ATP/GTP binding protein; in addition, calmodulin binding activity was demonstrated in vitro for the C terminus of the DRL1 protein. Phenotypic and genetic data position DRL1 relative to regulatory loci for leaf development, in which it acts early. We identified Arabidopsis homologs for the six Elongator components and hypothesize that DRL1 regulates transcription elongation through a putative plant Elongator. Upregulation of the ANGUSTIFOLIA transcript in the strong drl1-2 allele supports this model.

Adaptor Proteins, Signal Transducing↗