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Biomedical subjects

Peter Waterhouse

Publications and source records attributed to Peter Waterhouse.

6 recordsLinked to original sources
PubMed2005

A high-throughput inducible RNAi vector for plants.

We describe here a vector system that allows dexamethasone-inducible RNAi against plant genes. The system utilizes a modified pHELLSGATE vector, under the control of the pOp6 promoter, and the synthetic transcription factor, LhGR. We demonstrate that the production of RNAi-inducing hairpin RNA from this system can be regulated by the application and removal of dexamethasone. Silencing of a target gene encoding phytoene desaturase was highly effective 24 h after application of dexamethasone. In the presence of the hormone silencing was maintained for at least 5 days while removal of the inducer resulted in significant recovery within 24 h. A transgene encoding luciferase was silenced with similar speed and efficiency following application of dexamethasone but unlike phytoene desaturase, mRNA levels did not recover within 10 days after dexamethasone was removed. Insertion of target gene sequences into this vector is mediated by Gateway recombination, facilitating its use for high-throughput applications, such as gene discovery or validation. The inducibility of RNAi from this system may be useful in helping to identify the functions of genes which when constitutively silenced give embryo lethality or pleiotropic phenotypes. A modified version of this system may also be used for tissue-specific hairpin RNA expression.

Journal Article↗
PubMed2004

A plant orthologue of RNase L inhibitor (RLI) is induced in plants showing RNA interference.

RNase L inhibitors (RLIs) correspond to a group of soluble proteins from the large ATP binding cassette (ABC) family of proteins. Structurally, RLIs have an N-terminal Fe-S domain and two nucleotide binding domains. Orthologous RLI sequences with more than 48% identity have been found from Archea to Eukaryota, but have not as yet been identified in Eubacteria. Some organisms, like Arabidopsis thaliana and human, have paralogous genes with differential expression patterns, the function of which remains to be determined. Expression of Arabidopsis RLI2 was slightly increased in transgenic plants showing RNA interference, suggesting a role in this pathway.

ATP-Binding Cassette Transporters↗
PubMed2004

Posttranscriptional gene silencing in plants.

Double-stranded RNA when introduced into cells results in severe reduction of the target mRNA. This phenomenon is known as posttranscriptional gene silencing in plants and RNA interference in animals. Hairpin RNA-mediated gene silencing exploits this cellular mechanism. A convenient way of generating hairpin constructs is to use generic vectors such as pHANNIBAL and pHELLSGATE, vectors based on the Gateway technology. These vectors are suitable for high-throughput gene silencing, and the silencing effect is stably inherited over many generations.

Cloning, Molecular↗
PubMed2003

Constructs and methods for high-throughput gene silencing in plants.

Gene silencing can be achieved by transformation of plants with constructs that express self-complementary (termed hairpin) RNA containing sequences homologous to the target genes. The DNA sequences encoding the self-complementary regions of hairpin (hp) RNA constructs form an inverted repeat. The inverted repeat can be stabilized in bacteria through separation of the self-complementary regions by a "spacer" region. When the spacer sequence encodes an intron, the efficiency of gene silencing is very high. There are at least three ways in which hpRNA constructs can be made. The construct may be generated from standard binary plant transformation vectors in which the hairpin-encoding region is generated de novo for each gene. Alternatively, generic gene-silencing vectors such as the pHANNIBAL and the pHELLSGATE series can be used. They simply require the insertion of PCR products, derived from the target gene, into the vectors by conventional cloning or by using the Gateway directed recombination system. In this article, we describe and evaluate the advantages of these vectors and then provide the protocols for their efficient use.

Gene Silencing↗
PubMed2002

A branched pathway for transgene-induced RNA silencing in plants.

In plants, RNA silencing can be induced by highly transcribed sense transgenes (S-PTGS) or by transgene loci producing double-stranded RNA (dsRNA) due to the presence of inverted repeats (IR-PTGS). Both phenomena correlate with accumulation of 21-25 nt sense and anti-sense RNA homologous to the silent gene and with methylation of the coding sequence. We have challenged IR-PTGS with four viruses known to inhibit S-PTGS: CMV, TuMV, TVCV, and TCV ( this work) and in sgs2, sgs3, and ago1 mutants impaired in S-PTGS. Surprisingly, whereas the four viruses inhibit IR-PTGS, IR-PTGS and methylation of a GUS trangene and IR-PTGS of three endogeneous genes occur in the sgs2, sgs3, and ago1 mutations. Based on these results, we propose a branched pathway for RNA silencing in plants. RNA silencing would occur via the action of dsRNA produced either via the action of SGS2 (also known as SDE1), SGS3, and AGO1 on the S-PTGS branch or by transgenes arranged as inverted repeats on the IR-PTGS branch. Moreover, transgene methylation would result from production or action of dsRNA, since it does not require SGS2/SDE1, SGS3, and AGO1.

Arabidopsis Proteins↗