Biomedical subjects

Helen Townley

Publications and source records attributed to Helen Townley.

3 recordsLinked to original sources

PubMed2005

New pOp/LhG4 vectors for stringent glucocorticoid-dependent transgene expression in Arabidopsis.

To facilitate glucocorticoid-inducible transgene expression from the pOp promoter in Arabidopsis the ligand-binding domain of a rat glucocorticoid receptor (GR LBD) was fused to the amino terminus of the synthetic transcription factor LhG4 to generate LhGR-N. Fusions bearing the GR LBD at other positions in LhG4 exhibited incomplete repression or inefficient induction. LhGR-N was stringently repressed in the absence of exogenous glucocorticoid but was fully activated by addition of 2 microm dexamethasone which resulted in 1000-fold increase in GUS reporter activity. Half maximal induction was achieved with 0.2 microm dexamethasone. Reporter transcripts were detectable within 2 h of dexamethasone application and peaked 4-10 h later. Neither LhGR-N nor dexamethasone affected seedling development although ethanol retarded development when used as a solvent for dexamethasone. The efficiency of the pOp target promoter was improved 10- to 20-fold by incorporating six copies of the ideal lac operator with sufficient inter-operator spacing to allow simultaneous occupancy. Introduction of the TMV Omega sequence into the 5'UTR resulted in a further 10-fold increase in dexamethasone-inducible reporter activity and an increase in the induction factor to 10(4). Although promoters containing the TMV Omega sequence exhibited slightly increased basal expression levels in the absence of dexamethasone, stringent regulation of the cytokinin biosynthetic gene ipt was achieved with all promoters. Despite the severity of the induced ipt phenotypes, transcripts for the KNOX homoeodomain transcription factors BREVIPEDICELLUS and SHOOTMERISTEMLESS were not significantly increased within 48 h of dexamethasone application to seedlings.

Arabidopsis↗

PubMed2005

Improved transcriptional activators and their use in mis-expression traps in Arabidopsis.

The synthetic transcription factor LhG4 has been used in numerous mis-expression studies in plants. We show that the sequence encoding the LhG4 activation domain, derived from Saccharomyces cerevisiae GAL4, contains several cryptic polyadenylation signals in Arabidopsis. The GAL4-derived sequence was modified according to preferred Arabidopsis codon usage, generating LhG4AtO which was faithfully transcribed in Arabidopsis plants. In protoplasts, LhG4AtO achieved maximum transactivation of the pOp promoter with 10-fold less input DNA than LhG4. The same methods were used to compare 10 other LhG4 derivatives that carried alternative natural or synthetic activation domains. Lh214 and Lh314, which contain synthetic activation domains comprising trimers of a core acidic activation domain, directed threefold more GUS expression from the pOp promoter with 20-fold less input DNA than LhG4. In contrast, when expressed from the CaMV 35S promoter in transgenic plants carrying a pOp-GUS reporter, Lh214 and Lh314 yielded transformants with substantially lower GUS activities than other constructs including LhG4AtO and LhG4 which performed similarly. When incorporated into an enhancer-trapping vector, however, LhG4AtO and Lh314 yielded enhancer traps with approximately twice the frequency of LhG4, suggesting that the modified activation domains offer improved performance when expressed from weaker transcription signals. To increase the number of LhG4 patterns available for mis-expression studies, we describe a population of enhancer-trap lines obtained with LhG4AtO in a pOp-GUS background. We show that enhancer-trap lines can transactivate an unlinked pOp-green fluorescent protein (pOp-GFP) reporter in the pattern predicted by staining for GUS activity.

Amino Acid Sequence↗

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↗