SummaryThe phas promoter displays stringent spatial regulation, being very highly expressed during embryogenesis and completely silent during all phases of vegetative development in bean, Phaseolus vulgaris. This pattern is maintained in transgenic tobacco and, as shown here, Arabidopsis. Dimethyl sulphate in vivo footprinting analyses revealed that over 20 cis-elements within the proximal 295 bp of the phas promoter are protected by factor binding in seed tissues whereas none are bound in leaves. The hypothesis that this complex profile represents a summation of several module (cotyledon, hypocotyl, and radicle)-specific factor-DNA interactions has been explored by the incorporation of site-directed substitution mutations into 10 locations within the À295phas promoter. Only 2.6% of À295phas promoter activity remained after mutation of the G-box; the CCAAAT box, the E-box and the RY elements were also found to mediate high levels of expression in embryos. Whereas the CACA element has dual positive and negative regulatory roles, the vicilin box was identified as a strong negative regulatory element. The proximal (À70 to À64) RY motif was found to bestow expression in the hypocotyl while all the RY elements contribute to expression in cotyledons but not to vascular tissue expression during embryogenesis. RY elements at positions À277 to À271, À260 to À254, and À237 to À231 were found to orchestrate radicle-specific repression. The G-box appears to be the functional abscisic acid responsive element and the E-site may be a coupling element. The results substantiate the concept that autarkical cis-element functions generate modular patterning during embryogenesis. They also reflect the existence of both redundancy and hierarchy in cis-element interactions. Importantly, the virtually identical expression patterns observed for the two distantly related plants studied argue strongly for the generality of function for the observed factor-element interactions.

We have shown previously that a rotationally and translationally positioned nucleosome is responsible for the absence of transcriptional expression from the phaseolin (phas) gene promoter in leaf tissue and that the repressive chromatin structure is disrupted on transcriptional activation during embryogenesis. To investigate how the chromatin structure is modified, we ectopically expressed PvALF, a putative seed-specific phas activator, in leaf tissue of a tobacco line transgenic for a chimeric phas/uidA construct. DNase I footprinting in vivo revealed that the ectopic expression of PvALF resulted in remodeling of the chromatin architecture over the TATA region of the phas promoter but did not lead to transcriptional activation in the absence of abscisic acid (ABA). Treatment of the transgenic tobacco leaves with ABA in the absence of PvALF neither alleviated the repressive chromatin architecture nor activated transcription. However, in the presence of PvALF, high levels of ␤-glucuronidase expression were obtained on exposure of leaves to ABA. These results reveal that expression from the phas promoter involves at least two discrete steps: chromatin potentiation by PvALF followed by ABA-mediated transcriptional activation.

Elucidating the mechanisms by which the transcription machinery accesses promoters in their chromatin environment is a fundamental aspect of understanding gene regulation. The phas promoter is normally constrained by a rotationally and translationally positioned nucleosome over its TATA region except during embryogenesis when it is potentiated by the presence of Phaseolus vulgaris ABI3-like factor (PvALF), a plantspecific transcription factor, and activated by an abscisic acid (

The promoter for the phaseolin (phas) bean seed protein gene adopts an inactive chromatin structure in leaves of transgenic tobacco. This repressive architecture, which confers stringent spatial regulation, is disrupted upon transcriptional activation during embryogenesis in a process that requires the presence of both a transcription factor (PvALF) and abscisic acid (ABA). Toward determining the need for de novo synthesis of proteins other than PvALF in transcriptional activation we explored the effect of several eukaryotic protein synthesis inhibitors. Surprisingly, cycloheximide (CHX), emetine, and verrucarin A were able to induce transcription from the phas promoter in tobacco and bean leaf tissue in the absence of either PvALF or ABA. This induction was decreased by the replication inhibitors hydroxyurea and aphidicolin but not by genistein or mimosine. Since protein phosphatases and kinases are essential components of the ABA signal transduction pathway, it is conceivable that CHX is also capable of inducing phosphorylation of proteins usually involved in ABA-mediated activation. Interestingly, okadaic acid, an inhibitor of serine/threonine phosphatase, also strongly activated transcription from the phas promoter. In contrast, the protein synthesis inhibitors anisomycin and puromycin did not activate transcription from the phas promoter, nor did the tyrosine phosphatase inhibitors phenylarsine oxide and sodium orthovanadate. These discrete but different results on transcriptional activation may reflect specific modes of action of the inhibitors, or they may reflect differential interactions of the inhibitors or of downstream events resulting from inhibitor activity with presently unknown components of the transcriptional activation system.

Background: Virulent and avirulent isolates of Rhodococcus equi coexist in equine feces and the environment and are a source of infection for foals. The extent to which plasmid transfer occurs among field strains is ill-defined and this information is important for understanding the epidemiology of R. equi infections of foals.Objectives: To estimate the frequency of transfer of the virulence plasmid between virulent and avirulent strains of R. equi derived from foals and their environment.Animals: None. Methods: In vitro study; 5 rifampin-susceptible, virulent R. equi isolates obtained from clinically affected foals or air samples from a farm with a history of recurrent R. equi foal pneumonia were each mixed with 5 rifampin-resistant, avirulent isolates derived from soil samples, using solid medium, at a ratio of 10 donor cells (virulent) per recipient cell. Presumed transconjugates were detected by plating on media with rifampin and colony immunoblotting to detect the presence of the virulence-associated protein A.Results: Three presumed transconjugates were detected among 2,037 recipient colonies, indicating an overall estimated transfer frequency of 0.15% (95% CI, 0.03-0.43%). All 3 transconjugates were associated with a single donor and 2 recipient strains. Genotyping and multiplex PCR of presumed transconjugates demonstrated transfer of the virulence-associated protein A-bearing plasmid between virulent and avirulent R. equi.Conclusions and Clinical Importance: Transfer of the virulence plasmid occurs with relatively high frequency. These findings could impact strategies to control or prevent R. equi through environmental management.