Further examination of the Xist promoter-switch hypothesis in X inactivation: Evidence against the existence and function of a P0 promoter

The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P0) produces an unstable transcript, while the known downstream promoter (P1) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P0 but retaining P1. We confirm the previous finding that P0 is dispensable for Xist expression in undifferentiated cells and that P1 can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P1 is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P0 does not represent the 5′ end of a distinct Xist isoform. Instead, P0 is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P1 originates from the DNA strand opposite Xist and represents the 3′ end of the antisense Tsix RNA. Thus, these data do not support the existence of a P0 promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.

Evidence for the existence of a sulfonylurea-receptor-like protein in plants: Modulation of stomatal movements and guard cell potassium channels by sulfonylureas and potassium channel openers

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of compounds known for their ability to modulate ATP-sensitive potassium channels (K-ATP) in animal cells. By using epidermal strip bioassays and whole-cell patch-clamp experiments with Vicia faba guard cell protoplasts, we describe a pharmacological profile that is specific for the outward K+ channel and very similar to the one described for ATP-sensitive potassium channels in mammalian cells. Tolbutamide and glibenclamide induced stomatal opening in bioassays and in patch-clamp experiments, a specific inhibition of the outward K+ channel by these compounds was observed. Conversely, application of potassium channel openers such as cromakalim or RP49356 triggered stomatal closure. An apparent competition between sulfonylureas and potassium channel openers occurred in bioassays, and outward potassium currents, previously inhibited by glibenclamide, were partially recovered after application of cromakalim. By using an expressed sequence tag clone from an Arabidopsis thaliana homologue of the sulfonylurea receptor, a 7-kb transcript was detected by Northern blot analysis in guard cells and other tissues. Beside the molecular evidence recently obtained for the expression of ATP-binding cassette protein transcripts in plants, these results give pharmacological support to the presence of a sulfonylurea-receptor-like protein in the guard-cell plasma membrane tightly involved in the outward potassium channel regulation during stomatal movements.

On the existence and implications of an inverse folding code in proteins.

The existence of a code relating the set of possible sequences at a given position in a protein backbone to the local structure at that location is investigated. It is shown that only 73% of 4-C alpha structure fragments in a sample of 114 protein structures exhibit a preference for a particular set of sequences. The remaining structures can accommodate essentially any sequence. The structures that encode specific sequence distributions include the classical "secondary" structures, with the notable exception of planar (beta) bends. It is suggested that this has implications as to the mechanism of folding in proteins with extensive sheet/barrel structure. The possible role of structures that do not encode specific sequences as mutation hot spots is noted.