Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics.

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down

Intragenomic 16S rRNA gene heterogeneity in Lactococcus lactis subsp. cremoris

Partial 16S rRNA gene sequencing of Lactococcus lactis subsp. cremoris strains from our collection identified strains containing sequences diagnostic of both subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris together in the same strain. The presence of a plasmid-encoded 16S rRNA pseudogene partly explained this result. Twenty-four out of 46 L. lactis subsp. cremoris strains tested by PCR contained this pseudogene. However, further analysis showed that five of these 24 strains also contained chromosomal 16S rRNA genes with sequences typical of L. lactis subsp. lactis. Genetic and phenotypic tests indicated these strains were otherwise normal L. lactis subsp. cremoris strains. Past recombination events between the 16S rRNA pseudogene and chromosomal 16S rRNA genes may explain this phenomenon. Genomic heterogeneity for both 16S rRNA and other gene sequences was observed for L. lactis subsp. cremoris SK11 from different laboratories, indicating caution is needed when integrating data from diverse sources for nominally the same strain

Neuronal ensembles sufficient for recovery sleep and the sedative actions of α2 adrenergic agonists

Do sedatives engage natural sleep pathways? It is usually assumed that anesthetic-induced sedation and loss of righting reflex (LORR) arise by influencing the same circuitry to lesser or greater extents. For the α2 adrenergic receptor agonist dexmedetomidine, we found that sedation and LORR were in fact distinct states, requiring different brain areas: the preoptic hypothalamic area and locus coeruleus (LC), respectively. Selective knockdown of α2A adrenergic receptors from the LC abolished dexmedetomidine-induced LORR, but not sedation. Instead, we found that dexmedetomidine-induced sedation resembled the deep recovery sleep that follows sleep deprivation. We used TetTag pharmacogenetics in mice to functionally mark neurons activated in the preoptic hypothalamus during dexmedetomidine-induced sedation or recovery sleep. The neuronal ensembles could then be selectively reactivated. In both cases, non-rapid eye movement sleep, with the accompanying drop in body temperature, was recapitulated. Thus, α2 adrenergic receptor-induced sedation and recovery sleep share hypothalamic circuitry sufficient for producing these behavioral states