The ‘Standard European Vector Architecture’ database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

Meiotic recombination initiates from DNA double-strand breaks (DSBs) generated by SPO11 topoisomerase-like complexes. Meiotic DSB frequency varies extensively along eukaryotic chromosomes, with hotspots controlled by chromatin and DNA sequence. To map meiotic DSBs throughout a plant genome, we purified and sequenced SPO11-1-oligonucleotides. SPO11-1-oligos are elevated in gene promoters, terminators, and introns, which is driven by AT-sequence richness that excludes nucleosomes and allows SPO11-1 access. A positive relationship was observed between SPO11-1-oligos and crossovers genome-wide, although fine-scale correlations were weaker. This may reflect the influence of interhomolog polymorphism on crossover formation, downstream from DSB formation. Although H3K4me3 is enriched in proximity to SPO11-1-oligo hotspots at gene 5' ends, H3K4me3 levels do not correlate with DSBs. Repetitive transposons are thought to be recombination silenced during meiosis, to prevent nonallelic interactions and genome instability. Unexpectedly, we found high SPO11-1-oligo levels in nucleosome-depleted Helitron/Pogo/Tc1/Mariner DNA transposons, whereas retrotransposons were coldspots. High SPO11-1-oligo transposons are enriched within gene regulatory regions and in proximity to immunity genes, suggesting a role as recombination enhancers. As transposon mobility in plant genomes is restricted by DNA methylation, we used the DNA methyltransferase mutant to investigate the role of heterochromatin in SPO11-1-oligo distributions. Epigenetic activation of meiotic DSBs in proximity to centromeres and transposons occurred in mutants, coincident with reduced nucleosome occupancy, gain of transcription, and H3K4me3. Together, our work reveals a complex relationship between chromatin and meiotic DSBs within genes and transposons, with significance for the diversity and evolution of plant genomes.

The flowering of Arabidopsis thaliana winter annuals is delayed until the subsequent spring by the strong floral repressor FLOWERING LOCUS C (FLC). FRIGIDA (FRI) activates the transcription of FLC, but the molecular mechanism remains elusive. The fri mutation causes early flowering with reduced FLC expression similar to frl1, fes1, suf4, and flx, which are mutants of FLC-specific regulators. Here, we report that FRI acts as a scaffold protein interacting with FRL1, FES1, SUF4, and FLX to form a transcription activator complex (FRI-C). Each component of FRI-C has a specialized function. SUF4 binds to a cis-element of the FLC promoter, FLX and FES1 have transcriptional activation potential, and FRL1 and FES1 stabilize the complex. FRI-C recruits a general transcription factor, a TAF14 homolog, and chromatin modification factors, the SWR1 complex and SET2 homolog. Complex formation was confirmed by the immunoprecipitation of FRI-associated proteins followed by mass spectrometric analysis. Our results provide insight into how a specific transcription activator recruits chromatin modifiers to regulate a key flowering gene.

Multiple populations of wake-promoting neurons have been characterized in mammals, but few sleep-promoting neurons have been identified1. Wake-promoting cell types include hypocretin and GABA (γ-aminobutyric-acid)-releasing neurons of the lateral hypothalamus, which promote the transition to wakefulness from non-rapid eye movement (NREM) and rapid eye movement (REM) sleep2,3. Here we show that a subset of GABAergic neurons in the mouse ventral zona incerta, which express the LIM homeodomain factor Lhx6 and are activated by sleep pressure, both directly inhibit wake-active hypocretin and GABAergic cells in the lateral hypothalamus and receive inputs from multiple sleep–wake-regulating neurons. Conditional deletion of Lhx6 from the developing diencephalon leads to decreases in both NREM and REM sleep. Furthermore, selective activation and inhibition of Lhx6-positive neurons in the ventral zona incerta bidirectionally regulate sleep time in adult mice, in part through hypocretin-dependent mechanisms. These studies identify a GABAergic subpopulation of neurons in the ventral zona incerta that promote sleep.