Identification of 15 New Bypassable Essential Genes of Fission Yeast.

Every organism has a different set of genes essential for its viability. This indicates that an organism can become tolerant to the loss of an essential gene under certain circumstances during evolution, via the manifestation of 'masked' alternative mechanisms. In our quest to systematically uncover masked mechanisms in eukaryotic cells, we developed an extragenic suppressor screening method using haploid spores deleted of an essential gene in the fission yeast Schizosaccharomyces pombe. We screened for the 'bypass' suppressors of lethality of 92 randomly selected genes that are essential for viability in standard laboratory culture conditions. Remarkably, extragenic mutations bypassed the essentiality of as many as 20 genes (22%), 15 of which have not been previously reported. Half of the bypass-suppressible genes were involved in mitochondria function; we also identified multiple genes regulating RNA processing. 18 suppressible genes were conserved in the budding yeast Saccharomyces cerevisiae, but 13 of them were non-essential in that species. These trends suggest that essentiality bypass is not a rare event and that each organism may be endowed with secondary or backup mechanisms that can substitute for primary mechanisms in various biological processes. Furthermore, the robustness of our simple spore-based methodology paves the way for genome-scale screening.Key words: Schizosaccharomyces pombe, extragenic suppressor screening, bypass of essentiality (BOE), cut7 (kinesin-5), hul5 (E3 ubiquitin ligase).

Unexpected Photo-instability of 2,6-Sulfonamide-Substituted BODIPYs and Its Application to Caged GABA.

Investigation of the unexpected photo-instability of 2,6-sulfonamide-substituted derivatives of the boron dipyrromethene (BODIPY) fluorophore led to the discovery of a photoreaction accompanied by multiple bond scissions. We characterized the photoproducts and utilized the photoreaction to design a caged γ-aminobutyric acid (GABA) derivative that can release GABA upon irradiation in the visible range (>450 nm). This allowed us to stimulate neural cells in mouse brain slices.

Fluorometric assay of integrin activity with a small-molecular probe that senses the binding site microenvironment.

We have developed a small-molecular probe, consisting of cyclic RGD pentapeptides bearing a nitrobenzoxadiazole fluorophore at the 4'-residue, which detects integrin αVß3 activity in terms of fluorescence intensity decrease due to quenching of the fluorophore by interaction with tyrosine-122 at the binding site of the protein. This probe appears to be suitable for a practical, high-throughput fluorescence assay to screen small-molecular modulators of integrin activity.

Rational design of boron dipyrromethene (BODIPY)-based photobleaching-resistant fluorophores applicable to a protein dynamics study.

We studied the photobleaching of a library of boron dipyrromethene (BODIPY) derivatives with a range of electron densities, and found that the photobleaching rate is influenced by the electron-withdrawing capacity of the substituents. Electron-deficient BODIPYs generated less singlet oxygen, were less reactive to singlet oxygen, and were highly resistant to photobleaching. We confirmed the utility of one of these fluorophores, 2,6-diCO(2)R-BDP, for visualizing EGF receptor dynamics in cells expressing an SNAP-tagged EGF receptor.