Surrogate reporters for enrichment of cells with nuclease-induced mutations.

Zinc-finger nucleases (ZFNs) and TAL-effector nucleases (TALENs) are powerful tools for creating genetic modifications in eukaryotic cells and organisms. But wild-type and mutant cells that contain genetic modifications induced by these programmable nucleases are often phenotypically indistinguishable, hampering isolation of mutant cells. Here we show that transiently transfected episomal reporters encoding fluorescent proteins can be used as surrogate genes for the efficient enrichment of endogenous gene-modified cells by flow cytometry.

Regiochemical and stereochemical evidence for enzyme-initiated catalysis in dual positional specific maize lipoxygenase-1.

Dual positional specific maize lipoxygenase-1 catalyzed the formation of racemic mixtures of four possible regioisomers and was strongly inhibited by the radical scavenger, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinoxy radical. Molecular modeling studies indicated that the oxygen-binding cavity is segregated from the substrate-binding cavity. The data suggest that a bis-allylic radical reaction intermediate is generated enzymatically, released from the enzyme active site, and subsequently oxygenated outside of the enzyme active site by a nonenzymatic mechanism.