Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2)) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH(2) supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies

Nitrogen fertilization has differential effects on N allocation and lignin in two Populus species with contrasting ecology

Black cottonwood (BC, Populus trichocarpa) and hybrid aspen (HA, P. tremula 9 tremuloides) differ in their ecology of being adapted to wet and drier conditions as riparian and early successional forest species, respectively. We tested the hypothesis that these ecological differences were reflected in higher nitrogen (N) use efficiency in HA than in BC and that HA would allocate more resources belowground than BC in the presence of high and low N availability. We expected that responses of wood properties to elevated N would be more pronounced in the species with higher wood formation in response to N supply. HA showed higher belowground biomass partitioning than BC in the presence of low (0.2 mM) and high (2 mM) N supply, but in contrast to our expectation wholeplant nitrogen use efficiency and the stem carbon-tonitrogen balance were lower than in BC. In response to elevated N, HA exhibited stronger stimulation of biomass production than BC, especially of the stem, which showed significant increases in biomass and volume but decreases in density. Lignification, especially the production of guaiacyl (G)-compared to syringyl (S)-lignin, was delayed in HA compared with BC wood. Since G lignin leads to stronger crosslinking than S lignin, its delayed formation may have enabled stronger expansion and higher volume increment of HA than of BC stems. Our results suggest that BC, a poplar species adapted to fluctuating N supply, is less responsive to differences in N availability than aspen that occurs in low N environments.peerReviewe