01 Oct 2002

Currently, researchers have at their disposal a wide range of promoter systems that can be used to turn on and off the expression of selected genes. Most of these depend on incubating engineered organisms, cells, or tissues in a non-physiological environment, be it a bath of chemicals or of heat. In this month’s Nature Biotechnology, researchers from Peter Quail’s lab at the University of California, Berkeley, report an expression system that requires just a flick of a switch.

First author Sae Shimizu-Sato and colleagues, have married the well-known yeast two-hybrid technology with plant phytochrome-dependent transcription regulation components, to produce a promoter that can be turned on and off by red (664nm) and far-red (748nm) light, respectively.

Plant phytochrome B (phyB) can bind to the protein PIF3, but only when activated by red light. This binding is upon irradiating phyB with far-red light. This reversible, light-induced binding serves as the basis for Quail’s light switch. The authors coupled phyB to the DNA-binding domain of the GAL4 transcription factor (GBD), and coupled PIF3 to the GAL4 activating domain (GAD). In darkness the PhyB-GBD hybrid will seek out and bind the LacZ promoter but will not recruit the PIF3-GAD activating hybrid until PhyB is activated by red light.

Shimizu-Sato et al. tested their system in yeast. It behaves exactly as theory predicts. Red light turns on the promoter, leading to expression of the LacZ gene, while far-red light turns off gene expression. The authors were also able to regulate expression by titrating the yeast cells with light. This ability to fine-tune the expression system may be extremely useful, eliminating problems commonly associated with protein overexpression.

Curiously, however, the method suffers from the same disadvantages of those it is purported to replace. Namely, the yeast must be incubated with an exogenous chemical, a closed tetrapyrrole that is the phytochrome chromophore. The authors suggest that organisms may be engineered to synthesize their own chromophores, but this would require the expression of a whole suite of foreign proteins. For now, at least, it seems there is no way around the chemicals.

A Green Reporter to Envy
Another use of light to improve reporter systems is in tomorrow’s Science by George Patterson and Jennifer Lippincott-Schwartz from the National Institute of Child Health and Human Development, Bethesda, Maryland. They report a new variant of green fluorescent protein (GFP). This protein, from the bioluminescent jellyfish Aequorea victoria, has proved to be an extremely useful reporter, as its green fluorescence is visible to the naked eye and can be readily resolved by the light microscope.

Patterson and Lippincott-Schwartz have boosted the fluorescence output of GFP by over 100-fold. Their method is based on a technique known as photoactivation, whereby molecules can be rapidly converted to a fluorescent state by intense radiation. In the case of GFP, this intense radiation results in the conversion of the chromophore, normally a neutral phenol, to a phenolate. This effectively shifts the major absorption peak of GFP from 397 nm to 475 nm and triples the fluorescence yield when the protein is irradiated at 488 nm.

The authors searched for a mutant GFP with a poor absorbance at 475 nm, and hence poor fluorescence, in the hope of increasing the contrast when the protein is photoactivated. Indeed, one such mutant, with a threonine replaced by a histidine, at 475 nm has poor absorbance, which improves dramatically after intense radiation, resulting in over a 100-fold increase in fluorescence.

The beauty of this new variant of GFP is that it cannot fluoresce until it is photoactivated. This property allowed the authors to activate GFP in a cell nucleus and then trace its fate by following the fluorescence, without having newly synthesized GFP interfere-very much like a real-time pulse-chase experiment. They were able to follow GFP-tagged lysosomal proteins as they moved through the organelles. This new tool should be very useful for studying protein movement and stability.

Fluorescent Reporters-Will that Be Red or Green?
An alternative to the GFP variant is a protein that has just been isolated from the stony coral Trachypyllia geoffroyi by Ryoko Ando and coworkers at Atsushi Miyawaki’s lab, The Institute of Physical and Chemical Research (RIKEN), Wako, Japan. This fluorescent protein, named Kaede, meaning maple leaf in Japanese, changes its emission from green to red upon irradiation with violet or ultraviolet light. The conversion was serendipitously discovered when a sample of the green fluorescent protein was left exposed to sunlight. Properties of the protein are reported in the online early edition of PNAS.

The protein is remarkably different from fluorescent “timer” proteins previously described (see Terskikh et al), which have green fluorescing chromophores that mature slowly to fluoresce red. Kaede, in contrast, will continue to emit green light until zapped with uv light, only then does the chromophore rapidly undergoes a conformational change that results in the red emission.

The benefit of this is obvious. A miniscule amount of the protein, that falling under a narrow beam of light for example, may be converted while keeping the rest of the protein in the native state. Ando et al exquisitely demonstrated this using primary hippocampal neurons transfected with the Kaede gene. By photoconverting a small amount of the protein in the cytosol of a single neuron, the authors could measure the diffusion of the red emitting protein against a green background. The red emitting Kaede was seen to diffuse down axons that made contact with other green neurons in the densely packed cell culture.

Red Kaede is stable and emits with about the same intensity as the green form making it an ideal tool for such tracking experiments. It should prove extremely valuable, particularly for delineating cell-cell contacts and measuring protein-protein interactions.—Tom Fagan

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References

Paper Citations

1. Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, Zhao X, Lukyanov S, Matz M, Kim S, Weissman I, Siebert P. "Fluorescent timer": protein that changes color with time. Science. 2000 Nov 24;290(5496):1585-8. PubMed.

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