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GENETICS Versatile Switch Designed for Protein Studies Imagine a light switch that turns on, but not off. When it's flipped, the light stays on forever. Most gene expression systems operate in such a limited one-way fashion. In this month's Nature Biotechnology, HMS professor of genetics and Howard Hughes investigator Norbert Perrimon and colleagues report a temperature-dependent switch for controlling protein activity--one that goes on and off with the ease of a light switch. Hot spots. A versatile temperature switch for protein studies is illustrated in the image. Cells in drosophila wing imaginal discs, identified by nuclei staining (blue), produce green fluorescent protein (green) in response to Gal4, expressed through a promoter for the apterous protein. The process occurs in the dorsal section (right side). Gal80, restored at lower, permissive temperatures from a Gal80/intein chimera expressed through a promoter for the decapentaplegic protein, inhibits Gal4 and turns off expression of green fluorescent protein in a small region (center) on the anterior side of the anterior/posterior divide. This is exactly where expression of decapentaplegic and apterous overlap. For clarity, an antibody to the protein engrailed (red) stains the posterior side of the disc. (Image adapted from original from Nature Biotechnology) Perrimon, with joint first authors and research fellows Martin Zeidler and Change Tan, has created a temperature-sensitive intein. Naturally found in unicellular organisms, inteins are peptide stretches endowed with the catalytic ability to splice themselves out of a larger protein. Normally, inserting an active intein into a key regulatory protein, such as a transcription factor, would have little consequence because the intein would splice itself out, leaving the protein intact and functional. But what if the catalytic activity of that intein were exquisitely sensitive to temperature? By controlling temperature, one could control splicing, and by controlling splicing, one could regulate protein expression. This is exactly what the Perrimon lab has done. "The beauty of the system is that it can easily be used to achieve both temporal and spatial control of protein expression," Tan said. On Again, Off Again To obtain a useful temperature-sensitive intein, Zeidler and Tan started with the well-characterized vacuolar ATPase (VMA) intein from yeast. They strategically placed the code for the intein into the gene for Gal4, choosing a site that would likely inactivate the widely used transcriptional activator. After confirming--with a mutant, inactive intein--that Gal4 activity was indeed abolished in unspliced chimeras, they randomly mutated the VMA portion and tested the constructs for splicing at different temperatures. This was done by expressing the chimeras in Gal4-negative yeast growing on galactose as the sole carbon source. In the absence of Gal4, these cells are not viable, so for survival they depend on the intein being spliced out of the chimera. Zeidler and Tan found several intein/Gal4 mutants that allowed yeast to grow at 18 degrees Centigrade, but not at 29 degrees Centigrade. This suggested that splicing and restoration of native Gal4 was temperature dependent. They confirmed this by Western blotting, which showed that splicing did take place at the permissive lower temperature, but not at the restrictive 29 degrees Centigrade. By shuttling the temperature-sensitive intein into Gal80, a Gal4 inhibitor, they were also able to confirm that it behaves as a universal temperature switch that is independent of its protein host. At the permissive temperature, yeast expressing the Gal80 construct failed to grow because splicing activity restored Gal80, which then inhibited Gal4. At the restrictive temperature, however, the yeast thrived. Glowing Hot and Cold Though temperature-sensitive switches cannot be used in most warm-blooded animals, they can be used in many model organisms, including insects, zebrafish, frogs, plants, and virtually any unicellular organism. Perrimon and colleagues demonstrated the versatility of their system in Drosophila melanogaster by using three expression constructs: a Gal80/intein chimera under the control of the promoter for decapentaplegic, a protein that plays a key role in morphogenesis; a Gal4 expression sequence under control of the promoter for the apterous protein, involved in wing patterning; and a green fluorescent protein (GFP) reporter under control of Gal4. Temperature-sensitive switches have proven extremely useful as a means for controlling protein activity, but very few proteins lend themselves to such mutation. Now, Norbert Perrimon (left), Change Tan, and colleagues have engineered a temperature-sensitive intein that can be fitted to almost any protein. (Photo by Steve Gilbert) In these flies, cells that normally express apterous should fluoresce the green of GFP. However, in cells where both apterous and decapentaplegic are expressed, the presence of GFP should be temperature dependent, being turned off at permissive temperatures at which splicing should restore native Gal80, which should then prevent Gal4 from turning on expression of GFP. This is exactly what Zeidler and Tan found, as demonstrated in the figure (left). The image shows a dearth of GFP on the anterior side of the anterior/posterior divide in wing imaginal discs. The location of this void is where expression of decapentaplegic and apterous overlap. "Although several methods of gene silencing or gene activation exist, few have similar flexibility in switching a gene on or off," writes Francine Perler, of New England Biolabs Inc., in an accompanying "News and Views." Perler, a pioneer of intein research, also noted that the system developed in the Perrimon lab has other unique advantages. For example, temperature-sensitive mutations can be introduced directly into proteins, but there is always the niggling doubt that the mutation has unforeseen effects. This is not an issue with intein mutations because they are spliced out. And while finding temperature-sensitive mutations in yeast may be easy, in higher organisms such as fruit flies, it has proven difficult, explained Tan. The universal intein switch, however, can be inserted into any protein. More immediately, the switch can be used to regulate any Gal4-driven gene. There are more than 7,000 well-characterized Drosophila lines that utilize this system, and crossing any of these with the Gal80/temperature-sensitive intein would, in effect, retrofit the lines with a temperature switch. Because many Gal4 constructs are targeted to specific cells, they would then become temporal and spatial regulators of protein activity. --Tom Fagan