GAL4/UAS system

The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It has also been adapted to study receptor chemical-binding functions in vitro in cell culture. It was developed by Hitoshi Kakidani and Mark Ptashne, and Nicholas Webster and Pierre Chambon in 1988, then adapted by Andrea Brand and Norbert Perrimon in 1993 and is considered a powerful technique for studying the expression of genes. The system has two parts: the Gal4 gene, encoding the yeast transcription activator protein Gal4, and the UAS (Upstream Activation Sequence), an enhancer to which GAL4 specifically binds to activate gene transcription. The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It has also been adapted to study receptor chemical-binding functions in vitro in cell culture. It was developed by Hitoshi Kakidani and Mark Ptashne, and Nicholas Webster and Pierre Chambon in 1988, then adapted by Andrea Brand and Norbert Perrimon in 1993 and is considered a powerful technique for studying the expression of genes. The system has two parts: the Gal4 gene, encoding the yeast transcription activator protein Gal4, and the UAS (Upstream Activation Sequence), an enhancer to which GAL4 specifically binds to activate gene transcription. The Gal4 system allows separation of the problems of defining which cells express a gene or protein and what the experimenter wants to do with this knowledge. Geneticists have created genetic varieties of model organisms (typically fruit flies), called GAL4 lines, each of which expresses GAL4 in some subset of the animal's tissues. For example, some lines might express GAL4 only in muscle cells, or only in nerves, or only in the antennae, and so on. For fruit flies in particular, there are tens of thousands of such lines, with the most useful expressing GAL4 in only a very specific subset of the animal—perhaps, for example, only those neurons that connect two specific compartments of the fly's brain. The presence of GAL4, by itself, in these cells has little or no effect, since GAL4's main effect is to bind to a UAS region, and most cells have no (or innocuous) UAS regions. Since Gal4 by itself is not visible, and has little effect on cells, the other necessary part of this system are the 'reporter lines'. These are strains of flies with the special UAS region next to a desired gene. These genetic instructions occur in every cell of the animal, but in most cells nothing happens since that cell is not producing GAL4. In the cells that are producing GAL4, however, the UAS is activated, the gene next to it is turned on, and it starts producing its resulting protein. This may report to the investigator which cells are expressing GAL4, hence the term 'reporter line', but genes intended to manipulate the cell behavior are often used as well. Typical reporter genes include: For example, scientists can first visualize a class of neurons by choosing a fly from a GAL4 line that expresses GAL4 in the desired set of neurons, and crossing it with a reporter line that express GFP. In the offspring, the desired subset of cells will make GAL4, and in these cells the GAL4 will bind to the UAS, and enable the production of GFP. So the desired subset of cells will now fluoresce green and can be followed with a fluorescence microscope. Next, to figure out what these cells might do, the experimenter might express channelrhodopsin in each of these cells, by crossing the same GAL4 line with a channelrhodopsin reporter line. In the offspring the selected cells, and only those cells, will contain channelrhodopsin and can be triggered by a bright light. Now the scientist can trigger these particular cells at will, and examine the resulting behavior to see what these cells might do. Gal4 is a modular protein consisting broadly of a DNA-binding domain and an activation domain. The UAS to which GAL4 binds is CGG-N11-CCG, where N can be any base. Although GAL4 is a yeast protein not normally present in other organisms it has been shown to work as a transcription activator in a variety of organisms such as Drosophila, and human cells, highlighting that the same mechanisms for gene expression have been conserved over the course of evolution. For study in Drosophila, the GAL4 gene is placed under the control of a native gene promoter, or driver gene, while the UAS controls expression of a target gene. GAL4 is then only expressed in cells where the driver gene is usually active. In turn, GAL4 should only activate gene transcription where a UAS has been introduced. For example, by fusing a gene encoding a visible marker like GFP (Green Fluorescent Protein) the expression pattern of the driver genes can be determined. GAL4 and the UAS are very useful for studying gene expression in Drosophila as they are not normally present and their expression does not interfere with other processes in the cell. For example, GAL4/UAS-regulated transgenes in Drosophila have been used to alter glial expression to produce arrhythmic behavior in a known rhythmic circadian output called pigment dispersing factor (PDF). However, some research has indicated that over-expression of GAL4 in Drosophila can have side-effects, probably relating to immune and stress responses to what is essentially an alien protein. The GAL4-UAS system has also been employed to study gene expression in organisms besides Drosophila such as the African clawed frog Xenopus and zebrafish. The GAL4/UAS system is also utilized in Two-Hybrid Screening, a method of identifying interactions between two proteins or a protein with DNA.