Fig. 2. The Constructs A) The mGAL-mGFP5 HDEL 15 enhancer trapping construct (designed and tested by J Haseloff, Cambridge, UK) comprises a modified GAL4 transactivator (GAL4-VP16), an ER localised GFP under the control of a UAS site to which the GAL4 protein binds, and kanamycin resistance as a selectable marker. B) A modified version of the above enhancer-trapping construct,, in which GUS serves as an additional reporter to improve detection of weaker expression and basta resistance is used as the selectable marker. C) A constitutive GAL4 source. D & E) Two GAL4 response constructs. When transformed into the background of the constitutive GAL4 source these constructs drive misexpression of an adjacent gene. These constructs utilize hygromycin resistance as a selectable marker (distinct from basta resistance in the GAL4 source). F-I) An alternative transactivation system, designed and tested by Ian Moore (Oxford, UK). F) A constitutive inducible transactivation source. The chimeric transactivation protein (LhG4GR) comprises the GAL4 transcriptional activation domain fused to the modified DNA binding domain of LacZ. The site recognised by this binding domain is believed to be less susceptible to silencing problems encountered by some researches using the GAL4-UAS system. The transactivator protein is fused to GR permit inducibility. G) A responder construct for the LhG4 system containing a repeat of six operators recognised by the transactivator protein driving transcription of a GUS reporter on one side and any gene placed into the multiple cloning site on the other. H & I) The misepression constructs for the LhG4 system. In the constitutive inducible LhG4 background binding of the transactivator could drive transcription of adjacent genes.
