Transcription factor vector

Another important requirement for the chosen bait protein is that it should not yield autoactivation of transcription factor activity. This may occur in the GAL4 and LexA systems if the bait actually is a transcription factor or if the bait mimics the transcription factor activity. Autoactivation is evident if the DNA-BD bait construct is cotransformed into competent yeast with an empty AD vector and the resultant transformants show reporter gene enzyme activities and growth on SD media. Obviously, this needs to be avoided because cDNA library screening with such a bait will result in the detection of many false positives. 

Fig. 7.1. A planarian individual of a line transformed with a Hermes transposon-derived vector 12 months after microinjection and subsequent electroporation. Besides the transposon part, the vector contained the E(enhanced)GFP reporter gene under the control of an artificial promoter that responds to the transcription factor Pax6 (Gonzalez-Estevez et ai, 2003). Binding sites for this transcription factor are found in rhodopsin and other photoreceptor-specific genes. Reporter-gene activity was found in the eyes (arrows) of transformed individuals or in regenerated heads as a mosaic of EGFP-positive photoreceptor cells. Figure courtesy of Dr E. Salo.

Fig. 4. Schematic representation of yeast one-hybrid transcription factor screening. C. roseus cDNAs were cloned in a fusion with the GAL4-activation domain (GAL4-AD) in yeast/E.coli shuttle vector pACTII. Yeast reporter strains carrying a tetramer of the cis-acting elements of interest (indicated in the figure with 4xbait) were transformed with the cDNA library and selected for transformation and reporter gene activation on medium lacking leucine and histidine, respectively...

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