Two-Hybrid and Clontech

[wvcrieki at lmb1.rug.ac.be]

Dear ...,

In page 12 of the protocol (of the supplement Kit) there is marked that pAS2 
contains a wild-type ADH1 promotor driving the expression of yeast. This 
promotor is longer and stronger than the used in pGBT9. Introduction of pAS2 
alone into yeast reporter strain may result in slight activation of the 
reporter genes. This low level of reporter gene activation may (or may not) 
decrease below detectable (!?) levels when fusion proteins are cloned into the 
MCS. 

- Is there a molecular reason for activating reporter genes with only the 
DNA-binding domain ? 
- What is the molecular significance of adding 3-AT to the medium (where does 
it act upon) ? 
- Is there an influence of 3-AT on both reporter genes (lacZ and  HIS) ?   
- Do both reporter genes result in an equal amount of false positives ? And if 
not is there a molecular explenation ?
- Is there someone who has more information on the map of pAS2 ? 

Thanks for you time