Question

Assignment:

1) Describe the principle of the yeast-two hybrid system

2) A positive result for the yeast two-hybrid analysis means:

A. two fusion proteins, one containing the Gal4p binding site domain and one containing the Gal4p activation domain, have interacted through their Gal4p domains resulting in transcription of the reporter gene.

B. two fusion proteins, one containing the Gal4p binding site domain and one containing the Gal4p activation domain, have bound together, which disrupts the ability of the reporter gene to make a blue colony.

C. two fusion proteins interact with each other and because the Gal4p binding site domain on one fusion protein binds to the reporter gene and the Gal4p activation domain of the other fusion protein interacts with RNA polymerase, the reporter gene is activated.

D. two fusion proteins interact with each other and because of the Gal4p binding site domain and the Gal4p activation domain contained in each fusion protein, the reporter genes are activated and the cells die, resulting in no colonies. E. the colonies growing are expressing the two fusion proteins and the two proteins have functioned independently.

3) What could the reporter gene encode and how would you detect reporter gene expression?

Answer 1

A).Yesat to hybrid screen detects protein-protein or protein-DNA interaction. The method is based upon the principle that if two proteins ( which we want to know whether interact or not) interact with each other, a functional transcription factor will regenrate. This experiments are carried out on genetically yeast strains which have been modified genetically. The expression of transcription factor is monitores by a reported gene. So we take two proteins, and with one protein the DNA binding domain of a transcription factor is fused, this is called bait. The activation part of the ranscription factor is then bind with the second protein of interest and it is called prey. If these two constructs interacts they will produce the functional transcription factor.

2) A positive result for the yeast two-hybrid analysis means:

A. two fusion proteins, one containing the Gal4p binding site domain and one containing the Gal4p activation domain, have interacted through their Gal4p domains resulting in transcription of the reporter gene.

3) Generally the transcriptional activation of reporter genes give a phenotype. For an e.g the yeast might need to grow on a special selective medium or the yeast colonies might change colour. For instance, if HIS3 is used as reporter gene, which is responsible for histidine synthesis. If HIS3 is active, then yeast can grow on a medium without histidine.