13th Oct

Question 1

Which genes are regulated by Gal4?

Answer 1

Gal 1, Gal2, Gal 7, Gal 10 and MEL1

Question 2

How can Gal 4 activity be easily regulated?

Answer 2

By altering the galactose concentration in the medium

Question 3

What is the bait plasmid?

Answer 3

Gal4 DNA binding domain fused with gene X

Question 4

What is encoded on the prey plasmid?

Answer 4

Gal4 activation domain is fused with gene Y

Question 5

What are the two main ways of creating prey plasmids?

Answer 5

A Genome Library

A cDNA library

Question 6

How do you make a genome library?

Answer 6

1. Cleave human ds DNA w/ restriction nuclease
2. Insert DNA fragmetns into plasmids
3. Introduce plasmids to bacteris

Question 7

What are the disadvantages of a genome library?

Answer 7

Sheer size - would require >1000 plates

500kb probably is only part of a human gene

Question 8

How do you make a cDNA library?

Answer 8

1. Purify mRNA
2. Hybridise with poly T primer
3. Make complementary DNA with reverse transcriptase
4. Degrade RNA with RNase H
5. Synthesize a 2nd cDNA strand using DNAP

Question 9

What is the main disadvantage using cDNA?

Answer 9

Don’t know how many we need to make to cover the whole genome, due to varying mRNA expression levels

Question 10

What are the 2 main forms of screening Y2H?

Answer 10

LacZ

Auxotrophs

Question 11

What are the benefits of an auxotroph screen over lacZ?

Answer 11

It is much quicker and more efficient to identify successful clones

Question 12

What are the benefits of lacZ over an auxotroph?

Answer 12

Lacz in liquid can be quantatively measures, auxotroph screening can not

Question 13

What are the common problems of Y2H screens?

Answer 13

X could act as the Gal4 activating domain
Y can act as the gal4 DBD
Fusion to Fal4 may disturb the X-Y protein interaction

Question 14

How can you exclude that X is acting as the Gal4 AD?

Answer 14

By using only part of the X protein

Perform a screen without adding Y-AD, to see if self activation occurs frequently

Question 15

How can you exclude that Y is acting as the Gal4 DBD?

Answer 15

Gal4 can use a variety of upstream activator sequences, which have little consensus, therefore you can perform a screen using several different UAS with different reporter genes to see if the effect is universal or unique to one UAS

Question 16

What is the most effective was to select for interacting domains in a Y2H screen?

Answer 16

1st and 2nd screens should be auxotrophic i.e. HIS3 or ADE2
The plasmids should then be isolated from yeast cells, re-transformed and then confrim that both plasmids are required for HIS3+ of ADE2+ phenotype.
Then 3rd assay is the lacZ liquid assay

Question 17

What are the intrinsic problems of Y2H screening?

Answer 17

Difficult to identify false negatives
Can only use nuclear localised proteins
Can’t look at interactions between transcriptional regulators

Question 18

What is the split ubiquitin system?

Answer 18

Uses split Ub rather than Gal4, otherwise same concept as a Y2H screen

Ub us split into 2 halves, the N-terminal domain (Nub) and the C-terminal domain (Cub)

Cub is bound to a reporter TF

When Nub and Cub come together the reporter is released by Ub specific proteases. The free TF then enters the nucleus andactivates transcription of the reporter genes

Question 19

What are the key benefits of the split ubiquitin system over Y2H?

Answer 19

Can use TFs

Can use non-nuclear localised proteins