Rodgers 2-26

Question 1

Main assay for finding DNA-protein interactions

Answer 1

ChIP- Chromatin Immunoprecipitation assay: 1) crosslink DNA and protein with formaldehyde 2) digest with micrococcal nucleases the parts of DNA not bound to the protein 3) sequence the DNA to find where proteins associate. Can use Ab’s to associate with protein modifiers

Question 2

ENCODE

Answer 2

database for DNA, RNA modifications

Question 3

What are the three classes of Histone modifying/modulating proteins

Answer 3

Writing, Editing, Reading

Question 4

What role do histone modifications play in cancer

Answer 4

The vast majority of the >700 histone modifiers known have been identified as mutated in cancer cells

Question 5

Are proteins recruited to histone modification sites individually or combinatorially ? What kind of dynamic influences this?

Answer 5

proteins are recruited based on combinatorial modifications on histones that alter proteins’ affinities. Many proteins compete for the same site.

Question 6

lysine acetylation (3)

Answer 6

chromatin decondensation: for transcription and replication

Question 7

methylation (3)

Answer 7

heterochromatin, protein binding, stabilizes nucleosome

Question 8

phosphorylation (3)

Answer 8

decondensation, DNA repair, replication,

Question 9

ADP ribosylation (2)

Answer 9

decondensation, DNA repair

Question 10

glycosylation (1)

Answer 10

transcriptional regulation?

Question 11

Is transformation usually an efficient process

Answer 11

No

Question 12

What does each colony of bacteria represent?

Answer 12

clones of a single bacterium

Question 13

Why are white colonies usually desired over blue?

Answer 13

because it means that the gene was taken up in the lacZ gene, deactivating its product Beta-galactosidase

Question 14

Why is OriC not used in vestors

Answer 14

Because it has too many safeguards against rapid replication. Also 2 plasmids with the same replication origin are not usually allowed in same cell.

Question 15

MCS

Answer 15

multi cloning site- polylinker/ many cleavage sites for RE’s

Question 16

What is the point of placing protein thyrodoxin (Trx) in the vector?

Answer 16

It is native to prokaryotes and increases the solubility of the protein inserted in the vector, since eukaryotic proteins can become “tofu” when grown in prokaryotes

Question 17

rbs

Answer 17

ribosomal binding site- placed before gene of choice in plasmid

Question 18

What is the point of His tags

Answer 18

They are great at coordinating divalent metal ions, the metal ions can bind to other things to precipitate the protein of interest on resin column

Question 19

What is the point of S tags

Answer 19

Binds the protein to S protein from an endonuclease that can create a color

Question 20

What protease cleavage sites are used? Why?

Answer 20

thrombin, enterokinase. To remove tags mad on the protein.

Question 21

What are affinity tag examples?

Answer 21

His tag, S tag

Question 22

Where are tags usually found on the vector?

Answer 22

After the RNA Pol binding site, but before the gene insertion at the MCS (occasionally after)