Lecture 6

Question 1

What is an operon?

Answer 1

In bacteria and archaea genes are transcribed from a single promoter, have a linked function and produce a polycistronic mRNA

Question 2

What are repressors?

Answer 2

regulatory proteins that prevent transcription when bound to DNA

Question 3

What are activators?

Answer 3

regulatory proteins that activate transcription when bound to DNA

Question 4

What is an inducer?

Answer 4

activates activators or inactivates repressors

Question 5

What is a co-repressor

Answer 5

activate repressors or inactivate activators

Question 6

global regulation

Answer 6

multiple regulatory factors are swtiched on/off at the same time e.g. SOS response.

Question 7

regulon

Answer 7

a set of operons gontrolled by a single regulatory protein e.g PHO regulon

Question 8

2 mechanisms that control sugar metabolism

Answer 8

catabolite repression

induction by lactose

Question 9

what does induction of the lac operon require and why?

Answer 9

inactivation of the lac repressor bc it sts on the operator which stops expression of the gene
activation of CAP in order to remove catabolite repression

Question 10

What does repressor inactivation require

Answer 10

presence of lactose

Question 11

what does CAP activation require

Answer 11

absence of glucose

Question 12

what is a trans-acting element

Answer 12

diffuse away from the site they are encoding

Question 13

what is a cis-acting element

Answer 13

Other genes did not produce a product BUT regulated genes on the DNA molecule they were encoded upon

Question 14

cloning

Answer 14

The insertion of a DNA fragment into a self-replicating genetic element to:
Make many copies of the same piece of DNA
Isolate a particular piece of DNA from the rest of a cell’s genome for analysis or exploitation

Question 15

what is fosmid/cosmid

Answer 15

Large plasmids based on the F plasmid with cos-sites, lacZ and a T7 promoter added
The cos-sites allow the vector to be packaged into λ phage particles for transfection into E. coli

Question 16

Ti plasmid

Answer 16

The Ti plasmid is found in the plant pathogen Agrobacterium tumefaciens and causes tumours (crown galls) on plants

Question 17

BACs

Answer 17

can carry up to 300 kbp of cloned DNA

Based on the F plasmid they often carry selectable markers and the lacZ gene

Question 18

YACs

Answer 18

based on real yeast chromosomes and in order to replicate they must contain origins of replication, a centromere and telomeres

Question 19

What do YACs produce?

Answer 19

This YAC includes 2 selectable and one screenable marker.
The YAC is transfected into a mutant yeast cell line that is auxotrophic for tryptophan (trp1-) and uracil (ura3-) synthesis
The YAC encodes trp1+ on one arm and ura3+ on the other arm
For the transfected cell to grow the YAC MUST must contain both arms

Question 20

Where is the cloning site on a YAC?

Answer 20

within the sup4 gene. used for red/white screening.

can contain up to 600kbp inserts, new mega-YACs can carry up to 1.5 Mbp inserts.