Lecture 4.2 (reporter gene assay)

Question 1

Give an example of a promoter that contains a single transcription binding site.

Answer 1

cAMP response element (CRE)

Question 2

What kind of promoters reduce the potential interference seen with less selective promoters?

Answer 2

Promoters that contain a single transcription binding site

Question 3

What is the advantage of using promoters that contain a single transcription binding site?

Answer 3

reduce the potential interference seen with the less selective promoters

Question 4

How to overcome the problem of ‘promiscuity’ of endogenous promoters?

Answer 4

to choose a promoter that does not bind transcription factors native to the cellular system being studied

e.g. yeast Gal4 system

Question 5

Give an example of a promoter gene that does not bind to transcription factors native to cellular systems being studied

Answer 5

yeast Gal4 system

Question 6

The utilisation of yeast Gal4 system as a reporter gene assay requires the transfection of:

Answer 6

• Gal4 transcription factor (DNA binding domain coupled to regulatory or binding domain)

-reporter gene whose expression is regulated by Gal4 response elements

Question 7

Gal4 is a Zn2Cys6 binuclear cluster containing transcription factor that
binds DNA as a _________ .

Answer 7

homodimer

Question 8

What should be considered when choosing a reporter gene?

Answer 8

the product has unique
enzymatic activity or easily identified from the mixture of intra- or extracellular proteins.

Question 9

What is vital to the success of a reporter gene assay system?

Answer 9

the sensitivity of the detection procedure

Question 10

For reporter gene assay system, what may the detection method be based on?

Answer 10

photometry, colorimetry, radiometry, fluorescence or immunoassay

bioluminescence and chemiluminescence in recent years

Question 11

What are the upsides of using bioluminescence and chemiluminescence as detection systems for reporter gene assay system?

Answer 11

higher sensitivity and ease of detection

Question 12

Two types of reporter gene are commercially available:

Answer 12

1. Intracellular reporter genes
2. Extracellular reporter genes

Question 13

What is the full form of CAT?

Answer 13

Chloramphenicol O-acetyltransferase

Question 14

What is CAT?

Answer 14

a bacterial enzyme

Question 15

What is the reaction catalysed by CAT?

Answer 15

Acetyl-CoA + chloramphenicol = CoA + chloramphenicol 3-acetate

Question 16

The CAT gene protein product is easily assayed and detected even at __________ levels

Answer 16

attomole (10-18)

Question 17

What are the detection options using CAT?

Answer 17

1. Radiolabelled [14C]chloramphenicol and acetyl-CoA
   –> separate using TLC –> autoradiography or perform liquid scintillation counting
2. use [3H]acetate or [3H]acetyl-CoA
3. using fluorescent BODIPY 1-deoxychloramphenicol substrates

Question 18

Explain the principle of autoradiography

Answer 18

exposing photographic film or a photosensitive imaging plate to sample containing radioactive material

–> emitted radiation create image on film/plate

Question 19

Explain the principle of liquid scintillation

Answer 19

detection scintillation light produced when beta particles emitted by radioactive isotopes interact with a liquid scintillator

Question 20

What is a scintillator?

Answer 20

scintillator is a cocktail containing molecules that emit light when excited by the beta particles

Question 21

What is the downside of TLC?

Answer 21

development of the signal can take several hours

Question 22

using TLC for CAT assay, compare the distance travelled by unacetylated chloromphenicol, mono-acetylated chlorophenicol and di-acetylated chloramphenicol?

Answer 22

di-acetylated
chloramphenicol&raquo_space; mono-acetylated chlorophenicol&raquo_space; unacetylated chloromphenicol

Question 23

Why is florescent substrate a popular choice for CAT assay as opposed to using radiolabelled C or H?

Answer 23

simplified handling procedures and higher speed of sample processing

Question 24

What is the main disadvantage of CAT assays when scaling up to high throughput formats?

Answer 24

lack of simple separation and detection methods

Question 25

What gene is beta-galactosidase a product of?

Answer 25

bacterial lacZ gene

Question 26

What is the drawback of using beta-galactosidiase for assay development?

Answer 26

it is an enzyme endogenous to most mammalian cell lines --> experiments to distinguish between exogenous and endogenous enzyme

Question 27

Deficiencies in beta-ga;actosidiase can lead to which disease?

Answer 27

galactosialidosis or Morquio B syndrome

Question 28

What is Morquio B syndrome?

Answer 28

inherited autosomal recessive mucopolysaccharide storage disease --> leads to dwarfism

Question 29

What is the substrate for beta-galactosidase?

Answer 29

1. ONPG (o-nitrophenyl-beta-D-galactopyranoside) for colorimetric assay 2. phenylgalactose-substituted dioxetane or AMPGD for chemiluminescence detection 3. firefly luciferin beta-D-galactopyranoside substrate for bioluminescence

Question 30

What is ONPG converted to by the action of beta-galactosidase?

Answer 30

ONP anion --> can be quantified colorimetrically

Question 31

What substrate for beta-galatosidase shall be used if the assay is to be adapted to chemiluminescence detection? What advantage does it serve over ONPG colorimetric assay?

Answer 31

phenylgalactose-substituted dioxetane --> 20-fold improvement in sensitivity over the ONPG colorimetric assay.

Question 32

For detection using beta-galactosidase assay, what is the advantage of using AMPGD over ONPG or phenylgalactose-substituted dioxetane?

Answer 32

50,000-fold more sensitive than the ONPG --> able to detect as little as 2 fg of beta-galactosidase

Question 33

What is luciferin beta-D-galactopyranoside substrate converted into under the action of beta-galactosidase?

Answer 33

firefly luciferin --> measured using firefly luciferase assay

Question 34

What is Aequorin?

Answer 34

photoprotein isolated from jellyfish Aequorea Victoria

Question 35

What are the 3 components of Aequorin?

Answer 35

1. apoaequorin 2. coelenterazine chromophore 3. oxygen

Question 36

What is the detection method of using Aequorin based reporter gene based on?

Answer 36

In presence of calcium ions, the photoprotein complex emits a flash of blue luminescence (max wavelength 469nm)

Question 37

What are advantages to using an aequorin based reporter gene assay?

Answer 37

high sensitivity and low backgrounds

Question 38

What is the downside of using an aequorin based reporter gene assay?

Answer 38

1. requires the exogenous application of coelenterazine 2. bioluminescent flash triggered by Ca2+ lasts less than 5 seconds --> use luminometer with ability to initiate and rapidly detect the reaction

Question 39

Aequorin stably expressed in the cells detects the increased [Ca2+]i and emits luminescence. True or False

Answer 39

true