Studying gene expression

Question 1

What is acridine orange?

Answer 1

A fluorescent dye used in cell cycle determination.

Question 2

What is Bismarck brown?

Answer 2

Stains acid mucins (mucus) yellow.

Question 3

What is Carmine?

Answer 3

Intensely red dye used to stain glycogen.

Question 4

What is Carmine alum?

Answer 4

A nuclear stain.

Question 5

Carmineen stains require a mordant. What is a mordant?

Answer 5

A fixing agent.

Question 6

What is Coomassie Blue?

Answer 6

A brilliant blue dye that stains proteins.

Question 7

What is crystal violet?

Answer 7

Stains cell walls purple.

Question 8

What does crystal violet require?

Answer 8

A mordant.

Question 9

What is DAPI?

Answer 9

A fluorescent nuclear stain.

Question 10

What conditions are needed for the use of DAPI?

Answer 10

Ultraviolet light, causing it to fluoresce when bound to DNA. It does not react under visible light.

Question 11

What kind of DNA regions does DAPI prefer?

Answer 11

AT-rich.

Question 12

What is eosin?

Answer 12

It stains cytoplasm, cell membranes and some extracellular structures pink.

Question 13

What colour does eosin stain red blood cells?

Answer 13

Red.

Question 14

Eosin is often used as a counterstain to what?

Answer 14

Haematoxylin derivatives.

Question 15

What is the central dogma of molecular biology?

Answer 15

DNA makes RNA makes protein,

Question 16

What evidence is there if a gene is being expressed in a particular cell?

Answer 16

The relevant RNAs will be present, plus polypeptide products.

Question 17

How can we detect mutants? Give 2 methods.

Answer 17

1. By phenotype

2. Using antibodies in IHC

Question 18

Explain how immunohistochemistry (IHC) works?

Answer 18

1. Use ABs complementary to particular antigen
2. ABs bind to antigen
3. Unbound ABs washed off
4. ABs have been conjugated with indicator molecules for recognition, e.g. dyes OR secondary ABs with recognition molecules are applied to bind to the first ABs
5. Certain molecules intensifying this effect

Question 19

How does IHC identify mutants?

Answer 19

ABs can be created to bind to particular gene products: if they become fixed or not will tell you whether the gene is being expressed.

Question 20

What is horseradish peroxidase (HRP)?

Answer 20

An alkaline phosphatase enzyme that is used as an indictor molecule. When DAB (diaminobenzidine) is applied a coloured precipitate is formed that binds to the surrounding tissue.

Question 21

Define a fluorophore.

Answer 21

A fluorescent chemical compound that re-emits light upon excitation.

Question 22

How do different fluorophores produce different colours?

Answer 22

The absorb and re-emit various wavelengths of light.

Question 23

What produces phalloidin?

Answer 23

The death cap mushroom.

Question 24

What does phalloidin do?

Answer 24

Prevents actin depolymerisation. Phalloidin is highly toxic.

Question 25

How can phalloidin be used to identify muscle?

Answer 25

Polymerised actin is a component of muscles. Phalloidin prevents its depolymerisation.

Question 26

What method is used to detect mRNA and thus genes that are being actively transcribed?

Answer 26

In-situ hybridisation (ISH).

Question 27

How does ISH work?

Answer 27

It uses labelled complementary mRNA/DNA strands that bind to mRNA in cells. N.B. you have to wash off unbound probes.

Question 28

Define a hapten.

Answer 28

A small molecule recognised by an antibody (particularly when bound to a larger molecule).

Question 29

Give 3 examples of haptens.

Answer 29

1. Biotin 2. Fluorescin 3. Digoxygenin

Question 30

How can haptens be used in ISH?

Answer 30

1. Haptens are added to the probes 2. Enzyme-conjugated AB (AB with an enzyme stuck to it) binds to the hapten 3. Enzyme catalyses the production of a coloured precipitate

Question 31

What does WMISH stand for?

Answer 31

Multiplex Whole Mount In-Situ Hybridisation.

Question 32

What is WMISH?

Answer 32

Where a whole embryo is used, not just a section of tissue etc.

Question 33

What is a reporter gene?

Answer 33

A gene that is inserted into an organism, usually next to a regulatory sequence. It is expressed when genes are switched on or can be used to show whether a certain gene has been uptaken, e.g. in transformation experiments. Basically a marker gene.

Question 34

What is 'fruitless'?

Answer 34

A gene expressed in the brain of drosophila. Expression has been found to differ between males and females.

Question 35

We can stain cells and gene products to see where they end up. True or false?

Answer 35

True.

Question 36

What is the difference between a) fluorophores and b) fluorescent proteins?

Answer 36

a) Are chemicals that are applied | b) Are proteins that produce fluorescence