Studying gene expression Flashcards
What is acridine orange?
A fluorescent dye used in cell cycle determination.
What is Bismarck brown?
Stains acid mucins (mucus) yellow.
What is Carmine?
Intensely red dye used to stain glycogen.
What is Carmine alum?
A nuclear stain.
Carmineen stains require a mordant. What is a mordant?
A fixing agent.
What is Coomassie Blue?
A brilliant blue dye that stains proteins.
What is crystal violet?
Stains cell walls purple.
What does crystal violet require?
A mordant.
What is DAPI?
A fluorescent nuclear stain.
What conditions are needed for the use of DAPI?
Ultraviolet light, causing it to fluoresce when bound to DNA. It does not react under visible light.
What kind of DNA regions does DAPI prefer?
AT-rich.
What is eosin?
It stains cytoplasm, cell membranes and some extracellular structures pink.
What colour does eosin stain red blood cells?
Red.
Eosin is often used as a counterstain to what?
Haematoxylin derivatives.
What is the central dogma of molecular biology?
DNA makes RNA makes protein,
What evidence is there if a gene is being expressed in a particular cell?
The relevant RNAs will be present, plus polypeptide products.
How can we detect mutants? Give 2 methods.
- By phenotype
2. Using antibodies in IHC
Explain how immunohistochemistry (IHC) works?
- Use ABs complementary to particular antigen
- ABs bind to antigen
- Unbound ABs washed off
- 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
- Certain molecules intensifying this effect
How does IHC identify mutants?
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.
What is horseradish peroxidase (HRP)?
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.
Define a fluorophore.
A fluorescent chemical compound that re-emits light upon excitation.
How do different fluorophores produce different colours?
The absorb and re-emit various wavelengths of light.
What produces phalloidin?
The death cap mushroom.
What does phalloidin do?
Prevents actin depolymerisation. Phalloidin is highly toxic.
How can phalloidin be used to identify muscle?
Polymerised actin is a component of muscles. Phalloidin prevents its depolymerisation.
What method is used to detect mRNA and thus genes that are being actively transcribed?
In-situ hybridisation (ISH).
How does ISH work?
It uses labelled complementary mRNA/DNA strands that bind to mRNA in cells.
N.B. you have to wash off unbound probes.
Define a hapten.
A small molecule recognised by an antibody (particularly when bound to a larger molecule).
Give 3 examples of haptens.
- Biotin
- Fluorescin
- Digoxygenin
How can haptens be used in ISH?
- Haptens are added to the probes
- Enzyme-conjugated AB (AB with an enzyme stuck to it) binds to the hapten
- Enzyme catalyses the production of a coloured precipitate
What does WMISH stand for?
Multiplex Whole Mount In-Situ Hybridisation.
What is WMISH?
Where a whole embryo is used, not just a section of tissue etc.
What is a reporter gene?
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.
What is ‘fruitless’?
A gene expressed in the brain of drosophila. Expression has been found to differ between males and females.
We can stain cells and gene products to see where they end up. True or false?
True.
What is the difference between a) fluorophores and b) fluorescent proteins?
a) Are chemicals that are applied
b) Are proteins that produce fluorescence
