Gene Regulation

Question 1

What does the Wnt signalling pathway control?

Answer 1

Pluripotency, differentiation and proliferation.

Question 2

Give a brief description of how the Wnt signalling pathway operates.

Answer 2

• It is a paracrine signalling pathway (short distance).
• There is continuous turnover of beta catenin in the cytosol.
• When Wnt protein is free to bind to receptor (not bound to another complex) it binds causing downstream signalling that prevents the phosphorylation and subsequent ubiquination of beta catenin.
• Beta catenin can enter the nucleus and bind to TCF transcription factor.

Question 2

What are epigenetics?

Answer 2

Modifications of DNA that do not affect base sequence (primary structure). Predominantly DNA methylation and histone modifications.

Question 3

What is the role of epigenetics in ‘normal’ cells?

Answer 3

• Compartmentalising the genome into active and repressed regions, regulating gene expression.
• It is reversible.

Question 4

How does DNA methylation repress transcription?

Answer 4

Modifies chromatin to more condensed form so that transcriptional factor cannot access binding sites.

Question 5

What effect does acetylation of histone proteins have on transcription?

Answer 5

More transcription as it causes chromatin to take its more open form.

Question 6

What do DNA Methyltransferases (DNMTs) do? Which 2 types do I need to know?

Answer 6

Catalyse addition of methyl group to cytosine bases. Reversible reaction.
- DNMT1 - maintenance DNMT copies methylation pattern to other strand of DNA.
- De Novo DNMTs - establish methylation patterns during embryonic development.

Question 7

What is the consequence of methylation of lysine 9 and 27 in histone tails?

Answer 7

Formation of heterochromatin (condensed chromatin, blocking transcription factors - gene silencing).

Question 8

What is the consequence of methylation of lysine 4 and acetylation of lysine 9 in histone tails?

Answer 8

Gene expression (chromatin more open and active).

Question 9

How might epigenetic modifications of tumour supressors genes leads to cancer?

Answer 9

Many cancers caused by hyper-methylation of these genes and homozygous deletion. This can repress expression of these proteins.

Question 10

What links have been found between hypermethylation and mutations?

Answer 10

• Can lead to silencing DNA repair genes.
• Spontaneous deamination (methylated C can mutate to T).
• Methylated CpGs can absorb more UV (leads to CC to TT mutations).
• Methylated CpGs have enhanced carcinogen binding (increase in G to T mutations).

Question 11

There are 8

What are the requirements for cell culture?

Answer 11

1. Tissue Sample
2. Method to digest tissue (e.g. trypsin is a protease which breaks cell: cell contacts).
3. Sterile conditions
4. Tissue culture medium: growth liquid containing amino acids, vitamins, glucose (energy source).
5. Blood serum containing growth factors.
6. Antibiotics
7. Surface to grow cells on: normal cells (e.g. Keratinocytes in skin) require attachment using agents such as collagen, fibronectin.
8. Controlled environmental conditions.

Question 12

Give 8 clinical uses of cell culture.

Answer 12

• Skin grafting (must contain stem cells below basement membrane).
• Amniocentesis
• Carcinogen testing
• IVF
• Cloning
• Expanding of biological material (e.g. proliferation of small tumour for further analysis).
• Testing new anti-cancer drugs
• Chromosome analysis

Question 13

What is amniocetesis?

Answer 13

Checking of developing foetus for genetic abnormalities. Amniotic fluid taken from pregnant mother containing a few foetal cells which can be cultured and analysed.

Question 14

How can cell culture be used for chromosome analysis (in more detail)?

Answer 14

• Colchicine is used (drug inhibits the mitotic spindle leading to increased cells arrested in mitosis for chromosome analysis).
• Visualise chromosomes under microscope by staining with Giemsa (stain DNA blue/purple).