Cell and Molecular Biology

Question 1

What are the general properties of prokaryotes?

Answer 1

1 - 2 µm

No nucleus

Have a single, circular chromosome

Divide by binary fission

Internal membranes are rare

70S ribosome (50S + 30S)

No cytoskeleton

Move by a rotary motor driving bacterial flagellum

Question 2

What are the general properties of eukaryotes?

Answer 2

5 - 100 µm

Nucleus is bounded by a nuclear envelope

DNA is in the form of multiple chromosomes that are associated with proteins

Divide by mitosis or meiosis

80S ribosomes (60S + 40S)

Cytoskeleton consists of microtubules and microfilaments

Move by dynein driving cilia and eukaryotic flagellum, also have myosin

Question 3

What is the cell cycle? Describe each stage.

Answer 3

► G0 → non-dividing cells rest

G1 → RNA and protein synthesis

S phase → DNA inside nucleus doubles

G2 → cell mass doubles

M phase → cell division by mitosis

S and M phases must only occur once per cell cycle and in the correct order. To ensure this, the phases are separated by gaps where everything is checked

Question 4

What is the difference between mitosis and meiosis?

Answer 4

Mitosis → produces 2 x identical daughter cells

The parent cell is 2n, the daughter cells are 2n

Meiosis → produces gametes

Meiosis 1 produces 2 x 2n daughter cells

Meiosis 2 produces 4 x n naughter cells

Question 5

What is the difference between ploidy, euploidy and aneuploidy?

Answer 5

Ploidy → the number of sets of chromosome in a cell

Euploidy → having the correct number of chromosome in the cell

Aneuploidy → having the incorrect number of chromosomes in the cell

Question 6

What is the function of the nucleolus, the nucleus, the SER and the RER?

Answer 6

Nucleolus → within the nucleus; has no membrane; site of RNA processing and ribosomal assembly

Nucleus → has a double membrane; contains the genome; site of mRNA and tRNA synthesis

SER → synthesise steroid hormones (in certain tissues); storage and release of Ca²⁺

RER → site of protein synthesis at ribosomes; ER is continuous with outer nuclear membrane

Question 7

What is the function of the plasma membrane, lysosomes and peroxisomes?

Answer 7

Plasma membrane → regulates movement of materials in and out of the cell; facilitates electrical and hormonal signalling between cells

Lysosomes → low pH; contain enzymes that digest cellular components

Peroxisomes → contain over 50 enzymes, including catalase which converts H₂O₂ into H₂O and O₂

Question 8

What is the function of mitochondria, the Golgi body, the cytoskeleton and chloroplasts?

Answer 8

Mitochondria → has a double membrane; has its own DNA; principal site of ATP synthesis

Golgi body → protein sorting, modification and transport

Cytoskeleton → forms networks of microtubules and microfilaments; supports membranes and organises organelles; important for cell movement

Chloroplasts → has a double membrane and own DNA; has thylakoids which are made up of grana; site of photosynthesis

Question 9

What is the advantage of plasma membranes being able to have different compositions?

Answer 9

Different compositions will enable the membrane to do different things

eg. more cholesterol in the membrane will make it less fluid, or can make the membrane thicker as the fatty acid tails are more stretched out
eg. some tails will have wider ends (due to double bonds) that enable the membrane to form corners

Question 10

How does the technique of centrifugation work?

Answer 10

1. Break open membrane with detergent and filter
2. Spin at 600g for 10 minutes → nuclei sinks to the bottom
3. Spin at 15,000g for 5 minutes → mitochondria, chloroplasts, lysosomes and peroxisomes sink to bottom
4. Spin at 100,000g for 1 hour → plasma membrane, ER fragments, large polyribosomes sink to bottom
5. Spin at 300,000g for 2 hours → ribosomal subunits, small polyribosomes sink to bottom
6. Remaining is the soluble part of the cytoplasm ie. the cytosol

After each spin, remove the centrifuged bits.

Density gradient centrifugation → put cells in a test tube which has an increasing density of sucrose from top to bottom and spin. This can be done for each of the portions removed in differential centrifugation (above).

eg. Lysosomes 1.12gcm^-3, mitochondria 1.18gcm^-3, peroxisomes 1.23gcm^-3.

Question 11

What is electrophysiology?

Answer 11

By using radioactive tracers to follow the ion flow in/out of the cell, the activity of an isolated ion channel can be determined.

Question 12

What is the advantage of using fluorescent or phase-contrast microscopy compared to light microscopy?

Answer 12

Fluorescent → easier to see organelles and other components this way, as different things can be stained with different fluorescent molecules that will be different colours

Phase-contrast → light travels at different speeds in regions with different compositions, so differences in the refractive index will produce differences in contrast. This gives the same more detail, so it is easier to see

Question 13

What is fluorescence activated cell sorting, FACS?

Answer 13

Cells are fluorescently marked, identified with a laser and then sorted

Alive and dead cells can be sorted - enable us to find out different percentages of a population

Question 14

What is flow cytometry analysis?

Answer 14

Enables us to measure the levels of various markers on a large number of cells.

Can use Ca²⁺ sensitive dyes, cAMP dyes, reactive oxygen species dyes, etc.

Question 15

What is fluorescence recovery after photobleaching?

Answer 15

This measures the mobility of proteins in cells.

Cells are inserted with the gene for green fluorescent protein (GFP). This means the cells will be fluorescently marked.

The cells are the partially bleached, and the time taken to regain fluorescence is measured.

Different proteins can be investigated using different colours.

Mutations to GFP will cause a change in the colour.

Question 16

What is the difference between a TEM and an SEM?

Answer 16

TEM → An electron gun fires a beam of electrons through the sample. The fluorescent screen emits light when struck by electrons. However, the beams are highly destructive.

SEM → produces a 3D image by firing a beam of electrons at the sample and detecting where they bounce off. The sample must be coated in resin and carbon to allow the electrons to bounce off, or it’s not dense enough.

The electron must be under vacuum otherwise air particles will diffract the electrons and slow them down.

Question 17

What are the differences between light, TEM and SEM microscopes?

Answer 17

Light TEM SEM

Resolution 200nm 1nm 10nm

Depth of focus Low Medium High

Field of view Low Medium High

Ease Easy Requires Skill Easy

Speed Rapid Slow ~Rapid

Cost Low High High

Question 18

What is genetic engineering and recombinant DNA?

Answer 18

Genetic engineering → direct manipulation of the genome of an organism using biotechnology

Recombinant DNA → combining 2 or more pieces of DNA that would not normally be found together, using artificial means rather than genetic recombination

Question 19

Why might bacteria or bacteriophages be used to clone recombinant DNA?

Answer 19

Bacteria grow exponentially, so the DNA will be replicated very quickly.

Bacteriophages, although they don’t replicate as quickly as bacteria, can be used to insert plasmids in the desired bacteria.

Question 20

How can a DNA fragment be cloned in bacteria?

Answer 20

1. Both the plasmid vector and DNA fragment are cut using the same restriction enzymes. The sticky ends of the DNA and plasmid can then anneal together using DNA ligase, which catalyses the formation of the phosphodiester bond between the nucleotides.
2. The plasmid is also inserted with a marker gene, such as antibiotic resistance or GFP, using restriction enzymes. This enables transformed cells to be identified.
3. The bacteria are then mixed with the plasmids in the presence of CaCl₂, which makes the cell walls more permeable, and so more likely to take up the plasmids. The cells could also be heat-pulsed or electrically shocked to give the same effect.
4. Bacterial culture is grown on nutrient agar plates containing the marker, so only transformed cells survive.
5. The transformed plasmids are then cultured so they replicate, producing a clone library.

Question 21

How does electrophoresis work?

Answer 21

1. DNA is cut into fragments using restriction enzymes. Fragments may be stained with a marker eg. fluorescence or radioactivity, enabling them to be detected.
2. Place DNA fragments in the well of an agarose or polyacrylamide gel, and cover with a buffer solution that can conduct electricity.
3. An electric field is applied, so because DNA is negatively charged, the fragments will move towards the positive electrode. They do so at a rate inversely proportional to their size.
4. Gel is then subject to autoradiography, UV detection or may be stained with a fluorescent dye at this stage.

Question 22

How does Southern blotting work?

Answer 22

1. DNA is isolated and is cut with restriction enzymes. The fragments are separated by electrophoresis and stained with ethidium bromide (fluorescent).
2. The gel is soaked in alkali to break apart the double helix. A nitrocellulose gel is placed on top of the agarose gel, and capillary action of the alkali transfers the DNA from the agarose to the nitrocellulose gel. This produces an exact replica of the DNA fragment pattern on the nitrocellulose gel.
3. Nitrocellulose is then incubated with a cloned DNA fragment, aka DNA probe, which is tagged with a label. The probe is heated beforehand to ensure that it is a single strand.
4. The probe will hybridise to its complementary sequence, and this is detected with UV or autoradiography.

Question 23

How does mammalian cell transfection work?

Answer 23

This requires a circular vector to get past the phospholipid membrane; a liposome can be used to get the vector inside the cell.

Transient transfection → the plasmid is left in the cytoplasm and is eventually removed due to an immune response. The protein is expressed via cDNA from the plasmid DNA.

The plasmid vectors are engineered to carry: an origin of replication derived from a virus that infects mammalian cells; a strong promoter recognised by mammalian RNA polymerase; the cloned cDNA encoding the protein to be expressed adjacent to the promoter.

Stable transfection → cDNA is integrated into the host DNA. The DNA can be integrated anywhere in the genome, so it may disrupt an important gene and cause disease.

To see where the protein is expressed a fluorescent marker is used.

Question 24

How can a protein’s function be proven using transgenic mice?

Answer 24

The gene for a protein is ‘knocked out’ and a marker gene is put in. The gene can be knocked out either by disrupting the base pair sequence or by deleting the entire gene.

The genome with the transgene is then inserted into a blastocyst, and all the normal genes will be replaced by the transgene via homologous recombination.

This method could be used to remove the gene for cancer or genetic diseases.

Question 25

What is somatic gene therapy and how does it work?

Answer 25

The correction or alleviation of a genetic disorder by the introduction of a normal gene copy into an affected individual via somatic cells.

Direct delivery → the transgene is packaged into a virus and administered. Where the gene is inserted into the genome cannot be controlled, but CRISPR can be used to give more precise targeting.

Cell-based delivery → the virus containing the transgene infects stem cells.

Every single body cell must be changed in order for the patient to be cured.

Question 26

How does the CRISPR-Cas9 system work?

Answer 26

It enables genes to be cut out of the genome.

It is found in archaea and bacteria and acts as an adaptive immune system against viral infection.

CRISPR → Clustered Regularly Interspersed Short Palindromic Repeats

After a bacterium has survived viral infection, the viral DNA is inserted in between a CRISPR repeat. After transcription of the CRISPR region, the mRNA is cut up into small fragments containing the CRISPR repeat and the viral fragment. The CRISPR region forms a complex with complementary transactivating CRISPR RNA (tracrRNA). This complex then binds to the enzyme Cas9.

When the bacterium is infected by the same virus, the inserted viral DNA will bind to the complementary crRNA, while the rest of the viral genome remains a double helix.

Cas9 then produces a double-strand break in the viral genome, after the complementary fragment. This means the viral DNA cannot take over the bacterium.