Cell fractionation application

Question 1

The scientists treated cells in a culture with small interfering RNA (siRNA). This siRNA causes RNA interference of expression of the CENP-W gene. The scientists took samples of cells at 0, 48 and 72 hours after adding the siRNA. They then used gel electrophoresis to separate CENP-W and tubulin from these samples.

The diagram shows the results of the electrophoresis. The size of each band is proportional to the amount of CENP-W or tubulin present.

Suggest how the siRNA produced these results.

Answer 1

siRNA binds to / destroys mRNA for CENP-W;

Reject if siRNA binds to gene / DNA

Context is important, siRNA acts on mRNA for CENP-W, not tubulin.

Ref. to CENP-W required once for MP1 and MP2

2. Prevents translation of CENP-W;

Context is important, siRNA acts on mRNA for CENP-W, not tubulin.

Ref. to CENP-W required once for MP1 and MP2

Accept reduces translation of CENP-W

3. (After / as) CENP-W reduces so does tubulin production;

Question 2

Explain how ultracentrifugation separates CENP-W from other molecules

Answer 2

Spin (liquid / supernatant) at (very) high speed / high g;

Need context of high, not just ‘faster’, ‘higher’ in context of use of bench centrifuge

Accept high centrifugal force

Award 1 max if ref. to organelle separation

Question 3

Describe and explain how centrifuging the culture allowed the scientists to obtain a cell-free liquid.

Answer 3

Large / dense / heavy cells;

2. Form pellet / move to bottom of tube (when centrifuged);
3. Liquid / supernatant can be removed.

Question 4

Some of the catalase produced by Aspergillus niger is intracellular and some is extracellular.

Intracellular enzymes stay inside the cells that produce them. Extracellular enzymes are secreted from the cells that produce them.

Another group of scientists grew a different strain of A. niger.

• A. niger grows from tiny structures called spores. The scientists kept the spores in an isotonic medium at a low temperature until they needed them.
• They put spores of A. niger into a 500 cm3 flask containing a sterile medium. The medium contained starch.
• They measured the total amount of catalase and the amount of extracellular catalase produced by the fungus over a period of 100 hours.

The graph shows their results.

(a) (i) The scientists kept the spores in an isotonic medium until they were needed.
Suggest why it was important that the medium was isotonic.

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(2)

(ii) The scientists kept the spores at a low temperature until they were needed.
Suggest why.

Answer 4

1. Water potential same (inside and outside) / no
   water potential gradient;

Accept symbol Ψ or abbreviation WP as alternatives to water potential.

2. Water does not enter / leave spores;
3. By osmosis / prevents osmotic damage;

Answer must refer to osmosis.

2 max

(ii) Prevents growth (before ready) / stops growth of (other) microorganisms / slows enzyme action / prevents enzymes being denatured;

Question 5

To get reliable results in this investigation, the medium must be sterile.
Explain why.

Answer 5

Prevents contamination by (other) microorganisms;

Accept alternatives such as microbes, bacteria, other fungi.

2. Which also produce the enzyme / catalase / which would produce substances that affect catalase;

Question 6

(e) Technologists prefer to manufacture extracellular enzymes rather than intracellular enzymes. This is because intracellular enzymes are more expensive to purify than extracellular enzymes. Suggest why intracellular enzymes are more expensive to purify.

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Answer 6

Extra steps (with intracellular enzymes);

2. Cells have to be broken open;
3. Cell walls / bits of cells have to be removed / separated from enzyme;
4. Needs to be separated from all the other enzymes in the cell;