Unit 2 KA7

Question 1

Mutagenesis

Answer 1

The creation of mutations by deliberately exposing an organism to mutagenic agents such as UV light, other types of radiation or chemicals, which alter their DNA.

Question 2

Wildtype

Answer 2

The original strain of the micro-organism.

Mutants can revert to wildtype by repairing mutations in their DNA, so improved strains used in industry need constant monitoring.

Question 3

Mutant strain

Answer 3

An improved strain of a micro-organism created by mutagenesis.

It may lack a key enzyme, which may stop or divert a metabolic pathway, leading to accumulation of a desired product.

Mutant strains tend to be unstable and can revert to wildtype.

Question 4

Recombinant DNA technology

Answer 4

Genetic engineering.

A selected gene can be transferred into a host organism using a vector such as a bacterial plasmid or artificial chromosome.

The host organism is then able to copy and express the new gene, producing the desired protein.

Question 5

Recombinant DNA

Answer 5

Host DNA and donor DNA combined together.

eg. a bacterial plasmid with a human gene inserted into it.

Question 6

Restriction endonuclease.

Answer 6

DNA cutting enzymes, used to cut genes out of donor DNA and used to cut plasmids open.

There are many different types eg. EcoR1 (named after E.coli where it is found)

Each type recognises and cuts at a different specific DNA sequence.

Cuts may be straight or staggered, creating ‘sticky ends’

Question 7

DNA ligase

Answer 7

An enzyme which is used to join fragments of DNA together. Seals the donor gene into the plasmid.

Question 8

Vector

Answer 8

A bacterial plasmid or artificial chromosome.

Used to carry the donor gene into the host organism.

Question 9

Artificial chromosomes.

Answer 9

Constructed by scientists.

Have the same features as a plasmid vector but can carry much longer DNA sequences into the host organism.

Question 10

Features of vectors

Answer 10

Origin of replication

Marker gene

A restriction site

Question 11

Restriction site

Answer 11

Contains target (recognition) sequences for specific restriction endonucleases.

The same restriction endonuclease must be used to cut the donor DNA and the plasmid, so that the sticky ends are complementary and the 2 pieces of DNA can be joined together.

Question 12

Marker gene

Answer 12

Allows scientists to select only the cells that have taken up the recombinant plasmids, to avoid wasting resources.

eg. Ampicillin (antibiotic) resistance gene.

When the culture is treated with an antibiotic, only the cells containing the plasmid survive, as they have obtained the resistance gene along with the required donor gene.

Cells which don’t have the plasmid are destroyed.

Question 13

Origin of replication

Answer 13

Essential for the replication of the recombinant plasmid in the transformed host cell.

Contains genes that control the self replication of the plasmid or artificial chromosome.

Also contains regulatory sequences that control expression of existing and inserted genes.

Question 14

Limitations of prokaryotes

Answer 14

Prokaryote DNA does not contain introns, so bacterial cells cannot splice eukaryotic DNA (which does contain introns).

Eukaryotic polypeptides (such as insulin) may not be cut or folded correctly after they have been translated, as prokaryotes lack the machinery to do this.

Question 15

Recombinant yeast

Answer 15

Yeast contains plasmids which can be modified using recombinant DNA technology.

Yeast is harder to grow than bacteria, but it is eukaryotic and so is able to carry out RNA splicing and modification of polypeptides.

Question 16

Handling micro-organisms

Answer 16

Ethical considerations and safety need to be taken into account when altering micro-organisms.

Additional genes may be added to the plasmid as a safety mechanism to prevent the GM organism surviving outside the lab.

Aseptic technique should always be used, and additional containment steps may be needed to prevent escapes.