Cloning beyond E.coli

Question 1

What is yeast used for?

Answer 1

Brewing and baking, and biotechnology in pharmaceuticals.

Question 2

What is the naturally occuring yeast plasmid?

Answer 2

The 2um plasmid - one of very few plasmids found in eukaryotes.

Question 3

What is the copy number of the 2um plasmid?

Answer 3

70-200.

Question 4

What are some of the genes in the 2um plasmid?

Answer 4

REP1 and REP2 that are involved in replication as well as host enzymes. FLP flips the gene order, and the function of D is unknown.

Question 5

What is a problem with the 2um plasmid?

Answer 5

There is no selectable marker. You can’t use an antibiotic resistance gene as most antibiotics work against bacteria and not eukaryotes.

Question 6

What can be used as a selectable marker?

Answer 6

An auxotrophic mutant yeast host that has a mutant Leu2 gene - the mutant cannot synthesise leucine and must be grown on a medium containing leucine in order to grow.

Question 7

What does auxotrophic mean?

Answer 7

A mutant that must be given an extra nutrient in order to survive.

Question 8

How can the LEU2 gene be used to select transformed yeast cells?

Answer 8

If the 2um plasmid contains a LEU2 gene, only transformed cells will be able to grow on a medium without leucine present as the functional LEU2 gene in the plasmid will complement the mutant LEU2 in the host.

Question 9

What is YEp13?

Answer 9

A yeast episomal vector that is a mixture (amalgam) of pBR322, LEU2 gene and the ori from the 2um plasmid. It is a shuttle vector that can be used in E.coli and yeast.

Question 10

What are the benefits of YEp13?

Answer 10

It can replicate as an independent plasmid in yeast and can integrate into the yeast genome via homologous recombination between LEU2 and leu2 - the mutant gene in the yeast and the leu2 in the plasmid.

Question 11

What are most of the vectors used for in cloning plants based on?

Answer 11

The Ti plasmid of Agrobacterium tumefaciens, a soil bacteria that infects wounds woody plants and produces a tumour.

Question 12

What are cloned plants used for?

Answer 12

Research and crops for food, feed and products.

Question 13

How large is the Ti plasmid?

Answer 13

200kb and circular (big!).

Question 14

What is the action of the Ti plasmid?

Answer 14

T-DNA is transferred into the plant cell and integrates into the plant’s chromosomal DNA.

Question 15

What do the genes on the T-DNA cause?

Answer 15

Rapid cell division and the synthesis of opines (exotic amino acids) that are a carbon and nitrogen source for the A. tumefaciens in order to grow - not used by the plant.

Question 16

How can Ti be used as a cloning vector?

Answer 16

Oncogenes are removed from T-DNA and replaced by selectable markers, restriction sites and useful promoters to control the expression of introduced genes.

Question 17

What is the Ti cloning vector used for in cloning?

Answer 17

Used to transform plants cells and regerenate whole plants.

Question 18

How does the complexity of plant vectors compare to prokaryotic vectors?

Answer 18

Much more complex.

Question 19

What is the purpose of cloning in animals?

Answer 19

Research, production of recombinant proteins (pharming) and gene therapy.

Question 20

What significance does the Drosophila melanogaster have?

Answer 20

It is an important model organism for genetics and development and vectors have been based on the Drosophila P element.

Question 21

What is a transposable element?

Answer 21

A DNA sequence that can change its position within a genome. Part of a plasmid or chromosome that can be cleaved out and put to another part of the chromosome/plasmid.

Question 22

What is required for transposition?

Answer 22

Genes to code for transposases, Terminal inverted repeats (TIRs).

Question 23

How many kb is the P element?

Answer 23

2.9kb.

Question 24

What percentage of the human genome to transposable elements make up?

Answer 24

40%.

Question 25

What is the difference between the two P elements a the plasmid carries?

Answer 25

The first has a restriction site for cloning and the second lacks TIRs - wings clipped.

Question 26

Why do we need eukaryotic cloning vectors?

Answer 26

For large scale production

Question 27

Give a brief overview of cloning into Yep13 to create the desired recombinant vector.

Answer 27

Insert the gene into the YEp13 vector, transform E.coli and select the correct recombinants based on replica plating with ampicillin. Purify the DNA from the E.coli and insert into yeast (auxotrophic mutant) and grow on a medium without leucine.

Question 28

Where in the world produces the most GM crops?

Answer 28

USA, Brazil, Argentina etc

Question 29

What happens when the plasmid DNA is microinjected into fly embryos?

Answer 29

Transposases from the wings clipped element recognise the TIRs from the first element and excise the first P element - with the gene inserted in. These transpose into a fruit fly chromosome and if it is in the germline nucleus, the resulting adult fly wll have all the gene in its cells.

Question 30

Why might we clone in mammals?

Answer 30

To generate gene knock-outs (inactivate a gene and observe the changes in phenotype), for pharming (making important proteins) and for gene therapy (to study or treat human disease)

Question 31

What are mammalian vectors based on?

Answer 31

Viruses such as the SImian virus 40 and adenoviruses.

Question 32

How can adenoviruses be used as vectors?

Answer 32

Insert the gene of interest into the viral vector - it will integrate into the host DNA. Infect mammalian cells or tissue. The cells infected with encode for a reporter gene (beta galactosidase) that will show a blue colour if there is successful gene expression.

Question 33

How is the adenovirus modified to protect the public?

Answer 33

It is modified so that it is replication deficient and unable to lyse the the cell so it can’t be transmitted.

Question 34

How else can the adenovirus be used aside from the normal method of infection into mammalian tissue?

Answer 34

It can be injected into the tail of the mouse and the adenovirus will be taken up by hepatocytes. The protein will be secreted into the bloodstream.

Question 35

What does chimeric mean?

Answer 35

A mixture of transformed and non-transformed cells.

Question 36

How can transgenic animals be made?

Answer 36

Microinject bacterial plasmids or linear DNA copies of genes into a fertilised egg cell or embryonic stem cell. The DNA will be inserted into the chromosomes.

Question 37

Why would transgenic animals be made inside of viral infection?

Answer 37

It can avoid secondary effects that may arise due to the viral infection.