PMS L2

Question 1

Outline the essential properties of binary plasmid vectors. How is T-DNA marked and what does it contain?

Answer 1

• Vectors contain a backbone with origins of replication, a bacterial selection marker and T-DNA marked by 25 base-pair left and right border sequences for plant transfer.
  -The T-DNA includes
  genes of interest and a selection marker for transformed plant rescue.

Question 2

What must the foreign DNA inserted include for proper expression? What can also influence gene activity?

Answer 2

• Control sequences compatible with plant transcription factors, RNA polymerase, and regulatory proteins.
• Plant chromatin structure around the insertion site

Question 3

How must a synthetic gene be analysed to check its functioning?

Answer 3

• In situ analysing is needed after introduction to the plant.

Question 4

Outline the transcription mechanism in eukaryotes.

Answer 4

• Protein-encoding genes are transcribed by RNA polymerase II
• This binds upstream of the
  transcribed sequence at the TATA box
• Enhancers or silencers help initiate molecular contacts between the core RNA polymerase and the gene via mediator proteins.
• Enhancers and silencers mediate DNA looping and can be positioned anywhere
• Appropriate sequences in synthetic gene needed adjacent to coding sequence for these contacts with RNA.

Question 5

Outline the Post-Transcriptional Processing of Plant Genes

Answer 5

• Addition of (7-methylguanylate) cap at 5’ end of the RNA.
• Addition of a polyadenylate tail at 3’ end of the RNA.
• Intron removal by splicesomes.
• Synthetic gene design must ensure this is all accurately mediated.

Question 6

Explain the implications of the modular architecture of plant genes

Answer 6

• Conserved sequences in plants allow a modular design approach.
• Elements can be exchanged between different genes if sequence and position within the gene is respected.
• Allows use of standardised plant DNA parts for synthetic genes.

Question 7

When were GM crops first commercially released and for what purposes?

Answer 7

• Mid 1990s
  -Insect, herbicide and virus resistance

Question 8

Give a common GM modification for insect resistance. Explain how it works.

Answer 8

• Bacillus thuringiensis (Bt) gene introduced, produces a Cry protein toxin.
• Toxin effective against insects but not mammals.
• Toxin binds to gut receptors in insects, causing pore formation and cell leakage.
• Used for organic farming.
• Pests can develop resistance.

Question 9

Give a common GM modification for herbicide resistance. Explain how it works.

Answer 9

• Glyphosate (a herbicide) resistance.
• Glyphosate normally inhibits the shikimate pathway in plants but transgenic crops express resistant enzymes.
• Allows no-till practices, reducing soil erosion.
• Herbicide resistance seen in weeds.
• Multiple herbicide resistance traits needed in crops.

Question 10

What is trait stacking?

Answer 10

• Modern crops stacked with multiple traits, e.g. herbicide resistance and Bt toxin.

Question 11

Give an example of a non-fluorescent reporter gene. Explain how it works. Give Pros + Cons.

Answer 11

• Gene encoding for bacterial enzyme β-glucuronidase (GUS)
• Addition of X-gluc substrate causes a reaction catalysed by GUS.
• Reaction produces insoluble indigo product at reaction site.
• PROS: Allows simple detection of expression patterns in whole mounts.
• CONS: Is usually lethal, not that high resolution (can’t resolve sub-cellular location)

Question 12

Give an example of a fluorescent reporter gene. Explain how it works. Give a Pro

Answer 12

• Green fluorescent protein (GFP), from jellyfish Aequoria victoria
• Emits green light upon excitation.
• Visualised using fluorescent microscopy.
• Different coloured fluorescent proteins available.

Question 13

Explain the Pros of fluorescence microscopy.

Answer 13

• Allows visualisation of living cells (non-lethal)
• Confocal microscopy gives high-res imaging by focusing laser light, excluding blur.

Question 14

Give applications of Confocal Microscopy

Answer 14

• Detailed examination of plant tissues (visible sub-cellular features)
• Can track gene expression and movement of tagged viruses, e.g. by use of a fluorescent reporter gene