PMS L2 Flashcards
Outline the essential properties of binary plasmid vectors. How is T-DNA marked and what does it contain?
- 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.
What must the foreign DNA inserted include for proper expression? What can also influence gene activity?
- Control sequences compatible with plant transcription factors, RNA
polymerase, and regulatory proteins. - Plant chromatin structure around the insertion site
How must a synthetic gene be analysed to check its functioning?
- In situ analysing is needed after introduction to the plant.
Outline the transcription mechanism in eukaryotes.
- 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.
Outline the Post-Transcriptional Processing of Plant Genes
- 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.
Explain the implications of the modular architecture of plant genes
- 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.
When were GM crops first commercially released and for what purposes?
- Mid 1990s
-Insect, herbicide and virus resistance
Give a common GM modification for insect resistance. Explain how it works.
- Bacillus thuringiensis (Bt) introduces, produces a 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.
Give a common GM modification for herbicide resistance. Explain how it works.
- 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.
What is trait stacking?
- Modern crops stacked with multiple traits, e.g. herbicide resistance and Bt toxin.
Give an example of a non-fluorescent reporter gene. Explain how it works. Give Pros + Cons.
- 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)
Give an example of a fluorescent reporter gene. Explain how it works. Give a Pro
- Green fluorescent protein (GFP), from jellyfish Aequoria victoria
- Emits green light upon excitation.
- Visualised using fluorescent microscopy.
- Different coloured fluorescent proteins available.
Explain the Pros of fluorescence microscopy.
- Allows visualisation of living cells (non-lethal)
- Confocal microscopy gives high-res imaging by focusing laser light, excluding blur.
Give applications of Confocal Microscopy
- Detailed examination of plant tissues (visible cellular features)
- Can track gene expression and movement of tagged viruses.
