Midterm 2 - Notes 6 (Part 6) Flashcards
What does VirE1 do?
It is a small protein that interacts with and stabilizes VirE2
- molecular chaperone
What does VirE2 do?
It transcribes and translates in the bacterium
Where is VirE2 unstable?
In the bacterium
- would be degraded quickly if VirE1 and that acts as a molecular chaperone and it binds to it and stabilizes it in the bacterium
What does VirE2 interaction with VirE1 prevent?
T-DNA binding in bacteria
What is not needed in bacteria?
VirE2
What initiates the nik?
VirD1
What does VirE2 have?
Protein protein function domains that only work in eukaryotes
What does VirE2 bind to in plant cells?
T-DNA and creates a coat that protects the T-DNA
What does VirE2 contain?
Nuclear localization signal targets T-DNA to the nucleus
What does VirE2 interact with?
Plant proteins
- promote nuclear translocation
What does VirD2 act as? (2)
- DNA ligase to integrate into the plant genome
- needs a DNA break present
- thats when ligase integrates the tDNA - Unspecific cutting site
- only happens at the L side sequences
What does VirD2 bind to?
Plant DNA repair proteins
- so it highjacks the repair mechanisms that plant has to fix DNA strand breaks by binding VirD2 and the location
Are nicking reactions reversible?
Yes
What does VirD2 do?
It catalyzes ligation into plant genomes
What does nuclear localization signal interact with?
Importin alpha
What is VirD2 simulated by?
Phosphorylation
- plant CDK
What does VirD2 recruit?
T-DNA to damage DNA
What does VirD2 initiate?
Integration via ligase activity
What do mature plant cells not send?
Dont send signals anymore unless they are wounded
What are the 6 steps in T-DAN transfer?
1a. Specific attachment of bacterium to plant cell wall
1b. Plant derived signals are recognized via ChvE/VirA, resulting in high levels of VirG-P
2. VirG-P activates transcription by Vir genes resulting in…
3. … Production of transported substates and VirB complex
4. Substrates are recognized by VirB complex and transported to the plant cell
5. A T-complex assembles and moves into the nucleus
6. T-DNA integrates into the host chromosome
How is tumour formation in animals and plants dissimilar? (3)
- Morphology
- both are uncontrolled outgrowths - Plant tumours are caused by over production of of growth hormones
- animal tumours are caused by alterations in growth factor perception/signal transduction - Plant oncogenes are of bacterial origin that cause production of phytohormones
- animal tumours are caused by multiple mutations in endogenous genes, resulting in oncogenes
What was the only case of the inter-kingdom lateral gene transfer that was understood on the molecular level?
Agrobacterium mediated gene transfer
What can be used as a shuffle system?
Anti-bacterium
What are 3 key features of Agrobacterium mediated gene transfer?
- T-DNA is stable once transferred to the plant genome
- Only T-DNA borders and Vir genes are required for transformation
- Wide range of host plants
YGI
Your gene of interest
What can you do with Ti-plasmid in E.Coli?
You can manipulate it
What is removed and added from the Ti-plasmid in the binary vector system?
Removed = T-DNA region Added = antibiotic resistance marker
What does disarmed Ti-plasmid provide machinery for?
Transformation
- still has the machinery that allows transformation into the plant
What are 2 examples of resistance markers?
- KanR
2. BASTAR
What are resistant markers driven by?
A eukaryotic promoter close to L border
What can the binary vector be propagated/manipulated by?
E.Coli
What can be cloned and transformed into Agrobacterium?
YGI
What is modified stain used for?
Plant transformation
