Midterm 2 - Notes 6 (Part 6)

Question 1

What does VirE1 do?

Answer 1

It is a small protein that interacts with and stabilizes VirE2
- molecular chaperone

Question 2

What does VirE2 do?

Answer 2

It transcribes and translates in the bacterium

Question 3

Where is VirE2 unstable?

Answer 3

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

Question 4

What does VirE2 interaction with VirE1 prevent?

Answer 4

T-DNA binding in bacteria

Question 5

What is not needed in bacteria?

Answer 5

VirE2

Question 6

What initiates the nik?

Answer 6

VirD1

Question 7

What does VirE2 have?

Answer 7

Protein protein function domains that only work in eukaryotes

Question 8

What does VirE2 bind to in plant cells?

Answer 8

T-DNA and creates a coat that protects the T-DNA

Question 9

What does VirE2 contain?

Answer 9

Nuclear localization signal targets T-DNA to the nucleus

Question 10

What does VirE2 interact with?

Answer 10

Plant proteins

- promote nuclear translocation

Question 11

What does VirD2 act as? (2)

Answer 11

1. DNA ligase to integrate into the plant genome
   - needs a DNA break present
   - thats when ligase integrates the tDNA
2. Unspecific cutting site
   - only happens at the L side sequences

Question 12

What does VirD2 bind to?

Answer 12

Plant DNA repair proteins

- so it highjacks the repair mechanisms that plant has to fix DNA strand breaks by binding VirD2 and the location

Question 13

Are nicking reactions reversible?

Answer 13

Yes

Question 14

What does VirD2 do?

Answer 14

It catalyzes ligation into plant genomes

Question 15

What does nuclear localization signal interact with?

Answer 15

Importin alpha

Question 16

What is VirD2 simulated by?

Answer 16

Phosphorylation

- plant CDK

Question 17

What does VirD2 recruit?

Answer 17

T-DNA to damage DNA

Question 18

What does VirD2 initiate?

Answer 18

Integration via ligase activity

Question 19

What do mature plant cells not send?

Answer 19

Dont send signals anymore unless they are wounded

Question 20

What are the 6 steps in T-DAN transfer?

Answer 20

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

Question 21

How is tumour formation in animals and plants dissimilar? (3)

Answer 21

1. Morphology
   - both are uncontrolled outgrowths
2. Plant tumours are caused by over production of of growth hormones
   - animal tumours are caused by alterations in growth factor perception/signal transduction
3. Plant oncogenes are of bacterial origin that cause production of phytohormones
   - animal tumours are caused by multiple mutations in endogenous genes, resulting in oncogenes

Question 22

What was the only case of the inter-kingdom lateral gene transfer that was understood on the molecular level?

Answer 22

Agrobacterium mediated gene transfer

Question 23

What can be used as a shuffle system?

Answer 23

Anti-bacterium

Question 24

What are 3 key features of Agrobacterium mediated gene transfer?

Answer 24

1. T-DNA is stable once transferred to the plant genome
2. Only T-DNA borders and Vir genes are required for transformation
3. Wide range of host plants

Question 25

YGI

Answer 25

Your gene of interest

Question 26

What can you do with Ti-plasmid in E.Coli?

Answer 26

You can manipulate it

Question 27

What is removed and added from the Ti-plasmid in the binary vector system?

Answer 27

Removed = T-DNA region 
Added = antibiotic resistance marker

Question 28

What does disarmed Ti-plasmid provide machinery for?

Answer 28

Transformation

- still has the machinery that allows transformation into the plant

Question 29

What are 2 examples of resistance markers?

Answer 29

1. KanR

2. BASTAR

Question 30

What are resistant markers driven by?

Answer 30

A eukaryotic promoter close to L border

Question 31

What can the binary vector be propagated/manipulated by?

Answer 31

E.Coli

Question 32

What can be cloned and transformed into Agrobacterium?

Answer 32

YGI

Question 33

What is modified stain used for?

Answer 33

Plant transformation