RNA

Question 1

Why is it strange that a mild strain of TMV (tobacco mosaic virus) would give immunity to plants?

Answer 1

Plants do not have an immune system with antibodies hence our immunization logic should not be applicable here.

Question 2

What do they now think was the primary reason for this immunity in tobacco plants?

Answer 2

Coat proteins being produced by mild strain (could be genetically engineered to produce coat protein though)

Question 3

What method was primarily used to identify plants producing high levels of coat proteins and what would a positive result look like?

Answer 3

The western blot- a larger blob would mean more protein present.

Question 4

By which year had the Ringspot Virus wiped out all of which fruit except in Puna?

Answer 4

1960, papaya

Question 5

When was it first found in Puna and why was this such a problem?

Answer 5

1992 once Ringspot virus is found papaya can no longer grow:(

Question 6

How did they solve the problem of the ringspot virus?

Answer 6

They genetically engineered the papaya plants to produce coat proteins that then made them resistant to disease.

Question 7

How does Coat Protein Mediated Resistance work?

Answer 7

The coat protein surrounds the long thin virus RNA genome and stops it being able to dissassemble and hence be translated. (Literally a coat over the viral genome)

Question 8

Why would it be of value to shut down the enzyme chalcon synthase (CHS)?

Answer 8

This would stop colour production in the petals which, in chrysnthumums, is valuable as a wedding flower.

Question 9

What is the biochemical pathway to the production of anthocyanins (the red pigment in plants)

Answer 9

Malonyl-coA is converted by chalcone synthase to Chalcone -> Naringenen -> Anthocyanins

Question 10

What colour is chalcone and where can it be found?

Answer 10

Yellow, corn or maize

Question 11

What method was taken to deactivate CHS?

Answer 11

Rotate gene for CHS into antisense position.

Question 12

What are the three expected outcomes of this petunia experiment?

Answer 12

one) endogenous/wildtype petunia - purple in colour
two) transgene introduce extra copy of gene (CONTROL) - extra protein being translated so darker purple colour
three) extra transgene antisense construct - white

Question 13

What were the actual outcomes?

Answer 13

Control showed complete blocking of CHS production in some areas and normal function elsewhere
Antisense - purple all over reduced but still present

Question 14

Why does white appear in the control of the petunia experiment?

Answer 14

One copy of a gene can silence another (occurs in corn paramutation) hence introducing a new copy of gene can stop CHS production

Question 15

How come it was found later that coat proteins were not actually required to give resistance?

Answer 15

Instead RNA was the driving factor (post transcriptional gene silencing)

Question 16

Describe the setup of Herves grafting exeriment

Answer 16

Genetically mutated tobacco plants to have non functioning nitrate reductase (gene silenced) resulting in chlorosis (yellow leaves). Put together bottom of GM tobacco with top of non GM.

Question 17

What are the outcomes of Herves experiment and what does it show?

Answer 17

The top of the plant became chlorotic despite being wildtype showing that the factor resulting in gene silencing (RNA) must be motile and can move throughout the plant!

Question 18

How can you genetically modify c.elegans?

Answer 18

Inject DNA directly into gonads

Question 19

Describe the outcome of making transgenic worms in both a sense and an antisense position.

Answer 19

Both showed targetted gene silencing effects

Question 20

Describe the outcome of injecting RNA into worm gonads?

Answer 20

Showed targetted gene silencing right into nex generation meaning it is acting like a contaminant.

Question 21

What did the experiments of Fire with sense, anti sense and mixed RNA show in worms?

Answer 21

Only once sense and antisense are introduced simultaneously (double-stranded) is gene silencing (uncontrolled muscle twitching) effective.

Question 22

What happens when you feed worms bacteria expressing this dsRNA?

Answer 22

It finds a way to move out of the gut and into the nucleus of cells to cause gene silencing.

Question 23

Describe a hairpin transgene for gene silencing

Answer 23

This is a stretch of single stranded RNA made up of a sense and antisense sequence with a short buffer region in the middle. It can then fold back on itself to create double stranded RNA for effective gene silencing.

Question 24

What does a positive result for the silencing of green flourescence protein look like?

Answer 24

Shines red under UV light.

Question 25

How can gene silencing spread through a leaf?

Answer 25

Diffusion of dsRNA through the plasmodesmata between cells.

Question 26

How does RNA tend to spread?

Answer 26

It systematically follows phloem pathway.

Question 27

Describe two important enzymes in the process of gene silencing

Answer 27

Dicer - cuts lengths of double stranded RNA into small 25 bp sections called siRNA
Argonaute - One strand is thrown away and the other strand joins to argonaute enzyme that can assist with the degradation of proteins.

Question 28

Where were the first miRNAs located and what were they?

Answer 28

Lin-4, let-7 involved in the developmental process of c.elegans.

Question 29

Do miRNA have to be introduced?

Answer 29

No, they occur endogenously often conserved across many species, encoded by nuclear genes. They can be tissue specific and expressed constitutively.