RNA Flashcards
Why is it strange that a mild strain of TMV (tobacco mosaic virus) would give immunity to plants?
Plants do not have an immune system with antibodies hence our immunization logic should not be applicable here.
What do they now think was the primary reason for this immunity in tobacco plants?
Coat proteins being produced by mild strain (could be genetically engineered to produce coat protein though)
What method was primarily used to identify plants producing high levels of coat proteins and what would a positive result look like?
The western blot- a larger blob would mean more protein present.
By which year had the Ringspot Virus wiped out all of which fruit except in Puna?
1960, papaya
When was it first found in Puna and why was this such a problem?
1992 once Ringspot virus is found papaya can no longer grow:(
How did they solve the problem of the ringspot virus?
They genetically engineered the papaya plants to produce coat proteins that then made them resistant to disease.
How does Coat Protein Mediated Resistance work?
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)
Why would it be of value to shut down the enzyme chalcon synthase (CHS)?
This would stop colour production in the petals which, in chrysnthumums, is valuable as a wedding flower.
What is the biochemical pathway to the production of anthocyanins (the red pigment in plants)
Malonyl-coA is converted by chalcone synthase to Chalcone -> Naringenen -> Anthocyanins
What colour is chalcone and where can it be found?
Yellow, corn or maize
What method was taken to deactivate CHS?
Rotate gene for CHS into antisense position.
What are the three expected outcomes of this petunia experiment?
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
What were the actual outcomes?
Control showed complete blocking of CHS production in some areas and normal function elsewhere
Antisense - purple all over reduced but still present
Why does white appear in the control of the petunia experiment?
One copy of a gene can silence another (occurs in corn paramutation) hence introducing a new copy of gene can stop CHS production
How come it was found later that coat proteins were not actually required to give resistance?
Instead RNA was the driving factor (post transcriptional gene silencing)
Describe the setup of Herves grafting exeriment
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.
What are the outcomes of Herves experiment and what does it show?
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!
How can you genetically modify c.elegans?
Inject DNA directly into gonads
Describe the outcome of making transgenic worms in both a sense and an antisense position.
Both showed targetted gene silencing effects
Describe the outcome of injecting RNA into worm gonads?
Showed targetted gene silencing right into nex generation meaning it is acting like a contaminant.
What did the experiments of Fire with sense, anti sense and mixed RNA show in worms?
Only once sense and antisense are introduced simultaneously (double-stranded) is gene silencing (uncontrolled muscle twitching) effective.
What happens when you feed worms bacteria expressing this dsRNA?
It finds a way to move out of the gut and into the nucleus of cells to cause gene silencing.
Describe a hairpin transgene for gene silencing
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.
What does a positive result for the silencing of green flourescence protein look like?
Shines red under UV light.
How can gene silencing spread through a leaf?
Diffusion of dsRNA through the plasmodesmata between cells.
How does RNA tend to spread?
It systematically follows phloem pathway.
Describe two important enzymes in the process of gene silencing
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.
Where were the first miRNAs located and what were they?
Lin-4, let-7 involved in the developmental process of c.elegans.
Do miRNA have to be introduced?
No, they occur endogenously often conserved across many species, encoded by nuclear genes. They can be tissue specific and expressed constitutively.
