15 - microRNAs and small RNAs

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What is chalcone synthase (CHS)? why was it used?
is the enzyme at the start of the biosynthetic pathway for anthocyanins = the regulaing, limiting enzyme - wanted to see if could make more intense colour
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What was the hypothesis for CHS experiments?
sense RNA production enhances pigmentation (if chalcone synthase is rate limiting, maybe we should insert more genes or another RNA of chalcone synthase so that we have more copies of the genes so the petunia produces more anthocyanins)
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What did the CHS experiments observe?
antisense RNA production blocks pigmentation (a silencing effect. Realised an antisense RNA dimerises with the mRNA -> mRNA is not transcribed and chalcone synthase is not produced -> white colours (hybrid)
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What did they conclude from CHS experiments?
Silenced tissues do not express endogenous or introduced CHS = co-supression (both the introduced gene and the endogenous gene are silenced)
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How can pigment production be inhibited?
Surprisingly, both antisense and sense gene constructs can inhibit pigment production
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How can plants recover from viral infection and become resistant?
Inoculated older leaves (at bottom of plant) with viruses  found that a later inoculation of younger leaves resulted in no disease symptoms in younger leaves which developed after initial virus infection = plants become resistant
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What is the principle of viral infection and replication?
influenza virus becomes attached to a target epithelial cell->cell englufs virus by endocytosis->viral contents released. viral RNA enters nucleus where is replicated by viral RNA pol->viral mRNA is used to make viral proteins->new viral particles
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What is the Principle of local and systemic viral infection?
Virus should move up the plant and end up in other leaves, systemically infecting the plant
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How do you inoculate a plant for viral resistance?
Day 1: Inoculate leaf with virus or water. Day 22: Inoculate younger leaf with virus or water. Day 32: Isolate RNA from leaf inoculated on Day 22. Measure viral RNA level.
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What do low RNA levels on day 32 show?
show that the first viral infection has induced RNA silencing, preventing subsequent viral replication.
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Why are Small RNAs are correlated with viral-induced gene silencing?
A small RNA homologous to viral RNA is present in inoculated leaves and distal, “systemic” leaves, but not mock-infected leaves.
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2 key C. elegans experiments?
1)injected small RNAs into C. elegans to try to silence unc22 gene-> observed (by worm twitch) when ds smallRNA used most 2)Transformed gonads of C. elegans->do not get colour any longer if you use ds small RNA do not get colour any longer w ds small
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Significance of the discovery of RNAi?
protects against viral infection, genome stability, repress protein synthesis and regulate development, keep chromatin condensed and suppress transcription, experimtental tool to repress genes specifically, useful in gene therapy
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Steps at which eukaryotic transcription can be controlled?
Histone modification: Acetylation, methylation/Transcriptional control: RNA polymerase Transcription factors/RNA processing control/RNA transport and Localisation control/mRNA degredation/Translational control/Protein activity control
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What are small RNAs?
Small noncoding RNA molecules (20-30 nt)/Different types found in all euks except yeast/>60% of human protein-coding genes contain at least one conserved miRNA-binding site->most are under control,of miRNAs
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How are small RNAs made?
Polymerase II produces small RNAs Plants do have in addition Pol IV and V involved in the production of small RNA and in RNAi
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What is an argonaut?
Form the RNA-Induced Silencing Complex (RISC) Passenger strand is removed in pre-RISC miRNA loading is energy-dependent and mediated by HSC70-HSP90
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Processes regulated by small RNAs?
RNAi is a strategy to avoid viral infection, contribute to genome stability (transposon degradation) and (post-) transcriptional gene silencing, Disturbance or misactivation of small RNA activities can affect development, support diseases (e.g. Cance
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Adaptation of small RNAs?
Are highly mobile, Systemic resistance strategy againt virus
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Card 2

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What was the hypothesis for CHS experiments?

Back

sense RNA production enhances pigmentation (if chalcone synthase is rate limiting, maybe we should insert more genes or another RNA of chalcone synthase so that we have more copies of the genes so the petunia produces more anthocyanins)

Card 3

Front

What did the CHS experiments observe?

Back

Preview of the front of card 3

Card 4

Front

What did they conclude from CHS experiments?

Back

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Card 5

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How can pigment production be inhibited?

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