Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

z:Cell 300 > Lecture 11: RNA interference > Flashcards

Lecture 11: RNA interference Flashcards

1
Q

RNA interference is a type of _____

A

Reverse genetics

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Reverse genetic techniques

A

-Gene ablation/knockout by recombination
-Gene ablation using CRISPR
-Antisense RNA (for transient inhibition of gene expression)
-RNA interference (RNAi)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Gene ablation/knockout by recombination summary

A

-to figure out what a gene is for, you take it out and see what doesn’t work
-permanent insertion of marker (usually drug resistance) into chromosome to create knockout organism

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Gene ablation/knockout by recombination drawbacks

A

-difficult and expensive
-time-consuming
-may not be able to study essential genes cause knock-out organism would die

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

CRISPR stands for ____

A

clustered regularly interspaces short palindromic repeats

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

CRISPR is derived from ____

A

bacterial antiviral system

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Gene ablation using CRISPR summary

A

-“Guide” RNAs target the nuclease Cas9 to homologous DNA sequences after which cuts are
made in the DNA.
-Can be used to delete, modify or activate genes
-permanent

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Antisense RNA (for transient inhibition of gene expression) summary

A

-addition of antisense rna binds to mRNA and prevents translation
-simple but not efficient
-controls: sense RNA (without 5’ cap and 3’ tail)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

loss of unc22 gene in C.elegans led to ___

A

twitching

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

insertion of dsRNA in C.elegens found that _____

A

Found that dsRNA was much more potent than
anti-sense RNA for inducing unc22 phenotype

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

detection of mRNA by in situ hybridization

A

-Digoxigenin is a small molecule to which there are good antibodies available to it (a way to mark
the cDNA probe for detection with secondary antibody
-Digoxigenin label on uridine + alkaline phosphate and colourless chemical –> purple dye

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

RNA interference (RNAi) can be applied to ____ and ___ cells

A

-plants and mammalian cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

RNA interference (RNAi): provided that DNA sequence is known, _____

A

it is now possible to “knock-down” the activity of any gene rapidly

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

extreme benefit of RNAi

A

-can study function of essential genes (not knocking them out completely, just toning down expression)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Dicer (def.)

A

RNase cleaves dsRNA molecules

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Dicer products are ____

A

21-23 bp RNAs termed small interfering RNAs (siRNAs)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Argonautes (def.)

A

RNA-binding proteins that interact with siRNAs to form RNA induced silencing complex (RISC)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

RISC is targeted to ____ by _____

A

mRNAs by siRNA or miRNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

microRNAs (miRNAs) (def.)

A

similar to siRNAs but generally result in translational repression

20
Q

-miRNAs control expression of ____ of human genes

A

~70%

21
Q

siRNA cause ____ while miRNA causes _____ because ____

A

-RNA cleavage
-translation inhibition
-siRNA base pairing is complementary while miRNA is generally not fully complementary

22
Q

what encodes for miRNA?

A

the genome

23
Q

miRNA blocks translation because of the ___ and can work on ____

A

-bulge in base-paring
-many targets as a result

24
Q

long dsRNA are ___

A

usually not found in euk. cells (sign of danger)

25
Q

LNP stands for

A

lipid nanoparticles

26
Q

LNP is used to ___

A

deliver siRNA into cells

27
Q

LNP are

A

3-4 lipids that bind to RNA + help it cross cell membrane

28
Q

LNP morphology

A

-single lipid bilayer
-lipid carrier (+ charged lipid at lower PH), structural lipid (cholesterol + phospholipid) and shielding lipid (PEG-lipid)

29
Q

protein levels _____ with different kinetics than mrna in response to RNAi

A

decrease (it can take more than 1 day for protein expression to decrease while mrna decreases in hours)

30
Q

Northern blot is a laboratory analysis method used to study ____

A

RNA

31
Q

P bodies are sites of ___

A

RNA degradation (processing bodies) + contain mRNA decapping enzymes (Dcp1a,2)

32
Q

Resolution power of conventional light microscope is ___ nm so objects less than ___ nm apart cannot be resolved

A

250 nm

33
Q

Western blot is often used in research to ____

A

separate and identify proteins in a complex mixture of proteins

34
Q

western blot + co-immunoprecipitation (co-IP)

A

IP-Western (immunoblotting); used to analyze specific protein-protein interactions

35
Q

Co-Immunoprecipitation (co-IP) procedure

A

-Cells lysed with nondenaturing detergent (does not disrupt protein-protein interactions)
-Incubate with antibody to protein X and
Protein A agarose
-Wash away unbound proteins
-Analyze complex by Western blotting

36
Q

western blot procedure

A

-electrophoresis(SDS)/transfer
-antibody detection (1/2, wash excess)

37
Q

To confirm interaction between X and Y, ___

A

reciprocal co-IP Western blot should be
performed.
* i.e. Antibody to protein Y should be able to
co-IP X.

38
Q

Depending on substrate
(DAB or ECL), IS used, _____

A

HRP catalyzed oxidation produces a colored
precipitate (from DAB, as shown here) or light (ECL)
on membrane where protein
Y is located

39
Q

Argonautes ____complexes with Dcp1a

A

do form

40
Q

If Ago and Dcp form complex, both
proteins should be ____

A

co-IP’d by antibodies to either protein

41
Q

Argonaute proteins are ____

A

Modular proteins with PAZ and PIWI domains

42
Q

PAZ domain of argonaute

A

-si/miRNA binding

43
Q

PIWI domain of argonaute

A

-nuclease (slicer and binding to DICER)

44
Q

when nuclease function of argonaute is knocked out, it goes to ___

A

P bodies

45
Q

PAZ mutants of argonaute don’t ____

A

associate with P-bodies

46
Q

____ of Ago2 is required for Pbody association

A

siRNA/miRNA-binding activity

z:Cell 300 (17 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions