Edgell Final Flashcards

Question

Which polymerase transcribes miRNAs?

Answer 1

RNAP II therefore they have a 5' cap (it is needed for them to be exported out of the nucleus)

Answer 2

a stem-loop structure

Answer 3

pri-miRNA is the primary transcript generated by transcription pre-miRNA has been processed by microprocessor and is what gets exported from the nucleus to the cytoplasm (is the precursor stem loop miRNA)

Answer 4

DICER cleaves ds pre-miRNA at the loop end into 20-30nt unit lengths this generates a 22-nt darn product with a 2nt overhang at the 3' ends passenger strand gets degraded and the miRNA strand that is complementary to the target RNA is incorporated into RISC Ago2/Slicer is what cleaves the target RNA molecule if there is perfect complementarity (is an RNaseIII type endoribonuclease) if there is not perfect complementarity then RISC functions in translational repression and/or deadenylation of the mRNA Note: the miRNA or siRNA can also be called the guide RNA/gRNA

Answer 5

perfect base pairing puts the phosphate of the target RNA into the active site of Ago2/slicer it gets cleaved 10nts from the 5' end of the guide RNA Note: only the target RNA is cleaved

Answer 6

the first 8 nts at the 5' end of the miRNA it is required to have perfect complementarity (the 3' region can be variable)

Answer 7

yes because only the first 8 nucleotides i.e. the seed region need to have perfect complementarity

Answer 8

prevent the assembly of the 40S ribosome and circularization of the mRNA may also prevent the 60S subunit from joining the 40S it is also thought that they may mediate an elongation block or catalyze proteolysis of the nascent polypeptide strand miRNPs bind to them and can help mediate these sort of things

Answer 9

the molecule has different stabilities at each end because of base stacking RISC has 5' to 3' helicase activity the end with the less stable interactions will get pulled apart first whichever 5' end becomes free first will be docked in argonaut

Answer 10

a DNA transposon that move by cut-and-paste

Answer 11

targeting mobile elements for degradation

Answer 12

have a transposase in the middle with inverted repeats on either side transposes cuts out the sequence and inserts it somewhere else in the genome insertion can be specific or random, depends on the element

Answer 13

are from viruses i.e. HIV

Answer 14

a non-LTR retrotransposon 21% of our genome is made of them however, 99.9% of them are inactive

Answer 15

there is an RNAP II promoter or 2 in the 5'UTR that drives transcription in either direction transcript encodes ORF1 and ORF2, it is capped, polyadenylated and exported ORF1 and ORF2 are translates ORF1 coats the mRNA, ORF2 binds to the polyA tail now have a RNP complex Note: these are cis acting elements! the entire thing is imported back into the nucleus ORF2 endonuclease activity loosely targets AT-rich areas which allows the polyA tail to anneal to the T-rich area ORF2 makes a ss nick generating a 3'OH ORF2 then reverse transcribes itself i.e. makes a cDNA copy of itself second strand synthesis and strand cleavage is not yet understood

Answer 16

has RNA binding and chaperone activity

Answer 17

has endonuclease and reverse transcriptase activity

Answer 18

reverse transcriptases don't have sliding clamps and are thus not very processive so they fall off and will result in a truncated 5' end often (synthesize DNA 3' to 5') haven't evolved to be more processive because they need to keep their host alive

Answer 19

cis event if RNAP doesn't stop transcribing at the polyA tail and includes downstream info, this extra info will be exported, imported etc along with the LINE-1 element and could end up in other exon etc

Answer 20

trans event ORF2 could bind to the polyA tail of a different mRNA this other mRNA could then be imported back into the nucleus, reverse transcribed and incorporated into a new region Note: in this case ORF1 is not binding

Answer 21

need dsRNA for RNAi depending on which way LINE-1 is incorporated into the genome there can be a LINE-1 and an antisense strand they can anneal then you have darn that can go into RISC and can then go back and act on itself

Answer 22

clustered regularly interspersed short palindromic repeats

Answer 23

RNases restriction enzymes abortive infection blocking uptake

Answer 24

when a phage infects a cell the bacterial cell transcribes the segments of DNA that match the virus these little segments of RNA act in a way similar to siRNA and cause the cleavage of viral DNA Note: this can also work on plasmids because technically plasmids are foreign DNA

Answer 25

there are CRISPR associated genes (Cas genes) they are the protein parts of the mechanism (have the catalytic activity) adjacent to them there is a leader sequence that has a promoter etc and a series of alternating spacers and repeats the repeats are identical, the spacers can be identical or different, the spacers are derived from phages i.e. are exact complements to parts of the viral genome the spacers, once transcribed are the guide RNA (crRNA)

Answer 26

because recombination is very efficient and if they get too long they will end up getting deleted

Answer 27

viral DNA gets chopped up by proteins NOTE: this dicing step is DNA not RNA!! this DNA that is chopped up is called the protospacer, it needs to be adjacent to a PAM sequence Cas proteins chop out the protospacers and integrate it into the CRISPR array i.e. the protospacer becomes the spacer Note: PAM is specific for each bacteria

Answer 28

it is a trans-activatin element for CRISPR it is needed for the processing of CRISPR repeats it is expressed separately from the rest of the CRISPR part of it is identical to the repeats

Answer 29

Cas9 is inactive until it binds tracrRNA and crRNA, which causes it to undergo conformational changes Cas9 has 2 active sites, one on the HNH domain and one on the RuvC domain, they are endonuclease that will nick a single strand crRNA (guide RNA) base pairs with the target and tracrRNA base pairs with the repeated part of the crRNA this C-terminal of Cas9 recognizes the PAM motif in the target DNA this complex is only stable enough for cleavage if the there is a PAM motif adjacent to where it is trying to bind the endonuclease domains make nicks and cause a DSB

Answer 30

because there isn't a PAM motif beside them so the complex won't be stable enough for cleavage

Answer 31

no, need perfect complementarity for the complex to be stable enough to be cleaved

Answer 32

by making a DSB because when there is a DSB the DNA gets chewed back to make 3' overhangs which are very good at strand invasion however, it is hard to target a DSB to a specific location

Answer 33

in classic there is minimum DNA loss, it is repaired in cis and gives genome stability in alternative there is DNA loss, can be translocation and causes genome instability

Answer 34

can be used to knockout a gene break in a protein coding region, some info is removed, causes change in reading from, mRNA stability, a non-functional protein etc Note: you still need to target the DSB though

Answer 35

at the same time as DSB is introduced, provide a correct sequence on donor DNA template to be used for repair

Answer 36

at the same time as DSB is introduced, provide donor DNA template that has flanking homology with extra genes in the middle

Answer 37

CCR5 is a co-receptor for HIV entry into T cells use a Zn finger nuclease that targets the human CCR5 gene and makes a DSB rely on the NHEJ pathway to repair and knockout the gene ex vivo use CD4+ cells transfect an adenovirus that expresses the nuclease make sure the gene has been knocked out, expand the cells and then infuse them back into the patient need to keep doing it also very expensive

Answer 38

they have a Zn-finger DNA binding domain and a non-specific DNA nucleus domain from a type IIS restriction enzyme called FokI each Zn finger contacts 3bp of DNA, can put a few in a row to target more specifically however, you can't put too many or else the non-specific binding constant will become the same as the specific one Foci functions as a dimer so you need to make Zn finger domains on each side which is hard (in order to generate a DSB) also FokI can dimerize with one monomer that is floating in solution and still cleave DNA so you can get off-target DSBs

Answer 39

proteins that are made in bacteria that infect rice they are made in the bacteria and imported into the nucleus of the plant cells there they act as transcription factors for genes that make the plant more susceptible to infection by the bacteria

Answer 40

very repeated structure 30-35 AAs that are repeated called the repeat variable domains the repeated domains are identical except for 2 amino acids, these 2 amino acids specify contact to a specific base each TAL repeat contacts a single base can use them to target 18-20bps, are more specific than Zn fingers

Answer 41

TAL repeats fused to FokI domains | a problem is that they are huge proteins and have a lot of repeated units, so for practical concerns it is difficult

Answer 42

you can make a synthetic guide RNA (crRNA) and tracrRNA (on a single vector) the guide RNA specifies where Cas9 goes can cleave any sequence you want, just need to order an oligonucleotide for it

Answer 43

no, they will cleave things that are 2, 3 or 4 nts different from the guide RNA with the same efficiency as the on-target sites (sometimes even more efficiently) partially due to the high frequency of PAMs because they are so short also because it is occurring at 37 degrees so off-target sites can have stability that is just as good or even better than the on-target sites also it depends on whether or not the DNA is accessible to breakage and repair

Answer 44

biology is hierarchical and uses small modular parts to create more complex systems

Answer 45

DNA is used to make parts parts are used to make devices devices are like regulatory on/off circuits systems use devices to make circuits can report things etc

Answer 46

DNA sequences which conform to a restriction-emzyme assembly standard i.e. there are promoters, terminators, coding sequences etc are used to assemble synthetic biological circuits and then are transplanted into living organisms "cut and paste cloning" all plasmids have the same restriction sites, you cut out the parts that you want and ligate them into a destination plasmid uses classic type IIP restriction enzymes and put things together using compatible overhangs

Answer 47

when you put everything together you destroy some of the cleavage sites would need another enzyme to cleave here i.e. you are limited by the enzymes that you have available you also need to cleave and ligate the reaction separatey which takes time

Answer 48

both sides of the piece you want to insert use the same restriction sequence so it can be ligated in either direction therefore you need to screen for the right orientation

Answer 49

each side of the piece you want to insert is cleaved using a different restriction enzyme so that it can only be inserted in one direction

Answer 50

PCR type IIS restriction enzymes Gibson assembly in vivo cloning

Answer 51

take DNA fragments that have overlapping sections of homology and put them in yeast yeast will recombine them for you so you don't need to do any cloning steps

Answer 52

have a symmetric recognition site cut within their recognition site cut specific sequences

Answer 53

assymmetric recognition site cleavage site is distant from the binding site do not cut specific sequences binding site is destroyed so it can't be cleaved again ie Dsa1 and FokI

Answer 54

have a vector with BsaI sites on it and a PCR product with them as well make it so that the overhangs that are generated are identical in both (but different on each side) PCR product and vector are cut at the same time and since there is only one way for it to be ligated and the substrate is not regenerated, it can all happen in a one-pot reaction

Answer 55

(either use PCR or restriction digest so that the DNA fragments have identical ends) use DNA fragments that have overlapping/compatible ends isothermally amplify them 5' exonuclease cuts back the ends making an overhang so that they are compatible they anneal DNAP extends the 3' end to put it back together ligase seals the nick do this 10-15 times result is a longer piece of DNA that is sewn together at homology can then put this together with another one etc

Answer 56

don't need restriction enzymes etc reaction can be performed in a single reaction with all of the buffers, enzymes etc can also use it for cloning i.e. put fragments into plasmids and then transform them into bacteria etc no restriction "scar" sit left

Answer 57

5'-3' exonuclease DNA polymerase DNA ligase DNA fragments with at least 15-40 bases of overlap

Answer 58

8 linear ssRNA fragments, each encoding a gene | NA and HA are the ones we care about (major antigenic determinants)

Answer 59

Day 1- get sequence Day 2- get oligos and start synthesizing gene Day 3- ship synthetic gene to NV&D and order PCR primers for cloning Day 4- clone gene into SAM vector Day 5- DNA scale up Day 6- purify DNA construct and linearize it for transcription Day 7- in vitro transcription/capping of SAM vector and transfect it into BHK cells Day 8- confirm RNA integrity, expression of proteins etc use Gibson assembly to make the HA/NA gene correct errors clone into a vector that has promoters, terminators etc needed for expression in mammalian cells do large scale transcription with T7 RNA polymerase mix sRNA with lipids and cholesterol have a little RNA virus, this is what you use as the vaccine

Answer 60

derived from an alpha virus | it contains the genes needed for viral replication but the structural genes have been replaced by NA and HA

Answer 61

does not require cell culture and is amenable to automation robust, generic process, can manufacture against any influenza strain reproducibly small manufacturing footprint with standard, disposable equipment you can stockpile the raw materials and equipment in a single facility, which allows you to rapidly makes the vaccine after getting the gene sequence

Edgell Final Flashcards

(86 cards)