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[Music]

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[Applause]

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you know talk about expansion sequences

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in 5s RNA well this this is a very

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interesting story to me anyway we've

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published this paper back in 1981 and

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that means that it was published before

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the first 16s RNA complete sequence was

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available which means this was the first

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insertion sequence discovered and

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ribosomal RNA however that turns out not

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to be true because was in its infinite

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wisdom had done a catalogue of our

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ribosomal RNA fragments from 5s RNA from

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a clostridium and that actually had an

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insert as well so it's kind of

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interesting now the way this insert

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works is what's happening is you have

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extra material right here right and you

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come around and what happens if you

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cleave this out if you break this off

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and throw it away you can just reconnect

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this U and G and you have a perfectly

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normal secondary structure of the 5s RNA

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okay so so that's that's the key thing

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that you these insertion sequences of

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many of them you you can swittel remove

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them and you're right back to where you

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were and so they're not causing problems

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in particular okay so this is a it's

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more or less the secondary structure of

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5s RNA there's basically four and a half

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helical regions there's the fifth helix

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here which involves a non-standard base

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pairs okay so let's see how we do here

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erased the message managed to push the

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wrong button okay so this is what you've

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seen already today multiple examples of

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our insertion sequences that have been

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associated with with the large ribosomal

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RNAs and what we see here on the top is

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in fact the the thing growing as you go

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through a variety of organisms starting

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with E coli and ending up with human

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this grad graduate gets bigger as you

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keep inserting within the insert okay

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and so you know so the ribosomal RNA has

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grown over time

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well so what we wanted to do is to find

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out how common this insertion was in 5s

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RNA and what were some of the properties

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of this insertion so we did something

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which in retrospect was sort of stupid

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what we decided to do was to look for

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more examples okay and so this is an

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interesting thing you should be warned

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about if you if you probably know this

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but you really ought to be aware of it

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these databases are not very helpful if

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you want accurate information okay so

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there's a database here I'm sorry to

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name it specifically but this particular

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database is a database of 5s RNA s5s RNA

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sequences nothing else it doesn't

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contain the hallo caucus 5s RNA with the

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insert which is interesting since the

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hewa caucus 5s RNA was actually probably

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among the first ten that were actually

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even sequenced but it's not it's not

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even in the database in fact none of no

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large 5s RNA is in that particular

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database so they just simply have a

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prejudice against large they don't like

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inserts but we then we went to other

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more traditional places like like refseq

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and we looked at one hundred and twenty

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seven thousand genomes we found 157

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candidates for possible 5s rnas wood

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inserts and then there were then when

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you go through this it turns out that

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most of a lot of them are junk and 24

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would incorrect gene boundary so when

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people annotate these things they have a

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hard time finding where it starts and

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where it ends and you know that kind of

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thing and and there there's some

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problems like people have like with

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insertions in like it like there were a

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lot of 5s RNA is just sort of had a

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duplication in the middle of the 5s RNA

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and of course there were a lot of them

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in which various portions were just an

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so so the sequence was not complete so I

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can continue forever

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but it was so we ended up creating

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specialized search tools looking

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specifically for the sorts of 5s RNAs

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that we thought were interesting so in

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the end we found a bunch of them but I

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guarantee you we have not found them all

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ok but I would just like to say most of

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the one

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we found they're actually real and so we

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found 79 examples with 13 to 108

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residues as the insert and so we had put

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a boundary I think of 10 the in a

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minimum insert we would consider to be

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interesting was 10 so if so the smallest

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was 13 the largest was in fact the halo

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caucus one that we started with in the

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hundred and eight residues got 79 in the

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bacteria and then among the Archy we we

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have 10 examples here and 25 bacterial

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species so a lot of different organisms

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have this insertion of 5s RNA this is

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kind of a summary there is one rkl group

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which is the halo bacteria Alys which

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includes inhale caucus and hey will have

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OBM and a bunch of things and then a

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bunch of mainly anaerobic organisms in

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the Clostridium E's and the hail will

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have rallies how we say that the thermal

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era of bacteria always and alter

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modalities which is actually a

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gram-negative group and we were very

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upset we hadn't found any gram negatives

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until at the very very end of the search

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we and we found this guy here and so

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there's there's a fair number of these 5

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srna's containing insertions so this is

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a slide this has been previewed by the

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CIA and they don't want you to get

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access to this information so so if the

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print is very very small so that you

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know him if you're not in the first or

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second roll you can't read it however

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this is a summary this is a summary of

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how big the insert is so here's an

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organism name over here which you can't

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read and you don't care about anyway and

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and it has an insert of 61 residues okay

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and these down here this guy has 32

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residues this one here has 3 inserts

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that in the same organism has 3 inserts

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one of life 29 one of 39 in one of 85

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just sort of interesting and so we have

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there's also this is a grouping of the

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organisms that have the large inserts

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and it's basically what we have is that

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we have the

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certs I'm not going to show it to you

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but we have them categorized into kind

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of a structural grouping as to where

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they are and this kind of thing and so

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this is telling you the structure will

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do grouping and we have 17 groups

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distinct structural groupings and then

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this is showing you where which grouping

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it is in the well this is showing you

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the position I think okay the other

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thing anyway so there's turns out to be

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then 16 locations in which you find 5s

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RNA insertions and interesting thing

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here is if you go start here at the 5

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prime end and you walk around the 5s RNA

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when you come to these he oversees the

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insertion is always on the 3 prime stand

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always on the second half of the helix

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you know coming around 5 prime to 3

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prime is e to the to the second half of

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the helix that's where you get your

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insertions and the only exception to

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that is is right here I don't know if

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that means anything but it's kind of

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kind of strange and interesting now some

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of these insertions occur with greater

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frequency so there seems to be hotspots

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for insertion in different things so

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this is a subset of what was on the

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previous slide now we we have hot spots

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for insertion yes ok and some of these

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hot spots I mean we've been looking at

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the structure of the 5s RNA in this area

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right here is one of the most conserved

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parts of the 5s RNA so I'm kind of

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shocked to see you know insertions

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occurring here we had expected them over

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here from some prior mutation or work

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that we've done well I should tell you

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about 5s RNA and in particular 5s RNA is

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typically part of an operand 5s 16s

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2016's 23 s 5s is a common operon in

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Blunk bacteria and archaea and when

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they're frequently in many organisms

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there's multiple copies of the operon so

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e---coli has something like seven up

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seven or six or seven copies of the

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operon each of which has a 5s RNA and so

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in some of the cases

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these operons some of the organisms we

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looked at have in fact insertions and

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more than more than one copy of the

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insertion and one of the more

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interesting ones there's an organism

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which has six copies of the 5s RNA gene

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3 with the insertion and 3 without the

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insertion ok now if you only have one

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copy of the 5s RNA we assume it's

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functional if you have 6 copies of the

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identical 5s RNA it's probably also

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still functional but you know if there's

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only one copy we're pretty confident

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it's going to be functional because 5s

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RNA is kind of important in most

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organisms Anton will disagree with that

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note that ok so sometimes multiple genes

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exist but not all have the inserts so

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this is in the same organism you see

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you've got the insert here in this case

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and you it's not present here ok and so

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then that raises the question when

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there's more than one copy is this

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really functional we suspect so but we

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haven't done any experiments to indicate

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that so here's another case sometimes

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there's more than one example of the

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intron and so here's a case where we

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have in the same organism write it with

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two different versions of the 5s RNA one

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one of which has the well it has the

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insertion here right and this other one

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has the insertion in the same place as

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over here but it has an extra insertion

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out here small insertion here so that

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that's sort of there's lots of strange

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things that show up I should mention to

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you I suppose you know how is the

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insertion accommodated in the ribosome

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that's sort of a fundamental question

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and this is the predicted secondary

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structure of the hewa caucus insert and

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we have up here the 5 prime end of this

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thing ok and and the 3 goes all the way

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around back to the 3 prime end and so

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what we have are two major HeLa C's we

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have a helix here above the insert and

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we have this huge helix here below the

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insert and

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have predicted the predicted structure

250
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but it's probably pretty good because

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you got lots of base pairs and now

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there's we had a poster downstairs some

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of you may have noticed it and which we

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actually have been doing some cryo-em

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work with collaborators to understand

256
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the structure of the 5s RNA and we have

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how the inserts accommodated and it

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turns out that there's a little when you

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we have like a seven angstrom resolution

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structure and when you look at it

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there's two bulges there's a little very

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small bulge which apparently corresponds

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to this top part here and then there's a

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larger bulge which appears the core

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which is probably corresponds to this

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but what's likely happening is sort of

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speculative we're just saying this has a

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very extensive secondary structure so

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it's pretty much a rod and so what you

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can do is if you put that rod in the

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right place in the ribosome it'll just

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go out in the middle of nowhere and

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cause no problem so I suspect that's how

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they get accommodated by just a little

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bit of these asymmetrical loops along

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the way there's all these logical roofs

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that allow it to instead of being a

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totally straight thing it's probably

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bending a little bit and then it ends up

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going off in a direction which is not

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getting anybody's way and so that's sort

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of okay now before I talk about this

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there there's as they're gonna say I

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forgot so I won't talk about that

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these 5s RNA expansions are actually

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rare so I mean we found a lot right now

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so we finally 75 of them but the reality

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is that 75 out of hundred thousand

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genomes I mean it's they're pretty rare

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actually and only occurring in four

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general okay I don't know if it means

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anything but even the bacterial example

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sent me I mean the are actually the are

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actually hallo Felix so not all of them

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are hail effect but a lot of the

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organisms that have this insert or halo

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philic there appear to be for short

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locations which is not in the hot spots

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okay and frequently or sometimes

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multiple copies exists and we suspect

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most of them are actually functional

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okay and

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there's a question which comes up which

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is kind of the origins question why are

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they mostly recent now when you take

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when you have examples from the same

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organism from different species of the

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same organism so you get the genus and

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different species it's the same organism

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they have the 5s RNA and you compare the

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sequences what you find out is there are

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some sequence differences and those

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sequence differences do you know they

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take care of the structure okay they

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keep the structure valid and so the

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question we have is since then some of

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the some of them are old in the sense of

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you know a hundred thousand years but

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none of them are old in the sense of the

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four million years that we would like

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okay and some of the earlier talks and

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so I don't know why you don't have any

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old 5s RNA expansions but we don't and

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Jessica you she wants me to answer

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do we have any questions I was just

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wondering if there's been any rt-pcr

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work that demonstrates whether these

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insertions are actually edited out of

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the the ribosomal RNA are they edited

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out there they're simply immature cells

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they're not added at all I mean we

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haven't we have an ongoing experiment

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where we tried trying to insert them

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into the 23 s RNA institution so they

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interact with the 23 s RNA and we're a

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characteristic place and we want to we

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want to lock it in and see if the 5s RNA

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has to be separate from the 23 s RNA and

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that experiment has kind of ongoing for

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the last five years yeah I was just

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wondering whether there are any cases in

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which these sequences of RNA are ever

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translated I'm not sorry translated into

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proteins we're not supposed to be I

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guess but you know they're not supposed

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to be we haven't specifically

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specifically looked in the case of 5s

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RNA people have looked in the case of

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the large ribosomal RNAs and there's one

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area in the 23 s RNA that some people

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are convinced is coding

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even though it's in the 23 snrna gene

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and think might be coding something so

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they're happening but there hasn't been

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anything like that found for 5s RNA yeah

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yeah the other thing one thing I didn't

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mention is I showed you the way the

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insertion occurs you you cut it out and

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every goes away and there's no problem

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but occasionally some of them actually

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you can't just cut it out and put it

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back together you have to do some kind

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of insertion or deletion event along

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with it

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but the vast majority I mean it's a

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simple cut and it goes away and you just

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seal it back up thank you I allowed