Molecular Biology #2 2020

okay so uh before we get going i just want to mention that you know jim estes gave us a class in ecology and he has organized another series of lectures for january for an online only course and it's going to be about the ecology of disease he's lined up a series of really super research people from all around the country so this is going to be a really international event so uh watch watch for your only newsletter you you'll get the timing on it but it's basically going to be the four weeks in january so that's that's got a a little ways to go all right so let's get started so our speaker today is josh era bear and josh do i remember right you you grew up in san jose isn't that yeah san jose gilroy do you know where that is senator gilroy so if you want to look at a stellar cv in terms of training it would be hard to beat josh so josh got a bachelor's degree at berkeley then he went on to get a phd at mit and then did a postdoctoral fellowship at stanford university and just to make sure he had the best people in the best places his mentor at stanford was andy fire who won the nobel prize in 2008. so we were quite pleased to be able to attract josh to a faculty position at santa cruz and i should say that despite all of this josh is just a regular guy all right and he and i have shared a lab for a number of years uh so um uh just one other uh tidbit to add in here just to keep josh on his toes he's one of the many power member one of the many power couples up at uclc since josh's wife is an internal medicine doctor at stanford university i want to say that the the faculty at the university are all really pretty stressed out these days and under under a lot of pressure because they're trying to teach online they're trying to keep their labs going their provisions to do that but under highly restricted rules trying to keep the research going and then of course many of them i mean have kids at home so they're being primary school teachers at the same time so we really appreciate the fact that josh has taken out the time to to talk to us this morning so josh it's it's all yours thank you thank you barry can everyone hear me yes all right um i have a second screen up so if you want to interrupt me at any point you're welcome to unmute yourself or you can just do this in front of the camera and i'll stop and call on you um so as barry said um i have a lab up here at santa cruz um i started at santa cruz in 2017 with my own lab and we've been very fortunate to get some really solid phd students and techs and undergrads during that time that have contributed to some of the work that i'm going to show you today i just like starting out with this picture because i think it's pretty i'll give you a little bit more information on what this picture shows in a couple slides this is an artist's depiction of the inside of a cell it's not coming up yet josh uh-oh the screen share can you what can you see i'm seeing the gallery view okay let's fix that then how about that that's it yep good perfect and i can still see everyone all right so this is uh an artist's depiction of the inside of the cell i like showing this just because i think it's visually stunning and pretty and the more you stare at it actually a lot of these molecules are based on the actual three-dimensional structure of these molecules that we now know and so it's a fairly accurate picture as far as the crowdedness and the number of molecules that are inside of our cells oh all right so that was the slide that i thought we were all staring at for a while but apparently it was just me um so i wanted to start out with a slide which is shamelessly stolen from barry's introduction which is on the size and scale of biology so uh each one of us is made up of trillions of cells hundreds of trillions of cells and each one of those cells is made up of thousands tens of thousands hundreds of thousands of molecules each one of which has thousands of atoms and the ability of the cell to replicate is a huge feat of biosynthesis because each one of those molecules that it has those hundreds of thousands of molecules it has to make again so that it can make a perfect copy of itself and divide and so for example in every one of your cells there is a genome present in two copies and each single copy of that genome contains three billion bases or three billion letters and it copies it every time it wants to divide it copies that into two separate copies and you can imagine the incredible fee that that is to copy a billion letters and not make many mistakes um but our cells are not perfect at this if you look at them very closely you can appreciate that they do in fact make mistakes and it's a frequency of about one in a billion or so but they do make mistakes if you look however at the prevalence of errors it's actually much lower than that and so that gets into an area that is what my lab studies which is how the cell helps mitigate the consequences of errors and help mitigate the occurrence of errors to try and keep things at a low and tolerable level and the analogy that we use is quality control and manufacturing you can imagine if you have an assembly line and you're producing cars if you're just taking that car and then putting it on the road without checking whether or not it works you're going to have a disaster a certain frequency of the time so you have to be checking your products as they come out of the assembly plant and verifying that there is no appreciable amount of errors because you don't want to be having the issues that emerge from those errors and so as cars come out of a factory they undergo rigorous quality control testing to make sure that they do in fact work that the brake actually works and the gas allows you to accelerate the steering wheel is hooked up right and so on and so forth that's about the extent of my understanding of manufacturing i've never worked an honest day in my life so that's quality control as you know we can imagine it and as we can visualize it and quality control during molecular biology works very similar um but it's a little bit different so as barry told you in the introduction um dna which is what where your genome is gets copied into rna and then that is used to make proteins and so you can think of this as the assembly line that's going on in each of your cells all the time so your cells are constantly making rna they're constantly making protein and each one of your cells has to make something like tens of thousands of rna molecules each one of them is thousands of letters long and so there's a huge amount of space of things that it has to do molecules that it has to make and it needs to be very careful about the error frequency of this the twist though is that we have the benefits of eyes and brains and we can think very rationally and we can test a car but it's very dark inside of a cell and there's nothing that has an eye there's nothing that has a brain so how do you build a set of rules to ensure that the molecules that you make are accurate and functional but you can't use your eyes so everything's blinded so everything happens by chemical reactions and by molecules binding to other molecules in a very dark place so that's the sort of challenge of quality control so you can think of quality control as cars coming out of a factory that's pretty straightforward to imagine because that's in our world but in the cells world everything is done by binding and chemical reactions and kinetics any questions so far i'm not seeing any hands waving so i've seen a couple people talking but i assume they're talking to all right and this is the uh complex that my lab studies a lot this is one that very introduced to you and this is the ribosome so the ribosome is a giant machine that takes rna in and then outputs protein and these proteins are what make up the bulk of your cells they're they help you store memories for example they help give your skin elasticity they help pull cells apart during cell division and so on and so forth the proteins really carry out a lot of the molecular functions so again ribosome is what carries out this step of rna going to protein and so the cell has a very roundabout set of rules it uses to ensure the quality of what it makes and i'm going to list some of those rules here so some of the rules that cells adhere to is that a ribosome has to stop at a certain point and that point cannot be too early and cannot be too late if it is the cell gets concerned that that ribosome might have made something that was inappropriate ribosomes also have something of a lower speed limit there is no real slow lane when it comes to making proteins if you're going too slow the cell will perceive that as being problematic and will try and prevent that ribosome from completing its task as barry mentioned in the introduction a protein also needs to fold so it has to fold up into a three-dimensional shape in order to carry out its function and if a protein doesn't do that the cell perceives that something has gone awry and that it's going to junk that and then there's other rules as well um rnas need to be sort of linear kind of like a piece of spaghetti rather than fold it up into something um they also need to have seals of approval on both ends and these are commonly called a cap and a tail and if an rna doesn't have a cap and a tail it's perceived as being bad and lastly rnas need to be made inside of the nucleus which is this structure which houses the genome in the heart of the cell and the cell has a very low tolerance for anything that breaks these rules so the cell has an ability to cut up either an rna or a protein that violates one of these rules and so it does not hesitate to junk those and anything that fails to adhere to these rules to help illustrate why these are important i've highlighted over here that there's examples where cells break these rules or entities break these rules and they give rise to disease and i put those with a blue d over here and so i'm going to talk today about this one in particular this is a major focus of study in my lab what happens when a ribosome stops too early and there's plenty of examples of diseases that are caused by cells or genes violating these rules and wreaking havoc on the organism also of relevance to our current world is viruses often will attempt to break these rules and in this sense you can think of quality control as a first line of defense against invasive things like viruses so if sometimes viruses will have rna and it will exist in a form that's folded up and that can be a hallmark of a virus and so if a cell perceives that rna is folded up into something it can then attack it because it might mistake it for a virus so we study this using what's called a model organism so our model organism is cinerad ditus elegans or c elegans for short it is a worm it's found in soil it's probably out in your soil right now if you have a compost bin it is certainly hanging out in there they feed on bacteria and microorganisms for a living and while it may not look very much like a human at the cellular level it is very similar to a human so everything that goes on inside of worm cells is very similar to what goes on inside of human cells now the advantage here is that worms are considerably cheaper than it is to raise an entire human and also the ethical considerations so we can grow up millions tens of thousands of worms and do experiments on them in a very fast time frame in a time frame that wouldn't be doable for humans so unfortunately i can't see you all in person but i can do a little bit of show and tell because um i'm in the lab right now so this is a plate and you can see sort of how big it is it's about the size of one of my eyeballs and this has on it tens of thousands of worms so each one of the worms is about a millimeter long and you can't really see it with that but that's where they live i'm gonna give me a sec i'm gonna hook so i hooked my phone up to a microscope i'm going to log in with my phone and then try and do screen share so that you can see what these things actually look like in the real world and if you have questions while i'm doing that just fire off you can kind of see them now so this is what they look like that is a right in the middle there is a fully grown adult worm and then the let's see can i do this or is this too gonna be too problematic yeah how big is that adult worm that is a millimeter okay now i'm gonna terrorize it sorry sorry little guy so that right there is an adult worm and then all the little dots around it are its eggs and then the really small thing right there that it's crawling towards is a baby and so it takes about 48 hours for that really small one to become one of those really big ones and then start cranking out more eggs so by monday the small worms on this plate will be the size of big worms and we'll have tens of thousands of more of them and that is one of the huge strengths of this system so we can grow those worms incredibly quickly and do experiments in a much faster time frame than you obviously ethically could with a human or even with something like a mouse did the worms have sex for the most part no so they're an interesting system they're uh mostly hermaphrodites and that is a really tricky thing to think about initially because we're used to thinking about male and female species and animals having to have sex in order to reproduce because they're hermaphrodites they fertilize internally and they can make both sperm and eggs at the same time one of the real strengths of the system historically has been to study muscle function and that's for the somewhat obvious once you say it but difficult to sort of derive from first principles fact that if you can't move you can't have sex if you have a male and a female species but if you have a hermaphrodite because it fertilizes internally it even if it's incapable of moving it can self reproduce and then generate more of itself so actually a lot of what we know about basic muscle function as far as what molecules are involved was actually worked out with this animal um not with things like flies or mice um it was it was generated with this animal for that reason so does that make them essentially clones of each other uh since they're not mixing chromosomes with anybody yeah it's really interesting from a sort of species individual level standpoint they are the these animals on this plate are essentially what's called isagenix meaning they all have the exact same genome so their genomes a hundred million base pairs and barring small errors that they make um it's essentially identical down to every single base for that reason this has been a really useful system because you can actually clone at them out and isolate several individuals that are exactly identical down to you know just about every single atom that's not the case obviously with humans or even with things like flies um so it's been a really powerful system attractively to discover a lot of basic biology because you can eliminate a lot of the variables of like what causes me to have you know lighter hair and my wife to have black hair and you can't really see it but there is some bacteria on here that they're munching on you can kind of see the the ripples around like the the waves it kind of looks like a sand dune that is uh indentations in the bacteria so they're munching as they go along and they're making ripples in it and then you can kind of see here the left side is off the bacteria and the right side is the bacteria so you can see the left side looks more like a barren desert and the right side you see more indentations more waves do these show up at all in the fossil record oh that's a good question um i don't think so because i don't think there's any structures to fossilize but i i actually don't know i've never i've never heard of fossilized nematodes there might be evidence indirect evidence for them but i don't know of any fossil record for them so when when you speak of nematodes is that a general term or or c elegans one type of nematode yep so silicon is one type of nematode and there's there's all sorts of different species of nematodes they're all related to each other they're sort of an interesting branch of the invertebrate lineage uh c elegans just happens to be the one that someone picked out of the compost pile when they were doing there's other related ones called cigarettes briggsia i see japonica so on and so forth um c elegans has a lot of traits that are really nice um among them hermaphroditism uh not all nematodes even have that okay my second question is you you referred to musculature and these these um nematodes are moving and you can see that they move you know they're not segmented but but what what did you mean when you referred to when you referred to musk muscle function studying so they have um uh they don't you know obviously have a bicep like you or i do um but they have um along either sides of them they sort of have uh what's called body wall muscles and i'm showing it here as where's a i can i can build a simple muscle if i had a pen where's a pen if you can see my video hopefully it won't destroy this so this is a proxy of what a word is basically a couple muscles stretched out on either side of a pen are you doing a drawing uh because when you can't see that uh josh yeah let's do um i'm gonna we have to unpin it yeah i'm gonna stop the worms if everyone's okay with that okay all right and then you should be able to see in one second you see the picture of the worm again yeah my video is up on top right you're back to your slides yeah so this that i'm holding up in front of the camera is a pen with a rubber band strapped around it yeah and it's actually can you see that no no we see your slide okay let's do this um oh but barry everyone will have to unpin the the worm picture and how to do it um so you go back to gallery view and and again click on those double dots and you should get the unpin option okay so you heard that go to gallery view and then go to those three dots and then the windows that show up uh let's see on pin is the your fourth choice it says on pin video actually i might be able to do this by okay and that worked okay let me force it thank you brian again you can't do your own tech support yeah um can you see me am i appearing on people's the question was about muscle and you know what what muscles does a worm have this is basically a crude model of what the worm is um so it's a pen and i've strapped a rubber band on both sides let me just interrupt josh so so if you know you had to go to gallery view to on pin and then go to speaker view click on speaker view and now you can see josh full screen go ahead um and the worm has two sets of muscles one that's on one side of its body another words on the other side of the body and when you're looking at it from the top down it kind of looks like this and the way the worm moves is by basically flexing this half and then flexing this half and going back and forth and by flexing those two muscles it can sort of wiggle and by flexing up here it can move its head by flexing back here it can move its tail [Music] so that's how um but you don't actually don't need those those muscles on either side that doesn't have anything to do with its reproductive system and so it can still be reproducing internally and these muscles can be completely defunct and it'll still be fine it'll just lie there on the plate flaccid so that's one example of of how they've been used to study muscle function they also actually use their mouths to pump too so you can study muscles in their mouth as well and now i hope my slides are back up it's good perfect um so that's you know a brief little uh description of of why we uh know and love this organism um a lot of basic biology has been worked out in this organism as uh barry alluded to my postdoc advisor won a fancy prize for biology that he discovered in this organism which actually is also biology that's true in our cells as well it's also very economical which is nice when you're working at a public university so you know that plate that i showed you of the animals um you know i don't know costs a dollar or something to make and so you can grow up you know hundreds of those plates and you can hand them out to your students and your students can play around with them and learn how to manipulate the system without breaking the bank which is nice um and another really nice feature of this going back to the sort of what i study is that you can actually break their quality control processes and they still live so if you break a lot of quality control in humans uh we die it's embryonic lethal and so you it's very difficult to study with humans or with any sort of mammal for that reason but a worm can get by without its quality control processes and that allows us to do experiments that we couldn't do in other animals so here's an example of a experiment that we do we can grow up a large number of animals worms and then we can engineer them in a way where they will light up if their quality control processes are compromised so they'll start literally start just producing light in this case glowing green so we can grow up hundreds of thousands millions of these animals and then look for a rare animal that suddenly lights up and we know that that animal is broken in some way that's interesting to us that's um an example of a suppressor mutant and we've got a freezer full of suppressor mutants that are all broken in various ways pertaining to quality control and we use that to help identify how that quality control works here's an example of what these actually look like so these are some strains that were made in my lab that were engineered to fluoresce green when their quality control processes are compromised and so starting up here you can sort of ignore the names um the name's a sort of boring genetic nomenclature for c elegans but the starting strain doesn't really glow at all but uh you can make mutations that cause them to glow um and that's shown here as well there's a bit of a gruesome aspect of these animals which is as i mentioned they are hermaphrodites and they are capable of fertilizing internally sometimes if the mother does not succeed in laying the progeny fast enough it will actually hatch inside of her and it's pretty gruesome just like a horror movie alien it can actually hatch inside and eventually they'll crawl their way out sorry to spoil some of your breakfast it is it is a sort of uh it's it's kind of humorous for the for us warm people because i don't know it's the first time you see it it's horrible and then the second time you see it it's like oh and then it's sort of just a running joke amongst worm people does that kill the mother eventually yes yeah um but it's a trade-off that's while it doesn't seem like it's in her interest it's in her genetic interest because she sacrifices herself and then these 10 animals get to survive and there are 10 animals that are all directly from her genetic material and so from a genetic perspective it's in an it's a great idea it's not a great idea as far as her being her being passes on rather quickly um but she gets 10 clones of herself and from a genetic perspective that's a great idea other questions what is her lifetime uh it depends um generally these animals live for only about two weeks uh there's some variability you know depending upon their environment but most of them don't make it past three weeks some of them will poop out after a week and a half they're reproductively competent in about 48 hours which again goes back to the fast generation time so those worms that i showed you the small ones will be cranking out eggs by monday but by december they'll all be toast can you tell who is who do you tag them in any way you can um there's a couple ways of doing that um one is genetically so if you've um because animals on a plate are essentially exactly the same as one another you can introduce an animal that's marked in somehow that's different from all of those and then you can pull it back out if you need to you have to mark it usually genetically you can uh it's it's really difficult to actually like make a mark on the animal um like use a laser to ablate some cell or something you can do that but that's that's much more difficult and is um beyond me to be honest thank you how many rounds of egg laying does an individual make about three to four hundred so one hermaphrodite gives rise to three to four hundred eggs and each of those can give rise to three to four hundred so that's really nice because if you have one worm that's really precious for some reason you can put it on a plate and it will clone itself 300 times within 48 hours and then you've got all sorts of versions of it that you can then study and so it's really nice for mutations and screens like what we do because if you find one worm that's incredibly precious it will clone itself basically overnight what is the mechanism for dying i mean what happens what what doesn't work that is a great question um it's an area that i know a little about but i think we are collectively still learning about it there is a signal that goes throughout the entire organism when it does die or like the split second right before it dies um that is some small molecule of some kind we don't yet know what where that signal comes from or what exactly the purpose of that signal is but it's actually it causes them to glow blue for a split second so it's kind of i don't i don't know you know what that means spiritually but there's this split second right before they die where the whole thing will just glow blue and it's it's cause it's called death fluorescence um it is associated with the process of them dying and it is an organism-wide signal uh i i last i read about that was 2015 and to be honest i haven't kept up on that literature but there is a there is a organism dying process joshua you use the term several times if we find one that's incredibly precious what do you mean by incredibly precious yeah so you know um this plate that i showed you has you know 10 000 animals on it we have plates that are the size of my head that might have hundreds of thousands or millions of animals on them sometimes we find one worm on there that we care that we that we need for some reason and it could be for example a worm that now glows green that's in this background that worm presumably glows green because something has been altered about its quality control machinery we would like to study that and so you want to get that worm isolated and we and the sort of term that we use is that worms incredibly precious because if you lose it you don't know what was wrong with it we would like to study what was wrong with it what went wrong with it and so you might have you know quite literally a needle in a haystack but it's it's a needle that will you can put on a plate and will clone itself a hundred times um they're good at reproducing but once they run out of e coli uh the gig is up so they have to eat for a living and they need to eat e coli for a living and so when e coli runs out they go into a starvation mode and they stop their development there's actually this whole study of um there's an alternate form of development that they can go through called dower and the dower form is sort of like the tank version of uh a c elegans it has a much harder outer shell and it can survive even rough chemical treatments and it can live even for months so it's you know i someone asked me how long do they live for they typically live for a couple weeks but dour which is a specialized form can live for a couple months and it's basically designed to like hunker down and wait for e coli to come back since we have uh e coli in our gut i think do these things worms live inside humans also um they don't but there are versions of nematodes that do parasitize humans um so they're the basis of a lot of um diseases that predominantly affect tropical areas um for example i think elephantitis is based on um the growth of a nematode brugi mla um that parasitizes humans um for the most part they don't these these ones are benign which is another reason why i can safely hand them out to undergrads luis you had a question yes what do you do with these worms what do i do with these worms um so we use them to study quality control and we use them to try and identify the machinery that actually does that quality control so i sort of use the analogy of you know a car coming off an assembly line and like someone doing quality control can look at the car and be like yeah no that that break doesn't work like and that's pretty obvious but what the cell has to do is it has to basically feel up the molecules as it makes them and say yeah i know that that feels about right or that doesn't feel about right and we don't actually know what that hand is that's essentially grabbing onto the molecules as they come off the assembly line to check them and so an example of a work that just did that was from a phd student in my lab marissa and she actually used these animals right here to discover one of those genes that was required for doing that and so she got to name that gene as well and she called it nonu1 because she thought that was a cute name there's a molecular biology story behind why she thought nonu was a good name but she got to name it because she discovered that gene and this turns out no new is a gene which is conserved between worms and humans and yeast and so it's a very ancient uh protein that no one had discovered before but she found because she found one very precious worm that happened to be a mutant for that factor and then she was able to show that that factor was sort of one of the things that was grabbing onto the ribosome as it was trying to translate things yeah josh it might it might be worth mentioning that even though we know how many genes these all these creatures yeah we don't know the function of most of these genes we're still watching the stage of trying to figure out what all those genes do yeah yeah and so um you know the worm is a great system for that uh you know so we have um humans we have 25 000 or so genes and we know what a fair number of them do but we don't know what all of them do and this was an example this gene that my student marissa found uh was a gene that was known to exist but no one could actually figure out what it did before she sort of discovered its function and it's known to exist you know in all eukaryotes so going all the way back to yeast um worms humans all of us uh and she finally was able to ascribe a function to it but there's still you know i don't actually know what percent of genes we we have no idea on fair do you know that is it like 10 20 well one of the most interesting numbers i've heard is that e coli bacterium which is simple and probably one of the best understood organisms in the world has 6 000 genes and as of a few years ago we only knew the function of 3 000 of those genes so it's generally half at least half unknown so that um gene uh that produces that produces a protein that does that check does the check and so you figure there's probably like a dozen or more of those or how many of those are have you discovered yeah um so we've got about a dozen or so um acting um so it's it it depends on which of these sort of rules that you're talking about so each one of these rules has a different set of effectors that carry it out and so marissa's gene that she found was actually involved with rule number four here of ribosomes moving too slowly um and we have about three or four genes that we know act in this pathway um we've got seven to nine that act up in this pathway um up here uh we don't know any of them that act in this pathway yet that's a that's a big unknown we know about four to five of the ones that act in this pathway that act in this in enforcing this rule and there is some overlap between them uh but there is also some distinctness so some of the machinery that carries out this enforces this rule uh is actually different from the machinery that carries out this rule all right um so what i want to talk about in the last bit here is actually um rule number one which relates to what happens if a ribosome finishes its job too soon and this is a substantial focus of my lab and also a big tie-in with disease so again shamelessly stealing from professor bowman's slides um so you have dna up here which is a t c and g and that gets copied into rna and the t gets swapped out for you and then that gets translated into a protein so what happens if you have a mutation in here that changes the nature of this information so if you change this one base here you can kind of see this g goes to a u that actually changes the meaning of this protein so uag can be interpreted as a stop signal just like how taa can be interpreted as a stop and so when that happens when the ribosome translates this it terminates right here and so you only have half of the protein getting made so you have one very simple error you just changed one letter to another letter and it basically cut off the entire latter half of this protein you no longer get to make that protein it's not really an academic consideration so when molecular biology came around and we started applying those techniques to human patients we discovered that there were people walking around that had mutations like that so this is an abstract from a paper and based upon the molecular biology that barry's taught you so far i think you can understand this abstract so this was published in 1979 and this was one of the first instances of someone walking into a clinic and very obviously being the result of a genetic disease so the abstract reads we determined the complete sequence of the five prime region of the first amino acids of the beta globin mrna so there's a gene that encodes for the beta globin mrna in a patient with beta thalassemia so this is someone who walked into the clinic and had beta thalassemia and they decided to get the dna sequence of their gene explain beta thalassaemia oh ah does anyone here know or have beta thalassemia would be comfortable sharing that so it's a deficiency in one of your hemoglobin subunits that causes you to not be able to carry oxygen as efficiently and so this is someone who presented uh as not being able to carry oxygen and they decided to look at their gene and so they identified the defect as a single nucleotide substitution in the coding region of the beta-globin gene and so it turned out that this person had a mutation like this which caused one of those stop signals to happen too soon and as time has gone on we've sequenced more and more people thousands of uh thousands of genomes at this point everyone you know on this zoom call myself included has some of these types of mutations lurking in our genome and over the years we've identified around 1 in 10 or so genetic diseases are caused by mutations like this and so this is a major pathway of of focus for the lab uh because we're interested in understanding what the consequences are for the cell when this happens and why are they called nonsense mutations they're called nonsense because of um it's because this used to be called nonsense a a stop signal used to be called a nonsense signal because each of these makes sense to the cell because the cell can make sense of it and read it out as an amino acid and make a protein from it but something like taa does not make sense to the cell because it can't read it out as an amino acid so it's nonsense so it's kind of a historical thing that that's that's they were originally identified as a kind of mutation that happened in bacteria actually until it was realized that nonsense happens all the time um all sorts of nonsense related puns in the lab related to that so there's two things that happen the first is relatively obvious which is that because you have nonsense or you have a stop signal you no longer get to make the second half of this protein so you've only got half of a machine that's a potentially really bad thing so imagine you made a car but you left off the brake or you left off the steering wheel or you made a door and you never bothered to give it a handle those sorts of machines are kind of the stuff of nightmares and so the cell doesn't want you to be making half of something it says you have to finish the entire job or you get nothing because in the analogy it's better to have no car than it is to put a car out on the road give it a gas and not give it a break and so one of the things that the cell does is if it determines that a protein is only half of a protein it will actually start attacking the mrna and get rid of that because again in the analogy it's better to not have a car than to have 100 cars on the road and give one of them no brake so that's that's a very uh fundamental principle and that's true of all of our cells so and this is this is true of worms this is true of humans as well and so what what my substantial focus of my lab is studying how the cell identifies nonsense and how it decides to attack that mrna so if you replace the protein that's only half made in the cases of the people that had that deficiency you were talking about does that correct their deficiency sometimes yes and so that's the million dollar question so you know going back to this person right here who walks into a clinic and can't carry oxygen in their cells through their veins that's for two reasons one they only have half of a beta globin but also their beta globin protein is actually dampened down so you might be able to help them if you could give them back their half of beta globin that might be better than having no beta globin and there are some pretty clear examples where that's the case and so one of the hopes of this line of research is that if we could understand how the mrna gets attacked we may be able to shut that down in some genetic diseases and potentially give those patients some reprieve from the genetic disease that they're suffering from josh i have a question so this this could be caused by a mistake in the dna and in that paper that person did in fact have a dna mutation isn't that correct yeah yeah i assume that where this happens at a much higher rate is when the rna is synthesized the mistake is made and so there's a small number of these being made kind of at a low level in the background is that correct yeah that is um it's a about one in a million at the level of rna which sounds low but you know when you think that you know there's tens of thousands of rna molecules in a cell at a given time and each one of those is a thousand you've got tens of millions of bases so you know you've got about 10 mrnas per cell that have a mistake like this yeah so it's it's pretty and you know that's every single one of our cells so so that is uh the bulk of what i wanted to talk about today i'm going to jump down to these are the people who actually do this work um while i sit in my office and talk to you all and teach students and so on and so forth um this is the lab uh this is our lab meeting um which of course has to happen on zoom these days um this is marissa the one who looks like a ninja um this was her in lab the other day um she has to wear a face mask obviously um this was the one who discovered that uh new gene the one that she got to name dhoni and then these are some other phd students an undergrad and a tech these are organizations that provide money for us to do this amongst them are the nih uh paid for by your tax dollars so thank you all um your tax dollars contribute to research uh like what you're seeing here okay so another question would be in your paper uh the gene that was identified is an endonuclease so you might explain what this gene does yeah so the gene that marissa found um this one no new that you got the name is a class of gene that's known to attack and cut rna so it's called an endonuclease and what we think it actually does is binds inside of the ribosome and then cuts the rna and that elicits its own host of red flags for the cell because the cell says you're trying to translate something which has no stop signal that's a really bad idea um and that's in response to the ribosome moving too slow so you can kind of imagine that if you're not doing this fast enough if you're not shunting these things through and trying to make peptide bonds too quickly enough marissa's gene no new one hops in here and says now that's too slow you're done finish off with it okay other questions for josh luis i can see yeah um would you mind if i took a picture of that cell i think that's a fascinating picture [Music] which one the one that you started with oh yeah yeah that one yeah i've never seen a picture like that i really love it yeah so if you google this person and then probably molecular biology you'll find out you'll find all sorts of uh examples of this um so this is uh so now now that i've talked a little bit about the molecular biology this kind of light blue spaghetti that's coming out of here is an rna and this is leaving the nucleus so this is the dna in here and the rna is getting moved out and then this is his depiction of a ribosome which is trying to translate the rna and make a protein from it gosh josh i have a i have a question when you say uh the the process is going too slow how do you measure there's a concept of time and and progress per time so how does that how's that measurement yeah that that is a great question and it took people 11 years to answer that so how do you measure speed um you know is there you know there's there's no radar in the in the cell right it's not like you know everything's going down the highway and you're just trying to measure so it turns out and this was work that was done by honey zaire who's uh a assistant professor at washu in st louis he came up with a really cool idea which is speed is measured by traffic jams if you have a traffic jam you know that things are going too slow and if you don't have a traffic jam who cares and that was the idea that he came up with and it turns out that's right and so basically what happens is if so you have an rna like this rna or this rna and it can actually be translated by two ribosomes at the same time so one ribosome can hop on start making protein and another ribosome can hop on start making protein and what hani found was that if that first ribosome goes too slow you get a collision and it's like bumper cars and it's actually that collision of having a traffic jam that the cell uses as the red flag and that's where marisa mrs protein binds it turns out is it actually binds at the interface of two collided ribosomes and so that's how you can sense speed it's indirect a question i have about that is so i guess one one kind of mutation you could get too is to get these uh relatively rare codons that would as i understand that's one way to slow down the speed at which things move along because some three-letter codes are abundant and some are rare and the rare ones have few trnas to recognize them is that a part of the story yeah that's how we that's how we elicit traffic jams in the cell so we give them a run of uh codons that are very rare but they shouldn't be able to translate quickly um analogy it's sort of like i don't know giving them you know 10 speed bumps in a row on the freeway um if they're going into the if the molecules going into that you know 100 miles an hour by the time it hits the third speed bump it's going to be complete chaos and so that's that's how we elicit that um problem we we yeah yeah uh in looking up and you know introduction for your for your intro i saw that there are some specific diseases that that you're focusing on that are related what what are those specific diseases yes um there are diseases that are uh like this one where actually like this one um i'm interested in situations where you have an early stop signal that causes loss of the rna even though the protein is perfectly fine and that's a minority of all what's called nonsense diseases so it's a minority of this 11 percent but it is a area where if we could find some way of shutting down the attack on the mrna we might be able to offer some reprieve to those patients so i have a question about something you just said a moment ago you said that um you find the traffic jams because is it you have two uh you have two proteins being created at the same time with through different ribosomes but they're following the same our piece of rna through is that normal to have that more than one at a time being produced yeah our we're very good at multitasking on the cellular level um it's you can have uh you know 10 20 ribosomes all stacked up competing on the same bit of rna trying to churn out protein that is one advantage of this system being this way so you have one copy of your genome you can make you know 10 hundreds of copies of your rna and each one of those rnas can be bound by several ribosomes tens of ribosomes and you can potentially get thousands or tens of thousands of copies of proteins so it allows for an amplification um to get a lot of protein and you know things like myosin what we have in our muscles that allows us to flex that undergoes a huge amplification like this you still only have one myosin gene but because you have so many rna molecules and so many proteins made from each of those you get a huge amplification of the amount of that gene and allows you to get you know protein to where you can actually build an entire muscle makes up your entire arm off of that okay so it just makes as much as as it can always or is there some place where it stops it because it doesn't really want all that much whatever it's making yeah and that's an area of a study called uh gene regulation and that was actually what i did a lot of my phd work in um so how does a cell know how much of a given protein to make you need to make a lot of myosin because you need to build an entire muscle and that's going to be most of the cell but something like marissa's protein nonu you don't actually need that much of nonu because you don't need one molecule of nonu for every myosin you only need you know one per ribosome or so so there's a whole field that's focused on understanding how the cell does that and it turns out there's a lot of waste and a lot of the excess that gets made actually gets funneled through quality control pathways thank you yeah so josh you also uh i understand use the crispr technique quite a bit in your lab and this is of course something we're all hearing about now so you might just say a little bit about crispr and how you use it study worms um crispr was also this is a little bit of a little bit of history so in science we refer to our um some people refer to their lineage of like who they got trained by um so like you know barry mentioned that i was trained by andy fire and my postdoc my so the science nomenclature for that is sort of you know andy would be my my father scientifically because he trained me so jennifer doudna is the scientist who won the nobel prize this year for discovering crispr um she is in scientific terms my grandmother because she trained my phd advisor so it's a little bit of science uh culture there um so let's show actually we'll stick with this one so one of the things that so crispr allows you to edit the dna in a cell and that's very powerful for the kinds of things that we do because we can create mutations like uh this and we can create them wherever we want so if we wanted to study this process of nonsense we could use the crispr system to make a cut here and then offer the cell a different version that hard codes a stop signal right here that allows us to engineer the worms to have a mutation at an exact site that we want it to that's actually what went into engineering these worms right here we engineered them to have a specific lesion and then we also engineered them with a protein that lights up green and so normally they attack that because they perceive it as it being a lesion or as it being problematic because it violates the rules of quality control but by breaking something like no new they know they lose the ability to attack that and then they start making the green protein so we use crispr daily it's actually what i was doing this morning before this i was sitting at the uh microscope trying to put crispr into some worms to make a mutation add a specific protein that we study that i didn't talk about today and when you put it in you actually inject it is that right yeah we inject it so um you can kind of see their structure here so actually this little blob right here this little series of blobs these circles are there are the oocytes so the eggs developing inside of the mother and they pass through the spermatheca right here and then they start go undergoing development and then they get laid through this opening right here the vulva this whole arm of the animal is essentially an egg producing machine and we can using a very fine needle inject dna into there to do something uh molecular biology wise and so what i spent this morning doing was actually um i had a needle hooked up to a microscope and i inject 25 worms that look like this worm each with a mix of components to do the crispr and some of those will get taken up into the dna of the cells and cause mutations and today is saturday so [Music] 48 hours monday tuesday i'll be able to start looking at those animals okay any any further questions for for josh here i'm gonna stop your screen share put everybody on uh so i can see gallery view there's all the any any further questions further questions okay i'm not seeing any well and from mark was that or is that just saying hi oh okay oh they were applauding oh okay all right silent applause here well thank you very much for for spending your saturday morning with us josh and uh i can kind of see out the window here the waves do not look very good this morning is there a good swell two o'clock they're supposed to be decent surf there's a low tide around two or three okay okay well that's good to know all right well thank you and so next week our speaker is going to be martha zuniga martha is an immunologist you know this coronavirus pandemic is in many ways all about the immune system and uh she's going to talk about how our immune system recognizes viruses and also about how the fact that our immune system can be turned on to too high a level such that it actually starts to attack ourselves and that's a serious problem in some people who get infected with the coronavirus so i hope to see you all again next saturday and we will have lecture three so thank you very much josh uh all right and and we will see you all in a week thank you josh sure thank you all you know it was fun being in your lab yeah you can kind of do a field trip yes thank you thank you barry okay bye have you josh recording uh

Channel: OLLI UCSC

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Id: OkvLrSQSKcY

Length: 68min 15sec (4095 seconds)

Published: Wed Nov 25 2020